HYALURONAN Volume 1 - Chemical, Biochemical and Biological Aspects
Editors: JOHN F. KENNEDY BSe, PhD, OSe, EurChem CChem FRSc, CBiol FIBiol, FCIWEM, FCMI, FIFST Director of Birmingham Carbohydrate and Protein Technology Group, School of Chemical Sciences,The University ofBinningham, BirminghamB15 2IT, England, UK, Director of Chembiotech Ltd, University of Birmingham Research Park, Birmingham B15 2SQ, England, UK, Director of Inovamed Ltd, Chembiotech Laboratories, University of Birmingham Research Park, Vincent Drive, Birmingham B15 2SQ, England, UK, and Professor of Applied Chemistry, The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd, LUI 2AW, Wales, UK
GLYN O. PHILLIPS BSe, PhD, OSe, HODOSe, HODLIB, CChem FRSC Chairman of Research Transfer Ltd, Newtech Innovation Centre, Professorial Fellow, The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd, LUI 2AW, Wales, UK, and Professor of Chemistry, The University of Salford, England, UK
PETER A. WILLIAMS BSe, PhD, CChem FRSC Director of the Centre for Water Soluble Polymers, The North East Wales Institute of Higher Education, Plas Coch, Mold LUI 2AW, Wales, UK, Director of the Centre for Advanced and Renewable Materials at Institute and University of Wales, Bangor, The North East Wales Institute of Higher Education, P1as Coch, Mold LUI 2AW, Wales, UK Professor of Polymer and Colloid Chemistry, The North East Wales Institute of Higher Education, Plas Coch, Mold . LL11 2A W, Wales, UK
Road, Wrexham, Clwyd,
the North East Wales Road, Wrexham, Clwyd,
Road, Wrexham, Clwyd,
Guest Editor: VINCE C. HASCALL
PhD
Co-Direetor of the Orthopaedic Surgery Musculoskeletal Research Center, Department of Biomedical Engineering ND-20, Lerner Research Institute. Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA Adjunct Professor Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA Adjunct Professor Department of Biochemistry, Rush Presbyterian S1. Lukes Medical Center, Chicago, Illinois, 60612 USA
WOODHEAD PUBLISHING LIMITED
Published by Woodhead Publishing Ltd, Abington Hall, Abington, Cambridge CB I 6AH, England www.woodhead-publishing.com First published 2002 © 2002, Woodhead Publishing Ltd The authors have asserted their moral rights Conditions of sale This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from the publisher. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN I 85573 5709 (2 volume set) Printed in Great Britain by MFK Group Ltd
CONTENTS Preface GO Phillips
xvii
Introductory remarks E A Balazs
xix
PART 1: OVERVIEW OF THE HISTORY AND DEVELOPMENT OF HYALURONAN l.
Hyaluronan before 2000 T C Laurent
2.
Karl Meyer - Discoverer of hyaluronan Biomatrix Inc
3.
17
Alexander G ("Sandy") Ogston (1911-1996) T C Laurent
4.
3
25
Albert Dorfman N R Schwarz and L Roden
29
PART 2: CHARACTERISATION AND SOLUTION PROPERTIES OF HYALURONAN S.
Predictive and experimental behaviour of hyaluronan in solution and solid state K Haxaire, E Buhler, M Milas, S Perez and M Rinaudo.
6.
37
Aqueous SEC, light scattering and viscometry of ultra-high molar mass Hyaluronan R Mendichi and A G Schieroni
7.
47
Molecular characterisation of hyaluronan and hylan using GPC MALLS and asymmetrical Flow FFF-MALLS SAl-Assaf, P A Williams and G 0 Phillips
8.
55
Force measurements on surfaces bearing covalently linked hyaluronan M Morra and C Cassinelli
67
iv
Contents
9.
The intrinsic viscosity of hyaluronan M K Cowman and S Matsuoka
10.
75
Viscosity of polymer solutions revisited S Matsuoka and M K Cowman
79
11. Conformational and rheological properties of hyaluronan K Nishinari, Y Mo, R Takahashi, K Kubota and A Okamoto 12.
89
The heat dependence of hyaluronan conformation G Armand, K Fan and E A Balazs .
99
13. Temperature effect on the dynamic rheological characteristics of hyaluronan, hylan A and Synvisc J M Hoefling, M K Cowman, S Matsuoka and E Balazs
14.
Tapping mode atomic force microscopy of hyaluronan and hylan A M K Cowman, M Li, A Oyal and S Kanai
15.
103 109
Biological properties of hyaluronan are controlled and sequestered by tertiary structures J E Scott and F Heatley
16.
117
Analysis of the concentrated solution properties of hyaluronan by confocal-FRAP show no evidence of chain-chain association T Hardingham, B CHeng and P Gribbon.
17.
Hyaluronic acid self-association in the presence and absence of salts T M McIntire and 0 A Brant.
18.
123 137
Comparison of the reactivity of different reactive oxidative species (ROS) towards hyaluronan B J Parsons, SAl-Assaf, S Navaratnam and G 0 Phillips
19.
141
Polysaccharide fragmentation induced by hydroxyl radicals and hypochlorite M 0 Rees, C L Hawkins and M J Davies
20.
151
Getting to grips with HA-protein interactions C.O Blundell, J 0 Kahmann, A Perczel, 0 J Mahoney, M R Cordell, P Teriete, I 0 Campbell and A J Day
161
Contents
v
PART 3: RHEOLOGICAL BEHAVIOUR OF HYALURONAN 21
Effect of metal ions on the rheological flow profiles of hyaluronate solutions C J Knill, J F Kennedy, Y Latif and D C Elwood
22.
. 175
Rheological behaviour of hyaluronan, healon and hylan in aqueous solutions M Milas, M Rinaudo, I Roure, SAl-Assaf, G 0 Phillips and P A Williams
23.
181
Rheological creep experiments utilizing mixtures of 1 % hylan A solution and 0.5 % hylan B gel slurry J .M Hoefling, S Matsuoka and E A Balazs
24.
Rheology of hyaluronan products
o Wik, B Agerup and H B Wik 25
. 195
201
Structural change in hydrogelation of hyaluronan induced by annealing the solution in sol state M Takahashi, T Iseki, H Hattori, T Hatakeyama and H Hatakeyama
26
. 205
The roles of extensional and shear flows of synovial fluid and replacement systems in joint protection C Backus, S P Carrington, L R Fisher, J A Odell and D A Rodigues
. 209
PART 4: BIOSYNTHESIS AND BIOLOGICAL DEGRADATION OF HYALURONAN 27
The production of hyaluronic acid from Streptococci D C Ellwood
28.
. 221
Hyaluronan synthases: mechanistic studies and biotechnological applications P L DeAngelis
29.
. 227
Hyaluronan synthase expression in human endometrium during the menstrual cycle M Tellbach, LA Salamonsen, G Brownlee, T Brown and M-P Van Damme
237
vi
Contents
30.
In vivo investigation of hyaluronan synthase function during vertebrate embryogenesis J Y Lee and A P Spicer
31.
. 245
Influence of substrate and enzyme concentrations on hyaluronan hydrolysis kinetics catalysed by hyaluronidase T Asteriou, B Deschrevel, F Gouley, and J C Vincent
32.
· 249
Human hyaluronidase polymorphism and evidence for conserved hyaluronidase potential N-glycosylation sites in mammalians and non mammalian species B Fiszer-Szafarz, A Litynska and L.Zou
· 253
PART 5: NOVEL MODIFIED FORMS OF HYALURONAN
33.
Hyaluronan linear and crosslinked derivatives as potential/actual biomaterials V Crescenzi, A Francescangeli, D Renier and D Bellini
34.
· 261
Novel biomaterials based on cross-linked hyaluronan structural investigations L Michielin, C Bevilacqua, S Paoletti, A Gamini R Toffanin and F Micali
35.
A novel crosslinking process for hyaluronan X Zhao, J Fraser and C Alexander .
36.
293
Hyaluronan DNA matrix for gene transfer W Chen, D Checkla and P Dehayza
39.
285
Derivatized hyaluronan for gels and nanochemically patterned surfaces R Barbucci, D Pasqui and G Leone.
38.
· 277
A biocompatible gel of hyaluronan A Okamoto and T Miyoshi
37.
· 269
· 305
Thermal properties of hyaluronic acid-based polyurethane derivatives associated with water H Hatakeyama, Y Asano, T Hatakeyama and J F Kennedy
40.
· 313
Phase transition of sodium hyaluronate, hylan and polyurethanes derived from hyaluronic acid in the presence of water T Hatakeyama and H Hatakeyama
· 323
Contents
VB
PART 6: CELL SURFACES AND HYALURONAN RECEPTORS 41.
CD 44: The link between hyaluronan and the cytoskeleton C.B Knudson, G A Nofal, G Chow and R S Peterson
42.
. 331
Hyaluronan binding by cell surface CD44 J Lesley, N English, V C Hascall, M Tarnrni and R Hyman
43.
. 341
Inhibition of tumor growth in vivo and anchorage-independent growth in vitro by perturbing hyaluronan-cell interactions B P Toole, R M Peterson and S Ghatak
44.
.
. 349
Novel Endothelial Hyaluronan Receptors D G Jackson, R Prevo, J Ni and S Banerji
45.
. 355
An insight into cellular signalling mediated by hyaluronan binding protein (HABPl) T B Deb, M Majumdar, A Bharadwa], B K Jha and K Datta
46.
RHAMM (CD168) co-associates with and regulates ERK kinase R Harrison, F S Wang and E A Turley
47.
. 365
. 373
Poly I:C induces mononuclear leukocyte-adhesive hyaluronan structures on colon smooth muscle cells: Icd and versican facilitate adhesion C A de la Motte, V C Hascall, J A Drazba and S A Strong .
48.
The generation of hyaluronan-dependent pericellular matrix in vitro J R E Fraser
49.
. 381
. 389
Purification and characterization of the hyaluronan receptor for endocytosis (HARE) PH Weigel, C McGulary, B Zhou and J A Weigel
50.
.
. 401
Identification of hyaluronan as crystal binding molecule at the surface of migrating and proliferating MDCK cells C.F Verkoelen, B G vd Boom, M S J Schepers and J C Romijn
•
. 411
Vlll
Contents
PART 7: THE ACTION OF HYALURONAN IN CELLS
51.
Anti-cancer activity of hyaluronan Me Filion, S Menard, B Filion, J Roy, S Reader and N C Phillips
52.
. 419
Pro-inflammatory activity of contaminating DNA in hyaluronan preparations M C Filion and N C Phillips .
53.
. 429
Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72 H Xu, T Ito, A Tawada, H Maeda, H Yarnanokuchi, K Isahara, K Yoshida, Y Uchiyama and A Asari
54.
. 435
The impact of hyaluronan on the in vitro invasive properties of human breast cancer cell lines with CD44 expression A Herrera-Gayol and S Jothy
55
. 443
Identification of a novel intracellular hyaluronan-bfndlng protein, IHABP4 L Hung, N Grarnmatikakis, M Yoneda, S D Banerjee and B P Toole
56.
. 447
The presence and processing of intercellular hyaluronan in proliferating cells S P Evanko and T N Wight
57.
451
Low molecular weight oligosaccharides of hyaluronan potently activate dendritic cells C C Termeer, P Prehm and J C Simon
58.
. 457
Signal transduction pathways in hyaluronan induced proliferation of endothelial cells M Slevin, S Kumar and J Gaffney..
59.
. 469
Control of hyaluronan (RA) generation in renal proximal tubular epithelial cells G Stuart, S Jones, M Jones and A 0 Phillips
60.
. 473
Mechanical injury of human peritoneal mesothelial cells (HPMC) is accompanied by an increase in hyaluronan synthesis S Yung , G J Thomas and M Davies
. 481
Contents
61.
IX
Apoptosis and hyaluronan-enriched extracellular matrix degradation in cumulus cell-oocyte complex: implication in fertility M D Giacomo, A Camaioni and A Salustri
62.
489
Proteoglycan enhances the formation of the SHAP-Hyaluronan complex and its effect in hyaluronan-rich matrix M Zhao, M Yoneda, L Zhuo, L Huang, H Watanabe, Y Yamada, S Nagasawa, H Nishimura and K Kimata .
63.
. 497
Enhanced thromboxane synthesis through the induction of cycleoxygenase-2 by hyaluronan in renal cells L KSun
. 501
PART 8: KERATINOCYTES AND HYALURONAN
64
Hyaluronan metabolism and distribution in stratified differentiated cultures of epidermal keratinocytes S Pasonen-Seppanen, R Tammi, M Tammi, M Hogg, V C Hascall, and D K MacCallum
65.
. 51 I
Intracellular Hyaluronan in epidermal keratinocytes R Tammi, K Rilla, J P Pieairnaki, M Hogg, 0 K MacCallum, V C Hascall and M Tammi
66.
517
Evaluation of the influence of hyaluronan and hyaluronan fragments on human keratinocytes during UV irradiation D Gerlach, C Huschka and W Wohlrab .
67.
525
Effect of hyaluronan on matrix metalloprotease expression in fibroblasts and keratocytes N Isnard, J. M Legeais, G Renard and L Robert
68.
531
Aging and regulation of hyaluronan biosynthesis comparative studies on human skin fibroblasts and corneal keratocytes L Robert, I Fodil, N Isnard, F Dupuy, A M Robert and G Renard.
69.
537
Effects of KGF and TGF-b on Hyaluronan synthesis and distribution in extra-, peri-, and intra-cellular compartments of epidermal keratinocytes S Karvinen, M Tammi and R Tammi
. 545
x
70.
Contents
Hyaluronan stimulates keratinocyte migratiou and activates the transcription factor AP·l in keratinocytes through the JNK pathway K Torronen, M Yabal, K Rilla, K Kaamiranta, R Tammi, M J Lammi and M Tammi
71.
. 551
Hyaluronan synthase 2 (HAS2) regulates migration of epidermal keratinocytes K Rilla, M Lammi, R Sironen, V C Hascall, R Midura, M Tammi and R Tammi. .
72.
. 557
EGF regulates HAS2 expression controls epidermal thickness and stimulates keratinocyte migration M Tammi, J P Pienirnaki, K Rilla, C Fullop. M J Lammi, R Sironen, R Midura, V C Hascall, M Luukkonen, K Torronen, T Lehto and R Tammi . 561
THE CELLUCON TRUST Incorporating
Cellucon Conferences International Educational Scientific Meetings on Wood and Cellulosics and Other Carbohydrate Polymers
Cellucon Conferences as an organisation was initiated in 1982, and Cellucon '84, which was the original conference, set out to establish the strength of British expertise in the international field of cellulose and its derivatives. This laid the foundation for subsequent conferences on carbohydrate etc. polymer topics in Wales (1986), Japan (1988), Wales (1989), Czechoslovakia (1990), USA (1991), Wales (1992), Sweden (1993), Wales (1994), Finland (1998), Japan (1999), and Wales 2000. These conferences have had truly international audiences drawn from the major industries involved in the production and use of cellulose pulp and fibre derivatives of cellulose, plus representatives of academic institutions and government research centres. This diverse audience has allowed the cross-fertilisation of many ideas, which has done much to give the field of cellulose in its diverse forms the higher profile that it rightly deserves. More recently other carbohydrate polymers have been the centre of focus, particularly hya1uronan, with the conference in 2000 - Hyaluronan 2000 - being the first major international conference on this majorly important carbohydrate polymer. Cellucon Conferences are organised by The Cellucon Trust, an official UK. charitable Trust with world-wide objectives in education in wood and cellulosics. The Cellucon Trust is continuing to extend the knowledge of all aspects of cellulose, lignin, hyaluronan and other national polymers world-wide. At least one book has been published from each Cellucon Conference as the proceedings thereof This volume arises from the 2000 conference held in Wrexham, Wales and the conference planned to be held in the USA in 2003 again on hyaluronan, will generate further useful books in this area. THE CELLUCON TRUST TRUSTEES AND DIRECTORS Prof G.O. Phillips (Chairman) Prof. IF. Kennedy (Deputy Chairman and Treasurer) Prof P.A. Williams (Secretary General)
Research Transfer Ltd, UK. The North East Wales Institute, UK, and The University of Birmingham, UK. The North East Wales Institute, UK.
The Cellucon Trust is a registered charity, UK. Registration No: 328582 and a company limited by guarantee, UK. Registration No: 2483804 with its registered offices at Chembiotech Laboratories, The University of Birmingham Research Park, Vincent Drive, Birmingham, B15 2SQ, UK
The 12th International Cellucon Conference
YA L URONA N
H 2000 An International Meeting Celebrating the
80'" Birthday of Endre A Balazs
ACKNOWLEDGEMENTS This book is one of the two volumes arising from the International Conference - HYALURONAN 2000 - which was held at The North East Wales Institute for Higher Education, Wrexham, Wales, UK. This meeting owes its success to the invaluable work of its Executive Committee, Scientific Committee and International Advisory Board, and its Generous Supporters and Exhibitors.
SUPPORTERS AND EXHIBITORS Acordia, USA Anika Therapeutics Inc, USA Bingham Dana LLP, USA Biomatrix Inc, USA Fermentech Medical Limited, UK Fidia SpA, Italy Fidia Advanced Biopolymers SrI, Italy Genzyme Corporation, USA I-Med Pharma Inc, USA Lifecore Biomedical, USA Orquest Inc, USA Q-Med, Sweden Seikagaku Corporation, Japan Vitrolife UK Ltd Wyeth-Ayerst Laboratories,USA
EXECUTIVE COMMITTEE G. O. Phillips (Chairman) J. F. Kennedy (Deputy Chairman & Treasurer) P .A Williams (Secretary General) S. AI-Assaf M. Davies T. Hardingham H. Hughes (Administration Secretariat) C. J. Knill (Scientific Secretariat)
Research Transfer Ltd, Wales Univ of Birmingham Res Park, UK The North East Wales Institute, Wales The North East Wales Institute, Wales College of Medicine, Wales University of Manchester, UK The North East Wales Institute, Wales Univ of Birmingham Res Park, UK
SCIENTIFIC COMMITTEE AND INTERNATIONAL ADVISORY BOARD V.c. Hascall (Chainnan) C. Abetangelo P.A Band TJ. Brown B. Caters on M.K. Cowman AJ. Day J.L. Delinger M. Ferguson K. Harding H. Hatakeyama N.E. Larsen T.C. Laurent K. Moore K. Nishinari B.J.Parsons A Okamoto M. Rinaudo S. Takiami B.P.Poole C. Weiss
Cleveland Clinic Foundation, USA University of Padova, Italy Biomatrix Inc, USA Monash University, Australia University of Wales College of Medicine, UK Polytechnic University, USA Oxford University, UK Biomatrix Inc, USA University of Manchester, UK University of Wales College of Medicine, UK Fukui University of Technology, Japan Biomatrix Inc, USA University of Uppsala, Sweden. University of Wales College of Medicine, UK Osaka University, Japan The North East Wales Institute, Wales Denki Kagoku Kogyu Japan University of Grenoble, France Gunma University, Japan Tufts University, USA Mount Sinai Medical Centre, USA
THE CELLUCON CONFERENCES 1984 Cellucon '84 UK.
CELLULOSE AND ITS DERIVATIVES Chemistry, Biochemistry and Applications
1986 Cellucon '86 UK.
WOOD AND CELLULOSICS Industrial Technology, Biotechnology, Structure and Properties
1988 Cellucon '88 Japan
CELLULOSICS AND WOOD Fundamentals and Applications
1989 Cellucon '89 UK.
CELLULOSE: SOURCES AND EXPLOITATION Industrial Utilisation, Biotechnology and Physico-Chemical Properties
1990 Ce11ucon '90 Czechoslovakia
CELLULOSE New Trends in the Complex Utilisation of Lignocellulosics (phytomass)
1991 Cellucon '91 USA
CELLULOSE A Joint Meeting of: ACS Cellulose, Paper and Textile Division, The Cel1ucon Trust, and 111h Syracuse Cellulose Conference
1992 Cellucon '93 UK.
SELECTIVE PURIFICATION AND SEPERATION PROCESSES
1993 Ce11ucon '93 Sweden
CELLULOSE AND CELLULOSE DERIVATIVES Physico-Chemical Aspects and Industrial Applications
1994 Cellucon '94 UK.
CHEMISTRY AND PROCESSING OF WOOD AND PLANT FIBROUS MATERIALS The Chemistry and Processing of Wood and Plant Fibrous Materials
1998 Ce11ucon '98 Finland
PULP AND PAPER MAKING Fibre and Surface Properties and other Aspects of Cellulose Technology
1999 Ce11ucon '99 Japan
RECENT ADVANCES IN ENVIRONMENTALLY COMPATffiLE POLYMERS
2000 Hyaluronan 2000 UK.
HYALURONAN 2000 An International Meeting Celebrating the 801h Birthday of Endre A Balazs
2003 Hyaluronan 2003 USA
HYALURONAN 2003
The proceedings of each conference were formerly published by Ellis Horwood, Simon and Schuster International Group, Prentice Hall, Campus 400, Maylands Avenue, Hemel Hempstead, Herts, HP2 7EZ, UK. and from 1993 are published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CBl 6AH, UK.
PREFACE
Together, these two volumes are the most comprehensive account of the chemistry, biology and medical aspects ofhyaluronan now available. They are based on the international congress "HYALURONAN 2000" held at The North East Wales Institute. Wrexham, Wales, which attracted 350 specialists from 23 countries, and who delivered 221 presentations. The timing was deliberate to enable the achievements of one century to be evaluated and the opportunities ofthe new to be identified. A principal objective was to celebrate the so" birthday ofDr Endre A Balazs and his unique contribution to the subject. There is not a worker in the hyaluronan field who is not familiar with his name. For the past 50 years he has pioneered both basic hyaluronan research and its clinical use in a spectrum of medical applications. In this meeting we honoured his gigantic contribution to the field and in his continuing to innovate even after his so" birthday. Friends, scientific and medical colleagues as well as commercial competitors, when they had notice of this Meeting, immediately signified that they wished to be present to mark his contribution to the subject. Chemists, cell biologists, clinicians and a range of medico-scientific researchers are now building on the foundations, which he has laid. It is fitting, therefore, that in both of these volumes there is an invited Introduction and Final Evaluation from Dr Balazs. The papers presented by his research group in these volumes testify also to the breadth ofhis current contribution. All aspects ofthe science ofhyaluronan are included. The initial contributions on the chemistry, rheology, chemical modification and characterisation provide a foundation for existing and potential new applications. These depend on the basic structure, its physical and solution properties, such as viscoelasticity, intermolecular interactions and conformation, as well as the specific participation of functional chemical groupings. The recognition that hyaluronan can induce cell-signalling functions has led to an explosion in associated cell biology investigations. This information is of vital importance because it points to mechanisms for wound healing and tissue regulating functions. There are already products entering the market based on these observations and the clinical contributions cover these aspects also. The tissue supplementation and application in osteoarthritis is now an established clinical application, and rightly is given due prominence in these volumes, along with the associated control of and interaction with pain receptors. After years of basic research into a material which seemed only to have shock absorbing and protective functions within the matrix, and was little more than a scientific novelty, the subject has now exploded. Those who doubt the link between basic research and practical application should scrutinise the now ubiquitous role of hyaluronan, as exemplified in the following subjects which were covered at the Meeting:
xviii
Preface
•
Hyaluronan receptors and signalling
•
Inhibition oftumor progression by perturbing hyaluronan-tumor cell interactions
•
Mechanoprotective actions of elastoviscous hylans on articular pain receptors
•
Hyaluronan as a drug vehicle in breast cancer
•
Anti-cancer activity ofhyaluronan
•
Hyaluronan in corneal wound healing
•
The role ofhyaluronan in inflammation and repair oflung injury
•
Functions ofhyaluronan in wound repair
•
HA based dermal and epidermal grafts for diabetic foot ulcers
•
Hyaluronan and hylans in the treatment of osteoarthritis
•
Hyaluronan and rheumatoid arthritis
•
Hyaluronan based medical formulations for control of post surgical adhesion development.
•
Clinical application of serum hyaluronan for liver diseases and its significance
There can be little doubt, therefore, that these volumes will constitute a landmark in hyaluronan science and application. My thanks go to my co-Editors, the Chairs and members of the Scientific, Executive, Industrial and International committees for their hard work and vision. Haydn Hughes, the Organising Secretary, bore the main burden and deserves all our appreciation for his quiet efficiency.
Glyn O. Phillips Chair, Executive Committee
Introductory Remarks Endre A. Balazs Matrix Biology Institute. 65 Railroad Avenue. Ridgefield, New Jersey 07657 USA
It is wonderful to see so many friendly and familiar faces in this audience -friends and colleagues who have been with us, forming this community of scientists, for the past half century. We grew up together scientifically and learned this science as we went along, with very little help from the outside. Many of us grew old together, sharing friendship, fellowship and success. I greet you all with the warmth of many pleasant memories. It is also encouraging to see so many new faces in the audience, young scientists whom we will know better during the next week. I greet them with the warmth of great expectations, because they represent the future, the fulfillment of many of our dreams. I must sadly remind you that since the last meeting in Stockholm, we lost a great friend and a great scientist, Sven Gardel!. He was not only a brilliant and very influential researcher, but also a great teacher and educator. Many of us learned from his teaching and counsel during half a century. Our thanks and appreciation to the organizers of this meeting for the work they did. We know that organizing a meeting with more than 300 participants and more than 200 presentations of lectures and posters is not an easy task. The diversity and richness of the program clearly reflects their successful work. I am here to introduce Torvard Laurent's presentation. To put this in perspective, I have to take you back to a sunny September day fifty-two years ago in 1948 in the Karolinska Institute of Stockholm. On this day Torvard entered my laboratory, my life and, most importantly, the life and adventures of hyaluronan and the intercellular matrix. I had already been hooked on this subject for ten years, because, like Torvard, I was also introduced to the intricacies of the "intercellular substance" at the age of 18 as a first-year medical student. Little did we know on that September day in 1948 that a lifelong friendship and scientific fellowship was born. We only knew that at that time very few people in the world were interested in our subject. What was happening in the world of hyaluronan research in 1948? Haddian and Pirie at the Worcester Foundation in Massachusetts had just published a paper on the preparation of hyaluronan. Some years later this would trigger Roger leanloz' definitive work at the same institution on the primary structure of hyaluronan. Three years earlier, Gunnar Blix (with Snellman) published the first determination of intrinsic viscosity of hyaluronan prepared from bovine synovial fluid. They concluded that the behavior of hyaluronan solution was Newtonian. This conclusion was the result of the low molecular weight and low intrinsic viscosity of their preparation.
xx
Introductory remarks
Karl Meyer's pioneering research on hyaluronan was interrupted by the war effort. His work was concentrated on penicillin and Iysosyme during that time. In the late 1940s he had just started to pick up his work on hyaluronidase. But his work in the late 1930s triggered the interest of several prominent chemists to determine the primary structure of hyaluronan by methylation and periodate oxidation methods: M.A.G. Kay, in Stacey's laboratory in Birmingham, UK; 1. Felling in Kurt Mayer's laboratory in Geneva; and Roger Jeanloz in the Worcester Foundation in Massachusetts started their work in 1948-49. Lars Sundblad at G. Blix' laboratory in Uppsala and J.E. Stanier in A. G. Ogston's laboratory in Oxford would soon start their doctoral research work on synovialfluid hyaluronan. The hot topics of the day were Duran-Renal's spreading factor that was already identified as hyaluronidase. In 1948 Albert Dorfman published the first study on the kinetics of enzymatic hydrolysis of hyaluronan. Between 1935 and 1948 it was widely reported that most human and animal tumors contained hyaluronidase, and yet others also contained hyaluronan. The role of hyaluronidase in tumors was implicated, but it was noted that some invasive tumors had high hyaluronidase content while others had none. I also contributed to the confusion at that time by publishing that the necrotic part of invasive tumors contained higher levels of hyaluronidase activity than the living parts and that plant cells also have hyaluronan-degrading activity during mitosis. Bengt Sylven, a histopathologist at Karolinska Institute, working with histochemical methods, was convinced that sulfated glycosaminoglycans of the intercellular matrix and mast cells were part of the body's early defense system against growth and spread of tumors. This was the scientific environment in which Torvard started his work with me in 1948. I am very proud that during the years we worked together closely in Stockholm and Boston, I succeeded in infecting him with the spiritual virus of hyaluronan and intercellular matrix curiosity, which yielded a wealth of new knowledge though his own research activity and through that of two generations of his students. As a chain reaction, the number of scientists interested in the field grew logarithmically, and under his tutelage the research on hyaluronan and other macromolecular components of the intercellular matrix and its function in health and diseases flourished. We all must be grateful for his loyalty to this molecule and its role in the life of multi-cellular organisms. It is not an exaggeration to say that without his highly significant contribution to our field of science, the coming week's events could not have occurred.
PARTl OVERVIEW OF THE HISTORY AND DEVELOPMENT OF HYALURONAN
HYALURONAN BEFORE 2000 Torvard C Laurentt llnstiuae ofMedical Bioc1u!mistry and Microbiology, University of Uppsala.
BMC. Box582. SE·752 23 Uppsala; Sweden.
ABSTRACT In a review of the history of hyaluronan the author has tried to call attention to the key discoveries which have lead to paradigm shifts in the research on this unique polysaccharide. The selection is no doubt subjective.
KEYWORDS Hyaluronan, hyaluronate, hyaluronic acid, Endre Balazs
INTRODUCTION It is a great honour to be asked to give a key note lecture at the symposium "Hyaluronan 2000". However, realizing that I have nothing new to contribute to the field I have chosen to speak about "Hyaluronan before 2000". At the present pace of science the active researcher seldom has time to go back in literature to look for key discoveries and therefore I will try to make a summary.The selection is based on my own experience and may not agree with the view of other observers. However, first I would like to tell you how I entered the field. At this conference we are honouring Endre A. Balazs, who became 80 years in January. Endre - or Bandi as we always have said - was my teacher.
HOW IT STARTED In 1948 I entered Medical School in Stockholm and studied Anatomy and Histology for a year. In September 1949, at the age of 18, I got the opportunity to be unpaid instructor at the Department of Experimental Histology parallel to my medical studies. An Hungarian scientist, Bandi Balazs, immediately engaged me in research. He had left Hungary in 1947, before the communists took over, and had come to Stockholm to work on the biological role of extracellular polysaccharides and especially hyaluronan. My first assignment was to prepare hyaluronan from umbilical cords to be used in fibroblast cultures. Cell culturing was then performed very differently from what you are
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Overview of the history and development ofhyaluronan
used to now. Before antibiotics everything had to be done under strictly sterile conditions as in an operating theater. Cells were grown in hanging fibrin clots. Fibro-blasts were obtained from pieces of embryonic chicken hearts, which were placed in the fibrin clots, and the cellular growth rates were determined by planimetry of enlargements of the cultures, which estimated how far the cells had migrated. Hyaluronan was extracted from umbilical cords and precipitated by alcohol. It was freed from proteins by shaking the extracts with chloroform and isoamyl alcohol (Sewag technique). We were looking for techniques to sterilize the highly viscous hyaluronan solutions. They could not be ultraftltered. In the end we used autoclaving. This work led to three early observations, which should tum out to be of rather basic character. Firstly, when we extracted hyaluronan from the umbilical cords at different ionic conditions we got material of very different viscosities. The highest viscosity was obtained when we extracted with distilled water. We then realized that the viscosity of hyaluronan changed with pH and ionic strength. Today this is common knowledge but at that time it had only been observed for synthetic polyelectrolytes by Raymond Fuoss. We wrote a note in Journal of Polymer Chemistry with the title "The viscosity function of hyaluronic acid as a polyelectrolyte"(l). This started my interest in the physical chemical properties of hyaluronan. Secondly, when we tried to sterilize hyaluronan by UV-irradiation, it lost all its viscosity. It was later shown that irradiation with electrons also degraded hyaluronan and other polysaccharides (2). We now know that we observed one of the first examples of free radical degradation of hyaluronan. The third observation concerned the biological effects of hyaluronan and some sulphated polysaccharides, heparin, heparan sulphate (which at that time was called heparin monosulphuric acid) and synthetically sulphated hyaluronan (3). We compared effects on cell growth, anticoagulant activity, antithrombine activity and antihyaluronidase activity. The main purpose was to clarify if heparin actually was a sulphated hyaluronan, as had been stated by Asboe-Hansen, but we concluded that it was not so. However, hyaluronan promoted cell growth, in contrast to the sulphated polysaccharides, and this was probably one of the first observations that hyaluronan interacts with cellular functions - today we know that this occurs via cellular receptors. Interestingly, this was probably also one of the first studies of the biological activity of heparan sulphate. The work was performed in a short time between September 1949 and December 1950, i.e, during slightly more than a year. Then Bandi moved to Boston, the head of our department died and I moved to the Chemistry Department where I continued with Physical Chemistry. Also, more of my time was taken up by clinical training. In retrospect the year with Bandi more than 50 years ago dramatically changed my life. I met a charismatic person who induced enthusiasm in the work and who looked upon things in an unorthodox way far away from what you could read in the text books. Bandi and I were going to work together for another four years during two periods in Boston between 1953 and 1961.
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DISCOVERY OF HYALURONAN AND HYALURONIDASE Karl Meyer discovered hyaluronan in 1934 when he worked in the eye clinic of Columbia University (4). He isolated the compound from the vitreous body of bovine eyes under acid conditions and gave it the name hyaluronic acid after hyalos - a greek word for glassy - and uronic acid, a constituent of the polymer. It should be noted that other polysaccharides (chondroitin sulphate and heparin) had previously been isolated. Furthermore, already in 1918, Levene and Lopez-Suarez (5) isolated a polysaccharide from vitreous body and umbilical cord which contained glucosamine, glucuronic acid and some sulphate. They named the polysaccharide mucoitin sulphuric acid but with our present knowledge it must have been hyaluronan with some sulphate impurity. During the next ten years hyaluronan was isolated from various tissues by Karl Meyer and others. For example, it was found in joint fluid, skin, umbilical cord and rooster comb. Most notably it was discovered in the capsules of streptococci by Kendall et al. (6) in 1937. Since then it has been found in practically every tissue in vertebrates. Independently and preceeding the discovery of hyaluronan a factor was described by Duran-Reynals in testis (7). It was later named spreading factor. Similar activities were found in bee venom, extracts of leeches etc. When the spreading factors were injected in skin together with India-ink they caused a rapid spread of the black stain. The factors were identified as enzymes, which degrade hyaluronan, and were named hyaluronidases (8). Even in mammalian blood a hyaluronidase was present but it only acted at acid pH.
PREPARATION OF HYALURONAN
The original preparation procedures for hyaluronan followed what was conventional for polysaccharides, i.e, proteins were removed by the Sewag-technique or by proteolytic digestion. The polymer was then fractionally precipitated by alcohol. A great step forward in separating differently charged polysaccharides was taken by John Scott when he developed fractionated precipitation with a cationic detergent (CPC, cetylpyridinium chloride) by varying the salt concentration (9). Hyaluronan could be efficiently separated from sulphated polysaccharides. The method could also be used for molecular weight fractionation. In principle similar results can be obtained by ion exchange chromatography. THE STRUCTURE AND CONFORMATION OF HYALURONAN The chemical structure of the polysaccharide was essentially solved by Karl Meyer and associates in the 1950's (see e.g. 10). We now know that hyaluronan is a long polymer built from disaccharides consisting of N-acetyl-D-glucosamine and D-glucuronic acid linked by Bl-4 and Bl-31inkages. Karl Meyer did not follow the conventional approach to analyze the intact polysaccharide. Instead he obtained by hyaluronidase digestion
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Overview of the history and development ofhyaluronan
specific disaccharides or oligosaccharides which he characterized and from this information he could deduce the structure of the intact polymer. A conformational analysis of hyaluronan 'fibers' was first attempted by X-ray crystallographers. There was a heated debate between different groups at a meeting in Turku in 1972, regarding the helix structure of hyaluronan. Apparently different types of helices can be formed by hyaluronan dependent on counter ion composition and water content Various structures were published during the 70' s and 80' s. A break-through was made by John Scott when he on the basis of lack of reactivity to periodate oxidation suggested a structure in solution containing intra-chain hydrogen bonds (11). He later verified this hypothesis by NMR analysis (12) and the conformation could be accommodated in a two-fold helical structure described by Atkins et al. already in 1972 (13). PHYSICAL CHEMICAL CHARACTERIZATION
Fifty years ago we did neither know the chemical structure of hyaluronan nor its macromolecular properties, i.e. molecular weight, homogeneity, molecular shape, hydration, charge and interaction with other molecules. This became the interest in the following 10 years of A.G. Ogston and his collaborators in Oxford, of Balazs and coworkers in Boston, of myself in Stockholm and of a few other laboratories. Our main difficulty was to prepare hyaluronan free of proteins and other components before physical measurements. There was always a risk of degrading the polymer during the purification process. Ogston used the technique of ultrafiltration assuming that free proteins went through the filter and that proteins bound to hyaluronan were retained in the top solution. He studied a 'complex' which contained 30% protein (14). Other investigators used various physical, chemical and enzymatic means which removed proteins down to a few percent However, the general results of the physical chemical analyses gave a consistent picture of the hyaluronan molecule. The molecular weight was usually several millions, although many samples were polydisperse. Light-scattering showed the molecule to behave as a randomly coiled, relatively stiff, chain molecule with a radius of gyration of the order of 200 nm (15). The chain stiffness is due to the intrachain hydrogen bonds mentioned above. The random coil structure was later confirmed by the molecular weight/viscosity relationship. Ogston and Stanier (14) using sedimentation, diffusion and shear rate dependence of viscosity and birefringence drew the conclusion that the molecule behaved as a large hydrated sphere which was compatible with a random coil configuration. ANALYTICAL TECHNIQUES
The only way of analysing hyaluronan quantitatively was from the beginning to isolate the polysaccharide in pure form and measure its content of uronic acid and/or N-acetyl
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glucosamine. The methods of choice were the Dische carbazole technique for uronic acid (16) and the Elson-Morgan reaction for hexosamine (17). The importance of the carbazole technique for routine analysis cannot be overestimated. The analyses of hyaluronan required milligram quantities. The next step in the development came with the introduction of specific enzymes. The streptomyces hyaluronidase is specific for hyaluronan (18) and produces unsaturated hexa- and tetrasaccharides. This can be utilized to analyze the hyaluronan content in the presence of other polysaccharides and impurities and the 'unsaturated' state of the uronic acid can be used to lower the detection limit of the product. The enzymatic technique increased the sensitivity to microgram quantities of hyaluronan. The final step came with the use of affinity proteins recognizing hyaluronan. Tengblad (19) used hyaluronan binding proteins from cartilage and Delpech (20) subsequently used hyaluronectin from brain. These proteins could be used in assays similar to immunoassays and now nanogram quantities of hyaluronan could be measured directly in tissue fluids. Tengblad's technique formed a basis for much of the work performed in Uppsala since then.
VISUALIZATION OF HYALURONAN The detection of hyaluronan in tissue sections is closely related to analysis of the polymer in tissue fluids. From the beginning unspecific staining with basic dyes were used. John Scott increased the specificity by the same principle he had used in fractionation of anionic polysaccharides with detergents. He stained with Alcian Blue at different ionic concentrations (21) and could thereby differentiate between polysaccharides. Lately he has gone over to use Cupromeronic Blue. However, hyaluronan can also with advantage be localized by specific affinity proteins in tissue sections. The first such reports came in 1985 (22,23). The technique has been used with great success and has given us a detailed information of hyaluronan distribution in various organs. Hyaluronan can also be visualized by electron microscopy. The first picture was published by Jerome Gross (24) but did not show any details. The paper by Fessler and Fessler (25) can be regarded as the first interpretable study. It showed hyaluronan as a long extended single chain. Another ingenious way of visualizing pericellular hyaluronan was described by Robert Fraser (26). He added a suspension of particles to fibroblast cultures. The particles were excluded from a thick layer surrounding the fibroblasts. This pericellular coat turned out to be hyaluronidase sensitive.
ENTANGLEMENT AND RHEOLOGY From the dimensions of the largest hyaluronan molecules one can estimate that they
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Overview of the history and development ofhyaluronan
should fill the solution completely at concentrations of the order of 1 gil. At high concentrations the molecules will entangle and the solution consists of a continuous network of chains. The entanglement point is clearly visible as a point, where the specific viscosity increases dramatically when the concentration is further increased. Another property which changes dramatically with concentration is the shear dependence of the viscosity. The latter was first described by Ogston and Stanier (13). Also the elastic behaviour of the solution increases with increasing concentration and molecular weight Flow elasticity of pure hyaluronan was first shown by Jensen and Koefoed (27) and a thorough analysis of the viscoelastic behaviour was made by Gibbs et al. (28). Is this dramatic behaviour only a function of mechanical entanglement or could it also be due to chemical interactions between the chains? Already in early papers by Ogston it was discussed if some kind of interactions via proteins could occur. Clear evidence that there occurred a chain-chain interaction was obtained by Welsh et al (29) when they showed that the elasticity could be counteracted by addition of short hyaluronan chains (60 disaccharides) to the solution. Apparently these competed with the interactions between longer chains. In the more recent work by John Scott it has become clear that the conformation of hyaluronan, which displays hydrophobic patches along the chain, is well suited for forming helices with neighbouring molecules which are stabilized by hydrophobic forces (30). It is therefore most probable that chain-chain interaction to a large extent contributes to the rheological properties of hyaluronan.
PHYSIOLOGY OF HYALURONAN NETWORKS The discovery that hyaluronan chains entangle at concentrations, which may occur in many tissues, raised the hypothesis that hyaluronan actually exercised its physiological activity via the properties of a continuous three-dimensional chain network. Various properties of the networks were discussed: Viscosity. The very high visco-elasticity of concentrated high-molecular weight hyaluronan solutions, as well as the shear dependence, were connected with lubrication of joints and other tissues. Hyaluronan seems always to be present in spaces separating mobile tissues, e.g. in joints and between muscles. Osmotic pressure. The osmotic pressure of hyaluronan solutions is strongly concentration dependent and at higher concentrations the colloid osmotic pressure is larger than that of an albumin solution (31). This property was assumed to be of importance for the water homeostasis in the tissues. Flow resistance . A tight chain network exerts a very high resistance towards water flow. That hyaluronan really forms flow barriers in tissues was first shown by Day (32). Excluded volume. A three-dimensional network removes space for other macromolecules. The available volume can be measured by equilibrium dialysis between a hyaluronan solution and a buffer solution and it turns out that the effect corresponds to what one can calculate that it should be (33) according to a theoretical relationship
Hyaluronan before 2000
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deduced by Ogston (34). The exclusion effect has been discussed in connection with protein partition between the vascular space and extracellular tissue space but it has also been discussed in connection with deposition of physiological or pathological material in connective tissues. Polymer exclusion decreases the solubility of proteins (35). Diffusionbarrier . The movement of macromolecules through a hyaluronan solution can be measured by sedimentation or diffusion analyses. The larger a particle is the more it becomes retarded in its movements (36). This effect has been connected with formation of diffusion barriers in the tissues. For example, the pericellular layer of hyaluronan could protect cells from large macromolecules or other cells.
HYALURONAN·BINDING PROTEINS (HYALADHERINS)
Proteoglycans. Until 1972 it was believed that hyaluronan was an inert compound in tissues that did not specifically interact with other macromolecules. In this year Hardingham and Muir (37) showed that hyaluronan can aggregate cartilage proteogtycans. Thorough studies by Hascall and Heinegard (38) documented that there is a specific binding between hyaluronan, the N-terminal globular part of the proteoglycan and a link protein. This is a very firm association and many proteoglycans bind to the same hyaluronan chain forming large aggregates in cartilage and other tissues. Hyaluronan receptors. Also in 1972, Pessac and Defendi (39) and Wasteson et al. (40) demonstrated that certain cells in suspension aggregate when hyaluronan is added. This was the first report that hyluronan interacts specifically with cell surfaces. Subsequently, Underhill and Toole (41) described in 1979 that hyaluronan actually binds to cells and the responsible 'receptor' was purified in 1985 (42). In 1989 two groups reported that the lymphocyte homing receptor, CD44, was a hyaluronan binding protein with homology to the link protein in cartilage (43,44). It was soon shown that the receptor of Underhill and Toole was identical to CD44' Another hyaluronan binding protein was isolated from the supematant of cultures of 3T3 cells by Turley et al. in 1982 (45) and named RHAMM (receptor for hyaluronan mediating motility). Following these initial discoveries a number of other hyaladherins have been defmed. CELL BIOLOGICAL ROLE OF HYALURONAN Until the discovery of hyaladherins, hyaluronan was thought to influence cell behaviour entirely through physical interactions. Evidence that hyaluronan might playa role in biological processes was purely circumstantial and to a large extent built upon the presence or absence of hyaluronan during biological processes. Much of the speculations were based on unspecific histological investigations. A very successful research line started in Boston in the beginning of the 1970' s. Bryan Toole and Jerome Gross. (46) showed that during regeneration of the newt limb hyaluronan was first synthesized and subsequently removed by hyaluronidase, when
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Overview of the history and development of hyaluronan
instead chondroitin sulphate was formed, A similar pattern was seen in the developing chick cornea (47). Toole has pointed out that accumulation of hyaluronan coincided with periods of cellular migration in the tissues. As mentioned above Toole also pioneered studies on membrane-bound hyaladherins and with the introduction of hyaluronan 'receptors' we have a much firmer basis for the concept that hyaluronan plays a role in regulating cellular activity, e.g, motility (48). We have seen an explosion of publications dealing with the role of hyaluronan in cellular migration, mitosis, inflammation, cancer, angiogenesis, fertilization etc. in the last decade.
BIOSYNTHESIS OF HYALURONAN Research on the biosynthesis of hyaluronan has gone through three phases. The dominating person in the first phase was Albert Dorfman. He and his collaborators described during the early 1950's the origin of the monosaccharides to be incorporated into the hyaluronan chain in streptococci. However, it was Glaser and Brown in 1955 who for the first time demonstrated hyaluronan synthesis in a cell free system (49). They used a particulate enzyme from Rous chicken sarcoma and incorporated 14<: labelled UDP-glucuronic acid into hyaluronan oligosaccharides. The Dorfman group subsequently isolated the precursors UDP-glucuronic acid and UDP-N-acetyl glucosamine from extracts of streptococci (50) and synthesized hyaluronan with a streptococcal enzyme fraction (51). In the second phase it became apparent that hyaluronan must be synthesized by a different mode than other glycosaminoglycans. Hyaluronan production did not require an active protein synthesis (52) in contrast to sulphated polysaccharides. The synthase was located in the protoplast membrane of bacteria (53) and plasma membrane of eucaryotic cells (54,55) and not in the Golgi. The synthesizing machinery was presumably located on the internal side of the membrane as it was insensitive to external proteases, but the hyaluronan chain was apparently extruding through the membrane since extracellular hyaluronidase treatment enhanced hyaluronan synthesis (54). A couple of unsuccessful attempts to isolate the synthase from eucaryotic cells were made in the 80's. In the early 90's it was realized that hyaluronan synthase was a virulence factor for group A streptococci and utilizing this infonnation two groups could deflne a gene locus responsible for the production of the hyaluronan capsule (56,57). Soon the synthase was cloned and its sequence determined, Homologous vertebrate enzymes were discovered and a wealth of information had accumulated in a few years. An important field for future research will be the mechanism by which the activity of the synthase is regulated.
TURNOVER AND DEGRADATION OF HYALURONAN The discovery that hyaluronan is present in blood and is carried from peripheral tissues to the circulation by lymph (58) started a collaborative investigation between Robert
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Fraser in Melbourne and our laboratory in Uppsala on the turnover of hyaluronan. Trace amounts of the polysaccharide labeled with tritium in the acetyl group were injected in the circulation of rabbits (59) and humans and the label disappeared with a half-life of a few minutes. It was soon realized that the major part of the radioactivity had been taken up by the liver, where the polymer was rapidly degraded. Tritiated water appeared in the circulation within 20 minutes. Autoradiography revealed that uptake also occurred in spleen, lymph nodes and bone marrow. Through cell fractionation it could be demonstrated that the cells responsible in the liver were the sinusoidal endothelial cells and it was confirmed by uptake studies in vitro (60) and by in situ autoradiography. These cells carry a receptor for endocytosis of hyaluronan which is very different from other hyaluronan binding proteins. The polysaccharide is degraded in lysosomes. Studies on hyaluronan were also extended to other tissues and we now have a fair view of the general tum-over of the polysaccharide in the organism (61). Another aspect of hyaluronan degradation has gained recent attention. Through the work of Gunther Kreil in Austria and Robert Stem and associates in San Francisco the structure and properties of various hyaluronidases have been described which has led to interesting observations on the biological roles of these enzymes (for ref.see 62,63).
PATHOLOGY OF HYALURONAN There was an early interest in the properties of hyaluronan of joint fluid in joint disease and it was known that hyaluronan was overproduced in some malignant diseases, e.g. mesothelioma, but it was not until the new analytical tools for hyaluronan were developed in the 1980's that it became clinically interesting to study variations in hyaluronan levels. The normal concentration in blood was determined (64) and pathological levels were noted especially in liver cirrhosis (65). In rheumatoid arthitis the blood level rose during physical activities in the morning giving an explanation to the symptom 'morning stiffness' (66). High levels were found both locally and in blood during various inflammatory diseases (for ref. see 67). Organ dysfunctions could be explained by local accumulation of hyaluronan followed by interstitial edemas.
CLINICAL APPLICATIONS The dramatic development in the medical use of hyaluronan is entirely due to Balazs. He derived the main concepts, he was the first to prepare hyaluronan samples which were tolerated, he promoted the industrial production of hyaluronan and he popularized the use of the polysaccharide. During the 1950's Balazs concentrated his research on the composition of the vitreous body and started to experiment with vitreous substitutes to be used in surgery for retinal detachment (68). One of the crucial obstacles for using hyaluronan in implants was to prepare hyaluronan free of impurities causing inflammatory reactions. Balazs solved this
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Overview of the history and development ofhyaluronan
problem and his final preparation was called NIF-NaHA (noninflammatory fraction of sodium hyaluronate) (69). In 1970 hyaluronan was injected in arthritic joints of track horses (70) with a dramatic positive effect on the clinical symptoms. Two years later Balazs convinced Pharmacia AB in Uppsala to start production of hyaluronan for veterinary and human use. Miller and Stegman, after advice from Balazs, started to use hyaluronan as a device in the implantation of plastic intraocular lenses (71) and hyaluronan became a major product for ophthalmic surgery under the trade name of Healon®. Numerous other applications have since been suggested and tested (for ref. see 72). Also derivatives have been used clinically, e.g. cross-linked hyaluronans. It is notable that Balazs reported on the biological activity of the first derivative ever made from hyaluronan in 1951 (3). CONCLUSIONS It has only been possible to mention some of the high-lights of the hyaluronan story. You will be reminded of many more during the symposium. The program gives ample evidence for the rapid growth and importance of hyaluronan research. Today between 300 and 400 articles on hyaluronan are published every year. The first international conference entirely devoted to hyaluronan was held in St. Tropez in 1985, followed by meetings in London (1988), Stockholm (1996) and Padua (1999). The present conference is by far the largest one. This growth in interest is to a great extent due to the vef¥ successful work by Endre Balazs, who has contributed so much to our basic knowledge of hyaluronan, who first of all showed the practical use of hyaluronan in medicine and who has constantly inspired the scientific community. REFERENCES 1.
2. 3. 4. 5. 6.
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E.A. Balazs & T.C. Laurent, Viscosity function of hyaluronic acid as a polyelectrolyte. J. Polym: Sci., 1951, 6, 665-667. E.A. Balazs, T.e. Laurent, A.F. Howe & L. Varga, Irradiation of mucopolysaccharides with ultraviolet light and electrons. Radiation Res., 1959, II, 149-164. E.A. Balazs, B. Hogberg & T.e. Laurent, The biological activity of hyaluron sulfuric acid. Acta Physiol. Scand., 1951, 23, 168-178. K. Meyer & J.W. Palmer, The polysaccharide of the vitreous humor. J. Bioi. Chem., 1934, 107, 629-634. P.A. Levene & J. Lopez-Suarez, Mucins amd mucoids. J. Bioi. Chem., 1918, 36, 105-126. F.E. Kendall, M. Heidelberger & M.P. Dawson, A serologically inactive polysaccharide elaborated by mucous strains of Group A hemolytic streptococcus. J. Bioi. Chem., 1937, U8, 61-69. F. Duran-Reynals, The effect of extracts of certain organs from normal and
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immunized animals on the infecting power of vaccine virus.J. Exptl. Med., 1929, 50, 327-340 K. Meyer, G.L. Hobby, E. Chaffee & M.H. Dawson, Relationship between spreading factor and hyaluronidase. Proc.Soc.Expti.Biol.Med. 1940, 44, 294-296 J.E. Scott, Solubility of cetylpyridinium complexes of biological polyanions in solutions of salts. Biochim. Biophys. Acta, 1955, 18,428-429. K. Meyer, Chemical structure of hyaluronic acid. Fed. Proceed., 1958, 17, 1075-1077. J.E. Scott & M.J. Tigwell, Periodate oxidation and the shapes of glycosaminog1ycuronans in solution. Biochem. J.,1978, 173, 103-114. F. Heatley & J.E. Scott, A water molecule participates in the secondary structure of hyaluronan. Biochem. J., 1988, 254, 489-493. NMR E.D.T. Atkins, D. Meader & J.E.Scott, Model for hyaluronic acid incorporating four intramolecular hydrogen bonds. Int. J. Bioi. Macromol., 1980,2,318-319. A.G. Ogston & J.E. Stanier, The dimensions of the particle of hyaluronic acid complex in synovial fluid, Biochem. J., 1951, 49, 585-590. T.e. Laurent & J. Gergely, Light scattering studies on hyaluronic acid. J. Biol. Chem; 1955, 212, 36-40 Z. Dische, A new specific color reaction of hexuronic acids. J. Bioi. Chem., 1947, 167, 189-198. L.A. Elson & W.T. Morgan, A colorimetric method for the determination of glucosamine and chondrosamine. Biochem. J., 1933, 27, 1824 -1828. T. Ohya & Y. Kaneko, Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta, 1970, 198,607-609. A. Tengblad, Quantitative analysis of hyaluronate in nanogram amounts. Biochem. J., 1980, 185, 101-105. B. Delpech, P. Bertrand & C. Maingonnat, Immunoenzymoassay of the hyaluronic acid-hyaluronectin interaction: Application to the detection of hyaluronic acid in serum ofnormal subjects and cancer patients. Anal. Biochem., 1985, 149, 555565. J.E. Scott, J. Dorling & G. Quintarelli, Differential staining of acid glycosaminoglycans by Alcian Blue in salt solutions. Biochem. J., 1964, 90, 4-5. C.B. Knudson & B.P. Toole, Fluorescent morphological probe for hyaluronate. J. Cell Biol., 1985, 100, 1753-1758. J.A. Rippelino, M.M. Klinger, R.D. Margolis & R.K. Margolis, The hyaluronic binding region as a specific probe for the localization of hyaluronic acid in tissue sections. J. Histochem. Cytochem., 1985, 33, 1060-1066. J. Gross, Electron microscope studies of sodium hyaluronate. J. Biol. Chem., 1948, 172,511-514. J.H. Fessler & LJ. Fessler, Electron microscopic visualization of the polysaccharide hyaluronic acid. Proc. Nat. Acad. Sci-U.S., 1966, 56, 141-147.
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Overview ofthe history and development ofhyaluronan
26. B.l. Clarris & 1.RE. Fraser, On the pericellular zone of some mammalian cells in vitro. Exp. Cell Res., 1968,49, 181-193. 27. C.E. Jensen & J. Koefoed, Flow elasticity of hyaluronate solutions. J.Colloid Sci., 1954,9,460-465. 28. D.A Gibbs, E.W. Merril, K.A. Smith & E.A Balazs, Rheology of hyaluronic acid. Biopolymers ,1968, 6, 777-791. 29. E.J. Welsh, D.A Rees, E.R Morris & J.K. Madden, Competitive inhibition
30.
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evidence for specific intermolecular interction in hyaluronate solutions. 1. MoL Bioi., 1980, 138, 375-382. IE. Scott, C. Cummings, A Brass & Y. Chen, Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient networkforming polymer. Biochem. J., 1991, 274, 699-705. T.C. Laurent & AG. Ogston, The interaction between polysaccharides and other macromolecules. 4. The osmotic pressure of mixtures of serum albumin and hyaluronic acid. Biochem. J., 1963, 89, 249-253. T.D. Day, Connective tissue permeability and the mode of action of hyaluronidase. Nature, 1950, 166,785-786. T.C. Laurent, The interaction between polysaccharides and other macromolecules. 9. The exclusion of molecules from hyaluronic acid gels and solutions. Biochem.J., 1964, 93, 106-112. AG.Ogston, The spaces in a uniform random suspension of fibers. Trans. Faraday Soc., 1958, 54, 1754.1757. T.e. Laurent, The interaction between polysaccharides and other macromolecules. 5. The solubility of proteins in the presence of dextran. Biochem. J., 1963, 89,
253-257. 36. T.C. Laurent, I. Bjork, A Pietruszkiewicz & H. Persson, On the interaction between polysaccharides and other macromolecules. II. The transport of globular particles through hyaluronic acid solutions. Biochim. Biophys. Acta, 1963. 78,
351-359. 37. T.E. Hardingham & H. Muir, The specific interaction of hyaluronic acid with cartilage proteoglycans. Biochim. Biophys. Acta, 1972, 279, 401-405. 38. V.C. Hascall & D. Heinegard, Aggregation of cartilage proteoglycans I, II and III. J. BioI. Chem., 1974, 249, 4232-4256. 39. B. Pessac & V. Defendi, Cell aggregation: role of acid mucopolysaccharides. Science, 1972, 175, 898-900. 40. A. Wasteson, B. Westermark, U. Lindahl & 1. Ponten, Aggregation of feline lymphoma cells by hyaluronic acid. Intern. J. Cancer, 1913. 12, 169-178. Presented at the meeting "Biology of fibroblast" in Turku, 1972. 41. C.B. Underhill & B.P. Toole, Binding of hyaluronate to the surface of cultured cells. J. Cell Biol., 1979, 82, 474-484..
Hyaluronan before 2000
15
42. C.B. Underhill, A.L. Thurn & RE. Lacy, Characterization and identification of the hyaluronate binding site from membranes of SV-3T3 cells. J. Bioi. Chem., 1985, 260, 8128-8133. 43. I. Stamenkovic, M. Amiot, 1.M. Pesando & B. Seed, A human lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family. Cell, 1989, 56, 1057-1062. 44. L.A. Goldstein, D.F.H. Zhou, L.I. Picker, C.N. Minty, R.F. Bargatze, I.F. Ding and E.C. Butcher, A human lymphocyte homing receptor, the Hermes antigen, is related to cartilage proteoglycan core and link proteins. Cell, 1989,56, 1063-1072. 45. E.A.Turley, Purification of a hyaluronate-binding protein fraction that modifies cell social behavior. Biochem. Biophys. Res. Comm., 1982, 108, 1016-1024. 46. RP. Toole & 1. Gross, The extracellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation. Dev. Bioi., 1971, 25,57-77. 47. RP. Toole & R.L. Trelstad, Hyaluronan production and removal during corneal development in the chick. Dev. Biol., 1971,26,28-35. 48. B.P. Toole, Glycosaminoglycans in morphogensis. In: Cell Biology of the Extracellular Matrix, E.n. Hay (ed.), Plenum Press, New York, 1981, pp.259-294. 49. L. Glaser & D.H. Brown, The enzymic synthesis in vitro of hyaluronic acid chains. Proc. Natl. Acad. Sci. U.S., 1955, 41, 253-260. 50. I.A. Cifonelli & A. Dorfman, The biosynthesis of hyaluronic acid by Group A Streptococcus. V. The uridine nucleotides of group A streptococcus. J.BioLChem., 1957, 228, 547-557. 51. A. Markowitz, I.A Cifonelli & A. Dorfman, The biosynthesis of hyaluronic acid by group A streptococcus VI. Biosynthesis from uridine nucleotides in cell-free extracts. J. Bioi. Chem., 1959, 234, 2343-2350. 52. AC. Stoolmiller & A Dorfman, The biosynthesis of hyaluronic acid by streptococcus. J. Bioi. Chern.• 1969,244, 236-246. 53. A Markowitz & A. Dorfman, Synthesis of capsular polysaccharide (hyaluronic acid) by protoplast membrane preparations of group A streptococcus. J. Bioi. Chem., 1962, 237, 273-279. 54. L.H. Philipson & N.R Schwartz, Subcellular localization of hyaluronate synthetase in oligodendroglioma cells. J. BioI. Chem., 1984,259,5017-5023. 55. P. Prehm, Hyaluronate is synthesized at plasma membranes. Biochem. J., 1984, 220, 597-600. 56. B.A. Dougherty & I. van de Rijn. Molecular characterization of a locus required for hyaluronic acid capsule production in group A streptococci. J. Exp. Med. 1992, 175,1291-1299. 57. P.L. deAngelis, I. Papaconstantinou & P.H. Weigl, Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants in heterologous bacteria. J. Bioi. Chem., 1993, 268, 14568-14571.
16
Overview of the history and development ofhyaluronan
58. U.B.G. Laurent & T.C. Laurent. On the origin of hyaluronate in blood. Biochem. Int., 1981, 2, 195-199. 59. I.R.E. Fraser, T.C. Laurent, H. Pertoft & E. Baxter, Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit. Biochem. J., 1981, 200, 415-424. 60. B. Smedsred, H. Pertoft, S. Eriksson, I.R.E. Fraser & T.C. Laurent. Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells. Biochem. J., 1984, 223, 617-626. 61. T.C. Laurent & I.R.E. Fraser, Catabolism of hyaluronan. In: Degradation of bioactive substances: Physiology andpathophysiology. I.H. Henriksen ( ed.), CRC Press, Boca Raton, 1991, pp.249-265. 62. G. Lepperdinger, B. Strobl & G. Kreil, HYAL2, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity. J. BioL Chem., 1998,273,22466-22470. 63. T.B. Csoka, G.I. Frost & R. Stem. Hyaluronidases in tissue invasion. Invasion Metastasis, 1997,17,297-311. 64. A. Engstrom-Laurent, U.B.G. Laurent. K.. Lilja & T.C. Laurent. Concentration of hyaluronate in serum. Scand: J. Clin. Lab. Invest., 1985, 45, 497-504. 65. A. Engstrorn-Laurent, L. Loof, A. Nyberg & T. Schroder, Increased serum levels of hyaluronate in liver disease. Hepatology, 1985, 5, 838-642. 66. A. Engstrom-Laurent & R. Hallgren, Circulating hyaluronic acid levels vary with physical activity in healthy subjects and in rheumatoid arthritis patients.Relationship to synovitis mass and morning stiffness. Arthr. Rheum.; 1987, 30, 1333-1338. 67. T.C. Laurent, U.B.G. Laurent & I.R.E. Fraser, Serum hyaluronan as a disease marker. Ann. Med., 1996, 28, 241-253. 68. E.A. Balazs, Physiology of the vitreous body. In: Importance of the vitreous body in retina surgery with special emphasis on reoperations. C.L. Schepens (ed.) C.V. Mosby, St. Louis 1960, pp.27-48. 69. E.A. Balazs, Ultrapure hyaluronic acid and the use thereof, United States Patent, 1979, No. 4,141,973. 70. N.W. Rydell, I. Butler & E.A. Balazs, Hyaluronic acid in synovial fluid. VI. Effect of intraarticular injection of hyaluronic acid on the clinical symptoms of arthritis in track horses. Acta Vet. Scand; 1970, 11, 139-155. 71. D. Miller & R. Stegmann (eds.), Healon (sodium hyaluronate). A guide to its use in ophthalmic surgery. Wiley, New York, 1983. 72. T.C. Laurent (ed.), The chemistry, biology and medical applications ofhyaluronan and its derivatives. Wenner-Gren International Series vol. 72, Portland Press, London, 1998.
Karl Meyer - Discoverer of Hyaluron Karl Meyer was born September 4, 1899. In 1917, at the age of 17, Karl Meyer was drafted into the German army and served the last year of the war on the Western Front in Flanders and Central France. It is quite possible that his experiences during this cataclysmic conflict were important factors in his decision to redirect his studies from the classics (panel 1) toward medicine. 1. BEGINNINGS
I was born on September 4, in Kerpen, Cologne, Germany as the fourth child and only son of Ludwig and Ida Meyer. Kerpen was then a village ofabout 4,000 people. I grew up in a simple rural household where from early childhood on, I, with the rest ofthe family had my assigned duties in the house, garden and fields. My first reading instruction at 4 years of age was in Hebrew. At 5·112 years I joined the Jewish School of Kerpen and at 10 transferred to the Hiihere Schole in Kerpen. This was a private Catholic gymnasium with almost exclusive emphasis on Latin and Greek. National Academy of Sciences, 1967
ABOUT THE AUTHOR Dr. Karl Meyer was born in Kerpen (Cologne) Germany in 1899. He was awarded his M.D. degree from the University of Cologne in 1924 and his Ph.D. degree in chemistry m Berlin in 1927. Dr. Meyer came to the United States In 1930 as an As· sistant Professor of Experimental Biology at the University of California. From 1933 to date he has been Assistant Professor of Biochemistry and Chemist to the Institute of Ophthalmology. Associate Professor and Professor of Biochemistry. Columbia University. Since 1948 he has been assigned to the Department of Medicine. Among the many honors bestowed on Dr. Meyer are: the Lasker Award. 1956. the T. Duckett Jones Award. 1959. the Gairdner Award (Canada) 1960. the Award of the New York Medical College. 1961 and a Fellowship In the American Academy of Arts and Sciences. 1965.
After demobilization he entered medical school and received his M.D. in 1924 from the University of Cologne (panel 2). He worked as a clinician for the last time during his final months of internship at Cologne in the division of Infectious Diseases, where he treated women terminally ill with tuberculosis and was at considerable risk for contracting this, at the time, dread disease. Dr. Meyer then went to Berlin to take a one-year course in medical chemistry. There he met several promising young scientists embarking on distinguished careers, including Hans Krebs, Fritz Lippman, and Ernst Chain, among others. At this major crossroad in his life, Meyer decided to obtain further training in chemistry, eventually enrolling as a graduate
18
Overview of the history and development ofhyaluronan
student in Otto Meyerhofs laboratory at the Kaiser-Wilhelm Institute. His thesis work on the enzymatic formation of lactic acid in muscle tissue and in yeast fermentation showed that the reaction required a heat-stable "co-enzyme", later identified as ADP, and launched him on his research career path. In 1927, Dr. Meyer received a Rockefeller Foundation Fellowship to study with Professor Kuhn at the Federal Swiss Institute of Technology in Zurich, where he spent almost 3 years studying the ability of heme complexes to catalyze the oxidation of unsaturated compounds. In 1930, he accepted an offer from Herbert Evans to work on anterior pituitary hormones as an Assistant Professor at Berkeley. In April, he and his new bride, Martha, whom he had met in Zurich, embarked on an ocean liner for New York City. At Ellis Island, they found that he had been issued a tourist visa, rather that a work permit. However, a sympathetic immigration officer suggested that they have a nice vacation on their way to California, and instructed him to go to a United States Consulate in either Mexico or Canada to obtain the appropriate documents. After a scary 2 days in Tijuana, Dr. Meyer succeeded in doing so and was then able to accept his position at Berkeley.
Dr. Meyer attended a conference in Europe in 1932 and faced another major crossroad. He learned at the conference to his dismay that Dr. Evans was terminating his position at Berkeley and recommended that he stay in Germany. However, Dr. Meyer decided to return to the States, perhaps sensing the storm clouds of World War II on the horizon. After his arrival in New York, Hans Clarke at Columbia University provided him with an interim fellowship until he received a position as Assistant Professor in the Department of Ophthalmology at Columbia in 1933. Under some pressure to work on relevant tissue, Meyer initiated studies on lysozyme in tears and sought another source for a "mucoid" substrate for the enzyme. He considered the highly viscous vitreous humor as a likely candidate. The discovery of hyaluronan quickly followed (panel 3).
3. BIRTH ANNOUNCEMENT December 1934 J. BioI. Chern. 107:629-634 The Polysaccharide of the Vitreous Humor By Karl Meyer and John Palmer
"From the vitreous humor of cattle eyes a polysaccharide acid of high molecular weight has been obtained...As constituents there have been recognized a uronie acid, an amino sugar...It appears to be a substance unique in higher animals, and may be best compared with some ofthe specific polysaccharides ofbacteria... ... we propose, for convenience, the name "hyaluronic acid," from hyaloid (vitreous)
+ uronic acid...
Karl Meyer - Discoverer of hyaluronan
19
4.
It would take almost 25 years (panel 4) before his studies would link the two sugars identified in the classic 1934 paper together correctly to form the polymer that captures our interest this week. Along the way, a series of classic studies with hyaluronidases would prove essential in defining the structure.
s. . . . . . r...T. . I01I'u... OI'~C.&lUllnT \·... It1,N..I.~.lUI
THE HYDROLYSIS OF HHLt:RO:'\IC ACID BY PXEnrOCOCCAL HYALL"ROXIDASE'
o. ~URlCE M. ILIPPORT, ALFRED LINKER, AN' KARL ~IEYER
The experimental results depicted in panelS, for example, led to the correct interpretation that the limit digest by testicular hyaluronidase (unfilled circles) yielded mainly tetrasaccharides that could be cleaved to smaller disaccharides by the bacterial hyaluronidase (dashed line). 2. 4 "12·
,4
30
48
nilE If< HOURS
6.
The structure of the disaccharide (panel 6) was defined in the gem of a small paper published in Nature in 1954 clearly indicating that the bacterial enzymes are eliminases.
Production of Unsaturated Uronides by Bacterial Hyaluronidases OR
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,I
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'
_
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:
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()
i
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20
8.
Overview of the history and development of hyaluronan
Those of us who have worked hard to isolate hyaluronan oligosaccharides of defmed sizes can only admire the profile in panel 7 showing baseline resolution through l8-mers in fractions from an ion exchange column, collected without the benefit of a fraction collector in 1954. By the late 1950s (panel 8), Meyer's work was gaining recognition, no doubt prompting the comment in panel 9.
7. ........ f",.
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\'<>1.-''''''1.''''""" 180L~TIOl' OF OLIGOSACCHARIDES El'ZnL~ TlCALLY
PRODt"CED FROM HYALLlWl'!C ACID· 81' BERXARD WEISSMA~'"X.t KARL ~lEYER. PHYLLIS s.nfPSOX, ASO .\LFR.ED LIXKER
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iz
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9. 1958 American Societyof Biological Chemists
Symposium on Acid Mucopolysaccharides of Animal Origin Chairman: Karl Meyer It is my opinion that the mucopolysaccharides will never be a highly popular field in biochemistry, but they probably will not be relegated again to the insignificance and disregard in which they were held not so long ago.
Karl Meyer - Discoverer of hyaluronan
21
Hyaluronan was his first love, but Dr. Meyer by no means ignored other glycosaminoglycans. Early work published in 1937 (panel 10) used calcium chloride solutions to extract chondroitin sulfate from cartilage.
10. J. Bioi. Chern. 119:507-510,1937 On Glycoproteins VI. The Preparation of Chondroitinsulfuric Acid By Karl Meyer and Elizabeth M. Smyth It was observed that the chondroitinsulfuric acid salt of gelatin was soluble in a concentrated solution of calcium, barium, or strontium chloride. This observation was utilized in the extraction of chondroitinsulfuric acid from cartilage in neutral solution. Hitherto extraction with strong alkali has been employed for the preparation of chondroitinsulfuric acid. Since treatment with alkali might easily lead to decomposition, the present method of extraction by a neutral solution of CaCh seems advantageous. The major portion of the cartilage is a protein salt of chondroitinsulfuric acid.
This led to the hypothesis that the extracellular matrix was primarily a protein salt of "chondroitinsulfuric acid", a concept which prevailed until the mid-l 950s. However, as noted by Dr. Meyer in 1958 (panel 11), work primarily in Max Schubert's laboratory refuted this hypothesis by unmasking the core protein and laying the foundation for research on proteoglycans.
11. Josiah Macy Jr. Foundation, Conference 4, 1958 Chondroitin Sulfates Karl Meyer Meyer: It was known for quite some time that most ofthe chondroitin sulfates ofthe tissues do not occur as free polysaccharides, but rather as protein complexes. There have been many contributions to the literature of the protein complexes, including some of my own in 1936 which have now been proven wrong, namely, that the polysaccharide was bound to protein only by polar bonds. Dr. Schubert started some of the most fundamental studies on the protein complexes of chondroitin sulfate of cartilage.
22
Overview of the history and development ofhyaluronan
While chondroitin sulfate had been known for almost a century by this time, keratan sulfate remained to be discovered. Turning once again to tissue from the eye, this time cornea, Meyer isolated an unknown glycosaminoglycan. Initially he thought it might be a sulfated form of hyaluronan. However, it became clear that the sugar partner was galactose and not glucuronic acid, and he proposed the name keratosulfate (panel 12).
12. J. BioI. Chem., 205:611-616,1953 The Mucopolysaccharides of Bovine Cornea By Karl Meyer, Alfred Linker, Eugene A. Davidson, and Bernard Weissmann From bovine cornea, three distinct mucopolysaccharide fractions were obtained. They have been identified as (1) chondroitin sulfate, (2) a fraction resembling hyaluronic acid, and (3) a sulfated mucopolysaccharide, composed of equimolar quantities of glucosamine, acetyl, galactose, and sulfate, for which we propose the name keratosulfate. The last represents approximately half of the total mucopolysaccharide fraction of the cornea.
The same study proposed that hyaluronan was also present in cornea, a conclusion based on the observation that some of the glycosarninoglycans were undersulfated (chondroitin, as it turned out). By 1981, the time of the photograph in panel 13, Meyer had received many awards, including election to the National Academy of Sciences in 1967 (panel 14), and his reputation as the father of glycosarninoglycan chemistry was firmly established. At this time, he was back in the Department of Ophthalmology at Columbia as an emeritus Professor, having returned there in 1976 at the invitation of Endre Balazs after a 9-year stint as Professor of Biochemistry at Yeshiva University. He continued to work in the laboratory for a few more years, into the late 1980s, before failing health made this impossible.
13.
Karl Meyer - Discoverer of hyaluronan
23
14.
Looking back on my scientific career I have often wondered whether it was worthwhile to stick so tenaciously to a technicaUy di.f]U:ult and, conceptuaUy, apparently unexciting field, while my colleagues and friends shifted over to more fashionable and rewarding areas. The reasons for my persistence are manifold: among them a distaste for jumping in on ground broken by others. Besides, I felt committed to problems such as the biological functions of the mucopolysaccharides of connective tissues, to their role in differentiation, in cell membranes and in inherited diseases. National Academy of Sciences, 1967
15.
Fittingly, his last paper (pane) 15), published in 1983, returned to the eye, in this case a study of the glycosaminoglycans in the vitreous humor of a fish.
ISOLATION AND CHARACTERIZATION OF ICHTHYOSAN FROM TIJNA VITREOUS GEI\ARD ARMAND, ENDRE A. BALAZS. KARLMEYER.aadMORAIMA REYES Meix Biology LaIJt1raIory. Dqarmterfl t!f OpluMltMiogy. CoJu,e oj PJrylidan., fJNJ S~ Colambia UTrivu1i1y, HIM' yort. New York /0031 f~~Ul.lWl:iI'-'i_"..,.J""',
19lI2,
Karl Meyer died May 18, 1990, at the age of90.
DRY FILM MADE OF HYLAN TO PREVENT ADHESION BETWEEN TWO HEALING TISSUE SURFACES 'Endre A. Balazs, 2Nancy E. Larsen, 2Edward A. Leshchlner", 2John D. Boney, 'Vadirn Mitlitski, 2Edward G. Parent and 'Julie L. Whetstone /Matrix Biology Institute, 65 Railroad Avenue. Ridgefield. New Jersey, 07657, USA 2Biomatrix. Inc" Ridgefield, New Jersey. 07657, USA
ABSTRACT When the epithelial cell layer covering two adjacent tissues is removed accidentally or intentionally during surgical procedures, the underlying connective tissue will grow together during the wound- healing process. Similarly, when two connective tissue surfaces not covered by endothelium but separated by elastoviscous fluid containing high molecular weight hyaluronan are wounded by trauma or during surgical procedures, they can grow together during the healing process. Such adhesion between two tissue surfaces may interfere with function and the excessive new connective tissue formed (scar tissue) may exert pressure on adjacent nerves, causing chronic pain. This paper describes the use of new formulations of dry films containing only hylan. In animal models, this film prevented adhesion formation between two tissue surfaces denuded from their mesothelial or epithelial cell cover. The most important property of this film after it is hydrated by tissue fluids was that it still adhered to the tissue surface, ensuring its stay in place. Thus, it functions as a barrier material, separating the healing tissues. The films do not cause inflammation or foreign body reaction and they do not interfere with the healing of adjacent tissues. These films successfully prevented adhesions between tissue surfaces in liver and cecal abrasion models in rat and uterine horn abrasion models in rabbits. Such films can also be used as delivery vehicles for various drugs, influencing them by combining their physical barrier effect with regulation effects on the healing process. BACKGROUND The first use of the highly purified fraction of hyaluronan (non-inflammatory fraction of Na-hyaluronan, NIF-NaHA) to reduce adhesions around injured tendons in rabbits was reported in 19711• This hyaluronan (average molecular weight 2-3 million) was used in the form of elastoviscous solutions containing 8 mg/ml polymer. The foreign body reaction around subcutaneously implanted polyethylene tubing was also reduced by this hyaluronan solution '. The same hyaluronan preparation surpressed graft-versus-host reaction in mice and prolonged survival time of allogenic skin grafts in mice'. In the form of a viscoelastic paste (20 mg/ml), the same hyaluronan preparation was applied to the lacerated profundus tendons of the third and fourth fingers of owl monkeys. After 4 - 5 weeks of immobilization the hyaluronan-treated fingers showed significantly less flexion deformity than the saline controls. Hyaluronan did not interfere with the healing process of the tendorr', In the 1980s hylan B gel slurry was also shown to diminish adhesion formation in surgical models of rabbit extensor hallucis longus tendon 4 .5, In all these studies the
8
Adhesion formation and hyaluronan
elastoviscous hyaluronan fluid or the viscoelastic hylan gel did not interfere with the healing of the tendon and did not cause chronic inftamrnation''".
REQUIREMENTS One of the approaches to adhesion prevention uses polymer solutions, gels, or films which act as a physical barrier to adhesion formation. Regardless of surgical application, all barriers must be: safe, effective, resorbable, non-inflammatory, quick and easy to administer, compatible with normal healing, compatible with minimally invasive surgery, and potentially suitable as pharmaceutical carrier.
MATERIAL Success of the material used is determined by such factors as the residence time of the barrier at the applied location and the reaction it may cause. Hyaluronan is the material of choice due to its exceptional biocompatibility. However, when used as dry film or as concentrated paste, hyaluronan (with any molecular weight of less than 4 million) will disappear from the target site rather quickly and the reliable elimination of adhesions would be difficult. Films can be made of hylan with any average molecular weight of more than 4 million in such a way that it hydrates and subsequently dissolves very slowly, consequently providing a long but limited residence time. Such a film provides a temporary barrier that prevents the formation of fibrin clots connecting two tissue surfaces. Films used in this study were made from sodium salt if HY -A dissolved in water (Hylafilm®). Such a dry film is shown on Figure 1. This film in dry form is flexible, does not crack and slowly re-hydrates in physical salt solution.
Figure I
ANIMAL STUDIES CECAL ABRASION STUDY IN RATS Pathogen-free rats were used to evaluate the efficacy of hylan films (Hylafilm") in preventing peritoneal adhesions. Surgically created defects were produced on the abdominal wall and the proximal end of cecum. The control animals not treated with anything after surgery showed adhesions. All but one of the Hylafilrn'" treated animals were free of adhesions'r" ( Figure 2).
Dry film
10
prevent adhesion
9
UVER ABRASION STUDY IN RATS Hylafilm'" was effective in preventing adhesion formation in the rat liver abrasion model. The membranes were found to adhere tightly to the liver surface without the use of sutures and were not readily displaced. 92% of animals with applied Hylafilm" did not develop adhesions. In contrast, only 8% of the control animals were free of adhesions. The surface wounds in all animals were completely healed indicating that Hylafilm®did not interfere with the wound healing process (Figure 3). RABBIT UTERINE HORN MODEL The ability of Hylafilm®to reduce the development of post-surgical adhesions was investigated in the rabbit uterine horn model (reference). Hylafilm® reduced adhesion formation compared with controls. 46% of the control animals showed adhesion formation, while only 20% of the Hylafilm@ treated animals showed comparable adhesions. The difference is significant.
Figure 3
Figure 2
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o
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Rat Cecal Abrasion
Hylafilm4ll
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Rat Liver Abrasion
PHYSICAL TESTING OF HYLAFILM® The physical properties of Hylat1lm® can be controlled by various factors such as molecular weight of hylan and starting concentration. The following physical characteristics of the test film formulations included: time required for films to break up in excess saline (Figure 4), force required for breakage of wet film using a TA-XT2i Texture Analyzer, Texture Technologies Corp., with lI8" ball probe (Figure 5), swell rate in excess saline (Figure 6), % elongation (strain) at breakage using a TA-XT2i Texture Analyzer with 118" ball probe (Figure 7).
10
Adhesion formation and hyaluronan Figure 5
Figure 4
Time Required lor a Filmto Break UpWhen In Excess Saline with Increasing Molecular Weight
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Comparison of '10 Elongation (Strain) at Breakage for Bacterial HA (HAl andHylan (HY) at 1'10 and2%Polymer Content
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HYLAFILM@ FOR TARGETED DRUG DELIVERY Sustained delivery of antineoplastic drugs was investigated by making a Hylafilm@ sample and loading a drug into the sample using the appropriate solvent. As a control, Whatman® filter paper was loaded with the drug in the same manner. The samples were placed in vials with physiological salt solution and separated from the delivery compartment by a 0.45 Jlm filter. The samples were agitated for the whole duration of the experiment to assure proper mixing. Samples from the delivery compartment were removed for testing for drug concentration at various times (Figure 8).
Dry film to prevent adhesion
11
Figure 8 DrugDelivery from Hylafllm'"
8 7 Time (hours) Required to Release 50%of the Drug
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MEDICAL USES OF HYLAN FILMS Adhesion prevention and/or reduction may be attained using Hylafilm" in the following applications: orthopedic surgery to prevent adhesions between tendon and tendon sheaths and after joint replacement surgery to prevent post-surgical adhesions, cardiac and pleural surgery to decrease pericardial adhesions, neurosurgery around peripheral nerves to prevent excessive scar formation which could exert pressure on the nerves and cause chronic pain. Hylafilm@ may also be used in ophthalmic surgery to maintain post-surgical exit channels for aqueous flow after glaucoma surgery.
CONCLUSIONS Dry films made of hylan were developed to be used as a therapeutic device to separate healing tissue surfaces after a broad range of surgical procedures in abdominal, pleural and pericardial locations. The interposition of such films may prevent or decrease postoperative connective tissue formation that may cause adhesion between a broad spectrum of tissue surfaces. Hylafilm@ can also be used to wrap around tendons and nerves to prevent adhesions and decrease scar formation. The major advantage of dry films made of only pure hylan is that they are made of the same hyaluronan molecule that is already present in most tissue spaces and therefore do not behave as foreign materials and do not interfere with the healing process of the wounds. Hylan film can be the carrier of a variety of drugs that will diffuse slowly from the hydrated film, assuring targeted delivery.
12
Adhesion formation and hyaluronan
REFERENCES 1. N. Rydell and E. A. Balazs, Effect of intra-articular injection of hyaluronic acid on the clinical symptoms and on granulation tissue formation, CUn. Orthop., 1971, 80, 25-32. 2. E. A. Balazs and Z. Darzynkiewicz, The effect of hyaluronic acid on fibroblasts, mononuclear phagocytes and lymphocytes. In: Biology of the Fibroblast (papers of the symposium held in Turku, Finland, 1972), E. Kulonen and J. Pikkarainen (eds.), Academic Press, London, 1973, 237-252. 3. R. St. Onge, C. Weiss, J. L. Denlinger and E. A. Balazs, A preliminary assessment of Na-hyaluronate injection into "No-Man's Land" for primary flexor tendon repairs, CUn Orthop., 1980, 146,269-275. 4. C. Weiss, H. J. Levy, J. L. Denlinger, J. Suros and H. Weiss, The role of Na-hylan in reducing postsurgical tendon adhesions, Bulletin of the Hospital for Joint Diseases, Orthopaedic Institute, 1986,46(1),9-15. 5. C. Weiss, J. Suros, A. Michalow, J. L. Denlinger, M. Moore and W. Tejeiro, The role of Na-hylan in reducing postsurgical tendon adhesions: Part 2, Bulletin of the Hospital for Joint Diseases, Orthopaedic Institute, 1987, 47(1), 31-39. 6. E. S. Harris, P. A. Foresman, G. T. Rodeheaver, N. E. Larsen and E. A. Balazs, Efficacy of a resorbable hylan barrier membrane in the prevention of adhesions in a rat cecal abrasion model, Fifth World Biomaterials Congress, Toronto, Canada, May 29-June 2,1996, 371(abstract). 7. N. E. Larsen, Management of adhesion formation and soft tissue augmentation with viscoelastics: hyaluronan derivatives, In: The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives (Proceedings of the Wenner-Gren Foundation International Symposium held in honor of E. A. Balazs, September 18-21, 1996, Stockholm, Sweden), T. Laurent (ed.), Portland Press, London, 267-281.
ALEXANDER G. "SANDY" OGSTON (1911-1996) FATHER OF BIOPHYSICAL CHEMISTRY OF THE MATRIX Some of Sandy Ogston' 5 collaborators:
Sandy Ogston worked at: Oxford University (1933-35) London Hospital (1935-38) Oxford University (1938-59) AND, Canberra (1959-70) Trinity College, Oxford (1970-78)
Baruch S. Blumberg Martin Davies 1. R. Dunstone Elizabeth Edmond Susan Farquhar John H. Fessler 1. P. Johnston Torvard C Laurent Laurie W. Nichol Charles F. Phelps John Philpot Barry N. Preston T. F. Sherman Panee Silpananta Oliver Smithies Jean E. Stanier John D. Wells Donald 1. Winzor E. F. Woods
CHARACTERISATION OF THE BYALURONAN MOLECULE In a series of papers Sandy Ogston and coworkers determined the molecular parameters of a hyaluronan preparation containing up to 30 % protein isolated from synovial fluid: A. G. Ogston & 1. E. Stanier, 'The dimensions of the particle of hyaluronic acid complex in synovial fluid', Biochem. J., 1951, 49, 585-590.
B. N. Preston, M. Davies & A. G. Ogston, 'The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma', Biochem. J., 1965, 96, 449-474. After using various physical chemical techniques they drew the conclusions that hyaluronan had a molecular weight in the order of 107 Da, that it had the configuration of a highly hydrated sphere, which is compatible with an extended, "possibly" crosslinked, random coil with a radius of gyration of200-250 nm.
26
Overview of the history and development ofhyaluronan
.............
i
.... ......
...........
.....
~~ l _ OH
~
-+4)-13-D-GlcpA-(1-.+3)-13-D-GlcpNAc-(l-.+
PHYSIOLOGICAL FUNCTIONS OF HYALURONAN Ogston discusses the biological function of hyaluronan: 1) He analyses the rheological properties of synovial fluid and solutions of hyaluronan (viscosity, elasticity, concentration- and shear dependence) and connect them with lubrication: A. G. Ogston & J. E. Stanier, 'The physiologica function of hyaluronic acid in synovial fluid; viscous, elastic and lubricant, properties', J. Physiol., 1953, ll2, 244-252.
2) He measures factors of importance for water homeostasis in the tissues, i.e. the osmotic pressure and flow resistance ofhyaluronan solutions:
Alexander G ("Sandy") Ogston
27
B. N. Preston, M. Davies & A. G. Ogston, 'The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma', Biochem. J., 1965, 96, 449-474. 3) He studies interaction of hyaluronan with other macromolecules - exclusion and restriction of movement. These properties are important for distribution and transport of plasma proteins in tissues: A. G. Ogston & C. F. Phelps, 'The partition of solutes between buffer solutions and solutions containing hyaluronic acid', Biochem. J., 1960, 78, 827-833. A. G. Ogston, B. N. Preston & J. D. Wells, 'On the transport of compact particles through solutions of chain-polymers', Proc. R Soc. Lond. A, 1973, 333, 297-316.
THE MOST CITED DISCOVERIES BY SANDY OGSTON ARE: 1) The Johnston-Ogston effect which explained a puzzling phenomenon observed in analytical ultracentrifugations of protein mixtures: J. P. Johnstone & A. G. Ogston, 'A boundary anomaly found in the ultracentrifugal sedimentation of mixtures', Trans. Faraday Soc., 1946, 42, 789-799.
2) The three-point attachment. Ogston explained how a symmetric substrate in the Krebs cycle could be handled by the enzyme as asymmetric through a three-point attachment: A. G. Ogston, 'Interpretation of experiments on metabolic processes, using isotope tracer elements', Nature, 1948, 162,963.
3) Available volume for a sphere in a random network of rods. The paper is frequently cited in connection with macromolecular transport through gels (gel electrophoresis; gel chromatography): A. G. Ogston, 'The spaces in a uniform random suspension of fibers',
Trans. Faraday Soc., 1958, 54,1754-1757.
THE CONCEPT OF EXCLUSION Ogston realised that the available fraction (K.,v ) in a random network of rods is limited for spherical particles. The larger the sphere and the higher the concentration of rods the less space. If the rods are regarded as infinitely long the equation takes the form: K av = exp [- L (r, + rril where r, and r, are the radii of the sphere and the rods, respectively and L is the concentration of rods expressed in ern/ern': A. G. Ogston, 'The spaces in a uniform random suspension of fibers',
Trans. Faraday Soc., 1958, 54,1754-1757.
28
Overview of the history and development ofhyaluronan
Kav can be determined by various techniques such as partition of proteins between hyaluronan and buffer solutions or by gel chromatography on hyaluronan gels. The results show that hyaluronan excludes proteins as expected if it forms a random network of single elongated polysaccharide chains: T. C. Laurent, 'The interaction between polysaccharides and other macromolecules,
9. The exclusion of molecules from hyaluronic acid gels and solutions', Biochem. J., 1964, 93, 106-112. At low hyaluronan concentrations one gram of hyaluronan excludes 50-100 mL solvent for albumin. This has been verified by osmometry on mixtures of hyaluronan and albumin as well as by light-scattering: T. C. Laurent & A. G. Ogston, 'The interaction between polysaccharides and
other macromolecules, 4. The osmotic pressure of mixtures of serum albumin and hyaluronic acid', Biochem. J., 1963, 89, 249-253. B. N. Preston, M. Davies & A. G. Ogston, 'The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma', Biochem. J., 1965, 96, 449-474. A. G. Ogston & B. N. Preston, 'The exclusion of protein by hyaluronic acid measurement by light scattering', J. Bioi. Chem., 1966, 241, 17-19.
Torvard C. Laurent
ADHESIONPREVENTION EFFECTSOFTOLMETIN COMBINED WITH HYALURONIC ACID Shaojin Wangl JThe
Peixue Lini
2
YanliBe
Tianmin Zhang'
FirstAffiliatedHospitalofShandongMedical University, Jinan250012, China: 2Shandong C.P.Freda Pharmaceuticals Co; Ltd, Jinan250014, China: 3 Shandong Biopharmaceuticals Institute,
Jinan250014, China.
ABSTRACT
This study was to investigate the effects of intra-sheath injection of tolmetin sodium plus sodium hyaluronate (folmetin-HA) on adhesion formation in chicken flexor tendon model. Seventy-five chickens were divided into three groups. The long flexortendon of centraldigit of all chickens was incised and sutured with a modified Kessler stitch. Tolmetin-HA (0.1% of tolmetin sodium and 1% ofHA in PBS) or HA (1% HA in PBS) or saline was appliedto each group respectively by injecting 0.2ml into intra-sheath space near the conclusion of the surgical procedure. The chickens were executed 2 weeks after the opemtion. The extent of adhesion formation in each chicken was evaluated grossly and histologically. The results indicated that tolmetin-HAsignificantly reduced the extent and severity of post-surgical tendon adhesion in comparison with HA or saline (P
Tolmetin, sodium hyaluronate, adhesion formation, fcxor tendon INTRODUCTION
Adhesionformation, a majorcause ofpostopemtive morbidity, depends upontwo factors: the leaking of serosanguinous fluid, including fibrinogen (leading to fibrin deposition), and the impairedlysisofthe fibrinmatrixthat does form. Periopemtive administration of nonsteroidal anti-inflammatory drugs (NASIDs), including ibuprofen and tolmetin, can reduce adhesion formation after peritoneal surgery. Previous studies' showed that administmtion of tolmetin in sodium hyaluronate (HA) solutionthrough injectionunderthe injuredsurfaceof the peritoneum or intra-sheath space nearthe conclusion of the surgical procedure significantly reduced the extent and the severity of post-surgical abdominal and tendon adhesion in the animal models. In this paper, the efficacy of tolmetin combinedwith HA in preventing post-surgical tendonadhesionin animal model was fonfirmed.
14
Adhesion formation and hyaluronan
MATERIALS AND MEmODS
Drug HA Injection: Sofust1M, 2ml contains20mgHA, which has a mean molecularweight rangeof (1.5-2.0)x 106Da, providedby ShandongC.P. FredaPhann. Co. Tolmetin-HA Injection: 2ml contains2mg oftolmetin sodiumand 20mg ofHA, provided by ShandongC'P FredaPhann. Co. Saline:purchasedfrom pharmacy
Animal model Seventy-five adult chickens of either sex, with a body weightrange 2.5-3.5kg were chosen. The surgical model was made accordingthe methods designed by Daley et all. The chickens were divided randomly into three groups, i.e. tolmetin-HA group, HA group and saline group. An incision was made in the plantar aspect of the long flexor tendon of central digit on the chicken's right foot, the tendonsheath was incisedand the flexor sublimis tendon was exposed and excised in order to increase the intra-sheath space. A incision was made in the flexor profundustendon and sutured with a modified Kessler stitchusing 5-0 Maxon, and the tendon sheath was closed with a simple continuous stitch using 9-0 single nylon thread. Following closureof the sheath,0.2mlof drug or saline was injectedintointra-sheath region. The skin was closed with simple interrupted stitches. The foot was immobilized and kept in a natura1 position withplaster, leavingthe plantaraspect exposed. All ofthe chickenswere returned to theircages and subjected to do flexionexercises 30 times passively, once a day sincethe day after operation. The plaster was removed2 weeksafterthe operation. All of the chickenswereexecuted. The skin sutures were removed and the toe was dissected along the original incision. Gross and histological evaluationsweremade on the extentand severity of adhesionformation.
Gross evaluation The semiquantitative grading scale (Table 1) 2 was used to evaluatethe extentand severityof adhesionformationwithinthe intra-sheath region.
Table 1. Semiquantitative grading scalefortendon adhesions Description Grade No adhesion 1 Filmy(separable) adhesion, tendon was able to glidefreely. 2 Mild (not separable), fibrousbandswere formed. Glidingfunction was interfered 3 slightly. Moderate (35....(j0% of area), densefibrousadhesionformed and glidingfunction 4 wasinterferedseriously. Severe(>60%of area),dense fibrous adhesion formed and coveringvirtuallythe 5 entire surgical field. The tendoncan'tglide.
Adhesion prevention effects of tolrnetin
15
Histologicalevaluation Hematoxylin-erosin stained sections was used to quantitate the number of neutrophils and maerophages. Masson's trichrome stained sections were used to evaluate the collagen organization. Toluidine-blue stained sections were used to evaluate fibroblasts. Samples for microscopic analysis werefixed, rinsedand stainedaccording to routine procedures. Statistical analysis Wilcoxon, Mann and Whitney SignedRank Test was usedto analyze the significance of the difference in the distribution of severity grades of adhesion between the treatment groups and the control group. Two-tailed statistical test was used and P=:{).05 was taken as the threshold abovewhichdifferences are reported as non-significant. RESULTS & DISCUSSION The incised flexor tendon of seven chickens in two treatment groups and four chickens in control groupruptured at the suturesite,and were excludedfrom statistical analysis. Gross evaluation Each specimen wascategorized basedon its maximumseverity of the adhesions present. The semiquantitative gross evaluations of the threegroupsare shown in Table2. Table 2. Group
The results of gross evaluation of the three grOUPS Number
1
2 Gr;cte
4
5
Tolmetin-HA* 22 5 9 6 2 0 HA** 21 0 2 5 8 6 Saline 21 0 1 2 9 9 *: P
O.05, in comparison withsalinegroup. Histological evaluation The results of histological evaluation of the three groups are shownin Table 3. Table3. The results ofhistologicalevaluation of the three grOUps* Numberof Collagen fabrin Fibroblasts macrophages Number Arrayorder Density Array order SaJine>HA==
Tolmetin-HA Saline>HA> Saline> HA?= Tolmetin-HA ?=HA>saline Tolmetin-HA tolmptin.HA =I-IA>saljne *: symbols: > indicates more or superior, ;:?:indicates the tendency for superior; ""'indicates equivalent. Iolmetin-HA
Significant increase of fibroblasts arrayed densely and disorderly was observed in the
16
Adhesion formation and hyaluronan
specimens of HA and saline group, but the situations in tolmetin-HA group were fewer and much milder. The newbornfinecollagen fibrils began to form in the initial stage. Even though the newborncollagen fibrils arrayed disorderly in saline group, theyarrayedas parallel bundles in good order in the samples of the HA group and tolmetin-HA group, and the situation was more evident in Tolmetin-HA group. Macrophages accumulated more significantly in saline groupthan in HA group,but the situation was not evidentin tolmetin-HA group. Gross observations indicated that significantly fewerand lesssevereadhesion occurredin the tolmetin-HA group than in the HA group and the saline group. The results of histological evaluation also indicated a beneficial tendency of tolmetin-HA over HA, even though these indices were not assessed quantitatively. HA showed a beneficial tendency of preventing adhesion in gross observation as well as in histological evaluation, but the difference was not significant in comparisonwith saline. A single dose of tolmetin sodium (20mg) was not effective in the reduction of adhesion formation, most likelydue to the rapidclearance from the injured site. Harris'demonstrated that adhesions continueto form in animalmodelsfor at least 36h after the initial injwy Rodgers et al2 also found that 2 to 3 days of delivery were necessary to elicit the beneficial effects of tolmetin Therefore, further studies were conducted to identify a suitabledrug deliverysystem allowing prolongedexposure of the injury surfaceto the drug, which wouldbe more effective. Many results indicated that the key issue in tolmetin delivery is how long the drug can be in contactwiththe injurytissue. HA itself has anti-adhesive effects. Balazs and many investigators' reported the effect of hyaluronan on adhesion formation between tendonand tendon sheaths. Solutions ofviscoelastic, high-molecular-weight hyaluronan were found to reduce the incidence of adhesion formation The molecular network of viscoelastic HA forms an effective barrierfor fibrinogen as well as cells, reducing the number of cellsthat migrate into the injury site and leading to less collagen deposition'. As a vehiclefor tolmetin, HA mayact to slowthe releaseof thedrugand to preserveits action over a prolonged period of time. The results of the this paper indicated that tolmetin-HA has high efficacy in preventing post-surgical tendonadhesion The mechanism may be based on the following: mechanical separation of the wound serosa from the other normal serosa by HA molecular network; supression of bleeding by tolmetin-Ha, which reduces the numberofblood clots that are the scaffolds of permanent adhesions; the pharmacological effectof tolmetin-HA on the function, migration, adherence, phagocytosis, and proteolytic enzyme secretion of post-operative inflammatory cells, which may enhance the degradation of the fibrinous adhesion.
REFERENCE I. RA. Daley, L.M. Cannistra, WR Walsh, et al, A chicken modelfor flexor tendon adhesion studies. Proc. .Orthop., Res., Soc., Annu, Mtg., 1992,38,103-110. 2. K Rodgers, W Girgis, GS. diZerega, D.B. Johns, Intraperitoneal tometin prevents post-surgical adhesionformation in rabbits. Int. J. Ferttl., 1990,35,40-45. 3. E.S. Harris,RE Morgan,GT. Rodheaver, Analysis of the kinetics of perionteal adhedion in the rat and evaluation of potential antiadhesive agents. Surgery, 1995, 117, 663-669. 4. N. Rydell, Decreasedgranulation tissue reaction after installment of hyaluronic acid, Acta. Orthop. Seand, 1970,11,307-311. 5. N.E. Larsen, The chemistry, biology and medical applications qf hyaluronan and its derivatives, PortlandPress,London, 1998, 267-281.
ALBERT DORFMAN
(6tb July, 1916 - 27tb July, 1982 Albert Dorfman's research on the biosynthesis and chemistry of bacterial and connective tissue polysaccharides, conducted over a period of more than 35 years, provided the basis for many medical advances in human biochemical genetics, including prenatal diagnostics of genetic diseases that cause mental retardation. One of his many scientific accomplishments was the discovery of the cause of Hurler's syndrome, a crippling genetic disease that affects most of the tissues and organs of the body and results in mental retardation. THE EARLY YEARS Albert Dorfman was born and raised in Chicago, the third child of Russian Jewish immigrant parents. His father was a manger of a metalware factory and his mother was a seamstress. Although his parents had received no formal education, they placed great emphasis on scholarship and instilled a love for learning in their children. Al's older sister was a pre-law and accounting student and an accomplished singer, which fostered a lifelong interest in music in her younger brother. His older brother Ralph I. Dorfman, who later became a well-known steroid biochemist and, like Al, a member of the National Academy of Sciences, had developed an interest in mathematics and science early in life and had a great influence on Al by emphasising high academic achievement and kindling an interest in chemistry. His younger sister, Florence, became a mathematician and married Professor Nathan Jacobson, also a member ofthe Academy. In 1940 Al married Ethel Steinman, and they had two daughters, Abby and Julie. Early schooling was a pleasant experience that Al pursued with an all-consuming energy. While AI was in high school, his interest in science gradually matured as a result of the stimulation received from his brother, who was then majoring in chemistry at the University of Illinois. Since his high school years were during the depths of the Great Depression, higher education seemed impossible. This was all changed by a high school teacher who convinced AI to take the competitive scholarship examinations at the University of Chicago, and success in obtaining a scholarship then opened a vista of higher education. When AI arrived at the University of Chicago, with the limited background of public schooling, the new college system was just getting underway. The intellectual stimulation was intoxicating, and during the first year, almost weekly, he was ready to change his career to such diversified fields as sociology, economics, and history. However, stimulation in the sciences was greatest, largely as a result of the teaching by many great scientists in the introductory courses at the college. Medicine or Chemistry, or Both? Gradually, it became clear to Dorfman that he wished to pursue a career in medicine or chemistry; the conflict between the two was never to be completely resolved.
30
Overview of the history and development ofhyaluronan
Accordingly, after first pursuing a curriculum in chemistry, he switched to biochemistry and also entered the University of Chicago School of Medicine during his senior year in college. However, he found biochemistry so interesting that he began work in this discipline and dropped out of medical school after two years. Early in his graduate work he came in contract with Felix Saunders, who was then interested in bacterial metabolism, a field yet in its infancy. This talented but unrecognised scientist played a most important role in Dorfman's scientific and personal development. Saunders foresaw the great advantages microorganisms offered for the study of metabolism and was also an accomplished carbohydrate chemist. This early training was to be of great advantage when Dorfman subsequently became interested in carbohydrate-containing macromolecules. His Ph.D. thesis research was concerned with the identification of nicotinamide as a growth requirement for Shigella dysenteriae and the synthesis of various nicotinic acid derivatives to correlate structure with biological activity (1939). The Ph.D. Degree - A Door to What? After receiving the Ph.D degree from the University of Chicago in 1939 (at the age of 23), Dorfman tried in vain to obtain a position or a postdoctoral fellowship to study elsewhere. During this period, he became intrigued with the beginning expansion of enzymology, resulting particularly from the studies of Warburg and von Euler. With stimulation and help from R. W. Gerard and E. Guzman-Barron, he learned some of the early techniques of enzymology and remained at the University of Chicago as a research associate, initiating studies on the role of bacterial growth factors in metabolism (by then known to be vitamins). These studies led to development of the technique of growing deficient cells to be used to determine the role of growth factors in metabolism. This short period was extremely productive and led to the discovery of the role of pantothenic acid in pyruvate metabolism (1942), the role of biotin in aspartic acid biosynthesis (1942), and one of the earliest suggestions that drugs may be competitive inhibitors in enzyme reactions (1942). Medicine Again With the advent of World War IT and lack of an academic position, Dorfman returned to medical school, graduating in 1944. Contrary to his expectations, exposure to clinical studies immediately rekindled an interest in medicine, in particular pediatrics. An early encounter with a child with rheumatic fever stimulated an interest in the mechanism of action of aspirin and profoundly affected Dorfman's subsequent career. Following completion of medical school and an internship at Beth Israel Hospital in internal medicine, he served a residency in pediatrics at the University of Chicago. Biochemistry in the U.S. Army Dorfman then served two years in the U.S. Army, and being assigned to the Army Medical School he was able to take up a career in biochemistry. Because of a publication at this time by Guerra claiming that aspirin exerted its antirheumatic effect by inhibition of hyaluronidase, he initiated studies on connective tissue polysaccharides, an area of research which he pursued for the next thirty years. In particular, his earlier experiences in bacterial metabolism and carbohydrate chemistry served as an excellent background to purse the biosynthesis of hyaluronic acid in Group A streptococci. These studies led to development of quantitative methods for assays of hyaluronidase (1948),
AlbertDorfman
31
discovery that chondroitin sulfate was a substrate for testicular hyaluronidase (1951), and recognition that hyaluronidase was unusually stable to heat and acid pH (1954), special properties that were later recognised as those oflysosmal enzymes. BIOSYNTHESIS OF HYALURONIC ACID Upon returning to the University of Chicago, Dorfman initiated studies on the synthesis of hyaluronic acid with the goal of determining the origins of the fourteen unique carbon atoms of the polysaccharide, using specifically labeled precursors. At that time, the biosynthetic reactions leading to formation of hexosamine and glucuronic acid were unknown. Together with Saul Roseman, [l-14C]glucose, [6_1 4C]glucose, and [1-14C]acetic acid were synthesised. it was then established that glucose was converted to the glucosamine and glucuronic acid portions of the molecule without scission of the carbon chain, that acetate was a precursor of the acetyl group ofN-acetylglucosamine, and that glucosamine but not N-acetylglucosamine served as a precursor of the N-acetylglucosamine residue in hyaluronic acid (1953. 1954, 1955). Besides Dorfman and Roseman, the participants in these investigations included Julio Ludowieg and his wife, Frances Moses, whose grandmother on her father's side was Anna Mary Robertson Moses, better known as Grandma Moses (1860-1961). In parallel with this work, similar studies were carried out on mammalian polysaccharides - hyaluronic acid and dermatan sulfate of rat and rabbit skin - with the added dimension that the turnover rates of these polysaccharides in vivo were also determined. In these studies, now part of the classical accomplishments in the field, it was established that the two polysaccharides have a surprisingly rapid turnover with half-lives of only a few days. Following the same pattern of experimentation, Sara Schiller and Dorfman subsequently carried out a number of studies on the effects of various hormones on the metabolism ofthe glycosaminoglycans. The discovery of uridine nucleotide sugars by Luis Leloir suggested that these compounds may be intermediates in polysaccharide synthesis. The identification of certain uridine nucleotide sugars and the appreciation of their role in monosaccharide interconversions and as glycosyl donors then occurred in rapid succession. Together with 1. A. Cifonelli, Dorfman established that streptococci contained the two uridine nucleotide sugars, UDP-N-acetylglucosamine and UDP-glucuronic acid, requisite for the biosynthesis of hyaluronic acid (1957). The chance observation oflarge amounts of UDP-glucuronic acid in one batch of streptococci made it possible to prepare substrate amounts of labeled nucleotide sugar by the Wilzbach procedure. With the labeled nucleotide, synthesis of hyaluronic acid in a cell-free preparation of streptococci was then quickly demonstrated together with Alvin, Markovitz and 1. A. Cifonelli (1959). This work followed earlier studies by Glaser and Brown, who had obtained evidence for the formation of small hyaluronic acid oligosaccharides in a cell-free preparation of the Rous sarcoma, but the investigation by Dorfman and coworkers represented the first conclusive demonstration of the formation of macromolecular hyaluronic acid. Together, these investigations were the first to show the cell-free synthesis of heterologous polysaccharide and established a basis for understanding the mechanism of synthesis of many other complex carbohydrates. In a farsighted prediction it was suggested that a single enzyme catalyses the assembly of the hyaluronic acid chain, and a mechanism was proposed which, however, subsequently has been revised. Attempts to solubilise the enzyme failed, but led to the important conclusion that the enzyme responsible for glycosyl transfer in contrast to those required for nucleotide synthesis »
32
Overview of the history and development ofhyaluronan
was located on the protoplast membrane (1962). This was one of the first observations relating macromolecular synthesis to membrane-associated enzymes. More than twenty years later, Nancy B. Schwartz and Louis Philipson localised the mammalian hyaluronic acid synthetase to the inner side of the plasma membrane and proposed a mechanism for membrane-bound synthesis of the hyaluronic acid polymer. Apart from his seminal contributions to our knowledge of hyaluronic aid metabolism, Dorfman made many other important discoveries in the field of connective tissue research, but a full account of his accomplishments is beyond the scope of his brief memoir.
ALBERT DORFMAN AS WE KNEW HIM AI Dorfman had an imaginative, inquisitive, well-trained, and disciplined mind. He taught his associates the self-evident lesson that is so easy to forget: learn the facts, formulate a problem, and design a logical solution. In one word: "Think!". And when AI showed you how, it seemed so easy as if we could all do it with just a little effort. What AI always strived to accomplish in his thinking was to predict the further consequences of a certain experiment or a certain action. He would advise: time is too short to do the obvious experiment, try to do more important and interesting one after that. This attitude was a hallmark of Ai's intellect: be it a matter of probing the ins and outs of a scientific problem, or considering diagnostic and therapeutic problems, or dealing with the personal and professional problems of his associates, he would always try to like the master chess player to anticipate the future consequences of his moves and decisions. But unlike the chess player, AI was dealing with human beings, and he was keenly sensitive and understanding of the souls of his fellow human beings. He might on occasion chastise a fellow scientist for faulty experimentation or sloppy thinking, if he thought that that individual ought to know better and be capable of doing better. He would be an aggressive protagonist for his own scientific accomplishments and those of members of his immediate laboratory family, but he would also rise to defend and encourage the insecure graduate student or the Far East foreigner giving his first presentation at the Federation Meetings in faltering English. Al's sensitivity to other people was particularly evident when he saw him in his role as pediatrician. The child that was scared of the hospital and all the white coats could often be soothed by Al's kind and reassuring words and manners. He was, as a niece of his said at a memorial symposium in 1982, "Uncle AI, all children's pal." Kajsa and Lennart Roden saw how their youngest daughter Madeleine, very shy, did not remain shy for long in Al's company and used to crawl up into his lap and sit quietly for a while and enjoy his company. When AI became a grandfather, the stories he told about his granddaughter Sara were legend, and one day he reported on her exceptional prowess in mathematics at an early age. He told us that Sara had been able to count to 214, and he had then asked her: "If you can count that far, how come you can't count any further?" Sara's reply: "I get tired at 214." It was also in his role as a pediatrician that AI unexpectedly, one day, had to display unusual courage in the face of clear physical danger. When Martin Luther King was assassinated and Chicago burned, Al went to the university's outreach clinic in the black ghetto in Woodlawn together with its director, Jack Madden, to keep the clinic open for business as usual, as at the same time a couple of not so brave graduate students were fleeing from the burning city. Like Martin Luther King, AI Dorfman had a dream of the betterment of humankind, and he worked with great sense of responsibility to make his dream become reality.
Albert Dorfman
33
Throughout Al' s life of service to others, however, an important reason that he was able to give so much was that he had his wife Ethel at his side, who also gave generously of herself, directly or indirectly through her support of Al. (The Rodens remember arriving at the airport in a snowy Chicago on January 3, 1957, where Ethel met them with two bags full of groceries and, especially thoughtfully, diapers for baby Ann-Sofi, a warm welcome for weary travelers, indeed.)
ALBERT DORFMAN, THE ADMINISTRATOR During his career, Dorfman held several important positions at the University of Chicago. He was director of the Kennedy Mental Retardation Center, chairman of pediatrics, Richard T. Crane distinguished service professor, and director of the La Rabida-University of Chicago Institute, a hospital for children with rheumatic fever and a connective tissue research center. Thus he was able to influence significantly clinical medicine, genetics, and developmental biology at the University of Chicago. He was instrumental in the construction of Wyler Children's Hospital and the establishment of the Joseph P. Kennedy Mental Retardation Research Center, one of the charter mental retardation research centers from the National Institute of Child Health and Human Development. After giving up most major administrative responsibilities in the late 1970's, he indulged more in his scientific efforts where his enjoyment and enthusiasm for research were infectious. This was also the period when he was most adventurous. Although never afraid to go beyond his own sphere of expertise to burrow and apply new ideas and methodology to his own field, he was even more ready to speculate on mechanisms of differentiation and molecular genetics. IN IDS OWN WORDS Dorfman cared deeply about the scientific enterprise and was very concerned about the changes that had taken place and other pending changes that he considered detrimental to the future ofthe biomedical research endeavor. In his last years Dorfman always took the opportunity to complain about the constriction of research funding and was among the first to discuss the scientific, ethical, and social implications of genetic engineering and screening. Notably, the Ryerson Lectureship at the University of Chicago in 1978 and the address he presented when inducted as president of the Pediatric Society in 1979 contained pleas concerning the demise of basic research controversies in these areas. The final paragraph from the Ryerson Lecture is particularly poignant:
It is possible that the technology that stems from curiosity will destroy mankind Perhaps the mutation that produced intelligence is indeed lethal. If so, there are more likely vehicles for man's demise than research on human genetics. I would prefer to believe that the mutation which produced intelligence will lead to a continuing increase ofwisdom and that the technology that results from curiosity will continue to enhance the quality oflife. Nancy B. Schwartz Lennart Roden
BIOCOMPATIBLE GEL OF HYALURONANMEDICAL APPLICATIONS Teruzoh Miyoshi and Akio Okamoto
Research Center, Denki Kagaku Kogyo KK. 3-5-1 Asahi-machi, Machida-shi, Tokyo, 194-8560, Japan KEYWORDS Hyaluronan, HA gel, adhesion prevention, adhesion preventative, gel sheet
INTRODUCTION Tissue adhesion, which often occurs after operations, leads to various surgical traumas and undesirable symptoms such as infertility by a uterine adhesion and chronic pain by acecum adhesion', These adhesions were thought to be induced by following mechanisms:vascular permeability in tissue increases after operation and inflammatory factors are secreted, which allow peripheral tissues to contact with each other. To reduce postoperative adhesion, one of the effective strategies is to introduce barrier materials between tissues. During the past few decades, large number of polymer materials such as dextrin, heparin, hyaluronan(HA), carboxymethylcellulose, and oxidized regenerated cellulose were widely tested as adhesion preventatives'". Recently some materials, which have decreased frequency of adhesion formation, have been developed for this purpose". One of the most effective of them is hyaluronan"". Unique rheological properties of hyaluronan
have
been
offering
ophthalmic
viscosurgery
and
orthopeadic
viscosupplementation based on the concept of E.A.Balazs. In recent years, by modifying hyaluronan's water solubility via chemically covalent derivatization, its solidity and in vivo residence time are able to be controlled to create materials more suitable for further medical uses.We ourselves have already succeeded in producing the novel HA gel by one-step freezing-procedure of acidified HA solution which is composed of HA alone without any chemical modification nor crosslinking reagent which is hardly soluble in neutral water. The gel is able to be provided in the form of a sheet, a film, a sponge, a transparent and fluid gel corresponding to medical
22
Adhesion formation and hyaluronan
demand. Such a gel film or sheet has so prolonged in vivo residence time that those excellently prevented postoperative intraperitoneal adhesion formation in the rat-cecal model. In this report, we evaluate our HA gel sheet in an attempt to reduce cecal adhesion with a rat model.
MATERIALS AND METHODS Hyalronan The sodium salt of HA, with an average molecular weight of 1.9 x 10·, was supplied by Denki Kagaku Kogyo Co. Ltd. ( Tokyo, Japan ). GPC analysis
In order to determine the concentration and the molecular weight of HA, a high performance liquid chromatography with a gel permeation mode was used.
HA gel and HA gel sheet HA solution adjusted to pH 1.5 by 1M nitric acid was frozen at _20°C to be gelated and then by thawing at room temperature sponge-like HA gel was obtained. After washing by PBS (pH 6.8), the neutralized HA gel was crushed with a rotary blade mixer to a gel slurry. The resulting gel slurry was put around a nylon mesh to remove excess water and then air-dried to obtain the HA gel sheet.
RESULTS & DISCUSSION The gelation of HA rapidly progressed as freezing period goes to almost complete in 65 hr (see Fig. 1'). In vitro solubility test of the gel was done by incubating in I
PBS (pH 7.4) under accelerating condition of 60°C.
Fig. 2 shows the effect of
freezing period on solubility of the HA gel. These data indicates that in vitro solubility could be controlled by regulating the freezing period. The gel's half life values introduced from the results of this solubility test were temporarily adopted as an indicator of adhesion preventive effectiveness of the HA sheet. The HA gel sheets having different solubility at 60°C were applied into mouse abdominalcavity to evaluate their in vivo residenncy. Both HA solution and freeze-dried HA sheet without gelation were also used as a contro!'
Biocompatible gel - medical applications 100 ~ c:
o
.'"
'.;J
OJ
....o ...'" 0:: '"
20
40
60
80
100
120
Freezing period (hr)
Figure 1. Effect of freezing period on HA gel formation
g
75
...E'"
Freezing period
"iii :l
"'iii '" 0::
25
c:
42 hr.
•
19 hr.
•
65 hr. V 113 hr.
01.-----'------'-----'-------'
o
10
5
15
20
Incubation time (hr)
Figure 2. In vitro solubility analysis for HA gel 100
"" ...'" '"
0::
80
•
60
"iii :l "C
'iii
'"
l::>.
•...
40
0::
20
2
4
6
1.0 % HA solution Freeze-dried HA sheet HA gel sheet(3 days frozen) HA gel sheet(5 days frozen)
8
Days
Figure 3. HA gel residency in the mouse cavity
23
24
Adhesion formation and hyaluronan Residual amount of HA gel sheet was measured with the time after 2 mg
administration of the sample.
The HA gel sheets showed longer residence time in
proportional to freezing period, although both HA solution and freeze-dried HA sheet without any gel component completely disappeared in 3 days.
Fig. 3 shows good
correlation between half-life value from in vitro solubility test and in vivo residency. In order to evaluate the property of the sheets as adhesion preventatives, we adopted a rat cecal abrasion model. 50 female Wistar rats weighing 200-220 g were used to establish a reliable model of the adhesion. After ventrotomy, the cecal surface was abraded with gauze which was attached to a motor-driven rotating shaft (a circular surface is 1.3 em I.D.). Ten days after the initial surgery, the rats were sacrificed to evaluate abdominal adhesion. Each cecal adhesion was scored by five-ranged scaling (0: No cecal adhesion, 1: Filmy adhesions with easily identifiable plane, 2: Mild adhesions with freely dissectable plane, 3: Moderate adhesions with difficult dissection of plane, 4: Dense adhesions with nondissectable plane). Then various HA gel sheets ( 4cm
x
4cm, 2.0 mg/cm') with different half-lives were tested
with these models to estimate their effectiveness. The application of HA gel sheets showed excellent adhesion preventive effects compared with average score 1.7 of surgical control. For example, the half-life of HA gel sheet with the highest efficacy was 4 hr., reducing an adhesion score to OJ (see' FigA ' ). To investigate the relationship between the in vitro gel solubility( i.e. half-life) and the adhesion preventive effect, various HA gel sheet having different half-life and HA density were prepared. Some definite relationships between the half-life and the effectiveness were summarized in Fig. 5 that shows the relationship in 3D expression. From these results, it was proved that the sheet which has physical properties of strongest colored central region showed most effective adhesion prevention. HA gel : 2.0 mg/Cnf Film size: 4 cm x 4 cm
2 ~
a o
1.5
c"' o
.,
'iii s:
"U
«
0.5
Surgical control
12
Half - life at 60°C, pH 7.4 (hr) 7 6 4 :1
Figure 4. Effect of HA gel sheet on adhesion prevention
Biocompatible gel - medical applications
25
Adhesion prevention 2
1 Hoenect
o
0.5
1 1.5
Adhesion acceleraUon
2~
Half life of HA gel at60 ClhrJ,pH7A
0
Gel amount lmg/cm
2
J
3
0
Figure 5. 3D expression of adhesion prevention efficacy of HA gel sheet Our newly developed HA gel hardly soluble in water was expected to have latent possibilities to substitute for not only native HA preparation but also a part of biopolymers being already used in a broad range of medical applications.
REFERENCES 1. D. Menzies, Peritoneal adhesions:incidence, cause and prevention, Surg
AliI/II.,
1992, 24, 27-45
2. G. Holtz & E. R. Baker, Inhibition of peritoneal adhesion reformation after lysis with thirty-two percent dextran 70. Fertil Steril., 1980, 34, 394-395. 3. T.E. Elkines, R.J. Bury, J.L. Ritter, F.W. Ling, R.A. Ahokans & c.A. Homsey, Adhesion prevention by solution of sodium carboxymethylcellulose in therat, I. Fertil.Steril. 1984,41,926-928. 4. T.E. Elkins, F.W. Ling, RA Ahokas, T.N. Abdella, CA Homsey & Malinak LR. Adhesion prevent ion by solution of sodium carboxymethylcellulose in the rat.Il,
Fertil.Steril., 1984,41,
929-932.
5. R. Kennedy, DJ. Costain, V.C. Mcalister & T.D.G. Lee, Prevention of experimental postoperational peritoneal adhesion by N,O-carboxymethyl chitosan,
Surgely,1996,120,866-870. 6. lL. Hill-West, S.M. Chowdhury, R.C. Dunn & lA. Hubbell, Efficacy of a resorable hydrogel barrier, oxidized regenerated cellulose, and hysluronic acid in
26
Adhesion formation and hyaluronan the prevention of ovarian adhesions in a rabbit model, Fertil. Steril., 1994, 62, 630-634.
7. AF. Haney, l Hesla, B.S. Hurst, L.M. Kettel, AA Murphy & JA Rock, Expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane) is superior to oxidized regenerated cellulose (Interceed TC7) in preventing adhesions, Ferlil. Steri/., 1995,63, 1021-1026. 8. B. Urman, V. Gomel, N. Jetha, Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model, Fertil. Steril., 1991, 56, 563-567. 9. D.A Grainger, W.R. Meyer, AH. DE Cherney & M.P. Diamond, The use of hyaluronic acid polymers to reduce postoperative adhesions, J. Gynecologic Surgery, 1991, 7, 97-101. 10. E.P. Goldberg, lW. Burns, Y. Yaacobi, Prevention of postoperative adhesions by procoating tissue with dilute sodium hyaluronate solutions, Gynecologic Surgery and Adhesion Prevention, 1993,381, 191-204. 11. lD. Mithcell, R. Lee, K. Neya, GJ. Vlahakes, Reduction in experimental pericardial adhesion using a hyaluronic acid bioabsorbable membrane, Eur. J. Cardiothorac Surg., 1994, 8, 149-52. 12. H. Yarali, B.F.H. Zahradka & V. Gomel, Hyaluronic acid membrane for reducing adhesion formation and reformation in the rat uterine horn, J. Reprod. Med.,
1994, 39, 667-70 13. lW. Burns, K. Skinner, l Colt, A Sheidlin, R. Bronsnon & Y. Yaacobi. Prevention of tissue injury and postsurgical adhesions by precoating tissue with hyaluronic acid solutions, J. Surg. Res., 1995, 59, 644-652. 14. lW. Bums, L. Burgess, K. Skinner, R. Rose, MJ. Colt & M.P. Diamond, A hyaruronate based gel for the prevention of postsurgical adhesions: evaluation in two animal species. Fertil. Steril., 1996, 66, 814-821. 15. P.A De Laco, M. Stefanetti, D. Pressato, S. Piana & M. Dona, A novel hyaluronan-based gel in laparoscopic adhesion prevention: preclinical evaluation in an animal model, Fertil. Steril. 1998,69,318-323. 16. AF. aney, E. Doty,
A barrier composed of chemically cross-linked hysluronic
acid (Incert) reduces postoperative adhesion formation, Fertil. Steril., 1998, 70, 145-151. 17. I. Kosak, C. Unlu, Y. Aksan & K. Yakin,
Reduction of adhesion formation with
cross-linked hyaluronic acid after peritoneal surgery in rats, Fertil. Steril. 1999, 72, 873-878
PART 2 CHARACTERISATION AND SOLUTION PROPERTIES OF HYALURONAN
PREDICTIVE AND EXPERIMENTAL BEHAVIOUR OF BYALURONAN IN SOLUTION AND SOLID STATE. Katia Haxaire, Eric Buhler, Michel Milas, Serge Perez, Marguerite Rinaudo*. CERMA V-CNRS affiliated with Joseph Fourier University. BP 53. 38041 Grenoble cedex 9. France.
ABSTRACT This paper concerns the molecular characterisation of hyaluronan polymeric chain. The main point discussed is the determination of the intrinsic persistence length Lp by different experimental techniques; Lp is equal to a value of about 70 A, in good agreement with the value obtained by molecular modelling. From this value, the intrinsic viscosity and the viscosities of aqueous solutions as a function of polymer concentration and molar mass in salt excess can be predicted at zero shear rate following the general development usually adopted. The role of the temperature on the chain mobility, as monitored by nmr, molecular modelling and viscosity experiments, is discussed. The stiffness decreases progressively when the temperature increases in relation with the destabilisation of the H bond network. The conformations obtained in the solid state are in good agreement with those corresponding to the low energy conformations derived from molecular modelling. KEYWORDS Hyaluronan, rigidity, conformation, molecular modelling, solution properties. INTRODUCTION Hyaluronan is a natural polysaccharide originally extracted from different sources 1-3 and now often produced by bacterial fermentation4.5. For a long time, our experimental work has been performed on bacterial samples free of proteins. Different experiments were developed to characterise this polymer and to be able to interpret its behaviour in aqueous solutions. In a first step, we will describe the behaviour of the polymer in solution. Then, some results obtained in the solid state will be exposed. These features will be discussed and compared to those derived from molecular modelling. MATERIALS & METHODS Bacterial hyaluronan is produced by Soliance (Pomade, France) and carefully purified under the Na salt form in our laboratory as described previously". Different molar masses were prepared by sonication (down to 300,000 g/mol.) or acidic hydrolysis (down to 85,000 g/mol.) as usually.
38
Characterisation and solution properties ofhyaluronan
The samples were characterised by proton nrnr in D20 using a Bruker AC300 at variable temperatures. The concentration adopted is 5 mg/mL but may also vary when required. The molecular weight distribution is determined by steric exclusion chromatography using a multiangle laser light scattering detector Wyatt Dawn DSP-F associated with a differential refractometer", The solvent used is 0.1 M NaN03 ; the columns are Shodex OH pak 805 and 806. The dnldc=0.142 mUg at 30°C in the solvent used. The viscosity of dilute solutions as a function of the shear rate is determined using the Low Shear 40 from Contraves in the range 104 - 128 S,I in different aqueous solvents. For higher viscosity, a controlled strain equipment is used; it is AR 1000 from TA Instruments. The temperature control allows to work in the range 25°C< T< 80°C. This equipment allows also dynamic measurements in the range of 10'3 to 100 Hz. For the molecular modelling, the usual protocols developed for polysaccharide modelling are used. The main results have been reported recently 8. Small Angle Neutrons Scattering (SANS) experiments were done on the spectrometer PACE (Orphee reactor, LLB Saclay, France) using three settings: in the first we explore a q-range of 3.10'3- 3.3.1O,2A-I(. distance to detector D=4.65m, wavelength A=13.14±l.OOA), in the second a q-range of 5.10'3_ 5.33.1O,2A-l (0 D=4.65m, A=8.09±O.50A) and in the third a q-range of 1.37.10'2- 1.45.10.1 A-I (e D=2.264m, A=6.06±O.50A). q is the scattering wave-vector and is equal to: q = 4n sin!!..
A.
2
where e is the scattering angle
[1]
Dynamic Light Scattering (DLS) experiments were performed by means of a spectrometer equipped with an argon ion laser (Spectra Physics model 2020) operating at A=488nm, an ALV-5000 correlator from ALV, Langen-FRG Instruments, a computer-controlled and stepping-motor-driven variable-angle detection system and a temperature-controlled sample cell. The temperature was (25±O.1)OC unless otherwise noted. The scattering spectrum was measured through a band-pass filter (488nm) and a pinhole (50 urn) with a photomultiplier tube (ALV). RESULTS & DISCUSSION Hyaluronan has been the subject of many investigations in our laboratory using bacterial samples with different molecular weights. This water-soluble polysaccharide is considered as representative of the wormlike chain polymers characterised by a persistence length as developed as followed"!'. Its hydrophilic character as well as its relative stiffness allows interpreting the behaviour in solution.
Hyaluronan chain characterisation. A. Theoretical approach
A model has been described previously in which the chains are characterised by an intrinsic persistence length Lp, characterising the local stiffness of the polymerI2.13. If Lp is determined for the neutral equivalent chain (screening of the electrostatic repulsions by salt addition), in aqueous solution for a charged molecule such as HA, the effective persistence length is increased by the electrostatic persistence length Le due to such effects as electrostatic repulsions between neighbour ionic sites. Then, the persistence length in our experimentalconditions is : Lt=Lp+Le. [2]
Predictive and experimental behaviour
39
with Le depending on the external salt concentration. It is known that there is a relation given by Benoit-Dory'? for wormlike chain in a-conditions between the radius of gyration Rg, the contour length Land Lp : [3] Rg2=L Lp/3 - Lp2+ 2 Lp3/L - 2(Lp4/L2)[1_ exp (-LlLp)] Then, in the model, for given thermodynamic conditions, two electrostatic contributions are added following the Odijk treatment'P: 1) an electrostatic excluded volume coefficient which can be calculated for each charge parameter of the polyelectrolyte, each M and each salt concentration; 2) the electrostatic persistence length contribution mentioned previously. B. Molecular modelling
At 2SoC, the intrinsic persistence length was calculated and found equal to 7sA in very good agreement with the experimental value'. The same treatment allows to calculate Lp(T) as shown in Figure 1. 80
-r--------------------,
75
70
s
.9-
65
60 55
50 +----...,-------r-------,.----.------l 40 100 20 80 60 .0
Figure 1. Evolution of the intrinsic persistence length Lp with temperature obtained by molecular modelling. C. Experimental characteristic
Recently, the analysis of the three traces obtained for SEC analysis using a differential refractometer and a multiangle laser light scattering detector yield the molecular weight distribution of the different samples and the average molecular weights without any calibration. Especially, the curve giving the radius of gyration as a function of the molecular weight was analysed using a model of wormlike chain including the electrostatic contribution. Following this treatment, using home made software, we are able to determine for each experimental Rg(M) curve obtained from the multidetector SEC analysis the characteristic intrinsic persistence length Lp. This approach was recently presented and we determined Lp = 7oA7 • This parameter also controls the viscosity of the solution as discussed later. Using neutron scattering, one determines directly the persistence length. In fact, one can say that if the contour length of the polymer chain, L, is much larger than the total persistence length, LT , the chain behaves as a gaussian coil whereas if L is smaller than LT the chain behaves as a rod. One can express the radius of gyration of such a chain using the following relations:
40
Characterisation and solution properties ofhyaluronan
R g2
-_
LLr
3
lif L»L'l'
[4]
and
There is a crossover in the scattered curve intensity, I, as a function of the scattering wave-vector q in the q-range where the transition between the asymptotic regime of a coil and a rod occurs. In the first case the intensity is q-2-dependent, whereas when we look at smaller distances than Lr the intensity is q-l-dependent. One can use a Kratky plot" to determine the intrinsic persistence length, !.p, of hyaluronan in the dilute regime at 25°C. SANS experiments were performed in the presence of a large excess of external salt. In this case the total persistence length is equal to the intrinsic one (Lp=Lr) , the electrostatic repulsions being screened. In such a plot we represent the product I (q)q2 as a function of q as it is shown in Figure2.
1.e-4 , . - - - - - - - - - - - - - - - - - - - - - - - - - - ,
1.e-5
1.e-6
q* 1.e-7 +----.---,---.---.--,.---,...,.,...,---,...---,-,...-,-,-,-.,..,-,-----1
0.001
0.010
0.100
q (A-1)
Figure 2. Kratky plot obtained using SANS experiments for a dilute solution of hyaluronan Mw= 85,000 in the presence ofO.1M of NaCI at 25°C.
The transition between the coil and the rod regime is occuring at q*. From this q* value we calculated the intrinsic persistence length Lp using the following equation": L = 1.91 P
q*
[5]
The high value of Lp=95A is characteristic of the semi-rigid character of hyaluronan. This value is slightly higher than that determined from modelling or SEC analysis. Indirect determination from Rg values, as well as direct determination from SANS experiments, give Lp values significantly higher than that reported. previously by Cleland17 ( 45A) and more recently by Norisuye et al.18-20 (Lp around 40 A).
Predictive and experimental behaviour
41
D. Influence of temperature on the rigidity The stiffness of the chain characterised by the intrinsic persistence length was shown by molecular modelling to decrease when the temperature increases. The same feature was also demonstrated by nmr experiments or viscosity measurements. The chemical structure of the HA molecule (in the -COO· form) is shown in Figure 3; the polymer is usually used in its sodium salt form.
0-
Figure 3. Hyaluronan chemical structure with the representation of H bonds existing in the dried state. One of the best techniques for characterisation the chemical structure of hyaluronan is the IH nmr. The ratio of the integrals for the -CH3 and the H-l equals 3/2 corresponding to the theoretical structure. But, if one introduces a standard to calibrate the signals ( as example, DMSO in well controlled amount), it can be concluded that the ratio between integral of -CH3 group and the standard can provide an apparent degree of acetylation. Actually, all the signals of the polymer are modified due to a reduced rate of relaxation of all the groups of atoms in direct relation to the temperature 17. Figure 4 gives the evolution of the apparent degree of acetylation of hyaluronan with the temperature; the signal corresponding to the acetyl substituent is increasing when temperature increases due to a destabilisation of the intrachain H bonds along with an increase of the flexibility of the chain. The decrease of the stiffness is also demonstrated by the decrease of the intrinsic viscosity when temperature increases (Figure 5).
Solution properties. The theory of wormlike chain for the intrinsic viscosity was developed by Yamakawa &Fujii22 ; from this model, in a-conditions, it comes: [T1]=¢(Lr,dr) (MIL Lpr312 M" 2
[6]
with Lr and dr the reduced length and the reduced diameter of the chain; Lr equals the number of Kuhn segments.
42
Characterisation and solution properties ofhyaluronan
Xo.9 0.8 0.7
•
0.6
• • •
0.5 0.4
•
•
0.3 0.2 0.1 0 0
20
40
60
80
Tee)
100
Figure 4. Influence of temperature on the apparent degree of acetylation of HA (X) determined by IH nmr in D20 (Mw=85,OOO; 5gIL i.e.1.25 monomoI./L). Reference is DMSO at the same molar concentration. 3000,---------------------, 2600 ~ 2200
:§.
:E 1800 1400 1000
-+-----,----.----,---,-----..,----1
o
10
20
30
40
50
60
T{OC)
Figure 5. Evolution of the intrinsic viscosity with the temperature 10• • Experimental values in NaCI O.IM, Mw= 1,350,000. So, if Lp is known, the intrinsic viscosity can be calculated for each thermodynamic conditions following the extension of Odijk23 and then, the viscosity at given polymer concentration in salt excess at zero shear rate, following the usual relation: l1sp= C[11]+ k' (C[l1]i + B (C[ l1])D
[7 ]
In figure 6, the general dependence of the specific viscosity at zero shear rate is given assuming that k'= 0.42 (good solvent), B = 7.77xlO-3 and n = 4.18 as obtained in our previous work 10. These experimental values were recently discussed by Cowman et aI.24 This curve is interesting as it shows that the overlap parameter C[11] imposed the viscosity of the solution independently of the molar mass or the polymer concentration.
Predictiveand experimental behaviour
43
6
4
'"i
EC) 2
S 0
-2 -1
-2
2
0
3
Log[C[Tl]]
Figure 6. Log nsp at zero shear rate as a function of log C[t'l] , C[t'll being the overlap parameter. eExperimental values in NaCI 0.1M, 25°C, for different Mw lO • In the Dynamic Light Scattering (DLS) experiments, the normalised time autocorrelation function g(2)(q,t) of the scattered intensity is measured. The latter can be expressed in terms of the autocorrelation function of the scattered electric field g(1)(q,t) (the dynamical structure factor) through: g
(2)(
) _
q.t -
(I
2 *((q,O)I(q,t») )2 -_ A + /3\ g (1)(q,t )1
[8]
I(q,O)
where A is the baseline and ~ the coherence factor which in our experiments is equal to 0.7-0.9. Figure 7 shows log-log plot of g(1)(q,t) for a semi-dilute solution of hyaluronan at a polymer concentration c= 4x104g/cm3 in the solvent 0.1M NaCI at 25°C. The scattering angle is equal to 90°. In this regime the autocorrelation function of the scattered electric field can be described by a sum of two relaxations widely separated in time: g (1) (q,t) = Ajast(q)ex { - - t T~t
J+ A'low(q) ex{-- t )
[9 ]
T MW
'erast and 'esloware respectively the fast and the slow relaxation times. Afast(q) and Aslow(q) are the corresponding amplitudes. To determine 'erast and 'eslow we used the Contin method based on the inverse Laplace transform of g(l)(q,t). Figure 7 shows also a typical example of results obtained by applying the Contin method to our data for a semi-dilute solution of hyaluronan. Fast and slow modes are diffusive with characteristic times 'erast and 'eslow inversely proportioned to q2. Thus, it is possible to calculate for each polymer concentration a fast and slow diffusion coefficient: and
[10]
44
Characterisation and solution properties ofhyaluronan
The fast mode is attributed to the relaxation of polymer concentration fluctuations characterised by a co-operative diffusion mechanism of the polymer network meshes, i.e., the correlation mesh of the semi-dilute network", The slow mode is attributed to the formation of physical polymer associations in the semi-dilute regime. The apparition of associations is usually not observed with solutions in good solvent (second virial coefficient A2>O) but rather requires a solution under theta conditions or a solution under low ionic strength conditions. We think the high rigidity of hyaluronan could be related, in some way, to the origin of these associations".
1.0
. ... .-.
0.1
10'2
10"'
10·
10'
lQ2
11)3
t(ms)
Figure 8. A log-log representation of g(l)(~,t) for 8=90° relative to a O.IM NaCI semidilute solution of Hyaluronan Mw=2xlO at 25°C and at a polymer concentration c=4xlO-4g/cm3. The normalised distribution function of decay times obtained using the Contin method is also represented. It should be important to develop this aspect of the structure of semi-dilute solution and the relationship with rheology. Solid State Organisation A. Molecular modelling
This technique was used to construct the different helical conformations predictable for hyaluronan; these structures were described previously" . The objective was to describe the ordered solid state conformations and to compare them with the crystalline structures reported in the literature26•28 • B. X ray diffraction pattern.
Films of hyaluronan in well-controlled ionic form were extended under controlled relative humidity to determine experimentally their structure and to compare with the
Predictive and experimental behaviour
45
data given in the literature as well as those predicted by molecular modelling. From previous experiences26 -28 , X-ray fibre diffraction studies and concomitant structure refinements have identified several allomorphs which are all left handed 3 and 4-fold, and 2-fold helices with axial rises per disaccharide corresponding to the lowest energy conformations calculated in our study. C. Hydration of HA.
This polymer is used in cosmetics for its hydrating power; the water interaction was investigated using DSC to dissociate water molecules in strong interaction with the specific groups of atoms in HA (first layer) and the more loosely interacting water molecules". From this work it was concluded that 17 water molecules per disaccharide are strongly interacting with HA at RH 100%. Complementary experiments are in progress using infrared spectroscopy. CONCLUSION Hyaluronan is an interesting water-soluble polymer; it is mainly characterised by the viscoelastic character of semi-dilute solution in presence of external salt. The rheology of these solutions was previously described. In this paper, the most recent data concerning the characteristic of HA chain were described. HA behaves as a semi-rigid polyelectrolyte characterised by an intrinsic persistence length around 70A. which directly imposes the overall dimensions of the chain as well as the physical properties such as the viscosity. This paper demonstrates that molecular modelling is able to predict these characteristics. REFERENCES RW.Jeanloz, In 'The Carbohydrates. Chemistry and Biochemistry. Vol. I/B' W.Pigman, D.Horton, AHerp (Eds.), 1970, Academic Press, New-York & London,pp.592-597 2
J.RE.Fraser, T.C.Laurent, U.B.G.Laurent, 'Hyaluronan: its nature, distribution, functions and turnover' J. Intern. Med., 1997,242,27-33
3
I.F. Radaeva, G.A Kostina, AV. Zmievskii, 'Hyaluronic Acid: Biological Role, Structure, Synthesis, Isolation, Purification, and Applications' Applied Biochem.Microbiol.1997, 33,111-115
4
P.L. DeAngelis, P.H.Weigel, 'Characterization of the recombinant hyaluronic acid synthase from Streptococcus pyogenes' In 'Genetics of Streptococci and Lactococci', J.J.Ferretti, M.S.Gilmore, T.R K1aenhamrner,F.Brown (Eds.), Dev.Bio1.Stand.N°85. Basel: Karger, 1995, pp.225-229
5
N.ltano, KKimata,'Expression cloning and molecular characterization of HAS protein, a eucaryotic hyaluronan synthase', J. Bioi. Chem.,1996,271, 9875-9878
6
M. Rinaudo,'Polysaccharide characterization in relation with some original properties', J. Appl. Polym. Sci. : Appl. Polym. Symp.,1993,52, 11-13
7
M. Rinaudo, I.. Roure, M. Milas,'Use of steric exclusion chromatography to characterize hyaluronan, a semi-rigid polysaccharide', Int. J. Polym. Anal. Charact,1999, 5, 277 - 287
8
K Haxaire, I.. Braccini, M. Milas, M. Rinaudo, S. Perez.Xonformational behavior ofhyaluronan in relation to its physical properties as probed by molecular modelling', Glycobiology, 2000,10,587594
9
E. Fouissac, M. Milas, M. Rinaudo, R Borsali,'Influence of the ionic strength on the dimensions of sodium hyaluronate', Macromolecules, 1992, 25, 5613 - 5617
46
Characterisation and solution properties of hyaluronan
10 E. Fouissac, M. Milas, M. Rinaudo,' Shear-rate, concentration, molecular weight and temperature viscosity dependences of hyaluronate, a wormlike polyelectrolyte' Macromolecules,1993, 26, 69456951 II I. Roure, M. Rinaudo, M. Milas, 'Viscometric behavior of dilute polyelectrolytes.Role of electrostatic interactions', Ber Bunsenges, Phys. Chem., 1996, 100, 703 - 706 12
W. Reed, 'Light-Scattering Results on Polyelectrolyte Conformations, Diffusion and Interparticle Interactions and Correlations' In 'Macroion Characterization from Dilute Solutions to Complex Fluids',K.Schmitz (Ed.), ACS Symp.Ser. 1994, 548,pp.297-314
13 L. Chazeau, M. Milas, M. Rinaudo,'Conformations of xanthan in solution: analysis by steric exclusion chromatography', Int. J. Polym. Anal. Charact., 1995,2,21-29 14 H. Benoit, P. Doty, 'Light Scattering from non-Gaussian Chains' J. Phys. Chem., 1953, 57, 958-963 15 T. Odijk, A C. Houwaart,'On the Theory of the Excluded-Volume Effect ofa Polyelectrolyte in a 1-1 Electrolyte Solution', J. Polym. Sci. Polym. Phys. Ed., 1978,16,627-639 16 O. Glatter, O. Kratky, In' Small angle x-ray scattering ',Academic Press, 1982 17 RL. Cleland, 'Viscometry and Sedimentation Equilibrium of partially Hydrolyzed Hyaluronate: Comparison with theoretical Models of Wormlike Chains', Biopolymers, 1984, 23, 647-666 18 K.Hayashi, K.Tsutsumi, F.Nakajima, T. Norisuye, ATeramoto,'Chain-Stiffness and ExcludedVolume Effects in Solutions of Sodium Hyaluronate at High Ionic Strength', Macromolecules, 1995, 28, 3824-3830 19 K.Tsutsumi, T. Norisuye, 'Excluded-Volume Effects in Sdium Hyaluronate Solutions revisited' Polym.J.,1998, 30, 345-349 20 N.Mizukoshi, T.Norisuye,'Small-angle X-ray scattering from sodium hyaluronate in aqueous sodium chloride' Polym Bull., 1998, 40, 555-562 21 N. Berriaud, M. Milas, M. Rinaudo, 'Characterization and Properties of Hyaluronic Acid (Hyaluronan)' .In 'Structural Diversity, Functional Versatility " S. Dumitriu (Ed.), M. Dekker Publ.,1998, pp.313 - 334 22 H. Yamakawa, M. Fujii, 'Intrinsic Viscosity of Wormlike Chain. Determination of the Shift Factor' Macromolecules, 1974, 7, 128-135 23 T.Odijk, ' On the Ionic-Strength Dependence of the Intrinsic Viscosity of DNA', Biopolymers,l979, 18,3111-3113 24 T.K. Kwei, M.Nakazawa, S. Matsuoka, M.K. Cowman, Y.Okamoto, 'The Concentration Dependence of Solution Viscosities of Rigid Rod Polymers'(in press) 25 E. Buhler, M. Rinaudo,' Structural and Dynamical Properties of semi-rigid polyelectrolyteSolutions: a Light-Scattering Study', Macromolecules, 2000,33, 2098 - 2106 26 lK. Sheehan, E.D.T. Atkins, 'X-ray fiber diffraction study of the conformational change in hyaluronate induced in the presence of sodium, potassium and calcium cations', Int. J. Bioi. Macromol., 1983,5, 215-221 27 A.K. Mitra, S. Arnott, RP. Millane, S. Raghunathan, lK. Sheehan, 'Comparison of glycosaminoglycan structures induced by different monovalent cations as determined by X-ray fiber diffraction', J. Macromol. Sci-Phys., 1985.1986, B24 (1-4), 21-38 28
J.M. Guss, D.W.L. Hukins, P.J.C.Smith, W.T. Winter, S.Arnott, R Moorhouse, D.ARees, 'Hyaluronic acid: molecular conformations and interactions in two sodium salts', J. Mol. Biol., 1975, 95, 359-384
29. N.Jouon, M.Rinaudo, M.Milas, J.Desbrieres, 'Hydration of hyaluronic acid as a function of the counterion type and relative humidity', Carbohydr. Polym., 1995,26, 69-73
AQUEOUS SEC, LIGHT SCATTERING AND VISCOMETRY OF ULTRA-HIGH MOLAR MASS HYALURONAN Raniero Mendicbi, Alberto Giacometti Scbieroni Istituto di Chimica delle Macromolecole (CNR), Via Bassim 15.20133 Milan, Italy
ABSTRACT
Present study concerns the problems encountered in the SEC fractionation ofUHMM HA samples. Problems in the SEC characterization of UHMM HA samples are substantially two: the fractionation in the columns and the calibration of the chromatographic system. We have overcome the calibration problem by the use of light scattering and viscometer detectors on-line to the SEC system that do not need calibration. Shear degradation, concentration effects, viscous fmgering, poor column resolution and in general low reproducibility are the main difficulties in the SEC fractionation. A successful characterization of UHMM HA polymers requires an optimization of the experimental protocol. Each step of the experimental protocol should be performed methodically to obtain reliable results. The critical point is the fractionation in the SEC columns. Commercially available SEC aqueous columns have not been optimized for UHMM polymers. Also with an optimized experimental protocol shear degradation and non-ideal SEC fractionation can occur when the Mw of the HA sample is approximately higher than 3'10 6 g/mol. KEYWORDS
Hyaluronan, ultra high molar mass, SEC, light scattering, viscometer INTRODUCTION
The complex biological functions of Hyaluronan (HA) are closely related to the viscoelastic properties and to the molar mass distribution (MMD). It is well known that the HA molar mass ranges from relatively low to high and ultra-high (up to 1'107 g/mol). In particular we were interested in the MMD characterization of high and ultrahigh molar mass (UHMM) HA samples. Various off-line methods such as light scattering, viscometry, osmometry and sedimentation could be used for the molecular characterization of UHMM HA samples. In a previous paper] we have presented the characterization by off-line light scattering and viscometry ofUHMM HA samples with the weight-average molar mass (Mw) up to 1'107 g/mol. However many HA specific functions depend on the whole MMD rather than an average molar mass value. Hence, the demand for the fractionation and an accurate characterization of the whole MMD ofUHMM HA samples is increasing considerably. Theoretically different methods could be used for the fractionation of UHMM HA samples; practically there are two alternatives: Size Exclusion Chromatography (SEC) and Field Flow Fractionation (FFF). For our goal probably a fractionation by means of a FFF system could be more effective. However SEC is the method more commonly used and we were interested to explore the extreme potentialities of the SEC technique for
48
Characterisation and solution properties ofhyaluronan
the fractionation of UHMM HA samples. Hence, in this study we will investigate the fractionation by means of various SEC columns. Every time SEC fractionation was applied to UHMM polymers severe problems were invariably reported. Main problems were shear degradation, concentration effects, viscous fingering, poor column resolution and in general low reproducibilltyi", In the characterization of UHMM HA we have used both light scattering and viscometer detectors on-line to the SEC system. These detectors are "absolute" and when are used on-line to a SEC system measure directly molar mass, dimension and intrinsic viscosity of each fraction of the sample. However, also the use of the more sophisticated on-line detectors doesn't resolve the problem of a poor fractionation. The goal of an adequate fractionation is the main concern in SEC ofUHMM HA. Substantially, this study is an overview of the problems in the fractionation of UHMM HA samples by SEC columns. MATERIALS AND METHODS Materials Several HA samples were obtained from Pharmacia & Upjohn (Nerviano, Milan, Italy). Seven samples were of extractive source (rooster comb) with molar mass ranging from 1'10 6 to 7.4'106 g/mol. Other samples were of bacterial source with molar mass ranging from 4'10 6 to H0 7 g/mol. Four HA samples, M w from 4.3.10 5 to 1.4'10 6 g/mol, were kindly supplied from Dr. Ladislav Soltes of the Institute of Experimental Pharmacology of the Slovak Academy of Sciences. All HA samples were highly purified, typically contained less than 0.2% of proteins. Bovine Serum Albumin (BSA) was obtained from Sigma (USA). Water solvent was MilliQ grade (Millipore, USA). All other chemicals were of analytical grade. SEC system The MMD of the HA samples was obtained by a 150CV multidetector SEC system from Waters (Milford, MA, USA). The multidetector SEC system consisted of: a single capillary viscometer (SCV), a concentration detector and an additional multi-angle laser light scattering (MALS) Dawn DSP-F from Wyatt (Santa Barbara, CA, USA). As concentration detector we have used a Differential Refractometer (DRI) or UV. This multidetector SEC system has been described in detail previously". The experimental conditions consisted of: O.l5M NaCI or O.5M NaCI as mobile phase at 35°C, 0.2 mL/min flow rate and 200 JlL injection volume. The concentration of the HA solutions was as low as possible (in general lower than 0.1 mg/mL). We have tested several column sets: i) 2 TSKGel (G6000PW and G5000PW) from TosoHaas (Germany); ii) 2 OHpak (KB-806 and KB-805) from Shodex (Japan); iii) 2 PL Aquagel-OH 60 from Polymer Laboratories (UK). Besides we have also individually tested three Ultrahydrogel columns (2000, 1000 and 500 A ofpore size) from Waters. Light Scattering The MALS photometer uses a vertically polarized laser of 632.8 om of wavelength and measures the intensity of the scattered light at 15 angular locations ranging in aqueous solvent from 14.5° to 158.3°. The calibration constant was calculated using Toluene as standard assuming a Rayleigh Factor of 1.406'10-5 em". The photodiodes normalization was made by measuring the scattering intensity in the solvent of a BSA
Aqueous SEC, light scattering and viscometry
49
globular protein assumed to act as an isotropic scatterer. Details of the MALS detector have been described elsewhere", The refractive index increment, dn/dc, for HA polymers in the solvent was assumed as 0.150 mLig. Viscometer The on-line viscometer was a home-made SCV. Details of this on-line detector have been described elsewhere", The usual shear rate (755 S·I at 0.2 mLimin of flow rate) of our SCV detector (0.014 inch ID and 6 inch length) was too high for non-Newtonian solutions like UHMM HA polymers. Hence, we have replaced the capillary tube of the SCV detector with a larger internal diameter tube (0.02 inch ID and 20 inch length). In this way the average shear rate approximately decreased to 250 S·I. RESULTS AND DISCUSSION In a previous paper' we have presented the characterization of UHMM HA samples by off-line methods. Specifically we have used both off-line light scattering and viscometry. M w of the HA samples ranged from 1.106 to 1.10 7 g/mol. Molar mass M w, dimension of the molecules R g and intrinsic viscosity lnl obtained by off-line methods will be used as reference for the on-line results. The present study concerns the fractionation by SEC of the UHMM HA samples to obtain the whole MMD. Our SEC system used absolute detectors, MALS and SCV, therefore it did not need calibration. Virtually, at each elution volume after the fractionation on the SEC columns the system provided Mi, Rgj and [llli of the fractions. Figure I shows the original setup of our multidetector SEC system. The setup of the detectors was serial in the following order: SCV-UV-MALS. MALS detector was located after the concentration detector because the internal volume of its cell is relatively large. In such way the concentration detector was not affected from the local band broadening in the MALS cell.
Columns Solvent
I I I I
I I
I I
U 1
I I
MALS Software
:
rIJ II1
L
L-._-.-_-'
SC'l Software
-j-
0.45 urn I
Li
r
Waste
Figure 1. Scheme of the SEC-MALS-SCV chromatographic system
J
50
Characterisation and solution properties ofhyaluronan
Usual SEC experimental conditions applied to the fractionation of UHMM HA samples present many drawbacks. A preliminary step of this study was the optimization of the experimental protocol. Each detail of the experimental protocol (flow rate, sample concentration, fluid path and column set) should be checked methodically to obtain reliable results. In general flow rate and sample concentration should be as low as possible. As flow rate we have used 0.2 mL/min. The concentration of the sample solution depended on the molar mass and in general was lower than 0.1 mglmL. The signal to noise ratio of the MALS and SCV detectors with UHMM HA samples was unusual high and theoretically it is possible to use sample solutions extremely dilute. Unfortunately problems arise from the concentration detector. The minimal effective concentration depends on the signal to noise ratio of the concentration detector. As concentration detector for HA there was two alternatives: DR! and UV. Despite the fact that the DR! response is relatively better than UV, for HA, we have preferred to use the UV detector (202 om) because the cell presents a smaller flow resistance. Last problem introduces the optimization of the fluid path. In general in the fractionation of UHMM polymers it is necessary to avoid each critical point in the fluid path of the chromatographic system. It is quite important to replace the capillary tubes, from the injector to the detectors, with semi-capillary tubes. Furthermore it is better, if possible, to replace the 2 IJ.m inlet and outlet frits ofthe columns with new 10 um frits, It is well known that the macromolecules assume a more compact conformation and lower hydrodynamic radius in a a-solvent. A possible strategy, suggested by some authors", could be the use of a a solvent as mobile phase. HA in aqueous solvent is a negatively charged polyelectrolyte. A polyelectrolyte to fulfil a-conditions needs infmite ionic strength. Practically we have increased the ionic strength of the mobile phase from O.15M NaCI to O.5M NaCI. In this way the dimensions of the HA macromolecules decreases notably' and the exclusion limit of the columns significantly increases. When the experimental conditions are optimized the calibration curves of the SEC system (M=f(V), Rg=f(V) and [l'\]=f(V», obtained from MALS and SCV detectors, assume the usual monotonous decreasing appearance. These experimental functions are deprived of oscillations and anomalous changes of the slope that are typical of shear degradation, viscous fmgering and in general poor fractionation. Besides, the polymeric peak becomes monomodal and substantially symmetrical. However the performances of the SEC columns are not unlimited. Figure 2 shows the M= f(V) experimental function, from the on-line MALS detector, using 1 Ultrahydrogel 500 A column. The particle size of this column was 10 um and the nominal pore size 500 A. In this experiment we have fractionated six HA samples with M, that ranged from 0.66M to 704M g/mol. Figure 2 clearly shows the problems of the SEC fractionation of UHMM HA samples. The M= f(V) plot of three HA samples with lower molar mass, Mw from O.66M to 1.65M g/mol, shows the macromolecules are partially excluded. First part of the M= f(V) plot, the steepest slope, concerns the macromolecules excluded from the pores. Second part of the plot concerns the macromolecules not-excluded from the pores, substantially the size exclusion part of the plot. It is also evident that the relative fraction of macromolecules excluded increases when the molar mass of the sample increases. Practically the fractionation of these HMM and UHMM HA samples is a mixture of hydrodynamic and size exclusion chromatography. The M=f(V) plot of the last three HA samples with higher molar mass, Mw from 35M to 704M g/mol, show again the fractionation of the excluded macromolecules but the second part of the plot becomes substantially flat.
Aqueous SEC. light scattering and viscometry
51
l.E+08 1 Ullrahydrogel 500 A
M
l.E+07
l.E+06
l.E+05 4
5 Elution Volume
7
6
Figure 2. M= f(V) experimental function, from MALS, using 1 Ultrahydrogel 500 A column (10 Jim of particle size). From bottom to up M w of the HA sample was respectively: 0.66M, 1.0M, 1.65M, 305M, 5.0M and 7.4M g/mol.
l.E+08 M
2 TSK-Gel (G6000PW-G5000PW)
l.E+07
l.E+06
l.E+05 8
10
12 14 Elution Volume
16
Figure 3. M= f(V) experimental function, from MALS, using 2 TSKGel PW columns (17 Jim of particle size, > 1000 A of pore size). From bottom to up M w of the HA sample was respectively: 0.66M, 1.0M, 1.65M, 3.5M, 5.0M and 704Mglmol.
18
52
Characterisation and solution properties of hyaluronan
Reconsidering the plot more carefully we can see that the slope of the second part of the M= f(V) plot becomes positive. In other words, the HA macromolecules with UHMM eluted in reverse order with regard to the size exclusion (polymer molar mass increase with increasing elution volume). Practically for the highest molar mass HA sample there is not a real fractionation and macromolecules of approximately the same size could be found in any part of the polymer peak. Obviously results reported in Figure 1 do not exclude degradation of the polymeric samples. Despite the low flow rate and ultra diluted concentration, lower than the overlap concentration, degradation of the UHMM HA sample can occur. Using Ultrahydrogel columns with higher pore size (1000 and 2000A of pore size) the relative fraction of excluded macromolecules clearly decreases. In particular using the 2000A column the two different slopes in the M= (V) plot, respectively hydrodynamic and size exclusion fractionation, apparently disappears. The M= (V) plot of the six HA samples assume the usual monotonous decreasing appearance with substantially constant slope. However, interestingly the six different plots are not superimposed like they should be. We have also tested three other column sets particularly suitable for UHMM polymers (for details see the materials and methods part). In this paper we show only the results obtained with the column set composed of 2 TSKGel (G6000PW and G5000PW). The particle size of these columns was 17 um and the nominal pore size was higher than 1000A, as were generically described from the manufacturer. Figure 3 shows the M= f(V) plot, with the same HA samples, using the 2 TSKGel columns. Apparently Figure 3 shows that also for the higher molar mass HA samples there are not excluded macromolecules. However only the first three HA samples, with lower molar mass up to 1.65M g/mol, show relatively good superimposition of the plots. Besides the Mw averages of these samples obtained in the on-line SEC characterization are very closely to the off-line static values. Therefore we can surely assert that in the SEC fractionation of these three HA samples degradation does not occur. Results for the higher molar mass samples (Mw : 305M, 5.0M and 7.4M g/mol) were substantially different. Mainly for the higher molar mass samples the Mw averages obtained by the on-line SEC characterization was lower than the off-line static values. This result obviously means degradation of the sample in the columns. Furthermore, it was evident the retardation, entrapment, of the macromolecules in the columns. Under equilibrium conditions, that is ideal size exclusion, the elution volume should be independent from the molar mass of the unfractionated starting broad sample. In other words, at any elution volume, hydrodynamic volume, should elute macromolecules with the same size. As a result, for linear homopolymer all the M= (V) plot, from MALS, of the different HA samples should be superimposed. Figure 2 and 3 clearly show different behaviour. When the molar mass of the sample increases the elution of the macromolecules was delayed. This apparently unusual result is not new. Relatively new is the extent of the "retardation"; furthermore the Figures show experimental data not simulations. Other authors' reports and try to explain similar behaviours. The "retardation", Figure 2 and 3, is not related to the well-known concentration effect. This assertion derives from experiments performed to different concentration not here reported. Besides it needs to consider that the concentration of the solutions was ultra low (approximately 1/8 of the overlap concentration c*: from 0.02 mg/mL for 704M g/mol to 0.1 mg/mL for 0.66M g/mol). Our opinion is that both shear degradation and retardation may be explained with the partial penetration of the ends and/or loops of the macromolecules into relatively small pores of the packing with regard to the dimension
Aqueous SEC, light scattering and viscometry
53
of the macromolecules. The partial penetration of macromolecules in the pores of the packing decelerates their elution "retardation". Besides, the partially penetrated chains extend in the eluent flow and the probability of shear degradation increases. In this way, the degradation of UHMM polymers within porous column packing may rise even if narrow pore particles are used. The fractionation by SEC of UHMM HA samples present another intriguing problem. When the molar mass of the sample increases resolution decreases and band broadening increases. As a consequence at each elution volume the macromolecules are non-homogeneous in molar mass. In the presence of a polydisperse sample the MALS detector furnishes the Mw averages. We were interested to estimate the MMD of the UHMM sample, hence M, and the dispersity index D. Often the D value recovered from MALS is underestimated. Several authors associate this fact with the low sensitivity of the MALS detector to the low molar mass fractions of the sample. But this is not surely our case. The light scattering software calculates Mw and M, averages from the local values M; and Cj (concentration) with the usual formulae assuming homogeneous fractions. It is not difficult to demonstrate that if the fractions are nonhomogeneous, MWj instead of Mi, the global Mw average is correct but M, is overestimated hence the D value is underestimated. With UHMM sample the recovered D value requires always a critical evaluation. Concluding, despite the difficulties a successful fractionation and characterization of UHMM HA sample with Mw up to 2-3'10 6 g/mol was possible by a multidetector SEC system. When the experimental protocol, in particular the column set, is optimized the fractionation is effective and it is possible to obtain a good estimation of the MMD. Using a SEC-MALS-SCY system it is possible to estimate the whole distribution of molar mass, dimensions and intrinsic viscosity and the relative power laws. Figure 4 shows the Rg=f(M) power law, from SEC-MALS, obtained with four HA samples in 0.15M NaCI solvent at 37 DC. 1000 Rg
o 0.43M to 0.66M
n 1.06M
o 1.40M
100 0.15MNaCI, 37 DC a
Rg=KM K= 2.65' E-2 «=0.60 10 I.E+05
I.E+06 Molar Mass
I.E+07
Figure 4. Rg=f(M) power law, from on-line MALS, using four HA samples. Mw was respectively: 0.43M, 0.66M, 1.06M and 1.40M g/mol.
54
Characterisation and solution properties ofhyaluronan
Mw of the four HA samples was respectively: 0.43M, 0.66M, 1.06M and 1.40M g/mol. We can see the wide range in which the R,=f(M) power law was estimated. Furthermore the slope of the plot 0.6 is in a very good agreement with the value obtained for HA in similar conditions. CONCLUSIONS
Static off-line methods, light scattering and viscometry, furnish reliable Mw, Rg, A2' [11] results. However off-line methods are time-consuming and not suitable for the quality control of the samples. Besides these methods furnish only average values and not the whole MMD. A successful on-line SEC characterization requires an accurate optimization of the experimental protocol. Many aspects of the experimental protocol such as flow rate, sample concentration, columns, and fluid path that are not critical in the usual molar mass range, become crucial with UHMM polymers. The fractionation in the SEC columns is the critical point of the characterizationof the UHMM HA polysaccharides. Unfortunately the commercially available SEC aqueous columns have not been optimized for UHMM HA polymers. Despite the difficulties this study shows that a successful characterization of UHMM HA samples with Mw up to 2-3'10' g1mol is possible by means of a SEC-MALS-SCV system. ACKNOWLEDGEMENTS
The authors wish to thank Pharmacia & Upjobn and Dr. Ladislav Soltes of the Institute of Experimental Pharmacology of the Slovak Academy of Sciences that have provided the HA samples. REFERENCES
1. R. Mendichi, A. GiacomettiSchieroni,Characterization of ultra-high molar mass Hyaluronan: I. off-line static methods, Polymer, 1998, 39 (25), 6611-6620 2. T. Q. Nguyen, H. H. Kausch, Strategy for a reliably characterizationofUHMW polymers by GPC, Proceedings ofInt. GPCSymposium '91, S. Francisco CA, 1991, 373-397 3. R. Mendichi,A. Giacometti Schieroni, A strategy for a reliably characterizationof ultra-highmolar mass Polysaccharides, Proceedings ofInt. GPCSymposium '96. S. Diego CA, 1996, 183-200 4. R. Mendichi,A. GiacomettiSchieroni,Use ofthe single-capillary viscometer detector, on-line to a size exclusion chromatography system, with a new pulse-free pump, In: Hyphenated and Multidimensional Techniques. ACS SymposiumSeries; T. Provder Ed., ACS: Washington1999, Vol. 731, pp 66-83 5. P. J. Wyatt, Light scattering and the absolutecharacterization of macromolecules, Anal. Chim. Acta, 1993, 272, 1-40 6. E. V. Chubarova,V. V. Nesterov, Behaviourof macromolecules in non homogeneous hydrodynamic fields: degradationmechanismof macromolecules, In: Strategiesin Size Exclusion Chromatography, ACS SymposiumSeries; M. Potschka and P. L. Dubin Ed., ACS: Washington 1996, Vol. 635, pp 127-155 7. L. Soltes, D. Berek, D. Mikulasova, Characterizationofextremelyhigh molecular mass polystyrene by gel permeationchromatography, Colloid & PolymerSci., 1980, 258, 702-710
CELL ADHESION ON CROSSLINKED HYALURONAN SURFACES Martin Witt\ Michael Gellnsky", Ronald Mai\ Katharina Fladc 2 , Cora Rohlecke Eva Schulze", Richard H.W. Funk", Wolfgang Pompei I Department
1 ,
ofAnatomy, Technical University Dresden, Medical School, Fetscherstr. 74, D- Oi307 Dresden, Germany
'institute ofMaterial Science, Technical University Dresden, Hallwachsstr.S, 01069 Dresden, Germany J Nuffield
Department ofOrthopedic Surgery, Windmill Road, Headington, Oxford OX3 lLD, England
ABSTRACT Hyaluronan (HA), a high molecular weight glycosaminoglycan, is part of the extracellular matrix and involved in the regulation of cell/substrate interaction. HA has been reported to promote cell motility and proliferation. On the other hand, the non-adhesive properties of HA have been applied in clinical studies to prevent postoperative adhesions. In order to test the hypothesis that the probability of cells to adhere on a HA substratum depends on the amount of crosslinks present in HA, we coated glass slides with a watery HA solution and allowed the HA to dry out for 48 h. Subsequently, HA was crosslinked using a 0.2 % ethanolic N-(3dimethylaminopropyl)-N'-ethyl-carbodiimide (EDC) solution for 5 to 21 h. The degree of crosslinking was measured indirectly by the contact angle method that reflects the relative amount of ester bonds present in the compound and thereby the diminution of the polar carboxyl and hydroxyl groups. Osteoblast-like cells isolated and cultured from rat calvaria were placed on the HA-treated surface at various crosslinking conditions. As a parameter of adhesion, the formation of focal contacts together with re-establishment of the actin/vinculin complex was determined by means of immunofluorescence and laserscan microscopy. The results reveal that there is a direct dependence of cell adhesion on the extent of HA crosslinking, i.e., formation of focal contacts diminishes with the duration of crosslinking exposure. KEYWORDS Cell adhesion, carbodiimide cross linking, infrared spectroscopy, osteoblasts
INTRODUCTION Hyaluronan (HA) is a linear glycosaminoglycan consisting of repeating units of glucuronic acid and N-acetyl-glucosamine residues. As a major constituent of the extracellular matrix (ECM), it serves various mechanical and rheological (e.g., cartilage, synovial fluid, vitreous body) as well as more general biological functions by influencing cell adhesion, migration, or differentiation 1,2. HA influences not only the hydration state of tissues, it is involved in determining the structure of tissues by interaction with diverse other macromolecules, e.g. versican and aggrecan. These tasks arc accomplished by in-
28
Adhesion formation and hyaluronan
teractions with various binding proteins J that occur as cell surface receptor proteins (RHAMM, CD44) 4, extracellular and intracellular 5,6 binding proteins. In recent years, the biological properties of HA have lead to its widespread use as a biomaterial. Due to its biodegradability, HA is being exploited for numerous medical or pharmaceutical applications, for example, in wound healing 7, cataract surgery 8'"01' drug , 10 de IIvery . When used as a scaffold for tissue engineering, however, it is necessary to crosslink HA molecules in order to produce a water-insoluble gel II. The aim of this study was to test the hypothesis whether cell adhesion on chemically altered HA substrata is dependent on the amount of crosslinks present in the HA. We prepared glass slides which were coated with HA films with different degrees of crosslinking. As a chemical crosslinking agent, we used water-soluble carbodiimide, because it is not incorporated into the crosslinked polymer, to avoid possible cytotoxic. 12 ity .
MATERIALS & METHODS Measurement and Crosslinking of HA Round cover slips (11 mm in diameter) were coated with a solution of 0.1 mg/ml HA from streptococcus equi (F1uka, Heidelberg, Germany) and allowed to dry for 48h. Crosslinking with 0.2% N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide (EDC) dissolved in 80% ethanol was performed for 1, 5, 9, and 21h according to the method described by Tomihata and Ikada 13. (Crosslinking most probably occurs via the formation of ester bonds between carboxyl and hydroxyl groups ofHA molecules (Fig. 3). The degree of cross linking was determined indirectly by measurements of the contact angle using the Axisymmetric Drop Shape Analysis (ADSA) system. Simultaneous expansion of the water droplets during the measurements ensured that permanently new surfaces were wetted. To determine the presence of ester bonds formed by the crosslinking procedure, infrared spectra of thin HA films were recorded on a Perkin-Elmer FT-IR Spectrum 2000 spectrometer, equipped with an autoimage microscope". Cell adhesion tests Cultured osteoblasts of rat calvaria 14 were grown on HA-coated glass slides for 20 min. After short fixation in formaldehyde, the cells were processed for indirect fluorescence immunohistochemistry using an antibody against vinculin and texas red-labelled phalloidin for the demonstration of f-actin, Sections were examined with a confocal laserscan microscope (Leica L8M).
Cell adhesion on crosslinked surfaces
29
RESpLTS AND DISCUSSION We tested the adhesion properties of cultured osteoblasts on differently cross-linked HA films in an in-vitro model. Measurements using the contact angle method revealed a clear relationship between crosslinking time and hydrophobicity of the HA films. The contact angles increase with the time of crosslinking and diminution of carboxyl groups
/
--/
o
ro
crQulinkingtimc (h)
Fig.!. The contact angles increase with the time of crosslinking, demonstrating indirectly the relative degree of hydrophobicity of the HA surface.
Fig. 2a. Calvaria osteoblasts grown on glass slides do not adhere on spots of HA, which was crosslinked for21 h.
in favour of ester bonds. The hydrophobicity of the HA surface grows with the degree of crosslinking (Fig. 1). Accordingly, relatively few osteoblastic cells adhered to HA films, which had been crosslinked for 21 h (Fig.2), whereas clearly more cells adhered on HA films, which had been crosslinked to a lesser degree. Further evidence for the dependence of cell adhesion on the time of carbodiimide exposure is given by infrared spectra (Fig.3). To quantify the ester formation, the IR absorption at 2925 5h control cm- I was selected as a control, be€I cause this vibration is assigned to 00 .. 6 ( 04)0 ~!o the methylene groups (-CI-h-), ! () () Q G ;) which remain unchanged during the ~ 0 QO J ~ cross linking reaction, The semi e 0 0(1) linear correlation. (Fig.4). Waterinsoluble, chemically cross-linked dO r.1O '" .. Ul,,' \\ ' ..' 9h 21h HA (Hylan GF-20) has been used as a viscosupplementary agent in osFig. 2b: Osteoblasts grown onHAsubstrata which were theoarthritis 15. Its advantages concrosslinked for 5 to 21h with EDC. The laserscan imsist of higher viscosity and lower ages demonstrate that the preference of cells to adhere inflammatory potency compared on HA decreases withcrosslinking time. with low molecular weight HA fragments. Information on biological properties of HA is somewhat ambiguous. In some regards, the action of HA has been reported to be dependent on its molecular weight. Low molecular weight HA (in the range of 1-500x 103 daltons) induces inflammatory factors
•
30
Adhesion formation and hyaluronan
such as NF-kappa B fect.
16,
whereas high molecular weight (6000xl0 3) HA has no such ef-
3500
3000
2500
2000
1500
1000
500
Wave number [cm'"] Fig.3. Infrared spectra of both non-crosslinked (curve "0 h") and crosslinked (curves "1-21 h") thin HA films. The crosslinked samples show a new absorption at a wave number of 1700 cm· 1 (arrows), assigned to the carbonyl group most likely of ester bond. The non-crosslinked control (0 h) shows no additional peak. The intensity of the absorption increases with the crosslinking time.
3,5
o
3,0
~
s
2,5
«
2,0
~
FigA. Quantification of ester formation. The figure shows the intensity ratio of the absorbance at 2925 em" to that at 1700 ern" in correspondence to the cross linking time.
o
1,5
10
crosslinking time [hi
Also, angiogenesis is stimulated only by low molecular weight HA fragments, whilst high molecular weight HA suppresses angiogenesis 17. Generally, HA surfaces prevent mammalian cell adhesion 18, a phenomenon utilised in abdominal surgery to reduce postoperative adhesions 19. On the other hand, many tumour cells 20.21 as well as mast cells 22 adhere more easily on HA substrata. Malignant tumours express elevated levels of HA, and many tumour cells express elevated levels of the main HA binding protein, CD44 23. Since rat calvaria osteoblasts used in our study are known to express CD44 14, the impaired attachment of the cells indicates that CD44 or other related HA receptor proteins may not recognise modified HA, dependent on its degree of erosslinking. In summary, we have shown that chemical alteration of HA changes its cell adhesion properties. This might have important consequences for tissue engineering procedures,
Cell adhesion on crosslinked surfaces
31
especially when HA is used in multicomponcnt biomaterials, e.g. in mineralised collagen-hyaluronan membranes 24. The biocompatibility can be optimised particularly by the time of crosslinking of the HA component, until requirements of both favourable cell adhesion and water-insolubility are matched.
ACKNOWLEDGEMENTS We are indebted to the technical assistance of Mr Rene Born for infrared measurements. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG).
REFERENCES 1. T. C. Laurent, U. B. Laurent & J. R. Fraser. The structure and function ofhyaluronan: An overview.lmmunol Cell Biol, 1996,74(2), AI-7. 2. J. Y. Lee & A. P. Spicer. Hyaluronan: a multifunctional, megaDalton, stealth molecule. Curl' Opin Cell Biol, 2000, 12(5),581-586. 3. B. P. Toole. Hyaluronan and its binding proteins, the hyaladhcrins. Curl' Opin Cell BioI, 1990,2(5), 839-844. 4. P. W. Kincade, Z. Zheng, S. Katoh & 1. Hanson. The importance of cellular enviromnent to function of the CD44 matrix receptor. Curl' Opin Cell Biol, 1997, 9(5), 635-642. 5. S. P. Evanko & T. N. Wight. Intracellular Localization of Hyaluronan in Proliferating Cells. J Histochem Cytochem, 1999,47(10), 1331-1342. 6. 1. Huang, N. Grammatikakis, M. Yoneda, S. D. Banerjee & B. P. Toole. Molecular characterization of a novel intracellular hyaluronan-binding protein. J Biol Chern, 2000, . 7. M. Wiig, S. O. Abrahamsson & G. Lundborg. Effects ofhyaluronan on cell proliferation and collagen synthesis: a study of rabbit flexor tendons in vitro. J Hand Surg [Am}, 1996,21(4),599-604. 8. E. A. Balazs. Sodium hyaluronate and viscosurgery. In: Miller D, Stegmann R, eds. Healon (sodium hyaluronate). A guide to its use in ophthalmic surgery. New York: Wiley, 1983; 5-28. 9. T. J. Liesegang. Viscoelastic substances in ophthalmology. Surv Ophthalmoi, 1990, 34(4),268-293. 10. P. Bulpitt & D. Aeschlimann. New strategy for chemical modification of hyaluronic acid: preparation of functionalized derivatives and their usc in the formation of novel biocompatible hydrogels. J Biomed Mater Res, 1999,47(2), 152-169. 11. N. E. Larsen, C. T. Pollak, K. Reiner, E. Leshchiner & E. A. Balazs. Hylan gel biomaterial: dermal and immunologic compatibility. J Biomed Mater Res, 1993, 27(9), 1129-1134. 12. Y. S. Choi, S. R. Hong, Y. M. Lee, K. W. Song, M. H. Park & Y. S. Nam. Studies on gelatin-containing artificial skin: II. Preparation and characterization of crosslinked gelatin-hyaluronate sponge. J Biomed Mater Res, 1999,48(5),631-639. 13. K. Tomihata & Y. Ikada. Crosslinking of hyaluronic acid with water-soluble carbodiimide. J Biomed Mater Res, 1997,37(2),243-251. 14. E. Schulze, M. Witt, M. Kasper, C. W. Lowik & R. H. Funk. Immunohistochemical investigations on the differentiation marker protein Ell in rat calvaria, calvaria cell
32
Adhesion formation and hyaluronan
culture and the osteoblastic cell line ROS 17/2.8. Histochem Cell Bioi, 1999, 111(1),61-69. 15. M. E. Adams, A. 1. Lussier & J. G. Peyron. A risk-benefit assessment of injections of hyaluronan and its derivatives in the treatment of osteoarthritis of the knee [In Process Citation]. Drug Saj, 2000, 23(2), 115-130. 16. B. Oertli, B. Beck-Schimmer, X. Fan & R. P. Wuthrich. Mechanisms of hyaluronan-induced up-regulation ofICAM-l and YCAM-l expression by murine kidney tubular epithelial cells: hyaluronan triggers cell adhesion molecule expression through a mechanism involving activation of nuclear factor-kappa B and activating protein-I. J Immunol, 1998, 161(7),3431-3437. 17. M. Slevin, J. Krupinski, S. Kumar & J. Gaffney. Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation. Lab Invest, 1998, 78(8), 987-1003. 18. M. Morra & C. Cassineli. Non-fouling properties of polysaccharide-coated surfaces. J Biomater Sci Polym Ed, 1999,10(10), 1107-1124. 19. T. Sawada, K. Hasegawa, K. Tsukada & S. Kawakami. Adhesion preventive effect of hyaluronic acid after intraperitoneal surgery in mice. Hum Reprod, 1999, 14(6), 1470-1472. 20. D. Peck & C. M. Isacke. CD44 phosphorylation regulates melanoma cell and fibroblast migration on, but not attachment to, a hyaluronan substratum. Curr Bioi, 1996, 6(7), 884-890. 21. K. Takahashi, I. Stamenkovic, M. Cutler, A. Dasgupta & K. K. Tanabe. Keratan sulfate modification of CD44 modulates adhesion to hyaluronate. J Bioi Chem, 1996,271(16),9490-9496. 22. M. Fukui, K. Whittlesey, D. D. Metcalfe & J. Dastych. Human mast cells express the hyaluronic-acid-binding isoform of CD44 and adhere to hyaluronic acid. Clin Immunol, 2000, 94(3), 173-178. 23. Z. Rudzki & S. Jothy. CD44 and the adhesion of neoplastic cells. Mol Pathol, 1997, 50(2), 57-71. 24. M. Gelinsky, B. Knepper-Nicolai, K. Flade, W. Pompe, U. Hempel, C. Roehlecke & M. Witt. Mineralized collagen-hyaluronate membranes. Materials Week, Miinchen, Germany, Sept. 25-28, 2000.
CELL ATTACHMENT AND GROWTH ON SOLID HYALURONAN (RYLAN B GEL) Endre A. Balazs 2, lIana K. EUezer-Pye', Rita A. Dennebaum', Nancy E. Larsen! & Julie L. Whetstone2 I
Biomatrix, Inc.• 65 Railroad Avenue, Ridgefield. New Jersey 07657. USA
'Matrix Biology Institute, 65 Railroad Avenue. Ridgefield. New Jersey 07657. USA
ABSTRACT Rylan B is a water-insoluble hyaluronan produced by bis-ethyl sulfone covalent crosslinks. Rylan B gels containing 0.5% hyaluronan polymers are heat stable, but degradable by various hyaluronidase. They are more resistant to degradation by free radicals than high molecular weight (average MW > 4 million) hyaluronan of hylan A (avg. MW 6 million). Cells after trypsin treatment were seeded on the surface of hylan B gels imbibed with tissue culture media supplemented with fetal bovine serum. Cells from eight established cell lines originating from fibroblasts, epithelial or endothelial cells, chondrocytes, tumor cells and stem cells were used. All but the endothelial-origin cells attach to the gel, but only the L929 fibroblasts and stem cells multiplied. Fibronectins (plasma or cellular) added to the media-imbibed gel promoted the spread of the cells of some of these cell lines, while sulfated glycosaminoglycans inhibited the spread and growth of some of these cells. Some poly-lysines, on the other hand, promoted their growth. First explant chicken embryonic cells were also cultured on hylan B gels. Embryonic fibroblasts from the heart migrated and multiplied on the gel surface when homologous embryo extract was added to the culture medium. The results form these in vitro cell culture studies suggest that hylan B gel matrices may be modified by the addition of various types of cell attachment molecules as a means to promote cell attachment and growth.
INTRODUCTION Different cell types were cultured on a variety of materials to evaluate their potential as cell support systems. Hyaluronan (hyaluronic acid, HA) is a biocompatible, natural polysaccharide that may be modified to produce new materials. Hylan B gel' (produced by Biomatrix, Inc., Ridgefield, NJ) is a water-insoluble, crosslinked hyaluronan derivative that is heat stable and more resistant to degradation by free radicals than high molecular weight hyaluronan. A variety of cell lines were cultured on hylan B alone and on hylan B treated with various cell attachment molecules. First explant chicken embryonic cells were also cultured on hylan B gels. Using inverted-phase microscopy, the attachment, spread, and growth of cells on hylan B was monitored. The purpose of this study was to evaluate the potential of hylan B as a support to promote cell growth.
34
Adhesion formation and hyaluronan
METHODS CeU Lines Eight anchorage-dependent cell lines (L929 fibroblasts, BLO-II fibroblast-like cells, CPAE endothelial cells, CRFK and BSC-I epithelial cells, Monkey chondrocytes, LA-4 adenoma cells, and NE stem cells) were used in these experiments. Following confluency and subsequent trypsinization, cells were seeded on the surface of hylan B gel slabs. All slabs were equilibrated with tissue culture medium supplemented with 10% fetal bovine serum (medium for BLO-II cells was supplemented with 20% fetal bovine serum). 24 hours prior to the addition of cells, the hylan B gel slabs were treated in one of three ways: unaltered (10% or 20% EMEM only), 0.007 mg cellular fibronectin, or 0.007 mg plasma fibronectin. These substances were added to the surface of the gels and incubated for 24 hours at 37°C. One cell line was also seeded upon a gel slab coated with 0.007 mg poly-L-lysine. Cells were maintained at 37°C and monitored for attachment, spread, and growth using inverted-phase microscopy.
Chicken Embryonic CeUs Chicken embryos were sacrificed at 10 days. Heart muscle was obtained and cultured on hylan B gel slabs supplemented with homologous embryo extract. Using inverted-phase microscopy, cell behavior was monitored.
RESULTS Cell Lines Of the eight cell lines employed, all but the endothelial cells attached to untreated and plasma fibronectin-treated hylan B gel. Plasma fibronectin enhanced attachment of both BSC-l epithelial cells and L929 fibroblasts. Added cellular fibronectin did not affect the attachment of the cell lines tested. Spreading of cells on untreated gels was similar to fibronectin-treated samples, with L929 fibroblasts displaying an increased spreading with plasma fibronectin, and BLO-II spreading more readily on cellular fibronectin gel. BLO-ll cell spreading on cellular fibronectin gel was inhibited by the addition of various sulfated glycosaminoglycans (heparan sulfate, keratan sulfate). Slabs with poly-L-lysine facilitated the spreading of CRFK epithelial cells (Fig. 1). Two cell lines, L929 fibroblasts and NE stem cells, were found to proliferate on the gel slabs as determined by cell counts using a hemacytometer (Fig. 2). BLO-II cells appear to proliferate on cellular fibronectin gels as indicated by changes in cell morphology and increased gel surface coverage (Fig. 3).
Chicken Embryonic Cells Embryonic fibroblasts from the hearts of chicken embryos migrate and multiply on the gel surface, forming a reticulated network overriding the heart tissue (Fig. 4).
Cell attachment and growth
35
Figure 1. CRFK Epithelial Cells
48 hours
72 hours
With poly-L-lysine, 48 hours
With poly-L-lysine, 72 hours
36
Adhesion formation and hyaluronan
Figure 2. NE Stem Cells
48 hours
5 days
The effect of fibronectins on cell spreading and growth
Cell Spreading
CeUson hyianB gel
Cell Growth
cellular plasma fibronectin fibronectin
CPAE
.
NT
-
cellular plasma fibronectin fibronectin
-
NT
·
.
-
.
-
+
+
+
+
+
+
-
NT
-
-
NT
-
(fibroblastlike)
+
+
-
.
+
·
NE
. -
-
-
+
+
+
-
-
.
.
. -
(endothelial) Monkey I (chondrocyte)
L929 (fibroblast)
LA-4 (adenoma)
BLO-ll
(stem cell)
CRFK (epithelial)
BSC-l (epithelial)
- : no spread, no growth +: spread, growth NT: not tested
·
Cell attachment and growth
Figure 3. BLO-ll Fibroblast-like Cells with Cellular Fibronectin
24 hours
6 days
Figure 4. Chicken Embryonic Fibroblasts (From lO-day-old Embryo Heart Tissue)
18 days
32 days
37
38
Adhesion formation and hyaluronan
CONCLUSIONS The results from these studies indicate that hylan B gel provides a suitable scaffold for the growth of fibroblasts (L929), stem cell (NE), and embryonic cells (chicken). Other cell types such as the epithelial cells (CRFK) and fibroblast-like cells (BLO-II) grow on hylan B gel slab only when additional components are present (i.e. cellular fibronectin, poiy-L-Iysine). The results suggest that hylan B gel matrices may be optimized as cellular scaffolds by the addition of substances such as cellular fibronectin or poly-L-Iysine to enhance cell attachment. REFERENCES 1. E.A. Balazs & E.A. Leshchiner, Hyaluronan, its crosslinked derivative-hylan- and their medical applications, In: Cellulosics Utilization: Research and Rewards in Cellulosics (Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future), H. Inagaki & G.O. Phillips (eds.), Elsevier Applied Science, New York, 1989, pp 233-241.
MOLECULAR CHARACTERISATION OF HYALURONAN AND DYLAN USING GPC-MALLS AND ASYMMETRICAL Flow FFFMALLS S. AI-Assaf*, P.A. Williams and G.O. Phillips The North Hast Wales Institute, Centre for Water Soluble Polymers, Wrexham LLl I 24 W, UK. E-mail s.alassaf@ne.~i.ac.uk
ABSTRACT Gel permeation chromatography (OPC) and asymmetrical flow-FFF coupled to a multiangle laser light scattering detector have been used to determine the molecular weight and molecular weight distributions of hyaluronan, Healon and Hylan. It was shown that OPC-MALLS and FFF-MALLS gave comparable results for hyaluronan samples in the molecular weight range of9.0 x 104 to 2 X 106 • For Healon and Hylan it 6 was not possible to obtain molecular weight distribution by OPC but values of 5 x 10 7 and 1 x 10 were obtained using flow-FFF-MALLS and these values compare well to those obtained by static light scattering and low shear viscometry. It was concluded that FFF-MALLS is an effective method to determine the entire molecular weight range ofhyaluronan and Hylan. KEYWORDS Hyaluronan, characterisation, molecular weight distribution, OPC, FFF INTRODUCTION Due to the widening base of cosmetic, pharmaceutical and medical applications of hyaluronan and its derivatives I, macromolecular characterisation has become of great analytical importance. Balazs and co-workers 2 have developed a new family of hyaluronan derivatives named Hylan. Hylan can be produced, depending on the reaction conditions, over a range of molecular weight from 2 - 24 million. Hylan A (water soluble) is produced by cross-linking hyaluronan in situ in cock's comb with formaldehyde. Recently we reported static light scattering measurements on a range of Hylan samples with different molecular weights 3. The results were compared to capillary and low shear viscometry data and showed that successive filtration of Hylan low and high molecular weight did not influence the intrinsic viscosity value determined by capillary viscometry and were similar to that of the unfiltered sample. The intrinsic viscosity values, determined using low shear viscomet'J' for Hylan with Mw ca. 10 x 106 and 1.8 x 106 were found to be 8188 and 2070 em g-I. The comparative values, using capillary viscometry were 5146 and 2017 cm3 g-I demonstrating the effect of shear in the capillary viscometer. Filtration through a 1 um filter resulted in the average Mw of 10 x 106 for a range of hylan samples. The filtration showed pronounced effects on the results obtained by static light scattering where the Mw of Hylan (10 x 106 ) with RMS-radius of276nm was reduced when filtered through a 0.45 um filter to ca. 3 x 106 with RMS-radius of 167 nm. Repeated filtration through a
56
Characterisation and solution properties of hyaluronan
0.45~m filter did not reduce the Mw any further. On the other hand filtration through a O.2J.lm filter caused a further reduction but was not always possible for high molecular weight Hylan. There is a need for an absolute size characterisation method which can cover the wide molecular weight range of hyaluronan samples and its derivatives, with good resolution. This paper reports and compares results on the determination of molecular weight and molecular weight distribution of hyaluronan, Healon and Rylan using GPC-MALLS and asymmetrical Flow FFF-MALLS.
MATERIALS Hyaluronan HAl (Lot No. F17S0762) and HA2 (lot HA(P) were obtained from Dr Akio Okamoto, Denki Kagasku Kogyo K.K., Japan. HAl was autoclaved, for various times, and then freeze-dried to produce RAJ, HAS and RA6. HA4 (originally labeled Auto Back 2) was donated by Biomatrix Inc, (NJ, USA). The sample was dialysed against water for 5 days then autoclaved at 128C for 30 minutes and then freeze-dried. Rylan samples and Healon GV were kindly donated by Biomatrix Inc, (NJ, USA). All samples were dissolved in, a 0.22 lim filtered aqueous solution of, O.ISM NaCI and left to tumble at 4°C until complete dissolution.
EXPERIMENTAL GPC-MALLS Background Gel permeation chromatography (GPC) is a widely used separation technique for the determination of molecular weight and molecular weight distribution of polymers. It involves a column packed with a porous material with certain pore size and when a polydisperse polymer (consists of different size molecules) is passed through the column separation occurs based on hydrodynamic size. Molecules that are large are excluded from some of the pores, whereas small molecules can penetrate most of the pores. The large molecules, therefore, move quickly through the column. One of the major disadvantages of GPC is that it has limited application for high molecular weight polymer samples 4,5 since the molecules may be completely excluded from all the pores of the column packing material. In addition, high molecular weight polymers may degrade due to the high shear forces generated in the column. For some polymers, adsorption onto the packing material also presents a problem. Multi angle laser light scattering (MALLS) is one of the few absolute methods available for the determination of molecular weight and size over broad range. It utilises the principle that the intensity of light scattered elastically by a molecule (Raleigh scattering) is directly proportional to the product of the weight average molecular weight and concentration ofthe polymer 6 (equation below):
The term given between two brackets represents P( () which is a general form of a scattering function. K is an optical constant given by (K=21f 2 no (dn/dc)2 / A 4 NA), C is
Molecular characterisation
57
the concentration, RfJ is the excess Rayleigh ratio which is the measured quantity, fJ is the scattering angle, M w is the weight average molecular weight, A2 is the second virial coefficient, n, is the refractive index of the solvent, dn/dc the refractive index increment of the polymer in solution.Z is the wavelength of light, NA is Avogadro's number. When size exclusion chromatography is coupled to an on-line absolute molecular weight determining device (such as MALLS) and a concentration sensitive detector (refractive index or photometric) it is possible to measure the scattering intensity and sample concentration for each slice (fraction) in the fractionated peak. Thus information about the weight average molecular weight (Mw), number average molecular weight (M,,), molecular we~ht distribution, polydispersity index (MwlMn ) and radius of gyration can be obtained . GPC-MALLS System The system utilised a Waters (Division of Millipore, USA) Solvent Delivery System Model 6000A or P-500 dual piston syringe pump (Pharmacia Biotech). Two sets of columns were used. The first set employed a stainless steel column- Hemabio linear (lOl1m)packed with hydrophilic modified Hema gel (hydroxyethyl methacrylate copolymer) obtained from Polymer Standard Services, Germany. The second set employed two columns packed with composite crosslinked agarose called Superose" 6HR 10/30 (with bead diameter of 11-15 um) and Superose" 12HR 10/30 (with a bead diameter of 8-12 um) connected in series. According to the manufacturer (Pharmacia Biotech, Sweden) the separation range is from 1000 to 5 X 106, based on globular proteins. Injection into the GPC column was made with a manual Rheodyne Model 7125 syringe loading sample injector equipped with either a 100, 250 or 1000111 sample loop, a concentration dependent detector Wyatt OptilabDSP interferometric refractomter operated at 632.8 nm equipped with a Ia-mm PIOO cell (Wyatt Technology Corporation, USA). DAWN DSP laser light scattering photometer was equipped with a 632.8 nm He-Ne laser (Wyatt Technology Corporation., USA) with 15 detectors calibrated with filtered toluene and normalised with pullulan (23.8K) obtained from Polymer Standards Services. A value of 0.162 was used for the refractive index increment (dn/dc) 7. Data accumulation for the detectors used Wyatt Technology ASTRA 4.5 software. All measurements were performed at room temperature. Flow-field flow fractionation Background Field flow fractionation (FFF) is a family of fractionation techniques capable of separating particles and macromolecules according to their size. Asymmetrical flowfield flow fractionation (flow-FFF) is a variant of FFF that was first developed by Wahlund & Giddings 8. It separates molecules or particles according to the difference in diffusion coefficients and does not suffer from problems such as adsorption, molecular weight limit and shear degradation encountered by other techniques such as GPC. It has been shown recently that flow-FFF is an effective technique for the determination of the molecular weight distribution of polymers such as glutenin 9, amphiphilic graft copolymers 10 , carrageenan and xanthan 11, hydroxypropylmethyl
58
Characterisation and solution properties of hyaluronan
cellulose 12 and globular proteins, polystyrene latex beads and anionic polystyrene sulfonates 13. In this technique, the fractionation is achieved in a thin flat trapezoid shaped channel (see Figure 1). The channel consists of an upper (solid) wall made from polymethyl methacrylate and a lower porous wall, called the accumulation wall (Figure 1). An ultrafiltration membrane, permeable to the carrier liquid but not to the sample molecules, is placed on top of a porous frit. The membrane usually has a cut off of 104 and this determines the lower limit of molecular weight that can be fractionated. The top and bottom walls are clamped together. When an aqueous liquid is pumped through the channel it creates a secondary flow vector perpendicular to the primary axial flow vector (channel flow). The channel flow creates a parabolic flow velocity distribution across the thickness of the channel and transports the sample component down the channel to the outlet end where they are detected (Figure 1). The secondary flow, termed crossflow, drives any sample molecule or particle down to the membrane surface. This crossflow is counteracted by molecular diffusion caused by Brownian motion so that an exponential concentration distribution layer of polymer molecules is established. The thickness of this layer, therefore, depends on the magnitude of the diffusion coefficients. The larger polymer molecules tend to reside closer to the wall whereas the smaller ones diffuse towards the channel centre. Since the channel flow has a parabolic velocity profile the smaller species which are furthest from the accumulation wall move faster along the channel and elute first followed by the slowly diffusing component (high molecular weight). Therefore the elution profile in flow-FFF is opposite to that achieved by GPC. The basic theory of the asymmetrical flow-FFF and other retention modes are given in detailS, 14. Channel flow
Channel flow inlet
outlet,
':at..
'~~ - - - _ ~ I;;:~:""tec-~-'~-~--(Vout)
Focusing
poinl (z')
. ._ _•
~
c32f.l
Solid Wall
Cbaone
Width (W)
Figure 1. (top) Asymmetrical flow field flow fractionation channel and (bottom) separation principle in flow-FFF. For all FFF experiments a pre-experiment time was used in order to obtain a baseline for all detectors and was followed by activating the FFF software to start the fractionation procedure. A complete sample fractionation includes three necessary stages, namely injection/focusing/relaxation, elution and rinsing. The first stage is injection- focusing-relaxation mode (see Figure 2A). In this mode the liquid carrier, delivered by the main pump, enters the channel via the carrier outlet and exits the
Molecular characterisation
59
channel via the crossflow outlets (no flow at the carrier inlet). Following this the sample is injected into the channel, by a separate (injection) pump connected to the carrier reservoir, and enters at the injection point at a rate of 0.1ml/min. The sample is then concentrated at the focusing point (injection point) and left to relax for few seconds while the injection pump is off. The injection time was determined by the volume of the connecting tubes from the injection pump and the volume of the sample loop. At the end of stage 1 the elution (stage 2) starts by means of the control valve controlled by the software. In this phase, the liquid carrier enters the channel via the inlet and can exit through the channel outlet and crossflow outlet (see Figure 2B). For low molecular weight hyaluronan a crossflow of 3mVmin was used and lower cross flow (from 1.0 to 2.0 mVmin) were used for high molecular weight (> 3 million). Once the baseline for all detectors is back at the pre-determined baseline this is an indication that all the expected sample components are eluted, the flow direction is switched to the rinsing position (stage 3). In this stage, the carrier liquid enters the channel via the outlet and exit via the inlet to waste. This procedure is maintained for at least 10min at a flow rate of Iml/min. By switching to elution position the system is ready for the next run. All measurements were performed at room temperature unless otherwise stated. A
Carder
in
1
C,ossf:ow
ou11et
8
-_., ,.. ...
.
. : .... F r it: ., ~ ......... -
p.....-
.....,...-- MerTlbrane
I
Cro~sflow
outfet
Figure 2. Schematic representation ofthe flow in the asymmetrical FFF. (A) during injection/focusing/relaxation mode, (B) elution mode". FFF-MALLS System The flow-FFF system was supplied by ConSensus (Germany). The Channel has a trapezoidal geometry where the length of the channel is 28.6cm, the trapezoid breadths were 2.12 and 0.47cm respectively. The cut off area at the inlet end was 2.25cm2 and the total area of membrane enclosed by the spacer was 36.09cm2. The channel thickness was 190mm and the resulting channel volume 0.68ml. A Nadir UF-IOCIO ultrafiltration membrane of regenerated cellulose (Hoechst, Germany) was placed on the accummulation wall. Inlet and outlet holes are drilled in the upper wall to coincide with the tips of the cut out channel. Sample injection is carried out through an
60
Characterisation and solution properties ofhyaluronan
injection port that is 2.05 em downstream from the liquid carrier inlet port. The whole set (upper and lower wall) was clamped together with bolts tightened by approximately 4 Nm. The channel and crossflow were controlled using CSC V2.0 software supplied by ConSensus, Germany. The flow in the channel was generated by a Constant Metric 3200 pump and the injection of the sample was made with a Knauer pump (Microstar KlOO) connected to a rheodyne injector with an injection loop of 50, 100 or 250 ,.J.!. The solvent was filtered through using a 0.22 urn cellulose nitrate filter and was degassed before entering the channel using a ERC 3215a degasser. An in-line filter (0.22 um cellulose nitrate) was fitted between the pump and the flow-FFFchannel. The outlet end of the channel was connected to a DAWN-DSP (multiangle laser light scattering detector) and Wyatt OptilabDSP interferometric refractomter as described above. RESULTS AND DISCUSSION
Figure 3 shows the molecular weight distribution of different hyaluronan samples in the range of 1.8 x 104 - 2 X 106 obtained by GPC-MALLS. ~
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _----j .
't!,~:"'Ul9
......
,
Norm
~Lug
-~
~:~UlJ
_.
_u.-YF-o..:l
-e-
-,-
~~';:/'IJ
_YUK
~:~~11 Norm-LOll 1.orVer
Molecular weight (gfmol)
Figure 3. Differential molecular weight distribution of hyaluronan obtained by GPC-MALLS which employed superose 6HR + 12HR columns connected in series, flow rate 0.5mVmin. 6
A (HAl (Mw 2.0 x 106 , injected mass lOOJ1l of9 x 10-4 g/OO];B (HA2 (Mw 1.5 x 10 , injected 4 mass lOOJ1l of9 x 10-4 g/OO]; C [HA3 (Mw 8.0 x 106, injected mass 100J1lof9 x 10- g/OO];D (HA4 4 (Mw 4.4 x 106, injected mass 100J1lof 9 x 10-4g/OO]; E [HAS (Mw 9.0 X 10 , injected mass 250J1l 3 of 2 x 10-3 g/OO];F [HA6 (Mw 1.8 x 104, injected mass 250111 of 2 x 10- g/OO].
The results shown above agree well when compared to other techniques such as capillary viscometry and static light scattering. However, the GPC-MALLS technique is capable of covering only a limited range of hyaluronan molecular weight (up to 3 million). Above 3 million due to the size exclusion the high molecular weight is underestimated. Furthermore, the efficiency of a GPC column is markedly influenced by the carrier flow rate, column condition, sample concentration and pH. This clearly shown in Figure 4 for hyaluronan HA4 (Mw 4.4 x lOs). All runs shown in Figure 4 gave the same weight average molecular of 4.4 x io' ± 2%. However, the distribution is quite different and the polydispersity index increases with decreasing the flow rate which is an indication of a better separation.
61
Molecular characterisation 12.0
~
A
~-+-
1uloHler 6
..
. ~ - - - __.,._~-
-~
Nann'" I_og
~:,~~~;;':,:ag
C
Nonn = Log
1::Jt crder
440·QPC1
r:~:'~'r~e~ug
D O~O ._-~- ----.-~"""""......-.:;;;. 1.Oxlof' MolecuJar weight (gImol) Fi~ure
4. Molecular weight distribution obtained for hyaluronan HA4 (M, 4.4 x 10 ) by GPC-MALLS under different conditions.
A (column (Hemabiolinear, flow rate I mlImin, Cone. 2 x 10"" glml, Iml injected volume, at pH2; B, (Column-Hemabiolinear, flow rate I mlImin, Cone. 2 x 10-4J'ml, Iml injected volume, at pH6); C (Column-Hemabiolinear, flow rate 0.5 ml/min, Cone. 2 x 10 g/ml, Iml injected volume, at pH6; D (Superose 6HR and superose 12HR eonnected in series), flow rate 0.5 mlImin, Cone. 9 x 10"" g/ml, injected volume 1001-11.
In order to examine the applicability of flow-FFF to the separation of hyaluronan we used a hyaluronan sample of low molecular weight that can be easily separated by GPC. Figure SA shows the molecular weight distribution obtained by GPC and compares with the distribution of repeated runs (B and C) obtained by flow-FFF. Weight average molecular weight values of 9.08 and 9.41 x 104 were obtained and compare well with that of9.0 x 104 obtained by GPC (see Table 1).
I
J
I
3.0
2.0
1.0 -
1.0.10"
Molecular weight (gImol)
Figure 5. Differential molecular weight distribution for hyaluronan HA5. A, obtained by GPC-MALLS, conditions as in Figure 3E. B, obtained by FFF-MALLS, Cone. 1.77 x 10-3 glm!, 2501J,1 injected mass. 3m! erossflow during focusing. Elution mode- lmllmin ehannel flow and O.3ml/min erossflow; C as in B (repeated run).
Table 1. Weight average molecular weight and radius of gyration for hyaluronan sample HA5 obtained using GPC-MALLS and flow FFF-MALLS. Sample
Method
A
GPC-MALLS FFF-MALLS FFF-MALLS
B
C
Flow rate (ml/min) 0.5 1.0 1.0
Mwt 9.01 ±0.15 x 104 9.08 ± 0.15 x 104 9.41 ± 0.25 x 104
KgI Dm 30.0 ± 5.7 28.1 ± 6.8 26.1± 8.8
Figure 5 5 5
62
Characterisation and solution properties ofhyaluronan
The separation by GPC was less effective in comparison to that of flow-FFF. Furthermore, quite good reproducibility was obtained for the flow-FFF runs which 4 showed fractions from - 1 x 10 to -8 X 105.. In order to examine the quality of separation a plot of molecular weight as a function of the elution volume is usually used. Figure 6 clearly shows that good separation has been achieved. -------"'--'=---==,:::..J
1.0JC10~
1.0x10"
-,
1.0x10"
"'''
<,
.....
I
1'.0... 0::1
9.5
""-
--'~._.~2::--=~··
'10.0 VOlum_ (rnL)
'10.5
....J 11.0
Figure 6. Molecular weight plotted as a function of elution volume obtained using FFF-MALLS for repeated runs ofhyaluronan (HAS, Mw 9.0 x 104) . Conditions as in Figure 5B and C.
Figure 7 shows repeated runs of the best separation obtained by GPC for HA4 (4.4 5
x 10 ) and compares with that obtained for repeated runs using flow-FFF as shown in
Table 2. Both low and high molecular weight materials are separated better than GPC and the same weight average molecular weight value was obtained. By comparing the two values it is clear that FFF offers better separation and by adjusting the crossflow and channel flow one can obtain separation conditions similar or better than a GPC columns.
I
I"
I
D ".0.. 10· _ o l . r M • • • (",hngll
Figure 7. Comparison of molecular weight distribution ofhyaluronan HA4. (Mw 4.4 x 105) (A and B) obtained by GPC-MALLS; (C and D) by FFF-MALLS (crossflow and channel flow as in Figure 5). Conditions for GPC-MALLS as in Figure 3D.
Table 2. Weight average molecular weight and radius of gyration for hyaluronan sample HA4 obtained using GPC-MALLS and flow FFF-MALLS. Sample
Method
A B C
GPC-MALLS GPC-MALLS FFF-MALLS FFF-MALLS
D
Flow rate (mllmin) 0.5 0.5 1.0 1.0
Mw
4.44 ± 0.12 x 4.46 ± 0.25 x 4.41 + 0.27 x 4.35 + 0.32 x
105 105 105 105
Rg(nm)
Figure
52.3 ±2.6 51.7 ± 2.4 47.6±4.8 47.3 ± 5.2
7 7 7 7
Molecular characterisation
63
Up to date the highest molecular weight of hyaluronan, produced from animal sources, commercially available is produced by Pharmacia and marketed under the trade name of Healon GV. Healon GV was characterised and a molecular weight value of 4.7 x 106 obtained by static light scattering. The low shear viscometry gave an intrinsic viscosity value of 6400 cm3 g-I which corresponds to an identical Mw value of 4.8 x 106 . Using flow-FFF we also have obtained a value of 5.07 and 4.92 x 106 for this sample. GPC-MALLS gave scattered results for Hylan samples of 10 x 106 . A value of 5-6 x 106 was obtained which clearly shows the effect of exclusion. In our characterisation of Hylan of high molecular weight we used static light scattering and low shear viscometry. Both techniques gave a value of 10 x 106 . We have also shown rheologically IS that this sample has a molecular weight of 10 x 106 . Therefore, we used the same sample in our FFF-MALLS system. Figure 8 gives the fractogram showing the light scattering and refractive index response. Figure 9 is a plot of the molecular weight versus elution volume for the sample shown in Figure 8 and clearly shows a good separation for this high Mw Hylan. A molecular weight value of lOx 106 was reproducible when the sample was subjected to the same filter treatment as in static light scattering. However due to the ability of high molecular weight hyaluronan, even diluted solutions, to form a tertiary structure 16 the separation was often difficult and yielded a low polydispersity value «1.2). The formation of tertiary structure by hyaluronan may result in what is called a 'concentration effect'. This effect is usually explained by a decrease in molecular size and an increase in solution viscosity. The concentration effect was previously observed in GPC analysis of hyaluronan and was eliminated by increasing the temperature 17. The separation for this high molecular weight (Hylan) was greatly improved when the sample, carrier liquid and channel were maintained at 40°C. A polydispersity value of 4.5 was obtained. This is clearly shown in the molecular weight distribution given in Figure 10 which demonstrates the potential of flow-FFF in separating high Mw molecules where fractions of 1 x 106 up to ca. 40-50 x 106 can be fractionated. It is not possible to separate these high molecular weight Hylan samples on a GPC column due to their complete exclusion.
::b-"~-'
i ::
/ "~-
~._--.J
o,.
;'~ Av~ ""'00' :
Fowsing
~I-.::J
s,..pcha..-Hv:,O
~I
..---, i
"T
~
"" Volume (m1) Start Roo
Figure 8. Fractogram showing the light scattering at 90° response together with the refractive index response for Hylan (HYl) in aqueous O.15M NaCI. Concentration (2 x 10-4 glmI , 50~ injected mass, channel flow O.4m1/min (a crossflow of O.lm1/min was used during elution). Crossflow during focusing used 2m1/min. The carrier and the channel were maintained at 40°C.
64
Characterisation and solution properties ofhyaluronan
'.D.'U•. - - - - - - - - - - -
~~=
"v••·n, )
•. o:oc.o"
...o•
.,o·'::--"-~_........,...~~~c_L
.:&.0
__
_'_....__t....___l_ ........
4.0
4." Vol....... ( n i L )
Figure 9. Molecular weight as a function of elution volume for Hylan (HY1) obtained by flow FFF-MALLS, conditions as in Figure 8.
I
I
".0
I Figure 10. Molecular weight distribution for Hylan (HY1) obtained by flow FFFMALLS. Conditions as in Figure 8. CONCLUSION The study demonstrates the applicability of asymmetrical flow-FFF to the fractionation and characterisation of low and ultra-high molecular weight hyaluronan and its derivatives. We have confirmed that the separation can be performed using flow FFF in a relatively short run time in comparison to GPC. ACKNOWLEDGMENT We thank Dr Endre A Balazs for supporting this investigation. REFERENCES 1.
2.
3.
4.
5.
E.A Balazs, E.A. Leshchiner, N. Larsen & P. Band, 'Application ofhyaluronan and its derivatives', In: Biotechnological Polymers, C.G. Gebelein (ed.), 1993, Technomic Publishing Co., Inc., Lancaster, Basel, pp. 41-65. E.A Balazs, E.A Leshchiner, A Leshchiner, N. Larsen & P. Band, 'Hylan preparation and methods of recovery thereof from animal tissue' U.S. Patent # 5,099,013. March 24, 1992. S. AI-Assaf, 0.0. Phillips, AP. Gunning & V.l. Morris, 'Molecular interaction studies of the hyaluronan derivative, hylan using atomic force microscopy', Carbohydrate Polymers, 2001, (in press). S.E. Harding, K.M. Varum, B.T. Stokke & O. Smidsord, 'Molecular weight determination ofpolysaccharides', Advances in Carbohydrate Analysis, 1991, 1, 123-125. S. Nilsson, L.-O. Sundelof & B. Porsch, 'On the characterisation principles of some technical important water soluble non-ionic cellulose derivatives', Carbohydrate Polymers, 1995,28,265-275.
Molecular characterisation
6. 7. 8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
65
P.l Wyatt, 'Light scattering and the absolute characterisation of macromolecules', Analytica Chimica Acta, 1993, 272, 1-40. S. AI-Assaf, 'Rheological properties and free radical stability of cross-linked hyaluronan (Hylan)', Ph.D. Thesis, University of Salford, 1997. K-G. Wahlund, lC. Giddings, 'Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall', Analytical Chemistry, 1987, 59, 1332-1339. K-G. Wahlund, M. Gustavsson, F. MacRitchie, T. Nylander & L. Wannerberger, 'Size characterisation of wheat proteins, particularly glutenin, by asymmetrical flow field flow fractionation', Journal ofCereal Science, 1996, 23, 113-119. B. Wittgren, K-G. Wahlund, H Derand & B. Wesslen, 'Size characterisa-tion of a charged amphilic copolymer in solutions of different salts and salt concentrations using flow field-flow fractionation', Langmuir, 1996, 12,59996005. C. Viebke & P. Williams, 'Determination of molecular mass distribution ofkcarrageenan and xanthan using asymmetrical flow field-flow fractioonation', Food Hydroco//oids, 2000, 14,265-270. B. Wittgren & K-G. Wahlund, 'Size characterisation of modified cellulose in various solvents using flow FFF-MALS and MB-MALS', Carbohydrate Polymers, 2000, 43, 63-73. C. Tank & M. Antonietti, 'Characterisation of water-soluble polymers and aqueous colloids with asymmetrical flow field-flow fractionation', Macromol. Chem. Physic., 1996, 197,2943-2959. LC. Giddings, 'The field-flow fractionation family: underlying principles', In: Filed-Flow fractionation handbook, M.Schimpf, KCaldwell & lC.Giddings (eds.), Wiley Interscience, New York, 2000, pp3-30. M. Milas, M. Rinaudo, I. Roure, S. AI-Assaf, G. O. Phillips & P. A. Williams, 'Rheological behaviour ofhyaluronan, Healon and hylan in aqueous solutions', In: Hyaluronan 2000, IF. Kennedy, G.O. Phillips, P.A. Williams & V.C. Hascall (eds.), Woodhead Publishing, Cambridge. RH Mikelsaar & lE. Scott, 'Molecular modelling of secondary and tertiary structures of hyaluronan, compared with electron-microscopy and nmr data possible sheets and tubular structures in aqueous-solution', Glycoconjugate J , 1994, 11,65-71. E. Orvisky, L. Soltes & S. AI-Assaf, 'Concentration effect in hyaluronan analysis by size exclusion chromatography', Chromatographia, 1994, 39, 366368.
FORCE MEASlJREMENTS ON SURFACES BEARING COVALENTLY LINKED HYALURONAN Marco Morra*, Clara Cassinelli Xol»! Bill Riccrchc. .\'11' S. Rocco 31, /-IOUi iillnfrancn d Asti, n.u.v
ABSTRACT
The Atomic Force Microscope (AFM) was used to probe the aqueous interface of glass slides bearing a top layer of hyaluronan (HA) The effect of the surface linkage (ionic Versus covalent), of the surface density of HA, and of the HA molecular weight were investigated by the acquisition of force-separation curves between the sample surface and the AFM cantilever tip in saline Data show that this approach can yield useful information on the interfacial properties of HA coated samples In particular, the range of interaction between the tip and the surface is much lower when HA is covalently linked than when it is ionically coupled, suggesting a more compact surface structure in the former case Increasing HA surface density minimises the interaction force between the surface and the AFM tip, while the molecular weight of the surface linked HA apparently does not affect the shape and range of the interaction curve KEYWORDS
Hyaluronan, surface modification, atomic force microscopy, force-distance curves, interfacial interactions INTRODUCTION
Studies and applications of hyaluronan (HA) and hyaluronan derivatives in homogeneous solutions, or as a gel, or as fabrics are constantly growing. An intriguing sub-section of HA applications is the use of HA in the surface modification of medical devices I The covalent linking of a thin overlayer of HA to medical devices allows to impart to the substrate surface (ie to the surface of plastics or metals used to make a given medical device) several of the intriguing properties of HA A growing number of papers describe the application of surface-linked HA in different fields of the biomedical devices industry, from intra-ocular lenses-, to surgical rneshes-'. to blood contacting applications'[ An intriguing aspect of these studies is the attempt to correlate the unique properties and behaviour of HA in homogeneous solutions 5,6 with the performances of surface immobilised HA In this respect, it has been shown 2,7 that surface linked HA can prevent fibroblast cells adhesion and greatly reduce bacterial adhesion in ill vi/to experiments; and that this anti-adhesive behaviour is definitely affected by the surface density of HA chains 2,7,8 only a complete surface coverage by HA (HA fractional coverage close to 1) can effectively result in anti-adhesive surfaces
68
Characterisation and solution properties of hyaluronan
The understanding of the structure-properties relationship of surface-linked HA can greatly benefit from surface analytical techniques that can directly probe the interface between the aqueous environment and the coated device. The evaluation of the chemicophysical interfacial properties of medical devices in aqueous environment has long been hampered by the lack of suitable analytical techniques? The growth of direct force measurements 1';0 force-distance curves obtained, in particular, by the Atomic Force Microscope (AFM), has offered new ways to investigate the device/aqueous interface and to study the mechanisms that control bio-adhesive phenomena-. 10-12 Briefly, besides imaging applications 13. the AFM has evolved as a tool to measure the force exerted on the AFM cantilever tip as it approaches the sample surface l 0- 12 By the recording of the tip deflection induced by interfacial forces as a function of the separation between the tip and the sample surface, it is possible to gather interesting information on intramolecular forces operating at the interface. Since these measurements can straightforwardly be performed in aqueous environment, it is possible to check directly forces operating at aqueous interfaces The aim of this paper is to present our results on the measurement of force-distance curves between the AFM tip and surfaces coated by HA In particular, in this study, glass was used as a model substrate surface By using HA of different molecular weight, using different coupling strategies, and obtaining different surface densities of HA chains, we wanted to check whether the force-distance approach can distinguish between closely related HA coated samples, and ifit can yield information on the fine structure of the HA surface layer
MATERIAL & METHODS
Materials HA (Mw '" 200000) and high molecular weigth HA (Mw '" 2000000) were kindly supplied by Fidia Advanced Biopolymers All other chemicals were purchesed from Sigma-Aldrich
Samples preparation Surface modification was performed as follows 7. microscope cover glass slides were first coated by a hydrocarbon layer deposited from ethylene plasma, using a stainless steel, capacitively coupled parallel-plate plasma reactor, with the samples located on the water-cooled grounded electrode The reactor volume is about 3 dm 3, and the distance between the electrodes 10 cm Flow rate, controlled by a MKS mass flow controller, was 20 seem (standard cubic centimeters/minute), the pressure inside the chamber before the onset of the discharge was 2 Pa The power discharge was 30 W and the treatment time 60 s. The hydrocarbon surface obtained in this way was then oxidised by air plasma, in the same' reactor After treatment, samples were placed in polystyrene Petri dishes and dipped in 2 mL ofa 05% (W/v) aqueous solution of PEl for two hours After prolonged rinsing in doubly distilled water, samples were dried under a laminar flow hood Then, 5 mL of aqueous HA solutions were placed into the Petri
Force measurements on surfaces
69
dishes Ionic coupling (coded HAion) was obtained simply by leaving the aminated glass surface in the HA solution overnight (using 200 KDa HA), followed by extensive rinsing Chemical coupling between surface amino groups and carboxyl groups of the polysaccharides chain was performed by carbodiimide condensation To obtain different surface densities of polysaccharide different concentrations of polysaccharide were used 7,8 a 05% solution was used to achieve surface fractional coverage close to one (coded HAOS), while a 00 I (w/v) solution was employed to obtain a low fractional surface coverage (coded HAOO I) The just quoted experiments were performed using 200 KDa HA and surface composition was checked by XPS, as described previously7,8 A further set of samples were prepared using 2000 KDa HA, to evaluate the effect of HA molecular weight The same conditions used for the preparation of HAaS were used These samples will be coded HAHMW in the following After overnight reaction, samples were extensively rinsed and stored overnight in doubly distilled water. Before testing, samples were dried under a laminar flow hood.
AFM force-distance curves measurements All measurements were performed with a commercial AFM (Nanoscope 1II, Digital Instruments, California), using a conventional V-shaped silicon nitride cantilever (200 urn length, spring constant, as indicated by the supplier, 006 N/m) A 125xl25 11m scanner (J scanner) was used Force-distance curves were acquired in a 00 I M NaCI solution, using the fluid cell supplied by Digital. In the force measurement the sample is moved continuously up and down Deflection of the cantilever and height position of the sample are recorded. The force is obtained by multiplying the deflection of the cantilever times its spring constant In this case the spring constant was not measured and the value suggested by the producer was used, but this does not affect the meaning of the data. First of all, we are not interested in the absolute values of force but on the comparison between values measured on different samples Then, measurements were repeated using the same cantilever for the whole set of samples, or a new cantilever tor each sample no significant differences between the data obtained was detected To obtain force versus distance curves, the deflection of the cantilever must be subtracted to the piezo scanner position The point of zero distance was determined from the linear part of the contact line Force versus scanner position curves were continuously recorded with a typical frequency of about I Hz, the scan length was 200 rim. A minimum of20 curves in 5 different regions of the each sample were recorded RESlJLTS
Typical examples of the results obtained are reported in Figures I and 2 In particular, Fig I shows the force exerted on the silicon nitride AFM tip as it approaches the surface of HAOS in 0001 M NaCL Upon approaching, a monotonously increasing repulsive force is detected, whose range spans a few tens of nrn. This general behaviour is in agreement with results obtained on HA coated intra ocular lenses 2 The lack of any attractive jump is consistent with the hydrophilic nature of the HA coated surface. The range of interaction shows that a soft, compressible overlayer (HA) exists on the substrate surface, as recently described by Butt 14 Control experiments
70
Characterisation and solution properties ofhyaluronan 2
\
\
i \ \
l....,
\
'<..: ..._
approaching
o -..
retracting
-I
o
20
40
60
80
lOa
separation (nm) Fig. I. Force-separation curve obtained on HAaS in 000 1 NaCI 2
'\.
\\
\\ \
\
\"
~_._,--'-
-..,"---'--_.
.
\
a
approaching
__
..,:----..
----_..,---retracting
-I
a
20
40
60
80
100
separation (nm) Fig. 2 Force-separation curve obtained on HAOO 1 in 0.00 I NaCI
Force measurements on surfaces
71
performed on bare glass or aminated glass show a completely different behaviour: in the former case a "hard wall" repulsion, with a decay length much lower than in the HAOS case, is observed As to aminated glass, the curve is characterised by an attractive interaction between the tip and the surface Interestingly, measurements performed on HAOO I show a behaviour similar to that of HAOS when the tip approaches the sample Significant differences between samples, however, are observed when the tip is retracted from the coated glass. As clearly shown by Fig. I and 2, a certain adhesive force is detected upon tip retraction In both cases the retracting curve shows a "saw-tooth" appearance, which is generally attributed to the stretching of polymeric segments of different length 14 Clearly, the adhesive force is greater in the case of the sample with a lower surface coverage The figures reported show just one example of the experimental curves obtained: adhesive interaction in AFM measurements should be interpreted from a statistic point of view In the present case, the recorded adhesive force on retraction was constantly higher in the case of HAOO I, and in no case low forces such as those shown in Fig I were measured on HAOO I As to the comparison between ionically and covalently bonded HA, Fig 3 shows force-distance curves measured as the AFM tip approaches to the HAOS and the HAion surface in 0.001 NaCl
2
z: C
'-
<2
o
-I
o
20
40 60 separation (nm)
80
100
Fig 3 Approaching force-separation curves showing the different behaviour of HAOS and HAion aqueous interfaces in 0.001 NaCI
72
Characterisation and solution properties of hyaluronan
Clearly, the range of the interaction is significantly different in the two cases In the former case, the tip starts to "feel" the presence of the surface at a distance of about 4050 nm, while in the second case the monotonously increasing repulsive force starts at about 100 nm from the surface Clearly, these data suggest that the HA overlayer is much tighter or that the HA overlayer thickness is lower in the case of the covalent HA Surface chains are probably "looser" when it comes to ionic HA and the overall apparent thickness of the surface polyelectrolyte layer is greater than in the "covalent" case Finally, considering the comparison between the different molecular weights, approaching curves such at that shown in Fig I and those obtained in the case of HAHMW prepared in the same condition show a very similar range of interaction. Approaching curves were fitted using a double exponential, as shown by Butt 14 No significant differences were detected between the two samples, curve-fitting yielded decay lengths substantially identical in the two cases Apparently, from these measurements, the molecular weight of HA does not affect the thickness of the surfaceimmobilized HA layer.
DISCllSSJON The evaluation of the properties of aqueous interfaces play an important role in the understanding of the performances of biomedical devices coated by hydrophilic polysaccharides 15 In this study, the surface structure of a set of different HA coated samples was evaluated by force-separation curves obtained by the AFM From a general point of view, force-separation curves of surfaces bearing a hydrophilic overlayer show, on approach, a monotonously increasing repulsive force between the sample surface and the AFM cantilever tip I0,12,14 The range of this force is very different from that of repulsive forces detected on hydrophilic, but "rigid" surfaces, as recently discussed in the case of plasma treated Versus HA coated polymethylmethacrylate intraocular lenses 2 Data presented in this work clearly show that this approach can distinguish between the different interfacial nature of the samples. In particular, the covalent linking of HA leads to a significant decrease of the range of interaction (thickness of the surface layer) as compared to the simple ionic coupling (Fig 3), as expected also from the behavior of homogeneous polyelectrolye solutions These data indicate that the establishment of covalent bonding between the aminated surface layer and HA greatly reduces the mobility and freedom ofHA surface chains, increasing the "stiffness" of the surface layer as compared to simple ionic interaction. The nature of the coupling reaction involves a high frequency of interfacial bonds between HA and the aminated layer, resulting in multi-point attachment of the HA chain to the substrate surface Studies on the effect of the ionic strength and ion type on the interfacial behavior of covalent and ionic HA are in progress The effect of increasing fractional coverage by HA, which has been shown to control the cell-resistant behaviour of HA coated surfaces in in vitro experiments7,8,15, is clearly reflected in the retraction cUlve of HAOO I and HA05 In the former case a significantly higher adhesive force is detected, as shown in Fig I and 2 A likely reason for this behaviour is that the AFM tip can still interact, in the case of HAOO I, with the substrate or with available amino groups, while most of them are inaccessible when the
Force measurements on surfaces surface fractional coverage approaches 17,8
73
Experiments with surface-functionalized
AFM tips2,12, that allow to add a "chemical" dimension to force curves, could be of much help to clarify this point. Finally, the lack of difference of the interaction range between samples prepared, in the same conditions, using different molecular weights, could be due to the specitic coupling reaction as previously remarked, this approach leads to "multi-point" attachment of Hi\, which is coupled in a "side-on", as opposed to an "end-on" configuration Another possible explanation is that the coupling reaction has a higher yield with low or medium molecular weights, so that, irrespective of the actual nominal HA molecular weight, because of the polidispersity of the HA chains, the same chain lengths have more chance to be coupled This aspect could be clarified in experiments with samples having a narrow molecular weight distribution ACKNOWLEDGEMENTS We thank Davide Renier (Fidia Advanced Biopolymers) for helpful discussion REFERENCES
A Pavesio, D Renier, C Cassinelli, and M Morra, .Anti-adhesive surfaces through hyaluronan coatings', Med Dev Tech., 1997,8,20-28 2 C Cassinelli, M. Morra, A Pavesio, D Renier, 'Evaluation of interfacial properties of hyaluronan coated intraocular lenses', J Biomat. Sci Polim Ed, 2000, II, 961978 , .1 M Lise, C Belluco, S Pucciarielli, F. Meggiolaro, L Codello, D Pressato, M Dona, and E Bigon, In Redeftutng Hyaluronau, G Abatangelo, and P H. Weigel, (eds.), 2000, Elsevier, Amsterdam, pp 339-343 4 S Verheye, C P Markou, M Y Salame, B Wan, S B. King III, K A Robinson, N A F Chronos, S R Hanson, 'Reduced thrombus formation by hyaluronic acid coating of endo vascular devices', Arterioscler. Thromb. Vase Biol., 2000, 20, 1168-1172 The Biology of Hyaluronan, D Evered and J Whelan (eds.), 1989, Wiley, Chichester 6. The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives, T C Laurent (ed.), 1998, Portland Press Ltd, London 7 M. Morra, and C Cassinelli, 'Non-fouling properties of polysaccharides-coated surfaces' J Biomat Sci Polym Ed, 1999, 10, 1107-1124 8 M Morra, C Cassinelli, .Simple model for the XPS analysis of polysaccharide coated surfaces' Surf Interf Anal., 1998,26, 742-749 9 E A Vogler, 'On the biomedical relevance of surface spectroscopies' J. Electron. Spectr. Related Phenomenon, 1996,81,237-248 10 H J Butt, M Jaschke, W. Ducker, 'Measuring surface forces in aqueous electrolyte solution with the atomic force microscope', Bioelectrochemistry and Bioengineering, 1995,38, 191-201 II B Cappella, P Bascheri, C Frediani, P Miccoli, and C Ascoli, 'Improvements in AFM imaging of the spatial variation of force-distance curves on-line images' , Nanotechnology, 1997, 8, 82-87 12 H J Butt, and V Franz, In Water III Hiomateriuls Surface Science, M. Morra
74
Characterisation and solution properties ofhyaluronan
(ed.), 200 I, Wiley, Chichester, pp 245-266 13 M K Cowman, 1 Liu, M. Li, D M Hittner, and 1 S Kim, in The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T C Laurent (ed.), 1998, Portland Press Ltd, London, pp 17-24 14 H 1 Butt, M Kappl, H. Mueller, R Raiteri, W. Meyer, and 1 Ruhe, 'Steric forces measured with the atomic force microscope at various temperatures', Langmuir, 1999, 15,2559-2565 15 M. Morra, and C Cassinelli, In Wafer ill Biomaterials Surface Science, M Morra (ed.), 2001, Wiley, Chichester, pp. 353-387
PART 2
APPLICATION OF HYALURONAN IN TISSUE ENGINEERING
VISCOAUGMENTATION: A HISTORICAL PERSPECTIVE Biomatrix Inc. 65 Railroad Avenue, Ridgefield, New Jersey, USA
ABSTRACT
The biocompatibility and unique viscoelastic properties of hyaluronan (HA), a natural polysaccharide and major component of the intercellular matrix of all connective tissues, make it a polymer of choice for use as an implant to augment soft tissues. Native, unmodified hyaluronans were found to have residence times in soft tissue which were too short for practical use in augmentation procedures. On the other hand, the physical properties of HA are ideal: natural viscoelastic characteristics, high water content, and high permeability to macromolecules. Therefore a suitably modified HA molecule must possess similar or enhanced rheological properties and must have a significantly prolonged residence time in vivo. The first crosslinked, water-insoluble HA derivative with appropriate physical and biological properties for medical therapeutic use was developed in the early 1980s. Hylan B gel, as it is called generically, is produced by chemically crosslinking the HA chains via the hydroxyl groups using divinyl sulfone, which creates bis-ethyl sulfone crosslinks. Hylan B gel for viscoaugmentation was developed as a "bulking agent" that is physically and biologically compatible with various soft tissues, including subdermal and sphincter muscle tissues. Hylan B gel can be injected through a 30G needle as small, elastically deformable gel particles. This gel is available in some countries for soft tissue augmentation to correct dermal wrinkles and depressed scars. In addition, epoxy reagents have been used since the 1960s for crosslinking hyaluronan, based on the industrial experience of crosslinking dextran and other polysaccharides. In the late 1990s, gel preparations made from epoxy 'stabilized' HA became available in some countries for dermal tissue augmentation. Hylan B administered by injection into the urethral sphincter muscle is in clinical trials for the treatment of urinary stress incontinence and for the viscoaugmentation of vocal cords in functional inadequacy. DEFINITION Viscoaugmentation
The use of viscoelastic gels to provide augmentation of the intercellular matrix of the dermal and other soft tissues either as scaffolds for tissue regeneration or as inert, biocompatible elastic fillers. HISTORICAL REVIEW Viscoaugmentation with HA Derivatives
1984-1988: The first cross-linked, water-insoluble HA derivative with appropriate biological and physical properties is invented at Biomatrix developed for use in soft tissue augmentation'". Hylan B gel is produced via bis-ethyl sulfonyl cross-links between
42
Application of hyaluronan in tissue engineering
hydroxyl groups of the HA chain. using vinyl sulfone as a cross-linker. The invention is I patented. 1990-1994: The first publications on preclinical and clinical studies using hylan B gel (Hylaform") for soft tissue augmentation 4-6. . 1993: An HA gel preparation made from 'stabilized' HA developed for use in dermal augmentation (Restylane®); stabilized HA is prepared using epoxide chemical crosslinking methodology'; Restylane is tested in clinical trials in Europe'. 1996, October: Hylaform® is introduced in Europe for use in dermal augmentation for correction offacial wrinkles and scars. 1996, November: Restylane is introduced in Europe for use in dermal augmentation for correction of facial wrinkles and scars. 1997: The first publication on preclinical studies using hylan B gel for augmentation of the sphincter muscle for the treatment ofurinary stress incontinence". 1999: Perlane® is introduced in Europe, a modified Restylane product. 1999: The first publication on preclinical studies using hyIan B gel for augmentation of the vocal folds for treatment of glottic insufficiencyin laryngeal disorders 10. Hyaluronan Derivatives For Viscoaugmentation
Native, unmodified hyaluronan has insufficient residence time in tissue to be used for viscoaugmentation. Cross-linked hyaluronan derivatives with natural viscoelastic properties have prolonged residence time in tissues, therefore usable for viscoaugmentation (see Figure 1).
Viscoaugmcntation: A historical perspective
43
Bylan B Gel Hylaform®
Bis-ethyl sulfone cross-links between hydroxyl groups of hylan A or hyaluronan form viscoelastic, insoluble gels. These highly biocompatible water insoluble gels are injected to correct wrinkles and depressed scars. Figure 2. 100-.--------------------------,
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Epoxy cross-linked hyaluronan forms viscoelastic insoluble gels. It is available for use for correction offacial wrinkles and scars. (Figure 3) OiffE",ntiai Volume
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Figure 3. Particle Size Distributions for Restylane", Perlane" & Hylaform"
44
Application ofhyaluronan in tissue engineering
REFERENCES 1. 2.
3.
4. 5. 6. 7. 8. 9. 10.
E.A Balazs and A. Leshchiner, Cross-Linked Gels of Hyaluronic Acid and Products Containing Such Gels. Biomatrix File #701-29*. United States Patent #4,582,865. April 15, 1986 E.A. Balazs and E.A. Leshchiner, Hyaluronan, its cross-linked derivative - hylan- and their medical applications. In Cellulosics Utilization : Research and Rewards in Cellulosics (Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future) (Eds. Inagaki, H. and Phillips, G.O.), Elsevier Applied Science,New York., 1989,233-241. E.A.. Balazs, J.L. Denlinger, E. Leshchiner, P. Band, N. Larsen., A. Leshchiner, and B. Morales. Hylan: hyaluronan derivatives for soft tissue repair and augmentation. In Biotech USA 1988 (Proceedings of Fifth Int. Congo on Biotechnology, Nov. 14-16, 1989, San Francisco, CA) Conference Management Corp." 1989,442-451. N.E. Larsen, M.B. Kling, E.A. Balazs, and E.A. Leshchiner. Hylan gel for soft tissue augmentation. Society for Biomaterials 16th Ann. Meeting, May 20-23, 1990, Charleston, SC, 302 (abstract). N.E. Larsen, C.T. Pollak, K. Reiner, E. Leshchiner, and E.A. Balazs. Hylan gel biomaterial: dermal and immunologic compatibility. J Biomed. Mater. Res. 27, 1993,1129-1134. D.J. Piacquadio, Crosslinked hyaluronic acid (hylan gel) as a soft tissue augmentation material: a preliminary assessment. In Evaluation and Treatment ofthe Aging Face (Ed. Elson, M. L.), Springer-Verlag,New York, 1994,304-308. T.C. Laurent, K. Hellsing, and B. Gelotte, Cross-linked Gels of Hyaluronic Acid. Acta Chern. Seand., 1964, 18(1),374-375 . M. Olenius. The first clinical study using a new biodegradable implant for the treatment of lips, wrinkles, and folds. Aesthetic Plast. Surg., 1989,22:97, 1998 N.E. Larsen. How to use hyaluronan and hylan matrices for regulation of biological activities by therapeutic agents. New Frontiers in Medical Sciences: Redefining Hyaluronan, Meeting June 17-19, 1999, Padua, Italy. L. Hallen, C. Johansson, and C. Laurent. Cross-linked hyaluronan (hylan B gel): a new injectable remedy for treatment of vocal fold insufficiency -- an animal study. Acta Otolaryngol., 1999, 119, 107-111.
HYALURONAN AND TISSUE ENGINEERING Luis A. Solchaga', Victor M. Goldberg'' and Arnold I. Caplan' I Skeletal Research Center, Department ofBiology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106, USA.
2Department ofOrthopaedics, Case Western Reserve University School ofMedicine and University Hospitals ofCleveland, I lIOO Euclid Avenue, Cleveland, OH 44106, USA.
ABSTRACT
Hyaluronan (HA) can be fabricated into a number of different physical formats including liquids of various viscosities, strands, weaves, sponges and fleeces. All of these formats have uses as delivery vehicles for cells or bioactive molecules for implantation into in vivo sites. Moreover, the physical, chemical, and biological properties of these HA scaffolds can be altered to provide different breakdown kinetics, different cell and molecular attachment domains, and different porosities and hydrophobicities. Importantly, HA in high and low molecular weight configurations have been shown to have different inductive, inhibitory or stimulatory effects on certain cell types. Thus, HA delivery vehicles could be used in a variety of Tissue Engineering constructs. We have used HA sponges to investigate their intrinsic and extrinsic repair/ regeneration potential in a full-thickness osteochondral defect in rabbit knees. The results provide the basis for defining the optimal characteristics of this class of delivery vehicles for skeletal tissue regeneration. Specifically, multifunctional delivery vehicles are required and they must exhibit different signaling capabilities at different stages of the repair process. By controlling the intrinsic breakdown characteristics of these HA delivery vehicles, the kinetics and sequence of the repair process can be precisely controlled. In other words, the change in phase of the delivery vehicle from high to low molecular weight polymer can be the trigger for several biological events. Indeed, the release of HA oligomers has profound biological ramifications in in vivo sites. Both theoretical and practical information forms the basis for considering HA scaffolds tor use in Tissue Engineering strategies especially for the regeneration of skeletal tissues. KEYWORDS
Hyaluronan, tissue engineering, cartilage, repair, regeneration. EXTRACELLULAR MATRIX MOLECULES
Components of the embryonic or adult extracellular matirx (ECM) serve two major functions: structural and informational'. The structural role of ECM components in mesenchymal tissues is quite remarkable, especially when comparing the properties of different tissues, for example, cartilage, bone and tendon where resiliency, stiffness and tensile strength are all quite different. Common to all of these tissues, is the transition of the highly cellular progenitor tissue that has a very low ECM to cell ratio, to the differentiated tissue with a distinctly high ECM to cell ratio. The unique composition and
46
Application of hyaluronan in tissue engineering
molecular arrangement of the ECMs of cartilage, bone and tendon have formed by the sequential transition of an embryonic ECM to an adult ECM. Importantly, the initial components of the embryonic mesenchyme ECM provide the template for future events; this template is certainly structural", but, it may also be informational'. A variety of observations support this hypothesis. Some ECM molecules bind growth factors and cytokines. For example, heparin sulfate binds fibroblast growth factors as do chondroitin sulfates; Transforming growth factor-beta (TGF-f3) binds to the core protein of decorin; Bone morphogenetic proteins (BMPs) bind to collagen, heparin and apatite. Moreover, ECM molecules bind various dormant enzymes including metalloproteinases, collagenases and phosphatases. Importantly, the cells themselves all have cell-surface receptors which bind ECM components. For example, human mesenchymal stem cells (hMSCs) have CD44 receptors for HA 3, integrins which bind to arginine-glycine-aspartic acid (RGD)-containing matrix molecules including fibronectin", endoglin which binds to TGF-f35, not to mention receptors for FGFs, PDGF, BMPs and a variety of other growth factors/cytokines which are stored in differentiated tissue ECMs3 . Thus, the ECM itself and its interaction with the cells is both structural and instructive. We have shown that hyaluronan (HA) is instructive to embryonic mesenchymal progenitor cells (MPCs). In separate experiments, HA bound to petri tissues inhibited myogenic development and separately induced chondrogenic differentiation of embryonic limb MPCs6,7. This observed enhancement of chondrogenic development was controlled by a distinct size range of bound HA, 200-400 kD7 . These observations support the treatise of Toole and colleagues", in which HA is prominent during the morphogenetic phases of development and is then replaced by more complex glycosaminoglycans. The premise is that HA positions MPCs for their eventual commitment down a unique lineage pathway", in this case the chondrogenic pathway. In addition, high molecular weight HA was shown to be directly responsible for the avascular regions of limb mesenchyme", further supporting its role in inducing MPCs to form cartilage which is avascular. HA control of angiogenesis, as alluded to above, is a fundamental activity important in embryonic development, tissue repair/regeneration and tumoragenesis. High molecular weight HA inhibits angiogenesis and the entry of vessels into a region of mesenchyme 10. In distinct contrast, low molecular weight HA (ie, HA oligomers) is highly stimulatory to angiogenesis'", Thus, for tissue engineering purposes, high molecular weight HA could be used to deliver osteoprogenitor cells to an orthotopic site. If this fabric is broken down into oligomeric units, these HA oligomers could serve to rapidly stimulate angiogenesis which is obligatory to new bone formation. There are two engineering considerations in this regard: one is that it may be very useful to fabricate an hMSC delivery vehicle from oligomeric units so that when it dissolves, the angiogenic-optimal units will emerge; or two, that the HA delivery vehicle breakdown kinetics (fast or delayed) may be used to control bone formation by controlling angiogenesis'<". Fibronectin serves multiple roles during the embryonic development of skeletal tissue and the regeneration of damaged skeletal tissue in the post-natal organism. With multiple binding sites for cells, collagen, heparin and fibrin, fibronectin serves to organize and link the various cellular and matrix components of the tissue". In the case of bone and cartilage, fibronectin is one of the earliest ECM components expressed by the progenitor cells that eventually differentiate and express distinct profiles of tissue-specific ECM molecules. For example, fibronectin was recently shown to be the earliest matrix protein expressed during fetal rat calvarial bone formation'Y'". This expression coincided with the condensation of pre-osteoblasts and decreased once bone mineralization began. With the down regulation of fibronectin, other bone-associated matrix molecules including
Hyaluronan and tissue engineering
47
type I collagen, osteonectin and bone sialoprotein were dramatically upregulated. In addition to attaching to cells and linking them to other components of the tissue, fibronectin also provides instructions to the cells that are necessary for the future differentiation of the tissue. In the case of osteoblast differentiation, addition of antifibronectin antibodies or soluble fibronectin protein fragments have been shown to block bone nodule formation in vitro and inhibit gene expression of multiple cell surface and matrix proteins associated with the osteoblast phenotype4,16, 17. The signals from fibronectin are transmitted to cells by several different integrin heterodimers", including 0.5/31 and several a.v-containing heterodimers that can interact with either the RGD sequence of fibronectin!", or in the case of U4/31, attach to the non-RGD connecting segment sequence or CS-I site. Various signal transduction proteins have been shown to be activated by fibronectin-integrin interactions. These include focal adhesion kinase which is a tyrosine kinase that phosphorylated other signaling molecules involved in gene expression regulation. In addition, the transcription factor nuclear factor kappa-B (NFlCB) is also activated". This may explain why IL-I-stimulated osteogenic differentiation of human osteosarcoma cell lines MG-63 is blocked by antibodies against /31 integrins2o,21. Since Ils-I signaling is mediated through NFlC-B, and blocking integrin interaction/ECM interactions blocks IL-I effects, it can be inferred that integrin matrix interactions can regulate ll..-l's activation of the NFlC-B pathway. This logic is supported by recent data that cells plated onto fibronectin enhanced in a dose dependent manner IL-I-induced NFlC-B activity by 1.5 -Z-fold compared to cells on poly(l-lysine) or bare tissue culture plate. The instructive influence of fibronectin interactions with integrins on hMSCs has also been demonstrated, a substrate of fibronectin enhances the early osteogenic marker alkaline phosphatase when cells are exposed to osteogenic supplements. PRINCIPLES OF TISSUE ENGINEERING
Until recently, Tissue Engineering has been restricted in its experimental approach and the results obtained have, thus, been less than perfect. The experimentalists, us included, have had logics that have been somewhat simplistic for the complex processes involved in the regeneration of tissues. We have not adequately used logics available from embryonic development where tissues are being generated for the first time. With this in mind, we hypothesize that regeneration of a specific tissue requires the recapitulation of selected key formative events of embryonic development and tissue growth. Tissues initiallyform in embryos from a high cell to ECM rati022,23. The relatively high progenitor cell number further increases by mitotic expansion in ways which simultaneously allow for major tissue growth and subsequent specialization of the progeny of these primitive progenitor cells. The progenitor cells become specialized in a multi-step differentiation cascade which brings groups of cells into developmental states that result in the synthesis of site-specific specialized molecules". The initial ECMs of embryonic mesenchyme are high in water, HA, fibronectin and type I collagen. Particularly obvious examples of this are found in muscle, cartilage, bone, and heart development and differentiation1,24. A series of sequential signals must be provided to the developing tissue mass to allow for the appropriate differentiation cascade to take placel ,9,24-26 . If the wrong signal or an altered sequence of bioactive agents is provided, the end phenotypes will be affected. Tissues are continuously changing in molecular and cellular constituents as a function of the developmental stage and the age of the organism". The molecular and cellular
48
Application ofhyaluronan in tissue engineering
constituents at the beginning of the differentiation process will not be the same during the life span of the tissue and, especially, at its senescent end. These changes which are rapid in early events, (embryonic development) and slow during subsequent events (maturation and/or aging) are genomicallyprogrammed. In current Tissue Engineering approaches, the delivery vehicles in use are primarily mono-functional, used as mere vehicles for reparative cells or inductive cytokines and the question is whether they should be multi-functional? The important point here is that if sequential change is a key parameter in any formative process then this will imply that the optimal implant material must also change in both its characteristics and, in the end, its function as the multi-step regenerative process takes place. A direct example of multifunctional are biodegradable vehicles were the implant is totally replaced by the newly forming tissue during the course of these regeneration process. It would seem logical in this case that the design of delivery vehicles should account for inductive capacities during early events and, as this vehicle breaks down and is replaced by differentiation-specific materials, the breakdown products should themselves contribute to the control ofthese subsequent events. Sequential signaling is an important component in the fabrication of tissues. To engineer sequential signaling is a complicated process. We are reminded of the multilayered candy spheres called "jawbreakers" we consumed when we were younger. The spheres were constructed of layers of different colors. As you dissolved a layer your tongue would be colored by that layer. The rate of dissolution was controlled by a variety of factors including how rapidly you move the sphere in your mouth, the hydration level in your mouth, whether you were nervous or calm, etc. the sequential release of bioactive molecules to control de developmental cascade might be engineered in a comparable multi-layer. The current Tissue Engineering logic is to deliver single but potent factors (such as BMPs or FGFs) to the site as triggers for specific repair processes28•30 . The use of inductive or trigger molecules to tissue engineer the regeneration of a site-specific tissue assumes a cascade process will follow the initial events although it is now clear that other fators are required. Based on the above, the next generation of delivery vehicles should be engineered to sequentially release discrete components that will accomplish discrete and known functions to facilitate the regeneration of a specific mesenchymal tissue. Alternately, an absolutely uniform tissue can be regenerated into a particular location and the newly formed tissue must be primitive enough to respond to local cueing33•35 and mechanical signals to allow it to further differentiate into non-uniform but functionally discrete and site-specific tissue whose functions will be determined by the exact site in which the regeneration takes place. Although we can obtain biopsies of differentiated tissues and mitoticaly-expand cells from such tissues for later implantatiorr", experimentation is not currently being pursued in which progenitor cells are differentiated in vitro32,33 and then these newly in vitro differentiated cells are being incorporated into appropriate delivery vehicles for in vivo implantation". We have the knowledge and understand some of the triggering factors to induce MPCs into specific phenotypes yet we are just now exploring the use of in vitro differentiated cells as either tissue substitutes or implants to regenerate damaged tissues. As we have stated at the beginning of this treatise, ECM components are informational. Rather than focus our attention on the cytokine cascade that controls differentiation, we have investigated the use of native materials as delivery vehicles. This approach assumes that such native materials will have multiple signaling roles. As discussed below, this assumption is validated by our recent experiments and, more
Hyaluronan and tissue engineering
49
importantly, reflects on ECM-cell interactions as a trigger to developmental events. BYALURONAN IN TISSUE ENGINEERING As mentioned above, HA is a major component of the ECM of embryonic mesenchymal tissues. Thus, to help create a mimetic of the embryonic microenvironment, HA scaffolds could be of interest HA has a dual functionality as a delivery scaffold depending upon its molecular weight: one function in its fabricated (large molecular weight) mode and the other in small functional oligomers generated during its dissolution. Thus, it is possible to design HA-based materials which can initiate chondrogenic differentiation of MPCs and subsequently, with its timely breakdown, it can accelerate the endochondral replacement of that cartilage by woven bone. To explore this concept of multi-functionality in a delivery vehicle, we have used two HA-based sponges in subcutaneous implantations and in osteochondral defects. These two sponges were provided by Fidia Advanced Biopolymers, srl (Abano, Terme, Italy). One is a 100% benzylated derivative, called HYAFF®-11 that breaks down to oligomers in 8-10 weeks, the other one is only 75-80% derivatized (HYAFFiIll_ll p75HE) and breaks down in 7-10 days. Importantly, in a separate series of experiments, we have demonstrated that the optimal loading of MPCs is observed when these vehicles are coated with fibronectin; as discussed above, fibronectin is also important in the differentiation of embryonic tissues. MPCs were isolated from rabbit bone marrow and used at first passage. The MPCloaded sponges were implanted subcutaneously in nude mice and harvested 3 and 6 weeks after implantation. The HYAFF®-11 sponges retained their shape and size during the implantation; however, the size of the recovered HY AFF®-11p75HE sponges was significantly smaller than that of the starting material. At both time points, the implants appeared macroscopically as oblong, hard, white bodies.
Figure 1.
Light microscopy of the composites after implantation.
A, c: HYA~-llp75HE sponge. B, D: HYAFF"'-ll sponge. A, B: 3 weeks after implantation. C, D: 6 weeks after implantation.
Three weeks after implantation the HY AFFiIll_l1 p75HE sponge had dissolved and the tissue collected had the appearance of trabecular bone with loose undifferentiated
50
Application of hyaluronan in tissue engineering
mesenchyme filling the space between the trabeculae. Because of the presence of areas were chondrocytic morphologies were still recognizable, we concluded that the bone forms in a process of endochondral ossification. This interpretation is supported by the observation of specimens harvested after a l-week implantation were the HYAFF®llp75HE sponge was still present and the cellular component of the implant uniformly presented a chondrocytic phenotype. Six weeks after implantation, the bone had continued to mature. The implant had been remodel into an ossicle containing bone marrow-like tissue in its core. In a histologic cross-section of the implant, this is observed as a ring ofbone around a few trabeculae embedded in fatty stroma-like tissue. Three weeks after implantation the HYAFF®-11 sponge was present and the pores of the sponge were filled with various amounts of cartilage, fibrocartilage and fibrous tissue depending upon the quality of the cell preparation. Six weeks after implantation, the pores of the HYAFF®-II sponges were mostly filled with hypertrophic cartilage with some areas of bone. The sponge was still present and recognizable within the implants". Without discussing here the entire series of experiments or full time course studied, we created full thickness osteochondral defects in the medial femoral condyles of young adult rabbits and filled them with fibronectin-coated HYAFFoo-11p75HE or HYAFF
Figure 2.
Light microscopy of the defects after implantation.
A, c. HY~-llp75HE sponge. B, D: HY~-ll sponge. A, B: 4 weeks after implantation. C, D: 12 weeks after implantation.
I-1yaluronan and tissue engineering
51
following implantation with Figure 2 documenting the appearance of defects filled with empty sponges at 4 and 12 weeks. The 4 week images identified two very different cellular processes in the defects. In those filled with the relatively stable HYAFFQll-ll sponges, the implant has an outer sector filled with chondrogenic tissue in the delivery matrix that appears to be intact. Importantly, no new bone is observed in the osseous portion of the defect. In defects implanted with HYAFF®-llp75HE sponges, the delivery vehicle is not present and the chondrogenic tissue that was present earlier has been completely replaced in an endochondral process up to the tide mark with vascularized woven bone. The top of the implant appears to be hyaline cartilage that is impressively integrated with the host cartilage. By 12 weeks, the HYAFF®-11p75HEfilled defect has the same general morphology except that the woven bone has remodeled to more closely match the surrounding bone and the hyaline cartilage cover is about half as thick as the host cartilage. At this analysis time, the HYAFF®-l l-filled defect no longer has most of the vehicle although in some specimens some remnants are observed. The chondrogenic tissue in the osseous region of the defect appears to have been endochondrally replaced by vascularized woven bone. The top of the defect is filled with hyaline-like cartilage that is impressively integrated with the host cartilage.
Figure 3.
Pictorial representation of the sequence of events that take place in the repair of an osteochondral defect with HYAFF""-llp75HE and HYAFF"!'-ll sponges. A speculative panel in each suggests that HA-oligomers released into the synovial space may influence both the integration of nco-cartilage with host and the population and properties of the chondroprogenitor cells that are eventually responsible for the hyaline cartilage.
These observations document several important aspects: First, both delivery vehicles are fabricated from HA of 200-400kd coated with fibronectin and the initial tissue that forms in both is chondrogenic. This chondrogenic tissue in these vehicles appears to be similar independent of whether the vehicles were empty or initially loaded with bone marrow or cultured MPCs. The vehicles loaded with cells appear to develop chondrogenic tissue more rapidly than those which were implanted empty. We assume that the host MPCs from the marrow in the osseous portions of the defect enter the empty vehicles and eventually form the chondrogenic tissue observed at 2 to 3 weeks later. Second, the endochondral replacement of the initial cartilage in the osseous sectors of the defect is directly dependent on the dissolution of the vehicles; with HYAFF®Ilp75HE breaking down at about 7-10 days and HYAFF®-ll at 8-10 weeks. This is clear when comparing the two vehicles at 4 weeks and then comparing the HYAFF®-II at 4 and 12 weeks. Third, where neo and host cartilage meet there is impressive and consistent integration. Previously, we have shown that the enzymatic unloading of proteoglycans from the host cartilage at the edge of the defect facilitates the molecular integration of the neo tissue''". It may be that the HA oligomers also, either directly or
52
Application ofhyaluronan in tissue engineering
indirectly influence this host-nee tissue integration. HA oligomers have been shown to compete with the aggrecan-HA bind region-HA interaction". It is also possible that the oligomers affect angiogenesis and bring blood borne cells to the defect's edge where a local release of proteases could facilitate molecular integration. Lastly, it may be that the release of HA oligomers into the synovial fluid acts as a chemoattractant for a special class of MPCs originated from the synovium or edge of the condyle which are capable of 4o forming articular cartilage ,41. This suggestion implies that marrow-derived MPCs may not be able to stop at the articular stage as they progress down the chondrogenic lineage". In this regard, others have suggested that articular cartilage is exclusively derived from special chondroprogenitor cells which originate from the ring of LaCroix41,42 . THE PAST, PRESENT AND FUTURE HA is a repeating disaccharide that as been implicated in the control of morphogenesis, cell differentiation, cell division and more recently signal transduction into the cell's nucleus. The replacement ofHA by other more complex ECM molecules is a key component in the differentiation process of mesenchymal tissues like bone and cartilage. Clearly, HA can induce very different cellular and molecular responses from progenitors and their progeny depending upon its format; large molecular weight form versus oligomers of a variety of sizes. Its use as a Tissue Engineering scaffold can, therefore, provide material that can induce and modulate the sequence of events necessary for the repair/regeneration of damaged tissues. In the case of articular cartilage repair/regeneration, HA-based matrices and their breakdown products can function to attract progenitor cells, induce chondrogenesis, inhibit or stimulate angiogenesis, control cell division, and facilitate the integration of neo with host tissue. It is, therefore, possible that this molecule may serve as a very smart, multifunctional Tissue Engineering vehicle for mesenchymal tissue regeneration. REFERENCES 1. A. I. Caplan. Extracellular matrix is instructive. BioEssays., 1986, 5, 129-132, 2, H, B. Fell. The histogenesis of cartilage and bone in the limb bones of the embryonic fowl. J. Morph. Physiol., 1925,40,417-459. 3. M. Majumdar, M. Thiede, 1. Mosca, M. Moorman and S. Gerson. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells.. 1. Cell. Phys., 1988, 176,57-66. 4. R. W. Rupp, H. Reddy and S, E. Haynesworth. 131 integrin expression by human mesenchymal stem cells and their involvement in osteogenic differentiation in vitro. Trans. Or/hop. Res. Soc., 1997,22, 17, 5, R. E. Boynton, S. E. Haynesworth, 1. Zaia, 1. M. Murphy, A. McIntosh and F. P. Barry, A transforming growth factor-beta binding protein expressed on the surface of bone marrow-derived mesenchymal stem cells is recognized by the monoclonal antibody SH-2. In Press. 6. M. J. Kujawa and A. 1. Caplan. Hyaluronic acid bonded to cell-culture surfaces stimulates chondrogenesis in stage 24 limb mesenchyme cell cultures. Devel. Biol., 1986,114,504-518.
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7. M. 1. Kujawa, D. A. Carrino and A. I. Caplan. Substrate bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal meJls in culture. Devel. Bio/., 1986, 114, 519-528. 8. B. P. Toole. Hyaluronan in Morphogenesis. 1. Internal. Med., 1997,242,35-40. 9. K. Noonan,1. Stevens, R. Tammi, M. Tammi,1. Hermandez, and R. Midura. Spatial distribution of CD44 and hyaluronan in the proximal tibia of the growing rat. 1. Orthop. Res., 1996, 14,573-581 10. R. N. Feinberg and D. C. Beebe. Hyaluronate in vasculogenesis. Science, 1983,220, 1177-1179. II. T. Sasaki, and C. Watanabe. Stimulation of osteoinduction in bone wound healing by high-molecular hyaluronic acid. Bone., 1995 16,9-15. 12. P. Klokkevold, L. Vandemark, E. Kenney and G. Bernard. Osteogenesis enhanced by chitosan (poly-N acetyl glucosaminogylcan) in vitro. J. Peridon., 1996, 67, 11701175. 13. A. Pilloni, and G. Bernard. The Effect of Hyaluronan on Mouse Intramenbranous Osteogenesis In Vitro. Cell. &. Tiss. Res., 1998,294,323-333. 14. M. 1. Humphries,S. K. Akiyama, A. Komoriya, K. Olden and K. M. Yamada. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. 1. Cell. Biol., 1986, 103,2637-2647. 15. A. Moursi, C. Damsky, 1. LuJl, D. Zimmerman, S. Doty, S. Aota, and R. Globus. Fibronectin regulates calvarial osteoblast differentiation. 1. Cell Science, 1996, 109, 1369-1380. 16. A. Moursi, R. Globus and C. Damsky. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro. 1. Cell . Science, 1997 110,2187-2196. 17. G. Gronowicz and M. Derome. Synthetic peptide containing arg-gly-asp inhibits bone formation and resorption in a mineralized organ culture system of rat fetal parietal Bones. 1. Bone Min. Res., 1994, 9, 193-201. 18. S. Gronthos, K. Stewart, S. Graves, S. Hay and P. Simmons. lntegrins expression and function on human osteoblast-like ceJls. 1. Bone Min. Res., 1997, 12, 11891197. 19. P. Zhu, W. Xiong, G. Rodgers and E. Owarnstrom. Regulation of interleukin-l signalling through integrin binding and actin reorganization: disparate effects on NFkappa B and stress kinase pathways. Biochem. 1., 1998, 330, 975-981. 20. S. Dedhar. 1989. Signal transduction via the 131 integrins is a required intermediate in intcrleukin-l a. induction of alkaline phosphatase activity in human osteosacoma ceJls. Exper. Cell Res., 1989, 183,207-214. 21. S. Dedhar. Regulation of expression of the cell adhesion receptors, integrins, by recombinant interleukin-l a. in human osteosarcoma cells: inhibition of cell proliferation and stimulation of alkaline phosphatase Activity. 1. Cell Phys., 1989, 138,291-299. 22. T. F. Linsenmayer, B. P. Toole and R. L. Trelstad. Temporal and spatial transitions in collagen types during embryonic limb development. Dev. Bioi., 1973,35,232-239. 23.1. R. Hinchlifee and D. R. Johnson. The Development oj the Vertebrate Limb, Oxford University Press, New York, 1980. 24. A. I. Caplan. The Mesengenic Process. Clinics in Plastic Surgery, 1994, 21, 429435. 25. G. Maor, K. von der Mark, D. Heinegard and Silberman. Human growth hormone
54
Application ofhyaluronan in tissue engineering
enhances chondrogenesis and osteogenesis in a tissue culture system of chondroprogenitor cells. Endocrinology, 1989, 125, 1239-1245. 26. A I. Caplan and B. D. Boyan. Endochondral Bone Formation: The Lineage Cascade, In: Bone, Volume 8, B. Hall (ed), CRC Press, Inc., Boca Raton, Chapter I, 1994, pp. 1-46. 27. A 1. Caplan, M. Y. Fiszman and H. M. Eppenberger. Molecular and cell isoforms during development. Science, 1983,221,921-927. 28. M.R. Urist. The search for and discovery of bone morphogenetic protein (BMP), pp. 315-362. In: Bone Grafts, Derivatives and Substitutes. M. R Urist, B.T. O'Connor, & R G. Burwell (eds). Butterworth Heinemann, London, 1998, pp. 315-362. 29. E.A Wang, OJ. Israel, S. Kelly and D.P. Luxenberg. Bone morphogenetic protein-2 causes commitment and differentiation in C3HIOTl/2 and #T3 cells. Growth Factors, 1993,9, 567-571. 30. C. Deng, A Wynshaw-Boris, F. Zhou, A Kuo and P. Leder. Fibroblast growth factor receptor 3 is a negative regulator ofbone growth. Cell, 1996,84,911-921. 31. W.S. Kim, JP. Vacanti, L. Cirna, D. Mooney, J Upton, W.C. Puelacher and C.A. Vacanti. Cartilage engineered in predetermined shapes employing cell transplantation on synthetic biodegradable polymers. Plastic Reconstr. Surg., 1994, 94, 233-240. 32. RK. Khouri, B. Koudsi and AH. Reddi. Tissue transformation into bone in vivo. JAAdA, 1991,266,1953-1955. 33. M. Brittberg, A Lindahl, A Milsson, C. Ohlsson, O. Isaksson, L. Peterson. Treatment of deep cartilage defects in the knee with autologous chondrocytes transplantation. New Eng. J. Med., 1994,331,889-895. 34. B. Johnstone, T. M. Hering, V. M. Goldberg, 1. U. Yoo, and AI. Caplan. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res., 1988, 238, 265-272. 35. L. A Solchaga, 1. E. Dennis, V. M. Goldberg and A I. Caplan. Hyaluronic acidbased polymers as cell carriers for tissue engineered repair of bone and cartilage. J. Orthop.Res. 1999, 17,205-213. 36. L. Solchaga, 1. U. Yoo, M. Lundberg, B. Hubregtse, AI. Caplan. Hyaluronic acidbased polymers in the treatment of osteochondral defects. Trans. Orthop. Res. Soc., 1999, 24, 56. 37. L. Solchaga, J. U. Yoo, M. Lundberg, V. M. Goldberg and A I. Caplan. Augmentation of the repair of osteochondral defects by autologous bone marrow in a hyaluronic acid-based delivery vehicle. Trans. Orthop. Res. Soc., 1999,24, 801. 38. Y. Mochizuki, M. Elyaderani, R G. Young, V. M. Goldberg, and A I. Caplan. Mesenchymal stem cell implantation into a pretreated large full-thickness articular cartilage defect. Trans. Orthop. Res. Soc., 1995,20,367. 39. V.C. Hascall and D. Heinegard. Aggregation of cartilage proteoglycans. J. Bioi. Chem., 1974,249,4242-4249. 40. J. H. Kimura, T. E. Hardingham, V. C. Hascall and M. Solursh. Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the swarm rat chondrosarcoma..J. Biological Chem., 1980,25.7134-7143. 41. P. LaCroix. The Organization ofBones. J.E. Churchill, Ltd., London, 1951, pp 132133. 42. D. Robinson, A Hasharoni, N. Cohen, A Yayon, R Moskowitz and Z. Nevo. Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells. Clin. Orthop. & ReI. Res., 1999, in press.
THE INTRINSIC VISCOSITY OF HYALURONAN Mary K. Cowman l ,2 and Shiro Matsuoka' I Department
a/Chemistry and Chemical Engineering and 2Human F. Mark Polymer Research Institute Polytechnic University. Six Metrotech Center. Brooklyn, NY 11201 U<JA
ABSTRACT
The large hydrodynamic volume of high molecular weight hyaluronan in solution is primarily the result of the the large size of its smallest unit of independent motion (the sugar ring), and the decrease in chain segment density which is required for all polymers as a function of increasing molecular weight. A direct comparison of chain dimensions for hyaluronan and the synthetic polymer polyisoprene shows that both polymers have similar expansions, if the difference in conformer size is considered. KEYWORDS
Hyaluronan, polysaccharide, polymer, viscosity, hydrodynamics INTRODUCTION
The viscometric behavior of high molecular weight hyaluronan in dilute aqueous salt solution has been interpreted as that of a stiffened random coil or a worm-like chain. High values are obtained for the intrinsic viscosity, implying large hydrodynamic volumes, with considerable water included within the molecular domain. We re-examine the applicable theories for viscosity of dilute polymer solutions, and available data for the molecular weight dependence of the intrinsic viscosity for hyaluronan. We investigate the degree to which the behavior ofhyaluronan is typical of polymers in general, and whether any unusual stiffening influence is apparent. THEORY VS. EXPERIMENT FOR HYALURONAN
We consider two simple models for the hydrodynamic properties ofhyaluronan. The non-free-draining ball model is based on the statistical conformation of a polymer chain for which the root mean square (rms) end-to-end distance, 1/2, approximately equal to the diameter of the coil domain, increases as M0 5 to Mo. 6 • The volume, V, varies as the cube of that dimension, thus as M 1.5 to M 1.8. The density of chain segments within the ball varies as MN, or M-O.5 to M-O· 8 • The specific volume, Vs, is the inverse of the density, and it therefore varies as Mo. 5 to MO. s. The intrinsic viscosity, [11), may be considered directly proportional to the specific volume, which is the spatial domain occupied per unit weight of polymer segments. It is mostly filled with solvent, having only a low density of polymer segments. The second model we will use is the freedraining chain model. Here the chain is more extended, and its viscosity reflects the worm-like shape. The intrinsic viscosity varies as the square of the rms end-to-end distance, thus as MI.O-I2. The experimental 1.2 study ofhyaluronan intrinsic viscosity (Figure 1) shows that Sh0l1 chains act like free-draining chains, and long hyaluronan chains act like non-free-
76
Characterisation and solution propertiesof hyaluronan 10000 , - - - - - - - - - - - - - - - - ,
[11] = K Ma 1000 ~
-
__.,
Tumer, Un, & Cowman, 1988
---------
U>
8 U>
so
~
- - - - . - . -..
-Balazs, 1965 ._-,,~
--
."
_.
--
.1
a = 0.80
100
I
, I
'wc:
I
E
I I
S
j/M"37~
10
1+------.----.------,,-------1 1000 10000 100000 1000000 10000000 Molecular Weight
Figure 1. Experimental dependence of intrinsic viscosity on molecular weight for hyaluronan in 0.15 M NaCl solution. Low molecular weight hyaluronan behaves as a free-draining chain. Longer hyaluronan chains act like non-free-draining ball-like coils.
draining coils. The molecular weight at which the two types of behavior coexist is approximately 3.75 x 104• This size hyaluronan corresponds to the smallest ball-like hyaluronan. Using the equation [11] == 0.029 MO so, describing the behavior of high molecular weight hyaluronan', we can calculate the intrinsic viscosity for several molecular weights (Table 1). We also determine the chain contour length, L, as MIMI., where ML is the mass per unit length, approximately 401 urn'l. The specific volume is [11)/2.5, given by the Stokes-Einstein equation. The rms end-to-end distance is equal to ([Tj]M//2L). The apparent characteristic ratio, C', is lNf, where N is the number of conformers and 1 is the conformer length. We may use a monosaccharide conformer (1 == 0.5 nm) or a disaccharide conformer (1 == I nm). Both the persistence length and characteristic ratio are measures of the chain rigidity and thus its expansion. As a comparison, we may calculate the chain diameter expected Table 1.
Example Dimensions of Ball-Like Hyaluronan Chains in Saline Solution
M
L
(nm) 4
3.8 X 10 I X 105 5 x 10' 1 x lOb 3 x lOb 6 x lOb
94 250 1250 2500 7500 15000
[Tj) cm/g
132 290 1050 1830 4410 7670
Vs
cm3/g
53 116 420 732 1760 3070
II' (urn)
29 52 136 206 398 603
<00"' (nm) mono
di'
10 16 35 50 87 122
14 22 50 71 122 173
C'
q'
(nm) mono'
4.5 5.4 7.4 8.5 10.6 12.1
18 22 30 34 42 48
di'
9 11
15 17 21 24
Intrinsic viscosity
77
Figure 2. The specific volume of a hyaluronan chain depends on the molecular weight. Hyaluronan with a molecular weight of I million (at left) can be modeled as a ball with a hydrodynamic diameter of 200 nm; hyaluronan with a molecular weight of 6 million (at right) has a hydrodynamic diameter of 600 nm. The specific volume increases from 730 crrr'zg to 3100 cnr'zg.
if the bonds linking conformers were free of an?, hindrance to rotation, but had fixed tetrahedral bond angles, so that 01/2 = (2Nl2) 11 . The results in Table I show that the specific volume of a high molecular weight hyaluronan chain can be very large., and the corresponding density very low. This is schematically illustrated in Figure 2, where the ball-like domains of chains having molecular weights of I million and 6 million are shown. Because hyaluronan is naturally occuring at very high molecular weight, its domain volume will be extremely large. Another observation we may make from Table I is that the hydrodynamic diameter measured by viscometry is much larger than would have been predicted for a freely rotating chain. This is illustrated in Figure 3, which compares the experimental hydrodynamic size with that predicted for the freely rotating model.Thus there is some significant restriction to rotation, that expands the molecular domain. The apparent persistence length and the apparent characteristic ratio values presented in Table I increase with molecular weight. This is because the hyaluronan chains have a significant excluded volume contribution. Thus <~> depends on MI.2, so that q' and C' depend on MOl. There is no need to consider an excluded volume contribution for low molecular weight hyaluronan, so the hyaluronan with M of 3.75 x 104 , which can just barely form a hydrodynamic ball, gives the true, or intrinsic, value of these parameters. The parameters characterizing the smallest ball-like hyaluronan are provided in Figure 4. Because the length is related to the hydrodynamic diameter by the
Figure 3. The experimentally-determined hydrodynamic diameter (400 nm, at right) of a 3 million molecular weight hyaluronan chain is much larger than that calculated (90 nm, at left) for an unperturbed chain with fixed tetrahedral bond angles between monomers, but free rotation about the bonds.
78
Characterisation and solution propertiesofhyaluronan M approx. 37,500 L=94nm [111 = 132 cnr'zg 1/2 = 29 nm LID = 3.2 (close to rt) q = 4.5 nm Llq = 21
Figure 4. The smallest hyaluronan chain that can act hydrodynamically like a ball has a molecular weight of 3.75 x 104 . Its chain length is just great enough to form a circle with the experimental hydrodynamic diameter. factor of 3.2 (very close to the value of n), the hyaluronan may be usefully drawn as a circle. It takes about 20 persistence lengths to form the circle. The circle is, of course, only an instantaneous conformation. The end-to-end distance varies from 0, as in the circle shown, to L. Its time average is such that the approximate hydrodynamic diameter is that of the circle shown. As a comparison with hyaluronan, we have considered the hydrodynamic properties of the synthetic polymer, polyisoprene. The size of a conformer in polyisoprene is a single carbon in the polymer backbone. Polyisoprene with a molecular weight of 1 million has a hydrodynamic diameter of I 14 nm, in comparison with a predicted diameter of38 nm based on the freely rotating chain. The expansion factor is similar to that seen for hyaluronan with a molecular weight of 1 million (206 nm experimental vs. 71 nrn, using a disaccharide conformer). The smallest ball of polyisoprene contains 20 persistence lengths of 0.42 nm, which is 3.8 times the conformer length, vs. 4.5 disaccharide conformers per persistence length in hyaluronan. The characteristic ratio is 8 for polyisoprene, vs. 9 for hyaluronan using as disaccharide as the conformer size. Thus the expansion of hyaluronan is similar to that of polyisoprene, if the enormous difference in conformer size is taken into account. Simply stated, hyaluronan is highly expanded because the smallest independently moveable unit is a sugar ring, because there is restricted rotation about the glycosidic linkages, and because the chain length is so large that a low density coil domain is dictated by statistical considerations.
ACKNOWLEDGEMENTS We are indebted to Endre A. Balazs, for inspiration, support, and numerous helpful discussions. Financial support for this research was provided by Biomatrix, Inc. This publication includes images from WordPerfect® Office 2000 which are protected by the copyright laws of the U.S., Canada and elsewhere. Used under license.
REFERENCES 1. E.A.Balazs, 'Amino sugar-containing macromolecules in the tissues of the eye and the ear', In: The Amino Sugars: The Chemistry and Biology of Compounds Containing Amino Sugars, E.A. Balazs and R.W. Jeanloz (eds.), 1965, Academic Press, New York, pp. 401-460. 2. R.E. Turner, P. Lin, & M.K. Cowman, 'Self-association of hyaluronate segments in aqueous NaCI solution', Arch. Biochem. Biophys., 1988,265,484-495.
VISCOSITY OF POLYMER SOLUTIONS REVISITED S. Matsuoka and M. K. Cowman Polymer Research Institute, Polytechnic University, Six Metrotech Center, Brooklyn, New York 11201, USA.
ABSTRACT A rubbery sphere as a hydrodynamic model for the polymer molecule is useful in predicting many aspects of the solution behavior, but it has some inconsistencies in its physics. Instead of this model of hydrodynamic volume, we propose the Gaussian cloud with its average density as the characterization parameter. The Stokes-Einstein equation is applied, not to the molecules as solid spheres, but to the individual mass points that makes up the cloud. The cloud has no definitive boundary, but has an average internal density, Cj in g/cnr', which is equal to c· or 2.5/[1'\] at c~o. With increased concentration, the internal density of clouds increases, starting at 0 concentration. The increased density adds another term to the increase of viscosity. The Huggins equation is derived from this increased density affecting the internal viscosity in the cloud. Further increase in the concentration will cause the overlap among more than two neighbors, resulting in higher order terms. The resulting equation for the specific viscosity: 1'\sp = c[1'\]{ 1+kHC[1'\]+(kHc[1'\]iI2!+(kHC[1'\])3/3!}, is a four term polynomial of the parameter, c[1'\], which might be called the generalized Huggins equation. Each term is related to the number of the interacting neighbors. This equation seems to be applicable to all kinds of polymers, from rigid polyphenylenes to semi-rigid hexyl isocyanates to flexible vinyl polymers, provided the equation is fitted directly to raw data, rather than by first obtaining kH and [1'\] from an approximate straight line at small concentrations. The universal equation holds from the dilute to the concentrated solutions including the so-called entanglement regime, i.e., where 1'\-M3 to Nt, and to a polyelectrolyte such as hyaluronan with added salt ions to shield intramolecular coulombic repulsion. INTRODUCTION The well-known Stokes-Einstein equation deals with the flow of fluid around a spherical particle in suspension. The rate for the dissipation of energy of flow is considered from the two standpoints. First, the overall viscosity 1'\ is reduced by the presence of a particle. Second, the solvent viscosity 1'\0 is increased by the presence of the same particle. A Gauss integral was used over the surface of the sphere to calculate the effect. Equating these two effective viscosities, the equation v
ll- 2Vllo = llo + -llo 2
(1)
is obtained. Here v is the volume of the particle in the spherical form, but it can be an empirical effective volume if it is randomly oriented with respect to the flow line. When there are n such particles per cnr' of the solution, the flow lines for the particles are superimposed, and the equation for the specific viscosity 1'\sp is obtained:
80 n
, tsp
Characterisation and solution properties of hyaluronan
= 11- 110 = 25nv
(2)
110 In solution, polymer molecules change the conformation rapidly. The conformational changes take place through redistribution of bonds' rotational angles. The bonds rotate rapidly from one stable angle to another with rate of between MHz to GHz 1• A polymer molecule suspended in liquid can be pictured as a superposition of three-dimensional images of millions or trillions of these conformations that appears as a cloud of beads (also called repeat units, segments, or conformers). Because of the nature of the statistical distribution of shapes of the chain, the density of the cloud is not uniform, but decreases at points further away from the center. The fluid can easily pass through such a cloud, though more slowly than outside the cloud, i.e., the local viscosity is higher in the denser part of the cloud. According to the Stokes-Einstein formula, as applied to individual beads, the local viscosity is proportional to the density of that locality. Now, these clouds in suspension are replaced by the solid spheres that will obtain the same viscosity values as the viscosity of the solution. The volume of such a sphere is the definition of the empirical hydrodynamic volume. This hydrodynamic volume, therefore, is a mathematical parameter. It differs physically from the cloud of real polymer segments in some serious ways. The solid sphere is a non-draining body, whereas the cloud can pass fluid through it readily. Unlike a solid body, the cloud has no definitive boundary, so no definitive volume. Yet, so many aspects of viscosity behavior by polymers in solution have been successfully predicted by invoking the hydrodynamic volume of the polymer molecule. We will sort out reasons for this success by comparing the cloud model, which is realistic, with the solid sphere model, which is not.
The Gaussian Cloud As stated above, the volume of a Gaussian cloud cannot be defined. However, its density, being a decreasing function of the radius, has an average value. The average distance between many particles can be characterized by the RMS distance to the particles from the center of the cloud, ~Mos2=M S 2. The average density may be defined as the total mass divided by the volume of a sphere with the radius of s. Since the viscosity is proportional to the density, this average density is appropriate for the hydrodynamic calculations. 2 The mean square end-to-end distance of a chain with N segments of length I is NI 2 for a freely jointed chain, whereas it is 2Nl for a freely rotating chain with the tetrahedral bond angle'. We introduce a generalized segmental dimension, t , such that N £ 2=<~> for any kind of chains, including the freely rotating chains with £ = ~ I, even with bond angles that are slightly different from the tetrahedral bonds with some empirically determined value of e that mal be greater than ~ I. The volume of a sphere with the diameter of <~>l/2 is voN31 , where vo is the volume of a bead with diameter t . The radius of gyration s mentioned above is equal to <~>1/2 / J6 for the chain with N £ 2=<~>, according to Florl. Letting the density of one bead as po = MlNNAvo, (l/po is the partial specific volume, and it is 0.56 for hyaluronan.i"), the mass of each bead is defined as MoINA, which is MINNA. The average density is this mass over the volume with radius s,
Viscosity of polymer solutions _ M/N 41t -3 -s 3
_
M/N 41t < r >3/2
A A c· - - - - ----:;--"-:;-;,,2 I
63/2
3
41t(fl/ 2)3 23W/ 2 3 6 312
81 (3)
For the general case -112 is substituted by 1-3v, as will be explained later, and P*o = 1.8po, the average density is defined: (4)
The statistical conformational probability determines the average density c, of the cloud. In the limit of dilution, the cloud takes on the maximum conformational probability, and we define c* as the limit of c, for c~o, As the flow of the fluid through the cloud depends on the local concentration of beads within the cloud, the specific viscosity can be formulated from the sum of the flow around individual beads with the Stokes-Einstein equation, resulting in a formula, Tl sp
c cj
= 25-
(5)
When c-so, Cj is replaced by c*. The specific viscosity in an ideal dilute solution is written in two alternative forms: l1sp
c c*
c M
= 25- = 25-N A Vb
(6)
The last term is the same as eq.2, with Vb the hydrodynamic volume of a sphere having the average density c* of the cloud. Thus, the average of the non-uniform concentration of beads within the cloud is shown to be the key parameter for the viscosity of polymer solutions. The freely rotating chain means that there is no preferred rotational bond angle (with respect to the adjacent bond, e.g., trans or gauche configurations). A good solvent, on the other hand, means that the polymer segments would prefer being in contact with the solvent molecules as much as, or even more than, being adjacent to another segment of own kind. Good solvent molecules will enter the cloud more readily than poor solvent molecules, and making the cloud swell. The probability for the straighter configuration is increased. The expansion rate a. is not constant at all densities, so a. depends on the molecular weight. Thus, the density of the cloud in good solvents will have a higher dependence on the molecular weight than under a theta condition. The density dependence is generalized as M I-3v instead of W· s, and v>0.5 for good solvents. For a real polymer, there is always some excess volume even when v is 0.5. The excess volume is the difference between the average specific volume (lIc;) of the cloud and the partial specific volume of the solute (l/po where po is the density of a bead). Flory's treatment' takes a view that the excluded volume is the excess over the specific volume of the unperturbed chain conformation, with v of 0.5.) The persistence length, if calculated from the viscosity measurements, will also depend on the solvent type and molecular weight. Both are calculated de facto from the density of the cloud. The term "chain stiffness" is a phrase for describing the density of a cloud, regardless of what causes the cloud to swell. For example, a polyelectrolyte such as
82
Characterisation and solution properties ofhyaluronan
hyaluronan tends to exert intramolecular coulombic repulsive potential. This will tend to expand the cloud, unless such interaction is shielded by adding salt ions, as all of the data cited here were. The dependence of the persistence length on the ionic strength for the hyaluronan molecules has been demonstrated and analyzed by Rinaudo et al." Also, the chain stiffness may be enhanced through the intramolecular bonding. Analysis of interatomic potentials has led to a speculation that adjacent saccharides in the hyaluronan chain would favor a rigid straight configuration by hydrogen bonding. 5 However, the potential energy is only one side of the Helmholtz free energy for the conformational probability. The other is the entropic probability, stirred up by thermal agitation. One may consider the extended conformation a statistical rarity in an amorphous cloud of a dynamic chain. The Intrinsic Viscosity The intrinsic viscosity is defined as the low concentration limit of11sp Ie, so one obtains many kinds of expressions for it as below: (7)
The first and the second expressions describe the reciprocal of the average density of the cloud, 2.5 being 5/2 from the Stokes-Einstein formula. In the third expression, is the Flory constant. For several polymers, the empirical value of2.lxl023 is found that agrees with light scattering data. according to Flory. 3 This value of is compared with Kirkwood and Riseman's" theoretical value 00.6 x 1023 . If g2=/6 for the freely jointed chain is used, the value of 4.3xlif3 is obtained. If the internal density defined by Eq. 4 is used for [11], the value of2.3x1023 will be obtained. The last term in Eq. 7 is the well-known Mark-Houwink-Sakurada equation," With the theta solvent, the end-to-end distance is proportional to N I12, and a is 0.5. With a good solvent, the polymer coils expand and, as the rate of expansion a is molecular weight-dependent, the RMS radius,
(8)
Viscosity of polymer solutions
83
to cover the both regimes of molecular weight. The parameters A and B in the above equation involve the bond length and the molecular weight per bead. It is seen in the above equation that, for small N, tTl] is proportional to the molecular weight, while with high molecular weight, it is proportional to N1/2 • Turning to the high molecular weight range in hyaluronan, 112 is calculated from the intrinsic viscosity, using the Flory constant of 2.1xl023 • The results, as shown below, agree well with SEC data for hyaluronan." The persistence length'" is the projection of the end-to-end distance in the direction of the first segment, divided by the number of segments in the chain. It is a measure that indicates how much larger the experimental is than that of the freely jointed chain in the theta solvent. The freely rotating chain is characterized by the contour length L = N f , f being the generalized bond length, with persistence length q = f / (1 - cos e ). In general, is related to the persistence length q from the equation: (9)
The length of a disaccharide unit is assumed to be 1 nm. The molecular mass per unit length, ML, ofhyaluronan molecule is 40111 = 401 nm", Milas, Rinaudo and Borseli" have used 410.
M
3.75xl04 3.5xlO' l.oxl O" 3.0x1Ob 6.0xlOb 1.2xlO'
Table I Molecular size ofhyaluronan in salt solution ,12 qnm Lnm c* cgs L/q L/diameter [TI] cgs nm 4.5 3.26 94 29 21 132 1.8x1O,2 7.97 872 790 3.16x1O,3 110 6.9 126 14.6 429 3,990 2,664 9.38x1O-4 273 9.3 18.8 4,407 5.67xl0-4 10.6 706 7,481 397 24.8 14,963 7,672 3.26xlO-4 603 12.1 1237 32.8 29,925 13,358 1.87xlO-4 913 13.9 2153
Table I represent the persistence length and other parameters calculated for hyaluronan of various molecular lengths. The first row with molecular weight of 3.75 x 104 is at the border of the two regimes, each with its own set ofK and a. This molecular weight is, therefore, at the lower limit of the high molecular weight range in which a is 0.8. For this molecular weight, the length L is only 3.2 times the diameter. As a complete circle would require 7t times the diameter, this molecule can just make a complete circle. This is the minimum molecular weight necessary to form a loop. The radial distribution of local concentration in the cloud ought to be very different above and below this critical molecular weight. Above it, it is a Gaussian cloud, and the MarkHouwink-Sakurada formula is followed by a of 0.5 if in the theta solvent, or a higher value if in a good solvent. Below it, the density at a distance r from center is inversely proportional to 4xr, instead of 4m for the longer chains. The average internal concentration obtained is inversely proportional to the effective length of a molecular rod, rather than to ll2. So [TI] should be proportional to M. The short and wriggling molecules are actually better described as flexible rods. [TI] should be proportional to M2v , or M I for theta solvent, and M L2 for a good solvent. Recall the experimental value for the exponent was 1.16. 4 The persistence length q for hyaluronan of M=3.75xl0 has been calculated to be 4.5 nm, or 5 time as large as the length of disaccharide unit that is 1 nm. This ratio,
84
Characterisation and solution properties ofhyaluronan
however, is not limited to the stiff chain polymers. A similar set of quantities has been calculated for polyisoprene. The critical molecular weight and the absolute q are both an order of magnitude smaller than for hyaluronan, but the ratio of q to the bond length is about 4 for the critical molecular weight where L / diameter is 7t. The Semi-Dilute Solution By applying the Stokes-Einstein equation to the dilute solution of the "mass points" in a cloud of a dynamic polymer chain, the specific viscosity was shown to be equal to 2.5 c/c*. To arrive at this result, the cloud was allowed to pass the fluid around individual "mass points" with a normal laminar flow. No hydrodynamic interactions were assumed among the "mass points" other than, indirectly, from their Gaussian distribution in space. The Newtonian flow was restricted to be slow enough so the conformational probability was not altered by the flow field. The only serious assumption made has been that the concentration is low enough that no clouds were allowed to touch each other. However, a true Gaussian cloud would "feel" the presence of others at infinitesimal concentrations. Clouds of real polymer chains, too, can be considered to reach a great distance in excess of the length of a chain, as a conformation of the molecule far removed from the center of the cloud is a statistical possibility. If only the value of average internal density were of interest, this is perhaps a moot point, because the most significant part of the Gaussian distribution is limited to within some distance from the center of the cloud. However, whether to account or neglect intermolecular interaction at very low concentrations is a very serious question. The non-draining hydrodynamic sphere model, too, can account for the interaction to start at 0 concentration, by allowing the partial overlap as a possibility over the time. With the hydrodynamic volume model, various kinds of interactions between the spheres were speculated in the past, sometimes resulting in some polynomials without placing a physically significant meaning to each term, calling the polynomial a generalized hydrodynamic theory. 19 Initially at concentration of nearly 0, the internal density is at c*. The cloud is at the unperturbed state with the highest possible conformational entropy. When the concentration is increased to c, the internal density increases to a higher level than c* because, even though the concentration c is still below the average internal density, the outer region of the cloud, where the local density is lower than c*, will increase. Conformational rearrangement that follows will raise the average density higher than c*. If, for the sake of argument, the internal density were to remain unchanged at unperturbed c*, then Tlsp will always remain as c[Tl] at all concentration levels. Experimentally, this is found not to be the case. We now attempt to estimate that part of the viscosity increase, dTlsp, which is solely due to the increased internal density in the cloud. There are three kinds of concentrations to be dealt with: c, c; , and c*. c starts at 0, and so does the difference between c; and c*, that we define dc; = Cj - c*. All changes in these concentration values are a unique function of the respective relative volume. Thus, dc; = c. Now, the viscosity is proportional to the average concentration, even on local levels. dTlsp =_1=_ dc c __ c[ '1']] C* C* or,
(10)
Viscosity of polymer solutions
~11sp =C[11]·~=0.4c2[11]2
c* and for the total 11sp, one obtains:
85
(11)
which is the Huggins equation. The theoretical value of 0.4 for the Huggins constant, kH, has its origin in the value of2/5 from the Stokes-Einstein formula. This value should hold even for non-spherical particles, as long as they are randomly oriented. Actually, the values of k H ranging between 0.3 and 0.5 are found in the extensive compilation for many golymers by Stickler and Sutherlin. 20 The values for hyaluronan, obtained by Shimada, 2 are typically from 0.35 to 0.40 in the high molecular weight 21 range. For polyisoprene, the value of 0.42 was found by Patel and Takahashi throughout the molecular weight range of their investigation. From our experience, we find it difficult to determine the values of kH and [11] by assuming a linear equation from real data that actually curve upward with concentration. However, we were able to obtain kH nearly always of 0.4 if the eq.13 below was directly applied to raw data. The same Huggins formula can be derived from the model of hydrodynamic volume. Specifically, by considering the overlap of two hydrodynamic volumes to be the volume fraction of a volume fraction, the square term in the Huggins equation results. However, the actual value of the hydrodynamic volume fraction can be two orders greater than unity, which would be difficult to visualize physically. Also, in the overlapped region the hydrodynamic volume must be assumed to expand with increased concentration. The reason why the solid sphere model works in spite of these unreal operations is that it is the average density of the cloud that is being utilized for the calculation. Eq. 12 was derived, assuming the interaction to occur between two neighboring molecules. For one .molecule to interact with two neighbors at the same time, an additional increase in Cj must be included. In this case, the choice of two neighbors is combinatorial, so it must be divided by 2! to avoid redundancy. For this, ~211sp= 2/2. c[llH0.4c[1l]J For a greater number of interacting neighbor molecules, there will be a greater number of terms. Specifically, for one molecule interacting with up to three neighbors, the specific viscosity llsp is given by: (13) with value of 0.4 for k H. If the neighbor interaction were assumed to extend to all existing molecules, the polynomial is extended to infinite terms,
which is precisely the exponential function, (15)
86
Characterisation and solution properties ofhyaluronan
This is the Martin equation. 22 Previously, wei have subscribed to the Martin equation, based on the data that did not extend to the large enough value of c[n]. A critical examination for the choice between this equation vs an equation based on a limited number of neighbor interactions, such as Eq. 13, requires a set of very accurate Newtonian viscosity data obtained for high molecular weight polymers at relatively high concentration. Data obtained by Berriaud, Milas, and Rinaud0 27 extend to unusually high values of c[n], with added assurance that the Newtonian viscosity is being measured. These authors fitted their data with an empirical expression: (16)
7.0 6.0 I --eq.15 5.0 -eq.13 4.0 o Berriaud et al 3.0 --------- ---T I 2.0 1.0 0.0 -1.0 0.0 -1.0
i
+----------..~""'-"--------t--------.-----1
--ji --- -------1.0
2.0
log c[eta]
Fig. 1. Comparison ofeq. 13, eq. IS, and data [eq. 16, Ref. 27] fornsp vs c[n].
Eqs. 13, 15, and 16 are compared in Fig. I, indicating that Eq.l3, but not Eq, 15, is in better agreement with the data. In addition to Berriaud et al's data on hyaluronan, data on polyisoprene in hydrocarbon solvent, obtained by Patel and Takabashi;" data on polystyrene, polyisoprene, polybutadiene, by Raspaud et al,24 data on semi-rigid polyhexyl isocyanates by Ohshima et al,25 and on straight and rigid polyphenylenes by Kwei et al26 have been examined. Fit with eq. 13 is excellent with these data. In all these data, we fitted eq.13 directly to raw data, without going through the procedure of obtaining kH and [n] by drawing a straight line for Huggins and Kraemer equations for the low concentration range. We have shown that Eq. 13 is a universal curve for many types of polymers. It is based on the viscosity behavior of dynamic polymer chains modeled by clouds of mass points. These clouds allow for fluid to flow through. The flow around the points within the cloud was assumed to obey the Stokes Einstein law. No hydrodynamic interactions
Viscosity of polymer solutions
87
between these mass points were incorporated. The average density of the cloud is affected by the concentration. Overlapping of clouds increases the internal density of the cloud, thereby constraining the original unperturbed conformation, Although we have not discussed, the elastic modulus can be calculated based on the change in Cj such that G will depend on c2 for v=O.5, or a higher power if v>0.5. Eq. 13 can be applied to cc*, as clouds are touching their neighbors at all levels of concentration. The concept of molecular entanglement was not needed and not used in the development of the theory. The applicability of our model to rigid polyphenylene molecules endorses this contention.
Rigid, Sem i-rigid and Flexible Polym ers
7....--------------------, 6
-1-----
5-+---------------------~¥-
4
-+-------------------,.,.....IK-'~--~
3-+--------------2 - + - - - - - - - - - - - - - - -....1 ) 6 ' " - - - - - - - - - - - - 1 1 O-+------- ~'¥"-------------__i -1 -1
-0.5
o
0.5 log c[eta]
1
1.5
2
I I I ____-I
Fig. 2 Comparison of eq. 13 with experiment for many polymers.
ACKNOWLEDGEMENT Helpful discussions with Professors E. A. Balazs and T. K. Kwei are gratefully acknowledged. This work was partially funded by Biomatrix, Inc.
REFERENCES (1) Matsuoka, S. Relaxation Phenomena in Polymers, Hanser, Munich, New York, 1992. pp 170. (2) Eyring, H. Phys. Rev. 1932 39, 746. (3) Flory, P. J. Principles ofPolymer Chemistry, Cornell University Press, Ithaca, 1953 pp 601-621. (4) Milas, M.; Rinaudo, M.; BorseIi, J. J. Brazil AAS. 1993,45(11),46-48. (5) Arnott, S.; Mitra, A. K.; Raghnathan, S.; J. Mol. Bio!. 1983 169, 861-872. (6) Kirkwood, J. G.; Riseman, J. J. Chem. Phys. 1948,16,565. (7) Mark, H.; Tobolsky, A. V. Physical Chemistry of High Polymer Systems. Interscience, 1950, p.344
88
Characterisation and solution properties ofhyaluronan
(8) Turner, R.E., Lin, P., Cowman, M.K.; Arch. Biochem. Biophys. 1988,265,484-495. (9) Balazs, E.A (1965) in The Amino Sugars: The Chemistry and Biology of Compounds Containing Amino Sugars (Balazs, E.A & Jeanloz, R.W., eds) Vol. 2A, ppAO1460, Academic Press, New York. (10) Laurent, T. C.; Ryan, M.; Piertruszkiewicz, A. Biochim. Biophys. Acta. 1960,42, 476 (11) Cleland, R. L., and Wang, J. L., Biopolymers. 1970, 9, 799 (12) Shimada, E.; Matsumura, G.J. Biochem. 1975,78, 513-517 (13) Bothner, H.; Waaler, T.; Wik, O. Int. J. Bioi. Macromol. 1988, 10,287-291. (14) Yanaki, T.; Yamaguchi, M. Chem. Pharm. Bull. 1994,42 (8), 1651-1654. (15) Cleland, R. L. Biopolymers. 1970,9,811-824; ibid 1971,10,1925-1948. (16) Peterlin, A J. Polymer Sci. 1950,5,473. (17) Kuhn, H.; Kuhn, W. J. Polymer Sci. 1950,5, 519. (18) Fujita, H. Polymer Solutions, Elsevier 1990 pl40 (19) Frisch, H. L.; Simha, R. Rheology - Theory and Practice, Eirich, F. R., ed. 1956, Academic Press, New York, Chapter 14, pp525 - 614 (20) Stickler, M.; Sutherlin, N, in "Polymer Handbook", ed. Immergut, E. H.; Brandrup, J., John Wiley & Sons, New York, 1989. (21) Patel, S. S.; Takahashi, K.; Macromolecules 1992,25,4382-4391. (22) Martin Eq see for example, Tyrrell, Matthew, Rheology of Polymeric Liquids, C. W Macosko, Rheology Principles, Measurements, and Applications Wiley-VCH 1994 NYpp481 (23) Fouissac, E.; Milas, M.; Rinaudo, M. Macromolecules. 1993,26,6945-6951. (24) Raspaud, E, Lairez, D., Adam, M; Macromol. 199528, 978 (25)Ohshima, A Yamagata, A; Sato, T.; Teramoto, A; Macromolecules 1999, 32, 8645 (26) Kwei, T. K.; Nakazawa, M; Matsuoka, S; Cowman, M. K.; Okamoto, Y. Macromolecules 2000 33235 (27) Berriaud, N; Milas, M; Rinaudo, M; Int. J. Bioi. Macromol. 1994,16,137-142 (28) Davies, A.; Gonnally, J.; Wyn-Jones, E.; Wedlock, D. J.; Phillips, G. 0.; Int. J. Bioi. Macromol. 1982 4 436438..
CONFORMATIONAL AND RHEOLOGICAL PROPERTIES OF HYALURONAN Katsuyoshi Nishinari·), Yunqian Mo" Ryo Takahashr', Kenji Kubota 2 & Akio Okamoto3 I Department
ofFood and Nutrition, Faculty ofHuman Life Science,
Osaka City University, Sumiyoshi, Osaka 558-8585, Japan 2 Department
ofBiological and Chemical Engineering, Faculty ofEngineering, Gunma University, Kiryu, Gunma 376-8515, Japan 3Research
Center, Denki Kagaku Kogyo, Co.Ltd.,
Asahi-machi, Machidashi, Tokyo 194-8560, Japan
ABSTRACT
Effects of' urea, sodium chloride (NaCl), guanidine hydrochloride (GuHCI) or sucrose on the viscoelasticity of sodium hyaluronate (NaHA) solutions were studied. Urea did not change both storage and loss moduli so much, NaCl and GuHCI decreased both moduli, while sucrose increased both moduli. The critical overlap concentration C* was determined as an inflection point in the plot of zero shear specific viscosity vs concentration for NaHA solutions with and without urea, NaCI, GuHCI or sucrose. It is suggested that sodium ions or guanidinium ions shield the electrostatic repulsion of NaHA molecules, hence reduce the coil dimension, and C" shifted to higher concentrations. However, sucrose enhances the entanglement coupling between NaHA molecules and promotes the creation of hydrogen bonds, and then C* for NaHA solutions with sucrose shifts to lower concentrations. Both C* and magnitude of zero shear specific viscosity did not depend so much on the concentration of added urea either in the presence or in the absence of O.2M NaCI. This is in agreement with experimental results by light scattering. The radius of gyration Rg and hydrodynamic radius Rh were determined as a function of urea and sucrose concentration in the presence of O.2M NaCl by light scattering. Although the addition of sucrose reduces the coil dimensions of NaHA and stiffens NaHA molecules in dilute solutions, it increases both storage and loss moduli of concentrated NaHA solutions because of the enhancement of the formation of the temporary network via newly created hydrogen bonds. Both moduli of hyaluronan solution with urea were higher than those without urea in the lower angular frequency region, and tend to become nearly the same in the higher angular frequency region. The crossover angular frequency of both moduli decreases only a little with the addition of urea. It was found that the disruption of the hydrogen bonds due to urea, even if it occurs, does not affect significantly the rheological behavior of hyaluronan solutions. Intermolecular hydrogen bonds which lead to the formation of network in many gelling polysaccharides do not seem to exist in hyaluronan solutions. KEYWORDS
urea, guanidine hydrochloride, hydrogenbonds, rheology, light scattering
90
Characterisation and solution properties ofhyaluronan
INTRODUCTION
Hyaluronan (HA) is a major macromolecular component of the intercellular matrix of most connective tissues such as synovial fluid, cartilage, eye vitreous humor. Because of its unique viscoelastic properties, it is used as a medicine for arthritis and a surgical aid in ophthalmic surgery'", Hyaluronan is a linear polysaccharide consisting of disaccharide repeating sequence. The two saccharide residues are D-glucuronic acid and N-acetyl-D-glucosamine, which are linked P(l-3) and P(l-4) to each other4-6. The rheological characteristics of hyaluronan solutions have been widely studied, especially the relationships of its lubricating and shock-absorbing functionalities in synovial fluid to its viscoelasticity", The flow properties of normal synovial fluid extracted from human and cattle joints have been extensively studied8, JO, and are shown to be shear-thinning at high shear rates. By comparison of dynamic viscosity and static viscosity of synovial fluid at infinite time scales, Myers et al found that the viscosity at zero frequency and zero shear-rate agreed with each other and synovial fluids act only as viscous liquids in human joints under very slow motion 7 • It is reported that in osteoarthritis joints, the viscoelasticity of synovial fluid decreases and low molecular weight HA appears. It is important, therefore, to study the effect of the addition of the low molecular weight HA on high molecular weight HA solutions, Morris et al found that the addition of segmented chains of HA prepared by enzymatic degradation reduces drastically storage and loss moduliII, whilst Fujii et al. did not observe the similar phenomenon using segmented chains prepared by physical methods such as ultrasonic degradation or pyrolysis'f. It was suggested that surviving enzyme continued to degrade long molecular chains ofHA in the previous experiment", Several authors have reported that hyaluronan molecular coil expands at low ionic strength and high pH causing the increase in intrinsic viscosityI4.16. However, Preston et al. were unable to verify the expansion of the hyaluronan molecule with decreasing ionic strength by light-scattering measurements'". Cleland reported that the intrinsic viscosity and the radius of gyration of a hyaluronan preparation were 1400ml/g, 453ml/g and 75.8nm, 62.5nm in O.OlM and 2.0M NaCI solution, respectively 16. Morris et al. showed that increasing ionic strength of hyaluronan solutions is analogous to lowering pHII. A reduction in viscosity results from the suppression of electrostatic repulsion between the dissociated carboxyl groups, Scott proposed that the carboxyl group in hyaluronan forms a hydrogen bond with one of the hydroxyl groups in the uronic residue l 8 ; and it was confirmed!'. This concept "super hydrogen-bond" may be used to explain the extended coil dimensions at physiological pH and ionic strength. In the present work, the influence of urea on viscoelasticity of HA solution was examined to understand the role of hydrogen bonds because urea is known to be a hydrogen bond breaker. Then, the effects of urea on the rheological properties of hyaluronan solutions were compared with those of guanidine hydrochloride, which is also known to be a hydrogen bond breaker, and is also an electrolyte like sodium chloride. EXPERIMENTAL
Preparation of sample solutions Three samples of powdered HA, extracted from the culture medium of Streptococcus equi and purified were kindly supplied by Denki Kagaku Kogyo Co., Ltd. and Hoya Co.,Ltd.. Molecular weights of those samples determined from intrinsic viscosity using
Conformational and rheological properties
91
the Mark-Houwink parameters'" are 1.6x10 6 and 2.02x10 6 and 4.83 x10 5, respectively. NaC1, GuHC1, and urea of the reagent grade were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and ICN Biomedicals, Inc. (Ohio,USA) respectively and were used without further purification. HA solutions of various concentrations were prepared by dissolving in NaCl, GuHCI, and/or urea by weighing and were stirred for one day at room temperature. Special care toward the contamination of bacteria was taken. Light scattering measurements Light scattering measurements were carried out using a homemade spectrometer" for the NaHA solution in the presence of urea in 0.2M NaCI solution. Weight average molecular weight for the light scattering measurements is 4.83 x105 • Sample solutions were dialyzed against the respective urea and 0.2M NaCI solutions thoroughly before the measurements. Light source was Ar ion laser operated at 488.0 nm. Temperature of the sample solution was regulated within ±O.Ol°C. Correlation functions of the scattered light intensity were obtained by ALV-5000JE multiple-tau digital correlator. The radii of gyration were determined by the conventional angular dependence of scattered intensity. The hydrodynamic radii of HA were determined from the average decay rate obtained by using the third cumulant expansion method and the Stokes-Einstein equation. Corrections for the refractive index and the solvent viscosity were made by use of an Abbe's refractometer at 589 nm and an Ubbelohde capillary viscometer. It was not possible to carry out light scattering measurements without NaCI with sufficient accuracy because of the too low scattered intensity as is normally the case for an aqueous polyelectrolyte solution without salt. Intrinsic viscosity measurement The intrinsic viscosity [11] was determined using an Ubbelohde capillary viscometer at 25°C. The flow time for water was about ~141s at 25°C. A unit-thermalbath (Yamato Science, Inc. Tokyo, Japan) was used to regulate the temperature at 25±0.02°C. Dynamic and steady viscoelastic measurements Dynamic and steady viscoelastic measurements were performed by a RFSn (Rheometries Fluids Spectrometer; Rheometries, Inc., NJ, USA). The diameter and angle of the cone were 2.5cm and O.lrad, respectively. The strain was 5% and angular frequencies ranged from 0.01 to 100 radls in the dynamic measurements. The shear rates ranged from 0.01 to 1000 S·l in the steady state measurements. Temperature was fixed at 20°C. RESULTS AND DISCUSSION Light scattering The coil dimensions of NaHA solution with urea in 0.2M NaCl solutions were studied by light scattering. Radius of gyration (Rg), hydrodynamic radius (Rh) and the ratio (RglRh) in the presence of urea are shown together with those in the presence of sucrose in Fig. I.
92
Characterisation and solution properties ofhyaluronan 2.5 ~
• • •
2.0
l!:",
0
II:: 1.5
Oct
l!:co 1.0 co
• 0
II::
0
• • •
• • •
ir,"'" 0.5 -co
II::
•
•
s
•
••
0
0
0.5
1.0
• •
sucrose RgfRh
0
• e (mol)
sucrose RgfRg,o sucrose RhfRh,O
0
0.0 0.0
•
~ 3.0 3.1
urea RgfRg,o
0
urea RhfRh,O
0
urea RlRh
Figure 1. Radius of gyration and hydrodynamic radius of NaHA as a function of concentration of added urea and sucrose in the presence of O.2M NaCl.
The results suggest that urea does not affect significantly the coil dimensions of HA molecules within experimental error, although a slight decrease in Rg and Rg/Rt, was observed. These results show a clear contrast with our recent results for NaHA with sucrose in O.2M NaCI solutions, where both Rg and Rn decrease and Rg/RJ. increases appreciably with the increase of sucrose concentration". The magnitude of Rg and Rn and the slight decrease of Rg/Rt, by the addition of urea indicate that HA molecules are in the form of an extended coil affected by the excluded-volume effect and the solubility of HA becomes worse with the addition of urea causing a reduction in the excluded-volume effect. No intrinsic interaction between urea and HA molecules other than the excluded-volume effect might work at least in the experimental concentration region, and less solubility simply results in an increase in intermolecular interaction. Therefore,.the concentration dependence of 11spO would be stronger with the addition of urea in the dilute region, and the behavior in the entangled region is not affected so much by the presence of urea (especially without NaCl). It has been known that HA molecules have both the hydrophilic portions consisting of equatorial OH groups of saccharides and the hydrophobic portions consisting of axial CH groupS22. Hydrogen bonds are formed intramolecularly between the continuing monosaccharides and work to stiffen HA molecular chains. Such a stiffuess was evaluated quantitatively by use of wormlike chain model recentlY3,24. It was suggested that the stiffness of HA chains in solution originates from intramolecular hydrogen bonding by NMR measurements'". It was also suggested that the majority of stiff chains survive even after the change in ionic strength, temperature or by the addition of urea; that is, the addition of urea can not disrupt effectively the intramolecular hydrogen bonds between HA molecules, but no experimental data in the presence of urea was shown2s. Less solubility and larger intermolecular interaction should be due to the destruction of intramolecular hydrogen bonds, and chain dimensions must decrease much more than the experimental observations. The presence ofNaCI might lower this effect of urea. Intrinsic viscosity For dilute solutions (C
Conformational and rheological properties
93
viscosity did not give a straight line at low urea concentrations (less than 1M) because of the strong electrostatic repulsion, but obeyed a linear relation with HA concentration and decreased substantially at high urea concentrations showing linear dependence on the HA concentration (1,3, and 6M urea). This should result from the partial shielding action of urea of the electrostatic repulsion. Intrinsic viscosities in water are much larger than those in 0.2M NaCI suggesting an elongated molecular structure and remaining appreciable electrostatic interaction. The intrinsic viscosity in 0.2M NaCI decreased with the addition of urea, but did not depend so much on the concentration of urea. Huggins constant k' for flexible molecules is reported as 0.3-0.6. When k' is larger than 1, bulky aggregated structures should exist in the solution.
Table 1.
Intrinsic viscosity [1')] and Huggins constant k' for NaHA solutions with urea in the presence of 0.2M NaCI (Temperature, 25°C; Molecular weight, 1.6 x 106)
urea
none 24.8 0.373
[1')] (dl/g) k'
IMurea 18.6 0.322
6Murea 21.8 0.722
3Murea 19.0 0.613
Dynamic and steady viscoelasticity The specific viscosity TJsp of 1wtOlo HA solutions in the presence of urea of various concentrations, and with and without 0.2M NaCl, as a function of shear rate is shown in Fig.2. The specific viscosity of HA solution without 0.2M NaCI increased with increasing concentration of urea up to 3M urea but that in the presence of 6M urea showed almost the same value in the absence of urea, whilst it decreased by the addition of 0.2M NaC!. This decrease results from the shielding effect of the intermolecular electrostatic repulsion and shrinkage of the elongated chain ll ,14,16,26. It should be noted that the magnitude of 1')sp did not depend so much on the urea and NaCI concentrations in the region of shear thinning, which is different from the behavior in the Newtonian region. 10"'r--------~---____.
'lsp,O
0
HA
e
1M urea
0
3M urea
~
6Murea
• •
O.2MNaCI+ IMurea O.2MNaCI+3Murea
O.2MNaCI+6Murea 10- 1 Y(s -1) Figure 2. Shear rate dependence of specific viscosity for 1wtOlo NaHA solutions withor withouturea(Temperature, 20°C;Molecularweight, 1.6 x 1O~
10-2
94
Characterisation and solution properties ofhyaluronan
lOS 10 4 10 3
1-
';!
• • • •
O.2MNaCI
HA
3Murea 3Murea
10 2
HAC*
0
O.2MNaCIC*
e
IMureaC*
b.
6MureaC*
100 10- 2
10- 1 C (wt%)
Figure 3.
•
6Murea
0
IMureaC* 3MureaC*
10 1 10° 10-2
b
IMurea
IMurea
10- 1 C (wt%)
10°
(a) Zero shear specific viscosity for NaHA solutions with or without urea as a function of NaHA concentration. (b) Zero shear specific viscosity for NaHA solutions with or without urea in the presence of 0.2MNaCI as a function ofNaHA concentration (Temperature, 20°C; Molecular weight, 1.6 x 10i).
Fig.3 shows the concentration dependence of zero shear specific viscosity T)spo for HA solutions in the presence of urea of various concentrations without NaCI(a) and with 0.2M NaCI (b). The viscosity ofHA solution without NaCI is much higher (more than one order) than that with 0.2M NaCl due to electrostatic interaction. Fig.3(a) shows that T)spO decreased in the less concentrated region on adding urea. and T)spO was more dependent on urea concentration. However, in the concentrated entangled region T)spO coincided with each other, and the HA concentration dependence of T)spO seems almost independent of the concentration of urea. These features are also observed in Fig.3(b) in the presence ofO.2M NaCI. In both cases of (a) and (b), it is obvious that the addition of urea did not change so much C* (or slight decrease of C*) and the value ofT)spO at C*, where C* is defined as the concentration at the flection point ofT)spO. Fig.4 shows the critical shear rate c of the onset of shear thinning as a function of concentration for HA solutions with urea without NaCI and with 0.2M NaCI. As shown for the case without NaCI, two regimes were observed and they are in agreement with the result in Fig.3. In the case of HA solutions with urea and NaC!, the plots of reVS. concentration were shown in the concentrated region, because revalues in the dilute region were beyond the experimental shear rate range. The smaller chain dimension ofHA molecules in the presence ofO.2M NaCI than that without NaClled to larger revalues. Fig.5 shows the pseudoplasticity index n as a function of coil overlap parameter C[T)] for HA solutions in the presence of urea of various concentrations with 0.2M NaC!. The value n was close to 0.8 or lar§er than 0.8 for C[T)] > 20. The limiting value of the pseudoplasticity index is reported 7 as about 0.85 when C[T)] > 20. In the case of weak gels, n is found to be higher than 0.928 . The present result is in parallel with the picture that HA solutions in the presence of urea behave like entangled flexible coils, although it has non-negligible chain stiffness.
r
95
Conformational and rheological properties
100
10-
• • •
1:2
.~
0.1 0.01 Figure 4.
0
O.2MNACI+6Murea
I I
Q
... 1
A
O.2MNACI+3Murea ~
0
,-.. ";'
$
O.2MNaCI O.2MNACI+1Murea
~
~ ~
HA
o
IMurea
0 A.
3Murea 6Murea
•I,
B § !f! g DO 0 0 ~!
I
0.1 C (wt%)
1
The critical shear rate of the onset of shear thinning behavior as a function of concentration of NaHA with and without urea in the presence and in the absence of 0.2MNaCI (Temperature, 20°C; Molecular weight, 1.6 x 106) .
0 . 8 5 , . - - - - - - - - - - - - - - - -__- - - - - .
0.7-
=
0.55-
Figure 5.
• ••••• •• •• • ••• •
•• • ••• •• •
Pseudoplasticity index n as a function of the coil-overlap parameter C[11] for NaHA solutions with urea of various concentrations with and without 0.2MNaCl (Temperature, 20°C; Molecular weight, 1.6 x 106) .
Zero shear specific viscosity llsp,o as a function of the coil-overlap parameter showed a fairly good superposition for various urea concentrations in 0.2M NaCl solution as shown in Fig. 6. The slopes in the dilute (C < C*) and concentrated (C > C*) regions are 1.6 and 4.0, respectively, and C*[l1] and Tlspo at C = C" is evaluated ae- 3.2 and 24
96
Characterisation and solution properties ofhyaluronan
105 10 4 10 3 <:>
• • •
IMurea 3Murea 6Murea
., 10 2 Q,
I='
10 1
slope=1.6
C*[ f)J=3.2
10° 10- 1 10- 1
10°
C[ f)J
10 2
10 1
Figure 6. Zero shear specific viscosity for NaHA solutions with urea in the presence of 0.2MNaCI as a function of the coil overlap parameter C[TJ] (Temperature, 20°C; Molecular weight, 1.6 x 106) .
no urea 0"
........<>........
O.2MNaCIG"
+ IMurea 0"
........<>........
+lMUrea G"
········0·····..·
+3MureaO"
········0········
+3MUrea GOO
········6·····..·
+6Murea 0"
········6········
+6MUrea GOO
10 -2 -f-'.............-........""t-........""r-..........................""" 10- 3 10-2 10- 1 100 10 1 10 210-3 10- 2
10- 1 100
10 1
10 2
w (rad/s) w (rad/s) Figure 7. (a) The angular frequency dependence of G' and G" for lwt% NaHA solutions with and without urea of various concentrations (b) The angular frequency dependence of G' and G" for Iwt% NaHA solutions with and without urea of various concentrations in the presence of 0.2MNaCI (Temperature, 20°C; Molecular weight, 2.02 x 106) . respectively. These values are in agreement with our previous results". Any intrinsic characteristics of the addition of urea were not observed. Fig.7(a) and (b) show the angular frequency dependence of storage shear modulus G' and loss shear modulus G" for 1wt% HA solutions in the presence of urea without NaCI and with 0.2M NaCl, respectively. At low frequency, both G' and Gil increased, and the crossover frequency of G' and G" shifted to lower angular frequencies with increasing concentration of urea. The similar shift was observed by the addition of
Conformational and rheological properties
97
sucrose", however, it was more pronounced than the shift by urea; the shift by urea was less than half by sucrose. Although the decrease of crossover frequency with adding urea might correspond to the strengthening the transient network structure formed by the chain entanglements, this is not so pronounced as in the case of sucrose. The increase in G'(Fig.7) and T1sp (Fig.2) of 1wt"10 HA by the addition of urea might be attributed to the breaking of intermolecular hydrogen bonds between the groups separated by many intervening segments along the chain, which have been called long range hydrogen bonds, used to explain the similar increase in the intrinsic viscosity of poly(vinyl alcohol) solutions in the presence of urea 29. It was, however, not possible to detect the change in the radius of gyration without adding NaCI by light scattering, and this should be explored in the future. CONCLUSION The effect of urea on the viscoelastic properties of HA solution was examined. The addition of urea decreases the expansion of HA molecular coils and promotes the intermolecular interaction in dilute solution presumably by a reduced excluded-volume effect due to poorer solvent conditions. The presence of NaCI could lower this effect of urea On the contrary, in concentrated and entangled solutions, urea has almost no effect on the rheological behavior at all. The addition of urea does not affect the alleged hydrogen bonds in the HA molecules significantly. This finding is in a sharp contrast with the results of the addition of sucrose in HA solution. REFERENCES 1. E. A. Balazs, In: Disorders of the Knee, A. Helfet, (ed.), 1974, T. B. Lippincott Company, Philadelphia, pp.63-75. 2. D. J. Tate, P. D. Oliver, M. V. Miceli, R. Stem, S. Shuster & D. A. Newsome, 'Age - dependent change in the hyaluronic acid content of the human chorioretinal complex', Arch. Ophthalmol., 1993, HI, 963-967. 3. N. Yerushalmi, A. Arad & R. Margalit, 'Molecular and cellular studies of hyaluronic acid - modified liposomes as bioadhesive carriers for topical drug delivery in wound healing', Arch. Biochem. Biophys, 1994, 313, 267-273. 4. R. L. Cleland, 'Ionic polysaccharides. IV. free - rotation dimensions for disaccharide polymers. Comparison with experiment for hyaluronic acid', Biopolymers, 1970,9,811-824. 5. W. T. Winter, P. J. Smith & S. Arnott, 'Hyaluronic acid: structure of a fully extended 3-fold helical sodium salt and comparison with the less extended 4-fold helical forms', J. Mol. Bioi., 1975, 99,219-235. 6. J. K. Sheehan, K. H. Gardner & T. E. D. Atkins, 'Hyaluronic acid: a double- helical structure in the presence of potassium at low pH and found also with the cations ammonium, rubidium and caesium',J. Mol. Bioi., 1977, H7, H3-135. 7. R. R. Myers, S. Negami & R. K. White, 'Dynamic mechanical properties of synovial fluid', Biorheology, 1966, 3, 197-209. 8. V. Tirtaetmadja, D. V. Boger & J. R. E. Fraser, 'The dynamic and steady shear properties of synovial fluid and of the components making up synovial fluid', Rheol. Acta, 1984,23,311-321. 9. J. E. Gomez & G. B. Thurston, 'Comparisons of the oscillatory shear viscoelasticity and composition of pathological synovial fluids' Biorheology, 1993,30,409-427. 10. J. Schurz, 'Rheology of polymer solutions of the network type', Prog. Polym. Sci.,
98
Characterisation and solution properties ofhyaluronan
1991, 16, 1-53. 11. E. R. Morris, D. A. Rees & E. J. Welsh, 'Conformation and dynamic interactions in hyaluronate solutions', J. Mol. Bioi., 1980, 138,383-400. 12. K. Fujii, M. Kawata, Y. Kobayashi, A. Okamoto & K. Nishinari, 'Effects of the addition of hyaluronate segments with different chain lengths on the viscoelasticity of hyaluronic acid solutions', Biopolymers, 1996, 38, 583-591. 13. E. R. Morris, Private communication 14. E. A Balazs & T. C. Laurent, 'Viscosity function of hyaluronic acid as a polyelectrolyte', J. Polym. Sci. Lett. Ed, 1951, 6, 665-668. 15. T. C. Laurent, 'Studies on hyaluronic acid in the vitreous body', J. Biol. Chem., 1955,216,263-270. 16. R. L. Cleland, 'Ionic polysaccharides. II. Comparison of polyelectrolyte behavior of hyaluronate with that of carboxymethyl cellulose', Biopolymers, 1968, 6, 1519-1529. 17. B. N. Preston, M. Davies & A G. Ogston, 'The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma', Biochem. J., 1965,96,449-471. 18. J. E. Scott, 'Periodate oxidation, pKa and conformation of hexuronic acids in polyuronides and mucopolysaccharides', Biochim. Biophys. Acta, 1968, 170, 471-473. 19. T. C. Laurent, M. Ryan & P. Kizwica, 'Fractionation of hyaluronic acid: the polydispersity of hyaluronic acid from the bovine vitreous body', Biochim. Biophys. Acta, 1960,42,476-485. 20. D. Ito and K. Kubota, 'Solution properties and thermal behavior of poly (N-n-propylacrylamide) in water', Macromolecules, 1997,30, 7828-7835. 21. Y. Mo, T. Takaya, K. Nishinari, K. Kubota & A. Okamoto, 'Effects of sodium chloride, guanidine hydrochloride, and sucrose on the viscoelastic properties of sodium hyaluronate solutions', Biopolymers, 1999, 50, 23-34. 22. B. Weissmann & K. Meyer, 'The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord',J. Am. Chem. Soc., 1954, 76,1753-1757. 23. K. Hayashi, F. Tsutsumi, T. Nakajima, T. Norisuye & A. Teramoto, 'Chain-stiffaess and excluded-volume effects in solutions of sodium hyaluronate at high ionic strength', Macromolecules, 1995,28, 3824-3830. 24. R. Takahashi, K. Kubota, M. Kawata & A Okamoto, 'Effect of molecular weight distribution on the solution properties of sodium hyaluronate in 0.2M NaCI solution', Biopolymers, 1999,50,87-98. 25. A Darke, E. G. Finer, R. Moorhouse & D. A Rees, 'Studies of hyaluronate solutions by nuclear magnetic relaxation measurements. Detection of covalently-defined, stiff segments within the flexible chains', J. Mol. Biol., 1975, 99, 477-486. 26. Y. Kobayashi, AOkamoto, & K. Nishinari, 'Viscoelasticity of hyaluronic acid with different molecular weights', Biorheology, 1994, 31, 235-244. 27. W. W. Graessley, 'The entanglement concept in polymer rheology', Adv. Polymer Sci, 1974, 16, 125-179. 28. S. B. Ross-Murphy, V. J. Morris & E. R. Morris, 'Molecular viscoelasticity of xanthan polysaccharide', Faraday Symp. Chem. Soc., 1983, 18, 115-129. 29. H. Maeda, T. Kawai & S. Sekii, 'Intra-and intermolecular hydrogen bonds in polyvinyl alcohol solutions', J. Polymer Sci., 1959, 35,288-292.
CARTILAGE REPAIR WITH BONE MARROW IN A HYALURONAN-BASED SCAFFOLD Luis A. Solchaga', Victor M. Goldberg2 and Arnold I. Caplan' ISkeletal Research Center, Department ofBiology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106, USA. 2Department
ofOrthopaedics, Case Western Reserve University School ofMedicine and University Hospitals ofCleveland. 11100 Euclid Avenue, Cleveland, OH 44106, USA.
ABSTRACT The repair of osteochondral defects can be enhanced by providing structural support for the reparative activity. We hypothesize that the impregnation of specialized materials with autologous bone marrow will provide progenitor cells and cytokines to accelerate the healing response and, therefore, improve the quality of the repair. New Zealand White rabbits received bilateral, osteochondral defects on the femoral condyle. One leg received fibronectin-coated ACP""', a hyaluronan-based polymer, and the other flbronectin-coated, bone marrow-loaded ACp™. Rabbits were sacrificed 3, 4, 12 and 24 weeks after surgery. The reparative tissue was assessed by histology. Both treatment groups presented similar appearance at 4, 12 and 24 weeks after surgery. Four weeks after surgery, the defects presented new bone filling the defect with a layer of hyaline cartilage on top that integrated well with the adjacent cartilage. At the 12 and 24-week time points the defects presented bone filling the defect area beyond the level of the tidemark and a layer of hyaline cartilage about half as thick as the adjacent normal cartilage. At the 3-week time point , the defects that received bone marrowloaded implants presented more bone and the surface layer contained more cartilage. The early events of the regeneration process are accelerated by the inclusion of bone marrow; however, because the access to the marrow space is open in osteochondral defects, the medium- and long-term results obtained appeared to be similar. KEYWORDS Hyaluronan, tissue engineering, cartilage, bone marrow, repair, regeneration. INTRODUCTION The natural repair process of osteochondral defects can be enhanced through the use of biocompatible, biodegradable materials to serve as scaffolding for the regeneration process I. These materials provide structural support for the reparative activity of mesenchymal progenitor cells recruited into the area, presumably from the underlying bone marrow. The failure or success of the reparative process can be determined by the acquisition of mechanical stability in the damaged area through synthesis of extracellular matrix by the cells recruited into the defect. We hypothesize that impregnation of the implant material with bone marrow at the time of surgery will provide progenitor cells and bioactive factors required for the regeneration and accelerate the healing response.
64
Application ofhyaluronan in tissue engineering
METHODS - Preparation ofthe implants: The ACpTM sponge was cut into 2-mm diameter cylinders and then pre-coated by immersion into solution of fibronectin. After a I-h incubation at 4°C, the implants were removed from the fibronectin solution and dried overnight'. During the surgical procedure, autologous bone marrow, obtained as described below, was combined with the ACpTM sponges in a 5-mL tube. Negative pressure was applied to the tube to allow complete infiltration of the delivery vehicles with the cell suspension. The composites were incubated at room temperature for 20 min prior to implantation. Control implants were hydrated in sterile saline solution prior to implantation. - Surgical procedure: A total of 33 4-month-old New Zealand White rabbits were used in this study. All procedures followed an Institutional Animal Care and Use Committeeapproved protocol. Under anesthesia, the knee was exposed trough a skin incision; the capsule was incised and the medial femoral condyle exposed after luxation of the patella. A full-thickness defect (3-mm diameter x 3-mm deep) through articular cartilage and into the subchondral bone was prepared on the center of the medial femoral condyle. The implants were then placed into the defect and, after reducing the patella, the capsule, muscle and skin were closed with 4-0 dexon suture. Each rabbit received ACpTM sponge in one knee and BM-loaded ACpTM sponge in the contralateral knee. - Procurement ofbone marrow: In the same surgical act, the proximal medial surface of the rabbit tibia was exposed through a small incision. Subcutaneous tissue and periosteum were incised to expose the bony surface. The tibia was perforated with a 16gauge needle, and bone marrow (4 - 5 mL) was then aspirated from the tibial shaft through a pre-heparinized plastic tubing affixed to a 1O-mL syringe containing heparin. - Histologic processing: Rabbits were killed at 3, 4, 12and 24weeks after surgery. The knee joint was approached as described above and the distal femoral condyles dissected. The specimens were fixed in formalin, demineralized with RDO, and embedded in paraffin. Sections of 6 urn were cut and stained with Toluidine Blue. Representative sections were scored independently and blindly by 4 investigators with a 29-point scale. - Statistical analysis: The histologic scores were compared with a Wilcoxon signed rank test. P values minor than 0.05 were considered significant.
RESULTS At the 3-week time point (Fig. lA, E), the ACpTM-treated defects presented bone formation in the bottom half of the defect with a superficial layer of non-mineralized tissue composed of loose fibrous tissue, hypertrophic and hyaline-like cartilage. The 8M-loaded ACpTM-treated defects exhibited bone formation in the bottom 3/4 of the defect with a superficial layer composed of hypertrophic and hyaline-like cartilage. Four weeks after surgery (Fig. IB,F), the repair tissue in defects treated with ACpTM did not fill the defect area up to the level of the surrounding cartilage. Most of the defects presented new bone filling the defect with a layer of hyaline cartilage that integrated well with the adjacent cartilage. In some cases, hypertrophic cartilage was present between the bone and the hyaline cartilage. The defects treated with BM-Ioaded ACpTM presented a very similar appearance to those of the ACp''M-treated group.
Cartilage repair with bone marrow
Figure 1.
65
Light microscopy of the defects after implantation. A, B, C, D: ACP"" sponge. E, F, G, H: Bone marrow-loaded ACP"" sponge. A, E: 3 weeks after implantation. B, F: 4 weeks after implantation. C, G: 12 weeks after implantation. D, H: 24 weeks after implantation. Table I. Histologic scores by category (median (range». 4 weeks
Catezorv % hyaline cartilaae surface reaularftv degenerative changes structural
ACI)~
~CP~+8M
12 weeks ACP~
4.7 3.3 2.7
ACP~+8M
5.7 0.3-7.3) 2.7 (0.3-3.0) 2.0 (1.7-2.3) 1.7 (1.0-2.0)
24 weeks ACP~
ACP~+8M
5.3 6.0 (2.7-6.7) 0.3-7.3) 2.3 2.7 (0.3·-3.0) (0.3-3.0) 2.0 1.7 (I.D-2.7) ( 1.0-2.3) 1.7 1.7 (0.0-2.0) (0.3-2.0) 1.3 1.3 1.0 (0.7-2.0) (0.7-1.3) (0.7-2.0) 4.0 3.0 4.0 (1.0-4.0) ( 1.7-4.0) (0.0-3.7) 2.2 2.0 2.0 (2.0-2.7) <2.0-2.3) (2.0-2.3) 0.8 1.0 1.0 (0.3-1.0) (0.3-1.0) (1.0-1.0) 3.0 3.0 3.0 (2.7-3.0) 1(2.37-3.0) (3.0-3.0) 23.0 20.7 21.7 16.3-25.0) 1'14.7-24.7) If(4.7-25.0)
66
Application ofhyaluronan in tissue engineering
At the 12-week time point (Fig. lC,G), the overall appearance of the defects was similar in both groups with bone filling the defect area beyond the level of the tidemark and a layer of hyaline cartilage well integrated with the host tissue but that was about half as thick as the adjacent normal cartilage. The superficial layer in the 8M-loaded ACp™-treated defects was thicker than in the ACp™-treated group. The integration of the repair tissue with the normal cartilage was also superior in the BM-loaded ACpTM_ treated group. Twenty-four weeks after surgery (Fig. lD,H), the histologic appearance of the defects was similar to that of the twelve-week defects. Bone filled the defect area beyond the level of the tidemark and the cartilaginous surface layer was well integrated with the host cartilage although it was thinner than the normal cartilage. Again, the superficial layer in the 8M-loaded ACpTM-treated defects was slightly thicker than in the ACpTM_ treated group. The integration of the repair tissue with the normal cartilage was also superior in the BM-loaded ACpTM-treated group. The overall histologic scores of the specimens did not reveal any statistical differences between the treatment groups at any time point. The overall histologic scores remained unchanged between 12 and 24 weeks. The only category where statistical differences were found between the treatment groups was in the integration of the repair with the adjacent cartilage 12 and 24weeks after surgery (Table I).
CONCLUSIONS •
Hyaluronan-based scaffolds can focus and organize the natural repair capability of young-adult rabbits.
•
Pre-loading of the scaffold with fresh autologous bone marrow accelerates the early events of the repair process.
•
The differentiation of the mesenchymal progenitor cells implanted with the scaffold or mobilized into it from the bone marrow takes place between days 14 and 28.
•
Pre-loading of the scaffold with fresh autologous bone marrow does not have a dramatic effect on the medium- and long-term outcome ofthe repair process.
•
The more uniform and earlier differentiation observed in bone marrow-loaded implants facilitates a more effective integration of the neocartilage with the host.
•
The repair tissue does not degenerate overtime within the time frame considered. in these experiments.
REFERENCES 1. L. Solchaga, J. U. Yoo, M. Lundberg, B. Hubregtse, A.I. Caplan. Hyaluronic acidbased polymers in the treatment of osteochondral defects. Trans. Orthop. Res. Soc., 1999,24,56. 2. L. A. Solchaga, J. E. Dennis, V. M. Goldberg and A. I. Caplan. Hyaluronic acidbased polymers as cell carriers for tissue engineered repair of bone and cartilage. J. Orthop. Res. 1999, 17,205-213.
THE HEAT DEPENDENCE OF BYALURONAN CONFORMATION Gerard Armaad': Kesuo Fan2 and Endre A. Balazs3 IGlycoMed Research. Inc.• 137 Southside Ave.• Hastings-on-Hudson. New York 10706. USA 2Biomatrix. Inc. 65 Railroad Ave., Ridgefield. New Jersey 07657. USA 3Matrix Biology Institute. 65 Railroad Ave.• Ridgefield. New Jersey 07657, USA
ABSTRACT Hyaluronan preparations from bacteria (Lifecore hyaluronan, Average MW 0.66 million), rooster combs (Pharmacia, Healon®, Average MW 3.5-4 millions) and a formaldehyde crosslinked hyaluronan (Biomatrix, hylan A, average MW 5-6 millions) were applied to an ion exchange resin column (DEAE-Sephacel) at 6° C, 25°C and 65° C and eluted with salt gradients ranging from 0.20 M to 0.55 M NaCl. When hyaluronan and hylan A were applied to the column at 65° C, it was observed that significantly higher concentrations of NaCI were required for their elution than at 6° C, suggesting the occurrence of a conformational change with a concomitant increase in the binding affinity and capacity of these polymers to the DEAE resin with increasing temperature. This phenomenon was found to be reversible, since hyaluronan samples first fractionated at 65° C, and subsequently rechromatographed at 6° C required much lower concentrations of NaCI for their elution from the DEAE-Sepacel column. A comparison of the chromatographic profiles between hyaluronan and hylan A revealed some interesting observations. At 6° C, the binding affinity of hyaluronan to the DEAE-Sephacel is weaker than that observed with hylan A under identical experimental conditions. However, at 65° C, the reverse phenomenon is observed; hyaluronan is bound stronger to the DEAE than hylan A. Presumably, the presence of intramolecular cross-links in the hylan A molecule limited its ability to fully undergo conformational changes. When hyaluronan and hylan A were subjected to gel permeation chromatography (OPe) in 0.4 M NaNO) buffer using the Waters Alliance system at 35°, 50°, and 65° C, the retention time significantly decreased at higher temperatures. Based on the OPC and ion-exchange chromatographic data. it is suggested that in aqueous media, hyaluronan and its derivatives undergo reversible changes in hydrophobic interactions as a function of temperature.
INTRODUCTION It is generally accepted that in solution, hyaluronan (HA) molecules undergo selfassociation leading to the formation of large aggregates or networks that can reversibly be dissociated with a change in the pH, ionic strength or temperature of the solvent. While the mechanism responsible for the conformational transitions that bring about the formation of these aggregates remains to be firmly established, the prevailing view is that they are primarily controlled by hydrophobic interactions as described by Scott and his associates 1-3. There is also evidence that these aggregates or networks are formed in dilute HA solution. Studies on the effect of temperature on the viscosity of different preparations of HA in solution revealed significant decreases in their limiting viscosity values (11) with increasing temperature 4-5. Here we have initiated these column chromatographic studies on HA and hylan A at 6° C and 65° C in order to gain insights
100
Characterisation and solution properties ofhyaluronan
into those temperature dependent conformational changes observed with hyaluronan in solution. MATERIALS & METHODS Bacterial hyaluronan (average MW 0.66 million) was supplied by Lifecore Biomedical, Inc) Minneapolis, Minnesota. Healon was a rooster combs hyaluronan preparation (MW 3-4 million) supplied by Pharmacia AB, Sweden. Hylan A (MW 5-6 million), a cross-linked hyaluronan, was prepared and supplied by Biomatrix, Inc., Ridgefield, New Jersey. Ion exchange chromatography was carried out on DEAE-Sephacel resin (pharmacia Fine Chemical, Piscataway, New Jersey). The resin was washed extensively with deionized distilled water and packed into a 1.2 x 120 em jacketed column connected to a temperature-controlled circulating water system. Prior to the application of the samples, the column was equilibrated and maintained at the temperature used throughout each run, i.e., 6° C, 25° C or 65° C. Samples of HA and hylan ( 15-20 mg ) in a total volume of 150 ml of water were applied to the column at a rate of 8 - 10 mllhr., using a LKB peristaltic pump. Elution was carried out with linear salt gradients ranging from 0.20 M to 0.55 M NaCI. The flow rate was adjusted to 8 mVhr. and fractions of 1.0 ml were collected. The salt concentration was monitored with a Leica refractometer. The fractions were analyzed for their HA content by using the carbazole assay. Gel permeation chromatography (GPe) was performed with a Waters Alliance System 2690 coupled with a differential refractometer. Two Shodex Ohpak SB806 GPe columns ( 8 mm x 300 mm) were used for the application of standards and samples. The samples were diluted with the elution buffer (0.40 M NaNO) to a concentration of 0.1 0.2 mg/ml, and aliquots of 100 J.LL were injected into the columns at different temperatures. The flow rate was adjusted to 0.4 mllmin., and the duration of the runs was usually 55 - 60 minutes. RESULTS In Figure I, curves A and B illustrate typical chromatographic flow profiles obtained for low MW bacterial hyaluronan chromatographed on a DEAE-Sephacel column at 6° C and 65° C, respectively. It is found that higher concentrations of NaCI are required to elute this material from the column at 65° C than at 6° C. (see Table 1) In another experiment, when bacterial hyaluronan is first chromatographed on DEAE-Sephacel at 65° C, the eluate collected, dialyzed against distilled water and re-chromatographed at 6° C, its elution profile reverts closely to that normally obtained for bacterial HA fractionated at that temperature (see Table 1). In Figure 2, curves A and B, display typical chromatograms obtained for the fractionation of high MW rooster comb hyaluronan on the DEAE-Sephacel column at 6° C and 65° C, respectively. The concentrations of NaCI needed for eluting Healon from the DEAE resin at 65° C are also seen to be significantly higher than those required at 6° C (see Table 1). Similarly, when hylan A, a very high MW HA, is chromatographed on the same column at 6° C and 65° C as shown in Figure 3, curves A & B, the same trend continues. Hylan A is eluted with higher concentrations of NaCI at 65° C than at the lower temperature (see Table 1). Gel permeation chromatography showed that high MW HA experienced significant decreases in its retention time with temperature increasing fro~ 35° C to 6~0 C, suggesting an increase in the molecular volume of the polymers at 65 C, as seen in Figure 4.
Heat dependence '.6 r-----------,
g
8
1.4 1.2
)
.s!.0.6 0.4 0.2
..
10I
'.8 1.4 '.2
8 J::; ,
J- o ,
t'
'1-1 0.8.
8
08
I
0.4 0.2 0
o +-.L.....J-4-..:..,..-...--4--_~
~
200300400500600700600900 EIUlIon Volume (mL)
~
~
§ ~ § § ~ ~
Elution Volume (mL)
Figure 1. Fractionation of Low MW Bacterial HA on DEAF-Sephacel Column. Curve A: at 6° C; Curve B: at 65° C
Figure 3: Fractionation of Hylan A on DEAE-Sephacel. Curve A: at 6° C; Curve B: at 65° C
Table 1: Effect of Temperature on the Elution Profile of Hyaluronan and Hylan A 3.5.,.---HA MWiuMlDlou 0.660 3-4'5-6 0 0 0 0.66=
*
•• •••
NaCi Molarity atS" C 0.39-0.43 0.39-0.45 0.37 0.43
-----
Bacterial HA Rooster comb hyaluronan Cross-linked rooster comb hyaluronan
0.9 0.8 0.7 ~:r 0.6 c.E 0.5 .!! 1ft 0.4 Ii.a. 0.3 !j 0.2
~
8
I
NaCi Molarity at 6°C 0.26-0.28 0.27-0.31 0.29 0.32 0.28-0.31
-r-----------::--, A
0.1
o -I-L....-.....,.....~__.-....._..._.I,_........-I
~ ~
§
~ ~ § § ~ ~ ~ Elution Volume (mL)
Figure 2: Fractionation of High MW Rooster Comb HA on DEAESephacel. Curve A: at 6° C; Curve B: at 65° C
---------,
3
12.5
t ,;
I
1 0.5
o• •: : . - -..........--=.:~~~ 25
30
35
40
RetMItIon Tim. (min)
Figure 4: Gel Permeation Chromatography (GPC) of Hylan A at Different Temperatures
102
Characterisation and solution properties ofhyaluronan
DISCUSSION Hyaluronan molecules require higher concentration of NaCI when eluted from DEAESephacel column at 65° C than eluted at 6° C, which could indicate the occurrence of a conformational change. The conformational change of HA molecules in turn could result in an increase in the binding capacity of HA to the ion-exchange resin with increasing temperature. Although the precise nature of the molecular transformation, which is responsible for the observed increase in the binding affinity of hyaluronan to the resin at 65° C, is unknown, a probable explanation would be that in solution HA forms aggregates that are stabilized primarily by hydrophobic bonds, which dissociate at 65° C. If it is accepted that their dissociation initiates some conformational changes resulting in making additional charges available for binding with DEAE, this possibility can be envisaged. Comparing the elution profiles of different HAs, it was found that at both temperatures, 6° C and 65° C, the low MW bacterial HA tends to be eluted from the column at lower molarities of NaCI than high MW Healon® as it would be anticipated in view of their differing molecular weights and charge densities. Also, it is interesting to note the differences between hyaluronan and hylan A. At 6°C it is noted (see Table 1) that Hylan A is eluted from the resin at higher NaCI concentrations than the low MW bacterial HA and Healon. This would be anticipated in view of the size of the cross-linked hylan A molecule compared with that of HA. By contrast, at 65° C, a different picture emerges. Hylan A is eluted with lower concentrations of NaCI (see Table 1) indicating that the molecules had not fully undergone the same conformational transitions observed with native HA with increasing temperature.
CONCLUSION From the results obtained from the gel permeation chromatographic (GPC) and ion exchange chromatographic experiments, it appears that in solution, hyaluronan and its derivatives undergo definite conformational transitions with a concomitant increase in charge densities with increasing temperature.
REFERENCES 1. J.E. Scott, C. Cummings, A. Brass & Y. Chen, Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by shadowing-electro microscopy and computer simulation, Biochem. J., 1991, 274,699-705. 2. J.E. Scott, Chemical morphology of hyaluronan, In: Chemistry, Biology and Medical Applications of Hyaluronan and 1ts Derivatives, T.C. Laurent (ed.), Venner-Gren International Series, Portland Press, Ltd., London, 1998, 72,3-15. 3. J.E. Scott & F. Heady, Hyaluronan forms specific stable tertiary structures in aqueous solution: A 13C-NMR study, Proc. Natl. Acad. Sci. USA, 1999,96,4850-4855. 4. R.L. Cleland, Effect of temperature on the limiting viscosity number of hyaluronic acid and chondroitin 4- sulfate, Biopolymers, 1979, 18, 1821-1828. 5. E. Fouissac, M. Milas & M. Rinaudo, Shear-rate, concentration, molecular weight, and temperature viscosity dependences of hyaluronate, a wormlike polyelectrolyte, Macromolecules, 1993, 26,6945-6951.
TEMPERATURE EFFECT ON THE DYNAMIC RHEOLOGICAL CHARACTERISTICS OF HYALURONAN, HYLAN A AND SYNVISC~
Joanne M. Hoefting 1*, Mary K. Cowman JBiomatrix,
2,3,
Shiro Matsuoka3 and Endre Bala7S1
Inc. and Matrix Biology lnstituie, 65 Railroad Ave. Ridgefield. New Jersey 07657 USA.
2Department of Chemistry and Chemical Engineering and J Herman F. Mark Polymer Research IflStirule Polytechnic University. Six Metrotech Center, Brooklyn. New York 11201 USA
ABSTRACT
This study examines the effect of temperatures in the range 25-65 "C on the dynamic rheol~cal behavior of aqueous salt solutions containing hyaluronan, hylan A or Synvisc . Increasing temperature significantly reduced the modulus and complex viscosity for all three samples. This change is qualitatively understood to result from the increasing population of higher energy conformations as the temperature is raised, resulting in a decrease in the persistence length of the polymer chain. The data were found to be compatible with the viscosities predicted using a general relation proposed by Matsuoka and Cowman for polymer solutions. KEYWORDS
Hyaluronan, viscosity, elasticity, hydrodynamics INTRODUCTION
The rheological properties of hyaluronan solutions are known to be sensitive to changes in temperature". In this report, we examine the extent to which the rheological changes are predictable on the basis of an increased flexibility and smaller hydrodynamic volume of hyaluronan as temperature increases. We also examine whether hylan A solution (MW 6 million) and Synvisc·, a mixture of hylan A and hylan B, show the same dependence of rheology on temperature. MATERIALS AND METHOD
The materials used were various average MW hylan A solutions (l % polymer content), Synvisc'", an 80:20 mixture of hylan A solution (l % polymer content, average MW 6 million) and hylan B gel slurry (0.5% polymer content), and Healon· GV (average MW 5 million, 1.4% polymer content). All samples were in 0.15N NaC!. The study was done using a Bohlin couette rheometer, Model VOR, a C14 concentric cylinder measuring system in dynamic oscillation mode. The T1.p values reported are the complex viscosities at 0.01 Hz. G' values are reported at 5 Hz.
104
Characterisation and solution properties ofhyaluronan
RESULTS AND DISCUSSION The hydrodynamic volumes of hyaluronan chains decrease as temperature is increased, because higher energy linkage conformations become more accessible. This reduces the rigidity of the polysaccharide backbone. The intrinsic viscosity [ll] measures the specific hydrodynamic volume of hyaluronan in solution. Data from Cleland' and Fouissac, Milas, and Rinaud04 show that the intrinsic viscosity of high MW hyaluronan is decreased by about 25% as the temperature is increased from 25° to 65°C.
The temperature effect on solution viscosity at low shear. The zero shear specific viscosity of a polymer solution depends on the weight concentration, c, and the intrinsic viscosity, Inl. of the polymer. The product, c [ll], is called the coil overlap parameter, because it reflects the degree to which the polymer chains can interact hydrodynamically with each other. We have found that the specific viscosity of hyaluronan solutions at 25°C may be expressed in terms of the coil overlap parameter, using the equation below with k'=O.4.
(k,)3 (k,)2 1]sp =c[1]]+ k'(c[1]])2 +_(C[1]])3 +_(c[1]])4 2!
3!
Equation 1.
This is in agreement with the published data by Berriaud, etal', See Figure 1.
1000ooo
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Figure 1.
Comparison of the four-term interaction equation proposed by Matsuoka and Cowman" with experimental data for hyaluronan 7 published by Berriaud, Milas and Rinaud0 •
Temperature effect on rheological characteristics
105
Because the intrinsic viscosity decreases at higher temperatures, the specific viscosity of the solution should also decrease, according to equation 1. Data calculated using equation 1 and our experimental results are compared in Table 1 and illustrated in Figure 2. Table 1.
Sample
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Temperature Effect on the Specific Viscosity: Prediction vs. Experiment.
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ca. 6000 ca. 3100
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1.9
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ca.356 ca. 200
1.9
1.8
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ca. 13 ca. 9
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25
77
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58
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55
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3.6
3.1 ·Cannot be calculated because MW cannot be determined.
106
Characterisation and solution properties ofhyaluronan
The temperature effecton solution elasticity at high shear At high rates of deformation, the elastic modulus measured for a polymer solution is close to the plateau value. At the plateau, the elastic modulus is dependent on polymer concentration to the second power, but is not dependent on polymer molecular weight or intrinsic viscosity. At somewhat lower frequencies of deformation, there is weak dependence of the elastic modulus on molecular weight and/or intrinsic viscosity. Thus, we expect the following equation to apply: G'(5 Hz)
(X
X
C
[T1]Y
Where x is close to 2, and y is close to O. We therefore predict that G' at 5 Hz will be only slightly lower at 65°C than at 25°C. Results are found in Table 2 and illustrated in Figure 2.
Table 2.
Temperature Effect on Elasticity.
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Complex Viscosity
Dynamic Elastic Modulus (G')
(n*)
....+:---~--_----l
,+----...----..--~ ..
&1
107
,
Frequency (Hertz)
Frequency (Hertz)
1.4 % Hyaluronan (MW 4.8 million)
~4.. --_---~_-l
....+---_..--_~, - - - I Frequency (Hertz)
Frequency (Hertz)
1% HyianA (MW 6 million)
~t-.---------l
Frequency (Hertz)
Frequency (Hertz)
Synvisc@ Figure 2.
Experimental dependence of complex viscosity, 11* and dynamic elasticity, G', on frequency (Hz) at two different temperatures, 25 DC and 65 DC for 1.4% hyaluronan, 1% hylan A and Synvisc~.
108
Characterisation and solution properties ofhyaluronan
CONCLUSIONS The magnitude of the change in the low shear viscosity of hyaluronan solutions as temperature is increased from 25°C to 55 °c to 65°C is correctly predicted by applyinli a 4-term interaction equation relating 11sp to c [11J. Hyaluronan, hylan A and Synvisc all have similar temperature dependence of the low shear viscosity. The elastic modulus at high shear rate is only weakly dependent on temperature because it is insensitive to changes in the hydrodynamic volume of the chains.
REFERENCES 1.
2. 3. 4.
5.
6. 7.
D. A. Gibbs, E.W. Merrill, K.A Smith & E.A. Balazs, Rheology of hyaluronic acid, Biopolymers, 1968,6,777-791. R.L Cleland, Effect of temperature on the limiting viscosity number of hyaluronic acid and chondroitin 4 sulfate, Biopolymers, 1979, 18, 1821-1828. E.R Morris, D.A Rees & EJ. Welsh, Conformation and dynamic interactions in hyaluronate solutions, J. Mol. Bio., 1980, 138,383-400. E. Fouissac, M. Milas & M. Rinaudo, Shear-rate, concentration, molecular weight, and temperature viscosity dependence of hyaluronate, a worm-like polyelectrolyte, Macromolecules, 1993, 26. 6945-6951. M. Milas,. I. Roure & G. C. Berry, Cross-over behavior in the viscosity of semi-flexible polymers: solutions of sodium hyaluronate as a function of concentration, molecular weight, and temperature, J. Rheol., 1996, 40, 11551166. S. Matsuoka & M. K. Cowman, Viscosity of polymer solutions revisited, In: Hyaluronan 2000, Woodhead Publishing, Cambridge, publication in progress. N. Berriaud, M. Milas, and M. Rinaudo, Rheological study on mixtures of different molecular weight hyaluronates, Int. J. Biol. Macromol., 1994, 16, 137-142.
A NOVEL HYALURONAN BIOMATERIAL (HYAFF®-ll) AS SCAFFOLD FOR CHONDROCYTES AND BONE MARROW STROMAL CELLS ".I , M . F"IOrlm, ".I L • Scorzona, .I B . G" 1 G • L"" . .1 .., A . P'iacentmi rlgo I0, ISlgno1.1 I, A . F acchini 2 2 3 3 P. Gobbi , G. Mazzotti , M. Duca , A. Pavesio J
Laboratorio di Immunologia e Genetica, Istituto di Rieerea Codivilla Putti, Istituti Ortopedici Rizzoli, Via di Barbiano l/LO, 40136 Bologna,ltaly. 2 Unita
3
Complessa di Scienze Anatomiehe Umane e Fisiopatologia dell 'Apparato Locomotore, Via Irnerio 48. 40126 Bologna, Italy.
Fidia Advanced Biopolymers. Via Ponte della Fabbriea 3/11, 35031 Abano Terme (padova), Italy.
ABSTRACT Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage and bone repair. Such scaffolds should provide a preformed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells, and stimulate the phenotype of transplanted cells. Hyaffll> -II is a recently developed hyaluronic-acid based biodegradable polymer, that has been shown to provide succesful cell scaffolds for tissue-engineered repair. The aim of this study was to evaluate in vitro the potential of Hyaftl>-11: a) to maintain a chondrocyte phenotype when seeded with human chondrocytes; b) to induce osteoblast differentiation when seeded with rat bone marrow stromal cells (BMSC) in a mineralizing medium. Our data indicate that human chondrocytes seeded on Hyaftl>-11 express and produce collagen type 11 and downregulate the production of collagen type I. BMSC seeded on Hyaffll>-11 differentiate into cells positive for bone-specific markers. These results provide an in vitro demostration of therapeutic potential of Hyaffll>-II as a delivery vehicle in tissueengineered repair of articular cartilage and bone defects.
KEYWORDS Chondrocytes, bone engineering.
marrow stromal cells, hyaluronic acid derivative, tissue
INTRODUCTION Joint pain is a major cause of disability in middle-aged and older people. Pain usually results from degeneration of the joint's cartilage due to primary osteoarthritis or from trauma causing loss of cartilage'. Different techniques have been used to facilitate repair of articular cartilage such as debridemenr', drilling of subchondral bone'' and periosteal and perichondrial transplantation':'. On the other hand, bone injuries often require implantation of grafts, and although autogenous bone is the elective graft material, it is limited in supply and necessitates traumatic harvesting procedures". Allogenous bone is
72
Application ofhyaluronan in tissue engineering
inherently limited by risks of rejection and/or disease transmission, whereas permanent synthetic grafts are limited by osteolysis and inflammatory reactions to wear debris/". Recent advances in biology and materials have pushed tissue engineering to the forefront of new treatments also for cartilage and bone repai?·IO. This technique combines isolated cells with scaffold/cell carriers in order to promote cartilage or bone formation and repair. A vast amount of research has been focused on the design of new scaffolds to improve the success of such therapeutical strategy. The ideal matrix should prevent cells from floating out ofthe defect, provide mechanical strength, make uniform celI spreading possible and stimulate in this case, the chondrogenic and osteogenic phenotypes ofthe transplanted celIs. Furthermore, it should be viscous enough to alIow three-dimensional trapping of celIs and adhesive enough to secure their fixation to the implantation site. Fibrin, polymers and polyglycolic and polylactic acids, alginate and collagen gels, are some examples of three-dimensional scaffolds used for cartilage repair ll - I S. On the other hand, poly-lactic acid, poly-glycolic acid, poly-propylene fumarate, and apatite are some examples of three-dimensional scaffolds which have been used for bone tissue engineering 16- 18 . Aim of the study was to evaluate in vitro the potential of Hyaff'll-ll, a recently developed hyaluronic acid-based biodegradable polymer, to maintain chondrocyte phenotype and facilitate mineralization of bone marrow stromal cells. MATERIALS & METHODS
Test material The scaffold used in this study was made of HyaffO-11 , a polymer derived from the total esterification of sodium hyaluronate (80-200 kDa) with the benzyl alcohol on the free carboxyl groups of glucoronic acid along the polymeric chain. The configuration used was a non-woven mesh that is a pad composed ofa random array of polymer fibers having a diameter of 40 urn and kindly provided by FAB. S.r.I. (FIDIA Advanced Biopolymers, Abano Terme, Italy).
Human chondrocytes isolation and seeding on the biomaterial Human articular cartilage specimens were obtained from the knees of3 patients aged 13, 18, 32 years undergoing joint replacement surgery. Fragments of the excised cartilaginous tissues were put in Dulbecco's Modified Eagle's Medium (DMEM) (GlBCO BRL, Grand Island, NY, USA) and chondrocytes were isolated by sequential enzymatic digestions: 30 min with 0.1 % hyaluronidase (Sigma, St. Louis, MO, USA», I hour with 0.5% pronase (Sigma) and 1 hour with 0.2% collagenase (Sigma) at 37°C in DMEM with 25 mM HEPES (Sigma), 100 units/ml penicillin (Biological Industries, Kibbutz, Israel), 100 ug/rnl streptomycin (Biological Industries), 50 ug/ml gentamicin (Flow Laboratoires, Biaggio, Switzerland), 2.5 ug/ml amphotericin B (Biological Industries). The isolated chondrocytes were filtered by 100 urn and 70 urn nylon meshes, washed, and centrifuged. The cell number and viability were assessed by the Tripan Blue dye exclusion method and the celIs were cultured under conventional monolayer culture conditions. Once sufficient celIs were available, usualIy after the 3'd th to 4 passage, chondrocytes were seeded onto 2x2 em HyaffB'-11 non-woven meshes at a density of Ixl0 6 celIs/cm 2 in 35 mm Petri dishes. Four different chondrocyte preparations were used and cultures were harvested at 1 h, 24 hrs, 7 days, and 14 days.
A novel hyaluronan biomaterial
73
For each experimental time point one sample was snap-frozen for immunohistochemical analysis and another one was processed for electron microscopy. Electron microscopy
Sterilized silicon wafer chips of 3 x 5 mm utilized as specimen holder were coiled up with a thin layer of sterile Hyaffll'-11 and on each device 2 x lOScells were deposed. The samples were then cultivated for 1 day in DMEM medium at 37° C and 5% C02. At the end of the growth period, the specimens were fixed with 1% glutaraldehyde in O. I M phosphate buffer pH 7.2 for 45 min., post-fixed in 1% osmium tetroxide in Veronal buffer for 30 min, dehydrated in an increasing ethanol series and critical point dried (Critical point dryer CPD 030, Bal-Tee AG, Lichtenstein). Before the electron microscopy analysis, all the samples were coated with a 1.5 nm thick Platinum - Carbon film (pt 80%; C 20%) by means of a multievaporation device Balzers MED 010 (BalTee). The observations were performed with a Field Emission In lens Scanning Electron Microscope (FEISEM) Jeol JSM 890 (Jeol LTD., Tokyo, Japan) at 7 kV accelerating voltage and 1 x 10-I 1 A probe current. Histochemistry and immunohistochemistry
Snap-frozen biomaterial scaffolds were sliced into 5 urn sections and stained with Aldan blue and Safranin-O. For immunohistochemistry, sections were predigested with O. I % of hyaluronidase (Sigma) and incubated with monoclonal antibody anti-collagen type II (Chemicon, Temecula, CA) for 1 hour at room temperature. Slides were then rinsed and incubated for 30 minutes at room temperature with secondary goat antimouse/rabbit antibody (Dako, Glostrup, Denmark), rinsed again and treated with newfucsin substrate detection Kit (Dako). Finally, samples were counterstained with hematoxylin. Negative control sections were obtained by omitting the primary antibody. Analysis ofmRNA expression by RT-PCR
Scaffold cultured cells were analyzed by semiquantitative RT-PCR in order to investigate temporal changes in collagen type I and II mRNA expression. mRNA was extracted by RNAzol B reagent (Biotecx Laboratories, Huston, TX) and reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Perkin Elmer, Norwalk, CT) and oligo dT priming. A1iquots of cDNA were then amplified with collagen I, II and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers in a Gene Amp PCR System 9600 thermocycler (Perkin Elmer) in conditions allowing a linear amplification range. Amplified products were electrophoresed on 2% agarose gel stained with ethidium bromide and compared with a 123 bp DNA ladder (Life Technologies Ltd, Paisley, UK) to confirm the predicted size. Relative levels of PCR products were quantified by densitometric analysis of gel photographs and normalized to the signal from the housekeeping gene GAPDH. Rat BMSC cultures and seeding on biomaterial
Bone marrow aspirates from inbred Fisher 344 rats femur (Charles River Laboratories, Wilmington, MA) were diluted and layered over Ficoll-Hypaque density gradient and centrifuged at 1300xg for 20 min. The nucleated cells were collected, washed twice and resuspended in a-J\1EM containing 15% heat inactivated FCS,
74
Application ofhyaluronan in tissue engineering
freshly-prepared ascorbic acid (50/lg/ml) and antibiotics (lOO/lg/m1 penicillin G, 50/lg/ml gentamicin sulphate and O.3/lg/m1 Fungizone) (standard medium). They were then seeded on petri dishes at a concentration of 3xI04/cm2 . The media were changed twice a week and BMSC were allowed to grow until confluent. Cells were then trypsinized, tested for viability by eosin exclusion dye and finally seeded on non-woven Hyaftl' -11 mesh (2 x 5 x 2 mm ) at the density of 2x106 cells/em' in 200/l1 of standard medium supplemented with IOmM f3-GP (f3-glicerophosphate) and 1O·8M Dex (Dexametasone) (hereafter referred to as Dex + f3-GP medium) in 6-well petri dishes. After 5, 10, 20 and 40 days scaffold BMSC cultures and supernatants were collected and analysed for cell growth by MTT mitochondrial reduction test and differentiation by light and electron microscopy as above reported.
RESULTS & DISCUSSION Histochemistry and immunohistochemistry Chondrocytes grown on HyafFl9-ll appeared round in shape and most chondrocytes seem to adhere to the fibers as confirmed by FEISEM evaluation. A1cian blue and Safranin-O stain revealed the production of metachromatic extracellular matrix particularly at 14 days. Immunohistochemical analysis for collagen type II showed the re-expression of this molecule through the different incubation time evaluated, suggesting that chondrocytes grown on the biomaterial underwent to a progressive redifferentiation process.
Figure 1. Chondrocytes after the seeding (24 h) on Hyaff'-ll at FEISEM analysis. The cell adhesion on the bio-thread is evident. In correspondence to the nucleus, the cell shape is remarkably raised on the thread surface and the cell membrane is characterized by close and fine ondulopodia. On the contrary, other membrane areas are flattened on the thread and smooth. Not frequently, large ondulopodia are detectable at the border of the cell and the thread.
A novel hyaluronan biomaterial
75
mRNA expression of collagen type I and II RT-PCR was performed on scaffold cultured samples in order to follow up condrocyte re-differentiation at the mRNA level. Samples analyzed in the linear phase of amplification and visualized on agarose gel showed an evident signal modulation at the different time points. Collagen I mRNA expression decreased after 24 hours and was significantly down-regulated on days 7 and 14 (p
:c c 140 0« C)
120
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QI
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UI
)(
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e
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Figure 2. mRNA expression of collagen type I in chondrocytes grown on HyafP!9-11 scaffolds at different experimental times. (Data obtained from samples from a representative patient). Collagen II 360 ~
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Figure 3. mRNA expression of collagen type II in chondrocytes grown on Hyaffll'-ll scaffolds at different experimental times, (Data obtained from samples from a representative patient).
76
Application ofhyaluronan in tissue engineering
Proliferation and differentiation of rat bone marrow stromal cells The MTT test, which was used to evaluate cell growth, indicates that cell proliferation peaked as early as day 10 (Fig.4). On day 40, mineralised areas of Dex + f3-GP medium cell cultures were strongly positive to von Kassa staining. Alkaline phosphatase showed a stronger staining only on the outer layer of the cells. Electron microscopy analysis showed that starting from day 20 the extracellularmatrix presented mineralised areas localised around the cells and also between the cells and the Hyatf'-ll fibres. These cells showed -osteoblastic features: a large ovoid nucleus and extensive granular endoplasmic reticulum. Large mineralised areas were observable on day 40 between osteoblasts and Hyatf'-II fibres (Fig. 5). Lacunae containing cells with typical osteocytic morphology could also be seen. Cell proliferation (MTT test)
>.
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o
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Figure 4. Cell proliferation of rat BMSC cultured on Hyatf'-II in Dex + f3-GP medium for 5, 10, 20 and 40 days analysed by MTT test.
Figure 5. Electron microscopy of rat BMSC cultured on Hyatf'-11 for 40 days. H = Hyatf'-11 fiber, OB = osteoblast, OC = osteoclast. Magnification x6000.
A novel hyaluronan biomaterial
77
CONCLUSIONS In this study we showed that Hya~-11 scaffolds are suitable delivery vehicles for chondrocytes and bone marrow stromal cells. These cells are able to re-express their differentiated phenotype as in the case of chondrocytes or to differentiate in osteoblasts expressing osteogenic markers once they are grown in this three dimensional configuration. In fact, phenotypic instability of chondrocytes when they are removed from their cartilage matrix has been consistently observed in different animal species I9 ' 21. It has been shown that when chondrocytes are grown in monolayer cultures, they proliferate but they cease to express the specialised ~roteins of cartilage, like collagen type II, and become fibroblastic in appearancev" 3. This situation can be reversed using suspension cultures which promote the re-expression of cartilage phenotype24,2s . When rat BMST were cultured on Hya~-11 light and electron microscopy clearly demonstrated that after at least 20 days all the stages characteristic of the mineralization processes occurred. These in vitro data demonstrate that the association of osteoprogenitor cells with Hya~-11 can provide an appropriate carrier vehicle for repairing small bone losses such as nonunions and cavitational defects that can readily be filled.
ACKNOWLEDGEMENTS This work was supported by grants from IRCCS "Istituti Ortopedici Rizzoli", Bologna, C.N.R. Progetto Finalizzato "Materiali speciali per tecnlogie avanzate II" and Progetto Finalizzato Ministero della Sanita.
REFERENCES 1. lA. Buckwalter, J. Martin & H.l Mankin, 'Synovial joint degeneration on the syndrome of osteoarthritis', Instr. Course Lect., 2000,49,481-489. 2. M.R. Baumgaertner, W.D. Cannon, lM. Vittori, E.S. Schmidt & R.C. Maurer, 'Arthroscopic debridement of the arthritic knee', Clin. Orthop., 1990,253,197-202. 3. T. Furukawa, n.R Eyre, S. Koide & MJ. Glimcher, 'Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee', J Bone Joint Surg. (Am), 1980,62, A79-89. 4. S.W. O'Driscoll, F.W. Keeley & RB. Salter, The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-tickness defects in joint surfaces under the influence of continuous passive motion: An experimental investigation in the rabbit', J. Bone Joint Surg. (Am), 1986,68, AIOI7-1035. 5. G.N. Hornminga, S.K. Bulstra, P.S.M. Bouwrneester, & A.l van der Linden, 'Perichondral grafting for cartilage lesions of the knee', J. Bone Joint Surg. (Br), 1990,72, BI003-1007. 6. R.C. Schultz. In: Bone defects and autogenous reconstruction, RC. Schulz (ed.), 1988, pp 564-601. 7. WM. Tomford, S.H. Doppelt, H,l Mankin & G.E. Friedlander, 'Bone bank procedures', Clin. Orthop. ReI. Res., 1983,174,15-21. 8. P.A. DeLuca, RW. Lindsey & P.A. Ruwe, 'Refracture of bones of the forearm after removal of compression plates', J. Bone Joint Surg. (Am), 1988, 70, 1372-1376. 9. C.A. Vacanti & lP. Vacanti, 'The science of tissue engineering', Orthop. Clin. North. Am., 2000, 3], 351-356.
78
Application or hyaluronan in tissue engineering
10. B.D. Boyan, C.H. Lohmann, 1. Romero & Z. Schwartz, 'Bone and cartilage tissue engineering', Clin. Plast. Surg., 1999,26,629-645. II. D.A Hendrickson, A1. Nixon, D.A. Grande, R.I. Todhunter, R.M. Minor, H. Erb & G. Lust, 'Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects', 1. Bone Joint Surg., 1994, 12,484-497. 12. L.E. Freed, 1.C. Marquis, A Nohria, J. Emmanual, AG. Mikos & R. Langer, 'Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers', 1. Biomed. Mat. Res., 1993,27, 11-23. 13. 1.L.c. Van Susante, P. Burna, G.I.V.M. van Osch, D. Versleyen, P.M. van der Kraan, W.B. van der Berg & G.N. Homminga, 'Culture of chondrocytes in alginate and collagen carriers gel', Acta Orthop. Scan., 1995,66,549-556. 14. I Bonaventure, N. Kadhom, L. Cohen-Solal, KH. Ng, 1. Bourguignon, V. Lasselin & P. Freisinger, 'Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads', Exp Cell Res, 1994, 212, 97-104. 15. IS. Stanton, V. Salih, G. Bentley & S. Downes, 'The growth ofchondrocytes using Gelfoam as a biodegradable scaffold', 1. Mater. Sci. Mater M, 1995, 6, 739-744. 16. K Whang, C.H. Thomas & KE. Healy, 'Fabrication of porous biodegradable scaffolds', Polymer, 1995,36,837-845. 17. S.L. Ishaug, G.M. Crane, M.I. Miller, AW. Yasko, & M.I. Mikos, 'Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds', J Biomed. Mat. Res, 1997, 36, 17-28. 18. S.L. Ishaug-Riley, G.M. Crane-Kruger, M.I. Yaszemski & AG. Mikos, 'Threedimensional culture of rat calvarian osteoblasts in porous biodegradable polymer scaffolds', Biomaterials, 1998, 19, 1405-1412. 19. K Von der Mark, V. Gauss, H. Von der Mark & P.K MOller, 'Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture', Nature, 1977,267,531-532. 20. R. Mayne, M.S. Vail, P.M. Mayne & E.J. Miller, 'Changes in type of collagen synthesized as clones of chick chondrocytes grow and eventually lose division capacity', Proc.Natl. Acad Sci., 1976,73, 1674-1678. 21. P.O. Benya, S.R. Padilla & 1.0. Shaffer, 'Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture', Cell, 1978, 15,1313-1321. 22. C. Archer, J. McDowell, M. Bayliss, M. Stephens & G. Bently, 'Phenotipic modulation of sub-populations of human articular chondrocyets in vitro', 1. Cell Sci., 1990,97,361-371, 23. AL. Aulthouse, M. Beck, E. Griffey, 1. Sanford, K Arden, M.A. Machado & W.A. Horton, 'Expression of the human chondrocyte phenotype in vitro', In Vitro Cell Dev. Bioi., 1989, 25, 659-668. 24. P.O. Benya & 1.0. Shaffer, 'Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels', Cell, 1982, 30, 215-224. 25. H.I. Hauselmann, R.I. Fernandes, S.S. Mok, T.M. Schmid, IA Block, M.B. Aydelotte, K.E. Kuettner & E.1. Thonar, 'Phenotipic stability of bovine articular chondrocytes after long-term culture in alginate beads', 1. Cell Sci., 1994, 107, 1727.
TAPPING MODE ATOMIC FORCE MICROSCOPY OF HYALURONAN AND RYLAN A Mary K. Cowman*'1,2, Min Lit, Ansil Dyal', and Sonoko Kanai' /Department ofChemistry and Chemical Engineering and]Herman F. Mark Polymer Research Institute Polytechnic University, Six Metrotech Center, Brooklyn, NY 11201 USA
ABSTRACT Hyaluronan and hylan A have been examined by tapping mode atomic force microscopy. Isolated polymeric chains were deposited on mica from dilute aqueous solution and imaged in air. Long extended chains could be observed when sample preparation favored attachment to the surface, and the chains were uncoiled by the force of a moving air-water interface. The chain dimensions were compatible with the known covalent structure, but with an unlikely extended conformation. When chains were less fully extended by the moving interface, we observed more condensed molecular conformations. Images corresponding to chain conformations typical of hyaluronan in dilute solution were obtained by altering the hydration of the polymer on the mica surface and reducing adhesion of the hyaluronan chains. The chains adopted more relaxed structures, with substantial twisting and coiling, and a possible helical bias in the coiling patterns. These images were consistent in curvature and overall dimensions with instantaneous conformations adopted by hyaluronan molecules in dilute solution. KEYWORDS Hyaluronan, polysaccharide, atomic force microscopy INTRODUCTION On the basis of hydrodynamic, spectroscopic, and theoretical investigations, the structure of high molecular weight hyaluronan in physiological salt solution may be characterized as typical of a semi-flexible coil polymer. The chain is stiffened by dynamically formed and broken hydrogen bonds, but segmental motions on the nanosecond time scale result in a continually changing overall chain conformation. Selfassociation has been shown to exist for short, relatively extended, chain segments in solution, but its contribution to the properties of high molecular weight hyaluronan is not obvious in any of its hydrodynamic properties. Electron microscopic I and atomic force rnicroscopicv' studies have indicated a strong tendency for high molecular weight hyaluronan to form networks of chains, when deposited and dried on a mica surface. Recently, we4-6 have undertaken studies of hyaluronan and hylan A (a water-soluble hyaluronan derivative with enhanced molecular weight attributed to protein-mediated crosslinks) by tapping mode atomic force microscopy (TMAFM). TMAFM allows hydrated macromolecules to be imaged on a mica surface. We have observed isolated chains in extended and intramolecularly associated forms, as well as intermolecularly associated networks. In the present work, these TMAFM studies were extended to
110
Characterisation and solution properties ofhyaluronan
conditions under which isolated hyaluronan chains adopt relaxed coil conformations, which appear to be consistent with the solution properties. MATERIALS AND METHODS Rooster comb hyaluronan (HA) was obtained from Pharmacia and Upjohn. Hylan A was obtained from Biomatrix. All sample preparation procedures were as previously describedi". Briefly, a few microliters ofa dilute (1-5 ug/ml) HA or hylan A solution containing 10 mM MgCh was deposited on a mica surface and allowed to interact for a short time «I min). The surface was rinsed with water, then briefly dried with a stream of nitrogen. This leaves a thin layer of water on the surface. The surface was imaged by TMAFM, using a Digital Instruments Multimode AFM. with a type EV scanner, and Nanoscope IlIa controller. Details of the procedures have been presented previously'r'. RESULTS AND DISCUSSION Figure I shows a hylan A chain imaged by TMAFM. This image is typical of HA or hylan A molecules which have been subjected to molecular combing. In molecular combing, the molecule initially becomes attached to the mica surface over only a short portion of its length. The remaining portion of the molecule remains in the aqueous phase. As the surface is rinsed and blown dry, the liquid droplets are forced to recede. The HA chain(s) in the droplet are unwound like a ball of string, becoming fully stretched in the process. If the chain becomes attached to the mica surface during this procedure, it remains in the extended conformation. If the chain is incompletely unwound before the droplet dries, a lacy residue is observed on the surface, with extended HA chains leading from the residue. The extended HA or hylan A molecules we have observed are as long as 12 urn. This corresponds to a molecular weight of
Figure 1.
TMAFM image ofhylan A extended by molecular combing. Height image, 1.5 urn x 1.5 urn, with gray scale covering 2 nm.
Tapping mode atomic force microscopy
III
Figure 2. High resolution TMAFM image of hyaluronan extended by molecular combing. Height image, 500 nm x 500 nm, with gray scale covering 2 nm.
about 5-6 million for a fully stretched chain, in which the disaccharide length is about 0.8-1 nm. The chain diameter is determined from the height of the chain in the 3dimensional AFM image, and is best measured in a high resolution scan with at least about 0.5 points/nm, such as Figure 2. The average height measured is 0.5-0.6 nm, for both HA and hylan A. This is in good agreement with the expected diameter of a single chain of HA. Thus the extended chain images represent stretched single chains of HA. The conformation of such a chain is expected to be similar to those found by X-ray fiber diffraction studies, and can be well visualized as a tightly pulled telephone cord. In agreement with the work of other laboratories, we often see HA or hylan A chains with some degree of self-association present. The association can be intramolecular, for which the simplest form is a hairpin turn. The height of the hairpin stem is twice that of the single chain, suggesting the chain segments associate as a double helix, presumably similar to that seen by X-ray fiber diffraction. The hairpin turn can also be a mode of topological strain relief, arising when chains become twisted and then attached to the surface, thus limiting the ability to untwist. More extensive intramolecular or intermolecular association results in network-like structures. Figure 3 shows a portion of a large molecularly-combed aggregate containing five nearly parallel strands of hylan A. The aggregate itself was pre-existing prior to AFM studies, either as a result of chain entanglement and knotting, or by virtue of covalent crosslinks in the hylan A sample. It was isolated by filtering the sample. In the aggregate, the strands show intermittent association into double stranded segments. It is impossible to know whether the double-stranded regions of association were preexisting in solution, or created on the surface. The AFM studies should be viewed as a way to image different possible structural forms of a macromolecule. Thus we find that HA has a tendency to associate, which may be relevant in very concentrated solutions or molecularly crowded physiological environments. The viscoelastic properties of HA solutions at concentrations up to 8% can be well modeled without association't".
112
Characterisation and solution properties ofhyaluronan
Figure 3. TMAFM image of entangled and/or crosslinked hylan A molecules with extensive self-association. Height image, 1 urn x 1 J.Lm, with gray scale covering 2 nm. In our early studies, we occasionally observed chains that appeared to lie on a rugged background, rather than a smooth surface. These chains were usually less extended than the molecularly combed chains described above. An example of this is the image in Figure 4. The HA chain has not been strongly elongated in a specific direction, and shows evidence of one hairpin tum. The background has an appearance which is like that seen when ice forms on a cooled mica surface in a low humidity environment", We also noted that there sometimes appear to be very low height condensed chain structures in images such as Figure 3, which contain clear images of extended HA or hylan chains.
Figure 4.
TMAFM image of incompletely extended hyaluronan on mica surface with corrugated appearance. Height image, 700 nm x 700 nm, with gray scale covering 1.5 nm.
Tapping mode atomic force microscopy
113
In interpreting these results, we reconsider the location of the extended HA chains with respect to the mica surface and the thin (about 1-2 ron) layer of water on its surface. It now seems likely that the extended chains may lie on the surface of an ordered, partially ice-like, water layer, rather than directly on the mica surface. The chains which appear to be low in height may lie below the water surface, or partially within the water layer. Those chains which appear to be lying on a rugged background are presumably imaged in the midst of the partially ordered water layer, with its islands of ice-like structure. An observation which supports this interpretation is the experimental technique which favors one type of image over the other. Predominantly extended chains are seen on a smooth background when the mica is cleaved and allowed to equilibrate with ambient humidity for a time period longer than perhaps an hour, before sample application. This may favor formation of a uniform layer of water on the surface, on which the HA chains can be subsequently layered. In contrast, mica which is cleaved and used immediately for sample application gives images of relaxed coiled HA or hylan A chains, frequently on a background with corrugated appearance. These chains are more difficult to image with clear contrast between the chain and the background, because the tapping motion of the tip is strongly affected by the structure within the water layer, and/or because the HA or hylan chains do not lie on an atomically flat surface, but on and around islands of ice-like water. We have been able to selectively image these chains using hard tapping to ensure penetration of the tip into the water layer, and contrast enhancement of the images, to allow the chains to be more clearly seen without complication from the rugged landscape on which they lie. As an example of this approach, Figure 5 shows hylan A chains which have been stretched by molecular combing but show evidence of relaxation into partially coiled conformations. The fuzzy appearance of the picture reflects the difficulty of imaging the chains within the water layer.
Figure 5.
TMAFM image of partially extended hylan A chains showing loose coiling within the water layer on the mica surface. Contrast-enhanced height image, 2 urn x 2 um, with gray scale covering I ron.
114
Characterisation and solution properties of hyaluronan
Figure 6. TMAFM image of isolated hylan A chain in relaxed coil form. Contrastenhanced height image, 1.6 urn x 1.6 J.U11, with gray scale covering 1 nm.
When the HA or hylan A molecules have been more poorly extended by molecular combing and/or have relaxed to a greater extent because of reduced adhesion to the surface when fully immersed in the water layer, the molecular conformation is that of a randomly coiled polymer chain. Figures 6 and 7 show hylan A molecules in the relaxed forms. The molecules show evidence of loose coiling, with a suggestion of a weak helical bias.
Figure 7. TMAFM image of a field of relaxed hylan A molecules, showing loosely coiled conformations. Contrast-enhanced height image, 4 urn x 4 J.Lm, with gray scale covering 1.9 nm.
Tapping mode atomic force microscopy
115
The relaxed coil forms of HA and hylan A imaged by TMAFM, when the molecules appear to be fully immersed in the thin water layer on the mica surface, can be viewed as relatively realistic representations of the solution conformation, Based on our analysis' of its hydrodynamic properties, HA or hylan A with a molecular weight of about 6 million would have an rms end-to-end distance (approximately equal to the diameter of the hydrodynamically equivalent sphere) of about 600 nm. The field of hylan A chains shown in Figure 7 is compatible with this dimension. It is useful to note that none of the molecular domains is spherical in appearance, with a Gaussian distribution of segment density, so the hydrodynamic sphere model should not be construed as an accurate representation of individual chain conformations at any given instant in time. Rather, it is the time-average of the conformation for a given chain, or the ensemble-average for all chains at a given instant in time. In fact, if we would take the images for a large number of the relaxed molecules and overlay them so that we achieve maximum overlap of the chains, the density of the resulting composite would be greatest at the center and decreasing with distance, which is exactly the statistical model of a randomly coiled chain. Thus we see that HA chains in solution can have instantaneous conformations which are far from spherical, and which generally can be visualized as the relaxed form of a telephone cord model. The tendency for slow coiling is simply a reflection of the inherent twist in the cord, just as HA has a preferred left-handed helical bias. In HA solutions of increasing concentration, the domains of HA molecules overlap and interfere with each other, contributing additional density to their respective domains. The viscosity of polymer (including HA) solutions can be accurately predicted'' using a model for this density increase, without any consideration of specific intermolecular association. This brings into question the common observation by EM or AFM of networks of interacting HA molecules. We have found that, ifHA solutions are allowed time for molecular disentanglement following dilution from high concentration to low concentration, the chains imaged are predominantly separate and independent, as seen in Figure 8. Here the sample was hylan A at a concentration of
Figure 8. TMAFM image of hylan A deposited on mica at 500 ug/ml in physiological NaCI solution. Height image, 3 urn x 31lm, with gray scale covering 3 nm.
116
Characterisation and solution properties of hyaluronan
500 ug/ml, in physiological salt solution. The hylan A chains appear to be somewhat condensed coil forms, whose wormlike conformations accommodate to each other. This image is compatible with the hydrodynamic properties of semi-dilute HA or hylan A solutions.
ACKNOWLEDGEMENTS We are indebted to Endre A Balazs, for his inspiration, support, and numerous helpful comments. Financial support for this research was provided by Biomatrix, Inc., and all AFM work was performed on site at Biomatrix.
REFERENCES 1. J.S. Scott, C. Cummings, A. Brass, & Y. Chen, 'Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing - electron microscopy and computer simulation', Biochem.J., 1991, 274, 699-705. 2. AP. Gunning, V.I. Morris, S. AI-Assaf, & G.O. Phillips, 'Atomic force microscopic studies ofhylan and hyaluronan', Carbohydr. Polym., 1996,30, 1-8. 3. I. Jacoboni, U. Valdre, G. Mori, D. Quaglino Jr., & I. Pasquali-Ronchetti, 'Hyaluronic acid by atomic force microscopy', J. Struct. Bioi., 1999, 126,52-58. 4. M.K. Cowman, J. Liu, M. Li, D.M. Hittner, & 1.S. Kim, 'Hyaluronan interactions: self, water, ions', In: The Chemistry. Biology and Medical Applications of Hyaluronan and its Derivatives, T.C. Laurent (ed.), 1998, Portland Press, London, pp.17-24. 5. M.K. Cowman, M. Li, & E.A Balazs, 'Tapping mode atomic force microscopy of hyaluronan: extended and intramolecularly interacting chains', Biophys. J., 1998, 75, 2030-2037. 6. M.K. Cowman, M. Li, A Dyal, & E.A Balazs, 'Tapping mode atomic force microscopy of the hyaluronan derivative, hylan A', Carbohydr. Polym., 2000, 41, 229-235. 7. M.K. Cowman & S. Matsuoka, 'The intrinsic viscosity of hyaluronan', In: Hyaluronan 2000. Woodhead Publishing, Cambridge. 8. S. Matsuoka & M.K. Cowman, 'Viscosity of polymer solutions revisited', In: Hyaluronan 2000. Woodhead Publishing, Cambridge. 9. K. Ogawa, Z. Shi, J. Lai, & A Majumdar, 'Molecular-level imaging of thin film ice crystal growth by atomic force microscopy', Proc. ASME Heat Transfer Div., 1995, 317-2,483-488.
BIOLOGICAL PROPERTIES OF BYALURONAN ARE CONTROLLED AND SEQUESTERED BY TERTIARY STRUCTURES John E. Seott! and Frank Heatley2 lChemical M0T-hology, Manchester University Medical School, Oxford Rd, Manchester M13 9PT Chemistry Dept. Manchester University, Manchester MI3 9PL
ABSTRACT Hyaluronan (HA) is the characteristic polysaccharide component of vitreous humor (from which it was first purified I), Wharton's jelly and synovial fluid. The unusual mechanical properties of these gels and viscoeleastic fluids were attributed non-specifically to interactions between HA molecules, which are stiff in solution partly because they prefer to take up 2-fold helical secondary structures, stabilised by H-bonds2 (Fig IA). HA has since been found in many tissues from many species-', Probably all animals produce it. Unexpectedly, a new class of specific and potent biological activities shown by HA fragments (in angiogenesis, inflammation etc) emerged4-6. Highly polymerised HA does not share these properties. HA is vitally important during developmenr/, It is a pluripotent material with a simple strucure (FigIA). Is there a unifying concept behind this diversity? We suggest that physiological properties of HA are controlled and sequestered by reversible tertiary structures8. We introduce an NMR approach which can monitor their formation and behaviour. Biological properties may thereby be linked to specific chemical aspects of HA and HA supramolecular organisation. KEYWORDS {3 Sheets, twofold helices, angiogenesis, hyaluronidase, erythrocyte lysis.
INTRODUCTION
l3c NMR studies on HA in aqueous solution proved that acetamido and carboxylate groups were involved in NH....COO- H-bonds, probably in stacked antiparallel agregates formally similar to {3 sheets as seen in proteins 8 (Fig IB), but uncommon if not unique in the polysaccharide field. The l3C acetamido C=O signal is much broadened (unlike all the other 13C resonances) because rotation of the amide group is restricted by participation in an H-bond. This broadening can be used as a reporter of H-bond formation, the first specific spectrometric test so far in this field. DISCUSSION It is a necessary consequence of the tertiary structure model that the acetamido NH is oriented trans-to the C2H of the glucosamine ring, permitting the NH....COO H-
118
Characterisation and solution properties of hyaluronan
A
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.... --
Figure 1 (A) secondary structure of HA (a tetrasaccharide fragment) as a two-fold helix with hydrogen bonds (dotted lines). (B) tertiary structure of HA viewed from the side. showing three HA molecules in antiparallel array stacked above each other. Arrows to the left and right point to the reducing ends; cross hatched areas are the hydrophobic patches; vertical dotted lines delineate sugar units. Square symbols (0) denote acetamido groups. circles (0) carboxylate groups. Filled symbols are on the distal side and open symbols on the proximal side. Vertical arrows indicating H-bonds from donor NH to acceptor -COO- are in pairs alternately pointing up and down. linking each HA molecule with both its neighbours in a {3 sheetlike structure 8. Overlapping hydrophobic patches provide additional stabilisation by hydrophobic bonding. Four different environments in which the amide group can exist in HA are labelled I. II. III and IV. I and II are not H-bonded to carboxylates, I because there is no suitably placed carboxylate. and II because the relevant carboxylate has been converted into a poor receptor for H-bonding e.g.as a methyl esterS. III and IV are H-bonded either in secondary (twofold helices) or tertiary ({3 sheets) structures. Characteristic NMR signatures are associated with each environment (see refs 8 and 12).
Biological properties
119
bonds to form between stacked anti~arallel polysaccharide chains (Fig.IB). It was known, using IH NMR to measure JCH-:¥I coupling, that the trans- arrangement was present in monomers and oligomers of up to 27 disaccharides l 2 which, however, do not aggregate efficiently to tertiary structures'[- We tried to extend these data to high mol. mass HA, but all signals were broadened, as is usual in high polymers, and although the NH signal was broader than other peaks in the spectrum (Fig. 2), as expected if it was intrinsically two peaks with a large value of 3JCH-NH coupling, a precise value of the coupling constant could not be obtained. The broadness of this peak is probably also due to lack of rotation of the amide group, as already shown by l3C NMR8. If the sharpening of the l3C acetamido C=O resonance on warming is secondary to simple rupture of NH--+COO H-bonds, the trans- CH-NH orientation should persist at high temperatures and this is compatible with the persistent broadening of the NH signal up to -80 oC (Fig 2).
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Figure 2. Left, l3C NMR resonances of high mol. mass HA acetamido C=O (174.5 - 175 ppm, amide IV) at 23 0C (lower trace) and 80 oC (upper trace). The sharp signal at 173.5 -174ppm is the carboxylate C=O resonance'[. HA concentration lOmg/ml in D20 containing O.14M NaCl. Note the marked sharpening of the acetamido C=O resonance on warming. Right, IH NMR resonance of high mol. mass HA NH proton. HA concentration 2mg/ml in 4: 1 H20ID20 solution containing 0.15M NaC!. The broad resonance is of similar width at both 23 0C (lower trace) and 80 °C (upper trace).
120
Characterisation andsolution properties of hyaluronan
It would be predicted that any process that caused disaggregation of high mol. mass HA tertiary structure would bring about sharpening of the l3C acetamido C=O signal. Simple warming of HA solutions is one such process (Fig 2). The predicted spectral change was completely reversible on cooling. The cycle was repeated several times with identical results. HA aggregation is therefore specific and reversible. Marked decreases in viscosity of HA solutions on warming9 parallel the temperature dependant NMR changes. We suggest that rupture of intermolecular H-bonds in the 13 sheet is an important part of this phenomenon. 0·3
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Arrhenius plot [In( k, x 105 ) against 10','7"] ofreaction of periodate with the glycol-containing compounds methyl 4-0-methyl-,-o-glucopyranoside (A l. chondroitin 6-sulphate (.l and hyaluronate (el The vertical lines show the spread of values of Ink, at each temperature point. The plotted point is the arithmetical average. The plots are drawn by inspection. All solutions contained 0.2M-NaClO•. The glucoside concentrations were all OAmM in O.4m.....NaIO.. Hyaluronate concentrations were either 6.53mM or 8.7mM in either 8.0mM- or 10mMNaIO•• except for the readings at 364 and 358 K. which were 1.9mM in polymer and 2mM in NalO•. Chondroitin 6-sulphate concentrations were 7.7!mM in 8.0mM-periodate.
10 u.J...L.L..........u...L.L.J...L.L.u...u...L.L.J...L.L.u..........~ 0.0028 0.003 0.0032 0.0034 1ITfK"
Figure 3. Arrhenius plots (In. [variable] against l/oK) of (A) second order rate constants of periodate oxidation of HA, chondroitin 6 sulphate and methyl 4Osmethyl-o-Dcglucopyranosidel l, (B) viscosity of HA solutionsr', and (C) the widths of the high mol. mass HA acetamido C=O resonance (Scott and Heatley, submitted). All show a change in slope at -40 0C (see text).
Biological properties
121
Arrhenius plots of the NMR signal width showed a break (a melting) at 40-50 °C (Fig 3). Similar plots of viscosity also showed a break at about 40 oC9, as did those of periodate oxidation kinetics of HA1O(Fig. 3). Taken together these data suggest that at physiological temperatures HA is on the edge of a critical change in supra molecular organisation. On the upper side of the break glycol groups react more easily with periodate, amido groups rotate more freely and viscosity becomes a property of single chains which can entangle randomly with other long chains. Disaggregation of the tertiary structure on warming parallels a marked increase in a biological activity, viz. inhibition of complement-mediated lysis of erythrocytesl l. This effect, too, is reversible on cooling. Disaggregated HA chains interact with complement, thereby inactivating it. On reaggregation the HA active sites are masked by inter-HA liaisons. The aggregated form sequesters biological activity that is an intrinsic property of HA primary structure. This hypothesis implies that certain biological activities of HA would be expressed if aggregation was prevented. Indeed, angiogenesis (the formation of new blood vessels) is inhibited by HA oligosaccharides although highly polymerised HA is without effect 6. Angiogenesis is fundamental to the facilitation of tumor growth. The 13C NMR method showed that HA oligosaccharides do not aggregated, since they cannot form enough collaborative intermolecular bonds to produce stable aggregates at ambient temperatures. They act freely and independantly, deploying sites that would be masked or otherwise engaged in the tertiary structure. Similarly, ultrasonication of high mol. mass HA in solution produced fragments which activated an autoregulatory loop in murine macrophages, or induced chemikine expression in alveolar macrophages - activities which were not shown by undegraded HA4,5. Probably some fragments were too small to participate in stable tertiary structures and could hence exert an activity that was hidden in the high mol. mass HA tertiary structures. It was suggested that HA degradation products could potentiate inflammatory reactions in tissues'l. The NMR evidence provides proof of a tertiary HA structure, probably a (3. sheet-like array, which is stabilised by H-bonds and hydrophobic bonds at ambient temperatures, provided the HA chains are long enough for sufficient cooperative intermolecular bonds to form. Disaggregation of the tertiary structure exposes the hydrophobic patches in particular, opening up biological activities and potential control mechanisms that were hidden in the tertiary structure. Easy transitions between secondary and tertiary structures in physiological conditions, which the Arrhenius plots suggest is probable, offer convenient and economic mechanisms for switching between functions dependant on these structures (Fig.l). Conversely, the tertiary structure may present possibilities that are not present in the single stranded secondary structure. An intriguing finding by Kreil's group13 is that a hyaluronidase with specific tissue localisation patterns is nor able to break down HA of a size less than about 50 disaccharide units. This is around the size at which stable tertiary structures would be expected to form8. We suggest that the enzyme, termed HYAL 2, is specific for the aggregated form of HA but not able to hydrolyse the single stranded form. The more familiar hyaluronidases, e. g. from testicles, are able to work on single stranded HA. Serendipitously, one was termed HYAL 113.
122
Characterisation and solution properties ofhyaluronan
The viscoelastic properties of high mol. mass HA are very probably fundamentally determined by the ability to form tertiary structures. A new NMR technique (rheo-NMR) has been used in preliminary experiments to show that the effect of shear on HA solutions can be observed by monitoring for ~ sheets using the 13C carbonyl signal (Fischar, Callaghan, Scott & Heatley, submitted). Elasticity in connective tissue extracellular matrices, in which analogous tertiary structures involving glycosaminoglycans similar to HA (chondroitins, keratans) may exist 14, probably depends on similar reversible interactions (Scott, unpublished). REFERENCES I. K. Meyer, The biological significance of hyaluronic acid and hyaluronidase. Pbysiol. Rev. 1947,27, 335-359
2. J.E.Scott, F. Heatley, & W.E Hull,. Secondary structure of hyaluronate in solution: A IH n.m.r. investigation at 300 and 500 MHz in dimethyl sulphoxide d6 solution. Biochem.J. 1984,220, 197-205 3.T.C. Laurent, (ed.) The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives. 1998, Portland Press Ltd, London 4. C.M.McKee, M.B. Penno, M.K. Cowman, M.D. Burdick, R.M. Strieter, C. Bao, P.W. Noble, Hyaluronan (HA) fragments induce gene expression in alveolar macrophages I. Clin. Invest. 1996, 98, 2403-2413 5. P.W.Noble, C.M. McKee, M.K. Cowman, H.S. Shin, Hyaluronan fragments activate an NF-KB/I-K Bet autoregulatory loop in murine macrophages. I.Exp. Med.I996, 183,2373-2378 6. D.C West & Kumar S. Hyaluronan and angiogenesis. In Hyaluronan. Ciba Foundation Symposium No. 143, 1989, 187-207 7. B.P.Toole, Hyaluronan-cell interactions in morphogenesis. In The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives. (Laurent, T.C. ed.) 1998, Portland Press Ltd, London 8. J.E. Scott, & F. Heatley, Hyaluronan forms specific stable cooperative tertiary structures in solution. A l3C n.m.r. study. PNAS 1999, 96, 4850-4855 9. E.D.Morris, D.A. Rees, E.J Welsh,. Conformation and dynamic interactions in hyaluronate solutions J. Mol. Bio1.1980, 138, 383-400 IO.N.S. Chang, R.J. Boackle, & G. Armand, Hyaluronic acid-complement interactions. I. Reversible heat-induced anticomplementary activity. Molecular Immunology,1985 22,391-397 I Ll.E. Scott & M.J. Tigwell, Periodate oxidation and the shapes of glycosaminoglycuronans in solution Biochem. 1.1978, 173, 103-114. 12.M.K. Cowman, D.M. Hittner & 1. Feder-Davis.v-C NMR studies of hyaluronan. Conformational sensitivity to varied environments. A1acromoleculesl996, 29, 28932902 13. G. Lepperdinger, B. Strobl, & G. Kreil, 'HYAL2, a human gene expressed in many cells which encodes a lysosomal hyaluronidase with a novel type of specificity' IBiol. Chem.1998, 273, 22466-22470 14. J.E. Scott, Supramolecular organisation and the 'shape module' concept' in animal matrix biology. J.Biochem. Mol. BioI & Biophys.I999, 2,155-167
ANALYSIS OF THE CONCENTRATED SOLUTION PROPERTIES OF HYALURONAN BY CONFOCAL-FRAP SHOW NO EVIDENCE OF CHAIN-CHAIN ASSOCIATION Tim Hardingham*, BC Heng & Philip Gribbon Wellcome Trost Centre for Cell-Matrix Research, School ofBiological Sciences, Stopford Building, University ofManchester, Manchester Mi3 9PT UK
Abstract
The network and hydrodynamic properties of fluoresceinamine labelled hyaluronan (FA-HA) was investigated at up to 10 mg/ml by confocal fluorescence recovery after photobleaching (confocal-FRAP). The influence of electrolytes on self and tracer lateral diffusion coefficients showed that in Ca 2+ and Mn 2+, FA-HA (830 kDa) was more compact than in Mg 2+, Na+ or K+. These results correlated with changes in the hydrodynamic radius of HA, determined by multi-angle laser light scattering analysis (MALLS) in dilute solution, which was smaller in CaCh (36 nm) than in NaCI (43 nm). The permeability ofHA solutions «10 mg/ml) to FITC-dextran tracers (2000 kDa) was also higher in CaCho HA showed a reduced hydrodynamic size and increased permeability to FITC-dextran in urea (up to 6M) and in ethanol/water (up to 62 % vol/vol). Both solvents did not show the changes predicted if chain-chain association was disrupted by urea, or enhanced by ethanol. Oligosaccharides of HA (HA20.26) also had no effect on the self diffusion of high molecular weight FA-HA (830 kDa) solutions, or on dextran tracer diffusion, showing that there were no chain-chain interactions open to competition by short chain segments. The results suggest that the physical properties ofHA solutions are primarily determined by solvent effects on chain flexibility, with entanglement dominating the properties of concentrated solutions and no evidence for association between chain segments making a significant contribution. Introduction
Hyaluronan (HA) is a high molecular weight (105_107 Da) unbranched glycosaminoglycan, composed of repeating disaccharides of D-N-Acetylglucosamine and D-Glucuronic acid. It is a widely distributed component of the extracellular matrix of vertebrate tissues (Balazs and Gibbs 1970). HA is a space-filling molecule and acts as a scaffold for the binding of other matrix molecules including aggrecan and other members of the hyalectan family (Laurent 1995, Lapcik et al 1998). At neutral pH and physiological ionic strength, HA acts as a stiffened random coil in solution, due to hydrogen bonding between adjacent saccharides, and mutual electrostatic repulsion between carboxyl groups (Morris et al 1980, Wik and Comper 1982, Sheehan and Atkins 1983, Reed et al 1989, Almond et a11997, Hardingham et al 1999, Gribbon et al 1999, 2000). It has also been proposed that the solution properties of hyaluronan are contributed to by association between chains, which have been visualised in EM preparations and interpreted as anti-parallel double helices, bundles and ropes (Engel 1989, Scott et al 1991) and NMR spectra have also been interpreted to suggest that
124
Characterisation and solution properties of hyaluronan
chain-chain association occurs in solution (Scott and Heatley, 1999). However, the presence and stability of such structures in solution remains unclear and how they contribute to the solution properties ofhyaluronan has yet to be determined. The conformation of HA is sensitive to its electrolyte environment. In solution, it undergoes electrostatically induced coil contraction with increasing concentration of electrolyte (Sheehan and Atkins 1983, Reed et a11989, Almond et al1997, Gribbon et al 1999). X-ray fibre diffraction studies of stretched hydrated preparations of HA have shown semi-crystalline forms in a 43 helix in Na+and a 32helix in Ca2+. Interestingly, in the presence of excess Na+ the addition of even sub-stochiometric concentrations of Ca2+ caused HA to adopt a 32 helix, suggesting an influential role for Ca2+ in determining the molecular packing of the secondary structure (Sheehan and Atkins 1983, Sheehan et al 1983) and also demonstrating the lack of any dominant thermodynamically preferred conformation. In this study, evidence for inter-chain interactions in HA solutions were investigated with fluoresceinamine-labelled HA (Glabe et al 1983) by confocal fluorescence recovery after photobleaching (confocal-FRAP). This is a powerful method for determining concentrated solution properties in the absence of flow and shear forces and with no concentration gradients (Kubitscheck et al 1994, Gribbon and Hardingham 1998). Confocal-FRAP is an equilibrium method and provides measurements oflateral self diffusion, which thus reveal the extent of intermolecular interactions and this can be followed to high concentration when interactions are maximal. It also provides a method to analyse the network formed by a polymer in solution by determining its effect on the diffusion within the network of fluorescently labelled tracer molecules of known size (Gribbon et al 1999, Hardingham and Gribbon 2000). In the present studies the sensitivity of HA solution properties to a range of conditions that would perturb different modes of intermolecular and intramolecular interaction were investigated. Hyaluronan properties in concentrated solutions may be contributed to by: I) electrostatic interaction of the regularly placed carboxyl groups; 2) hydrogen bonding between adjacent saccharides; 3) domain overlap and polymer entanglement; 4) chain-chain association through mechanisms such as interaction of hydrophobic patches. An important aspect, and indeed the value of using confocal-FRAP is because it permits analysis at concentrations of hyaluronan up to and far exceeding the critical concentration at which there is predicted molecular domain overlap (Gribbon and Hardingham, 1998, Hardingham et al 1999, Hardingham and Gribbon 2000). This analysis is thus best suited to investigations of entanglement and intermolecular chainchain association, as these would be concentration dependant and would be strongly favoured at high concentration, whereas electrostatic interactions and hydrogen bonding of adjacent disaccharides occur at all concentrations. Electrostatic and ionic effects on the hyaluronan network and its sensitivity to counterion type and valency were determined as these are known to greatly affect rheological and hydrodynamic properties (Sheehan et al 1983). The role of hydrogen bonds was investigated by comparing concentration dependant solution properties in deionised water, in 0.5M NaCl and 0.5M NaOH, as this revealed the profound affect of alkali on HA chain stiffuess. The presence of inter-chain associations that might involve hydrophobic interactions were investigated under physiological conditions, in solvents of varying polarity and in the presence of chaotropic agents (Scott and Tigwell 1975, Scott et al 1991, Cowman et al 1996, Geciavo et al 1995, Scott and Heatley 1999,
Concentrated solution properties
125
Gribbon et al 1999, 2000). Intermolecular chain-chain associations were also investigated using HA oligosaccharides as low molecular weight competitors of such interactions (Morris et al 1980, Cowman et al 1984, Fujii et al 1996).
Concentration Dependence of Hyaluronan Self Diffusion In characterising the general behaviour ofhyaluronan solutions (Gribbon et al 1999) it was shown that the lateral translational self-diffusion coefficients of hyaluronan showed a progressive fall with increasing concentration as it approached and exceeded the predicted critical concentration for domain overlap, (Fig. 1). Similar smooth transitions between dilute, semi-dilute and concentrated regimes have been observed experimentally in many polymer / solvent systems, (Callaghan and Pinder, 1984; Philliies, 1989; Imhoff et al., 1994) including HA solutions (Wik and Comper, 1982). The lateral self-diffusion coefficients reduced steeply with concentration in a manner consistent with phenomenological descriptions of polymer self-diffusion in terms of a universal scaling equation, (Phillies, 1989). acV O = 0 0 exp (1) Where Do is the polymer free self-diffusion defined in the limit of zero concentration and a and v are empirically derived. The parameter a describes the strength of interpolymer hydrodynamic interactions and the deviation of v from unity arises from chain contraction at high concentrations. Data were fitted to Equation 1 using a non-linear least squares fit (non-weighted). Analysis of data from results with hyaluronan (830 8 kDa) gave Do> 5.6 x 10- em's", a = 0.63 ml/mg and v = 0.74. These measurements
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Figure 1. Concentration dependance of the lateral translational self-diffusion coefficient of hyaluronan. The lateral translational self-diffusion was determined by confocal-FRAP for hyaluronan (830 kDa) (0) at 0.5-8.0 mg/ml in PBS at 25°C. The vertical arrow marks the critical overlap concentration c·. The solid line shows the data fitted to Equation 1 and extrapolated to zero concentration to give Do. (Data from Gribbon et al 1999). Confocal-FRAP technique described in Gribbon, Hardingham 1998 and Hardingham, Gribbon 2000.
126
Characterisation and solution properties of hyaluronan
were insensitive to pH over a broad range (pH 4 - 8) and also to temperature between 5°C and 60°C, when changes in solvent viscosity had been accounted for. The hydrodynamic scaling model (Eq. 1) predicts that a is proportional to molecular weight, and v equals 0.5 for high molecular weight polymers (Phillies, 1989). This was not apparent in these results and although all data fitted the form of Eq. 1, and a and v fall within accepted values (Phillies, 1989), it suggests that Eq. I does not provide a complete description ofhyaluronan behaviour. Effects of Electrolytes on Hyaluronan Solution Properties Investigation of the effect of increasing electrolyte concentration on hyaluronan solution properties (Gribbon et al 1999) showed that the self-diffusion coefficient of hyaluronan was very low in the absence of any supporting electrolyte, but increased dramatically with small increases in NaCI concentration, showing a 2.8 fold increase in lateral self-diffusion coefficient from zero to 100 mM (Fig. 2). This is consistent with increased electrostatic shielding resulting in polyanion coil contraction and as this was largely complete at 100 mM NaC!, the contribution of electrostatic effects to macromolecular stiffness under physiological conditions of ionic strength and pH is suggested to be smalL The effects of different counterions on the self-diffusion of hyaluronan showed that Ca2+ caused a significant increase compared with Na+, with less increase with Mn, Mg and K (Fig 3). The effects of CaCh and NaCI on the concentration dependence of hyaluronan solution properties were examined at constant ionic strength (0.5) (Gribbon et al 2000). At all concentrations up to 10 mg/ml, the self-diffusion coefficients of HA were greater in CaCh than in NaCI (Fig 4), although the difference became smaller at high concentration. The intrinsic effects of these counterions on the conformation of
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Figure 2. Effect oflncreasing Concentrations of NaCI and NaOH on Hyaluronan Self-diffusion. The lateral translational self-diffusion was determined for hyaluronan..500 kDa, 0.2 mg/ml) in increasing concentration ofNaCI (0), or NaOH ( ). Ionic strength of solutions in NaOH was maintained at 500 mM by the addition ofNaCL All measurements at 25°C. (From Gribbon et al 1999).
Concentrated solution properties
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The effect of ci+ on the properties of HA were also found to be dominant over the effects of Na+, as in the presence of 0.15 M NaCI the addition of CaCh at low concentration « 1OmM) caused a further significant increase in the self diffusion of HA (Fig 5). The differences between the self-diffusion of HA in ci+ and in Na+ were also accompanied by differences in the tracer diffusion of FIrC-dextran (2000 kDa) in solutions of HA at up to 20 mg/ml and tracer mobility in 150 mM NaCl was also increased by the addition of CaCh (Gribbon et al 2000). The contraction of the HA domain in calcium solutions suggested that ci+ (and 2 Mn +) increased the flexibility of the chain by promoting a greater range of movement at each glycosidic bond. Recently, molecular dynamics simulations of HA have demonstrated that short lengths of HA (5 disaccharides) access a range of compact 2 configurations including hairpin loops (Sheehan et al 1999). Individual ci+ or Mn + ions may co-ordinate two carboxyl groups, on the same HA chain, and promote chain 2 contraction. However, if this mechanism occurred Ca + would also be able to stabilise inter-chain associations and the results show that it does not. Alternatively, ci+ and Mn2+ may alter the co-ordination of water molecules with HA chains, thereby disrupting 2 hydrogen bonds involving water bridges. The presence of Ca + may therefore cause less stability in the range of hydrogen bonds that bridge adjacent sugars in these linkages (Almond et al 1998a, b). Molecular dynamics simulations incorporating counterions would be required to further substantiate this model. Overall, the changes in HA . properties caused by Ca2+ and Mn2+ are small compared to the effects 0 f strong alka I'I (see below), which disrupts the hydrogen bonds between adjacent saccharides and causes a major reduction in chain stiffness (Gribbon et al 1999). However, it may be speculated that HA destiffening by ci+ may have a role in cell mediated matrix remodelling processes, and this could be analysed further using cs" sensitive probes (Glabe et aI1983).
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Figure 6. Comparison of the Concentration Dependance of Hyaluronan Self-diffusion in NaOn, NaCI and Water. The concentration dependence of the lateral translational self-diffusion coefficient of hyaluronan (830 kDa) at 0.5-10 mg/ml was determined in, O.SM NaOH (0), O.5M NaCl (e), and De-ionised water (0). All measurements at 2SoC. (From Gribbon et al 1999).
130
Characterisation and solution properties of hyaluronan
The Effects of Alkali pH on Hyaluronan Self Diffusion and on Tracer Diffusion in Hyaluronan solutions
The effect of high pH in NaOH also contracted the domain size of hyaluronan, but the effect (Fig 6) was much greater than the reduction found due to electrostatic shielding (Fig 2). This effect is consistent with previously reported reductions in Rg and intrinsic viscosity (Ghosh et aI., 1993). Changes in the RH and hydrodynamic volume of hyaluronan with NaCI and NaOH were calculated using the Stoke's Einstein approximation for the behaviour of a sphere:
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where K is Boltzmann's constant, T is temperature, 1'] is the solvent. Ifit is assumed that the self-diffusion coefficient at 0.2 mg/ml is approximately equal to the free diffusion coefficient (see Fig. 1), then for hyaluronan of 500kDa, from the Stoke's Einstein equation, Rh contracted from 95 nm to 33.5 nm, in going from de-ionised water to O.5M NaCI, reducing further to 17.5 nm in 0.5M NaOH (Fig. 7). These results show that in going from 0.5 M NaOH into de-ionised water, the apparent domains of hyaluronan chains are increased by more than 100 times and this is most likely to result from increased electrostatic interactions and hydrogen bond formation (Gribbon et al 1999). In the most compact configuration in alkali, the hydrodynamics ofhyaluronan (500kDa) became similar to those of the partly branched FITC-Dextran (2000 kDa, Rh = 19 nm), which is neither charged, nor is predicted to form comparable hydrogen bonds. For hyaluronan (500 kDa) in 0.5 M NaOH (Fig 3), domain overlap is predicted to occur at 37 mg/m1. This implies that at 2-10 mg/ml solutions are well below c* and this is entirely consistent with the comparatively greater network mobility observed in self diffusion experiments, including those with higher molecular weight HA (830 kDa, Fig 6). These effects in alkali were reversible and caused no significant depolymerisation under the conditions used.
Hyaluronan 500kDa In : de-ionised water 0.5M NaCI
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Figure 7. Comparison of the Hydrodynamic Radius (RH) of Hyaluronan (SOOkDa) in De-ionised water and in Salt and Alkaline solutions.
Concentrated solution properties
131
Tracer diffusion results at low hyaluronan concentrations (l-4mg/ml) (Fig. 8) Gribbon et al 1999) show analogous behaviour to the changes in self diffusion (Fig. 6). The network is both more permeable and more mobile in 0.5M NaCI than in de-ionised water and this supports a model involving contraction of the hyaluronan chain conformation in the presence of increasing electrolyte. However, as the concentration of hyaluronan approached 20 mg/ml (Fig. 8), tracer mobility became progressively independent of salt concentration, indicating that at high concentrations chain density is the major determinant of matrix permeability. The lack of a salt effect at high hyaluronan concentration is interesting, as it suggests a lack of hydrophobic interactions between chains, as by analogy with RNA and DNA, high salt would be expected to favour chain-chain association. Tracer studies also provide a measure of the major changes induced by NaOH. In hyaluronan (930 kDa), at 20 mg/ml, (Fig. 7) the FITC-dextran translational diffusion is independent of NaCI concentration, but not of NaOH concentration. This reflects, as noted above, that for 930 kDa hyaluronan, 20 mg/ml is likely to represent a semi-dilute regime in the presence of NaOH, whereas it is clearly a concentrated, entanglement dominated regime, both in NaCI and in de-ionised water. These results strongly suggest that the solution properties at higher concentration in various solvents are directly related to the hydrodynamic volumes of single chains in the same solvent. Results at high pH, showing high mobility and permeability of hyaluronan, clearly reveal the degree to which, at neutral pH, intra-chain hydrogen bonds profoundly affect chain stiffness, chain entanglement and inter-chain hydrodynamic interactions. 16 .'111
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Figure 8 Comparison of Tracer Diffusion in Hyaluronan Solutions of Different Concentration, in NaOH, NaCI and Water. Lateral translational diffusion coefficients of FITC dextran (2000 kDa) were determined in hyaluronan solutions (930 kDa) 0-20 mg/ml, in de-ionised watert"), 0.5M NaCI (II), and O.5M NaOH (0). Inset shows the correlation length parameter (~) versus hyaluronan concentration for: de-ionised water (long dash), O.5M NaCI (solid line), and 0.5M NaOH (short dash). All measurements in PBS at 25°C. (From Gribbon et al 1999).
132
Characterisation and solution properties ofhyaluronan
Effects of Urea and Ethanol on Hyaluronan Solution Properties In concentrated polymer solutions, if the network properties are determined solely by chain entanglements, then they should be independent of agents that disrupt other associative mechanisms. To investigate whether HA properties were determined by hydrophobic chain-chain interactions, self-diffusion properties were investigated in the presence of urea, a potent disrupter of hydrophobic association (Gribbon et al 2000). Initially, the effect of urea on individual chain hydrodynamics was investigated by analysing HA self-diffusion at low polymer concentrations. In a dilute solution of HA, if there is association between segments of chains this will tend to contract its hydrodynamic domain, whereas in concentrated solution it might serve to additionally make linkages between adjacent molecules. However, the self diffusion of HA (SOO kDa) in the presence of 6M urea was consistently higher than in de-ionised water (Figure 4). This showed that in urea the polymer domain ofhyaluronan became smaller and thus showed no evidence for the disruption of intramolecular chain-chain association. The increased free diffusion coefficient of hyaluronan in dilute solution in the presence of urea is therefore inconsistent with the presence of intramolecular chain associations. On the contrary, the reduced hydrodynamic domain size shows that urea increases the flexibility of HA chains. The primary intra-molecular chain stiffening mechanism for HA arises from hydrogen bonding and the effect of urea may therefore be to reduce the hydrogen bonding between adjacent saccharides. However, the destiffening caused by urea is substantially less than that caused by O.S M NaOH, (Gribbon et al 1999). Therefore, urea may, for example, disrupt only a sub-fraction of H-bonds, such as those involving a water bridge. Interestingly urea had no detectible effect on HA diffusion in the presence of O.SM NaCI as supporting electrolyte, which suggested that it did not affect the chain-stiffening hydrogen bonds present in O.SMsalt,
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Figure 9. Effects of Urea and EthanollWater on Hyaluronan Self-diffusion The concentration dependence of self-diffusion of HA (SOOkDa) was determined 0.S-10 mg/ml for solutions in SS% vol/vol ethanol/water (....), 6 M urea (D) and de-ionised water (0). All data at 2SoC and corrected for solvent viscosity. Solid lines show data fitted to the polymer scaling equation. (From Gribbon et al 2000).
Concentrated solution properties
133
but could affect those additionally present in de-ionised water. Further investigation would be required to confirm this interpretation. Urea appeared to have little effect on intermolecular interactions between HA molecules, as the concentration dependence of self diffusion, which is a measure of intermolecular interaction, follows a very similar form in urea and in de-ionised water (Figure 9). These results therefore suggest that there are no chain-chain associations in HA in aqueous solution that is sensitive to this chaotropic agent. To investigate other solvent dielectric effects on HA (830 kDa) at 0.5 mg/ml, selfdiffusion coefficients were measured in ethanol/water mixtures (0 - 65% vol/vol) (Figure 6) (Gribbon et al 2000). There was an increase in self-diffusion with increasing ethanol concentration, suggesting that chain stiffness is reduced (Figure 6). Concentration studies HA (500 kDa) at 0.05 - 1.0 mg/ml, 55% vol/vol ethanol) showed in the presence of ethanol HA was consistently more mobile than in de-ionised water, or in urea (Figure 9) suggesting that the HA chains are more compact in ethanol. However, this description is inconsistent with theoretical calculations based on models of polyion behaviour, which predict enhanced stiffness in the presence of ethanol (Eisenberg and King 1997). Electrostatic repulsion between polyion chain elements is inversely proportional to the bulk relative permittivity (E,), and E, is 25.3 in ethanol and 80.1 in water. The lack of agreement with predictions of the polyion models suggests that there are additional mechanisms causing changes in HA conformation in ethanol/water mixtures. Analysis of HA circular dichroism spectra has been interpreted to suggest that double stranded helical structures form in ethanol (Staskus and Johnson 1988). If helix formation were predominantly within intra-chain segments, then this would be consistent with increased HA mobility. The consequent reduction in hydrodynamic size would reduce inter-chain entanglement, but at high concentration the effects of the tendency for intermolecular double stranded structures to form should become
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134
Characterisation and solution properties ofhyaluronan
significant, but no transition to an interlinked network at high concentrations was apparent. As noted with salt and alkaline solutions ofHA, the concentration dependence of self-diffusion in urea and ethanol/water also followed a very similar form to that in de-ionised water, which again suggested that the concentrated solution properties in these solvents were similarly related to the dilute solution behaviour in the same solvent. Effects of Hyaluronan Oligosaccharides on Polymeric Hyaluronan Properties
The presence of chain-chain interactions was also investigated using another strategy by competition with HA oligosaccharides. However, the presence of increasing concentrations of HA20-26 had no significant effect on the self-diffusion of FA-HA (2mglml), even at an oligosaccharide concentration twice that of the full-length HA (Figure 10). If hyaluronan mobility at 2mg/ml was restricted by associations between chains, then HA 20-26 should compete to form oligosaccharide-chain associations and this would increase the diffusion coefficient of HA. These results therefore also suggest that intermolecular chain-chain associations are not important in determining the concentrated solution properties ofHA (Gribbon et aI2000). Conclusions
The data presented in these studies (Gribbon et al 1999, 2000) suggest that intramolecular hydrogen bonds and polyanionic properties of hyaluronan both contribute to provide a highly expanded macromolecular conformation. However, under physiological conditions of ionic strength the results predict the electrostatic effects to be modest. The major contribution to the large hydrodynamic volume ofhyaluronan and hence its other important non-Newtonian viscoelastic properties are due to hydrogen bonding between adjacent saccharides. This restricts rotation and flexion at the glycosidic bonds and creates a stiffened polymer chain. The flexibility and permeability properties of the hyaluronan network can then be accounted for in terms of inter-chain hydrodynamic interactions of this extended structure, with entanglement being especially important at elevated concentrations. However, even at high concentrations, under physiological conditions, individual hyaluronan chains remain mobile and at no stage do hyaluronan solutions undergo transition to a gel-like state. These observations are incompatible with any significant degree of intermolecular association that is stable or co-operative. The results suggest that even at high concentrations the properties can be directly predicted from the behaviour in dilute solution and together a simple hydrodynamic model with chains stiffened by hydrogen bonds with domain overlap and entanglement at high concentration accounts for the major solution properties of hyaluronan. Acknowledgement We are pleased to acknowledge the immense contribution made by Endre Balazs to the study of hyaluronan. His work has been an inspiration to us all. We are very grateful to The Wellcome Trust and Seikagaku Corporation (Tokyo, Japan) for support for these studies. References 1. Almond A, Brass A, Sheehan JK. (1998a) Deducing polymeric structure from aqueous molecular dynamics simulations of oligosaccharides: predictions from
Concentrated solution properties
135
simulations ofhyaluronan tetrasaccharides compared with hydrodynamic and xray fibre diffraction data. J. Mol. BioI. 284, 1425-1437 2. Almond A, Brass A. Sheehan JK. (1998b) Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data. Glycobiology 8, 973-980 3. Almond A, Sheehan JK. Brass A. (1997) Molecular dynamics simulations of the disaccharides ofhyaluronan in solution. Glycobiology 7, 597-604 4. Balazs EA, Gibbs DA (1970). The rheological properties and the biological function of hyaluronic acid. In Chemistry and molecular biology of the intercellular matrix, (Balazs EA ed.) Academic press, London, New York, pp1241-1254 5. Callaghan PT, Pinder DN (1984) Influence of multiple length scales on the behaviour of polymer self-diffusion in the semidilute region. Macromolecules 17,431-437 6. Cowman MK, Cozart D, Nakanishi K, Balazs EA. (1984) IH NMR of glycosaminoglycans and hyaluronic acid oligo saccharides in aqueous solutions: the amide proton environment. Arch. Biochem. Biophys. 230, 203-212 7. Cowman M K, Hittner DM, Feder-Davies 1. (1996) IC NMR studies of hyaluronan: conformational sensitivity to various environments. Macromol. 29, 2894-2902 8. Eisenberg A, King M. (1977) in Ion-Containing Polymers: Physical Properties and Structure, pp. 105-154, Academic Press, New York 9. Engel J (1989) Figurel and Discussion comment. The biology of hyaluronan. Ciba Foundation Symposium 143, 18-19 10. Fujii K, Kawate M, Kobayashi Y, Okamoto A. (1996) Effects of the addition of hyaluronate segments with different chain lengths on the viscoelasticity of hyaluronic acid solutions. Biopolymers 38, 583-591 11. Geciavo R, Flaibani A, Delben F, Liut G, Urbani R, Cesaro A. (1995) Physicochemical properties of hyaluronan and its hydrophobic derivatives: a calorimetric study. Macromol. Chern. Phys. 196,2891-2903 12. Ghosh S, Khobal I, Zanette D, Reed WF. (1993) Conformational contraction and hydrolysis of hyaluronate in sodium hydroxide solutions. Macromol. 26, 46844691 13. Glabe CG, Harty PK, Rosen SD. (1983) Preparation and properties of fluorescent polysaccharides. Anal. Biochem. 130,287-294 14. Gribbon P, Hardingham TE. (1998) Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching. Biophys.J. 75,1032-1039 15. Gribbon P, Heng B C, Hardingham TE. (1999) The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching. Biophys. J. 77, 2210-2216 16. Gribbon P, Heng BC, Hardingham TE. (2000) The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chainchain association. Biochem J 350, 329-335 17. Hardingham TE, Gribbon P (2000) Confocal-FRAP analysis of ECM molecular interactions. In Methods in Molecular Biology, vol 139 Extracellular Matrix Protocols (C. Strueli and M. Grant eds) Humana Press, Totowa, NJ, pp 83-93 18. Hardingham TE, Gribbon P, Heng, BC (19990 New approaches to the investigation ofhyaluronan networks. Biochem. Soc. Trans. 27, 124-127
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Characterisation and solution properties ofhyaluronan
19. Imhoff A, Van Blaadren A, Maret G, Mallema J, Dhont JKG (1994) A comparison between the long time self diffusion of and low shear viscosity of concentrated dispersions of charged colloidal silica spheres. J. Chern. Phys. 100, 2170-2181 20. Kubitscheck H, Wedekind P, Peters R. (1994) Lateral diffusion measurements at high spatial resolution by scanning microphotolysis in a confocal microscope. Biophys J. 67, 946-965 21. Laurent TC, (1995) Structure of the extracellular matrix and the biology of hyaluronan, in Intersitium, Connective Tissue and Lymphatics. (Reed RK, McHale NG, Bert JL, Winlove CP, and Laine GA, eds.) pp. 1-12, Portland Press, London 22. Lapcik L(Jr), Lapcik L, De Smedt S, Demeeste, J, Chabrecek P. HA: preparation, structure, properties and applications. (1998) Chemical Rev. 98, 2663-2684 23. Morris ER, Rees DA, Welsh EJ. (1980) Conformation and dynamic interactions in HA solutions. J. Mol. BioI. 138,383-400 24. Phillies DJ. (1989) The hydrodynamic scaling model for polymer self-diffusion. J. Phys. Chern. 93, 5029-5039 25. Reed WF. (1996) Data evaluation for unified multi-detector size-exclusion chromatography, molar mass, and radius of gyration. Macromol. Chern. Phys. 197, 1539-1575 26. Reed WF, Ghosh S, Medjahdi G, Francois J. Dependence of polyelectrolyte apparent persistence lengths on ionic strength and linear charge density. (1991) Macromol. 24, 6189-6198 27. Reed CE, Li X, Reed WF. (1989) The hydrodynamic scaling model for polymer self-diffusion. Biopolymers 28, 1981-2000 28. Scott JE, Cummings C, Brass A, Chen Y. Secondary and tertiary structures of HA in aqueous solution, investigation by rotary shadowing electron microscopy and computer simulation. (1991) Biochem. J. 274, 699-705 29. Scott JE, Heatley F. (1999) Hyaluronan forms specific stable tertiary structures in aqueous solution: A C-l3 NMR study. Proc. Natl. Acad. Sci. 96,4850-4855 30. Scott JE, Tigwell MJ. (1975) The influence of the intrapolymer environment on periodate oxidation of uronic acids in polyuronides and glycosaminoglycuronans. Biochem. Soc. Trans. 3, 662-664 31. Sheehan JK, Arundel C, Phelps CF. (1983) Effect of the cations sodium, potassium and calcium on the interactions of hyaluronate chains: a light scattering and viscometric study. Int. J. BioI. Macromol. 5,222-228 32. Sheehan JK, Atkins EDT. (1983) X-ray fiber diffraction study of the conformational changes in hyaluronic acid induced in the presence of sodium, potassium and calcium cations. Int. J. BioI. Macromol. 5, 215-221 33. Sheehan JK, Brass A, Almond A. (1999). The conformations of hyaluronan in aqueous solution: comparison of theory and experiment. Biochem. Soc. Trans. 27,121-124 34. Staskus PW, Johnson We. (Jr) (1988) Double stranded structure for hyaluronic acid in ethanol-aqueous solutions as revealed by circular dichroism of oligomers. Biochemistry 27,1528-1534 35. Wik KO, Comper WD. Hyaluronate diffusion in semi-dilute solutions. (1982) Biopolymers 21,583-599
SOFT TISSUE RECONSTRUCTION IN SEVERELY TRAUMATIZED PATIENTS USING HYALURONAN BASED DERMAL AND EPIDERMAL GRAFTS Dirk A. Hollander*, Susanne Kramer, Mobssen Y. Hakimi, Joacbim Windolf Department of Trauma and Reconstructive Surgery, Johann Wolfgang GoetheUniversity, Theodor Stern-Kai 7, 60590 Frankfurt/Main, Germany
ABSTRACT Background:
This report demonstrates the potential of two-stage autologous keratodermal grafting as a starting point for non-invasive reconstruction of extensive traumatic soft-tissue defects. Metbods: In severely injured patients skin biopsies for cell cultivation were taken. Cultured "neodermis" consisting of cultured autologous fibroblasts grown on biocompatible threedimensional scaffolds made up of benzylester of hyaluronan was grafted on conditioned defect areas. After ingrowth of dermal substitutes transplantation of cultured autologous keratinocytes on hyaluronan based laserperforated membranes was performed. 10 days later a 0.2 mm thin, 1:6 meshed autograft was overlaid. Clinical follow-up with standard photography was documented. Results: Grafting with cultured autologous fibroblasts revealed a suitable dermal tissue replacement. Epithelialization was evident after transplantation ofkeratinocytes. Final closure of the defects with normo-elastic tissue properties was achieved after thin mesh-grafting. Conclusions: Preliminary findings with the described method seem to be very promising. As in all fields of tissue engineering, long-term studies and further follow-up are required. KEYWORDS Soft-tissue reconstruction, hyaluronan, skin equivalents INTRODUCTION The increasing number of high-velocity traffic injuries with traumatic extensive soft-tissue loss require the search for materials that may serve as skin replacements for permanent wound closure. Skin substitutes should resemble functional and aesthetic properties of dermis and epidermis. During the last 10 years biologists and biomedical engineers have started to develop human tissue substitutes [1]. Despite tremendous advancements in understanding wound
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Application ofhyaluronan in tissue engineering
healing and tissue repair the failure to achieve rapid wound closure following soft tissue loss still can result, amongst many other reasons, in death or permanent invalidity ofthe victims. A breakthrough in the history of wound coverage was the method of culturing colonies of human epidermal keratinocytes into confluent sheets suitable for grafting, first described by Rheinwald and Green in 1974 [2J. In 1981 the first successful transplantation of cultured epidermal autografts (CEA) in bum patients was reported by O'Connor et al. [3J. The use of CEA proved to be live-saving in patients with massive burn wounds and subsequently became commercially available. Although established as a treatment option in the management of a variety of acute and chronic wounds, cultured keratinocyte grafting still is associated with practical limitations. The disadvantages of using cultured keratinocyte grafts in the traditional way are well known. These include slow expansion rates associated with labour- intensive and time-consuming procedures, extreme fragility and vulnerability of the grafts combined with difficult handling, poor take rates and graft contraction of around 50% [4-7J. Extensive research has focused on the development of new carrier biomaterials that are able to support growth and differentiation ofhuman keratinocytes. Recent efforts to make cultured grafts more convenient for clinical use are aimed at reducing the time required for culture and improving the handling properties of grafts [8-12J. Although many of the major problems concerning keratinocyte grafting have been solved, several investigators have shown that permanent full-thickness skin replacement requires a dermal component to ensure adequate long-term graft stability [13-18]. The presence of a dermal layer is essential for the regulation of growth and differentiation of cultured keratinocytes. A problem in culturing a dermal structure is the fact that cultured fibroblasts, like most cells, expand two-dimensionally. In the past many dermal substitutes have been proposed to overcome this difficulty: collagen-based materials were used by several authors with different outcomes [19-22J, fibronectin scaffolds [23J, fibrin glue [6,24J, allogeneic or xenogeneic dermis [25,26J as well as synthetic or semisynthetic polymers with non-satisfactory results [27,28J. Nevertheless, it is well-known that the outcome of epithelial grafting procedures depends, among other things, on the condition of the wound bed before grafting. The best results can be expected if a well-vascularized, non-infected dermal layer with extracellular matrix components is provided. In the present work we have used three-dimensional scaffolds made up ofthe benzylester of hyaluronan for culturing and transplanting autologous fibroblasts. Keratinocytes obtained from the same biopsies were cultured separately on laser microperforated membranes consisting of the same benzyl esterified hyaluronan material, which has already been described as a delivering vehicle for cultured keratinocytes as a treatment option in severe burn wounds [29J and chronic deep soft tissue defects [11, 13J. Hyaluronan (HA), also known as hyaluronic acid, is a non-sulfated glycosarninoglycan, a linear polymer of glucuronic acid N-acetylglucosamine disaccharide, a major carbohydrate component of the extracellular matrix and can be found in skin, joints, eyes and most other mammalian tissues (Fig. 1). The fact that hyaluronan is associated with tissue repair processes is generally accepted [30,31J. It plays different roles in the physiological environment. Present in the extracellular matrix, it is a soluble molecule forming highly viscous solutions in water and contributes to the regulation of water balance acting on osmotic pressure. On the molecular level, HA has a free radical scavenger function [32,33J. Many reports have been focused on the influence of exogenous hyaluronan on the process of wound healing and angiogenesis [34-37J. Following injury, a range of well-structured tissue repair proceedings, as inflammation, granulation tissue formation, reepithelialization and remodeling takes place. Hyaluronan is likely to play an important role as a mediator of the mentioned cellular and matrix processes [38J.
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In the following report we demonstrate tissue reconstruction of high biomechanical value with good aesthetic results in massive soft tissue defects by development and transplantation of cultured autologous dermal and epidermal substitutes cultured on hyaluronan based scaffolds in a two-step procedure. Extensive surgical procedures could be replaced in the future after improvement and establishment of the described technique.
MATERIALS & METHODS Biomaterials The biomateriaIs used were derived from total esterification ofHA with benzylester and are referred to as HYAFFill>-11 (Fidia Advanced Biopolymers srl, Abano Terme, Italy). Two different forms ofHYAFP®-11were used for cell-cultivation and grafting: 1. Three-dimensional non-woven fleece, made of 20J.1m-thick fibres with a specific weight of n 100 g/mz , (Hyalograft 3D '), used for cultivation of autologous fibroblasts (Fig. 1); 2. Transparent, 20J.1m-thick membranes with laser-drilled microperforations (40J.1m in diameter), (Laserskin"), used for cultivation ofautologous keratinocytes (Fig. 2). Mentioned materials were obtained from Fidia Advanced Biopolymers srl, Abano Terme, Italy.
Fig. 1 Fig.l
Fig.2
Fig.2 Scanning electron microscope view of fibroblasts attached to Hyalograft 3D™_ fibres, an ideal environment for adhesion, proliferation and subsequent production of dermal extracellular matrix Electron microscopic view ofkeratinocytes grown on Laserskinf-membrane
Cell culturing procedures Autologous fibroblasts
Human keratinocytes and fibroblasts were isolated from 2x2 cm skin biopsies taken with an electric dermatome. During transportation to the laboratory, the biopsy was kept in a nutritional medium (Dulbecco s Modified Eagles Medium, DMEM containing 5% fetal calf serum (FCS) and antibiotics). On arrival, the skin was rinsed in phosphate-buffered saline (PBS), the deep dermal layer was removed and the tissue was cut into small fragments, which were transferred into petri dishes containing 20 ml dispase (5 mg/ml). The de-epidermalized fragments of dermis were rinsed with PBS and further minced with a scalpel. Human fibroblasts were isolated by overnight digestion with a solution of 80 U/ml of type 1 collagenase (Worthington Biochemical Corp. NJ) in DMEM 10% FCS, at 37°C 5% COz. Cells were propagated in DMEM 10% FCS, the culture medium was renewed twice a week. Cells were trypsinized at 80% confluency and splitted 1:3 for subsequent passaging.
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Fibroblasts were seeded onto the Hyalograft 3D matrix at a density of 8,0-10,0 x 104 cells/em" (size unit: 8 x 8 em) in DMEM 10% FCS, culture medium was changed twice a week. Starting day 8 from seeding, the dermal grafts were ready for transplantation.
Autologouskeratinocytes Human keratinocytes were isolated from the same 2x2 em skin biopsies. After incubation at 37°C for I hour, the epidermal layer was gently peeled off from the dermis and the epidermal sheets were treated with 0,25% trypsin for 15 minutes at 37°C under gentle stirring. Cell suspensions were seeded at a density of2,0 x 104 keratinocytes/cm' on a feeder layer of lethally irradiated 3T3 mouse fibroblasts. Culture medium was composed 00 parts ofDMEM, I part of Ham's FI2 supplemented with 10% FCS, Sug/ml insulin, 10-10 M cholera toxin. 0,4 ug/ml hydrocortisone, and l Oug/ml rh-EGF (all reagents were purchased from Sigma Chemical Co. , S1. Louis, MO). Keratinocytes were used up to passage 4. When keratinocytes were seeded on the HYAFF@-laserperforated membrane, the following seeding densities were 4 employed: 2,0 x 10 keratinocytes/cnr', 2,5 x 104 feeder cells (size unit: 10 x 10 em), The medium was renewed every second day. Starting day 7 from seeding, all the microperforations were populated with colonies of basal cells and the membrane was ready for grafting. The epidermal sheets were rinsed in PBS several times in order to remove the FCS, transferred into suitable trays filled with nutritional FCS-free medium, sealed and double packaged under sterile conditions. Transplantation could take place the following day.
Patient history In patients with massive injuries, skin biopsies (2x2 cm) were taken during the first surgical session with the electric dermatome after the decision of performing dermal and epidermal cell transplantation had been made. Patient's or parent's consent declarations were obtained in written form. First the wounds were conditioned by regular surgical debridement prior to fibroblast-grafting. After approximately two weeks the cultured ,,neo-dermis" consisting of autologous fibroblasts grown on biocompatible threedimensional scaffolds made up of an benzylester ofhyaluronan (Hyalograft 3D"') was ready for grafting. One week after the initial fibroblast-transfer and ingrowth of the dermal substitute the transplantation of autologous keratinocytes on HYAFF@ membranes (Laserskin") was performed. Approximately 10 days after keratinocyte transplantation mesh-grafting (0.2 rom thin, ratio I :6) onto the formed epithelium was carried out. The described patient is a male, 65 years old; after a train accident he showed extensive traumatic soft tissue loss ofhis right arm from the axillar region down to the palm of his hand with comminuted fracture of the forearm, metacarpal fractures I-IV; at the local hospital he was treated with external fixator and plaster cast; massive superinfection with Pseudomonas Aeruginosa and Staphylococcus Aureus on arrival in our hospital was obvious (Fig. 3-8).
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Patient: male,65 years old, extensive soft tissue loss right ann from axilla down to the palm ofhis hand with comminuted fracture ofthe forearm and metacarpal fractures I-IV after train accident, treated with external fixator and plaster cast. Appearance ofthe previously superinfected tissue defect in our department after surgical debridement a. total right ann b. dorsal view of right forearm with external fixator
Fig.4a
Fig.4
Fig.Sa
Fig.S
Fig.4b Patient: day 20 after admission; 4 days after autologous fibroblast transplantation on Hyalograft 3DTM, first change of secondary dressings, appearance of dry scaffold surface a. total right ann b. right forearm, dorsal view, removed instable fixator
Fig.Sb Patient: day 26 after admission; 10 days after fibroblast transplantation; take rate of grafts approximately 95%, impressive equalization of levels between
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surrounding healthy skin and former tissue defect area; no adverse or immunogenic reactions; no signs of infection a. total right arm, complete integration of grafts b. right forearm, dorsal view, superficial rejection of two fibroblast-sheets, unknown reason
Fig.6a
Fig.6b
Fig.6
Patient: day 35 after admission; 5 days after autologous keratinocyte-grafting on Laserskinf-membranes, first change of secondary dressings, diminished fluidloss, signs of beginning epithelialization; almost complete biodegradation of hyaluronan sheet, no adverse effects, no rejection; manipulations possible without pain a. right arm, healthy, clean tissue appearance, thin re-epithelialization visible b. right forearm, same smooth tissue structure
Fig.7a
Fig.7b
Fig.7
Patient: day 45 after admission to our department, first change of secondary dressing 5 days after mesh-grafting (0.2 rom thin, mesh-ratio I :6), perfect take result, firm attachment ofgraft on underlying thin epithelium a. right arm, stable repair b. right forearm; even mechanically insufficient meshed skin shows strong adherence to underlying skin layer
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Fig.8b
Fig.8a Fig.8
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Patient: out-patient visit, 10 weeks after initial admission, 4 weeks after final mesh-grafting; complete tissue replacement with normo-elastic properties and good cosmetic result; shoulder-, elbow- and wrist-joints show good mobility a. right ann, dorsal view, tissue reconstruction complete b. right forearm; good final result
RESULTS Preparation of grafts Human fibroblasts obtained from the patient's skin biopsy proliferated during the primary culture phase which took approximately 8 days; subsequently seeded onto non-woven HYAFpGl'-11 fleeces where they were able to adhere and proliferate within the scaffold (Fig. I). Human epithelial celIs harvested from the same biopsy proliferated in medium during the primary culture phase which lasted approximately 8 days; they were subsequently seeded on the Laserskinf-membrane where they showed rapid proliferation (Fig. 2). After a few days the celIs reached a sub-confluent stage and were suitable for grafting. The composite keratinocyteLaserskinf-sheets remained stable throughout the culturing procedure and did not show any sign of contraction. The transparency of the membranes made cell observation using light microscopy possible at any time. Patients In the described patient clean and non-infected defect-areas were revealed after regular surgical debridement under sterile conditions in the operating theatre (Fig. 3). Skin specimen (2x2 ern; O,6mm thick) was taken with an electrical dermatome in one of the first surgical sessions without any problems after written permission was obtained. Hyalograft 3D""-transplantation was performed 16 days after initial hospital-admission. The performance of transplantation and handling of the grafts was very easy from a surgical point of view. To avoid shear- or pressure-forces we applied non-adhesive paraffin secondary dressings below ordinary sterile gauze dressings, put on careful elastic bandages and kept the patient in bed for the first three days. After covering the soft tissue defects with dermal equivalents, pain experienced in the treated regions was reduced. First dressing changes was performed 4-5 days after transplantation under sterile conditions in the operating theatre (Fig. 4). Firm adherence of the grafts onto the underlying tissue was remarkable in the patient. The most superficial layers had dried out due to reduced wound fluid-secretion after grafting. 8- IO days after transplantation, integration of the fibroblastseeded biomaterial was almost complete (Fig. 5). Equalization of the levels between healthy
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surrounding skin and regenerated defect-areas was visible. No signs of infection, allergic reactions or any other side-effects were evident. 14 days after transplantation of Hyalograft 3D"", Laserskinf-graffing was possible. Again handling of the grafts was uncomplicated. In order to prevent shear and pressure-forces, patients were immobilized until first change of secondary dressings during the critical period of cell-attachment. Afterwards intensive physiotherapeutic exercise was resumed. 5-10 days after keratinocyte transfer the membrane was biodegraded. Thin epithelialization with diminished wound fluid secretion was evident (Fig. 6). To achieve improved biomechanical tissue properties mesh-graft-transplantation (0,2 mm thick, ratio 1:6) was performed onto the layer of thin epithelium in the patient 10 days after Laserskinf-grafting (Fig. 7). Two weeks after mesh-graft application all the defects showed final and complete tissue regeneration with macroscopically normo-elastic properties. Mobility of underlying joints was not impaired. After patient dismissal, control visits were performed on an out-patient basis (Fig. 8). DISCUSSION Chronic skin ulcers and burn wounds as examples for acute skin defects represent the most promising clinical applications of soft-tissue engineering. By presenting our results, we would like to widen the indications for tissue engineering to the area of trauma and reconstructive surgery. The continuous search for improved methods of clinical application of grafts composed of autologous fibroblasts and keratinocytes as skin substitutes cultured in vitro is reflected in the intense search for innovative carrier systems, i.e, biomaterials that are able to support the growth of human skin cells [39]. Amongst the various materials like collagen [19-22], autologous [17,40-43] or allogeneic [44-50], fibronectin [23] and fibrin glue [6,24] hyaluronan seems to be a very promising substrate. HA has been proven to play a fundamental part in wound healing itself both in adult and fetal tissue [38,51,52]. In comparison with the other mentioned macromolecules HA derivatives are less immunogenic and reduce the potential risk of viral and prion infections [53,54]. Development of cultured dermis consisting of autologous fibroblasts on threedimensional HYAF~scaffolds for primary reconstruction of extensive soft tissue defects represents a promising innovation from a clinical as well as a morphological point of view. Autologous fibroblasts actively proliferated throughout the culturing procedure and as previously shown by Campoccia et aI. [55], morphological observations on paraffin-embedded specimens demonstrated that the cells migrate through the non-woven fleece, populate the inner space and both sides of the biomaterial to produce a fibrillar network. Furthermore, immunohistochemical examinations detected the production of extracellular matrix components such as collagen type IV, fibronectin and laminin [56]. Our report confirms the in vivo biocompatibility and complete incorporation of the living autologous dermal equivalent. In all patients there was excellent performance and near-total take rate of all the sheets. Very interesting findings were reduced pain sensation and clearly reduced loss of wound secretions which was evident by the appearance of dried upper surfaces of the neo-dermis already visible at first change of secondary dressings. Ten days after dermal grafting the deeply fissured and clefted tissue defects showed smooth equaIizing at the margins to healthy surrounding skin without any signs oflocal or general adverse effects. From our own point of view we can state that the described method of dermal replacement enables the creation of ideal preconditions for any subsequent epidermal grafting procedure. The important factor in the strategy of restoring damaged soft tissue in layers is the assumed concept that the dermal component constitutes a permissive and regulatory microenvironment for the growth and differentiation of cultured keratinocyte-grafts [26,55,57].
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The second step in building up normal soft tissue morphology in our patients was the transplantation of autologous keratinocytes, obtained from the same biopsy and cultured separately on Laserskin"-membranes. Positive results based on clinical as well as preclinical settings have been reported using keratinocytes cultured on laser-perforated HYAF~-membranes [11,12,58]. The microperforated structure of the sheet itself enables keratinocytes to populate the pores and colonies grow both above and beneath the membrane. Furthermore, the cells differentiate and exhibit features similar to physiological epidermis like the presence of hemidesmosomes, previously shown by Trabucchi and co-workers [59]. As soon as the keratinocytes are subconfluent, they are ready for grafting; time spent in culture can be reduced, earlier grafting is possible. Culturing keratinocytes to confluent stages causes transition from a highly proliferative state to one of irreversible growth arrest in the meaning of terminal differentiation [60]. If keratinocytes can be transplanted in the state of hyperproliferation, before differentiation is induced, the dermal wound bed has the possibility to function as a "culture system in vivo", allowing the cells to adhere, proliferate and later to differentiate and build up cornified layers. In the demonstrated tissue defect good vascularized stable tissue with proliferating fibroblasts and dermal extracellular matrix components was visible. The incorporation of dermal equivalents should create perfect conditions for taking of the autologous keratinocyte grafts. The Laserskinf-graft we used was peeled from the petri dish without dispase digestion and could be easily applied to the wounded sites. First change of secondary dressings revealed ongoing biodegradation of the membrane, thin epithelial coverage was visible 8-10 days following grafting. The lack of biomechanical stability ten days after the final cell-grafting procedure is not a problem in smaller or in chronic wounds. Our patients are mostly "healthy" human beings, who are violently tom from the community by trauma. The injuries have to be restored as quickly as possible with the aim of minimizing functional disabilities. Therefore we decided to follow a strategy that comprises extremely thin mesh-grafting with mesh-ratio I:6, to minimize the donor site lesion, 10 days after Laserskinf-transplantation. All patients have been discharged from the hospital within 8 weeks of the trauma. They are followed on a regular out-patient basis. All patients are reintegrated in their normal daily routine. CONCLUSION
The present study has shown that hyaluronan benzyl ester, both as non-woven fleece and as membrane structure, is easy to handle, biocompatible, biodegradable and non-immunogenic. The combined use of hyaluronan scaffolds, cultured autologous keratinocytes, fibroblasts and mesh grafts leads to rapid wound closure and to a mechanically stable tissue. In summary, first experiences with transfer of Hyalograft 3D'" with autologous fibroblasts in order to replace lost dermal tissue in deep and extensive soft tissue defects indicate that this procedure is a viable and very promising treatment option. However, due to the mechanical instability of autologous epithelial grafts, thin mesh grafts are still required. Further studies are needed to establish whether this form of treatment will be accepted as standard procedure.
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22. Lamme EN, De Vries HJC, Van Veen H, Gabbiani G, WesterhofW, Middelkoop E, Extracellular matrix characterization during healing of full-thickness wounds treated with collagen/elastin dermal substitute shows improved skin regeneration in pigs, J Histochem Cytochem., 1996,44,1311-1322. 23. Ejim OS, Blunn GW, Brown RA, Production of artificial-oriented mats and strands from plasma fibronectin: a morphological study, Biomaterials, 1993, 14,743-749. 24. Hundyadi J, Farkas B, Berteny C, Hugo NE, The effect offibrin glue on skin grafts in infected sites, Plast Reconstr Surg., 1988,89,268-272. 25. Prunieras M, Regnier M, Schlotter M, A new method to culture human epidermal cells on allogeneic or xenogeneic dermis: preparation of recombined grafts, Ann Chir Plast., 1979, 24,357-362. 26. Krejci NC, Cuono CB, Langdon RC, McGuire J, In vitro reconstruction of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis, J Invest Dermatol, 1991, 97, 843-848. 27. Koyano T, Minoura N, Nagura M, Kobayashi KI, Attachment and growth of cultured fibroblast cells on PVAlchitosan-blended hydrogeIs, J Biomed Mater Res., 1998, 39, 486490. 28. Cooper ML, Hansbrough JF, Spielvogel RL, Cohen R, Bartel R, Noughton G, In vivo optimization ofa living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh. Biomaterials, 1991, 12,243-249. 29. Andreassi L, Casini L, Trabucchi E et al, Human keratinocytes cultured on membranes composed of benzyl ester ofhyaluronic acid suitable for grafting, Wounds, 1991,3, 116126. 30. Toole BP. Proteoglycans and hyaluronan in morphogenesis and differentiation, In: Hay ED, editor. Cell biology ofextracellular matrix (2nd edition). New York: Plenum Press, 1991,305-341. 31. Toole BP, Hyaluronan in morphogenesis, J Intern Med., 1997, 242, 35-40. 32. Presti D, Scott J, Hyaluronan-mediated protective effect against cell damage caused by enzymatically produced hydroxyl radicals is dependent on hyaluronan molecular mass, Cell Biochem Function, 1994, 12,281-288. 33. Abatangelo G, O'Regan M, Hyaluronan: biological role and function in articular joints, Eur J Rheumatol Irif/ammation, 1995, 15(1), 9-16. 34. Abatangelo G, Martelli M, Vecchia RP, Healing of hyaluronic acid-enriched wounds: Histological observations, J Surg Res., 1983, 35, 410-416. 35. West DC, Hampson IN, Arnold F, Kumar S, Angiogenesis induced by degradation products of hyaluronic acid, Science, 1985,228, 1324-1326. 36. Burd DA, Siebert JW, Ehrlich HP, Garg HG, Human skin and post-burn scar hyaluronan: demonstration ofthe association with collagen and other proteins, Matrix Vol., 1989,9, 322-327. 37. Wiegel PH, Frost SJ, McGary CT, LeBoeufRD, The role of hyaluronic acid in inflammation and wound healing, Int J Tiss Reac., 1988, 10, 355-365. 38. Chen WYJ, Abatangelo G, Functions ofhyaluronan in wound repair, Wound Rep Reg., 1999, 7, 79-89. 39. Rue LW, Cioffi WG, McManus WF, Pruitt BA, Wound closure and outcome in extensively burned patients treated with cultured autologous keratinocytes, J Trauma, 1993, 34, 662-668. 40. Heimbach D, Luterman A, Burke J et al, Artificial dermis for major burns. A multi-center randomized clinical trial, Ann Surg., 1988,208, 313-320. 41. Murphy GF, Orgill DP, Yannas IV, Partial dermal regeneration is induced by biodegradable collagen-glycosaminoglycan grafts, Lab Invest., 1990,62,305-313.
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42. Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ, Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan
sub&rate,JAAdA,1989,262,2125-2130. 43. Boyce ST, Glatter R, Kittmiller WJ, Treatment of chronic wounds with cultured skin substitutes: a pilot study, Wounds, 1996, 7, 24-29. 44. Hull BE, Finley RK, Miller SF, Coverage of full-thickness burns with bilayered skin equivalents: a preliminary clinical trial, Surgery, 1990, 107, 496-502. 45. Nanchahal J, Otto WR, Dover R, Dhital SK, Cultured composite skin grafts: biological skin equivalents permitting massive expansion, The Lancet, 1989,2, 191-193. 46. Cuono C, Langdon R, McGuire J, Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury, The Lancet, 1986, 1, 1123-1124. 47. Cuono CB, Langdon R, Birchall Net al, Composite autologous-allogeneic skin replacement: development and clinical application, Plast Reconstr Surg, 1987, 80,626637. 48. Krant D, Eckhardt M, Patton ML, Combined simultaneous application of cultured epithelial autograft and Allodenn, Wounds, 1995, 7, 137-142. 49. Gentzkow GD, Iwasaki SD, Hershon KS et ai, Use of dermagraft, a cultured human dermis to treat diabetic foot ulcers, Diabetes Care, 1996, 19, 350-354. 50. Pollak RA, Edington H, Jensen JL, A human dermal replacement for the treatment of diabetic foot ulcers, Wounds, 1997, 9, 175-183. 51. Moriarty KP, Crombleholme TM, Gallivan EK, O'Donnal C, Hyaluronic acid-dependent pericellular matrices in fetal fibroblasts: implication for scar-free wound repair, Wound Rep Reg., 1996, 4, 346-352. 52. Siebert JW, Burd AR, McCarthy JG, Weinzweig J, Ehrlich HP, Fetal wound healing: a biochemical study of scarless healing, Plast Reconstr Surg., 1990, 85, 495-502. 53. Gallico GG, Biologic skin substitutes, Clin Plast Surg., 1990, 17,519-526. 54. Heck E, Bergstresser P, Baxter C, Composite skin graft: frozen dermal allografts support engraftment and expression ofautologous epidermis, J Trauma, 1985, 25, 106-112. 55. Campoccia D, Doherty P, Radice M, Brun P, Abatangelo G, Williams DF, Semisynthetic resorbable materials from hyaluronan esterification, Biomaterials, 1998, 19,2101-2127. 56. Zacchi V, Soranzo C, Cortivo R, Radice M, Brun P, Abatangelo G, In vitro engineering of human skin-like tissue, J Biomed Mater Res., 1998, 40, 187-194. 57. Coulomb B, Lebreton C, Dubertret L, Influence of human dermal fibroblasts on epidermalization, J Invest Dermatol., 1989, 92, 122-125. 58. Donati L, Marazzi M, Veronesi AM et al, Treatment of cutaneous wound with cultured human keratinocytes on hyaluronic acid membrane, Wound Rep Reg., 1995, 3, 363. 59. Trabucchi E, Andreassi L, Malcovati M et al, Ultramicroscopic observations of cultured epithelial sheets before and after grafting for major human burns, Wounds, 1991, 3, 83-88. 60. Poumay Y, Pittelkow MR, Cell density and culture factors regulate keratinocyte commitment to differentiation and expression of suprabasal KIIKIO keratins, J Invest Dermatol., 1995, 104(2),271-276.
HYALURONIC ACID SELF -ASSOCIATION IN THE PRESENCE AND ABSENCE OF SALTS Theresa M. Mcintire and David A. Brant* Department ofChemistry University of California Irvine, CA 92697-2025 USA
ABSTRACT
Carbohydrate polymers and oligomers are excellent candidates for molecular level storage of biological information through variations in the chemical composition, primary sequence, and branching pattern. Diversity of polysaccharide primary structure affords diversity in higher order structure, and information may be stored in the three dimensional spatial relationships of the sugar residues as well. 1 This allows carbohydrates to function as highly specific markers in biological recognition processes. 2 Carbohydrates operate at cell surfaces, where they often occur as components of glycoproteins, glycolipids, or proteoglycans anchored in the cell membrane. 3 Signaling and recognition usually involve carbohydrate-protein interactions. 4-5 Instances of carbohydrate-carbohydrate signaling interactions are likewise emerging. 6 Carbohydrate-carbohydrate interactions are also clearly important in stabilizing the three dimensional structures of many biologically active oligo- and polysaccharides. 7 Many polysaccharides, e.g., hyaluronic acid (HA), contain charged groups that can affect their conformation and thus their physicochemical and biological properties. The self-interactions these polymers undergo are clearly important in stabilizing the three dimensional structures that may be important for their function in signaling and recognition. In this report we show that the extent and nature of association of sodium hyaluronate (NaHA) in aqueous solution, observed using AFM, is sensitive to the presence of salts. KEYWORDS
Atomic force microscopy (AFM), hyaluronic acid (HA), scanning probe microscope (SPM), self-association INTRODUCTION
The scanning probe microscope (SPM) 8 has been a valuable tool for routine imaging of complex biological structures as well as individual molecules with nanometer-scale resolution. 9 Carbohydrate-carbohydrate interactions have been investigated for several polysaccharides using AFM. 10-15 In this paper we document an increase in the extent of chain association with increasing HA concentration and the formation of stiff HA fiber networks in the presence of added low molecular weight salts.
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Characterisation and solution properties of hyaluronan
MA TERIALS & METHODS
A culture broth of the bacterium Streptococcus equi was controlled to produce HA with a molecular weight of 2 x 106 g/moI. This high molecular weight HA was purified and heat degraded to produce a sample of lower molecular weight (NaHA, 3 x 105 g/mol). These NaHA samples were supplied by Vitrolife UK Ltd. The NaHA stock solutions were diluted with distilled water or a low molecular weight salt solution to a final polymer concentration of 1 - 30 ug/rnl., Aliquots of these diluted solutions were deposited by spraying a fine aerosol 10, 13, 16-18 onto freshly cleaved mica (Polysciences, Inc., Warrington, PA) and air dried. Mica is easily prepared by cleaving with tape and provides an atomically flat substrate free of artifacts found on other commonly used SPM substrates such as highly oriented pyrolytic graphite. After air drying for a few hours, samples were imaged by AFM. Specimens were examined using a ThermoMicroscopes AutoProbe ® CP Research (Sunnyvale, CA) scanning probe microscope equipped with an NCAFM probe head. A piezoelectric scanner with a range up to 50 urn was used for all images. The scanner was calibrated in the xy directions using a 1.0 urn grating, and in the z direction using several conventional height standards. 10, 12-13 The oscillation frequency (I) of the cantilever/tip was offset from (1)0 to higher frequencies by a few kHz. 10, 13, 19 The oscillation amplitude and z direction set point were adjusted to avoid tip-sample contact according to the operating procedures for noncontact mode imaging. All measurements were performed in air at ambient pressure and humidity. Images were stored as 256 x 256 point arrays and analyzed using AutoProbe® image processing software supplied by ThermoMicroscopes.
Figure I. Scale bar =200 nm.
Figure 2. Scale bar =500 nm.
Mesh-like networks were formed at a NaHA concentration of 10 Ilg/mL in aqueous solution in the absence of added salt, as seen in Figure I. The NaHA networks showed no molecular ends or tails. The mean thickness of these molecular sheets, 0.66 nm, is consistent with a monolayer of HA molecules, suggesting strong lateral association. Diluting the HA to 1-31lg/mL yields mostly single strand species as shown in Figure 2. In the presence of added low molecular weight salts various extents of chain association or aggregation were seen. With 0.10 M sodium acetate (Figure 3) or 0.10 M lithium acetate (Figure 4) stiff fiber aggregates were observed. The average height
Hyaluronic acid self-association
139
of the fibrillar polymer bundles measured normal to the mica plane is about 1.4 nm, suggesting that they are composed of an association of several chains. The lateral spacing between tightly packed fibrils is about 19 nm.
Figure 3. Scale bar =200 nm.
Figure 4. Scale bar = 250 nm.
Less lateral ordering was seen with higher sodium acetate concentrations of 1.5 M, as shown in Figure 5. The average height of the rod-like polymer bundles measured normal to the mica plane is greater than 1.7 nm. ACKNOWLEDGEMENTS The authors acknowledge financial support from the National Institutes of Health NIH Grant GM 33062 (DAB). HA samples were kindly supplied by Vitrolife UK Ltd. Scale bar = 250 nm. REFERENCES 1. Dwek, R. A., Glycobiology: Toward Understanding the Function of Sugars. Chemical Reviews, 1996, 96, 683-720. 2. Laine, R. A., The Information Storing Potential of the Sugar Code, in Glycosciences: Status and Perspectives, Gabius, H.-J., and Gabius, S., Eds., Chapman and Hall, London, 1997, pp. 1-14. 3. Varki, A., Biological Roles of OIigosaccharides: All of the Theories are Correct. Glycobiology, 1993,3,97-130. 4. Ryan, C. A., and Farmer, E. E., Oligosaccharide Signals in Plants: A Current Assessment. Annual Review of Plant Physiology and Plant Molecular Biology, 1991,42,651-674. 5. Jackson, R. L., Busch, S. J., and Cardin, A. D., Glycosaminoglycans: Molecular Properties, Protein Interactions, and Role in Physiological Process. Physiological
140
Characterisation and solutionproperties ofhyaluronan
Reviews, 1991,71,481-539. 6. Bovin, N. V., Carbohydrate-Carbohydrate Interaction, in Glycosciences: Status and Perspectives, Gabius, H.-J., and Gabius, S., Eds., Chapman and Hall, London, 1997, pp. 277-289. 7. Brant, D. A., Shapes and Motions of Polysaccharide Chains. Pure and Applied Chemistry, 1997,69, 1885-1892. 8. Binnig, G., Quate, C. F., and Gerber, Ch., Atomic Force Microscope. Physical Review Letters, 1986, 56, 930-933. 9. Morris, V. J., Biological Applications of Scanning Probe Microscopies. Progress in Biophysics and Molecular Biology, 1994,61, 131-185. 10. McIntire, T. M., Penner, R. M., and Brant, D. A., Observations of a Circular, Triple-Helical Polysaccharide Using Noncontact Atomic Force Microscopy. Macromolecules, 1995,28,6375-6377. 11. Brant, D. A., and McIntire, T. M., Cyclic Polysaccharides, in Large Ring Molecules, Semlyen, J. A., Ed., Wiley, Chichester, England, 1996, pp. 113-154. 12. McIntire, T. M., and Brant, D. A., Imaging Carbohydrate Polymers With Noncontact Atomic Force Microscopy, in Techniques in Glycobiology, Townsend, R. R., and Hotchkiss, A. T., Eds., Marcel Dekker, New York, NY, 1997, pp. 187208. 13. McIntire, T. M., and Brant, D. A., Imaging of Individual Biopolymers and Supramolecular Assemblies Using Noncontact Atomic Force Microscopy. Biopolymers, 1997,42, 133-146. 14. McIntire, T. M., and Brant, D. A., Observations of the (l~3)-I3-D-Glucan Linear Triple Helix to Macrocycle Interconversion Using Noncontact Atomic Force Microscopy. Journal ofthe American Chemical Society, 1998, 120,6909-6919. 15. McIntire, T. M., and Brant, D. A., Imaging of Carrageenan Macrocycles and Amylose Using Noncontact Atomic Force Microscopy. International Journal of Biological Macromolecules, 1999,26,303-310. 16. Tyler, J. M., and Branton, D., Rotary Shadowing of Extended Molecules Dried from Glycerol. Journal of Ultrastructure Research, 1980, 71, 95-102. 17. Stokke, B. T., Elgseeter, A., and Smidsred, 0., Electron Microscopic Study of Single- and Double-Stranded Xanthan. International Journal of Biological Macromolecules, 1986,8,217-225. 18. Stokke, B. T., and Elgsa:ter, A., Conformation, Order-Disorder Conformational Transitions and Gelation of Non-Crystalline Polysaccharides Studied Using Electron Microscopy. Micron, 1994, 25, 469-491. 19. Braunstein, D., Imaging an F-Actin Structure With Noncontact Scanning Force Microscopy. Journal of Vacuum Science and Technology A, 1995, 13, 1733-1736.
EMAIL ADDRESSES [email protected], [email protected]
COMPARISON OF THE REACTIVITY OF DIFFERENT REACTIVE OXIDATIVE SPECIES (ROS) TOWARDS HYALURONAN B.J. Parsons", S. AI-Assaf, S. Navaratnam & G.O. Phillips The North East Wales Institute, Free Radical Chemistry & Photochemistry Group, Wrexham, LLI I 2A W. UK. E-mail [email protected]
ABSTRACT
Hyaluronan is a linear biopolymer found in all connective tissues and amongst its many possible roles, it has the important functions of lubrication and shock absorbing. Its biological usefulness has been attributed to its molecular weight, shape and structure. In inflammatory diseases, oxygen-derived free radicals are produced which can participate in the degradation of hyaluronan. Different mechanisms have been proposed for the generation of reactive oxidative species (ROS) such as 'OH and peroxynitrite in a biological environment. Hyauronan has been found to be particularly susceptible to attack by ROS in comparison with other biopolymers. This paper describes the interaction of some ROS, including 'OR, Brz", Ch- and, peroxynitrite with hyaluronan by measuring molecular weight changes using gel permeation chromatography (GPC) coupled to multiangle laser light scattering techniques. Kinetic measurements using stopped-flow techniques have also been used to investigate the depolymerisation reactions of peroxynitrite with hyaluronan. KEYWORDS
Hyaluronan, degradation, ROS, hydroxyl radicals, peroxynitrite INTRODUCTION The role of hydroxyl radicals in the degradation of hyaluronan. One of the main secretory components of synovial fluid is hyaluronan (HA) which is produced by hyalocytes of the synovial membrane and is a major component of joint tissue. The concentration and molecular size of hyaluronan shows great variation between individuals'<. In normal synovial fluid, the weight average molecular weight is about 7 million. In rheumatoid synovial fluid, the weight average molecular weight decreases to 4.8 million. There is now a significant amount of evidence (see reviews in references 3-9) which supports the involvement of highly reactive intermediates such as o,'. HzOz, ocr, chloramines and the hydroxyl free radical, ·OR. It now seems likely that the production of superoxide radical anions from neutrophiles in the synovial fluid is central to the initiation of damage to HAlO. Superoxide radical anions (Oz'") are produced in regions of the phagocyte membrane that remain in contact with the external milieu, most of which tend to escape during the respiratory burst ll • IZ. The dismutation of the Oz" produces HZ02 which is also liberated by leucocytes'<". Although both Oz" and HzOz are unlikely themselves to degrade hyaluronan, both species can participate efficiently in reactions with transition metal ions such as copper (lIII) and iron (II/Ill) to
142
Characterisation and solution properties ofhyaluronan
produce the highly-reactive and damaging species, the hydroxyl free radical (-OR). The probability of these reactions occurring in rheumatoid synovial fluid is greatly increased since it has been established that the enzymes catalase and superoxide dismutase, whose functions are to scaven¥e R 202 and O2' - respectively, are barely detectable in rheumatoid synovial fluid 6,17, There have been a number of previous investigations of the reaction of -OR free radicals with hyaluronan. Some of these have involved detection of the products of the reaction by, for example, viscosity chanBes, end group analysis, oxygen consumption, peroxide formation and CO2 release 18-2. All of this work has been of a qualitative nature in the sense that the yields of HA degradation or degradation products have not been related to the yield or flux of -OR free radicals. In contrast, the main thrust in our laboratories has been to use radiation chemical techniques to produce precise, ~uantifiable fluxes of -OR and to measure the fluxes of products and HA chain breaks 232 . By relating the changes in viscosity or irradiated HA solutions to changes in molecular weight, it has been possible to calculate the rate of production of chain breaks caused by -OR free radicals. It was found that in nitrous oxide saturated solutions where the main reactive species is -OR, each -OR leads to one HA chain break. The corresponding proportion in oxygenated solutions was 54%. These were the first quantitative and reliable results on the yield of chain scission in HA caused by -OR radicals. The latest work in our laboratories involved free radical studies of a cross-linked form of hyaluronan called Rylan30 . This material is manufactured by Biomatrix Inc (USA) and is used as a viscosupplement in arthritic joints'". We have shown that these new materials are three times more resistant to 'OR-induced degradation compared with hyaluronan. Much of this work and other aspects of free radical damage to hyaluronan have been reviewed 6,28,32.
The role of NO and peroxynitrite in HA degradation Nitric oxide (NO) has been identified as a biologically-important molecule involved in a number of physiological processes including relaxation of vascular smooth muscle, neurotransmission, platelet inhibition and immune regulation 33-3S. An important part of the biological function ofNO is its ability to diffuse through most cells and tissues with little consumption or direct reaction. One of the significant reaction pathways for NO is considered to be its reaction with the superoxide anion radical (02). The bimolecular rate constant for this reaction is so high (6.7 x 109 M I S·I) 36 that nitric oxide is the only known biological molecule produced in high enough concentration under pathological conditions to outcompete endogenous superoxide dismutase for the superoxide anion radical. Such conditions may arise under ischemic conditions or, indeed in rheumatoid synovial fluid. Where such pathological conditions arise, it is the reaction product, peroxynitrite, (see equation 1) which is formed on reaction of 02'- with NO, that is considered to be the major agent of damage to biological substrates.
+
NO
ONOO'
(1)
Studies of the reactivity of peroxynitrite have involved the use of a variety of techniques with substrates such as phenolic compounds 37 , ascor bic IC acid aCI 38, met hi' omne 39 and sulphydryls 40, The protonated form of peroxynitrite (ONOOR, pKa = 6.8) is more reactive than its conjugate anion and can react by nitrating and hydroxylating substrates or by participating in one-electron oxidations of the substrate. The reaction of ONOOR with substrates can occur either through direct or indirect pathways 41, It
Reactive oxidative species
143
has also been proposed that an excited form of ONOOH, (ONOOH*), can lead to products identical to those obtained in reactions of hydroxyl radicals 42. Peroxynitrite is, therefore in principle, an alternative or perhaps supplementary reactive species which could cause the degradation of hyaluronan in the synovial fluid of rheumatic patients, particularly given the low levels of superoxide dismutase and catalase found in the synovial fluids of rheumatoid arthritic patients and despite the often profound hypoxia characteristic of rheumatoid synovial fluid. Evidence that peroxynitrite species play a significant role in the pathological development of chronic inflammation comes in part from the demonstration that fragments of hyaluronan induce nitric oxide synthase in murine macrophages 43. Thus, HA fragments produced in free radical processes can augment the production of peroxynitrite and lead therefore to the development of an ongoing inflammatory state. In addition, previous work by Blake has confirmed that serum nitrite concentrations in patients with rheumatoid arthritis and osteoarthritis were significantly higher than in controls implying nitric oxide synthesis by the synovium and thereby suggesting a role for nitric oxide as an inflammatory mediator in rheumatic diseases 44. Recently Li et al,45 used simple mixing techniques to study the degradation of hyauronan by peroxynitrite. The degradation was monitored using agarose gel electrophororesis and viscometric measurements. The current investigation is aimed at comparing the reactivity of 'OR, Ck and, peroxynitrite towards hyauronan. The reaction of Cls" may have relevance to the role of myeloperoxdiase in inflammatory diseases. Stopped-flow techniques with mixing 'dead times' of S ms or less in combination with gel permeation chromatography/ multiangle laser light scattering (MALLS) detection were used to ascertain which of the peroxynitrite species would react. The specific aim here was to assess the relative importance of peroxynitrite in the degradation in the synovial fluid of rheumatoid arthritic patients relative to the proposed role of hydroxyl radicals in the chain scission process. EXPERIMENTAL Materials
Hyaluronan (Lot Auto Back 2) was donated by Biomatrix Inc. The sample was dialysed against water for 5 days then autocIaved at l2SoC for 30 minutes. The sample was then freeze dried. Sample Preparation
Hyaluronan samples were prepared by weighing out accurately the fibre and adding to a known volume of solvent. The concentrations of the HA solutions were based on the molecular weight of the disaccharide unit. The samples were left to hydrate at 4°C overnight then tumble-mixed the following day for at least 6-Shrs. GPC-MALLS
A GPC-MALLS system was used for the determination of the molecular weight and molecular weight distribution of hyaluronan samples obtained from undegraded and degraded HA samples. The system utilised a solvent delivery system model 6000A (Waters Division of Millipore, USA) connected to a stainless steel column- Hemabio linear (lOmicron) packed with hydrophilic modified HEMA gel (hydroxyethyl methacrylate co-polymer), a manual Rheodyne Model 7125 syringe loading sample
144
Characterisation and solution properties ofhyaluronan
injector equipped with 1 ml sample loop, a concentration dependent detector Wyatt Optilab DSP interformetric refractometer operated at 633nm (Wyatt Technology Corporation, USA). The DAWN-DSP laser light scattering photometer was equipped with a He-Ne laser at a wavelength of 633nm (Wyatt Technology Corporation, USA). A value of 0.162 was used for the refractive index increment (dn/dc)30. Stopped-flow reactions The detection system consisted of a xenon arc lamp and Applied Photophysics pulsing unit and monochromator, fitted with a stepper motor and quartz optics. Optical transmissions (lcm path length) at various wavelengths selected with the monochromator (bandwidth 1-10 nm) were measured as a function of time before and after stopping the flow using photoelectric detection. The output of the photomultiplier (Hammatsu 1384) was displayed on a Hewlett-Packard 5451 A Digital Storage Oscilloscope. Data acquisition and processing were carried out using a Hewlett-Packard 9000 Series 300 computer. RESULTS AND DISCUSSION The purpose of this study is to compare the efficiencies of degradation ofhyaluronan by Clt-, Br2°- and peroxynitrite with the known and extraordinary efficiency of OH radicals, where it has been shown almost all (>90%) ofoOH produce a chain break Z8. Cho-is a highly oxidising free radical (Eo = 2.3V)46 which produces OCr in its reactions, also a possible candidate for hyaluronan degradation". OCr, formed by myeloperoxidase, may for example, participate in a redox reaction with transition metal ions, as follows, OCr + Fe(II) ~
CIOHo- + Fe(III)
(2)
CIOH"- is a highly oxidising species in its own right which also may result in the formation ofoOH and Ch o- 48 . The related species, Brzo- , is of interest to this study since it is known that it can abstract hydrogen atoms from simple carbohydrate 49. In these experiments, Ch o- and Brzo- were produced by the radiolysis of either nitrous oxide saturated solutions of bromide or deareated solutions of chloride ions at pH2. The relevant reactions are: ---MM~ HzO e·aq+NzO ~ "OH +cr ~ CI" + cr ~ "OH + Br' ~ Br" + Br' ~
"OH, -n, e·aq, Hz, HzOz "OH+-OH+Nz CI" + 011 Ch"' Br' + 011
(3) (4)
Brz"·
(8)
(5) (6) (7)
The yields of chain breaks in these experiments have been calculated as in our previous studies using multiangle laser light scattering (MALLS) to determine the number of chain breaks in hyaluronan molecule (Mo - Mi / Mi) Where M, is the original molecular weight, M, is the molecular weight measured for each concentration of free radical used in a degradation experiment. A value of 2.3 for the polydispersity, i.e. MwlMo=2.3 Z8, was used throughout. The concentration of .c~~in breaks was found by multiplying the chain breaks per molecule by the initial concentration of hyaluronan.
Reactive oxidative species
145
Figure 1 shows the relevant changes in the concentration of breaks with increasing concentration ofCh o- and Brzo - The data are compared to those obtained with °OH alone (using NzO saturated solutions of HA). From the initial slopes of these plots, the degradation efficiencies (per reacting free radical) were found to be 0.90 ("OH), OAO (Ch and 0.15 (Br2°). O
- )
Figure 1 A plot of concentration of breaks with increasing concentration of (0) Ch o - and (.) Brzo - and (e) °OH. The reactions of peroxynitrite species were investigated using stopped-flow apparatus with a deadtime of 8 ms. Deaerated solutions at pH II (prepared by the method of Hughes and Nickin 50 and described by Li et a145) were mixed with phosphate buffered solutions of hyaluronan at pH 4 and 6. The mixing ratio was (1: 10) and the resultant pH values were 6.6 and 8.3 respectively. In principle, there are three peroxynitrite species which may participate in the potential degradation ofhyaluronan as set out in the equations below; (9) k = 1.4s·1 pKa = 6.8 ONOOH* +- ONOOH B J, HA J, HA
ONOO· J, HA
(10)
where the participation of ONOOH* may involve the production of °OH itself rather than its direct reaction with HA 51. It is also clear from the above scheme that the conversion of ONOOH to ONOOH* is slow and rate-determining and thus the rate of disappearance of peroxynitrite by this pathway would not be influenced by changes in HA concentration. It has also been established 33 that the decay of peroxynitrite is strongly dependent on pH. To establish that there were no impurities which could
146
Characterisation and solution properties ofhyaluronan
scavenge the peroxynitrite species thereafter, the first order rate of disappearance of peroxynitrite absorption at 302 nm was measured over the pH range 5.1 to 7.3. The relevant plot is shown in Figure 2 which is consistent with the anticipated effect of pH33 . 2.0
pH
Figure 2. First order rate constant of peroxynitrite as a function of pH. Figure 3 shows the rate of disappearance of peroxynitrite absorption at 302 nm both in the presence and absence of HA at pH 5.5. It is clear that there is very little dependence of HA concentration on the decay kinetics. In more detailed investigation, however, at the same pH, it was found that there was a slight but distinct decrease in the observed first order rate constant as HA concentration was varied from zero to 10-2 mol dm" (see Figure 4).
.....
\
:
!
I SOOmsldiv
I
500msldiv
Figure 3. Kinetics of peroxynitrite reaction in the (A) absence and (B) presence of IOmM HA at pH5.5. It seems likely that this small change in the rate constant in Figure 4 was in fact attributable to the effect of increased viscosity at higher HA concentrations. This was confirmed by studying the direct second order reaction of ONOOH with tyrosine. The second-order rate constant for this reaction was measured with and without 10-2 mol dm' 3 HA, where a similar 30% decrease in rate constant was observed. On the assumption that all the mechanisms of peroxynitrite disappearance are dependent on diffusional processes, the observed changes in Figure 4 are consistent with a viscosity effect on diffusion processes. It, therefore, seems unlikely that the data in Figure 4 indicate the presence of second-order processes involving the reaction of ONOOH and ONOO- with HA.
Reactive oxidative species
147
1.6 1.5 1.4 ..,.",
-""
1.3 1.2 1.1 1.0
4
2
0
6
8
10
[HA]/mM
Figure 4. First order rate constant for the reaction of peroxynitrite with HA plotted as function ofHA concentration. To study the effect of peroxynitrite species on the yield of HA degradation, peroxynitrite and HA were mixed in a stopped-flow apparatus and the resulting product solution was analysed using the GPC-MALLS technique to measure the concentration ofHA chain breaks (see above discussion ofCh o- and Br2°- reaction with HA). Figure 5 shows the data so obtained at pH 6.6 and 8.3 for a range ofperoxynitrite concentrations upto about 1.4 x 10-3 mol drn".
1.5x10-'
• • 1.0x10
pH 8.3
•
pH 6.6
I
•
co >< OJ
e
m
•
5.0x10· 1
0.0
~ 0.0
• 50x10··
1.5x 1 o'
1.0x10·J
[ONOO
2.0)(10- 3
J
Figure 5. A plot of the concentration of HA chain breaks as a function of peroxynitrite concentration. (e) at pH 6.6 and (.) at pH 8.3. From this data, it appears that peroxynitrite is ineffective at degrading HA, with low yields of about 0.001. This contrasts sharply with a recent study by Li et al45 which involved simple mixing experiments, where the data indicated an efficiency of 0.14. One possible explanation for the difference between the two studies may be the absence of the transition-metal complexing agent, DTPA, in the previous study. It is known that the presence of trace, micromolar amounts of copper (II), for example, can cause the rapid disappearance of peroxynitrite, perhaps to form a species which may degrade HA more effectively than peroxynitrite.
148
Characterisation and solution properties ofhyaluronan
In conclusion, the HA chain scission efficiencies of Ch·-, Br2·- and peroxynitrite have been measured and compared with that already found for ·OH. These are summarised in the Table below Table. Relative efficiencies of some ROS in inducing chain breaks in hyaluronan.
ROS ·OH Br2·' Ch o, ONOOH* ONOOH
Chain scission efficiency 0.9 0.15 0.4 0.001 0.14
Reference This work This work This work This work Li et a1 4 '
It would appear from this preliminary study that it is probably ONOOH* which is the likely species involved in HA chain scission, rather than °OH produced by the conversion ofONOOH* since this would presumably yields a very high (-1) efficiency for HA degradation. The proposed low efficiency of HA degradation by ONOOH* would rule this species out as a candidate in the observed reduction of molecular weight in the synovial fluid of patients suffering with rheumatoid arthritis. However, the possibility that a very reactive species is produced perhaps in the reaction of peroxynitrite species with, for example, trace amounts of Cu(ll) and Fe(ll) complexes is worthy of further study.
ACKNOWLEDGMENT We thank Dr Endre A. Balazs for supporting this investigation. REFERENCES 1. 2. 3. 4. 5. 6.
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Characterisation and solution properties ofhyaluronan
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48
49 50 51
POLYSACCHARIDE FRAGMENTATION INDUCED BY HYDROXYL RADICALS AND HYPOCHLORITE Martin D. Rees, Clare L. Hawkins and Michael J. Davies* The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia
ABSTRACT Both metal ion / peroxide redox couples, which generate HO', and hypochlorite (HOCI) induce oxidation of mono- and poly-saccharides and bring about polymer fragmentation. In this article recent studies using electron paramagnetic resonance spectroscopy (both direct and spin trapping) to detect free radicals generated during these reactions are reviewed. Evidence is presented for essentially random attack of HO' on mono-saccharides, and indirect evidence is presented for the occurrence of similar processes with a number of poly-saccharides including hyaluronan. This results in the formation of a-hydroxyalkyl radicals that undergo both acid- and base-catalyzed reactions to give carbonyl-conjugated species. In contrast, reaction of HOCl with similar substrates results in selective initial damage to the N-acetyl ring via the formation of chloramides at the N-acetyl function. Subsequent decomposition of these species results in the formation of nitrogen-centered radicals that can abstract hydrogen atoms from other sites and bring about fragmentation.
KEYWORDS Hydroxyl radicals, myeloperoxidase.
sugar radicals,
hypochlorite, fragmentation, EPR,
INTRODUCTION Activation ofleukocytes both in vitro and in chronic inflammatory conditions results in the production of highly reactive oxidant species such as 0/ and H 20 2, and the release of the enzyme myeloperoxidase I. Myeloperoxidase catalyses the oxidation of
cr to HOCl via reaction
1 2. HOCl exists as an equilibrium mixture of HOCl and ·OCl at physiological pH values (reaction 2); HOCl is used below to refer to this mixture. MPO (1) - -......- HOCl + OW HOCl
.........
(2)
HP2 can be converted in to the highly reactive hydroxyl radical (HG') on reaction with redox active transition metal ions such as Fe(II) and Cu(I) 3. It is well established
152
Characterisation and solution propertiesofhyaluronan
that the hyaluronan (hyaluronic acid) present in rheumatoid synovial fluid has a lower molecular mass and a reduced viscosity compared to that in normal joints, and this has been ascribed to oxidative damage to the polymer by reactive oxidants 3. It has also been proposed that lower-molecular weight, possibly fragmented, glycosarninoglycans present in the sub-endothelial matrix of arteries contributes to the development of atherosclerosis 4. Both myeloperoxidase and redox active metal ions have been reported to be present in rheumatoid synovial fluid 5,6 and early, middle and late stage atherosclerotic plaques 7, and in vitro studies have demonstrated that hyaluronic acid and glycosarninoglycans can be depolymerised upon exposure to HO" and HOCI3, 8-13. Whilst there is considerable evidence to support a role for radicals in the depolymerisation of hyaluronic acid and related polymers, direct evidence regarding the selectivity of initial attack and the nature of subsequent strand cleavage mechanism(s) is lacking. Whilst there is also evidence for a role for HOCI in poly-saccharide depolymerisation the intermediates generated during these reactions are poorly characterized. In this article we summarize recent EPR (electron paramagnetic resonance spectroscopy) studies which have allowed direct evidence to be obtained on the identity of intermediate radical species 12-14.
RESULTS Hydroxyl radical mediated attack: rapid-flow EPR experiments The direct detection of unstable substrate-derived radicals can be achieved by use of a three-stream rapid-flow system inserted in to the cavity of an EPR spectrometer 15. In these studies, HO' was generated using Ti(III) - HPz redox couple, with the substrate included in the third stream. This allows defined, steady-state, concentrations of HO' to be reacted with a substrate at a fixed time point after initiation of the reaction. Alteration of the pH of the reactant streams allows the occurrence of both acid- and base-catalyzed processes to be probed 12,14,16,17,
Monomer subunits The selectivity of HO' attack on the monomer sub-units of hyaluronan (glucuronic acid and N-acetylglucosamine) has been investigated together with the possible rearrangement of the initial radicals in to secondary species at high and low pH values (i.e. base- and acid-catalyzed rearrangements respectively). Assignment of the signals detected with these materials has been made by comparison of the spectral parameters with previous data obtained for other sugars. 16- 19 At pH 4, where rearrangement of the initial radicals is slow, a-hydroxyalkyl radicals arising from hydrogen abstraction by HO' from nearly all the possible C-H bonds in the two monomers have been identified 12, 16, 18, 19. No radicals were detected from hydrogen atom abstraction from N-
Polysaccharide fragmentation
153
acetylglucosamine at the C-2 position, and from the methyl group of the N-acetyl function 12. Formation of the C-2 radical with N-acetylglucosamine may be inhibited by the neighboring N-acetyl function, while the absence of radicals from the terminal methyl radical of the acetyl group can be explained by the powerful deactivating effect of the neighboring carbonyl function. When analogous reactions were carried out at pH values < 4, all of these radicals disappear consistent with the occurrence of acid-catalyzed reactions which remove these species. However not all of these radicals rearrange at equal rates as indicated by the observed pH profiles. Thus the order of stability for glucuronic acid-derived radicals is: CS> C4> C2b> C3> 2a> Cl 12. With N-acetylglucosamine the observed radicals are more persistent, with no significant loss of signal intensity until pH values < 2 were employed; these radicals therefore undergo slower rearrangement reactions than those from glucuronic acid. With both monomers, experiments carried out at pH values> 4 also resulted in the loss of these initial radicals 12. Under these conditions basecatalyzed reactions are believed to occur to give serni-dione species 16. These secondary species have been directly detected in the case of glucuronic acid 12.
Scheme 1. Acid- and base-catalyzed rearrangement of 1,2-dihydroxyalkyl radicals The variable rate of loss of the glucuronic acid and N-acetylglucosamine-derived radicals (with the exception of the C-6 radical on N-acetylglucosamine which cannot undergo such a reaction), is consistent with the intermediacy of a radical-cation (see top part of Scheme 1), as the stability of this type of species is known to depend on the nature of the neighboring groups 20. Thus the presence of a powerful electronwithdrawing group -COO' in glucuronic acid would be expected to slow down the loss of the C-S radical, and the presence of the N-acetyl group to decrease the rate of loss of all the radicals in N-acetylglucosamine. Similar effects have been reported with other 1,2-diol radicals 20. The base-catalyzed rearrangement of these radicals which is believed to occur via a radical-anion species (see lower part of Scheme 1) is known to be less sensitive to substituent effects 16, and this accords with observed loss of primary radicals from both monomers at pH 7. Studies with an equimolar mixture of the monomers indicate that these are approximately equally susceptible to attack by HO'. Poly-saccharides In analogous experiments with hyaluronic acid and chondroitin sulfate A polymerderived carbon-centered radicals have been detected. These polymer-derived radicals
154
Characterisation and solution propertiesofhyaluronan
exhibited the same sensitivity to changes in pH as the monomer-derived radicals, implying that analogous rearrangement reactions of the type already described (see Scheme 1) also occur 12, 14, 17. The spectra obtained in the polysaccharide experiments have two predominant features that are consistent with the presence of the C-5 radical from glucuronic acid and the C-6 radical from N-acetylglucosamine 14. The predominance of these features has been proposed to reflect either the stability of these particular species or an unexpected selectivity of reaction of HO' with hyaluronan and other polysaccharides 14. Both possibilities are consistent with radiolysis studies (e.g. 8, 11) which have proposed that the yield of chain breaks approximates to that of the initial HO', and that this is due to two processes: a fast process and a slower, thermal process. The first explanation implies that essentially random attack is occurring at both monomer subunits, but that many of these primary radicals are undetected as a result of their rapid rearrangement to secondary species with concomitant fragmentation of the polymer. The predominance of the C-5 (glucuronic acid) and C-6 (Nacetylglucosamine) derived radicals in the observed spectra would then arise from the less rapid rearrangement of these species compared to all the other radicals (see above). This interpretation appears to be the most likely explanation, as it is difficult to rationalize firstly, the high yield of strand breaks if attack occurs predominantly at C-5 on glucuronic acid and C-6 on N-acetylglucosamine, and secondly, this selectivity in the light of the unselective attack of HO' on the corresponding monomers (see above).
Hydroxyl radical mediated attack: EPR spin trapping experiments EPR spin trapping allows unstable radicals to be detected via the formation of stable adduct species with an added organic agent, the spin trap. Structural information about the initial radicals present can be inferred from the EPR parameters of the adduct species. This methodology is therefore complementary to direct EPR studies, and can be advantageous when only small amounts of material are available and / or when the rate of radical generation is too low or too slow for direct detection. In these studies both nitroso spin traps (DBNBS and MNP) and nitrones (DMPO) have been employed.
Monomers, oligomers and homopolymers Experiments performed at pH 4 with both monomer subunits of hyaluronan resulted in the observation of signals consistent with trapping both the initial a-hydroxyalkyl radicals and secondary, rearranged radicals 12. At neutral pH values the signals from the primary radicals (assigned to a-hydroxyalkyl radical adducts) were suppressed, indicating that rapid, base-catalyzed rearrangement of these radicals is occurring 12. The initial radicals generated on N-acetylglucosamine have been shown to be more stable than those formed from glucuronic acid consistent with the rapid flow data reported above. In experiments with N-acetylchitotriose (a trimer of N-acetylglucosamine) similar behavior was observed 12.
Polysaccharidefragmentation
155
Hyaluronic acid and related poly-saccharides Broad signals from carbon-centered, polymer-derived, adducts were observed in experiments with all the polymers tested 12. Under anaerobic conditions, these radicals were present at higher concentrations. These observations are consistent with reaction of HO' with the polymer by hydrogen atom-abstraction to yield carbon-centered
(J..-
hydroxyalkyl radicals that subsequently react with Oz to form (undetected) peroxyl radicals (Rees et al unpublished data)12. With hyaluronan, two substrate-dependent signals were observed consistent with the trapping of both primary and secondary (rearranged) carbon-centered radical adducts 12. Raising the pH from 4 to 7.4 resulted in a change in the ratio of these species consistent with the occurrence of base-catalyzed rearrangement reactions, and the formation of low-molecular-weight fragments. Use of a Fe(II)-EDTA, in place of aquo-Fe(IT), in the redox couple with HzOz resulted in the observation of new features consistent with the presence of multiple radicals 12. At pH values> 4, these features were better resolved, and additional lines were observed, consistent with the presence of higher concentrations of low-molecular-weight species and hence a greater extent of fragmentation. This behavior probably arises from the higher fluxes of HO' formed with the Fe(IT)-EDTA / HzOz system. The major signals detected in the presence of EDTA are identical to those seen with the two monomers, consistent with the attack on both sugar rings. The additional species seen at pH values> 4 have also been detected with poly-N-acetylglucosamine, poly-galacturonic acid, and chondroitin sulfate A, and assigned to rearranged radicals. If this interpretation is correct, the observation of identical species with each polymer implies a common fragmentation pathway for all three polysaccharides, The changes in the spectra, particularly in the increased yield of low-molecular-weight material, as the ligand is changed and the pH raised from 4 to 7, suggest that both pH-dependent (base-catalyzed) and pH-independent processes give rise to fragmentation (strand scission) 12. Further information on the radicals generated from these polysaccharides, has been obtained by enzymatic digestion of the spin-adducts 21. Incubation of the adducts detected with hyaluronic acid with either bovine testicular or leech hyaluronidase (which cleave the [~-I,4] bond between glucuronic acid and N-acetylglucosamine, and the [~-1,3] bond between N-acetylglucosamine and glucuronic acid, respectively) resulted in the loss of the features of the polymer-derived radicals and the detection of low-molecular-weight radicals. The species formed as a result of this treatment were similar to those observed with each monomer, confirming that attack occurs on both sugar rings 12. These data are consistent with an important role for acid- and base-catalyzed radical rearrangements in HO'
156
Characterisation and solution properties ofhyaluronan
suggests that non-pH dependent processes also playa role; these may involve ~-scission reactions 8. 11 of, for example, the C-I radical on the glucuronic acid ring, or analogous reactions involving radicals at C-4 on the glucuronic acid ring, or C-I or C-3 on the Nacetylglucosamine ring.
HOCI-mediated oxidation HOCI reacts slowly with mono-, and poly-saccharides in the absence of amine or amide functions 22. The presence of the N-acetyl function on the glucosamine groups in hyaluronan and related biopolymers increases the rate of reaction, and also alters the nature and selectivity of HOCI reaction. HOCI is known to react rapidly with amine groups to generate chloramines (RNHCl species) 23, and recent studies have suggested the formation of chloramides (RC(O)NClR') from amide groups (RC(O)NHR') can also occur 13. Chloramines and chloramides are well-established sources of radicals in synthetic organic chemistry as they can generate nitrogen-centered radicals by either homolysis or one-electron reduction of the N-Cl bond (reaction 3) 24. The role of such radical formation in aqueous solution under physiological conditions has received limited attention. Nitrogen-centered radicals are known to readily abstract hydrogen atom (either inter- or intra-molecularly) from C-H bonds, in a manner analogous to other radicals 24. The generation of chloramides on N-acetyl groups in mono- and polysaccharides might therefore be expected to give rise to radical formation and polymer oxidation.
Formation ofchloramides on mono- and poly-saccharides Studies on the reaction of HOCI with various saccharide derivatives, model amides and amines have shown that in the absence of the amine or amide function, reaction is slow. Thus glucuronic acid reacts much less rapidly than N-acetylglucosamine, Nacetylgalactosamine or N-acetylmannosamine. With N-acetylglucosamine HOCI reacts selectively at the N-acetyl function in ca. 70% yield. Glucosamine gives a similar yield of the analogous chloramine. The decay of these N-Cl species appears to be first order (within experimental error), with the chloramine decaying much rapidly than the chloramide (Rees et ai, unpublished data). The rate of reaction of HOCI with amides decreases with increasing steric bulk adjacent to the nitrogen center. Thus secondary acetamides (e.g. those present on N-acetylglycosamines) are intermediate in reactivity between acetamide and t-butylacetamide (Rees et ai, unpublished data) 13. Reaction of HOCI with chondroitin sulfate A, chondroitin sulfate C and hyaluronan is slower than with the N-acetylglycosamine, presumably as a result of steric factors, with the tertiary structure of the polymer modulating the reactivity of the N-acetyl group. The decay kinetics of the polymer-derived chloramides are also slower than those observed with
Polysaccharide fragmentation
157
the monomers (which decays in < 6 hours), with polymer-bound species still detectable after 5 days (Rees et aI, unpublished data).
EPR spin trapping experiments The formation of radicals on decay of chloramines and chloramides has been studied using EPR spin trapping. With model acetamides pretreated with HOCI, radical adducts
were observed which have been assigned, in the majority of cases, to carbon-centered radicals formed at the position adjacent to the amide nitrogen (Rees et aI, unpublished data) 13. These carbon-centered radicals are believed to arise via the initial formation of a nitrogen-centered radical that subsequently undergoes a rapid 1,2-hydrogen shift (i.e. migration of the hydrogen from the neighboring carbon to the nitrogen) (Rees et al, unpublished data) 13. With most substrates, the initial nitrogen-centered radicals were not observed, though these species have been detected with the chloramide from tbutylacetamide (as this compound cannot undergo a 1,2-shift reaction), and possibly ipropylacetamide, where absorptions tentatively assigned to the nitrogen-centered species were observed (Rees et al, unpublished data). With N-acetylglucosamine both the initial nitrogen-centered radical and a carboncentered radical have been detected. The carbon-centered radical is postulated to be the C-2 species, as couplings to both a single hydrogen and a nitrogen atom are detected, with this species formed via Scheme 2. Similar carbon-centered radicals have been detected in experiments with the C-3 and C-6 sulfated analogues of N-acetylglucosamine, and with N-acetylglucosamine-I-phosphate 13. The concentration of the radical detected with N-acetylgluocamine was not dependent on the concentration of unreacted substrate or on added glucose, both potential targets for inter-molecular hydrogen atom-abstraction, supporting the hypothesis that this secondary carboncentered radical is formed intra-molecularly. The concentration of this was radical was however enhanced in anoxic conditions, indicating that this carbon-centered radical undergoes ready reaction with O2 to give an (undetected) C-2 peroxyl radical (Rees et al, unpublished data). H
H
1,2-hydrogen shift OH,H
H
amidyl radical
.. C-2 radical
OH,H H
Scheme 2. 1,2-hydrogen shift reaction of the initial amidyl radical. Reaction of HOCI with N-acetylmannosamine (i.e change in stereochemistry at C-2) and N-acetylgalactosamine (change in stereochemistry at C-4) gave identical signals to those from the carbon-centered radical observed with N-acetylglucosamine. This suggests that changes in stereochemistry at C-2 and C-4 have no effect on the radicals generated (Rees et al, unpublished data) 13.
158
Characterisation and solution propertiesofhyaluronan
In contrast, N-acetylmuramic acid gave rise to three different radicals at approximately equal concentrations; one of these species is consistent with hydrogen abstraction from the C-3 side chain, possibly via a stereochemically favorable intramolecular 1,S-hydrogen shift. Different behavior was also observed with the open-chain compound N-acetyl-glucosaminitol were signals consistent with hydrogen-atom abstraction at sites other than the C-2 position were observed. The concentration of the radicals detected in this system was found to be dependent on the concentration of unreacted substrate, and was also found to be increased in the presence of added glucose. This is consistent the observed carbon-centered radicals being generated by inter-molecular hydrogen-abstraction by the initial nitrogen-centered species. These observations imply that the ring structure modulates the reactivity of the initial nitrogencentered radicals. This may be due to steric impedance of inter-molecular reaction, or a more rapid 1,2-shift of the C-2 hydrogen to the amidyl radical (Rees et al, unpublished data). Analogous experiments with glucosamine, where a chloramine is formed in place of the chloramide, resulted in the detection of carbon-centered radicals believed to be formed via inter-molecular hydrogen atom-abstraction from multiple sites on the sugar ring by the initial nitrogen-centered radical. At least some of the observed species are secondary, carbonyl-conjugated, species analogous to those detected on reaction of sugars with HO' (see above). With this substrate', oxygen-centered radicals were also detected, which were quenched by superoxide dismutase, suggesting that Oz-' is being formed. This radical is believed to arise via addition of o, to either an a-hydroxyalkyl or an a-aminoalkyl radical to give a peroxyl radical and subsequent rapid elimination of O,", This adduct was not detected under anaerobic conditions, and was not detected with N-acetylglucosamine (Rees et al, unpublished data). Treatment of hyaluronan or chondroitin sulphate A with low concentrations of HOCl, has been shown to give polymer-derived, carbon-centered, radicals 13. With higher HOCI concentrations signals from low-molecular-weight species were also observed consistent with an increasing extent of polymer fragmentation. The latter signals are identical to some of the species detected with N-acetylglucosamine, suggesting that reaction of HOCI with hyaluronan also occurs with the N-acetyl groups of the N-acetylglucosamine rings in the polymer with subsequent formation of carboncentered species and polymer fragmentation 13. CONCLUSIONS Both HO' and HOCI induce the formation of radicals on mono- and polysaccharides and bring about polymer fragmentation. EPR spectroscopy has been shown to be a powerful tool for the identification of radicals formed on these targets, and the elucidation of fragmentation mechanisms. Evidence has been presented for the
Polysaccharide fragmentation
159
occurrence of essentially random attack of HO" on mono-saccharides, and indirect evidence for the occurrence of similar processes with poly-saccharides. These reactions yield o-hydroxyalkyl radicals that undergo both acid- and base-catalyzed reactions to give carbonyl-conjugated species. In contrast, HOCI selectively oxidises the N-acetyl ring via the formation of chloramide, which subsequently decay to give a nitrogencentered radical. The latter can subsequently abstract hydrogen atoms from other C-H bonds, both intra- and inter-molecularly, and bring about polymer fragmentation.
ACKNOWLEDGMENTS The authors are grateful to the Australian Research Council for financial support.
REFERENCES 1. R. A. Miller & B. E. Britigan, 'Formation and biologic significance of phagocytederived oxidants', J. Invest. Med., 1995,43, 39-49. 2. A. J. Kettle & C. C. Winterboum, 'Myeloperoxidase: a key regulator of neutrophil oxidant production', Redox Report, 1997,3,3-15. 3. B. Halliwell & J. M. C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University Press, Oxford, 1999, 36-95. 4. H. A. Rowe & W. D. Wagner, 'Arterial dermatan sulfate proteoglycan structure in pigeons susceptible to atherosclerosis', Arteriosclerosis, 1985,5,101-109. 5. J. M. C. Gutteridge, 'Bleomycin detectable iron in knee joints from arthritic patients', Biochem. J., 1987,245,415-421. 6. S. W. Edwards, V. Hughes, J. Barlow & R. Bucknall, 'Immunological detection of myeloperoxidase in synovial fluid from patients with rheumatoid arthritis.', Biochem. J., 1988,250,81-85. 7. A. Daugherty, J. L. Dunn, D. L. Rateri & J. W. Heinecke, 'Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions', J. Clin. Invest., 1994,94,437-44. 8. C. von Sonntag, 'Free Radical Reactions of Carbohydrates as Studied by Radiation Techniques', Adv. Carbohydr. Chem. Biochem., 1980,37, 7-77. 9. M. S. Baker, S. P. Green & D. A. Lowther, 'Changes in the viscosity of hyaluronic acid after exposure to a myeloperoxidase-derived oxidant', Arthritis Rheum., 1989, 32,461-7. 10. S. P. Green, M. S. Baker & D. A. Lowther, 'Depolymerization of synovial fluid hyaluronic acid (HA) by the complete myeloperoxidase (MPO) system may involve the formation of a HA-MPO ionic complex', J. Rheumatol., 1990,17,1670-5. 11. B. J. Parsons, 'Chemical aspects of free radical reactions in connective tissue', In: Free Radical Damage and its Control. C. A. Rice-Evans, and R. H. Burdon (eds.), Elsevier Science, Amsterdam, 1994,281-300.
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Characterisation and solution propertiesof hyaluronan
12. C. L. Hawkins & M. J. Davies, 'Direct detection and identification of radicals generated during the hydroxyl radical-induced degradation of hyaluronic acid and related materials', Free Rad. Biol. Med., 1996,21,275-290. 13. C. L. Hawkins & M. J. Davies, 'Degradation of hyaluronic acid, poly- and monosaccharides, and model compounds by hypochlorite: evidence for radcial intermediates and fragmentation', Free Rad. BioI. Med., 1998,24, 1396-1410. 14. S. Al-Assaf, C. L. Hawkins, B.J. Parsons, M. J. Davies & G. O. Phillips, 'Identification of radicals from hyaluronan (hyaluronic acid) and cross-linked derivatives using electron paramagnetic resonance spectroscopy', Carbohydr. Polymers, 1999,38, 17-22. 15. B. C. Gilbert, 'Applications of electron spin resonance spectroscopy to the study of free radicals', Essays in Chemistry, 1972,4,61-89. 16. B. C. Gilbert, D. M. King & C. B. Thomas, 'Radical reactions of carbohydrates. Part 3. An electron spin resonance investigation of base-catalyzed rearrangements of radicals derived from D-glucose and related compounds', 1. Chem. Soc. Perkins Trans. 2, 1982, 169-179. 17. B. C. Gilbert, D. M. King & C. B. Thomas, 'The oxidation of some polysaccharides by the hydroxyl radical: an E.S.R. investigation', Carbohydr. Res., 1984, 125,217235. 18. B. C. Gilbert, D. M. King & C. B. Thomas, 'Radical reactions of carbohydrates: E.S.R. study of oxidation of D-glucose and related compounds with the hydroxyl radical.', J. Chem. Soc., Perkin Trans. 2,1981, 1186-1199. 19. B. C. Gilbert, D. M. King & C. B. Thomas, 'Radical reactions of carbohydrates: E.S.R. studies of radical induced oxidation of some aldopentoses, sucrose and compounds containing arabinose.', J. Chem. Soc., Perkin Trans. 2, 1983,675-683. 20. S. Steenken, M. J. Davies & B. C. Gilbert, 'Pulse radiolysis and E.S.R. studies of the dehydration of radicals from 1,2-diols and related compounds', J. Chem. Soc., Perkin Trans. 2, 1986, 1003-1010. 21. M. J. Davies, 'Detection and identification of macromolecule-derived radicals by EPR spin trapping', Res. Chem. Intermed., 1993, 19,669-679. 22. R. L. Whistler & R. Schweiger, 'Preparation of D-arabinose from D-glucose with hypochlorite.', J. Am. Chem. Soc., 1959,81,5190-5192. 23. C. C. Winterbourn, 'Comparative reactivities of various biological compounds with rnyeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite', Biochim. Biophys. Acta, 1985,840,204-210. 24. J. Fossey, D. Lefort & J. Sobra, Free radicals in organic chemistry, Wiley, Chichester, 1995,1-307.
PART 3 THE FUNCTION AND USE OF HYALURONAN IN WOUND HEALING
VISCOPROTECTION: A HISTORICAL PERSPECTIVE Biomatrix Inc. 65 Railroad Avenue. Ridgefield. NJ. USA
ABSTRACT
The protective effect of elastoviscous hyaluronan solutions on the surface of the eye was discovered during the late 1970s. This protective effect was evidenced by a decrease of pain and other senses of discomfort caused by desiccation of the corneal surface or by irritation from environmental factors. Later, when elastoviscous hyaluronan solutions became available for ophthalmic viscosurgery, such solutions were frequently used to prevent desiccation of the cornea during surgical procedures and for treatment of painful "dry eye syndrome". The availability of elastoviscous solutions of high molecular weight hyIan with high elastic properties made their use in dilute solutions (0.1 - 0.45%) for these purposes extremely attractive. Experiments showed that the predominately elastic behavior of hylan solutions at frequencies corresponding to the speed of the opening and closing of the eyelid results in a substantially longer residence time on the surface of the eye and consequently longer lasting protective effect than solutions with less elastic properties. In recent years, it was discovered that extended viewing of television or computer screens significantly decreased the frequency of blinking, causing a sensation of muscular fatigue and discomfort due to desiccation of the eye surface. This discomfort could be decreased by application ofhighly elastoviscous hylan solutions. DEFINITION VISCOPROTECTION: Elastoviscous fluids and viscoelastic gels shield and protect sensitive tissue surfaces, such as those of the eye, from dryness and noxious environmental conditions. HISTORICAL REVIEW
1974 - 1976: Highly purified high molecular weight hyaluronan (Healon", Biotrics, Inc., Arlington, MA, USA) is tested to protect the surface of the eye and provide comfort in severe dry eye conditions'. 1982: The first clinical study to evaluate a 0.1 % solution of Healon@ in patients with severe keratitis sicca. It was shown to relieve pain immediately after each application with a prolonged resolution of symptoms", 1984: Evaluation of 0.1% hyaluronan solution (Healon@, Pharmacia AB, Uppsala, Sweden) in dry eye syndromes and other ocular discomfort. Objective and subjective measurements showed improvement':".
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The function and use ofhyaluronan in wound healing
1990: Quantitative gamma scintigraphy showed that the mean ocular residence time for hyaluronan solutions was significantly increased as compared to buffered saline solutions. This improvement was dependent on the rheological properties of hyaluronan solutions'. 1993 - 1996: Studies showed the utility of 0.1% hyaluronan in the treatment of patients with dry eye conditions and patients wearing hard contact lenses. These studies confirmed improvement ofboth subjective and objective symptoms of discomforr'". 1994: Hylan A fluid with a very high molecular weight (avg. MW ~ 6 million), a derivative of hyaluronan, is tested for residence time on the corneal surface. Due to the high elasticity of the hylan A fluid at the frequency of blinking movement, its residence time is long, resulting in a less frequent need for application in dry eye conditions than commercial eye drops. Later it is marketed in Canada (Hylashield®, Biomatrix Medical Canada Inc., Point-Claire, Quebec, Distributor: I-MED Pharma Inc., Montreal, Quebec)8,9. 1995: Hyaluronan-containing (avg. MW < 2 million) comfort drops are available in Japan and Europe to patients for treating discomfort caused by wearing contact lenses and by dry eye syndrome. 1997: Elastoviscous hylan A fluid (HsS®, Biomatrix Medical Canada Inc., Point-Claire, Quebec) is tested as a corneal wetting agent during cataract extraction and lens implantation and found significantly more effective than physiologicalsalt solutions'", 1999: It was reported that the healthy blinking frequency (- 12 blinks per minute) significantly decreases (- 40%) during strong visual attention associated with video display terminals. This decrease of blinking frequency causes discomfort which is attenuated by such environmental conditions as dryness and high temperature. Eye drops containing highly elastoviscous hylan A reduce this discomfort more effectively than non-elastoviscous fluidII . 1999:
Elastoviscous eye drops containing hylan A are marketed in Europe
(Lcom" Comfort Shield, by MDLE, Medical Device Laboratories Europe GmbH,
Memmingen, Germany and Biomatrix, Inc., Ridgefield, NJ, USA). 2000: Hylashieldf CL (Biomatrix, Inc.), the first hylan-containing eye drops, become available in the USA, for patients wearing rigid gas permeable contact lenses.
Viscoprotection: a historical perspective
117
10
Hylasbield •
1. Lcom'IMComfortShldd
I I I I F~c:y
O.OJ
I I I
1
10
15
frequency, (Hz)
FIGURE 1: Comparison of the Elastic Modulus of Some Elastoviscous Eye Drops RHEOLOGICAL CONSIDERATIONS Figure 1 shows the elastic (G' storage modulus) as function of frequency from 0.1 to 10Hz. Note that the blinking movement of the eyelid corresponds to 5-10 Hz at which all hyaluronan- or hyian A-containing fluids have various levels of elastic properties. The elastic properties of these eye drops depend on the concentration and average molecular weight of the hyaluronan or hyian in the fluids. The greater the elasticity at the range of frequency that corresponds to the speed of the blinking movement of the eyelid, the more likely that the eye drop will not be removed by the blinking. Consequently the residence time of the elastoviscous fluid is longer on the cornea, meaning that fewer applications of the eye drop are required. Eye drops that contain hyaluronan or hylans are not artificial tears. They are elastoviscous fluids providing protection (by viscous lubrication), prolonged residence time (by elastic properties) and hydration (by water retention) to the corneal surface. SOME BYALURONAN-CONTAINING EYE DROPS AVAILABLE Vislube" by Chemedica AG Hylo-comod" by Ursapharm Arzneimittel GmbH & Co. KG Fermavisc'" by Ciba Vision RYLAN A-CONTAINING EYE DROPS AVAILABLE Hylashield'" distributed by I-MED Pharma Inc., Montreal, Quebec in Canada. i.com" Comfort Shield distributed by MDLE, Medical Device Laboratories Europe GmbH, Memmingen, Germany in Europe (developed and manufuctured by Biomatrix, Inc.)
118
The function and use of hyaluronan in wound healing
REFERENCES 1. J. L. Denlinger, Biotrics, Inc., Report, 1976. 2. F. M. Polack and M. T. McNiece, The treatment of dry eyes with Na-hyaluronate (Healon~, Cornea, 1982, 1, 133-136. 3. V. P. DeLuise and W. S. Peterson, The use of topical Healon" tears in the management of refractory dry-eye syndrome, Ann. Ophthalmol., ll984, 823-824. 4. J. C. Stuart and J. G. Linn, Dilute sodium hyaluronate (Healon) in the treatment of ocular surface disorders, Ann. Ophthalmol., 1985,17,190-192. 5. G. R. Snibson, J. L. Greaves, N. D. W. Soper, 1. L. Prydal, C. G. Wilson and A. J. Bron, Precorneal residence times of sodium hyaluronate solutions studied by quantitative gamma scintigraphy, Eye, 1990,4,594-602. 6. T. Hamano, K. Horimoto, M. Lee and S. Komemushi, Evaluation of the effect of sodium hyaluronate ophthalmic solution on corneal desiccation due to hard contact lens wear, J. ofthe Eye, 1993, 10,627-630. 7. M. Itoi, 0. Kim, T. Kimura, A. Kanai, T. Momose, K. Kanki, T. Yamaguchi. Y. Ueno, M. Kurokawa and S. Komemushi, The effect of sodium hyaluronate ophthalmic solution on corneal epithelial disorders in contact lens wearers, J. of the Eye, 1993,10,617-626. 8. N. E. Larsen and E. A. Balazs, Hylashield (2.0 Pa elastoviscous hylan fluid 0.15%) protective corneal shield: evaluation of biological and physical properties, Ophthalmic Practice, 1994, 12,137-140. 9. S. Arshinoff, S. Fichman, M. Laflamme, J. Rosen, S. Holland and I. Hofmann, Comparative, double-masked, crossover study evaluating the effectiveness of Hylashield" versus placebo eye drops in keratoconjunctivitis sicca, Presented at: ARVO, Fort Lauderdale, Florida, USA, May 1999. 10. S. A. Arshinoff and E. Khoury, HsS versus a balanced salt solution as a corneal wetting agent during routine cataract extraction and lens implantation, J. Cataract Refract. Surg., 1997,23, 1221-1225. 11. M. C. Acosta, 1. Gallar and C. Belmonte, The influence of eye solutions on blinking and ocular comfort at rest and during work at video display terminals, Exp. Eye Res., 1999,68,663-669.
GETTING TO GRIPS WITH HA-PROTEIN INTERACTIONS Charles D. Blundell l ,2, Jan D. Kahmann', Andras Perczel", David J. Mahoney', Martin R. Cordell l , Peter Teriete':', lain D. Campbelf & Anthony J. Day*l,2. MRC Immunochemistry Unit, 2Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OXI 3QU, UK.
J
J
Department of Organic Chemistry, Eotvos University, PO Box 32. Budapest 112, H-1518, Hungary,
ABSTRACT The interactions between HA and proteins are most commonly mediated by a domain termed a Link module. The Link module from human TSG-6, produced by expression in E. coli, has been used previously to determine its tertiary structure and identify the position of the HA-binding site by NMR spectroscopy in solution [1,2]. In addition, isothermal titration calorimetry (ITC) has been used to characterize the thermodynamics of this HA-protein interaction [2]. Microtitre plate assays have shown that the binding of the TSG-6 Link module to HA has a pH-dependency that is distinct from that of other hyaladherins; with maximal binding at pH 6.0 and a dramatic loss of function with increasing pH [3]. The NMR/ITC studies were carried out under low salt conditions (-2 mM NaCI), whereas the microtitre plate assays were performed in 100 mM NaCI. Here we show that the interaction of the TSG-6 Link module with HAg is saltstrength dependent, involving the formation of 1 or 2 salt bridges. However, the structure of the Link module, its folding and the position of the HA-binding surface are the same in the absence and presence of NaCl. Therefore, results from the microtitre plate assays and NMRIITC studies are comparable. The unusual pH-dependency of the HA interaction is probably mediated by the change of ionization state of one or more histidine residues, the pKa values of which are relatively unaffected by salt.
KEYWORDS TSG-6, Link module, HA-protein interactions, isothermal titration calorimetry, N:MR
INTRODUCTION The interactions of HA with HA-binding proteins are usually mediated by a domain of about 100 amino acids, termed a Link module or proteoglycan tandem repeat. This module has been described in extracellular matrix proteins (e.g., link protein and
162
Characterisation and solution propertiesof hyaluronan
A
B Y78
N
Figure 1. A) The Link module fold consists of 2 a helices and 613 strands [1]. B) Atomic representation of Link_TSG6 in which amino acids that are significantly perturbed on HAs binding are shown in black [2]. The majority of these are located on the loop (residues 61-74) between the 134 and 135 strands. aggrecan), the HA-receptor CD44 and TSG-6 (the product of the tumour necrosis factor-stimulated gene-6)[4]. TSG-6 is a 35 kDa protein, containing a single Link module, secreted by chondrocytes, monocytes and vascular smooth muscle cells in response to inflammatory mediators and growth factors [5]. Previously, we have expressed the Link module from human TSG-6 in E. coli [6,7] and used this recombinant material (denoted here as Link_TSG6) to determine its tertiary structure by NMR spectroscopy in solution [1]. As can be seen from Fig. 1 the Link module is comprised of two a helices and two triple-stranded B sheets and this has defined the consensus fold for this domain type. Microtitre plate assays revealed that the interaction between HA and Link_TSG6, in contrast to other hyaladherins, is highly pH-dependent [3]; we have hypothesized that pH-gradients may regulate TSG-6 function in vivo e.g., in inflamed cartilage. Fig. 2 shows there is maximal binding at pH 6.0 and a large reduction in Link_TSG6 activity at lower and higher pH. One-dimensional NMR spectroscopy has demonstrated that the Link module is unfolded at pH 3.5, with an increasing proportion of folded protein as the pH is raised (i.e., HA binding correlates with protein folding between pH 3.5 and 6.0 [3]). However, there is no gross change in the Link module structure between pH 6.0-8.5. Recently, we have used isothermal titration calorimetry (ITe) to characterize the interaction of Link_TSG6 with HA oligosaccharides of defined length [2]. This showed that oligomers of HA 6 to HA 16 all bind with K, values ranging from 0.2-0.5 ).l.M with favourable enthalpies and entropies at 2ye. In addition, the position of the HAs
HA-protein interactions
163
120
----TSG-6 OJ)
:::
100
:.a
- . - CD44 ----.-- Aggrecan --'1'--- Link protein
:.§ 80
E
E 60
';(
a
4-0
o
<s'<
40 20
0.1-11:--..,-------,..-----,------,.-----.-7 4 5 6 8
pH
Figure 2. The pH-dependency of the interaction between Link_TSG6 and HA is distinct from CD44 (A. A. Parkar, S. Banerji, D. G. Jackson & A. J. Day, unpublished data), aggrecan and link protein [3]. binding site on Link_TSG6 was localized by comparing NMR spectra from a 1: I complex and free protein (see Fig. I). The microtitre plate assays were carried out in 100 mM NaCl, whereas, the NMR and ITC studies were performed in -2 mM NaCI. Therefore, the results of these experiments may not be directly comparable. Here we have investigated the effect of salt on the structure and HA-binding activities of Link_TSG6.
METHODS Preparation of protein and HA oligosaccharides Link_TSG6 (with or without uniform 15N-labelling) was expressed and purified as described before [2,6,7]. HA oligomers were prepared by digestion of human umbilical cord HA with ovine testicular hyaluronidase and purified by gel filtration and ion exchange chromatography (D. J. Mahoney & A. J Day, manuscript in preparation).
Isothermal titration calorimetry The interaction between Link_TSG6 and HAs was investigated at different Na" concentrations (0-100 mM) on a MicroCal VP-ITC instrument at 25'C in 5 mM NaMES, pH 6.0 with NaCI at 0, 20, 45, 70 or 95 mM. HAs was added in 5 ul injections (28 in total) to Link_TSG6 in the 1.4 ml calorimeter cell. The protein and HA concentrations (determined as in reference [2]) were varied (see Table 1) in order to produce sigmoidal titration curves. Data were fitted to a one-site model by nonlinear
164
Characterisation and solution propertiesofhyaluronan
least-squares regression with the Origin software package, after subtracting the heats resulting from addition of HAs into buffer alone as described previously [2]. NMR spectroscopy
NMR spectra were recorded on a homebuilt/GE Omega spectrometer operating at a proton frequency of 500 MHz unless otherwise stated. All samples contained 1 mM 15N-labelled Link_TSG6 in 10% (v/v) DzO, 0.02% (w/v) NaN] and adjusted to the desired pH with NaOH (-2 mM NaCI final concentration). 10 spectra (at 3YC) were collected for Link_TSG6 in 100 mM NaCI at pH 3.5-8.5. lH_ 15N_HSQC datasets were acquired at 25°C in -2 mM (600 MHz) or 100 mM NaCI at pH 6.0-7.5 (in increments of 0.25 pH units). High-resolution lH_15N_HSQC experiments in 100 roM NaC!, pH 6.0 were performed at 2YC in the absence or presence of 1 mM HAs. Spectra were processed in Felix 2.3 (Biosym), referencing protons to Hp at 4.74 ppm as before [2].
RESULTS & DISCUSSION The effect of NaCI on the interaction between Link_TSG6 and HAs
ITC was used to investigate the interaction between HAs and Link_TSG6 over a range of NaCI concentrations (0-95 mM). Figure 3 shows the data derived from experiments at the lowest and highest salt-strengths. From Table 1 it can be seen that the free energy (L1bG) is NaCI-dependent and a plot of InKb vs In[Na+] shows a linear correlation (see Fig. 4); as the salt concentration increases there is a smaller entropic gain from the release of ions. These data can be fitted to a general model for the effects of interacting solutes on K, (see equation 15 in reference [8]), assuming that only Na' ions (and not Cl) are involved in the formation of the HA s/Link_TSG6 complex. This seems reasonable given that HA is a polyanion. The gradient of the InKb vs In[Na+] plot (-1.42) indicates that on average 1.4 Na" ions are displaced from HAs on binding Link_TSG6; the contribution from the release of water molecules is negligible at the salt concentrations used here. Therefore, the Link_TSG6/HAs interaction is likely to involve the formation of 1-2 salt-bridges. The y-intercept (Fig. 4) corresponds to L1bG at 1 M Na" concentration, where the ionic contribution is considered to be negligible [9]. Therefore, this value (L1bG =-19.8 kl/mol) represents the non-ionic (salt-independent) component of the binding. By extrapolation, it can be calculated that ionic interactions contribute 25% of the free energy of binding at physiological Na" ion concentrations (150 mM). Therefore, while it is clear that ionic associations are important, non-ionic interactions (e.g., hydrogen bonding and van der Waals forces) also contribute a significant proportion to the binding energy. This is consistent with our data derived from site-directed mutagenesis on Link_TSG6 showing that in addition to a lysine, 3 tyrosines and a phenylalanine are
HA-protein interactions
165
involved in binding to HA (D. J. Mahoney, 1. D. Campbell & A. J. Day, manuscript in preparation).
Tbne(min)
o
20
40
60
Time (min)
80
100
o
120
20
40
60
80
100
120
o
0.0
-0.1
~
-2
1-0.2 -0.3
o
•
j
-2
.:; -2
.a
'a
~ -4
i
-4
-6
0.0
0.5
1.0
1.5
2.0
2.5
-6 4---,----.,....-,..---.----,-..----,-----.----l 0.0 0.5 1.5 2.0 1.0
3.0
MoluRado
MolarRado
Figure 3. The top panels show the raw ITC data for the interaction between HAs and LinlcTSG6 at 0 roM (left) and 95 roM (right) NaC!. The lower panels
show the corresponding titration plots for the integrated raw data and the leastsquares best fit (X2 of 15,030 and 777, respectively).
Table 1. Thermodynamiccharacterization of the interaction between Link_TSG6 and HAs at different Na' ion concentrations.
(Nal [LinkJSG6] [HAs]
Stoichiometry
x, xI0
4M'!
mM
mM
mM
n
5
0.010
0.25
1.04±O.01
25
0.010
0.25
0.88±O.02
6I.53±5.99
50
0.020
0.34
1.03±O.01
25.21±1.42
75
0.061
1.03
0.98±O.01
12.98±0.52
100
0.183
3.09
1.00±O.00
5.61±0.11
483.20±46.35
x,
~bG
x10-7M kJ/mol 2.07
~bH
-T~bS
kJ/mol
kJ/mol
-38.1
-25.9±O.3 -12.2
16.25
-33.0
-29A±O.9
39.67
-30.8
-25.I±OA
-5.7
77.04
-29.2
-25.6±O.2
-3.6
178.25
-27.1
-25.0±O.1
-2.1
-3.6
166
Characterisation and solution properties ofhyaluronan 16.0 15.0 14.0 13.0 12.0 11.0
InKb =7.99 - 1.42 x In[Nal
•
10.0 +-----r---,---.-----r----r---.....,.-----,
-5.5
-5.0
-4.5
-4.0
-3.5
-3.0
-2.5
-2.0
In[Nal Figure 4. A plot of
ln~
vs In[Na+] for the interaction of HAgwith LinlcTSG6.
The effect of NaCI on the Link_TSG6 structure and its HAs binding surface One-dimensional NMR spectroscopy was used to investigate the effect of pH on the protein structure in the presence of 100 roM NaCI (data not shown). At pH 3.5 the Link module is unfolded, but as the pH is raised there is an increasing proportion of folded protein that becomes maximal at about pH 6.0. Between pH 6.0 and 8.5 the protein remains fully folded. Therefore, the increase in HA-binding activity between 3.5 and 6.0 (Fig. 2) correlates with the protein folding, whereas the loss of activity seen on altering the pH from 6.0-8.0 is unlikely to be due to a gross structural change in the protein structure. These results are essentially identical to those reported previously in the absence of NaCl [3]. High-resolution IH-1sN-HSQC spectra (at pH 6.0) were used to detect chemical shift differences (~8), in the amide resonances of the free protein or the Link_TSG6/HAg complex, between samples in either -2 roM NaCl (as determined previously [2]) or 100 mM NaCl. The only significant perturbations observed are for the NH of Tyr-33 that exhibit ~8s of 0.92 ppm and 0.59 ppm in the free and complexed protein, respectively. This may be due to the differential binding of a Cl' ion to a basic amino acid (e.g., Arg56) in the proximity of Tyr-33. However, this is unlikely to be of functional relevance since Tyr-33 is on the opposite face of the protein from the HA-binding surface [2]. In this regard, these experiments show (Fig. 5) that the amino acids perturbed on HAg binding in 100 roM NaCl are essentially identical to those we have identified previously under low salt conditions [2]. Therefore, we can conclude that the structure of Link_TSG6, its folding (between pH 3.5-6.0) and the position of the HA-binding surface are not significantly affected by the presence of 100 roM NaCl.
HA-protein interactions
167
I ~~C_~~J_:_~a~:: II ; ;':~__;;I:__; ~ ,.~ r~' ~~,~",~" ~r"r ',•.",. ~r'•.~ .' ,~r'~, itlrJ . l____________
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11
21
31
41
51
61
71
81
91
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51
61
71
81
91
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r----------------------------,-,
0.0
I ••
11
21
31
1
41
r-r-r-r-r-r-r-
51
r-r- r-r-r-
61
71
81
• •"
91
Residue number Figure 5. Chemical shift differences (ilo) between free protein and the Link_TSG61HAg complex, plotted against residue number, determined for backbone NH and RN in either -2 mM [2] or 100 mM NaC!. Dashed lines represent cut-off levels chosen to indicate a significantly perturbed residue (R N ;;:: 0.2 ppm, NH ;;:: 1.0 ppm); see Kahmann et al., 2000 [2] for further details.
168
Characterisation and solution properties of hyaluronan
The effect of pH on the Link_TSG6 structure IH_ 15N_HSQC datasets were acquired between pH 6.0-7.5 (in -2 roM and 100 mM NaCl) to see if a subtle change in Link_TSG6 structure could account for the loss of HA-binding activity over this pH-range. This NMR experiment monitors NH resonances (of backbone amides and some amino acid sidechains) which are very sensitive to changes in chemical environment. In Figure 6, equivalent regions from the spectra are superimposed to illustrate the effect of pH on NH chemical shifts. The positions of the indole proton resonances from Trp-51 and Trp-88 are unaffected by pH. Since these amino acids have an important role in stabilising the Link module structure [1] they would be influenced by any conformational change that affected the protein core. In addition, the majority of NH resonances have very similar chemical shifts, in both IH_ and 15N-dimensions, at all the pH values investigated (e.g., see Ala-31, Met-52
II
-2mMNaCI
M52 Y93
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o
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o ()')
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o C\l
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10.0 9.5 'H (ppm)
10.0 9.5 'H (ppm)
Figure 6. An overlay of IH_ 15N_HSQC NMR spectra acquired for 15N-labelled Link_TSG6 at pH values ranging from 6.0-7.5 in the presence of -2 roM and 100 roM NaCl. These spectra were acquired at 600 and 500 MHz, respectively, and this is the principle cause of the differences in line shapes observed.
HA-protein interactions
169
Table 2. Amino acid residues that exhibit significant amide chemical shift perturbations with increasing pH (6.0 to 7.5) in -2 roM and 100 roM NaCl.
Amino acid"
Val-2 Tyr-3 His-4 Arg-5 Glu-6 Ala-7 Lys-ll Tyr-12 Lys-13 Leu-14 Thr-15 Cys-23 Gly-28 His-29 Leu-30 Gln-35 (sc) Ala-48 Ala-49 Asn-67 (bb) Asn-67 (sc) Ala-90 Tyr-91 Tyr-93 Asn-94 (bb) Asn-94 (sc) His-96 Ala-97
Chemical shift change" Secondary (-2 roM NaCl) (100 roM NaCl) structure
++++ ++ ++++ ++ +++ ++ ++ ++++ ++ + + ++ +++ +++ +++
++++ +++ ++++ + ++ +++ + ++++ ++ ++ + +++ +++ ++++ +++ +
abb = backbone, sc = sidechain.
~1
His-4 His-4
~1 ~1
His-4
~1
His-4 His-4 His-4 His-4 His-4 His-4 His-29 His-29
~2 ~2
His-29 His-29 His-4 His-4
~3 ~3
+
+ +++ + ++ +++ ++++ +
~1
Proximity to His residues Space" Sequence
++ +++ + +++ ++ +++ ++++ + b chemical
~6
~6
His-96 His-96
His-4 His-4 His-29 His-29 His-29
His-96 shift perturbation categorized in comparison
to the strongest observed change (++++). cbackbone amide is :5 6 Afrom the specified His residue, apart from Lys-13 and Leu-14 which are -8 Aaway.
170
Characterisation and solution properties of hyaluronan
and Tyr-78 in Fig. 6). Therefore, it can be concluded that there is no measurable alteration in the Link module conformation between pH 6.0 and 7.5; this is consistent with the results of one-dimensional NMR experiments described above. Increasing the pH from 6.0 to 7.5 does, however, cause some large chemical shift changes for certain amide protons. As shown in Fig. 6 these include the backbone amide resonances of His-4 and Tyr-12; shift changes of similar magnitude occur for backbone NHs of Val-2, His-29 and His-96 (see Table 2). The large shift changes of His-4, His-29 and His-96 are most easily explained by a change in ionization state of their sidechains between pH 6.0 and 7.5. In fact, the chemical shift perturbations of all the other amino acids (in - 2 mM NaCl) can be attributed to their proximity in sequence and/or space to these three titrating histidines (see Table 2). For instance Tyr-12, which exhibits a large shift perturbation with pH (Fig. 6), is < 5 A from His-4. Therefore, the only 'structural' change that occurs in Link_TSG6 between pH 6.0 and 7.5 is the deprotonation of His-4, His-29 and His-96. Thus, it is likely that the unusual pHdependency of the Link_TSG61HA interaction is mediated by the change of chargestate of one or more of these histidine residues. From Figure 6 and Table 2 it can be seen that the lH-15N-HSQC titration is similar under both conditions of salt; this is further illustrated in Figure 7 which shows the chemical shift changes for the titrating histidines at -2 mM and 100 mM NaCI.
0.2
~H4
g
---......H4+ --~--H29-
~
0.15
'"o
......'
--.--H29+ ···B···H96···.···H96+
•
..c
.::: ~
.'
C]
..' .'
0.1
0 . .Fi-=;,-,r-r-.............-r-.--r-r-r-"T""11"'T".............,....,.-r-r-r"T""1r""T"'T"'T........., 7.6 7.2 7.4 6.8 7.0 6.0 6.6 6.4 6.2
pH
Figure 7. Chemical shift changes (M) with pH for His-4, His-29 and His-96 in -2 mM NaCI (-) or 100 mM NaCI (+). The curves indicate that the pK. values for these histidines are similar under both salt conditions.
HA-protein interactions
171
Significant differences in chemical shift are only observed for Asn-67 and Asn-94 (see Table 2). Both the backbone and sidechain amides of Asn-67 are perturbed by pH in the presence but not the absence of NaCI (see Table 2). This amino acid, which is in close proximity to the HA-binding site [2], is more than 10 A from the nearest titrating histidine. This perturbation may arise from the interaction of a Na" ion (in high salt) with the sidechain amide group of Asn-67. In the case of Asn-94 only its sidechain amide was affected by pH in the absence of salt. Asn-94, which is on the linker between the Link and CUB modules, is in close spatial proximity to both His-29 and His-96. It is possible that the difference observed results from altered conformational flexibility of the linker region at these salt-strengths. CONCLUSIONS The interaction of the TSG-6 Link module with HAg is salt-strength dependent, involving the formation of 1 or 2 salt bridges. However, the structure of the Link module, its folding and the position of the HA-binding surface are the same in the absence and presence of NaC!. Therefore, NMR and ITC experiments conducted in low salt are comparable with microtitre plate HA binding assays performed in 100 mM NaCI. The unusual pH-dependency of the HA interaction is probably mediated by the change of ionization state of one or more histidine residues, the pKa values of which are relatively unaffected by salt. ACKNOWLEDGEMENTS We thank Dr John E. Ladbury for helpful advice on interpretation of the ITC data and Dr Caroline M. Milner for critical review of the manuscript. This work was supported by the Arthritis Research Campaign (D0540, D0525, D0569) and the Wellcome Trust (052830/Z/97). NMR spectroscopy was performed at the Oxford Centre for Molecular Sciences which is funded by the BBSRC, EPSRC and MRC. CDB acknowledges the ARC and Yamanouchi Research Institute (Oxford) for their generous support. REFERENCES 1. D. Kohda, C. 1. Morton, A. A. Parkar, H. Hatanaka, F. M. Inagaki, I. D. Campbell & A. J. Day, Solution structure of the Link module: a hyaluronan-binding domain involved in extracellular matrix and cell adhesion, Cell, 1996, 86, 767-775. 2. 1. D. Kahmann, R. O'Brien, J. M. Werner, D. Heinegard, J. E. Ladbury, I. D. Campbell & A. J. Day, Localization and characterization of the hyaluronan-binding site on the Link module from human TSG-6, Structure, 2000, 8,763-774.
172
Characterisation and solution properties ofhyaluronan
3. A. A. Parkar, 1. D. Kahmann, S. L. T. Howat, M. T. Bayliss & A. J. Day, TSG-6 interacts with hyaluronan and aggrecan in a pH-dependent manner via a common functional element: implications for its regulation in inflamed cartilage, FEBS Len., 1998,428,171-176. 4. A. J. Day, The structure and regulation of hyaluronan-binding proteins, Biochem. Soc. Trans., 1999, 27, 115-121. 5. H. G. Wisniewski & J. Vilcek, TSG-6: an IL-l/TNF-inducible protein with antiinflammatory activity, Cytokine Growth Factor Rev., 1997, 8,143-156. 6. A. J. Day, R. T. Aplin & A. C. Willis, Overexpression, purification and refolding of link module from human TSG-6 in Escherichia coli: effect of temperature, media, and mutagenesis on lysine misincorporation at arginine AGA codons, Protein Expr. Purif., 1996,8, 1-16. 7. 1. D. Kahmann, R. Koruth & A. 1. Day, Method for the quantitative refolding of the link module from human TSG-6, Protein Expr. Purif., 1997,9,315-318. 8. M. T. Record, J. H. Ha & M. A. Fisher, Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA, Methods Enzymol. 1991,208, 291-343. 9. J. H. Ha, M. W. Capp, M. D. Hohenwalter, M. Baskerville & M. T. Record, Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA, J. Mol. Biol., 1992, 228, 252-264.
THE USE OF OPHTHALMIC VISCOSURGICAL DEVICES IN
CATARACT SURGERY Steve A. Arshinoff York Finch Eye Associates, 2115 Finch Ave. W. #316, Toronto, Ontario, Canada M3N 2V6.
ABSTRACT In the late 1970's the advent of the intraocular lens and the move from intracapsular to extracapsular cataract extraction and then phacoemulsification caused a dramatic increase in the incidence of postoperative corneal endothelial decompensation, which could only be treated by corneal transplantation. The pioneering work of Endre Balazs and the subsequent licensing of the noninflammatory fraction of hyaluronan to Pharmacia led to the development of ophthalmic viscosurgery using Healon®. Since then numerous other ophthalmic viscosurgical devices have appeared, most of them fitting into a few classes of copies and modified copies of Healonf and Healon®OV, a newer, more viscous variant of Healon", More recently, a new class of "viscoadaptive agents", exemplified by Healorr'S, has appeared and begun to demonstrate novel applications and advantages. This paper reviews the development of ophthalmic viscosurgery specifically with respect to cataract surgical techniques, and the evolution, uses, classification, advantages and disadvantages of different ophthalmic viscosurgical devices. KEYWORDS hyaluronan, hyaluronic acid, viscoelastic, ophthalmic viscosurgical device, viscosurgery INTRODUCTION This paper will review the way ophthalmic surgeons have come to look at and understand ophthalmic viscosurgical devices (OVDs - the new term to replace ''viscoelastics'', chosen by the recent international standards organization (ISO) OVD committee'. The work that I will discuss is almost entirely my own, having been performed over the past 20 years, often relying on the preceding efforts of others. The possibility of using hyaluronan for ophthalmic surgery (the first material successfully used as an OVD) came about as a result of many years of pioneering work by Endre Balazs, who was finally able to patent the noninflammatory fraction of hyaluronic acid, NIF NAHA, in 1979, and license it to Pharmacia for further development into a convenient surgical device for ophthalmologists to use". It was then ave Wik, and his student and later wife, Hege Bothner Wik, who took Balazs's NIF NAHA and tested various formulations, finally to arrive at the one that became Healon" and began to be used in ocular surgery', Miller and Stegmann were the first to use Healon" in human cataract surgery. Healon® came into fairly common ophthalmic use, around the world, in 19804 •
120
The function and use of hyaluronan in wound healing
Georg Eisner, of Switzerland, was the first ophthalmologist to ask "What are the physical strategic effects to consider in our use of viscoelastics in cataract surgery?". and therefore asked one of the principle questions that my work, and this paper, seek to answer'. A few others have played pivotal roles in the development of our understanding of OVDs. Dan Hofmann, an undergraduate classmate of mine at McGill University, in Montreal, went on to a PhD in Polymer Chemistry when I chose to study Medicine. We met again in 1979, when he had become an employee ofPharrnacia, while I was just completing my residency in Ophthalmology. That discussion resulted in my research over the next 20 years, his company, and our collaboration on numerous projects related to hyaluronans. John Alpar is an American ophthalmologist who has contributed considerably to the viscoelastic literature, and was the first to recognize that we needed a US and world standard to analyze these devices in a uniform and comparative manner. He first assembled a huge team under the banner of the American Academy of Ophthalmology (AAO). As we grew, we moved on, under his guidance, to the American National Standards Institute (ANSI), and finally to ISO, which, over the summer of 2000 voted to approve the ophthalmic viscosurgical device document as a world standard. John and I jointly recruited John Emes, of British Columbia, Canada, to act as chair of this committee, and John, over the 5 years of the committee's deliberations, acted as editor, arbitrator, and wise counselor, without who's efforts the standard never would have been completed I. Healon" was first launched commercially at the 1980 spring meeting of the American Intraocular Implant Society (AlOIS - now the American Society of Cataract and Refractive Surgery - ASCRS). Its use was rapidly found to be associated with fairly frequent severe post-operative intraocular pressure spikes. This hurdle was overcome with further research, instead of abandoning it, because of the fact that Healon® enabled eye surgeons to do things that we had considered impossible before. Subsequently, viscosurgery ofthe eye blossomed. One serious problem existed that challenged us all. The physical and polymer chemists who gave us Healon" did not understand the needs of eye surgery, and the ophthalmologists had never needed to understand their surgical manoeuvres in terms of physics instead of biology, and had absolutely no understanding of polymers. It is that gap that has been my research interest throughout my career, and serves as the principle subject of this paper. CATARACT SURGICAL TECHNIQUES, NEW OVDs & PROPERTIES From the mid 1970's to the mid 1980's cataract surgery underwent a dramatic change. In the early 1970's the predominant form of cataract surgery was the intracapsular method (ICCE) by which the lens is removed in its entirety, including its capsule. This results in an unstable anterior segment of the eye, with high risk of vitreous loss leading to retinal problems. Consequently, Healon® was initially envisioned as a device to create space, permitting the implantation of either an iris supported or anterior chamber-angle supported, intraocular lens into the eye, without causing too much damage to the corneal endothelium and preventing vitreous disruption. Over the next decade cataract surgery moved away from ICCE toward extracapsular methods (ECCE)), that permitted the retention of the posterior lens capsule in the eye and thereby stabilized the anterior segment, preventing vitreous loss, and permitting the implantation of the intraocular lens behind the iris, in the posterior chamber, where the normal human lens is located. As surgeons moved from manual ECCE into mechanized small incision ultrasonic phacoemulsification (phaco), where the eye was now often pressurized by constant, gravity fed, balanced salt solution infusion and a tight incision, rather than the viscoelastic, our concepts of what the viscoelastic was required to do needed
Ophthalmic viscosurgical devices
121
reevaluation. Phaco is a far more technically demanding procedure, but permits incisions to shrink to 3-4 millimeters (from over a centimeter). Additionally, phaco allows retention of as intact a capsular bag as desired, to permit implantation of the intraocular lens within the capsular bag - in other words - back into the exact same optical position as the normal human lens. Furthermore, the phaco hand piece emits powerful ultrasonic energy which, if allowed to get too close to the cornea, is destructive to the endothelium, and also requires constant cooling by irrigation and aspiration of balanced salt solution through the anterior chamber. Protection of the corneal endothelium and posterior capsule from these new dual dangers of fluid turbulence and ultrasonic energy were soon recognized to be new important roles for the viscoelastic that had not been previously considered. In the early 1980's two competitors for Healon® appeared in the marketplace (Viscoat'" and 2% hydroxypropylmethylcellulose - Table 1), further confusing surgeons, but allowing us to compare the rheological properties of all three OVDs and correlate those properties to surgical experience, yielding the first early comparative studies of ophthalmic viscoelastics'', Work then commenced to collect information on more new ophthalmic viscosurgical devices, and to relate the laboratory rheological data to surgical behaviour. It soon became apparent that the chemical properties reported in the package inserts of OVDs (concentration, pH, osmolality, etc.) were already checked by government regulatory bodies, as these related to the safety of the manufactured solutions, similar to what is normally checked in pharmacologic preparations. Ophthalmologists had assumed that approved surgical devices were safe solutions, once they were passed by the national regulatory bodies, but rapidly became aware that these chemical properties were essential for basic safety, but played no role in the physical behaviour, and therefore relevance to surgical utility, of OVDs. The more relevant physical properties, which are predictive of surgical behaviour of OVDs (viscosity, elasticity, pseudoplasticity, cohesion, etc.) were not being checked by anybody, and there was no requirement to disclose them in the package inserts, as these properties were not customarily considered to matter in drug submissions that regulatory bodies were accustomed to seeing. Consequently none of the companies (except Pharmacia) was willing to divulge the rheological properties of their product(s) to the surgeons. The situation was analogous to being asked to use a drug for an illness, with the manufacturer being unwilling to divulge how the drug might work, or indeed if it worked at all. My research interest then turned to solving this problem. Considerable progress has been made in the field, and the recently passed (Aug. 2000) ISO OVD standard requires that henceforth, all OVD manufacturers divulge the rheological properties of their material in a standard fashion in the package insert'. Table 1. The first marketed ophthalmic viscoelastics. Name
Date
Company
Content
Healon Viscoat
1980 1983
Pharmacia Cilco
HPMC
1985
Shah & Shah
1.0% 3.0% 4.0% 2.0%
M.W. (Daltons)
NaHa NaHa + CDS HPMC
Vo(mPas) 240 K 50K
4M 500K 25K 86K
4K
NaHa = Sodium Hyaluronate, CDS = Chondroitin sulfate, M.W. molecular weight, Va zero shear dynamic viscosity, M million, K thousand. Cilco was purchased by Coopervision, who were in turn acquired by Alcon. Shah & Shah (Calcutta) were the first to commercially market 2% HPMC. Many others followed.
=
=
=
=
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The function and lise ofhyaluronan in wound healing
CLASSIFYING OPHTHALMIC VISCOSURGICAL DEVICES Phacoemulsification requires different attributes of the viscoelastic during different phases of surgery. During capsulorhexis (creating a round opening in the anterior capsule to permit extraction of the lens material through it) and intraocular lens implantation, the OVD must maintain surgical space and stability, while the surgeon manipulates instruments in the anterior chamber. However, during the phacoemulsification of the lens nucleus and the irrigation/aspiration of residual cortex, the anterior chamber space is maintained by the height of the infusion bottle, while the OVD is required to protect the corneal endothelial cells from fluid turbulence and ultrasonic energy. Proceeding beyond a very elementary understanding of intraocular OVD behaviour required that we develop a classification scheme for OVDs based upon the rheological parameters found to be most important in surgery, and then analyze whether or not the theory developed matched surgical reality. Of the commonly measured physical properties of OVDs: viscosity, elasticity, rigidity, pseudoplasticity, surface tension and cohesion, I believed that zero shear viscosity and cohesion related most to our surgical use of OVDs and consequently developed the currently accepted classificationscheme on that basis", During cataract surgery, the surgeon performs various tasks at different operative rates of shear. During capsulorhexis and maintenance of an open capsular bag for IOL implantation, the OVD is stationary, and the operative shear rate is close to zero sec". High stability and space maintenance ability are required for these tasks, and so a viscoelastic with high zero shear viscosity is preferable. The act of IOL implantation, itself, results in the IOL moving through the OVD at a rate of shear close to 1 sec", The surgeon requires that the OVD viscosity at this point be considerably decreased from shear rate zero, in order to avoid excessive drag on the corneal endothelial cells. During injection of the viscoelastic into the eye, the viscoelastic must traverse a small bore canula, and it is important for the surgeon to Figure 1: Pseudoplasticity curves of common OVDs
OVD VISCOSITY IN DIFFERENT SHEAR RATE RANGES Microvis c Plus Healon GV Microvisc Provisc Healon Blolon Amvisc
MIO SHEAR
LOWSHEAR
7
HIGHSHEAR
6
Viscoal Cellugel Vilrax Occucoat
PSEUDOPLASTICITY .= Decreasing Viscosity with Increased Shear, with limiting viscOSity, at lov:r shea,r. -3
·2
·1
0
3
....,._ 1
.f-
2
Log Shear Rate (sec")
*Use zero-shear viscosity for classification.
2
3
Ophthalmic viscosurgical devices
123
feel the inflation pressure of the eye and not the resistance of the OVD traversing the canula. Therefore at shear rate 103 sec", the operative shear rate during injection, the OVD must have very low viscosity. Ophthalmic surgeons therefore require an OVD to have high zero shear viscosity as well as high pseudoplasticity (Figure 1). Cohesion is the second important parameter used in OVD classification. Cohesive OVDs are more easily removed from the eye by irrigation and aspiration at the end of the surgical procedure, but dispersive (the physical opposite of cohesive) OVDs are retained better in the anterior chamber, adjacent to the corneal endothelial cells in a protective layer, in the presence of irrigation and aspiration. This leaves us with a dilemma, in that we would like an OVD to be dispersive for one part of the surgical procedure, but cohesive for other parts 8,9 . To complicate matters, when I first proposed that we use OVD cohesion as a classification parameter, no applicable method of cohesion measurement was known, and the degree of importance of OVD cohesion to retention ofthe OVD in the anterior chamber (AC) during surgery was only conjectural. John Poyer, Kwan Chan and I devised an method to measure OVD cohesion", and showed that dispersive nature contributed roughly equally with negative OVD charge and the presence of hyaluronic acid (to bind to previously discovered specific binding sites) to determine OVD retention in the AC during phac08, lO. It is now generally accepted that zero shear viscosity and cohesion are the two physical parameters most applicable for classification of OVDs. The current classification system (Table 2), divides OVDs into higher viscosity cohesives (with the subcategories of super viscous cohesives and viscous cohesives), and lower viscosity dispersives (with the subcategories of medium viscosity dispersives and very low viscosity dispersives). It was later found that viscoadaptives technically fit into the classification of higher viscosity dispersivcs, but have important differences (see below). A major advantage of this classification of OVDs, is that it allows us to identify consistent best uses and liabilities of each OVD typical ofthe group within which it is classified. Higher viscosity cohesive OVDs are best at creating and maintaining surgical spaces, and for displacing and stabilizing tissues. They are optically very clear in the eye, because they coalesce into a single cohesive mass, and being elastic, they can pressurize spaces. Pressurization of the anterior chamber is very important in capsulorhexis and IOL implantation. While tearing a capsulorhexis, there is always posterior vitreous pressure, which tends to encourage the tear to migrate peripherally on the anteriorly convex lens surface. The injection of elastic higher viscosity cohesive OVDs into the AC permit "pressure equalized cataract surgery", in that the posterior vitreous pressure can be neutralized, thus allowing the capsulorhexis to proceed circumferentially without any tendency to extend outward. Similarly the capsular bag can be kept open, stretched and stable, with these OVDs, allowing safe implantation of even the most clastic foldable IOLs. However, higher viscosity cohesive OVDs tend to leave the anterior chamber too quickly during prolonged or difficult cases, as a consequence of prolonged irrigation, and higher viscosity cohesive OVDs are also incapable ofpartitioning the AC. Lower viscosity dispersive OVDs tend to be retained adjacent to the corneal endothelial cells longer during surgery, in the presence of irrigation, and can be used to selectively move one tissue in the anterior chamber, without moving everything. Furthermore, they can be used to partition spaces, permitting surgical fluid flow in one part of the AC (in order to achieve a surgical goal), while protecting another area from the detrimental effects of irrigation. This attribute is particularly useful in the management of complicated cases, such as traumatic cataracts with broken zonules and protruding vitreous, Fuchs' endothelial dystrophy, pieces offrayed iris, etc.
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The function and use of hyaluronan in wound healing
Table 2: Ophthalmic viscosurgical devices: Content, Molecular Weigbt & Zero-shear Viscosity HIGHER VISCOSITY - COHESIVE OVDs OVD
Content
MW(D) Vo(mPas)
WWER V.ISCOSfIY - DISPERSIVE OVDs OVD
Content
MW(D) Vo(mPs)
Viscoadaptives lIealon5
2.3%NaHa
4.0M
7.0M
Super Viscous-Cohesive OVDs (Yo> 1M): Micro'Visc Plus (iVisc Plus) Healon GV
Medium Viscosity Dispersive OVDs (lOOK>Vo > 10K):
1.4% NaHa
7.9M
4.8M
1.4% NaHa
5.0M
2.0M
Viscoat
3.0% NaHa 4.0% CDS
500K 25K
50K
Cellugel
2.0% chemically modified HPMC
lOOK
40K
3.0% Ha
500K
25K
Viscous-Cohesive OVDs (lM>Vo > lOOK): Vitrax MieroVisc (iVisc)
1.4% NaHa
6.IM
1.0M
Allervisc Plus (Viscorneal Plus) Provise
1.4% NaHa
5.IM
500K
1.0% NaHa
2.0M
280K
Healon
1.0% Nalla
4.0M
230K
Biolon
1.0% NaJ-Ia
3.0M
215K
Allervisc (Viscomeal) Amvisc
1.0% NaHa
5.IM
200K
1.2% NaHa
I.OM
lOOK
AmviscPlus
1.6% NaHa
1.0M
lOOK
Very Low Viscosity Dispersive OVDs (IOK>V o> lK):
MW(D) - molecular weight (Daltons) Vo(mPs) = zero shear viscosity (milli Pascal-seconds) M = million,' K = thousand
I-Cel
2.0% HPMC
90K
6.0K
Ocuvis
2.0% HPMC
90K
4.3K
Occucoat
2.0% HPMC
86K
4K
Hymccel
2.0%HPMC
86K
4K
Adatocel
2.0% HPMC
86K
4K
Visilon
2.0%HPMC
86K
4K
NaHa = sodium hyaluronate HPMC = hydroxypropylmethylcellulose CDS = chondroitin sulfate
However, lower viscosity dispersive OVDs do not maintain surgical spaces very well, they break up and cause irregular fracture boundaries (which obscure the surgeons view of the operative field), and require much more time, effort and irrigation to remove at the end of the surgical procedure. Many question whether the benefits derived from their routine use outweigh the drawbacks of reduced visibility and possible corneal endothelial damage caused by the increased irrigation required for their removal. The classification system for OVDs, and the subsequent analysis of the best uses and drawbacks of each group, made it clear that no OVD had lived up to its manufacturer's hopes of becoming "the best" for all circumstances occurring in cataract surgery. Surgeons were therefore faced with the dilemma that the OVD they chose for a surgical case may be excellent tor some steps of phaco, but had unavoidable drawbacks for others. We were forced to identify the surgical steps that caused each of us the most difficulty, and choose as our routine OVD, one that offered considerable assistance in the performance of that task, while accepting the fact that it may make other parts ofthe procedure more difficult.
Ophthalmic viscosurgical devices
125
The Viscoelastic Soft-Shell Technique It was into the above scenario that I introduced the concept of the "Dispersive-cohesive viscoelastic soft-shell technique". It is a programmed method of using a dispersive and a cohesive OVD together, to take advanta~e of the optimal properties of each class and minimize their corresponding drawbacks'!' 2. As long as two OVDs do not mix, different physical effects can be achieved in adjacent areas of the AC simultaneously. The Soft-shell technique is applicable to all facets of cataract surgery, but is particularly useful in managing complications, which previously were not well handled by other surgical techniques'<. Softshell has also encouraged ophthalmologists to look at how we could take better advantage of the physical properties of all the fluids that we use in surgery. Thomas Neuhann and I have introduced the related idea of cleaning the posterior capsule, after phacoemulsification, with a stream of balanced salt solution emitted tangentially from a small bore canula (like cleaning your driveway with a hose). This has led to a dramatic reduction in the incidence of post-operative posterior capsular opacification requiring YAG laser capsulotomy. But, like everything else surgical, the soft-shell technique also has drawbacks. The most obvious one is the additional cost of using two OVDs for each case, instead of one. The second problem is that many surgeons use the soft-shell technique improperly, injecting the two OVDs in the wrong sequence, or wrong relative amounts, thus yielding sub optimal results. So the question perpetually arises: "Can we do better?"
In September of 1996, I became involved in a project with Pharrnacia & Upjohn to develop a new OVD. This new OVD was to retain the best super viscous cohesive properties of Healon®GV, but was to be retained in the anterior chamber, throughout phacoemulsification, and irrigation/aspiration of cortex, as well as the best dispersive viscoelastics, without having any of the drawbacks of dispersive viscoelastics. The problem, of course was how to achieve these apparently mutually exclusive goals. The solution we chose, and the secret to "viscoadaptivity", was to increase the zero shear viscosity of the OVD to the point that it begins to adopt more physical characteristics of a solid, thus becoming fracturable. A useful analogy is to consider what happens when we make chocolate pudding. At first it is too "runny" for young children to be able to eat it without spilling most of it over their clothes. If we place the pudding in the refrigerator for progressively longer periods of time, we arrive at a point where the pudding is still liquid enough to pour, but just solid enough to take a piece out of it with a spoon, and to have that piece stay intact as it travels to the child's mouth, despite the spoon being tipped on undesirable angles. It is this degree of rigidity that we wanted to achieve with Healon®S. The properties and advantages of a viscoadaptive are best explained by reference to the Healon'fS. Figure 2 illustrates the behaviour characteristics of Healon®S, compared to Healon®GV and Viscoat", when exposed to the ranges of fluid turbulence normally encountered in phaco. Whereas Viscoat", behaves as a dispersive over this entire range of flow, and whereas Healon®GV behaves as a cohesive over the same range, Healon®S is unique in that in the middle of that flow rate range, about 2S cc/rnin, the molecular mass begins to fracture, and therefore behaves in a pseudo-dispersive fashion under conditions of high turbulence. Therefore Healon®S behaves as a super viscous cohesive under low shear, but mimics a dispersive under high shear, despite the fact that the zero shear viscosity (Table 2), and pseudoplasticity curve of Healon®S, make it look simply like "super Healon®GV (Figure 3).
126
The function and use ofhyaluronan in wound healing
Figure 2: Viscoadaptive behaviour of Healon®S
RESPONSE OF HEALON®5 TO TURBULENCE Property
Haalon5 Haalon GV Viscoat
•
10
36
Flow nile (cclmln)
Fluid turbulence In Immediate
viscoelastic vicinity
In order to test our hypothesis that Healon®S would indeed alter its rheologic behaviour under the different conditions encountered in phacoemulsification, we arranged for ten well known cataract surgeons to perform mock phaco procedures on human eye bank and porcine eyes, while blinded to the OVD being used, and to score the performance of the OVD (0-10) for each of the S steps of phaco that utilized OVD (Injection, Capsulorhexis, Phacoemulsification, IOL implantation, and Removal). Cumulative scores from hundreds of procedures were used to yield Figure 4. Figure 3: Pseudoplasticity of Healon®S compared to other common OVDs
RHEOLOGY OF HEALON 5
•
Ophthalmic viscosurgieal devices
127
Figure 4: Mock Phaco results with Healon®5, Healon®GV and Viscoat®
HANDLING TESTS: HEALON 5 vs HEALON GV & VISCOAT
•
It is apparent from Figure 4 that Viscoat® performed better than Healon®GV with respect to retention in the anterior chamber during the high turbulence ofphacoemulsification (more dispersive), and that Healon®GV performed better than Viscoat® for injection (more pseudoplastic), for IOL implantation (higher zero shear viscosity) and for removal (more cohesive). However, Healon®S was either rated best, or very close to best, for every step of surgery, and achieved the only excellent overall rating. This was objective confirmation that viscoadaptivity indeed worked. If the world is going to accept Healon®S and the concept of viscoadaptivity, it needs to offer what Healon" offered when it was first developed: The ability to perform surgical maneuvers that could not be done previously. Many examples ofthis have already appeared.
Figure 5: A new technique unique to Healon®5
THE ULTIMATE SOFT SHELL •
BSS injected over lens capsule, under the mass of Healon5.
• AC remains pressurized due to Healon5 blocadlng wound.
•
•
Capsulorllexls Is very easy, in a pressurized, but low viscosity environment.
128
The function and usc of hyaluronan in wound healing
Among the most technically difficult cataract procedures are those on mature cataracts. The cataracts in these patients are bright white, obscuring the surgeon's perception of contrast between the capsule and the lens contents, and the anterior lens is full of loose fluffy cortex, which clouds the surgical view as soon as the capsulorhexis is begun. Healon®S allows performance of the "ultimate soft shell technique", because the soft shell can be done with Healon®S and an aqueous solution. The anterior chamber is 7S% filled with Healon®S, and the aqueous solution is then injected into the distal angle, remote from the incision. This causes the Healon®S mass to rotate upwards, towards the incision blockading it, while the aqueous solution dissects along the lenticular surface (Figure S). When the aqueous fluid used is trypan blue, which stains the anterior capsule to greatly enhance visibility, mature cataracts become a very simple matter". Healon®S is the first OVD, with which a viscoelastic effect can be achieved, due to its rigidity, without completely filling the target space. Many other examples exist to suggest that viscoadaptives will become the new standard OVDs. Summary
By the age of 80, the incidence of cataracts in humans approaches 100%. All of us can expect to be cataract patients. I began my practice of ophthalmic surgery in 1980, the year Healon® appeared. Since then I have performed over 10,000 cataract procedures, every one of them with the assistance of a hyaluronan OVD. Tens of thousands of ophthalmologists, world wide, have done similarly to millions of patients. Cataract surgery has been made infinitely better and safer due to hyaluronan OVDs. This is a wonderful legacy that Endre Balazs has given to the world. Ophthalmic viscosurgery is moving faster now than ever before. Almost every major cataract meeting has a session devoted to viscosurgery and OVDs. As one who is intimately involved in OVD research, cataract surgery, and many of those meetings, On behalf of every ophthalmologist in the world, and humanity: Thank you Endre, and happy so" birthday. ISO/TC 172/SC 7/WG 7. Ophthalmic Implants - Ophthalmic Viscosurgical Devices. ISO/DIS 15798, 1999. Balazs EA. Ultrapure hyaluronic acid and the use thereof. U.S. Patent # 4,141,973. Oct. 17, 1979. 3 Bothner H, Wik O. Rheology of intraocular solutions. in Rosen ES, ed, Viscoelastic materials: basic science and clinical applications. Vol 2 Vision and Vision Health Care. Pergammon Press, 1986. 4 Balazs EA, Miller D, Stegmann R, Viscosurgery and the use ofultrapure Na-hyaluronate in intraocular lens implantation. Paper presented at the International Congress and First Film Festival on IOL Implantation, Cannes, France. 1979. 5 Eisner G. Use of viscosurgical tools in ophthalmic surgery. In Eisner G. ed.Ophthalmic Viscosurgery, Mcdicopca, Montreal, Canada 1986. 6 ArshinoITSA: Viscoelastic substances: Their properties and usc when placing an IOL in the capsular bag. Current Cdn. Ophthal. Pract. 1986; 4: 2, 64-5,72,74. 7 Arshinoff S: Dispersive and cohesive viscoelastic materials in phacoemulsification. In Solomon L, Ed: Ophthalmic Advisory Panel at the ASCRS, Boston, Mass. 1994. Medicopea international, Montreal: 1995; 28-40. 8 Poyer John F., Chan Kwan Y., Arshinoff Steve A: A new method to measure the retention ofviscoelastics on a rabbit corneal endothelial cell line after irrigation and aspiration. J. Cat. Refract. Surg. January 1998: 24.1, 84-90. 9 Poyer JF, Chan KY, Arshinoff SA: A quantitative method to determine the cohesion of viscoelastic agents by dynamic aspiration. J. Cat. Refract Surg. August 1998; 24: 8; 1130-1135. 10 Madsen K, Stcnevi U, Apple D, Harfstrand A. Histochemical and receptor binding sites of hyaluronic acid and hyaluronic acid binding sites on the corneal endothelium. Ophthalmic Practice 7:3; 2-8, 1989. II ArshinoffSteve. The Dispersive/Cohesive viscoelastic soft shell technique for compromised corneas and anterior chamber compartmentalization. Winner. Surgical Techniques category. American Society of Cataract and Refractive Surgery Film Festival, Seattle, Washington. June 1-5, 1996. 12 ArshinoffSteve A: Dispersive-cohesive viscoelastic soft shell technique. 1. Cataract Refract Surg. Feb. 1999. 25: 2; 167-173. 13 ArshinoffSteve. Using viscoelastics to solve problems in cataract surgery. Video. American Society of Cataract and Refractive Surgery, Annual meeting April 16-22, 1998 video film festival. Runner up award - Cataract complications. 14 ArsbinoffSteve. The ultimate soft - shell technique. Ophthalmic practice. 18: 6; Oct. 2000. I
2
PART 3
RHEOLOGICAL BEHAVIOUR OF HYALURONAN
EFFECT OF METAL IONS ON THE RHEOLOGICAL FLOW PROFILES OF HYALURONATE SOLUTIONS Charles J. Knill t, John F. Kennedy t, Yasmin Latif 1 & Derek C. Ellwood J
2
2
Chembiotech Laboratories, Institute a/Research & Development, University 0/ Birmingham Research Park, Vincent Drive, Edgbaston, Birmingham, Bl5 2SQ, UK.
Department a/Microbiology, Medical School, University a/Newcastle, Newcastle Upon Tyne, UK
ABSTRACT
The effects of various metal ions on the rheological flow profiles of hyaluronate solutions were investigated, by controlled incubation of metal chloride salt solutions with sodium hyaluronate (NaHA) solution under ambient conditions. Results from application of the Williamson model to the flow profiles of incubated solutions showed a progressive decrease in Williamson zero shear viscosity (110) with increasing atomic number / atomic mass of the metal ion, which, with the exception of data for u', could be related to a power equation (y = cx b) . Such reductions in viscosity were not a result of hydrolysis (determined by GPC), but may be due to diffusing ions disrupting hydrogen bonding and shielding electrostatic repulsions between carboxylate groups. INTRODUCTION
Hyaluronan is a biocompatible / biodegradable, linear, water-soluble, ionic polymer composed of repeating (1-H) linked disaccharide units consisting of ~-D-GlcpA and ~-D-GlcpNAc, linked together by (l~3) glycosidic bonds 1. It has a high molecular weight (around 105_107 Da), depending on source, giving a DP range of - 250-25000 2. It is found in all vertebrates, being present in almost every tissue as a component of the extracellular matrix and is distributed throughout the mammalian body, especially in synovial fluid, loose connective tissue, umbilical cord and the vitreous body of the eye 3. The largest amount of hyaluronan (7-8 g per average human, 50 % of the total in the body) is in the skin tissues (both the dermis and epidermis) 4. The most commonly isolated / utilised (in vitro) forms of hyaluronan are the free acid (hyaluronic acid, HA) and its sodium salt (sodium hyaluronate, NaHA). In solution the hyaluronan backbone is stiffened by the chemical structure of linked disaccharide units, internal hydrogen bonds, mutually repelling anionic groups and solvent interactions, making it a rigid and highly hydrated molecule. It adopts an expanded random coil structure in physiological solutions, occupying a large domain. Small molecules, e.g. water and electrolytes, can freely diffuse through the domain, whilst large molecules are partially excluded due to their hydrodynamic size. At low concentrations, individual chains entangle forming a continuous network, giving viscoelastic and pseudoplastic properties, which is unique for a water-soluble polymer at low concentration. At higher concentrations entangled networks can be formed, which can resist rapid, short duration fluid flow, thus exhibiting elastic properties. However, short fluid flow of longer duration can partially separate and align molecules, allowing movement and thus exhibiting viscous properties 5.
176
Rheological behaviour of hyaluronan
The viscoelastic properties of hyaluronan solutions are ideal for use as a biological shock absorber and lubricant, which is why it is present in synovial fluid, where it lubricates the cartilage between joints. The cartilage provides a cushion between the bones allowing the joint to move smoothly. However, in an arthritic joint the elasticity / viscosity of the joint fluid is reduced, diminishing the shock absorbing and barrier properties 6. Highly viscoelastic hyaluronan solutions can be injected into joints (viscosupplementation) in order to restore the rheological environment of the joint and thus improve joint function. This is used in osteoarthritic joints to provide instant protection and shock absorption, thus decreasing pain associated with mobility. The aim of this investigation was to see how the interaction of hyaluronan with a range of metal ions (of differing ionic size, valency, etc.) affected the rheological flow characterisitics (especially the zero shear viscosity, 110) and molecular weight profile. This is of particular interest with respect to localised in vivo applications of hyaluronan, since there are numerous metal ions in the body which could interact with administered hyaluronan resulting in significant changes in desired physical properties. MATERIALS & METHODS Preparation of boiled, nitrogen flushed, deionised water Deionised water was boiled (~ 5 minutes, under vacuum) to remove dissolved air, cooled to ambient temperature, and flushed with nitrogen (- 15 minutes) to displace any residual air, and prevent air redissolving. The resultant boiled, nitrogen flushed, deionised water was refridgerated (4°C) until required, and was used for the preparation of all subsequent solutions (the NaHA and metal salt solutions detailed below). Boiling / nitrogen flushing was used to exclude dissolved oxygen from the water to try and minimise any oxidative hyaluronan degradation effects. Preparation of sodium hyaluronate solution Sodium hyaluronate (NaHA, 6 g, produced by microbial fermentation using Streptococcus equi) was dispersed in boiled, nitrogen flushed, deionised water (600 mL) in a conical flask (1 L), and the flask headspace was flushed with nitrogen. The flask was stoppered and the NaHA allowed to dissolve slowly over a period of - 48 hours (at 4°C), with ocasional gentle agitation / swirling to assist solubilisation, resulting in a viscous, homogenous NaHA solution (1 % w/v). Care was taken to avoid the use of any metal equipment in the production of the hyaluronate solution (especially items containing iron / stainless steel), to minimise potential degradatory effects. Preparation of metal ion solutions Metal chloride salt solutions (0.25 M) were prepared by dissolving the necessary amounts of anhydrous lithium chloride (LiCI, M, 42.39), sodium chloride (NaCl, M, 58.44), potassium chloride (KCI, M, 74.55), anhydrous calcium cloride (CaCh, Mw 111.0), manganese (II) chloride tetrahydrate (MnCh.4H20, Mw 197.9), cobalt (II) chloride hexahydrate (CoCh.6H20, M, 237.9), and cerium (ill) chloride heptahydrate (CeCh.7H20, Mw 372.6), in boiled, nitrogen flushed, deionised water (50 mL) in volumetric flasks (50 mL). All salt solutions were filtered (using 0.45 11m pore size, 25 mm diameter Titan nylon membrane filters), and flushed with nitrogen (to remove any dissolved air / oxygen), before use in hyaluronate incubation experiments.
Effect of metal ions on now profiles
177
Incubation of hyaluronate with metal ions
Aliquots of NaHA solution (1 % w/v, 5 mL) were transferred into individual headspace vials (20 mL, Merck). Aliquots of the metal chloride salt solutions detailed above (0.25 M, 5 mL) were transferred into the individual headspace vials containing the NaHA solution (in duplicate) to give overall concentrations of 0.5 % w/v NaHA and 0.125 M metal ions. The pipette tip used for individual NaHA solution dispensing was also utilised for subsequent metal ion solution dispensing, so that the latter ensured any residual NaHA was washed out ofthe pipette tip. Duplicate controls were also prepared using boiled, nitrogen flushed, deionised water aliquots (5 mL). The resultant solutions were flushed with nitrogen (to remove any dissolved air / oxygen, which also facilitated mixing), and the vials sealed with butyVPTFE lined (3.0 mm) plain aluminium crimp caps (20 mm diameter, Merck). The solutions were incubated at ambient temperature, and the rheological flow profiles determined exactly 1 hour after metal ion solution addition (as detailed below). [This incubation test procedure had been previously validated by performing replicate rheological analyses on test solutions (NaHA and metal salts), which resulted in Williamson infinite shear viscosity (110) values with % variation values of < 10 %]. Determination of rheological flow profiles
All rheological flow profile measurements were performed using a TA Instruments AR 1000 'Rheolyst' controlled stress rheometer, equipped with 'Rheology Solutions' software (v. 1.2.2). The software is split into two modules, the 'ARI000' module controls the instrument itself and enables the operator to set up experimental procedures and perform the actual experiments, whilst the 'Data' module manipulates and presents the collected data. Duplicate rheological flow profile measurements were performed using the test parameters detailed below. Approximately 5 mL of sample was used for each flow test (the remainder being stored at 4 °C until molecular weight determinations were performed). The 'Data' module was used to apply the Williamson model to the resultant flow profiles (shear rate vs viscosity) in order to determine the Williamson zero shear viscosity (110) values. The Williamson model describes the low shear viscosity behaviour and is derived from the Cross model when 11 »1100 7. Geometry: Geometry gap: Geometry inertia: Instrument inertia: Temperature: Pre-experimental shear stress: Test stress range: Number of Points:
parallel plate (4 em diameter) 500 urn - 1.5 J.lNms2(calibrated before each test) -14.2 IlNms2(calibrated daily) 20°C (controlled by Peltier plate) 1.768 Pa (for 10 s) 0.5 - 500 Pa (log ramp) 3I (max. point time 1 min)
Determination of molecular weight profiles
The molecular weight profiles of incubated solutions (0.5 % w/w NaHA, 0.125 M metal salt, & controls) detailed above were determined - 12-24 hours after incubation, by GPC analysis using the isocratic HPLC system detailed below.
178
Rheological behaviour ofhyaluronan
Instrumentation:
GPC Columns: Eluent: Flow rate: Calibration: Injection volumes: Data collection: Data manipulation:
Knauer HPLC pump 64 Waters 712 WISP autoinjector Knauer column oven & control unit (set to 30°C) Knauer differential refractometer Dionex ill 20 Universal interface (for PC data link) Progel™TSK G5000, G4000 & G2500 PW (131J.IIl, 300 x 7.5 mm ill) columns (& guard) linked in series 0.05 M phosphate buffer containing 0.25 M NaCl, pH 7.0 (prepared using 18 Mil UHQ water with helium sparging) 0.5 mL/minute Pullulan standards with M p values of 853, 380, 186, 100, 48,23.7, 12.2 & 5.8 kDa (0.1 Ilg/J.J.L in UHQ water) 200 J.J.L (solutions diluted 1 in 10 with UHQ water) Dionex Chromeleon software (v. 6.11) PL Caliber Reanalysis software (v. 7.04)
RESULTS & DISCUSSION Mean flow profiles (of duplicate analyses, shear rate vs viscosity) for incubated NaHA / metal ion solutions (and controls) are displayed in Figures I & 2, and show that incubation with metal ions results in a reduction in viscosity. GPC analysis showed that the control sample (NaHA + water, i.e. no additional metal ions added) had M p and polydispersity (Mw/Mn ) values of2.5 x 106 Da and 1.6, respectively. Incubated samples had Mp values in the region of 1.9-2.5 x 106 Da and polydispersity values in the region of 1.7-2.0, i.e. no significant degradation had taken place. The Williamson model was applied to the flow profiles to determine the Williamson zero shear viscosities (Tlo) 7. This is the region where increasing shear stress / shear rate has little or no effect on viscosity (i.e. Newton's law ofliquid flow is obeyed). 1.00 __ control -.-Na
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1
10
100
1000
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Mean flow profiles (shear rate vs viscosity) for NaHA solutions incubated with water (control), Na+, u: and Mn 2+ ions (all cr salts).
Effect of metal ions on now profiles
179
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Mean flow profiles (shear rate vs viscosity) for NaHA solutions incubated with K+, C~+, C02+ and CeJ+ ions (all cr salts).
Figure 2.
The Williamson zero shear viscosities (110) were related to the atomic number and atomic mass of the associated metal ions used in the incubation (Figure 3). The data (with the exception of could be satisfactorily modelled using the power equation b y = cx (Figure 3). No obvious correlation could be obtained between the Williamson shear viscosity (Tjo) and ionic radii / ionic volume of the metal ions used in the incubation.
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The relationship between the Williamson zero shear viscosity (Tjo) and the atomic number / atomic mass of the metal ion used for incubation.
180
Rheological behaviour of hyaluronan
These results are in agreement with previous published data, where zero shear viscosity (110) was found to decrease for the series Na+, K+ and Ca2+, and Na+, Lt and K+ 8,9. It has been suggested that these observed reductions in viscosity are a result of ions diffusing between the hyaluronan chains and shielding electrostatic repulsions (between nearby carboxylate groups) and disrupting hydrogen bonding interactions between chains 8. This would result in a collapse of expanded stiffened coil structure to a more compact structure where chains could slide over each other more easily (i.e. flow), thereby leading to a reduction in viscosity. CONCLUSIONS As the atomic number / atomic mass of the metal ion increased, a progressive Contact with metal decrease in Williamson zero shear viscosity (110) was observed. ions thus alters the rheological characteristics of hyaluronan, which may in tum adversely affect its functional application properties. The body contains an abundance of metal ions (especially K+, Na+, Ca2+ and Mg2+), which therefore have the potential to alter the viscosity of administered hyaluronan-based materials. The effect of Li+ seemed to be more pronounced than expected (based solely upon its charge, size, etc), which is of particular interest since a considerable number of pharmaceutical products contain lithium. The effects of a broader range of metal ions of higher valency should be investigated (e.g. Al3+, Ti4+ & Sn4+) to see if any further correlations can be made. The effects of such metal ions on the viscoelasticity ofhyaluronan materials (using creep & oscillation measurements) also merits investigation. REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9.
A. Linker & K. Meyer, Production of unsaturated uronides by bacterial hyaluronidases, Nature (London), 1954, 174, 1192-1193. T. C. Laurent, Structure of hyaluronic acid, In: Chemistry and Molecular Biology of the Extracellular Matrix, Vol. 2, E. A. Balazs (ed.), 1970, Academic Press, New York, pp.703-732. K. Meyer & J. W. Palmer, The polysaccharide of the vitreous humor, J. Bioi. Chem., 1934, 107,629-634. J. R. E. Fraser & T. C. Laurent, Turnover and metabolism ofhyaluronan, In: The Biology of Hyaluronan, Ciba Foundation Symposium 143, D. Evered & J. Whelan (eds.), 1989, John Wiley & Sons, Chichester, pp. 41-53. R. E. Turner, P. Lin & M. K. Cowman, Self-association of hyaluronate segments in aqueous NaCl solution, Arch. Biochem. Biophys., 1988, 265, 484-495. M. F. McCarty, A. L. Russel & M. P. Seed, Sulphated glycosaminoglycans and glucosamine may synergize in promoting synovial hyaluronic acid synthesis, Med. Hypotheses, 2000, 54 (5), 798-802. H. A. Barnes, J. F. Hutton & K. Walters, An Introduction to Rheology, Rheology Series Vol. 3, 1989, Elsevier, Amsterdam, pp. 16-23. Y. Kobayashi, A. Okamoto & K. Nishinari, Viscoelasticity of hyaluronic acid with different molecular weights, Biorheology, 1994,11 (3), 235-244. K. Ogino, Viscoelastic properties of hyaluronic aqueous solutions, Kobushu Ronbunshu, 1998, 55 (12), 736-748.
THERAPEUTIC EFFECT OF 1% NA-HYALURONAN ON CORNEAL WOUND HEALING Jang-Hyun Chung Department a/Ophthalmology, Mokdong Hospital, College a/Medicine, Ewha Women's University, 911-1, Mokdong, Yangcheon-ku, 158-710, Seolll, Korea.
ABSTRACT The effect of topically applied 1% Na-Hyaluronan (Na-HA) on corneal wound healing was evaluated in n-heptanol- and alkali-induced corneal injuries. Central corneal wounds were produced in one eye of each rabbit by applying a 5.5-nun round filter paper soaked in 1M NaOH or n-heptanol onto the central cornea for 60 seconds. Eyes were treated with either 1% Na-HA or phosphate buffered saline (PBS) 4 times per day for 3 weeks. Epithelial healing rate measurement, hemidesrnosome counting, stromal polymorphonuelear leukocyte (PMN) and keratocyte counting, and endothelial wound morphmetry were performed after treatment. In n-heptanol wounded eyes the number of hemidesmosome in the re-surfaced epithelium was significantly increased in the Na-HA treated group. During the early healing period of alkali damaged eyes the epithelial healing rate was significantly increased in the Na-HA treated group. The stroma treated with Na-HA has less PMNs than the control group. The size of the endothelial defect area was significantly smaller in the Na-HA group than in the control. The present findings indicate that topically applied 1% Na-HA may enhance the early repair proccss after corneal injury. KEYWORDS Na-hyaluronan, cornea, alkali, n-heptanol, wound healing, rabbit INTRODUCTION Na-HA has been used in the treatment of ocular surface disorders, such as recurrent epithelial erosion, keratitis sicca and alkali burns!", and was reported to accelerate the epithelial healing rate in wounded cornea'", Hyaluronan, which is widely distributed in connective tissues as an important constituent of the extracellular matrix, was previously regarded as a biologically inert viscous substance but was subsequently shown to be involved in cell protection, control of cell migration and growth, cell differentiation and tissue morphogenesis':". In the previous experiment we showed that topically applied 1% Na-HA enhanced epithelial healing and appeared to have minor effects on stromal and endothelial healing in alkali-induced wounds". However, the mechanisms by which topically administered Na-I'IA influences wound healing following corneal alkali injury have not been elucidated. In the recent serial studies 10, II the effect of topically applied 1% Na-HA has been examined on the morphogenesis of epithelial hemidesmosome in a n-heptanol induced corneal wound and on stromal and endothelial healing in a standardized alkali wound.
130
The function and use ofhyaluronan in wound healing
In the present study, the therapeutic effects of 1% Na-I-IA on corneal wound healing have been summarized based on the results of previously performed experiments'v"!', MATERIALS & METHODS Corneal wounding & treatment
New Zealand white female rabbits, each weighing 3.0 kg were used. All experimental procedures were performed in accordance with the ARVO Resolution on the Use of Animals in Research. Animals were anesthetized with intravenous sodium pentobarbital, and corneal wounds were induced in one eye of each animal by placing a round 5.5-mm filter paper soaked in IM'NaOHor n-heptanoJ onto the central cornea for 60 seconds. The cornea was then rinsed with balanced salt solution (BSS®, Alcon, USA) for 2 minutes. The details of this technique have been described previously". After wounding, eyes were treated topically 4 times per day with 1% Na-I-IA (I-Iealon®, Pharrnacia-Upjohn, Uppsala, Sweden, i.e. the treatment group) or phosphate buffered saline (PBS, i.e. the control group) Measurement of epithelial healing rate
After initial wounding the wounded area was photodocumented at 6-to l2-hour interval for up to 48 hours. The defect areas were stained with one drop of 2% sodium fluorescein and photographed using a Nikon MFI0 with a Micro-Nikkor 105 mm 1:4 objective. A Kodak Wratten 47B filter was always used on the flashlight and gave adequate resolution to discriminate the defect area. Epithelial defect areas were measured planimetrically using a computer-assisted image analyzer (Cambridge, Mass., USA). Individual healing rates were calculated from defect area at different time intervals by using a linear regression analysis. Transmission electron microscopic examination & hemidesmosome counting
Eight animals (8 corneas) in each group were sacrificed 3 days and 7days after nheptanol injury. Central cornea, 1 x I mm, was fixed in a 2% glutaraldehyde solution for 24 h at 4°C. The specimen was rinsed with PBS, dehydrated in graded alcohol and propylene oxide, and postfixed in 2% osmium tetraoxide for 2 h. After dehydration the specimen was embedded in Epon and 3 thin sections from each cornea were cut on an ultramicrotome. The sections were stained with uranyl acetate and lead citrate, and were examined in a Hitachi-6000 transmission electron microscope. The number of hemidesmosome in basal epithelial cells was counted by an observer blind to the sample treatment using photographs (original magnification, x 30,000) in a 2 micrometer length of basement membrane. The mean of 3 determinations in each cornea was used for statistical comparison. Differential cell counting in the stroma
Eight corneas (8 animals) in each group were examined 2 days and 7 days after alkali damage. The specimens were fixed with 4% paraformaldehyde, dehydrated in a graded series of alcohol and the embedded in paraffin. Six-micrometer sections were cut and stained with hematoxylin and eosin for light microscopic examination. PMNs and
Therapeutic effect of 1% Na Hyaluronan
131
kcratocytes were counted both in the central and marginal areas of the stroma using a minor modification of the method of Campos ct al.", The whole histological specimen was photographed and the area (0.49 mrrr') for cell counting was randomly chosen in the 100-fold magnified photographs. Differential cytology was performed in a blind manner by light microscopy at a magnification of x400. The mean of 3 independent determination from each specimen was used for each cornea.
Endothelial wound morphomertry Eight corneas (8 animals) in each group were excised 5 days and 3 weeks after treatment and stained in alizarin red for 2 minutes. After rinsing, the specimen were immersed in 99% ethanol for 30 seconds and then stained with 0.25% trypan blue for 60 seconds. Immediately after staining, the endothelium was examined under the light microscope. Standardized photographs were taken for morphometric evaluation of the endothelial defect area. The endothelial defect area was determined from 10 times magnified photographs using a computer-assisted image analyzer (Cambridge, Mass., USA).
Statistical analysis Student's t test was applied for the comparison of the epithelial healing rates and for the endothelial defect areas between the Na-HA treated and control groups. Data obtained from the differential cell counting showed a skew distribution and were analyzed by Wilcoxon's two-sample rank test. The level of statistical significance used was 0.05.
RESULTS Epithelial healing The epithelial defects induced by both n-heptanol and 'alkali were resurfaced completely within 48 h of the damage. After the initial 48 h, recurrent epithelial defects occurred in all alkali wounded eyes. In alkali wounded eyes the initial epithelial healing rate in the Na-HA group was significantly increased compare to the PBS group. However, no significant difference was shown in n-heptanol damaged eyes between groups treated with Na-HA and PBS (Fig. I). In n-heptanol wounded cornea the number of hemidesmosome in the groups treated with Na-HA and PBS for 3 days were 10.0 ± l.l and 6.5 ± 2.5 and in those treated for 7 days 9.3 ± 1.9 and 6.6 ± 1.7, respectively (Fig. 2).
Stromal healing Two days after the alkali damage, a few cells, mostly PMNs, were observed in the central stroma of both control and Na-HA-treated groups (not determined). In the wound margin the number of keratocytes was significantly increased in the Na-HA treated eyes, while there was no significant difference in the number of PMNs between the two groups. After 7 days, the number ofPMNs was decreased in the Na-HA treated corneas. However, the number of keratocytes did not show any significant differences between the two groups (Table 1).
132
The function and use ofhyaluronan in wound healing ~
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Number of hemidesmosome in the basal epithelium. The number of hemidesmosomes was counted 3 and 7 days after n-heptanol injury. Data are plotted as the mean and S.D. from 8 separate corneas. * p
Table 1. The number of PMNs and keratoeytes
2 days 7 days
Center PMN n.d.
Keratocyte n.d.
Na-HA
n.d.
PBS
58.9 ± 24.5
Na-HA
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PBS
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n.d.
20.2 ± 7.7
225.1 ± 18.0"
125.7 ± 50.2
26.7± 30.1
177.0 ± 66.4
116.9 ± 56.5
9.8 ± 5.8'
183.2 ± 56.7
Data are expressed as the mean ± S.D. from 8 separate alkali damaged corneas. n.d.= not determined. * p<0.05, ** p
Therapeutic effect of 1% Na Hyaluronan
133
Endothelial healing Five days after the alkali damage only 2 out of 8 corneas in the Na-HA treated group showed any endothelial defects (0.61 and 0.74 mm'), while all eyes in the cont~ol group showed endothelial defect with a mean defect size of2.5 ± 2.0 mm' (p0.05, Fig. 3).
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The size of the endothelial defect area 5 days and 3 weeks after alkali wounding. Data are plotted as the mean and 95% confidence intervals in each group. ** p
DISCUSSION
In the present study two different types of corneal wounds, i.e. n-heptanol for the epithelial damage and alkali (1M NaOH) for the penetrating injury, were used for the evaluation of the therapeutic effects ofNa-HA on corneal wound healing. HA was initially introduced as a biologically inert substance and was used mainly as a supportive material to prevent tissue damage from physical insults during the surgical procedure". However, HA was found to be synthesized in the cell membrane and to have a widespread distribution in the extracellular matrices of various basement membrane I 5. 16. Several eell types, such as those of the corneal epithelium, keratocytes, endothelial cells, iris pigmented epithelium or lens epithelium, have been suggested to be capable of producing endogenous HA. Immunohistochemical studies also have identified I-lA-specific receptors in the cornea". HA has been shown to control cell migration and growth, cellular differentiation, and tissue morphogenesis. These effect of HA should facilitate wound healing and topically applied Na-HA has been reported to protect the surface structure of the corneal epithelium from chemical insult", increase the stability of the tear film" and enhanced the migration of corneal epithelial cells': 5. 20. Although Na-HA does not alter the epithelial healing rate of n-heptanol wounds, the result of the present study indicates that topically instilled Na-HA enhanced the formation of hemidesmosome in the basement membrane of the resurfaced epithelium. In chemically insulted cornea, hemidesmosome morphogenesis occurs after the regrowth of epithelial cells on the damaged cornea, and thus is dependent on the growth
134
The function and usc ofhyaluronan in wound healing
rate of the epithelium. Hemidesmosome act as adhesion molecules connecting the intermediate filament cytoskeleton network with components of the basement membrane zone": 22. Reduction in the number of hemidesmosome may influence cytoarchitecture remodeling and extracellular matrix interaction, and their recovery following injury may be associated with recovery of normal cytoarchitecture. Therefore, the Na-HA induced enhancement of hemidesmosome morphogenesis may play an important role(s) in recovery of normal epithelial cell function after corneal injury. Following corneal alkali injury (5.5mm, 11M NaOH, 60s-modelI 2) , the damaged epithelium resurfaced at around 2 days after the initial wounding. A long-standing recurrent epithelial erosion started immediately after the initial healing". The two main cell types, i.e. keratocytes and PMNs, were postulated as the main cellular components involved in the repair process of stromal healing. Although keratocytes normally synthesize collagen and proteoglycans, both keratocytes and PMNs are capable of releasing protolytic enzymes during the wound healing for the purpose of removing dead cells and cellular debris 23•2s • PMNs are known to be the predominant inflammatory cells that cause stromal ulceration and melting in the cornea following alkali injury. Hence, the prevention of stromal PMN infiltration may be crucial for the management of corneal alkali wounds. Mechanical barriers, such as a therapeutic contact lens, soft bandage lens, glue and artificial epithelium, have been tested to prevent PMN infiltration into the stroma, but the results were not satisfactory. In the present study, the eyes treated with Na-HA showed faster epithelial healing rate and had fewer PMNs but more keratocytes in the stroma than the control group during the early period of corneal alkali wound healing. These findings appeared to correlate with the endothelial healing, since the endothelial wound morphometry performed 5 days after injury showed a smaller defect area than the control cornea. These results seem to imply that topically applied Na-HA may act as a barrier, either directly because of its viscous property or indirectly by strengthening the resurfaced epithelium to stay on the stroma, to suppress PMN infiltration from the tear film into the stroma during the early healing period. Therefore, Na-HA may reduce the inflammatory reaction in the stroma and thus enhance the stromal keratocyte repopulation and endothelial healing during the early repair process of the alkali damaged cornea. In summary, topically applied Na-HA showed various therapeutic effects on the repair process of corneal wound healing. Further investigation is, however, needed to determine whether Na-HA retains any direct pharmacological action on both cellular and extracellular compartments during corneal wound healing.
ACKNOWLEDGEMENTS The author acknowledges financial supports from the Korean Ministry of Education (BK 21), Samsung Corp., DaeWoong Pharma. Co. and Yoohan Pharma. Co ..
REFERENCES 1. 2. 3.
F. M. Polack & M. T. McNiece, The treatment of dry eye with Na hyaluronate (Healon): A preliminary report. Cornea, 1982,1,133-136. 1. C. Stuart & J. G. Linn, Dilute sodium hyaluronate (Healon) in the treatment of ocular surface disorders. Ann. Ophthalmol. 1985, 17, 190-192. D. Saric & M. Reim, Behandlung von Veratzungen des vorderen Augenabschnitts mit hochpolymerem Na-Hyaluronat (Healon).Fortschr. Ophthalmol. 1984, 81,
Therapeutic effect of 1% Na Hyaluronan 4. 5. 6. 7.
8. 9.
10.
11.
12. 13.
14.
15. 16.
17.
18.
19. 20.
21.
135
588-591. .J.-H. Chung, P. Fagerholm & B. Lindstrom, Hyaluronate in healing of corneal alkali wound in the rabbit. Exp. Eye Res. 1989,48, 569-576. T. Nishida, M. Nakamura, H. Mishima & T. Otori, Hyaluronan stimulates corneal epithelial migration. Exp. Eye Res. 1991,53,753-758. M. Reim & V. Lenz, Behandlung von schweren Veratzungen mit hochpolymerer Hyaluronsaure (Healon). Fortschr. Ophthalmol. 1984,81,323-325. B. P. Toole, Morphogenetic role of glycosaminoglycans (acid mucopolysaccharides) in brain and other tissue, In: Neural Recognition, S.H Baronders(ed), Plenum Press, New York, 1976, pp275-329. M. Inoue & C. Katakami, The effect of hyaluronic acid on corneal epithelial cell proliferation. Invest. Ophthalmol. Vis. Sci. 1993,34,2313-2315. T. D. Fitzsmmons, N. Molander, U. Stenevi, P. Fagerholm, M. Schenholm & A. von Malmborg, Endogenous hyaluronan in corneal disease. Invest. Ophthalmol. Vis. Sci. 1994,35, 2774-2782. 1.-H. Chung, W.-K. Kim, J.-S. Lee, Y.-S. Pae & H.-J. Kim, Effect of NaHyaluronan on hemidesmosome formation in n-heptanol-induced corneal injury. Ophthalmic Res. 1998,30,96-100. J.-H. Chung, Y.-K. Park, S.-M. Paek, Y.-H. Chong & W.-K. Kim. Effect of NaHyaluronan on stromal and endothelial healing in experimental corneal alkali wounds. Ophthalmic Res. 1999, 31,432-439. J.-H. Chung, Experimental corneal alkali wound healing. Acta Ophthalmol. 1988, 66 (Supplement 187), 1-35. M. Campos, K. Szerenyi, M. Lee, J. M. McDonnell, P. F. Lopez & P. J. McDonnell, Keratocyte loss after corneal deepithelialization in primates and rabbits. Arch. Ophthalmol. 1994, 112,254-260. E. A. Balazs, Sodium hyaluronate and viscosurgery, In: Healon, A Guide to Its Use in Ophthalmic Surgery, D. Miller & R Stegman (eds.), Chichester, Wiley & Sons, 1983, pp 5-28. A. M. Alho & C B Underhill, The hyaluronate receptor is preferentially expressed on proliferating epithelial cells. J. Cell Biol., 1989, 108, 1557-1565. C. B. Underhill, The interaction of hyaluronate with the cell surface: The hyaluronate receptor and the core protein, In: The Biology of Hyaluronan. Ciba Found Symp. D. Evered & 1. Whelan (eds), Chichester, Wiley & Sons, 1989, 143, pp87-106. A. Tengblad, Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding proteins from cartilage. Biochem. Biophys. Acta, 1979,578,281-289. Y. S. Wysenbeek, N. Loya, I. Ben Sira, I. Ophir & Y. Ben Shaul, The effect of sodium hyaluronate on the corneal epithelium: An ultrastructural study. Invest. Ophthalmol. Vis. Sci. 1988,29,194-199. J. C. Stuart & J. G. Linn, Dilute sodium hyaluronate (Healon) in the treatment of ocular surface disorders. Ann. Ophthalmol. 1985, 17, 190-192. M. Nakamura, N. Sato, T-I. Chikami, Y. Hasegawa & T. Nishida, Hyaluronan facilitates corneal epithelial wound healing in diabetic rats. Exp. Eye Res. 1997,64, 1043-1050. M. C. Madigan & B. A. Holden, Reduced epithelial adhesion after extended contact lens were correlates with reduced hemidesmosome density in cat cornea. Invest. Ophthalmol. Vis. Sci. 1992,33,314-323.
136 22.
23.
24.
25.
The function and use ofhyaluronan in wound healing E. L. Stock, M. A. Kurpakus, B. Sambol & J. C. Jones, Adhesion complex formation after small keratectomy wounds in the cornea. Invest. Ophthalmol. Vis. Sci. 1992,33,304-313. M. S. Hibbs, K. A. Hasty, 1. M. Seyer, A. H. Kang & C. L. Mainardi, Biochemical and immunological characterization of the secreted forms of human neutrophl gelatinase. .J. Bioi. Chern. 1985, 260, 2493-2500. J. R. Chin, G. Murphy & Z. Werb, Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. .J. Biol. Chern. 1985,264, 1367-1376. D. Brown, M. Chwa, M. Escobar & M.e. Kenney, Characterization of the major matrix degrading metalloproteinase of human corneal stroma: Evidence for an enzyme/inhibitor complex. Exp. Eye Res. 1991,52,5-16.
RHEOLOGICAL BEHAVIOUR OF HYALURONAN, HEALON AND HYLAN IN AQUEOUS SOLUTIONS Michel Milas·, Marguerite Rinaudo', lsabeUe Roure· 1 Saphwan AI-Assaf, Glyn O. Phillipsl,' and Peter A. Williams E-mail: phil!ipsglynialaol.eom 'Centre de Recherches sur les Macromolecules Vegetates. CNRS, B.P.53, affiliated to Joseph Fourier University, 38041 Grenoble Cedex 9, France. 2Centre for Water Soluble Polymers, The North East Wales Institute, Wrexham, LL11 2.4 W, Wales, UK.
ABSTRACT Rheological properties of hyaluronan solutions are related not only to the molecular weight or concentration, but also to the origin of the samples. Here we present a comparative rheological study of hyaluronan, in aqueous solutions, from bacterial and animal sources with a cross-linked hyaluronan (hylan). Using a variety of rheological techniques, the behaviour of hyaluronan (M, 0.8 - 2.2 x 106 ) , crosslinked hyaluronan (hylan) (M; 1.8 - 12.5 x 106) and Healon (M, ~ 5 X 106) (a proprietary hyaluronan) was studied over a large range of molecular weights. The object was to study the effect of the cross-links in hylan on the various rheological parameters, in comparison with linear hyaluronan. There are significant differences. The Huggins constant and the critical overlap parameter C*[l1] are considerably lower for hylan and an increase in moduli at low frequencies was observed for hylan compared with the hyaluronan samples at all molecular weights studied. The results point to a difference in structure in dilute solution for hylan due to the ability to form networks, which can be removed by pressure filtration. In contrast, we do not find an increase of the steady shear viscosity and elastic modulus at higher concentrations when a homogeneous entangled network is reached. In i-dilute regime, imejhe ' VISCOSity , . scales as C 4.1 to C 3.6 b . t he t he semie N ewtornan y 'mcreasmg polymer concentration. The critical shear rate and the cross-over frequency (IDp) vary as 2 3 15 C- M . The plateau modulus G'- GO scales as cl· . We attribute this behaviour, which agrees well with the de Gennes prediction in a good solvent, to the semi-rigid character of the hyaluronan chain and to the predominance of entanglements over the cross-link points present in hylan in the semi-dilute domain. Due to the higher apparent molecular weights which are possible with hylan structures but not with the hyaluronans currently available, a wider range of applications can be achieved with hylans when viscoelasticity is required, particularly for the viscosupplementation of synovial fluid damaged by osteoarthritis.
KEYWORDS Hyaluronan, hylan, rheology, molecular weight, viscoelasticity
INTRODUCTION Hyaluronan occurs naturally in vitreous humour synovial fluid and umbilical cord and
182
Rheological behaviour of hyaluronan
in many animal tissues in smaller concentrations. It has been reported that the molecular 5 7 weight of naturally occurring hyaluronan varies within the range 10 to 10 1,2. Hyaluronan can be produced from biological sources such as bovine vitreous, umbilical cord and rooster comb. The highest molecular weight of hyaluronan produced from 6 animal sources commercially available is ca. 5 x 10 produced by Pharmacia and marketed under the trade name of Healon GV. Hyaluronan can also be produced from certain strains of Streptococcus bacteria, but the molecular weight of bacterial hyaluronan is less, despite of an easier production process of fermentation, extraction and purification from the broth. The highest molecular weight produced by this method is about 2.5 x 106 . Balazs and co-workers 3-6 have developed a family of cross-linked hyaluronan derivatives called hylans (HY) which can be produced either as a water soluble form (hylan A) or as viscoelastic gels (hylan B) 7. In the first procedure, formaldehyde is used at neutral pH to produce a permanent bond between the C-OH group of the polysaccharide and the amino group of a protein with relatively small molecular size and specific affinity to the hyaluronan chain. The protein forms a bridge between two polysaccharide chains. Under appropriate conditions, the cross-linking process will yield a molecular network consisting of permanent association of two to eight HA molecules 6 4.7. The weight average molecular weight of HY molecules is 2 - 24 x 10 with a protein content of 0.4-0.8% of the total polysaccharide weight 4. The second cross-linking process utilises vinyl sulfone. The HY molecules obtained from this technique are insoluble but are produced in the form of a viscoelastic gel 3,4. The two cross-linking procedures retain the biocompatibility and physical functionality of the unmodified hyaluronan, but physicochemical parameters such as molecular weight, molecular size and rheological properties, of the polymer solution or suspension, on hydration are substantially affected 3.4. Thus HY can be used in applications for which high molecular weights are needed such as viscosupplementation for the treatment of osteoarthritis of the knee joint 8,9. In these applications the viscoelastic properties of hyaluronan solutions play an important role and were first studied by Balazs and co-workers 11).12. It was shown that at low frequencies (long periods of deformation) HA macromolecules have enough time to readjust themselves to the original conformation through the disentanglements of chain segments. Under this condition the material response is essentially viscous. On the other hand, when HA is subjected to rapid deformation, at high frequencies, the entangled network structure can readjust itself very quickly and reform other entanglements giving rise to the overall elastic response of the material. As an example, from the values of the critical shear rate, for the onset of non-Newtonian flow, we can estimate the longest relaxation times in the solutions 13. They vary, according to the results given in this work from about 10.3 sec in diluted solutions to more than 10 sec in semi-diluted solutions. Welsh et al 14 investigated the effect of mixing high molecular weight HA with low molecular weight chains on the transient network structure formed by HA in solution and found that the low molecular weight HA (short chains containing 60 disaccharides) resulted in the disruption of intermolecular network formation. On the other hand, addition of segments with a degree of polymerisation -400 and 3500 enhanced the transient network structure which was attributed to increasing the molecular association by hydrogen bonding. Bothner and Wik 15 studied solutions of high molecular weight HA at a concentration of 1% and showed that the rheological properties indicate the formation of a highly
Behaviour in aqueous solutions
183
entangled dynamic network structure. This dynamic network structure is enhanced by the chain-chain interaction which induces transient development of tertiary and higher level structures 16,17. Kobayashi et al. 17 studied the effect of concentration and molecular weight on the transient network structure formed by 1% HA solutions and showed that increasing the molecular weight rather than concentration was more effective in enhancing the network structure ofHA solutions. Yanaki and Yamaguchi 18 studied the effect of temperature (5-25°C) on the viscoelastic properties (G', Gil) of a range of 1% HA samples at physiological conditions 6 (Mw 0.12 x 10 to 2.2 X 106) . The temperature shift factor was utilised to show the reduction in the temporary network structure as a result of the reduction in the entanglement density with decreasing molecular weight. HA concentration and its degree of polymerisation decrease in the synovial fluid as a result of osteoarthritis, a degenerative disease of the hyaline articular cartilage of the synovial joint, which often culminates with the total loss of cartilage. The influence of joint disease on the viscoelastic parameters was studied 19-23, and it was demonstrated that the osteoarthritic synovial fluid is viscous, not elastic, over most of the frequency range studied, due to the degradation of the HA component. Thurston and Greiling 24 also examined several pathological fluids over a wide range of oscillation frequencies relevant to physiological function and showed that enzymatic degradation of synovial fluid virtually eliminated the elasticity of synovial fluid (SF), which then exhibited Newtonian viscous properties. Thus, HA appeared to be the central molecular species responsible for the viscoelasticity of SF. Nuki and Ferguson 23 showed that SF exhibited a constant zero shear viscosity at low shear rates and, shear thinning behaviour at higher shear rates. The transition from the Newtonian region to the nonNewtonian region occurred over a wide range of shear rates. This broad transition was attributed to the broad molecular weight distribution with a polydispersity ranging from 3-4. Schurz and Ribitsch 25 proposed that in the first stages of the disease the conformation ofHA changed from loose (free draining) to tight (non-draining) coiling of molecular chains, thereby reducing the number of entanglements and, as a result, SF loses its viscoelasticity and behaves as a Newtonian fluid. Thus, hyaluronan biomaterials play an important rheological role and are, nowadays, used in many medical applications 6,26,27
The aim of this study is to compare the behaviour of the solutions of HA, from bacterial and animal sources and HY obtained from formaldehyde cross-linking (hylan A) in relation to the molecular size of their chains and the influence of the crosslinking on the rheological behaviour.
EXPERIMENTAL The rheological and static laser light scattering methods have been previously described 28. Bacterial hyaluronan samples with different molecular weights were produced by ARD Co (Pomade, France). The protein content was determined to be less than 0.1% by weight. Sodium hyaluronate NAHA (lot No. ECI1693), Healon (lot No. 10594, 032795) and Healon (GV) were obtained from Pharmacia via Dr. E.A. Balazs (Biomatrix Inc). Hylan samples with different molecular weights were produced from rooster comb following the formaldehyde cross-linking procedure, and were kindly donated by Biomatrix (Ridgefield, NJ, USA). The associated number designations correspond to the company code for a particular sample. For example. Hylan (H49) was
184
Rheological behaviour ofhyaluronan
extensively used in this study. For ease of presentation in this text we shall use HY, HE and HA to refer to Hylan (H49), Healon (GV) and hyaluronan unless otherwise stated. RESULTS AND DISCUSSION Intrinsic viscosity and molecular weight determinations of hylan In general, for the HY samples with the lowest apparent molecular weights (M; < 1.5 6 x 10 ) , the effects of the cross-linking do not perturb the measurements. Their dilute solutions can be filtered through a 0.2 J.IlIl filters and the shear thinning character appears at sufficiently high shear rate to allow measurements with a capillary viscometer. In contrast for the higher molecular weight samples (Mw > 1.5 X 106) some difficulties appear during the filtration even through 0.45 um filters. Moreover, it is necessary to work at very low shear rates to reach the Newtonian plateau ( < 100 S·l). The Huggins constant k', as well as the product ofC and intrinsic viscosity (C*['Il]) characterizing the end of the linearity of the reduced viscosity against polymer concentration plots are lower than the value found for HE and HA (Table 1). Table 1. Values of the intrinsic viscosity, Huggins constant k' and the critical overlap parameter C*[ '11] over which the Huggins law no longer applies. Sample Rylan 49 Healon Hyaluronan
(t)) (mUg) 8100 6400
k' 0.2 0.4 0.4
C*(!))
1.2 3.5 3.8
In Table 1 the values of k' and C*[Tl] obtained for HA, HE and HY are compared. There are significant differences between HA, HE and HY. For HY both k' and C*[Tl] values are considerably lower and can be attributed to the presence of large size aggregates and cross-link points in the high molecular weight hylan. These differences cannot be detected from a log-log plot of the relative viscosity versus C['11] due to the lack of sensitivity in this representation. The values ofk' and C*[Tl] have been directly deduced from reduced viscosity versus C['11] curves in a linear representation. Molecular weight determination has been performed using multiangle laser light 6 scattering. For hylan samples with molecular weight higher than about 1.5 x 10 , the weight average molecular weight (Mw) depends on the pore size ofthe filter used to treat the solution. Large aggregate retention on the filters and pressure disaggregation during 6 the filtration process could be factors for the decrease ofMw from 10 x 10 to ca 1.5 x 106 . From ['11] and M w measurements, the Mark-Houwink parameters have been determined for the hylan samples from unfiltered solutions. We have confirmed the K and a values of 0.033 ern' g" and 0.77 which were found previously 29. These values are very close to K and a values of 0.0336 and 0.79 found for linear hyaluronan 13. The small difference in the 'a' parameter may be explained by the difference in the solvent concentration used (0.15 M NaCI for HY and 0.1 M for HA). Filtration, despite reducing Mw, exerted no significant effect on the intrinsic viscosity. This shows the presence of high molecular weight material, such as large compact aggregates, which did not contribute to the intrinsic viscosity but did do so to light scattering. This is not the
Behaviour in aqueous solutions
185
case for samples containing large viscoelastic aggregates and / or high molecular weight molecules such as hylan 49. We have previously reported the presence of such aggregates using atomic force microscopy 27. The comparative behaviour in extensional viscosity has also been compared for both HA and HY 30. When these parameters are applied to the results from the viscometric measurements at low shear rate using the Contraves rheometer, the M w ofHY (hylan 49) corresponds to 8 to 10 X 106 in good agreement with the value estimated from light scattering (Table 2). This also confirms the high molecular weight values found by light scattering for HY. Thus the special cross-linked structural features of HY, while involving networks provide a viscometric behaviour associated with a HA equivalent to Mw ~ lOx 106 in dimensions. Larger or smaller molecular sizes of HY can be produced showing the same overall characteristics and equivalent molecular weights from both methods. In the same way, the intrinsic viscosity obtained for HE, 6400 mUg at 250C in O.IM NaCI corresponds to an equivalent weight average molecular weight of 4.8 ± 0.3 x 106 determined using the Mark-Rouwink equation and a value of 4.7 ± 0.2 x 106 determined by static light scattering. Table 2. Weight average molecular weight (Mw x 106 ) for hylan under different filtration regimes. Samples Rylan (1065) Rylan (49) Rylan (50) Rylan (20) Hylan (50301)
10 10 12.5 1.9 1.78
0.45 1lm 3.1 3.0 3.0 1.6 1.47
0.2 J.1m 2.4
1.34
Shear flow measurements The shear flow behaviour of different hylan samples of different molecular weights have been compared with that of linear hyaluronans and Healon, For example, Figure Ia shows the viscosity of hylan and Figure I b for Healon solutions as a fimftion of the shear rate. It can be clearly seen that even at relatively high viscosities (> 10 mPa.s) the -2 -I Newtonian plateau is reached at lower than lOs . Knowing the respective intrinsic viscosities we are able to compare the behaviour of HA, HE and HY using a master curve representing the relative viscosities as a function of the overlap parameter C[TIl (Figure 2). From Figure 2 it can be seen that HY 49 lies on this master curve and the Newtonian viscosity for HY and its variation with the concentration corresponds to that expected for a linear hyaluronan having an intrinsic viscosity equal to that of hylan. The Newtonian viscosity, 110, equals to about 5 x 105 mPas for HE solution at 10gIL in O.IM NaCI at 25°C (Figure Ib). Then, from the master curve (Figure 2) this value corresponds to C[ TIl of 64 which characterises the HE solution. Consequently, Newtonian viscosities ofHA, HE and HY solutions are directly related to their apparent intrinsic viscosity irrespective of the presence of aggregate structures. 4 1.and the viscometric activation In the hylan semi-dilute regime, Tlsp scales as C . energy, E. ,(TI = A expEJRT), after reaching a maximum, decreases with concentration (C) in a similar manner to that observed for HA. This decrease was interpreted for HA as
186
Rheological behaviour ofhyaluronan
an increase in the stiffness of the chain, corresponding to a chain expansion 31,32. This can explain the larger viscosity exponent 4.1 found in this domain compared with the theoretical predictions, as for example in the reptation model of de Gennes, which scales as C3.75 in a good solvent 33,34. At larger concentrations when E. tends towards a plateau for hyaluronans 31, the viscosity exponent decreases to about 3.6 in good agreement with the de Gennes predictions. a
•
<:-O.BS e-1.77 C-3.51 C=5.16 e-7.06
·· 0
""boo
- ..
lOs
0
n
i
gil gil gil gil gil
0
1000
~""o ....
no
oo~o
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.-......
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100
1
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1000
j
100
i:-
.S
.
!!
.~
10
1 0.01
0.001
100
IO
0.1 shear ....te (s")
Figure 1. Shear viscosity as a function of shear rate for a range of (a) hylan HY 49 concentrations ( 0.85 - 7.06 gIL) and (b) Healon at 10g/L in 0.1 M NaCI, T = 25°C.
.
10· 7
· ·•
10
a
10·
u
lOS
~~
o
4 10
;/
~
HA Mw-20oo000 HA M_22oo000 Hylan
,.,. ~
~
3
10
't>
s
10' 10'
,,'
HA __BOOOoo HA __1000000 HA __1300000
...
,p~
_.~..,~ 10
100
C[Tl]
Figure 2. Relative viscosity as a function of the overlap parameter C[ '1]] for different molecular weight hyaluronans and HY49.
Behaviour in aqueous solutions
187
It is interesting to compare the different regions defined by the variation of E. with C with those described by Yanaki et al based upon the molecular weight dependence of the characteristics of the temporary network formed in hyaluronan solutions 18. The regions I and II ofYanaki et al., characterized by a CM product lower than 104 g2 mL- l mol-I, correspond to the region showing the large increase of'E, with C. In this region a homogeneous network is formed from initially molecularly dispersed HA molecules at the lowest concentrations 31,32. The domain in which we observe the large decrease in E. corresponds well with region ill observed by Yanaki et al, in which they observe the existence of the entanglement network through 110 and Joe, the zero shear viscosity and the steady shear compliance respectively and the presence of a stress over-shoot phenomenon. At its extremity, the region IV is very similar in its limit to that obtained at the highest concentrations used in this work and corresponds to a CM product larger 4 2 ·1 ·1 than 3 x 109 mL mol and a plateau for E, versus C. Then obviously there is a correlation between the regions defined by Yanaki et al. and those obtained from the variation of E, with C. This clearly relates the dependence of E. to an evolution of the transient network before reaching a saturated entanglement network. Two other parameters can be deduced from the flow curves the critical shear rate, ie' for the onset of non-Newtonian flow, and the slope p corresponding to the linear variation of log 11 vs log in the power law region. From the log-log plot of vs C for HY 49 and a series of HA samples of different molecular weights, an average slope of - 2 was obtained for HY and for the different HA 28. This comparison was not possible for HE due to lack of sample quantity. In Figure 3, the critical shear rate, r c- is represented in a double logarithm plot as a function of the average molecular weight at a constant polymer concentration equal to 10 gIL. The critical shear rate found for HY 49 corresponds again to the value expected for a linear hyaluronan with M, near 8 x 106, the molecular weight which compared well with that found for HY 49 from viscometry and light scattering measurements. For HE, is found to be O.ls· lwhich again in good agreement with M; estimated from the intrinsic viscosity value for this sample 28. The curve also shows that rescales as M". From these
32
·2
·3
results we find r c - C M for the behaviour ofboth HA and HY. Yo
(s")
103 , . . - - - - - - - - - - - - - - . . . . ,
10' 10'
·3
10°
Figure 3. Critical shear rate, r c- as a function of molecular weight for hyaluronan, Healon and hylan 49 at a polymer concentration equal to 10 gIL in O.IM NaCl.
188
Rheological behaviour ofhyaluronan
r
For comparison, if we assimilate c -1 to the longest relaxation time in the solution, the 5 3 de Gennes reptation theory, in a good solvent, predicts 'tr _ C1. M 34 The correspondence is quite good and again the largest exponent for the concentration obtained experimentally could be due to the increase of the chain stiffness as described previously in this domain 31. Moreover, as for 110 at higher concentrations a decrease of the slope is observed in the domain where a plateau is reached in E. 31,32. In this domain the theory of de Gennes can be applied without correction, because of our interpretation ofthe chain stiffness with C. In the power law region of the viscosity versus shear rate, it is well known that the slope p depends on the overlap parameter C[11] 35. For C[11] values larger than about 50, p tends towards a limit which as been predicted in the entanglement concept of Graessley to be equal to -0.818 35. The results obtained for HA, HE and HY representing a wide range of molecular weights correspond to this behavior (Figure 4) and all samples studied approach this limit.
• • • •
"'().818
HylDn (19) HylaD (1065)
o
Hytan (l':HDP) Hya..(3O) Hylan (H49) HylaD (50301) Hea1un (10054)
• .... III
NIIHA (ECl1t'N3)
o
A
HA(200K) HA(J.3M)
A
HA(2M)
.OM C[1l ]
Figure 4. Slope p in the power law region determined on flow curves of different molecular weight hyaluronans and hylan in 0.1 M NaCl at 25°C. Dynamic measurements Dynamic measurements performed on HY 49 at various concentrations (from 3.4 to 43 gIL) have been compared with.those obtain~d for HA ft concentratiops from 10 to 83 gIL and a range of molecular weights (2 x 10 , 1.3 x 10 and 0.8 x 10) and for HE at 10gIL. From these measurements a master curve was obtained taking as reference the G' and Gil curves obtained from HA solutions at the concentration equal to 40 gIL (Figure 5 a). The three HA samples give rise to the same master curve in the frequency range studied (not shown). In contrast, at a frequency lower than the cross-over point of the G' and Gil curves, HY 49 shows higher values for the modulus. This difference is attributed to the presence of large aggregate structures in solution and the higher polymolecularity of this sample. Figure 5 b shows a master curve obtained from HA and HE in reference to HE curve at 10 gIL. Here we observe no difference in the shape of these curves. The higher moduli observed in HY curves at low frequency does not exist with HE, indicative of a difference in the structures in solutions of hylan compared to HE At higher frequency G' and Gil curves tend to form a plateau which is a consequence of the existence of a network in the solution.
Behaviour in aqueous solutions
189
(A) G'(ro) • O"(ro) x a y (Pa) 10" 10' 10' 10' 10'
10" 10-' lO-:Z
~~VAIii"~""vv
r
oioto°~~t.'
-... - .-. r.r
_
....
0
..........=:." •.~~
XotoH =--x x
-
o~ . .
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...
r 10. 2
"-
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an-
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x
..
I(i'
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MYO' e-3.4$ RIlBY 0" C=3.4S gil KYO' e-8.CS9
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10"
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oo x a, (rad.s")
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--
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-
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....
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. ca
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0
..
~
,
10"
ax
* w
.. ro'
100
(rad/s)
..
10
2
,
Figure 5. Master curves for the reduced viscous (G ) and elastic (G) moduli versus reduced frequency for (A) hyaluronan (HA) (Mw = 1.3 x 10 6) and hylan 49, reference HA at 40 gIL. (B) for Healon (HE) and HA (Mw = 1.3 x 106) reference HE at 10 gIL in 0.1 MNaCl at 25°C.
The shift factors used to obtain the master curves can represented as a function of concentration for the five samples ofHA, HE and HY (at lOgL-J). In order to determine the influence ofMw we have represented the characteristic of the cross-over point (G p and G"p moduli and angular frequency rop) as a function of M w in Figures 6 and 7 respectively. For a better precision we have taken as a reference the value obtained at 40gIL, then it was not possible to include the HE results only obtained at 10gIL. The general behavior ofHY and HE (only at 10gIL) cannot be differentiated from that ofHA -2 ·3 and the following relations can be deduced from these curves: COp scales as C M with perhaps a decrease in the concentration dependence at the highest concentrations. This behavior is similar to that observed for i c and not too far from the theoretical variation 3 C·J.S M· expected in good solvents according to the reptation theory, if we assume that Olp and i c vary as "t/ 34 and an increase of the chain stiffuess (decrease in E.) with C in this domain. Gp scales as C2.15 MO. This variation applies to that of the plateau modulus Geo too. Again, these variations agree quite well with those predicted for a semi-dilute solution in good solvent by de Gennes, Geo - C2.25 MO 36.
190
Rheological behaviour of hyaluronan G'P. G"p (N/m')
103
, 0' l..-.........................a._ _-'-_~...o-................~.L-_.......J
, o·
7 10
M
Figure 6. Molecular weight shift factor applied to the moduli. Reference: the moduli at the cross-over point for C = 40 gIL in 0.1 M NaCI at 25°C, as a function ofthe weight average molecular weight. "'p
1 0' ~----------------.
..
rad.s")
1 0'
- 3
, o·
'0
7
Figure 7. Molecular weight shift factor applied to the frequency. Reference: the frequency at the cross-over point for C = 40 gIL in 0.1 M NaCI at 25°C, as a function ofthe weight average molecular weight.
From the above results and by assuming a homogeneous network solution and the validity of the rubber elasticity theory on viscoelastic liquid it is possible to calculate Me, the average molecular weight of the polymer segments between two entanglements using the following equation 37:
This relation is valid for Me « Mw . The dependence of Me with the polymer Ll5 concentration is given in the figure 8 and Me scales as C· . This variation is quite different from the results obtained by de Smedt et al. 38. The difference can be possibly due to the lower concentrations, frequencies and molar masses used in their work and then the difficulty that arises in reaching the plateau modulus. Using the same processes that de Smedt et al. applied to their results, it is possible from the equivalent network model of Schurz 39 to determine the average distance between entanglement points DN and their variation with the polymer concentration. DN = (6 Mel C Na)1l3 where Na is the Avogadro number.
Behaviour in aqueous solutions
191
The results given in Figure 9 show that DN scales as C-o·72, which again is very close to the de Gennes prediction, if we assume that the concentration dependency of D N approximates the concentration dependency of the mesh size ~ in the blob model of de Gennes which for a semi-dilute solution in a good solvent is ~ - C-O·75 36. The values of DN range between 40 nm at 10 gIL to 8 nm at 100 gIL. No difference was observed between HA and HY samples. The low Me and DN values obtained need to be correlated with the semi-rigid character of the hyaluronan chains compared with flexible chains where larger values of Me and DN are usually found in the same range of polymer concentration. Me (glmol)
-1.15
10'
. x
10"
10'
C (gil)
Figure 8. Molecular weight between entanglement points Me as a function ofHA and HY concentration in 0.1 M NaCI at 25°C. D,.(nnt)
10" . . . . - - - - - - - - - - - - - - - - - , .. • •
x
10'
HA. Mw=!If)()()()() HA Mw=/JOOQOO HA MM.=200()(J(J(} Rylan
C (gil)
Figure 9. Average distance DN between entanglement points as a function ofHA and HY concentration in 0.1 M NaCI at 25°C. CONCLUSIONS In flexible polymers, a small amount of cross-linking, associated with an increase of the polymolecularity, does not greatly modify the intrinsic viscosity but leads at low polymer concentrations to a lower viscosity compared with the parent uncross-linked polymer. In contrast a larger viscosity is usually obtained at higher polymer concentrations. In dynamic measurements the increase of polymolecularity can have a large effect on the moduli at low frequencies and corresponds to an increase of the
192
Rheological behaviour of hyaluronan
steady-state shear compliance when a small amount of cross-linking is present. At higher frequencies, the presence of cross-link points contribute to an increase in the elastic modulus. For hylan, a cross-linked hyaluronan, the expected differences are found only at low concentrations, giving a decrease of the critical concentration C·, the Huggins constant k' and an increase of the viscous and elastic moduli at low frequencies. In contrast, we do not find an increase of the steady shear viscosity and elastic modulus at higher concentrations when a homogeneous entangled network is reached. We attribute this behavior to the semi-rigid character of the hyaluronan chain and to the predominance of the entanglements on the cross-link points present in hylan in this semi-dilute domain. In this domain the scaling laws proposed by de Gennes from its reptation model apply quite well to the molecular weight and concentration dependence of 110 and if we assume that t c - . / The slightly higher concentration exponents found experimentally can be
iP
attributed to an increase in the stiffness of the ch~ this regime based on our previous 1 assumption 38. The elastic modulus G' scales as C· MO again in good agreement with the prediction of de Gennes in a good solvent, including the hylan behaviour. To achieve the same rheological functionality as hylan-49, for example, HA would need to have a molecular weight of ca. lOx 106 which cannot be reached with the available HA preparations. Consequently hylan is able to offer a much wider range of applications, particularly in viscosupplementation, and other areas where viscoelastivity is called for.
ACKNOWLEDGMENT We thank Dr Endre A Balazs for initiating and supporting this investigation and for his stimulating discussions throughout and ARD for the bacterial HA samples.
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9. 10.
T.e. Laurent, In: Chemistry and Molecular Biology of the Intercellular Matrix, vol. 3. E.A. Balazs (Ed.) Academic Press, London, 1970, pp. 703-732. E.A Balazs; D. Watson; I.F. Duff & S. Roseman. Arthritis Rheum. 1967, 10, 357-376. E.A Balazs; E.A Leshchiner; A Leshchiner & P. Band, United States Patent 1987,4, 713-448. E.A Balazs & E.A Leshchiner, In: Cellulosic Utilizations Research and Rewards in Cellulosic, H. Inagaki and G.O. Phillips Eds., Elsevier, New York, 1989 pp. 233-241. E.A. Balazs; P.A. Band; J.L. Denlinger et aI., Blood Coagulation and Fibrinolysis 1991, 2, 173-178. E.A. Balazs and J.L. Denlinger. J. Rheum. 1993,20, 3-9. E.A Balazs, A Leshchiner, AE. Leshchiner, N.E. Larsen & P.A Band, European Patent Office, 1990, EP 0 320 164 A2. M.E. Adams, In: Chemistry, Biology and Medical Application of Hyaluronan and its derivatives, Ed. Laurent, T.e., Postland Press, London, 1998, 243-253. M.E. Adams; M.H. Atkinson; A Lunier; J.I. Schuly; K.A. Siminovitch; J.P. Wade & M. Zummer, Osteoarthritis and Cartilage 1995, 3, 213-226. E.A Balazs, University of Michigan Med. Center J., Special Issue 1968, 255259.
Behaviour in aqueous solutions
11.
12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
27. 28. 29 30 29. 32. 33. 34. 35. 36. 37. 38. 39.
193
E.A. Balazs,. & D.A. Gibbs, In: Chemistry and Molecular Biology of the Intercellular Matrix, Vol. 3, Ed. E.A. Balazs, Academic Press, London and New York, 1970, 1241-1254. D.A. Gibbs, E.W. Merril; KA. Smith & E.A. Balazs, Biopolymers 1968, 6, 777791. N. Berriaud, M. Milas, M. Rinaudo, In: Polysaccharides in Medicine and Biotechnology, S. Dimitriu Ed. Marcel Dekker Inc., New York, 1998, pp.313334. E.l Welsh, D.A. Rees, E.R Morris, 1.K Madden, 1. Mol. BioI. 1980, 138,375. H. Bothner, & O. Wik, Acta Otolaryngol (Stockholm) 1987,442,25-30. E.R Morris, D.A. Rees, & E.1. Welsh, 1. Mol. Biol. 1980, 138,383-400. Y. Kobayashi, A. Okamoto & K Nishinari, Biorheology 1994, 31, 235-244. T. Yanaki & T. Yamaguchi, Biopolymers 1990, 30, 415-425. E.A. Balazs, G.D. Bloom & D.A. Swann, Fed Proc.1966, 25, 1813-1817. E.A. Balazs, In: Disorders of the Knee, Ed. A. Helfet., 1. B. Lippincott Co., Philadelphia, 1972, pp 63-75. lE. Gomez & G.B. Thurston, Biorheology 1993, 30, 409-427. M. Mensitieri, L. Ambrosio, L. Iannace, L. Nicolais, & A1. Perbellini, Mat. Sci.: Materials in Medicine 1995, 6, 130-137. K Nuki, & 1. Ferguson, Rheol. Acta 1971, 10,8-14. G.B. Thurston & H. Greiling, Rheol. Acta 1978, 17,433-445. 1. Schurz & V. Ribitsch, Biorheology 1987, 24, 385-399. E.A. Balazs, E. Leshchiner, N.E. Larsen & P. Band, In: Handbook of Biomaterials and Application, Ed. D.L. Wise, Marcel Dekker Inc., New York, 1995,2719-2741. E.A. Balazs, S. AI-Assaf, G.O. Phillips, In: Advances in Tissue Banking, (Ed. G.O.Phillips), 1999, 3, 357-397. S. AI-Assaf, Rheological Properties and Free Radical Stability of Cross-linked Hyaluronan (Bylan). 1997, Ph.D. Thesis, Universityof Salford, England. S. AI-Assaf, G.O. Phillips, D.J. Deeble, B.1. Parsons, H. Starnes & C. von Sonntag, Radiat. Phys. Chem. 1995,46,207-217. S. AI-Assaf, J. Meadows, G.O. Phillips & P.A. Williams, Biorheology 1996, 33, 319-332. M. Milas, L Roure, & G.c. Berry, 1. Rheology 1996, 40,1155-1166. I. Roure, N. Berriaud, M. Milas & M. Rinaudo, Les Cahiers de Rheologie XV 1997,4,458-466. P.G. de Gennes,Macromolecules 1976, 9, 587-593. P.G. de Gennes, Macromolecules 1976, 9, 594-598. W.w. Graessley,Adv. Polym. Sci. 1974,16,1-179. P.G. de Gennes, Scaling Concepts in Polymers Physics, Cornell University Press, Ithaca and London, 1979. PJ. Flory, Principles ofPolymer Chemistry, Cornell University Press, New York 1953. S.C. de Smedt, P. Dekeyser, V. Ribitsch, A Lauwers & 1. Demester, Biorheology 1993, 30, 31-41. 1. Schurz, Prog. Polym. Sci. 1991, 16, 1-53.
RHEOLOGICAL CREEP EXPERIMENTS UTILIZING MIXTURES OF 1% HYLAN A SOLUTION AND 0.5% HYLAN B GEL SLURRY Joanne M. Hoeffingl *, Shiro Matsuoka' and Endre Balazs' I
Biomatrix, Inc. and Matrix Biology Institute. 65 Railroad Ave. Ridgefield, New Jersey 07657 USA 2Hennan
F. Mark Polymer Research Institute. Polytechnic University. Six Metrotech Center. Brooklyn. New York 11201 USA
ABSTRACT This study utilized several rheological parameters measured during controlled stress creep experiments to characterize mixtures of hylan B gel slurry and hylan A solution. The study parameters were I} the characteristic ringing effect on the percent strain (deformation) response versus time observed after the instantaneous stress is applied. 2} the viscosity calculated from the slope of the retardation compliance curve during steady motion and 3} the percent recovery (relative return to zero compliance over the relaxation period) as a measure of elasticity.
KEYWORDS Hylan, hyaluronan, viscosity, elasticity, creep
INTRODUCTION In prior studies' using a controlled strain rheometer, no significant rheological differences were found when small ratios (20% or less) of hylan B gel slurry were mixed with hylan A solution. The rheology of a product used for viscosupplementation, Synvisc'", made of 80% per volume hylan A solution and 20% per volume hylan B gel slurry closely resembled the rheology of hylan A solution. By applying a very low shearing stress during a creep test we were able to find significant differences in both elasticity and viscosity.
MATERIALS AND METHODS The materials used in this investigation are mixtures of two hylans. The first is hylan A solution (I % polymer content. average MW 6 million). The second material is hylan B gel slurry which is an infinite network insoluble in water (average particle size 1 mrn, 0.5% polymer content). The solvent for both hylan A and B is phosphatebuffered physiological saline at pH 7. This study was done using a TA Instruments AR 100 Controlled Stress Rheometer2• Creep tests were performed on 11 sets of hylan A and hylan B mixtures. The samples were prepared by mixing various volume ratios of the two components with a glass-stirring rod for approximately 3 minutes. Both the retardation and the recovery step were done using a 3- minute period and a 5% acceptance tolerance. During the retardation step, a 0.5 Pa stress was applied.
196
Rheological behaviour of hyaluronan
RESULTS ANDDISCUSSION During the retardation step in a creep experiment, a stress ao. is applied to the sample, in principle instantaneously, and held constant, the resulting strain yet) is monitored over time. The creep compliance, J(t) is then defined as yet)! ao . The stiffer the material, the lower the compliance. During the recovery step, the stress is released and the strain (deformation) recoil is monitored over time. A solid material will immediately recover its original dimensions. A liquid sample would cease to deform and will not show any recovery. Viscoelastic materials such as hylan, will exhibit characteristically retarded deformation and recovery curves. From the slope of the retardation spectrum versus time, we can calculate the zero shear viscosity. By calculating the relative return to zero strain in the recovery step, we are able to measure elasticity. An increase in zero shear viscosity of 50% was observed with the addition of 20% hylan B gel slurry to hylan A (1 %) (see Table 1 and Figures I and 2). By further increasing the percentage of hylan B gel slurry, the zero shear viscosity substantially increased. When the mixture contained 80% hylan B gel slurry, the zero shear viscosity increased 460%. Rylan B (0.5% cross-linked gel slurry) exhibited 23 times higher zero shear viscosity than hylan A (1%) alone (see Table 1 and Figures 1 and 2). An 8% increase in elasticity was noted with the addition of 20% hylan B gel slurry to hylan A (1%) (see Table 2 and Figures 3 and 4). Rylan B gel slurry exhibited a 21% increase in elasticity over hylan A, which itself is a very elastic fluid with a creep recovery of 83% (see Table 2 and Figures 3 and 4). All samples including hylan A (1%) showed a characteristic ringing strain response versus time when the strain was initially applied, indicative of a very viscoelastic material struggling to exhibit both solid and fluid-like behavior (see Figure 5).
Creep experiments
197
hylan A solution (1 % polymer) 80% hylan A + 20% hylan B
50% hylan A + 50% hylan B 20% hylan A + 80% hylan B hylan B gel slurry (0.5% polymer) 250.0
200.0
300.0
Time (s) Figure 1.
The typical retardation spectrum for hylan A solution and hylan B gel slurry mixtures. A 0.5 Pa stress was applied.
% hylan A solution 100
90
80
70
10
20
30
60
50
40
30
20
10
0
40
50
60
70
80
90
100
100000
~
~
....
';:'. 1l1OOO
'in Q OJ til
;;
100
-=C'-l= 1000 e ~
~
100 0
% hylan B gel slurry
Figure 2.
The zero shear viscosity of hylan A solution and hylan B gel slurry mixtures. The zero shear viscosity is calculated using the slope of the retardation spectrum in the creep compliance curve versus time. A constant stress of 0.5 Pa was applied.
198
Rheological behaviour of hyaluronan
0'17500~ 0.15000
_ W
0.12500
.5
•
W
M W
'M ' • • • • • • • • •IMI.lMllMll...
bylan A solution (1 % polymer)
0.10000
~
80% bylan A + 20% bylan B
I:J.J 0.075000 0.050000 0.025000
~~~~
50% bylan A + 50% bylan B
• • • • • • • •_ " " - " " "
20% bylan A + 80% hylan B bylan B gel slurry . _ .(0.5%polymer)
50.00
100.0
150.0
200.0
250.0
300.0
Time (s) Figure 3.
The typical strain (deformation) recovery for hylan A solution and hylan B gel slurry mixtures. A 0.5 Pa stress was applied during the retardation step.
% bylan A solution 100
90
80
70
60
50
40
30
20
10
0
10
20
30
40
50
80
70
80
90
100
95
to
c:.l
> Q
90
~
85
.....
-E=
...
~
~
80
75
70
% bylan B gel slurry Figure 4.
The percent strain recovery of hylan A solution and hylan B gel slurry mixtures. The stress was held constant at 0.5 Pa during the retardation step, then released at the start of the recovery step. The percent strain recovery is calculated at 180 seconds after the release of the strain.
100
Creep experiments Table 1.
199
Viscosity Results of Creep Experiments For Mixtures of Hylan A Solution (1% polymer) and Hylan B Gel Slurry (0.5% polymer).
Average % Increase Mixture Comoosition Zero Shear in Viscosity hylanA hylan B over 1 % Viscosity* solution gel slurry hylan A (Pa.s) (1 % polymer) (0.5% polymer) (%) (volume %) (volume %)
100 80
0 20
1200 + 600 1850±800
50 20
50 80
3200+ 800 7000± 1700 29,OOO± 10,000
-
50
Medical Applications
Synvisc" for viscosupplementation
160 460
Hylagel"" Neuro for viscoseparation
Hylagel(g) Uro Hylaform@ for vtscoausmentatlon *The zero shear viscosity is calculated from the slope of the compliance versus time curve generated during the retardation step.
0
Table 2.
100
2200
Elasticity Results of Creep Experiments For Mixtures of Hylan A Solution (1 % polymer) and Hylan B Gel Slurry (0.5% polymer).
Mixture Comnesltien hylan A hylan B solution gel slurry (1 % polymer) (0.5% polymer) (volume %) (% volume)
Average % Increase in Percent Elasticity over Strain 1% Hylan A (%) Recovery*
Medical Applications
(%)
-
100 80
0 20
83+7 89±.5
8
50 20
50 80
95+2 98±1
15 18
Synvisc@ for viseosunnlementation Hylagel(g) Neuro for
viscesenaration
Hylagef8'Uro Hylaform@ for viscoauzmentatien *The higher the percent strain recovery, the more elastic solid-like IS the material. An ideal elastic material will have a 100% strain recovery. An ideal viscous liquid will have 0% strain recovery.
0
100
99±0.3
21
200
Rheological behaviour of hyaluronan
1. 50% hylan A + 50% hylan B 2. 20% hylan A + 80% hylan B 3.80% hylan A + 20% hylan B 1.5000
4. hylan A solution (1 % polymer) 5. hylan B gel slurry (0.5% polymer)
1.2500
";=
-.
1.0000
r:J:J 0.75000
~
0.50000
Figure 5.
Time (s) The characteristic nngmg effect observed when 0.5 Pa stress is initially applied during the retardation step.
CONCLUSIONS These creep experiments using very low shearing stress demonstrated the rheological efficacy of mixing hylan B gel slurry and hylan A solution to obtain a material which is both more elastic and viscous than hylan A alone, even at a 20% hylan B + 80% hylan A ratio. Much higher zero shear viscosity values were extrapolated using this method for these high MW materials than previously calculated using controlled strain rheometers. The addition of hylan B gel slurry to hylan A solution in such therapeutic devices as Synvisc" (hylan G-F 20) and HylagelfNuro (hylan G-P 80) substantially increases the viscous and elastic properties of these products.
REFERENCES I. E.A. Balazs, & E. A. Leshchiner, Hyaluronan, its crosslinked derivative -hylan- and their medical applications, In: Cellulosics Utilization: Research and Rewards in Cellulosics (Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future), H. Inagaki & G.O. Phillips (eds.), Elsevier Applied Science, New York, 1989, pp 233-241. 2. Advantage Software Help Files, TA Instruments, Inc., New Castle, DE, PIN 92571O.0TOI Version 1.1.
FUNCTIONS OF HYALURONAN IN WOUND REPAIR W. Y. John Chen ConvaTec Wound Healing Research Institute, First Avenue, Deeside Industrial Park, Flintshire, United Kingdom, CH52NU
ABSTRACT Hyaluronan (HA), a major extracellular matrix macromolecule, has a repeated disaccharide structure that is completely conserved throughout a large span of evolution, indicating a fundamental biological importance. It has unique hygroscopic, rheological and viscoelastic properties, binds to many other extracellular matrix molecules, to body cells through cell surface receptors, and has a unique mode of synthesis in which the molecule is extruded immediately into the extracellular space upon formation. HA has many roles in biology, including skin wound healing. HA and its various chemical derivatives have already been used in wound healing or related tissue repair applications. This article aims to review how HA may function in wound healing through the utilisation of its general physicochemical and biological properties. The challenges of elucidating how the many functions of HA interact in tissue repair, and the possible functions of externally applied HA in modulating the wound healing response, are also discussed.
INTRODUCTION HA is a major component of the extracellular matrix. Most cells in the body have the capability to synthesise HA during some points of their cell cycles, implicating its function in some fundamental biological processes. It is generally accepted that HA is associated with the tissue repair process, as first elucidated by studies in morphogenesis and oncology [1-3]. Although HA may participate in tissue repair processes, on the whole, the detailed mechanisms of how it functions are not entirely clear and are only beginning to be elucidated. Some of these functions may be attributed to its role as an integral part of the extracellular matrix. Because of its unique hygroscopic, rheological and viscoelastic properties, HA may also affect cellular behaviour by affecting the extracellular macro- and microenvironment through its complex interactions with cells and other connective tissue components. HA and its oligosaccharides may also directly affect cell function through receptor binding events that directly lead to alteration of specific gene expression. Because of its unique physicochemical properties, and in particular its nonimmunogenicity, HA has found medical applications for many years, primarily in ocular and joint surgery [4-6]. More recently, the reported benefits of exogenously applied HA in tissue repair have resulted in HA-based biomaterials being developed for wound healing purposes.
148
The function and use of hyaluronan in wound healing
EXPERIMENTAL STUDIES OF HYALURONAN IN WOUND HEALING The function of HA in tissue repair is complex. It is recognised that HA has a dynamic role in connective tissue activation and inflammation [7]. The role of HA in tissue homeostasis can also facilitate the many biological functions that contribute to tissue repair. For a comprehensive review of the functions of HA in wound healing, please refer to the recent review of Chen & Abatangelo [8]. Many reports have attested to the effects of exogenous HA in influencing a beneficial wound healing outcome. In animal experiments, topically applied HA has been shown to accelerate skin wound healing in rats [9,10] and hamsters [11]. Similar results have been observed in the healing of perforated tympanic membranes in rats [12]. Corneal epithelial wound healing is also reported to be stimulated by applied HA [13]. In a study using the porcine dermal wound model, Navsaria et al. [14] showed that a benzyl alcohol ester of HA promotes healing and resulted in a better-organised wound bed, in comparison to the placebo group. HA has also been reported to affect beneficially the quality of tissue repair. The lack of fibrous scarring in foetal wound healing has been attributed, at least in part, to HA, the levels of which remain high for longer periods than in adult wounds. This leads to the suggestion that HA may, at least in part, reduce collagen deposition and therefore results in reduced scarring [4,15,16]. HA may also have a protective effect on chronic wounds, many of which have been shown to be highly inflammatory [17-21]. Foschi et al. [10] showed that HA prevents free radical damage to granulation tissue in rats. Ialenti & Di Rosa [22] also demonstrated the inflammation-moderating effect ofHA.
CLINICAL STUDIES OF HYALURONAN IN WOUND HEALING Exogenous HA has been used successfully for many years in ophthalmologic applications, joint conditions and post-surgical wounds [4-6]. An early paper by Vilesov et al. [23] has described the use of HA in burn wound bed preparation. Trabucchi et at. [24] carried out a clinical study, using topical treatment with HA, through the drains of the laparotomy suture. HA treatment reduced the incidence and degree of dehiscence macroscopically, increased the maturation of granulation tissue during the first post-operation days, and stimulated fibroblasts to synthesise procollagen shortly after the operation. Ortonne [25] reported a multicentre controlled clinical study on 50 patients with venous leg ulceration. The efficacy and safety of HA in comparison to Dextranomer, the product of choice for this indication in France, was evaluated. Both groups recorded significant wound improvements, but there was a faster and greater reduction in ulcer dimensions following treatment with HA, as well as significant reduction of oedema compared to the control group. Edmonds and Foster [26] reported encouraging results using a benzyl ester of HA for treatment of diabetic foot ulcers when used as an adjunct to the standard treatment that consists of sharp debridement, pressure relief and infection control. This was in comparison to the control group of patients who received the standard treatment only. Overall, the amount of experimental and clinical data available on the beneficial effects of HA is limited. However, the available data, although anecdotal, has already stimulated the exploration of HA and HA-derived materials as wound healing products. Some of these are already available for clinical practice.
functions of hyaluronan in wound repair
149
Table 1: A summary of tissue repair events involving hyaluronan. Stase Process Inflammatory phase lnllammation Activation
Granulation phase
Reepithelisation
Remodelling
Mechanism • Enhancement of cell infiltration • Increase of proinflammatory cytokines TNF-<X. IL- II} and IL-8 via a CD44-mediated mechanism • Facilitates primary adhesion of cytokine-activated lymphocytes to endothelium Inflammation • Free radical scavenging and antioxidant properties Moderation • TSG-6 and lui mediated inhibition of inflammatory proteinases Cell prnlilcralion • Hyaillfonan synthesis facilitates cell detachment and mitosis Cell migration • Increased hyaluronan synthesis • Hyaluronan-rich granulation tissue provides open. hydrated matrix that facilitates cell migration • Receptor mediated cell migration, e.g. CD44, RHAMM Angiogenesis • Angiogenic properties of low molecular weight byaluronan 01igosuccharidcs Kcratinocyte • Hyaluronan-rlch matrix is associated with proliferating functions basal keratinocytes • Facilitates keratinocyte migration via a CD44-mediated mechanism Scarring • Hyaluronan-rich matrix may reduce collagen deposition leading to reduced scarring as seen in foetal wound healing
Reference 29 30 31 47-49 50 44-46 35.39·41 2 32,33 54-61 28
64 4.15.16
ROLE OF HYALURONAN IN WOUND HEALING PROCESSES Following injury, wound healing follows a series of tightly regulated sequential events. These are inflammation, granulation, reepithelialisation and remodelling. HA is likely to have a multi-faceted role in mediation of these cellular and matrix events. This is summarised in Table I. The putative roles of HA in this sequence of wound healing events are described in more detail in the following sections. Inflammation Inflammation is the important first step that occurs shortly after injury. Inflammation cleanses the wound of damaged tissue, combats infection and recruits the necessary cell populations to rebuild the tissue. Wound tissue in the early inflammatory phase of wound repair is rich in HA, probably reflecting increased synthesis [27,28]. HA can act as a promoter of early inflammation. HA has been shown to enhance cellular infiltration into sites of inflammation [29]. Kobayashi & Terao [30] have shown a HA-dose-dependent increase of the proinflammatory cytokines TNF-a, IL-I~ and lL-8 production by human uterine fibroblasts, via a CD44 mediated mechanism. Microvascular endothelial cells, in response to inflammatory cytokines such as TNF-a and IL-l~, and bacterial lipopolysaccharide, also synthesise HA and this has been shown to facilitate primary adhesion of cytokine-activated lymphocytes expressing the HAbinding variants of CD44 [31] under laminar and static flow conditions. In a somewhat contradictory role, HA can also be a moderator of inflammation. This may contribute to the stabilisation of granulation tissue matrix. Granulation and organisation of the granulation tissue matrix Granulation tissue matrix is rich in HA [27,28] and may contribute in a variety of ways to essential tissue repair functions. HA and cell migration • Cell migration into the wound site is essential for the
150
The function and use ofhyaluronan in wound healing
formation of granulation tissue. An HA-rich extracellular matrix, characteristic of early granulation tissue, is regarded as a conducive environment for cell migration into this provisional wound matrix because of an open, hydrated matrix [2]. At the same time, via cell surface HA receptors, directed migration and control of the cell locomotory mechanisms can be mediated. The principal HA receptors include CD44, ICAM-I and RHAMM. RHAMM, in particular, forms links with several protein kinases associated with cell locomotion [32,33]. During foetal development, the migration path through which neural crest cells migrate is rich in HA [1]. Increased cell movement in response to HA can also be demonstrated experimentally in other cell types [32,33], whereas cell movement can be inhibited, at least partially, by HA degradation or blocking HA receptor occupancy [34-38]. HA synthesis has also been shown to correlate with cell migration [35,39-41]. HA synthesis may itself provide the dynamic force to facilitate cell migration, as it is synthesised at the plasma membrane and released directly into the extracellular environment [42,43]. This may provide the hydrated microenvironment, at sites of synthesis, to facilitate cell detachment essential for cell migration. Cell proliferation - cell proliferation is also an essential part of tissue repair. It has been shown that increased HA occurs, and is essential for fibroblast detachment from the matrix and mitosis [44]. HA has been shown to facilitate cell detachment [45,46], but has not displayed any direct mitogenic activity. However, through facilitating cell mitosis in response to mitogenic factors which are abundant during the early phases of tissue repair, HA may also have an important, albeit indirect, role in cell proliferation. HA and inflammation - Although inflammation is an integral part of granulation tissue formation, for normal tissue repair to proceed, inflammation needs to be moderated. The initial granulation tissue formed is highly inflammatory, with a high rate of tissue turnover mediated by matrix degrading enzymes and reactive oxygen metabolites that are products of inflammatory cells. Inflammation needs to be moderated in order to allow stabilisation of the granulation tissue matrix. HA protects against free-radical damage to cells [47-49]. This is probably mediated through a free-radical scavenging property. In a rat model of free-radical-induced inflammation, HA has been shown to reduce damage to the granulation tissue [10]. In addition to the free-radical scavenging role, HA may also function in the negative feedback loop of inflammatory activation. For example, HA binding to tumour necrosis factor stimulated gene-6 protein (TSG-6) and inter-n-inhibitor (IaI) has been reported to form a potent proteinase inhibitor complex that may attenuate the high levels of proteinase activity associated with inflammation [50J. As shown in a murine air pouch model of inflammation, where HA has been shown to have a proinflammatory property, administration of TSG-6 results in reduction of inflammation comparable with systemic dexamethasone treatment [51]. Angiogenesis - HA may also have a role in the control of angiogenesis. High molecular weight HA has been shown to inhibit angiogenesis [52,53], but low molecular weight HA oligosaccharides have been shown to promote angiogenesis in several experimental models [54-56], and to enhance the production of collagens by endothelial cells [57]. This phenomenon may be related to HA oligosaccharides inducing expression of several inflammatory genes including TNF-a. and IL-113 [58-61J. Observations of angiogenesis coinciding with increase of hyaluronidase and degradation of matrix HA have been made in several in vivo systems [53,62]. Hyaluronidase digestion of foetal wound HA leading to fibroplasia and capillary formation [63] are in general agreement with the hypothesis of a physiological role HA and its oligosaccharides have in the control of angiogenesis.
Functions of hyaluronan in wound repair
151
Reepithelialisation HA has important functions as an integral patt of the extracellular matrix of basal keratinocytes. Its free-radical scavenging function suggests an important role in normal epidermal biology, whereas its role in keratinocyte proliferation and migration strongly implies an important role in the reepithelialisation process. In wound healing, epidermal HA is particularly implicated in the control of keratinocyte proliferation. In healing wounds, HA is expressed in the wound margin, in the connective tissue matrix, and co-locates with CD44 expression in migrating keratinocytes [28]. Kaya et at. [64] showed that suppression of CD44 expression by an epidermis-specific antisense transgene resulted in animals with defective hyaluronate accumulation in the superficial dermis, accompanied by distinct morphologic alterations in basal keratinocytes and defective keratinocyte proliferation in response to mitogen and growth factors. Also observed are decrease in skin elasticity, and impaired local inflammatory response and tissue repair. These observations are strongly supportive of the important roles HA and CD44 have in skin physiology and tissue repair.
Foetal wound healing and scarring Foetal wound healing is characterised by lack of fibrous scarring. HA content in foetal wounds remains high for longer periods than in adult wounds, leading to the suggestion that HA may, at least in part, reduce collagen deposition and therefore reduce scarring [15,16]. These suggestions are in agreement with the data which showed that applied HA resulted in reduced scarring of healed tympanic membranes that had been perforated [12], and with Balasz & Denlinger [4] who hypothesised that aHA-rich environment inhibits the matrix cells responsible for fibrous scars. In a recent paper, West et at. [16] showed that in adult and late gestation foetal wound healing, removal of HA results in fibrotic scarring. As HA is a multi-functional molecule, it has many other functions that may well also contribute to the scarless healing quality of foetal wounds. A persistent HA-rich environment may affect cell-cell and cell-matrix interactions differently from that of an environment in which elevated HA is transient, such as that of the post-natal wound environment. This may lead to different activation and control of various cell populations in comparison to the post-natal wound environment.
CHRONIC WOUND PATHOLOGY AND HEALING CHALLENGES Much of the information on the role of HA in wound repair has been elucidated from the healing of acute wounds. With recent advances in the study of chronic wounds, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers, it is becoming clear that the chronic wound pathology is quite different from that of acute wounds [17-21]. Circulatory abnormalities, leading to chronic inflammation, arc the main features of these wounds (Figure I). The main causes for generation of these wounds and the impairment of their healing are likely to be the excessive tissue breakdown processes that are characteristic of severe inflammation. These are primarily reactive oxygen metabolites and matrix degrading enzymes, which, working in concert, can lead to very rapid tissue turnover, further activation of matrix proteinases, impairment of proteinase inhibitor functions and continuous promotion of inflammation. Whether HA has any function in chronic wound pathology is at present not known.
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The function and usc ofhyaluronan in wound healing
Ischaemia-reperfusion _ . . . . cycles
Tissue Ischaemia
'-_--'
Peripheral venous hypertension
Vascular Activation Platelet
~
actlval/on
Endothelial damage II
,
~
activation
Leukocyte
activation
-.
Leukocyte
extravasation
I Fibroproliferation
Tissue Breakdown
Figure 1: Schematic diagram showing the pathological events that are likely to occur during pathogenesis of the common chronic wounds - venous ulceration, pressure ulceration and diabetic foot ulceration. Recent clinical studies have reported some benefits of externally applied HA or HAbased materials on the healing of chronic wounds [25,26]. Even though these results are from small studies and should be considered to be anecdotal, they nevertheless raised the interest as to whether, and to what extent, HA may playa role in the modulation of the pathology of chronic wounds. In particular, it is interesting to speculate whether the inflammation modulation role, and angiogenic property of HA oligosaccharides, may function in promoting chronic wounds to heal. CONCLUSIONS It is clear that HA has many functions in biology. Some of its functions may be attributable to its biophysical properties and some to its biological properties as an extracellular matrix molecule as well as a biological signalling molecule. Much work still needs to be done in order to elucidate the biological mechanisms of HA in tissue processes. Although many of the HA functions are known individually, how these function interact with each other, and how they are related to other tissue factors to mediate the complex biological processes that are necessary for wound repair, remains poorly understood. Elucidating these complex mechanisms will undoubtedly be as a scientific challenge for the future. Because of its unique physicochemical properties, HA has already seen biomedical applications. In tissue repair, its physicochemical properties, the promising results shown in in vivo experimental studies and early clinical studies, and its known biological properties, strongly indicate applications in mediation of the wound healing process as well as a biomaterial for bioengineering purposes. Already in this field, products have been developed for anti-adhesion, wound healing, tissue implants and for
Functions ofhyaluronan in wound repair
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moisturising purposes [4,5]. These products are either based on pure HA, or derivatives using various chemical modification techniques to improve their physical handling and stability characteristics. Currently, HA-based medical products are mostly classified as medical devices, utilising the physical attributes of HA to achieve their intended functions. No doubt when the biology of HA becomes better known, applications will also be developed to utilise its biological functions.
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The function and use of hyaluronan in wound healing H. G. Wisniewski & J. Vilcek, TSG-6: an IL-l/TNF-inducible protein with antiinflammatory activity. Cytokine Growth Factor Rev. 1997,8, 143-56. H. G. Wisniewski, J. C. Hua, D. M. Poppers, D. Nairne, J. Vilcek & B. N. Cronstein, TNF/IL-I-inducible protein TSG-6 potentiates plasmin inhibition by inter-alpha-inhibitor and exerts a strong anti-inflammatory effect in vivo. J. Immunol. 1996, 156, 1609-1615. H. F. Dvorak, V. S. Harvey, P. Estrella, L. F. Brown, J. McDonagh & A. M. Dvorak, Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab. Invest. 1987,57,673-86. D. C. West & S. Kumar, The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Exp. Cell Res. 1989, 183, 179-196. V. C. Lees, T. P. Fan & D. C. West, Angiogenesis in a delayed revascularization model is accelerated by angiogenic oligosaccharides of hyaluronan. Lab. Invest. 1995, 73, 259-66. Sattar, P. Rooney, S. Kumar, D. Pye, D. C. West, 1. Scott & P. Ledger, Application of angiogenic oligosaccharides of hyaluronan increases blood vessel numbers in rat skin. J. Invest. Derm. 1994, 103, 576-579. D. C. West & D. M. Shaw, Tumour hyaluronan in relation to angiogenesis and metastasis. In: The Chemistry, Biology and Medical Applications (Jj' Hyaluronan and its Derivatives. T. C. Laurent (ed.) London: Portland Press, 1998, pp 227-33. P. Rooney, M. Wang, P. Kumar & S. Kumar, Angiogenic oligosaccharides of hyaluronan enhance the production of collagens by endothelial cells. J. Cell Sci.
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P. W. Noble, F. R. Lake, P. M. Henson & D. W. H. Riches, Hyaluronate activation of CD44 induces insulin-like growth factor-I expression by a tumor necrosis factor-a-dependent mechanism in murine macrophages. .1. Clin. Invest. 1993,91, 2368-2377. C. M. McKee, M. B. Penno, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao & P. W. Noble, Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J. Clin. Invest. 1996, 15, 2403-13. P. W. Noble, C. M. McKee, M. Cowman & H. S. Shin, Hyaluronan fragments activate an NFKB/IKBa autoregulatory loop in murine maerophages. J. Exp. Med. 1996, 183,2373-8. P. W. Noble, C. M. McKee & M. R. Horton, Induction of inflammatory gene expression by low-molecular-weight hyaluronan fragments in macrophages. In: The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T. C. Laurent (ed.), Wenner-Gren International Series 72, Portland Press, London, 1998, pp 219-225. D. C. Liu, E. Pearlman, E. Diaconu, K. Guo, H. Mori, T. Haqqi, S. Markowitz, J. Willson & M. S. Sy, Expression of hyaluronidase by tumor cells induces angiogenesis in vivo. Proc. Natl. Acad. Sci. USA, 1996,93,7832-7837. B. A. Mast, R. F. Diegelmann, T. M. Krummel & 1. K. Cohen, Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts. Matrix, 1993, 13,441-446. G. Kaya, 1. Stamenkovie, P. Vassalli, 1. L. Jorcano & 1. Rodriguez, Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 trans gene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation. Genes Dev. 1997, 15,996-1007.
THE ROLE OF PERICELLULAR MATRIX FORMATION DURING WOUND HEALING IN RENAL STONE DISEASE Marieke SJ Schepers", Burt G vd Boom & Carl F Verkoelen Department of Urology, Erasmus University Rotterdam. iN! Be330, P. a.Box 1738, 3000 DR Rotterdam, The Netherlands.
ABSTRACT The adherence of crystals to the surface of renal tubular cells is considered one of the earliest events in kidney stone formation. Since crystal-cell interaction is difficult to study in vivo, we developed a model to study crystal retention using Madin Darby Canine Kidney cells (MDCK). Cultured on permeable supports MDCK cells form functional epithelial monolayers with a high transepithelial electrical resistance (TER>5000 Q"'cm2) that are non-adherent to calcium oxalate (CaOx) crystals. The epithelium becomes susceptible to crystal adherence, however, during its recovery from mechanical created wounds. During repair the cells produce a cell coat enriched with hyaluronan. This polysaccharide in the pericellular matrix of mobile cells has been identified as a binding molecule for CaOx crystals. The association of the hyaluronan enriched cell coat is mediated by specific receptors. The main cell surface receptor for hyaluronan is CD44. It is possible that CD44 also serves as hyaluronan receptor at the surface of MDCK cells. Studies with anti CD44 antibodies demonstrated the co-localisation of CD44 and hyaluronan at the surface of mobile cells. Pericellular matrices are structurally stabilised by hyaluronan binding proteins (HASP), like members of the inter-u-trypsin inhibitor (IT!) family of proteins. Studies with specific antibodies showed that ITI-related proteins are indeed present in the hyaluronan-rich cell coat surrounding proliferating MDCK cells. The possibility that the association of crystals with pericellular matrix constituents along the urinary tract somehow is involved in the pathophysiology of renal stone disease is supported by the fact that the organic kidney stone matrix is enriched with hyaluronan and IT! proteins and by the finding that urine of stone forming men is enriched with IT! proteins. Collectively these observations suggest that tissue repair plays an important role in the aetiology of nephrolithiasis.
KEYWORDS Nephrolithiasis, MDCK strain I, crystal-cell interaction, pericellular matrix, inter-a -trypsin-inhibitor, hyaluronan, tissue repair.
INTRODUCTION The accumulation of crystalline material in the kidney sooner or later leads to stone formation. Calcium crystals that are occasionally present in anyone's urine normally are eliminated unhindered with the urine. Binding to renal tubular epithelium may turn harmless urine crystals into a stone nidus l •3 , Considering the potential hazard of crystal attachment it is reasonable to assume that physiological mechanisms exist to prevent
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The function and use of hyaluronan in wound healing
it. Evidence has been provided that, at sites in the urinary tract where crystals can be formed, the tissue indeed is resistant to crystal adherence', Tissue injury triggers an inflammatory response followed by re-epithelization and remodelling. Cell culture studies showed that calcium oxalate crystals adhered to the hyaluronan-rich pericellular matrix expressed by proliferating and migrating cells in the wound. The present study was undertaken to study the role of tissue repair in crystal retention more in detail.
MATERIALS & METHODS Cell culture, Preparation of CaOx crystals, Crystal binding, Wounds made in confluent monolayers These studies were performed with MDCK strain I cells. All these methods are described in detail elsewhere'<.
Confocal laser scanning microscopy (CLSM) Cells were fixed and permeabilized in 70 % ethanol for 15 minutes. Subsequently, the inserts were washed with PBS, cut out and incubated on both sides with rat a canine CD44 antibody (DAKO), diluted 1:50 in PBSI 0.1 % Tween! 5 % non-fat dry milk, overnight at 40 C. Secondary fluorescently (FITC) labelled anti-rat antibody was used (1:50) for 30 minutes at room temperature. To monitor the location of the cells in the sample, nuclei were counterstained for 15 minutes with propidiumiodide (l :500) in PBS. Inserts were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). Images were made with a Zeiss LSM 4110 confocal microscope (Oberkochen, Germany). A 488 urn Ar-laser was used to excitate the FITC.
Visualisation of pericellular matrix The method to visualise the PCM is adopted from Knudsen et al. 9 and based on the ability of the otherwise invisible matrix that surrounds a number of cell types in vitro to exclude the penetration of particles. MDCK-I cells are plated at a low seeding density (lx104 cells) in 24 well plates. After 24 hours, 750 p.L of a suspension of fixed and washed mouse red blood cells ( - 108 cells/ml) was added to the cells and allowed to settle for 15 minutes, after which the PCM was inspected and photographic images were made by phase-contrast light microscopy. To demonstrate the structural dependence of the PCM on HA, 1 hour before visualisation with the particle exclusion assay the cells were treated with 1 Ulml hyaluronate lyase (EC 4.2.2.1). The adherence of crystals to PCM was also visualised with the particle exclusion assay. The cells were seeded at low densities on 24 well plates and 50 p.L COM crystal suspension (1.46 mg COM/ml) was added in a physiological saline solution. The crystals were allowed to adhere for 15 minutes after which all non-adhered crystals were removed by extensive washings. Subsequently, the particle exclusion assay using fixed mouse red blood cells was performed as described above.
Isolation of pericellular matrix proteins MDCK-l cells were seeded at a density of 5 x 106 cells in plastic tissue culture
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flasks in DMEM + 10 % FCS. Two days post-seeding the cells were treated for 1 hour in serum-free DMEM with hyaluronidase after which the enzyme-releasable material was collected and Iyophilised. SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting SDS -PAGE was performed with a 1.0-mm thick 10 % mini-slab gel using MiniProtean II apparatus (Bio Rad). Samples of 20 ug protein and molecular weight standards were electrophoresed under non-reducing conditions. Proteins were visualised using silver staining or blotted on nitro-cellulose. The nitro-cellulose membranes were equilibrated in the blotting buffer (16.5 mmoIlI TRIS, 150 mmol/I glycine, and 200 ml methanol). Proteins were transferred for 1 h at 4° C in the same blotting buffer. Immuno-chemical staining was performed using polyclonal antibody rabbit-a-human inter-alpha-inhibitor (DAKO). Non-specific protein binding sites were blocked by incubating the nitro-cellulose membrane for 60 min in PBS/O.l % Tween/5 % non-fat dry milk. The membranes were incubated overnight with the primary antibody (4° C). Polyclonal antibody was used at 1:1000 dilution. The membranes were then incubated for 60 min. with the secondary antibody, goat-arabbit-HRP (DAKO) and used at a dilution of 1:1000. Finally, the membranes were allowed to develop for 1 minute by using a substrate kit (Bio Rad).
RESULTS & DISCUSSION Pericellular matrix formation by renal tubular cells in culture The particle exclusion assay was applied to visualise pericellular matrix formation by the MDCK-I cell line. This method demonstrated that proliferating cells were surrounded by clear areas up to 10 urn in size, which formed a barrier to the diffusion of fixed mouse red blood cells. This pericellular matrix disappeared after Hdasetreatment (30 min., 25 D/ml) indicating a functional role for hyaluronan in matrix formation. Phase-contrast LM-images showed that COM crystals appeared to have particular affinity for the clear zone around the cells. Cell surface expression of CD44 CD44 is the most common cell surface receptor for hyaluronan. The expression of CD44 at the surface of MDCK cells was studied with specific CD44 antibodies and visualised by confocal microscopy. CD44 appeared to be expressed randomly at the plasma membrane of proliferating subconfluent cells, but exclusively lateral (between cells) by quiescent cells in confluent monolayers. Western blotting showed the presence of CD44 in cell lysate, but not in PCM of subconfluent cells. ITI proteins in the pericellular matrix ITI and especially its heavy chains (HC) form stable covalently linked complexes with hyaluronan. ITI-HC are therefore known as hyaluronan-binding proteins (HABP). To investigate whether proliferating subconfluent MDCK-I cells produce
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The function and lise ofhyaluronan in wound healing
I'I'l-related HABP, PCM was isolated from developing cultures with hyaluronidase. Anti-ITI antibodies revealed that I'I'I-related proteins indeed were present in the PCM and in luminal culture supernatant. Although it is possible that these proteins were derived from serum in the growth medium, it was found that these cells were also capable to form relatively stable pericellular matrices under serum-free conditions. We are currently investigating the presence of I'I'l-related proteins or other PCM-associated HABP in the absence of foetal calf serum. The co-localisation of hyaluronan and CD44 at the surface of cells in subconfluent cultures suggests that CD44 is involved in the hyaluronan binding attachment of hyaluronan to the surface of MDCK-I cells in locomotion.
ITI proteins and kidney stones An important function of the kidney is the production of concentrated urine by the conservation of essential nutrients and the reduction of the quantity of salt and water excreted in the final urine. One of the side effects of this process is that the tubular fluid frequently becomes oversaturated with calcium salts. This oversaturated state can lead to the nucleation of crystals in the renal tubules. The continuous retention of crystalline material in the kidney ultimately leads to the formation of a stone. Members of the ITI family of proteins have our special attention because certain family members seem to playa role in nephrolithiasis. IT! is a glycoprotein composed of three polypeptide-chains: two heavy chains (HCI and HC2) and one light chain, also known as bikunin. IT! or its HC are normally only in trace amounts present in the urine. Bikunin can pass the glomerular filter and high levels are observed, for example, in urine of patients with various malignant tumours or infectious diseases. For several reasons members of the IT! family of proteins are considered nephrolithiasis-related, (I) Bikunin has been recognised as a potent inhibitor of crystallisation-''-'! (2) Hyperoxaluria is a common finding in renal stone disease. Renal tubular epithelial cells in culture produce bikunin as a response to high oxalate». (3) In rats, hyperoxaluria and crystalluria resulted in elevated levels of renal bikunin mRNA expression, which was accompanied by the urinary excretion of ITI-related proteinstt. (4) Only a minority (20 %) of the urine samples derived from normal males was found to contain IT! and its HC, whereas these proteins were found in all urine specimens derived from active male stone formersl-. These results can be interpreted that at least in males the synthesis of ITI proteins in the kidney is upregulated as a response to stone formation. However, it is also possible that the production of ITI proteins is a response to tissue damage.
Crystal retention The retention of crystalline material in the kidney is an absolute prerequisite for renal stone formation. Crystal growth and aggregation could lead to crystal clumps that are too large to freely pass the renal tubules. On the other hand, crystals may adhere to the surface of the epithelial cells lining the renal tubules. The latter possibility is repeatedly supported by histological inspection of renal tissue derived from hyperoxaluric humans and animals. We found that an intact and functional
Pericellular matrix formation
161
epithelium formed by renal tubular cells with characteristics of the late nephron is non-adherent to crystals 5. 7. 8. These non-adherent properties are lost after epithelial injury and during repair". Recently, we presented evidence that crystals most likely bind to hyaluronan (HA) in the pericellular matrix (PCM) synthesized by proliferating and migrating cells during wound healing.
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Possible mechanisms of stone formation in the kidney Although many risk factors have been identified, it is still difficult to outline the exact sequence of events leading to stone formation in the kidney. The finding that crystals adhere to the HA-enriched PCM surrounding wounds suggests that tissue damage in the kidney precede crystal retention and stone formation. It is conceivable that relatively small areas in the renal tubules are frequently subjected to injury-repair. Crystals passing these areas are continuously retained and released again into the tubular fluid after the wounds are healed. During this process the crystals are provided with a coat of sticky PCM which further retards their transit through the nephrons. Stones are cumulating crystalline/PCM material in corners that are not directly subjected to the force of the rapidly flowing fluid. Because of the limited and local nature of the epithelial damage this process may often proceed unnoticed. On the other hand, it is remarkable that the highest levels of nephrolithiasis-related proteins are found in urine of patients that actually actively are forming stones, suggesting that the inflammatory response is provoked by the stones themselves. In this scheme it is conceivable that newly formed crystals in the tubular fluid adhere to areas of injuryinduced reorganizing epithelia surrounding an existing stone leading to further stone
162
The function and use ofhyaluronan in wound healing
enlargement. In the latter situation, the stone-induced renal lesions may accelerate the stone forming process. Finally, it is possible that epithelial injury-repair processes in the kidney not only initiate but also aggravate stone formation.
REFERENCES 1. 2. 3. 4.
5.
6. 7.
8. 9. 10. 11.
12.
13.
Kok DJ, Khan SR. 'Calcium oxalate nephrolithiasis, a free or fixed particle disease'. Kidney Int 1994;46:847-854. Mandel N. 'Mechanism of stone formation'. Semin NephrolI996;16:364-374. Lieske JC, Toback FG. 'Interaction of urinary crystals with renal epithelial cells in the pathogenesis of nephrolithiasis'. Semin Nephrol1996; 16:458-473. Verkoelen C, Boom B vd, Romijn 1. 'Identification of hyaluronan as binding molecule for calcium oxalate crystals at the surface of mobile renal tubular cells in culture'. Kidney Int 2000;58:1045-1054. Verkoelen CF, van der Boom BG, Houtsmuller AB, Schroder FH, Romijn JC. 'Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture'. Am J PhysiolI998;274:F958-965. Verkoelen CF, Van Der Boom BG, Kok OJ, Schroder FH, Romijn JC. 'Attachment sites for particles in the urinary tract'. J Am Soc Nephrol1999; 10 Suppl 14:S430-435. Verkoelen CF, van der Boom BG, Kok DJ, Houtsmuller AB, Visser P, Schroder FH, Romijn JC. 'Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture'. Kidney Int 1999;55:1426-1433. Verkoelen CF, van der Boom BG, Kok OJ, Romijn JC. 'Sialic acid and crystal binding' . Kidney Int 2000;57:1072-1082. Knudson CB, Toole BP. 'Changes in the pericellular matrix during differentiation of limb bud mesoderm'. Dev BiolI985;112:308-318. Atmani P, Khan SR. 'Role of urinary bikunin in the inhibition of calcium oxalate crystallization'. JAm Soc Nephroll999;10 Suppl 14:S385-388. Medetognon-Benissan J, Tardivel S, Hennequin C, Daudon M, Drueke T, Lacour B. 'Inhibitory effect of bikunin on calcium oxalate crystallization in vitro and urinary bikunin decrease in renal stone formers'. Urol Res 1999;27:69-75. Marengo SR, Resnick MI, Yang L, Chung JY. 'Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone forming men'. J UrolI998;159: 1444-1450. Atmani P, Glenton PA, Khan SR. 'Role of inter-alpha-inhibitor and its related proteins in experimentally induced calcium oxalate urolithiasis. Localization of proteins and expression of bikunin gene in the rat kidney' Urol Res 1999;27:63-67.
RHEOLOGY OFHYALURONAN PRODUCTS Ove Wik, Bengt Agerup and Hege Bothner Wik Q-Med AB. Seminariegatan 21. S-752 28 Uppsala, Sweden
ABSTRACT
Various modified (stabilized or cross-linked) hyaluronan products used for tissue augmentation were examined by rheometry. Five products - Restylane Fine Lines, Restylane, Perlane, Hylaform and Dermalive - exhibited typical gel-like behaviour to varying degree after examination of the viscoelastic response as a function of frequency. This suggests that all products contain hyaluronan with permanent linkages between polysaccharide chains. One product (Rofilan) claimed to be a 'Hylangel' containing 'cross-linked' hyaluronan at a concentration of 20 mg/ml exhibited a behaviour typical of hyaluronan solutions. The results demonstrate that this product contains free hyaluronan chains with a molecular weight of 2 million. KEYWORDS
Rheology, viscoelasticity, cross-link, tissue augmentation. INTRODUCTION
Hyaluronan is intimately linked with rheology. The remarkable viscous and elastic behaviour of hyaluronan solutions in general and of body fluids such as synovial fluid in particular has been studied for decades. After the pioneering work by Balazs 1-2, modem rheometers were utilised for the subsequent development of hyaluronan products for use in e.g. ophthalmology and joint disorders. Fittingly, the multifaceted use ofhyaluronan in medicine has been described by Balazs as "viscosurgery" 3. In recent years hyaluronan has been modified by means of various types and varying degree of cross-linking 4-6. As a consequence of these modifications, products with quite different rheological behaviour have been marketed. We have performed basic rheological studies on some commercial products containing modified, gel-like hyaluronan derivatives, and report data on the viscous and elastic properties as a function of frequency. MATERIALS AND METHODS Samples
The following samples of modified hyaluronan products used in tissue augmentation were used. Lot numbers are given in parenthesis. Restylane Fine Lines (6083), Restylane (5922) and Perlane (6106) were obtained from Q-Med AB, Uppsala, Sweden. Hylaform (A709) was obtained from Biomatrix Inc., Ridgefield NJ, USA. Dermalive (VR26060) was obtained from Dermatech, Paris, France. Rofilan (4991) was obtained from Rofil Medical Nederland B.V., Breda, Netherlands. The products manufactured by Q-Med AB (Restylane Fine Lines, Restylane and Perlane) contain non-animal stabilised hyaluronan (NASHA) at a concentration of 20
202
Rheological behaviourofhyaluronan
mg/m!. These products are specifically designed for tissue augmentation in different layers of the skin. Information on the other products was obtained from the packaging inserts. Hylaform contains Rylan B at a concentration of5.5 mg/m!. Dermalive is a suspension of non resorbable fragments of acrylic hydrogel and a solution of slightly cross-linked hyaluronan. The product contain 200 mg/ml of acrylic fragments and 14.4 mg/ml ofhyaluronan. Rofilan is stated to be a 'Hylangel' containing 'cross-linked' hyaluronan at a concentration of 20 mg/m!. Rheological characterisation Bohlin VOR Rheometer System (Bohlin Reologi AB, Sjobo, Sweden) with the Windows compatible Millenium software was used for rheological characterisation. The samples were more or less gel-like and therefore all samples were studied in the oscillation mode by recording the response to varying frequency and strain. All experiments were performed at 25°C. When sufficient amount of sample (about 3 m!) was available the cup and bob measurement system C14 was used. Otherwise the cone and plate system CP5/30 (diameter 30 mm, cone angle 5°) was used. Precautions were taken to exclude possible effects of the formation of a dry hyaluronan film on surface layers. The strain-dependent response was recorded to ascertain determination of the viscoelastic response within the linear region. RESULTS AND DISCUSSION The viscoelastic response is shown in Fig. 1 where the elastic modulus (0') and viscous modulus (0") are plotted as function of frequency. All data were recorded at low enough strain to ascertain recordings in the linear region. Elastic modulus, G' (Pa) Viscous modulus, G" (Pa) ••••••.
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Rheology ofhyaluronan products
203
The classical fashion of describing the viscoelastic properties shown in Figure 1 is, though proper from a rheological point of view, somewhat complicated when discussing the rheological properties with the end user of products. Therefore, EA Balazs introduced a simple, yet very illustrious way to present and describe the viscoelastic properties of hyaluronate solutions and products. In most instances the interesting aspect of the viscoelastic properties as shown in Figure 1 is the proportion between elasticity and viscosity. A simple relationship describing the viscoelastic properties is obtained by calculation the percentage elasticity (Elasticity, % in graphs below) as follows: Elastic modulus· 100 G' Elasticity ('Yo) = Elastic modulus + Viscous modulus = G' + G" • 100
The same information is, of course, also obtained from the frequency dependence of the phase angle. However, the response for a viscoelastic sample changing from viscous to elastic gives a change in the phase angle from 90° to 0°, whereas the introduced parameter Elasticity (%) changes from 0 to 100. These data are plotted in Fig. 2 demonstrating that most products are predominantly elastic at all frequencies, whereas Restylane Fine Lines change behaviour in a somewhat complicated fashion and Rofilan is viscous at low frequencies and elastic at high frequencies. From the viscoelastic response the dynamic viscosity - 11' - may be calculated. The frequency dependence of the dynamic viscosity coincides with the shear rate dependence of the shear viscosity according to the Cox-Merz rule (Fig. 3). The results demonstrate a gel-like behaviour with a continuously increasing viscosity even at low Elasticity ("!o)
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10
204
Rheological behaviourofhyaluronan
frequencies for all samples except Rofilan. For the latter product a Newtonian, constant zero shear viscosity at low frequencies was recorded. The zero shear viscosity of hyaluronan is dependent on the concentration and molecular weight 7. Using the published formulas Rofilan was found to contain hyaluronan with a molecular weight of 2 million using the zero shear viscosity obtained (80 000 Pa • s) and the concentration (20 mg/m1) stated. CONCLUSIONS
The viscoelastic data obtained demonstrated that most products exhibit a gel or gellike behaviour to varying degree with an almost constant response independent on frequency. A slight variation in response was observed for Hylaform. Restylane Fine Lines showed a mixed behaviour changing from gel-like to solution-like indicating that the product is a pseudo-gel with a complicated mixture of solution- and gel-like response. Rofilan changed behaviour from predominantly viscous at low frequencies to elastic at high frequencies typical of a solution containing non-modified, separate hyaluronan molecules with molecular weight 2 million. REFERENCES
1.
D.A. Gibbs, E.W. Merrill, K.A. Smith & E.A. Balazs, The rheology of hyaluronic acid, Biopolymers, 1968,6,777-791.
2.
E.A. Balazs & D.A. Gibbs, D.A. The rheological properties and biological function of hyaluronic acid, In Chemistry and Molecular Biology of the Intercellular Matrix, E.A. Balazs (ed.), Academic Press, London and New York, 1970, pp. 1241-1254. E.A. Balazs & J.L. Denlinger, Clinical uses of hya1uronan, CIBA Foundation Symposium, 1989, 143,265-275.
3. 4.
E.A. Balazs et aI., Chemically modified hyaluronic acid preparation and method of recovery thereof from animal tissues, U.S. Patent No 4,713,448, 1987.
5.
B. Agerup, Polysaccharide gel composition, PCT/SE/96/00684, 1996.
6.
G.D. Prestwich et ai, Chemical modification of hyaluronic acid for drug delivery, biomaterials and biochemical probes. In The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T.e. Laurent (ed.) Portland Press, London and Miami, 1998, pp. 43-65.
7.
H. Bothner, Rheological studies of sodium hyaluronate in pharmaceutical preparations, Thesis, Uppsala University, 1991.
STRUCTURAL CHANGE IN HYDROGELATION OF HYALURONAN INDUCED BY ANNEALING THE SOLUTION IN SOL STATE Masato 'Takahashi', Takahiro Isekl', Hirotsugu Hattoril , Tatsuko Hatakeyama'' and Hyoe Hatakeyama' 'Department ofFine Materials Engineering, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, JAPAN 2Department of Textile Science, Faculty ofHome Economics, Otsuma Women's University, 12 Sanbancho, Chiyoda -ku; Tokyo 102-8357, JAPAN. 2Department ofApplied Physics and Chemistry, Faculty ofEngineering, Fukui University of Technology, 3 -6-1 Gakuen, Fukui 910-8505, JAPAN.
ABSTRACT Structural change of hyaluronan (HA) aqueous solutions in gelation induced by annealing in the sol state was investigated by differential scanning calorimetry (DSC). Melting enthalpy of water (.1 H m ) in the system was used as an index of structural change of HA molecules . .1 H m values remained constant when 3 wt% sol annealed at 60°C for 1 hr was maintained at the gelation temperature of 5 °C for 40 hours. On the other hand, .1Hm values fluctuated in an oscillatory manner during gelation at 5 °C when the solution was previously annealed at 60°C for 15 and 18 hr. This fluctuation (variation of .1 Hm) was not observed when the sol was annealed for 48 hours. The results suggests that a conformational change of HA molecules proceeds continuously during gelation when homogenization of the sol state is insufficient.
KEYWORDS Hyaluronan, gelation, annealing, differential scanning calorimetry
INTRODUCTION The gelation of polysaccharides is induced when aqueous solutions are annealed in the sol state [1-3]. By the annealing, hyaluronan (HA) and xanthan gum (XA), which are known as non-gelling polysaccharides, were found to form hydrogels [1-4]. It is thought that the molecular conformation of these polysaccharides changes in aqueous solution during annealing. The structural change of polysaccharides during the annealing process is revealed by the experimental results obtained by the falling ball method (FBM), differential scanning calorimetry (DSC), viscoelastic measurements and small angle x-ray scattering (SAXS) using a rotator anode and synchrotron radiation as the x-ray source [1-4]. When the junction zone formation of physical gels is investigated, not only the structural change of the polysaccharides in the sol state but also the structural change in the gelation process is an important subject to be considered. In this study, structural change of HAlwater systems in the hydrogelation process induced by annealing the
206
Rheological behaviour ofhyaluronan
solution in the sol state was investigated by differential scanning calorimetry (DSC). By DSC, the amount of bound water restrained by the saccharide network was estimated by calculating the melting enthalpy (f1Hm ) of water in the system. The annealing time dependency on the structure formation of HA aqueous solutions in the gelation process is discussed through variation of AHm • EXPERIMENTS
The HA used was supplied by Kibun Food Chemical Co. The nominal molecular weight was 2 x 106 • The glassware used in sample preparation was sterilized before use in order to avoid biological contamination. In DSC measurements, a Seiko DSC200 was used. The enthalpy of melting (f1Hm ) of water was measured at the heating rate 10°C/min. The experimental procedure is reported in detail elsewhere
[2,4]. RESULTS AND DISCUSSION
Annealing of solution As reported in our previous study [4], HA 3 wt % solutions formed gels when the aqueous solution was annealed at T = 60°C for more than 6 hr. Gelation was confirmed by FBM. It was revealed that the gel-sol transition temperature T g•s initially increases with the increase of annealing time. As shown in Figure 1, the enthalpy of melting f1Hm per 1 mg of water in the system measured by DSC showed oscillational change with repeated increases and decreases, and then approached a constant value in the annealing process. The final value of f1Hm was slightly smaller than that of the initial value. These experimental results suggested the following structural change occurs in the annealing and subsequent cooling processes. In the non-annealed solution, HA molecules form molecular assemblies and the solution is regarded as the emulsion or suspension of such assemblies. These assemblies seem to be metastable
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Structuralchange in hydrogelation
207
and easily destroyed by annealing the solution in sol state. If
Effect of annealing on gelation Figure 2 shows the relationship between IJ.Hm of 3 wt % samples and gelation time at T = 5 "C. The sample annealed in the sol state (T = 60°C) for 1 hour before maintaining the sample at 5 °C did not form a gel even when the sample was maintained at the same temperature for more than 50 hr. Similar relationships are shown in Figures 3-5 whose annealing times at sol state were varied at 15, 18 and 48 hr, respectively. As shown in Figures 2 and 5, IJ.Hm of sols annealed for 1 and 48 hr. remains constant at 5 "C. On the other hand, IJ.Hm varies in an oscillatory ways as shown in Figures 3 and 4. In particular, the change of IJ.Hm in Figure 4 is marked, i.e. IJ.Hm decreases in the initial stage, reaches a minimum point at around 10 hr. and then increases. This variation is very similar to that of IJ.Hm in the annealing process in the sol state, although IJ.Hm in the annealing process shows an increase at the initial stage. The gel was not formed for the sample shown in Figure 2. However, gels were formed for the samples shown in Figures 3,4 and 5. In spite of the similarity of the relationships of Figures 2 and 5, gels were formed in the former case and not in the latter. As described above, gelation was not observed when solutions were annealed for a period shorter than 6 hr suggesting that it takes a sufficiently long time in order to homogenize solutions by the destruction of molecular assemblies. Therefore, IJ.Hm of sols annealed for 1 hr. remain almost constant (Figure 2). On the other hand, sols annealed for longer than 6 hr. form hydrogels. The behaviour of IJ.Hm shown in Figures 3 and 4 suggests that further structural change is needed to form hydrogels in
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208
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order to complete the homogenization of the systems. In contrast to this, when well homogenized sol is used, LllIm during gelation remains almost constant (Figure 5). It is noteworthy that the value of Lllim shown in Figure 5 is slightly smaller than that in Figure 2, although the ,1Hm variation in both figures is similar. This fact suggests that the amount of non-freezing water in the sol that can form gels is slightly larger than that of the sol that can not form gels. Figure 6 shows the annealing time dependence of the equilibrium value of Lllim at 5 "C. The results suggest that the conformational change of HA molecules proceeds continuously during gelation when homogenization of the sol state is insufficient.
ACKNOWLEDGEMENTS This work was supported by Grant-in-aid for COE Research (No. 10CE2003) and that of (C) (No. 11650925) by the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. F. X. Quinn, T. Hatakeyama, M. Takahashi & H. Hatakeyama, 'The effect of annealing on the conformational properties of xanthan hydrogels', Polymer, 1994, 35, 1248-1252. 2. T. Yoshida, M. Takahashi, T. Hatakeyama & H. Hatakeyama, 'Annealing induced gelation of xanthan/water systems', Polymer, 1998, 39, 1119-1122. 3. J. Fujiwara, T. Iwanami, M. Takahashi, R. Tanaka, T Hatakeyama & H. Hatakeyama, 'Structural change of xanthan gum association in aqueous solutions', Thermochimica Acta, 2000, 352-353, 241-246. 4. J. Fujiwara, M. Takahashi, T. Hatakeyama & H. Hatakeyama, 'Gelation of hyaluronic acid through annealing', Polymer International, 2000, 49, 1604-1608. 5. M. Takahashi, T. Hatakeyama & H. Hatakeyama, 'Phenomenological theory describing the behaviour of non-freezing water in structure formation process of polysaccharide aqueous solutions', Carbohydrate Polymers, 2000, 41, 91-95.
PART 4
THE ROLE OF HYALURONAN IN TISSUES
IS HYALURONAN DEGRADATION AN ANGIOGENIC/ METASTATIC SWITCH? David C. West" and Haijuan Chen 1 Departments ofImmunology and I Haematology, Faculty ofMedicine, University ofLiverpool, The Duncan Building, Daulby St, Liverpool L69 3GA. UK.
ABSTRACT High-molecular-weight hyaluronan (HA) is inhibitory in both in vivo and in vitro models of angiogenesis. However, a discrete fraction of HA-degradation products have consistently been found stimulate angiogenesis in these same models. Recent studies in wound healing models revealed a close temporal relationship between tissue hyaluronidase activity, tissue HA-degradation and neovascularization. It appears that HA-degradation is a prerequisite for the induction of wound angiogenesis. On going studies, with transplantable tumours and tumour cell-lines, indicate that both metastatic properties and angiogenesis are associated with elevated levels of hyaluronidase and HA degradation. RT-PCR analysis suggests that this is due to an aberrant, or elevated, expression of the GPI-anchored cell-surface hyaluronidase PH-20 and, in some celllines, the HYALl hyaluronidase. PH-20 expression increases with both the angiogenic and metastatic potential of these cell-lines i.e. tumour progression. The role of HYALl is more controversial, but the present study suggests that in some cell-lines this may also playa role in the progression of tumours to a metastatic/ angiogenic phenotype. INTRODUCTION In common with other glycosamninoglycans, small amounts of HA are present in the extracellular matrix of most animal tissues, but the greatest concentrations of HA are found in the avascular adult connective tissues, such as cartilage, synovial fluid and the vitreous humour. Its common localisation to connective tissues, combined with its high water-binding capacity and chetnical simplicity, led to the general belief that its main function was that of an inert viscoelastic lubricant, or space-fillingmolecule. However, over the last ten years a growing number of reports have appeared indicating that HA can have profound effects on the behaviour of numerous cell- types, through its interaction with several surface receptors'<. These effects differ depending on the celltype concerned and appear dependent on both the size and concentration of the HA. A transient HA-rich matrix also develops during embryogenesis, and adult tissue remodelling, coincident with rapid cell proliferation and tnigration. Subsequently, a decrease in tissue HA occurs concotnitantly with tissue differentiation, vasculogenesis and angiogenesis 3. A localised accumulation of HA is also seen in association with tissue damage, organ rejection and many inflammatory diseases, such as psoriasis and scleroderma, and it is a major component of many tumours". The localisation and timing of HA accumulation during development and tissue repair suggests that the synthesis and degradation of HA plays an important regulatory role in these processes, especially vascularization, and many pathological conditions, including tumour growth and metastasis'.
166
The role of hyaluronan in tissues
HYALURONAN REGULATION OF ANGIOGENESIS. Size-dependent regulation of angiogenesis in vitro. Studies with cultured endothelial have consistently shown that macromolecular HA (;:::103 kDa), at HA concentrations found in adult avascular connective tissues and transiently during tissue remodelling and repair, significantly inhibits endothelial cell proliferation and migration" and disrupts cell/cell interactions in newly formed endothelial cell monolayers. This HA- mediated inhibition can not be reversed by the addition of exogenous growth factors, in vitro, unless the HA is degraded'. Recent studies, using a three-dimensional angiogenesis model in which a section of rat aorta is cultured in a collagen-gel matrix, under serum-free conditions', have confirmed and extended these original findings. Preliminary data using this model suggests that macromolecular HA selectively inhibits endothelial capillary formation in a size-dependent manner. In contrast, fibroblast numbers increase as the size of the HA increases. Watanabe et al. (1993l and Fournier & Doillon (1992)9 have independently reported similar inhibition, with isolated endothelial cells cultured in 3-dimensional collagen and fibrin gels. Our finding that a discrete fraction ofHA-degradation products (OHA: 2-8 kDa or 4-20 disaccharides in length) was able to induce angiogenesis in the chick chorioallantoic membrane (CAM) assay'", prompted a series of in vitro studies. These showed that OHA (1-10 0 g/ml) directly and specifically stimulated both the proliferation and the migration of cultured endothelial cells from several sources'r". Recently, OHA has been found to stimulate three-dimensional microvessel formation maximally at 0.1 flg/ml, compared with 1 DgimI in the two-dimensional models. Angiogenesis in this model is thought to be dependent on endothelial migration, rather than proliferation'f. Several reports, using alternative in vitro assays, have independently confirmed the angiogenic nature of OHA. Hirata et al. (1993)13 and Rahrnanian et al. (1997)14 reported that OHA markedly stimulated endothelial tube formation, in collagen gels and on "matrigel", Furthermore, two recent studies have shown that OHA can induce endothelial cell invasion of 3-dimensional collagen and fibrin matricesl 5,16, In addition, Trochon et aI. (1996,1997)16,17 both confirmed the lower size-limit of the angiogenic activity as the HA octasaccharide and that the effects were due to the HA-oligosaccharides, as they could be neutralised by the addition of specificHA-binding proteins. 10) Endothelial cells have about 105 high affinity Ha-receptors (Kd 2 X 10 on their cell 5 1 25]_ labelling, HA-affinity surface, that can both bind and internalise HA ,G. [1 chromatography, immune-blotting and RT-PCR analysis of cell-surface proteins from both human and bovine endothelial cells has identified non-variant CD44H as the major, if not the only HA-receptor, on non-hepatic endothelial cells. Although CD44 appears to play an important role in the angiogenic process 1G,18, it also binds the HA- hexasaccharidc'F", implicating an as yet uncharacterised endothelial cell HA-receptor in the "angiogenic" activity of OHA I9 However, Slevin et al (1998)20 have reported that OHA induces rapid phosphorylation of CD44 in bovine aortic endothelial cells. Also, CD44 mediates a similar size-dependent activation ofmurine macrophages and tumour celllines 21,22. Studies into the metabolic effects of OHA on cultured endothelial cells indicated that OHA could induce the expression of angiogenesis- associated proteins I5,23, The induction of E-selectin and IL-8 mRNA suggested that OHA rapidly induced NFkB activation. Electrophoretic mobility shift analysis (EMSA) and immune-blotting of cultured human umbilical vein endothelial cell proteins confirmed that OHA rapidly activates NFkB, but showed that macromolecular HA significantly down-regulates NFkB activation over 23 several hours . A similar size- dependent CD44-mediated activation of NFkB has been
Hyaluronan degradation
167
reported for murine macrophages'" and tumour celllines22. In contrast, Deed et al (1997)25 reported that OHA induces the immediate early response genes c-fos, c-jun, jun-B, Krox20 and Krox-24 in bovine aortic endothelial cells, mediated by the activation of protein kinase C20. However, Laniado-Schwartzman et al (1994i 6 reported that an angiogenic arachidonic acid metabolite, 12(R)-hydroxyeicosatrienoic acid, rapidly activated of both NFkB and, to a lesser extent, the AP-l transcription factor in cultured endothelial cells. Thus, OHA appears to bind to CD44 and activate both the NFkB and AP-l transcription factors, by a mechanism that may involve protein kinase C. HA metabolism and angiogenesis in vivo. In vitro studies suggest that the high levels of HA in avascular tissues, such as cartilage and vitreous humour, and at relatively avascular sites, such as the desmoplastic region of invasive tumours, are not coincidental and that extracellular matrix HA also inhibits angiogenesis in vivo', The finding that prior degradation of its HA matrix must precede cartilage neovascularization, gives support to this hypothesis". In addition, high concentrations of exogenous macromolecular hyaluronan have been shown to inhibit neovascularization during granulation tissue formation and induce regression of the immature capillary plexus in the developing chick limb bud . In contrast, exogenous OHA has consistently stimulated angiogenesis in the chick chorioallantoic membrane assay"; both wound healing and graft models, and after subcutaneous implantation or topical application (reviewed in refs 5,28). Hirata et al (1993)23 have independently confirmed the angiogenic activity ofthis range ofHA oligosaccharides, in the rabbit corneal assay. In a series of studies we examined the relationship between angiogenesis and tissue HA metabolism, using a rat sponge-implant wound healing model, a freeze-injured rat skingraft model, and sheep foetal wounds". In all three models there was an apparent close temporal association between tissue angiogenesis and the degradation of matrix hyaluronan, as indicated by rapid falls in both tissue HA size and level, concomitant with increased hyaluronidase activity. Mast et al (1992i 9 have shown that the addition of exogenous Streptomyces hyaluronidase to foetal wounds, decreases wound HA content and increases capillary formation. To date, we have not detected HA-degradation products as small as OHA in healing wound tissue, suggesting that merely removing the inhibitory effect ofmacromolecular HA may be sufficientto allow vascularization. TUMOUR HYALURONAN METABOLISM AND ANGIOGENESIS Increased hyaluronan levels have often been reported to be associated with human and animal malignancies, particularly in connection with tumour invasion", In addition, most tumours also exhibit increased angiogenesis (vessel density), notably metastatic tumours'", Given the anti-angiogenic properties of HA, these two statements appear contradictory. However, morphological investigations on the localisation of HA within tumours have shown that in many tumours hyaluronan accumulates within the tumourassociated connective tissue, with the tumour cells themselves essentially negative for hyaluronan". Although, Wilm's tumour is a notable exceptiorr'". Analysis of the level and size of serum HA in patients with Wilm's tumour or a bone-metastasising form of Wilm's tumour, Bone Metastasising Renal Tumour of Childhood (BMRTC) gave us the first insight into this apparent enigma. The study found that, whilst both groups of patients had extremely high levels of circulating HA (50 mg! L), the HA in the sera of metastatic BMRTC patients was very small, similar in size to OHA. In contrast, Wilm's tumours, which are rarely metastatic, produced high levels of high molecular weight HA 33 . Similar
168
The role ofhyaluronan in tissues
results were obtained when cultured cells from these two forms of kidney tumour were compared. Analysis of the HA contentJsize and hyaluronidase activity of allogeneic murine and rat tumours, and human tumour xenografts, showed that HA levels were generally increased, but there was a significant reduction in HA size, compared with normal tissues. Thus the tumour environment may be more receptive to vascularization than HA levels alone would suggest. Also, those tumours with low HA content (100 Dglg or less), or (ow molecular weight HA, were generally metastatic. Furthermore, the size of HA in this series of tumours also exhibited a loose inverse relationship to the relative levels of tumour HA and hyaluronidase activity". Others have also shown a correlation between hyaluronidase activity and tumour progression or metastasis (reviewed in refs 12, 34), supporting the hypothesis that tumour HA metabolism may playa part in regulating tumour angiogenesis. It is generally accepted is that HA degradation occurs exclusively intracellularly, in the lysosomal system, after receptor mediated internalisatiorr'". Although this may be the case for circulating HA, our analysis of HA in wound tissues and tumours suggests that a large scale, sequential degradation of the whole population of HA molecules takes place. Furthermore, the extent of HA depolymerization appears to increase with increasing extracellular "acidic" hyaluronidase activity. The most likely explanation of these data is that, in many tissues, HA degradation is mainly extracellular and is catalysed by extracellular hyaluronidase(s). Until recently, only one such enzyme had been reported, the glycosylphosphatidylinositol (GPI) -anchored sperm PH-20 hyaluronidase, whose distribution is normally restricted to the testes'", However, the recent finding that a second hyaluronidase (HYALI) present in normal serum also contains the GPI-anchor, indicating that cell-surface hyaluronidase(s) may be present on many cell-types'". Two other hyaluronidase genes have recently been identified on chromosome 3p21 (HYAL2 and 3), HYAL2 appears to be essentially lysosomal and both may be upregulated by exogenous cytokines (see 36,37). Two other poorly characterised putative hyaluronidases have been reported in the literature, HYAL4, found clustered with PH-20 ~SPAMI) on chromosome 7q31, and MEGAS, which maps to chromosome IOq24.13 •38 . Thus, of the known hyaluronidases, the two best candidates for extracellular matrix degradation are PH-20 and RYALL Recently, we have examined several tumour cell lines in respect of their HA production, HA size, hyaluronidase activity and angiogenic activity. The most metastatic, usually those with the highest angiogenic activity, generally produce lowmolecular- weight HA, both in the medium and on the their cell- surface, and expressed high hyaluronidase activity. A more detailed study of this relationship examined three closely related human colon carcinoma cell-lines, HCTll6 A(+) and HCTll6 B(+), originally isolated from the same colorectal carcinoma by Dr Brattain (Houston), and 2010-1 cells, a subclone of the more invasive HCTll6 B(+) line, selected by Dr Kinsella (Liverpool) for its increased invasiveness in vitro". However, in a caecal metastasis model the greatest number of liver metastases were found in animals bearing HCTll6 A(+) tumours (Dr Kinsella, personal communication). Analysis of these celllines for HA production and hyaluronidase activity gave some interesting results. HCTll6 A(+) cells produced much less HA than either of the other cell-lines, and this was also much smaller than that produced by the other two cell-lines. The HCTl16 A(+) cells expressed much higher levels of hyaluronidase, mostly cell-associated, than the other cell-lines, su g a causative relationship between high enzyme levels and the reduced HA size l l • . The size distribution ofHA secreted by HCTll6 A(+) cells « 30- 300 kDa), in common with other metastatic cell lines, suggests that angiogenic OHA are probably present. In common with earlier studies with transplanted tumours,
9festin
Hyaluronan degradation
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there is a loose correlation between HA size and hyaluronidase activity. Interestingly, the relationship is improved if the degree ofHA synthesis is also taken into account (i.e. the enzyme/ substrate ratio), again supporting the hypothesis that HA degradation is mainly extracellular. Comparison of the angiogenic activity of conditioned serum-free media from the three cell-lines, in the CAM assay, showed that only HCTl16 A(+)- conditioned medium was angiogenic. Interleukin-8 (IL-8) was found to be the main angiogenic cytokine in the HCT116 A(+) and HCTl16 B(+) cell lines", with HCTl16 A(+) cells secreting three times that of the HCTl16 B(+) cells. Although the difference in angiogenic activity between the cell lines was potentially due to their IL-8 concentration, removal of macromolecular HA from the HCT116 B(+)- conditioned medium removed both its macromolecular HA and the inhibitory activity". Sodium dodecylsulphate (SDS)-HA substrate-gel electrophoresis of the cell and medium proteins, from tumour cell lines, revealed two distinct patterns of hyaluronidase expression in the most active tumour cell-lines. One group, including the Hep2 (laryngeal) and Bu25 (cervical) cell lines, expressed a single 57 kDa hyaluronidase, active at pH3.5. A second group, including D98 (cervical), HeLa (cervical), MCF7 (breast), HT116A (colorectal) and weakly in HT116B (colorectai) cells, expressed two isoforms at 64 and 50 kDa. These patterns resemble those reported for BYALI and PH-20, respectively. Phospholipase C (pLC) digestion of cultured cells released hyaluronidase activity from the surface of the second group of tumour cell-lines, but not the putative HYAL1 group, consistent with their expressing PH-20 and BYALl, respectively'". PCR analysis of the mRNA isolated from these cell lines showed a range of PH-20 expression, with only 3 cell lines showing no PH-20 expression. Interestingly, the Hep2 cell-line was one of these, confirming that the 57 kDa hyaluronidase is not a form of PH20 hyaluronidase. In most cases PH-20 expression and hyaluronidase activity showed a good correlation. Some cell lines, including the Hep2 and Bu25 cell-lines, expressed less PH-20 mRNA than would have been predicted by their hyaluronidase activity. PCR analysis for BYAL141 confirmed that the Hep2 cell line expressed high levels of BYAL1 mRNA It is interesting that only a single hyaluronidase protein is expressed when multiple mRNA species were detected by PCR, in agreementwith Csoka et al (1997)41. Liu et al. (1996)42 have reported expression of the glycosylphosphatidylinositol (GPI)anchored PH-20 sperm hyaluronidase human colon carcinoma tissue, but not normal colon. Furthermore, in agreement with our own data, they found that the angiogenic activity of colon carcinoma cell lines was associated with their PH-20 expression. Addition of apigenin, a non- specific flavanoid inhibitor of hyaluronidase, to the cultured cells, prior to application, greatly reduced their angiogenic activity. However, apigenin is antiangiogenic and cytotoxic to tumour cells in vitro, but appears ineffective in vivo'". Furthermore, Madan et al (1999t4,45 have reported that PH-20 expression is related to breast and prostate adenocarcinoma invasiveness. However, BYALl and BYAL2 expression have also been implicated in tumour progression and aggressiveness'Y". CONCLUSIONS Macromolecular HA inhibits angiogenesis in both in vitro and in vivo models, whereas OHA stimulates angiogenesis in these same systems. Data from adult and foetal wound healing models indicate that high-molecular-weight HA degradation is a necessary prerequisite to tissue vascularisation. Related studies with transplantable tumours and cultured tumour cell lines, suggests that hyaluronidase degradation of matrix and cellassociated HA is related to the angiogenic activity and hence metastatic nature of tumours.
170
The role of hyaluronan in tissues
The high hyaluronidase activity in metastatic and angiogenic tumour cell-lines is due to aberrant PH-20 expression and! or overexpression ofHYALl. Both are thought to be GPIlinked cell-surface hyaluronidases, their increased expression coinciding with a greater degree of matrix and cell-surface HA degradation. It is probable that this abnormal upregulation in the expression of one, or more, of these hyaluronidases may represent a significant step in tumour progression i.e. "an angiogenic! metastatic switch". Nevertheless, not all cells in a particular tumour will express this phenotype, as exemplified by the three human coloreetal carcinoma cell-lines mentioned above which were all isolated from the same tumour. Thus it seems unlikely that such extensive HA degradation is present throughout the tumour, but may be localised to vascular "hot spots" 30
ACKNOWLEDGEMENTS DCW acknowledges the support ofthe NorthWest Cancer Research Fund. REFERENCES 1. J. Entwistle, C. L. Hall & E. A. Turley, 'HA Receptors: Regulators of signalling to the cytoskeleton'. JCellular Biochem., 1996,61,569-77. 2. J. Lesley, In: The Chemistry, Biology, and Medical Applications ofHyaluronan and its Derivatives, T. C. Laurent & E. A. Balazs (eds), 1998, Portland Press, London, pp. 123-134. 3. B. P. Toole, In: Cell Biology of the Extracellular Matrix, E. D. Hay (ed), 1982, Plenum Press, New York, pp. 259- 294. 4. W. Knudson, In: The Chemistry, Biology, and Medical Applications ofHyaluronan and its Derivatives, T. C. Laurent & E. A. Balazs (eds), 1998, Portland Press, London, pp. 169-79. 5. D. C. West & D. M. Shaw DM, In: The Chemistry, Biology, and Medical Applications ofHyaluronan and its Derivatives, T. C. Laurent & E. A. Balazs (eds), 1998, Portland Press, London, pp. 227-233. 6. D. C. West & S. Kumar, In: The Biology of Hyaluronan, D. Evered & J. Whelan (eds), 1989, Ciba Foundation Symposium 143, John Wiley and Sons, Chichester, pp. 187-207. 7. M. F. Burbridge, D. C. West, G. Atassi & G. C. Tucker, 'The Effect ofExtraceIIular pH on Angiogenesis In Vitro'. Angiogenesis., 1999,3,281-8. 8. M. Watanabe, K. Nakayasu & S. Okisaka, 'The effect of hyaluronic acid on proliferation and differentiation of capillary endothelial cells'. Nippon Ganka Gakkai Zasshi., 1993, 97, 1034-9. 9. N. Fournier & C. J. DoilIon, 'In vitro angiogenesis in fibrin matrices containing fibronectin or hyaluronic acid'. Cell Biol Int Reports., 1992, 16, 1251-63. 10. D. C. West, I. N. Hampson, F. Arnold & S. Kumar. 'Angiogenesis induced by degradation products of hyaluronic acid'. Science., 1985,228, 1324-8. 11. A. Sattar, S. Kumar & D. C. West, 'Does hyaluronan have a role in endothelial cell proliferation of the synovium'. Semin Arth Rheum., 1992,21,43-49. 12. D. C. West & M. F. Burbridge, 'Specific size dependent effects ofhyaluronan in the in vitro rat aortic angiogenesis model'. Int J Biochem Cell Bioi., 2000, In press. 13. S. Hirata, T. Akamarsu, T. Matsubara, K. Mizuno & H. Ishikawa, Arth Rheum., 1993, 36, S247. 14. P. Rahmanian, H. Pertoft, S. Kanda, R. Christofferson, L. Claesson-Welsh & P.
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Heldin. 'Hyaluronan oligosaccharides induce tube formation of brain endothelial cell line in vitro'. Exp Cell Res, 1997,237, 223-30. 15. R. Montesano, S. Kumar, L. Orci & M. S. Pepper, 'Synergistic effect ofhyaluronan oligo saccharides and vascular endothelial growth factor on angiogenesis in vitro' . Lab Invest., 1996, 75, 249-62. 16. V. Trochon, C. Mabilat, P. Bertrand, Y. Legrand, F. Smadia-Jotfe, C. Soria, B. Delpeche & H. Lu, 'Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro'. lnt J Cancer., 1996,66,664-668. 17. V. Trochon, C. Mabilat-Pragnon, P. Bertrand, Y. Legrand, 1. Soria, C. Soria, B. Delpeche & H. Lu, 'Hyaluronectin blocks the stimulatory effect of hyaluronanderived fragments on endothelial cells during angiogenesis in vitro'. FEBS Letts., 1997,418,6-10. 18. S. D. Banerjee, & B. P. Toole, 'Hyaluronan-binding protein in endothelial morphogenesis'. J Cell Bioi., 1992, 119, 643-52. 19. E. Tsifrina, N. M. Ananyeva, G. Hastings & G. Liau, ' Identification and characterization of three cDNAs that encode putative novel hyaluronan-binding proteins, including an endothelial cell-specific hyaluronan receptor', Am J Pathol., 1999, 155, 1625-33. 20. M. Slevin, 1. Krupinski, S. Kumar & J. Gaffney, 'Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation'. Lab lnvest., 1998, 78,987-1003. 21. M. McKee, M. B. Penno, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao & P. W. Noble, 'Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages - The role ofHA size and CD44', J Clin Invest., 1996, 98, 2403-13. 22. K. A. Fitzgerald, A. G. Bowie, B. S. Skeffington & L. A. 1. O'Neill, 'Ras, protein kinase C zeta, and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation ofNF-kappa B by hyaluronic acid fragments in T-24 carcinoma cells', J Immunol, 2000, 164,2053-63. 23. D. C. West, 1. Wilson, G. Lagoumintzis & M. Joyce. In: Vascular endothelium: Mechanisms of cell signaling, 1. D. Catravas, A. D. Callow & U. S. Ryan (eds), 1999, lOS Press, Amsterdam, pp. 233-41. 24. P. W. Noble, C. M. McKee, M. Cowman & H. S. Shin, 'Hyaluronan fragments activate an NFkB/IkBa autoregulatory loop in murine macrophages'. J Exp Med., 1996, 186,2373-2378. 25. R. Deed, P. Rooney, P. Kumar, J. D. Norton, J. Smith, A. 1. Freemont & S. Kumar, 'Early-response gene signalling is induced by angiogenic oligo saccharides of hyaluronan in endothelial cells. Inhibition by non-angiogenic, high-molecular-weight hyaluronan', lnt J. Cancer.,1997, 71, 251-6. 26. M. Laniado-Schwartzman, Y. Lavrovsky, R. A. Stoltz, M. S. Conners, 1. R. Falck, K. Chauhan, N. G. Abraham, 'Activation of nuclear factor kB and oncogene expression by 12 (R)-hydroxyeicosatrienoic acid, an angiogenic factor in microvessel endothelial cells', J Bioi Chem., 1994, 269, 24321-7. 27. S. A. Fenwick, P. 1. Gregg, S. Kumar, 1. Smith & P. Rooney, 'Intrinsic control of vascularisation in developing cartilage rudiments', Int J Exp Pathol., 1997, 78, 18796. 28. D. C. West & T-P. D. Fan, In: The New Angiotherapy, R. Auerbach & T-P. D. Fan (eds), 2001, Humana Press, in press. 29. B. A. Mast, 1. H. Haynes, 1. M. Krummel, R. F. Diegelman & I. K. Cohen, 'In vivo degradation of fetal wound hyaluronic acid results in increased fibroplasia, collagen
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The role of hyaluronan in tissues
deposition, and neovascularization'. Plastic Reconstructive Surgery., 1992, 89, 503-9. 30. G. Gasparini, In: Tumour angiogenesis, R. Bicknell, C. E. Lewis & N. Ferrara (eds), 1997, OUP Press, Oxford, pp. 29-44. 31. C. Wang, M. Tammi, H. T. Guo & R. Tammi, 'Hyaluronan distribution in the normal epithelium of esophagus, stomach, and colon and their cancers', Am .JPathol., 1996, 148, 1861-1869. 32. M. T. Longaker, N. S. Adzick, D. Sadigh, B. Hendin, S. E. Stair, B. W. Duncan, M. R. Harrison, R. Spendlove & R. Stem, 'Hyaluronic acid-stimulating activity in the pathophysiology of wilms- tumors', J Natl Cancer Inst., 1990, 82, 135-9. 33. S. Kumar, D. C. West, 1. Ponting & H. R. Gattamaneni, 'Sera of children with renal tumour contain low molecular mass hyaluronic acid,. Int J Cancer., 1989,44,445-8. 34. D. C. West, In: 'Cancer metastasis, molecular and cellular biology and its clinical aspects', W. G. Jiang & R. E. Mansel (eds), 2000, Kluwer Pub, 55-71. 35.1. R. E. Fraser, T. 1. Brown & T. C. Laurent, In: The Chemistry, BiologyandMedical Applications ofHyaluronan and its Derivatives, T. C. Laurent & E. A. Balazs (eds), 1998, Portland Press, London, pp. 85-92. 36. T. B. Csoka, G. L. Frost & R. Stem, 'Hyaluronidases in tissue invasion', Invasion Metastasis, 1997, 17,297-311. 37. T. B. Csoka, S. W. Scherer & R. Stem, 'Expression analysis of six paralogous human hyaluronidase genes clustered on chromosomes 3p21 and 7q31', Genomics., 1999, 60, 356-61. 38. D. Heckel, N. Comtesse, N. Blin, K. D. Zang & E. Meese, 'Novel immunogenic antigen homologous to hyaluronidase in meningioma', Human Mol Genet., 1998, 7, 1859-72.R. 39. R. Brew, 1. S. Erikson, D. C. West, B. F. Flanagan & S. E. Christmas, 'Interleukin-8 as an autocrine growth factor for human colorectal carcinoma cells in vitro', Cytokine.,2000, 12, 78-85. 40. D. C. West & H. Chen, In: 'New Frontiers in medical sciences: Redefining hyaluronan', G. Abartangelo & P. H. Weigel (eds), Excerpta Medica International Congress Series (ICS), 2000, Elsevier Science. pp. 77- 86. 41. T. B. Csoka, G. L. Frost, T. Wong & R. Stern, 'Purification and microsequencing of hyaluronidaseisozymesfrom human urine', FEES Letts., 1997, 417, 307-10. 42. D. Liu, E. Pearlman, E. Diaconu, K. Guo, H. Mori, T. Haqqi, S. Markowitz, 1. Wilson & M-S Sy, 'Expression of hyaluronidase by tumor cells induces angiogenesis in vivo', Proc Nat! Acad Sci USA., 1996,93, 7832-7. 43. Engelmann, E. Blot, C. Soria, P. M. Schlag & Y. Panis, 'Inhibitors of angiogenesis in surgical oncology efficacy in vitro versus efficacy in vivo', Langenbecks Arch Chir., 1999, SI, 343-7. 44. A. K. Madan, K. Yu, N. Dhurandhar, C. CulIinane, Y. Pang & 1. Beech, 'Association of hyaluronidase and breast adenocarcinoma invasiveness', Oncol Rep., 1999,6,6079. 45. A. K. Madan, Y. Pang, M. B. Wilkiemeyer, D. Yu, & 1. Beech, 'Increased hyaluronidase expression in more aggressive prostate adenocarcinoma', Oncol Rep., 1999,6,1431-3. 46. V. B. Lokeshwar, M. 1. Young, G. Goudarzi, N. Iida, A. I. Yudin, G. N. Cherr & M. G. Selzer, c Identification of bladder tumour-derived hyaluronidase: its similarity to HYAL1', Cancer Res., 1999, 59, 4464-70. 47. U. Novak, S. S. Slylli, A. H. Kaye & G. Lepperdinger, 'Hyaluronidase-2 overexpression accelerates intracerebral but not subcutaneous tumour formation of murine astrocytoma cells', Cancer Res., 1999, 59, 6246-50.
THE ROLES OF EXTENSIONAL AND SHEAR FLOWS OF SYNOVIAL FLUID AND REPLACEMENT SYSTEMS IN JOINT PROTECTION C. Backus, S.P. Carrington, L.R. Fisher, J.A. Odell* & D.A. Rodrigues H.H. Wills Physics Laboratory, University ofBristol, Tyndall Avenue, Bristol BS8ITL, UK.
ABSTRACT Extensional flows occur as components of most real flows and, unlike simple shear, can significantly stretch macromolecules, providing orders of magnitude increases in elastic forces and extensional viscosity. We examine the role of non-Newtonian rheology in the performance of synovial fluid in joints. We report the response of hyaluronic acid (HA) molecules, the major polymeric component of synovial fluid, to extensional and shear flows, and to the complex, but physiologically relevant, squeeze flow of a thin film. Extensional and shear flow experiments, which assess molecular orientation and its effect on the flow field, have been performed. HA molecules stretch at strain rates of biomechanical relevance, resulting in high extensional viscosity and elasticity. This mechanism can provide shock absorption and recovery in the knee joint. The effectiveness of the fluid is dependent upon the HA molecular weight. Degradation of HA molecules, as occurs in osteoarthritis, reduces the overall extensibility of the HA molecules, and so reduces the ability of the fluid to absorb a compressive shocks in the knee joint The complex stretching flow arising from squeezing a thin film is comparable to some flow regimes experienced in a knee joint, and we have developed apparatus to model such squeeze flows. Studies include linear and branched HA and systems with cross-linked gel particles to improve elasticity in prosthetic viscosupplementation. KEYWORDS Hyaluronic acid, synovial fluid, shear flow, extensional flow, squeeze flow, Hylan INTRODUCTION The role of synovial fluid in the protection of human joints is complex and demanding. In the work that follows we will discuss the human knee joint, both as a paradigm of a complex joint and because it is the one joint most affected by arthritic deterioration and amenable to mechanical replacement and viscous supplementation. The joint consists of the bones of the femur and tibia, both surfaces of which are protected by a layer of elastic cartilage (Figure la). The cartilage provides shock absorbtion and makes the synovial fluid which lubricates and protects the cartilage surface. The most important component of the synovial fluid is a very high molecular weight HA (of order 10 Daltons), which results in the fluid having complex viscoelastic rheological properties.
210
Rheological behaviour of hyaluronan
In the healthy joint, the fluid lubricates the joint and provides protection against shocks to prevent cartilage erosion. In the diseased joint (Figure lb) the synovial fluid is characteristically of much lower molecular weight.' The protection of the cartilage fails leading to erosion, initially of cartilage and later of bone. The healthy knee is principally a "hinge joint" as shown in Figure lc. This type of movement produces mainly simple shear flow and the role of the synovial fluid is primarily that of lubrication. This it performs well, since HA is highly shear thinning/ and the movement of cartilage on cartilage lubricated with synovial fluid has very low friction. There are more complex demands put upon the fluid in compression of the joint, such as occurs in running and jumping as shown schematically in Figure ld. Compression results in a "squeeze flow" consisting of a superposition of both simple shear and uniaxial compression (biaxial extension) flows. By symmetry, at the centre of compression there is a "stagnation point" where the velocity is zero. Linear high molecular weight synthetic polymers in extensional and shear flows can produce dramatically different responses, particularly in the presence of stagnation points. Under conditions of high Deborah number and persistent extension, high molecular weight, flexible chain polmers "unravel" resulting in massively increased extensional viscosity and normal forces.' Under simple shear, typically, the polymers are strongly shear thinning. Although synovial fluid is strongly shear-thinning, it is thought to produce a rapid stress rise during the final stages of parallel plate compressive flow ('squeeze flow,' i.e. under uniaxial compression in a narrow gap, where the fluid can flow sideways). This is certain to be of physiological importance in joint protection. It is likely that the resistance of the fluid is due to its polymeric nature, and specifically to the ordering of the polymer molecules in solution. The squeeze flow is complex, containing strong shear and extensional components that increase as the gap narrows. The origin of the stress rise has been attributed to transient shear flow effects", but our results lead us to believe that extensional flows are important in joint protection at narrow gaps.
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Role of synovial tluid in joint protection
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The force required to compress a Newtonian fluid at a constant rate can be calculated using the quasi-steady-state approximation, where the flow at any time t is treated as steady-state. Analysis of a fluid of viscosity ~ compressed between two parallel plates of radius R and separation 2h which close with constant velocity V2 dhldt, yields a 1/h3 dependence for the force F on the plates': (1)
Similar analyses have been made for a non-Newtonian "power law" fluid'', but these cannot realistically represent extensional components of the flow. Our aim has been to separate and understand the roles of shear and extensional flows in the non-Newtonian behaviour of HA in joints. We have developed a cell to model the complex flows in the knee joint, and have investigated separately idealised shear and extensional flows and their contribution to squeeze flow. The response and performance of a replacement synovial fluid has also been assessed. RESULTS AND DISCUSSION Squeeze Flow
To investigate squeeze flow in the knee joint we have developed a compression cell or squeeze-flow rheometer (Figure 2). The fluid is placed between two glass plates and compressed. The apparatus permits well-defined closing rates and excellent normal force resolution between 19 and lOOkg. The mirrors in the cell allow a laser beam to be passed through, so that birefringence can be detected. Constant stress or constant strain rate experiments can both be performed.
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As the closing rate (and hence strain rate) increases for a Newtonian fluid between the plates in the compression cell, the normal force increases. If the force-separation data is plotted as force to the power -113, a linear relationship is obtained, as predicted by equation (1). Figure 3 shows schematically the expected behaviour of Newtonian, thinning and thickening fluids on such a plot. The flow behaviour of a high molecular weight Hylan A sample (from Biomatrix) and Healon GV (from Kabi Pharmacia) have been studied. Both these HA samples are of similar molecular weight (approx. 5 x10 6 Daltons), but differ in that Hylan A is lightly cross-linked, whereas Healon GV consists of linear molecules.
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Role of synovial fluid in joint protection
213
Force-separation curves for a 1.4% (14000ppm) solution of branched chain Hylan A in the compression cell are almost superimposed at the lowest closing rates (1-100 mm/min) and largest gaps, implying that the fluid is strongly shear thinning. At higher closing rates and narrower gaps, the force increases as the fluid thickens. The flow regime at high closing rates is likely to be one in which the extensional properties of the fluid start to dominate over the shear properties i.e. the fluid is extension thickening. The same trend is apparent for a Healon GV solution of the same concentration. Plotting the data for Hylan A as force to the power -113 (see Figure 4), a deviation from Newtonian behaviour is apparent. • At low closing speeds, the curves show an increasing gradient as the gap closes i.e. predominantly shear thinning. • At higher closing rates, the curves begin to show a change in gradient, going from a shear thinning regime (gradient steepening) to a thickening regime (gradient reducing) as the gap decreases. The same trend is followed by the curves for Healon GV. The transition between these regimes occurs at strain rates of order of 20s· 1• We believe that this behaviour, if typical of high molecular weight HA, may be of biological relevance to the role played by healthy synovial fluid in joint protection. Simple Shear Behaviour A parallel plate optical shear cell was used where the sample is placed between two glass plates, one of which rotates. The cell was illuminated by a laser through crossed polars. The alignment of the molecules can be assessed from the flow-induced optical retardation. HAs were investigated at shear rates between 2 and 650s· 1•
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214
Rheological behaviour of hyaluronan
Figure 5 shows the retardation at different strain rates for 1.4% solutions of branched and linear material. There is a significant difference between the behaviour of linear and branched HA. The branched material, Hylan A, shows birefringence from the lowest strain rates, increasing rapidly to 100 S·1 and then more gradually increasing up to 650 S·I. In marked contrast the linear HA, Healon, shows little or no shear induced birefrin~ence until 200 s'. then increases rapidly to be comparable to the branched material at 650 s . Extensional Flow Behaviour
Conventional rheology fails to address the importance of extensional flows, which occur as components of almost all real complex flows e.g. in compression (as in joints), around obstacles, at surfaces and in contractions. They represent the class of "strong" flows, which, unlike simple shear, are capable of significantly stretching molecules, providing orders of magnitude increases in elastic forces and extensional viscosity.' Sufficiently strong extensional stresses can break molecules, and it is now recognised that extensional flow dominates the thermo-mechanical stability of dilute polymer solutions.? Flows which are purely extensional can be realised by uniaxial compression and pure shear. Uniaxial compression is generated in an opposed jets apparatus, where two symmetric jets facing each other with a small separation, are immersed in a solution. The jets used have a diameter of 0.6mm and a separation of 0.35mm. By blowing the solution into both jets simultaneously, a strong uniaxial compressive flow field is created, with cylindrical symmetry about the axis of the jet apertures. The opposed jets apparatus allows both optical birefringence and flow resistance measurements to be made, enabling the correlation of molecular orientation and its effect on the flow field. Birefringent intensity profiles are monitored, using a cooled back-thinned CCD camera. Figure 6 shows a 3000ppm solution of Hylan A in O.l5M NaCI in a uniaxial compressive flow. Such a flow corresponds to the extensional components of a squeeze flow (like a knee joint). The vertical birefringent line corresponds to a disc of stretched material seen edge on. We are confident that the high extensional elasticity and viscosity of this disc would result in shock absorbing and elasticity of such solutions.
Figure 6
Birefringence profile of Hylan A solution in uniaxial compression in the opposed jets.
Role of synovial t1uid in joint protection
Figure 7
The development of birefringencewith strain rate for a 500ppm Healon GV solution in the cross-slots.
Figure 8
The development of birefringence with strain rate for a 500ppm Hylan A solution in the cross-slots.
215
In order to assess the progressive response of HA to extensional flows, 500ppm (0.05%) solutions of both linear and branched hyaluronans have been subjected to the stretching flow field of the cross-slots. The cross-slots apparatus has a stagnation point at the centre of symmetry, where the molecules experience a high residence time and can approach steadystate stretched conformations. The stretching flow field in the cross-slots is twodimensional pure shear, whereas the opposed jets create uniaxial compression. Birefringence measurements, to assess the state of molecular orientation, as well as flow resistance measurements, to assess the effective extensional viscosity compared to the ideal (Newtonian) solvent, can be made in this apparatus. Figure 7 shows the development of birefringence with strain rate for a 500ppm Healon GV solution. At low strain rates (up to 40s·'), a highly localised birefringent line appears along the central outflow axis. This response is typical of a polymer which exists in solution as a flexible coil.' As the strain rate increases to 100s·', this birefringent line broadens and develops a dark central band, or 'pipe'. The dark central area indicates that the stretched molecules in this region are able to collapse back to coiled (isotropic) conformations i.e. the strain rate in this region is dropping. The area around the stagnation point is being 'screened' by the extending molecules, which create high extensional viscosity locally and hence modify the flow field. As the strain rate increases further, the birefringent line broadens even more, until there is evidence that the flow field becomes unstable (at around 400s·1) .
216
Rheological behaviour of hyaluronan
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Effective extensional viscosity versus strain rate for 500ppm Healon GV (.) and Hylan A (.) solutions in the cross-slots.
Figure 8 shows the development of birefringence with strain rate for a 500ppm Hylan 50 solution. The pattern of birefringence with strain rate is similar to that exhibited by the Healon GV solution, although there are higher intensities at the lowest strain rates (up to 40s· l ) and the flow field appears to become unstable at lower strain rates (around 3OOs· 1) . Figure 9 is a plot of effective extensional viscosity versus strain rate for 500ppm Healon GV and Hylan A. This viscosity response is relative to that of the Newtonian solvent (water) in the cross-slots apparatus. Both traces show an initial increase in extensional viscosity, and a subsequent reduction as the strain rate increases, consistent with previous results.' The response of the linear Healon GV is higher than that of the branched Hylan A. We observe a progressive reduction in the stretching birefringence and an increase in the onset strain-rate, correlating with the duration and strength of previous stretching flow. The molecules probably degrade under flow-induced stress with a thermo-mechanical scission process", This has clear implications for the performance of diseased synovial fluid. These results illustrate that it is vital to visualise the local flow-field in extensional flow rheology in the presence of a stagnation point. Once the flow-field has become modified the purely extensional nature is lost. Specifically, the observation of extensional thinning is clearly due to the break down of the flow-field, and earlier results showing this phenomenon in apparatus where the flow-field cannot be visualised should be reinterpreted.
Interpretation of the Shear and Extensional Flow Results There are striking differences in the response of branched and linear HAs to shear and extensional flow. In shear, the branched material shows a greater orientation from much lower shear-rates compared to the linear material, indicating that the branched material has a very long coil relaxation time. It should be no surprise that the branched material has a
Role of synovial fluid in joint protection
217
dramatic response to simple shear, since this flow cannot produce significant extension of the molecules and simply probes the dynamics of the relatively unstretched coil. In extension at low shear-rates, the branched material again orients earlier due to its long relaxation time. At higher strain-rates, however, the linear material shows a greater degree of birefringence, molecular stretching and extensional viscosity. No doubt this is due to the inherent linearity permitting a high degree of coil extension. Comparison of the shear and extensional flow studies, strongly suggests that the origin of the performance of synovial fluid in joint protection lies in a transition in rheological behaviour, from a shear thinning simple shear regime, to an extension thickening regime as the squeeze flow proceeds to narrow gaps. This is anticipated from the geometry of the flow and the approximate shear and extensional strain-rates. The transition occurs at strain rates which are believed to be of biological relevance. I Squeeze Flow of a Replacement Synovial Fluid System Attempts to find replacement fluids are restricted by the difficulty of matching the ultrahigh molecular weight HA of healthy synovial fluid. Synvisc is a successful replacement fluid produced by Biomatrix, Inc. which uses HA gel inclusions to enhance elasticity. It consists of a suspension of Rylan B gel particles in high molecular weight Rylan A. Synvisc in squeeze flow shows a marked stiffening of the compressive force as the gap becomes comparable to the gel dimensions. This may help Synvisc to mimic the protective stress behaviour of healthy synovial fluid. Greater stiffening is observed for gel samples with a higher degree of cross-linking. Repeat runs of Synvisc show almost perfect recovery and reproducibility, even after compression to only 1/25th of typical gel dimensions. Figure lO shows the behaviour of a lOOllm Rylan B gel sample in squeeze flow as a function of closing rate, plotted as force to the power -1/3. The closing rate does affect the normal force, slowly at first, but the with force increasing markedly above 200mmlmin.
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218
Rheological behaviour of hyaluronan
We believe the non-Newtonian behaviour of the suspension fluid at high closing rates plays a vital role in the transfer of stress to the gel component. Further, as the gel system is compressed, the suspension fluid must be excluded from the compressing, increasingly close packed system. This gives rise to increasing forces, due both to the viscosity of the fluid and the effective bulk modulus of the system.
CONCLUSIONS Experiments in shear, extensional and squeeze flow of HA suggest that synovial fluid has a remarkable combination of properties which make it effective for the protection of joints. It is likely to be extremely shear thinning and yet provide protection against high normal forces through its extensional properties. We interpret the behaviour of Rylan gel systems as successfully mimicking, over long periods, the behaviour in joints of healthy synovial fluid. Synvisc provides the high normal forces through a combination of the elastic properties of the gel and the viscoelastic properties of the suspension fluid. The remarkable degree of elastic gel recovery provides for longevity as a replacement fluid. We are about to start experiments in extension, shear and squeeze flow on healthy and diseased (osteoarthritis) human synovial fluid. Of primary interest is how the behaviour compares in each case with the linear and branched HA's and the replacement fluids. Utilisation of the optics in the squeeze cell to both assess birefringence in HA solutions and to visualise the soft gel inclusions in replacement synovial fluids, should provide more information to further the ideas presented here.
ACKNOWLEDGEMENTS The authors would like to acknowledge the EPSRC for financial support and Biomatrix, Inc. for provision of samples and helpful discussion.
REFERENCES 1. J. Schurz and V. Ribitsch, 'Rheology of Synovial Fluid', Biorheology, 1987,24, 385399. 2. S. AI-Assaf, J. Meadows, G.O. Phillips and PA Williams, 'The Application of Shear and Extensional Viscosity Measurements to Assess the Potential of Rylan in Viscosupplementation', Biorheology, 1996, 33, 319-332. 3. S.P. Carrington, J.P. Tatham, J.A. Odell and A.E. Saez, 'Macromolecular Dynamics in Extensional Flows: 1. Birefringence and Viscometry', Polymer, 1997, 38, 4151-4164. 'Macromolecular Dynamics in Extensional Flows: 2. The Evolution of Molecular Strain' , Polymer, 1997, 38,4595-4607. 4. N. Phan-Thien, F. Sugeng and R.I. Tanner, 'The Squeeze Film Flow of a Viscoelastic Fluid', J. Non-Newtonian Fluid Mechanics, 1987,24,97-119. 5. R.B. Bird and PJ. Leider, 'Squeezing Flow Between Parallel Discs', Industrial & Eng.Chem. Fundamentals, 1974, 13,336-341. 6. J.A. Odell and M.A. Taylor, 'Dynamics and Thermomechanical Stability of DNA in Solution', Biopolymers, 1994, 34, 1483-1493.
DISRUPTION OF HYALURONAN SYNTHASE-2 ABROGATES NORMAL CARDIAC MORPHOGENISIS AND HYALURONANMEDIATED TRANSFORMATION OF EPITHELIUM TO MESENCHYME Todd D. Camenlsch'", Andrew P. Spicer2,3, Tammy Brehm-Gibson', Jennifer Beisterfeldt', Mary Lou Augustine', Anthony Calabro, Jr. 4, Steven Kubalak/.Scott E. Klewer", and John A. McDonald 1* JSC
Johnson Medical Research Center, Mayo Clinic Scottsdale, 13400 East Shea Blvd., Scottsdale, AZ 85259, USA
2 These
authors contributed equally to the work and should be considered co-first authors
3Current address: Rowe Program in Genetics, Department of Biological Chemistry, University of California-Davis, Davis, CA 95616, USA "Department of Bioengineering, The Cleveland Clinic, Cleveland, OH 44195, USA 5 Department
6 Department
of CeLL Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
of Pediatrics, University ofArizona School of Medicine, Tucson, AZ 85724, USA
KEYWORDS Hyaluronan synthase, gene targeting, Has2, heart
INTRODUCTION The molecular cloning of hyaluronan synthases (HAS) from prokaryotes t and vertebrates 2 has ushered in a new era in hyaluronan (HA) biology. The primary sequences and deduced membrane topology have been established. Moreover, distinct catalytic properties have been assigned to each of the three distinct mammalian Has enzymes 2,3. Finally, the genomic organization of each mammalian HAS gene has been elucidated 4. This knowledge provides a solid basis for advancing our understanding of the biological roles of HA. Biochemical and cell biological studies employing mutagenesis and domain swapping approaches yield insights into the role of Has in catalysis and cell autonomous behavior. For example, over expression of HAS increases malignant behavior in cell lines 5.6. We have taken a genetic approach, namely insertional mutation of the Has loci by gene targeting to create null alleles in each HAS gene 7. Here, we report the results of targeting Has2.
HAS genomic organization, catalytic properties and expression Three HAS genes located on different chromosomes in man and mouse encode three distinct Has proteins 4. When expressed recombinantly, each of these has distinct catalytic properties. The catalytic activity varies by -20-fold (Has 3>Has 2>Hasl) 2.3.
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Figure 1. The mouse Has2 locus, targeting vector, and resulting gene. (A) A portion of the Has2 locus with restriction sites, exons (filled boxes), the targeting vector, and resulting targeted locus. Homologous recombination replaces the end of intron 3 and the first 60 codons of exon 4 with PGKneo. The BamHl and EcoRI restriction fragments confirming the structure of PCR positive ES clones are indicated. Arrowheads indicate the direction of transcription of PGKneo and DTA. (B) PCR screening with the PGKneo and flanking primer revealing predicted amplicons of 1.8 kb in two ES clones. (C) Southern blot analysis of Bamlll genomic DNA digests from the parental ES line (control) and the two targeted ES clones, and a representative wild-type and heterozygous mouse. Probe 2 (Box 2) detected the 12.5 kb and 5.4 kb restriction fragments corresponding to the wild-type and targeted alleles, respectively. Has! and Has2 both produce high (mega Dalton) HA whereas Has3 produces lower molecular mass HA. Evaluation of Has expression by northern analysis of total mRNA from mouse embryos reveals that Hasl mRNA is present at E7.5, Has2 throughout gestation, and Has3 increases during the second half of gestation. Localization of Has2 mRNA by in situ hybridization (ISH) reveals widespread expression in the embryo from E8.5 7. Has2 expression was prominent in pathways of neural crest migration, the developing vasculature, and cardiovascular system. Interestingly, the expression of
Disruption of hyaluronan synthase-2
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Has2 mRNA was very similar to that of versican (PG-M), an HA binding proteoglycan". Gene targeting of Has2 results in embryonic lethality We selected the Has2 gene as a candidate for targeting based upon its widespread expression in vivo in hopes of elucidating the source and function of HA in embryonic development. A null allele in the Has2 gene was created by homologous recombination in ES cells (Fig. 1). The resulting gene contains the neomycin phosphotransferase (Neo) coding sequence inserted into the intracellular catalytic domain of the Has2 protein followed by a stop codon. Targeted ES cell clones were identified by PCR (Fig. IB), and verified by southern analysis (Fig. lC). The targeted ES cells were injected into blastocysts, and implanted in pseudopregnant foster females. Chimeric offspring were identified by coat color, and the mutant allele introduced into 129 and BL6/J mice by mating chimeras with wild type mice. Heterozygous intercrosses were performed, and the offspring genotyped. As shown in Table 1, no homozygous null offspring were identified. To determine when the Has2 null mutation was lethal, timed pregnant females from heterozygous intercrosses were sacrificed at varying time points, and embryos genotyped and phenotyped. Genotyping revealed Mendelian frequency of the Has 2 null allele prior to E9.5 (Table 1). No phenotypic abnormalities were noted at E8.5. At E9.5, a striking phenotype was observed (Fig. 2). The top panel shows the yolk sac from a heterozygous (Fig. 2A) and HasT" embryo (Fig. 2B). Note the absence of vitelline vessels (VV) in the Has2'" yolk sac. The inset shows an H&E stained section taken through the yolk sac. Note the separation between endoderm ("E") and mesoderm ("M"). Comparison of wild-type (Fig. 2C) with Has2'/' (Fig. 2D) embryos revealed exhibited growth retardation, incomplete turning, distorted somites, and a translucent, bloodless heart in the Has2·/·. Pericardial edema, often massive, was typically but not inevitably present. Whole-mount staining for PECAM (CD31) revealed the characteristic vascular system of wild type E9.5 embryos (Fig. 2E) was lacking in Has2'/' embryos (Fig. 2F).
Table 1. Genotypes of mice resulting from matings of heterozygous Has2+/' mice. Has2+ + Has2+' Has2" " Genotype: 9.5 days post coitum Observed 150 145 122 Expected I 31 162 I 31 11.5 days post coitunr' Observed 114 120 I 3T Expected 120 1 10 110 Live Births Observed 190 I 159 10 Expected I 166 183 183 'Genotyping of 7 embryos in the 9.5 dpc group was inconclusive. 2A total of 39 embryos were observed in the U.S dpc group. Genotyping of two embryos undergoinrreabsorption was inconclusive. tThe three embryos in this group that were Hasz genotype were in various stages of decomposition.
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The role of hyaluronan in tissues
E
F
Figure 2. Phenotype of HasZ-I - embryos. Panel A shows the yolk sac of a HasZ +1and B the yolk sac from a HasZ-I - littermate. Cross sections of the yolk sac stained with hematoxylin and eosin are shown in the inset. Note the presence of vitelline vessels (VV) containing nucleated red blood cells in the yolk sac of the HasZ +1I embryo. The endoderm and mesoderm are not fused in the HasZ· . embryo, and the red blood cells are free within this space. (Continued next page.)
Disruption of hyaluronan synthase-2
181
(C) and (D) show representative wild-type and HasZ-I- embryos at E9.5. Note the diminished size of the HasZ- I- embryo, the bloodless heart, and distorted somites. (E) and (F) show E9.5 wild-type and HasZ-I- embryo stained for the endothelial marker PECAM. Note the absence of an organized vascular network expressing PECAM in the HasZ,I- embryo. A, atrium; V, ventricle; P, pericardium; E, endoderm; M,. mesoderm; OpP, optic placode; OtP, otic placode; first and second pharyngeal pouches are numbered. Bar in C-F indicates 500 urn.
Has2 null mouse embryos lack HA Histological analysis revealed a contracted extracellular space, with striking paucity of alcian blue staining (data not shown). Specific histochemical detection of HA using a biotinylated link protein preparation (generously provided by Dr. Charles Underhill) demonstrated absent staininl? in a saggital section through an E9.5 HasZ'I' embryo (Fig. 3A, wild-type; 3B, Hasz "), To confirm this observation biochemically, we performed FACE® analysis on individual and pooled HasZ-/- embryos. This revealed a -97% reduction in detectable HA in pooled homozygous null embryos compared with wild-type. Based upon this, we conclude that HasZ is the major source of HA during mid-gestation in the mouse. Moreover, the striking similarity of the HasZ-I - embryo and that of the hdfmouse lacking versican", indicates that a composite matrix containing both HA and versican is essential in forming a stable, expanded extracellular matrix.
A
Figure 3. HA is abundant in a wild-type E9.5 mouse embryo (A), and essentially absent from a HasZ-I- embryo (B). A, bright field; B, Nomarski DIC. Both stained using biotinylated link protein.
182
The role of hyaluronan in tissues
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Figure 4. AV canal morphogenesis is deficient in HasZ·I - AV canal explants and is restored by exogenous HA or activated Ras. Top Panel. AV canal explant morphogenesis in vitro. AV canal explants from E9.5 wild-type (A-C) or HasT 1- embryos (D-H) were cultured on collagen gels. Explants from wild-type embryos exhibit abundant endothelial cell migration and invasion (Image A is focused the on surface of gel and B below surface). In contrast, there is no endothelial cell migration in AV canal explants from E9.5 HasZ-I - embryos (D). Transfection with dominant-negative Ras cDNA significantly reduces (p
Disruption ofhyaluronan synthase-2
183
Endothelial cell migration and transformation in the developing cardiovascular system are lacking in the HasZ-I - mice Many studies have implicated HA as a mediator of cell migration and transformation, However, the interpretation of these studies has been limited by the lack of specific inhibitors of HA biosynthesis, the ubiquitous distribution of HA and the biological activity of HA fragments accompanying its degradation by hyaluronidases. The availability of a biological system lacking HA biosynthesis allowed us to directly address the role of HA in a physiologically relevant context. During cardiac morphogenesis, a population of endothelial cells undergoes a dramatic transformation into mesenchyme. This requires loss of cell-cell contacts, invasion of the underlying HA-rich cardiac cushion tissue, and expression of a new repertoire of mesenchymal cell products. Because they lack cardiac cushions, this event could not be evaluated morphologically in Has2-1- embryos. Accordingly, we utiliz(jfi the well characterized collagen gel assay originated by Bemanke and Markwald to assess atrioventricular canal (AVC) morphogenesis in vitro. In this assay, a type I collagen gel replaces cardiac jelly, providing a substrate supporting endothelial cell migration and invasion. AVC explants taken at E9.5 from wild type mice exhibit endothelial cell activation (loss of epithelial morphology), migration over the collagen gel surface, and transformation (invasion into the collagen gel, assessed by optical sectioning). Fig. 4A and B show DIC images taken at the surface and below the surface of a wild-type AVC explant.
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Figure 5. Dominant-negative Ras inhibits HA-mediated endothelial cell invasion in Has2- 1- AV canal explants. These images were obtained by laser scanning confocal microscopy following staining for a-smooth muscle actin. Exogenous HA (0.75 mg/ml of medium) was added to the culture medium in both HasT1explants. Panel A is a collapsed Z-series of 100 urn showing the characteristic transformation to mesenchyme and invasion of the collagen gel in the presence of exogenous HA. The dotted line indicates the previous location of the myocardium, whieh was removed. Panel B shows the characteristic effect of transfection with dominant-negative Ras. In contrast to the rescued AV explant, an epithelial sheet has migrated over the surface of the collagen gel, but there are no invading mesenchymal cells. Similar results were obtained in three independent experiments.
L84
The role of hyaluronan in tissues
To our surprise, AVC from Has2- 1- explants exhibited a total lack of cell migration and invasion (Fig. 4D). AVC morphogenesis is a complex event, requiring appropriate release of signals from myocardium and receptor-mediated changes in endothelial cell behavior. Thus, there could be trivial explanations for the failure of Has2·1• AVC explants to undergo normal morphogenesis in vitro, e.g., lack of cell viability, immature myocardium, or lack of expression of appropriate endothelial cell receptors. To test this, we performed a number of add back experiments, including adding conditioned medium from wild-type AVC cultures (data not shown), purified HA (Fig. 4F, G), and finally, restoring Has2 activity by transfecting HasZ- /- AVC cultures with the wild-type cDNA (Fig. 4E). All of these manipulations resulted in restoration of normal AVC morphogenesis in the Has2-1- explants. Identification of an HA dependent pathway involving Ras in AVC morphogenesis Normal AVC morphogenesis involves the GTPase Ras 10. We asked if the requirement for HA in cell migration and transformation was mediated by Ras. First, we confirmed that transfection with dominant negative H Ras (SI7N) 11 inhibited AVC morphogenesis in wild-type explants (Fig. 4C). Next, Has2-1- AVC explants were transfected with a plasmid encoding an activated form of Ras (H-Ras Q61L) 11. This restored AVC morphogenesis to wild-type levels (Figure 4H). Of course, this could result from a parallel, Ras dependent pathway not requiring HA. To test this, we added HA to AVC cultures from Has2- 1• mice, and transfected them with a cDNA encoding dominant negative H-Ras. This revealed an intermediate phenotype, with endothelial migration, but total absence of transformation and invasion (Fig. 5). Thus, HA appears to be required for two distinct phases of outgrowth from AVC cultures: Formation of the endothelial sheet, and transformation into mesenchyme. Transformation, but not epithelial migration, is dependent upon Ras. SUMMARY We may draw the following conclusions from our studies of Has2-1• mice. First, Has2 is the principal source of HA during mid-gestation in the mouse. Hasl and Has3 do not compensate for the absence of Has2. Second, the very similar phenotypes of Has2 and versican deficient mice underscore the critical importance of a composite matrix in maintenance and stability of an expanded extracellular matrix. Third, there appears to be an absolute requirement for HA in endothelial cell migration and transformation in the developing cardiovascular system. This involves both Ras independent and Ras dependent pathways. Collectively, these observations underscore the pivotal importance of HA during embryogenesis and cell migration and transformation. The availability of HA deficient animals, tissues and cell lines will add greatly to our understanding of this important biomolecule. REFERENCES 1. 2.
P. H. Weigel, V. C. Hascall, and M. Tammi, Hyaluronan synthases, J. Biol. Chem., 1997, 272, 13997-14000. A. P. Spicer and J. A. McDonald, Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family, J Biol Chem, 1998, 273, 1923-32.
Disruption of hyaluronan synthase-2 3.
4.
5.
6.
7.
8.
9.
10.
11.
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N. Itano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M. Hamaguchi, Y. Yoshida, Y. Ohnuki, S. Miyauchi, A. P. Spicer, J. A. McDonald and K. Kimata, Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties, J. Bio!' Chem., 1999,274,2508525092. A. P. Spicer, M. F. Seldin, A. S. Olsen, N. Brown, D. E. Wells, N. A. Doggett, N. Itano, K. Kimata, J. Inazawa and J. A. McDonald, Chromosomal localization of the human and mouse hyaluronan synthase genes, Genomics, 1997,41,493-7. R. Kosaki, K. Watanabe, and Y. Yamaguchi, Overproduction of hyaluronan by expression of the hyaluronan synthase Has2 enhances anchorage-independent growth and tumorigenicity, Cancer Res, 1999, 59,1141-5. T. Ichikawa, N. Itano, T. Sawai, K. Kimata, Y. Koganehira, T. Saida and S. Taniguchi, Increased synthesis of hyaluronate enhances motility of human melanoma cells, J Invest Dermatol, 1999, 113, 935-9. T. D. Camenisch, A. P. Spicer, T. Brehm-Gibson, J. Biesterfeldt, M. L. Augustine, A. J. Calabro, S. Kubalak, S. E. Klewer and J. A. McDonald, Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme, J. Clinical Invest., 2000, 106,349-360. C. H. Mjaatvedt, H. Yamamura, A. A. Capehart, D. Turner, and R. R. Markwald, The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation, Developmental Biology, 1998,202,56-66. D. H. Bemanke and R. R. Markwald, Effects of hyaluronic acid on cardiac cushion tissue cells in collagen matrix cultures, Texas Reports on Biology & Medicine, 1979,39,271-85. M. M. Lakkis and J. A. Epstein, Neurofibromin modulation of ras activity is required for normal endocardial-mesenchymal transformation in the developing heart, Development, 1998, 125,4359-4367. L. A. Feig and G. M. Cooper, Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins, Mol Cell BioI, 1988, 8, 2472-8.
PART 4
BIOSYNTHESIS AND BIOLOGICAL DEGRADATION OF HYALURONAN
THE PRODUCTION OF HYALURONIC ACID FROM STREPTOCOCCI Derek C. Ellwood Department ofMicrobiology, Medical School, University ofNewcastle, Newcastle Upon Tyne, UK
ABSTRACT In the late 1980's it became clear that Hyaluronic Acid (HA) might have more clinical applications than its use in eye surgery. If this were so, then a larger supply of HA than that produced from the conventional extraction of rooster combs would be required. The use of bacteria known to produce HA was an obvious option. This paper describes the studies that led to the production of clinical grade HA from a bacterial source. MacLennon (working at Porton Down) had shown that a number of strains were available that produced HA in good yield. These strains related largely to pathogenicity in specific hosts only. The organism Streptococcus equi causes strangles in horses and strains of this organism produced HA in good yield. Streptococci bacteria were also known to release their cell wall components when grown in complex media in batch culture. For these reasons a study ofHA production by strains of S. equi in steady state culture was undertaken to establish optimum conditions and strain selection. These experiments were carried out in Porton type chemostats (2 L) using semi-defined media strains selected for good HA yield after over 100 generations of growth at a mean generation time of 7 hours. These results allowed the development of a production process for HA that has been accepted by the Medical Control Agency (MCA).
INTRODUCTION There is something quite quixotic about this paper, since in a review of the production of bacterial polysaccharides, Sutherland and Ellwood (1979) quoted the opinion that HA production from bacteria was unlikely to be required in the future 1. The highly successful use of HA in eye surgery (pioneered by Balazs) gave rise to a number of investigations into the production of HA by several methods including fermentation. The principal method is of course the isolation of HA from rooster combs, which gives material of high molecular weight and purity. However, because the value of the product was high and the demand increasing there was interest in developing the bacterial production route. There were doubts expressed as to the chance of success of such a route. This paper gives an account of investigations, which led to the objective of bacterial production of HA of sufficient molecular weight and purity being achieved.
BACKGROUND The presence of HA in a bacterium Streptococcus haemolyticus was first demonstrated by Kendall et al. 2. The presence of HA in bacteria was surveyed by
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Biosynthesis and biological degradation ofhyaluronan
Faber and Rosendal 3, who concluded that the presence of HA was a common trait in Streptococci. MacLennan (working at Porton Down) showed that Group C organisms such as S. zooepidermis and S. equi could produce HA in good yield in complex media in batch culture 4. These and other studies showed that HA was produced in good yield during the growth phase but during the stationary phase it was often degraded in some organisms by a hyaluronidase. However a number of strains were identified which did not degrade the HA produced after the growth phase. The field continued to attract attention and a number of publications were produced together with a number of granted patents in different countries throughout the world, e.g. USA, Japan and Israel. The Centre of Applied Microbiology and Research (CAMR) at Porton Down was composed of a number of laboratories. The Pathogenic Microbes Research Laboratory (PMRL) was asked in the mid 1980's to examine the potential of continuous culture techniques for the production ofHA. PMRL had the advantage of the collection ofHA producing strains set up by MacLennan, together with a strong background in the use of continuous culture techniques to produce xanthan polysaccharides. CHOICE OF STRAIN FOR HA PRODUCTION
Pathogenic Streptococci are differentiated by these attributes s: •
Action on Blood: J3 - haemolytic - lyse red cells a. - haemolytic - green colouration of red.cells non-haemolytic - no change (which allow sevalogical differation)
•
Lancefield antigens (polysaccharide antigens) are found in the cell wall ofthe organisms: Group A - human pathogens, Group B - related to neonatal disease in children, Groups C & D - infrequently related to human disease, but cause disease in animals. For example, S. suis causes disease in piglets, which is occasionally passed on, to man by frequent association with rearing pigs. S. zooepidemecies occasionally is associated with human disease.
The pathogenicity of Streptococci: • • •
Protected from Phagocytosis by M Protein in the cell wall. Secrete a lipid containing teichoic acid, which can attach to human/animal cells. Some strains form HA capsules, but this is not known to be a relevant factor.
Clearly if a strain of Streptococcus was to be used to produce HA for use in human medicine then it would be best to use strains not related to human disease. On this basis, a number of strains were examined in continuous culture. From the obtained results, a strain of S. equi that gave good yields was focused upon. This was passed on to the company Fermentech Medical, who went on to develop the strain and design a process to produce HA by continuous culture techniques (European Patent Application No 91918590.5). The strain is deposited as NCIMB 40327).
Hyaluronic acid from Streptococci
223
THE PRINCIPLES OF CONTINUOUS CULTURE IN A CHEMOSTAT The culture vessel is fitted with an overflow device to keep the culture volume constant and with pH and temperature control. The medium is made up with a limiting nutrient and with all other nutrients in excess. In a chemostat, the mean residence time is determined by the ratio of the flow rate to the culture volume. This ratio is called the dilution rate D, which is expressed in units of h-1, and this in a chemostat is the exponential growth rate of the cells. Hence, by simply changing the flow rate the growth is also changed. The concentration of cells in the culture is a function of the concentration of the limiting substrate, with all other substrates in excess. The limiting substrate may be any required growth substrate, allowing the effects of different growth substrates to be investigated. The doubling time of the organisms in the cultures is given by In 2 divided by D: = 0.693. When these principles were applied to bacterial cultures over 40 years ago it was soon realised that the chemical composition ofthe cells varied markedly with respect to both growth rate and growth limitation. For example the amount of RNA present in the cell when grown at a doubling time of 14 hours was 7%, this increased to 16% when the organism was grown at a faster rate (doubling time of 50 minutes), presumably because there is a requirement for rapid syntheses of the cell components. THE DEBATE: BATCH VERSUS CONTINUOUS CULTURE Batch Culture This is the method used widely to produce materials from bacterial cultures, and in many laboratory studies. However, when a culture is inoculated with a bacterial culture (say on a sterile loop), the organisms start to grow after some time (lag phase). The organisms then grow at their maximum growth rate (growth phase) until nutrients are exhausted. Growth ceases and the culture degrades (decline phase). This means that the culture first adapts to the growth medium. Having done so it then grows at its maximum rate but the composition of the medium is changing as the nutrients are used up. This means that the metabolisms of the cells are changing and thus the composition of the cells changes. The medium can be designed to minimise these changes but these cultures are in a state of metabolic flux. With respect to Streptococci, Shockman's group at Temple University had shown that cell wall turnover occurred in rapidly growing cultures 6. Furthermore, Knox's group in Sydney showed that lipoteichoic acid in Streptococci was released from the cells when the organisms were grown in batch culture 7 However, growth in continuous culture at steady states at low dilution rates did not allow release of lipoteichoic acids. The importance of this was that the streptococcal lipoteichoic acids playa role in pathogenicity and antigenicity. Finally, Ellwood and Tempest (1972) showed that the cell wall composition of bacteria could be changed by growth under different nutrient limitations 8. On this basis it was reasonable to assume that investigation of the growth physiology for the production of HA would have value. These studies gave some basic parameters for the production ofHA which are given below as a guide to the design of a production facility
224
Biosynthesis and biological degradation ofhyaluronan
BASIC PARAMETERS FOR BA PRODUCTION BY CONTINUOUS CULTURE These comments are based upon experience gained using continuous culture techniques. Fermenter
Most modem fermenters are designed to give maximum yields of aerobic cultures with high stirring rates and high air input. They are high and thin, i.e. they have a high aspect ratio. For polysaccharide production, where high shear rates would give some degradation of the product, it is better to use fermenters with low aspect ratio. This allows adequate mixing and reduces shear. The fermenter and its equipment are usually made of stainless steel and there should be a minimum of sharp edges inside the fermenter. This applies particularly to the stirrer. Air Quality
This has become important in recent years because in some industrial sites air is polluted with high levels of ozone and emissions from power stations. Of course, the air is filtered sterilised before being put into the fermenter. Temperature
The temperature of growth can be controlled by internal or external heating systems. Given adequate stirring the temperature can be controlled within ± 1°C. It is also useful to consider the organism's optimal growth temperature as those of animal origin often grow best at about 40°C. pH Control
Fermentation with streptococci and a sugar such as glucose produces lactic acid, and the pH of the culture must therefore be controlled. This is done by the addition of alkali. This is often sodium hydroxide, but consideration of the use of potassium hydroxide should be given, since the internal concentration of potassium in streptococci may be as much as 200 roM. Medium
A great deal could be said about the medium of choice to grow Streptococci and experimental work is required to devise the right medium for the organism of choice. In chemostat culture there is always a limiting nutrient and for polysaccharide production the best yields are found when the culture is limited by phosphorus or sulphur. There is also considerable debate over the use of defined media but it is possible to use complex media including some of the basic inorganic components. The medium should be sterilised by filtration (as glucose interacts with amino acids to give growth-retarding substances). The water used to make the media should be deionised so that inorganic nutrients can be kept in balance. The amount of ferrous ions present in the medium should be kept at a minimum as in some circumstances, streptococci can produce hydrogen peroxide and hydroxy radicals could be formed.
Hyaluronic acid from Streptococci
225
Culture Redox This can be maintained at the required value by a combination of the airflow and the stirring rate. The redox value of -200 mV allows good production ofHA.
CULTURE STABILITY There is the general assumption that the genome of bacteria are constant. However, in the production of materials by fermentation there are a number of aspects that need to be considered. Production of the seed for Ranger scale batch culture is very important. The number of generations from the single organism to a colony is about 10 generations and to get to large volumes about 80-100 generations are needed. In continuous culture the seed is also important and again after 80-100 generations colony variants start to appear (previous experience by the author).
YIELD In either batch or continuous culture the yield of product (HA) is a key parameter. In earlier studies the yield values were complicated by the apparent presence of a hyaluronidase that was secreted into the medium. However, yields of 3-4 gIL were found with some strains, but the presence of secreted cell wall and other bacterial products required a purification process that was costly and markedly reduced overall yield. In continuous culture it is possible to stabilise the culture and minimise the costs of purification.
CONCLUSIONS The study of the growth of a strain of Streptococcus equi in continuous culture gave rise to a production process for HA. The material produced could be isolated and purified with a molecular weight of - 2 million. The material was found to be acceptable by the Medical Control Agency (MCA) for use in ophthalmic surgery.
REFERENCES 1.
2.
3.
I. W. Sutherland & D. C. Ellwood, Microbial exopolysaccharides - industrial polymers of current and future use, In: Microbial Technology: current status, future prospects, Society for General Microbiology Symposium 29, A. Bull, D. C. Ellwood & C. Ratledge (eds.), University of Cambridge, Cambridge, 1979, pp. 107-150. F. E. Kendall, M. Heidelberger & M. H. Dawson, A serologically inactive polysaccharide elaborated by mucoid strains of group A haemolytic streptococcus, 1. Bioi. Chem., 1937, ill, 61-69. V. Faber & K. Rosendal, Streptococcal hyaluronidase II: studies on the production of hyaluronidase and hyaluronic acid by representatives of all types of haemolytic streptococci belonging to Group A, Acta. Path. Microbiol Scand., 1954, 35 (2), 159-164.
226
4. 5. 6.
7.
8.
Biosynthesis and biological degradation ofhyaluronan
A. P. MacLennan, The production of capsules, hyaluronic acid and hyaluronidase by Groups A and C Streptococci, J. Gen. Microbiol., 1956, ]A, 134-142. G. Colman, Pathogenic streptococci, In: Oxford Textbook of Medicine, ]'d Edition, Vol. 1, D. 1. Weatherall, 1. G. G. Ledingham & D. A. Warrell (eds.), Oxford University Press, Oxford, 1996, pp. 497-511. R. Joseph & G. D. Shockman, Synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species, Infection and Immunity, 1975, .lb 333-338. K. W. Knox & A. 1. Wicken, Effect of growth conditions on the surface properties and surface components of oral bacteria, In: Continuous Culture 8 biotechnology, medicine and the environment, A. C. R. Dean, D. C. Ellwood & C. G. T. Evans (eds.), Ellis Horwood, Chichester, 1984, pp. 72-88. D. C. Ellwood & D. W. Tempest, Effects of environment on bacterial wall content and composition, Adv. Microb. Physiol., 1972, L. 83-117.
HYALURONAN SYNTHASES: MECHANISTIC STUDIES AND BIOTECHNOLOGICAL APPLICATIONS Paul L. DeAngelis University ofOklahoma Health Sciences Center Dept. ofBiochemistry and Molecular Biology Oklahoma City, Oklahoma, U.S.A. 73104
ABSTRACT
Hyaluronan [HA] is found in the extracellular matrix of vertebrate tissues, in the surface coating of certain Streptococcus and Pasteurella bacterial pathogens, and on the surface of one particular virus-infected alga. Hyaluronan synthases [HASs], the enzymes that polymerize HA using UDP-sugar precursors, are found in the outer membranes of these organisms. The HAS genes from all the above sources have been identified. There appears to be two distinct classes of HA synthases based on differences in amino acid sequence, predicted topology in the membrane, and putative reaction mechanism. All HASs were designated Class I synthases with exception of the Pasteurella HAS. The catalytic mode of pmHAS, the only Class II synthase, was elucidated. This enzyme will elongate exogenous HA-derived oligosaccharide acceptors by addition of individual sugar units to the nonreducing terminus to form long polymers in vitro; no Class I HAS displays this capability. The mode and the directionality of HA polymerization catalyzed by Class I HASs remains unclear. The pmHAS enzyme has also been dissected into its two component activities, the GlcUA-transferase and the GlcNAc-transferase. Thus two active sites exist in a single pmHAS polypeptide in contrast to a widely accepted dogma of glycobiology, "one enzyme, one sugar transferred". Preliminary evidence suggests that the Class I enzymes may also have two sites. The catalytic potential of the pmHAS enzyme may be harnessed to create novel polysaccharides or designer oligosaccharides. Due to the multitude of potential HAbased medical therapies, this chemoenzymatic technology promises to benefit our pursuit of good health. KEYWORDS
Hyaluronan, chondroitin, glycosyltransferase, synthase, catalysis, mechanism, chimeric polysaccharides, monodisperse oligosaccharides INTRODUCTION
Hyaluronan [HA] is a very abundant glycosaminoglycan in the vertebrate body with both structural and signaling roles I. Certain pathogenic bacteria, namely Group A and C Streptococcus and Type A Pasteurella multocida, produce an extracellular HA coating called a capsule 2,3. In both microbes, the HA capsule is a virulence factor that confers the bacteria with resistance to phagocytosis and complement. Another organism that produces HA is chiorella algae upon infection with a certain large
228
Biosynthesis and biological degradation ofhyaluronan
SYNTHASE n UDP-GlcUA + n UDP-GlcNAc Mx 2+
Figure 1.
~
[GlcUA-GlcNAcJIl + 2n UDP
In
3
= 10 -4
I
HA biosynthesis reaction.
double-stranded DNA virus, PBCV-l 4. The role of HA in the virus lifecycle is not clear at this point. The glycosyltransferase enzymes that polymerize HA are called HA synthases (older terminology included HA synthetases) 5,6. In all known HA synthases, a single polypeptide species is responsible for the polymerization of the HA chain. UDP-sugar precursors, UDP-GlcNAc [UDP-N-acetylglucosamine] and UDP-GlcUA [UDPglucuronic acid] are utilized by the HA synthases in the presence of a divalent cation (Mn and/or Mg) at neutral pH (Fig. 1). All synthases are membrane-associated in the living cell and are found in the membrane fraction after cell lysis. Between 1993 to 1998, HA synthases from Group A and C Streptococcus [spHAS and seHAS, respectively], vertebrates [HAS1,2,3], algal virus [cvHAS], and Type A Pasteurella multocida [pmHAS] were identified and molecularly cloned. The first three types of HA synthases appear to be very similar in size, amino acid sequence, and predicted topology in the membrane. The Pasteurella HA synthase, in contrast, is larger and possesses a rather distinct sequence and predicted topology. Therefore, we proposed that two classes ofHA synthases exist (Table 1) 6. Class I enzymes include the streptococcal, vertebrate, and viral proteins while the Pasteurella protein currently is the only member of Class II. We also have some evidence that the catalytic process of Class I and Class II enzymes differ '.
Table 1.
Two classes ofHA synthases.
Class I
Members Polypeptide Size Predicted Topology Molecular Directionality Of Polymer Growth Acceptor Utilization
spHAS,seHAS, cvHAS, vertebrateHAS 1,2 & 3
Class II pmHAS
417-588 residues
972 residues
multiple interspersed transmembrane or membrane-associated regions
soluble with C-terminal attachmentsite to a membrane component
unclear
nonreducing terminus extension
not likely
yes
Mechanistic studies and biotechnological applications
229
Although the Pasteurella HA synthase was the last enzyme to be discovered, some the characteristics of pmHAS have enabled substantial progress to occur in comparison to some members of the Class I enzymes that have been under study for four decades. The key characteristic of prnHAS that has allowed the elucidation of the molecular directionality of polymerization and identification of its two active sites is the ability of prnHAS to elongate exogenously supplied acceptor oligosaccharides 7. Recombinant prnHAS will add single sugars in a repetitive fashion to HA-derived oligosaccharides in vitro. The intrinsic fidelity of each sugar transfer is responsible for forming the alternating disaccharide repeat of this glycosaminoglycan; simultaneous formation of the disaccharide unit is not required 7. On the other hand, no similar elongation of exogenous acceptors has been proven with any Class I enzyme 7. Through basic science research, we now have developed some biotechnological applications for the remarkable synthases of Pasteurella. MATERIALS & METHODS Reagents Molecular biology reagents were from Promega unless noted. Custom oligonucleotides were from The Great American Gene Company. All other reagents were the highest grade available from either Sigma or Fisher unless otherwise noted. pmHAS truncation and point mutants A series of truncated polypeptides were generated by amplifying the pPm7A insert by Polymerase Chain Reaction with Taq polymerase (Fisher) and synthetic oligonucleotide primers corresponding to various portions of the prnHAS open reading frame 8. The amplicons were then cloned into the expression plasmid pKK223-3 (tac promoter; Pharmacia). The resulting recombinant constructs were transformed into Escherichia coli TOP IOF' cells (Invitrogen) and maintained on Luria-Bertani media with ampicillin selection. Mutations were made using the QuickChange site-directed mutagenesis method (Stratagene) with the plasmid pKKJprnHAS I . 703 DNA as template. Enzyme preparation Membrane preparations containing recombinant full-length prnHAS, prnHA1-972, was isolated from E. coli as described 8. For soluble truncated prnHAS proteins, prnHAS!·703, pmHAS 1•650, and pmHAS1•703-derived mutants, cells were extracted with B_Per™ II Bacterial Protein Extraction Reagent (Pierce) according to the manufacturer's instruction except that the procedure was performed at 7°C in the presence of protease inhibitors. Enzyme assays for HA polymerization, GlcNAc transfer, or GleDA transfer Three assays were designed to detect either (a) the polymerization of long HA chains, or (b) the addition of a single GlcNAc to a GlcUA-terminated HA oligosaccharide acceptor [G1cNAc-Tase], or (c) the addition of a single GlcUA to a GlcNAc-terminated HA oligosaccharide acceptor [GlcUA-Tase]. The complete HAS
230
Biosynthesis and biological degradation ofhyaluronan
activity was assayed in 50 mM Tris, pH 7.2, 20 roM MnCh. 0.1 M (~hS04, 1 M ethylene glycol, 0.12 roM UDP-e 4C]GlcUA (0.01 J,tCi; NEN), 0.3 roM UDP-GlcNAc, and even-numbered HA oligosaccharides (1 I1g uronic acid) derived from testicular hyaluronidase digests [(GlcNAc-GlcUA)n ; n = 4-10] at 30°C for 25 minutes in a reaction volume of 50 111. GlcNAc-transferase activity was assayed for 4 minutes in the same buffer system with even-numbered HA oligosaccharides but with only one precursor sugar, 0.3 roM UDP-eH]GlcNAc (0.2 I1Ci; NEN). GlcUA-transferase activity was assayed for 4 minutes in the same buffer system but with only 0.12 roM 4C]GlcUA UDP-C (0.02 J,tCi) and odd-numbered HA oligosaccharides [GlcNAc(GlcUA-GlcNAc)n; n = 7-20] (3.5 I1g uronic acid) prepared by mercuric acetate treatment of Streptomyces HA lyase digests 9. Reactions were terminated by the addition ofSDS to 2% (w/v). The reaction products were separated from substrates by descending paper (Whatman 3M) chromatography with ethanol/1 M ammonium acetate, pH 5.5, development solvent (65:35 for the HAS and GlcUA-Tase assays; 75:25 for the GlcNAc-Tase assay). For the HAS assay, the origin of the paper strip was eluted with water and the incorporation of radioactive sugars into HA polymer was detected by liquid scintillation counting with BioSafe II cocktail (RPI). For the half-assay reactions, the origin and the downstream 6 cm of the strip was counted in 2em pieces. All assays were adjusted to be linear with regard to incubation time and to protein concentration. Gel fIltration chromatography The size of HA polymers was analyzed by chromatography on a Phenomenex PolySep-GFC-P 3000 column eluted with 0.2 M sodium nitrate The column was standardized with various size fluorescent dextrans. Radioactive components were detected with a LB508 Radioflow Detector (EG & G Berthold) and Zinsser cocktail. In comparison to the full HAS assay using paper chromatography described above, these 3 minute reactions contained twice the UDP-sugar concentrations, 0.06 J,tCi UDP_[14C]GlcUA, and 0.25 I1g even-numbered HA oligosaccharide. Also, addition of ethylenediamine tetracetic acid (final cone. 22 roM) and boiling (2 min) was employed to terminate the reactions instead of addition of SDS. RESULTS & DISCUSSION pmHAS acceptor utilization and specificity 1 972
A variety of oligosaccharides were tested as acceptors for recombinant pmHAS • (Table 2). HA oligosaccharides derived from testicular hyaluronidase digests are elongated by pmHAS when supplied with the appropriate UDP-sugars 7. Reduction with sodium borohydride does not destroy the acceptor activity. On the other hand, the oligosaccharides derived from HA lyase digests do nQ1 support elongation; the dehydrated unsaturated non-reducing terminus GlcUA residue lacks the hydroxyl group that would accept the incoming sugar from the UDP-precursor. Therefore, pmHAS-catalyzed elongation occurs at the nonreducing terminus. In parallel experiments, we have found the recombinant forms of two Class I synthases, spHAS and xlHAS1, do DQ1 elongate HA-derived acceptors 7. With respect to the directionality of Class I enzymes, conflicting reports have been made and further analysis is required 6.
Mechanistic studies and biotechnological applications
Table 2.
231
pmHAS acceptor oligosaccharide specificity
pmHAS elongation activity?
Sugar I Structure
YES
HA oligosaccharide (hyaluronidase-derived) [(~1,4)GlcUA(~1,3)GlcNAcb
HA oligosaccharide (HA lyase-derived) ~GlcUA[(~ I ,3)GIcNAc(~ I ,4)GlcUA]1.2GIcNAc
NO
reduced HA oligosaccharide (hyaluronidase, borohydride-treated)
YES
GlcUA[(~1,3)GlcNAc(~1,4)GlcUA]I.2GlcNAc-alditol
chitotetraose, chitopentaose [(~ 1,4)GlcNAc]4.S
NO
heparosan pentamer
NO
GlcNAc[(al,4)GlcUA(~ 1,4)GlcNAc] 2
chondroitin sulfate pentamer GaINAc-sulfate[ (~1,4 )GlcUA
YES (~1,3 )GaINAc-sulfate
h
Interestingly, chondroitin sulfate pentamer serves as a good acceptor for pmHAS. Other structurally related oligosaccharides, such as chitotetraose or heparosan pentamer, however, do not serve as acceptors for pmHAS. Overall, pmHAS appears to require l3-linked, GleDA-containing acceptor oligosaccharides. We hypothesize that an oligosaccharide-binding site mediates HA chain retention during polymerization.
Molecular dissection of pmHAS transferase activities: two active sites in one polypeptide The ability to measure the two component glycosyltransferase activities of a HA synthase, the GlcNAc-transferase and the GleDA-transferase, has allowed the molecular dissection of pmHAS. We noted that a short duplicated sequence motif, Asp-Gly-Ser [DGS], was present in pmHAS 8. A comparison of hydrophobic cluster analyses of many other glycosyltransferases that produce l3-linked polysaccharides or oligosaccharides suggested that, in general, two types of domain exist: "A" domains and "B" domains 10. pmHAS, a Class II synthase, is unique in that it contains two "A" domains (personal communication, B. Henrissat). It has been proposed that certain members of the Class I HA synthases (spHAS) contained a single "A" and a single "B" domain 10. Various deletion or point mutants of pmHAS were assayed for their ability to polymerize HA chains or their ability to add a single sugar to a HA acceptor oligosaccharide (Table 3). In summary, pmHAS contains two distinct active sites. Mutagenesis of the aspartate of the DGS motif (residue 196 or 477) at either site resulted in the loss of HA polymerization, but the activity of the other site remained relatively unaffected II. Thus a dual-action HA synthase was converted into two different single-action glycosyltransferases.
232
Biosynthesis and biological degradation ofhyaluronan
Table 3.
Activity of pmHAS deletion and point mutants Enzyme Activity *
Enzyme wild-type pmHAS 1.97i
HAS
GlcNAcTase
GlcUATase
+
+ + + + + +
+
pmHAS'-650 pmHAS I-703 pmHAS i -7OJ D477N
+
D477K D477E
+
pmHAS i.703 D196N
+ + +
Dl96K Dl96E
+
* Activity
active - inactive/weak
The deletion of the last 269 residues from the carboxyl terminus transformed a weakly expressed membrane protein into a highly expressed soluble protein 11. Inspection of the amino acid sequence of pmHAS in this region, however, does not reveal any typical secondary structure features that would mediate direct interaction with of the enzyme to the lipid bilayer. We hypothesize that the carboxyl terminus of the catalytic pmHAS enzyme docks with a membrane-bound polysaccharide transport apparatus in the living bacterial cell. The first "A" domain, AI, of pmHAS is the GlcNAc-Tase while the second "A" domain, A2, is the GlcUA-Tase (Fig. 2). This is the first identification of the two active sites for any enzyme that produces a heteropolysaccharide as well as definitive proof that one enzyme can indeed transfer two distinct sugars. A distinct enzyme from Type F P. multocida, called pmCS, was found that catalyzes the formation of an unsulfated chondroitin polymer 12. Chondroitin and HA are identical in structure except the former polymer contains N-acetylgalactosamine instead of GlcNAc. pmHAS and pmCS are 87% identical at the amino acid level. The majority of residue changes are in the Al domain consistent with the hypothesis that this region is responsible for hexosamine transfer.
residue# 1
I
Figure 2.
0196
Al
GlcNAc-Tase _
D477
-700
972
Membrane Assoc.
Schematic of pmHAS domains. Two independent transferase domains, A1 and A2, are responsible for catalyzing HA chain polymerization. Repetitive sequential addition of single sugars rapidly builds It is likely that the carboxyl-terminus of the HA chain. pmHAS interacts with the bacterial membrane-bound polysaccharide transport apparatus in some fashion.
f
Mechanistic studiesand biotechnological applications
c=-
synthase
233
reducing end " ••••
UDP-GIcUA
CD
:"'-_.1
UDP-GlcNAc UDP
Figure 3.
0 ..... ·····
Model ofHA biosynthesis by pmHAS. Single sugars are added by each "A" domain in a repetitive fashion to the nonreducing end of the HA chain. The intrinsic fidelity of each transferase activity maintains the repeating disaccharide structure of HA. The nascent HA chain is probably retained by pmHAS during catalysis via an oligosaccharidebinding site.
We have demonstrated efficient single sugar transfer in vitro by pmHAS by several types of experiments, therefore, we hypothesize that HA chains are formed by the rapid, repetitive addition of single sugars by the Class II synthase (Fig. 3). Thus far, one line of evidence suggests that a Class I enzyme also possesses two transferase sites. Mutation of mmHASI at leucine residue 314, a part of the tentative GlcUATase site, to a valine was reported to convert this vertebrate HAS synthase into a chito-oligosaccharide synthase 13. No site has been identified for the corresponding GlcNAc-transferase activity. Polymer grafting by polysaccharide synthases: adding HA to molecules or solids The study of pmHAS has transformed HA synthases from the realm of difficult, recalcitrant beasts in the research laboratory to potential biotechnological workhorses. Novel molecules may be formed using the ability of pmHAS to graft long HA chains onto short HA-derived or chondroitin-derived acceptors. For example, useful acceptors may consist of small molecules or drugs with covalently attached HA or chondroitin oligosaccharide chains (e.g. 4 sugars long). In another case, HA chains may be added to oligosaccharide primers immobilized on solid surfaces (Table 4). Thus long HA chains may be gently added to sensitive compounds or delicate devices. In another application, novel chimeric polysaccharides may be formed because the acceptor oligosaccharide utilization of pmHAS is not as strict as the sugar transfer specificity. Chondroitin sulfate and chondroitin are recognized as acceptors by pmHAS and are elongated with a HA chain of various lengths (Fig. 4). Conversely, pmCS, the very homologous chondroitin synthase, will recognize and elongate HA acceptors with chondroitin chains. Chimeric glycosaminoglycan molecules are formed which contain a natural, defined connection linkage. These grafted polysaccharides may serve to join a celI or tissue that binds HA to another cell or tissue that binds chondroitin or chondroitin sulfate. In certain aspects, the grafted glycosaminoglycans resemble the proteoglycans that are essential matrix components of vertebrate tissues 14. But since no protein linkers are present in the artificial chimeric polymers, the antigenicity and proteolysis concerns surrounding medical uses of proteoglycans are eliminated. The risk of transfer of infectious agents in animal tissue extracts to the human patient is also abated with chimeric polymers.
234
Biosynthesis and biological degradation ofhyaluronan
Table 4.
pmHAS-mediated HA grafting onto polyacrylamide beads. Reactions containing pmHAS, radiolabeled UDP[14C]GlcUA and UDP-eHlGlcNAc, and various immobilized primer sugars (acceptors coupled by reductive amination to amino beads) were performed. The beads were then washed and radioactivity incorporated onto beads was measured by liquid scintillation counting. HA chains were grafted onto the plastic beads when a suitable primer and pmHAS was used. pmHAS added?
Bead Type H~primer H~primer
chitotetraose primer no primer
Bound
Bound eH]GlcNAc (dpm)
YES
990
1140
NO
10
10
YES
25
20
YES
5
35
reducing end
~///////////~
Figure 4.
4
(dpm)
nonreducing end
~///h
eq GlcUA
_
polysaccharide HA
_
Chondroitin
rza
Chondroitin sulfate
0
Schematic of grafted polysaccharide structures. The Pasteurella HA synthase or chondroitin synthase will elongate certain other polymers at the nonreducing end in vitro to form novel chimeric glycosaminoglycans. Some examples are depicted.
Synthesis of monodisperse HA and HA-related oligosaccharides In addition to adding large polymeric HA chains to acceptor molecules, pmHAS will synthesize smaller defined HA oligosaccharides in the range of 5 to 24 sugars. Using wild-type enzyme and various reaction conditions, the relatively easily obtained HA oligosaccharides containing 4 or 5 monosaccharides were elongated by several sugars to longer versions that are often difficult to prepare in large quantities 7. We found that combining a soluble mutant GlcUA-Tase and a soluble mutant GlcNAcTase in the same reaction mixture allows the formation of HA polymer if supplied with acceptor 11. In 3 minutes, chains of -150 sugars (-30 kDa) were made. Either mutant synthase ~ will Wlt make a HA chain. Therefore, if further control of the reaction is made by selectively combining various enzymes, UDP-sugars, and acceptors, then defined, monodisperse shorter oligosaccharides may be prepared (Figure 5).
Mechanistic studies and biotechnological applications
235
starting acceptor UDP-sugars
repetitive cycling of reaction mixture through enzyme bioreactors for desired size & composition oligosaccharide
customized product Figure 5.
Preparation of defined oligosaccharides. In this example, a HA tetrasaccharide acceptor is elongated by a single chondroitin disaccharide unit using two steps with immobilized mutant Pasteurella synthases (signified with white arrows). The product depicted is a novel hexasaccharide. Repeating the cycle one more time produces an octasaccharide, two cycles forms a decasaccharide, etc. If the acceptor was previously coupled to another molecule (e.g. drug or medicant), then the new conjugate would be elongated with short HA, chondroitin, or hybrid chains as desired.
For example, in one embodiment, a mixture of UDP-GlcNAc, UDP-GlcUA and acceptor are repetitively circulated through separate bioreactors with immobilized mutant synthases that transfer only a single sugar. With each cycle of incubation with the bioreactor, another sugar group is added to the acceptor to form small defined HA oligosaccharides. The use of a similar pmCS mutant (e.g. a GaINAc-Tase) in one of the steps would allow the formation of mixed oligosaccharides if supplied with UDPGaINAc. The biological activities and the therapeutic potential of small HA oligosaccharides are emerging research areas that will require defined, monodisperse sugars for unambiguous interpretation. CONCLUSIONS Apparently two distinct classes of HA synthases exist. The most well characterized, pmHAS of Pasteurella, a Class II enzyme, elongates HA by repetitive addition of single sugars to the nonreducing end of the HA chain. The directionality and the mode of synthesis of the Class I synthases (streptococcal, vertebrate, and viral enzymes) remains unclear. With respect to applied sciences, the ability ofpmHAS to elongate exogeneously supplied acceptor molecules is useful for creating novel molecules and/or devices with potential medical utility.
236
Biosynthesis and biological degradation ofhyaluronan
ACKNOWLEDGEMENTS I thank the National Institutes of Health (grant GM56497) and the National Science Foundation (grant MCB-9876193) for supporting this research, and Wei Jing, Amy Padgett-McCue, and Annette Fleshman for their efforts in the laboratory.
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T. C. Laurent & 1. R. E. Fraser, 'Hyaluronan', FASEB J., 1992, 6, 2397-2404. E. H. Kass & C. V. Seastone, 'The role of the mucoid polysaccharide (hyaluronic acid) in the virulence of group A Streptococci', J. Exp. Medicine, 1944, 79, 319330. G. R. Carter & E. Annau, 'Isolation of capsular polysaccharides from colonial variants of Pasteurella rnultocida', Am. J. Vet. Res., 1953, 14,475-478. P. L. DeAngelis, W. Jing, M. V. Graves, D. E. Burbank, & 1. L. Van Etten, 'Hyaluronan synthase of chlorella virus PBCV-l', Science, 1997, 278, 18001803. P. H. Weigel, V. C. Hascall, & M. Tammi, 'Hyaluronan synthases', J. Bioi. Chem., 1997,272, 13997-14000. P. L. DeAngelis, 'Hyaluronan synthases: fascinating glycosyltransferases from vertebrates, bacterial pathogens, and algal viruses', Cell. Molec. Life Sciences, 1999, 56, 670-682. P. L. DeAngelis, 'Molecular directionality of polysaccharide polymerization by the Pasteurella rnultocida hyaluronan synthase', J. Bioi. Chem., 1999, 274, 26557-26562. P. L. DeAngelis, W. Jing, R. R. Drake, & A. M. Achyuthan, 'Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida'; J. Bioi. Chern., 1998,273,8454-8458. U. Ludwigs, A. Elgavish, J. D. Esko, E. Meexan, & L. Roden, 'Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-0- (beta-D-gluco-4enepyranosyluronic acid)-D-glucose', Biochem. J., 1987,245, 795-804. I. M. Saxena, R. M. Brown, M. Fevre, R. A. Geremia, & B. Henrissat, 'Multidomain architecture ofbeta-glycosyl transferases: implications for mechanism of action', J. Bacteriol., 1995, 177, 1419-1424. W. Jing & P.L. DeAngelis, 'Dissection of the two transferase activities of the Pasteurella rnultocida hyaluronan synthase: Two active sites exist in one polypeptide', Glycobiology. 2000, In press. P. L. DeAngelis & A.J. Padgett-McCue, 'Identification and molecular cloning of a chondroitin synthase from Pasteurella rnultocida Type F', J. Bioi. Chern. 2000,275,24124-24129. M. Yoshida, N. Itano, Y. Yamada, & K. Kimata, 'In vitro synthesis of hyaluronan by a single protein derived from mouse HAS 1 gene and characterization of amino acid residues essential for the activity', J. Bioi. Chern. 2000, 255, 497-506. B. P. Toole, 'Proteoglycans and hyaluronan in morphogenesis and differentiation', In: Cell Biology of the Extracellular Matrix, E.D. Hay (ed.), Plenum Press, NY, 1991, pp. 305-341.
TRANSCRIPTIONAL REGULATION OF HYAL-2 HYALURONIDASE BY DE NOVO METHYLATION OF CpG ISLANDS IN BRAIN Gunter Lepperdinger*, Birgit Strobl, Johannes Miillcgger & Giinther Kreil Institute ofMolecular Biology of the Austrian Academy ofSciences, Billrothstr. 11, A-5020 Salzburg, Austria
ABSTRACT
The Hyal-Z gene encodes a lysosomal hyaluronic acid-degrading enzyme. This protein is related to the testicular form known as PH-20 hyaluronidase. The gene is expressed ubiquitously, with the sole exception of adult brain. In this contribution, we show that methylation of CpG islands is responsible for the downrcgulation of the transcriptional activation of this gene in adult brain and astrocytoma cells. Furthermore, the physiological role of the polymeric species ofhyaluronan is discussed in detail. KEYWORDS
Hyaluronidase, CpG islands, de novo methylation, embryonic development INTRODUCTION
In the course of searching for a putative tumor suppressor gene in a region on chromosome 3, which frequently is abnormal in a variety of human cancers I, in particular, in overlapping homozygous deletions in the region 3p21.3 that have been found in several small cell lung cancer cell lines 2, genes have been discovered which clearly are related to a hum;; gene that is located on chromosome 7 3 and encodes for the sperm head hyaluronidase, PH-20, These new genes named LU(ng) CA(ncer)-I, LUCA-2 and LUCA-3 were thus expected to code for hyaluronidases. In this context, the studies of Dc Maeyer and coworkers are of particular interest. They have analyzed hyaluronidase activities present in the serum of mice 4. Two main strain variants have been characterized which differ in the amount and/or polymorphism of serum hyaluronidases. One type, termed "b", contains a single 60 kDa protein with hyaluronidase activity, whereas in the "a" type different isoenzymes are present. The overall hyaluronidase activity of the "a" type serum is about 3 times higher than the "b" variant on an otherwise identical genetic background. This same difference is also detectable in extracts from liver, kidney and spleen of respective animals. Careful segregation analysis assigned this polymorphism to a locus on chromosome 9, which De Maeyer called Hyall 5. The a and the b alleles of this locus correspond to the variants mentioned above. In subsequent studies, the same authors could show that the growth of transplanted tumors was retarded in mice with the Hyal l-a allele 6. These results suggested a correlation between higher hyaluronidase levels with enhanced resistance to tumor development in these mice. The Hyal-I gene is located in a region which is syntcnic with the 3p21 in the human genome. This implied that the Ilyal-I gene indeed corresponds to one of the LUCA genes. The term LUCA has therefore been replaced by HYAL for those genes that encode a hyaluronidase.
188
The role or hyaluronan in tissues
We have concentrated on studying the LUCA/HYAL-2 gene. As opposed to the other two genes, HYAL-2 is expressed in many tissues. The little understood role of HA and hyaluronidases in tumor growth and metastasis and in angiogenesis clearly merited further studies. In particular, it was of interest to find out if this ubiquitously expressed Hyal-2 gene product is involved in these processes. Hyal-2 hyaluronidase was molecularly characterized and its biochemical properties determined? It could clearly be shown by tagging Hyal-2 with EGFP that this protein is sorted into acidified compartments. Most interestingly, experimental data (unpublished results) indicate that this protein is sorted by a shuttle mechanism via the plasma membrane to the destined compartments and that this transport is not mediated by mannose-6-phosphate receptor interaction. Due to its wide distribution, its enzymatic activity at acidic pH and the subcellular localization, Hyal-2 is most likely the lysosomal hyaluronidase which can be found in most cells and can thus be called a classic house-keeping enzyme. Hyal-2 was expressed in cell culture and shown to have hyaluronidase activity. Surprisingly, this enzyme hydrolyzes HA of high molecular mass, as is found in umbilical cord, rooster comb and the coat of Streptococcus strains. The reaction product is a polysaccharide of about 20 kDa corresponding to 50-60 disaccharide units", which may be readily digested by other hyaluronidases. The results obtained with the Hyal-2 enzyme are of interest with respect to structural as well as functional aspects. Firstly, one may speculate that distinct, ordered domains are present in HA which are resistent to the action of the Hyal-2 enzyme. Conversely, only very specific structural configurations along the I-IA chains can be hydrolyzed by Hyal-2. HA chains can form a complex network containing numerous helical structures. Our results on the substrate specificity of the Hyal-2 enzyme thus provided the first biochemical data in support of defined structural domains in high molecular weight HA. The fragments generated by the Hyal-2 enzyme may simply be catabolic intermediates, or may have distinct biological function such as stimulation of angiogenesis, regulation of inflammatory responses or cellular differentiation and migration. The Hyal-2 gene is expressed in embryonic brain but, for unknown reasons, transcription is turned off soon after birth. In this contribution we describe the mechanism of this downregulation. Furthermore, the biological role of Hyal-Z expression in normal development and tumorigenesis will be discussed. METHODS Cell Culture, mRNA Purification, cDNA Synthesis and PCR DBT astrocytoma cells were grown in lxDMEM containing 10% FCS. Total cellular RNA was prepared as described previously''. cDNA synthesis was performed with Ready-To-Go(R) reagents from Pharmacia according to the manufacturer's recommendation. For PCR amplification of Hyal-2 cDNA, primers were designed which bind sequences on two different exons: Exon3-forward (E3f): GAC TCG GAA GAC GCT TCA AGT ATG G, Exon4-forward (E4f): CCT GCC AAT ACC TCA AGA ATT ACC, Exon-l-reverse (E4r): GTT CCG TTG GCA CTG CTC GCC ACe. For the normalization of the PCR template, the following primers which bind to GAPDH cDNAwere used: forward: GTG AAG GTC GGT GTG AAC G; reverse: GTG AAG ACA CCA GTA GAC TC. Primers were annealed for 30 seconds at 55°C and eDNA was amplified in the presence ofTaq polymerase.
Transcriptional regulation of Hyal-2 hyaluronidase
189
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Figure 1 A: Dot blot analysis of mouse against human genomic sequence using MegAlign v3.05, DNASTAR, Inc. Parameters: Percentage: 65, Window: 30, Minimum Quality: I. B: Region of putative CpG islands in the 5' end of murine Hyal-Z gene. CpG are highlighted, primers used for experimental determination of de novo methylation are indicated, cleavage sites for HpaIl and MspI are designated HIM'" the first exon is marked by a curled line and the protein coding region is typed in bold. Digested DNA was amplified by PCR using three primers in a twofold combination (MEl/ME3; ME2/ME3): MEI:CGG GCT TGG TTG GTA CCA GGA TGG C; ME2: CGGTGC CCG AGA CTA AGT CCT GAC G; ME3: CTT TCT AGA GCA GGG AGC TGC CCG. Primers were annealed at 62°C for 20 seconds and the reaction was cycled 35 times. DNA Preparation and epG Methylation Analysis Genomic DNA from the brain of newborn and adult C57Bl/By mice was extracted in 100 mM NaCI, 10 mM Tris/CI, pH 8, 25 mM EDTA, 0.5 % SDS, 0.1 gil Proteinase K. DNA was digested with restriction enzymes, M:>pI or lIpaIl (MBI-Fermentas, Vilnius).
RESULTS Promotor analysis of the murine Hyal-2 gene
The cDNA encoding the human Hyal-2 enzyme has also been used to clone the murine cDNA and the gene of the murine orthologue". It is a small gene encompassing without promotor about 3 kbp with two introns of 530 and 415 bp, respecively (Genebank Ace: AJ000059, AJ000060).
190
The role of hyaluronan in tissues
-- --
Recognitionsite for Mspl and Hpall:
Mspl
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Hpall embryo
adult
Figure 2: PCR analysis of methylated genomic DNA. DNA was prepared from newborn [1, 2] and adult [3, 4] mice. The putative CpG island was amplified by PCR in the presence of either MEl or ME2 together with ME3 primers (see also Fig. 1) using untreated (upper panel), MspI, or Hpall treated DNA as a template. In order to control DNA integrity after the restriction digestion, a region flanking the MspI/ Hpall recognition sequence was amplified by ME2/ME3 which gives rise to a fragment 331bps in length [1, 3]. ME1 used in lane 2 and 4 binds within the putative CpG island and amplifies together with ME3 a product of 7l8bps.
A
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Figure 3: A) Detection of Hyal-2 mRNA by Reverse-Transcription PCR using primer pair E3f (=0) and E4f (=i). B) Genomic DNA [1] and Hyal-2 cDNA [2] were ampified using E3f and E4r as primers. DBT cells [3] were treated with 1f!M 5-aza2'deoxycytidin [4], mRNA was prepared, subsequently reverse transcribed and amplified with E3f and E4r. Amplification of genomic DNA or the products obtained before [4] with E4f and E4r yieled a specific fragment of smaller size [5, 6]. Lane 7 shows a DNA molecular weight marker (pBR327/HinFI). cDNAs used in [3] and [4] were normalized for GAPDH transcripts [8, 9].
Transcriptional regulation of Hyal-2 hyaluronidase
191
Dot blot analysis of the murine and human genomic sequences, reveal long stretches of the 5' flanking region of the Hyal-2 gene of either species as highly homologous (Fig. I). The 5' flanking region, the first exon and intron is highly GC rich, a GC content of more than 65% and a ratio of expected versus observed CpG dinucleotides of 0.69 has been calculated. The 3' end of the island extends into part of the first intron. Moreover, the CpG content is also highly enriched upstream to the transcriptional start site of the human gene (%GC=69.9; -1630-400 bps). Cross and colleagues 10 have cloned hypermethylated DNA by affinity chromatography from adult human blood. Indeed, a partial sequence (222bps) has been reported to the DNA databases (Macdonald et al., 1995: Genbank: Z64462) which is derived from the human Hyal-2 gene. Transcriptional regulation of Hyal-2 Genomic DNA prepared from brains of newborn and adult mice was restricted with MspI or Hpall. Both endonucleases recognize the tetrameric sequence: CCGG. However, Hpall is blocked by CpG methylation. The Hpall and Mspl restricted DNAs were subjected to PCR analysis using specific primers which amplify either a DNA fragment surrounding Hpall/Mspi recognition sequences or, as a control, a region adjacent to the CCGG tetramers. In the case of DNA derived from adult brains, the portion investigated was inert to digestion by Hpall whereas DNA from newborn mice was cleaved with Hpall. Both DNAs were cleaved by MspI in the region of interest (Figure 2). This indicates that de novo methylation modifies the 5' region of mouse Hyal-2 gene. Therefore, mouse DBT cells which contain neither endogenous Hyal-2 transcripts nor hyaluronidase activity have been cultured in the presence of 5' -de-aza-cytidine which is known to inhibit CpG methylation II. mRNA was isolated and reversetranscribed. cDNA was amplified using a primer pair which binds within exon3 and 4 giving rise to a 337bp fragment. Genomic DNA containing the third intron results in a 797bp DNA product. As shown in Figure 3, clearly, the Hyal-2 gene is activated in cells treated with de-aza-cytidine. In addition, those cells exhibit hyaluronidase activity compareable to the enzymatic properties of human Hyal-2 (data not shown).
DISCUSSION In mice, the Hyal-2 mRNA is present in many different tissues. An interesting exception is the brain, which apparently does not contain this enzyme. Several ESTs encoding part of the Hyal-2 enzyme, however, have been isolated from infant human brain. The experimental data presented in this contribution suggest that transcription of Hyal-2 is generally regulated by de novo methylation of CpG island present in the promoter and 5' -untranslated region. Expression of Hyal-2 is thus detectable at embr~onic stages but is down-regulated after birth, and completely silenced in the adult brain. These results strongly suggest a role of Hyal-2 in developmental processes and possibly also in tumorigenesis and de-differentiation phenomena. Due to the wide distribution of Hyal-2 in animal tissues it seems likely that the product of this gene plays an important role in the catabolism of hyaluronan. Indeed, no Hyal-2 mutants have been described. A deficiency in Hyal-2 could prevent degradation of long chain HA as well as production of HA fragments that may have important physiological functions. As outlined above, we could provide the first biochemical evidence that in solution HA is not simply a random-coil but forms well-ordered structures depending on the length of the polysaccharide chain. Recently, we were also
192
The role or hyaluronan in tissues
able to show that HA-synthesizing enzymes Xhasl and 2 form products of different chain lengths 7. This clearly demonstrated that it is not irrelevant which "kind" ofI-IA is being synthesized or degraded in certain tissues, organs or body fluids. The steady-state of many physiological processes and/or the differentiation status of cells are certainly influenced by the highly complex extracellular network of proteins, proteoglycans and polysaccharides. It is evident that in many types of ECM, HA is not just a passive part of the network, but that it also exerts regulatory functions. It has clearly been shown that low-molecular HA is able to influence gene expression 12. In line with this, changes of the normal HA content of the ECM has often been observed during malignant aberration of cells or tissues. In particular, this phenomenon has been described in certain types of cancer 13. The factors controlling the invasive behavior of tumors are not well understood. Since there is evidence that HA plays an role in invasiveness of some tumors 14.15, we designed experiments aiming to test the role of Hyal-2 in process. We have transfected a cell line derived from an astrocytoma with Hyal-2. When inoculated in brain these cells formed tumors with enhanced growth rates, an effect not observed at subcutaneous sites 16. This actually may reflect different extracellular environments, with e.g. higher levels of HA being found in the brain than in the subcutaneous space. The transcription of Hyal-2 and consequently the production of intermediate-sized HA-chains are blocked in the adult brain. Due to their physico-chemical properties, these short chains may facilitate cell migration and invasiveness. We also found that the vascularization level is increased in these intracerebral tumors. Even though the developing embryo is rich in HA 17 very little has been found in the developing brain 18. This may be due to the fact that the level of expression of Hyal-2 is particularily high in the embryonic brain. In general, changes in either hyaluronan concentration and/or polymer length may regulate cellular responses during tissue and organ differentiation. Formation of hydrated pericellular matrices is known to facilitate cell-rounding during mitosis 19. Subsequently, hyaluronan concentration decreases and leads to a reduced volume of intercellular matrix and condensation of cells prior to differentiation. Thus the process may be divided into two stages: a primary I-lA-richphase in which undifferentiated stem cells proliferate and migrate, followed by removal of the HA and the onset of cellular differentiation and morphogenesis 20. These results are in agreement with a concept of exclusion of spherical macromolecules by a network of randomly associated rods 21. This hypothesis was specifically applied to polysaccharides 22 and later on tested experimentally for cells 23. According to this model of organogenesis 24, cells coated by a glycocalyx of a linear polysaccharide, should disperse and migrate down a viscosity gradient of matrix polymer. On the other hand, cells that internalize their surface coat should aggregate 25. For instance, during migration myoblasts retain a hyaluronan coat that prevents their fusion. Prior to the initiation of fusion this coat disappears 26. This indicates, that the exclusion of cells by a network of linear polymer is a function of its concentration and viscosity. Low molecular weight GAG such as chondroitin sulfate effectively promotes aggregation whereas highly viscous solutions of hyaluronan inhibit tins phenomenon. However, the bulk viscosity at the same concentration of hyaluronan decreases dramatically with the decrease of polymer lengths 27. Furthermore, no pericellular coats can be observed with low molecular weight polysaccharide. There is increasing evidence that hyaluronan indeed plays a crucial role in creating cell-free spaces 28, and in controlling cell proliferation 29, migration 30 and differentiation 31. In line with this, one can expect that the biosynthesis of HA-synthesizing and degrading enzymes are tightly regulated in space and time during embryogensis.
Transcriptional regulation ofllyal-2 hyaluronidase
193
ACKNOWLEDGEMENTS
The technical assistance of Anita Weber is gratefully acknowledged. The authors would like to express their gratitude to Christa Mollay for many fruitful discussions. This work was supported by grant 13001-Bio from the Austrian Science Foundation (FWF) . REFERENCES
1. Wei, M.H., F. Latif, S. Bader, V. Kashuba, J.Y. Chen, F.M. Duh, Y. Sekido, C.C. Lee, L. Geil, 1. Kuzmin, E. Zabarovsky, G. Klein, B. Zbar, ID. Minna&M.I. Lerman, Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG, Cancer Res., 1996 56. 1487-92. 2. Daly, M.C., R.H., Xiang, D. Buchhagen, C.H. Hensel, D.K. Garcia, A.M. Killary, J.D. Minna&S.L. Naylor, A homozygous deletion on chromosome 3 in a small cell lung cancer cell line correlates with a region of tumor suppressor activity. Oncogene, 19938,1721-1729. 3. Jones, M.H., P.M. Davey, H. Aplin&N.A. Affara, Expression analysis, genomic structure, and mapping to 7q31 of the human sperm adhesion molecule gene SPAMI. Genomics, 199529,796-800. 4. De Maeyer Guignard, J., A. Cachard Thomas&E. De Maeyer, Linkage analysis of the murine Hyal-I locus on chromosome 9, J Exp. Zool., 1991 258,246-8. 5. Fiszer Szafarz, B.&E. De Maeyer, Hyal-l, a locus determining serum hyaluronidase polymorphism, on chromosome 9 in mice, Somat. Cell. Mol. Genet., 1989 15, 79-83. 6. Dc Maeyer, E.&J. De Maeyer-Guignard, The growth rate of two transplantable murine tumors, 3LL lung carcinoma and B l6F 10 melanoma, is influenced by Hyal1, a locus determining hyaluronidase levels and polymorphism. Int. .J. Cancer, 1992 51,657-60. 7. Lepperdinger, G., B. Strobl&G. Kreil, HYAL2, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity. J BioI. Chem., 1998273,22466-70. 8. Lepperdinger, G., B. Strobl, A. Jilek, A. Weber, .I. Thalhamer, H. FlOckner&C. Mollay, The lipocalin Xlcpll expressed in the neural plate of Xenopus laevis is a secreted retinaldehyde binding protein. Prot. Sci., 19965,1250-1260. 9. Strobl, B., C. Wechselberger, D. Beier&G. Lepperdinger, Structural organization and chromosomal localization of Hyal2, a gene encoding a lysosomal hyaluronidase. Genomics, 1998 53, 214-219. 10.Cross, S.H., J.A. Charlton, X. Nan&A.P. Bird, Purification of CpG islands using a methylated DNA binding column. Nature Genetics, 19946,236-244. 11.Creusot, F., G. Acs&J.K. Christman, Inhibition of DNA rnethyltransferasc and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza2'-dcoxycytidinc, J BioI. Chem., 1982257,2041-2048. 12.Noble, P.W., C.M. McKee, M. Cowman&H.S. Shin, Hyaluronan fragments activate an NF-KB/I-KBa autoregulatory loop in murine macrophages, J Exp. Med., 1996 183,2373-2378.
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The role of hyaluronan in tissues
13.Knudson, W., Hyaluronan in malignancies, in The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives, T.C. Laurent, Editor. 1998, Portland Press Ltd: London. p. 161-169. 14.Culty, M., M. Shizari, E.W. Thompson&C.B. Underhill, Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: correlation with invasive potential..J Cell. Physiol., 1994 160,275-86. 15.zhang, H., G. Kelly, C. Zerillo, D.M. Jaworski&S. Hockfield, Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion In vivo .J. Neurosci., 199818,2370-6. 16.Novak, U., 8.S. StyIIi, A.H. Kaye&G. Lepperdinger, Hyaluronidase-Z overexpression accelerates intracerebral but not subcutaneous tumor formation of murine astrocytoma cells. Cancer Res., 199959,6246-50. 17.Fenderson, B.A, 1. Stamenkovic&A Aruffo, Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis. Differentiation, 1993 54,85-98. I8.Koprunner, M., J. Miillegger&G. Lepperdinger, Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhasl and 2 during early development of Xenopus laevis. Mech. Dev., 2000 90, 275-278. 19.Laurent, TC.&J.R. Fraser, Hyaluronan. Faseb L, 1992 6, 2397-404. 20.Toole, B.P., Proteoglycans and hyaluronan in morphogenesis and differentiation., in Cell Biology of Extracellular Matrix, E.D. Hay, Editor. 1991, Plenum Press: New York. 21.0gston, AG., The spaces in a uniform random suspencsion of fibers. Trans. Farady Soc., 1958 54,1754-1757. 22.Laurent, TC., The interaction between polysaccharides and other macromolecules. 9. The exclusion of molecules from hyaluronic acid gels and solutions. Biochem . .I, 196493,106-12. 23.Morris, J.E., Steric exclusion of cells. A mechanism of glycosaminoglycan-induced cell aggregation. Exp. Cell Res., 1979 120, 141-153. 24.Edwards, P.A W., Differential cell adhesion may result from nonspecific interactions between cell surface glycoproteins. Nature, 1978 271, 248-249. 25.Morris, J.E., Proteoglycans and the modulation of cell adhesion by steric exclusion. Dev Dyn, 1993 196, 246-51. 26.0rkin, R.W., W. Knudson&B.P. Toole, Loss of hyaluronate-dependent coat during myoblast fusion. Dev. Bioi., 1985107,527-30. 27.Bothner-Wik, H.&O. Wik, Rheology ofHyaluronan, in The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives, TC. Laurent, Editor. 1998, Portland Press: London. 28.Comper, W., D, &T.C. Faurent, Physiological function of connective tissue polysaccharides. Physiol. Rev., 197858,255-315. 29.Brecht, M., U. Mayer, E. Schlosser&P. Prehm, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem. .I, 1986239,445-50. 30.Poclmann, R.E., A.C. Gittenberger de Groot, M.M. Mentink, B. Delpech, N. Girard&B. Christ, The extracellular matrix during neural crest formation and migration in rat embryos. Anat. Embryol. Berl., 1990 182,29-39. 31.Toole, B.P.&J. Gross, The extracellular matrix of the regenerating newt limb: synthesis and removal of hyaluronate prior to differentiation. Dev. Bioi., 1971 25, 5777.
HYALURONAN SYNTHASE EXPRESSION IN HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLE Marianne Tellbach 1 *, Lois A. Salamonsen", Gary Brownlee", Tracey Brown! & Marie-Paule Van Damme' J
Department of Biochemistry and Molecular Biology, PO Box 13D, Monash University, Clayton, Victoria 3800, Australia.
2
Prince Henry's Institute ofMedical Research, PO Box 5152, Clayton, Victoria 3168, Australia.
ABSTRACT The human endometrium undergoes extensive remodelling during the menstrual cycle in preparation of a favorable environment for implantation. Biphasic fluctuations of hyaluronan (HA) in the cycling endometrium have been demonstrated with peaks occurring during the early proliferative (days 6-8) and mid secretory (days 19-23) stages of the cycle. The second, mid secretory, HA peak encompasses the time of embryo implantation suggesting a possible role for HA in this process. The aim of the present study was to investigate expression patterns of the 3-hyaluronan synthase (HAS) isoforms: HAS-I, HAS-2 and HAS-3. In situ hybridization was carried out on formalin fixed endometrial tissue across the cycle to determine whether these enzymes were expressed in human endometrium and if so, their cellular source. Riboprobes were constructed for each HAS: HAS-l (64bp), HAS-2 (60bp) and HAS-3 (550bp) and labeled with digoxygenin (DIG). The HAS protein was also localized immunohistochemically using a polyclonal antibody against extracellular HAS peptides. All three forms of HAS mRNA were expressed in the human endometrium. Expression was observed in both stromal and glandular epithelial cells with greatest intensity in epithelial cells and considerable cyclical changes. In all cases expression of mRNA was low during menstruation (days 1-5). Levels increased throughout the proliferative stage (days 6-14) of the cycle and for HAS-2 and HAS-3 remained elevated until the mid secretory (days 19-23) / implantation phase. HAS-l expression temporarily decreased during the late proliferative stage (days 12-14). Following implantation expression of all HAS types decreased dramatically. HAS protein localization was demonstrated on the plasma membrane of both glandular epithelial and stromal cells. The partial correlation between HA and HAS supports a role for these enzymes in endometrial HA regulation during the menstrual cycle and also suggests tight control of enzyme expression.
KEYWORDS Endometrium, hyaluronan, hyaluronan synthases
238
Biosynthesis and biological degradation ofhyaluronan
INTRODUCTION During the menstrual cycle, the architecture of the endometrium undergoes dynamic remodelling in preparation of a favorable environment for blastocyst implantation. Ovarian steroid hormones have been assumed as the principal orchestrators of this event and are responsible for proliferation and differentiation of epithelial and stromal cells in the creation of a receptive endometrium. The human endometrium is normally a hostile environment for embryo implantation but for a few days in each menstrual cycle it attains a unique state of receptivity when, if the appropriate remodelling has occurred, the embryo will implant and pregnancy will be established. In the absence of implantation, tissue regression occurs followed by the shedding of the functionalis layer at menstruation (day 1-5) and regeneration of the denuded tissue. The basalis layer is responsible for regeneration, which is maximal prior to ovulation (day 14), during the proliferative stage of the cycle (days 6-14). Re-epithelialization, re-vascularization, numerous cell divisions and the production of extracellular matrix components characterize the proliferative stage I. Following ovulation, the cells differentiate, there is an increase in the degree of hydration of the tissue and the glands increase secretions into the lumenal cavity. This latter part of the cycle is termed the secretory phase (days 15-28). Throughout the course of the cycle the endometrium is transformed from a thin dense tissue into a thick, highly permeable secretory tissue. HA, a major component of most extracellular matrices, has been identified in the human endometrial stroma during the menstrual cycle. Biphasic fluctuations of HA occur throughout the cycle with peaks during the early proliferative and mid secretory phases 2. High HA levels are a common component of tissues with rapid cell proliferation 3 and this may explain the elevated HA levels during the early proliferative stage of the menstrual crcle. Furthermore high HA levels have been positively correlated with mitosis 4, while inhibiting cell differentiation 6.7 and consequently create an environment promoting cell proliferation. The hydrated matrix also encourages the diffusion of growth factors such as TGF-~ and promotes cytokine protection from proteolytic enzymes 8. The second, mid secretory, HA peak encompasses the time of implantation. It has been suggested, that a HA rich matrix may facilitate invasion of the stroma during this process 9. This data supports a role for HA in the preparation of the endometrium for embryo implantation, a critical step in the establishment of pregnancy. However, nothing is known about how the peaks of HA in the endometrium are regulated. The aim of the present study was to investigate expression patterns of HAS, the enzymes responsible for HA synthesis. HAS are a multigene family with three known members HAS-I, -2 and -3. Each HAS has distinct enzymatic properties in terms of enzyme stability, the elongation rate and chain lengths of the HA synthesized 10. MATERIALS AND METHODS Tissue collection Endometrial tissue was obtained at curettage from 84 cycling women (3 complete cycles) with no apparent endocrinological problems and normal endometrial histology. None of the patients had received steroid treatment during the past 12 months. Informed consent was obtained from each patient and approval was given by the Human Ethics Committee at Monash Medical Centre, Melbourne, Australia. Endometrial dating was confirmed by independent histological examination.
Synthase expression in human endometrium
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RNA probes and in situ hybridization HAS-I (64bp) II, HAS-2 (60bp) 12 and HAS-3 (550bp) cDNA were cloned into the expression vector pGEM-T (Gary Brownlee, Monash University, Australia). HAS plasmids were cut with Not I or Nco I to generate sense and antisense probes which were then labeled by doxigenin (DIG) RNA-labeling 13. HAS mRNA was localized in serial sections (5 microns) of formalin fixed, paraffin embedded endometrial tissue by in situ hybridization as previously described 14. To aid in permeablization of the tissue, sections were incubated in proteinase K (12 l1g/ml) at 37°C for 30 min, and post fixed with 4% paraformaldehyde at 4 °C for 10 min. Sections were then treated with acetyl ate prior to prehybridization. Hybridization was performed overnight at 42°C for HAS-2, -3 and 48°C for HAS-l with probe concentrations of 20, 18, and 10 ng/111 respectively. Probes were diluted in hybridization buffer containing 10% dextran sulphate in addition to all components of the prehybridization solution. Sections underwent a number of stringency washes at the temperature of hybridization and, to reduce background, were incubated with RNase A (20 ug/ml) at 37°C for 30 min. Slides were blocked with 10% normal sheep serum, 10% fetal calf serum and 0.1 % triton X-loo for 30 min. Sections were incubated with Anti-DIG-AP (1 :750) in blocking solution at 4°C overnight. Nitroblue tetrazolium / 5bromo-4-chloro-3-indoyl phosphate was applied and sections were incubated in the dark for 1-5 hrs at room temperature. HAS mRNA was represented by the deposition of a blue / purple precipitate. Sections were not counterstained. Evaluation of in situ hybridization To determine the endometrial reactivity for HAS mRNA expression, the staining intensity of cells were evaluated. Two observers using a light microscope graded the relative intensity of the precipitant. The relative intensity was estimated semiquantitatively on a 5-level scale as follows: very weak, 1; weak, 2; moderate, 3; strong, 4 and very intense, 5. Immunohistochemistry Immunological detection of HAS in endometrial tissue was preformed using a polyclonal human antibody cross-reacting with external peptides of HAS-I, -2 and 3 (Tracey Brown, Monash University Australia). Hydrated 5 11m paraffin sections were pre-treated with 0.2% trypsin / PBS for lhr at 37°C. Heterophite proteins were blocked with 10% FCS / PBS followed by HAS detection with a HAS polyclonal antisera (1 :250) at 37°C for 1 hr. Endogenous peroxidase was eliminated by incubation with 0.6% hydrogen peroxide / methanol for 20 min. Final visualization was performed with an anti-sheep IgO / HRP (1:100) for I hr at 37°C, fol1owed by reaction with HAS protein localization was determined by the addition of the colourimetric substrate 3,3'diaminobenzidine tetrahydrochloride (DAB). Sections were counterstained with hematoxylin, dehydrated and mounted.
RESULTS AND DISCUSSION To identify which HAS isoforms the human endometrium expressed and their site, in situ hybridization of HAS-I, -2 and -3 was preformed (Fig 1). Formalyn sections were
240
Biosynthesis and biological degradation of hyaluronan
Figure 1. Cellular location of HAS mRNA in human endometrium during the mid proliferative phase (day 10) of the menstrual cycle. HAS-I, -2 and -3 mRNA (A, C and E respectively) were detected in the endometrium by in situ hybridization and localized predominantly in the glandular epithelial cells with less intense staining in the surrounding stroma. Serial control sections incubated with HAS-I, -2 and -3 sense riboprobes (B, D and F respectively) showed no specific staining. (Magnification X20)
Synthase expression in human endometrium
241
probed with DIG-labeled antisense RNA probes for each HAS and sense probes were used as controls for non-specific hybridization. All HAS types were expressed in the endometrium by both stromal and glandular epithelial cells however a greater expression was displayed in the glandular epithelium (Fig 1). This is somewhat surprising given the location of endometrial HA as predominantly stromal 2 and suggests that the HA produced by the glandular HAS may transverse the basement membrane. Immunohistochemistry was also conducted on the endometrial samples using a polyclonal antibody cross-reacting with HAS-I, -2 and -3 and revealed predominant HAS localization in the glandular epithelial cells, thus confirming the in situ localization studies (data not presented). Expression of HAS-I, -2 and -3 demonstrated considerable changes throughout the menstrual cycle (Fig 2). HAS-I levels in both the glandular epithelial and stromal cells were low during the menstrual phase (days 1-5) of the cycle. At this time tissue breakdown has occurred and the endometrial layer is shed. HAS-I levels increase during the early proliferative stage (day 6) in conjunction with an increase in HA probably due to the extensive proliferation required to rebuild the functional endometrial layer. During the mid proliferative stage (days 9-11) of the cycle HAS-1 expression remains elevated however HA levels have decreased. Around day 13 HAS-1 expression suddenly declines. The early secretory phase (days 15-18) of the cycle is marked by an increase in HAS-1 transcription, an event well correlated with the upcoming HA peak at implantation. HAS-1 levels decrease dramatically on day 19 and remain low throughout the remainder of the cycle. This seems appropriate given the events occurring in the endometrium at this stage. If the cycle is infertile, the endometrium regresses, probably due to loss of water from the tissue. HAS-2 and -3 expression during the menstrual cycle differ slightly to that of HAS-I. Low expression levels are apparent during menses (days 1-5) and the early proliferative phase (days 6-8). Expression does not increase until day 9, somewhat later than the increase in HAS-1 levels for this time of the cycle. Expression remains elevated until day 18 of the secretory phase (days 15-28), after which levels dramatically decrease. HAS expression is partially correlated with HA content during the menstrual cycle as demonstrated by the low HAS / HA levels during menses and the mid / late secretory phases. Between mensturation and mid secretion HAS levels remained elevated while HA levels revealed two peaks separated by a HA depression. This suggests that the endometrial HA is not solely regulated by HAS and suggest a possible role for the degradative hyaluronidases. A possible reason for elevated HAS levels during the HA depression could be related to the vascular remodeling and angiogenesis occurring within the endometrium. It has been found that partial degradation products of HA, between 4 and 25 dissacharides in length, induce angiogenic responses 15. Furthermore, angiogenesis in the endometrium is greatest during the mid to late proliferative phase and during the early secretory phase as determined by the levels of vascular endothelial growth factor, an angiogenic marker and its tyrosine kinase receptors, FIt-1 and KDRJFlk-1 16,17. CONCLUSION The results obtained support a role for all three HAS isoforms in endometrial HA production and a probable role for HAS in endometrial HA regulation although this does not appear to be the sole regulatory mechanism.
242
Biosynthesis and biological degradation ofhyaluronan
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Figure 2. HAS expression in the human endometrium during the menstrual cycle as determined by in situhybridization. Sections from eachday of the cycle(n=3) wereprobed with HAS-I (A), HAS-2 (B) and HAS-3 (C) DIG-labeled riboprobes. Expression was quantified visuallyin both the glandular epithelial ( ) and stromal (- - - -) cells. HA levels are also shown ( _ ) as determined by Salamonsen et al. 2 , to aid in correlation between HA and HAS.
Synthase expression in human endometrium
243
ACKNOWLEGMENTS The authors acknowledge financial support from Meditech Research Limited, Level 1, Sterling House, 8 Parliament Place, West Perth, 6005 Australia REFERENCES I. E. C. Wienke, F. Cavazos, D. G. Hall & F. V. Lucas, Ultrastructure of the human endometrial stroma cell during the menstrual cycle, Arn J Obstet GynecoL., 1968, 102(1),65-77. 2. L. A. Salamonsen, S Svetlana & R. Stem, Distribution of hyaluronan in human endometrium across the menstrual cycle, Cell Tissue Res., 2001, 306, 335-340. 3. B. P. Toole, Proteoglycans and hyaluronan in morphogenesis and differentiation, In: Cell biology of the extracellular matrix, E. D. Hay (eds.), Plenum Press, New York, 1991, pp 305-341. 4. M. Brecht, U. Mayer, E. Schlosser & P. Prehm, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis, Biochem., 1986, 239, 445-450. 5. N. Mian, Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma membrane fraction of cultured human skin fibroblasts, Biochem., 1986, 237:333-342. 6. M. J. Kujawa, D. G. Pechak, M. Y. Fiszman & A. I. Caplan, Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis, Develop. Biol., 1986,113, 10-16. 7. M. J. Kujawa & K. Tepperman, Culturing chick muscle cells on glycosaminoglycan substrates: attachment and differentiation, Dev. Biol. 1983,99,277-286. 8. T. Tanaka, T. Nakamura, H. Ikeya, T. Higuchi, A. Tanaka, A. Morikawa, Y.Saito, K. Takagaki & M. Endo, Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix, FEBS Lett., 1994, 347, 95-98. 9. D. D. Carson, A. Dutt & J-P. Tang, Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function, Dev. Biol., 1987, 120,228-235. 10. N. Itano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M. Hamaguchi, Y. Yoshida, Y. Ohnuki, S. Miyauchi, A. P. Spicer, J. A. McDonald & K. Kimata, Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties, J. Biol. Chem., 1999, 274, 25085-25092. 11. A. M. Shyjan, P. Heldin, E. C. Butcher, T. Yoshino & M. J. Briskin, Functional cloning of the cDNA for a human hyaluronan synthase, J Biol Chem., 1996, 271(38),23395-9. 12. K. Watanabe & Y. Yamaguchi, Molecular identification of a putative human hyaluronan synthase, J Biol Chem., 1996,271 (38),22945-8. 13. P. Komminoth, Digoxigenin as an alternative probe labeling for in situ hybridization, Diagn. Mol. Pathol., 1992, 1, 142-150. 14. G- Y. Nie, Y. Li, H. Minoura, 1. K. Findlay & L. Solamonsen, Complex regulation of calcium binding protein D9K (Calbindin-D9K) in the mouse uterus during early pregnancy and at the site of embryo implantation, Biol. Reprod., 2000, 62, 27-36. 15. D. C. West, I. N. Hampson, F. Arnold & S. Kumar, Angiogenesis induced by degradation products of hyaluronic acid, Science, 1985, 228, 1324-1326.
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Biosynthesis and biological degradation ofhyaluronan
16. M. Perrot-Applanat, Hormonal regulation of vascular cell function: angiogenesis, In: Comprehensive vascular biology and pathology - an encyclopedic reference, A. Bikfalvi (eds.), Springer-Verlag, Heidelberg, Germany, 1999, in press. 17. G. Meduri, P. Bausero & M. Perrot-Applanat, Expression of VEGF receptors in the human endometrium: modulation during the menstrual cycle, Biol. Reprod., 2000, 62,439-447.
IN VIVO INVESTIGATION OF HYALURONAN SYNTHASE FUNCTION DURING VERTEBRATE EMBRYOGENESIS Janet Y. Lee* and Andrew P. Spicer, Rowe Program in Genetics, Department ofBiological Chemistry, University ofCalifornia Davis, School ofMedicine, Tupper Hall, Davis, California 95616, USA.
ABSTRACT Hyaluronan (HA) is synthesized at the eukaryotic plasma membrane by anyone of three members of the HA synthase (HAS) family. Northern analyses also revealed unique expression patterns; Hasl and Has3 are primarily expressed at early and later stages of embryogenesis, respectively. In contrast, Has2 is expressed throughout embryogenesis from as early as E7.5. This predominance of Has2 is also observed in loss-of-function studies; Hasl and Has3 null homozygotes are healthy and viable, while Has2 null embryos die at EIO.5. Thus, Has2 is the major HA synthase involved in normal embryo development. We are continuing our in vivo investigations into HA synthase function by expression analyses and by conditional gene targeting. Whole mount in situ hybridization studies revealed expression of Has2 in neural crest cellderived cardiac and craniofacial structures, in addition to the developing limb. In particular, Has2 may playa pivotal role in limb and joint development, as its expression pattern closely mimics that of growth factors necessary for limb and joint development. Has3 expression was detected at the developing vibrissae and hair follicles, which may indicate a role for Has3-dependent HA synthesis in the formation of whiskers, hair follicles and/or their associated sensory neurons. Our conditional gene targeting of Has2 will follow an allelogenic approach in order to establish mouse lines from which multiple variant lines will be derived through crosses with recombinase expressing transgenic mice. Through this approach, we will generate mice with hypomorphic phenotypes, as well as those with tissue-specific deficiencies in HA biosynthesis. KEYWORDS Hyaluronan synthase, HAS, Has2, gene targeting, expression INTRODUCTION In vertebrates, HA is synthesized by anyone of three hyaluronan synthases (HAS), designated, HASI, HAS2 and HAS3, respectively 1.2. During embryogenesis, large amounts of HA are synthesized by many cell types particularly those that are proliferating and migrating. Through specific inactivation of the mouse Has2 gene 3, we have recently shown that HA is critical for embryogenesis and is required for the maintenance and possibly the creation of extracellular matrix-defined spaces throughout the embryo, in addition to the migration of endocardial cushion cells. We rely upon the mouse as our model system for investigation of HA biosynthesis and function during vertebrate embryogenesis. Our most recent strategy employs sitespecific recombinases to create conditional or tissue-specific deficiencies of Has2
246
Biosynthesis and biological degradation ofhyaluronan
function during embryogenesis 4. However, before embarking upon this approach, we reasoned that it would be extremely important to document the normal expression pattern of the mouse Has genes. Why is this an important consideration? Conditional gene targeting relies upon the use of cell-type or tissue-specific promoter/enhancer sequences to restrict expression ofa Cre recombinase transgene, the activity of which is required for inactivation of the gene of interest. Numerous transgenic mice have now been derived in which Cre recombinase is expressed in a cell-type or tissue specific manner. Additional promoter/enhancer sequences are continually being characterized in an effort to derive transgenic mouse lines expressing Cre in essentially any spatial or temporal pattern. In order to investigate Has2 function in the tissue of interest, the Cre transgene must be expressed in either the cell that expresses Has2 or its precursor. Thus, it is of paramount importance that we know which cells express Has2 under normal circumstances during embryogenesis. Furthermore, it is also important to know if any of these cells, or their neighbouring cells, also express Hasl and/or Has3. As HA is released into the extracellular matrix of synthesizing cells, it may clearly act in a noncell-autonomous fashion, rescuing any defects that may result from restricted inactivation ofHas2 in a single cell. Herein, we present our strategy to create a Has2 conditional knockout in the mouse, in addition to preliminary data on the expression patterns for the Has gene family during mouse embryogenesis. MATERIALS AND METHODS Creation of the mouse Has2 allelogenic gene targeting vector
Three contiguous mouse Has2 genomic DNA fragments of 4.7kb, 1.6kb and 3.3.kb were cloned into a vector designed for allelogenic gene targeting. The 4.7 kb fragment represents the 5' arm of homology, the 1.6 kb fragment was cloned between two 10xP sites and the 3.3 kb fragment, representing the 3' arm of homology was cloned downstream of the selectable PGKneo cassette, which is flanked by two fit sites. The structure of a targeted allele (Has2 hy'j is illustrated in figure 1. Whole-mount in situ hybridisation
Staged embryos [E7.5 - El5.5] were isolated from timed pregnant mice, fixed in 4% paraformaldehyde at 4°C for 2-12h, and subsequently dehydrated in methanol. Digoxigenin riboprobes were generated from plasmids containing the mouse Has2 or Has3 open-reading frames according to the manufacturer's instructions (http://biochem.roche.com). The procedure for whole-mount in situ hybridisation was performed essentially as previously described 5, with modifications. RESULTS AND DISCUSSION Generation of the mouse Has2 allelic series
A hypomorphic Has2 allele (HasiIY'j is predicted to result from insertion of the PGKneo cassette into intron 3. Through crossing with transgenic mice expressing Flp recombinase, the neo cassette will be deleted, resulting in a functionally wild-type Has2 allele (Has2Llneo ) 6. By a similar strategy, crossing with transgenic mice expressing Cre
Synthase function during vertebrate embryogenesis
247
recombinase will delete exon 3, converting the targeted allele or its Flp-recornbined descendants into a functionally null allele (Has2 L!ex3). Has2 Allelic Series HHH
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Design of the allelogenic targeting vector for mouse Has2. The basic structure of the wild-type Has2locus is illustrated at the top. Exon 1 (not shown) is located 12.5 kb upstream of exon 2 and contains the entire 5' UTR. The black filled boxes represent the open-reading frame. Arrowheads and ovals represent 34bp loxP and frt sites, respectively.
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Expression of Has2 and Has3 during aspects of craniofacial morphogenesis. (A) Has2 is expressed in regions of precartilage formation, as well as around the developing vibrissae. (B) A high level ofHas3 expression is also apparent in the developing vibrissae.
248
Biosynthesis and biological degradation ofhyaluronan
Whole-mount in situ hybridisation analyses of Has2 and Has3 During vertebrate development, Has2 is expressed in tissues where hyaluronan is abundant, including the developing limb and endocardial cushions ofthe heart (data not shown). In contrast, Has3 expression is restricted to specialized tissues such as tooth and hair.
CONCLUSIONS Has2 is the major HA synthase involved in HA biosynthesis during vertebrate embryogenesis. Has2 is expressed in multiple tissues in a distinct temporal and spatial pattern, which correlates closely with tissues that are undergoing rapid expansion, cell migration and/or proliferation. Conversely, Has3 is expressed in a restricted number of tissues, although its expression overlaps, in part, with that ofHas2. The allelogenic targeting approach would provide a particularly powerful tool in dissecting the role of Has2-dependent HA function during embryonic development and potentially through adult maturation.
ACKNOWLEDGEMENTS This work was supported by research grants from the American Heart Association National Office AHA 0030184N and the March of Dimes Birth Defects Foundation #1FYOO-361 to APS.
REFERENCES I. 2.
3.
4. 5.
6.
A. P. Spicer & 1. A. McDonald, 'Characterization and molecular evolution of a vertebrate hyaluronan synthase (HAS) gene family', J. Bioi. Chem., 1998, 273, 1923-1932. N. Itano, T. Sawai, M. Yoshida., P. Lenas, Y. Yamada., M. Imagawa., T. Shinomura, M. Hamaguchi, Y. Yoshida., Y. Ohnuki, S. Miyauchi, A. P. Spicer, 1. A. McDonald & K. Kimata., 'Three isoforrns of mammalian hyaluronan synthases have distinct eniymatic properties', J. Bioi. Chem., 1999,274,25085-25092. T. D. Camenisch, A. P. Spicer, T. Brehm-Gibson, J. Biesterfeldt, M. L. Augustine, A. Calabro, Jr., S. Kubalak, S. E. Klewer & J. A. McDonald, 'Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J. Clin. Invest., 2000, 106,349-360. E. N. Meyers, M. Lewandoski & G. R. Martin, 'An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination', Nature Genetics, 1998, 18, 136-141. R. A. Conlon & 1. Rossant, 'Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo', Development, 1992, 116,357368. S. M. Dymecki, 'Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice', Proc. Natl. Acad: Sci. USA, 1996,93, 6191-6196.
INFLUENCE OF SUBSTRATE AND ENZYME CONCENTRATIONS ON HYALURONAN HYDROLYSIS KINETICS CATALYSED BY HYALURONIDASE T. Asterion, B. Deschrevel, F. Gonley,
J.e. Vincent*
Laboratoire "Polymeres. Biopolymeres, Membranes" UMR 6522 Universite de Rouen - CNRS 76821 Mont-Saint-Aignan cedex, France
ABSTRACT
It has been shown that both hyaluronan (HA) and hyaluronidase (HAase) are present at high levels in the extracellular matrix (ECM) of cancer tumours. As high molecular weight HA (h-HA) is anti-angiogenic and low molecular weight HA (l-HA) is angiogenic, HAase may play an important role in regulating the h-HA/I-HA balance. A detailed study of the HA hydrolysis kinetics catalysed by HAase may thus be useful to understand its implication in cancer development and should be examined in vitro through a model system. Kinetics was monitored by the Reissig method (improved in our laboratory) which estimates the number of reducing ends formed by hydrolysis of 13(1-4) glycosidic bonds. We carried out the effects of both substrate and enzyme concentrations and of ionic strength on the kinetics. The most original results concern the non-linear shape of the time course and the atypical behaviour of the initial rate versus both substrate and enzyme concentrations. The substrate dependence curve seems to be of the Michaelian type only for low concentrations. A significant decrease in the initial rate is observed for higher concentrations, which suggests inhibition phenomena. In order to explain our experimental results, we have to consider that enzymatic degradation of a polysaccharide is a particular case of enzymatic reactions because of the polymeric nature of the substrate. Elaboration of a kinetic modelling, in agreement with the experimental results, allows us to suggest a few assumptions about the reaction mechanism. KEYWORDS
Hyaluronan, hyaluronidase, enzymatic hydrolysis, kinetics, reducing ends assay, substrate-dependence, enzyme-dependence. INTRODUCTION
Hyaluronan (HA) is a linear negatively-charged polysaccharide composed of Dglucuronic acid-13(l,3)-N-acetyl-D-glucosamine disaccharide units linked 13(1,4). HA is present in both microorganisms and vertebrates and is one of the main component of the extracellular matrix (ECM) of higher animals. Its size ranges from a few hundred thousand to ten million Daltons according to its origin. HA is the main substrate of hydrolytic enzymes, called hyaluronidases (HAases). Both lysosomal and testicular HAases (E.C. 3.2.1.35) hydrolyze 13(1,4) glycosidic bonds producing HA fragments with a N-acetyl-D-glucosamine at the reducing extremity [1].
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Biosynthesis and biological degradation ofhyaluronan
Several techniques [2-3] have been described to assay HAase activity: turbidimetry, viscosimetry, ELISA-like assay, HPLC-SEC chromatography and colorimetric assays [4-7]. The colorimetric method of Reissig et al. [7] has mostly been used to investigate HAase activity and most of the authors estimate the activity by measuring the total reducing ends produced by the reaction after a given incubation time ranging from 10 minutes to 2 hours [8-9]. However, due to the non-linear shape of the kinetics, the enzyme activity should be estimated by the initial rate of the reducing ends production. Here, we are interested in the kinetics of the HA-HAase reaction and the method of Reissig et al. is particularly suitable since it gives the rate of glycosidic bond cleavage directly. The determination of the initial reaction rate allows us to study the influence of enzyme and substrate concentrations. MATERIALS & METHODS
Bovine testicular HAase with a specific activity of 990 units per mg was obtained from Sigma (H 3884). Sodium hyaluronate (HA) from bovine trachea was purchased from Fluka (Nr. 366047/1). HA and HAase were used without any further purification. The Reissig method needs two solutions: i) a borate solution prepared by dissolving 4.94 g boric acid and 1.98 g potassium hydroxide in 100 mL Milli-Q water (Waters), and ii) a 0.1 giL DMAB solution prepared by dissolving 5 g DMAB (Sigma D 8904) in 6.25 mL hydrochloric acid 12 N completed with glacial acetic acid to a final volume of 50 mL. A 1110 dilution of this solution with glacial acetic acid was performed just before use. Absorbance was measured with an Uvikon 860 Kontron spectrophotometer equipped with a temperature-controlled chamber and connected to a Pc. Measurement of HAase activity is based on the Reissig method [7] which determines the concentration of liberated reducing ~-N-acetyl-D-glucosamine from HA. P-Nacetyl-D-glucosamine (Sigma A 8625) was used as a standard. The HA solution, containing 5 mM ammonium acetate pH 5, is placed in a reactor, adjusted to the chosen pH with either acetic acid or ammoniac, stirred and temperature controlled at 37°C. The reaction was started by adding concentrated HAase. At each time point, a 200 J.lL aliquot of the reacting mixture was removed from the reactor and added to 50 J.lL of borate solution in a glass tube. The tube is immediately vortexed, heated in a boiling water bath for exactly 3 minutes, then placed in a cold water bath (approximately 10°C) until 10 aliquots have been treated. Then, 1.5 mL of the diluted DMAB solution was added to each tube. The content is rapidly vortexed and placed at 37°C for exactly IS minutes. This was transferred to a plastic cuvette of I cm pathlength, immediately scanned between 400 and 700 nm and the spectrum saved on the computer. RESUL TS & DISCUSSION
By following the entire time course of the reducing ends concentration under various HA and HAase conditions, non-linear shapes were observed which have been fitted by a bi-exponential curve [10]. The HAase activity was then determined by measuring the reaction rate at time zero. Influence of the substrate concentration
The HAase activity was measured for different HA concentrations ranging from 0.1 to 3 mg/mL. An atypical substrate-dependence curve (fig. 1) was observed. At low HA
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1
HAase activity plotted against HA concentration (left curve) and Lineweaver-Burk linearisation (right curve). Reactions were performed at 37°C and pH 5. The HAase concentration was 2 mg/mL.
concentrations, the HAase activity increases classically whereas at high HA concentration it falls down and stabilises at a low level. The kinetics does not obey the Michaelis-Menten mechanism. However, apparent constants Vm and Km could be determined for the low HA concentrations but only KIn (0.25 mg/mL) may have a certain signification. Concerning high HA concentrations, three hypotheses can be proposed: i) solution viscosity hinders enzyme diffusion, ii) changes in HA conformation penalise HA-HAase interactions, and iii) inhibition phenomena exist. Influence of the enzyme concentration The HAase activity was then measured for different HAase concentrations ranging from 0.25 to 5 mg/mL. As for the substrate-dependence curve, an atypical enzymedependence was obtained. A sigmcidal shape (fig. 2) was observed with a very low activity below lmg/ml HAase. This behaviour may be interpreted as a transition between a low level and a high level regimes which can be used for regulatory mechanisms by the ECM. These observations are in agreement with the hypotheses proposed above. CONCLUSION Whereas most of the authors estimate the enzyme activity by measuring the total reducing ends produced by the reaction after a given incubation time, we followed the entire time course of the reducing end production and deduced the HAase activity from the rate at time zero. The most original results concern the atypical behaviour of the initial rate versus both substrate and enzyme concentrations. The substrate-dependence seems to be of the Michaelian type only for low concentrations. A significant decrease in the initial rate is observed for higher concentrations. The sigmoidal enzymedependence may be interpreted as a transition between low level and high level regimes. The enzymatic hydrolysis of HA is a particular case of enzymatic reactions because of the polymeric nature of the substrate: i) the structure and conformation of the substrate may greatly influence the geometry of the active site of the enzyme, and ii) non-specific
252
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HAase activity plotted against HAase concentration. Reactions were performed at 37°C and pH 5. The HA concentration was 1 mg/mL.
interactions can also occur between the enzyme (protein) and the substrate (polysaccharide). This atypical behaviour could induce regulatory phenomena in the ECM, in particular in the cancer tumour case.
ACKNOWLEDGEMENTS We acknowledge Dr B. Delpech and P. Bertrand for helpful discussions. We are grateful to the 'Conseil Regional de Haute Normandie' for fellowship.
REFERENCES I. K. Meyer, Hyaluronidases, in The Enzymes, Boyer Ed., 3'd Ed, 1971,5, pp 307-320. 2. J.K. Herd, J. Tschida & L. Motycka, The detection of hyaluronidase on electrophoresis membranes. Anal. Biochem. 1974,61(1),133-43. 3. W.L. Hynes & J.J. Ferretti, Assays for hyaluronidase activity. Methods. Enzymol. 1994, 235,606-16. 4. K.P. Vercruysse, A.R. Lauwers & J.M. Demeester, Kinetic investigation of the action of hyaluronidase on hyaluronan using the Morgan-Elson and neocuproine assays. Biochem. J, 1995,310(1),55-9. 5. L.C. Benchetrit, S.L. Pahuja, E.D. Gray & R.D. Edstrom, A sensitive method for the assay of hyaluronidase activity. Anal. Biochem. ,1977, 79, 431-7. 6. K.A. Homer, L. Denbow & D. 8eighton, Spectrophotometric method for the assay of glycosaminoglycans and glycosaminoglycan-depolymerizing enzymes. Anal. Biochem., 1993,214,435-41. 7. J.L. Reissig, J.L. Strominger & L.F. Leloir, A modified colorimetric method for the estimation ofN-Acetylamino sugars, J Bioi. Chem., 217, 1955, 959-966 8. P. Gacesa, MJ. Savitsky, K.S. Dodgson & A.H. Olavesen, A recommended procedure for the estimation of bovine testicular hyaluronidase in the presence of human serum. Anal. Biochem. , 1981, 118, 76-84. 9. C. Maingonnat, R. Victor, P. Bertrand, M.N. Courel, R. Maunoury & 8. Delpech, Activation and inhibition of human cancer cell hyaluronidase by proteins. Anal. Biochem., 1999,268,30-4. 10. T. Asteriou et al. (submitted).
HUMAN BYALURONIDASE POLYMORPIDSM AND EVIDENCE FOR CONSERVED HYALURONIDASE POTENTIAL NGLYCOSYLATION SITES IN MAMMALIAN AND NONMAMMALIAN SPECIES Berta FISZER-SZAFARZI, Anna LITYNSKA2, & Liming ZOU 3 1 Institut Curie-Biologie, Batiment l Itl, Centre Universitaire, 91405 Orsay Cedex, France. 2 Institute ofZoology. Jagiellonian University, Ingardena 6. 30060 Krakow. Poland. 3 Shenyang Medical College, Department ofBiochemistry, 110 031 Shenyang, China.
ABSTRACT
A number of properties of human hyaluronidases in somatic tissues and in body fluids were studied. When analyzed on a PAGE-hyaluronan gel, liver and placenta exhibited 7 hyaluronidase forms while plasma and synovial fluid presented 3 and 2 forms, respectively. Ovary, breast, myometrium, endometrium, skin, leukocytes and platelets displayed distinct patterns of enzymatic micropolydispersity. The most acidic forms are in synovial fluid and plasma. Sialidase treatment decreases the number of forms to 3 in liver, 2 in placenta and to a slow basic form in serum. Plasma and placental hyaluronidases remain fully active after thermal treatment but desialylated hyaluronidase is inactivated slowly in plasma, and fast in placenta suggesting a higher overall glycosylation of the plasma enzyme. Potential N-glycosylation sites were searched in the amino acid sequences in hyaluronidases in human and other mammalian and nonmammalian species. A potential N-glycosylation site was observed at the same position in human plasma, human lysosomes, human Hyal-4 and Hyal-pl. The same site was also present in mouse plasma, mouse lysosomes, rat lysosomes, frog liver, wasp, hornet and honeybee venom, and C. elegans. However this site was absent in human Hyal-3, in human meningioma and in all sperm hyaluronidases examined. A second potential Nglycosylation site is also present in all hyaluronidases with an identical pattern (AsnVal-Thr) except for those of lysosomal origin (Asn-Val-Ser), Site 2 is absent in honeybee venom and in C. elegans. Such conserved sites strongly suggest that they may represent N-glycosylation sites. KEYWORDS
Hyaluronidase, polymorphism, human, somatic tissues, body fluids, sialic acids, potential N-glycosylation-site, species. INTRODUCTION
Duran-Reynals and Stewart demonstrated that rabbit testicular extracts contain a factor that enhances the spreading of viral agents and that this factor was also present in epithelial organs such as liver, kidney and skinl. It was further named hyaluronidase. Hyaluronidases playa role in many biological processes, including changes observed in lysosomal and extracellular matrix during normal and tumoral cell division 1,2-4.
254
Biosynthesis and biological degradation ofhyaluronan
Hyaluronidases, constitute a large family of enzymes and are widely expressed in many species in tissues and body fluids 5-7. In a previous study, we have demostrated a hyaluronidase polymorphism in human, mouse, rat, hamster, dog and Triton cristatus sera 8,9. But no hyaluronidase activity was detected in the sera of horse, swine, cattle, goat, sheep, rabbit, chicken, Triton alpestris, Triton palmatus, and in a number of other species 8. Many acid hydrolases occur in multiple forms differing in their electrophoretic mobility depending on their sialic acid content 10 . This prompted the present study on the multiple forms of hyaluronidases in human somatic tissues and body fluids. This study also includes a search of the potential N-glycosylated sites in hyaluronidases in human and other mammalian and non-mammalian species.
MATERIALS & METHODS Human biological samples Normal blood and urine were provided by healthy volunteer donors. Surgical tissue specimens were from the Curie Hospital, Paris, France. Knee synovial fluids from polyarthritic patients were from the Unite 291 Inserm, Lyon, France. Chemicals Human umbilical cord hyaluronic acid H 1751, sialidase EC 3.2.1.18 type III from Vibrio cholerae N 7885, gelling agent and the MW-ND-SOO kit for the molecular weight markers for non-denaturing polyacrylamide gel electrophoresis were from Sigma Chemical (St Louis, MO, USA) Stains-all®, W 2718, was from Eastman-Kodak, (Rochester, NY, USA). All other chemicals were from Merck ( Darmstadt, Germany). Tissue extraction and biological Ouids Tissues from different organs, leukocytes and platelets were homogenized in 4 volumes of 0.1 % Triton X-I 00. The homogenate was left for 1 h at room temperature and centrifuged 3000 g for 15 min at 4°C. Supernatants were concentrated over Aquacid and the final concentration was reached with Minicon B15. These extracts were stored at -20°C. Knee synovial fluids were collected by knee arthrocentesis from patients with rheumatoid arthritis, centrifuged at 300 g for 10 min at 4°C, and stored at -80°C 11. Electrophoretic studies The multiple forms of hyaluronidase were studied by a zymogram technique, using 5 % polyacrylamide-20 pg/ml hyaluronan gel (PAGE-HA). In some cases we used other concentrations of polyacrylamide, but the HA concentration was always the same. The principle of the method is that the high molecular weight hyaluronan included in the gel cannot move in the electrical field. Electrophoresis was performed at pH 8.3, under which conditions, hyaluronidases are inactive. Gels were then incubated at pH 3.5 and at 37°C to allow hyaluronan degradation. Next, the gels were stained with "Stains-all" in the dark and washed with water. Hyaluronidase activity was revealed by transparent pink spots on the blue background of undegraded hyaluronan. A fast moving white band is observed in the serum electrophoretic pattern, corresponding to a sialic-acid-rich glycoprotein unrelated to hyaluronidase activity 5,11.
Human hyaluronidase polymorphism
255
Sialidase treatment of human serum and tissue hyaluronidase The desialylation method was performed as previously describedl l, The PAGEHA zymogram technique was then used. Hyaluronidases N-glycosylation potential sites The hyaluronidase amino acid sequences were aligned with the multiple alignment program CLUSTAL W (1.74). Potential N-glycosylation sites were searched with the PROSITE database. The human hyaluronidase sequences compared were Hyal-l (plasma), Hyal-2 (lysosomes), Hyal-3, Hyal-4, Hyal-pl, PH-20 (sperm) (GenBank accession Nos; U96078, AJOOO99, AF36035, AF09010, AF51769 and S67798 respectively) and meningioma (SwissProt accession No AF36144). Mouse hyaluronidases compared were Hyal-l (plasma) and Hyal-2 (lysosomes) (GenBank accession Nos. AFl1567 and AJOOO59) and PH-20 (sperm) (SwissProt accession No P48794), rat lysosomal hyaluronidase Hyal-2 (SwissProt accession No AF34218), frog liver hyaluronidase Hyal-2 (SwissProt accession No AF13498l). Cynomolgus monkey, guinea pig, rabbit, and fox PH-20 hyaluronidase sequences were SwissProt accession Nos. respectively P38568, P23613, P38566, U41412.The wasp, white-face hornet and honeybee venom hyaluronidases sequences compared were SwissProt accession Nos. respectively P49370, P49371 and Q08169) and Caenorhabditis elegans (SwissProt accession No. Z4907l). RESULTS AND DISCUSSION The nomenclature used here Hyal-t applies to hyaluronidases in somatic tissues. The different bands of hyaluronidase activity were numbered according to the decreasing anodic mobility of the liver enzyme bands with the highest anodic mobility being 1.
I
I I" a
a
b
Fig. 1
Fig. 2
b
c
d
Fig. 3
e
f
256
Biosynthesis and biological degradation of hyaluronan
Figure 1 shows samples analyzed on 5% PAGE-20 }lg/ml HA. Lanes: (a) liver exhibited 7 active forms, the most active being Hyal-tZ-4' (b) placenta exhibited a pattern similar to that of liver, but the most active forms were associated with the slower moving bands, Hyal-t 5-7 Ovary, breast, skin, leukocytes and platelets, exhibited a characteristic pattern for each tissue. Serum and synovial fluid exhibited a single band. With respect to urine, no clear conclusion could be drawn, as differences between individuals depending on physiological and pathological conditions might affect the results. Figure 2 indicates that serum displays three forms on 6% PAGE-20 }llml HA. The hyaluronidase polymorphism observed may be due to different degrees of sialylation. To investigate this point desialylation with Vibrio cholerae sialidase with it broad specificity on the terminal sialic acid of glycoproteins followed by zymogram analysis was performed. Figure 3 shows electrophoretic patterns on 7% PAGE-20 }lg/ml of human hyaluronidases from serum (a.b), liver (c.d) and placenta (e,f) after 5 and 180 min incubation with sialidase. The desialylated hyaluronidase forms from serum migrate more slowly than the corresponding desialylated forms from liver and placenta. Liver and placenta desialylated hyaluronidases exhibite three and two electrophoretic forms, respectively. The molecular mass of native human hyaluronidase in serum and in breast tissue is 67 kDa. Here we have shown that the molecular mass of desialylated serum and placental hyaluronidase was 110 kDa. The higher than expected molecular mass of the desialylated hyaluronidases could be explained by dimer formation, possibly due to aggregation in the absence of the repelling negative charges of sialic acids. The thermal stability profiles of native hyaluronidases from serum and placenta are identical. The activity of these enzymes was stable up to 46°C. However, after treatment with sialidase and heating at 45°C, serum hyaluronidase retains 80 % of its initial activity, while placental hyaluronidase only retains 10%. Since glycosylation protects glycoproteins from thermal denaturation, serum hyaluronidase might not only be more sialylated but may also have a higher overall sugar content.
Hyaluronidase conserved potential N-glycosylation sites Potential N-glycosylation sites were sought in the amino acid sequences in hyaluronidases from human and other species. The potential N-glycosylation sites in glycoproteins are specific to the consensus sequence Asn-Xaa-Ser/Thr, However the presence of this tripeptide is not a condition to conclude that an asparagine residue is glycosylated, since the folding of the protein plays an important role in the regulation of N-glycosylation. When comparing human hyaluronidases amino acid alignments of plasma (Hyal-I), lysosomal (Hyal-2), Hyal-4 and Hyal-pl the presence of a potential N-glycosylation site with similar tripeptide patterns in the same location (site 1) was observed. Table 1 the same site was also present in mouse serum and Iysosomes, in rat lysosomes, in frog liver, in wasp, hornet and honeybee venom, and in C. elegans. This site was absent in human Hyal-3 and human meningioma, and in all sperm (PH-20) studied. A second potential N-glycosylation site was observed at a location further in the polypeptide
Human hyaluronidase polymorphism
257
chain, the pattern is Asn-Val-Thr for all mammalian non-lysosomal hyaluronidases examined as well as for frog liver. In contrast hyaluronidases of lysosomal origin display a tripeptide pattern Asn-Val-Ser. This site is present in wasp and hornet venom and is absent in honeybee venom and in C. elegans. Table 1. Hyaluronidase conserved potential N-glycosylation sites
Hyaluronidases
Site 1
Site 2
Plasma (Hyal-l) Lysosomal (Hyal-2) Hyal-3 Hyal-4 Hyal-pl Meningioma Sperm (PH-20)
99Asn-Ala-Ser 103Asn-Val-Ser
350Asn-Val-Thr 356Asn-Val-Ser 348Asn-Val-Thr 348Asn-Val-Thr 356Asn-Val-Thr 358Asn-Val-Ser 368Asn-Val-Thr
Mouse
Plasma (Hyal-l) Lysosomal (Hyal-2) Sperm (PH-20)
127Asn-Ala-Ser I03Asn-Gly-Ser
378Asn-Val-Thr 356Asn-Val-Ser 368Asn-Val-Thr
Rat
Lysosomal (Hyal-2)
103Asn-Gly-Ser
356Asn-Val-Ser
Frog
Liver (Hyal-2)
104Asn-Glu-Thr
372Asn-Val-Thr
C. monkey
Sperm (PH-20)
372Asn-Val-Thr
Guinea pig
Sperm (PH-20)
368Asn-Val-Thr
Rabbit
Sperm (PH-20)
368Asn-Val-Thr
Fox
Sperm (PH-20)
368Asn-Val-Thr
Species Human
115Asn-Ile- Ser 103Asn-Val-Ser
Wasp
Venom
99Asn-Glu-Ser
325Asn-Val-Thr
Hornet
Venom
79Asn-Ile-Thr
325Asn-Val-Thr
Honneybee
Venom
115Asn-Leu-Thr
C. elegans
119Asn-Glu-Thr
CONCLUSIONS The differences between the multiple forms of human hyaluronidases are not only related to their amino acid sequences but also to their glycosylation structure.
258
Biosynthesis and biological degradation ofhyaluronan
Hyaluronidases from different species and organ origin possess on their amino acid sequences similar/identical conserved potential N-glycosylation sites. REFERENCES 1.
F. Duran-Reynals, & F.W. Stewart, 'The action of tumor extracts on the spread of experimental vaccinia of the rabbit', Amer. J. Cancer, 1931,15, 2790-2797.
2.
B. Fiszer-Szafarz, 'Acid hydrolases and tumor invasion: relationship between intracellular distribution of hyaluronidase, cathepsin D and acid phosphatase, and cell proliferation in rat liver and diethylnitrosamine-induced hepatoma', BioI. Cell, 1981, 42, 97-102.
3.
P. Bertrand, N. Girard, C. Duval, J. D'Anjou, C. Chauzy, J. F. Menard, & B. Delpech, 'Increased hyaluronidase levels in breast tumor metastasis', Int. 1. Cancer, 1997, 73,327-331.
4.
T. B. Csoka, G.!. Frost, & R. Stem, 'Hyaluronidases in tissue invasion', Invas. Metast., 1997,17,297-311.
5.
B. Fiszer-Szafarz, 'Hyaluronidase polymorphism detected by polyacrylamide gel electrophoresis. Application to hyaluronidases from bacteria, slime molds, bee and snake venoms, bovine testes, rat liver lysosomes and human serum', Anal. Biochem., 1984,143,76-81.
6.
G.!. Frost, T.B. Csoka, T.Wong, & R. Stem, 'Purification, cloning and expression of human plasma hyaluronidase', Biochem. Biophys. Res. Commun., 1997, 236,10-15.
7.
G. Lepperdinger, B. Strobl, & G. Kreil, 'HY AU, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity', J. BioI. Chem., 1998, 273,22466-22470.
8.
B. Fiszer-Szafarz , D. Szafarz, & P. Vannier, 'Polymorphism of hyaluronidase in serum from man, various mouse strains an other vertrebrate species revealed by electrophoresis', BioI. Cell, 1990,68,95-100.
9.
B. Fiszer-Szafarz, & E. De Maeyer, 'Hyal-L, a locus determining serum hyaluronidase polymorphism, on chromosome 9 in mice', Som. Cell Mol. Genet., 1989, 15, 79-83.
10.
A. Goldstone, P. Konecny, & H. Koenig, 'Lysosomal hydrolases: conversion of acidic to basic forms by neuraminidase', FEBS Letters, 1971, 13,68-72.
11.
B.Fiszer-Szafarz, A. Litynska & L. Zou, 'Human hyaluronidases: electrophoretic multiple forms in somatic tissues and body fluids. Evidence for conserved hyaluronidase potential N-glycosylation sites in different mammalian species', J. Biochem. Biophys. Methods, 2000,45,103-116.
INTRATRACHEAL INSTILLATION OF LIPOPOLYSACCHARIDE IN MICE RESULTS IN INCREASED HYALURONAN DEPOSITION IN THE LUNG Juanita H.J. Vernooyl"', Mieke A. Dentener', Wim A. Buurman", Emiel F.M. Wouters' Departments afl Pulmonology and/Surgery, Maastricht University, PO Box 6/6, 6200 MD, Maastricht, The Netherlands.
ABSTRACT
An increase in hyaluronan (HA) in bronchoalveolar lavage fluid from human lungs has been demonstrated in many pathologic conditions characterised by a pulmonary inflammation, including asthma and chronic obstructive pulmonary disease (COPD). The Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is suggested to be an important factor in the pathogenesis of these lung diseases. The aim of the study was to investigate the effect oflocal LPS exposure on HA deposition in the lung. To this end, male Swiss mice received 5 ug LPS (E.coli 055:B5) intratracheally (IT) and were sacrificed at different time points after LPS exposure. Control mice received either saline or no treatment. Neutrophil influx in the lungs was determined as a characteristic for acute inflammation. HA content was assessed by histolocalization using a biotinylated HA binding protein (b-HABP). Our data demonstrate that IT LPS instillation resulted in a strong pulmonary inflammation characterised by rapid accumulation ofneutrophils peaking at 24 hours postexposure. Histolocalization of HA revealed constitutive HA staining in the interstitium of airways and larger vessels of control mice. The lungs of LPS-treated mice demonstrated HA deposition around small vessels of the microvascular bed and in the subpleural area at 24 and 72 hours after LPS instillation, especially at sites of leukocyte infiltration. Since HA upregulation on endothelium is suggested to playa role in leukocyte extravasation at sites ofinflammation, HA deposition observed in the microvascularbed may contribute to LPS induced pulmonary inflammation. KEYWORDS
Hyaluronan, inflammation, lipopolysaccharide, lung disease INTRODUCTION
The extracellular matrix glycosaminoglycan hyaluronan (HA) is a normal constituent of loose connective tissue in the lung. An increase in HA in bronchoalveolar lavage fluid from human lungs has been demonstrated in many pathologic conditions characterised by a pulmonary inflammation, including asthma', adult respiratory distress syndrome (ARDS)2, farmer's lung", and chronic obstructive pulmonary disease (COPD)4. Lipopolysaccharide (LPS), a major glycolipid component ofthe Gram-negative bacterial cell wall, is suggested to be an important factor in the pathogenesis ofthese lung diseases. The respiratory system, which is continuously exposed to airborne particles containing bacteria and LPS, has
218
The role or hyaluronan in tissues
efficient defense mechanisms against these agents under normal exposure conditions>. However, long-term exposure to dust containing LPS, as occurs in grain handlers or pig farmers, results in pulmonary inflammation accompanied by respiratory symptoms, and is implicated in the development oflung diseasesv'. In vitro studies have demonstrated that LPS can induce HA production in different types ofcells, including lung fibroblasts and endothelial cells ofthe microvascular bed", The aim of the present study was to investigate the effect oflocal LPS exposure on HA deposition in the lung in vivo. To this end, we used a murine model of acute pulmonary inflammation, induced by a single intratracheal instillation of LPS. Neutrophil influx in the lungs was determined as a characteristic for acute inflammation. HA content was assessed by histolocalization using biotinylated HA binding protein (b-HABP).
MATERIALS & METHODS Experimental protocol Male Swiss mice (30-40 g) were obtained from Charles River Breeding Laboratories (Heidelberg, FRG). Animals were housed individually in standard laboratory cages and allowed food and water ad libitum throughout the experiments, which were carried out under a protocol approved by the Institutional Animal Care Committee of the Maastricht University, The Netherlands. Mice were instilled intratracheally (IT) by a non-surgical technique". Bromothymol blue dissolved in 50 1110.9% NaCI was instilled to check distribution ofthe solution in the lung. Macroscopic and microscopic analysis demonstrated that blue marker dye had spread throughout the whole lung. Mice (n=6 per group) were anaesthetised by intraperitoneal injection of 3 mg/kg xylazinc and 75 mg/kg ketamin (Nimatek, AUV Cuijk, the Netherlands). 5 ug of LPS (Escherichia coli, serotype 055:B5, Sigma, St. Louis, MO) dissolved in 50 IIIsterile 0.9% NaCl was instilled IT via a canule, followed by 0.1 5 mlofair. Sham mice were instilled IT with 50 IIILPS-free sterile 0.9% NaCl, whereas control mice received no treatment. After IT treatment, the mice were kept in an upright position for 10 minutes to allow the fluid to spread throughout the lungs. Mice were sacrificed at 4, 8, 24 or 72 hours after instillation. After thoracotomy, lungs were prepared for light microscopy and myeloperoxidase analysis.
Histology The left lung was inflated with 10% phosphate-buffered formalin (pH 7.4) at a pressure of 20 cm H 20 through the trachea for 15 min and subsequently fixed in formalin for 24 hours. After paraffin embedding, 4 11m sections were cut and stained with hematoxylin and eosin (HE) for histological analysis.
Determination of myeloperoxidase Myeloperoxidase (MPO) was isolated from snap frozen lung tissue of the right lung as previously described!", Enzymatic detection of MPO was performed in a 96-well plate (Greiner, Nurtingen, FRG) according to Daemen et al.!'. Briefly, assay mixtures consisted of 40 J..lI 0.75 roM H 202 in 80 mMPBS (pH 5.4) and 40 J..ll sample diluted in 50 mMPBS (pH 6.0), 0.5% hexa-l,6-bis-decyltrimethylammonium bromide (Sigma). The reaction was
Intratracheal instillation oflipopolysaccharide
2 I9
initiated by adding 20 III of 8 mM 3,3',5,5'-tetramethylbenzidine (TMB; Boehringer Mannheim, Mannheim, FRG) in dimethyl sulfoxide (Sigma) and stopped after 15 min by adding 100 Ill/well 1 M H 2S04 . Subsequently, optical density was determined at 450 nm. All samples were assayed in triplicate. MPO activity was calculated per mg lung tissue and corrected for wet/dry ratios. A titration curve of horseradish peroxidase was used for the calculation ofMPO activity, which is expressed in arbitrary units (mean ± SEM). Statistical analysis was performed by means ofMann-Whitney U test and probabilityvalues below 0.05 were considered to be statistically significant.
Histolocalization of HA Histolocalization of HA was determined on paraffin sections using biotinylated bovine nasal cartilage HABP, which was a kind gift from J. Melrose (University of Sidney, Australia). Sections were subjected to deparaffinization followed by rehydration. Sections were stained with b-HABP (50 ug/ml) at 4°C for 24 hours. After washing, the Vectastain avidin:biotinylated peroxidase complex (ABC) system was used according to the manufactor's instructions (Vector, Burlingame, CA). Enzymatic reactivity was visualised with 3-amino-9-ethylcarbazole. Sections were lightly counterstained with hematoxylin and mounted in faramount (DAKO, Glostrup, Denmark). No significant stainingwas detected in sections pre-treated with 50 U/mJ Streptomyces hyaluronidase (Calbiochem, San Diego, CA) at 37°C for 2 hours indicating that this HABP staining reaction was specific for HA.
RESULTS & DISCUSSION IT LPS instillation in mice results in a strong acute pulmonary inflammation Analysis of general inflammatory characteristics on HE stained paraffin sections demonstrated that IT LPS challenge resulted in a transient pulmonary inflammation. Strong infiltration of neutrophils into the alveolar area was observed, which was found to be timedependent. Presence of neutrophils in the alveolar spaces was evident from 8 hours after -,~ 60 C :l
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Figure 1. Quantification of pulmonary neutrophil infiltration after IT LPS instillation, MPO activity was measured in lung homogenates and expressed in arbitrary units (mean 'if SEM). * P<0.05 vs. saline (Mann-Whitney U test).
220
The role ofhyaluronan in tissues
LPS treatment and peaked at 24 hours (data not shown). MPO activity in lung homogenates was measured to quantify the relative neutrophil accumulation in the lung (Figure 1). 10 line with our histological observations, MPO activity was not detected in lung homogenates from LPS-treated mice at 4 hours postexposure, whereas MPO activity increased to 17, 44, and 26 units at 8, 24, and 72 hours after LPS instillation, respectively. MPO activity was not demonstrated in lung tissue of saline-treated mice or control mice. At 24 and 72 hours after LPS exposure, also infiltration of macrophages and lymphocytes was observed in the lung tissue ofLPS-treated animals. Taken together, these data demonstrate that IT LPS exposure resulted in a transient pulmonary inflammation characterised by a strong accumulation of neutrophils peaking at 24 hours postexposure. Time-dependent accumulation of HA after IT instillation of LPS Recent in vitro studies have demonstrated that stimulation of endothelial cells with LPS resulted in increased HA production after 4 to 72 hours of treatment", To investigate ifLPS exposure to the lung in vivo results in increased pulmonary HA deposition, HA accumulation in lung tissue was assessed at several time points after IT LPS exposure by histolocalization using a b-HABP. As shown in Figure 2, HA was expressed constitutively in the interstitium of airways and larger vessels of both control and LPS-exposed mice. In addition, the lungs ofLPS-treated mice showed HA deposition around small vessels of the microvascular bed and in the subpleural area at 24 hours after LPS instillation, especially at sites of leukocyte infiltration. Further increase ofHA deposition was observed at 72 hours.
Figure 2.
Histolocalization of HA in lung tissue using b-HABP. A: control. B: saline treated, 24 hours. C: LPS-treated, 24 hours. 0: LPS-treated, 72 hours. Original magnification: 200x. Note the accumulation ofHA in the subpleural area and around small vessels of the microvascular bed, as indicated by the arrows.
Intratracheal instillation of lipopolysaccharide
221
Since upregulation of HA on endothelium is suggested to play a role in leukocyte extravasation at sites ofinflammation via interaction ofHA with its primaryreceptor CD44 8, our data suggest that the HA deposition observed in the microvascular bed after pulmonary LPS exposure in vivo contributes to the observed pulmonary inflammation.
CONCLUSIONS In the present study, we show that IT instillation ofLPS results in a strong pulmonary inflammatory reaction, characterised by a time-dependent accumulation ofneutrophils in the lung tissue. Histolocalization ofHA revealed constitutive HA staining in the interstitium of airways and larger vessels ofboth control and treated mice. Local LPS exposure results in increased HA deposition in the subpleural area and around small vessels of the microvascular bed at 24 and 72 hours, especially at sites ofleukocyte infiltration.
ACKNOWLEDGEMENTS This work was supported by a research grant from Glaxo Wellcome BV, The Netherlands
REFERENCES I. S. Sahu, and W.S. Lynn, Hyaluronic acid in the pulmonary secretions of patients with
asthma, Biochem.l, 1978, 173, 565-568. 2. R. Hallgren, T. Samuelsson, T.C. Laurent, and J. Modig, Accumulation ofhyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome, Am Rev Respir Dis, 1989, 139, 682-687. 3. L. Bjermer, A Engstrom Laurent, R. Lundgren, L. Rosenhall, and R. Hallgren, Hyaluronate and type III pro collagen peptide concentrations in bronchoalveolar lavage fluid as markers of disease activity in farmer's lung, Br Med.l Clin Res Ed, 1987,295, 803-806. 4. W.D. Song, AC. Zhang, Y.Y. Pang, L.R. Liu, J.Y. Zhao, S.H. Deng, and S.Y. Zhang, Fibronectin and hyaluronan in bronchoalveolar lavage fluid from young patients with chronic obstructive pulmonary diseases, Respiration, 1995,62, 125-129. 5. T.M. Martin, Recognition of bacterial endotoxin in the lungs, Am. 1. Respir. Cell Mol. Biol., 2000,23, 128-132. 6. D.A Schwartz, P.S. Thome, SJ. Yagla, L.F. Burmeister, S.A Olenchock, lL. Watt, and TJ. Quinn, The role of endotoxin in grain dust-induced lung disease, Am J Respir Crit Care Med, 1995, 152,603-608. 7. P.F. Vogelzang, lW. van der Gulden, H. Folgering, lJ. Kolk, D. Heederik, L. Preller, MJ. Tielen, and C.P. van Schayck, Endotoxin exposure as a major determinant oflung function decline in pig farmers, AmJ Respir Crit Care Med, 1998, 157, 15-18. 8. M. Mohamadzadeh, H. DeGrendele, H. Arizpe, P. Estess, and M. Siegelman, Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HAdependent primary adhesion, J Clin Invest, 1998, 101,97-108. 9. B. Starcher, and I. Williams, A method for intratracheal instillation ofendotoxin into the lungs of mice, Lab Anim, 1989,23,234-240. 10.W.M. Kuebler, C. Abels, L. Schuerer, and AE. Goetz, Measurement of neutrophil content in brain and lung tissue by a modified myeloperoxidase assay, Int J Microcirc Clin Exp, 1996, 16,89-97.
222
The role ofhyaluronan in tissues
l1.M.A. Daemen, M.W. van de Yen, E. Heineman, and W.A. Buurman, Involvement of endogenous interleukin-l 0 and tumor necrosis factor-alpha in renal ischemia-reperfusion injury, Transplantation, 1999,67, 792-800.
THE DEGRADATION OF HYALURONAN DURING PERIODONTAL DISEASES: A POTENTIAL ROLE FOR REACTIVE OXYGEN SPECIES R Moseley"', RJ. Waddington, G. Embery Department ofBasic Dental Science, Dental School, University of Wales College ofMedicine, Heath Park, Cardiff. CF14 4XY. us:
ABSTRACT
Periodontal diseases are a group of chronic inflammatory conditions associated with the accumulation of bacterial plaque and the promotion of host inflammatory responses, resulting in extensive degradation of the periodontal extracellular matrix (ECM). Hyaluronan is one ECM component extensively degraded in such cases, while sulphated glycosaminoglycans (GAG) remain relatively intact. Host and bacterial enzymic activities have been proposed to be solely responsible for this degradation, although increasing evidence supports an additional role for polymorphonuclear leukocyte-derived reactive oxygen species (ROS). Studies by our group into GAG degradation by ROS in vitro have demonstrated that hyaluronan undergoes greater depolymerisation and residue modification following exposure to ROS, compared with sulphated GAG. ROS were derived from cell-free sources (the oxidation of hypoxanthine by xanthine oxidase and by the reaction ofH202 with Fe3"). As such findings coincide with the clinical manifestations observed in inflamed periodontal tissues, these results provide further evidence for a contributory role for ROS in the hyaluronan degradation associated with periodontal diseases. KEYWORDS
Hyaluronan, sulphated glycosaminoglycans, periodontal diseases, reactive oxygen species INTRODUCTION
Periodontal disease is a term encompassing a number of inflammatory conditions affecting the supporting tissues of teeth and classified into gingivitis and periodontitis I Bacterial plaque accumulation is the main aetiological factor in the initiation of gingivitis and its progression to periodontitis, promoting host inflammatory responses and resulting in extensive degradation to the extracellular matrix (ECM) of the periodontiunr'". Clinical investigations have identified that numerous ECM components undergo degradation during gingivitis, including the non-sulphated glycosaminoglycan (GAG), hyaluronan, while the sulphated GAG attached to proteoglycans remain relatively intact?". Host and bacterial enzymic activities have been proposed to be responsible for such observations'", although there is increasing evidence implicating polymorphonuclear leukocyte-derived reactive oxygen species (ROS), such as
224
The role or hyaluronan in tissues
superoxide radicals (0 2'"), hydrogen peroxide (H202) and the highly reactive hydroxyl radical species (OH), in these inflammatory conditions". The aims of these studies therefore, were to compare the in vitro degradation of hyaluronan and sulphated GAG associated with periodontal tissues by ROS, derived from cell-free systems. MATERIALS AND METHODS Exposure of GAG to cell-free sources ofROS in vitro
The GAG examined were chondroitin 4-sulphate (C4S) (whale cartilage, 46kDa), dermatan sulphate (DS) (porcine skin, 9-10kDa), hyaluronan (RA) (human umbilical cord, 3-6000kDa), heparan sulphate (HS) (bovine kidney, 1O-12kDa), all purchased from Sigma. GAG were subjected to both short term and more prolonged periods of ROS exposure. Short term exposure to 02'-1H20:zlOR was implemented via the oxidation of hypoxanthine by xanthine oxidase in the presence of a FeCh-EDTA chelate'". Reaction mixtures (l.Oml) consisted of GAG (0.5mg/ml), hypoxanthine (0.46mM), FeCh-EDTA (50).l.M) and xanthine oxidase (Grade III buttermilk, Sigma) (1.l25mU/ml) in 50 mM potassium phosphate buffer, pH 7.8, containing 10mM EDT A. Control reaction mixtures containing superoxide dismutase (SOD) (bovine erythrocytes, Sigma) (62.5U/ml) and catalase (bovine liver, Sigma) (200U/ml) were also established for each GAG. Reaction mixtures were incubated at 37°C for lh. Prolonged exposure to 'OR was implemented via the reaction ofH202 with Fe3+ 11. Reaction mixtures (l.Oml) consisted of GAG (0.5mg/ml), H202 (l80mM) and FeCh (5mM) in 25mM sodium acetate buffer, pH 5.6, containing 80mM sodium chloride. Control reaction mixtures containing GAG only (0.5mg/ml) or the 'OR scavenger, thiourea (IOOmM) were also established for each GAG. Reaction mixtures were incubated at 37°C for 24h. Analysis of GAG degradation
GAG were assessed for depolymerisation by gel filtration chromatography and for hexuronic acid and hexosamine modification by colorimetric assay. Aliquots of the reaction mixtures (500).l.1) were applied to a Superdex 75HR column incorporated into a FPLC system (Pharmacia), eluted with 2M guanidinium chloride-0.5M sodium acetate buffer, pH 6.8. Fractions (l.Oml) were collected and assayed for hexuronic acid content'". Total hexuronic acid and hexosamine contents of the GAG following ROS exposure and of the controls were quantitated according to methods 12- 13 . RESULTS GAG depolymerisation
Chromatographic profiles of GAG exposed to ROS for lh and 24h are shown in figures I and 2 respectively. GAG chain depolymerisation was observed as a reduction in parent peak height and the elution of a range of lower molecular weight hexuronic acid fragments, compared to controls. All GAG examined were demonstrated to undergo depolymerisation following ROS exposure, with parent peak height loss and fragmentation more prominent following prolonged exposure.
Degradation during periodontal diseases
225
Hyaluronan
",-------------
Elulion Volume (ml).
Chondroitin 4-sulphate
15
20
Il:lution Vulume (ml).
Dermatan sulphate
15
20
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Heparan sulphate
'/
ElutionVolumo (ml).
Figure 1.
Hexuronic acid profiles for GAG following l h exposure to the xanthine oxidase/hypoxanthine/FeCb-EDTA system. xanthine oxidase/hypoxanthine only, - - - SOD/catalase (adapted from Moseley et al 10)
226
The role of hyaluronan in tissues
Hyaluronan
IS
10
EluUOD Volume (ml).
Chondroitin 4-sulphate
"
ElutiOD Volume (ml).
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Figure 2.
Hexuronic acid profiles for GAG following 24h exposure to the H 20;JFeCb system. - - H202IFeCb only, - - - thiourea, - - - - GAG only
Degradation during periodontal diseases
227
Hyaluronan was depolymerised to a greater extent than sulphated GAG, as noted by greater reductions and displacement of hyaluronan peak heights following both lh and 24h exposure compared to sulphated GAG, suggesting that hyaluronan is more susceptible to ROS than sulphated GAG. The presence of the antioxidants SOD/catalase and thiourea in controls reduced the loss of peak height for each GAG, confirming ROS as being responsible for the degradation observed. Hexuronic acid/hexosamine residue modification
The total hexuronic acid and hexosamine contents of the GAG exposed ROS are shown in table 1. ROS exposure resulted in significant modification of both the hexuronic acid and hexosamine residues of each GAG, compared to GAG only controls, with the extent of residue modifications increasing following 24h exposure. Hexuronic acid residues demonstrated greater modification than hexosamine residues for each GAG examined, suggesting greater hexuronic acid susceptibility to ROS than hexosamine residues. The hexuronic acid and hexosamine residues of hyaluronan exhibited greater modification than sulphated GAG, following both 1hand 24h exposure, implying that hyaluronan is more susceptible to residue modification by ROS than sulphated GAG. The addition of SOD/catalase or thiourea to control samples significantly reduced the extent of modification, confirming ROS as being responsible for the loss of residue contents. DISCUSSION
The results of this investigation indicate that hyaluronan and sulphated GAG are susceptible to residue modification and depolymerisation by ROS, although sulphated GAG are more resistant to ROS attack than hyaluronan. Numerous studies into hyaluronan and sulphated GAG degradation by ROS have reported that hyaluronan and sulphated GAG undergo similar mechanisms of destruction, with the random destruction and modification of hexuronic acid and hexosamine residues followed by the hydrolytic cleavage of glycosidic bonds'"?". However, these studies also suggest that sulphated GAG undergo more selective ROS attack than hyaluronan, with ROS preferentially attacking the non-sulphated hexuronic acid regions compared to the more sulphated hexosamine regions of sulphated GAG chains, further corroborating the results of this investigation. Such studies, therefore, indicate that sulphate groups "protect" these GAG from ROS degradation'?' 18-19. It has been suggested that the actual mechanism by which sulphate groups achieve this is via their hydration spheres" or via the binding of 'Ol-l-promoting iron and copper ions, thereby preventing these ions enhancing 'OH production and GAG degradation20-2 1. As such findings concur with the GAG degradation observed in inflamed periodontal tissues" 6, these results provide further evidence implying an additional role for ROS in the hyaluronan degradation associated with chronic periodontal diseases. The effects of ROS on hyaluronan structure are also significant in considering other inflammatory conditions, such as rheumatoid arthritis and chronic wounds, where ROS are believed to play an integral role in the tissue destruction observed. As hyaluronan has been proposed to perform a number of functions in the remodelling of these tissues, the susceptibility of hyaluronan to ROS may also be important in relation to the mechanisms of tissue repair processes in such inflammatory conditions.
228
The role ofhyaluronan in tissues
Table 1. % loss of GAG hexuronic acid/hexosamine residues exposed to ROS for lh and 24h (adapted from Moseley et al 10 ) GAG
ROS
HA C4S DS HS
Hexuronic acid SODI ROS
(lh)
catalase (1h)
(24h)
Thiourea (24h)
18.44 ± 0.46 3.55 ± 0.81 9.81 ± 0.24 14.39 ± 0.17
6.21 ± 0.22 1.59 ± 0.57 7.15 ± 0.36 9.39± 0.47
87.12 ± 0.60 74.80 ± 1.56 65.13 ± 0.40 42.16 ± 1.66
36.69 ± 0.27 29.22 ± 1.46 26.95 ± 0.90 28.93 ± 1.62
Hexosamine SOD 1 ROS
ROS (lh)
catalase (th)
(24h)
Thiourea (24h)
15.86 ± 1.32 5.94± 0.17 7.24± 0.35 3.06± 0.93
11.13 ± 2.62 3.44± 0.36 3.16 ± 0.51 0.98 ± 0.51
80.61 ± 4.09 62.18 ± 0.63 51.25 ± 0.98 20.31 ± 1.70
28.69 ± 2.92 20.51 ± 0.53 23.46 ± 0.83 14.86 ± 1.82
CONCLUSIONS
These results suggest that hyaluronan is more susceptible to modification and depolymerisation by ROS than sulphated GAG. As such findings coincide with the clinical manifestations observed in inflamed periodontal tissues, these results provide further evidence for a contributory role for ROS in the hyaluronan degradation associated with chronic periodontal diseases. REFERENCES 1. RC. Williams, "Periodontal disease", N. Eng. Med. .I., 1990,322,373-382. 2. P.M. Bartold, AS. Narayanan, "Diseased periodontium", In: Biology of the Periodontal Connective Tissues, Quintessence Publishing Co., IIlinois, 1998, pp. 197-219. 3. K.S. Komman, R.C. Page & M.S. Tonetti, "The host response to the microbial challenge in periodontitis: assembling the players", Periodontol. 2000, 2000, 14, 33-53. 4. G. Embery, W.M. Oliver & 1.B. Stanbury, "The metabolism of proteoglycans and glycosarninoglycans in inflamed human gingiva", J. Periodont. Res., 1979, 14,512-519. 5. J.A Purvis, G. Embery & W.M. Oliver, "Molecular size distribution ofproteoglycans in human inflamed gingival tissue", Arch. Oral Biol., 1984, 29, 513-519. 6. P.M. Bartold, RC. Page, "The effects of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid",.1. Oral Pathol., 1986, 15,367-374. 7. S.S. Socransky, A.D. Haffajee, "Microbial mechanisms in the pathogenesis of destructive periodontal diseases: a critical assessment", J. Periodont. Res., 1991, 26, 195-212. 8. J.1. Reynolds, M.e. Meikle, "Mechanisms of connective tissue matrix destruction in periodontitis", Periodontol. 2000, 1997,14,144-157. 9. R.J., Waddington, R Moseley & G. Embery, "Reactive oxygen species: a potential role in the pathogenesis of periodontal diseases", Oral Diseases, 2000, 6, 138-151. 10.R Moseley, R.J. Waddington, P. Evans, B. Halliwell & G. Embery, "The chemical modification of glycosaminoglycan structure by oxygen-derived species in vitro", Biochim. Biophys. Acta, 1995, 1244, 245-252. II. S.D. Aust, L.A Morehouse & C.E. Thomas, "Role of metals in oxygen radical reactions", Free Rad. Bioi. Med., 1985, 1,3-25. 12.T. Bitter, H. Muir, "A modified uronic acid carbazole reaction", Anal. Biochem., 1962, 4, 330334. 13.R Gatt, E.R Berman, "A rapid procedure for the estimation of amino sugars on a micro scale", Anal. Biochem., 1966,15,741-752.
Degradation during periodontal diseases
229
14.H. Uchiyama, Y. Dobashi, K. Ohkouchi & K. Nagasawa, "Chemical change involved in the oxidative reductive depolymerization of hyaluronic acid", J Bioi. Chem., 1990, 265, 7753-7759. 15. K. Nagasawa, H. Uchiyama, S. Noriko & A. Hatano, "Chemical change involved in the oxidative-reductive depolymerization of heparin", Carbohydr. Res., 1992,236,165-180. 16. N. Volpi, I. Sandri & T. Venturelli, "Activity of chondroitin ABC lyase and hyaluronidase on free-radical degraded chondroitin sulphate", Carbohydr. Res., 1995, 279,193-200. 17. c.L. Hawkins, M.J. Davies, "Direct detection and identification of radicals generated during the hydroxyl radical-induced degradation of hyaluronic acid and related materials", Free Rad. Bioi. Med., 1996,21,275-290. 18.P. Bianchini, B. Osima, B. Parma, C.P. Dietrich, HK Takahashi & H.B. Nader, "Structural studies and "in vivo" and "in vitro" pharmacological activities of heparin fragments prepared by chemical and enzymic depolymerization", Thromb. Res., 1985, 40, 49-58. 19.R. Moseley, RJ. Waddington & G. Embery, "Degradation of glycosaminoglycans by reactive oxygen species, derived from stimulated polymorphonuclear leukocytes", Biochim. Biophys. Acta, 1997, 1362, 221-231. 20. D. Grant, W.F. Long & F.B. Williamson, "Complexation of Fe2 ' ion by heparin", Biochem. Soc. Trans., 1992, 20, 361S. 21. D. Grant, W.F. Long & F.B. Williamson, "Evidence from potentiometric titration for lack of reversibility in the interaction between heparin and Cu2+ and Ca2+ ions", Biochem. Soc. Trans., 1992, 20, 362S.
EFFECT OF VENTILATION ON HYALURONAN PRODUCED IN THE PLEUlU.L SPACE OF RABBITS Ping M. Wang & Stephen J. Lai-Fook Center for Biomedical Engineering, University ofKentucky, Lexington, KY 40506, USA
ABSTRACT The goal of these studies was to elucidate the effect of pleural hyaluronan on the mechanical interaction between the lung and the chest wall. In anesthetized rabbits, a 2-fold increase in ventilation for 6 hours resulted in a doubling of the hyaluronan present in pleural liquid. This behavior was reproduced in conscious rabbits with increased ventilation induced by breathing a 3% CO2-air mixture in a box for 24 hours. We postulated that the ventilation-induced hyaluronan serves as a boundary lubricant. Its role as a hydrodynamic lubricant was minimal because of its low concentration measured by radioassay (1 ug/ml), The increased hyaluronan was accompanied by an increased filtration, as indicated by arterial pressure and protein kinetics. Thus, it was unclear whether the increased hyaluronan was caused by a sliding-induced increase in pleural liquid shear stress or by a washout of hyaluronan from the pleura by the filtrate. To eliminate the latter, we ventilated rabbits postmortem for three hours. Compared to control rabbits, pleural liquid volume decreased by 30%, while pleural liquid protein concentration increased by 80%, which indicated absorption of protein-free liquid. We used an Alcian blue assay to measure hyaluronan produced in the pleura. Pleural tissue hyaluronan doubled after 3 hours and was considerably greater than pleural liquid hyaluronan. The increased pleural hyaluronan was consistent with an increased shear stress-induced production of hyaluronan from mesothelial cells as pleural volume was reduced. This was supported by a deeper blue stain on the parietal pleura of the chest wall in the postmortem rabbits. Pleural hyaluronan may serve as an effective lubricant when pleural liquid is depleted.
KEYWORDS Pleural lubrication, hyaluronan, radioassay, Alcian blue, Evans blue fluorescence, ventilation, protein kinetics, microvascular filtration, edema, conscious rabbit.
INTRODUCTION The pleural space consists of an extremely thin (6-30 11m) layer of liquid that separates the lung from the chest wall. Lubrication of the pleural surfaces by the pleural liquid is one of the major functions of the pleural liquid 1-4. However, the type of lubrication that is relevant to the pleural space is still in question. Two types of pleural lubrication have been postulated. The first is based on the existence of a thin continuous layer of pleural liquid that transmits forces directly from the respiratory muscles to the lung surface 3,4. This implies the identity between pleural surface and pleural liquid pressure. Here the viscosity of pleural liquid together with lung velocity and pleural liquid thickness determines the shear forces that act on the pleural surfaces during ventilation according to Newton's law of viscosity. The mathematical theory of hydrodynamic lubrication can be applied to the study of pleural liquid flow. The second
232
The role ofhyaluronan in tissues
theory of pleural lubrication is based on pleural contact. Contact between the two pleural surfaces results from the absorption of pleural liquid by pleural capillary blood I. The question of pleural contact has long been debated with no direct experimental evidence for its existence 5.7. In this communication, we summarize the result of recent studies related to pleural lubrication, and in particular the role of hyaluronan in the lubrication of pleural surfaces. PLEURAL LIQUID THICKNESS AND SHEAR STRESS The forces that are generated by the sliding between the lung and chest waIl during breathing are given by Newton's law of viscosity: cr = vV/h
[1]
Here cr is the shear stress generated in pleural liquid as a result of the relative velocity between the lung and the chest wall (V), v is the pleural liquid viscosity, and h is the pleural liquid thickness. Pleural liquid thickness measured by a variety of techniques was very thin, 6-30 urn 5.9. GeneraIly pleural liquid thickness was uniform with respect of height in the thorax, except for a slight gradient in the sheep 6, and in the lobar margins and lung base where it was much thicker 7. In the latter study, pleural liquid thickness during apnea increased with animal size, ranging from 6 urn in the rat to 23 11m in the dog. The aIlometric relationship between pleural liquid thickness and animal mass (M) was 8: h ex: 1f. 20 . This relationship was consistent with a microvascular filtration rate that was proportional to body mass, based on a pleural liquid flow driven by the net effects of gravity and pleural pressure. Recent results using the imaging of fluorescent dye injected into the pleural liquid of rabbits showed that pleural liquid thickness increased with ventilation 8,9. In this technique, a smaIl amount of FITC labeled dextran was injected into the pleural space of anesthetized mechanically ventilated rabbits. The thickness of the pleural space was measured from the fluorescent light emitted by the pleural liquid through a thin transparent parietal pleural window made in the intercostal space. Pleural liquid thickness measured from the 4-5 intercostal space was 11 11m during apnea and increased by 40 % from 22 11m to 35 11m as tidal volume increased 3-fold from 6 to 20 ml at a frequency of 20 breaths/min 8. A similar behavior was observed with a 5-fold increase in ventilation frequency from 8 to 40 breaths/minute at 20 ml tidal volume. 60
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Figure 1. Pleural liquid thickness (A), lung velocity amplitude (B), and pleural liquid shear stress amplitude (C) versus cranial-caudal distance. Reproduced from ref 9.
Effectof ventilation in the pleural space
233
This behavior was qualitatively consistent with a ventilation pump that drives pleural liquid from the lobar margins to the flat costal surfaces, opposite to the behavior expected during apnea 10. Subsequently we showed that pleural liquid thickness was not uniform throughout the pleural space, but increased along the cranial-caudal axis from a minimal values of 5 ~m in the second intercostal space to a value of - 30 urn in the fifth intercostal space (Fig. IA). This cranial-caudal gradient in pleural liquid thickness increased with ventilation, primarily due to an increased thickness in the caudal regions. Lung velocity relative to that of the rib cage also showed a similar gradient that increased with ventilation frequency (Fig. IB). Since the increase in pleural liquid thickness offset the increase in velocity (Equation 1), the calculated pleural liquid shear stress was constant with cranial-caudal distance but increased with ventilation frequency (Fig. 1C). The regional variation in pleural liquid thickness and shear stress posed several questions related to the regional variation of pleural liquid filtration and the effects of pleural liquid shear stress and ventilation on this variation.
EFFECT OF VENTILATION ON PLEURAL FILTRATION OF ALBUMIN AND PRODUCTION OF HYALURONAN Regional variations in pleural filtration was studied by EBA (Evans blue dyed albumin) fluorescent imaging of frozen slices of the rib cage of rabbits and b;V measuring the fluorescence emitted from the parietal pleural surface of the rib cage 1 . In these studies, EBA was injected into the circulation of anesthetized mechanically ventilated rabbits and after 6 hours the chest wall was dissected postmortem for fluorescent imaging. Both measurements showed an increased cranial-caudal gradient in filtration with filtration increasing towards the diaphragm, similar to the behavior observed for pleural liquid thickness. This filtration gradient was most likely caused by a greater blood flow in the caudal regions resulting in a larger capillary surface area and a higher capillary pressure. Both of these effects increased microvascular filtration. The greater caudal blood flow in the intercostal muscles of the lower rib cage and diaphragm was consistent with the greater muscle activity producing a larger chest wall expansion in the caudal regions. Table 1. Effect of ventilation frequency (f, breaths(b)/min) on EBA, protein and hyaluronan concentrations in pleural liquid after 6 hours of mechanical ventilation in anesthetized rabbits. Reproduced from ref 12 with permission. f= 20 b/min (n = 5)
EBA Cliq/Cpl Total protein (gldl) Total protein Cliq/Cpl Hyaluronan (ug/ml) 1\ Mean ± SD (n).
groups.
0.006 ± 0.00341\ 1.24 ± 0.28 * 0.23 ± 0.04 0.95±0.19
f= 40 b/min (n = 5)
0.016 ± 0.008 2.27 ± 0.53 0.47 ± 0.11 * 1.84 ± 0.89 *
n, number of experiments.
*
Normal Control
* 2.35 ± 0.58 (21) 0.34 ± 0.06 (12) 0.73 ± 0.27 (14)
Significantly different than other
234
The role ofhyaluronan in tissues
In conjunction with the histological studies, we injected EBA into the circulation of anesthetized and conscious rabbits to study the effect of ventilation on the rate of albumin filtration into the pleural space 12. In the anesthetized rabbits, after 6 hours of mechanical ventilation with 100% O2, postmortem the pleural liquid was collected, and its EBA and protein concentration measured and compared to values measured in the blood plasma. Table 1 shows the effect of increasing ventilation frequency from 20 to 40 breaths/min on these parameters. Both the pleural liquid-to-plasma EBA and protein concentration ratio doubled with the increased ventilation frequency. This suggests a ventilation-induced change in pleural membrane permeability to protein. Hyaluronan concentration in pleural liquid also doubled with the increased ventilation with no change in plasma. Because pleural membrane permeability might have increased as a result of O2 toxicity induced by the ventilation with 100% 02 and of the anesthetic, we repeated the studies using conscious rabbits. The animals were allowed to breathe either room air or room air with 3% CO2 in a box for periods of 6, 12 and 24 hours. Table 2 shows the values of pleural liquid-to-plasma protein concentration ratio (Cliq/Cpl) measured at the time periods. Note that there was no change in the protein kinetics between the animals breathing room air and the animals breathing 3% C02. Thus the increased pleural liquid protein concentration associated with the increased ventilation in the anesthetized rabbits was probably caused by an 02 toxicity- or anesthetic-induced increase in pleural membrane permeability. By contrast, pleural liquid hyaluronan concentration increased with time for both the group breathing room air and the group breathing 3% CO2, but the increase was greater in the group with the hypercapnicinduced increased in ventilation (Fig. 2). An increased ventilation above that of normal control rabbits most likely caused the increased pleural liquid hyaluronan concentration in the group breathing room air in a box. The protein kinetics data also showed that Cliq/Cpl decreased by 6 hours indicating an initial increase in microvascular pressure which recovered to normal control values by 24 hours. This was evidence that microvascular filtration increased with time for both groups. Thus it was unclear whether the increased hyaluronan in pleural liquid was caused by the increased ventilation or an increased filtration, in light of studies showing that an increased lung filtration in sheep results in a washout ofhyaluronan in the lymph 13. Table 2. Pleural liquid-to-plasma protein concentration ratio (Cliq/Cpl) in conscious rabbits placed in a box. Reproduced from ref. 12 with permission.
Group
6hr
12 hr
24hr
Normal Control
Cliq/Cpl
Room air 3% CO2-air
0.24 ± 0.06 (5) ,..,," 0.31 ± 0.06 (5) 0.31 ± 0.06 (5) 0.31 ± 0.04 (5) 0.36 ± 0.11 (6) 0.21 ± 0.05 (6)
" Mean ± SD (n). n is number of animals.
0.34 ± 0.06 (12)
* Significantly less than control values.
Effect of ventilation in the pleural space
235
Table 3. Body weight; pleural tissue hyaluronan (HA); pleural liquid volume, protein concentration and protein mass; and lung wet-to-dry weight (WID) ratio of3 groups of rabbits studied postmortem. Reproduced from ref. 14 with permission. Ventilated
Normal Control
Nonventilated
BodyWt (kg) Lung WID ratio HA(mg/kg)
3.6 ± 0.23 (8)" 4.8 ± 0.26 (5) 0.21± 0.04 (8)*
3.7 ± 0.23 (8) 4.6 ± 0.11 (8) 0.12 ± 0.07 (7)
3.6 ± 0.30 (7) 6.1 ± 1.4 (7)* 0.11 ± 0.05 (7)
Pleural liquid volume (ml/kg)
0.057 ± 0.022 (8)*
0.085 ± 0.025 (7)*
0.18 ±0.11 (7)*
Pleural liquid protein cone. (g/dl)
3.6 ± 1.2 (7)
2.0 ± 0.96 (8)*
3.5 ± 0.39 (6)
Pleural liquid protein mass (mg)
6.5 ± 2 (7)
5.3 ± 2 (7)
19± 9(6)*
/\ Mean ± SD (n). n is number of animals.
* Significantly different from other groups.
EFFECT OF POSTMORTEM VENTILATION ON PLEURAL TISSUE BYALURONAN PRODUCTION To determine whether an increase in ventilation per se would result in an increase in the production of hyaluronan from ~leural tissue, we studied the production of pleural hyaluronan in postmortem rabbits 4. In these experiments, rabbits were ventilated postmortem for 3 hours, the pleural liquid collected, and the pleural tissue hyaluronan measured using an Aldan blue assay. Ventilating rabbits postmortem increased pleural tissue hyaluronan in conjunction with a 30% reduction of pleural liquid volume (Table 2). The 3 hours of postmortem ventilation caused a reduction in pleural liquid protein concentration with a constant protein mass, indicating that the ventilation-induced decrease in pleural liquid volume was due to absorption of protein-free liquid into the microvasculature. An increased pleural filtration was not responsible for the increased pleural tissue hyaluronan for two reasons. First, absorption of pleural liquid occurred rather than pleural filtration. Second, in rabbits that were not ventilated postmortem, pleural tissue hyaluronan concentration was normal 3 hours postmortem even though pulmonary edema and pleural edema occurred, as indicated by the increased wet-to-dry weight ratio (WID) and the increased pleural liquid volume above normal control values (Table 2). Thus we attributed the increased hyaluronan with postmortem ventilation to the increase in pleural liquid shear stress as pleural liquid volume was reduced. The amount of hyaluronan measured in the pleural membrane (0.12 mg/kg) was ~ 1OOO-foid greater than that present in the pleural liquid (0.1 ug/kg). The increase in pleural hyaluronan after 3 hours of postmortem ventilation was equal to the amount normally present in normal control rabbits. Accordingly only a tiny fraction of the total hyaluronan produced by the pleural mesothelial cells is found in pleural liquid. Thus
236
The role ofhyaluronan in tissues
hyaluronan measured in pleural liquid is not an accurate marker for the hyaluronan produced by mesothelial cells. PLEURAL LUBRICATION BY HYALURONAN The amount of hyaluronan present in pleural liquid is not sufficient to change pleural liquid viscosity significantly from that of water IS. Even a 100-fold increase in the normal pleural liquid hyaluronan concentration (0.7 ug/ml) will increase its fluid viscosity by less than 5% 12. How does the hyaluronan present in pleural tissue help in the lubrication of the pleural surfaces? Hyaluronan has been identified as a coating on pleural mesothelial cells 17.18 and has been postulated as a contact lubricant I. We speculate that some hyaluronan is trapped in the microvilli (1-3 urn) and that the microvilli act as a thin lubricating layer to facilitate lung sliding in the event that the pleural liquid is depleted. This is likely to be most important during quiet breathing at end expiration when gravity would deplete pleural liquid. Two phase model of the pleural liquid space We envision the pleural space to consist of a layer of pleural liquid of low hyaluronan concentration that lies between hyaluronan-rich layers that are attached to mesothelial cells covering the parietal and visceral pleural membranes. Microvilli are densely packed in the caudal region of the diaphragm because lung velocity is maximal so that any depletion of pleural liquid would result in high shear rates. In this location, a hyaluronan-rich layer adjacent to the mesothelial cells would act as a lubricant that lowers fluid viscosity 0 as the shear rate increase with ventilation, thereby protecting the mesothelial cells from prohibitively high shear stress. By contrast, microvilli are sparsely distributed in the apical region of the chest cavity 19 because here lung velocity is relatively low so that the shear stress generated by the depletion of pleural liquid is relatively small. Alcian blue bound to hyaluronan (and other glycosaminoglycans that bind to A1cian blue) in the parietal pleura of the rib cage was most prominent in the caudal regions after postmortem ventilation 14. This suggests that most of the decrease in pleural liquid observed with postmortem ventilation occurred in the caudal regions.
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Figure 2. Pleural liquid hyaluronan concentration versus time measured in conscious rabbits breathing room air and 3% C02. Reproduced from ref. 14 with permission.
Effect of ventilation in the pleural space
237
This might have been caused by a rigor mortis-induced decrease in the caudal rib cage compliance. By contrast, increased ventilation in vivo did not result in any obvious regional differences in Alcian blue staining most likely because the shear stress-induced production of hyaluronan was uniform. A thin hyaluronan-rich layer of high viscosity at low shear rates would resist the compression forces 20 due to breathing and prevent the depletion of the layer. Thus the primary function of pleural microvilli might be to act as a hyaluronan-rich lubrication layer that would resist compression forces at low shear rates at end expiration, yet would lower viscosity and shear stress at high shear rates during inspiration and expiration. CONCLUSIONS Our studies showed that an increase in ventilation produced an increase in pleural liquid hyaluronan concentration. The amount of hyaluronan in pleural liquid was too small to serve as an effective hydrodynamic lubricant, and was only a tiny fraction of the amount produced in the pleural membrane. The increase in pleural liquid hyaluronan was attributed to a ventilation-induced increase in pleural liquid shear stress that stimulated the mesothelial cells to produce hyaluronan, rather than to a washout of hyaluronan by microvascular filtrate. The specific role of the hyaluronan produced by ventilation in pleural lubrication needs to be defined. We speculate that the hyaluronan produced by ventilation is trapped in microvilli and forms a thin lubricating layer to effectively reduce shear stress in the event that pleural liquid is de~leted. A similar function has been assigned to surfactants measured in pleural liquid 21. 2. ACKNOWLEDGEMENTS This research was supported by research grants HL 36597 and HL 40362 and a postdoctoral fellowship HL 10142 (PMW) from the Heart, Lung and Blood Institute of the NIH. REFERENCES 1.
2.
3. 4.
5.
E. Agostoni, Lung and chest wall: mechanical coupling, In: The pleural in Health and Disease, 1. Cretien & J. Bignon & A. Hirsh (eds.), Marcel Dekker Inc., NewYork, 1985, pp. 141-149. G. Miserocchi & D. Negrini, Pleural space: pressures and fluid dynamics, In: The Lung Scientific Foundations, Volume 1, 2nd Edition, R. G. Crystal, P. J. Barnes PJ, 1. B. West & E. R. Weibel (eds.), Lippincott-Raven, Philadelphia, 1997, pp. 12171726. S. 1. Lai-Fook, Mechanics of the pleural space: fundamental concepts, Lung, 1987, 165, 249-267. S. J. Lai-Fook & 1. R. Rodarte, Pleural pressure distribution and its relationship to lung volume and interstitial pressure, Brief Review, J. Appl. Physiol., 1991, 70, 967-978. E. Agostoni, G. Miserocchi & M. V. Bonanni, Thickness and pressure of the pleural liquid in some mammals, Respir. Physiol., 1969,6,245-256.
238 6.
7, 8. 9.
10.
11. 12.
13.
14. 15.
16.
17, 18, 19,
20,
21. 22.
The role of hyaluronan in tissues K. H. Albertine, J. P. Wiener-Kronish, J. Bastacky & N. C. Staub, No evidence of mesothelial cell contact across the costal pleural space in sheep, 1. Appl. Physiol. 1991,70,123-134. S. 1. Lai-Fook & M. R. Kaplowitz, Pleural space thickness in situ by light microscopy in five mammalian species, J Appl Physiol. 1985,59,603-610. P. M. Wang & S. J. Lai-Fook, Effect of ventilation frequency and tidal volume on pleural space thickness in rabbits, 1. Appl. Physiol., 1993, 75, 1836-1841. P. M. Wang & S. J. Lai-Fook, Effect of mechanical ventilation on regional variation of pleural space thickness in anesthetized rabbits, Lung, 1997, 175, 165173. J. P. Butler, H. Huang, S. H. Loring, S. J. Lai-Fook, P. M. Wang & T. A. Wilson, Model for a pump that drives circulation of pleural liquid, 1. Appl. Physiol., 1995, 78,23-29. P. M. Wang & S. J. Lai-Fook, Regional pleural filtration and absorption measured by fluorescent tracers in rabbits, Lung, 1999, 177,289-309. P. M. Wang & S. J. Lai-Fook, Effect of ventilation on hyaluronan and protein concentration in pleural liquid of anesthetized and conscious rabbits, Lung, 1998, 176, 309-324. L. Lebel, L. Smith, B. Risberg, B. Gerdin & T. C. Laurent, Effect of increased hydrostatic pressure on lymphatic elimination of hyaluronan from sheep lung, 1. Appl. Physiol., 1998,64, 1327-1332. P. M. Wang & S. J. Lai-Fook, Pleural hyaluronan produced postmortem by ventilation in rabbits, Lung, 2000, 178, 1-12. S. J. Allen, J. R. E, Fraser, U. B. G. Laurent, R. K. Reed & T. C. Laurent, Turnover of hyaluronan in the rabbit pleural space, 1. Appl. Physiol. 1992, 72, 1457-1460, V. C. Broaddus, J. P, Wiener-Kronish, T, C. Laurent & N. C. Staub, Clearance of hyaluronan into the pleural space during high pressure pulmonary edema in sheep, Abstract, FASEB 1.,1988, 2, A170. T. C, Laurent, J. R. E. Fraser, Hyaluronan, Abstract, FASEB 1., 1992, 6, 23972404. J. R. E. Fraser & T. C. Laurent, Hyaluronan, In: Extracellular Matrix, Volume 2, W, Comper (ed.), Hardwood Academic Publishers, 1996, pp. 141-197. N, S, Wang, Mesothelial cells in situ, In: The pleural in Health and Disease, J. Cretin, J. Bignon & A. Hirsh, (eds.), New-York, Marcel Dekker Inc" 1985, pp. 2342 A. G. Ogston & J. E. Stanier, The physiological function of hyaluronic acid in synovial fluid: viscous, elastic and lubricant properties, 1. Physiol. (London), 1953, 119, 244-252. B. A. Hill, Graphite-like lubrication of mesothelium by oligolamellar pleural surfactant, 1. Appl. Physiol., 1992,73, 1034-1039. B. A. Hill, B. D. Barrow & R. E, Barrow, Boundary lubrication imparted by pleural surfactants and their identification, 1. Appl. Physiol. 1982,53,463-469,
PARTS NOVEL MODIFIED FORMS OF HYALURONAN
BY ALURONAN LINEAR AND CROSSLINKED DERIVATIVES AS POTENTIAL/ACTUAL BIOMATERIALS.
•
Crescenzi' V., Franeeseaageli' A., Renier.2D., Belliniz D. 'Department a/Chemistry. University "La Sapienza". P.le A. Mora 5. 00185 Rome. Italy; 2FidiaAdvanced Biopolymers, srl, Via Ponte deIIa Fabbrica 3/A. 35031 Abano Terme , Padua. Italy
ABSTRACT The synthetic routes leading to a few novel hyaluronic acid' (HA) derivatives recently obtained in our laboratories - and their main physical properties and potential or actual applications in the biomedical sector are briefly described. HA derivatives considered include linear, water soluble species and hydrophilic networks and membranes. The latter are biomaterials particularly suitable for controlled drug release formulations and for avoiding post-surgical adherence phenomena, respectively. KEYWORDS Hyaluronan derivatives, HA-based hydrogels and membranes, biomaterials. INTRODUCTION In our laboratories work is being devoted to the synthesis and characterisation of new hyaluronan (HA) derivatives consisting in either products soluble in water with special functional characteristics or materials (i.e. chemical hydrogels, which obviously only swell in aqueous media) with functional-structural properties of interest in the biomedical area. In Section A, the following HA-based products will be introduced: A-I, partial amides with alkyl side-chains of different lengths (HYADDlM) ; A-2, partially deacetylated derivatives; A-3 partially/totally C-6 oxidised HA and esters therefrom (new HYAFF® series). HA-based materials which will be briefly considered in Section B consist in: B-1, hydrogels obtainable from the cross-linking of partially deacetylated HA with glutaraldehyde or by means of multi-component condensation processes; B-2, gels and membranes via y-rays promoted reticulation of unsaturated HA esters. In the following, methods for the synthesis of the above mentioned products/materials and some of their characteristic properties as well as their possible application in the biomedical sector will be briefly described.
262
Novel modified forms of hyaluronan
RESULTS AND DISCUSSION A - linear HA derivatives
1. HA amides (HYADD™) Water soluble amides/ of HA have been prepared by letting HA (tetrabutylammoniurn salt form) to react with different aliphatic or aromatic amines (in excess) in dimethylsulfoxide (DMSO) after partial protonation of HA carboxylate groups with anhydrous, pure methane sulfonic acid (0.3 moles/moles of HA repeating unit) and the addition of l,t'-carbonyldiimidazole (amidation activator: stoichiometric with respect to the desired degree ofHA amidation (d.a.)). The reaction mixtures were left overnight at 30° C, and the products were recovered by precipitation and washings with non-solvents (e.g, ethanol) and fmally air-dried. The ensuing HA-derivatives, in the case of aliphatic amides, are water soluble (depending on the nature of the side chains and on the degree of amidation ) giving rise to very viscous solutions with zero-shear viscosities orders of magnitude higher than that of the starting HA sample, stable over time (days) even after steam sterilization. Interestingly, the same amides exhibit a marked increase in stability towards enzymatic degradation (bacterial hyaluronidase) with respect to native HA: this is clearly shown by the chromatpographic data reported in Fig. 1. These easily obtainable, HA-based amides thus appear as novel, high performance rheological agents.
0.4
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Figure 1. HPLC data for HA (A), HA dodecylamide (d.a.=12) (B) and HA hexadecylamide (d.a.=8) (C) after hyaluronidase degradation (60°C for 5 h)
Linear and cross-linked derivatives
263
2. Partially deacetylated HA In order to increase the reactivity of HA in a number of chemical transformations, we found it expedient to hydrolyse the amide groups of given fractions of the Nacetylglucosamine residues' along the polysaccharide chains thus endowing the latter with primary amine functionalities. The hydrolysis requires rather harsh conditions which we tried to minimise in order to control the concomitant chain degradation. In brief, working conditions, selected through a series of trial experiments, involve the use of hydrazine monohydrate as solvent with the addition ofhydrazine sulphate (1-2%) and controlled amounts ofHI03 and HI at 50°C. The degree ofdeacetylation depends on time of reaction and can range from about 5 to 20 %. The trend of samples molecular weight and degree of deacetylation (dd) (Fig. 2) as a function of reaction time (h) at three different temperatures (20, 50 and 80 0 C) indicates that the middle one is a reasonable compromise in that, after 72 h, dd values around 20% can be obtained although a dramatic decrease in HA molecular weight (from 750 kDa to 40 kDa) takes place. This unavoidable phenomenon is however not crucial for our purposes In fact, the partially deacetylated HA samples have been found useful in the preparation of, respectively: I) new amides soluble in aqueous media (patent pending); 2) N-sulfated derivatives (heparin like species: patent pending); 3) new crosslinked derivatives which will be briefly discussed in the following section B.
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50
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Figure 2.
Influence of time and temperature on the HA deacetylation process
264
3.
Novel modified forms ofhyaluronan
(6) oxidised HA and esters therefrom
N-acetyl glucosamine residues along HA chains can be quantitatively oxidised at position 6 in dilute aqueous solution employing the now well established TEMPOmediated regioselective oxidation process''" b, c. The oxidation reaction scheme and the experimental working conditions are summarised in Fig. 3a. Using such conditions, we have obtained from partially to totally - as desired - C(6) oxidised HA (HYOXX: Fig. 3b) samples with minimal chain degradation. The ensuing polyelectrolytes - with a charge density up to twice that ofHA - exhibit high affmity for divalent cations, especially Zn(II) ions, thus acting as selective, chelating agents as well as potential ionic drugs controlled delivery agents'". In addition, the new HA derived polycarboxylates are suitable substrates for the synthesis of a novel class of esters'" - new HYAFFiI) - with physico-chemical properties paralleling and in some cases challenging those of the already well known HA esters (HYAFF
1. Deacetylated HA reticulation processes based on multicomponent condensation reactions or on cross-linkingwith glutaraldehyde. As already reported, Ugi four-component condensation reactions can be conveniently employed in facile, one-pot reticulations in aqueous solution of synthetic and natural polyelectrolytes bearing either negative (e.g HA) or positive fixed charges",
A) Selective (partial) oxidation of primary alcobols to aldehydes - NaBr : 0.4 moVmol of -eH,OH - TEMPO: 7 mmoVmol of -CH,OH - NaCIO : 0.1 moVmolof ·CH,OH - NaHCO,: 5 % wN; pH = 8.6 = canst. - Temp. : 0° C; under Ar
Figure 3a.
B) Selective (partial to total) oxidation to carboxylic group - NaCIO : in excess (ca 3 mol/molof -CH,OH) - pH = 9.3 = const, (addition ofNaOH 0.5 M); in air - TEMPO. NaBr. and temnerature as in A)
Scheme of the TEMPO-mediated selective C(6) oxidation of carbohydrates
Linear and cross-linked derivatives
265
~~w ......
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_ N.
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Figure 3b. TEMPO mediated oxidation ofHA
The four basic components of such reactions must exhibit the following functionalities, respectively: carboxyl, amine (primary), aldehyde, and isocyanide. A generic Ugi condensation reaction, of the type schematically shown in Fig. 4, once chosen the appropriate reagents, is both kinetically and thermodynamically favoured so that, basically, only one fmal product is rapidly obtained in good yield. This explains the success that such Ugi reactions have recently encountered in "combinatorial chemistry". In our case, taking advantage of the double functionality exhibited by partially deacetylated HA (see A-2 above), i.e. amino and carboxyl groups, networks based on HA have been easily prepared via Ugi condensations involving addition of only formaldehyde and cyclohexyllsocyanide'". The hydrogels are stable, highly hydrophilic and can swell extensively in contact with aqueous media (depending of course on crosslinking degree) thus providing new products suitable for controlled drug delivery formulations (patent pending)",
Figure 4.
Representative Ugi's reaction product
An even easier route to hydrogels based on partially deacetylated HA consists in cross-linking the latter with glutaraldehyde in acid aqueous solution. In practice, 140 mg
266
Novel modified forms of hyaluronan
of deacetylated HA (dd = 20%) are dissolved in 1.75 mL of water: the solution is then added of 1-2 drops of 6 M Hel and of 100 mL of glutaraldehyde. In a few minutes a pink colored gel is formed which eventually turns light orange. After 10 days dialysis against water the swelling of the sample is close to 50 (weight of equilibrium swollen gel/weight of freeze-dried gel). The hydrogels do not need stabilization via reduction in as much as glutaraldehyde is in the form of unsaturated oligomers (in our working conditions) whose aldehyde groups form Schiff-bases with the primary amino groups of deacetylated HA which are stabilised by resonance" (Fig.5). 2. Reticulation ofunsaturated HA esters
As shown in Fig. 6a, unsaturated partial esters of HA can be prepared by applying the reaction conditions optimised for the synthesis of HA classical esters (HYAFF
OR 0'-
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Figure S. Representative structure of hydrogels obtained using partially deacetylated HA and glutaraldehyde.
Linear and cross-linked derivatives
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267
268
Novel modified forms of hyaluronan
CONCLUDING REMARKS Hyaluronan is an ideal substrate for a variety of chemical derivatization processes leading to new linear products as well as to polymeric networks. In our experience, this is a fertile field of basic and applied research. In fact, a number of novel reaction routes can be applied and the ensuing HA-based materials are of interest in the biomedical area where some of them have already found or are just fmding a remarkable success.
AKCNOWLEDGEMffiNTS Activities outlined above have been financially supported by Fidia Advanced Biopolymers, FAB, srl, and by the Italian Ministry for the Universities and Research, MURST, Rome, Italy.
REFERENCES 1. T. C. Laurent, The Chemistry, Biology and Medical Applications of .Hyaluronan and its Derivatives, Portland Press Ltd, London, 1998. 2. D. Bellini, L. Callegaro, Amides of hyaluronic acid and their derivatives, Italian Patent, No. WO 00/733. 3. D. Renier, V. Crescenzi, A. Francescangeli, Nuovi derivati cross-linkati di acido ialuronico, Italian Patent Application Pending. 4. a) V. Crescenzi, D. Delicato, M. Dentini, Synthesis and preliminary characterization of new uronans, J. Carbohydr. Chem., 1997, 16,697-701. b) A.EJ de Nooy, A. C. Besemer, H. Van Bekkem, Highly selective nitroxyl radical mediated oxidation of primary alcohol groups in water soluble glucans, Carbohydr.Research, 1995, 269, 89-98. c) B. Jiang, E. Drouet, M. Milas, M. Rinaudo, Study on TEMPO mediated selective oxidation of hyaluronan and the effects of salt on reaction kinetics, Carbohydr. Research, 2000, 327, 455-461. d) D. Bellini, V. Crescenzi, A. Francescangeli, Polisaccaridi percabossilati, processo per la loro preparazione e loro impiego in campo farmaceutico e biomedico-sanitario, Italian Patent Application Pending. 5. a) A.E.J de Nooy, G. Masci, V. Crescenzi, Versatile synthesis of polysaccharide hydrogels using the Passerini and Ugi multicomponent condensations, Macromolecules, 1999, 32,1318-1320. b) A.E.J. de Nooy, D. Capitani, G. Masci, V. Crescenzi, Ionic polysaccharide hydrogels via the Passerini and Ugi multicomponent condensations: synthesis, behaviour, and solid state NMR characterization, Biomacromolecules, 2000,1,259 - 267. 6. A. Jayakrishnan, S. R. Jameela, Glutaraldehyde as a fixative in bioprostheses and drug delivery matrices, Biomaterials, 1996, 17,471-484. 7. D. Bellini, L. Callegaro, Polysaccharide hydrogel materials: a process for its preparation and its use in medicine, surgery, cosmetic and for the preparation of health care products, Italian Patent, No. WO 96/37519.
NOVEL BIOMATERIALS BASED ON CROSS-LINKED HYALURONAN: STRUCTURAL INVESTIGATIONS. Luigi Michielin", Carla Bevilacqua', Sergio Paoletti", Amelia Gaminr', Renato Toffanin 2, and Fulvio Micali 2 J
Fidia S.p.A., Abano Terme, Padova (Italy)
2Department 0/ Biochemistry, Biophysics and Macromolecular Chemistry, University a/Trieste. Via L. Giorgieri I, 34127- Trieste, (Italy)
ABSTRACT
Structural properties of several cross-linked hyaluronan derivatives, obtained by electron microscopy, monodimensional NMR microscopy and low angle X-ray scattering by synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid used as reference material. The experimental results, obtained in different media by the different techniques, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of more dense polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover the presence of polymer junction zones gave rise to the appearance of an additional correlation peak in the pattern of the intensity of the scattered X-radiation. KEYWORDS
Cross-linked Hyaluronan, SAXS, lD-NMR microscopy, SEM INTRODUCTION
Biodegradability and biocompatibility are the fundamental requirements that determine the possible therapeutical and surgical applications of a polymeric biomaterial having appropriate chemical and physical properties [1]. The desired goal is the possibility of tailoring the stability of the biomaterial depending on the required application needs. The unique ability of hyaluronic acid to form a highly hydrated polymer matrix with peculiar viscoelastic properties, rendered its use feasible as a therapeutical agent in viscosurgery and for the treatment of arthritis through intraarticular injections[2-4]. From a purely chemical point of view, an increase of the hyaluronic acid stability in solution may be obtained by partially transforming the physical network into a chemical network through cross-linking. The structure of hyaluronan samples, chemically cross-linked via interchain ester linkages, was analyzed by electron microscopy, lD-NMR microscopy and SR-SAXS. The obtained results, which give a uniform and consistent picture of the novel biomaterial, are compared with those of a native hyaluronan sample and with those of the commercial cross-linked hyaluronan Synvisc®.
270
Novel modified forms ofhyaluronan
MATERIALS & METHODS Scanning Electron Microscopy
Polymer samples for SEM measurements have been prepared following standard procedures generally used for biological structures. A 2.5% glutaraldheyde in phosphate buffer solution has been used to fix the sample (2 hrs). The suspension has been then passed through 0.2 11m membranes (Isopore , Millipore) and the unfiltered solid repeatedly washed with buffer solution. The filter-supported material was then postfixed in osmium tetraoxide (I % in buffered solution) for I hr., washed three times for 5min. in buffer and dehydrated through graded ethanols. Dried to the critical point through liquid C02 and coated with Au. ID-NMR microscopy
NMR measurements have been performed using a Bruker AM300 spectrometer equipped with a standard microimaging apparatus and coupled with a Spectrospin vertical wide-bore superconducting magnet operating at 7.05 Tesla. 5mm NMR tubes were used for sample solutions. A standard microimaging probe was used with a RF coil diameter of 5mm. The relaxation times (T1 and T2) were measured by using a spin-echo pulse sequence modified to minimize diffusion effects [5]. T2 values were calculated from the signal integral obtained at 11 different echo times (TE) in the 10+800 ms range. The integrated data were fitted to a single exponential decay, according to the equation: 1=loExp[-TE IT 2 ] where 10 represents the proton density, and hence the relative distribution of water protons within the sample. The longitudinal relaxation times T I were calculated from projections measured at 11 different repetition times (TR) after the integrated data were fitted according to: Ln[1 - 10 ]
=
TR IT I
where 10 refers to the signal measured using the highest repetition time. Diffusion experiments were performed using a pulsed gradient spin-echo sequence. The diffusion coefficients D were calculated from 7 projections obtained by applying along the z-axis a field gradient g with intensity up to 0.38 Tm'l. The diffusion coefficients were calculated from a linear fit of the data according to: Ln[ I ] = Ln[ 10
] -
(gy8)2 (t1-8/3)D= Ln[ 10
] -
bD.
Saturation transfer experiments were performed in order to measure the magnetization transfer ratios (Ms/Mo) from protons of water molecules with low mobility to those of free water. Here, M, represents the equilibrium longitudinal magnetization in the absence of magnetization transfer. SR-SAXS
Cross-linked hyaluronan
271
The SAXS measurements have been carried out at the high-flux SAXS beamline 5.2. L-Small Angle X-ray Scattering Facility of the ELETTRA Synchrotron Light Source (Trieste, Italy). A flat double crystal monochromator and a double focusing toroidal mirror are used for the beamline optics. The sample-detector distance was 2.26 m, the wavelength of the incident beam was A. = 1.55 A and the range of momentum transfer, q = 4;rrsin BfA, covered 0.012 A-'
Solutions of native hyaluronan (from streptococcus equi, Mw = 6.8x10s g/mol) and of hyaluronan derivatives with different cross-linking amount (labeled as ACP) were provided by Fidia, S.p.A (Padua, Italy). The Synvisc® sample was from Biomatrix. The solvent was 5% mannitol in ImM PBS for all the samples except that for HA, Synvisc and ACP-515 samples for which 0.15 M NaCI was used. RESULTS & DISCUSSION Scanning Electron Microscopy
SEM analysis showed that substantially different morphologies characterize the ACP derivatives and that the synthesized biomaterials belong at least to two structural families of different topology.
Fig. I Electron micrograph of sample ACP-515 Comparing the micrographs reported in figures 1-3, a relatively homogeneous network of "necklace" fibers is observed for samples of the ACP-500 series; it finds its
272
Novel modified forms of hyaluronan
counterpart in the far less homogeneous matrices of the ACPW series, for which a disorderly aggregation of material of approximately spherical shape is observed.
Fig.2 Electron micrograph of sample ACP-555
Fig.3 Electron micrograph of sample ACPW-2000
ID-NMR microscopy
ID-NMR microscopy turned out to be a very useful tool to investigate the synthesized biomaterials in aqueous solutions. All the measured magnetic parameters are referred to water protons. and give an indirect picture of the polymer chain structure. The T[, T2, D and MsiM o values obtained for the ACP sample solutions are listed in Table I where also the magnetic parameters observed for native HA and for the commercial Synvisc® are reported. Among all the magnetic parameters the transversal
Cross-linked hyaluronan
273
relaxation time T 2 resulted to be the most sensitive to the structural organization of polymer chains in solution. In particular for highly viscous solutions a considerably low
Table 1. Magnetic parameters from ID-NMR microscopy for ACP samples in 5% mannitol and ImM PBS sample ACP-526 ACP-554 ACPW-2000 ACP-553 ACP-535 ACPW-2000 a ACP-508 ACP-555 ACP-508 b ACP-515 a SYNVlSC®a HA a
T 2 (ms.) 1.90 2.19 2.19 2.15 2.01 2.49 2.30· 2.70 2.30 2.46 2.50 2.35
93 124 127 130 134 398 489 560 637 757 767 768
MslMo (kHz) 1.66 1.78 1.74 1.8 I 1.77 2.06 1.94 2.10 2.08 2.05 2.04 2.05
0.88 0.93 0.94 0.94 0.94 0.93 0.94 0.99 0.97 0.98 0.97 0.96
a in 0.15 M NaCI hydrolyzed sample
b
relaxation time (,.,,100 ms.) was obtained. For the ACP-526 a T2 as low as 8 times the value measured for the HA and Synvisc® sample was observed. On the contrary, both T 1 and the diffusion coefficient D did not show to depend markedly on the viscosity of the medium. As far as the diffusion coefficient is concerned, these findings are a clear evidence that the water molecule diffusion is not much reduced by the polymer matrix. Only for the sample with the highest degree of cross-linking (i.e.: ACP-256) a 19% reduction of the diffusion coefficient with respect to the value measured for the reference HA solution is observed. The magnetization transfer data are in line with the above mentioned results. The lowest Ms/M, value is observed with solutions of the apparently highly cross-linked sample, ACP-526. It is worth mentioning that with this type of experiment a selective saturation of the "bound" water molecule protons with respect to those belonging to the "bulk" water is performed. A lower value of the M,IM o ratio indicates that a magnetization transfer from saturated protons of less mobile water molecules to the free water protons occurs. In addition, the magnetization transfer parameter is influenced also by the mobility of the solute molecules. For a polymer chain with a reduced tumbling rate the magnetization transfer process will be more effective and a lower Ms/Mo value will result.
Small Angle X-Ray Scattering
274
Novel modified forms ofhyaluronan
The intensity of the synchrotron radiation scattered solutions of high molar mass polymers is generally found to increase on decreasing q and to tend asymptotically to a maximum as q->O. As an example, the scattering pattern observed for the native HA in NaCI 0.15 M is reported in FigA together with the scattered intensity measured for the commercial Synvisc® in the same solvent. Conversely, for the other ACP samples with the only exception of the hydrolyzed one (ACP-508) an additional interference peak is found in the 0.2-1 nm-I q-range. In Fig. 5 the scattering intensity pattern measured for ACP·2000 and ACP-526 samples is reported as an example. The additional correlation peak is likely to arise from interactions between chain segments occurring in highly concentrated polymer solutions as well as in gels and in chemically cross-linked materials [6,7]. In this respect the position of the interference peak should be inversely proportional to the length within which those interactions do exist. Beyond this distance chain segments are no longer correlated. From the position of the interference signal it may be then deduced that the ACPW-2000, ACP-526 and ACP-535 samples are characterized by zones of dense material randomly distributed around an average distance of e 4 nm. In line with both SEM and NMR results this distance increases to '" 10 nm in the case of the ACP-508 sample due to the lower degree of cross-link that characterizes this particular polymer matrix. For the corresponding hydrolysed ACP-508
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with .the number of aquisitions. This finding, particularly evident for the native HA sample, indicates that chain degradation occurs, likely induced by the high energy of the beam. References
I. E. Sackmann & M. Tanaka, Supported membranes on soft polymer cushions: fabrication, characterization and application, TIBTECH, 2000,18, 58-64 2. L.M: Benedetti, E.M. Topp, V.J. Stella, A novel drug delivery system: Microspheres of Hyaluronic acid derivatives, In: Biomedical and Biotechnological Advances in Industrial Polysaccharides. V Crescenzi, 1.C.M. Dea, S. Paoletti, S.S. Stivala & 1. W. Sutherland (Eds),Gordon & Breach Sci. Publishers, 1989, pp 27-33 3. E.A. Balazs & P. Band, Hyaluronic Acid: its Structure and Use, Polymers in Cosmetics, 1984,99,64-72 4. l.A. Hunt, VJ. Stella & E.M. Topp, Characterization of Polymeric Films Prepared from Ester Derivatives of Hyaluronic Acid, In: Biomedical and Biotechnological Advances in Industrial Polysaccharides, V Crescenzi, 1.C.M. Dea, S. Paoletti, S.S. Stivala & I.W. Sutherland (Eds),Gordon & Breach Sci. Publishers, 1989, pp 55-62 5. V. Mlynarik, A. Degrassi, R. Toffanin, O. Jarh & F. Vittur, Magn. Res, Med, 1996, 35,423-425 6. K. Kajiwara, S. Kohjiya, M. Shibayama & H. Urakawa, Characterization of gel structure by means of SAXS and SANS, In: Polymer Gels, DeRossi et al. (Eds), Plenum Press, New York, 1991, pp 3-19
276
Novel modified forms ofhyaluronan
7. F. Yeh, E.L. Sokolov, T. Walter & B. Chu, Structure Studies of Poly(diallyldimethylammoniumchloride-co-acrylammide) Gels/Sodium Dodecyl Sulfate Complex, Langmuir, 1998, 14,4350-4358.
WHAT IS THE ROLE OF HYALURONAN IN UMBILICAL CORD? Max Meaneyl,2 & Ole Wiebkin l I Department 0/Medicine (RAH), University 0/Adelaide, Adelaide, South Australia, 5000. ; School o/Physical Education, Exercise & Sports Science, University o/South Australia, Underdale, Australia, 5032.
INTRODUCTION
Attention to the umbilical cord has been limited to its importance as a source of cord blood, rather than for its vital biological role. It is now valued for providing human umbilical vein epithelial cells and for its potential in the cloning of anatomical spare parts. Hitherto, it had been discarded, post-natally, generally without examination. This attitude was reflected in earlier defmitions "... a stalk from embryo to placenta" (A Dictionary of Biology, 1969) and "... a bundle of two arteries and a vein" (Penguin Medical Dictionary, 1972). This relative lack of attention may be credited to the remarkably rare instances of cord pathology leading to foetal morbidity and mortality I. A few studies have examined the biomechanics ofthe cord but very little attention has been accorded to its biochemistry, in comparison to other connective tissues. Notable exceptions are Frank Meyer's reports 2,3 on the diffusion ofmolecules within the organ and the work of Edward Bankowski's group on changes in composition within the cord in pre-eclampsia 4,5. In joining placenta and foetus, umbilical cord is truly a connective tissue. It functions to maintain viability and facilitate growth of the embryo and foetus. The ready transport of oxygen, nutrients and other growth factors to, and the removal of waste products from the foetus must be assured. Untrammelled movement ofthe foetus must be allowed while, at the same time, the umbilical blood vessels are protected against the tension, twisting and compression occasioned by this activity in utero. Umbilical cord, then, must exhibit tensile strength, elasticity, flexibility and the ability to resist compression. Despite these requirements, it is very rarely that foetal distress or death can be ascribed to umbilical cord defects or dysfunction, as noted earlier I. Analysis has shown umbilical cord to contain approximately 90% water 6, linking it with articular cartilage (95%) and intervertebral disc (80%). As with these latter tissues, water and the hyaluronic acid (HA) and proteoglycans which bind it, undoubtedly have a role in resisting the compression arising from "true knots and false knots" occasioned by foetal movement in utero, Despite the high water content and a cross-sectional area ofless than I.Ocm2 , umbilical cord exhibited breaking strains in the range 4.4-8.1 kg 6, figures in excess of normal birth-weights. The umbilical cord comprises a limited number of fibroblasts embedded in an extracellular matrix (ECM), known as Wharton's jelly which carries three blood vessels, the whole enclosed in epithelium which is an extension ofthe amnion, The foetoplacental circulation is an independent reticuloendothelial system. The umbilical vein obtains nutritional requirements from the maternal circulation by diffusion across the placental villi, The
240
The role ofhyaluronan in tissues
foetal heart retums blood from its circulatory system to the placenta via two arteries in the umbilical cord. The aim ofthis study is to examine the role of collagen and, particularly, HA in maintaining patency of these structures and viability ofthe cord proper. MATERIALS & METHODS Materials
Fresh umbilical cords were obtained, immediately following Caesarian section, from otherwise normal births. Ethical requirements of the hospital were fully observed. The cords were then measured for length and weight, and again after samples had been taken for histology and moisture content determinations. The remaining portions were used as described hereafter. Tensioning and histology
End-clamped cord lengths were stretched in a humid atmosphere at 37° for two hours, using a mass of 2.0 kg. The incubation chamber was drained and filled with buffered CPC-formalin. Following a month's fixation period and subsequent processing, longitudinal sections were cut and stained using the Masson trichrome procedure. Tissue extraction and gel chromatography
Single cord samples (20-30 g) were coarsely hand-chopped and subjected to 6-10 sequential associative extractions using five volumes of 0.5 M Na acetate (pH 5.8), in the presence of protease inhibitors. These were followed by one or two dissociative (4.0 M guanidinium chloride, GuHCI) extractions. The remaining tissue pieces, by now weighing more than the original, were exposed to one or two collagenase digestions. A small undigested residue remained. Collagen determinations of all fractions were made by hydroxyproline assay. Glycosaminoglycans (GAG) from a further series of extracts were isolated and purified by quaternary ammonium and repeated ethanol precipitations, respectively. Yields were determined by uronic acid (UA) assay. Aliquots ofthe isolated GAG were subjected to gel chromatography on Sepharose 4B, with UA analysis of each eluted fraction. Compression
Intact 20-25 em lengths of cord were sealed at each end with umbilical clamps and the protruding ends trimmed and covered with adhesive tape. Torsional compression as experienced in utero was duplicated by twisting the sealed lengths in their natural direction of coiling. Compression was carried out, variously, in a humid atmosphere, in distilled water and in saline at 37°, for periods of one to four hours. A further sealed sample was stirred in saline, in a manner more resembling gentle foetal movement. GAG levels in the substantial exudates were determined by UA assay. Diffusion
Short (8-10 em) lengths ofsealed umbilical cord were immersed in 0.5% starch solution,
Role ofhyaluronan in umbilical cord
241
for various times and temperatures. After briefly washing with saline, the samples were immersed in a solution of 0.05% iodine in 1.0% potassium iodide KIJ The samples were then washed, blotted and photographed. Alternatively, identical cord lengths were exposed to starch solution, washed and cut into transverse sections by hand. The coarse slices were then transferred to KI 3 solution, as before. In a further variation on the above treatments, the order of reagents was reversed. Similar experiments were conducted with Trypan blue, alone. Perfusion
The umbilical veins of short (10-15 em) cord lengths were cannulated at each end with blunted 18 gauge needles and the ends clamped. The pieces were installed in a humid chamber at 37° and perfused with starch solution for one hour. This was followed by a saline washout and a one hour KI 3 chase. The samples were coarsely sliced and photographed. Alternatively, the cord pieces were sliced after the starch perfusion and washout, and the coarse sections immersed in KI 3 solution. The reverse order of reagent application was also undertaken. RESULTS
The water content of umbilical cord was found to be 90.38 ± 2.66% (n=25). Histological examination of normal, relaxed umbilical cord showed occasional fibroblasts associated with short, wavy bundles of collagen fibres, stained blue by the Masson trichrome procedure (Figure 1). In contrast, the collagen of stretched, fixed cord appeared as long, thick, relatively straight bundles with many stained red by the Masson procedure (Figure I) and closely resembling Achilles tendon.
Figure 1.
Relaxed and stretched umbilical cord stained by the Masson procedure.
242
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Percentages of collagen and GAG from all associative and dissociative extractions and in collagenase digestions of umbilical cord.
The sequential associative extractions of chopped umbilical cord· yielded 28% of the total collagen content. Three dissociative extractions removed a further 5%. Two collagenase digestions were required, to release a further 58% while the balance remained in a very small undigested residue. Figure 2 illustrates the extraction profile. The same figure also outlines the pattern of extraction of GAG from umbilical cord. Six sequential associative extractions removed 45% ofthe total tissue GAG, in a time-dependent fashion. That is, the amount of GAG dissolved was proportional to the number of hours of stirring in each fresh batch ofNa acetate. A single GuHCI treatment extracted a further 14% of total GAG while one collagenase digestion released the balance (40%), leaving less than 1% in the undigested residue. When GAG isolates from all extracts and the collagenase digest were subjected to gel chromatography, peaks suggesting the presence ofthree hydrodynamic species ofHA were obtained, identified as I, II and III. Figure 3 illustrates the elution pattern of the first associative extract, the dissociative extract and the collagenase digest GAG isolates. The other five associative GAG isolates showed similar elution profiles with peaks II and III becoming progressively smaller. Compressing umbilical cord by twisting in a humid atmosphere resulted in a loss of 30% of its weight. A significant amount of GAG was detected in the substantial exudate. Similar quantities ofGAG were extruded when umbilical cord was compressed in aqueous media. Table 1 lists these data and also the amount of GAG released when a cord sample was simply stirred iIi. saline. (The GAG expressed from compressed cord was assumed to be HA rather than immobilized proteoglycan and is reported as such). Exposing sealed cord pieces to starch solution and then to KI 3 solution, after an intermediate saline wash, resulted in the appearance of the classic dark blue starch-I, complex, inside of the sample. Reversing the order 'of the reagents gave the same picture.
Figure 3.
Gel chromatography of GAG isolates from the first associative extract, dissociative extract and collagenase digest of umbilical cord.
244
The role ofhyaluronan in tissues
Table1. Amount of HA expressed when umbilical cord is compressed or stirred in various environments. Treatment
Medium
Compression Compression Compression Stirring
Humid atmosphere Physiological saline Distilled water Physiological saline
Cord wt (g) lUI 10.32 12.11 18.53
HA(mg) 4.86 4.15 2.00 0.20
When, following exposure to starch and saline wash, the cord piece was sliced and the coarse sections placed in KI] solution, the starch-I, colour was observed around the hiller circumference of the cord and radiating inwards towards each of the three blood vessels (See Figure 4). Perfusing cannulated umbilical vein with starch solution or with KI] solution resulted in no penetration into the perivascular areas under all conditions.
DISCUSSION The result ofmoisture content determinations on umbilical cord confirmed a preliminary report 6 of a 90% water content. Despite this degree of hydration, the organ can sustain tensive forces well in excess ofnormal birthweights 6. Over 60% ofcord collagen resisted repeated mild and rigorous extraction procedures which is in accord with the findings of Sobolewski, Bankowski et af5 that 50% ofumbilical cord collagen resisted removal from Wharton's jelly, using different methods of extraction. These results indicate that the
Figure 4.
Starch diffusion into umbilical cord followed by iodine treatment oftransversc sections.
Role of hyaluronan in umbilical cord
245
collagen of umbilical cord is highly cross-linked and, therefore, of considerable tensile strength. Given the Achilles tendon-like appearance and staining characteristics 7 of stretched umbilical cord, it is probable that the collagen bundles are responsible for the ability of the organ to resist tension, rather than the intima of its blood vessels which, hitherto, has been the unsupported assumption. Molecules without a purpose rarely occur in living organisms, raising questions as to the functions ofthe three hydrodynamically heterogenous species ofHA found in umbilical cord. Amniotic fluid is essentially a filtrate of plasma. However, it has been shown to contain HA of two different MW ranges which the authors 8 claim to be of foetal origin. Given the permeability ofthe umbilical epithelium to HA, even under conditions simulating gentle foetal movement in utero, it seems likely that the umbilical cord contributes HA to the amniotic fluid. When extensive twisting of the cord occurs naturally, the amounts of HA entering the amniotic fluid could increase dramatically, as suggested in Table I. It is our view that the smaller MW species of HA are able to move freely within the collagen framework of the ECM, giving flexibility to the organ. It is equally likely that these smaller, more mobile molecules constitute the HA which is extruded from umbilical cord under pressure. Furthermore, movement ofthese small hydrated molecules may allow the umbilical cord to accommodate to pressure changes in the foetoplacental circulation or in the amniotic sac. The largest molecules ofHA will remain trapped in the Wharton's jelly, physically entangled in the network of fibres', Together with bound water, they would contribute to resisting compression and the maintenance of umbilical cord architecture. That the smaller HA fractions, II and III are not artefactual degradation products of the largest MW species, I, is shown by the smaller molecules tending to be removed first during repeated extractions rather than continuing to accumulate. The ready diffusion ofsmall, medium and large molecules (KI 3, Trypan blue and starch, respectively) into umbilical cord showed that the amniotic epithelium enclosing the tissue is surprisingly permeable. That this permeability is not uniform throughout the ECM but funnelled from the circumference to blood vessels nearby (Figure 4) suggested a role for these porous zones. The umbilical vein carries nutrients from placenta to the foetus. Perfusion experiments have shown that both arteries and vein are impermeable to large and small molecules. The nutritional needs of the smooth muscle cells of these blood vessels and of the fibroblasts in the Wharton's jelly must, therefore, be met by diffusion into the cord from the surrounding amniotic fluid. Foetal movement, giving rise to tension, twisting and compression ofthe umbilical cord, would act to augment the diffusion process and the transfer of metabolites. MacMillan et a/ 9 have proposed that the water flow during flexion of intervertebral discs enhances nutrient transport, in an analogous manner. In the umbilical cord, the diffusion of metabolites into the organ may be balanced, electrostatically and osmotically, by the egress of corresponding quantities of HA into the amniotic fluid. The relationship between HA in the umbilical cord and in amniotic fluid remains to be resolved. An elucidation may throw some light on the conditions of oligohydramnios and polyhydramnios. A case study has been reported in which a reduced amount of Wharton's jelly has been ascribed as the cause ofcord constriction resulting in foetal death 10. Given the demonstrated extrusion of HA from the cord under pressure, it is our opinion that the reduced ECM in that pathologically constricted umbilical cord was a result, and not a cause, ofthe condition.
246
The role ofhyaluronan in tissues
Summary 1. Umbilical cord collagen is highly cross-linked and rcsemblesAchilles tendon, providing resistance to considerable tension. 2. The HA of umbilical cord occurs as three fractions of differing molecular size, permitting organ flexibility whilst resisting compression and maintaining structural integrity. 3. Blood vessel impermeability and umbilical epithelial permeability determines that cord fibroblasts and vascular cells must obtain their nutrients from the amniotic fluid rather than the foetoplacental circulation. 4. Trans-epithelial nutrient flow may be balanced electrostatically and osmotically by a reverse movement oflow MW HA from the cord into the amniotic fluid. 5. Foetal movement would facilitate trans-epithelial diffusion of nutrients.
REFERENCES 1. J. T. Maher & J. A Conti, 'A comparison of umbilical cord blood gas values between newborns with and without true knots', Obstet. Gynecol., 1996,88,863-866. 2. F. A. Meyer, 'Macromolecular basis of globular protein exclusion and of swelling pressure in loose connective tissue (umbilical cord)', Biochim. Biophys. Acta, 1983, 755, 388-399. 3. J. Klein & F. A. Meyer, 'Tissue structure and macromolecular diffusion in umbilical cord immobilization of endogenous hyaluronic acid', Biochim. Biophys. Acta, 1983, 755,400-411. 4. E. Bankowski, K. Sobolewski, L. Romanowicz, L. Chyczewski & S. Jaworski, 'Collagen and glycosaminoglycans of Wharton's jelly and their alterations in EPHgestosis', Eur. .I Obstet. Gynecol., 1996, 66, 109-117. 5. K. Sobolewski, E. Bankowski, L. Chyczewski & S. Jaworski, 'Collagen and glycosaminoglycans of Wharton's jelly', BioI. Neonate, 1997, 77, 11-21. 6. M. F. Meaney & O. W. Wiebkin, 'The collagen and hyaluronic acid of umbilical cord may contribute much to its function', ./mmunol. Cell BioI., 1996,74 (Suppl.), A41. 7. M. H. Flint, M. Lyons, M. F. Meaney & D. E. Williams, 'The Masson staining of collagen -an explanation of an apparent paradox', Histochem . .1,1975,7,529-546. 8. J. R. E. Fraser, T. C. Laurent, H. Pertoft & E. Baxter, ' Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit', Biochem .I, 1981, 200, 415-424. 9. D. W McMillan, G. Garbutt & M. A. Adams, 'Effect ofsustained loading on the water content of intervertebral discs: implications for disc metabolism', Ann. Rheum. Dis., 1996,55, 880-887. 10. L. A. Virgillio & D. E. Spangler, 'Fetal death secondary to constriction and torsion of the umbilical cord', Arch. Pathol. Lab. Med, 1978, 102, 32-33.
PARTS
THE USE OF HYALURONAN IN DRUG DELIVERY
THE USE OF HYALURONAN IN TOPICAL DRUG DELIVERY Marc B. Brown, Ben Forbes, Manita Hanpanitcharoen and Gary P. Martin MedPharm, Dept ofPharmacy, King's College London, 150StamfordSt, LondonSE1 9NN, UK
ABSTRACT Dermal delivery for the treatment of skin disorders offers numerous potential advantages over conventional therapies including avoidance of hepatic first-pass metabolism, improved patient compliance, lower systemic absorption and reduced side effects. Previous studies by the authors have shown that hyaluronan (HA) is more effective than other gel-forming materials in localising the delivery of radiolabelled diclofenac within the epidermis of human skin. Such phenomena have also been reported in vivo in both mice and humans and have helped to facilitate the regulatory approval of a topical HAldiclofenac formulation for the treatment of actinic keratosis. However, a mechanism of action to explain the topical delivery properties of HA remains to be elucidated. The aim of this study was to compare the effect of HA with other glycosaminoglycans and pharmaceutically relevant polysaccharides on the thermodynamic activity and percutaneous penetration of diclofenac and ibuprofen. The dermal partitioning of diclofenac and ibuprofen in various concentrations of HA, chondroitin sulphate (CS), heparin (HP), sodium carboxymethyl cellulose (NaCMC) and pectin were determined. The results from these studies were then compared to Franz cell skin deposition studies. The studies demonstrated that HA significantly enhanced the partitioning of both diclofenac and ibuprofen into human skin when compared to an aqueous control, pectin and CMC (pO.05). Results from the Franz cell diffusion studies showed that HA (1% w/w) significantly enhanced the amount of drug localising within the epidermis after 24 h when compared to an aqueous control (p
KEYWORDS Hyaluronan, polysacharides, skin, drug, dermal delivery
INTRODUCTION As the medical and pharmaceutical community become increasingly aware of the importance of safety and the effective use of drugs, investigation into novel delivery methods and techniques is fast becoming one of their major areas of research. The exploitation of drug delivery via the skin offers numerous potential advantages over more extensively utilised oral or parenteral routes. These include avoidance of hepatic
250
The use ofhyaluronan in drug delivery
first-pass metabolism, improved patient compliance, and the possibility for sustained and/or controlled release. The skin can be utilised for drug delivery in two ways, transdermally and dermally (the latter is sometimes referred to as intradermal delivery). Transdermal delivery involves percutaneous delivery of the drug across the skin into the systemic circulation (i.e. the blood stream). In dermal delivery the skin itself is the target organ, such delivery cannot be efficiently achieved systemically because of the avascularity or lack of blood supply to this layer. However, the targeted delivery of drugs for the treatment of topical disorders is not trivial. An ideal delivery profile for a therapeutic active agent applied topically to treat conditions such as eczema, psoriasis or skin cancer should involve diffusion from the skin surface through the stratum corneum to accumulate at the site of drug action before subsequent metabolism to inactive products. Such delivery is however often rendered difficult due to the low permeability and lipophilic nature of the stratum corneum. Relatively few investigations have been conducted into the development of delivery systems to optimise drug targeting to the skin. However, the incorporation of hyaluronan (HA) within a topically applied vehicle containing drug has been shown, on the basis of in vitro studies, to be effective in the targeting and localisation of radiolabelled diclofenac, within the epidermis of human skin when compared to an aqueous control [1,2]. In addition, these data have been supported in vivo where radiolabelled HA was found not only to penetrate the skin of nude mice but also to aid the transport of diclofenac to the epidermis [3]. Such targeting has also been reported to occur in human subjects and has helped to facilitate the regulatory approval of a diclofenac/hyaluronan topical formulation in Europe and North America for the treatment of actinic keratosis. However, the mode of action for the hyaluronan vehicle remains unclear. Hence the aim of the current study was to investigate the effect of HA on the partitioning and diffusion of diclofenac into human skin. To assess whether any effects were specific to either the drug or vehicle, comparitor experiments were also performed using ibuprofen and a range of polysaccharides including two other glycosaminoglycans, chondroitin sulphate (CS) and heparin (HP), and other pharmaceutically relevant polysaccharide excipients, including sodium carboxymethylcellulose (NaCMC) and pectin. MATERIALS AND METHODS Materials
Sodium hyaluronate (Viscosity average Mwt. 600,000) was kindly donated by SkyePharma (London, UK). Chondroitin sulphate was obtained from Sigma (Poole, UK). Pectin, sodium carboxymethyl cellulose (high viscosity), disodium hydrogen phosphate, sodium dihydrogen phosphate, 1,9-dimethylene blue, sodium formate and formic acid were obtained from BDH (Poole, UK). All solvents used for chromatography were analytical grade. Partition cells were made on site at King's College London and were constructed from polypropylene in two parts. The lower part provided a base to support the skin sample during the partition studies, whilst the upper part, comprising a hollow threaded cylinder, was employed to constrain the skin sample in situ. Franz cells were also made on site and made from glass. They comprised a sampling port and a donor (internal diameter 0.85 em) and receptor compartment (volume 1.80 ml) which were fixed together using a clip.
Use in topical drug delivery
251
Skin preparation Human skin was obtained from patients undergoing elective abdominoplastic surgery, the donors being female aged between 25-40 y. Excised skin was frozen at 20°C within 2 h of surgery and stored until use. Full thickness skin sections (dermis and epidermis layers) were prepared by carefully removing subcutaneous fat and other debris using forceps and scalpel [1]. The skin was then cut into small circular pieces using a template of similar dimensions to the partition cell or Franz cell. Skin from the same section was used for each drug study enabling comparison of the formulation effect for the same drug but not between drugs. Skin-partitioning studies Donor solutions containing drug (10 ug/ml for diclofenac, 20 ug/ml for ibuprofen) and 1% w/w polysaccharide were prepared in deionised water (control was deionised water alone, DW) and allowed to hydrate for 24 h. The skin was mounted in the partition cells with the epidermis side up, and the unit was then placed into an air tight 50 ml glass jar containing 20 ml of the donor solution such that the layer of skin was completely immersed. The bottle was then transferred to a water bath (32°C) and shaken for 48 h. Preliminary experiments showed that this time was required for equilibration to be established. Samples were then removed and assayed for drug or content by HPLC assay respectively. The amount of drug which partitioned into the skin was measured indirectly by the loss of compound from the donor solution. The percentage of skin partitioning was calculated from concentrations of compound in the donor solution before and after equilibrium. Control experiments were performed with no skin in the partition cell to determine any loss due to adsorption to the partition cell or glass. Results are expressed as mean ± s.d for n = 4 experiments. Franz cell experiment The small sections of full thickness skin were mounted, stratum corneum side up, in the Franz cells. The receptor compartment was filled with Hank's balanced salt solution pH 7.4, after which the diffusion cell was placed on a stirring plate in a water bath maintained at 32°C. The receptor fluid was continuously stirred using a small tefloncoated magnetic bar to ensure complete mixing of the receptor fluid. The diffusion cells were allowed to stand for 12 h in order to equilibrate the skin and also to wipe any visceral debris that may have remained on the dermal side of the skin. After equilibration, the receptor fluid was changed by replacing with fresh Hank's balanced salt solution and any air bubbles accumulated inside the receptor compartment or at the skin/receptor interface, were removed by tilting the cell. Subsequently, 50 ug of each formulation containing 1.75% w/w diclofenac or 20% w/w ibuprofen in 1% w/w polysacharides (or deionised water) unless otherwise stated, was applied to the surface of the skin (n = 4 for each formulation) and rubbed in with a circular motion both clockwise and anti-clockwise, using a pre-weighed glass rod. Any losses of formulation on the rod were compensated for in the final mass balance calculations. Throughout the 48 h of the experiment, the receiver fluid was stirred to ensure homogeneity, and was also maintained at the same level as the skin. After 48 h, the experiment was terminated and the skin removed from the diffusion cell to enable the mass balance study to be performed.
252
The use ofhyaluronan in drug delivery
Mass balance After completion of the diffusion experiment, a mass balance experiment was performed in order to quantify the amount of drug on the skin, in the skin and in the receiver fluid. The extraction techniques, which involved homogenisation, collagenase digestion, ultracentrifugation and solvent extraction, were all validated and proved to be 100% efficient in recovering diclofenac and ibuprofen from all of the sample matrices. Drug concentration was measured using HPLC analysis as follows: HPLC analysis of drug concentration The HPLC analysis of diclofenac and ibuprofen was performed using a 15 em x 4.6
mm J.D. Spherisorb RP-C18 (5 urn) column (Hichrom Ltd., Reading, Berkshire, UK), with a 10 mm Cl8 guard column (S50DS2-1OC5) (Hichrom Ltd., Reading, Berkshire, UK) using a CM 4000 pump and a CI-4100 integrator connected to a SpectroMonitor 3100 UV detector (all LDC Analytical, Florida, USA). Analysis was performed isocratically with a mobile phase comprising 73% phosphate buffer (45 mM pH 7) and 27% acetonitrile:tetrahydrofuran (7:3 v/v). All samples were made up in mobile phase, containing internal standard (20 ug/ml), to concentrations within the predetermined calibration curve. For diclofenac analysis, ibuprofen was used as the internal standard, and vice versa. An injection volume of 100 ul was used, the samples were eluted at a flow rate of 1.10 ml/min and monitored at 273 om. RESULTS AND DISCUSSION Partition studies The skin absorption of drugs is a partition-diffusion process. First the drug partitions from the formulation into the stratum corneum and then, after diffusing across the stratum corneum, it partitions into and diffuses across the epidermis and the processes are repeated as the drug moves to deeper skin layers. The ability of a drug to partition into the skin is dependent upon a number of physicochemical properties of the drug 20 18 16 14 12 10 8 6
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Use in topical drug delivery
18 16 14 12 10 8 6 4 2
253
(0.001)
o control Figure 2
HA
CS
HP
NaCMC pectin
The effect of 1% w/w polysaccharides on the dermal partitioning of 20 ug/ml ibuprofen, n=4, mean±SD (P-value compared to control)
including its solubility in the applied vehicle, lipophilicity, ionisation state and partition coefficient between the vehicle and the skin all of which influence the thermodynamic activity. The effects of 1% w/w polysaccharide concentrations on the skin partitioning of diclofenac and ibuprofen are given in figures 1 & 2 respectively. The results demonstrate that all three glycosaminoglycans: HA CS and HP significantly increased the partitioning of diclofenac and ibuprofen into the skin compared to the aqueous control. The extent of the increase induced by HA however was greater than that for both CS and HP (although not significantly) at the same w/w concentrations. In contrast, NaCMC, commonly used as a viscosity enhancer in transdermal formulations, effected a decrease in diclofenac partitioning and had no effect on the partitioning of ibuprofen. Pectin was found to have no significant effect on the partitioning of either diclofenac or ibuprofen. The effect of such partitioning characteristics on dermal drug delivery was then investigated in vitro using human skin mounted in Franz cells. Franz cell studies The amount of drug on the surface of the skin and diffused through to the receptor compartment was determined 48 h after application of 1.75% w/w diclofenac or 20% w/w ibuprofen formulated in all the 1% w/w polysaccharide formulations. For diclofenac none of the polysaccharides had a significant effect on the amount of drug remaining on the surface compared to the control (data not shown). However for ibuprofen, HA significantly reduced the amount of drug remaining (pCS>NaCMG"DW;:::pectin. Significantly more diclofenac and ibuprofen was delivered into the skin when applied in HA or CS, when compared to the other polysaccharides and the aqueous control. In addition, it was observed that HA could deliver significantly more drug to the skin when compared to CS (p
254
The use ofhyaluronan in drug delivery
25 (<0.001)
OJ
«l
s::
~ 0"0 -u .... Q) .- Q) "0
>
Od)
20
(0.01) (>0.05) (0.001)
15
Q)"O
'"
;;.., 0_
"Oc;j
10
] § -0.."0 Q)
5
~
0
g.
----,
control
Figure 3:
HA
CS
NaCMC
pectin
The effect of 1% w/w polysaccharide on the percentage of the applied dose of diclofenac (1.75% w/w) remaining in the skin after 48 h, n=4, mean±SD (P-value compared to control)
trends were observed. The driving force for passive transport through a membrane is the chemical potential gradient across the membrane [4]. To create the gradient necessary to deliver a drug across the skin, it is necessary to dissolve a drug in a solvent or vehicle to establish a certain concentration, and thus activity of the drug at the outer surface of the skin. Since different vehicles have different capacities to dissolve drug, at any fixed level of concentration, different levels of thermodynamic activity of the drug will be obtained. In principle, the simplest way to maximise delivery of a drug is to formulate it at maximum solubility to achieve the highest thermodynamic activity [5] (e.g. saturated
s::
~ 0
l5."g ....
;:l
,s
~ ._
o~
Q)"O '" _;;.., 0 "O«l
]0.. ~ 0..
«l
~
'0
16 14 12
(0.001)
10 8 6 4 2
ocontrol
Figure 4:
HA
CS
NaCMC
pectin
The effect of 1% w/w polysaccharide on the percentage of the applied dose of ibuprofen (20% w/w) remaining in the skin after 48 h. n=4, mean±SD (P-value compared to control).
Usc in topical drug delivery
255
systems). However the results depicted in Figures 3 & 4 were obtained at concentrations of the drugs equivalent to the lowest maximum solubility in any of the vehicles i.e. the limit of solubility NaCMC. Thus, further studies were performed in which the drugs were formulated at their maximum solubility in HA, NaCMC and DW i.e. at a thermodynamic activity of unity. The results obtained (data not shown) showed that a significantly higher proportion of diclofenac and ibuprofen was delivered to the skin (p<0.05) and significantly less to the receiver fluid (p<0.05) when applied in HA when compared to CMC and DW. It is apparent that the tendency to promote the localisation of the drugs within the skin was more apparent when HA was incorporated in the topical vehicle that when other glycosaminoglycans or other pharmaceutically relevant polysaccharides were utilised. It is also too simplistic to explain such enhanced dermal delivery by a change in drug thermodynamic activity in the presence ofHA. It is known that the degree of hydration of the stratum corneum influences skin permeability. Increased hydration opens the compact substance of the stratum corneum by loosening the dense, closely packed cells thus increasing the permeability to many drugs [6]. Hydrophobic patches and oleaginous formulations are based on such a principle in that they occlude the skin, increasing stratum corneum hydration resulting in enhanced percutaneous delivery of the formulated drug. Polysaccharides are regarded as moisture-control agents [7] which is the reason for their incorporation in many topical and cosmetic preparations. However, the skin hydration properties of HA are considered to be much higher than other polysaccharides because of its considerable capacity to bind water [8]. Thus, the topical application of HA may result in increased hydration of the stratum corneum and enhanced dermal delivery of a concomitantly applied drug. However, such properties do not account for drugs formulated in HA being retained in the skin with little systemic absorption, as demonstrated in previous studies [2, 9]. Brown et al [3] have already examined the movement ofHA into the keratin, epidermal and dermal layers of mouse and human skin and found that radiolabelled HA is apparently absorbed rapidly from the surface of the skin and into the epidermis. Thus, underlying skin HA receptors [10] and the hydrophobic patch within the hydrated structure ofHA [11] may both playa role in the dermal drug delivery properties ofHA although this remains to be elucidated. CONCLUSIONS
Hya1uronan is a naturally occurring polysaccharide which is biocompatible and nonimmunogenic, rendering it an ideal formulation aid. The results in this study demonstrate that the inclusion of hyaluronan as a vehicle excipient offers clear potential in the dermal delivery and localisation of drugs. Such localisation would be desirable for the dermal use of many drugs e.g. corticosteroids, immunosuppressants, antihistamines, anaesthetics, retinoids, sex hormones, pediculicides, rubifacients, antifungal, antibacterial, antiparasitic and antiviral agents, for a variety of skin disorders. This is especially true in the case of the delivery of cytotoxic agents for the treatment of skin cancer or psoriasis, where the adverse side effects of the drugs when delivered systemically can cause considerable problems. In addition, although the mode of action of HA still remains to elucidated it is likely, in part, to involve changes in the hydration state of the stratum corneum which could effect the pharmacodynamic disposition of the concomitantly applied drug.
256
The use of'hyaluronan in drug delivery
ACKNOWLEDGEMENTS The authors would like to thank SkyePhanna for their support of this work. REFERENCES M. B. Brown, C. Marriott and G. P. Martin, 'The effect ofhyaluronan on the in vitro deposition of diclofenac within the skin', Int. J. Tissue Reac., 1995, XVII, 133-140 2. G. P. Martin, F. C. Bennett, C. Marriott and M. B. Brown, 'An in vitro study of the diclofenac delivery properties ofl-IA in hwnan skin', Drug De/., 1999,6,39-43 3. T. I. Brown, D. Alcorn and R I. E. Fraser, 'Absorption ofhyaluronan applied to the surface of intact skin',J. Invest. Dermatol., 1999, 113,740-746 4. I. Hadgraft, 'Recent developments in topical and transdennal delivery', Eur. J. Drug Metab. Pharmacokin., 1996,21,165-173 5. K. L. Smith, 'Penetrant characteristics influencing skin absorption', In: Methods for Skin Absorption, B. W. Kemppainen & W. G. Reifenrath (eds.), CRC Press, Florida, 1990, pp24-34 6. D. Bucks and H. I. Maibach,'Occlusion does not uniformly enhance penetration in vivo' In: Percutaneous absorption, Drugs, Cosmetics, Mechanisms, Methodology, R. L. Bronaugh & H.I. Maibach (eds), Marcel Dekker, New York, 1999, pp 81-108 7. R L. Whistler, 'Introduction to industrial gums', In: Industrial Gums: Polysaccharides and Their Derivatives, R L. Whistler and J.N. BeMiller, (eds.) Academic Press, San Diego, 1993, pp 1-3 8. M. K. Cowman, I. Liu, M. Li, D.M. Hittner, and I.S. Kim, 'Hyaluronan interactions: salt, water, ions', In: The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T.C. Laurent, (ed.) Portland Press Ltd, London, 1998, pp. 17-25 9. W. Lin and H. I. Maibach, 'Percutaneous absorption of diclofenc in hyaluronic acid gel: in vitro study in human skin', In: Hyaluronan in Drug Delivery, D. Willoughby (ed) RS.M. Press, London, 1996, pp 167-174 10. S. Gustafson, 'Hyaluronan in drug delivery', In: The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T.C. Laurent, (ed.) Portland Press Ltd, London, 1998, pp 291-304 11. I.E. Scott, 'Secondary structures in hyaluronan solution: chemical and biological implications', In: The biology of hyaluronan, D. Everend & I. Whelan (eds), I. Wiley & Sons, Cirencestor, 1989, pp 6-15 1.
A NOVEL CROSSLINKING PROCESS FOR HYALURONAN Xiaobin Zhao *, Jane Fraser, Catherine Alexander Vitrolife UK Ltd.• Heriot Watt Research Park. Edinburgh EH14 4AP, UK
ABSTRACT
Hyaluronan has great potential in medicine as a biomaterial. However, in its native form, hyaluronan is rapidly metabolized in vivo by free radicals and enzymes such as hyaluronidase, and it is highly soluble. Various methods have been adopted therefore, to modify the physicochemical properties of hyaluronan, while maintaining biocompatibility, and thereby widen its spectrum of therapeutic applications. Hyaluronan has four reactive groups (acetamido, carboxyl, hydroxyl and the reducing end) available for crosslinking to itself or other polymers. Using a variety of crosslinking agents, researchers have developed a host of crosslinked hyaluronan derivatives with an increased in vivo residence time. This chemical modification has enabled the production of gels and films, which can be used in applications such as the prevention of post-surgical adhesions, wound healing and dermal augmentation. We have found that if hyaluronan is crosslinked to itself, or to other polymers (either synthetic or biopolymer), in two stages, then a high degree of crosslinking is achieved with improved biostability. In each of the two stages, the same crosslinking agent is used, but different functional groups are bound by altering the reaction conditions. The novel process can be tailored to yield water insoluble gels and films with a broad range of physical and chemical characteristics, and greater resistance to degradation by hyaluronidase and free radicals. These derivatives are currently undergoing biocompatibility testing, and should ultimately lead to a series of innovative secondgeneration medical products. KEYWORJl)S
Hyaluronan, crosslin king, biostability, hyaluronan derivatives, hyaluronan-polymer matrix INTRODUCTION
Hyaluronan (hyaluronic acid; sodium hyaluronate), abbreviated as HA, was first discovered by Meyer and Palmer in 1934 I. Hyaluronan is a long linear polysaccharide comprising of repeating disaccharide units of N-acetylglucosamine and D-glucuronic acid. The molecule has unique rheological properties, which allow it to behave as a viscoelastic gel even at low concentrations. HA is widely distributed throughout the body and forms the capsule of some bacterial species. It is found in particularly high concentrations in the vitreous humor of the eye, synovial fluid of joints and umbilical cord. As a general constituent of the extracellular matrix, HA in combination with collagen, chondroitin sulphate and other glycosaminoglycans is able to support cell and tissue growth. The viscoelasticity and the hydrophilic nature of hyaluronan enables it to retain the tonicity and elasticity of the
278
Novel modified forms ofhyaluronan
tissue in which it is incorporated. In addition it acts as a lubricant when present in body fluids and can maintain the shape of a body cavity when compromised. These physicochemical properties coupled with the biocompatibility and non-immunogenic nature of the molecule enable its use in a wide range of clinical applications. As a biocompatible hydrogel, HA has received increasing attention for use in the controlled delivery of drugs 2, wound healing 3, prevention of post-surgical adhesions 4, the correction of dermal deformities such as depressed scars and wrinkles and for the augmentation of soft tissues." HA is bioresorbable and is easily degraded in vivo due to the action of the enzyme hyaluronidase and free radicals. This restricts its use in some medical applications, which require a long-lasting effect", Therefore, in order to increase the residence lifetime of HA to extend its potential applications it is essential to modify HA whilst retaining its biocompatibility. Modification of HA can be achieved via chemical modification. This can be divided into chemical derivatisation and chemical crosslinking. Partial or total esterified HA can be produced by chemical derivatisation of the carboxyl group of the polymer with an alcohol 7. Through such modification, the residence time of the material is increased and the properties can be tailored by the esterification degree", Chemical crosslinking is commonly used to produce strong h~drogels, using crosslinkers such as divinyl sulfone", glycidyl ether'", polyepoxide I, and glutaraldehyde'f. The biostability and other physical properties of crosslinked HA have been improved by crosslinking the hydroxyl groups on the HA polymer. The use of water-soluble carbodiimide has also been reported to crosslink HA through the reaction between the carboxyl and hydroxyl groups of HA or available amino groups13,14. HA has been reported to react with synthetic polymers and biopolymers to form conjugates". We have found that a crosslinked HA network can be built through crosslinking between both carboxyVcarboxyl and hydroxyl/hydroxyl groups of HA or HAJpolymer combinations, which we call double crosslinking technology, as shown in Figure 1. According to this technology, in the first step, stable ether-linkages are obtained by crosslinking through hydroxyl groups. The second step involves the synthesis of esterlinkages obtained by crosslinking through carboxyl groups. We selected other polymers such as non-ionic synthetic polyvinyl alcohol (PV A) and ionic biopolymer sodium alginate, for combination with HA to perform our double-crosslinking technology. This novel process has allowed the generation of HA and HAJpolymer derivatives with improved biostability.
--r---
first step
~
"",ond"ep.
~ ....-+-......--
HA Intermediate (First Crosslink)
HA
---- Ether linkage
Double Crosslinked HA
••••• Ester linkage
Figure 1 Scheme of novel process of double crosslinked HA.
Cross-linkingprocess MATERIAJ~
279
& METHODS
Synthesis of crosslinked HA A solution of HA (10%) was prepared in 0.25M NaOH. Crosslinking agent epichlorohydrin or 1.2.7,8-diepoxyoctane was added and the reaction was carried out in a petri dish. After 72 hours drying in a fume-cupboard at room temperature, a dry sheetlike material was obtained. This material was referred to as HA intermediate. The dried sheet was neutralised with HCI solution and purified by washing the film with acetoneldeionised H20 (7/3(v/v», IPAI deionised H20 (7/3(v/v», and IPA. Thus the first crosslinked HA intermediate was obtained. The intermediate was subjected to a second crosslinking step by suspending it in acetoneIHCI (7/3(v/v» at pH < 4.0. To this crosslinking agent,I,2,7,8-diepoxyoctane, was added. The crosslinking conditions are shown in Table 1. After crosslinking for 24 hours, the product was washed with IPA. IPAideionised H20 (7/3(v/v». IPA and acetone. The samples were dried to obtain a constant weight. Table 1 Synthesis of crosslinked HA (CHA) from HA solution via HA intermediate
First crosslink Sample CHA-l CHA-2 CHA-3 CHA-4
Crosslinker E-l E-l E E
Feeding ratio 0.375/1
0.37511 0.375/1 0.375/1
Time (hr) 72 72 72 72
Second crosslink pH OIr OIr OIr OIr
pH
Feeding ratio
Time I 24
1
1 0.5/1 1
1
1
E
0.511
24
W
Crosslinker 1
E
(hr) 1
W
E-l: epichlorhydrin (Aldrich); E: 1.2.7.8-diepoxyoctane(Aldrich); Feeding ratio: the weight ratio ofHA to crosslinker. All reactions were performed at room temperature.
Synthesis of crosslinked PVAIHA (CPR) A 1% HA solution and a 5% PVA aqueous solution were prepared respectively. The solutions were mixed together to give a homogeneous HAlPVA solution with varied HA compositions as depicted in Table 2. The solution was cast in a petri dish and dried in a fume cupboard for 4 days. The resulting film was suspended in a mixture of CHCh 1 acidic or alkaline solution I crosslinker (1,2,7,8- diepoxyoctane or glutaraldehyde). The crosslinking reaction was performed at room temperature for a fixed time period of 24 hours. A further amount of crosslinking agent was added, and if necessary the pH was adjusted. The second crosslinking reaction was allowed to proceed for 24 hours at room temperature. The detailed crosslinking conditions are shown in Table 2. After crosslinking, the samples were washed with IPA and acetone three times and immersed into IPAideionised H20 (3/2 (v/v» overnight at room temperature. The film was finally washed with acetone and dried in an oven at 37°C until a constant weight was obtained.
280
Novel modified forms ofhyaluronan
Table 2 Formation of crosslinked PVA-HA (CPR) Samples
HA (%)
CPH-2 CPH-3 CPH-4 CPH-6 CPH-7 CPH-8 CPH-13 CPH-l CPH-5 CPH-9 CPH-1O CPH-ll CPH-12
10% 10% 10% 20% 20% 20% 50% 10% 20% 30% 50% 50% 50%
Crosslinker Istl2nd FJE FJE FJE GIE FJE FJE FJE G G G G FJE FJE
pH 1st/2nd OHW/Olf OlflW
W W/Olf OlflW OlflW
W W W W Olf
W
Time 1st/2nd 24h124h 24h124h 24h124h 24h124h 24h124h 24h124h 24h124h 24h 24h 24h 24h 24h124h 24h124h
WAC (%)
280 340 250 600 580 480 258 300 dissolved dissolved dissolved 3117 1084
E: I,2,7.8-diepoxyoctane; G: glutaraldehyde; H+ represents a pH of about 4; OH represents a pH of about 10; CPH-I, 5,9,10,11,12 were all prepared using a single crosslinking step for comparative purposes; WAC(%): Water absorption capacity (%) Synthesis of crosslinked HA.Alginate film A 2% sodium alginate aqueous solution was mixed with a 1% HA in phosphate buffered saline to give a HAlAlginate solution. 10ml of the above homogeneous solution were cast into a petri dish and CaCh solution added. The resulting HAlalginate slab was washed three times with deionised H20 and was allowed to dry in a fumecupboard for 72 hours. The resulting film was suspended in a solvent/alkaline solution and 1,2,7,8-diepoxyoctane was added for the first crosslinking proceeded at room temperature. The resulting film was washed with IPAideionised H20, suspended in acidic solvent and a further volume of crosslinker 1,2,7,8-diepoxyoctane added to carry on the second crosslinking. The detailed crosslinking conditions are shown in Table 3. The crosslinked film was washed with acetoneldeionised H20 (3/2(v/v» solution three times followed by three washes with IPAideionised H20 (3/1(v/v». The film was immersed in IPAldeionised H20 overnight. The film was allowed to dry in a fume cupboard. Table 3 Fonnation of double-crosslinked Alginate I HA (CAB) Samples HA(%) CAH-l CAH-2 CAH-3 CAH-4
90 90 50 50
Crosslinker 1st 12nd FJE FJE FJE FJE
eaZ+ConcD (M)
0.25 0.5 0.25 0.5
Times (hr) 1st/2nd 24/24 24/24 24/24 24/24
WAC (%)
2543 3342 908 1449
Cross-linking process
281
Characterisation of Crosslinked HA A. Solid-state
/3 C-NMR
characterisation
The solid-state 13C_NMR analysis of the uncrosslinked HA and double crosslinked HA was carried out at 50MHz using an advance 200 spectrometer. The spectra were obtained using a contact time of 1ms in the standard cross polarisation (CP) pulse sequence. B. Water absorption capacity measurement
20mg (Wd) of each dried crosslinked sample were immersed in phosphate buffered saline (PBS) solution for 24 hours to obtain a fully swollen gel. The wet gel was filtered and the residual water at the surface removed using fiber-free tissue paper. The wet gel was weighed to obtain Ws. Thus the water absorption capacity (WAC%) was calculated according to formula (1): WAC (%)=(Ws-Wd)lWd x 100
(1)
C. In vitro biostability assessment of crosslinked HA
I. Resistance to Hyaluronidase
20mg of crosslinked HA was suspended in 6ml PBS (pH=7.4) containing 1000D hyaluronidase and incubated at 37°C for 24 hours. The film was removed and rinsed using PBS. The rinse solution and incubation solution were made up to a known volume and boiled for 30 minutes to precipitate the hyaluronidase. The solution was centrifuged and the supernatant made u~ to 25ml using PBS. The HA concentration was measured using the Carbazole assay 6. Hyaluronidase solution without crosslinked HA was selected as ~L control. The HA weight loss (%) due to hyaluronidase digestion was calculated using formula (2): HA weight loss (%)
={HAj x 25/ {HAjo x 100
(2)
in which, [HA] (mg/ml) is the concentration of HA, [HA]o is the original HA content (mg).
2. Resistance to free radicals
Fenton's reagents were used to create free radicals, which were formed by the addition of 25~1 O.lM HzOz and 25~1 O.lM ascorbic acid to 5ml PBS solution. The digestion was allowed to proceed for 24 hours at 37°C. After which, the film was removed and rinsed using PBS. The incubation and rinsing solutions were collected to obtain a lOml solution. The HA concentration was measured using the Carbazole assay". Fenton's reagentlPBS solution without crosslinked HA was selected as a control. The HA weight loss (%) due to free radical degradation was calculated using the above formula (2).
RESULTS AND DISCUSSION
282
Novel modified forms of hyaluronan
Water absorption capacity (WAC%) of crosslinked HA
As a hydrogel, the measurement of water absorption capacity of crosslinked HA is often used as an index for the assessment of the degree of crosslinking. Normally, an increase in the degree of crosslinking will lead to a reduction in the water absorption capacity". To evaluate the effectiveness of our double crosslinking technology, the water absorption capacity of crosslinked HA or HAJpolymer was measured. Figure 2 and Table 2 give the WAC (%) results of single crosslinked and double crosslinked samples. 3 0 0 0 2 5 0 0 2 000
~
U
1 500
~
1 000
-e
500 0 CHA-1
CHA·2
CHA-3
Crosslinked
H
CHA·4
A
Figure 2 Water absorption capacity (WAC%) of crosslinked HA
12
12
~ '" .,g
~ ~ .s
10
8 6
1: OIl
1: "'l 4
~
~
2
o
10
8 6 4 2 0
CHA-3
CHA-4
Crosslinked HA
Figure 3 Resistance to hyaluronidase
CHA·3
CHA-4
Crosslinked HA
Figure 4 Resistance to free radicals
Figure 2 shows that the water absorption capacity is reduced after the second crosslinking of the single crosslinked HA. The same trend is observed when HA is combined with PVA. As shown in Table 2, the single crosslinking can only produce either water-soluble products (CPH-5, 9,10) or high water-uptake products (CPH-ll and 12). In contrast, double crosslinking can produce water insoluble hydrogels with varied water absorption capacities, which are related to the HA composition and crosslinking conditions. In comparison with their single crosslinked counterparts the water-uptake of the double crosslinked gels is considerably lower. The combination of HA and sodium alginate, was found to be influenced by the presence of calcium ions, which increased the water absorption capacity of the gel formed (Table 3). The increase in calcium ion concentration may reduce the carboxyl group reactivity due to ionic complexation, which may leave the carboxyl group free for absorbing more water (Table 3).
Cross-linking process
283
In vitro biostability study
The hyaluronidase and free radical digestion of crosslinked HA and HNpolymer were selected for determining the in vitro stability. The results shown in Figures 3 and 4 indicate that double crosslinking technology can produce a significantly more stable crosslinked HA network. The incorporation of other polymers within the HA network can produce materials with various physical properties and biodegradability. Solid State 13C.NMR characterisation of crosslinked HA Figures 5 and 6 depict the solid state 13C_NMR. spectra of uncrosslinked HA and double crosslinked HA. This reveals the presence of a long-chain methylene bridge (2040 ppm) in the crosslinked HA network. In addition, the chemical shift at 165-170 (shoulder) indicates the formation of an ester-linkage.
Figure 5 Solid state 13C_NMR. spectrum of HA !i " II
Ii
I·i • I.
, ., I I.
I
I: , I
.~~
100
...
_---~_._-~---~~
Figure 6 Solid state 13C_NMR spectrum of crosslinked HA
284
Novel modified forms of hyaluronan
CONCLUSIONS This novel double crosslinking technology enables the production of various HA derivatives with different degrees of crosslinking. The technology has also been shown to be suitable for crosslinking HA with other polymers. We have shown using this novel process that hyaluronan whether crosslinked to itself or other polymers produces HA derivatives, which have a high degree of crosslinking and significantly improved biostability. Thus a range of water insoluble films and gels can be produced with properties tailored to meet the needs of their application. Biocompatibility testing of various biomaterials produced using this method is currently under investigation.
REFERENCES l. K. Meyer & J.W.Palmer, 'The polysaccharide of the vitreous humor', 1. BioI. Chern.,
1934,107,629-634 2. K.P. Vercruysse, G.D. Prestwich, 'Hyaluronate derivatives in drug delivery', Crit Rev Ther Drug Carrier Syst., 1998,15(5),513-55 3. M.T. Longaker, N. Adzick, 'The biology and therapeutic implications of fetal wound healing' , Clinical Materilas, 1991, (8) 223-227 4. D.B. Johns, T.e. Kiorpes, K.E. Rodgers, G.S. diZerega et al, Reduction of adhesion formation by postoperative administration of ionically crosslinked hyaluronic acid, Fertility and Sterility, 1997,68,137-42 5. F. Duranti, G. Salti, B. Bovani, M. Calanda, M.L Rosati, 'Injectable hyaluronic acid gel for soft tissue augmentation: A clinical and histological study' , Dermatol Surg, 1998,24,1314-1316. 6. E. Bell, 'Strategy for the selection of scaffolds for tissue engineering'. Tissue Eng. 1995,1(2), 163-79. 7. A Rastrelli, M. Beccaro, F. Biviano, G. Calderini, A. Pastorello, 'Hyaluronic acid esters, a new class of semisynthetic biopolymers: Chemical and physico-chemical properties'. Clin Implant Mater 1990, 9,199-205. 8. AD. Campoccia, P. Doherty, M. Radice, P. Brun, G. Abatangelo, D.F. Williams, 'Semisynthetic resorbable materials from hyaluronan esterification', Biomaterials 1998,19,2101-2127. 9. E.A Balazs, A. Leshchiner, 'Crosslinked gels of hyaluronic acid and products containing such gels', US Patent, No 4,582,865,1986. 10. N.Yui, T.Okano, S.Yasuhisa, 'Inflammation responsive degradation of crosslinked hyaluronic acid gels', J Controlled Release 1992,22,105-116 11. T.C. Laurent, K. Hellsing, B. Gelotte, 'Crosslinked gels of hyaluronic acid', Acta Chern Scand. 1964,18,274-275 12. K'Tomihata, Y.Ikada, 'Crosslinking of HA with glutaraldehyde', J Polym Sci Part A-Polym Chern 1997,35(16),3553-3559 13. P. Bulpitt, D. Aeschlimann, 'New strategy for chemical modification of hylauronic acid: Preparation of functionalised derivatives and their use in the formation of novel biocompatible hydrogels' , J Biomed Mater Res 1999,47,152-169 14. K. Tomihata, Y. Ikada, 'Crosslinking of hyaluronic acid with water-soluble carbodiimide', J. Biomed Mater Res 1997,37,243-251 15. W.M. Rhee, R.A Berg, 'Glycosarninoglycan-synthetic polymer conjugates', USP 5,510,121, 1996. 16. T. Bitter and H. M. Muir, 'A modified uronic acid carbazole reaction' , Analytical Biochemistry, 1962,4,330-334.
A BIOCOMPATIBLE GEL OF HYALURONAN Akio Okamoto* & Teruzoh Miyoshi Research Center, Denki Kagaku Kogyo KK, 3-5-1 Asahi-machi, Machida, Tokyo 194-8560, Japan
ABSTRACT Herein we report on a novel biocompatible gel of hyaluronan. The novel gel is prepared from acidic solutions of hyaluronan by freezing without any cross-linking reagents or chemical modification. The degree of gelation or the stability of the gel formed depends on pH, temperature for freezing, time of freezing period. In other words, the stability of the gel or the sustainability of hyaluronan release is controllable. Although the exact nature of the cross-linking has not been identified yet, the branched-chain structure was detected by GPC-MALLS measurements for the fragment of the hyaluronan gel. This novel material consisting only of hyaluronan provides opportunities to substitute for naturally occurring hyaluronan being used already in a broad range of applications as well as to develop new applications such as controlled release of pharmacologically active compounds, cell encapsulation matrices, and certain aspects of wound treatment.
KEYWORDS Hyaluronan, freezing, gel, stability, sustainability, biocompatible material
INTRODUCTION Interest in hyaluronan (HA) has increased dramatically since about 1980, when major clinical applications in ophthalmology and in the treatment of joint disease were introduced in cooperation with industrial production of the polymer. The utilization of HA in medicine has also created considerable interest in methods to derivatize HA and thereby expand its therapeutic applications'. The ubiquitous distribution in nature is one of the unique advantages of HA as a starting point for medical products. Among other unique advantages of HA in medicine are its extremely viscoelastic properties" and the magnitude and pathways available for systemic HA metabolism 3. Although diverse reactions have been applied to HA for chemical derivatization", none of the materials synthesized has been used in clinical medicine except a class of derivatives generally referred to as Hylan polymers which are cross-linked by treating HA with aldehydes
286
Novel modified forms of hyaluronan
and/or divinyl sulphone'. The primary goal of the extensive recent efforts to derivatize HA is to customize HA for specific medical needs by improving rheological properties or by creating new physical forms, such as solids or gels, while maintaining its natural biocompatibility". Modification of the native HA should be limited to the minimum change required to bring about the customization, and ultimately customization without chemical modification is the ideal. An example we describe here is a novel gel of HA prepared without any cross-linking reagents or chemical modification.
EXPERIMAENTAL Gel preparation, Degree of Gelation and Half-life Period
Typical conditions for the gel preparation of HA are as follows. Sodium hyaluronate with a molecular weight of 2 million Da was dissolved in distilled water to give 1 wt % aqueous solution. The pH of the HA solution thus obtained was 6.0. The pH of the aq. HA solution was adjusted to 1.5 with IN hydrochloric acid and was placed in a refrigerator set at -20 °e for 63 hours and then thawed at 25 °e. Thus obtained HA gel was immersed in a phosphate buffer-physiological salt solution for neutralization at 5 °e for 24 hours and then washed thoroughly with distilled water. The degree of gelation was calculated from the weight of the dried HA gel. Half-life period was obtained from the solubility test for the HA gel. A piece of HA gel was immersed in 50ml of the buffer-physiological saline based on 20mg of dry weight. The solution mixture was slowly stirred at 60 °e for the prescribed time. The buffer-physiological saline solutions of pH 6, 7 and 8 were prepared by phosphate buffer at a concentration of 50mM in room temperature, then those of pH 4 and 5 were adjusted by acetic acid/sodium acetate at the same condition of pH6, 7 and 8. The degree of dissolution of HA in buffer-physiological saline at 60 °e was obtained from the concentration of HA in a part of the supernatant solution measured by OPe. Half-life period was determined from the plots of the residual amount of gel against time. Fragmentation of HA Gel and Molecular Weight Measurement
A freeze-dried HA gel sheet of 15 mg was immersed in 15 ml of aqueous hydrochloric acid solution at pH 1.5. The solution mixture was left in an oven set at 60 °e. After 12 hours, the HA gel sheet had disappeared completely. Molecular weights of both the fragment of HA gel and a reference HA are measured by OPC-MALLS. The fragment of HA gel or the reference linear HA was dissolved in an eluent of 0.2M aqueous sodium nitrate to 0.05 wt % and filtered through a membrane filter of 0.2 J,Lm and 0.1 ml portions of them were injected. The measurements were carried out by using a OPC column SB806HQ (Showa Denko), a differential refractometer 830-RI (lASCD Co.), and a MALLS DAWNDSP-F (Wyatt) at a flow rate of 0.3 ml/rnin at «rc.
Biocompatible gel
287
Preliminary in vivo Persistency Test A dried HA gel sheet and a freeze-dried HA sheet as a control were cut into 1 cm x 1 cm squares. Five female Sprague-Dawley rats were housed for a minimum of 1 week before surgery. Each rat was anesthetized with a single intramuscular injection of 85 mg/kg ketamine hydrochloride and 5 mg/kg xylazine hydrochloride. A rotary abrasion device with a motor-driven rotating spine shaft was used to induce abrasion injury. Then a HA gel sheet was applied onto the one abraded site (the right site in Figure 4) and a freeze-dried HA sheet was to the other site (the left in Figure 4). After 1,4, 7, 10 and 13 day(s), the condition of each sheet was evaluated. Residual HA was dyed with Aldan Blue saline solution.
RESULTS AND DISCUSSION A novel HA gel is prepared from acidic solution of HA by freezing (or repeating freezing-thawing cycles) without any materials such as cross-linking reagents, polymers with different charge, chemical modifiers, etc. The gel is obtained in the form of a sponge or a wad of cotton with the total shape depending on the vessel used. It is noteworthy that HA solution is known to form a viscoelastic putty gel at pH 2.5 and to become a viscous liquid below pH 2.0 or above pH 2.7 and this viscoelastic change is instant and reversible", In contrast to the putty gel, our HA gel formed at pH 2.5 remains unchanged both below pH 2.0 and above pH 2.7 for a certain period of time and is restored to a viscous liquid very slowly depending on the conditions of gel formation and/or of standing. Table 1.
Conditions of Gel Formation and the Degree of Gelation.
Freezing Period (hours) 15 39 63 63 63 63 63 63 63 63 63 63
Freezing Temperature COC) -20 -20 -20 -80 -40 -20 -10 -20 -20 -20 -20 -20
pH
Degree of Gelation
(%) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 2.8 2.0 1.6 1.1 0.9
67 95 96 14 77 95 96 13 82 95 83 60
288
Novel moditied forms of hyaluronan
The degree of gelation depends on pH, the temperature for freezing, and the time of the freezing period (see Table 1). For example, the longer the time of the freezing period the more gel is stabilized. These results imply that the stability of HA gel, in other words the sustainability of HA release, can also be controlled by different kinds of conditions. Figure 1 shows the pH dependence of the half-life period of the gel. The HA gel is most stable at around pH 5 and the half-life period decreases more than two orders at pH 8. It has been speculated that the hydrolytic degradation of HA occurs in acid solution on the glucuronic acid residue and the hemiacetal ring remains unchanged, while the destruction of the N-acetylglucosamine residue takes place in basic solution". Although the molecular weight of HA remains almost constant throughout the experimental period at pH 8, the molecular weight of HA decreases abruptly under acid conditions especially at pH 4. It seems reasonable to assume that the HA gel breaks up by loosening the cross-linking structure at pH 8, while under acid conditions it decomposes by main chain scission. 1000
100
10
3
4
5
6
7
8
9
pH Figure 1.
The pH dependence of half-life period of HA gel. The times of the freezing period for HA gel formation were 3days(+), 5days(.) and 7days (A).
Figure 2 (in the next page) shows a photograph of typical HA gels by TEM taken after rapid freezing followed by solvent substitution (Rapid Freezing and Substitution Fixation Technique"), The gel looks fibrous and a surface of the fiber is covered with numerous fibrils. The inner part of the fiber looks homogenious and any clear domain structure large enough cannot be detected. This implies that the cross-linking point is neither crystal like structure nor large bundles of HA chains.
Biocompatible gel
289
Figure 2. TEM photograph of HA gel after a rapid freezing and substitution fixation.
1 .Ox1 04L_...L_--'-".d:;:;::......=_____'~L.-..l.-...l____L_.L._J._L_...L___'____i____'______'______'_____J
6.0
7.0
8.0
9.0
10.0
Volume (mL)
Figure 3. Elution chromatograph and molecular weight of a fragment of HA gel and native HA. Two curves represent molecular weight distribution (in arbitrary units) of the fragment of HA gel and native HA, respectively. Two lines represent molecular weights of every fraction corresponding to each chromatograph.
290
Novel modified fOnTIS ofhyaluronan
(a)
(b) Figure 4. Photos of rat implantation sites during the preliminary in vivo persistency test. Residual HA on the surface of cecum was dyed with 0.5 % Aldan blue saline solution. Immediately after the sheet application, both HA gel sheet (right) and freeze-dried HA sheet (left) were stained with the dye (a). One day after the same operation, only the HA gel sheet was observed by the dying (b).
Biocompatible gel
291
We have tried to identify the cross-linking structure in the HA gel. The overall cross-linking structure was detected as a branched chain structure by OPC-MALLS measurements for the fragment of the HA gel as follows. The HA gel can be segmented into soluble fragments through degradation by treatment under accelerated conditions for acid hydrolysis of HA 6,s. When the fragment of HA gel retains the branched chain structure (or the branch point), it is easy to distinguish the branched HA chain from the linear HA chain with the same molecular weight according to the difference in the molecular size in solution. The OPC chromatographs of the fragment of HA gel together with the reference linear HA are shown in Figure 3. The molecular weight for the fragment of HA gel is clearly higher than that for the reference HA at the same elution volumes (or the same molecular size) within the elution volume range of at most 8.6ml. This indicates that the HA gel contains a cross-linking structure and further the structure is stable under accelerated conditions for acid hydrolysis of HA gels. Incidentally, the increase in the difference in molecular weight between the fragment of HA gel and the reference HA as decreasing the elution volume (or increasing the molecular size) can be accounted for by the increment of the branch points. Although the exact structure of the branch point has not been identified yet, tentatively suggested is a cooperative phenomenon resulting from the interaction of hydrophobic and H-bonds between HA molecules which was proposed for a secondary structure of HA in dimethyl sulphoxide by Heatley and co-workers", Their proposal has been supported by the conformational analysis using computational methods'", Possible explanation of the occurrence of this cooperative intermolecular interaction is that during the freezing period electrostatic repulsive forces between HA molecules are suppressed at low pH and also the HA molecules are concentrated'! and hence packed close to each other to facilitate the formation of the cross-linking structure. In vivo persistency test on the HA gel was also performed preliminary. Figure 4 demonstrates some examples of the observation of the states of the implantation sites. Although all the reference freeze-dried HA sheets were disappeared within a day, almost all the HA gel sheets were observed until 7 to 10 days, depending on the preparation conditions of the gel sheets. Throughout several repeating experiments of the similar in vivo persistency tests, we have never detected any symptom questioning about biocompatibility of both HA gel and freeze-dried sheets. These results clearly show that in vivo persistency is improved and controllable in the HA gel sheets as compared to the freeze-dried HA sheets and is useful as biocompatible material.
CONCLUSION In this study, we could demonstrate a novel biocompatible gel of hyaluronan without any cross-linking reagent nor chemical modification. The stability of the gel or the sustainability of hyaluronan release could be controlled by the conditions of the gel formation: pH, temperature for freezing, and time of the freezing period. Although the exact cross-linking structure has not been identified yet, a stable branched chain
292
Novel modified forms ofhyaluronan
structure was detected by GPC-MALLS measurements for the fragment of the hyaluronan gel. This new material provides some opportunity to substitute for naturally occurring HA being used already in a broad range of biomedical applications as well as to develop materials for new applications such as controlled release of pharmacologically active compounds, cell encapsulation matrices, mammary implants and certain aspects of wound treatment.
REFERENCES AND NOTE E.ABalazs & J.L.Denlinger, Clinical uses of hyaluronan, In: The biology of Hyaluronan, D.Evered & J.Whelan (eds.), Ciba Foundation Symp. No. 143, Wiley, New York, 1989, pp 265-280. 2. Y.Kobayashi, AOkamoto & K'Nishinari, Viscoelasticity of Hyaluronic acid with different molecular weight, Biorheology, 1994, 31, 235-244. 3. V.C.Hascall, C.FuIop, ASalustri, N.Goodstone, ACalabro, M.Hogg, R.Tammi, M.Tammi & D.MacCallum, Metabolism of hyaluronan, In: The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, T.C.Laurent (ed.), Portland Press, London, 1998, pp 67-76. 4. P.ABand, Hyaluronan derivatives: chemistry and clinical applications, In: ibid.; pp 33-42. 5. E.ABalazs, Sediment volume and viscoelastic behavior of hyaluronic acid solutions, Federation Proc. 1966, 25,1817-1822. 6. Y.Tokita & AOkamoto, Hydrolytic degradation of hyaluronic acid, Polymer Degradation and Stability. 1995, 48, 269-273. 7. Y.Tokita & AOkamoto, Degradation of hyaluronic acid -Kinetic study and thermodynamics, Polymer J. 1996, 132, 1011-1014. 8. KAmako, ATakade, The Fine Structure of Bacillus subtilis Revealed by the Rapid-Freezing and Substitution-Fixation Method, J. Electro. Microsc., 1985, 13-17. 9. F.Heatley, J.E.Scott, RW,Jeanloz & E.W-Nasir, Secondary structure in glycosaminoglycuronans: N.M.R spectra in dimethyl sulphoxide of disaccharides related to hyaluronic acid and chondroitin sulphate, Carbohydrate Research. 1982, 99,1-11. 10. M.Moulabbi, H.Broch, L.Robert & D.Vasilescu, Quantum molecular modeling of hyaluronan, J. Molecular Structure (Theochem), 1997,395-396, 477-508. 11. According to our preliminary DSC measurements, 95-97 % of water molecules are frozen and hence the concentration of HA is estimated to be 20-30 wt % under the normal condition of the gel preparation as described in the experimental section. It was confirmed that the same stable HA gel could be prepared from the concentrated (20-30 wt %) acidic solution of HA on standing at moderately low temperature without freezing. "To whom correspondence should be addressed. E-mail: [email protected]. 1.
EFFICACY PROMOTING EFFECT OF HYALURONAN ON PILOCARPINE NITRATE EYE DROPS Peixue Ling', J Shandong
Xueping Gu02 ,
Tianmin Zhang2 ,
Lijun Hou3
C.P. Freda Pharmaceuticals Co.Ltd., Jinan 250014. China.
2 Shandong J
Biopharmaceutical Institute. Jinan 250014, China.
Jinan Central Hospital, Jinan 250014, China.
ABSTRACT The influence of hyaluronan on the efficacy of pilocarpine nitrate eye drops was evaluated by comparing the residence time and miotic effect of the eye drops containing hyaluronan with the eye drops not containing hyaluronan in rabbits. The result indicated that hyaluronan could significantly prolong the residence time of pilocarpine nitrate eye drops, postpone the preparation entering nasal cavity, enhance miotic effect and increase the bioavailability of pilocarpine nitrate. KEYWORDS hyaluronan, pilocarpine, eye drops, rabbit INTRODUCTION Hyaluronan (HA) is a natural occuring biopolymer belonging to a family of mucopolysaccharide, widely exists in human and vertebrates. Due to its high molecular weight and long linear molecular chain with thousands of anions, hyaluronan solution is very viscous and lubricious. The unique physical properties and high biocompatibility with tissues of human, make it suitable for various medical and pharmaceutical applications. Hyaluronan was first used as a viscoelastic agent in ophthalmological surgery in 1970's, to maintain operative space (depth of the anterior chamber) and protect the endothelial layer of the cornea. Later, it was injected into cavity ofjoints as a lubricating agent for the treatment of osteoarthritis. Today, hyaluronan is found more and more medical applications not only based on its physical properties but also on its biochemical, biological profiles. A very important development of application of hyaluronan is its use in eye drops. Because of the outstanding characteristics of high viscosity and lubricity, hyaluronan is used as an ideal vehicle in ophthalmic preparationsl'l. It is now widely used to increase viscosity and lubricity of eye drops, decrease their irritation, and make them more effective and comfortable. Hyaluronan solutions adhere well to mucin layer of the precorneal tear film. As a result, cye drops containing hyaluronan coat the cornea effectively, and the coatings are long-lasting. Hyaluronan's high capacity for taking up and retaining water decrease the evaporation of water from the preconcal coating formed by the eye drops containing hyaluronan, and would be beneficial for ocular dryness. It is found that hyaluronan can enhance the healing of wounded cornea l2.3j . In this paper, we reported the influence of hyaluronan on residence time and miotic
258
The use ofhyaluronan in drug delivery
effect of pilocarpine nitrate eye drops in rabbits. MATERIALS Eye drop preparations
Pilocarpine-hyaluronan eye drops, containing pilocarpine nitrate (5mg/ml) and hyaluronan (3mg/ml), manufactured by Shandong C.P. Freda Pharmaceuticals CO.,Ltd; Pilocarpine eye drops, containing pilocarpine nitrate (5mg/ml), prepared in the laboratory with the same formulation as above, but without hyaluronan. Animals
New Zealand white rabbits, University.
2.0~3.0
kg, purchased from Shandong Medical
METHODS Evaluation of residence time of the eye drops in eyes
This test was carryed out according to Saettone and Giannaccini's methods I4,5J. Pilocarpine-hyaluronan eye drops and pilocarpine drops were used in test groups, saline (NS) was used as blank control. Fluorescein(lmg/ml), a fluorescent tracer, was added to two preparations and NS for detecting residence in rabbits' eyes with a slit lamp under UV light (366nm) illumination. 30 rabbits were randomly divided into 3 groups. 50 l.l I of pilocarpine-hyaluronan eye drops, pilocarpine drops and NS were instilled respectively into the right eye of each rabbit. The time maintaining integrated liquid film on cornea (Tc); the time maintaining sodium fluorescein in eyes (Te); and the time required for fluorescein appear in nasal cavity (Tn) were observed at every 5 minutes. Comparison of miotic effects of the two eye drops
Test the miotic effects of pilocarpine-hyaluronan eye drops and pilocarpine eye drops according to the methods of Saettone and Torracca I6,7J. 20 rabbits were randomly divided into two groups. 50 l.l I pilocarpine-hyaluronan eye drops and NS were instilled into right and left (self control) eyes of one group respectively, 50 l.l I of pilocarpine eye drops and NS were instilled into right and left eyes of another group respectively. Examine and calculate the following items:CD Diameter of pupil; ® I max, maximum miotic effect;@ Peak time, the time required to reach I max;@ Duration, time required for pupil diameter to come back to original value before instillation; @Draw the time-effect curves, and calculate AUC values. RESULTS Residence time of the eye drops in rabbit eyes
The group of pilocarpine-hyaluronan eye drops significantly prolonged the time maintaining integrated liquid film on cornea (Tc), the time maintaining fluorescein in eyes (Te), and the time required for the fluorescein appear in nasal cavity (Tn),
Effect on pilocarpine nitrate eye drops
259
compared with groups ofpiloearpine eye drops and NS (p< 0.01), see table 1.
Miotic effects Pharmacological test showed that pilocarpine-hyaluronan eye drops have higher I max value, longer duration, and higher AUC value (p<0.01), see table 2 and fig. 1.
Table 1. Residence time of the eye drops in rabbit eyes Groups
Number of ra bbitIS
T c/mi mm
T e/mi mm
Tn/mi mm
Pilocarpine-HA*
10
108±5.4
171±4.6
23.6±4.8
Pilocarpine
10
29.5±2.8
96±2.1
<5
NS
10
28.6±5.9
99±5.2
<5
*HA: hyaluronan
Table 2. Comparison ofmoitic effects of the two eye drops Peak time Imax/mm . Duration/min AUC Relative /min
Groups
Pilocarpine-HA 3.26 ±0.08*
20
301.2±3.7*
435.7
Pilocarpine
20
151.5±2.41
158.3
2.32±0.23
2.75
*p
:1.5
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:HJO
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260
The use ofhyaluronan in drug delivery
CONCLUSIONS Hyaluronan could significantly prolong the residence time of pilocarpine nitrate eye drops, postpone the preparation entering nasal cavity, enhance miotic effect and increase the bioavailability of pilocarpine nitrate. REFERENCES 1. A. Ludwig, M. Van Ooteghem, Evaluation of sodium hyaluronate as viscous vehicle for eye drops, J. Pharm. Belg., 1989,44(6),391-397. 2. H. Stiebel-Kalish, D.D. Gaton, D. Weinberger, et al, A comparison of the effect of hyaluronic acid versus gentamicin on corneal epithelial healing, Eye 1998, 12, 829-833. 3. J. H. Chung, H. J. Kim, P. Fagerholmb, B. C. Cho, Effect of topically applied Na-hyaluronan on experimental corneal alkali wound healing, Korean J. Ophtha/moi., 1996, 10(2),68-75. 4. P. Band, Effective use of hyaluronic acid, Drug Cosmo Ind., 1985, 100,54-99. 5. M. F. Saettone, B. Giannaccini, P. Chetoni, et al. Evaluation of high-and low-mol weight fractions of sodium hyaluronate and an ionic complex as adjuvants for topical ophthalmic vehicles containing pilocarpine. Int. J. Pharm., 1991, 72, 131-139. 6. M. F. Seattone, B. Giannaccini, A. Guiducci, et al . An evaluation of four organic hydrogels containing pilocarpine. Int. J. Pharm., 1986b, 31,261-270. 7. M. F. Saettone, M. T. Torracca, A. Pagano, et ai. Controlled release of pilocarpine [rom coated polymeric ophthalmic inserts prepared by extrusion. Int. J. of Pharm., 1992,86, 159-166.
PREPARATION OF HYALURONAN GEL AND THE APPLICATION TO DRUG DELIVERY Isao Kaetsu l ,2, Kouichi Sutanr', Kumao Uchida', Glyn O. Phillips3 Hidetoshi Matsumura/ & Yoshio Matsubara 2 I Faculty a/Science and Technology, Kinki University, Kowakae 3-4-1, Higashi-Osaka, Osaka, 577-8502 Japan.
2Interdiscip/inary Graduate School a/Science and Technology, Kinki University, Kowakae 3-4-1, Higashi-Osaka, Osaka, 577-8502 Japan. 3 The
North East Wales Institute, Wrexham, LLl I 2A W, UK.
ABSTRACT The stable gelation of hyaluronic acid by entrapping with other networked polymers was investigated. One method is the entrapping polymerization with hydrophilic vinyl monomers such as hydroxyethyl methacrylate and acrylamide. The other is the entrapping with natural polymers such as agarosc. Both methods gave the stable hydrogel products. Some model drugs were added to a hyaluronan solution and included into the gel by entrapping. The hyaluronan gel showed an intelligent function, showing reverse volume changes (shrinkage and expansion) with the on-off switching of signal input. Intelligent controlled release of model drugs in response to the changes of environmental conditions such as pH changes and on-off switching of electrical field was also studied. Those results suggested hyaluronan entrapped hydrogel acts as a polyelectrolyte. KEYWORDS Gelation, entrapping polymerization, pH-responsive gel, electro-responsive gel, drug delivery, controlled release INTRODUCTION Hyaluronan has been noticed as one of the most biocompatible natural polymer ever known and the most promising material for the biomedical appllcations". However, as it is hydrophilic, the method of gelation into a stable gel is the key technology for the use of wide and practical biomedical applications such as artificial organs and drug delivery systems. One of the important purpose of this work is to find the method of effective gelation for hyaluronan. In spite of trials, the gelation by means of direct crosslinking of hyaluronan molecule has not succeeded. Irradiation and the addition of chemical crosslinker such as glutaraldehyde were not effective for crosslinking, and radiation caused degradation of hyaluronan. Therefore, the authors studied and developed another
262
The usc off hyaluronan in drug delivery
gelation method of hyaluronan by entrapping with other networked polymers. Both various synthetic vinyl polymers and agarose as a natural polymer could be used for the matrices of entrapping gelation. The second purpose of the research is the clarification of stimuli-sensitivities of hyalronan. In recent years, a lot of work has been concentrated on the stimuli-sensitive and responsive functions of specific hydrophilic polymer gels (hydrogels). The authors have studied polyelectrolytes as the pH and electrically sensitive polymers'v'". Not only synthetic polyelectrolytes but also natural polyelectrolytes such as polysaccharides might show the same pH and electric sensitivity and responsiveness. Therefore, the stimuli-sensitive and responsive function of hyaluronan entrapped hydrogels was investigated, in this paper. The authors have studied and developed the drug delivery systems (DDS)12.13. Recently, the intelligent drug delivery system as a new generation of DDS has become one of the most interesting research targets in this field. Intelligent DDS is the system to control the drug release from a polymer with an on-off switching mechanism in response to signal inputs from the environment such as pH change, temperature change and electric signal. Therefore, an intelligent DDS is a combination of a sensor component and a drug delivery device. A stimuli-sensitive hydrogel is expected to fit to a sensor and actuator component in the intelligent system. Therefore, the final purpose of the present report is the study of the intelligent drug delivery (controlled release) function of hyaluronan entrapping hydrogels as a natural polyelectrolyte. It is expected that a biomedical material having both excellent biocompatibility and useful intelligent function would be developed based on those research results and directions. MATERIALS & METHODS Hyaluronan Hyaluronic acid(HA) was obtained from Denki-Kagaku Ltd. Vinyl monomers, crosslinker monomers All monomers (Extra-pure grade) were obtained from Wako Chemical Ltd. and used without further purification. Those are acrylamide(AAm), 2-hydroxyethyl methacrylate(HEMA), diethyleneglycol dimethacrylate(2G), polyethyleneglycol #400 dimethacrylate(9G), polyethyleneglycol #600 dimethacrylate(14G). Agarose, Drugs Agarose and model drugs were obtained from Wako Chemical Ltd and used. Entrapping polymerization Hyaluronan was dissolved into distilled water and mixed with monomers. The mixture was charged into a test tube and irradiated under nitrogen atmosphere with ultraviolet light from 400W Mercury Lamp at room temperature to be polymerized.
Hyaluronan gel and drug delivery
263
Entrapping with polysaccharide Hyaluronan and other polysaccharide were dissolved into an aqueous solution. Then the mixture was tested for gelation by heating and cooling or by changing pH into acidic or alkaline conditions. Gel fraction and swelling degree Gel fraction and swelling degree of the gel were measured and determined according to the following equations occasionally at certain intervals. Weight of gel x 100 Weight of total polymer (sol + gel)
Gel fraction [%]
' d Swe 11mg egree
= Weight
of swelled gel (water + dried gel) Weight of dried gel
Stimuli-responsiveness pH responsiveness was tested and evaluated as follows. Hyaluronan entrapped gel was immersed in water to swell into an equilibrium state. The pH value of aqueous medium was varied between acidic and alkaline conditions by the addition of hydrochloric acid or sodium hydroxide. Then the volume or weight changes of swelled gel with pH changes were measured. For electro-responsiveness test, a cubic form (about 1cm X 1cm X 1cm) hyaluronan entrapped gel was sandwiched between two Pt plates as electrodes, which were connected with a battery. Then the sample with the electrodes was put in water. Electric field was introduced by the on-switching of battery and then repeated the on-offswitchings. The volume or weight changes of the gel in each on and off switching time were measured and determined. Drug release To test for drug release, a model drug was mixed with aqueous solution of hyaluronan and monomer or polysaccharide preliminarily. Then the mixture was converted into a gel by polymerization and gelation. The formed gel was put in water alone at various different pHs or in a sandwiched state with Pt electrodes. 1ml of aqueous medium was picked up occasionally at intervals, and analyzed calorimetrically to determine the drug concentration (amount of released drug) in the medium. RESULTS & DISCUSSION Gelation by entrapping polymerization of vinyl monomers Gelation of hyaluronan is an essential problem to be solved for the extended
264
The use offhyaluronan in drugdelivery
applications. However, the authors have found that irradiation by v-ray and chemical crosslinking by glutaraldehyde were not effective for the purpose. Then the method of entrapping ofhyaluronan with other polymers was studied. This method is simple and generally applicable one, though the product is not pure hyaluronan. It was found that various kinds of hydrophilic vinyl monomers and polymers could be used as the matrix of entrapping. Fig.l showed an example of gelation with vinyl monomers by entrapping polymerzation. 100
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en
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20
1000
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Gelation by entrapping with natural polysaccharide
Vinyl polymers are generally non-biodegradable and sometimes not so biocompatiblc as natural polymers. Then, the authors investigated the use of hydrophilic natural polymers such as polysaccharides for the matrix of entrapping. However, most of aqueous solutions of natural polymers showed no gelation behaviour. It was found that agarose was the most effective one as an entrapping matrix and the mixture ofhyaluronan and agarose could be gelated effectively. Table I showed the results of gelation screening test using various kinds of natural polymers including agarose. Stimuli-responsiveness - - pH responsive volume changes of hyaluronan entrapping gel
As already mentioned, hyaluronan is expected to show the stimuli-responsive volume changes to cause shrinkage and expansion reversiblly as a natural polyelectrolyte. The authors investigates pH responsive and electro-responsive volume changes of hyaluronan entrapped hydrogels which are reverse with pH changes between acidic, alkaline conditions and also with on-off switchings of electric field.
Ilyaluronan gel and drug delivery
265
An example of the result on pH-responsiveness is shown in Fig. 5. According to the result, hyaluronan entrapped gels clearly showed pH responsiveness similarly to synthetic polyelectrolytes such as poly(acrylic acid). Table I Gelation of polysaccharide and hyalurona-polysaccharide system polysaccharide
gelation by cooling
o
Agarose Arabic gum i\. -Carrageenan Curdlan Dextan Dextrose Gelatin Gluco mannan (from konjac) Locust bean gum (from seeds of ceratonia siliqua) Pectin (from citras) Sodium Alginate
gelation of polysaccharide with hyaluronic acid
o X
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o
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o
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X
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o X
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field
X: not ok
responsiveness
of
hyaluronan
Figs. 2 and 3 were the results of electro-responsive volume changes of hyaluronan entrapped hydrogels with the on-off switching of electric field. It is obvious that those hydrogels also showed electro-responsiveness.
~5[
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6
266
The use ofhyaluronan in drug delivery
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- e - HA+AAm+14G+H2 0 : 0.05+0.015+0.015+0.5(g)
Spontaneous slow release of drug from hyaluronan entrapped gel
The authors studied the application of hyaluronan entrapped gels to drug delivery systems. A model drug was dissolved into a monomeric or polymeric aqueous solution and then included into the crosslinked matrix by gelation. As the hyaluronan entrapped gel is stark hydrophilic, the drug diffused out spontaneously from the swollen matrix even under the off-switching state by an osmotic pressure due to the concentration difference between inside and outside gel matrices. Then a spontaneous automatical release behavior was observed. An example of the spontaneous release of model drug from the hyaluronan entrapped gel was shown in Fig. 4. Intelligent controlled release of drug --pH responsive drug releases
Hitherto-developed drug delivery systems had only the spontaneous and automatic release functions. The rate and period of drug release could be controlled mainly by the variety of hydrophilicity of polymer matrix and the control of timing of drug release with on-off switchings could hardly be realized. However, in recent years, the needs for the intelligent drug release with an on-off switching mechanism in response to the sensing of environmental signals are increasing more and more. The authors have studied the application of stimuli-responsive hydrogels as a sensor and actuator component in the intelligent system to catch an environmental signal and transduce it into any mechanical energy to initiate the on-off switching of drug releases. The intelligent controlled release device and system was constructed according to the method described in the Materials
Hyaluronan gel and drug delivery
267
and Methods. An example of the results is shown in Fig. 5. According to this result predominant drug release was observed in acidic condition though some spontaneous release proceeded in alkaline condition.
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acid
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268
The use ofhyaluronan in drug delivery
Intelligent controlled release of drug --electro-responsive drug releases Some results of electric field responsive controlled releases of model drug are shown in Fig. 6. As shown in those results, pulsatile drug release occurred remarkedly in response to the on-off switchings of electric field. on
I
off 400
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Fig.6 Electro-responsive change of swelling ratio and release of drug from hyaluronan agarose system hyaluronic acid + agarose + distilled water : 10 + 5 + 2000 voltage: 6V
CONCLUSIONS Hyaluronan can be gelled into a hydrogel by entrapping with a crosslinked matrix of vinyl polymers and natural polysaccharide such as agarose. The hyaluronan entrapped gel has the stimuli-sensitive and responsive functions such as pH responsive and electro-responsive volume changes. The hyaluronan entrapped gel including a model drug shows intelligent controlled release function of the drug in response to the on-off switchings of environmental signals. REFERENCES
1. S. Takigami, M. Takigami & G. O. Phillips, 'Hydration characteristics of the cross-linked hyaluronan derivative hylan', Carbohydrate Polymers, 1993,22, 1-8. 2. M. G. Cascone, B. Sim & S. Downes, 'Blends of synthetic and natural polymers as drug delivery systems for growth hormone', Biomaterials, 1995, 16,569-574. 3. N. E. Larsen & E. A. Balazs, 'Drug delivery systems using hyaluronan and its derivatives', Advanced Drug Delivery Reviews, 1991, 7, 279-293. 4. I. Kaetsu, K. Uchida, Y. Morita & M. Okubo, 'Synthesis of electro-responsive hydrogels by radiation polymerization of sodium acrylate', Radiat. Phys. Chem.,
I-Iyaluronan gel and drug delivery 5.
6. 7. 8. 9.
10.
11. 12.
13.
269
1992,40(2),157-160. I. Kaetsu, K. Uchida, Y. Murai, Y. Morita & K. Sutani, 'Study on actuators for sensor-connected drug delivery systems', Polymer Preprints, Japan, 1993, 42(8), 3174-3176. I. Kaetsu, 'Signal responsive chem.ical delivery systems by radiation techniques and the use for brain research', Radiat. Phys. Chern., 1995,46(2),247-256. I. Kaetsu, 'Biocompatible and biofunctional membranes by means of radiation teclmiques', Nucl. Instr. and Meth. in Phys. Res. B., 1995, 105,294-301. I. Kaetsu, 'Biomedical materials, devices and drug delivery systems by radiation techniques', Radiat. Phys. Chern., 1996,47(3),419-424. I. Kaetsu, K.Uchida, H.Shindo, S. Gomi & K.Sutani, 'Intelligent type controlled release systems by radiation techniques' Radial. Phys. Chem., 1999, 55(2), 193-201. I. Kaetsu, K. Uchida & K. Sutani, 'Intelligent feedback release systems and the application to neuron network model research', Radiat. Phys. Chern., 1999, 55(5-6),673-676. I. Kaetsu, K. Uchida, K. Sutani & S. Sakata, 'Intelligent biomembrane obtained by irradiation techniques', Radiat. Phys. Chern., 2000, 57,465-469. I. Kaetsu, 'Radiation techniques in the formulation of synthetic biomaterials', In: Radiation processing of polyrners, A. Singh & 1. Silverman(eds.), Hanser Publishers. Munich Vienna New York Barcelona, 1992, pp 150-185. I. Kaetsu, 'Radiation synthesis of polymeric materials for biomedical and biochemical applications', Advances in Poly. Sci., 1993, 105,81-97.
DERIVATIZED HYALURONAN FOR GELS AND NANOCHEMICALLY PATTERNED SURFACES Rolando Barbucci, Daniela Pasqui, Gemma Leone C.R.I.S.MA. and Department of Chemical and Biosystem Sciences and Technologies, University ofSiena, Siena, Via Ettore Bastianini n. 12, 53100 Siena, Italy
ABSTRACT Hydrogels of hyaluronic acid (Hyal) and its sulphated derivative were synthesed, using several cross-linking agents of different length and hydrophilicity. The gels were characterised by TGA and DSC analysis. Their capability to adsorb plasma Proteins (HSA and Fbg) was studied in dynamic conditions by FT-IR ATR spectroscopy with a flow cell. A variation in the quantity and conformational change of the adsorbed protein was observed according to the hydrophilicity of the cross-linker. These hydrogels are also able to coordinate metal ions, such as Cu 2 +, Zn2+ and Ag+, and their coordination strongly influences the swelling degree. Ag+ complex shows antibacterial properties towards Staphylococcus Epidermidis. Micropatterned structures of Hyal and its sulphated derivative (HyaIS) polymers on glass and poly(ethylenterephthalate) with different stripes dimension were realised. The adhesion and polarisation of endothelial cells were studied.
KEYWORDS Hyaluronan hydrogel, thermal and mechanical properties, plasma proteins and adsorption, Cu 2+, Zn2+ Ag+coordination, micropatterned surfaces, endothelial cells.
INTRODUCTION Hyaluronan shows very interesting chemical and biological properties, making it the ideal building polymer for biomaterials in tissue engineering. In this paper we will present an overview of our current work on Hyal concerning: 1) synthesis and characterisation of Hyal hydrogels. 2) Hyal hydrogel-protein interactions. 3) coordinating ability of hydrogels towards Cu 2+, Zn2+, Ag+metal ions. 4) the role of micropatterned structures of the Hyal and its sulphated derivative in cellular signalling.
MATERIALS AND METHODS Hydrogel synthesis The first step in hydrogel synthesis was the conversion of the sodium salt to tetrabutylammonium to make Hyal soluble in dimethylformamide (DMF). Briefly, the procedures consists of sodium-hydrogen ion exchange of sodium Hyal solution using Amberlite CG50 resin to obtain the protonated form of Hyal. The solution was then titrated by TBA+ OH' to obtain the TBA-Hyal salt.
294
Novel modi tied forms of hyaluronan
The Hyal-TBA was dissolved in DMF under stirring and nitrogen flow, and the solution was kept at about O°c. The stoichiometric amount of chloromethyl-pyridine iodide (CMPI) was added to the solution to activate 50% of the carboxylate groups'. An excess of different crosslinking agents [1,3 diamine propane (1,3), 1,6 diamine hexane (1,6), 1,12 diamine dodecane (1,12), PEG 600, 800 and 900] was then added together with a small amount of triethylamine as catalyst. The gel formed immediately, but the process was completed, leaving the mixture under stirring for 3-4 h at room temperature. The gel was then washed several times with EtOH and water until no more solvents or reagents were found in the washing solutions'. Samples of gels were sulphated using a solution of sulphur trioxide-pyridine complex (S03-Py) in DMF, as previously described 3. Thermal analysis The thermal properties of the hydro gels were analysed by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). DSC analysis was performed with a DuPont differential scanning calorimeter (model 2910). All the samples were subjected to two scans at a rate of 10°C min" in a nitrogen atmosphere, the first scan was run through the temperature range _lOo+180°C, the second through the range lOo+250°C. Thermogravimetric data were evaluated by means of a TGA DuPont model TA2IOO at a heating rate of lOoC min" in a nitrogen atmosphere, in the temperature range 25°+300°C. Rheological analysis The rheological characterisation was performed on the cross-linked polymers prepared by adding 1 ml of bidistilled water to 20 mg of cross-linked polymers. A Bohlin VOR rheometer (Bohlin Reology AB, Lund, Sweden) was used at a controlled temperature of 25°C. This technique has been used successfully to determine the structure-mechanical properties relationship of materials. Water uptake measurement The swelling degree (S.D.) was calculated with the formula: S.D. (%)= (Ws- WdIWd)xlOO where Ws and Wd are the weight of the swollen and the dried gels respectively. In practice, established amounts (Wd) of cross-linked polymers were enclosed in small bags made of hydrophobic water-permeable net (nylon) and immersed for 24h at 25°C in 50 ml of distilled water. They were then placed between two pieces of dry filter paper to wipe off the excess water. The bags containig the swollen gels were weighed in order to calculated to get Ws. Water uptake measurements were performed at different pHs in the following solutions: pH 2, 6.5 ml of 0.2 M Hel mixed with 25 ml of 0.2 M KCl; pH 7.4 PBS (phosphate buffer solution); pH 9, 50 ml of 0.1 M TRIS with 5.7 ml of 0,1 M HC!.
Derivatized hyaluronan Cor gels
295
HSA and Fbg adsorption tests Tests were performed on gels swollen for 24 hours in saline solution using FT-IR ATR spectroscopy and a flow cell in hydraulic circuit, as previously described 4.
Complex formation reaction An aliquot of dried hydrogel was kept in contact with a solution of the different metal ions (CuS040.1 M, ZnS04 0.1 M, AgN03 0.1 M) for 24h. The gels were then washed several times till no release of metal ions was observed. Micropatterned structures of Hyal and HyalS polymers Hyal and its sulphated derivative were photoimmobilised on glass and poly(ethylenterephthalate) (PET) disks using a photomask. The process needed the conjugation of HyallHyalS with a photoreactive molecule (4-azidoaniline hydrochloride) through the formation of amide bound using a water soluble carbodiimde as activating agent of the carboxylates of the polysaccharide (lethyI3(3(dimethylamino)propyl(carbodiimmide)]. An aqueous solution (100 ul) of photoreactive HyallHyalS (1 mg/ml) was dropped on the surfaces and air dried at room temperature. Subsequently, the surfaces were irradiated with UV lamp at a distance of 5 em for 90 seconds in the presence of a photomask with a pattern having stripes of 5-100 urn width 5. The disks were washed with distilled water to remove the unbound polymer; in the end the surfaces showed the same pattern of the mask. The structures were observed with a contrast phase microscope, atomic force microscope (A.F.M.) and scanning electron microscope (S.E.M) to check the dimension of the stripes. The stripes on PET are 100, 50, 25, IOum width; the stripes on glass disk are 100, 50, 25, 10 um width and Ium high. In order to observe cell behaviour human endothelial cells (HGTFN) were seeded on micropatterned glass, and rat endothelial lung cells on micropatterned PET and incubated at 3rc. In order to measure the dynamics of cell-movement and their shape, time lapse videos were taken using a contrast phase microscope connected to a CCD camera at the magnification of lOX on S-VHS recorder. The surfaces were viewed using a fluorescence microscope. RESULTS AND DISCUSSION Synthesis and characterisation of Hyal hydrogels The creation of stable hydrogels from HA is challenging. We have synthesised new materials which exhibit good resistance to the physiological environment maintaining the excellent biological properties of Hyal. Our approach consists in the cross-linking of Hyal using the carboxylate groups as the point of attack of the reaction while maintaining an incomplete utilisation of all the carboxylate groups, which are necessary for the expression of the biological characteristics of the polysaccharide 2. The crosslinking agents used are hydrophobic or hydrophilic diamine chains with different lengths (Fig. 1). The gel may then be subjected to a heterogeneous sulphation reaction using a solution of S03-Py in DMF. The gels have been characterised by N.M.R., FTIR and potentiometric analysis. In the typical mechanical spectrum of the cross-linked polymers, both moduli are virtually frequency independent and both show some slight
296
Novel modified forms of hyaluronan
increase at a higher frequency. The storage moduli are one order of magnitude greater. than the loss moduli. The elastic modulus of the sulphated gels is always higher than that of the non sulphated one. The average and standard deviation of the elastic modulus calculated from the mechanical spectra are reported in Table 1. Polymers C.L.Hyal-l,3 C.L.HyaIS-l,3 C.L.Hyal-l,6 C.L.HyaIS-l,6 C.L.Hyal-PEG600 C.L.HyaIS-PEG600 C.L.Hyal-PEG900 C.L.HyaIS-PEG900
Elastic modulus G' (Pa) 450.0 ± 82.0 2225.0 ± 240.0 890.0 ± 97.0 1600.0 ± 90.0 712.0 ± 87.0 1215.0 ± 110.0 604.0 ± 48.0 998.0 ± 141.0
Table 1. Average elastic modulus (G') from the mechanical spectra of C.L.Hyal and C.L. Hyals polymers (T= 25°C, 20 mg ml" )
These materials possess the features of gel-like materials; under small deformation conditions they exhibit the typical behaviour of viscoelastic solids. The elastic modulus of these materials depends on the M.W. of the cross-linking agent. It tends to decrease with increasing of M.W. of the cross-linking agent.
-
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-
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= CHzCH·p for P600and
j
Derivatized hyaluronan for gels
297
The increase of G', when sulphated groups are present, is due to the fact that these groups are anionic, thus forming stronger hydrogen bond with water and leading to an increase of the elastic modulus. In fact, the sulphated polymers cross-linked with P600 possess better mechanical properties than the non-sulphated polymers cross-linked with 1,3 and 1,6 diamines. The thermal properties 6 are summarised in Table 2, where ~Ha is the heat related to the endothermic peak present in the first scan due to the evaporation of water, and (~Hb is the heat of the exothermic peak present in the second scan, due to the degradation. Polymers C.L.Hyal-l,3 C.L.HyaIS-l,3 C.L.Hyal-l,6 C.L.HyaIS-l,6 C.L.Hyal-P600 C.L.HyaIS-P600 C.L.Hyal-P900 C.L.HyaIS-P900 Hyal-TBA
~Ha (1 g - ) 276,7 357,1 327,0 465,6 239,0 384,0 179,0 206,0 164,0
TgCC) 51,5 64,5 64,2 64,3 45,7 69,0 73,1 65,7 Not present
~Hb (1 g -') 42,1 53,6 58,6 65,2 72,3 113,4 30,9 130,0 130,4
Table 2. Thermal properties of cross-linked polymers and Hyal-TBA The water content of the samples was evaluated by thermogravimetric tests. The water content calculated from the dehydration heat obtained by DSC analysis is higher than that by TGA analysis. The water content is always higher for the sulphated hydrogels because of the presence of OS03- groups, which make the polymers more hydrophilic. The difference between the water content of sulphated and non-sulphated samples is less remarkable on account of the polymer cross-linked with hydrophilic P600 and P900 (Table 3). Polymers C.L.Hyal-l,3 C.L.HyaIS-l,3 C.L.Hyal-l,6 C.L.HyaIS-l,6 C.L.Hyal-P600 C.L.HyaIS-P 600 C.L.Hyal-P900 C.L.HyaIS-P900 Hyal TBA
Water Content (%) From TGA 4.4 8.5 8.0 12.0 8.0 11.0 5.3 6.6 8.0
Water Content(%) formDSC 12.0 15.8 16.1 20.2 10.5 16.5 9.5 10.3 7.8
Table 3. Water content (calculated from TGA and DSC) of cross-linked polymers and HyalTBA The hyaluronic based hydrogel reaches the maximum water uptake value only after 48 hours. This behaviour depends on the pore size in the gel and the swelling rate can be changed modifying their width.
298
Novel modified forms of hyaluronan
The cross-linked gel (Fig. 2) shows an increment in water uptake as a function of pH. The trend of water uptake as a function of the pH remains constant. The behaviour of the water uptake versus pH can be explained by the involvement of the carboxylic groups in hydrogen bonds. Carboxylic acid groups would be fully ionised at pH 7.4 , but the effect shows up only at pH 9 where the swelling reaches its maximum value. This can be explained by the high number of hydrogen bonds giving rise to a hard Hbond network which can be broken only at pH 9. This has been confirmed by FTIR characterisation.
Figure 2. Swelling degree of Hyal gels as a function of time at different pHs Hyal hydrogel-protein interaction HSA and Fbg adsorption tests on gels swollen in saline solution have been performed using FT-IR and a flow cell in a hydraulic circuit. HSA and Fbg kinetics on the four 50% cross-linked gels by different cross-linking agents are shown in Figs. 3a and 3b, respectively. They are plots of the 1550 cm-l absorption band (Amide II region correlated with protein increase) versus time.
Fibrinogen kinetics on Hyal gel 6,00 -
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Kinetics of Fbg adsorption on Hyal hydrogels with different cross-linking agents
Derivatizedhyaluronan for gels
299
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300
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Figure 4. FT-IR spectra ofFbg adsorbed on Hyal hydrogels Coordinating ability of hydrogels towards Cu 2+, Zn 2+, Ag+
The coordinating ability of the Hyal gels towards metal ions was investigated to correlate the type of the metal ion-hydrogel complex with its biological properties. Cu2+ and Zn2+ ions were chosen because they have showed chemotactic activity when coordinated with the linear soluble Hyal macromolecule 7 and Ag" because it presents antibacterial properties 8. All three metal ions modify the morphological structure of the native gels. When the amount of Cu2+ and Zn2+ ions taken up by the gel was increased, the structure becomes more compact and shows small plates collapsed together. On the contrary, with Ag" the morphology of the gel remained open with a small increasing in the pore sizes. This pattern was reflected in the swelling degree in water (Fig. 5). ,-~ I 14000
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o HA
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Derivatized hyaluronan for gels
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Ag(I)A: complex with 0.06.10-4 moles Ag+ Ag(I)B: complex with 0.08.10-4 moles Ag+ Ag(I)C: complex with 0.1.10-4 moles Ag+ Ag(I)D: complex with 0.12.10-4 moles Ag+ Ag(I)E: complex obtained adding an excess of Ag+ Figure 5. Swelling degree of Hyal-Zn amount of metal ions
2+
and Hyal Ag" complexes as function of the
The swelling degree decreased with increasing number of Zn2+ and Cu2+ ions. The coordination around these ions shrank the polysaccharide chains to a more compact structure, even hindering the water uptake. With Ag+ we observed an opposite effect: the swelling degree increased with increasing number of metal ions. This pattern was particularly evident at pH 2. FTIR spectra showed that in the coordination reaction Ag+ ions deprotonated the carboxylic groups which are fully present at pH 2 and partially at pH 7.4. In the overall reaction, the gel with Ag" increased in charge number. Each Ag" ion formed linear complexes by coordinating two COO- units. [2COOH] gel + Ag" - - . . [COOAgOOCr gel +2H+ Both the factors favour the stiffening of the chains with their consequent removal. The hydrogel complex with Ag+ showed strong bacterial antiadhesive properties. The role of mieropatterned structures of the Uyal and its sulphated derivative in cellular signalling Cell-surface interaction is an important aspect in the production of biomaterials for tissue engineering. Studies have shown that chemical and topographical surfaces can affect cell behaviour differently in terms of adhesion, proliferation. migration and differentiation. Actually, most features and the bioactivity of micro and nanopatterned structures depend on domain size being similar to cell sizes (cells measure 5-10 lim). Manipulation of the two signals (cell-cell and cell-substrate) using structures with a defined morphology makes it possible to produce a "pattern" of oriented cells. In order to prepare micropatterned structures of Hyal and its sulphated derivative (HyalS), Hyal and HyalS were photoimmobilised on different kinds of substrates: glass disks (13,5 mm diameter) and polyethylene terephthalate (PET) using photomasks
302
Novel modified forms of hyaluronan
and photolithography. The stripes 100 um, 50 !J.ID, 25 urn, 10 um wide and 1 urn high were etched on PET and on glass disks (Fig. 6).
.....si_ Soan
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Figure 6. AFM image of a micropattemed surface of HyalS on glass with stripes of 10 um wide and 1 !J.ID high Human endothelial cells (HGTFN) were seeded on the micropatterned glass structures and rat lung endothelial cells (BIOD2) on the etched HyalS-PET. The cells aligned on the surfaces with an orientation matching the stripes . Time lapse video demonstrated that cells adhere and spread on HyalS after about an hour whereas on the glass substrate they maintained the spherical shape associated with their phenotype even after 24 hours (Fig. 7). Analysis of the trend of movement showed that cell spend most of their time on the polymer. In HyalS samples with 100 um stripes, they entered the grooves of the stripes and spread in a circular mode. In HyalS-glass samples with 50 !J.ID and 25 urn stripes, the cells aligned along the edge of the stripes between the two materials. In samples with 10 um stripes, the cells aligned on the polymer taking an elongate form, moved along HyalS stripes or jumped from one stripe to another avoiding the glass substrate (Table 4). Hence, the percentage of cells in movement on the polymer and the distance run by the time increased with decreasing stripe width. Cell count showed that the number of cells spread on HyalS stripes increased with time. Sample Glass-HyalS100!J.m Glass-HyatSduum Glass-Hya1S25!J.ID Glass-HyaIS10!J.m
HyalS 60% 5% 55% 91%
Substrate 4% 19% 25% 9%
Edge 36% 76% 20%
Table 4. Arrangement of human endothelial cells (HGTFN) on different disposals For 100 !J.ID stripes HyalS -PET micropatterned surfaces, the number of rat lung endothelial cells on PET was similar to that on HyaiS. As the stripes narrowed down to 10 um, most cells moved from PET to HyalS and split. Similar behaviour has been observed with human endothelial cells on glass.
Derivatized hyaluronan for gels
Figure 7. Adhesion
of human endothelial microstructures (10 um)
cells
(HGTFN)
on
303
glass-HyalS
In order to observe other types of cell behaviour, ovine chondrocytes were seeded on HyalS micropattemed in 100 urn stripes on PET. Cell count showed that cell density of ovine chondrocytes on the polymer stripes increased with time, whereas the cell density of endhotelial cells did not change.
ACKNOWLEDGEMENTS The authors thank Consiglio Nazionale delle Ricerche (Rome)/ Progetto Finalizzato: Materiali Speciali per Tecnologie Avanzate II, for financial support.
REFERENCES 1. T. Mukaiama,Angew. Chem. Int. Ed. Eng., 1979,18,707. 2. R. Barbucci, L. Ambrosio, A. Borsacchiello & R. Rappuoli, 'Synthesis and chemical and rheological characterisation of new hyaluronic acid-based hydrogels', Journal ofBiomaterials Science - Polymer Edition, 2000,11,383-399. 3. A. Magnani, S. Lamponi, R. Rappuoli & R. Barbucci, 'Sulphated hyaluronic acids: a chemical and biological characterisation', Polymer International, 1998,46,225-
240.
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Novel modified forms ofhyaluronan
4. F. Dousseau, M. Therrien & M. Pezzolet, 'On the spectral subtraction of water from the FTIR spectra of acqueous solutions of proteins', Appl. Spectrosc., 1989, 40, 339-344. 5. G. Chen, Y. Ito, Y. Imanishi, A. Magnani, S. Lamponi & R. Barbucci, 'Photoimmobilization of sulphated hyaluronic acid for antithrombogenicity'. Bioconjugate Chemistry, 1997,8,730-734. 6. A. H. Clark & S. B. Ross-Murphy, Advances Polymer Sci; 1987, 83, 236. 7. R. Barbucci, A Magnani, S. Lamponi, S. Mitola, M. Ziche, L. Morbidelli & F. Bussolino, 'Cu (II) and Zn (II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells', Journal of Inorganic Biochemistry, 2000, 81(4), 229-237. 8. A Magnani, R. Barbucci, L. Montanaro, C. R. Arciola & S. Lamponi, 'In vitro study of haemocompatibility and effect on bacterial adhesion of a polymeric surface with immobilised heparin and sulphated hyaluronic acid' , Journal of Biomaterials Science - Polymer Edition, 2000,11,801-815.
HYALURONAN DNA MATRIX FOR GENE TRANSFER Weiliam Chen1,2*, Daniel Checkla1 & Philip Dehazya' JClear
Solutions Biotech, Inc., 50 East Loop Road, Stony Brook, NY11790-3350
2Department Biomedical Engineering, Center for Biotechnology, 348 Psychology A Building, State University ofNew York, Stony Brook, NY1l794-2580
ABSTRACT Gene therapy appears to be an attractive modality for accelerating wound healing, as only temporary gene expression is required to achieve a desirable therapeutic outcome. We describe a crosslinked hyaluronan (HA) biocompatible and biodegradable matrix for sustained gene transfer. Results from in vitro DNA release kinetics studies indicate that this hyaluronan-DNA bioconjugate is capable of prolonged release of DNA. Electrophoretic motility studies indicate that the DNA released from this delivery vehicle is intact. The DNA samples collected during the course of the release study were successfully used for transfecting cell cultures. DNAHA matrix with a 0.5% DNA (encoding a ~-galactosidase gene) loading was implanted into pig subdermal (endodermis) biopsy wounds. Extensive gene transfer was observed after 12 days of implantation. This gene delivery matrix system could be used for sustained delivery of plasmid DNA encoding a cytokine (e.g., Platelet Derived Growth Factor) as a single application therapeutic for accelerating chronic wound healing. KEYWORDS Hyaluronan, DNA, gene transfer, biodegradable, INTRODUCTION Hyaluronic acid (HA) is a natural high molecular weight glycosaminoglycan synthesized in the plasma membrane of fibroblasts and other cells.(1-3) HA has been used extensively in both drug delivery and surgical applications.(4-11) HA is used as an adjuvant for ophthalmic drug delivery(12) and has been found to enhance mucosal absorption of drugs.(l3) The efficacy of many pharmacological agents such as nonsteroidal antiinflammatory compounds (14-15) or cyclosporin (16) is markedly enhanced after being combined with HA. Derivatization of HA with dihydrazides such as adipic dihydrazide (ADH) provides multiple, pendant hydrazido groups for further conjugation of drugs,(l7) biochemical probes,(18) or for crosslinking (19-20). Drugs (for promoting wound healing) could be dispersed in a crosslinked HA matrix to form a biodegradable sustained release drug delivery system that could be applied topically to chronic wound sites. The rate of delivery of these pharmacological entities could be controlled
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Novel modified forms of hyaluronan
by the rate of HA degradation. This therapeutic system could achieve a site-specific therapy for accelerating the healing of chronic wounds. Gene transfer as an approach to wound healing renders transfected cells capable of producing therapeutic proteins (such as PDGF) locally. This circumvents the issues of frequent administration, and (e.g., denaturation) associated with utilizing recombinant proteins. In this paper, we describe a biocompatible and biodegradable, crosslinked hyaluronan (HA) matrix gene delivery platform. This device delivers plasmid DNA in a protracted manner, and achieves sustained gene transfer. It could be used for sustained delivery of plasmid DNA (encoding a cytokine) for accelerating wound healing. MATERIALS AND METHODS Fabrication of HA-DNA matrix Approximately one milligram of DNA was mixed with 25 ml of 1% Hyaluronic Acid solution, which was then deposited in a mold. A DNA-Hyaluronic Acid matrix was formed by lyophilization. The matrix was subsequently crosslinked in a solution of adipic dihydrazide and ethyl-3-[3-dimethyl amino] propyl carbodiirnide dissolved in a solvent mixture of dimethyl formamide (DMF) and water (a 9:1 mixture) for 6 hours. Afterwards, the DNA-HA matrix was extracted with several portions of isopropyl alcohol to remove the DMF and unreacted reagents. Isopropyl alcohol was removed by aspiration. The matrices prepared were observed under a scanning electron microscope. Characterization ofDNA-Hyaluronan (DNA-HA) matrix The DNA-Hyaluronan matrices (3 formulations with different levels of crosslinking: 1,2 and 3) were incubated with a 0.05% ethidium bromide solution. All samples were then observed under UV light at different time intervals (1, 4 and 13 days). DNA-HA matrices were hydrated in a Tris-EDTA buffer, pulverized and loaded into a 0.8% agarose gel; electrophoretic motility analysis was performed. The kinetics of DNA release from a representative matrix formulation (which resisted degradation in phosphate buffer) was examined in a medium of phosphate buffer containing hyaluronidase (11.2 units/ml). The DNA content of all samples collected was determined by a Thiazole Orange fluorescence assay. Electrophoretic motility analysis Was also performed on the DNA samples collected during the course of the controlled release study. Transfection of CHO cells The biological activity of plasmid DNA encoding a ~-galactosidase reporter gene incorporated into a DNA-HA matrix was assessed with a gene transfer study using Chinese Hamster Ovary (CHO) cells. Briefly, DNA-HA matrices were placed in cell culture wells for 48 hours. Cytochemical analyses (X-GaI™ staining) were performed thereafter. DNA samples (encoding a ~-galactosidase)collected during the course of a controlled release study were also used to transfect CHO cells (using Lipofectamine" as a transfection aid).
DNA matrix for gene transfer
307
In vivo gene transfer in a porcine excisional wound model
DNA-HA matrices with DNA encoding a ~-galactosidase reporter gene (0.5% loading) was implanted into pig excisional wounds. After 12 days, the implant sites were excised and assessed for ~-galactosidase expression. RESULTS AND DISCUSSIONS Scanning Electron Microscopy
The appearance of a typical DNAHA matrix under a scanning electron microscope is depicted in Figure 1. The surface of the DNA-HA matrix is very porous.
Figure 1 Interaction of DNA-Hyaluronan (DNA-HA) Matrix with Ethidium Bromide
DNA-HA matrices (1, 2 and 3, with different levels of crosslinking) were incubated in an ethidium bromide solution and observed under UV light. This is illustrated in Figure 2A. The fluorescence of the matrices indicates that DNA is dispersed throughout them. In contrast, the control (containing no DNA) shows little fluorescence. The same samples were observed under UV light after 4 and 13 days. This is illustrated in Figures 2B and 2C, respectively. In Figure 2B, Matrix 1 is clearly disintegrating (the presence of numerous tiny fluorescent fragments). Matrix 2 is swollen and Matrix 3 remains largely intact. In Figure 3C, Matrix 2 has completely dissolved (the bright fluorescence in the liquid and disappearance of the matrix), the disintegration of Matrix 1 accounts for the presence of numerous fluorescent fragments in the ethidium bromide solution. DNA is clearly bound to these HA fragments.
Figure2A
Figure 2B
Figure 2C
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Novel modified forms ofhyaluronan
Sustained Release of DNA from DNA-HA matrix The result of DNA release study (from a DNA-HA matrix) is depicted in Figure 3. The DNA-HA matrix system is capable of DNA release for a prolonged period of time. Approximately 6.5 ug of DNA was eluted after 33 days.
RELEASEOFDNA. . - DNA-HAMA1lUX
Figure 3. Electrophoretic Motility Analysis of DNA-HA fragments and DNA eluted from DNA-HA matrices Figure 4 depicts a representative result of electrophoretic motility analysis of DNA-HA fragments. Lanes I and 2 are DNA standards and the plasmid DNA used to prepare the DNA-HA matrix, respectively. Lanes 3 to 6 are matrix fragments. Only a small fraction of DNA migrated into the gel under the electric field. DNA appears to be conjugated to the Hyaluronan. Figure 5 depicts a representative result of electrophoretic motility analysis performed on the DNA released from matrices during the controlled release study. Lanes I and 2 are the DNA marker and the plasmid DNA used to prepare the DNA-HAmatrices, respectively. Lanes 3 to 6 are DNA samples collected during the course of the study. The distinct double bands indicate that DNA eluted from the matrix during the course of the controlled release study is structurally intact.
Figure 4. Electrophoretic motility of DNA-HA matrix fragments in a 0.8% Agarose gel
Figure 5 Electrophoretic motility of DNA released from a matrix in a 0.8% Agarose gel
DNA matrix for gene transfer
309
Transfection of eHO cells using DNA-HA matrix Plasmid DNA encoding ~galactosidase was incorporated into DNA-HA matrices. Figure 6 depicts a representative area in a cell culture dish incubated with a DNA-HA matrix sample (after 48 hours). Overall, approximately 5% of the cells were transfected (expressing the enzyme ~ galactosidase as indicated by the dark colored cells in the figure).
Figure 6. Transfection of CHO cells using a DNA-HA matrix
Longevity of DNA release and cell transfection Table 1
The DNA samples collected during the course of the controlled release study were used in CHO cell transfection studies. The relative levels of transfection are depicted in Table I.
Time (Days)
2 5 9
14 19
24 30 35 39 46
Relative Levels of Transfeetlon ++++ +++/++ ++++ +++++ +++++/++++
++++1+++ +++++ +++
++++1+++ +
Hlgh-+++++ Moderate = +++
Low=+
In Vivo Gene Transfer by DNA-HA Matrix A representative tissue section of a pig excisional wound implanted with a DNA-HA matrix for 12 days is depicted in Figure 8. The blue spots (gray areas in the black-and-white figure) in the endodermis of the ~ specimen indicate galactosidase expression and this has significant implications for the gene transfer capability of the DNA-HA matrix.
Epidermis ?
$10M2
Endodermis-s-
"'00....
Figure 8
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Novel modified forms ofhyaluronan
CONCLUSIONS We have shown that a DNA-HA matrix could be fabricated by crosslinking an expression plasmid with HA using bifunctional dihydrazide crosslinkers. The rate of DNA release is controlled by the rate of degradation. The results from the in vitro studies indicate that the DNA-HA matrix system is capable of sustained delivery of DNA and the DNA released from it is intact. Results from the in vivo study show that the matrix system is highly effective in gene transfer. The gene delivery matrix described here could be used for sustained transfer of plasmid DNA encoding a cytokine (e.g., Platelet Derived Growth Factor) as a single application therapeutic for accelerating chronic wound healing. This Hyaluronan matrix could be mated to wound dressings for covering wound sites; to maintain a moist and occlusive environment, which is known to be vital for promoting wound healing. It is also conceivable that this gene delivery device could be used in other medical applications. Studies for evaluating these potential applications are underway. REFERENCES I.
2. 3. 4. 5. 6.
7. 8. 9. 10. II. 12.
13.
14. IS.
K. L.Goa, P. Benfield, 'Hyaluronic acid. A review of its pharmacology and use as a surgical aid in ophthalmology and its therapeutic potential in joint disease and wound healing,' Drugs, 1994,47,56-566. T. C. Laurent, J. R. Fraser, 'Hyaluronan,' FASEB, 1992,6,2397-2404. B. P. Toole, 'Hyaluronan and its binding proteins, the hyaladherins,' Curro Opn. Cel/. BioI., 1990, 2,839-844. . J. Drobnik, 'Hyaluronan in drug delivery,' Adv. Drug. Delivery Rev., 1991,7:295. E. A. Balazs, J. L. Denlinger, In: The Biology of Hyaluronan, 1989, D. Evered & J. Whelan (eds), Chichester, Sussex, UK; Wiley, p265. E. A. Balazs, E. Leschiner, N. Larsen, P. Band, In: Encyclopedic Handbooks of Biomaterials and Bioengineering, Part A: Materials, 1995, D. L. Wise, D. J. Trantolo, D. E. Altobelli, M. J. Yaszemski, J. D. Gresser, E. R. Schwartz (eds), New York, p2719. D. A. Wil\oughby (ed), First International Workshop on Hyaluronan in Drug Delivery, 1992, London. D. A. Wil\oughby (ed), First International Workshop on Hyaluronan in Drug Delivery, 1994, London. D. A. Willoughby (ed), First International Workshop on Hyaluronan in Drug Delivery, 1995, London. D. A. Willoughby (ed), First International Workshop on Hyaluronan in Drug Delivery, 1996, London. D. Gustafson, In: The Chemistry, Biology, and Medical Applications ofHyaluronan and its Derivatives, 1997, T. C. Laurent & E. A. Balazs (eds), London. M. F. Saettone, B. Giannaccini, P. Chetoni, M. T. Torraca, D. Monti, 'Evaluation of high- and low-molecular-weight fractions of sodium hyaluronate and an ionic complex as adjuvants for ophthalmic vehicles containing pilocarpine,' Int. J. Pharm., 1991. 72, 131. K. Morimoto, H. Yamaguchi, Y. Iwakura, K. Morisaka, Y. Ohashi, Y. Nakai, 'Effects of viscous hyaluronate-sodium solutions on nasal absorption of vasopression and an analogue,' Pharm. Res., 1991, 8,471. A. R. Moore, D. A. Willoughby, 'Hyaluronan as a drug delivery system for diclofenac: A hypothesis for mode of action,' Int. J. Tissue. Reac., 1995, 17, 153. J. A. Miller, R. L. Ferguson, D. L. Powers, J. W. Bums, S. W. Shalaby, 'Efficacy of hyaluronic acid / nonsteroidal anti-inflammatory drug systems in preventing postsurgical tendon adhesions,' J. Biomed. Mater. Res. (Appl. Biomater.), 1997,38,25.
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16. G. Gowland, A. R. Moore, D. Willis, D. A. Willoughby, 'Marked enhanced efficacy of cyclosporin when combined with hyaluronic acid: Evidence from two T cell-mediate models,' Clin. Drug Invest., 1996, 11:245. 17. T. Pouyani, G. D. Preswich, 'Functionalized derivatives of hyaluronic acid oligosaccharides: Drug carriers and novel biomaterials,' Bioconj. Chern., 1994, 5, 339. 18. T. Pouyani, G. D. Preswich, 'Biotinylated hyaluronic acid: A new tool for probing hyalutonate-receptor interactions,' Bioconj. Chern., 1994, 5, 370. 19. T. Pouyani, G. S. Harbison, G. D. Preswich, 'Novel hydrogel of hyaluronic acid: Synthesis, surface morphology, and solid state NMR, , J. Am. Chern. Soc., 1994, 116, 7515. 20. K. P. Vercruysse, D. M. Marecek, J. F. Marecek, G. D. Prestwich, 'Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels for hyaluronic acid,' Bioconj. Chem., 1997,8,686.
HYALURONIC ACID HYDROGEL FILM: A NEW BIOMATERIAL FOR DRUG DELIVERY AND WOUND HEALING Yi Luo', Kelly R. Kirker", & Glenn D. Prestwich',t,2 'Department of Medicinal Chemistry, The University of Utah, 30 South 2000 East, Room 201, Salt Lake City, Utah 84112-5820 USA 'Department of Bioengineering, The University of Utah, 20 South 2030 East, Room 506, Salt Lake City, Utah 841 ]2-9458 USA
ABSTRACT A new hyaluronic acid (HA)-based hydrogel film was developed and evaluated for use in drug delivery and wound healing. This biocompatible material crosslinks and gels in minutes, and the dried film swells and rehydrates to a flexible hydrogel in seconds. HA was first converted to the adipic dihydrazide (ADH) derivative and then crosslinked with the macromolecular homobifunctional reagent poly(ethylene glycolj-propiondialdehyde (PEG-diald) to give a polymer network. After gelation, a solvent casting method was used to obtain an HA hydrogel film. The dried film swelled sevenfold in volume in buffer, reaching equilibrium in less than 100 sec. Scanning electron microscopy (SEM) of the hydrogel films showed a condensed and featureless structure before swelling, but a porous microstructure when hydrated. The thermal behavior of the hydrogel films, characterized by differential scanning calorimetry, indicated that the crosslinking of the two polymers clearly produced a new material having a microstructure different from either of its two components. The in vitro enzymatic degradation of the HA hydrogel films by hyaluronidase (HAs e) was also studied using SEM. Drug release from the hydrogel film was also evaluated in vitro using selected anti-bacterial and anti-inflammatory drugs. This novel biomaterial can be employed for controlled release of therapeutic agents at wound sites.
KEYWORDS Hyaluronic acid, biocompatible, poly(ethylene glycol), hydrogel, biodegradable, drug release, wound healing
INTRODUCTION Hyaluronic acid (HA), is a component of extracellular matrix (ECM) of all higher animals. This anionic linear polysaccharide is comprised of P-I A-linked D-glucuronic acid (P-I,3) N-acetyl-D-glucosamine disaccharide units with a range of naturally-occurring molecular sizes from 1-10,000 kDa. HA has unique physicochemical properties and distinctive biological functions '. HA has been implicated in water homeostasis of tissues, in the regulation of the permeability of other substances by steric exclusion phenomena, and in the lubrication of joints", HA also binds specifically to proteins in the ECM, on the cell surface, and within the cell cytosol, thereby having a role in cartilage matrix stabilization'", cell motility':", growth factor action", morphogenesis and embryonic development", and inflammation". HA is an attractive building block for new biocompatible and biodegradable polymers with applications in drug delivery, tissue engineering, and viscosupplementation"". Chemical modification" allows the physicochemical properties and in vivo residence time of HA to be tailored to specific applications while retaining its natural biocompatibility, biodegradability, and lack of immunogcnicity'".
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The use ofhyaluronan in drug delivery
We describe herein a new HA hydrogel well-suited to drug delivery, prepared using a macromolecular crosslinker under neutral aqueous conditions. The hydrogel thus formed can be used directly in virtually any biological system, as both the h and its two 1drogel macromonomer components are biocompatible and biodegradable'r. It has significant advantages for purification and drug loading in comparison to previously reported HA hydrogels":'", since alkaline conditions, high temperatures, and small toxic crosslinkers can be avoided. This HA hydrogel film has been found useful in drug delivery.
MATERIALS & METHODS Fermentation-derived hyaluronan (HA, sodium salt, M, = 1.5 x 106) was provided by Clear Solutions Biotechnology, Inc. (Stony Brook, NY). PEG-diald (M; = 3400) was obtained from Shearwater Polymers, Inc. (Huntsville, AL).
Preparation of HA-PEG-diald hydrogel films Several modifications of published procedures 15·19 were implemented to give better control of the degree of modification and to allow for more complete purification of the HA-ADH product. The carbodiimide-mediated coupling of HA to dihydrazide compounds (adipic dihydrazide, ADH) was performed in water at pH 4.75 and was monitored by the increase of the pH of the reaction mixture (Figure 1). The degree of substitution by ADH was determined by lH-NMR I5 • The degree of ADH substitution of the HA-ADH used in this study was 55%, based on the number of available glucuronates modified. HA-ADH was effectively crosslinked with PEG-diald, which produced a bis-hydrazone functionality as the covalent crosslink (see Figure 1). A hydrogel began to form within 60 sec after mixing the HA-ADH and PEG-diald solutions. The mixture was agitated on an orbital platform for an additional 24 hr to obtain a solid, uniform hydrogel. Hydrogels were stored in an open dish overnight at 37 DC to allow solvent evaporation and thus provided a flexible, hydratable HA hydrogel film.
HA-ADH
HA
HA-ADH-PEG-dial d Crosslinked Hydrogel
Figure 1.
Preparation of HA hydrogel by crosslinking HA-ADH with PEG-diald.
Swelling behavior of the HA hydrogel film Swellin:fi can be quantified by microscopic observations for isotropically-cxpanding systems'" . To visualize with a microscope the change in diameter size during swelling, cationic dye acridine orange was used to stain the HA hydrogel. The swelling ratio (SW) was defined as the weight of absorbed water per weight of dried disk, and was calculated using the formula SW = (Ds/Dd)3.
Hyaluronic acid hydrogel film
273
Morphology characterization by SEM The HA hydrogel films were gently rinsed with H20 and air-dried in an incubator at 37 DC for 24 hr to give dried hydrogel films. Swollen film samples were obtained by immersing HA hydrogel film disks in distilled H20 for 15 min, freezing quickly on dry
ice, and lyophilizing. HA hydrogel films subjected to enzymatic degradation were prepared by immersing the films in pH 7.4 PBS buffer containing HAse (lOa U/mL) for 3 days. The films were removed from the HAse solution, rinsed gently with H20, and dried on PTFE surface at 37 "C. For comparison, control HA hydrogel films were immersed into pH 7.4 PBS buffer without HAse at 37°C for 3 days, and processed similar to the enzyme treated films. All samples were gold-coated for conductance, and the surfaces of hydrogel films were examined via SEM.
In vitro drug release To evaluate the drug delivery characteristics of HA hydrogel for controlled drug delivery, several anti-inflammatory agents in clinical use were selected for drug release studies. Drug molecules were loaded into the hydrogel films via the in situ polymerization method. That is, the drug was initially mixed with one of the two macromonomers, and then the crosslinking protocol was followed. Dried hydrogel films loaded with drug molecules were cut into 6-mm diameter disks. Each disk was placed in a cuvette suitable for an UV-Vis instrument, and the in vitro drug release in the stirred cuvette was measured in 3 mL PBS buffer at 37 DC. The drug concentration released into the PBS buffer was detected by UV as a function of time. The remaining drug content in the film was determined by optical absorbance after complete degradation of the HA hydrogel film in IN NaOH. RESULTS & DISCUSSION Swelling behavior The average (n =3) equilibrium SW of the HA hydrogel film in PBS at 37 DC was 7.11
± 0.56. The equilibrium SW in 50 mM pH 7.0 phosphate buffer at 37 DC was 7.83 ±
1.75. No significant difference was observed among the equilibrium SW values in the different buffers, indicating that the SW was largely unaffected by the changes in ionic strength 22. The swelling kinetics of HA hydrogel film was also studied, and the HA hydrogel film swelled rapidly and reached equilibrium within 100 sec. Moreover, the rate of swelling was independent of the buffer composition. SEM Characterization of HA hydrogel film morphology The flat and featureless images of the surfaces and cross-sections (Figure 2a and 2b) indicated that the HA films have a condensed structure when dry, Figure 2c and 2d show the appearance of a highly-porous structure of the surface and cross-sectional images of HA hydrogel films after hydration. . Degradation of the HA hydrogel films by HAse was also examined by SEM. The control sample of HA hydrogel film immersed in enzyme-free PBS buffer for 3 days retained an intact, condensed surface structure (Figure 3a). In contrast, addition of 100 U/mL of HAse to the buffer produced significant surface erosion of the HA hydrogel film (Figure 3b). The surface differences of the HA hydrogel films in buffer, with or without enzyme, clearly demonstrated that HAse can recognize and process the crosslinked HA, and that the HA hydrogel films would be expected to be bioerodable in vivo.
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The use of'hyaluronan in drug delivery
Figure 2.
SEM images of HA hydrogel films. Surface image (a) and cross-sectional image (b) of dried HA hydrogel film; surface image (c) and cross-sectional image (d) of a hydrated and lyophilized HA hydrogel film.
(a)
Figure 3.
(b)
SEM analysis of I-lAse-degraded HA hydrogel films. Surface image (a) of I-IA hydrogel film immersed in PBS buffer for 3 days; surface image (b) of HA hydrogel film immersed in 100 U HAase/ml PBS for 3 days.
In vitro drug release Drug release rate % 100
r-------------------..,
o
80
HYD
60 40
20
PDS
o
o o
0.5
1.5
2
2.5
Log P Oct/water
Figure 4.
Correlation between the drug release rate and the drug hydrophobicity. The drug release rate is the slope of the plot of percentage cumulative release vs. (time) 112. Key: HYD, hydrocortisone; 6MP, 6a-methylprednisolone; PDL, prednisolone; COR, cortisone; CST, corticosterone; DEX, dexamethasone; PDS, prednisone.
Hyaluronic acid hydrogel film
275
Some of the drugs, e.g., pilocarpine, hydrocortisone, prednisolone, and cortisone, were rapidly released from the hydrogel film. Similar release profiles were obtained with diclofenac sodium, indomethacin, 6a-methyl-prednisolone, and corticosterone. These drugs were almost completely released from hydrogel film in 10 min following first order kinetics, which is consistent with diffusion from the gel during hydration and concomitant swelling of the hydrogel film". In contrast, slow first-order release kinetics were observed for dexamethasone and prednisone. In the in vitro release assay, dexamethasone showed sustained release for 1 hr, and prednisone showed sustained release for almost 24 hr (data not shown). HA in aqueous solution exhibits a random coil-coil structure with hydrophilic and hydrophobic strands. Figure 4 demonstrates a linear relationship between the hydrophobicity (the octanol-water partition coefficients (log p/4) of the drug molecule and its release rate from the HA hydrogel film, with the more hydrophobic drugs being released more slowly. These results indicated that this HA hydrogel film has excellent potential for controlled drug release based on drug hydrophobicity, thus allowing this new biomaterial to act as a local delivery device at wound sites.
CONCLUSIONS Two potential applications of the newly-developed HA hydrogel films include wound healing and the prevention of post-surgical adhesions. In both cases, drug release could be a valuable addition to reduce local pain, infection, or inflammation. Current investigation indicates that in a mouse model, the HA hydrogel film has been found to accelerate wound healing through the re-epithelialization proeess.
ACKNOWLEDGEMENTS We thank the Center for Biopolymers at Interfaces, The University of Utah, and the U.S. Department of Defense (DAMD17-98-1-8254) for financial support. We thank Clear Solutions Biotech, Inc. (Stony Brook, NY) for providing HA for these studies and for cooperating in the development of practical uses for the hydrazide technology.
REFERENCES I. T. C. Laurent, U. B. G. Laurent, & J. R. E. Fraser, Functions of hyaluronan, Ann. Rheum. Dis., 1995,54,429-432. 2. J. R. E. Fraser, T. C. Laurent, & U. B. G. Laurent, Hyaluronan: Its nature, distribution, functions and turnover, J. Intern. Med., 1997,242,27-33. 3. G. P. Dowthwaite, J. C. W. Edwards, & A. A. Pitsillides, An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development, J. Histochem. Cytochem., 1998,46,641-651. 4. C. Hardwick, K. Hoare, R. Owens, H. P. Hohn, M. Hook, D. Moore, V. Cripps, L. Austen, D. M. Nance, & E. A. Turley, Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility, J. Cell Biol., 1992, 117, 1343-1350. 5. L. Collis, C. Hall, L. Lange, M. R. Ziebell, G. D. Prestwich, & E. A. Turley, Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action, FEBS Lett., 1998,440,444-449. 6. W. F. Cheung, T. F. Cruz, & E. A. Turley, Receptor for hyaluronan-rnediated motility (RHAMM), a hyaladherin that regulates cell responses to growth factors, Biochem. Soc. Trans., 1999,27,135-142. 7. B. P. Toole, Hyaluronan in morphogenesis, J. Intern. Med., 1997,242,35-40. 8. B. Gerdin, & R. Hallgren, Dynamic role of hyaluronan (HY A) in connective tissue activation and inflammation, J. Intern. Med., 1997,242,49-55. 9. G. D. Prestwich, & K. P. Vercruysse, Therapeutic applications of hyaluronic acid and hyaluronan derivatives, Pharmaceut. Sci. & Technol. Today, 1998, 1,42-43. 10. K. P. Vercruysse, & G. D. Prestwich, Hyaluronate derivatives in drug delivery, Crit. Rev. Therapeut. Carrier Syst., 1998,15,513-555.
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11. G. D. Prestwich, D. M. Marecak, J. F. Marecek, K. P. Vercruysse, & M. R. Ziebell, Chemical Modification of Hyaluronic Acid for Drug Delivery, Biomaterials, and Biochemical Probes, In: The Chemistry, Biology, and Medical Applications of Hyaluronan and its Derivatives, T. C. Laurent (eds.), Portland Press, London, 1998, pp 43-65. 12. S. P. Zhong, D. Campoccia, P. J. Doherty, R. L. Williams, L. Benedetti, & D. F. Williams, Biodegradation of hyaluronic acid derivatives by hyaluronidase, Biomaterials, 1994, 15, 359-365. 13. N. Yui, T. Okano, & Y. Sakurai, Inflammation responsive degradation of crosslinked hyaluronic acid gels, J. Controlled ReI., 1992,22, 105-116. 14. K. Tomihata, & Y. Ikada, Preparation of cross-linked hyaluronic acid films of low water content, Biomaterials, 1997, 18, 189-195. 15. T. Pouyani, & G. Prestwich, Functionalized derivatives of hyaluronic acid oligosaccharides: drug carriers and novel biomaterials, Bioconjugate Chem., 1994, 5, 339-347. 16. T. Pouyani, G. Harbison, & G. Prestwich, Novel hydrogels of hyaluronic acid: Synthesis, surface morphology, and solid-state NMR, J. Am. Chem. Soc., 1994, 116, 7515-7522. 17. K. P. Vercruysse, D. M. Marecak, J. F. Marecek, & G. D. Prestwich, Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid, Bioconjugate Chem., 1997, 8, 686-694. 18. Y. Luo, & G. D. Prestwich, Synthesis and selective cytotoxicity of a hyaluronic acidantitumor bioconjugate, Bioconjugate Chem., 1999, 10,755-763. 19. Y. Luo, K. R. Kirker, & G. D. Prestwich, Crosslinked hyaluronic acid hydrogel films: New biomaterials for drug delivery, J. Controlled ReI., in press. 20. O. Wichterle, & R. Chromecek, Polymerization of ethylene glycol monomethylacrylate in the presence of solvents, J. Poly. Sci.: Part C, 1969, 16,4677-4686. 21. C. Wang, R. J. Stewart, & J. Kopeeek, Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains, Nature, 1999, 397, 417-420. 22. D. Papini, V. J. Stella, & E. M. Topp, Diffusion of macromolecules in membranes of hyaluronic acid esters, J. Controlled ReI., 1993, 27,47-57. 23. L. M. Benedetti, E. M. Topp, & V. J. Stella, Microspheres of hyaluronic acid estersFabrication methods and in vitro hydrocortisone release, J. Controlled Rel., 1990, 13, 33-41. 24. A. Leo, C. Hansch, & D. Elkins, Partition coefficients and their uses, Chem. Rev., 1971, 71,525-616.
THERMAL PROPERTIES OF HYALURONIC ACID-BASED POLYURETHANE DERIVATIVES ASSOCIATED WITH WATE~ Hyoe Hatakeyama
1
* , Yasuhiro Asano 1
•
1
,Tatsuko Hatakeyama 3 & John F. Kennedy
2
Department ofApplied Physics and Chemistry, Fukui University of Technology, 3-6-1 Gakuen, Fukui, Fukui 910·8505, Japan 2
Department of Textile Science, Otsuma Women's University, 12 Sanbancho, Chiyoda-ku, Tokyo 102-8357, Japan 3
Birmingham Carbohydrate & Protein Technology Group, Chemibiotecb Laboratories, University ofBirmingham Research Park, Vincent Drive" Birmingham 815 2SQ, UK.
ABSTRACT Hyaluronic acid (HA) - based polyurethane derivatives (HAPU's) were prepared by polymerization with isocyanate with the presence of ethylene glycol (EG) and a small amount of catalyst. The HNEG ratio (g g-l) was varied from 1/99 to 1/2.5. Polymerization was carried out for more than 12 hr at room temperature with the presence of a solvent. Structural change of water of HAPU in the water content [We = (mass of water)/(mass of dry HAPU)] range from 0 to 6.0 g g' was monitored by differential scanning calorimetry. Based on the results obtained in this study, the following characteristics are found in HAPU by the introduction of crosslinking points to the HA molecular chain via urethane linkage; (1) water holding capability can be controlled by a certain molecular design, (2) the samples with a large amount of bound water, especially freezing bound water can be obtained.
KEYWORDS Hyaluronic acid, polyurethane, thermal properties, water
INTRODUCTION In our previous studies, we reported that various types of polyurethanes (PU's) are derived from mono-, oligo- and polysaccharides 1,2,3. Hydroxyl groups of saccharides are used as the reaction site and the degree of crosslinking is regulated by changing experimental conditions, such as the amount of saccharides. By introduction of saccharide core groups in the PU's, it was found that not only mechanical properties but also hydrophilicity of the PU's can easily be controlled over a wide range. Among numerous kinds of polysaccharides, sodium hyaluronate (HA) shows unique properties, such as high water holding capability and biocompatibility. HA is an amino
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Novel modified forms ofhyaluronan
polysaccharide "and synthesized in a living organ. At the present, HA produced by bacteria is widely utilized in fields, such as medicine and cosmetics. We reported that HA forms physically cross-linked hydrogels when the molecular chains of aqueous solution adopt a thermodynamically equilibrated conformation 5. The gel sol transition of HA hydrogel is observed at around 40°C 5. Since physical HA gels thus prepared are not thermally stable over a wide temperature range, it is necessary to prepare chemically bonding HA gels in order to obtain stable characteristics for practical applications. It is known that there are several chemical cross-linked HA hydrogels, for example hylan 6. In this study, the aim is to prepare HA hydrogels linked via urethane bonding. Polyurethanes (HAPU's) are derived from HA using lysine-based isocyanate. In order to study the characteristics of hydrogels, the structural change of water restrained in crosslinking networks has also been investigated. HAPU-water interaction has been examined based mainly on the amount of bound water 7 which is tightly restrained by the HAPU matrix.
EXPERIMENTAL
Materials HA in the powder form was supplied by Kibun Food Chemical. Co. Molecular weight 6 was 1.8 x 10 - 2.2 X 106 , calculated from intrinsic viscosity using the equation [fJ]=3.6 x 10-1 78 Mo. . HA-based PU's were synthesized by the polymerization of HA with isocyanate with the presence of ethylene glycol (EG) and a small amount of dibutyltin dilaurate. The HNEG ratio (g g'!) was varied from 1/99 to 1/1.25. Polymerization was carried out for more than 12 hr at room temperature with the presence of a solvent such as dimethyl sulfoxide (DMSO). The obtained polymer was filtered and was air-dried. Fig. 1 shows a scheme of the sample preparation The HA content in the polyol (= HA + ethylene glycol (EG» was 5%,20%,40%,60% and 80%. NCO/OH ratio was 0.4, 0.5, 0.6, 0.8, 1.0, 1.2 and 1.4. HAPU samples were obtained in powder form. The schematic chemical structure of prepared HAPU is shown in Fig. 2.
Methods Water content determination The samples with various water contents were prepared as follows. The samples with water were weighed, sealed in an aluminium pan and maintained for several days for equilibration. After measurements, the pan was pierced, placed in an oven at 110°C tor 2 hours. The dried sample was quickly weighed and water content was calculated. Water content (We) of gel samples was defined as follows. We= (mass of water)/(mass of dried gel) , g
s'
[1]
Hyaluronic acid-based derivatives Hyaluronic acid
Ethylene glycol
<£D1&DMSO
I
PU
Figure 1. Preparative scheme of polyurethane derived from HA
Figure 2.
Chemical structure of HAPU
315
316
Novel modified fOnTIS of hyaluronan
Differential scanning calorimetry A Seiko differential scanning calorimeter DSC 220 equipped with a cooling apparatus was used. Approximately 5 mg samples containing various amounts of water were hermetically sealed in an aluminium pan. The temperature was varied as follows (1) the sample was cooled from 40 to -150°C at a cooling rate of 10°C min"l. (2) the sample was maintained at -150°C for 5 minutes and heated at 10°C min'l to 40°C (3) the processes (1) and (2) were repeated. Phase transition temperatures were defined as reported previously 7. When melting endothermic peaks were observed, the low temperature side peak was designated as Tml and the high temperature peak Tmh- Enthalpy of melting and crystallization was calculated using peak area. Non-freezing water content (Wnf) 7,8 was calculated using the following equation and the enthalpy of melting of water (334 J g") was used for calculation. In this study, values obtained from heating curves were used for calculation.
[2] Where !illm is enthalpy calculated from melting peak of the gels and of dry gel.
mdry gel
is the mass
RESULTS & DISCUSSION In order to investigate the effect of water on the molecular motion of HAPU, phase transition behaviour of HAPU with various amounts of water was investigated by DSC. As stated in the experimental section, We of HAPU-water systems was varied from 0 to 6 g it. Fig. 3 shows representative DSC curves of HAPU-water systems having various water contents. As clearly seen, a baseline deviation due to the glass transition is observed. The glass transition temperature (T J shifts to low temperatures with increasing W", reaches the characteristic minimum temperature and increases slightly. This Tg variation is similarly observed in hyalurnonic acid-water systems 9.10. When We exceeds 0.6, endothermic peaks due to melting of water can be observed. In the We range 0 to 2.5 g s". a small peak or shoulder can be observed at a temperature lower than main melting peak. When We exceeds ca. 3.0, the low temperature peak was masked by the main large peak and disappeared. In this study, peak temperature was used as a criteria of melting temperature when two peaks were separately observed, it is considered that two types of water exist in the system. The high temperature peak is designated as Tgh and that of the low temperature Tgl- The enthalpy of each melting peak was calculated as described in the experimental section. The melting enthalpy of the low temperature peak (/},}{ml) was calculated using melting enthalpy of pure water. This causes errors of several per cent, since the melting enthalpy of irregular ice is not the same as normal ice. Using the values of Tg, Tmb and Tmh phase diagrams of HAPU-water systems with NCO/OH ratios 0.6, 0.8 and 1.0 were established. Fig. 4 shows the representative phase diagram of HAPU systems with NCO/OH ratio = 0.6 (HA content 80 %).
Hyaluronic acid-basedderivatives
317
Fig. 5 shows the three-dimensional relationship between T g, We and NCO/OH ratio. Tg decreases dramatically at low We and reached a critical point at around 1 g g' and then gradually increased. If a large amount of water is added to the system, Tg can not be observed due to formation of a large amount of ice. Fig. 6 shows the relationship between TmI and We' TmI'S can be categorized into two groups as shown in this figure. TmI values of the samples with NCO/OH ratio larger than 0.8 are found in the temperature range from -10 to 3 °C (curve I in Fig. 6) and those of the samples with NCO/OH ratio lower than 0.6 are in the range -20 to -14°C (curve II). Enthalpies of melting (MImi) of the samples located on curve II are less than 0.3 g g-l. These values are larger than reported values of other saccharides, for example MImi cellulose with different polymorphic structure and crystallinity ranges from 0.01 to0.08 g g-l. In contrast, Mlmh at a high We range were markedly large, more than 10 g g-l. The details of this MJml will be described in the latter section
~
Trm
~
o ~
"0
c::
1-
W
I
-70
_
~'
-50
~~
T.I"~ ~
~Tni
Figure 3.
2.81
0.23
"--
-120
3.80
1.95
-=== " -
we/gg-\
0.04
" Tg -60
T i-c
o
60
Representative DSC curves of HAPU HA content in polyol = 80%, NCO/OH ratio = 0.6 Numerals in the figure show water content (We) Tmh; high melting peak temperature Tml ; low melting peak temperature Tg; glass transition temperature
318
Novel moditied fOnTIS ofhyaluronan
-80
o
2
4 Wei 9 gol
6
8
Figure 4.
Repersentative phase diagram of HAPU-water system HA content in polyol = 80%, NCOIOH ratio =0.6 Tmh; high melting peak temperature TmJ; low melting peak temperature Tg; glasss transition temperature
Figure 5.
Three-dimensional diagram of glass transition temperature (T J, water content (We) and NCOIOH ratio.
Hyaluronicacid-basedderivatives
319
5.-------------, 1.2
•
I
11-
o
0.8
1.0
-5
!oJ
...... -10
. .:S
·15
-25
L-----I._--L._.....L-_....L...-_.l.------'
o Figure 6.
1
5
6
Relationship between low temperature side melting (T m1) and water content (We) of HAPU with various NCO/OH ratios HA content in polyol = 80% Numerals in the figure show NCO/OH
Fig. 7 shows the relationship between Tmh and We' All T mh values fall on a line. It is evident that the structure of free water in the systems with different NCO/OH ratios is the same. It is known that a characteristic amount of water restrained by hydrophilic groups shows no first-order phase transition and that this kind of water is designated as nonfreezing water (Wnf) 10. In our study, the amount of non-freezing water was obtained by subtracting the amount of water calculated from the enthalpy of melting from the amount of added water as stated in the equation (3). Fig. 8 shows relationships between Wf and We' Wnf values can be obtained where the Wf line intersects the x-axis. As shown in Fig. 8, Wnf values are almost the same regardless of NCO/OH ratio. However, the Wf line of the samples with NCO/OH ratio 0.8 and 1.0 is curved low We' This indicates the existence of freezing bound water. As described in our previous paper", the number of water molecules categorized into non-freezing water corresponds to the number of hydrophilic groups attached to linear molecular chains. However, when matrix polymers are non-linear, the above rule can not always be applied. At the same time, the amount of freezing bound water is markedly affected by the complex matrix structure. In this study, we estimated the amount of freezing bound water (WIb ) using the following equation. (3)
320
Novel modified formsof hyaluronan
As shown in Fig. 3, 4 and 5, the characteristic feature of the HAPU samples is that Wfb is distinctly observed. Fig. 9 shows a three dimensional diagram between Wfb , We and NCO/OH ratio. The Wfb values of HA are similar to the other polysaccharides, however when NCO/OH ratio increases, Wfb increases dramatically. In the low We region, the amount of water in the system is insufficient to form irregular ice. When We exceeds ca. 3.0 g g'l, the sudden increase of Wfb is observed. In the previous reports, molecular functionality, such as biocompatibility, molecular separability, etc. are strongly related with the amount of freezing bound water in the water-polymer systems 6, 11. In HA-water systems, it was found that the amount of freezing bound water depends markedly on thermal history 12. In HAPU samples, it is noted that the amount of bound water is controllable by changing the synthetic conditions. In this study, structural change of water of HAPU at low We was investigated in order to ascertain the initial stage of water swelling. HAPU samples have a water holding capability larger than We estimated in this study. In order to establish the water holding capability over a wide range, further investigation is necessary. Based on the results obtained in this study, the following characteristics are found in HAPU by the introduction of crosslinking points to the HA molecular chain via urethane linkage; (1) water holding capability can be controlled by certain molecular design, (2) the samples with a large amount of bound water, especially freezing bound water can be obtained.
6
0.8
~ if'
1.2
4
0
•
0
0
2
0
?....
• •
t-
0.6
.co E
0.4
0
-2
-4 -6'-----1._....1--..1...---1.----'---' 6 5 o 1
Figure 7.
Relationship between high temperature side melting (Tmh) and water content (We) of HAPU with various NCO/OH ratios HA content in polyol = 80% Numerals in the figure show NCO/OH
Hyaluronicacid-based derivatives
321
6r----------7'-"1
... '0'1 0'1
-
4
2
OL....o~....:.L_--l...
_ _...J.__~
o Figure 8.
Relationship between amount of freezing (Wr), water content (We) HA content in polyol = 80% Numerals in the figure show NCO/OH
1.6
1,0 -:'01 01
~
:li= 0.5
Figure 9.
Relationship between Wfb, We and NCO/OH ratio HA content in polyol = 80%
1.6
1.0
0.6
322
Novel modified forms ofhyaluronan
REFERENCES
1.
2. 3.
4. 5. 6. 7. 8. 9. 10
11
12.
P. Zetterlund, S. Hirose, T. Hatakeyama and H. Hatakeyama, Thermal and mechanical properties of polyurethanes derived from mono- and disaccharides, PoLymer InternationaL, 1997,42, pp.1-8. S. Hirose, K. Kobashigawa, Preparation and physical properties of polyurethanes derived from molasses, Sen-i Gakkaishi, 1994, 50, 538-54. H. Hatakeyama, S. Hirose, K. Nakamura and T. Hatakeyama, New type of polyurethenes derived from lignocellulose and saccharides, in CeLLuLosics, ChemicaL BiochemicaL and MateriaL Aspects, J. F. Kennedy, G. O. Phillips and P. A Williams (eds.), Ellis Horwood, 1993, pp.525-536. S. Aronott, A K. Mirta and S. Raghunathan, Hyaluronic acid double helix, J. Mol. BioI. 1983,169, 861-872. Jyunji Fujiwara, Masato Takahashi, Tatsuko Hatakeyama and Hyoe Hatakeyama, Gelation of Hyaluronic Acid by Annealing, Carbohydrate Polymers, accepted S. Takigami, M. Takigami and G. 0 Phillips, Hydration characteristics of the crosslinked hyaluronate derivative hylan. Carbohydrate Polymers, 1993,22,153-160. H. Hatakeyama and T. Hatakeyama, Interaction between water and hydrophilic polymers, Thermochirnica Acta, 1998, 130, 3-22. T. Hatakeyama and Liu Zhenhai (eds.), Handbook of ThermaLAnaLysis, John Wiley, Chichester 1998, pp.107-112. H. Yoshida, T. Hatakeyama, K. Nakamura and H. Hatakeyama, Glass transition of hyaluronic acid hydrogel, Kobunsi Ronbunshu, 1989, 46, 597-602. H. Yoshida, T. Hatakeyama, and H. Hatakeyama, Effect of water on the main chain motion of polysaccharide hydrogels, in Viscoelasticity of biomalerials, W. Glasser & H. Hatakeyama, ACS Symp. Ser. 489 Am. Chern. Soc. Washington DC. 1992, pp.217-230 T. Hatakeyama, and H. Hatakeyama, Thermal properties of water around the crosslinked networks in cellulose pseudo hydrogels, ChemicaL Biochemical and Material Aspects, J. F. Kennedy, G. O. Phillips and P. A Williams(eds.), Ellis Horwood, 1993, pp.225-230. T. Hatakeyama, H. Yoshida, K. Nakamura and H. Hatakeyama, DSC studies on ice in polysaccharide hydrogels, in Physics and Chemistry of Ice, N. Maeno and T. Hondoh(eds.), Hokkaido Univ. Press 1992, pp.262-269.
PHASE TRANSITION OF SODIUM HYALURONATE, HYLAN AND POLYURETHANES DERIVED FROM HYALURONIC ACID IN THE PRESENCE OF WATER Tatsuko Hatakeyama ' & Hyoe Hatakeyama" I
Otsuma Womell's University, 12 Sanbancho, Chiyoda-ku, Tokyo 102-8357, Japan , Fukui Institute of Technology, 3-6-1 Gakuen, Fukui, Fukui 910-8505, Japan
ABSTRACT Phase transition behaviour of sodium hyaluronate and two different types of hyaluronic acid derivatives was investigated in the presence of water. Water content ( mass of water)/(mass of dry sample, g g") of the systems was varied from 0 to 10 g g'. Sodium hyaluronate was supplied by Kibun Food Chemifer with molecular weight ranged from 1.8 x 106 to 2.2 X 106 • Polyurethanes derived from hyaluronic acid derivatives with various crosslinking densities were synthesized in our laboratory. Phase transition temperatures and enthalpy of phase transitions were measured by differential scanning calorimetry (DSC). Phase transition data of hylan reported by Takigami et al., (Carbohydrate Polym., 1993, 22,153), which was obtained under similar experimental conditions, was recalculated and compared with those obtained in this study. Glass transition (T g ) , cold crystallization (Tc ) and melting of water (Tm ) were observed for the above three series of the samples in the presence of water. The characteristic feature of the samples was detected as different melting behavior, i.e. two to three melting peaks of water can be observed in a temperature range from -20 to 0 'c. These facts indicate that freezing bound water, as well as free water, exist in the systems. The amount of freezing bound water (Wfb ) was calculated from the melting enthalpy of water in the systems. Hylan contains the largest amount of Wrb among the three samples. The amount of W rb of the chemically cross-linked sample is stable. On the other hand, Wfb varied according to thermal history.
=
KEYWORDS Hyaluronate, polyurethanes, hylan, phase diagram, water, bound water, DSC
INTRODUCTION Water holding capability is an unique property of hyaluronic acid (HA) and its derivatives. It is thought that the structural change of water restrained in hyaluronic acid takes an important role in biocornpatibility. In the previous reports concerning phase transition behaviour of hyaluronic acid in presence of water, the following characteristics are observed, i.e. (I) in a water content from to 004 to 2.0 g s''. glass transition is observed in a temperature range from -70 to -100 'C. Glass transition temperature depends upon water content'. Glass transition is attributed to the cooperative motion of HA and amorphous ice in the system, (2) cold crystallization caused by reorganization of amorphous ice in the system is observed in a water content ranging from 004 to 1.2 g g' I. (3) melting of ice in HA occurs in two or three steps in a temperature from -30 to 3 'C, suggesting the presence of not only normal ice but also irregular ice'. It is thought that irregular ice corresponds to freezing bound water and that the amount is affected by thermal history in a complex manner. The melting behaviour of water restrained by HA derivatives, such as hylan, varied in a complex manner' and a large amount of freezing bound water was observed. Polyurethanes derived from HA also show the existence of characteristic amount of freezing bound water 3.
324
Novel modified forms ofhyaluronan
In order to understand the nature of water restrained by the hyaluronic acid and its derivatives, it is important to make comparison the characteristic feature of phase transition behaviour measured by the same experimental technique. In this study, differential calorimetry data of three kinds of samples are compared in a water content ranging from 0 to 10 g s'.
EXPERIMENTAL
Materials Hyaluronic acid in the powder form was supplied by Kibun Food Chemiferl. Co. Molecular weight was 1.8 x 106 - 2.2 X 106 , calculated from intrinsic viscosity using the equation 1111=3.6 x 10,4 MO,7S. Hyaluronic acid (HA) - based polyurethane derivatives (HAPU's) were synthesized by the polymerization of hyaluronic acid with isocyanate with the presence of ethylene glycol (EO) and a small amount of catalyst. Hydrophilicity was varied by changing HA content in polyol (= HA + ethylene glycol (EG» which ranged from 5 to 80. Crosslinking density was varied by changing NCO/OH ratio which ranged from 0.4 to 1.2. The sample preparation and chemical structure of HAPU are shown elsew here",
Methods Water content determination The samples with various water contents were prepared as follows. The samples with water were weighed, sealed in an aluminum pan and maintained for several days for equilibration. After measurements, the pan was pierced, placed in an oven at 110 ·C and maintained for 2 hours. The dried sample was quickly weighed and water content was calculated. Water content (We) of gel samples was defined as follows. We
=( mass of water)/(mass of dried gel) , g g'
[II
Differential scanning calorimetry A Seiko differential scanning calorimeter, DSC 220 equipped with cooling apparatus and a Perkin Elmer DSC lIe with cooling apparatus, was used. The ca. 5 mg samples containing various amounts of water were hermetically sealed in an aluminium pan. The temperature was varied as follows (I) the sample was cooled from 40 to -ISO ·C at a cooling rate of 2 to 4O·C min'. (2) the sample was maintained at -150·C for 5 minutes and heated at 2 to 4O·C min" to 40 ·C (3) annealing of the sample was carried out in a DSC sample holder and heating/cooling runs were carried out at various rates after annealing. Phase transition temperatures are defined as reported previously". As shown in the schematic DSC curve in Fig. I, temperature indicated by the arrow is defined as glass transition temperature (Tg ) . The heat capacity difference between the glassy state and rubbery slate at Tg was designated as I'J.CI" Cold crystallization is designated as T"e When melting endothermic peaks were observed, the low temperature side peak is designated as Tmt and the high temperature peak Tmh. When more than two Tm,'s were observed, peak temperatures were numbered from the high to low temperature side. If the peaks were not clearly separated, the peaks were divided by a straight line from the lowest point of the curve as shown in Fig. I. Enthalpy of melting (I'J.Hmh, I'J.Hml) and cold crystallization
(I'J.H ) was calculated using each peak area. Ne~n-freezing water content (W nf)4.5 was calculated using the following equation. Enthalpy of melting of water (334 J g'l) was used for calculation. In this study, values obtained from heating curves were used for calculation.
Sodium hyaluronate, hylan and polyurethanes
325
T Figure 1. Shcematic DSC heating curve T ; glass transition temperature Sc; heat capacity difference at T: Teo; cold crystallization temperature Tmh Tm, ; melting temperatures !1Hee ; cold crystallization enthalpy !1Hmh • !1Hml ; melting enthalpy
W nr = 1 -!1Hmh13341 In dry samp Ie ' g g' where m dry sample is mass of dry sample. The amount of freezing bound
l2J water (Wrh) is
calculated using the values of!1Hml .
Wfb =!1H ml 1 In dry sample
'
g
s:'
[3]
The amount of bound water (W b ) is defined as follows.
Wb
=W
nf
+ W fb
t
g g'
l4J
RES ULTS & DISCUSSION Fig. 2 shows the representative DSC heating curves of HA (curve I), HAPU (curve II) and hylan (curve III) containing similar We (ca. 1.0 g g'). For HA and HAPU samples, T g , Teo' Tm' s are observed. T g of hylan is not reported", although the temperature range of measurement was almost the same as those of HA and HAPU. When three DSC curves are compared, the largest difference is the size and number of the low temperature melting peak. As clearly seen in the curve III of hylan, the low temperature melting is marked. When We increases, T"'I was not only large but also separated into several sub peaks. It is thought that ice whose melting temperature is lower than a 'C is not the hexagonal crystalline structure. The fact indicates that a large amount of irregular ice having a broad size distribution is formed in hylan. Cold crystallization was observed in a narrow We range for HA (We = 0.6 - 1.4 g g') and HAPU (We = ca. 1.0 g go' depending on NCOIOH ratio). In contrast cold crystallization is not reported for hylan in the same temperature ranges. Fig. 3 shows the relationships between Tm, of three samples and We' In the case of hylan, two Tm.'s, Tml l and Tm12 , were observed (see curve III in Fig. 2), Tml I values are used in Fig. 3. As has already been reported, Tml of HAPU is separated into two groups, high and low crosslinking groups). T nll of hylan is located between two groups of HAPU. Tml values of HA are shown in a broad range due to the fact that the value
326
Novel modified forms ofhyaluronan 5 0 ·5
-go
~
II
-.
w
...."
t
·10 -15
III -20 -25
0
·100
·50
T 1°C
o
Figure 2. DSC heating curves of HA (curve I), HAPU (curve II) and hylan (curve III). HA W e= 0.84 g g'. HAPU W e= 0.88 g g", hylan We=0.74 g s'.
2
4
6
We Igg-'
Figure 3.
Relationships between Tml and water content (We) O;HA o ; HAPU (NCOIOH=0.4--0.6) . ; HAPU (NCOIOH=0.8~1.2) t::,. ; hylan
varies according to the thermal history 4. Fig. 4 shows the amount of freezing bound water (Wrb) calculated from the enthalpy of melting (I1Hm, ) as a function of We' I1Hml of hylan is marked compared with that of HAPU. I1Hml of HA with a We ranging from 1.0 to 1.5 g s' scatters, since the amount varies depending on thermal history. Fig. 5 shows variation of Tmh of the samples. In contrast to Tm" the melting temperature of free water (T mh) of HA and HAPU samples is observed in the similar temperature range, in contrast, that of hylan maintains a constant value. Tmh of HA and HAPU slightly increases with increasing We . As already reported, the amount of water showing no first order phase transition is designated as non-freezing waters. The W nr calculated from equation 2 is 0.5 g g' for HA, 0.6 g g' for hylan and 0.5 g g-I for HAPU. The amount of non-freezing water mainly depends on the chemical structure of the samples, and it is reasonable that the three samples show no large differences. On this account, the differences of bound water among the three samples come from the WIb from equation 4. From the above results, it can be said that Wrb of HA and HA derivatives, especially hylan, is unusually large compared with other hydrophilic polymers", On this account, it is necessary to discuss how freezing bound water is formed in the HA and HA derivatives. In the case of HA, it is thought that freezing bound water is formed in the following manner. Absolute I1c values are affected by the chemical constituents of the sample and at the same time, the Pintermolecular interaction and presence of the side chain groups. As already slated in our previous papers on I1Cp of water-polysaccharide systems, I1Cp values show the maximum point, when free water starts to be formed in the system. I1Cp
Sodium hyaluronate, hylan and polyurethanes
327
at T g (see Fig. 1) of linear polymeric materials decreases with increasing T g or be, is constant if the repeating unit is decomposed in a certain unit of molecular motion. However, in the case of HA-water systems, the above general rule is not applied and large values are obtained. In our previous reports, we especially paid attention to the role of amorphous ice in the system. The homogeneous mixture of HA molecules and amorphous ice is thought to be mobile at T g • During the heating process, amorphous ice is transformed to irregular ice which melts at a temperature lower than a ·C. It is thought that irregular ice transformed from amorphous ice consists as a part of W f b • On this account, W fb , of HA is not a constant value, but the amount depends on thermal history as shown in Fig. 4, especially in a temperature range showing Tee' This result is supported by the variation of T g shown in Fig. 6. T g of HA at around We of 0.5~1.5 is exceptionally low due to the presence of amorphous ice. When HA molecules are chemically linked, the above phenomenon is thought to be changed. When be, values of HAPU in the presence of water are compared with those of dry HA, no large difference was found. The fact that the Sc, value is ca. 0.05 kJ kg' K 1 suggests that molecular enhancement at T g is restricted. Freezing bound water of chemically crosslinked HA derivatives is formed not from amorphous ice but from the network structure itself. This situation is quite similar to the structural change of water restrained in hollow fibre and separation membranes. In those cases, the water mobility is controlled by geometrical freedom. The above DSC results indicate that water restrained by HAPU having low crosslinking densities is similar to HA in which Wrb depends on thermal history. In contrast, hylan and HAPU having high crosslin king densities contain permanent Wrb , although the Wfb of hylan is marked.
sc,
8
'",
--'"
/ .16 L..-_..L..-_...I-_--,-_....L._.....1 o 10 8 2 4 6
Figure 4. Relationships between amount of freezing bound water (W fb )and water content (W) 0; HA, 0; HAPU, t::,.; hylan
Figure 5.
Relationships between Tmh and water content (W) 0; HA, 0; HAPU, t::,.; hylan
328
Novel modified forms ofhyaluronan
-40
P ..... C
1-'"
cO
-80
0
120 L.. _1..--1---''---'-_'---' 0
We /99"
Figure 6. Glass transition temperature and heat capacity difference at Tg (/).Cp ) of HA and HAPU as a function of We 0; HA (cooled at lO·C min" from 50 to -150·C • ; HA (quenched), 0; HAPU
REFERENCES
1.
2. 3. 4. 5.
H. Yoshida, T. Hatakeyama & H. Hatakeyama, Effect of water on the main chain motion of polysaccharide hydrogels, in Viscoelasticity of Biomaterials, W. Glasser & H. Hatakeyama (eds.), ACS Symp. Ser. 489 Am. Chern. Soc. Washington DC., 1992, pp. 217-230 S. Takigami, M. Takigami & G. O. Phillips, Hydration characteristics of the cross-linked hyaluronate derivative hylan. Carbohydrate Polymers, 1993,22, 153-160 H. Hatakeyama, Y. Asano, T. Hatakayama, & J. F.Kennedy, Thermal Properties of Hyaluronic acid-based polyurethane derivatives associated with water, in this book T. Hatakeyama and Liu Zhenhai (eds.), Handbook of Thermal Analysis, John Wiley, Chichester,1998, pp.107-112 H. Hatakeyama & T. Hatakeyama, Interaction between water and hydrophilic polymers, Thermochimica Acta., 1998, 130, 3-22
PART 6 ASPECTS OF HYALURONAN IN JOINTS
SYNTHESIS AND DEGRADATION OF HYALURONAN IN ARTICULAR CARTILAGE Carl R. Flannery*, Duncan R.R. Hiscock and Bruce Caterson Connective Tissue Biology Laboratories. Cardiff University CardiffSchool of Biosciences, Museum Avenue. Cardiff CF103US. Wales UK
ABSTRACT The glycosaminoglycan hyaluronan (HA) is an integral component molecule of articular cartilage, contributing to the specialized structural and functional properties of this connective tissue. In the studies reported herein, we have documented the mRNA expression profiles of three mammalian HA synthase (HAS) enzymes (HAS I, HAS2 and HAS3), four hyaluronidases (HYALI, HYAL2, HYAL2 and MGEAS) and the HA receptor CD44 in chondrocytes from normal (un diseased) animal and human cartilage and osteoarthritic human cartilage maintained in experimental culture systems exposed to catabolic or anabolic stimuli provided by cytokines, retinoic acid and growth factors.
KEYWORDS Cartilage, CD44, hyaluronan, hyaluronan synthases, hyaluronidase
INTRODUCTION Articular cartilage is a specialized connective tissue. Within cartilage, hyaluronan (HA) polymers serve as essential anchors which bind monomers of the large sulfated proteoglycan aggrecan, together with a stabilizing link protein, to facilitate the formation of macromolecular aggregates. Maintenance of these multi molecular hydophilic aggregates thus enables cartilage to reversibly deform during loading and is crucial for the low friction articulation provided by healthy joints. Several independent studies have demonstrated that HA is both synthesized and turned over in articular cartilage by the resident chondrocytes'", although the precise molecular mechanisms involved in such metabolism have not been elucidated. Recently, a family of enzymes (designated HAS for HA Synthase) responsible for the synthesis of HA have been identified", as has a group of at least six paralagous hyaluronidase (HY AL) genes" as well as one unrelated hyaluronidase designated MGEAS 6 • In recent studies we have examined several aspects of the expression of enzyme isoforms involved in the synthesis (i.e. HA synthases") or degradation (i.e. hyaluronidases") of HA in articular cartilage under conditions of steady state or cytokine-modulated metabolism. Four hyaluronidases are expressed by chondroeytes: HY ALl, HY AL2 and HYAL3 are conserved gene products with significant homology to the testicular hyaluronidase PH-20, while the meningioma expressed antigen MGEA5 represents a divergent mammalian enzyme which exhibits homology to a C. elegans hyaluronidase orthologue (GenBank Accession Number U28742) and the Clostridium perfringens nagH protein (see Fig. I). Of these hyaluronidase isozyrnes, HYAL3 mRNA expression appears to be upregulated by the proinflammatory cytokine IL-l,
292
Aspects of hyaluronan injoints
thereby implicating cartilage-derived hyaluronidase activity as a factor contributing to matrix degradation during synovial joint disease. In addition, we have found that mammalian chondrocytes from a number of species can express two of the three known HA synthases, HAS2 and HAS3, but do not synthesize HAS 1 mRNA. From these studies, it appears that HAS2 is constitutively expressed and is therefore likely to be primarily involved in homeostasis of HA levels in cartilage. HAS3 expression on the other hand is much more sensitive to changes in the cellular environment, being altered in response to factors such as culture conditions, growth factors/cytokines and cellular origin from within the cartilage matrix (i.e. superficial versus deep zone). PH-20
129.3% 22.3% 1
25.9%
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MATERIALS AND METHODS Cartilage specimens Human articular cartilage was from the femoral condyles of 68- to 79-ycar old patients obtained following arthroplasty for advanced osteoarthritis of the knee or from undiseased distal humerus of a 74 year old patient. Bovine and porcine cartilage was obtained from the metacarpophalangeal joints of immature (<10 days old) or mature (>18 months old) cattle and from 3-6 month old pigs.
Cell and tissue culture Chondrocytes from full-depth cartilage slices were isolated by pronase/collagenase digestion and maintained in monolayer or agarose cultures as described". Cultures were maintained for 4 days in DMEM containing the ascorbate analogue phosphatin-C (25 mg/ml) in the presence or absence of 10 ng/ml IL-1, 1J.lM all-trans retinoic acid, 40 ng/ml TNF-a, 2 ng/ml TGF-~ 1 or 10 ng/ml IGF-I. For some experiments, bovine chondrocytes were isolated from the superficial or deep zones of articular cartilage from immature or mature cattle and maintained in monolayer or agarose cullures. Explants of human distal humerus cartilage slices were cultured in DMEM/lO% FBS for 3 days, then maintained for four days in DMEM (without FBS) ± 10 ng/ml
Synthesis and degradation in articular cartilage
293
recombinant human IL-I, 100 ng/ml recombinant human TNF-a or I J-lM all-trans retinoic acid (RA).
RNA extraction and RT-PCR analyses RNA was extracted from chondrocyte cultures and from freshly excised cartilage and cultured explants as described", RNA samples were subsequently treated with RNasefree DNase I and re-purified by phenol extraction. RT-PCR was performed on the RNA samples using oligonucleotide primers specific for HAS, hyaluronidase, CD44 or GAPDH cDNAs. All PCR product sequences were validated using an ABI 310 Genetic Analyzer.
RESULTS AND DISCUSSION Hyaluronan synthase expression In order to determine which of the HAS enzymes were being expressed in vivo in articular cartilages, we first examined RNA samples extracted directly from freshly excised bovine and porcine tissues. Of the three mammalian HAS enzymes identified to date, only HAS2 and HAS3 mRNAs were detected in native bovine or porcine cartilage (Fig. 2A). Chondrocytes from immature or mature bovine monolayer cultures expressed approximately equal levels of HAS2 mRNA, however the expression of HAS3 mRNA was significantly diminished in immature bovine chondrocytes treated with cytokines and growth factors (Fig. 2B). Additionally, HAS2 and HAS3 mRNA expression was observed in immature and mature bovine chondrocytes derived from both the superficial and deep cartilage zones (data not shown). When human chondrocytes were maintained in monolayer cultures, they also expressed HAS2 and HAS3 mRNAs (Fig. 2e). Human chondrocytes also expressed both HAS2 and HAS3 when cultured in the presence of two different catabolic stimuli, IL-I or retinoic acid (Fig. 2e). As for the other animal species, the human chondrocytes did not express HAS I mRNA under any of the culture conditions, a phenomenon which has also recently been observed by other investigators 10. Collectively, these data indicate that only HAS2 and HAS3 mRNAs are expressed in several mammalian cartilages. HAS2 mRNA appears to be constitutively expressed while HAS3 mRNA expression may be differentially regulated within the cartilage tissue in response to local and/or systemic catabolic or anabolic stimuli.
Hyaluronidase expression In parallel studies, hyaluronidase and CD44 expression was examined in cartilage derived from undiseased human articular cartilage. Expression of mRNA for HY AL 1, HY AL2, HY AL3 and MGEA5 was readily detected in direct tissue extracts (Fig. 3). For the explant cultures, HYAL3 mRNA levels were upregulated by cytokine (IL-I) treatment, while HY ALI, HYAL2 and MGEA5 transcipts remained relatively constant. The observed upregulation of CD44 expression in response to IL-I stimulation (Fig. 3) may implicate a coordinate mechanism of HA degradation involving receptor mediated endocytosis 11 and intracellular degradation by acid-active hyaluronidase(s) such as HYAL3.
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Synthesisand degradation in articular cartilage
295
CONCLUSIONS The current investigations into HA metabolism compliment other studies which are furthering our understanding of the mechanisms which regulate and modulate the steady state balance between expression of synthetic enzymes such as HAS2 [which appears to play an essential role in chondrocyte matrix assembly and retention"], and degradative enzymes (such as HYAL3). Elucidation of such events will provide critical new insights into these important aspects of articular joint metabolism.
ACKNOWLEDGEMENTS This research was funded by grants from the Arthritis Research Campaign (ARC), UK. Dr. Carl R. Flannery is an ARC Research Fellow.
REFERENCES 1. G.c. Gillard, B. Caterson and D.A. Lowther, 'The synthesis of hyaluronic acid by sheep and rabbit articular cartilage in vitro', Biochem. J., 1975, 145,209-213. 2. T.r. Morales and V.C. Hascall, 'Correlated metabolism of proteoglycans and hyaluronic acid in bovine cartilage organ cultures', J. BioI. Chern., 1988, 263, 36323638. 3. C.K. Ng, C.l. Handley, B.N. Preston and H.C. Robinson, 'The extracellular processing and catabolism of hyaluronan in cultured adult articular cartilage explants', Arch. Biochem. Biophys., 1992,298,70-79. 4. P.H. Weigel, V.c. Hascall and M. Tammi, 'Hyaluronan synthases', J. BioI. Chem., 1997,272,13997-14000. 5. A.B. Csoka, S.W. Scherer and R. Stern, 'Expression of six paralogous human hyaluronidase genes clustered on chromosomes 3p21 and 7q31', Genomics, 1999, 60,356-361. 6. D. Heckel, N. Comtesse, N. Brass, N. BEn, K.D. Zang and E. Meese, 'Novel immunogenic antigen homologous to hyaluronidase in meningioma', Hum. Mol. Genet, 1998,7, 1859-1872. 7. D.R.R. Hiscock, B. Caterson and C.R. Flannery, 'Expression of hyaluronan synthases in articular cartilage' , Osteoarthritis Cartilage, 2000, 8, 120-126. 8. CR. Flannery, C.B. Little, C.E. Hughes and B. Caterson, 'Expression and activity of articular cartilage hyaluronidases', Biochem. Biophys. Res. Comm., 1998, 251, 824829.
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Aspects or hyaluronan in joints
9. C.R. Flannery, C.B. Little, B. Caterson and C.E. Hughes, 'Effects of culture conditions and exposure to catabolic stimulators (IL-l and retinoic acid) on the expression of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs) by articular cartilage chondrocytes, Matrix Biol., 1999, 18,225-237. 10.Y. Nishida, C.B. Knudson, J.J. Nietfeld, A. Margulis and W. Knudson, 'Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly', J. Biol. Chem., 1999, 274, 2189321899. II.Q. Hua, C.B. Knudson and W. Knudson, 'Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis, J. Cell Sci., 1993, 106,365375.
PART 6 CELL SURFACES AND HYALURONAN RECEPTORS
CD44: THE LINK BETWEEN HYALURONAN AND THE CYTOSKELETON Cheryl B. Knudson, Ghada A. Nofal, Geraldine Chow & Richard S. Peterson Department ofBiochemistry, Rush Medical College, 1653 West Congress Parkway Chicago, Illinois 60612 USA
ABSTRACT
Matrix receptors direct the assembly and retention of the pericellular matrix and provide a linkage to the cytoskeleton. Modulating the link between the cytoskeleton and CD44 might regulate the function of this hyaluronan receptor. Binding of hyaluronan to CD44 retains the hyaluronan/aggrecan aggregates in the pericellular matrix. Also, CD44 turnover is modulated by occupancy with hyaluronan, suggesting that enhanced matrix assembly increases CD44 stability at the cell surface. Using differential detergent extraction, CD44 was localized predominantly in the cytoskeletal-associated pool from matrix-intact chondrocytes. In matrix-depleted chondrocytes, a decrease in this pool with a parallel increase in the detergent soluble pool was detected. CD44hyaluronan binding may stabilize the chondrocyte cell shape by the interaction of CD44 with the cortical cytoskeleton. Conversely if cytoskeletal elements regulate CD44hyaluronan binding, disruption of the cytoskeleton may perturb matrix retention. Treatment with either cytochalasin or latrunculin reduced binding of fl-HA and pericellular matrix assembly, but did not reduce CD44 expression by bovine chondrocytes as monitored by flow cytometry. In addition, there was a decrease in the safranin 0 staining of cytochalasin- and latrunculin-treated cartilage explants indicating the loss of proteoglycan from these tissues as compared to cultured controls. Thus, destabilization of the cytoskeleton may induce changes in the hyaluronan-dependent matrix of chondrocytes. KEYWORDS
Hyaluronan, CD44, cytoskeleton, chondrocyte, pericellular matrix INTRODUCTION It has long been known that in cartilage, the extracellular matrix exerts effects on chondrocyte cell shape, phenotype and metabolism. Conversely, chondrocyte cell shape changes exert effects on matrix organization, assembly and retention. The primary function of chondrocytes is to maintain the complex extracellular matrix of cartilage, and in particular, to maintain the soluble, hydrophilic components such as hyaluronan and aggrecan. Chondrocytes likely respond to changes in, or needs of the extracellular matrix via cell-matrix interactions, in tum mediated by specific receptors. Evidence now suggests that the transmembrane matrix receptor CD44 1 may be at the heart of many of these cell-matrix interactions. Maintenance of the chondrocyte phenotype, as well as initial cartilage differentiation, may depend on the association of the cell with its matrix as well as on cell shape as modulated by the actin cytoskeleton-. In addition to integrins, CD44 represents another class of receptors that can participate in these matrix-ceil-cytoskeleton interactions. The spatial organization of CD44 at the cell surface, controlled via cytoskeletal interactions, may function to establish or regulate the structure of the pericellular matrix dependent on an hyaluronan scaffolding for aggrecan. The interaction of CD44 with the cytoskeleton and the matrix allow us to speculate that both inside-out and outside-in communication patterns are occurring within the chondrocytes via CD44. Chondrocytes may exhibit metabolic and physical changes through the interaction with the matrix and also able to bring about a change in
332
Cell surfaces and hyaluronan receptors
the matrix, such as remodeling or repair. The interaction between CD44 and the cytoskeleton may bring about a change in cell-matrix interactions. Hyaluronan 3 is a key component of the extracellular matrix of cartilage, functioning as the central filament of the cartilage proteoglycan aggregate. The most abundant pr~teoglycan in cartilage, aggrecan, is ordered principally in this aggregate structure. This glycosaminoglycan-rich component of the matrix is organized within a specific collagen network 4. The swelling pressure of the proteoglycans in combination with the tensile strength of the network of collagen fibers comprise the key biochemical components that generate the biomechanical properties of cartilage. Assembly of the chondrocyte extracellular matrix most likely begins with the organization of the pericellular matrix at the cell surface. The chondrocyte pericellular matrix also is a crucial zone for cartilage matrix turnover 5-7. The interactions between matrix components and receptors allow the chondrocytes to establish a cell-associated pool of extracellular matrix molecules 8. These associations have the potential to signal changes in cell behavior, such as cell migration, proliferation and matrix biosynthesis or turnover. A change in the capacity for interaction between chondrocytes and the pericellular matrix can bring about a decrease in the retention or turnover of the matrix components. MATERIALS AND METHODS Reagents
Aggrecan was isolated from 4M GuHCl extracts of rat chondrosarcoma tumors or bovine articular cartilage by cesium chloride gradient ultracentrifugation 9. Oligosaccharides of hyaluronan were generated by testicular hyaluronidase digestion and characterized by chromatography and an ELISA 10. Fluorescein-labeled hyaluronan (fl-HA) was prepared as described previously 6. Cell and organ culture
Full thickness adult bovine articular cartilage slices were subjected to sequential pronase/collagenase-P, and these isolated chondrocytes were then cultured in alginate beads, which maintains the chondrocyte phenotype over an extended time period 5, II. To release the chondrocytes into a single cell suspension, the alginate beads were depolymerized using 55 mM sodium citrate in 150 mM NaCI. Pericellular matrices were visualized using a particle exclusion assay 12. The chondrocytes, untreated or treated with either Streptomyces hyaluronidase (4 Uzml, 60 min, 37°C) or 0.5 ug/ml latrunculin A (Calbiochem) or 10 ug/ml dihydrocytochalasin B (Sigma) or incubated in the presence of hyaluronan oligosaccharides, were released from alginate beads and transferred to six-well plates. The cells were "splatted" onto the substratum by centrifugation at 500 g for 15 min in a microtiter plate holder 13. To test the ability to bindfl-HA, chondrocytes released from alginate beads and pretreated with 5U/ml Streptomyces hyaluronidase were incubated with either 10 ug/rnl dihydrocytochalasin B, 1 ug/ml latrunculin A or vehicle buffer. The cells were then with incubated with 200 ug/ml fl-HA in the continued ~resence of the above agents. The cells were rinsed, resuspended in DMEM at 3.3xlO cells/ml, transferred to a 96 well plate, and then analyzed on a fluorescent plate reader. Full thickness articular cartilage slices of -1 em' in size were cultured in separate 16 mm wells. Following one day of culture, latrunculin (2 ug/ml), cytochalasin (10 ug/ml) or hyaluronan oligosaccharides (50-250 ug/ml) were added for up to 7 days. Cryostat sections were stained with safranin 0 and counter-stained with fast green 14. CD44 antisense oligonucleotides were designed as 16-mer phosphorothioate oligonucleotides complementary to a region within the 5' untranslated region of the CD44 sequence IS. Controls included a sense oligonucleotide and a scrambled sequence complementary to the 5'untranslated CD44 mRNA as well as a nonsense
CD 44
333
oligonucleotide. Following dose-dependent optimization, chondrocyte cultures were incubated for 1 to 4 days in 4 flM antisense oligonucleotide and cartilage explant cultures were incubated for 5 days in the presence of 10 flM antisense oligonucleotide. Flow cytometry Chondrocytes were fixed for 10 min in 0.5% formaldehyde in PBS, pH 7.4 and rinsed in 0.2 M glycine in PBS. CD44 was detected with a biotin-conjugated antibody IM7.8.1 (Pharmingen) and phycoerythrin-conjugated streptavidin 16, Chondrocytes were released from alginate and cell surface proteins were enzymatically removed by a 30 min 0.25% trypsin treatment. Cells were rinsed, cultured in complete medium and at various time points, aliquots of cells were rinsed and fixed for flow cytometric analysis of CD44 re-expression. Other aliquots were used to determine the capacity of these fixed cells to assemble exogenous hyaluronan and aggrecan into a pericellular matrix 9. Cell surface CD44 turnover in the presence of cycloheximide was detected by flow cytometric analysis. Chondrocytes cultured for 5 days in alginate beads were incubated 1 h with 200 flM cycloheximide, followed by release into a single cell suspension. Three conditions were examined: a) matrix-intact chondrocytes, b) Streptomyces hyaluronidase matrix-depleted chondrocytes, and c) Streptomyces hyaluronidase matrix-depleted chondrocytes which were subsequently incubated in complete medium in the presence of 1 mg/ml hyaluronan (Sigma, Grade I). Chondrocytes were either fixed at 0 hour or incubated in complete medium containing 200 flM cycloheximide for 2,4 or 6 h and then fixed. To detect CD44 turnover over a timecourse, chondrocytes within alginate beads were incubated with 200 flM cycloheximide in the absence or continued presence of Streptomyces hyaluronidase, added at multiple intervals. Chondrocytes were released from the beads and fixed at 0, 6 or 26 h. Chondrocyte Lysis and Western Blotting Equivalent aliquots of matrix intact and matrix-depleted (Streptomyces hyaluronidase-treated) bovine chondrocytes released from alginate beads were extracted with 0.2% NP-40 in Tris lysis buffer with protease inhibitors for 30 min, and the residual proteins pelleted at 16,000g. The pellet was treated with 1 mg/ml DNA'ase I and extracted with 0.5% NP-40/0.5% DOCIl % Empigen BB (Calbiochem). Both the NP-40 soluble and insoluble proteins were then used for immunoprecipitation of CD44 with the IM7.8.1 antibody, using goat anti-rat IgG Sepharose 4B beads (Zymed). RESULTS AND DISCUSSION Chondrocytes express the hyaluronan receptor CD44 11-16, which is responsible for the more than the binding of native hyaluronan. Since hyaluronan serves as the backbone of aggrecan/hyaluronan/link protein aggregates, CD44 is also responsible for aggregate retention as well. In fact, the assembly of the glycosaminoglycan-rich pericellular matrix can be inhibited by antibodies to CD44 6-7; pericellular matrix assembly as well as the displacement of the majority of the glycosaminoglycan-rich pericellular matrix of matrix can be affected by the addition of exogenous hyaluronan oligosaccharides 7-9. Thus the use of reagents that compete with CD44 - hyaluronan binding, uncouple CD44 - hyaluronan binding or decrease the capacity of chondrocyte CD44 - hyaluronan binding will be discussed herein. Native Chondrocyte Pericellular Matrices The visualization of pericellular matrices on living cells in culture can be facilitated by the particle exclusion assay, first developed by Clarris and Fraser and used to distinguish a hyaluronan network surrounding synovial fibroblasts 17. A halo or "cell coat" can be revealed which cannot be penetrated by the particles. The removal of this
334
Cell surfaces and hyaluronan receptors
coat by Streptomyces hyaluronidase treatment demonstrates that there is a scaffold of hyaluronan within this pericellular matrix. This assay represents the most useful method of visualizing the full extent of the pericellular matrix, since conventional staining of the matrix often leads to significant collapse of this hydrated structure. Several cell types in culture, including embryonic and adult chondrocytes, exhibit large pericellular matrices or coats extending from the plasma membrane 9, 12. Treatment of chondrocytes with Streptomyces hyaluronidase removes the pericellular matrix. Since chondrocytes are typically liberated from cartilage via enzymatic treatment, the strategy to study the re-assembly of the pericellular matrix in vitro is straightforward. Reagents that compete with the binding of newly synthesized hyaluronan to the chondrocytes inhibit matrix re-growth - these are hyaluronan oligosaccharides (with HA 6 being the minimal effective size), chondroitin and antibodies to CD44, including IM7.8.1 and KM201 6,7,9. Even after treatment with HA6 for five days, chondrocytes synthesize aggrecan, which is not retained in the pericellular matrix but recovered in the conditioned medium. Following the removal of these reagents, pericellular matrix re-growth can be detected. Nonetheless, these shortterm experiments are unable to address the assembly and maintenance of the pericellular matrix within the cartilage tissue, an entity most clearly evident by transmission electron microscopy. Thus our approach includes the use of adult bovine cartilage slices in explant culture, and attempts to uncouple or reduce chondrocytematrix interactions, which are discussed below. An earlier attempt was to isolate chondrocytes with a modified approach, such that the pericellular matrix remained cell-associated; this was most successful with embryonic cartilage. We have also used hyaluronan hexasaccharides to "disengage" chondrocytes from their matrix 9, thus allowing us to study the interaction of a matrix assembled in vivo with its chondrocyte 13. Embryonic chick chondrocytes retain a cellassociated matrix after direct isolation from tibial cartilage by a brief treatment with collagenase P in DMEM containing 20% horse serum. The matrix surrounding these cells was sensitive to in vitro Streptomyces hyaluronidase treatment and also was displaced by a 90 min incubation with HA6 • However, when tibial explants were cultured for 48 h in the presence of HA6, and then the chondrocytes were released from the explants, the chondrocytes appeared "matrix-free". These chondrocytes had become "disengaged" from the endogenous matrix yet all exhibited the capacity to assembly exogenous hyaluronan and aggrecan into a pericellular matrix. Thus, disruption of native hyaluronan-chondrocyte interaction in cartilage with HA6 inhibited matrix retention. Using an alginate bead system for adult articular chondrocyte culture has the advantages of providing a scaffold for maintaining cells in a spherical morphology, promoting extracellular matrix deposition as well as the capacity to depolymerize this alginate scaffold releasing, into a suspension, cells that retain matrix 18. Chondrocytes released after 2 or more days in alginate bead culture exhibit a pericellular matrix (Fig. lA). However if the alginate beads are pre-treated with Streptomyces hyaluronidase or incubated in the presence of HA6 prior to depolymerization, the chondrocyte pericellular matrix is depleted (Fig. IB). The Streptomyces hyaluronidase treatment removed the pericellular matrix and the HA6-treatment uncouples the hyaluronan from the chondrocytes. Thus, although the mature chondrocyte pericellular matrix is composed predominantly of aggrecan, its structure depends on both a scaffold of hyaluronan and the anchorage ofhyaluronan to the plasma membrane 9. CD44 Expression and Turnover on Articular Chondrocytes
CD44 is a ubiquitous transmembrane glycoprotein. The predominant form of CD44 is the hemopoietic/standard isoform (CD44H or CD44s), which has an apparent molecular weight of 85 kDa and lacks all of the variant exons 19. It appears that the main function of this isoform is to act as a cell surface receptor for hyaluronan 1, 8, 20. Through this interaction with hyaluronan, cells that express CD44 are able to assemble
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a pericellular matrix around them in the presence of hyaluronan and hyaluronanaggregating proteoglycans such as aggrecan 21-22. By flow cytometry, the level of expression of CD44 on bovine articular chondrocytes was similar to that on a mouse lymphocyte T-cell line 16. Treatment of chondrocytes with Streptomyces hyaluronidase did not alter CD44 expression, however trypsin treatment eliminated CD44 detection by flow cytometry 18. The turnover/loss of cell surface-localized CD44 was measured by flow cytometry in the continued presence of cycloheximide (Fig. 2). Following 4 or 6 h of incubation, matrix-intact control chondrocytes exhibited 6% or 8% loss of cell surface CD44. Following treatment with Streptomyces hyaluronidase to deplete the pericellular matrix, the chondrocytes exhibited a 20% depletion of CD44 by 4 h, with 22% loss at 6 h. The addition of exogenous hyaluronan to these matrix-depleted chondrocytes partially reduced this loss of CD44 at 4 or 6 h to 11-12%, thus restoring turnover levels closer to those seen on matrix-intact chondrocytes. In the constant presence of Streptomyces hyaluronidase there was an even more dramatic loss of CD44 by 6 h, in comparison to only a single enzyme treatment at time equals zero. After 26 h there was -60% loss of CD44 with constant depletion of endogenous hyaluronan, compared with a 12% loss on matrix-intact chondrocytes. As an alternative approach, the loss or turnover of cell surface CD44 was also monitored in the absence of cycloheximide. For this approach, the depletion of monoclonal antibody pre-tagged CD44 was monitored 16. Matrix-intact chondrocytes were pre-labeled for 1 h with biotinylated-IM7.8.1, and then chased in complete medium minus monoclonal antibody. There was no dramatic loss of bound epitope at 0.5 or 1 h timepoints, but approximately 6% of antibody-tagged CD44 was lost after 4 h, correlating with the 6% loss of epitope on cycloheximide-treated matrix-intact chondrocytes by 4 h. The addition of fresh antibody to 6 h cultures resulted in recovery of98% of the initial level ofCD44 expression. Thus, addition of monoclonal antibody IM7.8.1 did not induce rapid loss (e.g., shedding) of CD44, or a change in total cell surface CD44 expression, so the loss of antibody-tagged CD44 on matrix-intact chondrocytes could be followed over time. When the chase analysis was continued over a 48 h incubation, mean channel fluorescence decreased by 40%. Thus, by this method, an estimation of ~ 48 hours for the half-life of CD44 on matrix-intact chondrocytes is indicated. These data suggest that CD44 on matrix-intact chondrocytes is relatively stable at the cell surface. However, following matrix depletion with
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CD44 Expression and Pericellular Matrix Assembly Using a specific phosphorothioate oligonucleotide, antisense treatment of chondrocytes in alginate beads resulted in a 60-75% reduction of cell surface expression of CD44 as detected with the IM7.8.1 monoclonal antibody 15. The capacity of these chondrocytes to retain endogenous pericellular matrix showed These variability within the population of antisense-treated cells (Fig. I C). observations led to the hypothesis that the level of cell surface CD44 expression is directly related to the capacity of chondrocytes for matrix assembly. Following trypsinization, re-expression of CD44 was observed by immunocytochemistry and flow cytometry. The function of CD44 was tested in parallel with CD44 epitope detection. Chondrocytes were analyzed in light of our previous observations that nonviable cells, following brief fixation retain the capacity to bind 3H-hyaluronan 23-24 and can serve as nucleating sites for matrix assembly in the presence of purified exogenous aggrecan plus hyaluronan 21. Even after only 2 h of recovery from trypsin, with 30-35% of cells exhibiting positive re-expression of cell surface CD44, a similar percentage of chondrocytes were capable of pericellular matrix assembly with exogenous aggrecan plus hyaluronan. The correlation of r = 0.98 was found between the percentage of cells positive for CD44 by flow cytometry and the capacity for exogenous matrix assembly 18. Mean fluorescence intensity of only 25% of normal cell surface CD44 expression on bovine chondrocytes is necessary for the assembly of hyaluronananchored pericellular matrix - correlating well with the observations on antisensetreated chondrocytes. Thus, CD44 density may be critical in regulating proper chondrocyte matrix assembly. The specificity of the interaction between CD44 and hyaluronan-mediated matrix assembly was shown with the use of COS-7 cells. These cells do not normally express CD44 and lack the ability to form an hyaluronan dependent pericellular matrix 22. CD44H is by far the most common isoform expressed by adult connective tissue cells, with no variant exons for the extracellular domain and with the use of exon 20 to encode 67 of the 70 amino acids of the cytoplasmic tail 19. Chondrocytes primarily express the CD44H isoform, but we have recently shown that human articular chondrocytes express varying levels (5-35%) of another alternatively spliced isoform of
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~.
-
Figure 4. Cytoskeletal CD44 after NP-40 extraction Control chondrocytes exhibit prominent CD44 staining (panel A), which is reduced upon NP-40 detergent extraction (panel B). The staining of the residual cytoskeletal CD44 following NP-40 extraction was greatly reduced on the cytochalasin-treated chondrocytes (panel C) and the latrunculin-treated chondrocytes (panel D).
more CD44 was detected in the NP-40 soluble pool of the matrix-depleted chondrocytes as compared with the matrix intact chondrocytes. These data suggest a correlation with CD44 occupancy and the interaction of CD44 with the cytoskeleton. Using an alternative approach, chondrocytes incubated in the presence or absence of latrunculin or cytochalasin were extracted with 0.2% NP-40 buffer and the residual CD44 detected by immunocytochemistry. Control chondrocytes exhibit prominent CD44 staining, which is reduced upon NP-40 detergent extraction. However, although the staining of intact chondrocytes by this method or by flow cytometry is not altered by latrunculin or cytochalasin treatment (see Fig. 3), the staining of the residual CD44 following NP-40 extraction was greatly reduced on the latrunculin and cytochalasin treated chondrocytes (Fig. 4) suggesting a linkage of CD44 with the cortical actin cytoskeleton.
Hyaluronan-Chondrocyte-Cytoskeleton Interactions in Cartilage To expand these studies to intact cartilage, three approaches were used. Addition of HA6 to cartilage explants was performed with the hypothesis that the oligosaccharides would uncouple chondrocyte-hyaluronan interactions. Addition of antisense oligonucleotides to cartilage explants was performed with the hypothesis that reduction in cell surface expression levels of CD44 would reduce CD44-hyaluronan interactions. And finally, latrunculin and cytochalasin treatments of explant cultures were evaluated with the hypothesis that hyaluronan-CD44 binding would be reduced upon cytoskeletal disruption. Hyaluronan oligosaccharides induced a dose-dependent state of chondrocytic chondrolysis, including a loss of stainable proteoglycan-rich matrix in the cartilage explants. The conditioned medium of these cultures showed activation of gelatinolytic activity, while the tissue exhibited aggrecan neoepitope expression 10. Similarly, antisense-treated cartilage slices displayed a near-total loss of stainable proteoglycanrich matrix, as well as intense staining for the aggrecanase-generated neoepitope 15. The chondrocytes within the latrunculin-treated or cytochalasin-treated cartilage appeared more compacted. By 48 h of treatment with cytochalasin and 5 days of incubation with latrunculin, a decrease in safranin 0 staining was detected, indicating the loss of proteoglycan from these tissues as compared to cultured controls. To determine the long-term effects of these cytoskeletal-disrupting agents on CD44 expression or turnover, flow cytometry was performed on chondrocytes released from alginate beads after 0 to 7 days of incubation. The levels of cell surface expression of CD44 after 7 days of latrunculin treatment was 76% of control while after 7 days of cytochalasin treatment, the expression was decreased to 92% of control. These results show that destabilization of the actin network in chondrocytes results in decrease matrix assembly or retention. The spatial organization of CD44 at the cell surface, controlled via cytoskeletal interactions, may function to establish or regulate the structure of the pericellular matrix dependent on a hyaluronan scaffolding for aggrecan.
CD 44
339
SUMMARY In osteoarthritis, proteoglycan retention is poor 29, and proteoglycan degradation is increased 30 - resulting in impaired cartilage function. Chondrocytes may exhibit metabolic and physical changes following modifications of the matrix, and through the interaction with the matrix initiation of matrix remodeling or repair. The interaction of CD44 with the cytoskeleton and the matrix allow us to speculate that both inside-out and outside-in communication patterns are occurring within the chondrocytes via CD44. Chondrocytes may exhibit metabolic and physical changes through the interaction with the matrix, which might induce matrix degeneration, remodeling or repair. Our results indicate that destabilization of the cytoskeleton may bring about a change in the matrix. Besides artificial cytoskeletal disrupting agents, such as cytochalasins and latrunculins, natural agents such as NO have also been shown to inhibit actin polymerization 31. Maintenance of the interaction between hyaluronan and CD44 and the cytoskeleton sustains chondrocyte phenotype, whereas disruptions of these interactions may initiate changes in the cartilage matrix.
ACKNOWLEDGMENTS The authors thank Warren Knudson, Ph.D. for his helpful discussions, Dean 1. Aguiar, Ph.D., for his assistance with the flow cytometry and Carita T. Constable for her excellent technical assistance. Supported in part by NIH grants P50-AR39239, AR43384, AR39507 and the Arthritis Foundation. REFERENCES 1. C. B. Underhill, 'CD44: The hyaluronan receptor', 1. Cell Sci., 1992, 103,293-298. 2. K. Daniels & M. Solursh, 'Modulation of chondrogenesis by the cytoskeleton and extracellular matrix', .I. Cell Sci., 1991, 100,249-254. 3. T. C. Laurent & R. E. Fraser, 'Hyaluronan', FASEB .I., 1992,6,2397-2404. 4. H. Muir, 'The chondrocyte, architech of cartilage: Biomechanics, structure, function and molecular biology of cartilage matrix macromolecules', BioEssays, 1995, 17, 1039-1048. 5. H. J. Hauselmann, M. B. Aydelotte, B. L. Schumacher, K. E. Kuettner, S. H. Gitelis & E. J.-M.A. Thonar, 'Synthesis and turnover ofproteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads', Matrix, 1992, 12, 130-136. 6. Q. Hua, C. B. Knudson & W. Knudson, 'Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis', 1. Cell Sci., 1993, 106, 365-375. 7. W. Knudson, D. 1. Aguiar, Q. Hua & C. B. Knudson, 'CD44-anchored hyaluronanrich pericellular matrices', Exp. Cell Res., 1996,228,216-228. 8. C. B. Knudson & W. Knudson, 'Hyaluronan-binding proteins in development, tissue homeostasis and disease', FASEB 1.,1993,7,1233-1241. 9. C. B. Knudson, 'Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix', .I. Cell Bioi., 1993, 120,825-834. 10. W. Knudson, B. Casey, K. E. Kuettner, W. Eger & C. B. Knudson, 'Hyaluronan oligosaccharides perturb cartilage matrix homeostasis and induce chondrocytic chondrolysis.', Arthritis & Rheumatism, 2000, 43, 1165-1174. II. G. Chow, C. B. Knudson, G. Homandberg & W. Knudson, 'Increased CD44 expression in bovine articular chondrocytes by catabolic cellular mediators', .I. Bioi. Chern., 1995, 270, 27734-27741. 12. C. B. Knudson & B. P. Toole, 'Changes in the pericellular matrix during differentiation oflimb bud mesoderm', Develop. Bioi., 1985, 112,308-318. 13. M. P. Maleski & C. B. Knudson, 'Matrix accumulation and retention in embryonic cartilage and in vitro chondrogenesis', Connect. Tis. Res., 1996, 34, 75-86. 14. 1. M. Williams, D. R. Ongchi & E. J.-M. A. Thonar, 'Repair of articular cartilage injury following intra-articular chymopapain-induced matrix proteoglycan loss', 1. Ortho. Res., 1993,11,705-716.
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15. G. Chow, 1. Nietfeld, C. B. Knudson & W. Knudson, 'Antisense inhibition of chondrocyte CD44 expression results in cartilage chondrolysis', Arthritis & Rheumatism, 1998, 41,1411-1419. 16. D. J. Aguiar, W. Knudson & C. B. Knudson, 'Internalization of the hyaluronan receptor CD44 by chondrocytes', Exp. Cell Res., 1999,252,292-302. 17. B. J. Clarris & J. R. E. Fraser, 'On the pericellular zone of some mammalian cells in vitro', Exp. Cell Res., 1968,49, 181-193. 18. C. B. Knudson, G. A. Nofal, L. Pamintuan & D. J. Aguiar, 'The chondrocyte pericellular matrix: a model for hyaluronan-mediated cell-matrix interactions', Biochem. Soc. Trans., 1999,27, 142-147. 19. G. R. Screaton, M. V. Bell, D. G. Jackson, F. B. Cornelis, U. Gerth & J. I. Bell, 'Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons', Proc. Nat!. Acad. Sci. USA, 1992, 89, 1216012164. 20. C. B. Underhill & B. P. Toole, 'Receptors for hyaluronate on the surface of parent and virus - transformed cell lines', Exp. Cell Res., 1981,131,419-423. 21. W. Knudson & C. B. Knudson, 'Assembly of a chondrocyte-like pericellular matrix on non- chondrogenic cells', J Cell Sci., 1991, 99,227-235. 22. W. Knudson, E. Bartnik & C. B. Knudson, 'Assembly of pericellular matrices by COS-7 cells transfected with CD44 homing receptor genes', Proc. Natl. Acad. Sci. USA, 1993,90,4003-4007. 23. R. L. Goldberg, C. B. Underhill & B. P. Toole, 'Affinity chromatography of hyaluronate glutaraldehyde-fixed SV-3T3 cells', Anal. Biochem., 1982, 125,59-65. 24. R. E. Nemec, B. P. Toole & W. Knudson, 'The cell surface hyaluronate binding sites of invasive human bladder carcinoma cells', Biochem. Biophys. Res. Commun., 1987, 149,249-257. 25. H. Jiang, C. B. Knudson, H. Koepp, W. Eger & W. Knudson, 'Alternative expression of cytoplasmic tail-less isoform of CD44 (CD44exonI9) by human articular chondrocytes', Orthopaedic Trans., 1998,23,913. 26. C. M. Isacke, 'The role of the cytoplasmic domain in regulating CD44 function', J Cell Sci., 1994, 107,2353-2359. 27. P. D. Brown & P. D. Benya, 'Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B- induced reexpression', J Cell Bioi., 1988, 106, 171-179. 28. I. Spector, N. R. Shochet, Y. Kashman & A. Groweiss, 'Latrunculins: Novel marine toxins that disrupt micro filament organization in cultured cells', Science, 1983,219,493-495. 29. M. T. Bayliss, In: Articular cartilage and osteoarthritis, K. E. Kuettner, R. Schleyerbach, J. G. Peyron and V. C. Hascall, eds., Raven Press, New York, 1992, 487-500. 30. M. W. Lark, J. T. Gordy, J. R. Weidner, J. Ayala, J. H. Kimura, H. R. Williams, R. A. Mumford, C. R. Flannery, S. S. Carlson, M. Iwata & J. D. Sandy, 'Cell-mediated catabolism ofaggrecan', J Bioi. Chem., 1995,270,2550-2556. 31. S. R. Frenkel, R. M. Clancy, 1. L. Ricci, P. Di Cesare, J. J. Rediske & S. B. Abramson, 'Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly', Arthritis & Rheumatism, 1996,39, 1905-1912.
HYALURONAN BINDING BY CELL SURFACE CD44 Jayne Lesley'>, Nicole English" Vincent C. Hascall1 , Markku Tammr', Robert Hyman' /Cancer Biology Laboratory. The Salk Institute. P.O. Box 85800. San Diego, CA 92186. USA 2
Department of Biomedical Engineering. Cleveland Clinic Research Institute. 9500 Euclid Avenue. Cleveland. Ohio 44195. USA 3
Department ofAnatomy. University of Kuopio, FlN-70211 Kuopio, Finland
ABSTRACT The interaction of unlabeled HA preparations with cell surface CD44 was assayed by their ability to inhibit the binding of high M, fluorescein-conjugated HA (FL-HA). Using unlabeled low M, hyaluronan oligomers of defined sizes, we observed monovalent interaction of CD44 with oligomers between 6 and 18 sugar residues. Above 20 sugars, there was a 2-4-fold increase in blocking activity of the oligomers, suggesting that divalent binding was occurring. Pretreatment of the cells with IRAWBl4, a CD44specific inducing mAb, resulted in a more dramatic increase in blocking activity of HA oligomers of lengths above 18 sugars compared to their activity on non-induced cells, suggesting that the mAb greatly enhanced divalent binding and/or promoted more multivalent interactions. FL-HA fragments of <100 kDa, obtained by digestion of high M, FL-HA, could not be detected by direct binding to untreated CD44+ cells. Pretreatment of the cells with IRAWB 14 mAb allowed stable binding of FL-HA fragments. Measurement of the dissociation of FL-HA from the surface of CD44+ cells revealed that: 1) Treatment with mAb IRAWB 14 reduced the rate of dissociation of bound high M,. FL-HA compared to untreated cells. 2) FL-HA fragments of >100 kDa dissociated rapidly from mAb induced cells in the presence of excess unlabeled HA. 3) In the absence of a large excess of unlabeled HA, bound FL-HA dissociated very slowly from both mAb induced and non-induced cells. These observations support the conclusion that CD44 mediated HA binding at the cell surface is the result of multiple weak and transient binding interactions. INTRODUCTION CD44 is a major cell-surface receptor for hyaluronan. It is involved in extra-cellular matrix assembly, in cell adhesion and migration, and in tumor cell invasion and metastasis 1-4. CD44 shares an -100 amino acid region of homology with other HA binding proteins termed a "link module" or "proteoglycan tandem repeat (PTR)" (see review"). Though CD44 uses a link module domain to bind HA, it differs in important ways from other HA binding proteins. While the link modules of TSG-6 and link protein are capable of binding HA on their own 5-7, HA binding by CD44 requires sequences outside the link module 8, is regulated by cell specific factors 1.2.9-12, and probably requires multiple CD44/HA engagements to achieve a functional avidity. Depending on the type and activation state of the cell in which it is expressed CD44 may be inactive (unable to bind HA), inducible (able to bind HA upon treatment with certain
342
Cell surfaces and hyaluronan receptors
CD44-specific mAb or inducers of cell activation such as phorbol ester) or constitutively active (able to bind HA without any treatment) 9. Our laboratory's study of CD44 has focused on the mechanisms regulating its hyaluronan binding function including the role of CD44 cytoplasmic and transmembrane domains, cell specific glycosylation of CD44 and receptor dimer-/oligomerization 9. 13-1S. The activation state seems to be determined, at least in part, by posttranslational modification (especially glycosylation) of the CD44 molecule itself, because CD44-Ig fusion proteins display the same activation phenotype as the cell surface CD44 of the cells in which they are made 10.11.15.16. The most significant feature that distinguishes CD44 from other HA binding proteins is that CD44 binding to HA takes place at the cell surface where multiple closely arrayed CD44-receptor molecules interact with the highly multivalent repeating disaccharide chain of HA. The affinity of a single CD44-HA binding domain for HA is likely to be very low. CD44-Ig fusion proteins (which are at least dimeric) were estimated to have K, in the range of 10,4 to 10-5 IS. Thus, binding of a CD44-positive cell to an HA substrate or of a soluble HA molecule to the surface of a CD44-positive cell involves multiple weak receptor-ligand interactions. These features of CD44 in conjunction with the unique properties of the HA ligand, which presents a very long, repeating chain of linked CD44 binding sites, create a system that is highly dependent on multiple cooperating interactions in order to achieve functional binding.
RESULTS Binding of HA to a CD44+ cell requires a critical density of CD44 molecules
Evidence for cooperativity from the CD44 side includes the observations that: 1) A 'threshold' level of CD44 expression is required for detectable binding of FL-HA (or adhesion to an HA substrate), and once that density is reached, binding activity increases with increasing CD44 expression 13-15. 2) Clustering of CD44 on the cell surface, either as covalent dimers or larger aggregates, increases the HA binding activity without an increase in the number of CD44 molecules 13.14.17. CD44 clustering lowers the threshold at which HA binding can be detected. Length Requirements for HA binding to cell surface CD44
What are the requirements on the HA side of the binding reaction? For example, what length of HA chain is needed to: 1) occupy a single CD44 binding site? ; 2) .. .link two CD44 receptors in a dimeric CD44 complex with HA, and 3) ... achieve stable binding of HA at the cell surface? To explore some of these questions we have looked at the interaction of HA oligomers of defined sizes with cell surface CD44. HA binding was detected by flow cytometry using a fluorescein-conjugated HA probe (FL-HA) made from rooster comb HA with a M, of about 106 • For most binding studies we used a CD44-negative cell line that was transfected with wild type CD44H and selected for high levels of CD44 expression. CD44 expressed in this line is binds HA constitutively. Binding of small, unlabeled HA oligosaccharides of defined M. (prepared as described in 18) was detected by their ability to block binding of FL-HA. Cells that had been pre-incubated with serial dilutions of unlabeled oligomers were subsequently labeled with a standard dilution of FL-HA (still in the presence of the unlabeled competitor) and assayed for FL-HA binding by flow cytometry 17. Sets of blocking
Binding by cell surface CD44
343
curves were used to determine the concentration of unlabeled HA giving 50% inhibition of FL-HA binding as a measure of the relative avidity of the unlabeled HA for CD44. About 5x more HA4 was required to achieve 50% blocking than HA6 or HAs. Therefore, it appeared that 6 sugars was the minimum size of HA chain to occupy the CD44 binding site. Chains of 6 and 8 sugars blocked almost equally, but there was an increase in blocking efficiency with oligomers between 10 and 18 sugars. This difference was small, but was seen repeatedly. We suggest that six sugars is the minimal size to occupy the CD44 binding site, in agreement with previous studies of Underhill et al and Knudson et aI 19•2o, but that an oligomer of at least 10 sugars is optimal. A series of larger oligomers was used to look for an HA chain length that would engage two CD44 molecules on the cell surface. We expected that there would be an increase in blocking activity at this point, because binding to two CD44s would reduce the probability of dissociation of the sugar chain. Each preparation of > 18 sugars was a mixture of oligomers of several sizes with mean sizes of 22, 26, 30, 34, and 38 sugars. All together they encompassed a range between 20 and over 40 mono-saccharides. The micromolar concentration required for 50% inhibition of FL-HA binding was plotted against oligomer chain Iength!". Between HAlO and HA 1S ' there was little change in the concentration needed for 50% inhibition with increasing oligomer size. Above HA ,s, there was a 2-4-fold increase in blocking efficiency. The increase in avidity at HA_20 suggests that divalent binding occurs at this chain length. It is noteworthy that there was no evidence for a further increase in avidity between oligomer preparations beyond HA20 up to over 40 sugar chains with non-induced cells. The significance of this observation is unclear. If linked binding of 3 or 4 CD44 molecules were occurring, one would expect some increase in binding avidity with the larger oligomer preparations. One possible explanation is that CD44 has an inherent tendency to spontaneously dimerize in the membrane and thus allow dimeric binding. (The possibility of CD44 dimerization has been suggested in several publications 2 1•23 ) . Higher order aggregation may not be favorable without mAb induction (see below). If the cells were pretreated with the inducing mAb IRAWB 14, which enhances FLHA binding, the blocking curves with lower M, oligomers, below HA 20 , were very similar to the curves with non-induced cells. But with oligomers of 20 sugars and above, there was a much more dramatic increase in blocking efficiency than with non-induced cells, and there was a steady increase in blocking avidity (decrease in the concentration needed to achieve 50% blocking) with each increase in HA chain length. This suggested that the mAb specifically enhanced divalent (and multivalent?) CD44 binding to the longer HA chains. IRAWB 14 mAb may promote larger aggregations of CD44 and/or orient neighboring CD44 molecules in a configuration that optimizes the possibility of dimeric or more multivalent binding. Though we observed quite efficient blocking of FL-HA binding to IRAWB 14 induced cells with oligomers containing chains of 30-40 sugars, we did not obtain detectable binding with AMAC- conjugated oligosaccharides of -22 or -38 sugars by microscopy or flow cytometry (Lesley, Tammi and Hascall, unpublished). It was concluded that binding of these small oligomers was too weak and too transient to detect by direct binding methods. In order to determine what chain length was needed to obtain stable binding, we digested high M, FL-HA with Streptomyces hyaluronidase (Fluka). Digests were then passed through Centricon filters (Millipore) of different molecular weight cut off (MWCO) to estimate their sizes. Results of digestion and filtration are shown in Figure 1. Sample D, which was incubated along with the others, but with no hyaluronidase added, was retained above the 100 kDa MWCO filter, while
344
Cell surfacesand hyaluronan receptors
all the digested samples passed through. Sample A, digested with the highest concentration of hyaluronidase (1.25 TRU/ml final concentration) readily passed through 50 kDa and 30 kDa MWCO filters, but was mostly retained above the 10 kDa MWCO filter. Digests Band C, two- and four- fold dilutions, respectively, of the enzyme concentration used in A, both concentrated between the 30 and 50 kDa MWCO filters, but B, digested with the higher hyaluronidase concentration, had more material below 30 kDa, while C had more material above 50 kDa. Hence, we have assigned estimated sizes of -30 kDa and -50 kDa to B and C, respectively. A is assigned a size of >30 kDa. Significantly, none of the digested samples bound to non-induced CD44+ cells, while all bound to IRAW14 treated cells. Table 1 summarizes the size estimates and binding properties of the FL-HA fragments. FL·HA digests separated by Centricon Filtration 80
«
:c ...:. u.
• III 1.1 E3
60
A, <30 kDa S, -30 kDa C, -50 kDa D (no enzyme)
'C
~
Cl)
40
> 0
o ~
'15
20
0~
0 >100 kD
30-50 kD
50-100 kD
10-30 kD
<10 kD
MWCO Figure 1. Fluorescein-conjugated high M, HA from rooster comb was digested with serial 2x dilutions of Streptomyces hyaluronidase (Fluka) starting at 1.25 TRU/ml in digest A. Sample D was incubated without enzyme. Digests were then diluted and passed through a series of Centricon filters (Millipore) of different molecular weight cut off (MWCO). The fraction of the FL-HA that was retained or passed through each filter was determined by measuring the absorbance of the fluorescein at 492 nm. Table 1. Binding of FL-HA fragments to CD44+ cells Approximate M, were estimated by Centricon filtration as shown in Figure 1. Approximate Mr
# of sugar residues
# of dimeric binding sites (20 sugars) 2-7
10-<30 kDa
50-150
-30kDa
-150
-8
-50kDa
-250
-12
>lOOkDa
>500
>25
Binding to Non-induced cells
Binding to IRAWBI4 induced cells
+
+ + + +
Binding by cell surface CD44
345
Dissociation of FL-HA from Induced CD44+ cells -0-
___ -
100
c
'E
~
1il
High MW FL-HA -50 kDa FL-HA -30 kDa FL·HA T,,1I2
80
60
Cl
c
'0 c
z
40
'0
20
~
00
00
100
1~
140
160
time of dissociation (min)
Figure 2. CD44+ cells were pretreated with mAb IRAWB 14 and then incubated with 1 ug/ml of FL-HA (undigested FL-HA, or digested fragments of FL-HA of -30 kDa or -50 kDa) for 45 min at 4° C. Aliquots of 50 IJ.I of labeled cells were washed in 1.0 ml of buffer, resuspended in 150 IIIof buffer containing 200 ug/ml unlabeled HA and incubated for the indicated time before a final wash and analysis by flow cytometry. Dissociation of bound FL-RA from cell surface CD44 How does binding of the digested FL-HA fragments differ from binding of undigested rooster comb FL-HA? Blocking studies with unlabeled bovine trachea HA (Sigma) and with oligomers of 16 and -26 sugars gave results similar to the blocking curves for high M, FL- HA. That is, similar concentrations of these unlabeled HA preparations were required to achieve 50% inhibition of binding of FL-HA of -30 kDa, -50 kDa and high M, (undigested) FL-HA (data not shown). However, kinetic studies of the dissociation of the Fl-HA preparations from the cell surface of IRAWB 14 induced cells gave very different results, as shown in Figure 2. While undigested FL-HA stayed cell associated for over two hours, the lower M, FL-HA fragments dissociated very rapidly (T 2 of <5 min and -10 min for -30 kDa and -50 kDa FL-HA, respectively). '/ The dissociation data shown in Figure 2 was obtained by including an excess of unlabeled HA (bovine trachea, Sigma) during the dissociation period. This was done to prevent rebinding of partially, or completely dissociated FL-HA. Once a CD44/FL-HA interaction dissociated, it would be replaced by a CD44/unlabeled-HA combination, Another way to look at this binding interaction is shown in Figure 3. If no unlabeled HA was included in the dissociation medium, even low M, FL-HA (-30 kDa) remained bound to IRAWB14 induced cells for quite a long time (T1/2 greater than the period of this experiment). Undigested FL-HA bound to non-induced cells dissociated with a T II2 of about 25 min when unlabeled HA was included in the dissociation medium. In the absence of unlabeled competitor, over two hours was required for dissociation of half of the undigested high M, FL-HA from the surface of non-induced cells.
346
Cell surfaces and hyaluronan receptors Dissociation of FL-HA +/- unlabeled HA
120
T"------------------------, undigested+block -30 kDa+block undigested. No block -30 kDa. No block
20
40
60
80
100
120
140
160
time of dissociation(min)
Figure 3. CD44+ cells were untreated and incubated with undigested high M, FL-HA (circles) or pretreated with IRAWB14 rnAb and incubated with digested FL-HA at -30 kDa (squares). Aliquots of 50 j.11 of labeled cells were washed in 1.0 ml of buffer and then resuspended in 150 j.11 of buffer with (open symbols) or without (filled symbols) 200 ug/ml of unlabeled HA. After the indicated dissociation time, cells were washed and assayed by flow cytometry.
DISCUSSION These studies demonstrate that cooperativity is the primary feature of HA binding by cell surface CD44. This cooperativity is the result of multiple binding sites on the repeating disaccharide ligand and multiple closely arrayed receptors on the cell surface. The ligand factor shown to be relevant here is the length of the carbohydrate chain, which determines the number of physically connected binding sites. The longer the sugar chain, the more linked binding sites are present, which reduces the probability of the HA polymer dissociating from the cell surface. Though any single CD44/HA interaction may be very weak and transient, binding interactions along the length of the HA chain keep the molecule bound to the cell surface and increase the probability of rebinding and binding at adjacent sites. The CD44IHA interaction in the absence of mAb induction is characterized by "thresholds" - a minimum level of CD44 expression and a minimum HA chain length are both required to achieve stable HA binding. As we have shown in the studies presented here: 1) There is a requirement for quite high M, HA for stable binding. This threshold length is at least >100 kDa. 2) Divalent binding occurs at chain length >18 sugar residues. It is not clear at what chain length higher order binding occurs. 3) Binding of high M, HA is relatively stable in the absence of competition from other HA molecules (T "2 > 2 hr). 4) In the presence of competing HA molecules, binding of high M, HA is of "intermediate-term", with T 1/2 -25 min. It remains an open question whether there are physiological mechanisms that mimic the effects of mAb induction on HA binding by cell surface CD44. However, comparisons between the binding of untreated and rnAb induced cells emphasize the unique features of the "natural" CD44/HA interaction. On cells treated with the
Binding by cell surface CD44
347
inducing mAb IRA WB 14 several parameters of the CD44IHA interaction are altered. 1) HA binding is observed at lower CD44 levels 9. 2) HA binding is observed at much shorter HA chain lengths (this report). 3) Dissociation from the cell surface is slowed dramatically 14 and Figures 2 and 3). Preliminary studies suggest that the link domain of TSG-6 may have modulating effects on the CD44IHA interaction that resemble the effects of inducing mAb. As we will be learning from a number of presentations at this meeting, low M, degradation products of HA can have significant effects on a number biological systems. The implication of these observations is that cells can distinguish HA chains of differing lengths. Since CD44 is the major cell surface receptor for HA, one expects that many of these biological effects will be mediated through CD44. Our results suggest that small HA fragments do not themselves bind CD44 stably, but their presence can definitely influence the association of CD44 with high M, HA. Additionally, there may be physiological mechanisms by which low M, HA may have more stable associations with cell surface CD44. Though the specifics are far from understood, we can begin to see how cells might discriminate between HA chains of different lengths and respond differentially.
ACKNOWLEDGEMENTS This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-31613 (to R.H.) and Health Research Council, Academy of Finland, grant #40807 (to M.T.). Flow cytornetry facilities at the Salk Institute were supported by National Cancer Institute Core Grant CA-14195 and Frances C. Berger Foundation.
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2. 3. 4. 5.
6.
7.
8.
Lesley, 1., R. Hyman & P. Kincade, 'CD44 and its interaction with extracellular matrix', Adv. Immunol., 1993, 54, 271-335. Lesley, J., R. Hyman, N. English, J. Catteral & G. Turner, 'CD44 in inflammation and metastasis', Glyconjugate Journal, 1997, 14,611-622. Naor, D., RY. Sionov & D. Ish-Shalom, 'CD44: Structure, function and association with the malignant process'iAav, Cancer Res., 1997,71,241-319. Lesley, 1. & R Hyman, 'CD44 structure and function', Frontiers in Bioscience, 1998,3,616-630. Grover, J. & P.I. Roughley, 'The expression of functional link protein in a baculovirus system: analysis of mutants lacking the A, Band B' domains' , Biochem. 1.,1994,300,317-324. Kohda, D., C.l. Morton, AA Parkar, H. Hatanaka, F.M. Inagaki, LD. Campbell & AJ. Day, 'Solution structure of the link module: A hyaluronan-binding domain involved in extracellular matrix stability and cell migration', Cell, 1996, 86,767775. Varelas, J.B., I. Kollar, T.D. Huynh & T.M. Hering, 'Expression and characterization of a single recombinant proteoglycan tandem repeat domain of link protein that binds zinc and hyaluronate', Arch. of Biochem. and Biophys., 1995, 321, 21-30. Peach, R, D. Hollenbaugh, S. I. & A Aruffo, 'Identification of hyaluronic acid binding sites in the extracellular domain of CD44', J. Cell Biol., 1993, 122,257-264.
348 9.
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22.
23.
Cell surfaces and hyaluronan receptors Lesley, 1., N. English, A. Perschl, J. Gregoroff & R. Hyman,'Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation', J. Exp. Med., 1995, 182,431-437. Katoh, S., Z. Zheng, K. Oritani, T. Shimozato & P. Kincade, 'Glycosylation of CD44 negatively regulates its recognition of hyaluronan', J. Exp. Med., 1995, 182,419429. Zheng, Z., R. Cummings, P. Pummill & P. Kincade, 'Growth as a solid tumor or reduced glucose concentrations in culture reversibly induce CD44-mediated hyaluronan recognition by Chinese hamster ovary cells', J. Clin. Invest., 1997, 100, 1217-1229. Kincade, P.W., Z. Zheng, S. Katoh & L. Hanson, 'The importance of cellular environment to function of the CD44 matrix receptor', Curro Opin. Cell BioI., 1997, 9,635-642. Lesley, J., N. English, C. Charles & R. Hyman, 'The role of the CD44 cytoplasmic and transmembrane domains in constitutive and inducible hyaluronan binding', Eur. J. Immunol., 2000, 30, 245-253. Perschl, A., J. Lesley, N. English, I. Trowbridge & R. Hyman, 'Role ofCD44 cytoplasmic domain in hyaluronan binding', Eur J. Immunol., 1995,25,495-501. English, N., J. Lesley & R. Hyman, 'Site-specific de-N-glycosylation ofCD44 can activate hyaluronan binding and CD44 activation states show distinct threshold densities for hyaluronan binding', Cancer Research, 1998, 58, 3736-3742. Skelton, T.P., C. Zeng, A. Nocks & I. Stamenkovic, 'Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hyaluronan', J. Cell Biol., 1998, 140,431-446. Lesley, J., V. Hascall, M. Tammi & R. Hyman, 'Hyaluronan binding by cell surface CD44, ,J. Biol. Chem., 2000, in press. Tammi, R., D. MacCallum, V. Hascall, J.-P. Pienemaki, M. Hyttinen & M. Tammi, 'Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides', J. Biol. Chem., 1998,273,28878-28888. Knudson, W., E. Bartnik & C. Knudson, 'Assembly of pericellular matrices by COS7 cells transfected with CD44lymphocyte-homing receptor genes' , Proc. Nat. Acad. sa.. USA, 1993,90,4003-4007. Underhill, C.B., G. Chi-Rosso & B.P. Toole, 'Effects of detergent solubilization on the hyaluronate-binding protein from membranes of Simian virus 40-transformed 3T3 cells', J. Biol. Chem., 1983,258,8086-8091. Sleeman, J.P., W. Rudy, M. Hoffman, 1. Moll, P. Herrlich & H. Ponta, 'Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity', J. Cell Biol., 1996, 135, 1139-1150. Liu, D. & M.-S. Sy, 'Phorbol myristate acetate stimulates the dimerization of CD44 involving a cysteine in the transmembrane domain', J. Immunol., 1997, 159,27022711. Li, R, J.R. Walker & P. Johnson, 'Chimeric CD4/CD44 molecules associate with CD44 via the transmembrane region and reduce hyaluronan binding in T cell lines' , Eur.1. Immunol., 1998,28, 1745-1754.
HYALURONAN SYNTHESIS IN HUMAN ARTICULAR CARTILAGE Michael T Bayliss and Jayesh Dudhia
Royal Veterinary College, Royal College Street, London NWI OTU, UK. ABSTRACT
Hyaluronan is a component of the aggrecan/link protein/hyaluronan complex isolated from the extracellular matrix of human articular cartilage. The age of the individual has a significant effect on the composition and structure of this complex and it also influences the rate of hyaluronan synthesis relative to the other components. We have determined the age-related rate of incorporation of 3H_ glucosaminc into hyaluroman and shown that it follows that of aggrecan and is much higher in cartilage from immature individuals than from adults. Wc have also investigated the rate of incorporation of 3H-glucosamine into hyaluronan through the depth of the tissue and shown that this is much higher at the surface of the tissue. We showed that cartilage contains different pools of hyaluronan, some of which are not associated with aggrecan, some appear to be in metastable conformations and some are not associated with the aggrecan/link protein complex at all. Changes in the synthesis of hyaluronan have been compared to the distribution of the endogenous macromolecule using both immunohistochemical and biochemical techniques. We conclude that the concentration of hyaluronan in the tissue at any age is a poor reflection of the rate at which the chondrocytes can synthesise the molecule. KEYWORDS
Hyaluronan, human aggrecan, link protein.
articular
cartilage,
3H-glucosamine,
G I-domain,
INTRODUCTION
Articular cartilage is a specialised connective tissue, the major function of which depends on its state of hydration and the structural arrangement of a large extracellular matrix. Hyaluronan is a ubiquitous molecule and its role in the formation of proteoglycan aggregates cannot be disputed I. However, it is often assumed that all of the hyauronan in articular cartilage is incorporated into aggrccan/link protein/hyaluronan complexes. As well as contributing to the structural properties of the extracellular matrix, hyaluronan may also have important regulatory functions in cartilage. Very low concentrations ofhyaluronan inhibit cartilage nodule formation in differentiating cluck limb buds in vivo and addition of hyaluronan to the medium of chondrocytes also reduces proteoglycan synthesis 2,3. The extracellular concentration of hyaluronan may, therefore, have a role in regulating the synthesis of aggrecan during maturation of articular cartilage and in repair processes such as those that occur in degenerative joint disease. Considerable progress has been made in understanding the structure and synthesis of aggreean in human articular cartilage during ageing 4, but there is very
298
Aspects or hyaluronan in joints
little information concerning the way in which ageing affects the structure of hyaluronan. Previous analyses confirmed that the molecular weight of hyaluronan decreased dramatically with increasing age and that the concentration ofthe molecule increases to accommodate the increased concentration of aggrecan (hyaluronanbinding domain) that is present 5. However, the molecular weight of the newly synthesised hyalurornan is excluded from Sephacryl S-IOOO columns at all ages, suggesting that there must be a biological mechanism for reducing hyaluronan molecular weight in articular cartilage, or the large hyaluronan is not retained by the tissue. The latter explanation is unlikely and we propose that the newly synthesised hyaluronan is used for purposes other than the formation of aggregates. MATERIALS AND METHODS Source of tissue. Normal human articular cartilage was obtained from the femoral condyles of lower limbs amputated for bone tumours not involving the knee joint. They were obtained fresh (within I hr) from the operating theatre and the cartilage was removed as full thickness from the femoral condyle with a scalpel. Cartilage was then either cultured immediately with radioisotope or stored at -20°C until it was analysed for its hyaluronan content. Preparation of tissue for analysis, its culture, and gel chromatographic analysis of hyaluronan. Full thickness pieces of cartilage (-200 mg) were cultured overnight in 2 ml Dulbccco's Modified Eagles Medium (DMEM) containing 50 /lCi/ml 35S-sulphate and 100 /lCi/ml 3H-glucosamine. When the rate of sulphate incorporation by the cartilage was determined, triplicate samples (-50 mg) were cultured for 4 hrs in DMEM containing 10 /lCi/ml 35S-sulhate and the rate of incorporation of the isotope was determined as described by Bayliss et al. (1999) 6. Articular cartilage was either analysed as a full thickness piece of tissue or it was sectioned through its depth into 50 urn slices, pooling every four of these to obtain 200 urn zones of tissue. Total hyaluronan was determined in papain digests of the cartilage using the method described by Holmes et al. (1988) 7 and the molecular weight of hyaluronan was determined in these digests by size exclusion chromatography in a column of Sephacryl S-100 7. The papain digests were applied to a column (ISO em x 0.65 em). Fractions (1 ml) were analysed for their radioactivity and for their content of hyaluronan. In some cases the cartilage was extracted sequentially with 0.5 M, I M and 4 M guanidine HCl and the amount of hyaluronan in each extract was determined. ImmunohistochemicallocaIisation of hyaluronan. Hyaluronan was localised in tissue cryo-sections. The biotinylated GI domain of aggrecan can only bind to sites that are not already occupied by intact aggrecan. Histological cryosections (10 urn) were incubated overnight with
Synthesis in human articular cartilage
299
biotinylated-G I domain prepared from porcine laryngeal cartilage 8 and the sites of binding were localised using peroxidase labelled secondary antibody.
Fractionation of hyaluronan and proteoglycans. 3H-glucosamine labelled hyaluronan and 35S-sulphate labelled proteoglycans were separated on a MonoQ ion exchange column eluted with a NaCI (0-1 M) gradient) in Tris-HCI. The tritium counts associated with the newly synthesised hyaluronan were determined as a proportion of the hyaluronan plus proteoglycan radiolabel.
RESULTS AND DISCUSSION Age-related changes in byaluronan content. The concentration of hyaluronan (% total uronic acid) in full-thickness cartilage increased during normal ageing of articular cartilage 7. The same finding was obtained if the results were expressed as ug/ml wet wt. of cartilage or on the basis of its collagen content. Interestingly, the purified aggregate preparations, fractions Al from CsCI density gradients, had very similar concentrations of hyaluronan to whole tissue digests, confirming that this fraction contained most of the hyaluronan in the extracts. Moreover, a separate set of samples gave the same results (Fig. 1). Thus, the concentration ofhyaluronan increases in the tissue with advancing age as the concentration of aggrecan and its hyaluronan-binding fragments increase 5.
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300
Aspects of hyaluronan in joints
Age-related changes in molecular weight. Holmes et al. (1988) 7 also showed that although the concentration of hyaluronan increased, there was a considerable decrease in the molecular weight of the hyaluronan in whole tissue digests of cartilage. In marked contrast, the newly synthesised hyaluronan was excluded as high molecular weight chains from the size exclusion columns, regardless of the age of the tissue. This finding suggests that there must be some biological mechanism, either enzymatic or free-radical driven, that can reduce the molecular weight of hyaluronan and, therefore, the aggregates it forms with aggrecan in the extracellular matrix. An alternative scenario could involve agerelated changes in the expression ofHAS1:HAS2, but we have no data on the mRNA for these enzymes. Age-related changes in extractability. Attempts were made to show that the hyaluronan was present in different compartments in immature and mature cartilage and to use the findings as support for an hypothesis in which aggregates are envisaged as undergoing a destabilisation during normal ageing. A higher proportion of hyaluronan was extracted from immature articular cartilage with non-dissociating extractants (Fig. 2), which suggests that the aggregates were not destabilised during ageing, at least not to the extent that fragments of hyaluronan are produced. However, other studies do suggest that the stoichiometry of aggrecan and link protein in aggregates diverges from the I: I molar ratio that is expected and the tissue becomes deficient in link protein with age.
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Synthesis in human articular cartilage
301
Age-related changes in the localisation of hyaluronan.
The binding of biotinylated-G I domain of aggrecan to hyaluronan was assessed immunohistochemically. This technique will only localise hyaluronan that is not masked already by aggrecan. In immature cartilage the biotinylated-G I domain was localised in the deep zone of cartilage in a region where one would expect there to be a rapid growth of the tissue and, therefore, considerable matrix turnover. In the mature cartilage, this zone ofhyaluronan localisation was absent and there was a faint 'blush' throughout the tissue depth, but more importantly there was also strong staining around each chondrocyte. This finding suggests that there is a multifunctional role for hyaluronan in adult cartilage and that it probably functions in ways other than aggregate formation. Age-related variations in hyaluronan synthesis.
This was assessed by radiolabelling the newly synthesised hyaluronan with 31-1_ glucosamine, separating radiolabclled macromolecules from non-incorporated isotope and fractionating the hyalurornan from the proteoglycan by HPLC using a MonoQ column and a NaCI gradient. Figure 3 shows that the effect of age on the rate of incorporation of tritium into hyaluronan was similar to that observed by Bayliss et al. (1999) 6 for the incorporation of 35S sulphate into aggrecan. Our studies have also shown that the 3H and 35 S content of chondroitinase ABC-generated disaccharides is not altered during ageing, ruling out the possibility that the pool of 3H glusocamine was changing. Therefore, the increase in hyaluronan observed during ageing of cartilage is a consequence of accumulation and not of an increase in the synthesis of the molecule.
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302
Aspectsof hyaluronan in joints
CONCLUSIONS The concentration of hyaluronan in human articular cartilage increases with age, as does the heterogeneity of aggrecan and the complexes that it forms with hyaluronanllink protein. The molecular weight of the endogenous hyaluronan decreases with age, which contrasts with the newly synthesised molecule, which is a high molecular weight species at all ages tested. Furthermore, it is unlikely that there is an age-related change in the expression of the enzyme necessary for hyaluronan chain elongation, because the newly synthesised chains are high molecular weight. There must, therefore, be a biological mechanism for degrading the hyaluronan during its relatively long resident time in the tissue.
ACKNOWLEDGEMENTS The authors would like to thank the Arthritis Research Campaign, UK for their generous financial support.
REFERENCES 1.
2. 3.
4. 5.
6.
7.
8.
D. Heinegard & V.C. Hascall, 'Aggregation of cartilage proteoglycans', J Bioi. Chern., 1974,249:4250-4256. B.P. Toole, G. Jackson & J. Gross, 'Hyaluronate in morphogenesis: inhibition of chondrogenesis in vitro', Proc. Nat!. Acad. Sci. USA, 1972,69: 1384-1386. M. Solursh, T.E. Hardingham, V.C. Hascall & lH. Kimura, 'Separate effects of exogenous hyaluronic acid on proteoglycan synthesis and deposition in pericellular matrix by cultured chick embryo limb chondrocytes' , Dev. Bioi. 1980, 75:121-129. M.T. Bayliss, 'Proteoglycan structure and metabolism during maturation and ageing of human articular cartilage', Biochern. Soc. Trans. 1990, 18:799-802. A. Maroudas, M.T. Bayliss, N. Uchitel-Kaushansky, R. Schneiderman & E. Gilav, 'Aggrecan turnover in human articular cartilage: use of aspartic acid racemization as a marker of molecular age', Arch. Biochern. Biophys. 1998, 350:61-71. M.T. Bayliss, D. Osborne, S. Woodhouse, C. Davidson, 'Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition', J. Bioi. Chern. 1999,274:15892-15900. M.W. Holmes, M.T. Bayliss & H. Muir, 'Hyaluronic acid in human articular cartilage. Age-related changes in content and size', Biochern. J. 1988, 250:435441. F. Bonnet, D.G. Dunham & T.E. Hardingham, 'Structure and interactions of cartilage proteoglycan binding region and link protein', Biochern. J. 1985,228:7785.
MECHANOMODULATORY INFLUENCES UPON HA PRODUCTION IN JOINT DEVELOPMENT AND MAINTENANCE Osborne AC, Kavanagh E, Lamb KJ, Dowthwaite G, Archer C and Pitsillides AA * Department ofVeterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NWI OTU. UK Tel: 02074685245; Fax: 0207388/027; E-mail; [email protected]
ABSTRACT Joint mobility is established by the formation of a cell-free, hyaluronan (HA)-rich space (cavitation), within a non-chondrogenic interzone between adjoining skeletal elements during development. Many factors contribute to joint formation and movement is an essential requirement. Using either spastic or flaccid paralysis before cavitation, we have shown that both abrogate joint cavity formation. This mechano-dependence is also supported by our studies using isolated embryonic chick articular surface cells which show many changes (24 hrs) in response to a brief (10 min) period of dynamic mechanical strain, that are consistent with the notion that dynamic strain selectively promotes 'joint line-related' differentiation. In these cells, dynamic but not static strain also induces marked increases in ERK. activation. Furthermore, constitutively active ERK.-l/2, but not inactive ERK-2, shows a very clear joint line-selective localisation in developing limbs that is abolished by the influence of immobilisation in ovo. Therefore, we hypothesise that cells at developing joint lines exhibit HA-synthetic potential that is regulated by 'up-stream' mechano-dependent ERK-I activation. INTRODUCTION The origins of synovial joints are present within discrete masses of blastemal mesenchyme found between cartilaginous condensations of the developing skeletal elements (anlagen). These anlagen expand by perichondral apposition and by extracellular matrix (ECM) production and cell division. Concomitantly, the presumptive joint becomes increasingly more defined as a transverse region of persisting, densely-packed, flattened cells representing the primitive blastemal remains (interzone, IZ). As the anlagen expand further, this interzone becomes increasingly attenuated and flattened. Later, the peripheral presumptive joint capsule/synovium, initially continuous with this interzone, becomes vascularised and subsequently tissue separation occurs within its avascular central region by an, as yet, undetermined mechanism. This separation establishes the primordial synovial 'space' that contains particularly high concentrations of high molecular weight hyaluronan (HA) in adults I. This is consistent with the view that tissues rich in HA with little collagenous matrix are relatively soft, whilst tissues with higher tensile strength contain high proportions of sulphated GAGs and quantitatively predominant collagen fibrils 2 • Indeed, synovial fluid is completely liquefied and its functional properties are, for the most part, independent of collagen. Thus, the initial elaboration and subsequent conservation of joint cavities provides an ideal circumstance for the 'differentiation' events, associated with the acquisition of a cellular phenotype which contributes to the synthesis of such HA-rich matrices, to be defined. It has been shown that HA is indeed a major constituent of developing joint spaces
304
Aspects of hyaluronan in joints
and our studies sug~est that it is synthesised by cells directly bordering such presumptive cavities . We have also shown that these cells express HA-binding proteins (principally CD44) and appear to actively bind HA, via a moesin-mediated (CD44-associated actin-capping protein) interaction with polymerised cytoskeletal actin. Foremost, the inhibition of joint formation by HA oligosaccharide-induced blockade of HA:HABP interaction in ova, has demonstrated the crucial role for enhanced HA synthesis, and its association with CD44 during joint cavitation 4. Therefore, using the developing joint we aim to establish the 'up-stream' signalling events responsible for regulating HA synthesis and binding. It is clear that mechanisms exist which ensure that the structural characteristics of connective tissues are matched to the demands of their prevailing mechanical environment. In the fully developed skeletal system, it is evident that in conditions where mechanical demands, or signals, may modulate, such mechanisms normally act 'up-stream' in order to ensure that functional appropriateness is maintained. The dynamic nature of this relationship, however, is particularly evident during the morphogenetic events associated with limb development. Indeed, many studies have emphasised movement's essential role in the joint formation process 5-7. Depriving limbs of movement-induced stimuli by maintenance in organ culture or spastic immobilisation in ova, prevents both cavity formation and promotes the regression of previously cavitated joints. Nonetheless, such studies fail to provide a mechanistic basis describing movement's role in the cavitation process. Accordingly, using in ova hyperactivity or spastic or flaccid paralysis, together with novel antibodies raised against UDP-glucose dehydrogenase (UDPGD, rate-limiting in irreversible conversion ofUDP-glucose to UDP-glucuronate) and chick CD44, as well as an assessment of UDPGD activity/cell and HA content, we aim to establish whether local HA synthesis and binding are modulated by movement in ovo. Secondly, we have used an in vitro system to examine changes in HA synthesis and binding induced by the application of a single episode of mechanical stimulation to cells derived from such developing articular surfaces. In addition, we examine the role of pericellular HA in modulating these events, and fmally we present preliminary evidence that the MAPK cascade plays a key 'up-stream' role in the control ofthese events. METHODS In ovo immobilisation: This was a modification from Mitrovic (1982) 6. Briefly, 0.1-0.5 mg decamethonium bromide (DMB, spastic paralysis), or 0.5-0.8 mg pancuronium bromide (PB, flaccid paralysis), or neuromuscular-active agent inducing hyperactivity (AP) was administered in 100 III Tyrode solution (TS) at 24 hour intervals onto the chorioallantois for 3 days. On the fmal day, embryos were examined for spontaneous limb and beak movement and only those with consistent paralysis or hyperactivity and a normal heart beat were used. Articular surface (AS) cell isolation and application of mechanical strain:
Articular fibrocartilage was excised from tibiotarsal joints of stage 42 White Leghorn chicks and cells isolated by collagenase (type I) digestion 8. Only primary or 151 passage cells were used. Mechanical strain was applied by subjecting plastic strips, on which cells were plated, to 4-point bending in a calibrated jig 9, Confluent serum-deprived cells received dynamic strain (3800 Ill> at 1 Hz) for 10 mins. Controls received similar cyclic medium perturbation without strain (flow) or were otherwise unperturbed (static). In
Mechanomodulatory influences upon HA production
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some studies 10 min of 3800 us was also applied non-dynamically (static strain). Medium was collected prior to strain and at various times up to 24 hrs. Activity and expression of UDPGD:
UDPGD activity/cell was assessed in fresh sections and AS cells'", Briefly, sections or cells were reacted in nitrogen-saturated medium (37°C) containing 5.3 roM UDPglucose, 1.5 roM NAD, 3.7 roM NBT. UDPGD activity/cell was measured by scanning/integrating microdensitometry. For immunohistochemistry, air-dried serial cryosections were fixed in ice-cold acetone and incubated with rabbit anti-UDPGD enzyme antibody diluted I: lOin TBS. Sections were immunolabelled with the ABCGO enzyme system (see Vector, UK). Assay for medium HA and s-GAG and differential alcian blue (AB) staining:
HA and sGAG concentrations were assessed by well-established methods 11,12. Fixed sections or monolayers were stained with 0.05% AB8GX containing between 0.025-1.0 M MgCl2 at pH 5.8. Intensity/area was measured by microdensitometry. Some sections were pre-incubated in HAase, Chase ABC or KSase I and II and washed before AB. HA labelling with HA-BR and immunochemistry for s-GAGs:
Sections were incubated in biotinylated HABR (25 ug/ml) overnight and washed in TBS-T. Streptavidin added and detected using an alkaline phosphatase enzyme system. For sGAGs, serial sections were treated with appropriate enzymes and after washing, incubated with monoclonal antibodies to C4S, C6S, KS and undersulphated KS. Western blotting:
AS cell protein extracts, run on SDS-PAGE gels, transferred to PVDF blots, were blotted with rabbit anti-bovine UDPGD antibody (1:500) in TBS-T, developed with 2 ECL, scanned and profiles (OD/mm) measured (Molecular Analyst'?"), ERKl/2 expression (non-phosphorylated ERKI and ERK2 antibodies) and activation (antibodies specific for phosphorylated ERKl/2 (1:1000)) were assessed by Westerns (see above). Assessment of HAS expression:
HAS2 and HAS3 PCR was carried out as described
13.
RESULTS I Mechanomodulatory effects in ovo UDPGD expression and activity:
In normal joints and those from chicks with induced hyperactivity, UDPGD immunolabelling was stronger in cells bordering the presumptive joint line than adjacent chondrocytes. In contrast, this joint line-selective labelling was markedly diminished in immobilised limbs. After cavitation, cells bordering developing articular FC were also labelled strongly for UDPGD compared to underlying FC. Paralysed limbs did not exhibit such differential labelling where joint fusion had occurred.
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Aspects of hyaluronan in joints
During cavitation, a band of cells at the presumptive articular surfaces had significantly higher UDPGD activity than adjacent interzone and FC cells. This selective activation was lost in immobilised limbs. These elevated levels of UDPGD activity at the articular surface were maintained after cavitation. Histo- and immuno-chemical analysis ofGAG content:
Before cavitation, CS labelling was evident in epiphyseal cartilage, but not in the FCinterzone region. In contrast, KS labelling was prominent in a restricted zone at the epiphyseal-FC interface, with little IZ labelling. Undersulphated-KS (2D6) was undetectable. Selective enzyme digestion resulted in appropriate loss of label. Alcian blue staining prior to cavitation (stage 39) indicated that 'HA-like' staining predominated at the joint line, IZ and developing articular surfaces. 'CS-like' staining was mainly restricted to cartilage and whilst 'KS-like' staining was weaker, it showed similar distribution. These patterns were maintained after cavitation. HAase digestion markedly decreased staining in both FC and IZ (60%). In contrast, Chase had little effect on 'HA-like' staining intensity, but significantly decreased 'sGAG-like' staining (by 80%) in all regions. Keratanase reduced (~30%) 'HA-like' staining in IZ and FC, decreased 'sGAG-like' staining in deeper cartilage regions, but had no effect on 'sGAG-like' staining intensity in IZ and FC. Effect ofskeletal movement on GAG content: An overall 'representation' of the effect of immobilisation on relative content and distribution of GAGs is shown (Figure 1). In cavitating joints, epiphyseal cartilage is CS-rich, articular FC is KS-rich and the joint line and IZ are HA-rich. With immobilization, the HA-rich joint line is replaced by KS-rich tissue, and CS-rich matrix extends into the non-cavitating region.
Cavitating joint
Non-cavitating joint ................. HA
----- CS --KS
Arbitary Scale
PEC
PFC IZ DFC DEC Tissue
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PFC IZ DFC DEC Tissue
Figure 1. Effect ofimmobilisation on GAG content in developing joint. Proximal (P), distal (D), epiphyseal cartilage (EC), fibrocartilage (FC) and interzone (IZ). Local ERKl/2 expression and activation:
In cavitating joints (stage 39) from control and experimentally-induced hyperactive chicks, antibody against human phosphorylated ERK-l/2 (pERK-II2, activated) specifically labelled cells at the joint line. Up to 2 days before cavitation, pERK-112 was expressed in all interzone cells between the unlabelled anlagen. In contrast, non-
Mechanomodulatory influences upon HA production
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cavitating joints from spastically or flaccidly paralysed limbs, exhibited little labelling for pERK-I/2 at the presumptive joint line (Figure 2). In joints from short-term spastic paralysis (stage 41) very faint pERK labelling was present at the articular surfaces, but this was absent in cells at fusing articular surfaces in flaccidly paralysed limbs. Interestingly, non-phosphorylated ERK-I (inactive), but not inactive ERK-2, also demonstrated increased levels of expression at articular surfaces and this was also absent in immobilised limbs.
c Figure 2. Immunolocalisation of active (phospho) ERK-l/2 in normal (A and B) and immobilised (C) knee joints at stages before and during cavitation. Joint line-selective expression is evident (A and B) and this is lost in immobilisedjoints (C). CD44 expression:
Initial studies used anti-hamster CD44 antibody (Figure 3). Using a novel anti-chick CD44 antibody (AV6) we can confirm previous results and also show a markedly diminished labelling for CD44 at the joint line of immobilised limbs.
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.
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Figure 3. CD44 immunochemistry in control (A) and immobilised (B) developing chick metatarsal-phalangeal joints using anti-CD44 antibody (30189). II Effects of mechanical strain on AS cells in vitro Release ofHA and sGAGs, and AB staining ofGAGs in monolayer:
Application of dynamic strain caused both significant early increases and enhanced rates of HA release over 24 hrs. Release of sGAG was unaffected (at 24 hrs). AB staining showed 'HA-like', but not 'sGAG-like', content within AS cell monolayers was increased 24 hrs after application dynamic strain, compared to 'flow', 'static strain' or static controls. However, 'sGAG' staining was significantly increased only by 'flow'.
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Aspects of hyaluronan in joints
CD44 expression:
In Western blots, rat anti-hCD44 monoclonal (Hermes-I) and the novel mouse monoclonal AV6, raised against chick CD44, specifically recognized a non-reduced protein of approximately 97 KDa in AS cell extract. Slight increases in CD44 expression were evident 24 hrs after 'flow' or static strain, whilst cells subjected to dynamic strain showed the most marked increases in CD44 expression (using Hermes-l or AV6). HAS mRNA expression:
HAS2 mRNA shows constitutive expression in AS cells and appears not to be changed 24 hrs after mechanical stimulation. HAS3 mRNA however, is absent 'basally' and is induced 24 hrs after application of dynamic strain but not by fluid perturbation alone (control 'flow'). ERKI/2 expression and activation:
In Western blots (Figure 4), antibody against human pERK-I/2 specifically recognized a reduced protein of approximately 44 kDa (activated p44 MAP kinase; pERK). Cells subjected to static or dynamic strain had greatly increased pERK-I/2 expression after 24 hours compared to cells given fresh medium alone. However, AS cells subjected to medium 'flow' showed the greatest increase in pERK-I/2 expression. kO. 160= 1057550-
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DISCUSSION During morphogenesis, phenotypic cellular complexity is being established and it is clear according to the epigenetic view of differentiation, that during this time each cell makes a series of choices. Some of these may have no obvious phenotypic outcome and might therefore be considered components of cell fate 'determination', whilst others will be involved in establishing the differentiated cell phenotypes characteristic of particular connective tissues. Thus, it is convenient that a model for considering the process of joint formation should involve its division into two continuous phases: the initial establishment of a cartilaginous anlagen, and the subsequent formation of a cavity within an apparently continuous ECM. Although these two phases might be interrelated, most recent studies have examined the nature of the 'determinants' in this initial phase, and several regulatory genes expressed in presumptive joints appear to act as coordinators of such patterning 14,15. Indeed, it has been shown that growth/differentiation factor-S (Gdf5), which influences chondrogenesis, is selectively expressed in developing interzones prior to cavity formation, and as cavitation progresses and joint competence established, its
Mechanomodulatory intluences upon HA production
309
expression decreases. These studies do not however, directly address the 'differentiation' events that comprise the second phase of joint cavity formation. We have shown that joint line-related differentiation includes high levels of UDPGD activity and protein expression, enhanced levels of CD44 expression and an association with an ECM rich in HA. Further, our finding that all these factors are diminished at the presumptive joint in immobilised limbs, where cavitation fails, provides a link between this phenotype and successful cavity formation. Additionally, their co-ordinated enhancement in cultured cells derived from developing articular joint surfaces in response to dynamic mechanical strain in vitro, suggests that this HA-synthetic differentiated status is normally dependent upon mechanomodulatory influences. Embryonic movements in the chick follow an organised pattern and reach a maximum of up to 20 movements per minute between days 11 and 13. It has also been shown however, that cultured chick quodratojugal joints maintained a functional cavity with only a few daily flexures 16. This might provide the basis for interpreting our in vitro fmdings, which show the intriguing sustained induction of enhanced HA release 24 hrs after brief transient application of dynamic strain. Furthermore, increases in UDPGD protein expression and enzyme activity as well as changes in HAS mRNA expression may provide a basis for explaining the increases in HA synthesis required to facilitate this response. UDPGD activity, which converts UDP-glucose to UDP-glucuronate has been described as the irreversible, rate-limiting, step in the production ofUDP-glucuronate 17. Further since the availability of UDP-amino sugars (N-acetylglucosamine) does not appear to limit the rate of GAG synthesis 18, it is tempting to speculate that by controlling the supply rate of HA's essential monosaccharide, UDPGD activity will indirectly control the potential of local HAS activity at these sites. Nonetheless, HAS3 mRNA is also induced in response to dynamic strain, and whilst mRNA expression may not necessarily reflect protein expression or enzymatic activity, these results suggest that HAS3 together with constitutive HAS2 might contribute to the sustained straininduced increases in HA production by AS cells. This is supported by our observations that HAase treatment also induces HAS3 mRNA expression and interacts with dynamic strain application to synergistically enhance HA synthesis and release (Dowthwaite et aI, these proceedings). It is evident that co-ordination between such mechanically-induced changes in HAsynthesis, CD44 expression, ECM HA-content and cell-binding, in response to mechanical stimuli both in vivo and in vitro, emphasises their association. However, little regarding the mechanisms that control these events is known. Recently, it has been found that both internal and external signals that control proliferation and differentiation converge at kinases that activate extracellular signal regulated kinases-l and 2 (ERK-l and ERK-2). These mitogen-activated protein kinases (MAPKs; components of the 3kinase, Raf, MEKJERK, 'classical' cascade) mediate many diverse events, including the response of endothelial cells to mechanical stimuli. Moreover, the duration of ERK activation appears to defme whether such signals lead either to proliferation (transient) or differentiation (sustained). We have observed that constitutively active ERK shows joint line-selective localisation that is lost upon immobilisation, and that AS cells exhibit sustained ERK activation in response to mechanical stimulation. This suggests that joint cavity formation sites are specified by mechanically-dependent activation of ERK, which promotes the acquisition ofthe differentiated 'HA-synthetic' phenotype.
ACKNOWLEDGEMENTS The authors thank Drs S Tsukita (Japan), F Davison (AHT, UK), M Chambers
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Aspects of hyaluronan in joints
(IHMS, UK) and Caroline Wheeler-Jones (RVC, UK) for supplying primary antibodies, and Prof. Lanyon for the use of the cell-loading jig.
References 1. Balazs EA, Watson D, Duff IF, Roseman S, 'Hyluronic acid in synovial fluid I Molecular parameters ofHA in normal and arthritic fluids', Arth Rheum, 1967, 10, 357-376. 2. Laurent TE, In: The Biology ofhyaluronan, J Wiley & Sons, Chichester, 1989, 1-5. 3. Pitsillides AA, Archer CW, Prehm P, Bayliss MT, Edwards JCW, 'Alterations in hyaluronan synthesis during developing joint cavitation', 1. Histochem. Cytochem., 1995, 43, 263-273. 4. Dowthwaite GP, Edwards JCW and Pitsillides AA., 'An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development.', J Histochem Cytochem, 1998, 46, 641-51 5. Fell and Canti, 'Experiments on the development in vitro of the avian knee joint', Proc. Royal Soc., 1934,116,316-351. 6. Mitrovic D, 'Development of the articular cavity in paralysed chick embryos and in chick limb buds cultured on chorioallantoic membranes', Acta. Anat., 1982, 113, 313-324. 7. Ward AC and Pitsillides AA, ' Mechanodependent joint line-selective activation of ERK-l during embryonic joint development', Trans ORS., 1999, 24(1), 343 8. Dowthwaite GP, Ward AC, Suswillo RFL, Flanelly J, Archer CW and Pitsillides AA., 'The effect of mechanical strain on the metabolism of hyaluronan and hyaluronan-binding protein expression in embryonic chick fibrocartilage cells', Matrix Biology, 1999, 18,523-532. 9. Pitsillides AA, Rawlinson SCF, Suswillo RFL, Bourrin S, Zaman G & Lanyon LE, 'Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?', FASEB J.,1995, 9,1614-1622 10. Mehdizadeh S, Bitensky L, Chayen J, 'The assay of UDPGD activity: discrimination from xanthine dehydrogenase activity', Cell Biochem. Func. 1991, 9, 109-110. 11. Fosang AJ, Hey NJ, Carney SL, Hardingham TE, 'An elisa plate based assay for hyaluronan using biotinylated proteoglycan 01 domain', Matrix, 1990, 10,306-310. 12. Farndale RW, Buttle DJ, Barrett AJ, 'Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue', Biochim. Biophys. acta., 1986,883, 173-177. 13. Dowthwaite OP, Flannery CL, Flannelly J, Fern T, Archer CW and Pitsillides AA, Depletion of pericellular matrix synergistically enhances mechanical strain-induced HA release from articular surface cells (submitted). 14. Storm EE, Huynh TV, Copeland NO, Jenkins NA, Kingsley DM, Lee S-J, 'Limb alterations in brachypodism mice due to mutations in a new member of the TOFBsuperfamily', Nature, 1994,368, 639-643 15. Tickle C, 'Vertebrate limb development', Curr Opin Genet Dev , 1995, 5,478-84 16. Hall BK, Chapter 3: Origins of skeletal cell types. In Development and Cellular Skeletal Biology, Academic Press, New York, 1978, 304. 17. Molz RJ and Danishefsky I, 'Uridine diphosphoglucose dehydrogenase in rat tissue', Biochim Biophys Acta, 1971,250,6-13. 18. Handley CJ and Phelps CF, 'The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage', Biochem J., 1972, 126,417-432.
INIllBITION OF TUMOR GROWTH IN VIVO AND ANCHORAGE-INDEPENDENT GROWTH IN VITRO BY PERTURBING HYALURONAN-CELL INTERACTIONS Bryan P. Toole*, Rebecca M. Peterson and Shibnath Ghatak Department ofAnatomy and Cellular Biology, Tufts University School of Medicine. Boston, MA 02111, USA.
ABSTRACT
Crucial elements of tumor growth and metastasis are interactions between tumor cells and their pericellular environment, a major component of which is hyaluronan. Thus, we determined whether hyaluronan-eell interactions are important in tumor progression by perturbing these interactions in the pericellular milieu of tumor cells and inhibiting downstream signaling or other cellular events. Our first approach was to use hyaluronan oligomers to displace endogenous hyaluronan polymer bound to hyaluronan-binding proteins. As predicted, treatment of subcutaneous tumors with hyaluronan oligomers, using slow release mini-osmotic pumps, was shown to inhibit growth in vivo. Our second approach was to over-express soluble CD44 to competitively inhibit endogenous binding of hyaluronan to cell surface proteins, including CD44 itself. Thus stable transfectants of murine mammary carcinoma cells producing high levels of soluble CD44 were introduced into the peritoneum of syngeneic mice. Transfection with soluble CD44 was shown to inhibit growth of these cells in ascites, as well as invasion and growth in the peritoneal wall, whereas transfection with mutated soluble CD44 that does not bind hyaluronan had no effect. Growth characteristics of tumor cells treated as above were also compared in culture. Growth in monolayer culture revealed no significant differences between tumor cells over-expressing soluble CD44, tumor cells treated with hyaluronan oligomers and control tumor cells. However, dramatic reduction in anchorage-independent growth in soft agar, a characteristic of transformed cells, was observed for the soluble CD44transfected and oligomer-treated tumor cells, compared to control tumor cells. Thus we conclude from these studies that perturbation of hyaluronan interactions at the tumor cell surface has a direct effect on the transformed properties of tumor cells, leading to inhibition of anchorage-independent growth in vitro and tumor progression in vivo.
INTRODUCTION Hyaluronan is commonly found in extracellular matrices and at the cell surface, especially during dynamic cellular events such as embryonic development, tissue remodeling and tumor progression':', Hyaluronan influences movement, growth, and adhesion of cells in vitro'", and recent experimental evidence in animal models directly implicates hyaluronan in embryonic cell invasion", and tumor growth and metastasis7-12. Most malignant solid tumors contain elevated levels of hyaluronan":", and high levels of hyaluronan expression have been correlated with poor differentiation, tumor
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Cell surfaces and hyaluronan receptors
spread and poor survival rates in several types of human cancers":", Enrichment of hyaluronan in tumors is due to interactions between tumor cells and surrounding stromal cells that induce increased stromal hyaluronan production, as well as to increased production by tumor cells themselves. For example, elevated hyaluronan production by mouse mammary carcinoma" and melanoma cells" correlates with their metastatic capacity. Also, however, interaction of several types of malignant tumor cells with fibroblasts in culture upregulates production of hyaluronan by the latter13·20.21. In accord with this observation, hyaluronan accumulation occurs at the interface of tumor invasion into host tissues in various tumor types13.15.22. We have used two general methods to perturb hyaluronan-protein interactions in order to probe whether these interactions are important in tumor progression. First we postulated that, irrespective of downstream mechanisms whereby hyaluronanprotein interactions affect tumor cell behavior, the effects of endogenous hyaluronan are almost certain to depend on its polymeric nature. Thus hyaluronan oligomers, which bind to most hyaluronan-binding proteins, including the hyaluronan receptor CD44, and compete for hyaluronan polymer binding":", would be expected to act as antagonists of endogenous hyaluronan polymer interactions. Consequently we conducted a series of experiments to test the effect of hyaluronan oligomers on growth in vitro and in vivo. Second, over-expression of soluble hyaluronan-binding proteins perturbs hyaluronan-protein interactions on the tumor cell surface by acting as a sink for endogenous hyaluronan. Thus we produced stable transfectants of TA3/St murine mammary carcinoma cells that over-express soluble CD44 and compared growth of these cells with control tumor cells in vitro and in vivo. INlllBITION OF TUMOR GROWTH IN VIVO BY BY ALURONAN OLIGOMERS
We tested the effect of administering hyaluronan oligomers on subcutaneous growth of 816-FI0 murine melanoma", To maximize local delivery of the oligomers, we used ALZET osmotic pumps filled with hyaluronan oligomers (12-16 monosaccharides in length) at various concentrations. The pumps were surgically inserted subcutaneously in nude mice, then a day later 816 melanoma cells were injected at an adjacent site. We used 100 III pumps which, at an initial concentration of I mg/ml, release -0.5~ hyaluronan oligomer/0.5 IlIl hour over the course of 7 days. In animals carrying these pumps, the average tumor weight was found to be -20% of controls treated with PBS or chondroitin sulfate. In another series of experiments the 816 tumor cells were injected two days prior to pump insertion, thereby allowing a small tumor mass to form before delivery of hyaluronan oligomers. Once again the oligomers significantly reduced the rate of tumor growth". Similar results have now been obtained with TA3/St murine mammary carcinoma cells in syngeneic mice and LX-l human lung carcinoma cells in nude mice. INlllBmON OF TUMOR CELL GROWTH BY OVER-EXPRESSION OF SOLUBLE CD44
Stable transfectants of TA3/St murine mammary carcinoma cells over-expressing variants of soluble CD44 were injected intraperitoneally in syngeneic mice". These soluble CD44 transfectants grew at a reduced rate within the ascites, as compared to wild type and vector-transfected TA3/St controls, and then went into Gl arrest within
Inhibition of tumor growth
Table 1.
351
Over-expression of soluble CD44 in TA3/St mammary carcinoma cells inhibits intraperitoneal tumor formation Solid tumors'
Ascites"
Numbers of animals
CONTROLS: Wild type TA3/St Vector transfectant Soluble CD44 (R43A) transfectant"
6/6 6/6 6/6
6/6 6/6 6/6
OVER-EXPRESSION OF SOLUBLE CD44: Soluble CD44 transfectant clone #1 Soluble CD44 transfectant clone #2
0/6 0/6
0/6 0/6
'Extensive tumor growth and invasion in the mesentery and peritoneal wall occurred at 10-15 days after i.p.injection for the three types of control cells but not the soluble CD44 transfectants. "Grossly visible accumulation of ascites occurred at 10-15 days after i.p. injection for the controls but not the soluble CD44 transfectants. 'In this control TA3/St cells were transfected with mutant soluble CD44 that does not bind hyaluronan.
the ascites. Eventually these cells were cleared from the peritoneum, presumably as a result of cell death, thus resulting in the absence of ascites accumulation (Table 1). Stable transfectants of TA3/St cells over-expressing mutated soluble CD44 (R43A) that does not bind hyaluronan were also isolated. These transfectants exhibit ascites accumulation, growth rates and cell cycle profiles in vivo similar to wild type and vector-transfected TA3/St cells", Thus the altered growth characteristics of the soluble CD44-transfected cells are due to the hyaluronan-binding properties of the soluble CD44. Although the soluble CD44 transfectants grow at a reduced rate in ascites, the cells that accumulate prior to their demise would be sufficient in number for extensive attachment to and invasion of the peritoneal wall in the case of controls. However, the soluble CD44 transfectants failed to attach and form tumors in the peritoneal wall. In contrast, wild type TA3/St cells and transfectants carrying vector alone or mutated soluble CD44 that does not bind hyaluronan formed such tumors rapidly and consistently (Table 1)12. Our results imply strongly that, in this system, interaction of the tumor cells with hyaluronan is required for tumor progression at two levels: attachment and growth characteristics. Other investigators have also shown partial inhibition of attachment of human ovarian carcinoma cells to mesentery in vitro and in vivo by antibody to CD44, suggesting that hyaluronan-CD44 interaction is involved. The TA3/St transfectants used above were also injected intravenously into mice and formation of metastatic nodules in the lung was assessed", Very large numbers of nodules formed from wild type, vector-transfected or mutant soluble CD44 (R43A)transfected TA3/St cells but virtually none formed from the soluble CD44
352
Cell surfaces and hyaluronan receptors
transfectants. It was found that all the cell lines rapidly penetrated lung stroma with approximately equal efficiency. However, within 48 hours most of the soluble CD44 transfectants died by apoptosis whereas the control cells continued to grow and form nodules rapidly". Thus disruption of hyaluronan interactions apparently leads to apoptosis of the metastasizing tumor cells. TREATMENT OF TUMOR CELLS WITH BYALURONAN OLIGOMERS OR OVER-EXPRESSION OF SOLUBLE CD44 CAUSES INlDBmON OF ANCHORAGE INDEPENDENT GROWTH It was not clear from the results obtained above whether treatment with hyaluronan oligomers and overexpression of soluble CD44 have direct effects on tumor cell growth or whether their effects are an indirect consequence of other alterations in vivo. Thus we sought additional evidence to discriminate between these two possible explanations. First, the effects on proliferation of treatment with hyaluronan oligomers and of over-expression of soluble CD44 were analyzed in monolayer culture. Addition of hyaluronan oligomers, at concentrations of 10-100 ~/ml, to TA3/St cells in monolayer culture had little effect on their rate of proliferation. Likewise, all of the transfected cell lines grew at approximately the same rates and exhibited similar cell cycle profiles as wild type TA3/St cells". We then examined the effect of treatment with hyaluronan oligomers and over-expression of soluble CD44 on anchorage independent growth in soft agar, a characteristic that distinguishes transformed and tumorigenic from non-transformed and non-tumorigenic cells", Dramatic differences in size and number of colonies formed were observed between the soluble CD44 transfectants and control TA3/St cells (Table 2). The wild type, vector-transfected cells and mutant soluble CD44-transfected cells formed many times more colonies than the soluble CD44 transfectants, and the colonies formed by the control cells were much larger than those few colonies formed by the soluble CD44 transfectants". Addition of 100 ~/ml hyaluronan oligomers to soft agar cultures of T A3/St cells also led to significant inhibition of growth of colonies. Table 2.
Over-expression of soluble CD44 in T A3/St mammary carcinoma cells inhibits anchorage-independent growth in soft agar Number of colonies
CONTROlS: Wild type TA3/St Vector transfectant Soluble CD44 (R43A) transfectant OVER-EXPRESSION OF SOLUBLE CD44: Soluble CD44 transfectant clone #1 Soluble CD44 transfectant clone #2 Soluble CD44 transfectant clone #3
235 (±28) 108 (±21) 448 (±11)
1 (±O) 21 (±4) 8 (+1)
Inhibition of tumor growth
353
CONCLUSIONS In the above studies, we have shown that stable transfection of malignant TA3/St mammary carcinoma cells with cDNA encoding soluble CD44 prevents tumor cell proliferation, invasion and metastasis in vivo and that this inhibition is due to perturbation of hyaluronan interactions. We have also shown that hyaluronan oligomers interfere with tumor growth in vivo, presumably via displacement of endogenous hyaluronan polymer. Finally, perturbation of hyaluronan interactions in these ways has a direct effect on the transformed characteristics of the tumor cells rather than, or in addition to, an indirect effect on other events in vivo. Current work is directed towards determining the biochemical relationship between hyaluronan interactions and anchorage-independent growth. ACKNOWLEDGEMENTS Work from this laboratory was supported by grants from the National Institutes for Health (CA73839 and CA82867) and Anika Therapeutics to B.P.T. and by a US Army Breast Cancer Research Program Fellowship (DAMD 17-96-1-6060) to R.M.P. REFERENCES 1. B.P. Toole. Hyaluronan in morphogenesis and tissue remodeling. www. glycoforum. gr.jp/science/hyaluronan/HA08/ HA08E, 1999. 2. B.P. Toole, Hyaluronan, In: Proteoglycans: Structure, Biology and Molecular Interactions. R.V. Iozzo (ed.), Marcel Dekker, New York, 2000, pp 61-92. 3. M. Brecht, U. Mayer, E. Schlosser & P. Prehm, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis, Biochem. J.,1986, 239, 445450. 4. S.P. Evanko, I.e. Angello & T.N. Wight, Formation of hyaluronan- and versicanrich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells, Arter. Thromb. Vasco Biol. 1999, 19,1004-1013. 5. C.B. Underhill & B.P. Toole, Receptors for hyaluronate on the surface of parent and virus-transformed cell lines-Binding and aggregation studies, Exp, Cell Res.,1981, 131,419-424. 6. T.D. Camenisch, AP. Spicer, T. Brehm-Gibson, I. Biesterfeldt, M.L. Augustine, A Calabro, S. Kubalak, S.E. Klewer, & I.A McDonald, Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme, J. Clin. Invest. 2000,106, 349-360. 7. A. Bartolazzi, R. Peach, A. Aruffo, & I. Stamenkovic, Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development, J. Exp. Med., 1994, 180,53-66. 8. Q. Yu, B.P. Toole, I. Stamenkovic, Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function, J. Exp. Med. 1997, 186, 1985-1996. 9. C. Zeng, B.P. Toole, S.D. Kinney, l.W. Kuo, & I. Stamenkovic, Inhibition of tumor growth in vivo by hyaluronan oligomers, Int. J. Cancer, 1998, 77:396-401. 10. R. Kosaki, K. Watanabe, & Y. Yamaguchi, Overproduction of hyaluronan by expression of the hyaluronan synthase has2 enhances anchorage-independent
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growth and tumorigenicity, Cancer Res., 1999, 59, 1141-1145. 11. N. Itano, T. Sawai, O. Miyaishi, & K. Kimata, Relationship between hyaluronan production and metastatic potential of mouse mammary carcinoma cells, Cancer Res., 1999, 59, 2499-2504. 12. R.M. Peterson, Q. Yu, I. Stamenkovic, I. & B.P. Toole, Perturbation of hyaluronan interactions by soluble CD44 inhibits growth of murine mammary carcinoma cells in ascites, Amer. J. Pathol., 2000, 156,2159-2167. 13. W. Knudson, C. Biswas, X.Q. Li, R.E. Nemec, & B.P. Toole, The role and regulation of tumour-associated hyaluronan, Ciba Found. Symp., 1989, 143, 150159. 14. W. Knudson, Tumor-associated hyaluronan: providing an extracellular matrix that facilitates invasion, Amer. J. Pathol., 1996, 148, 1721-1726. 15. P.K. Auvinen, J.J. Parkkinen, R.T. Johansson, U.M. Agren, R.H. Tammi, M.J. Eskelinen, & V.M. Kosma, Expression of hyaluronan in benign and malignant breast lesions, Int. J. Cancer, 1997, 74, 477-481. 16. K. Ropponen, M. Tammi, J. Parkkinen, M. Eskelinen, R. Tammi, P. Lipponen, U. Agren, E. Alhava E, & V.M. Kosma, Tumor cell-associated hyaluronan as an unfavorable prognostic factor in colorectal cancer, Cancer Res., 1998, 58, 342347. 17. L.P. Setala, M.I. Tammi, R.H. Tammi, M.J. Eskelinen, P.K. Lipponen, U.M. Agren, J. Parkkinen, E.M. Alhava, & V.M. Kosma, Hyaluronan expression in gastric cancer cells is associated with local and nodal spread and reduced survival rate, Br. J. Cancer, 1999, 79, 1113-1138. 18. K. Kimata, Y. Honma, M. Okayama, K. Oguri, M. Hozumi, & S. Suzuki, Increased synthesis of hyaluronic acid by mouse mammary carcinoma cell variants with high metastatic potential, Cancer Res., 1983, 43, 1347-1354. 19. L. Zhang, C.B. Underhill, & L. Chen, Hyaluronan on the surface of tumor cells is correlated with metastatic behavior, Cancer Res., 1995, 55, 428-433. 20. W. Knudson, C. Biswas, & B. Toole, Interactions between human tumor cells and fibroblasts stimulate hyaluronate synthesis, Proc. Nail. Acad. Sci. USA, 1984, 81, 6767-6771. 21. T. Asplund, M.A. Versnel, T.C. Laurent, & P. Heldin, Human mesothelioma cells produce factors that stimulate the production of hyaluronan by mesothelial cells and fibroblasts, Cancer Res., 1993, 53, 388-392. 22. T.K. Yeo, I.A. Nagy, K.T. Yeo, H.F. Dvorak, & B.P. Toole, Increased hyaluronan at sites of attachment to mesentery by CD44-positive mouse ovarian and breast tumor cells, Amer. J. Pathol., 1996, 148, 1733-1740. 23. C.B. Underhill, G. Chi-Rosso, & B.P. Toole, Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40transformed 3T3 cells, J. Bioi. Chem., 1983, 258, 8086-8091. 24. R.E. Nemec, B.P. Toole, & W. Knudson, Hyaluronate binding sites are present on the surface of invasive bladder carcinoma cells, Biochem. Biophys. Res. Commun., 1987, 149, 249-257. 25. T. Strobel, L. Swanson, & S.A. Cannistra, In vivo inhibition of CD44 limits intra-abdominal spread of a human ovarian cancer xenograft in nude mice: a novel role for CD44 in the process of peritoneal implantation, Cancer Res., 1997, 57, 1228-1232. 26. V.H. Freedman, & S. Shin S, Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium, Cell, 1974, 3, 355-359.
NOVEL ENDOTHELIAL HYALURONAN RECEPTORS David G. Jackson', Remko Prevo" Jian Nil & Suneale Banerjl', /MRC Human ImmunoLogy Unit. Weatherall Institute of MoLecuLar Medicine. John Radcliffe HospitaL, Headington. Oxford OX3 9DS UK. 'Human Genome Sciences Inc.. Rockville Pike. MaryLand 20850. USA.
ABSTRACT. The extracellular matrix glycosaminoglycan hyaluronan (HA) is a fundamentally important substrate for the migration of cells during embryonic morphogenesis and during processes such as wound healing and inflammation. The majority of proteins that interact with HA belong to the Link superfamily, whose members contain an HA-binding protein module known as the Link domain. However most Link family members are structural proteins and until recently, only a single cell surface receptor - the CD44 molecule - was known. In this paper we describe the characterisitics of new Link superfamily HA receptors identified from a homology search of the EST database. One of these, termed LYVE-l, is expressed almost exclusively in lymphatic vessel endothelium. Two other candidates, FEL-l and FEL-2 are large multidomain receptors that have yet to be characterized at the protein level but appear to be expressed both in leukocytes and in vascular endothelial cells.
KEYWORDS. Hyaluronan, Link module, EST, CD44, LYVE-l.
INTRODUCTION. The vascular and lymphatic systems are two fundamentally important organs for the maintenance of tissue homeostasis. These anatomically distinct but interconnecting systems provide a conduit for leukocytes involved in immune surveillance as well as a route for the dissemination of tumour cells to distant sites. In the course of these processes, both entry and exit from the vasculature and to a certain extent the lymphatics requires migration across or between the endothelial cells that form a physical barrier enclosing both vessel types. There is now considerable evidence to suggest that transendothelial migration in the vasculature is regulated by adhesive interactions between leukocytes and the glycosaminoglycan hyaluronan - mediated by the CD44 HA receptor. The CD44 glycoprotein is expressed on a variety of cell types and is thought to mediate the extravasation of leukocytes by engaging HA induced on or below the surface of vascular capillary endothelial cells in inflamed tissues 1-4. In addition, CD44 is known to be present on leukocyte populations within the lymph nodes where it is thought to playa role in the migration of recently emigrated peripheral blood lymphocytes through interactions with HA in lymph node reticular fibres 5. In contrast to the vasculature, much less is known about the mechanisms regulating transendothelial migration in the lymphatic system. Indeed the lymphatic system has largely been ignored because of the lack of suitable markers for immunohistochemistry and the difficulty in isolating and culturing primary lymphatic capillary endothelial cell lines 6. Intriguingly, the lymphatic system is the main conduit for transporting tissue HA to its site of degradation in the lymph nodes 7.8. Hence it is quite likely that HA/HAreceptor interactions might also regulate lymphatic transendothelial or intraluminal cell migration. Althogh CD44 is not expressed on lymphatic endothelial cells themsleves, its expression on leukocytes appears to regulate entry to lymphatic capillaries 9.
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Cell surfaces and hyaluronan receptors
The central role in leukocyte migration and HA homeostasis previously ascribed to COM as outlined above has recently been called into question. Two independent reports have demonstrated that disruption of the COM gene in homozygous COM,I, mice results in few if any defects in either the vascular or lymphatic systems 10.11. These results underline the likelihood that receptors other than COM exist and have prompted us to search for new candidate HA receptors within the human genome. In common with the majority of proteins that bind HA (eg. aggrecan, versican, link protein and tsg-6) COM is a member of the so-called Link protein superfamily - defined by the presence of a conserved HA-binding domain known as the Link module 12.13. The Link module is a unit of approximately 100 amino acids that forms a compact fold comprised of two 13 sheets flanked by two short alpha helices and stabilized by two conserved di-sulphide linkages enclosing a central hydrophobic core 14. Our strategy has been to carry out homology searches of the EST databases with the C044 Link sequence to identify new superfamily members encoding likely cell surface polypeptides. Here we review the results of this strategy which has yielded a novel HA receptor LYVE-l 15.16 expressed on lymphatic endothelium and two candidate receptors FEL-l and FEL-2 expressed in leukocytes and vascular endothelial cells.
MATERIALS & METHODS. Hyaluronan and antibodies. High molecular weight hyaluronan (rooster comb) and l-ethyl-3-(3dimethylaminopropyl) carbodiimide (BOAC) were obtained from SIGMA. Biotin-LChydrazide was obtained from Pierce. Rabbit polyclonal antisera to human LYVE-l were generated against LYVE-l Fc fusion protein as previously described 14. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti human IgG for immunohistochemical staining were from Pierce. An ICAM-2 Fc fusion protein was kindly donated by Or. Sue Adams (Molecular Parasitology Group, University of Oxford).
Cloning and expression of full-length LYVE-l and soluble LYVE·l Fe fusion proteins. A full-length human LYVE-l cONA (1017bp) derived originally from an EST identified in a BlastSearch of the TIGR EST database was cloned into the HindIII / Xbal site of the eukaryotic expression vector pRcCMV as described previously 15. The cONA was used to transfect human 293T cells using Lipofectin," For expression in soluble form, a 684bp fragment encoding the N-terminal leader,truncating at Gly232 was amplified from the full-length cONA and the resulting product cloned into HindIIIlBamHl cut IgFc vector for transfection of human 293T cells 15. 48h after transfection, culture supernatants were harvested and the LYVE-IFc fusion protein isolated by chromatography on Protein A-Sepharose eluted with 0.1 M Glycine buffer pH 3.0. Fractions containing Fc fusion protein were neutralized with 0.1 vol 1M Tris-HCI buffer pH 8.0 and verified by SOS-PAGE.
HA-binding assays. Binding of LYVE-l fusion protein to immobilised HA was tested in 96 well ELISA plates (Nunc Maxisorp) as described previously 15. Plates were coated by overnight incubation with Img/ml HA in coating buffer (50 mM sodium bicarbonate, pH 9.3). Wells were blocked for 2 h in PBS 1 % (w/v) BSA, 0.05 % (v/v) Tween 20 and subsequently incubated with purified mouse LYVE-l Fe fusion protein (62.5-1000 ng/ml) in PBS 0.05 % Tween 20 for Ihr at room temperature. Human COM Fe and ICAM-2 Fe fusion proteins were used as positive and negative controls, respectively. After washing (4 times with PBS, once with PBS 0.05 % Tween 20), bound fusion protein was detected with HRP conjugated goat anti-human IgG (1:4000; Pierce) followed by 0phenylenediamine substrate (OPO; SIGMA). Subsequently, absorbance at 490 nm was measured in a BioRad microplate reader. For binding to LYVE-l transfected cells, these
Novel Endothelial Hyaluronan Receptors
357
were incubated (20min, 25°C) with 15~g/rnl FITC-conjugated HA 17 alone or in the presence of 1mg/ml unconjugated HA in PBS, 0.1 % azide, followed by washing (x3) in the same buffer. Cells were then fixed in 2% (w/v) formaldehyde, mounted with fluorescence mounting medium and viewed under a Zeiss Axioskop microscope using epifluorescent illumination.
Immunoperoxidase and immunofluorescent antibody staining. Tissues were fixed in PBS 4% (w/v) paraformaldehyde and embedded in paraffin wax. Prior to staining, sections were de-waxed and re-hydrated by successive incubation in Citroclear" (2 x 5 min) 100 % industrial methylated spirit (IMS,2 x 5 min), 50 % IMS (5 min) and water (5 min). Antigen retrieval was performed by microwave treatment (95100°C, 10 min) in 0.1 M Tris, 2mM EDTA, pH 9.0. Sections were then blocked by incubation in PBS 5% FCS for 5 min and treated with a peroxidase quenching agent (DAKO) for 5min prior to incubation with rabbit polyclonal LYVE-1 (1:400 diluted serum) for 45 min. After washing with PBS, slides were incubated with anti-rabbit Ig peroxidase conjugate (Envision kit, DAKO) for a further 45 min and developed with diaminobenzidine (DAB; DAKO) before counterstaining with hematoxylin. All incubations were performed at room temperature. For immunofluorescent staining, LYVE-1 transfected cells were incubated with polyclonal LYVE-1 Abs in PBS, 10% FCS, 0.1% azide for 30 min prior to washing in PBS and re-incubation with FITC-conjugated goat anti-rabbit Ig (11100). RESULTS & DISCUSSION.
Identification of novel integral-membrane proteins of the Link superfamily. Recent reports have indicated that disruption of the CD44 gene in homozygous CD44'/' knockout mice produces few if any biological consequences, despite evidence that HAprotein interactions are key during embryonic development and in maintaining adult tissue architecture 10.11. These and other considerations led us to search for additional HA receptors in the human genome that might compensate for the loss of CD44 as well as others that might play more tissue-specific roles in HA metabolism. To identify candiate receptors we initiated a search of the Human Genome Sciences / TIGR human EST (Expressed Sequence lag) database for sequences homologous to the CD44 Link domain, based on the assumption that all such receptors are likely to contain copies of this protein module. The search identified a number of ESTs exhibiting >30% similarity to the CD44 Link amino acid sequence, many of which corresponded to known superfamily members, but several of which were clearly novel. Following isolation and sequencing of the full-length cDNAs, three (termed LYVE-1, FEL-1 and FEL-2, see Figure 1) were found to encode potential transmembrane proteins that were considered likely to function as cell surface HA receptors. The first of these, LYVE-1 (LYmphatic Vessel Endothelial HA receptor), encodes a 322 residue polypeptide comprising a 26 residue N-terminalleader, a 212 residue extracellular domain containing an N-terminal Link module, a 21 residue hydrophobic transmembrane domain and a 63 residue cytoplasmic tail respectively (see Fig 2). The FEL-l (Fasciclin / EGF repeat / Link module) cDNA encodes a 2597 residue multidomain receptor comprising a 25 residue N-terminal leader, a 2477 residue ectodomain containing 3.5 tandem repeats of a -130 residue sequence similar to Drosophila Fasciclin I, the human beta IgH3 protein and the bone adhesion protein OSF-2, interspersed with EGF repeats and a single C-terminal Link module. This is followed by a 24 residue transmembrane domain and a 71 residue cytoplasmic tail. The FEL-2 sequence is currently incomplete but appears to encode a protein similar to FEL-l. This present manuscript describes characterization of LYVE-1 followed by a short description of our current knowledge of FEL-1 andFEL-2. Alignment of the human LYVE-1 amino acid sequence with that of human CD44 (Figure 2) illustrates the degree of homology between the two receptors which have an
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overall similarity of 41% . The Link module in LYVE-l is marked by four central cysteine residues (Cys 61, 85, 106 and 128), whose spacing ( Cl - ~3- C2 - X20- C3 - X 21 - C4 ) is almost identical in CD44, and whose intervening sequences share 57% similarity (see Figure 2). These cysteine residues are conserved in all members of the Link superfamily where they form essential di-sulphide bridges that stabilize the Link module fold, a structure comprising two a helices and two anti-parallel 13 sheets encapsulating a large hydrophobic core 13.14. In addition, the LYVE-l Link module in common with that of C044 is flanked by two further cysteine residues (Cys 36 and Cys 139, Fig 2a ) that are not found in other Link superfamily members. Another potential similarity between LYVE-I and C044 is the presence of a tract of basic residues downstream of the Link module (RI9,RKKI98 in LYVE-l and R.soDGTRYVKK158 in CD44). Since the additional disulphides (involving C 38 and C 129) and the downstream basicresidues 150158 are believed to contribute to an extended HA binding domain in the C044- molecule 18.19, it is tempting to speculate that similar elements (ie. residues peripheral to the Link module) may also form an extended HA-binding site in LYVE-l 15,16. We are currently exploring this possibility in studies using a series of soluble LYVE-l Fc truncation mutants. Comparison of the Link domains of human LYVE-l and CD44 for the conservation of
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functionally significant amino acids suggests only a small number of these are present. For example, of the nine key residues identified by site-directed mutagenesis 19.20 as critical for HA-binding within the human CD44 Link domain - K38 + 68, R 41+ 78, Y42, 79 + 105, and NlOO + 101, only three of these, K38, Y79 and NlOO are conserved in human LYVE-l (residues 46, 87 and 109 respectively, Fig 2a). Interestingly, these latter residues are conserved in the murine orthologue 16 and appear to participate in HA-binding as assessed by preliminary site-directed mutagenesis studies (data not shown). Nevertheless it is clear that the majority of residues involved in the predicted LYVE-l-carbohydrate interaction are different to those in the CD44 molecule. We are currently mapping the HA-binding domain using a model of the LYVE1 Link module to target appropriate residues for mutagenesis. Other notable features of the LYVE-l sequence are the presence of a seventh cysteine residue (C201) within the extracellular domain that is predicted to form a free thiol, and a conserved cysteine residue (C257) within the hydrophobic transmembrane anchor. In the case of CD44 an equivalent cysteine has been shown to influence HA-binding 21. Down stream of the Link module however, the region corresponding to the membrane- proximal domain of LYVE-l shows little similarity to CD44, with the exception of a moderately high proportion of serine and threonine residues (37% within residues 145-216). The L YVE·l molecule is an HA receptor expressed in lymphatic endothelium. We have expressed human LYVE-l
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full-length form and as a soluble Ig Fe fusion protein in transiently transfected 293T cells in order to characterize HA-binding. As shown in Figure 3 for the human orthologue, cells transfected with full-length cDNA express high levels of LYVE-l at the cell surface (as assessed by staining with LYVE-l Abs) and bind high molecular weight FlTC-conjugated HA. No such binding is observed with mock (vector) transfected 293T cells or transfectants incubated with a SOX-fold molar excess of unconjugated HA (not shown). Furthermore. soluble LYVE-l Fe fusion protein binds immobilized HA and soluble biotinylated HA in a similar concentration dependent manner to CD44 Fc suggesting the two receptors may have similar apparent binding constants for (high-molecular weight) HA15. Preliminary experiments (in collaboration with Dr. Markku Tammi, University of Kuopio, Finland) using HA oligosaccharides (4-22mer) as competitors in these assays suggest that the minimal HA-binding unit for soluble LYVE-l may be similar to the 68mer reported for CD44 in some conditions. These studies will also test whether the same holds true for LYVE-l at the cell surface. Lastly, binding of neither human nor murine LYVE-l to immobilized HA is blocked by other glycosaminoglycans such as chondroitin sulphate or heparan sulphate (ref 15 and data not shown). Thus LYVE-l is more specific for HA than either CD44 which binds chondroitin sulphate, or the liver endothelial cell HA receptor HARE which binds both chondroitin sulphate and heparin 22. It is not yet clear if the HA-binding properties of LYVE-l - like those of CD44 - are subject to regulation by factors such as glycosylation/de-glycosylation of the extracellular domain, by phosphorylation/de-phosphorylation of the cytoplasmic tailor by clustering within the lipid bilayer (reviewed in ref 23). We are currently exploring these possibilities. Comprehensive analyses ofLYVE-l expression at the RNA level using Northern blotting
Novel Endothelial Hyaluronan Receptors
361
Figure 4. Immunohistochemical staining for LYVE-l in human lymph node. Paraffin sections of human tonsil were stained for LYVE-l using LYVE-l Abs and an enhanced immunoperoxidase detection method as described in Materials and Methods. The Abs stain the endothelium lining both large and small lymphatic sinuses as shown by arrows. The lymphatic endothelial cells have no underlying basement membrane and show characteristic bulging of nuclei towards the vessel lumen.
and RT-PCR indicated that LYVE-l had a much more restricted expression pattern than CD44 15. This has been fully confirmed in studies using antibodies both to human and murine LYVE-l molecules during immunohistochemical analyses of normal 15,16 and neoplastic tissues. These have confirmed that expression of LYVE-I is almost entirely confined to the endothelia of lymphatic capillaries and collecting vessels (see ego Figure 4) in the majority of tissues where these are found. Furthermore, these studies have revealed that LYVE-l is expressed in endothelial cells of mouse lymphangiomas and have demonstrated the usefulness of LYVE-l Abs for studying tumour lymphatics in a number of different human cancers and in animal models of tumour metastasis 6,24-26 LYVE-l can mediate HA·internalization. We have investigated the capacity of LYVE-I to mediate the uptake of highmolecular weight FITC-HA by transfected 293T cells. These studies 16 indicate that LYVE-I transfectants internalize HA with a linear time-course at 3t>C, reaching a plateau after 3h. The mechanism of LYVE-I mediated HA-uptake, like that for CD44-mediated uptake is currently unclear. In common with CD44, the LYVE-I molecule does not contain motifs for endocytosis via the clathrin-mediated pathway (ie. the clathrin and AP-2 clathrin adapter binding motifs NPXY and YXX<j> respectively, see ref. 27). Indeed LYVEI also lacks the di-hydrophobic LV motif present within the CD44 cytoplasmic tail that mediates basolateral sorting in polarized epithelial cells 28. Ongoing experiments in our laboratory are currently directed towards characterizing the apparently unusual endocytic mechanism by which LYVE-I mediates the uptake of hyaluronan. What is the physiological role of LYVE·l ? The lymphatic system forms an extensive network of vessels, facilitating the migration
362
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Figure 5. Possible roles for LYVE-l in lymphatic vessel trafficking. The cartoon depicts a small lymphatic capillary underlying the dermis in skin. LYVE-l on the luminal and abluminal faces of lymphatic vessel endothelial cells is speculated to function as a receptor for the transport of HA and I or the migration of leukocytes or metastasizing tumour cells (expressing CD44) into the vessel lumen.
of leukocytes from the peripheral tissues to the lymph nodes for the purpose of immune surveillance. Moreover, the lymphatic system is known to be a major conduit for the transport and degradation of hyaluronan 7,8. During this process newly turned-over HA originating from the tissues enters the afferent lymphatics, is degraded in lymph nodes and exits to the vasculature via the thoracic duct, for uptake and terminal hydrolysis in the liver 8. Currently it is not known which receptor(s) are involved in HA-uptake and degradation in lymph node or in binding I transport of HA in lymph capillaries and vessels. Clearly, LYVE-l is a candidate for one or more of these processes. Given the finding that LYVE-l can mediate HA-uptake in vitro, and that LYVE-l appears to be expressed on both the luminal and ablurninal faces of the endothelium 15,24, it is tempting to speculate that LYVE-l plays a role in the transcytosis of tissue HA into the lumena of draining lymphatic vessels in vivo. Alternatively, LYVE-I through its capacity to bind HA might provide an adhesive surface for migration of cells bearing CD44 or other HA-receptors into the lymphatics. Examples might include dendritic cells such as epidermal Langerhans cells, macrophages or metastasizing tumour cells (see Figure 5). The availability of appropriate experimental models should allow these hypotheses to be tested in the near future.
FEL-l and FEL-2. As outlined above, EST database searches identified two further candidate HA receptors PEL-I and FEL-2 in addition to LYVE-I (see Figure 1). These encode large multidomain receptors containing a single Link module at the C-terminal end of the predicted extra- cellular domain - an unusual location for this module. The sequences of the PEL-I and 2 Link modules (not shown) correspond to those recently reported for two protein fragments termed WF-HABP and BM-HABP respectively 29 and are more similar to TSG-6 Link 30 than either LYVE-I or CD44 Link. In addition both the FEL-l and FEL-2 ectodomains contain ROD putative integrin-binding motifs and multiple EGF repeats together with tandem repeats of a protein module contained in the human TOF(3-
Novel Endothelial Hyaluronan Receptors
363
inducible matrix protein beta-Igh3 31 and the Drosophila cell surface Fasciclin I protein 32 - implicated in cell adhesion. Northern blot hybridization with cDNA probes for FEL-l and FEL-2 indicate both receptors are subject to extensive alternative splicing and are expressed in a variety of cells including both leukocytes and vascular endothelia. These properties raise the exciting possibility that FEL-l and/or FEL-2 may have some functional overlap with the CD44 molecule. However experiments with truncated ectodomain Fc fusion constructs in HAbinding ELISAs so far indicate the FEL Link modules are not constitutively functional. We
CONCLUSIONS. Searching the EST database for proteins containing the HA-binding Link module has yielded three novel receptors, LYVE-l, FEL-l and FEL-2. LYVE-l is expressed almost exclusively in the endothelium lining lymphatic vessels where it may play a role in the transport of HA and the migration of immigrant leukocytes. LYVE-l is also a powerful new marker for lymphatics that is currently being used to study tumour-associated lymphangiogenesis in human cancers and transgenic mouse models of neoplasia. FEL-l and FEL-2 are large multidomain integral membrane molecules likely to play roles in cell and extracellular matrix adhesion. Preliminary data suggest they are expressed in both leukocytes and endothelial cells and that they are not constitutively active as HAbinding molecules. Research in our laboratory is currently directed towards understanding the physiological functions of each of these molecules.
ACKNOWLEDGEMENTS. This work was funded by grants from the MRC, Cancer Research Campaign and The A.I.C.R. to Dr David Jackson. Dr.Suneale Banerji is a post-Doctoral Research associate and Remko Prevo a D.Phil. student in the MRC Human Immunology Unit, Oxford.
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P. J. Neame & F. P. Barry, 'The Link proteins', Experientia , 1993, 49, 393-402. A. J. Day, 'The structure and regulation of HA-binding proteins.', Biochem. Soc. Trans. , 1999, 27, 115-121. D. Kohda, C. J. Morton, A. A. Parkar, H. Hatanaka, F. M. Inagaki, 1. D. Campbell & A. J. Day, 'Solution structure of the link module: a hyaluronan binding domain involved in extracellular matrix stability and cell migration.', Cell, 1996,86,767-775. S. Banerji, J. Ni, S.-X. Wang, S. Clasper, J. Su, R. Tammi, M. Jones & D. G. Jackson, 'LYVE-l, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.', J. Cell Bioi. ,1999, 144, 789-801. Prevo, R., Banerji, S., Ferguson, D.J.P., Clasper, S. & Jackson, D.G. 'Mouse LYVE-l is an endocytic receptor for hyaluronan in lymphatic endothelium.' J. Bioi. Chern. 2001,276, 19420-19430. A. N. de Belder & K. O. Wik, 'Preparation and properties of fluorescein-labelled hyaluronate.', Carbohydrate Res. ,1975,44,251-257. S. Banerji, A. J. Day, J. D. Kahmann & D. G. Jackson, 'Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli.', Protein Exp. Purification. , 1998, 14, 371-381. R. J. Peach, D. Hollenbaugh,!. Stamenkovic & A. Aruffo, 'Identification of hyaluronic acid binding sites in the extracellular domain of CD44.', J. Cell Bioi. , 1993, 122, 257-264. J. Bajorath, B. Greenfield, S. B. Munro, A. J. Day & A. Aruffo, 'Identification of CD44 residues important for hyaluronan binding and delineation of the binding site.', J. Bioi. Chern. ,1998, 273, 338-343. D. Liu & M. S. Sy, 'A cysteine residue located in the transmembrane domain of CD44 is important in binding of CD44 to hyaluronic acid.', J. Exp. Med. , 1996, 183, 1987-1994. C. T. McGary, R. H. Raja & P. H. Weigel, 'Endocytosis of hyaluronic acid by rat liver endothelial cells.', Biochem. J. , 1989, 257, 875-884. P. W. Kincade, Z. Zheng, S. Katoh & L. Hanson, 'The importance of cellular environment to function of the CD44 matrix receptor.', Current Opin. Cell Bioi. , 1997, 9, 635-642. Mandriota, S. Jussila, L., Jeltsch, M., Compagni, A., Baetens, D., Prevo, R., Banerji, S., Huarte, J., Montesano, R., Jackson, D., Orci, L., Alitalo, K., Christofori, G. & Pepper, M.S. 'Vascular endothelial growth factor-Cvmediated Iymphangiogenesis promotes tumour metastasis.' EMBO. J. 2001, 20, 672-682. Stacker, S.A. Caesar, c., Baldwin, M.E., Thornton, G.E., Williams, R.A., Prevo, R., Jackson, D.G., Mishikawa, S., .Kubo, H., & Achen, M.G. 'VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.' Nature Med. 2001, 7, 186-191. Skobe, M, Hawighorst, T., Jackson, D., Prevo, R., Janes, L., Velasco, P., Riccardi, L., Alitalo, K., Claffey, K. & Detrnar, M. 'Induction of tumour Iymphangiogenesis by VEGF-C promotes breast cancer metastasis.' Nature Med. 2001,7, 192-198. J. S. Bonifacino & E. C. Dell'Angelica, 'Molecular bases for the recognition of tyrosine-based sorting signals.', J. Cell Bioi. ,1999, 145, 923-926. H. Sheikh & C. M. Isacke, 'A di-hydrophobic Leu-Val motif regulates the basolateral localization of CD44 in polarized Madin-Darby canine kidney epithelial cells.', J. Bioi. Chern. , 1996, 271., 12185-12190. E. Tsifrina, N. M. Ananyeva, G. Hastings & G. Liau, 'Identification and characterization of three cDNAs that encode putative novel hyaluronan-binding proteins, including an endothelial cell-specific hyaluronan receptor.', Am. J. Pathol. 1999, 155, 1625-1633,. T. H. Lee, H. G. Wisniewski & J. Vilcek, 'A novel secretory tumor necrosis factorinducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to CD44', J Cell Bioi, 1992, 116, 545-557. J. Skonier, M. Neubauer, L. Madisen, K. Bennett, G. D. Plowman & A. F. Purchio, 'eDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factorbeta.', DNA Cell Bioi. ,1992, 11,511-522. K. Zinn, L. McAllister & C. S. Goodman, 'Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila.', Cell, 1998, 53, 577-587.
MUTUALLY EXCLUSIVE DISTRIBUTION OF LINK PROTEIN AND HYALURONECTIN DURING CARTILAGE MORPHOGENESIS: A ROLE FOR FREE HYALURONAN P. Rooney'>, N. Girard 2, B. Delpeejr', .J. Ponting'' & S. Kumar'' JDepartment
ofBasic Dental Science, Dental School, University of Wales College ofMedicine, Heath Park, Cardiff, CF14 4XY, UK.
2Laboratoire d'Oncologie Moleculaire, Centre Henri-Becquerel, Rauen, France. 3Department
ofPathological Science, University ofManchester, Oxford Road, Manchester, MI3 9PT,
UK
ABSTRACT
Cartilage morphogenesis is a pre-requisite for skeletal development and maintenance and involves a combination of cell division, cell hypertrophy and extracellular matrix secretion. Matrix secretion can account for up to 57% of the increase in volume of a cartilage long bone rudiment, however, the shape is regulated via the differential structure of the perichondrium where a multilayered, "tight" structure is found around the central hypertrophic zone and an overlapping, "loose" structure, which merges with the mesenchyme, is seen around the rounded zones. Hyaluronan (HA) is found within cartilage extracellular matrix as a backbone for aggregating proteoglycans (PG) and as a free glycosarninoglycan within a pericellular coat. In this study, the distribution ofPG-bound-HA has been determined in developing rat cartilage by a combination of antibodies to the HA binding proteins, link protein (LP) and hyaluronectin (HN) and PG-free-HA via enzyme-linked, sheep-brain HN as a probe. A mutually exclusive pattern of staining was observed for LP and HN with intense LP staining within the cartilage matrix but not in the perichondrium or the surrounding mesenchyme. LP was most intense in the hypertrophic zone but little staining was observed pericellularly around hypertrophic cells. In contrast, HN and PG-free-HA was detected within the pericellular region, even during cartilage resorption, and was also present in both the perichondrium, particularly surrounding rounded zones and within the adjacent mesenchyme. At later stages, HA staining was detected at presumptive joint regions, separating the rudiments. We suggest that the LP antibody detects HA bound to the PO aggrecan, whereas the HN antibody and probe detects aggrecan-free-HA. PG-bound-HA is constrained within the extracellular matrix whereas free-HA would be capable of utilising hydrodynamic forces to expand and produce space which enlarging rounded zones can occupy. The observation that overlapping perichondrial cells are rich in free-HA indicates that these cells may regulate cartilage morphogenesis by synthesising free-HA, increasing the width ofthe zone and allowing expansion to occur. KEYWORDS
Cartilage development, hyaluronan binding proteins, perichondrium, free-hyaluronan
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INTRODUCTION Cartilage morphogenesis appears to follow a similar pattern in every vertebrate species. The vertebrate limb develops as a outgrowth from the lateral body wall and consists of an ectodermal pouch filled with underlying mesoderm. I Chondrogenesis begins as a condensation of mesenchymal cells which, in the case of long bones is usually a sausage shape.' Cells in the centre of the condensation secrete cartilage extracellular matrix and the cells begin to be pushed apart, however, at this stage, the early cartilage rudiment consists of only two cell types arranged into three cellular zones along the proximal-distal axis - rounded, flattened and rounded.' Flattened chondrocytes rapidly hypertrophy and this, accompanied by the accumulation of extracellular matrix causes cells at the periphery of the central region to become constrained and aligned perpendicularly. At the light microscope level, this early perichondrium can be seen to be directly derived from what were flattened chondrocytes, e.g., in the developing chick ulna, at the stage where cartilage extracellular matrix secretion begins, the sausage shaped rudiment consists of 25 flattened cells across its short axis but when cell hypertrophy begins, the hypertrophic zone contains 17 cells across its short axis and the presumptive perichondrium is four At this stage the rudiment consists of three types of cells thick on each side.3,4 chondrocytes arranged into five identifiable zones - rounded, flattened, hypertrophic, flattened and rounded. As the rudiment continues to develop, the perichondrium expands to cover more than the central hypertrophic zone and eventually surrounds the entire rudiment. The perichondrium is a variable structure which is multilayered and "tight" around the hypertrophic zone with many cell-cell contacts, overlapping around the flattened zone and "loose" with little or no cell-cell contact at the rounded zone." From this stage onwards, the rudiment increases in length more than width with radial expansion occurring primarily in the rounded and flattened zones, we have suggested that this shape is regulated bi the structure of the perichondrium, a process called "directed dilation" by Wolpert ..5 Although the perichondrium can influence the shape of the cartilage rudiment, it can only act on the intrinsic factors and pressures produced by the cartilage. Cartilage morphogenesis is associated with cartilage growth and involves cell proliferation, cell hypertrophy and extracellular matrix secretion. Extracellular matrix secretion is by far the largest factor involved in cartilage growth, accounting for 57% in developing chick cartilage. In contrast, cell proliferation accounts for 6% and cell hypertrophy, where the volume of the cell increases eight-fold, accounts for 37%.3 The components of cartilage extracellular matrix have been extensively studied, 6,7 the major macromolecules being collagens (primarily types II, VI, IX, X, XI and other minor collagens) and proteoglycans (PO - primarily aggrecan but also some nonaggregating PO) together with growth factors and glycoproteins. Large aggregating PO consist of many PO monomers, each bound to a hyaluronan (RA) backbone via the HAbinding protein, link protein (LP). However, cartilage extracellular matrix also contains the RA as a non-PG bound free glycosarninoclycan, located within the pericellular coat surrounding chondrocytes." HA is a non-sulphated glycosarninoglycan composed of a repeating disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosarnine which has long been linked with morphogenesis and differentiation of several cell types,"!' where it is thought to playa role either by its hydrodynamic properties or by cell-matrix interactions. 12.13
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In this study, we have investigated the distribution of HA in developing rat cartilage with a view to determining if HA plays a role in the morphogenesis of the tissue. Our specific aim has been to localise and evaluate the role of PG-free-HA using a combination of antibodies to the HA-binding proteins LP and hyaluronectin (HN - a glycoprotein isolated from sheep and human brain and thought to be related to the PG versican I4•17) and an enzyme-linked HN probe designed to bind PG_free_HA. 18 . The results indicate that LP and HN distribution are mutually exclusive and that PG-free-HA plays a role both in the increase in volume observed during cell hypertrophy and also in the radial expansion of rounded cell zones. PG-free-HA is synthesised by cells of the perichondriwn, once again implicating this tissue in morphogenesis. MATERIALS AND METHODS Tissues Embryos were removed from pregnant Sprague-Dawley rats at days 16, 17 and 18 of gestation. Individual fore and hind limbs were removed and the entire limbs were fixed and processed for wax histology. 5 urn sections were cut through the whole limb along the long axis. Sections were stained with Haernatoxylin and Eosin for visualisation and serial sections were utilised for immunohistochemistry and enzyme-linked histochemistry. Immunohistochemistry Following de-waxing, sections were pre-treated with H202 and exposed to the primary antibodies, polyclonal anti-mouse LP antibody, 8-A-4, (kindly donated by Professor B. Caterson, Cardiff University, Wales) or porclonal anti-rabbit human HN purified by immunoadsorption as previously described. 1 Peroxidase anti-mouse and anti-rabbit antibodies were used as secondary antibodies and visualisation was observed following DAB treatment. Specific y globulins absorbed out on insolubilised HN were used as a control. Enzyme-linked histochemistry HA localisation was performed by an affinity immunological technique with alkaline phosphatase - linked sheep brain HN as previously described." Enzymatic activity was detected with Fast Red in the presence of Naphtol -As-Mx as substrate and levamisole as endogenous phosphatase inhibitor. Nuclear counterstaining was performed with haernatoxylin. Controls were obtained by two pre-incubations with Streptomyces hyaluronidase, 20 TRU/ml for 2 hours at 37°C in a humidified chamber. RESULTS The results from this study show that the distribution of LP and lIN are mutually exclusive in developing cartilage (Figs. 1 and 3). At all stages of development, LP is restricted to cartilaginous regions with negative staining in the perichondrium and mesenchyme (Fig. 1). The most intense staining was observed in the hypertrophic cell zone but little LP was detected within the pericellular region (not shown). In contrast, HN was detected within both the perichondrium and mesenchyme adjacent to the
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Figure 1. Link protein restricted to cartilaginous regions. 16 day embryo. DAB/peroxidase staining.
Figure 3. HN staining of Figure 1 showing opposite distribution as LP. DAB/peroxidase staining.
Figure 2. HN staining around expanding cartilage zone and extending into the perichondrium. DAB/peroxidase staining.
Figure 4. HN staining in presumptive joint region of 18 day embryo. DAB/peroxidase staining.
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rounded cell zone (Figs. 2 and 3). Each limb contained rudiments of varying developmental age along the proximal-distal axis and consequently the distribution of HN at differing developmental stages could be assessed. In the most distal digits (i.e. the youngest stages), HN was found along the entire length of the perichondrium but was still most intense at the rounded zone perichondrium (Fig. 2). At later stages, HN was restricted to rounded zone perichondrium (Fig. 3) and in developmentally older rudiments, HN was only observed in the presumptive joint regions separating the rudiments (Figure 4). Within the hypertrophic cell zone, intense HN staining was observed at the periphery of hypertrophic cell lacunae and also in the immediate pericellular region, extracellular matrix outside the pericellular region was negative for HN (Fig. 5). When an enzyme-linked HN probe was used to detect PG-free-HA, it was found to co-localise with HN but was more extensive. PG-free-HA was present in the perichondrium and adjacent mesenchyme of embryos of all stages but was also present throughout the extracellular matrix of hypertrophic cells (Figs. 6, 7). Pre-treatment of the sections with hyaluronidase completely removed any positive staining indicating that HA was being detected by the probe (Fig. 8). DISCUSSION Due to the glycosaminoglycan nature of HA, direct immunolocalisation is difficult, consequently, in this report, HA localisation was assessed by the localisation of HA binding proteins and the idea that binding proteins indicate the presence ofHA. LP is a HA binding protein which is known to bind the aggregating PG aggrecan to its HA backbone, therefore, the restriction of LP to cartilage extracellular matrix is not unexpected (Fig. 1). lIN is also a HA binding protein which is thought to bind to HA at the same binding site and in the same manner as LP, 10 the binding regions both having two link modules arranged in tandem array;" consequently the ability of the HN antibody to detect HN suggests that the HA it is detecting is not bound to aggrecan. HN has a molecular weight of 40 - 70 kDa, it is considered to be a glycoprotein whose primary structure contains sequences which are identical with portions of the N-terminal domain of the PG versican and HN is often considered to be a breakdown product of versican splice variant Vl. 16•19 However, although versican is present in prechondrogenic tissues within developing limbs, it is believed to be absent in cartilage where it is replaced by aggrecan." The pericellular coat of chondrocytes is rich in aggrecan-free-HA, which is believed to be tethered at the cell surface by the HA receptor CD44. 8,20 Our data indicate that some of the aggrecan-free-HA is as a free glycosaminoglycan but some of it is bound to HN (Fig. 5). In addition, hypertrophic lacunae are also known to be rich in HA,21 our results suggest that once again, this HA is part free glycosarninoglycan and part bound to HN. Since hypertrophic chondrocytes increase in volume by up to eightfold, these observations suggest that HN-bound HA may still be able to swell in size and aid the increase in volume. The perichondrial region surrounding rounded cartilage zones demonstrated some of the most intense staining for both HN antibody and HN probe (Figs. 2, 3 and 7). We have already demonstrated that the perichondrium is this region consists of overlapping cells which offer little resistance to swelling pressure," We would suggest that perichondrial cells in these regions synthesise PG-free-HA and this HA swells allowing the rounded zone to expand. Once a tight perichondrium develops around the central
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Figure 5. HN staining within the hypertrophic zone of 17 day embryo. Intense staining is observed at the periphery of the lacunae and immediately pericellularly. DAB/peroxidase staining.
Figure 7. HN-detected free HA at perichondrium of 18 day rounded cartilage zone. Alkaline phosphatase detection.
Figure 6. HN-detected free-HA distribution in 17 day cartilage. Intense staining is observed in the hypertrophic region and in the perichondrium surrounding expanding cartilage. Alkaline phosphatase detection.
Figure 8. Hyaluronidase pretreatment totally removes any positive HN-detected free HA from 18 day embryos.
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hypertrophic zone, the cartilage rudiment increases in radial width mostly at the rounded cell zones.' Increase in width around the central zone is due mainly to appositional deposition of bone layers.' The presence of PG-free-HA around hypertrophic chondrocytes may also aid in the process of endochondral ossification where the hypertrophic cartilage zone is invaded by blood vessels, resorbed and replaced by bone. Breakdown products of HA, 3 - 10 disacccharide units in length are known to be angiogenic and stimulate the expression of transcription factors for metalloproteinases.J' It is conceivable that PG-free-HA may be more susceptible to degradation and thus allow/stimulate cartilage vascularisation.
CONCLUSION HA within developing rat cartilage appears to be present in three states, i) linked to the PG aggrecan via LP, ii) linked to the glycoprotein HN, iii) as a free glycosarninoglycan. lIN-linked HA and free HA are located in regions where expansion in chondrocyte or cartilage zone volume is observed. lIN-linked and freeHA utilise the ability of HA to swell via hydrodynamic forces to increase in volume and thus influence morphogenesis.
REFERENCES
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J. P. Iannotti, S. Goldstein, J. Kuhn, L. Lipiello, F. S. Kaplan & D. 1. Zaleske, The formation and growth of skeletal tissues, In: Orthopaedic Basic Science: Biology and Biomechanics of the Musculoskeletal System, Amer. Acad. of Orthopaedic Surgeons, J. A Buckwalter (ed.), 2000, pp 77 - 109. C. W. Archer, P. Rooney & L. Wolpert, The early growth and morphogenesis of limb cartilage. Prog. in Clin., Biol. Res., 1983, 11OA, 267-278. P. Rooney, C. W. Archer, & L. Wolpert, Morphogenesis of cartilaginous long bone rudiments, In: The role ofextracellular matrix in development, R. L. Trelsted (ed.), Alan R. Liss, New York, 1984, pp 305-322. P. Rooney & C. W. Archer, The development of the perichondrium in the avian ulna,1. Anatomy, 1992, 181,393-401. L. Wolpert, Cartilage morphogenesis, In: Cell Behaviour, R. Bellairs, A S. G. Curtis, & G. Dunn, 1982, Cambridge University Press, pp 359-372. A Serafini-Fracasini & J. W. Smith, The Structure and Biochemistry ofCartilage, Churchill-Livingstone, Edinburgh, 1974, pps 354. M.E. Grant, A P. L. Kwan, G. P. Bates, 1. T. Thomas & 1. McClure, The structure and synthesis of cartilage collagens, In: The Control of Tissue Damage, AM. Glauert (ed.), Elsevier Science Publishing Co. Inc., 1988, pp 3-28. C. B. Knudson & B. P. Toole, Changes in the pericellular matrix during differentiation of limb bud mesenchyme, Dev. Biol., 1985, 112:308-318. B. P. Toole, Proteoglycans and hyaluronan in morphogenesis and differentiation In: Cell Biology ofthe Extracellular Matrix, 2nd edn., E. Hay (ed.), Plenum Press, New York, 1991, pp 305-341. B.P. Toole, Hyaluronan, In: Proteoglycans: Structure, Biology and Molecular Interactions, R. N. Iozzo (ed.), Marcel Decker Inc, 2000, pp 61-92. P. Rooney & S. Kumar, Inverse relationship between collagens and hyaluronan in development and angiogenesis, Differentiation, 1993, 3-9.
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12. M. Brecht, U. Mayer, E. Schlosser & P. Prehm, Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochemical J.. 1986, 239: 445450 13. E. Ruoslahti & Y. Yamaguchi, Proteoglycans as modulators of growth factor activities, Cell, 1991, 64: 867-869. 14. B. Delpech & C. Halavent, Characterisation and purification from human brain of a hyaluronic acid-binding glycoprotein, hyaluronectin. 1. Neurochem 1981, 36: 855-859. 15. B. Delpech, Immunochemical characterisation ofthe hyaluronic acidhyaluronectin interaction, 1. Neurochem., 1982,38: 978-984. 16. B. Delpech, C. Maingonnat, A. Delpech, P. Maes, N. Girard, P. Bertrand, Characterisation of a hyaluronic acid-binding protein from sheep brain: Comparison with human brain hyaluronectin. Int. J. Biochem., 1991,23: 329-337. 17. D. R Zimmermann, Versican, In: Proteoglycans: Structure, Biology and Molecular Interactions, R.N. Iozzo (ed.), Marcel Decker Inc, 2000, pp327-342. 18. B. Delpech, Enzyme-linked hyaluronectin: A unique reagent for hyaluronan assay and tissue location and for hyaluronidase activity detection, Anal. Biochem., 1995, 229: 35-41. 19. R. V. Iozzo, Matrix proteoglycans: from molecular design to cellular function, Ann. Rev. Biochem., 1998, 67: 609-652. 20. W. Knudson, D. 1. Aguiar, Q. Hua & C. B. Knudson, CD44-anchored hyaluronan-rich pericellular matrices: An ultrastructural and biochemical analysis, Exp. Cell Res., 1996, 228:216-228. 21. P. Pavasant, T. Shizari, & C. B. Underhill, Hyaluronan contributes to the enlargement of hypertrophic lacunae in the growth plate, 1. Cell Sci., 1996, 109:327-334.
MAINTENANCE OF CARTILAGE EXTRACELLULAR MATRIX: THE PARTICIPATION OF HAS-2 AND CD44 Warren Knudson', Yoshihiro Nishida 2 & Richard S. Peterson! J
J
Department ofBiochemistry. Rush Medical College. Rush-Presbyterian-St. Luke's Medical Center, Chicago. IL 60612, USA
Department ofOrthopedic Surgery. Nagoya University School ofMedicine. Nagoya 466-8550, Japan
ABSTRACT
Cartilage is a tissue whose function is highly dependent on the maintenance of its extracellular matrix. Hyaluronan (HA) provides a unique role in cartilage, serving to sequester and retain proteoglycan within the tissue. A fraction of the HA-proteoglycan rich matrix remains anchored to the chondrocytes via interactions with the HA receptor CD44. We have also determined that chondrocytes utilize the same CD44 receptor to internalize HA, thus providing one mechanism for HA catabolism in cartilage. To better understand these processes, the expression of HA synthases (HAS) and CD44 was examined. Using quantitative-competitive RT-PCR we determined that human as well as bovine articular chondrocytes express primarily HAS-2, substantially less HAS-3 and no HAS-I. Antisense oligonucleotides directed against HAS-2 inhibited chondrocyte HA production in proportion to the level of inhibition of HAS-2 mRNA. It was therefore concluded that HAS-2 is the predominant gene involved in HA synthesis by articular chondrocytes. Incubation of chondrocytes with an anabolic cellular mediator, osteogenic protein-l , an agent that stimulates collagen type II and aggrecan production, resulted in a substantial increase in HAS-2 and CD44 mRNA copy numbers and, a pronounced accumulation of pericellular HA. No change in HAS-3 mRNA was observed. Interestingly, treatment of chondrocytes with the catabolic cytokine IL-la also resulted in an increase in HAS-2 and CD44. However in this case, less accumulation of HA within the pericellular matrix was observed. Visualization of intracellular HA suggested that the increased synthesis of HA due to IL-l was offset by enhanced CD44-mediated HA endocytosis. Thus, chondrocytes maintain their extracellular matrix composition, in part, by coordinating the expression of two components critically necessary for retention of proteoglycan, namely HAS-2 and CD44. KEYWORDS
Hyaluronan, HAS, CD44, antisense, chondrocytes, cartilage INTRODUCTION
The abundant extracellular matrix of cartilage is composed predominately of collagen type II and aggrccan proteoglycan (PG) 1. Aggrecan can be visualized within slices of normal human articular cartilage by staining of the tissue with the basic dye safranin O. Healthy human articular cartilage exhibits intense red staining throughout all the layers of cartilage with the exception of the uppermost layer of cells and matrix as shown in Figure
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l A, However, on some occasions we have received samples of normal human donor cartilages in which there is extensive loss of PG-with loss progressing in gradient fashion from the superficial to deeper layers of the tissue (Figure IB). Further, the loss of PG staining appears to originate within the pericellular matrix, the matrix closest in proximity to the cells of cartilage, the chondrocytes. Does this represent early osteoarthritis? The answer is as yet unknown. The tissue was derived from a donor with no known history of arthritis or joint pain and, by gross examination, the surface properties of the tissue were normal. However, osteoarthritis is characterized by an extensive loss ofPG. Further, the degenerative disease is thought to be due to inherent changes in the metabolism of the resident chondrocytes, a process termed chondrocytic chondrolysis 2. Therefore, the changes observed in Figure IB represent what one might expect to observe during the early stages of osteoarthritis. In other words, the cell-associated matrix of cartilage chondrocytes is where metabolism is most active and where changes will first be witnessed. How are PGs retained within cartilage? It has long been known that the principal PG of cartilage, aggrecan, forms strong, link protein stabilized interactions with filaments of another glycosaminoglycan, hyaluronan (HA) 3-6. Chondrocytic chondrolysis is thought to involve proteolytic cleavage of the core protein ofaggrecan and release of the PG from the tissue 2. We have determined that PG bound to filaments of HA are, in turn, bound to the plasma membrane of chondrocytes via the interaction of the HA with the HA receptor CD44 7-10. Chondrocytes removed from the tissue and grown in culture exhibit a prominent cell-associated matrix that we visualize using a particle exclusion assay (Figure 2A). This cell-associated matrix is rich in aggrecan yet sensitive to treatment with dilute Streptomyces hyaluronidase. The gel-like coat is retained even during centrifugation of the cells and as the assay suggests, able to resist the intrusion of small particles (in this case, glutaraldehyde-fixed red blood cells). Nonetheless, these cell-associated matrices can be readily released by the addition of HA hexasaccharides, oligosaccharides that compete for the binding of HA to the HA receptor, CD44. Further, when the coats are released by hyaluronidase treatment, the matrices are re-synthesized and re-assembled within 24 hours. However, re-assembly can be inhibited by the presence of HA hexasaccharides, nonsulfated chondroitin, or anti-CD44 antibodies 8-10. This, and other experiments have led us to develop a model for cartilage depicted in Figure 2B. Part of our long term goal is to determine how retention of HA and PG within the cell-associated matrix of chondrocytes participates in overall maintenance of healthy cartilage tissue. Although these cell associated coats can be displaced from chondrocytes via the addition of HA hexasaccharides, this does not mean that all of the HA and PG is displaced from the surface. Using 3H-acetate labeling, following hyaluronidase treatment, 3H-HA re-accumulates at the cell surface reaching semi-saturation between 4-6 hours. 3H-HA appears in the media after a short lag phase. So, of this cell surface bound HA, how much is displaceable? In separate studies by C. Knudson, >90% of the HA at the 2 and 4 hour time points was not displaceable 11. However, between 4 to 6 hours there was a switch. Now, at the 6 hour time point, 80% of the 3H-HA is displaceable. Similar percentages are observed for the retention ofPG. Thus with chondrocytes, a portion of the cell-associated matrix is represented by non-displaceable HA and PG. However, this residual matrix is not sufficient to support the assembly of a cell-associated matrix that can exclude particles as in Figure 2A. Most of the residual non-displaced PG of these chondrocytes can be removed by treatment with Streptomyces hyaluronidase and thus likely represents aggrecan bound to non-displaceable HA. What is this non-displaceable HA? We have always
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assumed that this represented synthase-bound HA (Figure 2B) and, that somewhere between 4-6 hours there is a transfer from the HA synthase to CD44. In some ceIls, coats are established that are non-displaceable with HA oligosaccharides and thus presumably represent a matrix assembled on a synthase-bound HA scaffold 12. Transmission electron micrographs of HA hexasaccharide-treated chondrocytcs stained with ruthidium hexamine trichloride to detect PGs reveal small patches of PG granuoles tethered to the ceIl surface 10. It is intriguing to speculate that these granuoles represent the PG bound to synthasebound HA as illustrated in Figure 2B. This cartoon figure illustrates that two components, namely CD44 and HA synthase, participate in the retention of HA and PG. As will be discussed below, these two proteins also share responsibility for the overaIl metabolism ofHA. Up until the last few years the nature and identity of the HA synthase (HAS) has been a mystery. Now it is known that a single protein is responsible for the synthesis ofHA 13. It is also now known that eukaryotic cells exhibit at least three separate but highly homologous HAS genes, each present on three different chromosomes 14. The HAS genes have been designated has-I, has-2 and has-3 13. HAS-I, HAS-2 and HAS-3 are purported to exhibit different elongation rates and synthesize HA of differing sizes 15. The HAS responsible for the synthesis of HA in cartilage was unknown and as such, represents the primary focus of this discussion. Three approaches were taken to determine the primary HAS gene used by chondrocytes. As anti-HAS antibodies were not readily available, the first approach was to document HAS mRNA expression levels. A second approach was to transfect chondrocytes or cartilage tissue slices with antisense phosphorothiate oligonucleotides directed against HAS mRNA and determine the effect on the synthesis of HA as well as other HA-mediated functions (e.g., periceIlular matrix assembly). The third approach was to examine changes in HAS mRNA expression that occur in response to ceIlular mediators/growth factors known to alter HA production. From these studies we determined that HAS-2 is likely the major HAS used by chondrocytes 16-19.
Figure 1. Human articular cartilage. Panels depict safranin 0 stained sections of normal articular cartilage derived from the talocrural joint of two human donors.
Figure 2. HAlPG cell-associated matrix of cartilage chondrocytes. Panel A depicts the cell-associated matrix as visualized by the use of a particle exclusion assay. Panel B is an illustration of the molecular components likely responsible for the matrix assembly shown in panel A.
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METHODS Tissue acquisition and culture Human articular cartilage was obtained from the talocrural ankle joint of normal human donors. Tissue was obtained within 24 hours of death through the Regional Organ Bank of Illinois (ROBI), and all donors were documented as having no known history of joint disease. Donors ranged in age from 33 to 58 years. Bovine articular cartilage was obtained from the metacarpophalangeal joints from 18-month-old steers obtained from a local slaughterhouse. Full thickness slices of articular cartilage were dissected under aseptic conditions and subjected to sequential pronase and collagenase digestion to liberate chondrocytes from the tissue 20. Isolated chondrocytes were cultured for 5 days in alginate beads with daily medium changes 20. In some experiments, the chondrocytes were treated with a cellular mediator for up to 14 days prior to release from the alginate using 55 mM sodium citrate 21. In other experiments, after 5 days of recovery, the chondrocytes were released from the alginate beads and used as monolayer cultures prior to analysis. For culture of intact cartilage tissue, full thickness slices (~I xlOxI0 mm) of human or bovine articular cartilage were cultured directly in 1.0 ml of DMEM-4.5 containing 10% fetal bovine serum (FBS). Typically, the slices were used in an experiment following I day of culture for recovery. At the end of the experiment, the slices were either (I) frozen in liquid nitrogen, ground to a powder and extracted for total RNA using the Trizol reagents 18, (2) resuspended in 100 mM Nf4Acetate, 0.0005% phenol red, pH 7.0 at 100 mg tissue wet weight per ml and incubated with 125 ug/ml Protease K for fluorophore-assisted carbohydrate electrophoresis (FACE) analysis 18 or; (3) embedded in Histo PrepTM freezing medium for cryostat sectioning (8.0 urn) for histochemical / immunohistochemical staining for PG, HA and CD44. For HA staining, the tissue sections were pretreated with 2 units of chondroitinase ABC (at pH 8.0) for 2 hour at 37°C for unmasking. Following this treatment the tissues were incubated with 2.0 ug/ml of a biotinylated HA binding protein (HABP) probe for 2 hour at room temperature followed by streptavidin peroxidase and color reagents for the detection of HA. In other experiments, human cartilage tissue was dissected into two layers (superficial/uppermiddle and lower-middle/deep layers) 22. Each of the dissected cartilage slices was cultured separately in the presence or absence of IL-I a. prior to analysis. RESULTS AND DISCUSSION Detection and quantification of chondrocyte HAS-2 and HAS-3 mRNA A quantitative competitive RT-PCR approach was taken to characterize HAS expression by human and bovine articular chondrocytes 16, Using this approach it was determined that human as well as bovine articular chondrocytes express both HAS-2 and HAS-3. However, the level of HAS-2 expression is -40 times greater than HAS-3 16. Neither bovine nor human chondrocytes expressed any detectable levels of HAS-I even at greater than 35 cycles ofRT-PCR. The same primers were able to amplify HAS-I mRNA derived from human dermal fibroblasts (positive control). Nonetheless, the possibility exists that HAS-I mRNA is expressed but at very low levels or only expressed under certain pathological conditions. However, given that HAS-2 and HAS-3 are readily detectable, it is not likely that a low abundance of HAS-I, if it exists, is physiologically
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relevant. The copy number for HAS-2 and HAS-3 varied slightly in human cartilage samples from donor to donor but the ratio of HAS-2 to HAS-3 remained quite constant. In all cases, HAS-2 represented the predominant enzyme. When total RNA was isolated [rom intact human cartilage slices, similar values for HAS-2 versus HAS-3 mRNA expression were observed 18-19. This 25-50 fold difference in copy numbers suggests that HAS-2 is the predominate HAS message expressed by articular chondrocytes.
The effect of HAS-2 antisense oligonucleotides on chondrocyte HA production Given the predominance of HAS-2 mRNA in chondrocytes, phosphorothioate oligonucleotides (16-mers) were prepared directed against sequence that included the translation start site of human HAS-2 mRNA 16. Antisense, sense and reverse sequence oligonucleotides were prepared and transfected into human chondrocytes using lipofectamine as a carrier. The protocol used for cultured chondrocytes used one transfection pulse of oligonucleotide for 5 hours under serum-free conditions followed by the addition of fresh medium containing fetal bovine serum. Total RNA was isolated from the cells at various times following transfection and characterized by quantitative competitive RT-PCR. HAS-2 mRNA was inhibited by -30% 8 hours, subsequently reaching a maximum inhibition of 60% by 24 hours as compared to cells treated with the sense control oligonucleotide. Forty to 48 hours following transfection, the levels of HAS-2 began to return to pre-transfection levels. The HAS-2 antisense or control oligonucleotides had no effect on the mRNA expression of HAS-3, aggrecan or GAPDH. Thus, the effect of the HAS-2 antisense oligonucleotide appeared to be specific but what about HAS-2 protein? HAS-2 antibodies were not available to determine changes in protein levels. So instead, changes in the levels ofHA accumulation were examined. One function of chondrocyte HA discussed above is to participate in cell-associated matrix assembly. At 24 hours post-transfection with HAS-2 antisense oligonucleotides, no coats were observed surrounding the chondrocytes. On the other hand, chondrocytes transfected with reverse and sense HAS-2 oligonucleotides displayed coats similar to control, untreated cells. At 48 hours post-transfection, small coats surrounding the chondrocytes began to reappear. To detect HA production directly, the transfected chondrocytes were incubated with a biotinylated HABP probe followed by peroxidase conjugated streptavidin and color reagents. Intense staining for HA was observed surrounding the sense HAS-2 treated chondrocytes with staining extending well beyond the limits of the plasma membrane. HABP staining intensity and extent were significantly reduced in the antisense treated cells. Quantification by image analysis suggested an -60% reduction in HABP staining intensity of antisense treated cells as compared to control cells. Thus, the change in HA staining matched the level of HAS-2 mRNA inhibition. Antisense or sense oligonucleotide-treated cells were also labeled with 35S-S04 and incorporation into PG determined. The total level of 35S_PG was equivalent in the sense and antisense treated chondrocytes. This was expected as no effect on aggrecan mRNA due to HAS-2 antisense oligonucleotide was observed. However, the antisense transfected cells exhibited a 30% reduction in the amount of cell-associated PG with a concomitant increase in mediumlocalized PG as compared to control, sense treated cells. This again confirms that the amount of PG retained by chondrocytes is proportional to the amount of cell-associated HA. In pilot studies we determined using rhodamine-tagged phosphorothioate oligonucleotides that these nucleotide probes could readily penetrate into intact cartilage tissue slices and accumulate within chondrocyte lacunae (likely within chondrocytes).
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We also found that this penetration and accumulation was facilitated by lipofectamine, just as it was for transfection of cultured chondrocytes. Therefore, human cartilage slices were incubated for 7 days with HAS-2 sense or antisense oligonucleotides (with daily medium changes) and then evaluated for accumulation of HA within the tissue. As with the cultured chondrocytes the antisense-treated slices displayed a substantially lower expression ofHA, throughout the matrix, as compared to sense oligonucleotide slices. The effect of cellular mediators I cytokines on HAS and CD44 expression. The bone morphogenic protein-7 (BMP-7) also known as osteogenic protein-I (OP-I) has been shown to stimulate aggrecan synthesis in cultured bovine and porcine articular chondrocytes as well as cartilage explants 23-24. Flechtenmacher et aI., also demonstrated stimulation of collagen type II as well as aggrecan in human articular chondrocytes 25. Since matrix retention also depends on HA and the HA receptors, the effect of BMP-7 on HAS and CD44 was examined. Treatment of bovine or human articular chondrocytes with BMP-7, resulted in a substantial increase in the exclusion size of the pericellular matrix 17-19. Many of the matrices grew to over one-cell-diameter-representing some of the largest coats we have visualized to date (larger than coats present on rat chondrosarcoma cells). Staining of these cells for HA revealed an increase in staining intensity and extent. Flow cytometry analysis demonstrated increases in CD44 as well 17. So, given that HA accumulation was dramatically increased under these conditions, what about HAS mRNA expression? Using the same quantitative RT-PCR approach described above, BMP-7 treatment resulted in 1.5 fold increases in HAS-2 and CD44 mRNA increasing to 2.0 fold by 24 hours. The ratio of HAS-2 copy numbers appears to peak at 3 days of BMP-7 treatment, ~3.7 times the level expressed by control cultures. Aggrecan mRNA copy numbers showed a significant upregulation at 3 days of treatment (2.3 fold difference) increasing to 4.2 fold by 7 days of treatment. No changes in HAS-3 or GAPDH were noted over the same time period. Slices of bovine articular cartilage were also treated BMP-7. Biotinylated HABP staining for HA revealed that control untreated cartilage slices were positive for HA, with definitive staining throughout the extracellular matrix. However, in the presence ofBMP7, the staining for HA was substantially increased, elevated both within the matrix and surrounding the chondrocytes. When the tissue slices were analyzed for mRNA expression, a 3.4 fold increase in copy number for HAS-2 was observed. As with the isolated chondrocytes, there was no change in HAS-3 copy number 17. The inflammatory cytokine IL-I has been shown to enhance chondrocyte catabolism inhibit aggrecan PG expression 27 yet induce an increase in CD44 expression 21. The effect on chondrocyte HA synthesis was unknown although D'Souza et al., had reported increases in 3H-HA in bovine articular chondrocytes due to IL-I 28. Following only two days of treatment with IL-Ia., human articular chondrocytes displayed 2.5 fold drop in copy number for aggrecan but no change in either GAPDH or HAS-3 mRNA. CD44 copy number as expected, was 4 fold higher than control, untreated cells. Interestingly, HAS-2 mRNA copy numbers were also -3 fold higher than control chondrocytes 18. Again, changes in HA expression are reflected by changes in HAS-2 mRNA with little effect on HAS-3. 26,
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Human articular cartilage slices were also treated with IL-la. Safranin 0 staining for PG was lost from the superficial to upper middle layers of the tissue. Given the increase in HAS-2 rnRNA in the chondrocytes, it was expected that HABP staining for HA would be enhanced in the IL-l treated slices. However, little to no change in HA was observed in the middle / deep layers. Further, the superficial/upper middle layers appeared devoid of HA staining. To determine if this was merely a staining artifact, the cartilage slices were carefully dissected (divided into -upper 1/3 and deeper 2/3), cultured separated with or without IL-la and then analyzed using FACE analysis. As with the staining, there was little to no change in the middle / deep layers but a substantial loss of HA disaccharide recovered from the superficial/upper middle layers of the IL-l treated tissue. To investigate whether the expression of HAS was different in the tissue or tissue layers as compared to isolated cells, RNA was extracted from the dissected tissue layers. HAS-2 mRNA copy number was still elevated 3 fold in both the upper and lower layers of cartilage. CD44 copy number was also elevated in the tissue, 5 fold in the upper layers and 3 fold in the deeper. One possible mechanism to explain this data was that HA synthesis is upregulated but, the increased CD44 induced by IL-l was now participating more in HA catabolism 21-29 rather than matrix retention. To investigate this aspect, we went back to the human articular chondrocyte culture system. In the IL-l treated cells, the size of the particle exclusion pericellular matrix was visibly reduced as compared to control cells. This however could be due to decreased PG expression (observed by mRNA as well as alcian blue staining) and/or a change in CD44 function. HABP staining for HA was more intense on the IL-l treated chondrocytes. To investigate HA internalization, the chondrocytes were trypsinized extensively, permeabilizcd and then stained using the biotinylated HABP probe. Both control and treated chondrocytes displayed positive staining, but staining was sequestered into round inclusions (i.e., vesicles). In the IL-l treated chondrocytes, the staining for intracellular HA was intense-substantially more than in the control untreated cells. Thus, it would appear that, by whatever mechanism, IL-I treated chondrocytes have an increased capacity to internalize HA. CD44-mediated HA internalization
The mechanism for the accumulation of endogenous intracellular HA at present is unknown. Does this represent HA synthesized from an intracellular site? Possibly, but not likely given the localization of HAS enzymes. Control experiments rule out the expression of intracellular endogenous biotin-containing proteins. Does this represent HA internalized via CD44? This, is at present also unknown. However, the appearance of intracellular HA within perinuclear vesicles looks identical to exogenous fluorescein- or 3H-labeled HA that we find is internalized by chondrocytes via a CD44-dependent mechanism 21, 29. The internalization of fluorescein-H'A or 3H-HA can be blocked by addition of unlabeled HA, HA oligosaccharides, or pre-incubation of the chondrocytes with anti-CD44 monoclonal antibodies. Fluorescein-labeled dextran was not internalized ruling out fluid phase pinocytosis for internalization ofHA. Internalized 3H-HA could be isolated in two pools, as 3H-counts voided on a Sepharose CL-2B column and as 3I-I-counts included on a Sephadex G-50 column. We speculated that these pools represent internalized HA within endosomes and within lysosomes. Further, the generation of small fragments of 3H-HA could blocked by the addition of the lysosomotropic agent chloroquine. Thus, exogenous, labeled HA can be bound to the cell surface of chondrocytes via CD44, internalized into
326
Aspects ofhyaluronan injoints
the cells and degraded to small fragments. We have also shown that IL-l treated chondrocytes, that exhibit a 5-fold increase in CD44 expression, accumulate 3-times as much fluorescein-labeled HA during a 24 hour incubation as compared to control untreated cells. All these data validates the potential that increased CD44-mediated internalization and catabolism of HA could be responsible for the lack of HA accumulation observed in IL-l treated cartilage slices. If anabolic agents such as BMP-7 increase CD44 expression and catabolic mediators increase CD44 expression, how do chondrocytes regulate thefunction ofCD44? We have begun to address this issue by a variety of approaches. We have shown previously that the transfection of COS-7 cells with human CD44 containing overexpression constructs led a capacity to assemble chondrocyte-like pericellular matrices in the presence of exogenous purified aggrecan and HA 9. In new preliminary work, we have found that these same CD44-transfected COS-7 cells have the capacity to bind and internalize fluorescein-labeled HA. This has allowed us to address whether interactions of different regions within the intracellular tail domain of chondrocytes are responsible for HA matrix retention and assembly versus HA internalization. Some intracellular domain truncation mutants of CD44 result in no coats, no fluorescein-HA binding and consequently, no fluorescein-HA internalization. However, we have found one truncation mutant, CD44H~54, in which 54 of the 70 amino acids ofthe CD44 intracellular domain have been deleted, has the capacity to bind fluorescein-HA to the cell surface (similar to control CD44Hwt-transfected cells) but, lacks the capacity to internalize the HA. This suggests that chondrocytes may use a similar mechanism that includes differential interactions with the proximal versus distal portions of the CD44 cytoplasmic tail, to regulate the function of various functions. The nature of these interactions are, at present, speculative. The interactions may include selective differential binding to different actin-binding-proteins or different interactions of CD44 with itself. In summary, our work suggests that two proteins in particular, HAS-2 and CD44, control much of the metabolism, retention and function of HA that occurs within cartilage tissues. Acknowledgements: Collaboration with the laboratory of Cheryl B. Knudson, Ph.D. as well as Allan Valdellon, M.D. of the Regional Organ Bank of Illinois and his staff; are gratefully acknowledged. Special thanks to Susan Chubinskaya, Ph.D., co-director of the in situ hybridization/histochemistry core of the Department of Biochemistry at Rush for use of the safranin 0 stained human donor tissue shown in Figure IB. This work was supported in part by NIH grants and ROI-AR43384 and P50-AR39239 as well as a grant from the National Arthritis Foundation.
REFERENCES 1. 2. 3.
W. Knudson & K. E. Kuettner, In: Primer on the Rheumatic Diseases, u" ed., R. L. Wortmann, ed., Arthritis Foundation, Atlanta, 1997,33-38. K. E. Kuettner, In: Rheumatology, J. H. Klippel and P. A. Dieppe, eds., Mosby-Year Book Europe Limited, St. Louis, MO, 1994, 6.1-6.16. D. Heinegard & V. C. Hascall, 'Aggregation of cartilage proteoglycans. III. characteristics of the proteins isolated from trypsin digests of aggregates', J Bio!. Chem., 1974, 249, 4250-4256.
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1. A. Buckwalter & L. C. Rosenberg, 'Electron micrographic studies of cartilage proteoglycans', J. Biol. Chem., 1982, 257, 9830-9839. 5. V. C. Hascall, T. R. Oegema, M. Brown & A. I. Caplan, 'Isolation and characterization ofproteoglycans from chick limb chondrocytes grown in vitro', J. Bioi. Chem., 1976, 251,3511-3519. 6. J. H. Kimura, T. E. Hardingham, V. C. Hascall & M. Solursh, 'Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma', J. Bioi. Chem., 1979,254,2600-2609. 7. W. Knudson & C. B. Knudson, 'Assembly ofa chondrocyte-like pericellular matrix on non- chondrogenic cells', J. Cell Sci., 1991, 99, 227-235. 8. C. B. Knudson, 'Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix', J. Cell BioI., 1993, 120,825-834. 9. W. Knudson, E. Bartnik & C. B. Knudson, 'Assembly of pericellular matrices by COS-7 cells transfected with CD44 homing receptor genes', Proc. Nat!. Acad. Sci. USA, 1993, 90, 4003-4007. 10. W. Knudson, D. J. Aguiar, Q. Hua & C. B. Knudson, 'CD44-anchored hyaluronan-rich pericellular matrices: An ultrastructural and biochemical analysis', Exp. Cell Res., 1996,228,216-228. 11. C. B. Knudson, In: The Chemistry, biology and medical applications of hyaluronan and its derivatives, T. C. Laurent, ed., Portland Press, London, 1998,216-228. 12. P. Heldin & H. Pertoft, 'Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells', Exp. Cell Res., 1993, 208, 422-429. 13. P. H. Weigel, V. C. Hascall & M. Tammi, 'Hyaluronan synthases', J. Bioi. Chem., 1997,272,13997-14000. 14. A. P. Spicer, M. F. Seldin, A. S. Olsen, N. Brown, D. E. Wells, N. A. Doggett, N. Itano, K. Kimata, J. Inazawa & J. A. McDonald, 'Chromosomal localization of the human and mouse hyaluronan synthase genes', Genomics, 1997,41,493-497. 15. N. Itano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M. Hamaguchi, Y. Yoshida, Y. Ohnuki, S. Miyauchi, A. P. Spicer, J. A. McDonald & K. Kimada, 'Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties', J. Bioi. Chem., 1999,274,25085-25092. 16. Y. Nishida, C. B. Knudson, J. J. Nietfeld, A. Margulis & W. Knudson, 'Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly', J. Biol. Chem., 1999,274,21893-21899. 17. Y. Nishida, C. B. Knudson, W. Eger, K. E. Kuettner & W. Knudson, 'Osteogenic protein-l stimulates cell-associated matrix assembly by normal human articular chondrocytes', Arthritis Rheum., 2000, 43, 206-214. 18. Y. Nishida, A. L. D'Souza, J. M. A. Thonar & W. Knudson, 'IL-la stimulates hyaluronan metabolism in human articular cartilage', Arthritis Rheum., 2000, 43, 1315-1326. 19. Y. Nishida, C. B. Knudson, K. E. Kuettner & W. Knudson, 'Osteogenic protein-l promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures', Osteoarthritis Cartilage, 2000, 8, 127-136. 20. H. J. Hauselmann, M. B. Aydelotte, B. L. Schumacher, K. E. Kuettner, S. H. Gitelis & E. J.-M. A. Thonar, 'Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads', Matrix, 1992, 12, 130-136. 21. G. Chow, C. B. Knudson, G. Homandberg & W. Knudson, 'Increased CD44 expression in bovine articular chondrocytes by catabolic cellular mediators', J. Biol. Chem., 1995, 270, 27734-27741.
4.
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22. M. B. Aydelotte, B. L. Schumacher & K. E. Kuettner, In: Articular Cartilage and Osteoarthritis, K. E. Kuettner, R. Schleyerbach, J. G. Peyron and V. C. Hascall, eds., Raven Press, New York, 1992,237-250. 23. P. Chen, S. Vukicevic, T. K. Sampath & F. P. Luyten, 'Osteogenic protein-I promotes growth and maturation of chick sternal chondrocytes in serum-free cultures', J. Cell. Sci, 1995, 108, 105-114. 24. S. A. Lietman, M. Yanagishita, T. K. Sampath & A. H. Reddi, 'Stimulation of proteoglycan synthesis in explants of porcine articular cartilage by recombinant osteogenic protein-I (bone morphogenetic protein-Z)', J Bone and Joint Surg, 1997, 79-A, 1132-1137. 25. J. Flechtenmacher, K. Huch, E. I. M. A. Thonar, I. Mollenhauer, S. R. Davies, T. M. Schmid, W. Puhl, T. K. Sampath, M. B. Aydelotte & K. E. Kuettner, 'Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes', Arthritis Rheum., 1996,39,478-488. 26. J. A. Tyler, S. Bolis, J. T. Dingle & J. F. S. Middleson, In: Articular Cartilage and Osteoarthritis, K. E. Kuettner, R. Schleyerbach, I. G. Peyron and V. C. Hascall, eds., Raven Press, New York, 1992,251-264. 27. M. B. Aydelotte, R. X. Raiss, R. Schleyerbach & K. E. Kuettner, 'Effects of Interleukin-l on metabolism of proteoglycans by cultured bovine articular chondrocytes', Trans. Ortho. Res. Soc., 1988, 13,247. 28. A. L. D'Souza, K. Masuda, L. Otten, S. Momohara, L. Wang & E.-I. M. A. Thonar, 'Effects of IL-l a on the metabolism of hyaluronan in different compartments of the matrix formed by adult articular chondrocytes in vitro', Trans. Ortho. Res. Soc., 1997, 22,470. 29. Q. Hua, C. B. Knudson & W. Knudson, 'Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis', J. Cell Sci., 1993, 106, 365375.
AN INSIGHT INTO CELLULAR SIGNALLING MEDIATED BY HYALURONAN BINDING PROTEIN (HABPl) T. B. Deb, M. Majumdar, A. Bharadwaj, B.K. Jha & K. Datta' School a/Environmental Sciences. Jawaharlal Nehru University. New Delhi-1 10067. India
ABSTRACT We have reported the characterization of a cell surface glycoprotein of 34 kDa on SDS-PAGE, having specific affinity for hyaluronan and this protein has been termed as HABPI by GDB (Ac. No. 9786126). The role of HABPI in cell adhesion and tumor invasion has also been confirmed. In continuation, the gene encoding hyaluronanbinding protein from human fibroblast was isolated and its localization on human chromosome 17p12-p13 has been reported. Sequence analysis shows the identity of HABPI with other proteins P-32, a protein co-purifying with splicing factor SF2; and gCIqR, the receptor for the globular head of C1q, indicating its multifunctional nature. Several reports suggest differential localization of this protein in various cell types. To confirm its role in signalling, the enhanced phosphorylation of HABPI is being reported in mitogen activated cells and in sperms induced with progesterone and Calyculin A, the acrosome reaction and capacitation inducers. Another interesting observation is oligomerization of HABPI, which enhances its affinity for hyaluronan, highlighting its regulatory role in hyaluronan mediated signalling. Hexasaccharide of hyaluronan is the minimum chain length required for interaction with HABPI. Attempts are being further made to address the intricate mechanism by which this protein is phosphorylated and to understand how HABPI phosphorylation is related to the signalling pathway and if there occurs any nuclear translocation after phosphorylation. INTRODUCTION Hyaluronan, the ubiquitous glycosaminoglycan present in extracellular matrix and pericellular matrixes is involved in structural organization of extracellular matrix and its level is regulated during rapid tissue proliferation and regeneration'. To establish the mode of cellular interaction, a number of ECM and cell surface hyaluronan binding proteins have been identified and grouped as the family of "hyaladherins'", since they share the common hyaluronan-binding motif. In our laboratory, we have been working on a novel member ofhyaladherin family, named HABPI 3 . We confmned its role in cell adhesion & tumor invasion" and sperm maturation & motility':", This protein is ubiquitously present in different cell types and is associated with diverse cellular signalling, as evident by the inhibition of HA-binding to lymphocytes and HA mediated lymphocyte aggregation by pretreatment of cells with anti-HABPI antibodies. Its role in sperm-oocyte interaction has also been established, since the blocking of sperm surface HABPI by its antibody inhibits zona binding'. Its role in cellular signalling is established by the observation on enhanced phosphorylation at serine/threonine residues of HABPI and increased IP3 and PLC-y formation in transformed and HA supplemented cells", which is inhibited by pretreatment of the cells with antibodies against HABPI 8. Enhanced phosphorylation of HABPI in HA supplemented motile sperm is also reported by our group", To further
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Cell surfaces and hyaluronan receptors
analyze the role of this protein in cellular functions, the cDNA encoding HABPI from human skin fibroblast was cloned and sequenced. The presence ofHA-binding motif was confirmed and subsequently, the recombinant protein has been shown to bind to HA9• The gene encoding this protein has been shown to localized on human chromosome 17pI2-p13 lO• Computer search analysis of the sequence encoding HABPI revealed its multifunctional characteristics11, since it was found to be identical with SF2/P-32, a protein copurified with splicing factor SF212 and gClqR, the receptor of complement subcomponent". Sequence analysis further supports the role of this protein in cellular signalling as it has five casein kinase II (CKII) phosphorylation sites, and one extracellular signal regulated kinase (ERK) site. Protein phosphorylation plays a major role in molecular signalling event in cellular proliferation in which phosphorylation by CKII is also involved". Rapidly transient increased activity of cytoplasmic CKII was reported by the addition of serum to cultured cells followed by a change in the properties of growth regulating proteins including oncogenes and transcription factors". Thus, in order to correlate phosphorylation of HABPI with cellular signalling, we made an attempt to investigate the in vivo phosphorylation of HABPI in sperms, treated with either progesterone or Calyculin A, the acrosome reaction and capacitation inducers respectively. Simultaneously we intend to examine whether HABPI acts as substrate for CKII in vitro. MATERIALS & METHODS Materials
Chemicals used in the study were purchased from Sigma Chemicals Co., S1. Louis, USA, unless otherwise mentioned. [y_32 p] ATP was purchased from BARC, India. Recombinant casein kinase II was purchased from Boehringer Mannheim, Germany. Carbobind plates were purchased from Corning Costar, Netherlands. HAhexasaccharide was a kind gift from Dr. B. P.Toole. Purification of recombinant 34 kDa HABPI
The E. coli BL21 (DE3) was transformed with expression vector construct containing the mature HABPIIP-32 cDNA insert and protein expression was induced by the addition of IPTG. The overexpressed protein was purified by HA-Sepharose-4B chromatography as described earlier" Differential affinity of HA-oligosaccharides with HABPI
The various oligosaccharides at different concentrations were covalently linked with carbobind costar plates according to the supplier's instruction. After coating, the solution was removed and the wells were rinsed with PBS containing 0.05% Tween-20 and blocked with 5% non-fat milk at room temperature. The plates were incubated with 100 III of biotinylated HABPI (10 ug/ml) at room temperature for I hour and probed with Extravidin-HRP and detected with ABTS. Optical density was measured at 405 run. Immunoprecipitation of hyaluronan-binding protein
e
I x 106 intact spermatozoa were incubated with I mCi of 2 p], in phosphate free Biggers-Whitten-Whittingham medium for 45 min at 37°C with and without the exogenous substances, Calyculin A, orthovanadate and progesterone. After labeling,
Cellular signalling
367
the sperm pellets were rinsed with ice-cold PBS and solubilized with radio-immuno precipitation (RIPA) buffer. The lysates were centrifuged at 15,000 x g for 3 min at 4°C and the supernatant was incubated with 1.5 mg of swollen protein A-Sepharose-4B conjugated to anti-HABPI antibodies overnight at 4°C with continuous rocking. Precipitates were washed thoroughly, three times with lysis buffer, twice with 0.15M NaCl and twice with 0.1% SDS, boiled in 1 x Laemmli sample buffer and resolved on 10% SDS-PAGE. For visualizing the labeled protein, the gel was stained with CBB, dried and analysed on a phosphoimage analyzer (Bio-Rad, USA). In vitro liver kinase assay
Liver extract was prepared by homogenizing the tissue in sucrose and then centrifuged and the extract was used as a source of protein kinases. Phosphorylation of recombinant HABPI/P-32 was carried out with the liver extract. The reaction mixture (60 fJ.l) contained 20 mM HEPES, pH 7.4; 3 mM MnCh, 5 fJ.Ci [y_32 p ] ATP, 50 fJ.M ATP and 5 ul of liver kinase (containing 5 ug total protein) and the reaction was carried at 37°C for 30 minutes. The reaction was stopped by the addition of Laemmli sample buffer, boiled, resolved on 4-20% gradient SDS-PAGE and analysed by autoradiography. In vitro casein kinase II assay
Casein kinase II in vitro kinase assay was performed according to the supplier kit protocol (Boehringer Mannheim). The reaction assay (50 ul) included 50 mM TrisHCI, pH 6.9; 130 mM KCI, 10 mM MgCh, 4.8 mM dithiothreitol, 5 fJ.Ci [y_32 p ] ATP (specific activity: 3000 Ci/mmole) and different amount of recombinant HABPI/P-32 in the presence ofCKII stimulator (spermidine) or inhibitor (heparin). The reaction was initiated with the addition of I fJ.I of diluted recombinant CKII (corresponding to 0.1 mU) and the reaction was performed at 37°C for 30 min. The reaction was terminated by 10% TCA (final concentration) and proteins were precipitated with 40 ug of carrier protein BSA. Pellet was washed with 5% TCA and ethanol.diethyl ether (1:1), solubilized in SDS-PAGE sample buffer and analyzed in 12.5% SDS-PAGE. For dephosphorylation of proteins, the dried pellet after ethanol:ether (1:1) wash was solubilized in a reaction mixture containing 50 mM Tris-HCI, pH 7.5; 50 mM MgCh and incubated with 7.3 units of alkaline phosphatase at 37°C for 15 min. Reaction was stopped by Laemmli buffer and analyzed on a 12.5% SDS-PAGE. The in vitro CKII assay was also done using tissue purified CKII enzyme. The reaction was performed in the same way as done with recombinant CKII (described above). Tissue purified CKII was used as kinase (I :20 dilution) and HA (5 ug/assay) was used in one ofthe reactions.
RESULTS Comparative affinity of HABPI to HA-hexasaccharide and HA-polymer
In continuation of our earlier studies, we report here that HABPI exhibits almost similar affinity towards HA-hexasaccharide as that shown for HA- polymer (Fig. 1). However, it does not interact with HA disaccharide suggesting a requirement for a critical chain length for HA-HABPI interaction.
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Hyperphosphorylation of HABPI with protein phosphorylation modulators To see the phosphorylation status ofHABPl, rat sperms were metabolically labeled with ezp] orthophosphate under stimulation with progesterone and Calyculin A and subsequently, immunoprecipitated with anti-HABPl antibody and protein Avsepharose 0.9 , . . . - - - - - - - - - - - - - - - - ,
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under the stimulation of Calyculin A (lane 1), orthovanadate (lane 2) and progesterone (lane 3) as compared to the control (lane 5). Pre-immune serum (PIS) control is seen in lane 4. This observation suggests the hyperphosphorylation of HABPI in the presence of progesterone (tyrosine kinase stimulator) and Calyculin A (serine/threonine phosphatase inhibitor), acrosome reaction and capacitation inducers, respectively and Na3V04 (tyrosine phosphatase inhibitor). Evidence for HABPI as a substrate of CKII The in vitro phosphorylation of recombinant HABPIIP-32 was carried out in three successive steps using kinase from different sources: (a) crude rat liver extract known to contain a variety in kinases including CKlI, (b) tissue purified CKlI and (c) commercial recombinant CKlI, free from any contaminating kinase. These experiments clearly established the 34 kDa HABP 1 as a substrate of CKlI. Initially, the crude liver extract was used to phosphorylate the recombinant HABPl. As shown in Fig.3A, recombinant HABPI did not undergo any autophosphorylation in vitro (lane 1), but was phosphorylated only when incubated in the presence of rat liver kinase (lane 3), raising the possibility of rHABPI being a substrate of protein kinase. Furthermore, the phosphorylation ofrHABPI was found to be concentration dependent as seen by an increase in its phosphorylation with an increase in concentration (Fig. 3B). The observation on the phosphorylation ofHABPl by crude liver kinase, suggested CKlI as one of the probable kinases in liver extract, responsible for the in vitro phosphorylation of HABPIIP-32. As shown in Fig. 4A, tissue purified CKlI also phosphorylated rHABPI (lane 6) which was significantly enhanced by the addition of
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Phosphorylation of purified rHABPl/P-32 protein by liver kinases in vitro. Purified rHABPI (10 ug) was incubated under standard assay conditions with [y_ 32p]_ATP (5 ~Ci/assay). rHABPI (lane 1, 10 ug) only, crude liver kinases (lane 2, 10 ug total protein), rHABPI (lane 3, 10 ug) in the presence ofliver kinases (10 ug total protein). B. Phosphorylation of rHABP I was carried out in a concentration dependent manner in the presence of crude liver kinases (10 ug total protein), 5 ug ofrHABPI (lane 1), 10 Ilg of rHABP I (lane 2),15 ug of rHABPl (lane 3) and 20 ug ofrHABPl (lane 4). Proteins were
separated in 4-20% gradient SDS-PAGE, dried and autoradiographed. Spermidine and heparin showed a stimulation (lane 5) and inhibition (lane 4) in HABPI phosphorylation by the tissue purified CKlI (Fig. 4A), substantiating the possibility of HABP 1 as the substrate ofCKII.
370
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rHABPI as CKII substrate was further confirmed by the use of commercially available recombinant CKII. As shown in Fig. 4B, recombinant CKII was shown to be autophosphorylated at the regulatory B subunit of 26 kDa and catalytic a. subunit of 42 kDa (lane 4) which was stimulated by spermidine (lane 3) and inhibited by heparin (lane 2), the known stimulator and inhibitor of CKII respectively. Substrate phosphorylation by CKII using histone as substrate was also studied (lane l ). Interestingly, CKII was shown to phosphorylate rHABP} at the concentration of 0.5 Ilg (lane 8) and I ug (lane 7) along with autophosphorylation of CKII subunits. However, higher concentrations of rHABP} at the level of 5 Ilg (lane 6) and 10 Ilg (lane 5) inhibited autophosphorylation of both the regulatory and catalytic subunit of CKII and
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Figure 4. A. Specific phosphorylation of rHABP I IP-32 by tissue purified casein kinase II. Phosphorylation by tissue purified CKII. CKII (lane I) is autophosphorylated, rHABPIIP-32 alone (lane 2), phsophorylated by CKII alone (lane 6), treated along with HA (lane 3), heparin (lane 4) and spermidine (lane 5). B. Phosphorylation by recombinant CKII. Autophosphorylation of the recombinant CKII (lane 4, 0.1 mO), enhanced CKII phosphorylation in presence of basic protein histone (lane I) and specific stimulator spermidine (lane 3). CKII phosphorylated rHABP1/P-32 at 0.5 ug (lane 8) and 1 ug (lane 7). rHABP1/P-32 at 5 /-lg (lane 6) and 10 ug (lane 5) inhibited autophosphorylation of CKII. Both CKII autophosphorylation (lane 2) and HABPI/P-32 phosphorylation (lane 9) are inhibited in presence of heparin. Alkaline phosphatase abolished both the phosphorylations (lane 10). finally reflected upon the phosphorylation of HABPI, suggesting a regulatory mechanism operating with HABP I. The data confirmed HABP 1 as CKII substrate. This finding was further supported by the inhibition of phosphorylation of CKII (lane 9) by addition of heparin and its enzymatic dephosphorylation by alkaline phosphatase (lane 10). DISCUSSION In this study, we have identified HABPIIP-32as a substrate of CKII and tried to unravel the mechanism of cellular signalling by HABPI. Our hypothesis is supported
Cellular signalling
371
by the following observations. Firstly, HA-hexasaccharide is equally competent in interacting with HABPI as HA-polymer. Secondly, stimulating the cellular phosphorylation level regulates the phosphorylation of HABPl. Finally, HABPI confirmed as a substrate of CKII. The recombinant HABPI was phosphorylated by liver kinase and then specifically by both tissue-purified and recombinant CKII, implying that HABP1/P-32 may be an endogenous substrate of CKII. Heparin inhibition, spermidine stimulation of the CKII and HABPI phosphorylation and dephosphorylation by alkaline phosphatase treatment prove the specificity of this phosphorylation. Higher concentration of HABPI was found to be inhibitory for CKII kinase activity, suggesting a regulatory effect of HABPI on CKII activity as seen in the substrate inhibition of certain regulatory enzymes. Significantly, the HABPI phosphorylation was found to be enhanced in the presence of HA. HA, though predominantly present in extracellular space, is also reported to be present in the cytosol and nucleus. Our observations of the affinity of HABPI to HA polymer as well as HA hexamer needs to be mentioned in this regard. CKII mediated phosphorylation of HABPI may be speculated to be regulated by nuclear/cytosolic level ofHA oligomer in cells. Specific phosphorylation ofHABPI/P32 by CKII may further explain its association with splicing factor SF2 in HeLa cells as reported by us. CKII is ubiquitously present in cytosol and nucleus of eukaryotic cells and can also behave as RNA binding protein kinase"; The C group hnRNP protein, implicated in splicing is known to get phosphorylated in vivo by a CKII type activity. So, the co-association of HABPI with SF2 and the phosphorylation of HABPI/P-32 may be an intricate mechanism involved in RNA splicing. SF2-P32 interaction leading to the inhibition of SF2 phosphorylation suggests a regulatory role of HABPI/P-32 in RNA splicing!". Recent report on compartment specific interaction of HABPIIP-32PKCIl leading to inhibition of its kinase activity either by steric blockage of the kinase domain or by inducing conformational change, disabling its kinase activity, further strengthen our idea of the involvement of HABPIIP-32 in cell signaling. Another interesting fact that needs to be mentioned about HABP1/P-32 is that it is reported to have diverse functions and subcellular localizations. It has been reported to be localized on cell surface', mitochondria", nucleus and cytoplasm'?' 21. The role of CKII participation in mitogenic signals by cytoplasmic nuclear translocation is well reported, which may lead to translocation of its substrate HABPlIP32 after phosphorylation and regulate the binding of splicing factor SF2 and finally the spliceosome reaction. ACKNOWLEDGEMENTS We thank the Department of Biotechnology (DBT) and the Department of Science and Technology (DST), Government of India for the financial assistance. REFERENCES 1. T.C. Laurent & l.R.E. Fraser. The properties & turnover of hyaluoman. In: elBA Found. Symp. 1986, 124,9-29. 2. B.P. Toole Hyaluronan & its binding protein, the hyaladherins. Curro Opin. Cell. Bioi. 1986,2,839-844. 3. S. Gupta, B.R. Babu & K. Datta. Purification, partial characterization of rat kidney hyaluronic acid binding protein & its localization on the cell surface. Eur. J of Cell Bioi. 1991, 56, 58-67. 4. S. Gupta & K. Datta. Possible role of hyaluronectin on cell adhesion in rat histiocytoma. Exp. Cell. Res. 1991, 195, 386-394.
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5. S. Ranganathan, AK. Ganguly & K. Datta. Evidence for presence of hyaluoman binding protein on spermatozoa & its possible involvement in sperm function. Mol. Reprod. Dev. 1994,38,69-76. 6. S. Ranganathan, A Bharadwaj, & K. Datta. Hyaluronan mediates sperm motility by enhancing phosphorylation of proteins including hyaluronan binding protein. Cell. Mol. Bioi Res. 1995,41(5): 467-476. 7. C.M. Rao, T.B. Deb & K. Datta. HA induced hyaluronic acid binding protein phosphorylation & inositol triphosphate formation in lymphocytes. Biochem. & Mol. Bio!. Inter. 1996,40,327-337. 8. C.M. Rao, T.B. Deb, S.B. Gupta & K. Datta. Regulation of cellular phosphorylation of HABPI & its role in the formation of second messenger. Biochem. Biophys. Acta. 1997, 1336,387-393. . 9. T.B. Deb, & K. Datta. Molecular cloning of human fibroblast hyaluronic acid binding protein confirms its identify with P-32, a protein copurified with splicing factor SF2. J. Bioi. Chem. 1996,269,2206-2212. 10. M. Majumdar, & K. Datta. Assignment of cDNA encoding hyaluronic acid binding protein 1 to human chromosome 17 pI2-13. Genomics 1998,51. 476-477. II. S. Das, T. B. Deb, R. Kumar, & K. Datta. Multifunctional activities of human fibroblast 34-kDa hyaluronic acid-binding protein. Gene 1997, 190, 223-225. 12. AR. Krainer, A. Mayeda, D. Kozak, & G. Binns. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, UI 70K, & Drosophila splicing regulators. Cell 1991, 66 (2): 383-94. 13. B. Ghebrehiwet, B. L. Lim, E.I.B. Peerschke, AC. Willis, & K.B. Reid. Isolation, cDNA cloning, & overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" ofClq.J. Exp. Med.1994, 179(6). 1809-21. 14. D.R. Marshak & L. Russo. Regulation of protein kinase CKII during the cell divisions cycle. Cell Mol. Bioi. Res. 1994,40,513-7. 15. R. Pepperkol, P. Lorenz, W. Ansorge & W. Pyerin. CKII is required for transition ofGO/GI, early GI & GI/S phases of cell cycle. J. Bioi. Chem. 1994,269,6986-91. 16. K.V. Knondron & AS. Stepanov. RNA-binding protein kinase from amphibian oocytes is a casein kinase II. FEBS Lett. 1984, 170, 33-37. 17. S.K. Petersen-Mahrt, C.E.C. Ohrmalm, D.A. Mathews, W.C. Russell & G. Akusjarvi. The splicing factor associated protein p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding & phosphorylation. EMBO J. 1999, 18(4), 10141024. 18. P. Storz, A Hausser, G. Link, J. Dedio, B. Ghebrehiwet, K. Pfizenmaier, & F-l. Johannes. Protein kinase CIJ is regulated by the multifunctional chaperon protein p32. J. Bioi. Chem. 2000 May 30. 19. J. Dedio, W. Jahnen-Dechent, M. Bachmann, & W. Mueller-Esterl. The multiligand-binding protein gClqR, putative Clq receptor, is a mitochondrial protein. J. Immunol. 1998, 160,3534-3542. 20. D.A Mathews & W.C. Russell. Adenovirus core protein V interacts with P-32 a protein, which is associated with both the mitochondria & the nucleus. J. Gen. Viro!. 1998, 79, 1677-1683. 21. B.J. Soltys, D. Kang and R.S. Gupta. Localization of P32 protein (gClq-R) in mitochondria and at specific extramitochondrial location in normal tissues. Histochem. Cell Bioi. 2000 114,245-255.
RHAMM (CD168) CO-ASSOCIATES WITH AND REGULATES ERKKINASE R. Harrison, F.-S. Wang and E. A. Turley London Regional Cancer Center Cancer Research Laboratories 790 Commissioners Road. East London. Ontario N6A 4L6 Canada
ABSTRACT RHAMM was originally described as a hyaluronan binding protein that is present on the cell surface as well as inside the cell and that regulates cell motility and cell cycle. It has been reported to occur as multiple protein forms, some of which are generated by alternative splicing of mRNA. RHAMM occurs in several sub-cellular compartments. RHAMM is first seen at the plasma membrane during cell attachment, then within lamellae as cell spreading occurs. Later, it is present in mitotic spindles as cells divide and along interphase microtubles in quiescent cells. The secondary structure of full length RHAMM is predicted to form a series of coiled coil regions flanked by globular C and N terminal sequences. RHAMM sequence encodes multiple putative protein kinase phosphoacceptor, several potential kinase docking sites, a putative dimerization site, as well as several SH2 and SH3 binding sites suggesting that intracellular protein forms may function to link signaling complexes to both the actin and microtubule cytoskeletons. In particular, RHAMM contains two putative erk phosphoacceptor sites and a rsk-like erk-docking site. Experimentally, RHAMM overexpression or HAIRHAMM interactions activate erk kinase. RHAMM co-immunoprecipitates with erk kinase and co-localizes with this map kinase in the mitotic spindle, cell lamellae and cell nucleus. A model is proposed where both cell surface and intracellular forms of RHAMM affect cell motility and proliferation by regulating erk kinase activity.
KEYWORDS Hyaluronan (HA), erk kinase HMMR, CD168, RHAMM, intracellular, cell surface
INTRODUCTION RHAMM 1 is a hyaladherin that is also known as HMMR (genomic classification), IHABP 2 and most recently as CD168 (2000 CD Harogate workshop, http:// gryphon.jr2.ox.ac.ukl), the latter reflecting its presence on leukocyte cell surfaces. In addition to the cell surface, RHAMM associates with other sub-cellular compartments including the nucleus, and the actin and microtubule cytoskeleton.l". RHAMM belongs to a sub-group of hyaladherins' that also include cdc37 7•8 and HABP-1. 9 These "itinerant" hyaladherins (www.glycoforum.gr.jp/science/hyaluronan/HAl1/HAllE.html) are characterized by their multiple locations within the cell and by the manner with which they bind to hyaluronan. For instance, HA associates with these proteins via basic amino acid clusters rather than "link" modules that mediate binding ofHA to hyaladherins such as CD44 lO• Furthermore although itinerant hyaladherins have been reported to occur on
374
Cell surfacesand hyaluronanreceptors
the cell surface, they do not encode either signal peptides or transmembrane domains. However, this group has consistently been shown to regulate cell motilityll-14 and, in the case of RHAMM, to promote protein tyrosine phosphorylation/dephosphorylation of intracellular kinases, notably FAK, 15.16 to promote protein tyrosine phosphorylation of actin binding proteins and to activate erk kinase in response to hyaluronan growth factors such as PDGF I6. 17 or bFGF l 8 and mechanical stimuli". The mechanisms by which RHAMM is exported out of the cell, how it associates with the cell surface and how it functions to regulate signaling, both outside and inside the cell, are not yet defined. Here, we review what is known about RHAMM structure and some of its known functions. We also propose a model for how RHAMM may regulate signaling cascades that impact upon cell motility.
RESULTS AND DISCUSSION RHAMM is expressed as multiple protein forms A partial murine RHAMM cDNA was originally isolated in 199z!, and a full-length human cDNA was cloned in 19962 The full-length sequence was confirmed in mouse in 199821. The entire murine gene was sequenced in 19992 and the human HMMR gene was cloned as part of the human genome project. Several RHAMM protein forms result from alternative splicing ofmRNA (Fig. 1). Smaller forms ofRHAMM proteinsI5.22.23, some that lack N-terminal sequence", are expressed most commonly in malignant cells. How these are generated is not yet clear but addressing this issue is important since a truncated
°.
Figure 1. RHAMM protein forms. Several RHAMM forms have
I) Standard RHAMM (v5) 01
02
03
D4
05
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I
j~'
_
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j
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I RHAMMv5(<:>4); h
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2) Alternatively spliced v5
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3) Short RHAMM forms
• - N-glycosylation sites h =human. m = murine
II
RHAMMv4(<:>1-5); m
II
RHAMMv2(<:> 1-9); m
been identified that include the full length and several alternatively spliced forms that are generated either by removal of exon 4 or exon 13. Additional truncated forms have been reported (termed here as v4 and v2) although whether these result from alternative splicing or other mechanisms has not yet been established.
form of RHAMM that lacks the N-terminal 160 amino acids is transforming when overexpressed in fibroblasts 15. Possibilities include alternate start codon usage, since the full length RHAMM cDNA contains multiple downstream consensus start codons, or proteolysis. It is unlikely alternative splicing ofmRNA24 generates these forms. Recent reports suggest that the various RHAMM protein forms can differ in both their sub-cellular location and their functional effects on cell behavior",
RHAMM(CDl68)
375
RHAMM is associated with multiple sub-cellular compartments The presence of RHAMM in multiple sub-cellular compartments is most readily observed over time after plating cells onto fibronectin-coated surfaces (Fig. 2). Within 1/2 to 1 hour after plating, RHAMM can be seen at the plasma membrane, then as cells initially spread, it can be seen in circular structures or podosomes detected by the presence ofthe marker protein, cortactin. When cells begin to divide, which can often occur as late as 24-48 hours after plating, RHAMM can be observed in the apical region of the mitotic spindle. Later when cells have completed division and are more confluent, RHAMM decorates interphase microtubules (Fig. 2).
Figure 2. RHAMM occursin multiplesub-cellular compartments infibroblasts.
A. RHAMM
occurs at the plasma membrane as cells are attaching to a fibronectin-coated substratum. J!. Several hrs after plating, when cells have spread, RHAMM occurs in circular structures called podosomes. £. In dividing cells, RHAMM localizes in the apical region of the mitotic spindle. ]!. 72-96 hr after plating and at culture confluence, RHAMM decorates interphase mierotubules.
RHAMM is a coiled coil protein The predicted secondary structure for RHAMM is a series of coiled coils flanked by non-helical regions", RHAMM therefore has weak homology with other coiled coil proteins such as myosin, and this secondary structure together with its association with the actin and microtubule cytoskeleton has lead to the proposal that RHAMM is a structural protein linking the actin and microtubule cytoskeletons'", in a manner similar to Clasp proteins'", However, RHAMM also contains a variety of kinase recognition motifs and 8H2 and 8H3 binding sites. The ability ofRHAMM proteins to co-associate with kinases such as src'" and erk 17.18 as well as the presence of docking/phosphoacceptor motifs suggest that intracellular RHAMM forms are able to associate with these kinases and therefore may perform adapter functions linking these kinases to the cytoskeleton, much like other actin binding proteins such as paxillin", vinculirr", vay28and Wasp prcteins'", The HA binding ability ofRHAMM and RHAMM antibodies 10 to affect activation of src and erk in response to either HA or PDGF 16,17 suggest that cell surface RHAMM also plays a role in activation of these kinases. Interestingly, the full length RHAMM cDNA encodes multiple di-leucine internalization signals that have been shown to be associated with rapid internalization of activated receptors and protein targetinlfO·32 and RHAMM may therefore also playa role in receptor trafficking/recycling. RHAMM is notable for protein domains that it lacks. The absence of a signal peptide and a membrane spanning sequence encoded in the RHAMM gene 2 does not equip this
376
Cell surfacesand hyaluronan receptors
protein for export and signaling via the conventionally accepted paradigm. This, and the absence of cell surface RHAMM in some cell types and/or culture conditions", has lead to questions regarding both its presence on the cell surface and its signaling function there 33. A number of independent laboratories have shown that cell surface RHAMM is detectable in sub-confluent adherent cells and in leukocytes I2-14,16,17,19. Clearly, understanding both the stimuli that permit cell surface expression ofRHAMM as well as identifying the mechanism{s)by which it is exported will clarify this issue. In this context, it may be useful to view RHAMM and other itinerant hyaladherins as a subclass of ''messenger proteins'f" that include bFGF, HN tat protein and homeobox transcription factors. These proteins also occur in multiple intra- and extra-cellular compartments and can associate with the cell surface, yet do not encode signal peptides for export through the golgi-ER or membrane spanning sequence. They appear to be exported out ofthe cell by a golgi-ER independent route that requires a leucine rich region resembling the dimerization domain ofRHAMM. They are picked up by other cells and gain direct access to the cytoplasm by a cell internalization signal (CIS) that contains basic and hydrophobic amino acid clusters, similar to sequence within the HA binding domains ofRHAMM.
RHAMM protein forms co-associate with erk kinase The presence of the erk phosphoacceptor sites, the ability of erk I kinase to phosphorylated recombinant RHAMM in vitro (unpublished data), reports that RHAMM regulates erk activation l6,17 and the co-immunoprecipitation of erk 1 kinase'" with RHAMM suggest that one function of intracellular forms ofRHAMM may be to regulate the activity of protein kinases such as erkl. This possibility is particularly intriguing in view ofrecent evidence that erk kinase is required for locomotion on fibronectirr" substrata and in response to growth factors such as hepatocyte growth factor", PDGp7 EGF 38 and bFGF 39. Erk was originally shown to translocate from the cytoplasm to the nucleus when its upstream activator, MKKl 40, phosphorylates it. However, active erk is also present in nonnuclear compartments41-44 and has recently been shown to phosphorylate previously unsuspected substrates". These last results suggest that little is known regarding the likely multiple functions of erk. Like ~6, erk kinase is known to play a role in progression through the cell cycle" and to associate with multiple sub-cellular compartments including the mitotic spindle, interphase microtubules, the cell nucleus, cell lamellae, cell-substratum adhesion sites known as focal contacts and the actin cytoskeleton41-44. Importantly, the association oferk kinase with non-nuclear compartments is required for extracellular matrix- and growth factor-mediated motility". Activated erk associates with microtubules via MAPs and it's activity enhances turnover of microtubules", a property required for lamellae extension during cell motility and for assembly and also disassembly of the mitotic spindle. Erk is also involved in actin cytoskeleton remodeling as a result of its binding to actin binding proteins such as cortactin", alpha actinin 42 and, likely, RHAMM 17• RHAMM and erk co-Iocalize in mitotic spindles, cell lamellae and the cell nucleus (Fig. 3). The association ofRHAMM with a protein kinase is reminiscent of the properties of the hyaladherin cdc37, which binds to src and rat9. Cdc37 has been proposed to stabilize raf and src in an active conformation and it is possible that intracellular RHAMM forms function in this capacity for erk kinase.
RHAMM (CDI68)
377
Figure 3. RHAMM co-localizes with erk kinase in several sub-cellular compartments. A.RH.AMM and erk 1 kinase co-localizeat the apex ofthe mitotic spindle in rastransformed IOTl/2 cells. .lI- RHAMM and erk also co-localize in cell lamellae of spreading 10Tl/2 cells and may represent either early podosomes or ruffles. £. RHAMM and erk also co-localize in the cell nucleus.
A model We propose that intracellular and cell surface forms ofRHAMM may act, in part, to regulate protein kinases, such as src and erk (Fig. 4). At the cell surface, RHAMM (CDI68) has been shown by antibody blocking to be required for activation of erk kinase by growth factors such as PDGF. Cell surface RHAMM may therefore function as a coreceptor to accomplish this. Co-immunoprecipitation assays suggest that intracellular RHAMM forms associate with both erk I kinase and MKKI kinase 17. Intracellular RHAMM proteins may act to chaperone, target and/or stabilize erk to various cytoplasmic substrates that are involved in cell locomotion. Recently, HA has been shown to occur within the cell nucleus, lamellae and along the mitotic spindle50•51• Whether or not itinerant, intracellular hyaladherins such as RHAMM and cdc3? require an association with this intracellular polysaccharide to exert their effects on the various kinase cascades that they regulate remains as an intriguing question that may help to clarify the precise role of these proteins.
i
III
RHA~lMI."'.
,_~ ----..... Cytoplasmic ~ Targets
Figure 4. A model ofthe association ofRHAMM with the erk kinase cascade. Multiple cell surface receptors modulate signaling throughkinase cascadessuchas the raserk kinase cascade including growth factor receptors, integrinsand hyaladherins(e.g. cell surface RHAMIM or CDl68 and CD44). Auto-phosphorylation of the growth factor receptors provide dockingsites for masterswitches such as the smallGTPase ras and adapterproteinsthat link receptors to other signaling molecules. Activation of ras brings raf to the cell membrane, which then activates MEKl. A complex of MKKI, erk I and RHA.'v1M is proposed to be required for the efficient activation of erk kinase. Activated erk kinase may translocate to the cell nucleus and/or to cytoplasmic compartments, where it phosphorylates substrates that impacton genetranscription and actin cytoskeleton, respectively.
ACKNOWLEDGMENTS Work was supported by CllIR. (Cardiovascular, Cancer) and NCIC of Canada. RH is
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Cell surfaces and hyaluronan receptors
a recipient ofa CIHR training fellowship. The authors thank C. Tolq for her suggestions and critique and J. Edwards for editorial assistance.
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33. 34. 35.
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hyaluronan receptor RHAMM regulates extracellular-regulated kinase, J Biol. Chem., 1998, 273, 11342-11348. B.D. Lynn, X. Li, P.A. Cattini & J.I. Nagy, Sequence protein expression and extracellular-regulated kinase association of the hyaladherin RHAMM (receptor for hyaluronan mediated motility) in PCI2 cells, Neurosci Lett, 2001, 306, 49-52. K. Aitken & D.J. Bagli, Stretch-induced bladder smooth muscle cell (SMC) proliferation is mediated by RHAMM-dependent extracellular-regulated kinase (erk) signaling, Urology 2001,57, 109. C. Wang, 1. Entwistle, O. Hou, Q., Li & E. A. Turley, The characterization of a human RHAMM eDNA: conservation of the hyaluronan-binding domains, Gene, 1996,174, 299-306. M. Hofmann, C. Fieber, V. Assmann, M. Gottlicher, J. Sleeman, R. Plug, N. Howells,O.von Stein, H. Ponta & P. Herrlich, Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein, J. Cell Sci., 1998, 111, 1673-1684. H. Li, L. Guo, J.W. Li, N. Lui, R. Qi & 1. Liu, Expression ofhyaluronan receptors CD44 and RHAMM in stomach cancers: relevance with tumor progression, Int J Onco12000, 17,927-932. T. Ahrens, V. Assmann, C. Fieber, C. Termeer, P. Herrlich, M. Hofmann & J.C. Simon, CD44 is the principal mediator of hyaluronic-acid-induced melanoma cell proliferation. J Invest Dermatol, 2001,116,93-101. J. Entwistle, S. Zhang, B. Yang, C. Wong,Q. Li, C. L. Hall, J. A, M. Mowat, A. .H. Greenberg & E.A. Turley, Characterization of the murine gene encoding the hyaluronan receptor RHAMM, Gene, 1995, 163,233-238. V. Assmann, D. Jenkinson, J. F. Marshall & 1. R. Hart, The intracellular hyaluronan receptor RHAMMlIHABP interacts with microtubules and actin filaments, J. Cell sa; 1999, 112, 3943-3955. A. Akhmanova, C.C. Hoogenraad, K. Drabek, T. Stepanova, B. Dortland, T. Verkerk, W. Vermeulen, B.M. Burgering, C.I. De Zeeuw, F. Grosveld & N. Galjart, Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts, Cell, 2001, 104,923-935. C.E. Tumer, Paxillin interactions, J Cell Sci, 2000, 113,4139-4140. J.S. Kennedy, M. Raab & C.E. Rudd, Signaling scaffolds in immune cells, Cell Calcium, 1999,26,227-235. R. Rengan & H.D. Ochs, Molecular biology of the Wiskott-Aldrich syndrome, Rev Immunogenet, 2000, 2, 243-255. R. M. Gibson, W. P. Schiemann, L. B. Prichard, J. M. Reno, L. H. Ericsson & N. M. Nathanson, Phosphorylation of human gp130 at Ser-782 adjacent to the Di-leucine internalization motif. Effects on expression and signaling, J Bioi. Chem., 2000,275, 22574-22582. L. Preisser, N. Ancellin, L. Michaelis, C. Creminon, A. Morel & B. Corman, Role of the carboxyl-terminal region, di-leucine motif and cysteine residues in signaling and internalization of vasopressin Via receptor, FEBS Lett, 1999,460,303-308. M. S. Marks, L. Woodruff, H. Ohno & 1. S. Bcnifacino, Protein targeting by tyrosine and di-leucine-based signals: evidence for distinct saturable components, J. Cell Biol., 1996,135,341-354. M. Hofmann, V. Assmann, C. Fieber, J. P. Sleeman, J. Moll, H. Ponta, I. R. Hart & P. Herrlich, Problems with RHAMM: a new link between surface adhesion and oncogenesis?, Cell, 1998,95, 591-2; discussion 592-593. A. Prochiantz, Messenger proteins: homeoproteins, TAT and others, Curro Opin. Cell Biol., 2000, 12,400-406. B. Anand-Apte, B. R. Zetter, A. Viswanathan, R. G. Qiu, 1. Chen, R. Ruggieri & M. Symons, Platelet-derived growth factor and fibronectin-stimulated migration are
POLY I:C INDUCES MONONUCLEAR LEUKOCYTE-ADHESIVE BYALURONAN STRUCTURES ON COLON SMOOm MUSCLE CELLS: ror AND VERSICAN FACILITATE ADHESION Carol A. de la Motte l *, Vincent C. HascaU 2, Judith A. Drazba'', & Scott A. Strong' 'Inflammatory Bowel Disease Research Group - NB3 Department ofColoreetaI Surgery 'Connecttve Tissue Biology Section - ND2 Department ofBiomedical Engineering J
Imaging Core Facility - NBI
Cleveland ClinicFoundation, Lerner Research Institute 9500 Euclid Avenue, Cleveland, OH 44195
ABSTRACT Inflammatory bowel disease (IBD) is a chronic, unremitting disorder whose etiology is linked to triggering events, including viral infections, that lead to immunoregulatory dysfunction in genetically susceptible people. The pathological changes characteristic of this disorder include increased mononuclear leukocyte influx into the intestinal mucosa as well as mucosal smooth muscle cell (M-SMC) hyperplasia. We have shown that virus infection or viral mimic (poly I:C) treatment of human colon MSMCs in vitro increases cell surface hyaluronan expression and that non-activated mononuclear leukocytes bind via CD44 to virus-induced hyaluronan structures. Confocal microscopy revealed that poly I:C-induced hyaluronan on the M-SMC surface is arrayed in patchy, coat-like structures and in lengthy cable structures that can span several cell lengths. The cables are primarily responsible for mediating leukocyte adhesion. Immunohistochemical staining shows that two hyaluronan-binding proteins, inter-atrypsin inhibitor (laI) and versican, are localized in both patch and cable structures. Treatment of hyaluronan on the surface of poly I:C-induced M-SMCs with a versican specific monoclonal antibody inhibits leukocyte adhesion -80%, suggesting a dual receptor-ligand interaction as the mechanism by which unstimulated mononuclear leukocytes adhere to the cable structures. We have confirmed the presence of upregulated hyaluronan in inflamed colon tissue of IBD patients as compared to patient matched noninflamed tissue. Immunohistochemical staining also identifies versican associated with hyaluronan in inflamed tissue sections, underscoring the relevance of our in vitro data specifically, and this novel mechanism of leukocyte recruitment in general. KEYWORDS: Inflammation, leukocyte adhesion, hyaluronan, CD44, Icd, versican INTRODUCTION The origin of inflammatory bowel disease (IBD) is multifactorial, where environmental and microbiological factors initiate and perpetuate an immune response in the intestine of genetically susceptible individuals, which results in the clinical
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Cell surfaces and hyaluronan receptors
manifestations of Crohn's disease and ulcerative colitis. Immune phenomena involved in this disease have been investigated extensively, and have underscored the differences between the responses of normal and affected individuals. However, much less is known about how microbial agents affect the disease process. Speculations that viruses may be involved in the pathogenesis of IBD have been advanced for some time due to the clinical association of respiratory virus infections with subsequent IBD flare-up. In an extensive study, I 40% of disease flares in a population of susceptible individuals were temporally associated with documented viral respiratory infections. Other studies demonstrated association of measles virus infection alone, or temporally associated with other virus infections, to Crohn's disease as compared with patients who did not have IBD. And in separate studies, the simultaneous presence of DNA from Herpes virus 6 and Epstein Barr virus was detected more frequently in ulcerative colitis than in Crohn's disease or in control tissues.v' Normally (Fig. 1), colonic mucosal tissue (lamina propria) contains a population ofleukocytes, including T- and B-Iymphocytes, plasma cells, histiocytes and mast cells, which are scattered in a network of collagen fibers and smooth muscle cell bundles. These leukocytes arrive to the area via regularly distributed capillaries in the lamina propria. They serve a surveillance function in the tissue, providing immune protection against the lumenal contents of the colon. Mucosal lymphocytes may reenter the blood stream, presumably via the lymphatic vessels located in close proximity to the muscularis mucosae, and are free to recirculate through blood and lymphoid organs until a specific antigenic challenge recalls them to an affected area." In IBD (Fig. I), the mucosal immune cell population increases dramatically, and the infiltrate is predominantly comprised of mononuclear leukocytes. Further, a hyperplastic thickening of the juxtaposed muscularis mucosae also occurs. This suggests that interactions between leukocytes and mesenchymal smooth muscle cells are important in the development ofIBD. We have recently shown that colonic mesenchymal cells can proliferate in response to leukocyte-derived inflammatory cytokines.l Increasingly however, investigators are finding evidence for bi-directional interaction and communication between smooth muscle cells and immune cells within tissues, events that can playa role not only in IBD, but in other chronic inflammatory diseases. We have previously shown that virus infection or poly I:C (viral mimic) treatment
Fig. I Schematic diagram of tissue layers of the colon (normal and inflammatory bowel disease).
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of M-SMCs increases mononuclear leukocyte adhesion via M-SMC-expressed hyaluronan interacting with leukocyte-expressed CD446 (Fig. 2). This mechanism is very different from the cytokine (TNF-a)-induced mechanism of leukocyte adhesion, which involves VCAM-l on the M-SMCs interacting with its integrin ligand, VLA-4, on the mononuclear leukocytes. Carbohydrate gel electrophoresis reveals an increase in total hyaluronan synthesis (-2 fold) and a proportionately larger increase (-3 fold) in cell associated hyaluronan by M-SMCs treated with poly I:C compared with untreated MSMCs. Specific mononuclear leukocyte adhesion to unstimulated M-SMCs is generally very low (-0.5-1 % of the added leukocytes) and increases substantially on poly I:C treated M-SMCs (10-20 fold in most experiments). In addition, we have shown that leukocyte activation is not required for binding to hyaluronan on virally-induced MSMCs, while others have found that leukocyte activation and subsequent CD44 activation are necessary for hyaluronan binding in other systems. Since the increase in leukocyte adhesion to M-SMCs induced by poly I:C is disproportionately higher than the increase in cell-associated hyaluronan and because suprisingly, CD44 on non-activated leukocytes is capable of binding to the hyaluronan on virally activated M-SMCs, we investigated the possibility that there are differences in the presentation or structure of hyaluronan between untreated and poly I:C (or virus)activated M-SMCs. MATERIALS AND METHODS
Cells
M-MSMs are isolated from the mucosal layer of human colon specimens by collagenase digestion as previously described," grown in culture and used in the first through third passages. Cultured M-SMCs routinely stain positively for a-smooth muscle cell actin. U937 cells, a monocytic cell tumor line, were procured from American Type Culture Collection, and are cultured in suspension as previously described." Monocyte Adhesion Assay
The binding of U937 cells to adherent cells has been demonstrated to be a valid model for mononuclear leukocyte adhesion,' including adhesion to M-SMCs. 6 Briefly, 5lCr-labelled U937 cells are incubated with appropriately treated cultures of M-SMCs for 1 h at 4°C. Non-adherent U937 cells are removed during the course of three washes with
384
Cell surfaces and hyaluronan receptors
mediwn, cultures solublized with 1% Triton, and aliquots removed for quantitation of radiolabel. The nwnber ofU937 cells bound is calculated from the initial specific activity (cpm/cell). Immunohistochemistry
Fixation of cultured cells - M-SMCs, grown on cover slips and treated appropriately for the described experiments, are rinsed with Hank's balanced salt solution, fixed in methanol at -20°C for 15 min and air dried. Tissue specimens - Samples of colon mucosa are fixed in Histochoice'I'", parafin embedded, sectioned into 5 um thick sections, and affixed to slides. Immediately before staining, the sections are deparifinized in Clear-rite™ and hydrated through sequential steps of decreasing concentrations of ethanol. Sections are stored in water until stained. Staining - The cover slips and tissue slides are blocked with Hank's balanced salt solution containing 1% fetal bovine serum, and incubated with a solution containing biotinylated hyaluronan binding protein (5 ~rnl) and an appropriate antibody [CD44 and versican monoclonals (10 ~g/rnl); Icd polyclonal at 1:100)] (-16 h, 4°C). The cover slips and slides are washed three times, and a solution containing flurosceinated streptavidin (1:500) or Texas red conjugated anti-Ig (H + L) directed to the native species of the primary antibody (l :500) in Hank's balanced salt solution containing I % fetal bovine serwn is incubated with the cover slips (I h,25°C). The cover slips and slides are washed and affixed to slides and cover slips, respectively, in mounting mediwn containing DAPI, and then sealed and stored at -20°C. Confocal images are obtained using a Leica TCS-SP laser scanning confocal microscope. RESULTS AND DISCUSSION
To address whether the in vitro mechanism of hyaluronan-mediated interaction of leukocytes with M-SMCs that we have reported is relevant to the inflammation associated with IBD, we collected tissue from grossly inflamed and non-inflamed areas of colon from IBD patients. Sections were stained for hyaluronan using the hyaluronanbinding proteins prepared from aggrecan aggregates (HA-BP) and a green fluorescent (FITC) indicator. Confocal microscopic observation (Fig. 3) shows that hyaluronan is detectable in uninflamed colon tissue, consistent with previous reports.i However, inflamed tissue sections from the same patient stained much more intensely for hyaluronan when imaged under identical lumination. Characteristic of most of the inflamed IBD colon tissue we have observed, hyaluronan appears to be arrayed in a reticulwn composed of long strands. When inflamed colon tissue is double stained for hyaluronan (green) and CD44 with a monoclonal CD44 antibody (Texas red indicator) (Fig. 4A), intimate contact between infiltrating leukocytes and the hyaluronan strands is observed. In the large intestine of another IBD patient, we compared mildly involved colon to severely inflamed tissue. In the mildly inflamed section (Fig. 4B), defined hyaluronan strands are most commonly located in and around the muscularis mucosae layer suggesting that they arise from the M-SMCs, as our in vitro studies suggest. In highly inflamed, ulcerated tissue where the crypt architechture has been destroyed (Fig. 4C), hyaluronan is seen abundantly throughout the section, and is enmeshed with CD44
Poly I:C
20 control
non-inflamed
385
inflamed
Fig. 3 Confocal micrograph demonstrating hyaluronan (green) in non-inllamed and inllamed sections of colon from a Crohn's disease patient.
Fig. 4 Confocal micrograph demonstrating hyaluronan (green), CD44 (red) and nuclei (blue). (A) Enlargement of Inllamed colon tissue demonstrating intimate contact between CD44 leukocytes and hyaluronan in between the intestinal villi. (8) Mildly Inflamed and (C) very inflamed sections of colon from another IBD patient. Arrows indicate examples of mononuclear leukocytes.
Time (h) Fig. 5 Confocal micrograph showing the time course of appearance of poly I:C induced hyaluronan (I!reenl on M-S:\ICs.
386
Cell surfacesand hyaluronanreceptors
Fig. 6 Confocal micrograph showing hyaluronan (green) and lal (red) staining of poly I:C treated M·SMCs.
Fig. 7 Confocal micrograph showing hyaluronan (green) and verslcan (red) staining of poiy I:C treated M-SMC.
Fig. 8 Confocal micrograph showing hyaluronan (green) and versican (red) staining of colon tissue from an ulcerative colitis patient. Versican Is localized on the hyaluroDan strands in vivo. Arrows indicate examples of monomudear leukocytes associated with regions of hyaluronan containing verslean,
Poly [:C
387
positive leukocytes. Further, reticular structure of the hyaluronan is no longer present. Previously, in our in vitro model, we had observed by phase contrast microscopy that hyaluronan-mediated leukocyte adhesion appeared to tether monocytes to the cell surface, and frequently in a chain formation. This is quite different from adhesion mediated by traditional leukocyte binding molecules (VCAM-l, I-CAM-l, and E- and Pselectin), where leukocytes bind singly and in close association with the membrane of the cell carrying the adhesion molecules. Consequently, we investigated histochemically how hyaluronan is arrayed on the surface of cultured M-SMCs with or without poly I:C treatment. Figure 5 demonstrates that unstimulated M-SMCs have some hyaluronan randomly scattered on their surfaces. However, within three hours of poly I:C stimulation, the cells form pericellular coats. By 11 hours, in addition to further coat formation, hyaluronan strands are visible on the apical surfaces of some cells. At 17 hours, the strands ofhyaluronan emanating from neighboring cells are organized into thick cables. In parallel cultures, where we have allowed mononuclear leukocytes to bind to MSMCs treated with poly I:C for 17 hours, almost all adhesion of the CD44 positive leukocytes is to the hyaluronan cables. This fmding agrees with our prediction from the phase contrast observation and correlates well with the peak in the time course of poly I:C-induced, hyaluronan-mediated leukocyte adhesion by M-SMCs. 6 The obvious question arose, 'Why do leukocytes bind to hyaluronan cables but not to the coat-like structures on M-SMCs?'; i.e. 'What is unique about the hyaluronan cables that permit CD44 receptors to bind, without prior activation?' Hyaluronan has a simple basic structure of repeating disaccharides of N-acetyl glucosamine and glucuronic acid that arrange into long polymers, and therefore tertiary structure is likely to be conferred by molecules that bind to hyaluronan. The involvement of hyaluronan-binding proteins has already been shown to be important in pericellular coat formation on a variety of cells. One binding molecule, a serum protein known as inter-n-trypsin inhibitor (luI),9 is investigated in this report. Confocal micrographs (Fig. 6) of poly I:C-treated M-SMCs histochemically stained for hyaluronan (green) and lui (red) indicate that lui is associated with both the coat and cable structures. Since Icd is contributed by the serum in the culture medium, we looked at poly I:C-induced generation of hyaluronan cables by M-SMCs in the presence or absence of serum. Under serum-free conditions, cables did not form, although long, thin strands of hyaluronan were present. In separate experiments where leukocyte adhesion was measured, treatment of poly I:C-stimulated M-SMCs with lui antiserum after cable formation, but before leukocyte adhesion, resulted in a modest decrease (-40%) in monocyte binding. However, inclusion of the lui antiserum during M-SMC stimulation was more effective (-60% inhibition) in reducing monocyte adhesion. Taken together, our results suggest that Icd is important for hyaluronan cable formation and therefore for adhesion. The role of another hyaluronan-binding protein, the large proteoglycan, versican, was also investigated in hyaluronan-mediated leukocyte adhesion by M-SMCs. Versican is known to be produced by smooth muscle cells, and a role for its involvement in another chronic inflammatory condition, atherosclerosis, has recently been advanced.l'' Confocal micrographs (Fig. 7) of untreated and poly I:C-treated M-SMCs stained for hyaluronan (green) and versican (red) indicate that versican is present on unstimulated as well as poly IC-treated M-SMCs. When cell surface hyaluronan is induced by poly IC,
388
Cell surfaces and hyaluronan receptors
versican is associated with both the patchy coat hyaluronan structures as well as the cables. Strikingly, when leukocyte adhesion was measured, treatment of poly I:Cstimulated M-SMCs with a monoclonal antibody to versican after cable formation but before leukocyte adhesion resulted in a substantial decrease (-80%) in monocyte binding. The data suggest that two co-operative molecular interactions, hyaluronan with CD44 and versican with another leukocyte receptor, are important for the actual binding interaction between mononuclear leukocytes and M-SMCs, and may help to explain why the requirement for CD44 activation before binding to hyaluronan is circumvented in this system. We also investigated whether versican is associated with the increased hyaluronan levels we observed in IBD colon tissue. Confocal micrographs of inflamed colon sections from an ulcerative colitis patient demonstrate fenestrated hyaluronan structures in the lamina propria, and in certain areas versican staining is colocalized with hyaluronan. With increased magnification (Fig. 8), versican (red) is seen to be located in patches on the hyaluronan (green) strands, and leukocytes appear intimately associated with the hyaluronan strands in regions where versican is located. Thus, the unique method of leukocyte-smooth muscle cell interaction we have described in vitro appears to be operative in vivo in inflamed colon.
ACKNOWLEGEMENT This work was supported of America (S.A.S.).
by
the
Crohn's
and
Colitis
Foundation
REFERENCES 1. H.O. Kangro, S.K. Chong, A. Hardiman, et al. A prospective study of viral and mycoplasma infections in chronic inflammatory bowel disease Gastroenterology 1990, 98, 549-553 2. C.N. Bemstein & J.F. Blanchard Viruses and inflammatory bowel disease: Is there evidence for a causal association? Inflammatory Bowel Diseases 2000, 6, 34-39 (review) 3. A.J. Wakefield, J.D. Fox, A.M. Sawyer, et al. Detection of Herpes virus in the large intestine of patients with ulcerative colitis and Crohn's disease using the nested polymerase chain reaction. J Med Virol1992 38, 183-190 4. M. Salmi & S. Jalkanen Endothelial ligands and homing of mucosal leukocytes in extraintestinal manifestations of ISO Inflammatory Bowel Diseases 1998,4, 149-156 5. SA Strong, T.T. Pizarro, J.S. Klein, et al. Pro-inflammatory cytokines differentially modulate their own gene expression in human intestinal mucosal mesenchymal cells. Gastroenterology 1998, 114, 1244-1256 6. C.A. de la Motte, V.C. Hascall, A. Calabro, B. Yen-Lieberman & SA Strong. Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on human intestinal mucosal smooth muscle cells after virus infection or treatment with poly I:C. J Bioi Chem 1999, 274, 30747-30755 7. P.E. DiCorieto, & C.A. de la Motte Characterization of the adhesion of the human monocytic cell line U937 to cultured endothelial cells. J Clin Invest 1985, 75, 11531161 8. M. Wang, M. Tammi, H. Guo & R. Tammi Hyaluronan distribution in the normal epithelium of esophagus, stomach, and colon and their cancers. Am J Pathol 1996, 148, 1861-1869 9. A. Blom, H. Pertoft & E. Fries Inter-a-inhibitor is required for the formation of the hyaluronan containing coat on fibroblasts and mesothelial cells J Bioi Chern 1995, 270, 9698-9701 10. S.P. Evanko, J.C. Angello & T.N. Wight Formation of hyaluronan and versican rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler. Thrornb. Vase. Bioi. 1999, 19, 1014-1013
ULTRASTRUCTURAL EVIDENCE FOR EXTRACELLULAR MATRIX DISORGANISATION FOLLOWING SPECIFIC REMOVAL OF HYALURONAN FROM SYNOVIUM Peter J. Coleman Department ofPhysiology, St George's Hospital Medical School, Cranmer Terrace, London, SW17 ORE, UK
ABSTRACT A 4 to 6 fold increase in rabbit synovial hydraulic permeability following the specific removal of synovial hyaluronan with Streptomyces hyaluronate lyase was investigated ultrastructurally using transmission electron microscopy. Bundles of type l/Ill/V fibrils appear to undergo an internal collapse following enzyme treatment in vivo with a reduction in interfibrillar volume fraction from 57.13 + 9.09 in the control tissue to 42.69 ± 5.87 in the enzyme treated tissue (n=lOO bundles each, p=7.13xl0-9) . Removal of hyaluronan appears to cause matrix disorganisation and collapse, contributing to the permeability increase previously observed. KEYWORDS Synovium, interstitium, hyaluronan, joint permeability, Streptomyces HA lyase, electron microscopy. BACKGROUND The lining of diarthrodial joints is a thin specialist layer of mesenchymal tissue known as synovium, consisting of macrophage derived A cells, fibroblast derived B cells, a rich vasculature of both fenestrated and continuous capillaries, and an abundant interstitial matrix. The synovial cell layer is highly discontinuous with abundant intercellular gaps of 1.1 to 5.5!J.m in the rabbit. Despite this, synovium offers substantial resistance to fluid loss from the joint, and plays a significant physiological role in the conservation of intra-articular fluid. The site of synovial hydraulic resistance is believed to be the complex network of interstitial biopolymers occupying the many intercellular gaps. This matrix contains bundles of types I, III, and V collagen fibrils, and a significant quantity of type VI microfibrillar collagen. Large polymeric molecules occupy the spaces within the collagen network (i.e, the "extrafibrillar" spaces), namely the large unsulphated glycosaminoglycan hyaluronan, and proteoglycans with sulphated glycosaminoglycan sidechains of chondroitin, keratan or heparan sulphate. Many smaller glycoproteins such as fibroncctin are also present, together with pericellular type IV collagen. (Revell et al 1996; Levick et al 1996; Coleman ct al 1998a). Hyaluronan is present within the rabbit synovial interstitial matrix at a concentration of 0.8 mg per ml extrafibrillar space (biotinylated G1 binding assay; Price et al 1996). It is the least concentrated of all interstitial glycosaminoglycans, with chondroitin
338
Aspects ofhyaluronan in joints
sulphate, (chondroitin-4-sulphate and chondroitin-6-sulphate combined), and heparan sulphate at 1.16 and 1.94 mg per ml extracellular space respectively (Price et al, 1996). Removal of individual interstitial glycosaminoglycans from rabbit synovium in vivo using specific enzymes resulted in increased synovial hydraulic permeability compared to control contralateral joints. The increase was greatest for hyaluronan with a 4 to 6 fold increase over control (Coleman et al, 1998b). The fact that hyaluronan is the least concentrated of all synovial interstitial glycosaminoglycans, yet its specific removal leads to the largest increase in synovial hydraulic conductivity gave rise to the question "does hyaluronan play a significant organisational role in the maintenance of synovial interstitial integrity?" In order to answer the question, the ultrastructure of rabbit knee synovium +/- Streptomyces HA lyase was studied by transmission electron microscopy.
METHODS The full protocol for enzyme treatment and preparation of pressure - flow relations is published in Coleman et al; 1998b. Briefly; New Zealand White rabbits (2.5 - 3kg) were anaesthetised, and two cannulae inserted into the suprapatellar joint space as described previously by Levick (1979). One, a 21 gauge hypodermic needle with perforations drilled at the tip was connected to a Validyne differential pressure transducer. The other, a conunercially available polypropylene "Medicut" cannula contained lOOOU Streptomyces HA lyase (Sigma) in Iml of sterile Baxter Ringer solution, which was injected intra-articularly and left for 1 hour. Following the enzyme digestion, the Medicut cannula was connected to a saline filled infusion system, and a pressure - flow relation prepared according to the protocol published previously by Coleman et al (1998b) to check for enzyme activity. The procedure was repeated on the contralateral control joint, but Ringer solution was used in place of the enzyme. The animal was then killed by anaesthetic overdose, and both joints fixed in situ with EM fixative (4% glutaraldehyde in 0.2M sodium cacodylate buffer) for 1 hour. Once fixed, the synovial tissue was carefully dissected out, and further fixed overnight in EM fixative at 4°C. The following day the tissue was prepared for resin embedding. Briefly; tissue samples were washed twice for 15 min in sodium cacodylate buffer, and then placed in 1% osmium tetroxide (in cacodylate buffer) for 4 hours to undergo secondary fixation. Samples were then washed in cacodylate buffer as above, then dehydrated in a series of graded ethanol solutions (30%, 70%, 90% and absolute x2; 1 hour each) followed by propylene oxide (1 hour). Samples were left overnight in a 50/50 mix of propylene oxide and Spurr resin. Tissue samples were embedded the next day following 2 changes of Spurr resin, and cured overnight at 50°C. 50nm ultrathin sections were cut on a Leitz "Ultracut" motor driven ultramicrotome, and caught on to 200 gauge copper grids. Ultrathin sections were stained with uranyl acetate and Sato's lead citrate, and viewed in a Zeiss EM900 transmission electron microscope. Digital images of individual collagen bundles were grabbed using a digital CCD video camera on the microscope, and dumped directly into a Macintosh personal computer. Digital image analysis was carried out using NiH Image image analysis software. The system was calibrated for use at 85,OOOx magnification using O.26408/lm diameter latex microspheres,
Extracellular matrixdisorganisation
339
RESULTS
Bundles of type I/IIIN fibrils undergo an internal collapse following hyaluronan removal with a reduction in extrafibrillar volume fraction from 57.13 ± 9.09 (sd) (control, n= 100 bundles, 5 animals) to 42.69 ± 5.87 (sd) \Streptomyces HA lyase, n= 100 bundles, 5 animals). This was significant; p=7.13x 10- , t-test, 2 tailed, samples of equal variance. The diameter of the individual fibrils was unchanged. Figure 2 shows typical examples of control and Streptomyces HA lyase treated type I/IlIN collagen bundles. DISCUSSION
The reduction in collagen extrafibrillar volume fraction appears to indicate that interstitial hyaluronan chains not only contribute directly to synovial hydraulic drag, but have a major organisational role in synovial extracellular matrix. It seems likely that hyaluronan acts as a "thread" that interacts with and organises other synovial matrix components. Enzymatic removal of hyaluronan appears to cause matrix disorganisation and collapse, and this probably contributes to amplifying the permeability as previously described. The nature of this interaction in synovial tissue is as yet unclear. Kielty et al (1992) demonstrated an interaction between type VI collagen microfibrils and hyaluronan, although Spissinger and Engel (1994) were unable to repeat their findings. Hyaluronan interacts with other matrix components such as aggrecan, link protein, versican and hyaluronectin (Mason et ai, 1989). Marked ultrastructural changes following Streptomyces hyaluronate lyase disruption of the hyaluronan-aggrecan-Iink protein interaction in cartilage have been reported by Poole et al (1982), and in umbilical cord, Meyer et al (1983) reported that digestion with Streptomyces hyaluronate lyase led to the release of sulphated glycosaminoglycans. Hyaluronan could also link matrix components to the cell surface via hyaluronan binding proteins such as CD44. It is possible there are many interactions between hyaluronan and interstitial matrix components, but this requires further study. ~
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340
Aspects of hyaluronan in joints
Figure 2. Transmission electron micrographs of type lIlIIN collagen bundle in control (A), and enzyme treated rabbit synovium (B). x50,OOO. Stained with uranyl acetate and Sato's lead citrate. REFERENCES. P. J. Coleman, E. Kavanagh, R. M. Mason, J. R. Levick, D. E. Ashhurst, The proteoglycan and glycosaminoglycan chains of rabbit synovium. Histochemical Journal, 1998a, 30, 519-524. P. J. Coleman, D. Scott, A. Abiona, D. E. Ashhurst, R. M. Mason, J. R. Levick, Effect of depletion of interstitial hyaluronan on hydraulic conductance in rabbit knee synovium. J.Physiol., 1998b, 509.3, 695-710. C. M. Kielty, S. P. Whittaker, M. E. Grant, C. A. Shuttleworth, Type VI microfibrils: evidence for a structural association with hyaluronan. J. Cell. Bioi, 1992, 118,979-990 J. R. Levick, The influence of hydrostatic pressure on trans-synovial fluid movement and of capsular expansion in the rabbit knee. J. Physiol, 1979,289,69-82 J. R. Levick, F. M. Price, R. M. Mason. Synovial matrix - synovial fluid system of joints. In: Extracellular matrix vol 1, W. D. Comper (ed), Harwood academic publishers, Amsterdam, 1996, pp 328-377. R. M. Mason, M. V. Crossman, C. Sweeney, hyaluronan and hyaluronan binding proteins in cartilaginous tissues. In: The Biology of Hyaluronan. D. Evered, J. Whelan, (cds). Wiley and Sons, Chichester, 1989, ppl07-120. F. A. Meyer, Z, Laver-Rudich, R. Tanenbaum, Evidence for mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton's jelly, Biochim. et Biophys. Acta, 1983, 755, 376-387. A. R. Poole, 1. Pidoux, A. Reiner, L. Rosenberg, An immunoelectron microscope study of the organisation of protcoglycan monomer. J.Cell.Biol., 1982,93,921-937. F. M. Price, J. R. Levick, R. M. Mason, Glycosaminoglycan concentration in synovium and other tissues of rabbit knee in relation to hydraulic resistance. J.Physiol, 1996,495,803-820. P. A. Revell, N. Al-Saffar, N. Fish, D. Osei, Extracellular matrix of the synovial intimal layer. Ann. rheum. dis. 1995,54,404-407. T. Spissinger, 1. Engel, Type VI beaded microfibrils from bovine cornea depolymerise at acid pH. Depolymerisation and polymerisation are not influenced by hyaluronan. Matrix. Biol., 1994,499-505.
THE GENERATION OF HYALURONAN-DEPENDENT PERICELLULAR MATRIX IN VITRO J.R.E. Fraser* Dept. ofBiochemistry and Moleculor Biology, Monash University, Wellington Road, Clayton, Victoria 3168. Australia..
ABSTRACT
In 1967, it was reported that human synovial cells, which secrete hyaluronan copiously, were surrounded in culture by optically clear zones that prevented contact by lymphocytes though the latter were unattached and motile'. Although these clear areas were very irregular in outline, they called to mind the hyaluronan-rich capsules of certain bacterial species, and it was shown that they could be quickly dispersed by hyaluronidase. They could also be effectively outlined by erythrocytes, heat-killed yeast and other kinds of small particle. Their resistance to other enzymes indicated that hyaluronan was the main component, and they were seen as the first stage in the formation of extracellular matrix. Time-lapse microcinematography showed that the margins of the cells moved freely within this material, giving rise to the lack of symmetry between the cells and their pericellular investments. When cells moved to another area, the clear area would persist until its content was dissipated in the growth medium. The investments thus showed quite strong cohesion within themselves but little adhesion to the cell surfaces. The pericellular investments were found to prevent cytotoxic allogeneic reactions with lymphocytes until removed by hyaluronidase, and likewise to moderate infection with virus. A limited comparison with other kinds of cell indicated that the formation of these pericellular coats depended initially on the capacity to synthesise hyaluronan. The main findings of these studies have since been confirmed and extended by other workers to a wide variety of cells, including fibroblasts from other tissues, some but not all sarcoma and carcinoma cell lines, and some other common cell lines. Protection against lymphocytotoxicity and moderation of virus infection have also been confirmed. The need for a critical level of hyaluronan synthesis to initiate the formation of a pericellular matrix has been repeatedly confirmed. Cells that have hyaluronan receptors but poor capacity for synthesis ofhyaluronan can acquire a pericellular matrix by addition of hyaluronan and aggregating proteoglycan together. Participation of receptors has been confirmed by blocking them with hyaluronan hexasaccharides or a monoclonal antibody. This kind of matrix simulates that of cartilage. Mesothelial cells also generate a large pericellular matrix that is not dependent on a hyaluronan receptor. Although there are some anomalies and apparent contradictions in recent work there appear to be two distinct kinds of pericellular matrix generated ill Vitro, which can be related in functional terms to the tissues from which they arise.
KEYWORDS Pericellular, extracellular, matrix, coat, generation, hyaluronan, initiation, ill vitro
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INTRODUCTION
During studies of interactions in cell culture between normal human lymphocytes and synovial cells of fibroblastic phenotype (Type B), to be described below, pale areas were seen beneath the lymphocytes, and with low proportions of lymphocytes, the intended target cells were found to be surrounded by an optically clear zone that prevented contact with the lymphocytes and resisted vigorous attempts to wash it away. Since synovial cells secrete hyaluronan prolifically, this phenomenon was immediately likened to the heteropolysaccharide capsules that surround monocellular organisms such as Cryptococcus neoformans, and in particular, those of Groups A and C Streptococcus pyogenes and Pasteurella multocida in which the capsule consists of hyaluronan. These capsules are similarly demonstrable as clear areas outlined by a particulate suspension such as India ink, necessarily finer because of their much smaller size. THE DEVELOPMENT OF A CONCEPT
Further investigations'f were undertaken with washed human Group 0 erythrocytes (Fig. I). The integrity of the pericellular synovial gel was shown to depend on its hyaluronan content by its dispersal with bacterial hyaluronidase. (The only other substrate for this enzyme is sulphate-free chondroitin which we have never identified in these cells, and which in any case is rarely found in nature, if ever.) This conclusion was later confirmed by ourselves' (Fig.2) and others after introduction of the monospecific Streptomyces hyaluronidase. We were unable to see it with phase-contrast or Baker interference optics, and it would not stain after fixation or with supravital fluorescent stains. These findings were attributed to its consisting mainly of highly hydrated hyaluronan. At this time there was virtually no evidence for the development of a structured pericellular matrix in cultured eukaryote cells. A fraction of the hyaluronan secreted by cultured fibroblasts had been shown to be cell-associated and released by trypsin .j..6 and this kind of cell was also found to form fibrous collagen on the cell surfaces" after secreting soluble collagen abundantly for several days after passage". Various sialoproteins, RNA and other materials had been identified on cell surfaces but not visualised in the manner seen here and rarely shown by staining (see"), The gels we saw were unaffected, however, by nucleases, neuraminidase, and EDTA or trypsin, unless the last two detached the cells from the culture surface. Low levels of trypsin did induce some swelling before detachment and we did not exclude the possibility that other components contributed to the structure or stability of the gels', though aggrecan and binding proteins were yet to be defined. Although our range of cells was limited, the development of the gels was related to their capacity to synthesise hyaluronan, a fact elegantly confirmed later by Underhill and Toole 1o who also showed a direct relation between the rate of synthesis and the amount retained at the cell surface. The synovial pericellular matrix invested single cells and small groups, extended as far as 20 to 30 ILm from cell margins and often presented an irregular outline lacking symmetry with cell margins (Figs. 1-3). It extended vertically as shown by entrapment of tiny protein aggregates or cell debris in a web floating above them. It resisted vigorous washing with culture medium, but was fully divested after cell detachment by trypsin, to be regenerated within 4-5 h after reattachment in fresh culture medium. If the culture
Generation of pericellular matrix
Figure 1.
Pericellular coat outlined in cultured human synovial cells A, by human erythrocytes; B, by suspension ofMoS z
powder, mean diameter O.Spm. Note asymmetry of coating and irregular protrusion well beyond cell margin which has probably retracted from that area (arrow in Panel B).
Reproducedfrom Ref 2 by courtesy ofAcademic Press.
391
392
Figure 2.
Cell surfaces and hyaluronan receptors
Pericellular coat outlined by heat-killed Saccharomyces cerevisiae, Mean diameter 4.2Jlm. Same field: A, before and B, after gentle addition of Streptomyces hyaluronidase solution to an adjacent area in the culture dish. This enzyme degrades hyaluronan only. Yeasts began to move over the cellular domains within 30 seconds. Bar represents lOOpm.
Reproducedfrom Ref 3 with permission ofthe Novartis Foundation.
Generationof pericellular matrix
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Figure 3. Pericellular coats of cultured human synovial cells outlined by erythrocytes
Time-lapse microcinematography. Intervals: Frames 5 to 6, 5 min; 6 to 7, 103 min; 7 to 8, 77 min. In Frame 6, cell initially at zone B has moved away, leaving residue of gel still just perceptible in Frame 8. Note also residues after cells move around in zone CD, Frames 5 to 8. Reproducedfrom Ref 12 with permission ofCSJRO Australia.
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Cell surfaces and hyaluronan receptors
medium was replaced without serum, however, the gel dissipated overnight. This was attributed to the requirement for serum to sustain HA synthesis", The gel was most conveniently outlined on these large cells by washed erythrocytes (D = 7.8 ,urn). Heat-killed Saccharomyces cerevisiae (D = 4.2 ,urn) and MoS z powder (D = 0.5 ,urn) were effective alternatives, although the latter, like colloidal carbon, would gradually penetrate it to be taken up by the cells. The random Brownian movement of the larger outlining particles was visibly restricted in the direction of the pericellular gel. In high magnification, cell margins showed rapid irregular ruftling movements. Within the pools of matrix, time-lapse microcinematography" recorded protrusions and retractions of cell margins that left behind an irregular outline of the matrix when they retracted (Fig. 3). The cells moved freely within the gels, and also migrated for relatively long distances, leaving an isolated patch of matrix that might take an hour or more to disperse. It was thus clear that at least in synovial cells, the bulk of the matrix was not firmly adherent to the cell membrane but certainly cohesive within itself, both characteristics of some significance. We initially reported the phenomenon as a "barrier?' because it prevented contact between synovial cells fixed in culture and lymphocytes. Synovial cells generally grew as well in the presence of lymphocytes as they did alone. On the addition of hyaluronidase, the synovial cells ceased to grow after about 48 h and their numbers then declined; presumably due to lysis, since they could not be found in the growth medium". The effect depended on the cell ratios, and presumably on the donor pairings. In most cases there was also a very marked increase in glucose consumption inversely related to the surviving numbers of synovial cells (Table I), providing further evidence for the protection of the target cells by hyaluronan.
Table 1. Effect of hyaluronidase on cultured synovial cells with and without exposure to allogeneic lymphocytes. Controls Ratios*, C+/C(a) for final cell counts, >1.0 9 <1.0 3 Lymphocyte treatments Ratios*, L+/L(a) for final cell counts, >10 2 <1.0 12 * +/- with or without hyaluronidase.
(b) for glucose consumption. Significance 7 5 N.S. (b) for glucose consumption. Significance 12 2 P < 0.01 Fisher exact test.
The passage of small particles (MoS z, C) through the gels showed that they acted merely as a hindrance to Brownian movement rather than a barrier. This was confirmed by the enhancement of cellular uptake of Chlamydia spp. in a gentle centrifugal field after hyaluronidase treatment of the cells (unpublished), and also by infection with Newcastle disease virus, a paramyxovirus which permits recognition of infected cells by haemagglutination". Eighteen hours after inoculation, twice as many cells were infected if pretreated with hyaluronidase (Table 2). There was no difference if the cells were treated with hyaluronidase immediately before adding the erythrocytes, indicating that virus
Generation of pericellularmatrix
395
infection either destroyed the gels, the virus haemagglutinin had penetrated it, or both. It was clear at least that the cell coats reduced the initial intensity of infection by impeding access of the virus to its specific cell-membrane receptors. There was no difference in susceptibility of rheumatoid or nonrheumatoid cell lines, a burning question of the day. Similar enhancement of virus infection in rheumatoid synovial cells by hyaluronidase was confirmed by others the following year. l~ The pericellular coating might even prevent infection in the case of exposure to viruses that are particularly labile in an extracellular environment.
Table 2. Influence of hyaluronidase on infection of cultured synovial cells with Newcastle disease virus. -------------------------------------------------------------------------------------------------------Before infection Before haemadsorption % infected cells Chi-square P 1. Hyaluronidase 36 2. Diluent <0.001 17 41.5 3. 4.
Hyaluronidase Diluent
19 20
0.31
N.S.
5. Hyaluronidase 6. Diluent
Hyaluronidase Hyaluronidase
40 19
69.9
<0.001
Although we did not undertake any studies to confirm it, the pericellular coating would potentially affect diffusion as it did Brownian movement. It would, for example, serve to maintain a higher concentration of the smaller secreted forms of collagen and thus promote the development of collagen fibres close to the cell with less loss in the soluble form, and in a similar manner promote the development of other kinds of complex matrix. It represented at the least a partial restoration of the pericellular matrix in vitro; and by its nature helped to rebut superficial criticisms levelled at the time against the use of cell culture on the grounds that the cells were stripped naked, and rapidly "dedifferentiated". It focussed our attention on important details that could compromise the use of cell culture in experimental pathology if ignored and it has since provided a model for the study of extracellular matrix now widely adopted in various contexts that could not be foreseen when it was first recognised. PERICELLULAR MATRIX IN OTHER KINDS OF CELL
The foregoing observations were made almost entirely with cells from human knee joints, which are a very prolific source of hyaluronan . In 1979, McBride and Bard" reported transparent zones around a variety of adherent sarcoma and carcinoma cell lines, and fibroblasts from mouse embryo and adult human skin, which were not penetrated by several kinds of particles or other cells. These zones developed in 2-4 h, extended up to 17 JIm from the cells, and averaged 8.8,um in thickness. They were ablated by testicular or Streptomyces hyaluronidase but like ours resisted nucleases or neuraminidase. They did not develop without serum in the culture medium and were not found on Vero cells, B- or T-lymphocyte lines or melanoma cells. Hyaluronidase treatment of fibrosarcoma cells rendered them susceptible to lysis by immune splenic lymphocytes but not by
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Cell surfaces and hyaluronan receptors
non-immune cells. The reaction was, however, limited to 7 h, insufficient to detect allogeneic reactions to the latter. Time-lapse records showed the clear zones of motile cells to be thinner on the leading edge and thicker on the trailing edge but no variations of the kind noted above were recorded. The same authors" succeeded in showing the pericellular layers in scanning electron microscopy after fixation, rapid freezing and freeze -drying but noted , in contrast to our own findings, that the material was very fragile and easily disrupted by robust pipetting in culture before fixation. This lead them to discount any structural role for it. Underhill and Toole" detected similar hyaluronan-dependent coatings, susceptible to Streptomyces hyaluronidase, in a rat fibrosarcoma cell line, and in the widely used 3T3 and BHK lines. In virus-transformed 3T3 and BHK cells (SV-3T3 and P¥-BHK) the coatings were very much smaller or undetectable. The total amount of hyaluronan released in the cultures, and the proportion of synthesised hyaluronan retained on the cell surface were very much less after transformation. SV-3T3 cells were shown to exhibit a high-affinity cell-surface binding ofhyaluronan 18.19, and later both SV-3T3 and PY-BHK were found to possess higher affinity and capacity for binding hyaluronan than their parent cells". These features were associated with ability to aggregate despite their lower hyaluronan synthesis and much smaller total surface coatings. Numerous other kinds of cell, such as skeletal myoblasts" and smooth muscle cells" have since been shown to develop similar coatings. It is likely that they will appear whenever a cell is engaged in a sufficiently high level of hyaluronan synthesis to form a coat by cohesion without the participation of a hyaluronan receptor as a necessary condition.
rne ROLE OF HYALURONAN RECEPTORS IN PERICELLULAR MATRIX With the recognition that hyaluronan is an essential component of the macromolecular aggregates of cartilage proteoglycans, that the pericellular matrix of chondrocytes collapses with hyaluronidase despite the dominance of the proteoglycans, and that chondrocytes are rich in the hyaluronan receptor, CD44, Knudson and Knudson" explored the contribution of the receptor to the initial generation of the pericellular matrix in a most ingenious manner in vitro. They took several cell lines without detectable coatings, with and without a capacity for hyaluronan binding, and showed that a visible coating could be generated by the addition of aggrecan and hyaluronan together but not individually, and only in those shown to bind hyaluronan. This occurred without serum in the medium, and also took place after glutaraldehyde fixation. The coats began to shrink after 3 h and disappeared in 6 h. After removal of the natural coatings of fibrosarcoma cells, this technique restored a coat within 2 h, whereas its spontaneous regeneration took 6 h. Hyaluronan tetralhexasaccharides (HA-6S), which in sufficient concentration will bind to hyaluronan receptors and displace polymers, could prevent these exogenous coatings from forming or disperse them after formation. After transfection with CD44 genes, COS-7 cells, a virus-transformed African green monkey kidney line, could be induced to form similar structures that were also dispersed with hyaluronan hexasaccharides or Streptomyces hyaluronidase". These findings have been replicated by use of a monoclonal antibody reactive with a hyaluronan-binding protein 2~. This could prevent the formation of cell coats or disperse them in rat fibrosarcoma, embryonic chicken chondrocytes or mesodermal cells. HA-6S did likewise. These studies have lead to the hypothesis that the formation of pericellular matrices might require participation of
Generation of pericellular matrix
397
hyaluronan receptors and one of the numerous forms of aggregating proteoglycans produced by various kinds of cell found in many tissues. A most informative comparison has been made between normal human mesothelial cells and human malignant mesothelioma cells". The former formed large hyaluronan-dependent coats within 5 h of passage into fresh cultures, which were also promoted in "starvation" medium (containing 0.5% instead of 15% serum) by adding platelet-derived growth factor BB and epidermal growth factor, both stimulants of hyaluronan synthesis. The size of the coatings corresponded broadly with the amount of hyaluronan synthesised. In contrast with the foregoing, hyaluronan dodecasaccharides (HA-12S) or larger oligosaccharides inhibited development of or dispersed the coatings without inhibiting hyaluronan synthesis, whereas HA-6S, a small amount of exogenous hyaluronan or aggrecan had no effect. Mesothelioma cells produced no surface coatings, and in further contrast with the normal mesothelium, exhibited a high-affinity, trypsin-sensitive CD44-related hyaluronan receptor". Induction of pericellular matrix ill vitro has been observed in cells with little intrinsic capacity for hyaluronan synthesis following their transfection with one of the three hyaluronan synthase genes, HASl, HAS2 or HAS3 (see":"). A similar study found that cell coats appeared only above a certain rate of hyaluronan synthesis, but below this level, coats could be induced by the addition of aggrecarr". Induction of hyaluronan synthesis by transfection was associated with a reduction of cell surface CD44 and reduced cell migration. All these studies have shown that HAS2 produces the largest hyaluronan polymers.
DISCUSSION Although there was earlier biochemical and electron microscopic evidence that hyaluronan and fibrous collagen, two major components of the extracellular matrix, were closely associated with the surfaces of cultured tissue cells and would be expected to be found there if synthesised by the cells, the visual portrayal of such material gave the phenomenon a cogency that has encouraged much fruitful investigation. Reason dictates that this should represent the first stages in the generation of pericellular matrix, but if such a view is accepted, it is still proper that we regard it as an experimental artefact and pay close attention to those conditions that are not reproducible in vitro, or might be overlooked. For example, cells are almost always grown at 37°C whether they come from birds or animals with higher core body temperatures, or from human tissues such as lung, skin or peripheral joints where the temperature is at least 2° to 7° lower than core temperature. it is difficult to reproduce the pOl, pH and metabolite gradients that prevail in tissues remote from blood vessels, such as articular cartilage. Yet we know that lowering pH will stimulate hyaluronan synthesis, and such a simple measure as plating chondrocytes at high density with attention to pH and nutrient control leads to formation of a tissue like normal articular cartilage in 2 to 3 days". It is even more difficult to mimic other physical influences on tissues, or the passage of extracellular fluid through the tissues which can affect, for example, the retention of'hyaluronan". Another peculiarity in the practice of diploid cell culture is the common use of serum, which sustains growth and stimulates hyaluronan synthesis. It can provide numerous growth factors, and osmotic activity. Yet few consider that 10% fetal bovine serum may contain only 4mg protein per ml compared with - IS mg per mI in extracellular fluid, and
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that its albumin content is proportionately much lower. These variables may compromise cell function and the level of hyaluronan synthesis necessary to induce matrix function. Although they might appear incompatible at first, the studies cited here indicate that the pericellular coatings represent the development of two distinct kinds of extracellular matrix whose integrity depends on the presence of hyaluronan. The more recent findings portray a complex of hyaluronan with an aggregating form of proteoglycan that also requires specific binding of the hyaluronan to a cell receptor, identified in some instances as CD44, notably in chondrocytes. These cells include some which naturally form pericellular matrices and secrete hyaluronan ;/1 vitro, and others that don't do either, but possess receptors naturally or induced by gene transfection. This kind of matrix is dispersed by hyaluronan hexasaccharides, an index of binding by hyaluronan receptors. The other kind of pericellular matrix is exemplified by the synovial and mesothelial cell cultures and possibly occurs in cells that require transfection with HAS genes. This type does not seem to require a hyaluronan receptor and its behaviour as seen in time-lapse microcinematography is also inconsistent with tight adhesion to cells. In the mesothelial example, the gels are dispersed with hyaluronan dodecasaccharides, which suggests that their cohesion relies on self-entanglement of hyaluronan or its association with other matrix macromolecules. These could still include proteoglycans without cell attachment, some collagens, or secreted binding proteins. Even in the absence of specific attachments, purely physical interactions with other macromolecules can alter the visco-elastic behaviour and the diffusional properties of hyaluronan and contribute to its cohesion and slow dissolution. Indeed these distinctions in the form of the pericellular matrix are consistent with the variations in extracellular matrix in the body, which range from that of bone to the liquid forms of synovial fluid and the fluids of the serous cavities. The cell-adhesive pattern has been designated as a cartilaginous kind of matrix, and might also represent that of glial tissue, or aorta, where it is retained for structural reasons and meets a range of visco-elastic requirements different from those applying in the fluids. Close binding to cell surfaces would, however, impede the egress of hyaluronan, which must be unhindered in tissues such as synovial joints, skeletal and cardiac muscle and the mesothelium-lined cavities (pleura, pericardium and peritoneum). In these sites, hyaluronan serves as a soft-tissue lubricant, and it must be constantly replenished as it is displaced. Moreover, its turnover is known to be high in many parts of the body where it is most abundant". Regardless of the kind of matrix that will ultimately develop, its initial development appears to depend on the presence of hyaluronan rn vitro, es in v;vo34 . REFERENCES l. B.J. Clarris & IR.E. Fraser, Barrier around synovial cells;/I vitro, Nature 1967,214, 1159. 2. B.J. Clarris & J.R.E. Fraser, On the pericellular zone of some mammalian cells ;'1 vitro. Exper. Cell Res., 1968,49, 181-93. 3. T.e. Laurent & IR.E. Fraser, The properties and turnover of hyaluronan. In: Functions of the Proteoglycans, David Evered & Julie Whelan (eds.), John Wiley & Sons Ltd., Chichester. Ciba Foundation Symposium 124, 1986, pp. 9-29
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4 C.W. Castor & F. F Fries, Composition and function of human synovial connective tissue cells measured in vitro, 1. Lab. Clin. Med., 1961, 57, 394-407 5. M.R. Daniel, 1.T Dingle & 1.A. Lucy, Cobalt-tolerance and mucopolysaccharide production in rat dermal fibroblasts in culture, Exper, Cell Res., 1961,24,88- 105. 6. c.c. Morris & Gc. Godman, Production of acid mucopolysaccharides by fibroblasts in cell cultures, Nature, 1960, 188,407-9. 7. C.W. Castor & K.D. Muirden, Collagen formation in monolayer cultures of human fibroblasts: the effects of hydrocortisone, Lab. lnvest., 1964, 13,560-74. 8 B Goldberg & H Green, An analysis of collagen secretion by established mouse fibroblast lines, 1. Cell Bio\., 1964,22,227- 58. 9. C.W Castor, Regulation of collagen and hyaluronate formation in human synovial fibroblasts, J Lab. Clin. Med, 1970, 75, 798-810. 10 c.B. Underhill & B.P. Toole, Transformation-dependent loss of the hyaluronate-containing coats of cultured cells, 1. Cell Physio\., 1982, 110, 123-8. II. JR.E. Fraser, G.S. Harris & B.J Clarris, Influence of serum on secretion of hyaluronic acid by synovial cells. Its possible relevance in arthritis, Ann. Rheum Dis, 1969,28,419-423. 12 JR.£. Fraser. B.J. Clarris & L.A. Kont, The morphology and motility of human synovial cells and their pericellular gels: a time-lapse microcinematographic study, Aust. 1. Bio\. Sci, 1970,23, 1297-303. 13. JR.E. Fraser & BJ Clarris, On the reactions of human synovial cells exposed to homologous leucocytes in vitro, Clin. Exper. lmmunol., 1970,6,211-225. 14. BJ. Clarris, J.R.E. Fraser & SJ Rodda, Effect of cell-bound hyaluronic acid on infectivity of Newcastle disease virus for human synovial cells in vitro, Ann. Rheum. Dis., 1974,33,240-42 15. R.L. Patterson, DA. Peterson, F. Dienhardt & G. Howard, Rubella and rheumatoid arthritis: hyaluronic acid and susceptibility of cultured rheumatoid synovial cells to viruses, Proc. Soc. Exp. Biol., 1975, 149,594-8. 16. W.H. McBride & JB Bard, Hyaluronidase-sensitive halos around adherent cells. Their role in blocking lymphocyte-mediated cytolysis, J. Exp, Med., 1979, 149,507-515. 17. JB. Bard, W.H. McBride & A.R. Ross, Morphology of hyaluronidase-sensitive cell coats as seen in the SEM after freeze-drying, J Cell Sci., 1983,62,371-83. 18. C.B Underhill & B.P Toole, Binding of hyaluronate to the surface of cultured cells, 1. Cell BioI, 1979,82,475-84. 19. C.B Underhill & B P. Toole, Physical characteristics of hyaluronate binding to the surface of simian virus 40-transformed 3T3 cells., J. BioI. Chern, 1980,255,4544-9. 20. C.B Underhill & B.P Toole, Receptors for hyaluronate on the surface of parent and virus-transformed cell lines, Exper. Cell Res., 1981, 131,419-23. 21. R.W Orkin, W Knudson, and B P. Toole, Loss of hyaluronate-dependent coat during myoblast fusion, Deve\. Biol., 1985, 107,527-30 22 PG McGuire, JJ. Castellot and R.W Orkin, Size-dependent hyaluronate degradation by cultured cells, J Cell Physiol., 1987, 133,267-76 23 W. Knudson & CB. Knudson, Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells. Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix, J Cell Sci., 1991,99, 227-35. 24. W Knudson, E Bartnik & C.B Knudson, Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing genes,
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Proc. Natl Acad. Sci., 1993,90,4003-7. 25. Q. Yu, S.D. Banerjee & B.P. Toole, The role of hyaluronan-binding protein in assembly of pericellular matrices, Devel. Dynamics, 1992, 193, 145-51. 26. P. Heldin & H. Pertoft, Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells, Exper. Cell Res., 1993,208,422-9. 27. T. Asplund & P. Heldin, Hyaluronan receptors are expressed on human malignant mesothelioma cells but not on normal mesothelial cells. Cancer Res., 1994, 54, 4516-23. 28. AP. Spicer & 1.A McDonald, Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family, J. BioI. Chern., 1998, 273, 1923-32. 29. N. ltano, T. Sawai, M. Yoshida, P. Lenas et aI., Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties, 1. BioI. Chern., 1999, 274, 25085-92. 30. 1. Brinck & P. Heldin, Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44, Exper. Cell Res., 1999, 252, 342-51. 31. B.W. Oakes, C.J. Handley, F.Lisner & D.A. Lowther, An ultrastructural and biochemical study of high density primary cultures of embryonic chick chondrocytes. 1. Embryol. Exp. Morph. 1977,38,239-263. 32. H. Onarheim, AE. Missavage, R.A. Gunther, G.c. Kramer, RK. Reed & T.C. Laurent, Marked increase of plasma hyaluronan after major thermal injury and infusion therapy, 1. Surg. Res., 1991, 50, 259-65. 33. 1.RE. Fraser & U.B.G. Laurent, The turnover of hyaluronan. In: Connective Tissue Biology: Integration and Reductionism, RK. Reed & K. Rubin (eds.), Portland Press, London & Miami. Wenner-Gren International Series, 1998, Vol.71, pp. 49-69. 34. B.P. Toole, Morphogenetic role of glycosaminoglycans (acid mucopolysaccharides) in brain and other tissues. In: Neuronal Recognition, S.H. Barondes (ed.), Plenum Press, New York. 1976, pp. 275-329.
EFFECT ON JOINT TISSUES OF INTRA-ARTICULAR TREATMENT WITH 500-730 kDa HYALURONAN: INSIGHT INTO THE MECHANISM OF ACTION Diego Guidolin 1 & Luigi Frizziero 2 I FIDIA Research Laboratories. v.Ponte della Fabbrica 3.4. 35031 Abano Terme (I'D), Italv 2 Department ofInternal Medicine, Rheumatology Unit, Ospedale Maggiore. Largo 13.Nigrisoli 2. -10133Bologna. Italy
INTRODUCTION
Osteoarthritis (OA) is presently regarded as a disease process affecting the entire joint I, originated by a variety of mechanisms and resulting in a progressive degradation of the articular cartilage. Current therapy for OA is capable of alleviating the symptoms but no agent has, as of yet, been definitively shown to blow down the progression of cartilage damage. Significant resources are now being dedicated to the search for drugs characterized by structure-modifying activity, i.e. able to slow down the rate of cartilage degeneration and/or enhance that of cartilage repair 2 Hyaluronic acid (HA) is the most characteristic substance of articular joints and its key role in the maintenance of the structural and functional characteristics of the joint has long been recognized. It is actively synthetized by the synoviocytes and is present in high concentrations in the synovial fluid, of which it determines the viscoelastic properties. In the cartilage HA is a major component of the extracellular matrix. and contributes to the formation of an amorphous layer about 0.6 um thick, covering the articular surface of the cartilage 3, contributing to the boundary lubrication mechanism in conditions of extreme loading and partially protecting the tissue from the penetration of inflammatory cells and lytic enzymes. It is well known that in pathological processes, such as OA and rheumatoid arthritis, the molecular weight of the HA in the synovial fluid can be reduced by an order of magnitude (from 106 to 105 Da) and its concentration is also reduced mainly due to the accumulation in the joint cavity, of liquid derived from the inflamed synovial vessels 4. The result is a reduction in the viscoelasticity of the fluid and an increased susceptibility of cartilage to breakdown. To counteract this process a therapeutic strategy based on the use of intra-articular injections of exogenous HA started in the late sixties 5 The original rationale was to replace the depolymerized endogenous HA and to improve joint lubrication. Since then various molecular weight (MW 250-2000 KDa) HA preparations, obtained by extraction from rooster combs or bacterial fermentation, have been injected into joints for the treatment of OA.. To obtain a prolonged increase in the viscoelasticity of the synovial fluid, preparations of chemically crosslinked HA molecules (hylans), characterized by a very high MW (>6000 kDa), have also been created and proposed for intra-articular (i.a.) therapy 6.
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The 500-730 kDa hyaluronan (Hyalgan®, Fidia, Italy) is a preparation widely used for La. treatment of OA and a large body of clinical studies exists demonstrating the efficacy on OA symptoms of this specific HA fraction and the long duration of the effect 7. Recently, combined arthroscopic and histomorphometric studies on the structural features of human joint tissues 8-10 provided some evidence that Hyalgan® treatment was effective in reducing structural alterations typical ofOA. The results obtained in these studies are reviewed in the present paper and discussed with particular reference to the mechanism of action of the 500-730 kDa hyaluronan. PATIENTS AND METHODS Patients This paper describes data coming from two combined arthroscopic and histomorphometric clinical studies 8-10 designed to study the effect of 500-730 kDa hyaluronan (Hyalgan®, Fidia, Italy) on joint morphology in patients fulfilling the criteria of the American College of Rheumatology for the diagnosis of knee OA. The first trial was a 6-month open study carried out on 40 patients, while the second one was a randomized controlled study on 99 patients comparing the effect of HA and methylprednisolone (MP) 6 month following the treatment. Study design At the admission visit, after a check of the selection criteria and an evaluation of the clinical and radiological severity of OA (Kellgren-Lawrence), patients judged uncontrollable or unreliable, those with severe concomitant diseases making assessment of the result difficult, those receiving therapies during the last six months (apart from FANS), women in pregnancy or breast feeding were excluded from the trial. Patients judged eligible for the study underwent a first knee arthroscopy during which biopsies of cartilage and synovial membrane were obtained. After 15-20 days from the arthroscopic inspection, all the patients had a baseline visit with collection of blood and urine samples for routine laboratory assessments. Patients included in the first study then started on a course of i.a. injections of 2 ml Hyalgan® (10 mg/ml 500-730 hyaluronan in saline) once a week for 5 weeks. In the second trial this treatment was administered to half of the patients, while half of them were treated with I ml MP (Depo-Medrol®, Pharmacia & Upjohn, 40 mg/ml 6methylprednisolone acetate in saline) once a week for 3 weeks. In both studies on day 180 each patient underwent a second arthroscopic examination and a second biopsy was performed. During the second trial a normal control group of tissue samples was also obtained from 19 subjects who underwent arthroscopy for pain, but did not reveal any sign of either OA or rheumatoid arthritis. Arthroscopic examination In both studies arthroscopic and rnicroarthroscopic assessments performed under local anaesthesia used Hamou-Storz and Microview-Wolf arthroscopes. Entry was always effectuated antero-laterally with the knee flexed at an angle of 30°. Intermittent irrigation with Ringer acetate was used to optimize the degree of distension of the joint
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cavity. Microartrhroscopy of the synovial membrane was carried out following the injection of a 1% aqueous solution of methylene blue (3 ml, pH 4.5), followed after 5 minutes by a saline wash. The entire arthroscopic examination was always performed by the same investigator and documented using a video camera. All the videotapes were analysed by a second investigator using a blinded procedure in which the investigator was unaware of patient identity and chronology. To assess the degree and extension of cartilage damage for each compartment of the knee Outerbridge and Noyes scales 11-12 were used. The scoring system proposed by Pasquali Ronchetti et at. 13 was used for the grading of synovitis. Biopsy sampling During arthroscopy biopsies of both synovial membrane and cartilage were taken according to a series of standardized procedures described by Frizziero et at. 8. Briefly, the synovial membrane was sampled in the suprapatellar pouch and in the antero-medial compartment. Biopsies of articular cartilage were only taken from those patients with arthroscopic grade II lesions (i.e. cartilage marked by fibrillation, fissures, and a velvety aspect). Samples were taken from the edge of the lesions, which was easily identified by the more intense staining with methylene blue of the damaged area compared to healthy tissue. Since the same tissue region had to be sampled again during the final arthroscopic examination on day 180, a graded needleprobe was inserted under continuous arthroscopic control, using a second route of access,depending on the tissue compartment involved, to determine the precise sampling area. After excision biopsy samples from both synovial membrane and articular cartilage were immediately divided into two blocks and fixed for light and trasmission electron microscopy respectively according to standard procedures. Histological evaluations In both studies the histological evaluation of the synovial membrane was carried out estimating, by using established criteria and scores, the following parameters: lining cell arrangement, cell appearance (size, shape, vacuolization) and type, matrix features (fibrosis, oedema, necrosis), vessel number and appearance. In the second study a semiquantitative description of the synovial cell ultrastructure was also reported. A global assessment of cartilage damage was obtained in the first study using the well known Mankin scale. A quantitative morphometric approach was followed in the second study by using a computer assisted image analysis system (IBAS, Kontron, Germany) to estimate a set of widely accepted parameters in literature: surface roughness I~, chondrocyte density 15 and ultrastructure 16 . The mean thickness of the 'superficial amorphous layer were also measured and its appearance estimated by a fourpoint scale: O=absent; I=fragmented; 2=discontinuous; 3=compact. All of the evaluations were performed under blind conditions.
RESULTS Arthroscopy In the open study 8 at the final control (day 180), the mean (±SD) total score for the synovitis grading was 48.5±13.4 compared with 62.3±9.7 at baseline. This was equivalent to an improvement of 22%, the difference being highly significant (p
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paired t-test). As far as the individual patients were concerned, 32 exhibited a decrease in their synovitis grading, 3 were unchanged and 5 showed an increase. After HA treatment there was a decrease in the extension of cartilage lesions in 30% of the cases and in the grading in 17.5%. At the final control deterioration was observed in only 3 of the 40 patients. These data were confirmed in the controlled study 9-10 At the level of the synovial membrane HA treatment appeared comparable to that with MP in inducing a significant decrease of the inflammatory and hyperplastic features of the osteoarthritic synovial membrane. As shown in Fig. 1, however, the reduction in the degree and extension of cartilage damage was higher in HA treated patients when compared to MP treated patients. Synovial membrane histology In both studies at the final control (day 180) there was a significant difference in the thickness of the lining compared with baseline (p
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Cartilage histology
A global evaluation of the cartilagineous tissue was carried out in the first study using the Mankin scale. In the 67.5% of the subjects the total score was reduced at the final control compared with baseline. The item exhibiting the greatest degree of change was the stainability with safranin, which retlects the proteoglycan content of the matrix. A more datailed analysis was obtained in the controlled study. At the final control (day 180) a reduction of the typical osteoarthritic lesions was observed in about 80% of patients. Defects of the articular surface are still present but the surface roughness resulted improved alter HA treatment. Six months after treatment the number of viable chondrocytes appeared significantly increased (Fig.2) and the cells showed a shift towards a more anabolic activity, as indicated by the increased extension of the synthetic cytocavitary network and mitochondria with respect to the structures having catabolic or storage functions (Fig.3). As reported in Table I, the superficial amorphous layer appeared improved in its structure after HA treatment. Since this structure is mainly composed of endogenous hyaluronic acid synthetized by the synoviocytes, this result is consistent with the abserved results at the level of the synovial membrane. Table 1. Change from baseline of the parameters describing the superficial layer Parameter Vallie Final vs. Basal (Sgn Rank test) Compactness (score) 0.70 ± 0.22 p = 0.0050 p = 0.0020 Thickness (u) 0.28 ± 0.06 Mean change from bascline±Slandard Error and significance level after HA treatment
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DISCUSSION A large number of clinical studies have been carried out to date to determine the effects of 500-730 kDa HA therapy in OA and there is a broad consensus that this treatment is well tolerated and results in sustained relief of pain and functional impairment in patients suffering from this disease. The data from the morphological studies reviewed in the present paper provide preliminary evidence that some improvement in joint tissue integrity induced by the treatment could be at the basis of the long-term effects observed in the clinical trials. They further support the suggestion, coming from animal studies 17. that i.a. treatment with this specific MW fraction of HA may represent not only an efficacious symptomatic treatment for OA of the knee, but also a candidate for structure modification. With this respect they are consistent with the findings recently reported by an arthroscopic study showing a significantly lower degree of cartilage deterioration in comparison with a control group after treatment with 500730 kDa HA 18.
Remarks on the mechanism of action The term "viscosupplementation" has been coined 19 to describe the mechanism of action of HA in OA and is based only on the mechanical properties of the molecule. According to this paradigm the i.a. injected HA, staying in the joint compartments for a
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suitable period of time, restores their normal rheological state and provides protection, lubrication and mechanical stability to the tissues. Viscosupplementation could be at the basis of the effect obtained with high MW hyaluronans and hylans 20. The long-term effects obtained with 500-730 kDa HA, however, cannot be explained simply by a temporary restoration of the viscoelasticity, since it has been reported that this specific fraction of HA has a metabolic half-life of approximately 20 hours when injected in healthy knee joints and of about 12 hours in inflammed joints 21 . An interesting finding of the morphological studies reviewed in the present paper are the significant changes following treatment of the cellular features indicating an improved metabolic activity of synoviocytes and chondrocytes. This result is consistent with the growing evidence that HA is not an inert space-fiIler but a molecule with specific pharmacological properties zz In particular, several in vitro studies 23-26 suggest that HA in the MW range here considered could achieve its therapeutical effect predominantly through a direct modulation of the behaviour of the cell populations present in the joint. The ability of 500-730 kDa HA to control the inflammatory process by exercising an immunomodulating effect on the inflammatory cells, which renders them less active, has been documented in a cytological and cytofluorimetric study on cell populations present in the synovial fluid of patients with OA 23. The authors reported a decrease in cellularity of the synovial fluid and a significant reduction in activated lymphocytes and monocyte-macrophage phenotypes after treatment. In chondrocytes the interaction with exogenous HA through the CD44 receptor induced a variety of stimulatory signals, such as c-myc and TGF-B mRNA expression, involved in the maturation or differentiation of chondrocytes and regulating chondrocyte proliferation as well as matrix synthesis 24. Chondrocytes regulate the cartilage homeostasis by secreting various substances. In particular the balance between the activity of MMPs and their inhibitor (TIMP) is thought important for the manteinance of cartilage matrix. The MMP/TIMP ratio is therefore an index of cartilage degradation. It has been demonstrated 25 that 500 kDa hyaluronan is able to reduce such a ratio in chondrocytes cultured for 8 days in presence of IL 1B, suggesting a possible mechanism for the maintenance of cartilage integrity after treatment. An interesting effect of 500-730 kDa HA is the stimulation of endogenous HA synthesis by synoviocytes in vitro 26 It has been shown that synovial fibroblasts obtained from knee joints of patients with OA synthetized hyaluronic acid at a lower rate than cells derived from normal synovia. These fibroblasts respond to the presence of 500-730 kDa HA by increasing the biosynthesis of hyaluronic acid in a concentration dependent way. Once initiated such a process may be self-sustaining and may explain the prolonged effect of the substance. The possibility that the mechanisms described by in vitro studies occurs also ill vivo and could explain the observed results revolves about the ability of i.a. administered HA to keep contact with the cells in the joint tissues. Of course this is true when inflammatory cells in the synovial fluid are considered, but some data exist indicating that an interaction between synoviocytes or chondrocytes and 500-730 kDa HA (the lower range of MW among hyaluronans showing therapeutical efficacy) can occur in arthritic synovial joints. It was reported that, due to tissue fibrillation, i.a. administered HA could penetrate into the cartilage where it was found concentrated in the pericellular matrix of chondrocytes 27-28 . A better accessibility to synoviocytes of HA in the MW range here considered with respect to higher MW HA preparations (2300 kDa) was also
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reported 29 The authors found that the lower MW HA was intensely distributed in the synovial lining layer, while HA having higher MW was almost undetectable in the tissue, indicating a certain difference in the accessibility to the lining cells between the two HA molecules. Further support to the hypothesis of a different mechanism of action between high MW hyaluronans and HA in the lower MW range among the preparations having therapeutical efficacy comes from recent in vivo studies performed in an ovine model of OA It was reported that HA ofMW close to that here considered (900 kDa) stimulated the synovial cell in vivo synthesis of HA to a greater extent than when a HA preparation of higher MW was administered by the same protocol 30. In conclusion, the short half-life of 500-730 kDa hyaluronan in the synovial fluid suggest that the long-term therapeutical efficacy observed in the clinical studies is not principally due to a simple replacement of the depolymerized endogenous HA. Due to its MW, however it could have a quite good accessibility to synoviocytes and chondrocytes and the possibility to modulate their metabolism as indicated by a large body of data in literature. Thus, a principal mechanism of action of pharmacological nature has to be taken into account for HA with MW in the lower range among therapeutical hyaluronans.
REFERENCES 1. P.Dieppe, 'Osteoarthritis: risk factors, process and outcome', Rheumatology Europe, 1995,24,66-68. 2. GREES (Group for the Respect of Ethics and Excellence in Science - Osteoarthritis section), 'Reccornendations for the registration of drugs used in the treatment of osteoarthritis', Ann.Rheum.Dis., 1996, 55, 552-557. 3. D.Guerra, L.Frizziero, M.Losi, B.Bacchelli, G.Mezzadri, I.Pasquali Ronchetti, 'Ultrastructural identification of a membrane-like structure on the surface of normal articular cartilage', .LSubmtcroscop.Cytol.Pathol., 1996,28,385-393. 4. L.B.Dahl, LM.Dahl, AEnstrom-Laurent, K.Granath, 'Concentration and molecular weight of sodium hyaluronate in synovial fluid from patients with rheumatoid arthritis and other arthropaties', Ann.Rheum.Dis., 1985,44, 817-822. 5. J.G.Peyron, E.A Balazs, 'Preliminary clinical assessment of NA-hyaluronate injection into human arthritic joint', Pathol.Biol., 1974, 22, 731-736. 6. M.E.Adams,' An analysis of clinical studies of the use of crosslinked hyaluronan, hylan, in the treatment of osteoarthritis', J.Rheumatol., 1993, 20 (Supp.39), 16-18. 7. E.Maheu, 'Hyaluronan in knee osteoarthritis: a review of the clinical trials with Hyalgan@', Enr.LRheumatol.Irflamm., 1995, 15, 17-24. 8. L.Frizziero, E.Govoni, P.Bacchini, 'Intra-articular hyaluronic acid in the treatment of osteoarthritis of the knee: clinical and morphological study', Clin.Exp.Rheumatol., 1998, 16, 441-449. 9. LPasquali Ronchetti, D.Guerra, F.Taparelli, F.Zizzi & L.Frizziero, 'Structural parameters of the human knee synovial membrane in osteoarthritis before and after hyaluronan tratment', In: New frontiers in medical sciences: Redefining hya/uronan,G.Abatangelo & P.H.Weigel (eds.), Elsevier, Amsterdam, 2000, pp 119127. 10. D.Guidolin, I.Pasquali Ronchetti, E.Lini, D.Guerra & L.Frizziero, 'Morphological analysis of knee cartilage biopsies from a randomized, controlled clinical study comparing the effects of 500-730 kDa sodium hyaluronate (Hyalgan®) and
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methylprednisolone acetate in osteoarthritis', In: Redefining hyaluronan-Abstract book, Consorzio Tissue-Tech, Padova, 1999,48. 11. R.E.Outerbridge, 'The etiology of chondromalacia patellae', .l.Bone Joint Surg., 1961, 43B, 752-757. 12. F.R.Noyes, C.L.Stabler, 'A system for grading articular cartilage lesions at arthroscopy', Am.JiSport Med., 1989, 17, 505-513. 13. I.Pasquali Ronchetti, L.Frizziero, D.Guerm, 'Aging of the human synovium: an in vivo and ex vivo morphological study', Semin.Arthrttis Rheum., 1992, 21, 400-414. 14. S.AHacker, R.M.Healey, M.Yoshioka & RD.Coutts, 'A metodology for the quantitative assassment of articular cartilage histomorphometry', Osteoarthr. Cartil., 1997,5, 343-355. IS. P.S.Eggli, E.E.Hunziker & R.K.Schenk, 'Quantitation of structural features characterizing weight- and less weight-bearing regions in articular cartilage: a stereological analysis of medial femoral condyles in young adult rabbits', Anat.Rec., 1988, 222, 217-227. 16. M.Annefeld, 'A new test method for the standardized evaluation of changes in the ultrastructure ofchondrocytes', Int.J. Tiss.Reac., 1985,7,273-289. 17. ASchiavinato, E.Lini, D.Guidolin, G.Pezzoli, P.Botti, M.Martelli, RCortivo, ADe Galateo & G.Abatangelo, 'Intra-articular spodium hyaluronate injections in the Pond-Nuki experimental model of osteoarthritis in dogs. II.Morphological findings', Clin.Orthop.Rel.Res., 1987,241,286-299. 18. V.Listrat, A Xavier, F.Paternello, 'Arthroscopic evaluation of potential structure modifying activity of hyaluronan (Hyalgan) in osteoarthritis of the knee', Osteoarthr.Cartil., 1997, 5, 153-160. 19. E.ABalazs, lL.Dehlinger, 'Viscosupplementation: a new concept in the treatment of OA'. .LRheumatol., 1993,20 (Supp.39), 3-9. 20. M.E.Adams, 'An analysis of clinical studies of the use of crosslinked hyaluronan, Hylan, in the treatment of Oat eoarthritis' , Lliheumatol., 1993, 20(Supp.39), 16-18. 21. G.Abatangelo, M.O'Regan, 'Hyaluronan: biological role and function in articular joints', Eur.J. Rheumatol. luflamm., 1995, IS, 9-16. 22. LEntwistle, C.L.Hall, E.A Turley, 'HA receptors: regulators of signalling to the cytoskeleton', .J.Cell Biochem., 1996,61,569-577. 23. E.M.Corrado, G.F.Peluso, S.Gigliotti, C.De Durante, D.Palmieri, M.Savoia, G.O.Oriani, G.F.Tajana, 'The effects of intra-articular administration of hyaluronic acid on osteoarthritis of the knee: a clinical study with immunological and biochemical evaluations', Eur.J.Rheumatot.Inflamm; 1995, 15,47-56. 24.0.Ishida, Y.Tanaka, I.Morimoto, M.Takigawa, S.Eto, 'Chondrocytes are regulated by cellular adhesion through CD44 and hyaluronic acid pathway', 1. Bone Miner.Res., 1997,12, 1657-1663. 25. T.Yasui, M.Akatsuka, K.Tobetto, lUmemoto, T.Ando, K.Yamashita & T.Hayakawa, 'Effects of hyaluronan on the production of stromelysin and tissue inhibitor of metalloproteinase-l (TIMP-l) in bovine articular chondrocytes', Biomed.Res., 1992, 13,343-348. 26. M.M.Smith & P.Ghosh, 'The synthesis of hyaluronic acid by human synovial fibroblasts is int1uenced by the nature of hyaluronate in the extracellular environment', Rhellmatol.!nt., 1987,7,113-122. 27. K.Fukuda, H'Dan, M.Takayama, F.Kumano, M.Saitoh & S.Tanaka, 'Hyaluronic acid increases proteoglycan synthesis in bovine articular cartilage in the presence of interleukin-l', Ll'harmacol.Exp. Ther., 1996,277,1672-1675.
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28. TSakamoto, S.Mizuno, TMaki, 'Hyaluronic acid and articular cartilage', Orthopedic Rev.Sci., 1984, 11,264-266. 29. AAsari, S.Miyauchi, S.Matsuzaka, THo, E.Kominami & Y.Uchiyama, 'Molecular weight-dependent effects of hyaluronate on the arthritic synovium', Arch.Histol.Cytol., 1998,125-135. 30. P.Ghosh, N.Adam & C.Little, 'Effects of intra-articular hyaluronan therapy on the molecular characteristics of OA synovial fluid', In: Australian Rheumatology Association Meeting- Abstract book, ARA, Sidney, 1999.
PURIFICATION AND CHARACTERIZAnON OF THE HYALURONAN RECEPTOR FOR ENDOCYTOSIS (BARE) Paul H. Weigel!·, Carl McGary2, Bin Zhou!, and Janet A. Weigel! / Department ofBiochemistry & Molecular Biology. BMSB 860 University ofOklahoma Health Sciences Center. Oklahoma City, OK 73104, U.S.A. !
Department ofPathology, University ofRochester Medical Center Rochester. NY 1464. U.S.A.
ABSTRACT Liver sinusoidal endothelial cells (LECs) express two HARE proteins of 175 kDa and -300 kDa that endocytically clear hyaluronan (HA) from the circulation. We have characterized eight monoclonal antibodies (mAbs) raised against the partially purified 175 kDa HARE. Seven mAbs recognize the 175 kDa HARE after nonreducing SDS-PAGE and in all cases also crossreact with the -300 kDa HARE. Two of the mAbs inhibit 12Sr_ HA binding and endocytosis by LECs at 37°C. We purified these two HAREs and showed that the 175 kDa HARE is a single protein, whereas the -300 kDa species contains three subunits at 260, 230 and 97 kDa (Zhou, et al. J. BioI. Chem. m. 33831-33834, 1999). Two mAbs recognized both the two nonreduced HARE species and three of the four proteins present after reduction. The 97 kDa subunit was not recognized by any mAbs in Western blots. Based on their cross-reactivity with the mAbs against the 175 kDa HARE, the 175, 230 and 260 kDa proteins are related to each other. Immunocytochemistry using these mAbs shows high protein expression levels in rat liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Little or no HARE expression was observed in muscle, heart, lung, kidney, brain, skin, eye, pancreas, thymus, testis, adipose, salivary gland, adrenal, thyroid, larynx, tongue, stomach or intestine. We propose the name HARE (HA Receptor for Endocytosis) because this receptor mediates the very rapid endocytosis ofHA and its tissue distribution is not unique to liver. KEYWORDS Coated pits, hyaluronic acid, hyaluronan receptor, monoclonal antibodies, receptor mediated endocytosis, recycling receptor INTRODUCTION Hyaluronan (HA) is an extracellular matrix component of all tissues, in particular in cartilage, skin and vitreous humor. The total body turnover of HA in humans is several grams per/day', Two major clearance systems are responsible for HA degradation and removal in the body': the lymphatic system, which accounts for -85% of the HA turnover, and the liver, which accounts for almost all the other -15%. HA is continuously synthesized and degraded in almost all tissues. Large native HA molecules (_10 7 Da) are partially degraded to large fragments (_10 6 Da) that are released from the matrix and enter the lymphatic system, flowing to lymph nodes. The lymph nodes completely degrade the majority ofHA (-85%) by unknown mechanisms. The responsible cell types, the receptor involved, and the location in lymph nodes at which HA uptake and degradation occurs had
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not been determined through 1998. The remaining HA (-15%) that escapes degradation in the lymph nodes ultimately flows into the blood at the thoracic duct. Clearance of circulating HA by liver is likely important for normal health. since HA in solution is exceptionally viscous. Mammalian liver sinusoidal endothielial cells (LECs) express a recycling endocytic receptor that removes HA and other glycosaminofycans, including chondroitin sulfate, 2.5. from the blood Using a photoaffinity derivative ofHA and a novel ligand blot assay' with I2S I_HA, we previously identified two specific HA-binding proteins of 175 kDa and 300 kDa in isolated rat LECs as candidates for HARE. In 1999 we reportedt the first purification of these two HAREs, using a specific monoclonal antibody (mAb). The 175 kDa HARE is a single protein, whereas the 300 kDa species contains three subunits, at 260, 230 and 97 kDa, respectively. Here we summarize some of the characteristics of a panel ofmAbs raised against the 175 kDa HA Receptor for Endocytosis. MATERIAL AND METHODS Materials
HA (human umbilical cord) from Sigma, was purified as described previously", 1251_ HA was prepared using Iodogen (Pierce) and a unique hexylamine derivative of HA, modified only at the reducing end to contain an iodinatable hydroxyphenyl group", Other chemicals, which were reagent grade, were from Sigma. TBS contains 20 mM TrisHCl, pH 7.0, 150 mM NaCl. Preparation and culture of LEes and assay for HA endocytosis Male Sprague Dawley rats were from Harlan, Indianapolis, IN. LECs were isolated by a modified collagenase perfusion procedure'", followed by differential centrifugation and then discontinuous Percoll gradient fractionation. The LECs, were collected from the 25/50% interface and were washed twice with RPMI-1640 (GIBCO) containin~ penicillin/streptomycin (100 units each) and 2 mM glutamine and plated at 1.5-2 x 10 cellslml on fibronectin-coated (50 f,1g/ml) tissue culture plates. After incubation at 37°C for 2 h in a 5% C02 atmosphere, the cells were washed 3 times with PBS, once with RPMI-1640 and put back in RPMI-1640 without serum. Endocytosis assays were performed as described", Cultured LECs were washed and incubated for 60 min at 37°C with 125I_HA (2 ug/ml) in MEM medium containing 5 f,1g/ml of an affinity-purified antibody, isolated from ascites fluid using Protein-G-Sepharose, or immobilized mannan binding protein (Pierce) in the case of#159. The plates were chilled on ice, the media were aspirated, and the wells washed 3-times. The cells were solubilized in 0.3 N NaOH and radioactivity and protein content determined. Specific uptake, the fraction of endocytosed ligand competed with excess unlabeled HA, is ~ 90% in this assay. Monoclonal antibody production The starting antigen was a highly purified fraction of the 175 kDa HARE II. Mice were immunized with nonreduced or reduced protein, and boosted twice at two-week intervals. Standard procedures'f were followed for cell fusion, limited dilution cloning, and mouse ascites production. The hybridoma supernatants were screened using an ELISA assay with the 175 kDa antigen. Antibody isotypes were determined with the ISO Strip kit from Boehringer Mannheim.
Receptor for endocytosis (HARE)
403
Immuncytocbemistry Rat tissues were dissected within 15 min after sacrifice and fixed in 10% neutral buffered formaldehyde at room temperature for 2 h, processed and paraffin embedded overnight on a Tissue Tek V.I.P processor. Tissue sections (5 1J.Il1) were collected on charged slides, and dried at 60°C overnight. The slides were dewaxed 3 times for 3 min each with xylene, followed by 4 washes for 3 min each with alcohol (100%, 95%, 90%, 70%), followed by a single 2 min wash in water at room temperature. The endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 6 min, followed by two 2 min water washes. The slides were digested for 15 min at 37°C in pre-warmed O.IN HCl containing 0.32 mg/ml pepsin, followed by a 2 min water wash and a 2 min PBS wash. The slides were washed with PBS and incubated with the appropriate primary antibody (1:500) at room temperature for 60 min. After a 1 min PBS wash, the slides were treated with secondary antiserum (biotinylated horse anti-mouse, 1:200) for 30 min at room temperature. After another PBS rinse, the slides were incubated with streptavidinhorseradish peroxidase (1:1000 from Jackson Labs) for 30 min, washed once with PBS and once with distilled water. Color development was for 5 min with 2.0% (v/v) aminoethylcarbazine and hydrogen peroxide according to the manufacturer's instructions (ScyTek, Utah), followed by counterstaining with hematoxylin. Slides were viewed with an Olympus BX-40 light microscope equipped with an Olympus DPIO digital camera for photography. General Protein was determined by the method of Bradford l 3 using BSA as a standard. Receptor protein content was assessed after precipitation with 5% trichloroacetic acid to l4 remove detergent. SDS-PAGE was performed according to the method of Laemmli . l5. Western blotting procedures were performed essentially as described by Burnette 1251 radioactivity was measured using a Packard Auto-Gamma Counting system. RESULTS Development and characterization of mAbs to the rat 17SkDa HARE Two very specific HA-binding proteins can be detected in LEes by ligand blotting? using I25 I_HA; a result that corresponds perfectly to the previous identification of HARE on intact LECs using an HA photoaffinity derivative that specifically photo-labeled proteins of 175 kDa and 300 kDa6 • A partially purified 175 kDa fraction was used to immunize mice against the nonreduced or reduced protein. We isolated eight different hybridomas that were consistently positive in ELISA screens with the nonreduced or reduced 175 kDa antigen. Except for mAb-159 (lgM) and mAb-30 (lgG2b) all of the HARE-specific mAbs are IgGl. MAbs were prepared from ascites fluid produced by these hybridomas and tested for the ability to immunoprecipitate active 175 kDa HARE from LEC extracts, which shows that a mAb recognizes the bone fide 175 kDa HARE. These mAbs were also tested for their reactivity with a 175 kDa species in Western blots using whole rat LEC extracts. A summary of these results is shown in Table I. Each of the mAbs recognizes the rat LEC 175 kDa HARE and/or 300 kDa HARE in blots after either nonreducing or reducing SDS-PAGE (Table I). MAbs 54, 159 and 174 recognize both reduced HAREs in Western blots. Most mAbs against the nonreduced
404
Cell surfaces and hyaluronan receptors
Table I. Characteristics of mAbs against the rat HA Receptor for Endocytosis. The eight mouse mAbs raised against the rat liver 175 kDa HARE were tested for their usefulness (+, yes; -, no) as reagents: for immunoprecipitation or Western blot (WB) analysis of either the rat 175 kDa or 300 kDa HARE proteins; for inhibition of HA binding to LECs or to either HARE in a ligand blot assay; and for immunocytochemistry of HARE expression in rat or human tissues.
Pronertv Immunoprecipitation of the rat I75kDa HARE Immunoprecipitation of the rat 300kDa HARE Recognizes nonreduced rat I 75kDa HARE in WB Recognizes nonreduced rat 300kDa HARE in WB Recognizes reduced rat I75kDa HARE in WB Recognizes 260 kDa subunit of rat 300kDa HARE in WB Recognizes 230 kDa subunit of rat 300kDa HARE in WB Recognizes 97 kDa subunit of rat 300kDa HARE in WB Blocks HA uptake in rat LEes at 37-dewees Blocks HA binding to I75kDa HARE in blots Blocks HA binding to 300kDa HARE in blots Immunocytochemistry of rat tissues Immunocytochemistry of human tissues
28
Mouse Monoclonal Antibody Number 235 54 154 159 174 30
+ + +
+ + +
-
+
+
-
-
467
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+ + +
+ + +
+ + +
+ + +
-
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+
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-
-
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+
-
-
-
+
+
+
-
-
+
-
-
+
+
+
+
+
-
-
-
+ -+
175kDa HARE do not react with either HARE after reduction. The exceptions are mAb-159 and mAb-174, which recognize both the 175 kDa HARE and 300 kDa HARE proteins in Western blots, whether they are reduced or nonreduced. MAb-54 recognizes only the reduced HAREs. Six of the mAbs immunoprecipitated both HARE proteins from detergent extracts of LECs (Table I). Surprisingly, all mAbs that bind to the 175 kDa HARE species, the original antigen, also recognize the 300 kDa HARE species. However, the 300 kDa s~ecies is not a dimer of the 175 kDa protein and does not contain a 175 kDa subunit. The two proteins, therefore, share common epitopes. Characterization of the purified 175 kDa HARE and 300 kDa HARE 8
We have purified the two rat LEC HAREs for the first time using mAb-30 • The reduced 175 kDa HARE yields no other proteins, but its apparent size increases to -185 kDa. After reduction, the 300 kDa HARE gives three proteins with MrS of 97, 230 and 260 kDa. The molar ratio of these three subunits of the 300 kDa HARE is 1:1:1. The
Receptor for endocytosis (HARE)
405
molar ratio of the 175 kDa to the 300 kDa HARE is 2: I. The reduced 175 kDa HARE and the 260 and 230 kDa subunits of the 300 kDa HARE complex are recognized by the three mAbs that bind the reduced proteins (Table I). The 97 kDa subunit of the 300 kDa HARE is the only HARE protein not recognized by any of the mAbs. Our model for the organization of the rat HARE (Fig. I) is that the 175 and 300 kDa HAREs are isoreceptors. Although it is possible that there is one large "super HARE complex" containing both species, our working hypothesis is that the two isoreceptors have different affinities for, or ability to internalize, HA of larger versus smaller sizes,I I. Recent results with stably transfected cell lines expressing only the 175 kDa HARE confirm that this protein is a functional receptor in the absence of the larger HARE. Figure 1. Scheme for organization of the rat HARE. The three subunits of the rat liver 300 kDa HARE complex are held together by disulfide-bonds. The 175 kDa HARE species may by noncovalently associated with the 300 kDa HARE to form a large complex but both species are likely to be independent isoreceptors for HA, perhaps with different specificity for HA, depending on whether the HA size is larger or smaller.
-
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W" 300 kDa HARE Complex MAb-174 and mAb-235 specifically block the endocytosis of 125I _HA by rat LEes Endocytosis and accumulation of l2SI-HA at 37"C by cultured LECs was completely inhibited by mAb-174 (Fig. 2). MAb-235 consistently inhibited 125I_HA endocytosis
406
Cell surfaces and hyaluronan receptors
partially (-50%). None of the other anti-175 kDa HARE mAbs, irrelevant mAbs, or mouse serum affected HA uptake (not shown). These results verify that the anti-175 kDa HARE mAbs are specific for the bone fide HARE present in LECs. Interestingly, mAb174 also blocked HA binding to both the 175 kDa HARE and 300 kDa HARE in the ligand blot assay (Table I). Since mAb-174 completely blocks HA uptake by LECs and recognizes both proteins in Western blots, then both the 175 kDa and 300 kDa proteins are probably independent HAREs capable of mediating HA binding and endocytosis. Although mAb-174 inhibits 125I_HA binding and endocytosis by LEes at 37°C, it is important to point out that it does not block HA binding to live LECs at 4°C. Figure 2. MAb-174 and mAb-235 inhibit endocytosis of HA by LECs at 37°C. Cultured rat LECs were washed and incubated for 60 min at 37°C with 125 1_ HA (2 J.1g/ml) in MEM medium containing 5 J.1g/ml of purified mAb from each of the 9 indicated hybridomas. MAb-117 is a control mAb that is not specific for HARE. Data are the mean of triplicates, expressed as a percent of the specific HA uptake (DPMlmg protein) in the absence of any antibody. ::=:-
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Tissue distribution and immunolocalization of HARE Immunocytochemical analysis of various rat tissues indicated that the 175 kDa HARE and 300 kDa HARE proteins are highly expressed in lymph node, liver, and spleen (Fig. 3). Using each individual anti-HARE mAb, no significant staining was observed for brain, lung, heart, muscle, kidney, pancreas, testes, thymus, adipose, salivary gland, adrenal, skin, eye, thyroid, larynx, tongue, stomach or intestine. As expected HARE is localized to the sinusoids in liver (Fig. 3B). No staining was observed in parenchymal cells. Consistent with this cellular distribution, the protein is not expressed in isolated hepatocytes in culture, but is strongly expressed in purified, cultured LECsII. HARE is present in LECs in a pattern typical for an endocytic, recycling receptor; it is at the cell surface, in pericellular vesicles (presumably endosomes) and in the perinuclearregion, presumably ER and Golgi (not shown).
Receptor for endocytosis (HARE)
407
Figure 3. Immunocytochemical localization of HARE in rat tissues. Sections of rat liver (A and B), spleen (C) or lymph node (D) were incubated with mAb-30 against the 175 kDa HARE (B, C, and D) or normal mouse serum (A), stained and analyzed as described in Methods. In this black and white version, the stained areas are essentially black. The bar represents -50 !J.1l1; all panels are at the same magnification.
In rat spleen, the HARE proteins are present in the venous sinuses of the red pulp (Fig. 3C). No staining was observed in the germinal centers or white pulp of the splenic nodules. In rat lymph nodes, HARE is localized to the medullary sinuses (Fig. 3D). It is not present in the spheroid nodules, their germinal centers or in medullary cords. Vascular endothelial cells were not stained in any of the tissues examined. DISCUSSION
HA, a ubiquitous component of vertebrate tissues, is a linear unbranched alternating polymer composed of the two monosaccharides; P(I,4)-N-acetyl-D-glucosamine and P(I,3)-D-glucuronic acid. HA is not sulfated, not covalently attached to a protein core, and is typically 100-700 times the size of other glycosaminoglycan chains attached to 6 proteoglycans. The molecular weight of native HA can exceed 7 x 10 , The physicochemical and rheologic properties ofHA, particularly its viscoelasticity, are ideally suited for its specialized roles in skin, cartilage, and fluids such as in the vitreous humor of eye and synovium ofjoints. HA-binding proteins and receptors HA-binding proteins, or hyaladherins'", can be categorized as: enzymes, matrix
408
Cell surfaces and hyaluronan receptors
components, cell surface receptors and soluble plasma or intracellular molecules. Cell surface HA receptors include CD44, RHAMM, ICAM-I, and the endocytic receptor in LECs. Based on the nonexclusive tissue distribution of this latter endocytic HA receptor, it should no longer be called the liver (or LEC) HA receptor. We, therefore, proposed the more general name HARE, HA. Receptor for Endocytosis II. The HARE proteins identified here are distinct from all of the other cell surface receptors for HA, which do not appear to mediate endocytosis ofHA via the coated pit pathway. CD44 is a large diverse family of transmembrane glycoproteins found in lymphocytes, epithelial and endothelial ceJls, hemopoietic cells and in many types of cancer cells 17. CD44 has structural homology to cartilage link proteins, and some forms of CD44, although not all, bind HA 18 • RHAMM is a cell surface" and intracellular20 protein that mediates a cell migration response in the presence of HA. ICAM-I is a ceJl surface adhesion molecule that binds HA21. ICAM-1 was incorrectly identified as the LEC endocytic HA receptor by McCourt et al. 22. Although later explained as an artifacr", this report has not been formally withdrawn and subsequent papers24 ,2S based on these erroneous findings may still confuse the field. Clinical uses of HA and HA turnover in humans Due to its physical properties and nonimmunogenicity, HA is used in a large number of clinical applications. For example, surgeons worldwide use sterile solutions of pure, pyrogen-free, high molecular weight HA in ophthalmological procedures'", Many patients with osteoarthritis or rheumatoid arthritis now receive intra-articular injections ofHA and experience significant improvement". HA has also been used to heal perforated tympanic 28 membranes and restore hearing • HA has been used topically to reduce postoperative pericardial adhesions and as an aerosol to prevent elastase-mediated injury in pulmonary emphysema'", HA is also used as a vehicle for drug delivery". Because of the wide use of HA in medical applications, it is important that we understand the biological effects of HA and how its synthesis and degradation are regulated in humans. HA turnover and metabolism in mammals, including humans, is weJl understood at the whole body and organ levee. Mice 3 ! and rabbits 32 rapidly remove injected 3H_HA from the blood and concentrate it in liver and, to a lesser extent, in spleen and lymph nodes. Based on our results 8•11, we conclude that the same HARE found in liver is present and functional in spleen and lymph node. HA is continuously synthesized and secreted by fibroblasts, keratinocytes, chondrocytes and other specialized cells throughout the body. Despite the daily turnover of up to 5 g of HA, the clearance systems in lymph and liver keep the normal steady-state concentration ofHA in blood very low (i.e. 10-100 ng/ml). Lymph node expresses multiple HA receptors Banerji et at. 33 recently discovered a lymph-specific homologue of CD44 that is able to bind HA. This lymph HA receptor, designated LYVE-I, is localized to the luminal face of lymph vessel walls in the lymphatic system. It is not present on blood vessels. Like CD44, LYVE-l does not appear to be a recycling endocytic receptor designed for the continuous and efficient internalization of HA. However, these two lymphatic HA receptors, HARE and LYVE-l, could work together to create an effective mechanism for the removal of HA from lymph, especiaJly if the HA levels in the entering lymph varied substantially. LYVE-l could bind HA from afferent lymph and create a reservoir of sequestered HA. Although HA bound to LYVE-I would only be internalized very slowly, it would nonetheless be removed from circulating lymph. When this bound HA
Receptor for endocytosis (HARE)
409
dissociated and reentered the sinusoids of the lymph node, it could then be bound and endocytosed by HARE. HARE would continuously clear HA from lymph but if the HA concentration saturated this system, the reservoir capability of LYVE-I would minimize the amount ofHA that escaped the node in efferent lymph and entered the blood. Our results now indicate that HA and chondroitin sulfate clearance by the two main systems in mammals, lymphatic tissues and liver, are mediated by the same HA receptor, HARE. Further molecular analysis at the eDNA, mRNA and genome levels is in progress to determine if there is a single HARE or a family of closely related proteins and genes. In summary, our results support the conclusion that mammals express an endocytic HA receptor in the sinusoids of liver and lymphatic tissues, which is a localization well suited to keep the systemic blood and lymph levels of HA very low. Liver, spleen and lymph node clearly express large amounts of HARE for this purpose. More sensitive methods, such as RT-PCR, will be required to determine whether other tissues express substantially lower, but significant, levels of HARE.
ACKNOWLEDGEMENTS We thank Anil Singh and Brian Duff for technical assistance. This research was supported by grant GM35978 (to PHW) from the National Institutes of Health.
REFERENCES 1. D. Evered & J. Whelan, The biology ofhyaluronan, Ciba Fnd. Symp.,1989, 143, 1-298. 2. T. C. Laurent & J. R. E. Fraser, Hyaluronan, FASEB J., 1992,6,2397-2404. 3. R. H. Raja, C. T. McGary & P. H. Weigel, Affinity and distribution of surface and intracellular hyaluronan receptors in isolated rat liver endothelial cells, J Bioi Chem., 1988, 263, 16661-8. 4. C. T. McGary, R. H. Raja & P. H. Weigel, Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling. Biochem J., 1989,257, 875-84. 5. C. T. McGary, J. Yannariello-Brown, D. W. Kim, T. C. Stinson & P. H. Weigel, Degradation and intracellular accumulation of a residualizing hyaluronan derivative by liver endothelial cells, Hepatology, 1993, 18, 1465-76. 6. J. Yannariello-Brown, S. J. Frost & P. H. Weigel, Identification of the Ca2+-independent endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells using a photoaffinity cross-linkingreagent, J Bioi Chem., 1992, 267, 20451-6. 7. J. Yannariello-Brown, B. Zhou & P. H. Weigel, Identification ofa 175 kDa protein as the ligand-binding subunit of the rat liver sinusoidal endothelial cell hyaluronan receptor, Glycobiology, 1997,7, 15-21. 8. B. Zhou, J. A. Oka, A. Singh & P. H. Weigel, Purification and subunit characterization of the rat liver endocytic hyaluronanreceptor, J Bioi Chem., 1999,274,33831-4. 9. R. H. Raja, R. LeBoeuf, G. Stone & P. H. Weigel, Preparation of alkylamine and 1251_ radiolabeled derivatives of hyaluronic acid uniquely modified at the reducing end, Anal Biochem., 1984, 139, 168-77. 10. J. A. Oka & P. H. Weigel, Monensin inhibits ligand dissociation only transiently and partially and distinguishes two galactosyl receptor pathways in isolated rat hepatocytes, J Cell Physiol., 1987, 133,243-52,257. II. B. Zhou, J. A. Weigel, L. Fauss & P. H. Weigel, Identification of HARE, the HA receptor for endocytosis, J Bioi Chem., 2000, 275, 37733-41. 12. E. Harlow & D. Lane, Antibodies: A laboratory manual, Cold Spring Harbor Laboratory. 13. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem., 1976, 72, 248-54. 14. U. K. Laernmli, Cleavage of structural proteins during the assembly of the head of
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bacteriophage T4, Nature, 1970, 227, 680-5. 15. W. N. Burnette, "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A, Anal Biochem., 1981, 112, 195-203. 16. B. P. Toole, Hyaluronan and its binding proteins, the hyaladherins, CU" Opin Cell Bioi.. 1990,2,839-44. 17. J. Lesley, R. Hyman & P. W. Kincade, CD44 and its interaction with extracellular matrix, Adv Immunol., 1993,54,271-335. 18. J. Bajorath, B. Greenfield, S. B. Munro, A. J. Day & A. Aruffo, Identification of CD44 residues important for hyaluronan binding and delineation of the binding site, J Bioi Chem., 1998,273,338-43. 19. E. A. Turley, A. J. Belch, S. Poppema & L. M. Pilarski, Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes, Blood•. 1993,81,446-53. 20. M. Hofinan, C. Fieber, V. Assmann, M. Gottlicher, J. Sleeman, R. Plug, N. Howells, O. Von Stein, H. Ponta & P. Herrlich, Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein, J Cell Sci., 1998, Ill, 1673-84. 21. J. S. Hayflick, P. Kilgannon & W. M. Gallatin, The intercellular adhesion molecule (ICAM) family of proteins. New members and novel functions, Immunol Res., 1998, 17, 313-27. 22. P. A. G. McCourt, B. Ek, N. Forsberg & S. Gustafson, Intercellular adhesion molecule-I is a cell surface receptor for hyaluronan, J Bioi Chem., 1994,269,30081-4. 23. P. A. G. McCourt & S. Gustafson, On the adsorption of hyaluronan and ICAM-l to modified hydrophobic resins, Int J Biochem Cell Bioi. 1997,29, 1179-89. 24. S. Gustafson, T. Bjorkman, N. Forsberg, T. Lind, T. Wikstrom & K. Lidholt, Accessible hyaluronan receptors identical to ICAM~1 in mouse mast-eell tumours, Glycoconj J., 1995, 12,350-5. 25. K. Fuxe, L. F. Agnati, B. Tinner, N. Forsberg, P. McCourt & S. Gustafson, Evidence for receptors for hyaluronan in discrete nerve cell populations of the brain, Brain Res., 1996, 736,329-37. 26. K. L. Goa & P. Benfield, Hyaluronic acid. A review of its pharmacology and use as a surgical aid in ophthalmology, and its therapeutic potential in joint disease and wound healing. Drugs, 1994,47,536-66. 27. J. P. Pelletier & J. Martel-Pelletier, The pathophysiology of osteoarthritis and the implication of the use of hyaluronan and hylan as therapeutic agents in viscosupplementation, J RheumatolSuppl., 1993, 39, 19-24. 28. C. Laurent, S. Hellstrom & E. Fellenius, Hyaluronan improves the healing of experimental tympanic membrane perforations. A comparison of preparations with different rheologic properties, Arch Otolaryngol Head Neck Surg., 1988, 114, 1435-41. 29. J. O. Cantor, J. M. Cerreta, G. Armand & G. M. Tutino, Aerosolized hyaluronic acid decreases alveolar injury induced by human neutrophil elastase, Proc Soc Exp Bioi Med., 1998,217,471-5. 30. S. T. Lim, G. P. Martin, D. J. Berry & M. B. Brown, Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan, J Control Release, 2000, 66, 281-92. 31. J. R. Fraser, L. E. Appelgren & T. C. Laurent, Tissue uptake of circulating hyaluronic acid. A whole body autoradiographic study, Cell Tissue Res. 1983,233,285-93. 32. J. R. Fraser, T. C. Laurent, H. Pertoft & E. Baxter, Plasma clearance, tissue distribution and metabolism ofHA injected intravenously in the rabbit, Biochem J.. 1981,200,415-24. 33. S. Banerji, J. Ni, S. X. Wang, S. Clasper, J. Su, R. Tammi, M. Jones & D. G. Jackson, LYVE-I, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan, J Cell Bioi., 1999, 144, 789-801.
EFFECTS OFINTRA-ARTICUIARINJECTION OFHYALURONAN ON PAPAIN-INDUCED HYDRARllIROSIS OF KNEEINRABBITS
lMedical research Pharmacology, Tokyo Research Institute, Seikagaku Corporation, 3-1253,Tateno, Higashi-yamatoshi, Tokyo, Japan.
ABSTRACT Intra-articular injection of sodium hyaluronan (Na-HA) is widely applied in the treatment of osteoarthritis. Na-HA reduces hydrarthrosis accompanied with improvement of a synovitis. However, the molecular weight dependence or the mechanism of the effect of Na-HA on the hydrarthrosis is unclear. The objective of the present study is to investigate an effect of various molecular weights of Na-HA on an experimental acute hydrarthrosis of knee in rabbits. Highly purified Na-HA preparations driven from chicken combs, their average molecular weight 30x10 4 (30Na-HA group), 84x104 (84Na-HA group) and 190x10 4 (190Na-HA group), were used. The experimental acute hydrarthrosis was induced by intraarticular injection of papain at 1.5mg/150 /.i I/joint. On the next day, Na-HA was injected one time intraarticuiarly at a dose of 1.5mg/150 u. l/joint, Animals were sacrificed on the seventh day after the intraarticular injection of Na-HA. Synovial fluid volume (SFY), total protein (TP), hyaluronan (HA) and chondroitin 4-sulfate (C4S) in the synovial fluids were determined. SFY (p
Hyaluronan, hydrarthrosis, synovitis, model INTRODUCTION Intraarticular injection of sodium hyaluronan (Na-HA, ARTZ®) , average molecular weight of 84 X 104, is widely applied in the treatment of osteoarthritis 1,2. Na-HA improves articular functions accompanied with the reduction of hydrarthrosis. Hydrarthrosis, a general symptom together with joint pain in osteoarthritis and other types of arthritis, may be due to synovitis. It has been reported the effects of Na-HA on the release of proteoglycan in cultured chondrocyte 3 and on denatured cartilage in the experimental arthritis models 4,5. Little is known, however, on the effect of Na-HA on
364
Aspects of hyaluronan in joints
hydrarthrosis and synovitis. In this study, we produced an experimental synovitis model induced by intra-articular injection of papain in the rabbit knee. This arthritis is a transitory acute inflammation of knee joint. The objective of the present study is to investigate the effect of various molecular weights of Na-HA on papain-induced acute hydrarthrosis of knees in rabbits.
MATERIALS & METHODS Hyaluronan preparations Hyaluronan (Na-HA) preparations were extracted from chicken combs and highly purified; average molecular weight of 30x104 (30 Na-HA group), 84x104 (84 Na-HA group) and 190x104 (190 Na-HA group). Na-HA was dissolved in a phosphate buffered physiological saline (PBS) at a concentration of 1%(10 mg/ml).
Experimental animals Thirty female Japanese White rabbits weighing 2.7",,3.3 kg were used in this study.
Experimental synovitis Experimental Synovitis, which is a transitory acute inflammation of knees, was induced by intra-articular injection of papain. Papain powder (2.8U/mg solid, Sigma chemical Co., St Lous, MO. USA) was dissolved in physiological saline containing 0.8mg/ml of L-Cysteine (Junsei Chemical co., Ltd, Japan) at a concentration of lOmg/ml. The solution was filtered using a membrane with a pore size of 0.45um, and 0.15 ml of the filtrate was injected with a 27gage-injection needle into each joint cavity of both knees. The dose of papain administrated was 1.5 mg/joint.
Experimental design One day after intra-articular injection of papain, a total of thirty rabbits, ten rabbits in each group, were injected intra-articularly with a corresponding Na-HA preparations. The Na-HA was injected into the left joint cavity at a dose of 50 Ili/ 0.5mg/kg/ joint. As a control, PBS was injected into the right joint cavity with the same volume. Seven days after the intra-articular injection of Na-HA, animals were sacrificed with intravenous injection of a fatal dose of pentobarbital sodium.
Specimens sampling Plasma was separated by centrifugation at the time of sacrifice. After the animals were sacrificed, both knee joints were isolated. The lavages of the joint cavity were collected using 2mlx3times of physiological saline. The volumes collected were measured accurately. Both knees were opened and the cartilage of the medial femoral was sliced with a blade.
Synovial fluid volume The synovial fluid volumes (SFV) were estimated by measuring the endogenous calcium concentration. The calcium concentration is maintained rigidly at a constant
Effects or injection on hydrarthrosis of knee
365
level in the body fluid. Calcium concentrations in the plasma and total calcium content of the lavage of the joint cavity were colorimetrically determined (Calcium C-Test Wako Kit, Wako Pure Chemical Industries Ltd., Osaka. Japan). SFV was estimated from the following formula. Total calcium content of the lavage of the joint cavity (fJ, gljoint) X 1000 SFV (fJ, l/joint) == Calcium concentration in plasma( u. g/rnl) Total protein in the synovial fluid Total proteins (TP) in the synovial fluid were determined by Lowry's method
6.
Hyaluronan (HA) and chondroitin 4-sulfate (C4S) in the synovial fluid HA and C4S in the synovial fluid were digested with chondroitinase ABC and AC- II (Seikagaku Co.) as previously described 7. After ultrafiltration of the digestive the mixtures, the LI Di-HA and zl Di-C4S in filtrates obtained were analyzed by high performance liquid chromatography. Chondroitin sulfate (CS) in cartilage After measurement of the dry weights, the cartilage slices were digested with Actinase-E (2.0ml mM sodium acetate buffered pH 6.0). CS concentrations in the cartilage were determined with HPLC in the same way as described above. Statistical analysis Statistical analysis of this study was performed using a statistics aid system SAS (SAS Institute Japan Inc., Tokyo). The mean value and standard deviation was calculated for each group. The comparison between each of the Na-HA groups and PBS group was carried out by paired Student's t-tcst with the level of significance at p<0.05. RESULTS & DISCUSSION 30 Na-HA and 84 Na-HA suppressed the increase of synovial fluid volume (fig.l, p
366
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Effects of injection on hydrarthrosis ofknee
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Figure 4. C4S in the synovial fluid. (values: mean±.S.D., n=lO, *: p<0.05 versus control by paired student's t-test, Normal range; 4.54±0.50nmolljoint) 30 Na-HA and 84 Na-HA preparations improved the hydrarthrosis, but not 190 Na-HA. The smaller sized molecule of Na-HA, 84xlO\ was more permeable in synovial tissue than the larger sized molecule, 230xlO4, in the experimental canine arthritic synovium 12. The results suggest that the accessibility of the Na-HA molecules differ to synovial cells depending on the molecule size. The smaller sized molecules, 30 Na-HA and 84 Na-HA, may be more effective on synovitis than the larger sized molecule, 190 Na-HA. No clear effect of Na-HA on denatured cartilage was observed in this study (fig.5). At the time of Na-HA injection, the level of CS in the cartilage completely disappeared. Under these severe conditions, the effect of a single injection of Na-HA may not be sufficient. It seems likely that a greater more number of Na-HA injections times are needed to recover the proteoglycan in denatured cartilage.
100 Ii ..; 80
.....
::::: 0
Ii
..5
60
U>
u
0
II cont Na-HA 30.10 ..
cont Ha-HA IblO ..
m ".-HA --,110.10
cont
Figure 5. CS in the cartilage. (mean ± .S.D., n=lO, Normal range; 174± 26nmollmg of dry weight, d.w.: dry weight)
368
Aspects of hyaluronan in joints
CONCLUSION 30 Na-HA and 84 Na-HA suppressed the increase of synovial fluid volume, the level of protein and C4S in the experimental papain-induced synovitis, but not 190. These results suggest that 30Na-HA and 84 Na-HA may directly act on the inflamed synovium and the optimal molecular size of Na-HA may be present in the improvement of hydrarthrosis in the experimental synovitis model. REFERENCES 1. Dixon ASJ, Jacoby RK., Berry H, Hamilton EBD, 'Clinical trial of intra-articular injection of sodium hyaluronate in patients with osteoarthritis of the knee', Curr Med Res Opin., 1988, 11: 205. 2. Peyron JP., Balazs EA. 'Preliminary clinical assessment of Na hyaluronate injection into human arthritic joints', Path. Biol., 1974,22, 731. 3. Shimazu A, Jikko A, Iwamoto M, et al,. 'Effects of hyaluronic acid on the release of proteoglycan from the cell matrix in rabbit chondrocyte culture in the presence and absence of cytokines', Arthritis Rheum., 1992,36: 247-53. 4. Yoshioka M, Shimizu T, Harwood FL., Coutts RD. and Amiel D., 'The effects of hyaluronan during the development of osteoarthritis'. Osteoarthritis Cart., 1997, 5: 251-260. 5. Shimizu T, Yoshioka M, Coutts RD., et ai, 'Long-term effects of hyaluronan on experimental osteoarthritis in the rabbit knee', Osteoarthritis Cart, 1998,6: 1-9. 6. Lowry, 0., Rosebrough, N. J., Farr & Raudall, 'Protein measurement with folin phenol reagent. Jouma of Biological Chemistry', 1951, 193: 265-275. 7. Shinmei M, Miyauchi S, Machida A, Miyazaki K, 'Quantitation of chondroitin 4-sulfate and chondroitin 6-sulfate in pathologic synovial fluid', Arthritis Rheum., 1992,35: 1304-8. 8. Goldberg RL and Toole BP, 'Hyaluronate inhibition of cell proliferation'. Arthritis Rheum., 1987,30: 769 -778. 9. Tamoto K, Nochi H, Tokumitsu Y, 'High molecular weight hyaluronic acids inhibit interleukin-I-Induced prostaglandin E2 generation and prostaglandin E2-elicited cyclic AMP accumulation in human rheumatoid arthritic synovial cells', Jap J Rheumatol., 1994,5: 227-36. 10. Mourao PAS, 'Distribution of chondroitin 4-sulfate and chondroitin 6-sulfate in human articular and growth cartilage', Arthritis Rheum., 1988,31: 1028-33 11. Huang Y, Tadano H, Toida T, Imanari T, 'Determination of chondroitin sulfates in human whole blood, plasma, blood cells by high-performance liquid romatography. Biomedical Chromatography, 1995,9: 102-105. 12. Asari A, Miyauchi S, Matsuzaka S, Ito T, Kominami E and Uchyama Y, 'Molecular weight-dependent effects of hyaluronate on the arthritic synovium', Arch. Histol. Cytol., 1998,61: 125-135.
IDENTIFICATION OF HYALURONAN AS CRYSTAL BINDING MOLECULE AT THE SURFACE OF MIGRATING AND PROLIFERATING MDCK CELLS Carl F Verkoelen*, Burt G vd Boom, Marieke SJ Schepers and Johannes C Romijn Depanment of Urology. Erasmus University Rotterdam, iNl Be330. P.O.Box 1738.3000 DR Rotterdam, The Netherlands.
ABSTRACT The adherence of calcium oxalate crystals to the renal tubule epithelium is considered a critical event in the pathophysiology of calcium nephrolithiasis. Calcium oxalate monohydrate crystals (COM) cannot adhere to the surface of a functional MDCK monolayer but they bind avidly to the surface of proliferating and migrating cells. To identify crystal binding molecules (CBMs) at the surface of crystal attracting cells we applied metabolical labeling protocols in combination with differential enzymatic digestion and gel filtration, which was compared with 4 C]COM crystal binding and confirmed by confocal microscopy. The indication that hyaluronan (hyaluronic acid, HA) might act as a CBM in subconfluent cultures came from studies with glycosaminoglycan (GAG)-degrading enzymes. Subsequently, metabolic labeling studies revealed that hyaluronidase cleaved significantly more radiolabeled glycoconjugates from crystal attracting cells than from cells without affinity for crystals. During wound repair crystal binding could be prevented by pretreating the healing cultures with hyaluronate lyase, an enzyme that specifically hydrolyses HA. Binding to immobilized HA provided evidence that COM crystals physically can become associated with this polysaccharide. Finally, confocal microscopy demonstrated that fluorescently labeled HA binding protein (HABP) adhered to the surface of proliferating cells in subconfluent cultures as well as to cells involved in closing a wound, but not to cells in confluent monolayers. These results identify HA as binding molecule for COM crystals at the surface of migrating and proliferating MDCK cells.
e
KEYWORDS Nephrolithiasis, MDCK, crystal-cell interaction, hyaluronan, wound repair. INTRODUCTION Urine is frequently supersaturated with respect to calcium salts, which occasionally leads to the spontaneous nucleation of crystals that are washed out
412
Cell surfaces and hyaluronan receptors
unhindered with the urine. Crystal adherence leads to the retention of crystalline material in the kidney and ultimately to stone formation'P:'. The mechanisms by which crystals adhere to the renal tubule epithelium are poorly understood and difficult to study in vivo. Cell culture4.s,6 and animal models':" have been employed to reconstruct the transitory and spatial events contributing to the pathogenesis of renal stone disease. The purpose of this study is to reveal the identity of binding molecules for crystals at the surface of renal tubular cells in culture.
MATERIALS & METHODS Cell culture, Preparation of CaOx crystals, Crystal binding, Wounds made in confluent monolayers, and Metabolic labeling protocols. Described in detail elswhere't". Enzyme treatment Cells were treated with enzymes dissolved in DMEM at 37°C. Hyaluronidases: hyaluronoglucosaminidase (EC 3.2.1.35), 25 Vlml, pH 5.5, Ih., hyaluronate lyase (EC 4.2.2.1), 5 Vlml, pH5.5, 1h, Heparinase: heparitinase I (EC 4.2.2.8),0.2 Vlml, pH 7.4, 4h, Chondroitinase: chondroitin ABC lyase (EC 4.2.2.4),0.1 U/ml , pH 7.4, 1h. All enzymes derived from Sigma-Aldrich Chemie, Zwijndrecht, the Netherlands. Hyaluronan staining using hyaluronic acid binding protein (HABP) Cells were fixed in 5% glacial acetic acid, 10% formalin and 70% ethanol (v/v), washed and incubated overnight at 4°C with 5 J.!g/ml bHABP in 3% BSA. The bHABP bound to cell surface hyaluronan which was visualized by confocal microscopy after a 1h incubation in avidin-FITC (1:250) and embedding into Vectashield. To monitor the location of the cells in the sample, the nuclei were counterstained with propidiumiodide which appears red in the images. Crystal binding to plastic surfaces precoated with HA The wells of 24-well tissue cell culture cluster plates were coated with HA by adding 0.5 ml/well of a solution containing increasing amounts of HA in PBS and incubating the plate at 4°C overnight. Unbound HA was removed by extensive washing with PBS and nonspecific binding sites were blocked with Iml 1% BSA. Approximately 150 J.!g [14C]COM crystals were then added to the well in a final volume of 0.5 ml. After an incubation period of 30 min, all non-adhered crystals were removed by extensive washings and the remaining adhered crystals were dissolved in 0.5 ml 1 N HCl. After neutralization with 0.5 ml I N NaOH the amount of surface associated radioactivity was determined in a scintillation counter. Crystal-cell interaction by confocal laser scanning microscopy Cells were fixed in 3.7% formaldehyde for 15 min. and then permeabilized for 15 min. with 70% ethanol. Subsequently, the inserts were washed with PBS, cut out
Crystal binding molecule
413
and incubated for 15 min. with 5 ug/ml fluorescein isothiocyanate conjugated phalloidin (FITC-phalloidin) at the apical site, washed 2x3 min. with PBS and mounted in Vectashield. Images were made with a Zeiss LSM 410 laser scanning confocal microscope. A 488 nm Ar-laser was used to excitate the FITC-phalloidin. COM crystals were detected by their reflection of the 633 nm (red) Kr-laser.
RESULTS & DISCUSSION Metabolic labeling and COM binding
Removal of [3H]glucosamine-labeled glycoconjugates with Hdase from the surface of MDCK cells in subconfluent and confluent cultures, and its effect on C4C]COM crystal binding (Fig. I). Closed bars are untreated controls and open bars are Hdase treated cultures. Expressed per cell, comparable amounts of radiolabeled glycoconjugates were extruded spontaneously 2 and 7 days post-seeding (A, closed bars). Hdase cleaved significantly more radioactively labeled material from the surface of cells in subconfluent cultures than from cells in confluent monolayers (A, open bars). The level of C4 C]COM crystal binding is much higher to proliferating cells in subconfluent cultures than to growth-arrested cells in confluent cultures (B, closed bars). Treatment of the cells with Hdase significantly reduced the adherence of crystals to undifferentiated cells in subconfluent cultures, whereas the enzyme did not affect the already low levels of crystal binding observed to differentiated cells in confluent cultures (B, open bars). These results clearly demonstrate that Hdasesensitive crystal binding molecules (CBMs) are expressed at the surface of proliferating undifferentiated cells, but not on the surface of differentiated cells in an intact epithelium. Release (dpmx10
3/10 6cells)
COM binding(JJg/cm
'0
A
2)
B '0
20
Days post-seeding
Dayspost-seeding
Fig.I Metabolic labeling and COM binding
414
Cell surfaces and hyaluronan receptors
Expression of hyaluronan at the surface of cells in subconfluent cultures The presence or absence of HA at the surface of MDCK cells was studied by CLSM using biotinylated hyaluronan binding protein (bHABP) after coupling to avidin-FITC (green). Cells are visualized by counterstaining the nuclei with propidiumiodide (red). FITC-bHABP was found abundantly attached to the surface of undifferentiated (crystal binding) cells in subconfluent cultures (2 days post-seeding), which could be avoided by pretreating the cultures with hyaluronidase. HABP did not or only hardly attach to the surface of differentiated (non-crystal binding) cells in confluent, 7 days post-seeding monolayers (not shown). Effect of GAG-degrading enzymes on [14C]COM crystal-cell interaction Table 1. COM binding expressed in J.Lg/cm2 to proliferating MDCK cells (two days post-seeding) in subconfluent cultures compared to untreated controls. COM binding (J.Lg/cm 2)
Control
7.09 ± 0.92
Hyaluronidase
2.64 ± 0.15
Hyaluronate lyase
3.16±0.09
Chondroitinase ABC
2.23 ± 0.09
Heparinase III
7.45 ± 0.24
Crystal binding to hyaluronan during repair We studied the possibility that the enhanced susceptibility of the cells for crystal binding during wound healing is mediated by surface exposed HA. Confluent monolayers were mechanically damaged by the removal of approximately 30% of the cells from the growth substrates. We reported earlier that during 1-3 days post-injury crystals can adhere to cells that are migrating and proliferating in order to close the wounds (9). The results obtained from C4C]COM crystal binding studies with GAGdegrading enzyme treated healing cultures were comparable to those obtained with cells in subcontluent cultures. The only remarkable difference was that HA degradation resulted a much more complete reduction of crystal binding capacity in wounded cultures than in subconfluent cultures (not shown). This suggests that crystals adhere exclusively to cell surface associated HA in wounded cultures, whereas also other types of CBMs are present at the surface of cells in subconfluent cultures.
Crystal binding molecule
415
Cell surface expression of hyaluronan during wound healing Intact MDCK-I monolayers were mechanically injured and allowed to recover in time. To demonstrate that HA is indeed expressed at the surface of MOCK cells during wound healing, confocal microscopy images of FITC-bHABP binding were made for a period of 0-4 days after injury. HA was found to be present in the crossshaped re-epithelialized zones but could not be detected in undamaged areas within the same cultures. bHABP was unable to adhere to bare growth substrates (not shown). Directly after removing cell strips from confluent cultures, bHABP could not bind to the cells at the edge of the wound. Although occasional binding was observed to the growth substrate from which cells were scraped, these binding patterns were rather vague. One day post-injury, HA was found associated with the surface of flattened cells at the border of the wound. Two days post-injury HA was expressed at the surface of cells in the center of the healing wounds and at the surface of cells that formed a scarr at sites where the wounds were closed. Four days post-injury the epithelium had completely re-established its functionality and bHABP could no longer or only sparsely bind to the cells in the monolayer (not shown). Crystal binding to plastic wells coated with hyaluronan The affinity of HA molecules for COM crystals was investigated by measuring the binding of C4C]COM crystals (70 J.l,g) to the surface of plastic wells that were coated with increasing concentrations of HA (ranging from 0 to 5000 J.l,g/ml). A linear relationship was found between the amount of radiolabeled crystals that was able to adhere and HA concentrations used to coat the wells. Maximal binding was observed to wells coated with approximately 500 J.l,g HA/ml. Prior to the addition of the crystals, the HA-coated wells were washed extensively with BSA-containing PBS to cover all non-specific binding sites. Crystals were practically unable to become associated with wells treated only with PBS/BSA. The adherence of the crystals to HA (500 J.l,g/ml) pre-coated wells could be prevented by treating HA-coated wells with Hdase shortly before the addition of [14C]COM, indicating that COM crystals become specifically associated with HA. Glycosaminoglycans such as chondroitinsulfate and heparansulfate are present at the cell surface as the polysaccharide side chains of surface-associated proteoglycans. Because of their negative charge and known affinity for calcium crystals, these polysaccharides have often been proposed as candidate CBMs. A potential role for HA in crystal-cell interaction was largely ignored possibly because this non-sulfated GAG is not incorporated into proteoglycans and is no common constituent of the epithelial cell surface. Nevertheless, our results clearly identify HA as CBM at the surface of developing MDCK-I cells in subconfluent cultures or during wound healing. HA is an anionic high-molecular-mass GAG composed of repeating disaccharides of N-acetylglucosamine and D-glucuronic acid. HA is an integral part of the extracellular (ECM) and pericellular matrix (PCM). This GAG is involved in several fundamental cell biological processes such as development, proliferation, migration, differentiation, metastasis, inflammation and wound healing13.16. During development or repair, migrating cells produce large amounts of HA. The expression
416
Cell surfaces and hyaluronan receptors
of HA by renal tubule epithelial cells is indicative for triggered enhanced cell motility, for example, as a reponse to epithelial injury. Although HA constitutes only a minor portion of total urinary GAGs it was found that HA is the major polysaccharide component in the organic kidney stone matrix. This is in agreement with the idea that somewhere along the urinary tract stone crystals become associated with this polysaccharide. Taken together, the identification of hyaluronan as binding molecule for calcium oxalate crystals at the suface of migrating and proliferating renal tubule epithelial cells is in agreement with a tissue repair mediated etiology of renal stone disease.
REFERENCES 1 2 3 4 5
N.S. Mandel, 'Mechanisms of stone formation', Sem Nephrol, 1996, 16:364-374. J.C. Lieske & F.G. Toback, 'Interaction of urinary crystals with renal epithelial cells in the pathogenesis of nephrolithiasis', Sem Nephrol, 1996, 16:458-473. DJ. Kok & S.R. Khan, 'Calcium oxalate nephrolithiasis, a free or fixed particle disease', Kidney Ira, 199446:847-854. C.F. Verkoelen, J.e. Romijn, W.C. de Bruijn, E.R. Boeve, L.C. Cao & F.R. Schroder, 'Association of calcium oxalate monohydrate crystals with MDCK cells', Kidney Int 1995, 48:129-138. M.W. Bigelow, I.H.Wiessner, J.G. Kleinman & N.S. Mandel, 'Surface exposure of phosphatidylserine increases calcium oxalate crystal attachment to IMCD cells', Am J Physioll997,272:F55-F62.
6
7 8
9
10
11 12 13 14 15 16
J.C. Lieske, R. Leonard, H. Swift & F.G. Toback, 'Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of epithelial cells', Am J Physiol, 1996, 270:F192-F199. S.R. Khan, 'Pathogenesis of oxalate urolithiasis: lessons from experimental studies with rats', Am. J. Kidney Dis, 1991, 17,398-401. R. de Water, C. Noordermeer, T.R. van der Kwast, H. Nizze, E.R.Boeve, D.J. Kok & F.H. Schroder, 'Calcium oxalate nephrolithiasis: effect of renal crystal deposition on the cellular compositionof the renal interstitium', Am J Kidney Dis, 1999, 33:761 C.F. Verkoelen, B.G. van der Boom, A.B. Houtsmuller, F.R. Schroder & J.C. Romijn, 'Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture', Am J Physiol, 1998, 274:F958-F965. e.F. Verkoelen, B.G. van der Boom, D.J. Kok, A.B. Houtsmuller, P. Visser, F.R. Schroder & J.C. Rornijn, 'Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture' Kidney Iru, 1999, 55: 1426-1433. C.F. Verkoelen, B.G. van der Boom, DiJ. Kok & J.C. Romijn, 'Sialic acid and crystal binding' Kidney Ini, 2000, 57:1072-1082. C.F. Verkoelen, B.G. van der Boom, D.l. Kok, F.H. Schroder & I.C. Romijn, 'Attachment sites for particles in the urinary tract', J Am Soc Nephrol, 1999, S430 T.e. Laurent & J.R.E. Fraser, 'Hyaluronan', FASEB J. 19926:2397-2404. B. Toole, 'Hyaluronan in morphogenesis', J Int Med. 1997 242 35-40. C.B. Knudsen & W. Knudson, 'Hyaluronan-binding proteins in development, tissue homeostasis and disease', FASEB J, 19937: 1233-1241. W.Y. Chen & G. Abatangelo, 'Functions of hyaluronan in wound repair', Wound Repair Regen, 1999, 7:79-89.
PART 7
THE ACTION OF HYALURONANINCELLS
ANTI-CANCER ACTIVITY OF HYALURONAN Mario C. Filion*, Sonia Menard, Benoit Filion, Julie Roy, Stephanie Reader & Nigel C. Phillips Bioniche Therapeutics Research Centre Montreal. Quebec, Canada. H4P 2R2
ABSTRACT
Although hyaluronan (HA) has been shown to modulate cellular proliferation in numerous cell types little is known about its effect on cancer cells. We have evaluated the anti-proliferative activity ofHA with a molecular mass of5.0-7.5 x 105 Da towards a wide range of cancer cell types. We have found that HA at low concentrations « 80 ug/rnl) inhibits, in a dose-dependent manner, the cellular proliferation ofprostate cancer cells (LNCaP, PC-3, DU-145), bladder cancer cells (HT-1376, RT-4, T24 and UMUC-3), breast cancer cells (MCF-7), melanoma cells (B 16-F 1) and fibrosarcorma cells (HT1080). The presence of a number of escape mechanisms associated with cancer progression such as p53/p21 mutations, Rb-mutations, pl6 deletion, Fas resistance, absence of caspase-3 and overexpression ofP-glycoprotein did not affect the ability of HA to inhibit cancer cell growth. The inhibition of cancer cell proliferation appeared to be independent of the level of expression of the HA receptor CD44. Furthermore, we found that HA potentiated the anti-proliferative activity of anti-cancer agents based on nucleic acids (mycobacterial cell wall complex and Mycobacterium phlei DNA) and of chemotherapeutic drugs (5-fluorouracil, cisplatin and tamoxifen). Our data indicates that HA having a molecular mass of 5.0-7.5 x 105 Da has considerable potential for development either as a chemotherapeutic agent or as an adjunct to anti-cancer agents. KEYWORDS
Cancer, proliferation, CD44 INTRODUCTION
Hyaluronan (HA) is a major non-structural component of connective tissue important for maintaining extracellular matrix structure and promoting cellular motility, adhesion and proliferation'. Low and high molecular mass HA have both been reported to modulate the proliferation ofsome normal (non-malignant) cell types. Low molecular mass HA of approximately 1.3 to 4.5 x 103 Da (3-10 disaccharide units), but not high molecular mass HA, has been shown to induce the proliferation of aortic endothelial
420
The action ofhyaluronan in cells
cells, an effect which was correlated with increases in the level of protein kinase'. In contrast, HA of approximately 1.3 to 7.2 X 103 Da (3-16 disaccharide units) specifically inhibited the in vitro proliferation of normal endothelial cells, but not of normal fibroblasts or normal smooth muscle cells'. High molecular mass HA also been reported to both induce and to inhibit the proliferation ofsome normal cells. HA with a molecular mass of 1.1 x 106Da appeared to stimulate the proliferation of human fibroblast cells suspended in a collagen matrix via an enhanced synthesis of'tubulin" and HA of8.6 x 10 5 Da stimulated the proliferation of corneal epithelial cells'. In contrast, HA of 4.0 x 10 5 to > 1.0 x 106 Da inhibited normal endothelial cell proliferation' and HA of> 1.0 x 106 Da has been shown to inhibit normal rabbit synovial cell and murine fibroblast 3T3 cell proliferation", Moreover, at < 1.0 mg/ml, HA of < 1.0 x 106 Da stimulated the proliferation ofsynovial cells and of3T3 cells". The disparate results observed with often similar HA preparations may be related to differences in HA receptor expression, to HA concentration or to the presence ofpro-stimulatory contaminants in the HA preparations. Although the effects ofboth low and high molecular mass HA on the proliferation of normal cells has been extensively studied ,little is known about the effect ofHA on cancer cells. In vitro, at> 0.32 mg/ml, HA of 1.3 to 5.6 x 103 Da (3-12 dissacharide units) and of> 1.2 x 106 Da inhibited the proliferation of highly tumorogenic, CD44H receptor expressing, BI6-FIO murine melanoma cells by 50 to 90%7. At < 0.16 mg/ml, HA slightly stimulated their proliferation. In vivo, at 1 mg/ml, HA of 1.3 to 5.6 x 103 Da administrated subcutaneously over 7 days by an Alzet osmotic pump into the intermediate vicinity ofa BI6-FIO murine melanoma tumor reduced tumor volume by 85%7. Administration of higher concentrations ofHA, up to 100 mg/ml, did not have a significantly greater inhibitory activity. The effect of this HA on the tumor cells was attributed to the disruption of the HA-CD44 complex 7. The effect of high molecular mass HA on B16FIO tumor growth in vivo has not been tested. In this report, we have evaluated the ability of a pharmaceutical preparation ofHA with a molecular mass of5.0-7.5 x 105 Da to directlyinhibitthe proliferation ofanumber of cancer cells or to potentiate the activity of anti-cancer drugs based on DNA and of chemotherapeutics drugs.
MATERIALS & METHODS Cells All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in the medium recommended by the ATCe. Table I shows the cell lines, their origins and their properties. Reagents HA of 5.0-7.5 x 105 Da was purified from Streptococci sp. and dissolved in sterile saline at 0.8 mg/ml (Cystistat®, Bioniche Life Sciences Inc, Belleville, Ontario, Canada).
Anti-cancer activity
421
MCC, a mycobacterial cell wall composition isolated from Mycobacterium phlei (M. Phlei) where mycobacterial DNA is preserved and complexed to the cell wall was prepared by Bioniche Life Sciences Inc. M. phlei DNA was purified as previously described", Cisplatin, 5-fluorouracil and tamoxifen were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada), dimethylthiazoldiphenyltetrazolium bromide (MTT) from Ameresco (Solon, Ohio, USA) and FITC-Iabelled antibodies to CD44 from BD Pharmingen (Mississauga, Ontario, Canada). Cellular proliferation assay Cancer cells were incubated in 6 well tissue culture plates at 1.0 x 105 cells/ml for 48 hours with different concentrations of HA, MCC, M. phlei DNA, cisplatin, 5fluorouracil and tamoxifen. Cell proliferation was measured using MTT reduction", Briefly, 100 lJ.l of 5 mg/ml ofMTT dissolved in PBS was added to each well. After 4 h. 1.0 ml ofacid-isopropanol (0.04 N HCI in isopropanol) was added and solubilized MTT was measured at 570 urn using a multiplate reader. Analysis of CD44 expression CD44 expression at the cell surface was measured by flow cytometry (FACScan, Becton-Dickinson, San Jose, CA, USA) using 1 x 105 cells with an FITC-labelled antibody to CD44 (clone G44-26) using the conditions described by the manufacturer.
Table 1 Cancer cells used in this study CELL MCF-7 PC-3 LNCaP DUc145
TYPE Human breast cancer Human prostate cancer Human prostate cancer Human prostate cancer
T-24 RT-4 UMUC-3
Human bladder cancer Human bladder cancer Human bladder cancer
HT-1376 HT-1080 B16-F1 JURKAT THP-1 HL-60 EL-4
Human bladder cancer Human fibrosarcoma Murine melanoma cancer Human T cell leukemia Human monocytic leukemia Human myelocytic leukemia Murine T cell leukemia
PROPERTIES Caspase-3 negative p53 mutated TGF -~ receptor-negative Rb, p16 and p53 mutated, Fas resistance p53 mutated pl6 deleted P-glycoprotein overexpression, p 16 deleted, p53 mutated p53 and p21 (Waf-I) mutated not determined not determined atypical multi drug resistance not determined p53 mutated not determined
422
The action ofhyaluronan in cells
RESUL TS & DISCUSSION Inhibition of cancer cell proliferation
The ability of HA with a molecular mass of 5.0-7.5 x 105 Da to inhibit the cellular proliferation of cancer cells was evaluated using various murine and human cancer cell lines (Table 1). As shown in figure 1, HA in the concentration range 0.08 to 80 ug/ml inhibits in a dose-dependent manner the proliferation of prostate cancer cells (PC-3, LNCaP, DU-145), breast cancer cells (MCF-7), melanoma cells (BI6-Fl) and fibrosarcoma cells (HT-1080). Bladder cancer cells (UMUC-3, RT-4, HT-1376, T24) were less sensitive to HA than the other cell lines tested and inhibition of their proliferation did not increase with increasingconcentrations ofHA (figure 1). Cancer cells from hematopoeitic origins, leukemia and lymphoma (J urkat T, THP-l, K562 EL-4), did not respond to HA treatment at the concentrations used (figure 1).Inhibition ofcancer cell proliferation by HA did not seen to be correlated with the presence of any particular escape mechanisms (Table 1).
40
--
--..-.
PC-3 LNCaP DU-145 HT-1080 -----..- MCF-7 -+ B-16Fl ~ T-24 c=--- UMUC-3 --<>--- RT-4 --e-- HT-1376 --8-- Jurkat T 0 THP-1 - -(a-HL-60 - -6--EL-4
-
-----
I::
.s:.E ~ ~
30 i 20
:2 d) u
~
10
~
!
I
I 0
I
0.1 HA (ug/ml)
Figure 1
Cancer cells were incubated at 1 x 105 cells/ml with different concentrations ofHA for 48 hat 37°C, 5% CO2 , Cellular proliferation was determined by MTT reduction as described in the Materials & Methods. The results shown are the mean of three independent experiments. S.D. were less than 10% and are not shown.
CD44 expression level
We have determined whether the anti-proliferative activity observed with HA
Anti-cancer activity
423
correlates with the expression level ofCD44. CD44 glycoprotein is the main extracellular receptor for HA 1• CD44 is associated with the malignant process in several types ofcancer and has been shown to promote the survival ofcancer cells". Disruption ofCD44 function in murine mammary carcinoma has been reported to induce apoptosis I o. CD44 expression by cancer cells was evaluated using an antibody (clone G44-26) that recognizes a domain common to all isoforms ofCD44 I I . As shown in Table 2, we have found that the cancer cells used in this study expressed variable levels of CD44 at the cell surface.
Table 2 CD44 cell surface expression CANCER CELL LINE HT-1376 RT-4 T-24 UMUC-3 PC-3 DU-145 MCF-7 LNCaP JURKAT THP-l HL-60
CD44 EXPRESSION* Cells + anti-CD44 Unstained cells 4 4
200 60
4
1800 1800
3 6
3 3
6 4 4 8
400 450 70 6 5
5
20
* mean fluorescence unit We found no correlation between the level ofCD44 expression and the ability ofHA to inhibit cellular proliferation at any concentration (p > 0.4). For example, LNCaP prostate cancer cells respond to HA in the absence of detectable amounts of CD44 at the cell surface, while T-24 and UMUC-3, which express high levels of CD44 at the cell surface, respond marginally to HA. However, since over ten isoforms of CD44 has been described", we can not exclude the possibility that a discrete CD44 isoform could be associated with the anti-proliferative activity observed with HA. This anti-proliferative activity could also be associated with other HA receptors. In particular, RHAMM and its isoforms have been implicated in the regulation of cell cycle progression 13. For example, overexpression of RHAMM by transfection into non-malignant fibroblasts has been shown to transform these cells into a fully metastatic fibrosarcoma".
HA potentiate the activity of anti-cancer agents based on DNA MCC, a mycobacterial cell wall preparation where mycobacterial DNA is preserved and complexed to the cell wall, and M phlei DNA possess anti-cancer activity against various cancer types':". More particularly, MCC has been shown to directly induce apoptosis in human bladder cancer cells. M ph lei DNA associated with MCC is responsible for its pro-apoptotic activity". MCC appears to mediate its anti-cancer activity
424
The action of hyaluronan in cells
by modulating the expression of a number of oncogenes, cell-cycle-related proteins and genes regulating apoptosis". We have tested whether HA can potentiate the activity of MCC or M. phlei DNA against bladder and prostate cancer cells. We have found that HA at various concentrations can act synergistically with MCC or M. phlei DNA As illustrated in figure 2, HA at 0.8 ug/ml potentiates the activity ofMCC and M. phlei DNA for both PC-3 prostate cancer cells (figure 2a) and RT-4 bladder cancer cells (figure 2b). Experiments are underway to understand why HA acts synergistically with MCC and M. phlei DNA. One possible explanation for this synergistic activity is the protection of the DNA from degradation by deoxyribonucleases present in the milieu (manuscript in preparation). Another possibility is the differential action of DNA and HA on cell cycle progression. HA has been shown to be present in the nucleus of cells, suggesting that HA may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells". HA potentiate the activity of anti-cancer drugs We have determined whether HA can also potentiate the activity ofchemotherapeutic drugs. These studies were carried out at an HA concentration which not have inhibitory activity. As shown in figure 3, we found that HA at 0.008 ug/ml can potentiate the activity ofcisplatin, an alkylating agent, and 5-fluorouracil, a DNA/RNA antimetabolite, against RT-4 bladder cancer cells. We have also tested the activity ofHA in combination with tamoxifen, an anticancer drug used in the treatment ofbreast cancer. Tamoxifen, an oestrogen receptor antagonist, can also directly induces apoptosis in breast cancer cells 17. We have found that HA at 0.008 ug/ml interacts synergistically with tamoxifen against MCF-7 cancer cells (figure 3). Our data suggest that the synergistic activity observed between HA and a number of anti-cancer drugs (MCC, M. phlei DNA, cisplatin, 5fluorouracil and tamoxifen) seems to be independent of their mechanism of action. Furthermore, it has been shown recently that the covalent linkage of HA to sodium butyrate, an anti-cancer compound, improves the anti-proliferative activity of butyrate towards MCF-7 breast cancer cells 18. CONCLUSIONS Our data show that HA purified from Streptococci sp. and having a molecular mass of 5.0-7.5 x 105 Da has a direct anti-proliferative activity on a number of cancer cells at low concentrations (0.8-80 ug/ml). In a number ofthe cancer cell lines tested, inhibition of proliferation by HA was significantly less at the highest concentration used. Cancer cells originating from bone marrow appear to be insensitive to HA at the concentrations tested. The activity of HA appeared to be independent of the presence of a number of escape mechanisms associated with cancer progression and of the presence of CD44 at the cell surface. HA also potentiated the activity of a number cancer drugs having different mechanisms of action. Our data indicates that HA having a molecular mass of 5.0-7.5 x 105 Da has considerable potential for development as a chemotherapeutic agent or as an adjunct to anti-cancer agents.
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PC-3 cells (a) or RT-4 cells (b) were incubated at I x l O'cells/ml with 0.8 ug/ml of HA alone or in combination with increasing concentrations of MCC or M phlei DNA for 48 h at 37°C, 5% CO 2, Cellular proliferation was determined by MTT reduction as described in the Materials & Methods. The results shown are the means of three independent experiments. S.D. were less than 10% and are not shown.
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REFERENCES 1.
2.
3.
4. 5.
J. Entwistle, C.L. Hall & E.A. Turley, HA receptors: regulators of signalling to the cytoskeleton, 1. Cell. Biochem., 1996,61,569-577. M. Slevin, J. Krupinski, S. Kumar & J. Gaffney, Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation, Lab. Invest., 1998,78,987-1003. D.C. West & S. Kumar, The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity, Exp. Cell Res., 1989, 183, 179196. R.M. Greco, J.A. Iocono & H.P. Ehrlih, Hyaluronic acid stimulates human fibroblast proliferation within a collagen matrix, 1. Cell. Physiol., 1998, 177,465-473. M. Inoue & C. Katakami, The effect of hyaluronic acid on corneal epithelial cell proliferation,Invest.Ophtamol. Vis. Sci., 1993,34,2313-2315.
Anti-cancer activity 6. 7. 8.
9. 10.
11.
12.
13.
14.
15.
16. 17. 18.
427
R.L. Goldberg & B.P. Toole, Hyaluronate inhibition of cell proliferation, Arthritis Rheum., 1987,30,769-778 C. Zeng, B.P. Toole, S.D. Kinney, I-W Kuo & I. Stamenkovic, Inhibition of tumor growth in vivo by hyaluronan oligomers, Int. J. Cancer, 1998, 77, 396-401. M.e. Filion, P. Lepicier, A Morales & N.e. Phillips, Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells, Br. J. Cancer. 1999, 79,229-235. D. Naot, R.V. Sionov & D. Ish-Shalom, CD44: structure, function, and association with the malignant process, Adv. Cancer Res., 1997, 71,241-319. Q. Yu, B.P. Toole & I. Stamenkovic, Induction ofapoptosis ofmetastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function, J. Exp. Med., 1997,186,1986-1996. N. Ohta, H. Saito, T. Kuzumaki, M.M. Ito, T. Saito, K. Nakahara & M. Hiroi, Expression of CD44 in human cumulus and mural granulosa cells of individual patients in in vitro fertilization programmes, Mol. Hum. Reprod., 1999, 5, 22-28. e.R. Mackay, H.J. Terpe, R. Stauder, W.L. Martson, H. Stark & U. Gunthert, Expression and modulation ofCD44 variant isoforms in humans, J. Cell. Bioi., 1994, 124,71-82. S. Mohapatra, X. Yang, I.A Wright, E.A Turley & AH. Greenberg, Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin Bl expression, J. Exp. Med., 1996,183,1663-1668. e.L. Hall, B. Yang, X. Yang, S. Zhang, M. Turley, S. Samuel, L. Lange, e. Wang, G.D. Curpen, R. Savani et al, Overexpression ofthe hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation, Cell, 1995, 82,19-28. S. Reader, M. C. Filion, V. Marie, B. Filion & N. e. Phillips, Mycobacterial cell wall-DNA complex (MCC) inhibits proliferation and induces apoptosis in androgendependent and independent human prostate cancer cells, Br. J. Cancer, 1999, 80S2, 76. S.P. Evenko & T.N. Wight, Intracellular localization ofhyaluronan in proliferating cells, J. Histochem. Cytochem., 1999,47, 1331-1342. R.E. Favoni & A. de Cupis, Steroidal and nonsteroidal oestrogen antagonists in breast cancer: basic and clinical appraisal, Trends in Pharm. Sci., 1998, 19,406-415. D. Coradini, e. Pellizzaro, G. Miglierini, M.G. Daidone & A Perbellini, Hyaluronic acid as drug delivery for sodium butyrate: improvment of the anti-proliferative activity on a breast-cancer cell line, Int. J. Cancer, 1999, 81, 411-416.
CONTROL OF HYALURONAN SECRETION INTO JOINT FLUID IN VIVO: ROLE OF PROTEIN KINASE C (PKC) C. L. Anggiansah', D. Scotr', A. Pout, P. J. Coleman", J. James', A. Houston', R. M. Mason z & J. R. Levick,,1 I Department ofPhysiology,
St George's Hospital Medical School, London SWI7 ORE, U. K.
2Molecular Pathology, Division ofBiomedical Sciences, Imperial College School ofMedicine, London W68RF, U. K.
ABSTRACT The rate of secretion of hyaluronan into the joint cavity of rabbit knees was measured in vivo over 6 h using a washout method and hyaluronan analysis by HPLC. Hyaluronan secretion in vivo was stimulated by joint distension, indicating the existence of a mechanosensitive regulatory pathway. The hyaluronan secretion rate increased >3-fold upon activation of protein kinase C (pKC) by phorbol ester. This effect was partially inhibited by cycloheximide, indicating that part of the response to PKC involves new protein synthesis.
KEYWORDS Synovium, secretion rate, stretch, protein kinase C, phorbol ester, cycloheximide.
BACKGROUND; HYALURONAN SECRETION INTO JOINTS IN VIVO Hyaluronan is secreted by synoviocytes into synovial fluid, where it not only serves as a lubricant but also has a profound buffering effect on fluid loss from the joint cavity during periods of raised pressure, as described by Levick et al. in this volume. The concentration of hyaluronan in the joint fluid is an important factor governing its actions'. Because joint fluid is continually being replaced by capillary filtration and lymphatic drainage', hyaluronan slowly drains out of the joint cavity', despite partial reflection of hyaluronan molecules by the synovial lining". As a result, the hyaluronan concentration can only be maintained by continuous secretion by the lining synoviocytes. Because reflection helps to retain hyaluronan in the cavity, the intraarticular half-life of hyaluronan is long, around 1Yz days in rabbit knees (cf. 2h for albumin or water), and a low secretion rate (3-5 ug hOi) suffices to maintain the physiological concentration 00.6 mg mr l . Since intra-articular concentration is stable, it is probable that hyaluronan secretion rate is a regulated variable and subject to physiological control processes. One such regulatory influence, identified recently in vivo, is stretch". As Figure 1 shows, the hyaluronan secretion rate into the cavity of a rabbit knee increase by ~20% within hours of distension of the joint lining by an acute volume expansion of 2 ml endotoxin-free physiological electrolyte solution. Control studies show that the increase is related to the joint expansion, not intra-articular cannulation. Thus the physiological secretion rate
370
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appears to be mechano-sensitive in vivo. This may also be relevant to the relation between joint embryogenesis and limb bud movement. Work by other groups has concentrated on stimulation of hyaluronan secretion by cultured cells in vitro, rather than in vivo, in response to growth factors and cytokinesr". In view of the findings in Figure 1 we asked 'What intracellular pathways might be involved in regulating h~aluronan secretion rate in vivo?' Studies in vitro by the groups of Heldin 7, 9 and Prehm show that protein kinase C (pKC) is often a key mediator of increased hyaluronan secretion. Our objective, therefore, was to test whether activation of conventional, phorbol ester-sensitive PKC isotypes could increase hyaluronan secretion rate into the joint cavity of rabbit knees in vivo. METHODS
New Zealand rabbits (2Y:z kg) were anaesthetized and a sterile cannula inserted into the cavity of both knee joints. The native synovial fluid hyaluronan was removed from the cavity by twenty 1 ml washes. This ensures complete removal of endogenous fluidphase hyaluronan (Figure 2). One joint was then injected with 1 ml of 200 ng mr' PMA (phorbol 12-myristate 13-acetate) to activate classical PKC isotypes. The contralateral joint received 1 ml sterile electrolyte solution as a control. Because PMA is a small solute that is quickly cleared by the fenestrated synovial capillaries (see later), 0.3 ml was aspirated from the cavity every half-hour and replaced by an equal 'top-up' volume of fresh PMA solution. The same procedure was followed on the control side using a sterile electrolyte solution. The joints were allowed to secrete hyaluronan into the cavity for 6 hours. The new hyaluronan is formed de novo, not by leaching from surrounding tissue, as shown by its
Control of secretion intojoint fluid
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RESULTS Stimulation of hyaluronan secretion rate by PKC The basal rate of hyaluronan secretion into the cavities of the control joints over the 6 hour period was 2.7 ± 0.5 ug per hour (n=5, mean ± s.e.m.). In joints treated with 200 ng mr l PMA the rate of hyaluronan secretion increased to almost 4 times the control value (p
372
Aspects of hyaluronan in joints 20
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Figure 3. Rate of secretion of hyaluronan into joint cavity and its stimulation by 200 ng mr!PMA. Dose-response relation The stimulatory effect ofPMA on hyaluronan secretion rate was dose dependent and reached a peak at 200 ng ml" PMA (Figure 4). At 500 ng mr! the stimulatory effect appeared to decline, but the difference between peak response and response to 500 ng mr l PMA did not reach statistical significance.
Intra-articular half-life of small solutes in vivo (Figure 5) The mean intra-articular half-life of acridine orange (370 Da) was short, 13.8 ± 2.9 min (n=5). Half-life increased with molecular mass, to 24.2 min for patent blue V (582 Da) and 55 min for Evans blue (963 Da). The molecular masses of PMA and cycloheximide are respectively 617 and 281 daltons. 16
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Figure 5. Intra-articular half life of small solutes (acridine orange, patent blue V and Evans blue) in rabbit knee in vivo Dependence of increased hyaluronan synthesis rate on protein synthesis de novo Evidence for regulation of HAS activity by direct phosphorylation is lacking''; but the stimulation of nuclear transcription leading to increased ribosomal synthesis of HAS-2 de novo is well documenred'", as is the short half-life of HAS in cultured cells'", We therefore tested the effect of the ribosomal translation blocker cycloheximide (CHX) on the stimulatory effect ofPKC. One joint received 200 ng mr l PMA and the contralateral joint PMA plus CHX at 100 IJ.g rnl" (> 10-times the concentration that totally blocks protein synthesis by cultured chondrocytes'"). In 3 successful paired experiments to date, CHX reduced hyaluronan secretion in every case, from 10.6 ± 4.2 IJ.g h-I in PMA-stimulated joints to 7.0 ± 4.3 IJ.g h-I in PMA+CHX treated joints. DISCUSSION The results show that PKC has a potential role in the pathways regulating hyaluronan secretion into joint fluid. Similarly, HAS gene transcription is increased by PKC in vitro:", Although the CHX results need further confirmation, they indicate that CHX only partially inhibits hyaluronan secretion in vivo, namely by 34%. Ifso, it follows that 1] synthesis de novo of new HAS-I/2/3 or an upstream, rate-limiting enzyme ll contributes to the upregulation of joint hyaluronan secretion in hours; and 2] some additional regulatory mechanism may exist, since CHX only partially blocked the stimulatory effect of PKC despite a very high CHX concentration. The possible existence of a regulatory membrane complex" or supply-limitation by uridine diphosphoglucose dehydrogenase (UDPDG)lI require clarification. A speculative transduction system for the stimulation of hyaluronan secretion by synovial stretch must begin with a mechanosensor, for which synoviocyte integrins and/or stretch activated ion channels are candidates, activating PKC throu~h the phosphoinositide cascade. PKC activation of the MAP kinase cascade is known' . This could lead to nuclear transcription of HASIUDPDG and, to account for the CHXresistant secretory component, possibly phosphorylation of other regulatory factors.
374
Aspects ofhyaluronan in joints
It is concluded that PKC can control HA secretion into the synovial fluid of rabbit knee joints in vivo; and that its action is partially dependent on new protein synthesis. ACKNOWLEDGEMENTS The research was funded by Wellcome Trust grants 039033/Z, 056983/Z and European Community Training & Mobility of Researchers grant ERBFMRXCT980219. REFERENCES 1. D. Scott, P. 1. Coleman, R. M. Mason & 1. R. Levick, 'Concentrationdependence of interstitial flow buffering by hyaluronan in joints', Microvasc. Res., 2000, 59, 345-353. 2. 1. R. Levick, R. M. Mason, P. 1. Coleman & D. Scott. In: Biology of the Synovial Joint. C. W. Archer, M. Benjamin, B. Caterson & 1. R. Ralphs (eds), 1999. Harwood Academic Publishers, London. pp. 235-252. 3. 1. R. E. Fraser, W. G. Kimpton, B. K. Pierscionek & R. N. P. Cahill, 'The kinetics of hyaluronan in normal and acutely inflamed synovial joints: observations with experimental arthritis in sheep', Seminars in Arthritis & Rheumatol., 1993,22 Suppl. 1,9-17. 4. D. Scott, P. 1. Coleman, R. M. Mason & 1. R. Levick, 'Direct evidence for the partial reflection of hyaluronan molecules by the lining of joints during transsynovial flow', 1. Physiol., 1998, 508, 610-623. 5. P. 1. Coleman, D. Scott, 1. Ray, R. M. Mason & 1. R. Levick, 'Hyaluronan secretion into the synovial cavity of rabbit knees and comparison with albumin turnover', J. Physiol., 1997,503,645-656. 6. A. P. Spicer & T. K. Nguyen, 'Mammalian hyaluronan synthase:investigation of functional relationships in vivo', Biochem. Soc. Trans. 1999,27, 109-115. 7. P. Heldin, T. Asplund, D. Ytterberg, S. Thelin & T. C. Laurent, 'Characterization of the molecular mechanism involved in the activation of hyaluronan synthetase by PDGF in human mesothelial cells', Biochem. J., 1992, 283, 165-170. 8. L. Klewes & P. Prehm, 'Intracellular signal transduction for serum activation of hyaluronan synthase in eukaryotic cells', 1. Cell. Physiol., 1994, 160,539-544. 9. M. Suzuki, T. Asplund, H. Yamashita, C-H Heldin & P. Heldin, 'Stimulation of hyaluronan biosynthesis by platelet derived growth factor BB and TGF -~ 1 involves activation of protein kinase C', Biochem. 1.,1995,307,817-821. 10. M. K. Bansal & R. M. Mason, 'Evidence for rapid metabolic turnover of hyaluronate synthetase in swarm rat chondrosarcoma chondrocytes', Biochem. 1., 1986,236,515-519. 11. A. A. Pitsillides, L. S. Wilkinson, S. Meydizadeh, M. T. Bayliss & J. C. W. Edwards, 'Uridine diphosphoglucose dehydrogenase activity in normal and rheumatoid synovium', 1. Exp. Path., 1993,74,27-34. 12. N. Mian, 'Characterisation of a high-Mr plasm-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex', Biochem. J., 1986,237, 343-357. 13. D. C. Schonwasser, R. M. Marias, C. 1. Marshall & P. J. Parker, 'Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel and atypical protein kinase C isotypes', Mol. Cell. BioI., 1998, 18, 790-798.
HYALURONAN METABOLISM IN RESPONSE TO MECHANICAL STRAIN IS MODULATED BY MATRIX DEPLETION. G. P. Dowthwaite, S.Thomas, C. R. Flannery, A. A. Pitsillides 1 & C.W. Archer*. Connective Tissue Biology Laboratory, School ofBiosciences, CardiffUniversity, Cardiff, CFlO 3US. JDepartment
of Veterinary Basic Sciences, The Royal Veterinary Col/ege, University ofLondon, Royal Col/ege Street, London, NWI Oro, U.K
ABSTRACT Many studies have highlighted the importance of movement-induced mechanical stimuli in the development of functional synovial joints. However, such phenomenological results have failed to provide a full explanation of the mechanism essential for the morphogenesis of fluid-filled joint cavities. We have previously demonstrated that the large glycosaminoglycan hyaluronan (HA) in association with its principal cell surface receptor CD44, playa major role during the morphogenesis of chick joints (see Osbourne et aI., in this volume). Here, we have taken cells from the surface of recently cavitated joints and subjected them to a brief period of dynamic mechanical strain (3800 f.lE for 10 minutes) and measured changes in HA synthesis/release and HA synthase gene expression. In addition, we have subjected cells to matrix-depletion prior to the application of mechanical strain in order to examine any potential modulatory function of the extracellular matrix during the cell's response to strain. Removal of the cell-associated HA-containing matrix with hyaluronidase significantly increases the release of HA into tissue culture media over 24 hours and is associated with alterations in HA synthase gene expression. Such changes in HA release are shown to be synergistically enhanced by the application of dynamic mechanical strain. These results show that cell-matrix interactions modify the response of embryonic cells to mechanical strain and provide further insight into mechanodependent mechanisms ofjoint cavity morphogenesis. KEYWORDS Hyaluronan, hyaluronan synthase, articular fibrocartilage, mechanical stimulation, matrix depletion. INTRODUCTION The role of movement-induced mechanical stimuli in joint morphogenesis has long been recognised using paralysis or in vitro culture 1-4. In particular, these immoblisationJparalysis studies directly showed that movement was required for joint cavitation between cartilage anlagen and for the maintenance of previously cavitated joints, thus indirectly implicating mechanical stimuli in the process. More recent efforts have centred on providing a cellular basis to the phenomenological experiments mentioned above. Previous work by us identified
376
Aspects ofhyaluronan injoints
hyaluronan (HA) as a major constituent of the early coalescing extracellular vesicles that merge to form the cavity' and the association of uridine diphospo-glucose dehydrogenase (UDPGD), an enzyme involved in HA slnthesis, with the fibrocartilaginous articular cells of the presumptive joint line 6- • Later studies also demonstrated the involvement of the HA receptors CD44, IVD4 together with the actin capping protein moesin within these fibrocartilage cells. Furthermore, the importance of functional binding of HA to its respective cell receptors was demonstrated by the intraarticular injection of oligosaccharides of HA that displaced nascent HA with a consequent inhibition of cavitation (as assessed by reduction in HA accumulation) and a down-regulation of the recerotor expression 9,10. Similarly, paralysis of embryos in ovo showed very similar results 0. In an attempt to defme the effects of mechanical strain on the relevant fibrocartilage cells, we have isolated them in culture, and subjected them to a known strain regime. In brief, cells were placed on a 4-point bending jig that subjected cells to 3,800 f.!E at 1 Hz for 10 minutes. Strain enhanced the accumulation ofHA in the medium by 10 fold at 24 hours post stimulation when compared with static controls. (For more details of these experiments see the chapter by Osbourne et al., in this volume). Thus, for the first time, we were able to demonstrate a direct effect of mechanical stimulation on the accumulation ofHA by fibrocartilaginous articular cells in culture, an effect that can be correlated with an in vivo morphogenetic event. MATERIALS & METHODS Fibrocartilage cell isolation and culture Articular fibrocartilage was aseptically excised from the tibiotarsal joints of stage 42 11 White Leghorn chicken embryos and fibrocartilage cells isolated as described previously 12. Briefly, cells were isolated from diced fibrocartilage by digestion in 300 units mI,l collagenase (type I; Sigma, UK) for 1 hour at 37°C in sterile PBS. Cells were centrifuged and resuspended in Dulbeccos modified eagle medium containing 2 mM Lglutamine, 50 ug mI- 1 Gentamycin, 50 f.!g ml' ascorbic acid, 1 mg ml" D-glucose and 5% chick serum (DMEM(+); Life Technologies Ltd. UK). Cells were then either seeded into 75 cm2 tissue culture flasks and expanded or plated out directly. In all experiments, only primary or passage I (PI) cells were used. Matrix depletion using hyaluronidase Fibrocartilage cells (I x 105 mI- 1 in 35 mm dishes) were incubated overnight in Thereafter, media were removed and DMEM without chick serum (DMEM( Streptomyces hyaluronidase (Sigma, UK) added at 25 ill ml" and incubated at 37°C for 60 minutes. Cells were then washed 3 times with PBS (with gentle agitation for 5minutes) and fresh DMEM(-) added to the dishes. In addition, some experiments involved supplementation of such matrix-depleted cells with 200 ug mI-\ hyaluronan (Sigma) diluted in fresh DMEM(-) which was added immediately after the final wash in PBS. Controls for these experiments included cells that were not treated with hyaluronidase but were supplemented with media containing 200l1g mI-] HA (HA only control), cells treated with hyaluronidase alone (HAase only control) and cells cultured in serum free media alone (no treatment control).
-».
Metabolism modulated by matrix depletion
377
Application of mechanical strain to cells in vitro
Application of controlled, dynamic mechanical strain was achieved by subjecting plastic strips (6 x 2 ern) on which the cells were plated to four point bending in a specially designed and calibrated jig 12,13. Briefly, primary or first passage cells were plated onto plastic strips and grown to confluence (2 x 105 cells/strip) in DMEM(+) in a humidified atmosphere of 95% air/5% CO2 at 37°C. Confluent cells were serumdeprived in DMEM(-) overnight and the strips placed in the jig in 12 mI of fresh DMEM(-) and allowed to equilibrate for 30 minutes. Strips were then dynamically strained using 4 point bending to engender 3800 IlE at 1Hz for 10 minutes (total of600 cycles). Controls comprised strips carrying cells subjected to a similar cyclic perturbation of the medium as strained cells but without the applied strain (flow), and strips that were unperturbed (static). Medium was sampled (400 Ill) immediately prior to the application of strain and at various time points up to 24 hours. In addition, some experiments incorporated pre-treatment of cells with 25 ill ml" hyaluronidase (as descried above) followed by 10 minutes of strain, and others examined the effect of exogenous HA supplementation of media after HAase treatment. Determination of media HA concentration.
Duplicate samples of undiluted medium were assessed for HA concentration uSin either a competitive ELISA plate-based assay 14 or a modified uronic acid assay I f• Comparison of the 2 assays revealed no significant differences in HA concentration from duplicate samples (data not shown). cDNA synthesis and polymerase chain reaction
Total RNA was isolated from hyaluronidase-treated and control cells (1 x 105 cells ml") at 0, 2, 6 and 24 hours after treatment using Tri reagent (Sigma; manufacturers instructions) and incubated with 10 units of DNase (Calbiochem, U.K.) at 37°C for 30 minutes. RNA was reverse transcribed into first strand cDNA using the Gene Amp PCR core kit (Perkin Elmer, UK) following the manufacturers instructions. Reverse transcribed mRNA was then amplified using the polymerase chain reaction (PCR) with gene-specific primers identified from partial DNA sequences of the chick HAS 2 and 3 genes 16: HAS 2 sense: 5'-ACC CGC TGG AGT AAA TCG TAT-3'; HAS 2 anti-sense: 5'-TAA GGA AGA AAG GAA AGA ATC-3'; HAS 3 sense; 5'-CCT ACT TTG GCT GTG TGC-3' and HAS 3 anti-sense: 5'-GCG GGT CTG ITG GTT GAG-3'. Conditions for 'hot start' PCR were as follows: a lower mixture containing 1.25 III lOx PCR buffer, 3 III MgCh (final concentration 1.5 mM), 4 III of dNTP (mix fmal concentration 200 1lM), I III of each primer and an Ampliwax Gem 100 (Perkin Elmer) bead were incubated at 70°C for 90 seconds and cooled to 20°C. An upper mixture containing 5 III lOx PCR buffer, 0.3 III Taq polymerase (final concentration 1.5 units), 0.1 ug mRNA and sterile water to a final volume of 50 III was laid over the solidified wax pellet. Samples were initially denatured at 94°C for 60s followed by 40 cycles of 95°C 30s, 48°C 45 s for HAS 2, 57.6°C 45s for HAS 3, noc 30s and a fmal extension step at ire: for 5 minutes. PCR products (10 Ill) were run on 3% agarose gels containing 1 ug mI -I ethidium bromide at 100 volts for 30-45 minutes and visualised under UV light. PCR products of the expected size were then purified using a Wizard PCR Prep kit (Promega, UK) following the manufacturers instructions. Purified PCR
378
Aspects of'hyaluronan in joints
products were then sequenced using the d-rhodamine dNTP cycle sequencing kit (ABI Prism) following the manufacturers instructions and sequenced using an ABI Prism 377 automated sequencer. Sequence homologies to published HAS gene sequences were confirmed using the BLAST search protocol 17. RESULTS & DISCUSSION
Effect of mechanical stimulation on hyaluronidase pre-treated fibrocartilage cells. A. Strain-related changes in medium HA concentration In order to determine the specific increases in HA concentration in the medium of cells exposed to a short (lOminute) period of dynamic mechanical strain or medium flow, with or without prior treatment with HAase, data have been converted to a logarithmic scale (1oglO). In addition to equalising variability, this allows simple additive, or more complex multiplicative (synergistic), models of interaction between two factors (mechanical stimuli and matrix involvement) to be examined. The statistical significance ofthese results was evaluated using ANOV A and r-tests. Without prior HAase treatment of cells, both 'flow-specific' and 'strain-specific' increases (p0.05) regardless of prior HAase treatment. In accord with our earlier results, at both 6 and 24 hours after treatment there was significantly more HA in the media of HAase-treated cells (p < 0.01 at 6h and p < 0.001
Metabolism modulated by matrix depletion
379
at 24h) compared with control cells. However, at these time points the medium HA concentration from cells supplemented with HA after HAase treatment were not significantly different from (p > 0.05) those measured in untreated controls (Fig 2). These results indicate that supplementation of medium with exogenous HA also significantly diminishes the HAase-induced increases in HA release for up to 24 hours 0.5
***
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's
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* +
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eu CI)
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0.1
*
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Oh
20 min
6h
24h
lime
Figure 1: The application of strain after hyaluronidase treatment synergistically increases media HA concentration over 24 hours. Cells were treated with HAase (251U mr! for 60 minutes) prior to 10 minutes of mechanical strain and media assayed for HA over 24 hours as described in the materials and methods. *, p < 0.01 compared with static control; ** p < 0.01 compared with non-HAase treated cells; ***, p < 0.001 compared with non-HAase treated cells; +, p < 0.01 compared with flow C. Changes in HAS-2 and HAS-3 mRNA expression in relation to HAase treatment Sequence analysis ofPCR products generated using HAS 2 and HAS 3 gene-specific primers each produced a positive match to the published gene sequences for HAS 2 and 3 16 in mRNA extracted from cultured primary fibrocartilage cells. Analysis of HAS 2 and HAS 3 mRNA expression by PCR at various times after HAase treatment, revealed markedly differing expression patterns. HAS 2 mRNA was constituitively expressed in untreated fibrocartilage cells at all times points examined. However, immediately after HAase treatment HAS 2 mRNA was undetectable but was apparent after a further 2 hours incubation (Fig 3). HAS 3 mRNA was undetectable in control cells at all time points, but was apparent at 2, 6, and 24 hours after HAase treatment (Fig 3). Using PCR, GAPDH mRNA expression was unaltered in both control and HAase pre-treated cells over the time course of the experiment.
380
Aspects of hyaluronan in joints
**
1000
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_HAase
+
~HAase+ HA
C
III
-~
~HA
750
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8 III
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=
** **
250
-*-
== = = ~
=n = o
1
n
1= 1=
f=
6
= = = == == ~
24
Time in hours
Figure 2: Effect of exogenous HA on media HA concentration (ug mI- l , mean ± sem) over 24 hours following hyaluronidase treatment. Cells were treated with HAase (25IU for 60 minutes) and fresh media containing 200llg ml" HA added to cells immediately after treatment and sampled at 0, 1, 6 and 24 hours as described in materials and methods. Immediately after HAase treatment unsupplemented samples contained significantly less HA than those supplemented with HA. At 1 hour, media HA concentration from unsupplemented cells treated with HAase had reached those in supplemented samples and was significantly increased by 6 and 24 hours compared with both supplemented and control samples. *, p < 0.05 compared with control; **, p < 0.001 compared with control; NS, p > 0.05 compared with control.
These experiments clearly demonstrate that cells isolated from the articular surface of developing synovial joints respond to mechanical stimuli, which are of known physiological relevance in other skeletal tissues 18, by differentially elevating the formation and release ofHA. We do not know the strains experienced by these celIs in OVO, and they are likely to vary between joints. Our choice of 3,800 IlE is based on those measured on bone surfaces during normal physiological use in a variety of species'f and it is likely that such magnitudes of strain will be less than that experienced by celIs lining the fibrocartilaginous surfaces of a developing joint cavity. We also show that depletion of the surrounding hyaluronan-rich matrix of these cells, prior to application of mechanical strain, synergistically potentiates elevated levels of cellular
Metabolism modulated by matrix depletion
381
HA release and that supplementation of HA depleted cells with exogenous HA abrogates these increases in HA release. Many previous reports have emphasised the role of mechanical stimulation/movement as a pre-requisite for normal joint formation and specifically during the cavitation event. These data have been accrued largely through in vitro culture of developing joints or the application of neuromuscular blocking agents to embryos 1-4, 19-21. However, these studies have not yet provided direct evidence illustrating the nature ofthe contribution oflocal mechanical stimuli to the relevant cells during joint morphogenesis. From a biological perspective, two particular facets of our findings are important. First, that the effect ofa brief IO minute period of mechanical stimulation is still evident after 24 hours indicates the transitory nature of the stimulus that may be required to achieve cavitation. Within the egg, embryo movement is often restricted by position and our findings may provide a mechanism whereby joints can cavitate normally with minimal mechanical input. Second, previous work by us has shown that exposure to the mechanical stimuli described resulted in the induction of HAS 3 which together with HAS 2 may partly explain the accumulation of HA within the medium. Here, we show that the effect of matrix depletion also results in differential HAS gene expression. Whereas HAS 2 is constitutively expressed by fibrocartilage cells, HAS 3 is expressed immediately after HAase treatment, concomitant with the disappearance of HAS 2 expression. Thus, it is apparent that at times of HAase-induced enhanced HA release, HAS 3 expression predominates and this suggests that upon HAS 2 recovery that the cells are again capable of elaborating a pericellular coat. Two hours after hyaluronidase treatment, both HAS 2 and 3 are expressed in matrix depleted cells whereas only HAS 2 is expressed in control cells. Whilst mRNA expression may not necessarily reflect changes in HAS activity, it may be responsible for the increased media HA concentrations seen at the later time points during the course of the experiment. It is known that HAS 2 and HAS 3 (at least in vitro) have differential enzyme kinetics in that HAS 3 has a higher V max than HAS 2 and that HAS 3 synthesises smaller HA chains than HAS 2 (Spicer and McDonald, 1998; Spicer and Ngyuen 1999). Although it is unlikely that the expression of HAS 3 mRNA, immediately after HAase treatment is a direct reflection of HAS enzyme activity, at 6 hours both HAS 2 and 3 protein elaboration at the cell membrane may contribute to the increased media HA concentrations. Earlier increases in media HA concentration may be due to release of HA from the cell surface or the activity of extant HAS 2. These experiments represent another step in our understanding of matrix metabolism occurring during joint formation. Previously we have shown that HA is not present to any great degree surrounding the cells of the developing articular surface itself but that HA is concentrated in the developing joint cavity 5,8. Additionally, the cells of the developin articular surface express Habinding proteins and have increased UDPOD activity 8,B 2. A similar situation exists after HAase-mediated depletion of pericellular matrix in vitro, where elevated CD44 expression, increased UDPOD activity per cell and increased media HA concentration are evident. It is, therefore, possible that the exclusion of HA from the articular surface of the developing joint, by an as yet unknown mechanism, increases HA synthesis in these cells allowing the joint cavity to develop where HA concentrations are highest.
382
Aspectsof hyaluronan injoints t= 0
t= 2 hours
t= 6 hours
t= 24 hours
HAS 2 HAS 3 GAPDH HAS 2 HAS 3 GAPDH HAS 2 HAS 3 GAPDH HAS 2 HAS 3 GAPDH
+
-+-+-+-+-+-+-+
+-+
-+
+
Figure 3: Changes in expression of HAS genes in fibrocartilage cells at various times (0-24 hrs) after HAase pre-treatment (25 IV rnl" Streptomyces HAase for 60 minutes). Cells were treated with HAase, washed with PBS and RNA extracted at various time points over 24 hours as described in materials and methods. HAS 2 is constitutively expressed in control cells (t = 0, HAS2-) but is not expressed immediately after HAase pre- treatment (t == 0, HAS 2+). HAS 3 is expressed immediately after HAase pre- treatment (t == 0, HAS 3+) but is not expressed in control cells (t == 0, HAS 3-). At 2 hours both control and HAase pre-treated cells express HAS 2 (t = 2, HAS2-, HAS2+), but HAS 3 is only expressed in HAase pre-treated cells (t == 2, HAS3+). This expression pattern is maintained at both 6 and 24 hours. GAPDH expression is not altered at any time point or by HAase pre-treatment. - == controls, + == HAase pretreated.
CONCLUSIONS Chick fibrocartilage cells that are largely responsible for the formation ofthe initial joint cavity respond to mechanical cues in vitro by selectively up-regulating HA synthesis. This effect is further potentiated by the depletion ofthe pericellular matrix an effect that can be considered synergistic. ACKNOWLEDGEMENTS The authors would like to thank the Arthritis Research Campaign for generous financial support ofthis work. REFERENCES l. Fell, H. B. and Canti, R. G.: Experiments on the development in vitro of the avian knee joint. Proc. Roy. Soc. Lon. 1934, B1l6: 316-351.
2. Hamburger, V. and Waugh, M.: The primary development of the skeleton in nerveless and poorly innervated limb transplants of chick embryos. Physiol. Zool. 1940, 13: 367-380.
Metabolism modulated by matrix depletion
383
3. Drachmann, D. B. and Sokoloff, L.: The role of movement in embryonic joint development. Dev. Bioi. 1966, 14: 401-420. 4. Murray, P. D. F. and Drachrnann, D. B.: The role of movement in the development of joints and related structures: the head and neck of chick embryos. J. Embryol. Expt. Morphol. 1969,22: 349-371. 5. Craig, F.M., Bayliss, M.T., Bently, G., and Archer, C.W. A role for hyaluronan in joint development. J.Anat.1990, 171, 17-23. 6. Archer, C. W., Morrison, H. and Pitsillides, A A: Cellular aspects of the development of diarthrodial joints and articular cartilage. J. Anat. 1994, 184:447456. 7. Edwards, J.C.W., Wilkinson, L.S., Jones, H.M., Soothill, P., Henderson, K.l, Worrall, J.G., and Pitsillides, AA. The formation of human synovial joint cavities: a possible role for hyaluronan and CD44 in altered interzone cohesion. J. Anal. 1994, 185,355-367. 8. Pitsillides, A A, Archer, C. W., Prehm, P., Bayliss, M. T. and Edwards, J. C. W.: Alteration in hyaluronan synthesis during developing joint cavitation. J. Histochem. Cytochem. 1995,43: 263-273. 9. Dowthwaite, G.P., Edwards, J.C.W., and Pitsillides, AA An essential role for the interaction ofhyaluronan and hyaluronan binding proteins during joint development. J. Histochem. Cytochem. 1998,46,641-651. 10. Pitsillides, A A: The role of hyaluronan in joint cavitation. In Biology of the Synovial Joint cd. by Caterson, B., Archer, C.W., Benjamin, M. and Ralphs, J.R, Harvard Academic Press. 1999, pp 41-62.
11. Hamburger, V., and Hamilton, H.L. A series of normal stages in the development of the chick embryo. .J. Morphol. 1951, 88,49-92. 12. Dowthwaite, G.P., Ward, AC., Flane1ly, J., Suswillo, RF.L., Flannery, C.R, Archer, C.W., and Pitsillides, AA The effect of strain on hyaluronan metabolism and hyaluronan binding protein expression in embryonic fibrocartilage cells. Matrix BioI. 1999, 18, 523-532. 13. Pitsillides, A A, Rawlinson, S.C.F., Suswillo, RF.L., Bourrin, S., Zaman, G. and Lanyon, L.B.: Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modelling? FASEB J. 1995,9,1614-1622. 14. Fosang, AJ., Hey, N.J., Carney, S.l, and Hardingham, T.E. An ELISA plate-based assay for hyaluronan using biotinylated proteoglycan G1 domain (HA binding domain). Matrix. 1990, 10, 306-310. 15. van den Hoogen, B.M., van Weeren, P.R, Lopes-Cardozo, M., van Golde, L.M.G., and van den Lest, C.H.A A rnicrotitre plate assay for the determination of uronic acids. Analytical Biochem. 1998,257, 107-111.
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Aspects ofhyaluronan in joints
16. Spicer, A.P., and McDonald, lA Charecterization and molecular evolution of a vertebrate hyaluronan synthase gene family. J. Bioi. Chem. 1997,273, 1923-1932 17. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, E.]. Basic local alignment search tool. J. Mol. Bioi. 1990,215,403-410. 18. Lanyon, L.E., Goodship, AE., Pye, c.J., and MacFie, lH. Mechanically adaptive bone remodelling. J. Biomech. 1982,15,141-145. 19. Mitrovic, D. R.: Vaisseaux sanguins au cours de l'arthrogenese et leur participation eventuelle a la cavitation articulaire. Zeitschrift fur Anatomie und Entwick. 1974, 144: 39-60. 20. Mitrovic, D.: Development of the articular cavity in paralysed chick embryos and in chick embryo limbs cultured on chorioallantoic membranes. Acta Anat. 1982, 113: 313-324. 21. Ruano-Gil, D., Nardi-Vilardaga, J. and Teiexidor-Johe, A: Embryonic mobility and joint development. Fol. Morph. 1980,28: 221-223. 22. Spicer, AP., and Nguyen, T.K. Mammalian hyaluronan synthases: investigation of functional relationships in vivo. Biochem. Soc. Trans 1999,. 27, 109-115
PRO-INFLAMMATORY ACTIVITY OF CONTAMINATING DNA IN HYALURONAN PREPARATIONS Mario C. Filion* & Nigel C. Phillips Bioniche Therapeutics Research Centre Montreal. Quebec. Canada. H4P 2R2
ABSTRACT Hyaluronan (HA) preparations are currently used in the treatment of inflammatory disorders. Low molecular mass HA fragments are however believed to induce inflammation. In this study we have evaluated whether low molecular mass fragments derived from HA preparations induce the synthesis of the pro-inflammatory cytokines IL-12 and TNF-lX by human monocytic cells. Since DNA from numerous species has been shown to possess proinflammatory activity, we have additionally tested for the presence of DNA in these preparations. We have found that low molecular mass fragments obtained from noninflammatory HA preparation by sonication (3 x lOs Da) or by hyaluronidase digestion (1 x 104Da) do not have the ability to induce IL-12 and TNF-lX by human monocytic cells. We unexpectedly found however that high molecular mass HA present in 2 out of 7 HA preparations tested was able to stimulate the synthesis of significant levels ofIL-12 and TNF -c; The pro-inflammatory cytokine-inducing activity was shown to be due to the presence of DNA, since treatment of the HA preparations with DNase I abrogated or significantly reduced the induction of cytokines. The presence of pro-inflammatory DNA in HA preparations should be evaluated before their use, not only for the treatment ofpatients with inflammatory disorders, but also before studying inflammatory processes. KEYWORDS Inflammation, cytokine, DNA INTRODUCTION HA preparations containing high molecular mass polymers (> 0.5 x 106 Da) have been reported to decrease inflammation in a number ofdiseases. The efficacy ofintra-articular HA administration has been demonstrated in patients with either osteoarthritis' and/or rheumatoid arthritis', HA preparations have also been reported to decrease the inflammatory symptoms in patients with interstitial cystitis'. Paradoxically, even although pharmaceutical grade high molecular mass HA has been shown to reduce inflammation in a number of diseases, it has been recently reported that inflammatory reactions occur following intraarticular administration of high molecular mass HA preparations':".
430
The action ofhyaluronan in cells
Low molecular mass fragments derived from HA preparations by either sonication or digestion with hyaluronidase have been reported to induce pro-inflammatory cytokine genes expression in mast cells, monocytes and macrophages, and could potentially exacerbate inflammation in patients with inflammatory disorders. The up-regulation by low molecular weight HA fragments of inflammatory mediators includes IL-l~, IL-12, TNF -a, MIP-l a, MIP-l~, MCP-l and RANTES 8•9 • Monocytes have been shown to be present in the joints of patients with ostoearthritis" and rheumatoid arthritis" and in the bladder wall" of patients having interstitial cystitis. We have therefore tested whether native high molecular mass HA preparations or low molecular mass fragments obtained from HA preparations by sonication ( "'3 x 105 Da) or by hyaluronidase digestion ( '"1 X 104 Da) have the ability to induce the synthesis IL-12 and TNF-a by monocytes. MATERIALS & METHODS HA analysis
The characteristics of the HA preparations used in this study are summarized in Table 1. All HA preparations were tested for endotoxin content using a colorimetric Limulus amebocyte lysate assay with a sensitivity of 0.01 EU/ml (QCL-1000, BioWhittaker, Walkersville, MD, USA), and for protein contents by a micro protein determination kit (Sigma-Aldrich Canada, Oakville, Ontario) using Coomassie Brillant Blue G. Low molecular weight HA was obtained by digestion with hyaluronidase type IV-S derived from bovine testes (Sigma-Aldrich Canada) for 60 min at 37°C or by sonication on ice using a Fisher model 550 sonifier (Fisher Scientific, Nepean, Ontario) for 20 min at maximal intensity. The molecular weight distribution of HA was analyzed by electrophoresis as described 13. Evaluation of pro-inflammatory cytokines
Human monocyte THP-l cells were obtained from the American Tissue Culture Collection (ATCC; Rockville, MD, USA). THP-I monocytes were incubated in six-well flat-bottomed tissue culture plates at I x 106 cells/ml with various concentration ofthe HA preparations in 1.0 ml ofRPMI-1640 supplemented with 10% fetal bovine serum (GibcoBRL, Burlington, Ontario, Canada) for 48 hours at 3rC in an atmosphere of5% CO 2 , IL-12 and TNF-a released into the supernatant were measured using commercial ELISA kits (BioSource, Camarillo, CA, USA). Detection and treatment of DNA
The presence of DNA in the HA preparations was determined by electrophoresis in agarose gels containing 0.7% agarose and 0.5 ug/ml ethidium bromide at 100 V for 3 h. DNA was visualized using an UV transilluminator (wavelength: 302 urn). HA preparations containing DNA were incubated with 10 U ofdeoxyribonuclease I (DNase I) (Gibco-BRL) for I hour at 37°C in 20 mM Tris-HCl (pH 8.4), 2 mM magnesium chloride and 50 mM potassium chloride. EDTA was then added to the reaction mixture (final concentration 2.5
Pro-intlammatory activityof DNA
431
nM) which was heated for 10 min at 65°C to inactivate the DNase 1.Digestion of the DNA
was confirmed by electrophoresis in agarose gels as described above. For experiments with inactivated DNase I, DNAse I was inactivated as described above prior to its addition to HA. HA preparations treated with DNase I or with inactivated DNase I were incubated with THP1 cells and IL-12 and TNF-cc synthesis was evaluated as described in the previous section. RESULTS & DISCUSSION It has been previously shown that low molecular weight fragments derived from HA are able to induce the expression of a number of pro-inflammatory cytokines, including IL-12 and TNF-a., by monocytes. The generation of low molecular weight HA fragments could arise following intravesical or intra-articular administration due to the digestion by inflammation-related hyaluronidase. We have determined whether low molecular weight HA fragments could induce the synthesis of the pro-inflammatory cytokines IL-12 and TNF-a. by human monocytes. Low molecular mass HA fragments obtained by sonication (,,3 x 105 Da) or by hyaluronidase digestion (" 1 x 104Da) of Suplasyn", a preparation ofHA with a molecular mass of 5.0-8.0 x 105 Da, do not induce the synthesis ofIL-12 and TNF-a. in the dose range 0.01 to 100 ug/ml. We have tested whether native high molecular mass HA preparations from different sources (Table 1) could induce pro-inflammatory cytokines. We unexpectedly found that native high molecular weight HA preparations "D" and "F"were able to stimulate the synthesis of significant levels ofIL-12 and TNF-a. in a dose dependent manner (Figure la) while other native high molecular weight preparations showed no activity.
Table 1 Characteristics ofHA preparations used in our study. Products
Molecular mass (Da)
Source
Suplasyn®
0.5-0.8 x 10"
Streptococci sp.
A
0.6-2.0
X
10"
Streptococci sp.
B
0.5-1.0
X
10"
Streptococci sp.
C
0.5-0.8 x 10"
Rooster comb
D
0.6-2.0 x 10"
Rooster comb
E
>2.0xl0"
Rooster comb
F
0.5-1.2 x 10"
Bovine trachea
HA preparations were provided by or purchased from several manufacturers. Suplasyn® was from Bioniche Life Sciences Inc., London, Ontario, Canada.
The ability of high molecular weight HA to induce the synthesis of IL-12 and TNF-a. by monocytes suggested that contaminating molecules could be responsible for the induction of these cytokines. Endotoxin contamination has been shown to be able to induce pro-
432
The action of hyaluronan in cells
a •
__ J
1000
=-
750
-E
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500
u
250
~
HA "D" (lL-12)
o
HA "D" (TNF-a)
•
HA "F" (IL-12)
M
HA "F" (TNF-a)
0.1
10
50
100
HA concentration (ug/ml)
b
No treatment
l ~
+treatment DNase
~.I
__ ,
o
'
200
•
HA "0" (IL-12)
o
HA "0" (TNF-a)
•
HA "F" (IL-12)
•
HA "F" (TNF-a)
400
600
800
Cytokine (pg/ml)
Figure 1
Monocytes were incubated at 1 x l06cellsJml for 48 h at 37°C, 5% CO 2 with (a) different concentrations ofHA preparation "D" and "F", and (b) with 10 ug/ml ofHA preparations "D" and "F" or with 10 ug/ml ofHA preparations "D" and "F" treated with 10 U of DNAse I as described in the Materials & Methods. IL-12 and TNF-ex levels in the supernatant were measured after 48 h by ELISA. Data are expressed as the mean +/- S.D. of three independent experiments.
Pro-intlammatory activity of DNA
433
inflammatory cytokines such as IL-12 and TNF -a by monocytes and to cause septic shock in mammals!", However, none of the HA preparations evaluated contained detectable endotoxin or protein. DNA from numerous species has been shown to induce pro-inflammatory cytokines including IL-12 and TNF-a I 5 • In vivo, DNA purified from either Gram-positive or Gramnegative bacteria has been shown to cause septic shock in D-galactosamine-sensitized mice". Administration ofbacterial DNA as well as synthetic oligonucleotides into the lungs ofmice has been found to cause severe inflammation in the lower respiratory tract", Furthermore, intra-articularly injection ofDNA containing CpG motifs has been shown to induce arthritis in mice". We therefore determined whether the different HA preparations contained DNA. We found that 3 out of the 7 HA preparations tested contained significant levels of DNA with molecular weights ranging from 500 b.p. to> 20 000 b.p.; HA preparation "A": 5% , HA preparation "D": 3% and HA preparation "F": 15%. Treatment ofthe HA preparations with DNase I, an endonuclease which digests both single- and double- stranded DNA molecules to small oligodeoxyribonucleotides, resulted in the abolition ofIL-12 and TNF-a induction by HA preparation "D" and in a reduction ofIL-12 and TNF-a synthesis by HA preparation "F", with reductions of 77% and 32% respectively (Figure Ib). The results obtained with the preparation "F" correlated with an incomplete digestion ofthe DNA by the DNAse I (data not shown). Inactivated DNase I had no effect on the cytokine-inducing activity ofHA preparations "D" and "F" (data not shown). The DNase I used in these studies does not possess hyaluronidase activity. The presence ofDNA in HA preparations could be explained by association or contamination during the isolation and purification ofHA from rooster comb or from bovine trachea. One HA preparation, HA preparation "A", isolated from streptococci sp. also contained DNA but did not elicited a cytokine response from monocytes. It would therefore appear that the nature of the DNA is important. Experiments are underway to determine the sequence motifs present in DNA associated with HA which are responsible for the induction of cytokines.
CONCLUSIONS In summary, we found that two out of seven HA preparations examined had the ability to induce pro-inflammatory cytokines. The ability ofHA to induce these cytokines was not related to the presence of low molecular weight HA fragments, but rather to the presence of contaminating DNA. It is perhaps advisable to determine whether pro-inflammatory DNA is present in HA preparations before their use in the treatment ofpatients with inflammatory disorders as well as the potential modulation ofpro-inflammatory cytokines or inflammation in animal models.
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2.
A. Lussier, A. Cividino, C. A. McFarlane, W. P. Olszynski, W. 1. Potashner & R. De Medicis, Viscosupplementation with Hylan for the treatment of osteoarthritis: findings from clinical practice in Canada, J. Rheumato/., 1996, 23, 1579-1585. H. Matsuno, K. Yudoh, M. Kondo, M. Goto & T. Kimura, T. Biochemical effect of
434
3. 4.
5. 6. 7. 8.
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15.
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18.
The action ofhyaluronan in cells intra-articular injections of high molecular weight hyaluronate in rhematoid arthritis patients, Inflamm. Res., 1999,48, 154-159. A. Morales, L. Emerson, J. Curtis Nickel & M. Lundie, Intravesical hyaluronic acid in the treatment of refractory interstitial cystitis. J. Urol., 1996, 156,45-48. M. P. Puttick, J. P. Wade, A. Chalmers, D. G. Connell & K. K. Rangno, Acute local reactions after intraarticular hylan for osteoarthritis ofthe knee. J. Rheumato/., 1995,22, 1331-1314. M. E. Adams, Acute local reactions after intraarticular hylan for osteoarthritis of the knee. J. Rheumato/., 1996,23,944-945. D. O'Hanlon, Acute local reactions after intraarticular hylan for osteoarthritis of the knee. J. Rheumato/., 1996,23,945-946. J. R. Kirwan & E. Rankin, Intra-articular therapy in osteoarthritis. Bail/ieres Clin. Rheumatol., 1997,11,769-794. C. M. McKee, M. B. Penno, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao & P. W. Noble, Hyalronan (HA) fragments induce chemokine gene expression in alveolar macrophages: the role ofHA size and CD44. J. Clin. Invest., 1996, 98, 2403-2413. J. Hodge-Dufour, P. W. Noble, M. R. Horton, C. Bao, M. Wysoka, M. D. Burdick, R. M. Strieter, G. Trinchieri & E. Pure, Induction ofIL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages. J. Immuno/., 1997, 159, 2492-2500. R. A. Dodds, J. R. Connor, F.H. Drake & M. Gowen, Expression of cathepsin K messanger RNA in giant cells and their precursors in human ostoarthritic synovial tissues. Arthritis Rheum., 1999,42, 1588-1593. F. Liote, B. Boval-Boizard, D. Weill, D. Kuntz & J. L. Wautier, Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression and cytokine release. Clin. Exp. Immunol., 1996, 106, 13-19. T. J. Christmas, & G. F. Bottazzo, Abnormal urothelial HLA-DR expression in interstitial cystitis. Clin. Exp. Immunol., 1992, 87,450-454. H. G. Lee & M. Cowman, An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal. Biochem., 1994,219,278-287. M. Astiz, D. Saha, D. Lustbader, R. Lin & E. Rackow, Monocyte response to bacterial toxins, expression of cell surface receptors, and release of anti-inflammatory cytokines during sepsis. J. Lab. Clin. Med., 1996, 128,594-600. D. M. Klinman, A-K. Vi, S. L. Beaucage, J. Conover & A. M. Krieg, A. M., CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon. Proc. Natl. Acad. Sci. USA, 1996,93,2879-2883. T. Sparwasser, T. Miethke, G. Lipford, K. Borschert , H. Hacker, K. Heeg & H. Wagner, Bacterial DNA causes septic shock. Nature, 1997, 386, 336-337. D. A. Schwartz, T. J. Quinn, P. S. Thome, S. Sayeed, A-K, Yi & A. M. Krieg, CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract. J. Clin. Invest., 1997, 100, 68-73. G-M. Deng, I-M. Nilsson, M. Verdrengh, L. V. Collins & A. Tarkowski, Intraarticularly localized bacterial DNA containing CpG motifs induces arthritis. Nature Med., 1999,5,702-705.
EFFECT OF HYALURONAN OLIGOSACCHARIDES ON THE EXPRESSION OF HEAT SHOCK PROTEIN 72 Heping xu·,J, Tomomi Ito" Akira Taweda', Hiroshi Maeda" Hiroko Yamanokuchi" Kyoko Isahara', Keiichi Yoshida" Yasuo Uchiyama", Akira Asari t 'Seikagaku Corporation. Tateno 3-1253, Higashiyamato-shi, Tokyo 207-0021 Japan. 2
Department of Cell Biology and Anatomy. Osaka University Medical School. Suita-shi, Osaka. Japan. 3
Present address: Department ofOphthalmology, University ofAberdeen Medical School. Foresterhill, Aberdeen AB25 2ZD. UK.
ABSTRACT We have previously shown that intra-articular treatment with a hyaluronan preparation (849 kDa), HA84 up-regulates heat shock protein 72 (Hsp72) expression and suppresses degeneration of synovial cells in an arthritis model. In that study, the HA administered was degraded into HA oligosaccharides in the synovial tissue, suggesting that HA84 or degradation products of HA may up-regulate Hsp72 expression. Thus, in the present study, we examined the effects of HA oligosaccharides of various molecular sizes on Hsp72 expression and/or cell death in stressed cells. Western blotting analysis showed that treatment of K562 cells with HA tetrasaccharides up-regulated Hsp72 expression after exposure to hyperthermia. On the other hand, treatment of the cells with HA of other sizes (di-, hexa-, deca-, dodeca-saccharides), HA84 or tetrasaccharides of keratan sulfate did not elicit any change in expression of the Hsp72 protein. Treatment of the cells with tetrasaccharides of HA up-regulated not only expression of the Hsp72 protein but also Hsp72 mRNA expression, and enhanced activation of HSFl, a transcription factor controlling Hsp72 expression, after exposure to hyperthermia. Since the level of Hsp72 protein was not affected by tetrasaccharides of HA when the K562 cells were kept at 37°C without any stress, it is evident that tetrasaccharides of HA did not act as a stress factor. In addition, tetrasaccharides of HA suppressed cell death in the case of K562 cells exposed to hyperthermia and of PC12 cells under serum deprivation. These results suggest that certain types of oligosaccharides i.e., the tetrasaccharides of HA, up-regulate Hsp72 expression by enhancing the activation of HSFI under stress conditions. KEYWORDS
436
The action of hyaluronan in cells
Cell death, heat shock factor I, heat shock protein 72, hyaluronan oligosaccharides
INTRODUCTION Heat shock proteins (Hsps) are induced to suppress cell damage when cells are exposed to environmental insult'. Hsp70 suppresses apoptosis by preventing processing of caspase 3 2• We have previously shown that intra-articular treatment with an HA preparation (840 kDa), HA84, suppresses degeneration of synovial cells in a canine arthritis model and up-regulates Hsp72 expressiorr'", In that study, we also examined the kinetics of HA84 degradation in synovial tissues by injecting fluorescent-labeled HA84 and found that some labeled HA particles could not be detected by means of an HA-binding protein that binds specifically to HA molecules larger than decasaccharides". These observations suggested that HA oligosaccharides formed through degradation of HA84 in the tissue may suppress cell damage by up-regulating Hsp72 expression. In the present study, we prepared HA oligosaccharides of various molecular sizes and treated cultured cells with them under stress conditions in an effort to determine the appropriate size of HA oligosaccharides required to up-regulate Hsp72 expression or to suppress cell death. Effects of HA molecules on Hsp72 expression were investigated by examining Hsp72 protein levels and Hsp72 mRNA levels, and the activation of heat shock factor 1 (HSFl), a transcription factor controlling Hsp72 expression, in K562 cells exposed to the stress of hyperthermia. HSFI is known to be transferred to the nucleus from the cytoplasm and it binds to a heat shock element in the DNA 5 ,6. Moreover, HSFI is phosphorylated, and its molecular weight thereby increases when activated, soon after heat shock treatment", In addition to Hsp72 expression and HSFI activation, the effects of HA molecules on cell death were examined using 1<562 cells exposed to hyperthermia and PCI2 cells under conditions of serum deprivation. It has been reported that HA fragments induce angiogenesis', and/or induce the expression of genes involved in the inflammatory responses. We show here a novel activity of HA oligosaccharides, i.e., up-regulation of Hsp72 expression through enhancing HSFI activation under stress conditions.
MATERIALS & METHODS Preparation of oligosaccharides HA oligosaccharides were prepared from the degradation products generated by treatment of HA (Seikagaku Corporation, Tokyo, Japan) with dimethylsulfoxide containing HCI or testicular hyaluronidase (Seikagaku Corporation, Tokyo, Japan). The degraded HA was divided into fractions containing HA oligosaccharides of each size by anion-exchange chromatography according to the modified method of Inoue et a1. 9 • Unsaturated HA oligosaccharides were prepared from the degradation products
Effect of oligosaccharides on heat shock protein 72
437
generated by treatment of HA with chondroitin AC-I lyase (Seikagaku Corporation) by the same chromatography method as mentioned above. The following oligosaccharides of HA were used in the present study: HA 2, unsaturated disaccharides of HA (MiA2) , HA 4 , hHA4 , HA6 , hHA 6 , HAs, HAlO' HA 12 • Keratan sulfate oligosaccharides, Gal(S03)-GlcNAc(S03) [IA] and Gal(S03)GlcNAc(S03)-Gal(S03)-GlcNAc(S03) [1A1A] , were prepared from a keratanase II (Seikagaku Corporation) digest of keratan sulfate (shark fin, Seikagaku Corporation) through sequential steps of gel-filtration and anion-exchange adsorption column chromatography. LA and LALA were used for comparison with HA oligosaccharides as the other kind of glycosaminoglycan oligosaccharides. Sizes of HA oligosaccharides were determined by HPLC. Culture of K562 cells for detection of Hsp72 and HSFI Western blotting was performed examining K.562 cells which had been incubated in the presence of 0, 1, 10 or 100 ng/ml HA 2 , MiA 4 , HA 6 , HAlO' HA 12, HA84 or LAU at 43°C for 20 min followed by further incubation at 37°C for 2 h. 1<562 cells incubated at 37°C for 2 hand 20 min without any treatment were used as the 'no heat shock' normal control. Moreover, 1<562 cells incubated in the presence of hHA4at 37°C for 2 hand 20 min were examined by Western blotting to investigate whether Hsp72 expression is induced by MiA4 under non-stress conditions. To detect Hsp72 mRNA expression by northern blotting, K.562 cells were incubated in the presence or absence of 1 ng/ml hHA 4 at 43°C for 20 min, with or without further incubation at 37°C for 30 min, 1 h or 2 h. To evaluate HSFI activation by Western blotting, 1<562 cells were incubated with 0, 1, 10 or 100 ng/ml MiA 4 , HA4 , HA6 , HAs or HA84. The K.562 cells were stressed at 42°C or 43°C for 20 min. K.562 cells incubated at 37°C for 20 min without any treatment were used as the 'no heat shock' normal control. Antibodies used in immunostaining for Hsp72 and HSFI For the detection of Hsp72, monoclonal anti-Hsp72 antibody (RNP1197, Amersham, Buckinghamshire, England) as the first antibody and horseradish peroxidase- or ATCconjugated goat anti-mouse IgG (Jackson Lab., West Grove, PA) as the second antibody were used. For the detection of HSF1, rabbit anti-HSFI polyclonal antibody (SPA-901, Stressgene, Victoria, B.C., Canada) as the first antibody and horseradish peroxidase- or ATC-conjugated goat anti-rabbit IgG (Jackson Lab.) as the second antibody were used. Northern blotting analysis of Hsp72 Total RNA was prepared from control 1<562 cells and each culture of hHA4-treated
438
The action ofhyaluronan in cells
cells. Each sample was fractionated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to a nylon membrane. For hybridization, the membrane was incubated overnight at 42°C in the presence of a denatured 32P-Iabeled human hsp72 oligonucleotide probe (Oncogene Science, Inc., Cambridge, MA, USA), added to the prehybridization solution. A labeled cDNA probe specific for glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as a hybridization control. The membrane was washed RT in SSPE, then subjected to autoradiography. Confocal laser scanning microscopy 1<562 cells were fixed with paraformaldehyde, then permeabilized by incubation in PBS containing Tween 80. The cells were incubated with the rabbit anti-HSFI polyclonal antibody followed by the ATC-conjugated goat anti-rabbit IgG. Then, they were observed using a confocal laser scanning microscope (Leica, Heidelberg, Germany). Detection of ceUdeath To evaluate the effect of MiA4 on cell death induced by hyperthermia, 1<562 cells were incubated with 1 nglml MiA4 for 20 min at 43°C followed by incubation for 2 or 4 h at 37°C. 1<562 cells incubated for 4 h at 37°C without any treatment were used as a 'no heat shock' normal control. Then, these cells were incubated with Annexin V (R&D Systems Europe Ltd., UK), which binds to phosphatidylserine exposed on the outer surface of the cell membrane of dead cells, just after cell culture as described above. Cell death was analyzed by flowcytometry (FACScan; Becton-Dickinson, Franklin Lake, NJ, USA). It has been reported that serum deprivation induces apoptosis in PCI2 cells". PCI2 cells were cultured under conditions of serum deprivation in the presence of HA oligosaccharides, HA84 or KS oligosaccharides at 100 nglml. The cell death assay was performed by the trypan blue exclusion method, 24 h after the start of culture. The survival rate of cells cultured in the absence of serum but in the presence of 100 nglml nerve growth factor (NGF) was taken to be 100%.
RESULTS EtTects of HA oligosaccharides on Hsp72 expression Hsp72 protein expression was detected even in non-treated 1<562 cells not exposed to hyperthermia. The results showed that treatment of the 1<562 cells with MiA4 upregulated Hsp72 expression 2 h after exposure to hyperthermia (Fig. la). The same result was obtained in the case of HA4-treated cells (data not shown). Hsp72 expression was not affected by MiA4 treatment in the case of cells not exposed to hyperthermia
Effect of oligosaccharideson heat shock protein 72
439
(Fig. lb). The Hsp72 protein level was not changed by treatment with HA 2, HA 6 , HA84, LALA (Fig. Ia), HA l 2 (data not shown) or HAlO (data not shown) in the case of K562
Figure 1. (a) Western blotting of Hsp72 in K562 cells exposed to hyperthermia in the
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presence or absence of HA 2 , AHA 4 , HA 6 , HAlO' HA84 or LALA. (b) Western blotting of Hsp72 in K562 cells incubated at 37°C in the presence or absence of AHA4 • bH: bacterial Hsp72 used as a control. cells exposed to hyperthermia. Northern blotting analysis showed that Hsp72 mRNA expression in K562 cells was up-regulated 30 min and 1 h after exposure to hyperthermia as a result of treatment with AHA4 (Fig. 2). Time after h IlDerthermia
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Figure 2. Northern blotting of Hsp72 in K562 cells incubated in the presence (+) or absence (-) of 1 ng/ml AHA 4 • Effects of HA oligosaccharides on HSFI activation Western blotting analysis showed that treatment of the K562 cells with HA 4 or AHA 4 up-regulated the level of phosphorylated HSFI (approx. 80 kDa), an activated form of HSFl, and diminished the level of non-phosphorylated HSFI (approx. 70 kDa), in a dose-dependent manner, in cells exposed to hyperthermia at 42°C (Fig. 3a). In addition, HA 4 or AHA 4 increased the levels of both phosphorylated and non-phosphorylated HSFI when the cells were exposed to hyperthermia at 43°C (Fig. 3b). Activation of HSFI was little influenced by HAs, HA84 (Fig. 3b) or HA 6 (data not shown).
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Figure 3. Western blotting of HSFI in K562 cells treated with AHA 4 , HA4 , HAs or HA84 immediately after exposure to hyperthermia at 42°C (a), or 43°C (b)
440
The action of hyaluronan in cells
for 20 min. bHF: bacterial HSFI used as a control. Immunodeposits of HSFI were detected in MiA4 -treated (Fig. 4b) as well as nontreated (Fig. 4a) 1<562 cells not exposed to hyperthermia. After exposure to hyperthermia, immunodeposits of HSFI were detected as granular structures in the 1<562cells incubated in the absence of MiA4 (Fig. 4c, e). The HSFl-positive granules in the cells incubated at 37°C for a further 2 h were slightly larger in size than those observed immediately after exposure to hyperthermia (Fig. 4c, e). The HSFl-positive granules in the 1<562cells incubated in the presence of MiA4 (Fig. 4d, f) were finer than those in the cells incubated in the absence of MiA4 (Fig. 4c, e) after exposure to hyperthermia. 37'C 2b 20min
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Figure 4. Immunostaining of HSFI in 1<562cells incubated in the presence (AHAi +); b, d, f) or absence (MiAk); a, c, e) of I ng/ml MiA4 • The 1<562cells were incubated at 37°C for 2 hand 20 min (a, b), at 43°C for 20 min (c, d), or at 43°C for 20 min followed by 37°C for 2 h (e, f). Bar = 10 urn, Effects of tetrasaccharides of HA on ceU death Treatment with AHA 4 suppressed cell death in the case of 1<562 cells, as determined 2 and 4 h after exposure to hyperthermia (Fig. 5).
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Effect of oligosaccharides on heat shock protein 72
441
Apoptosis of PCl2 cells under conditions of serum deprivation was prevented by treatment of the cells with tetrasaccharides of HA (Fig. 6). Among the oligo saccharides tested in the present study, HA4 and ~HA4showed high effectiveness. On the other hand, treatment with the other HA oligosaccharides, high molecular weight HA (HA84) or keratan sulfate oligosaccharides faintly suppressed the cell death (Fig. 6 ). 100
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-10
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00
20
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Figure 6. Survival rates of PC12 cells under conditions of serum deprivation in the presence or absence (None) of several kinds of saccharides (100 nglrnl). NGF: Nerve growth factor DISCUSSION A critical size of HA oligosaccharides was required to up-regulate Hsp72 expression including HSFI activation in K562 cells exposed to the stress of hyperthermia, and to suppress cell death in the case of PC12 cells under conditions of serum deprivation in the present study. HA 6 have been used as a tool for probing the cell surface in a study of HA receptor function, having the minimum size required to effectively compete with native HA in binding to its cell surface receptor!', HA4as well as LUiA 4 up-regulated the Hsp72 expression in the present study, suggesting the possibility that there may be as yet unidentified receptors for HA tetrasaccharides in the cells. ~HA4 treatment suppressed the formation of HSFI granules in cells exposed to hyperthermia. Sarge et at. have shown that the kinetics of appearance of HSFI granules in the nuclei of Hela cells during heat shock is very well correlated with the kinetics of HSF DNA binding and heat shock gene transcription'", Alternatively, they noted the possibility that the granules observed may represent large aggregated particles of inactive HSFI. This coincides well with our present findings that LUiA 4 treatment suppresses HSFI granule formation and up-regulates the HSFl activation as well as Hsp72 expression after exposure to hyperthermia. In conclusion, our results show that tetrasaccharides of HA up-regulates Hsp72 expression by an increase in the level of activated HSFI under stress conditions, but not non-stress conditions. When HA is natively depolymerized by hyaluronidases or radicals in vivo, HA may acquire a novel function, i.e., up-regulation of Hsp72 expression.
442
The action ofhyaluronan in cells
REFERENCES S. Lindquest, The heat shock response. Annu. Rev. Biochem.,1986, 55,1151-1191. D. M. Dick, W. e. Antoine, B. Lucie, D.-L. Claude, and M. Bernard, Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol. Cell. Bioi., 1997, 17,5317-5327. 3. A. Asari, S. Miyauchi, S. Matsuzaka, T. Itoh, & Y. Uchiyama, Hyaluronan on heat shock protein and synovial cells in a canine model of osteoarthritis. Osteoarthritis Cartilage, 1996,4, 213-215. 4. A. Asari, S. Miyauchi, S. Matsuzaka. T. Itoh, E. Korninami, & Y. Uchiyama, Molecular weight-dependent effects of hyaluronate on the arthritic synovium. Arch. Histol. Cytol., 1998,61,125-135. 5. 1.1. Cotto, M. Kline, & RI. Morimoto, Activation of heat shock factor 1 DNA binding precedes stress-induced serine phosphorylation. Evidence for a multistep pathway of regulation. J. Bioi. Chem., 1996,271,3355-3358. 6. W. Xia, & R Voellmy, Hyperphosphorylation of heat shock transcription factor 1 is correlated with transcriptional competence and slow dissociation of active factor trimers. J. BioI. Chem., 1997,272,4094-4192. 7. D.e. West, & D.M. Shaw, Tumour hyaluronan in relation to angiogenesis and metastasis. In: The Chemistry, Biology And Medical Applications Of Hyaluronan And Its Derivatives, T.e. Laurent (ed.) 1998, Portland Press Ltd., London, pp. 227233. 8. P.W. Noble, e.M. McKee & M.R Horton, Induction of inflammatory gene expression by low-molecular-weight hyaluronan fragments in macrophages. In: The Chemistry, Biology And Medical Applications Of Hyaluronan And Its Derivatives, T.e. Laurent (ed.) 1998, Portland Press Ltd., London, pp. 219-225. 9. Y. Inoue, & K. Nagasawa, Preparation, by chemical degradation of hyaluronic acid, of a series of even- and odd-numbered oligosaccharides having a 2-acetamido-2deoxy-D-glucose and a D-glucuronic acid residue, respectively, at the reducing end. Carbohyd. Res., 1985, 141,99-110. 10. K. Isahara, Y. Ohsawa, S. Kanamori, M. Shibata, S. Waguri, N. Sato, T. Gotow, T. Watanabe, T. Momoi, K. Urase, E. Kominami & Y. Uchiyama, Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases. Neuroscience, 1999,91,233-49. 11. e.B. Knudson, & W. Kundson, Hyaluronan-binding proteins in development, tissue homeostasis, and disease. FASEB J., 1993,7, 1233-1241. 12. K.D. Sarge, S.P. Murphy & R.I. Morimoto, Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNAbinding activity, and nuclear localization and can occur in the absence of stress. Mol. Cell. BioI., 1993, 13, 1392-1407. 1. 2.
VISCOSUPPLEMENTATION: A HISTORICAL PERSPECTIVE Biomatrix, Inc., Ridgefield, NJ, USA ABSTRACT The development of the therapeutic paradigm viscosupplementation for long-lasting pain relief in human and animal joints with osteoarthritis or traumatic arthritis was based on the finding that in arthritic conditions, the average molecular weight and concentration of hyaluronan decreased and consequently the elastoviscous properties of the synovial fluid are substantially reduced. Viscosupplementation is a therapeutic process in which the pathological synovial fluid or effusion is removed from the joint by arthrocentesis and is replaced with a highly purified hyaluronan solution which has a 16- to 30-times greater concentration than the pathological joint fluid, and a 2- to 5-times greater concentration than that of the hyaluronan in the healthy synovial fluid. In some preparations of hyaluronan used today for therapeutic purposes, the rheological properties (viscosity and elasticity) are low, and therefore the elastoviscosity of the fluid is similar to that of fluid removed from an arthritic joint. Another preparation available to patients worldwide is composed of hyaluronan derivatives (hylans) with substantially greater elastoviscosity than hyaluronan solutions and has comparable rheological properties to those of the fluid found in young, healthy individuals. The clinical benefit of viscosupplementation is longlasting pain relief in arthritic joints. DEFINITION VISCOSUPPLEMENTATION: The use of elastoviscous fluids and viscoelastic gels to replace or supplement body fluids and the extracellular matrix of tissues. In recent decades, viscosupplementation became a new therapeutic paradigm for the treatment of arthritis; in this therapeutic process the pathologic synovial fluid is replaced with elastoviscous fluid made from hyaluronan or from its derivative, hylan. This supplementation of synovial fluid results in long-lasting pain relief and improved function of the joint. HISTORICAL REVIEW 1966 - 1969: The concept of viscosupplementation for the joint was developed based on extensive studies of the rheology of synovial fluid and the physiochemical properties of hyaluronan in bovine, equine and human joints under normal and arthritic conditions 1-4. 1968 - 1971: Biotrics, Inc. (Arlington, MA, USA) developed a high molecular weight (23 million) purified hyaluronan from human umbilical cord and rooster comb and defined it as the non-inflammatory fraction of hyaluronan (NIF-NaHA) which was later patentedl". 1970 - 1971: The high molecular weight, non-inflammatory fraction of hyaluronan was first used as a viscosupplement in the joints of animals. In dogs, cartilage was artificially
386
Aspects of hyaluronan in joints
injured, and NIF-NaHA subsequently introduced; the cartilage wound healed, the inflammatory reaction of the synovial tissue decreased, and the capsular pain was reduced.v'' Similar observations were made in race horses, demonstrating that the pain of natural traumatic arthritis could be relieved by viscosupplementation't". 1972 - 1976: Biotrics, Inc. trademarked the name Healon® for their first highly purified hyaluronan (NIF-NaHA) and licensed it to Pharmacia AB (Uppsala, Sweden) for manufacturing and worldwide distribution for use in the treatment of arthritis in animals and humans and for ophthalmic viscosurgery. 1969 - 1981: Clinical studies in humans performed in Sweden, France, South Africa, Great Britain and the USA showed clinical utility of Healon® in osteoarthritic joints7.10-13. Healonf was never marketed for viscosupplementation of human arthritic joints. 1987 - 1988: Two NlF-NaHA products with an average molecular weight of 0.5 - 0.75 million, were marketed for viscosupplementation in human osteoarthritis: in Japan, Artz® (Seikagaku SPH) and in Italy, Hyalgan® (Fidia S.p.A.). Both were recommended to be injected 5-10 times on a weekly basis I4- 17• Hyalgan and Artzal are currently available worldwide. 1985 - 1992: Two crosslinked hyaluronan molecules were developed at Biomatrix, Inc. (Ridgefield, NJ, USA), generically called "hylans,,18. Hylan G-F 20 for treatment of osteoarthritis was formulated from two forms of hylan, a fluid and a geI19•2o • Hylan G-F 20 has physical properties that closely resemble those of healthy young synovial fluid, and has greater elastoviscosity and longer tissue residence time than hyaluronan". 1992: Hylan G-F 20 (S~ViSC®) was first marketed in Canada for viscosupplementation in human osteoarthritis/ ,23. Synvisc is currently approved for use in 58 countries, with a treatment regimen consisting of three injections administered over fifteen days. 1998: Another purified NIF-NaHA was marketed in Canada with an average molecular weight of one milliorr'". This product, Orthovisc® (manufactured by Anika Therapeutics, Inc., Woburn, MA), is presently also sold in Europe, Turkey and Israel, using three injections administered over two weeks.
Viscosupplementation: a historical perspective
387
RHEOLOGICAL CONSIDERATIONS 100
0 10 20 30
90 80 70
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Figure 1. Viscosupplemenation Basic Principle The elastoviscosity of osteoarthritic synovial fluid is diminished compared to that in a healthy joint. Viscosupplementation provides a long-lasting analgesic effect, improves the rheological environment in the joint, and restores protective lubricating and shockabsorbing functions.
10 20
80 70
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60
40 50 60
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FIGURE 2. Percent Elasticity of Synvisc®, Healon®, Orthovisc®, Artzal® and Hyalgan® The elasticity in % and viscosity in % express the elastic and viscous properties of the material. They are calculated by the following formulae. % Elasticity = 100 x [(90 - Phase Angle)/90] % Viscosity 100 - 100 x [(90 - Phase Angle)/90]
=
388
Aspects ofhyaluronan in joints
All viscoelastic materials exposed to faster movement wiII be increasingly elastic and less viscous. This means that more of the mechanical energy transferred to the viscoelastic material from its environment will be stored as elasticity, and less will be lost as heat. The consequence of this is that a material with a higher percent of elasticity is more protective (as a shock absorber and vibration isolator) for the tissues and cells of the joint than a material with lower elasticity. In conclusion, the higher the elastic properties, the more efficacious the material as a viscosupplementation device. REFERENCES 1. E. A. Balazs, Viscoelastic properties of hyaluronic acid and biological lubrication, (Symposium: Prognosis for Arthritis: Rheumatology Research Today and Prospects for Tomorrow, Ann Arbor, Michigan), Univ. Mich. Med. Ctr. J., 1968, (Suppl), 255259. 2. E. A. Balazs, D. Watson, I. F. Duff and S. Roseman, Hyaluronic acid in synovial fluid. I. Molecular parameters of hyaluronic acid in normal and arthritic human fluids, Arthritis Rheum., 1967, 10,357-375. 3. E. A. Balazs and D. A. Gibbs, The rheological properties and biological function of hyaluronic acid, In: Chemistry and Molecular Biology ofthe Intercellular Matrix (E. A. Balazs, ed.), Academic Press, London, 1970, 1241-1254. 4. E. A. Balazs, The physical properties of synovial fluid and the special role of hyaluronic acid, In: Disorders of the Knee (A. Helfet, ed.), JB Lippincott, Philadelphia, 1974,63-75. 5. E. A. Balazs, Ultrapure Hyaluronic Acid and the Use Thereof, US Patent No.4 141 973, February 27, 1979. 6. E. A. Balazs, Hyaluronic Acid and Matrix Implantation, Biotrics, Inc., Arlington, MA,1971. 7. N. Rydell and E. A. Balazs, Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of osteoarthritis and on granulation tissue formation, Clin. Orthop., 1971, 80, 25-32. 8. E. A. Balazs and J. L. Denlinger, Sodium hyaluronate and joint function, J. Equine Vet. sa; 1985, 5, 217-228. 9. N. W. Rydell, J. Butler and E. A. Balazs, Hyaluronic acid in synovial fluid. VI. Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of arthritis in track horses, Acta Vet. Scand., 1970, 11, 139-155. 10. J. G. Peyron and E. A. Balazs, Preliminary clinical assessment of Na-hyaluronate injection into human arthritic joints, Pathol. Biol., 1974, 22, 731-736. 11. A. J. Helfet, Management of osteoarthritis of the knee joint, In: Disorders ofthe Knee (A. J. Helfet, ed.), Lippincott, Philadelphia, 1974,175-194. 12. C. Weiss, E. A. Balazs, R. St. Onge and J. L. Denlinger, Clinical studies of the intraarticular injections of Healon® (sodium hyaluronate) in the treatment of osteoarthritis of human knees, Semin. Arthritis Rheum., 1981, 11, 143-144. 13. J. G. Peyron, Intra-articular hyaluronan injections in the treatment of osteoarthritis state of the art review, J. Rheumatol., 1993,20 (Suppl. 39), 10-15. 14. O. Namiki, H. Toyoshima and N. Morisaki, Therapeutic effect of intra-articular injection of high molecular weight hyaluronic acid on osteoarthritis of the knee, Int. J. ofClin. Pharm. Ther. and Tox., 1982, 20,11501-507. 15. W. Puhl, A. Bemau, H. Greiling, et al., Intra-articular sodium hyaluronate in osteoarthritis of the knee: a multicenter, double-blind study, Osteoarthritis and Cartilage, 1993, 1,233-241.
Viscosupplemcntation: a historical perspective
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16. G. U. Grecomoro, Martorana and C. DiMarco, Intra-articular treatment with sodium hyaluronate in gonarthrosis: A controlled clinical trial versus placebo, Pharmatherap., 1987, 5, 137-141. 17. R. D. Altman and R. Moskowitz, A randomized clinical trial of intra-articular sodium hyaluronate in patients with osteoarthritis of the knee: A summary, Am. J. Orthop., 1999, 28, 3-4. 18. E. A. Balazs and E. A. Leshchiner, Hyaluronan, its crosslinked derivative - hylan and their medical applications, In: Cellulosics Utilization: Research and Rewards in Cellulosics. (H. Inagaki and G. O. Phillips, eds.), (Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future), Elsevier Applied Science, New York, 1983, 233-241. 19. E. A. Balazs and A. Leshchiner, Cross-Linked Gels of Hyaluronic Acid and Products Containing Such Gels, US Patent No.4 636524, January 13, 1987. 20. E. A. Balazs, E. A. Leshchiner, A. Leshchiner and P. Band, Chemically Modified Hyaluronic Acid Preparation and Method of Recovery Thereof From Animal Tissues, US Patent No.4 713 448, December 15, 1987. 21. E. A. Balazs and J. L. Denlinger, Viscosupplementation: A new concept in the treatment of osteoarthritis, J. Rheumatol., 1993,20 (Supp\. 39), 3-9. 22. M. Wobig, A. Dickhut, R. Maier and G. Vetter, Viscosupplementation with hylan GF 20: A 26-week controlled trial of efficacy and safety in the osteoarthritic knee, CUn. Therapeut.1988,20,410-423. 23. A. Lussier, A. A. Cividino, C. A. McFarlane, W. P. Olszynski, Potashner and R. de Medicis, Viscosupplementation with hylan for the treatment of osteoarthritis: findings from clinical practice in Canada, J. Rheumatol., 1996, 23, 1579-1585. 24. K. D. Brandt, J. Michalski, J. A. Block and J. R. Caldwell, Clinical experience with Orthovisc'" (Sodium Hyaluronate) in the treatment of osteoarthritis of the knee, Anika Therapeutics, Inc., Woburn, MA, 1999.
THE IMPACT OF HYALURONAN ON THE IN VITRO INV ASIVE PROPERTIES OF HUMAN BREAST CANCER CELL LINES WITH HIGH CD44 EXPRESSION Andrea Herrera-Gayol'and Serge Jothy2* 'Department ofPathology, McGill University, 3775 University St. Montreal, Quebec, Canada, H3A 2B4. 2 Department
ofAnatomic Pathology, Sunnybrook and Women's College Health Sciences Centre, 2075 Bayview Ave., Toronto, Ontario, Canada, M4N 3M5.
ABSTRACT Invasion is a three step-process characterized by altered cell adhesion, cell motility and degradation of the extracellular matrix (ECM) by tumor cells. The ECM interacts with tumor cells facilitating or inhibiting invasion. Hyaluronan (RA), a component of the ECM, binds to cell membrane CD44. The tumor-stroma interface of breast cancer is enriched with HA. Its function is not well understood. Hypothesis: RA induces tumor behavioral changes related to invasion. Experiments: The effects of immobilized RA (iRA) and soluble RA (sRA) on CD44 expression, adhesion, haptotaxis and non-directed motility were studied in four human breast cancer cell lines: Hs578T (high CD44 expression); MDA-MB-231 (high CD44 expression); MCF-7 (low CD44 expression) and ZR-75-1 (CD44 negative). Results: iRA increased adhesion of Hs578T cells by 600%, haptotaxis by 110% and nondirected motility by 150%. iRA enhanced the CD44 expression of Hs578T cells by 140%. After two days in culture with sRA, CD44 expression remained unchanged while adhesion to and haptotaxis towards RA increased. After three days, cells expressed 60% more CD44, adhesion increased and haptotaxis decreased by 40%. Non-directed motility remained unchanged. Similar changes were observed with MDA-MB-231 cells. No changes were observed with ZR-75-1 and MCF-7 cells. Conclusions: Our results suggest that RA has an impact on the in vitro invasive properties of the high CD44 expressing cells, Hs578T and MDA-MB-231, by changing CD44 expression, adhesion, haptotaxis and motility. KEYWORDS Breast cancer, hyaluronan, adhesion, motility INTRODUCTION Both cell adhesion and migration are key players in the invasion of the extracellular matrix (ECM) by tumor cells. Hyaluronan (HA) is a component of the ECM, The intact polymer of HA participates in cell adhesion and motility 1-2 while its fragments can induce the production of cytokines and chemokines 3-4 and angiogenesis 5. HA binds to CD44 6. CD44 is a membrane glycoprotein playing a role in cell adhesion, migration and invasion of malignant cells 7. As human breast cancer cells can express CD44 8 and
444
The action ofhyaluronan in cells
the tumor-stroma interface is enriched with HA 9 we hypothesized that HA could induce tumor behavioral changes related to the invasiveproperties ofCD44 positive cells. MATERIALS & METHODS Cell culture Four human breast cancer cell lines from American Type Culture Collection (ATCC, Rockville, MD) were used: Hs578T (high CD44) MBA-MB-231 (mediumhigh CD44); MCF-7 (low CD44) and ZR-75-1 (CD44 negative) 8. All cells but ZR-75I, were cultured in regular culture medium (RCM): DMEM (Gibco, Burlington, Ontario, Canada), 5% FBS (Gibco), 2mM glutamine (Gibco) and gentamycin (Gibco). The ZR-75-1 cells were cultured in RPMI (Gibco) and 10% FBS. Cells were exposed to: 1- HA (Sigma, St Louis, MO) in solution (sHA) at Img/ml diluted in RCM for 1-6 days; 2- immobilized HA (iHA) where different surfaces were coated with HA; 3hyaluronidase Type I (Sigma), 500 U or 1000 U per ml of RCM. Antibodies and flow cytometry A pan-CD44 antibody (clone F.I0.44.2) was obtained from Novocastra Laboratories (Newcastle, U.K). A FITC labelled goat anti-mouse IgG (Cedarlane, Hornby, Ontario, Canada) was used as secondary antibody. 3 x 105 unfixed cells were resuspended in 0.05% NaN3 in PBS. Primary and secondary antibodies were diluted in NaN3/PBS at a 1/50 and 1/250 concentration respectively. Background fluorescence was evaluated by omitting the primary antibody. The analysis were performed on an EPICS® scan (Coulter Corporation, Hialeah, Fl). Adhesion assays Cells were cultured for different time points in RCM or in sHA (Img/ml) trypsinized and plated on 96-well plates which have been previously coated with HA at 500ug/ml, l mg/ml or 5mg/ml or bovine serum albumin (BSA, Sigma) 0.2%. After 1-2 or 24 hr of incubation at 37°C, wells were washed and the adherent cells were fixed with cold methanol. Cells were stained with Gill's hematoxylin and counted under an inverted microscope. Haptotaxis The underside of inserts' membranes (modified Boyden chambers) were coated with HA at l mg/ml or 5mg/ml or uncoated (controls). After 4 hr, cells in the upper surface were removed. Cells in the underside were fixed, stained and counted. Cell migration assays: "wound assay" Glass coverslips were coated with HA at 5 mg/ml or 0.2% BSA. Cells were plated on top. After reaching confluence, cells were wounded. The medium was replaced by DMEM with or without serum or with sHA. Cells were allowed to migrate for 24 hr Coverslips were washed, fixed with cold methanol and stained with Gill's hematoxilin. Cells inside the margins of the wound area were counted.
Impact on human breast cancer cell lines
445
Cell proliferation assay Cells were plated on 6-well plates containing RCM, sHA lrng/rnl in RCM or on top of HA-coated wells. Cells were allowed to grow for 1, 2, 3, or 6 days, trypsinized, and counted with a hemacytometer after trypan blue staining. RESULTS & DISCUSSION Table I. Effect of hyaluronan (HA) and hyaluronidase (HAdase) on the in vitro biiopat h0 Iogica I charactenstics 0 f 4 human breast cancer cell lines Cell Lines
CD44 CD44
Adh
Hap
Mot
CD44
sHA' Adh
Hap
CD44
iHA"
HAdase
Hs578T
++++
tl40%
t600%
tno%
t150%
t60%
t75%
.1.40%
t900/0**
MDA-MB231
+++
t40%
t20%
t30%
tlO%
t60%
300%
.1.50%
tSO%
MCF-7
++
ne
ne
ne
ne
ne
ne
ne
ne
-
ne ne ne ne ne ne ne ne ZR-75-1 . effect of coatmg the surfaces with HA, CD44 expression at 3 days 10 culture on .HA,. , sHA. data at 3 days 10 culture with sHA; Adh: adhesion; Hap: haptotaxis.; Mot: random motility; ne: no measurable effect. "at 3 days for Hs578T and 2 days for MDA-23I. In bold: statistically significant
iHA acts as a promoter of adhesion and migration for the Hs578T cell line. iHA induced CD44 expression after 3 days in culture. Small increases in adhesion, migration and CD44 expression were observed when using the MDA-23I cell line. No measurable effects were detected when the MCF-7 and ZR-75-1 cell lines were studied. The Hs578T and MDA-MB-231 cells cultured with sHA changed their CD44 expression and their adhesive and migratory patterns after 3 days in culture. No significant changes were observed in proliferation or random motility. CONCLUSIONS Immobilized and soluble HA has the ability to modulate membrane CD44 expression and the adhesive and migratory characteristics of tumor cells according to their CD44 status: Hs578T (high CD44 expressor) and MDA-MB-231 cells (mediumhigh CD44 expressor) respond to HA; but no effects were observed on the MCF-7 (low CD44 expressor) and ZR-75-1 cells (CD44 negative). In vivo, the concentration ofHA, its location within the tissue, the proportion of its soluble form and the relation between polymeric versus oligomeric HA, and the upregulation of CD44 would determine the adhesive and migratory behavior of tumor cells during the invasion process.
446
The action of hyaluronan in cells
REFERENCES 1. E. A. Turley, Hyaluronan and cell locomotion. Cancer Metastasis Rev., 1992, 11,
21-3. 2. L. Thomas, H. R. Byers, J. Vink and 1. Stamenkovic, CD44H regulates tumor cell migration on hyaluronate-coated substrate. J. Cell BioI., 1992, 118,971-7. 3. C. M. McKee, M. B. Permo, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao, and P. W. Noble, Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J. Clin. Invest., 1996, 98, 2403-13. 4. D. E. Boyce, A. Thomas, J. Hart, K. Moore, and K. Harding, Hyaluronic acid induces tumour necrosis factor-alpha production by human macrophages in vitro. Br. J. Plast. Surg., 1997, 50, 362-8. 5. D. C. West, 1. N. Hampson, F. Arnold, and S. Kumar, Angiogenesis induced by degradation products of hyaluronic acid. Science, 1985, 228, 1324-6. 6. 1. Entwistle, C. L. Hall and E. A. Turley, HA receptors: regulators of signalling to the cytoskeleton. J. Cell. Biochem., 1996,61,569-77. 7. A. Herrera-Gayol and S. Jothy, CD44 modulates Hs578T human breast cancer cell adhesion, migration, and invasiveness. Exp. Mol. Pathol., 1999,66,99-108. 8. M. Culty, M. Shizari, E. W. Thompson and C. B. Underhill, Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms ofCD44: correlation with invasive potential. J. Cell Physiol., 1994, 160,27586. 9. P. Bertrand, N. Girard, B. Delpech, C. Duval, J. d'Anjou, and J. P. Dauce, Hyaluronan (hyaluronic acid) and hyaluronectin in the extracellular matrix of human breast carcinomas: comparison between invasive and non- invasive areas. Int. J. Cancer, 1992,52, 1-6.
THE AVAILABILITY OF HIGHLY ELASTOVISCOUS HYLAN FOR VISCOSUPPLEMENTATION CAN DELAY KNEE REPLACEMENT IN PATIENTS WITH ADVANCED OSTEOARTHRITIS I'C. Weiss, 2 D. Waddell and 3E. Miller "Mount Sinai Medical Center of Greater Miami 6431 Pine Tree Drive Circle, Miami Beach, Florida 33141 USA 20 rthopedics Specialists of Louisiana, Shreveport, Louisiana, USA 3 Wellington
Orthopedic and Sports Medicine, Cincinnati, Ohio USA
ABSTRACT
An analysis was performed to determine how viscosupplementation influences the need for knee replacement surgery in orthopaedic practice. A total of989 patients (1366 knees), in three clinical practices were treated with hylan G-F20 over a two-year period. The patients had advanced osteoarthritis (predominantly Kellgren and Lawrence X-ray Grade IV) and severe symptoms/disability. Most patient knees (82%) received a single 3injection course of treatment. The majority of patients were clinically improved for 6 months or longer. No significant systemic adverse reactions were observed, and local reactions occurred at a rate of 2-3% per injection, similar to other intra-articular procedures. The response to the second course of treatment was found to be similar to that of the first course in terms of safety and effectiveness. The rate of knee replacement in patients at all three clinical sites was significantly reduced compared to historical controls. Interestingly, Health Care Utilization Data from the United States similarly reflect declined national rates of knee replacement since the introduction of viscosupplementation. These data demonstrate that viscosupplcmentation with hylan GF20 is an effective treatment for patients with advanced knee osteoarthritis that may postpone knee replacement surgery in certain patients. INTRODUCTION
Osteoarthritis (OA) is a chronic degenerative disease with serious societal consequences. It affects more than half the population over age 65 and consumes approximately 1% of the GDP in health care costs in the USA 1,2 . Though satisfactory treatments are available to reduce pain and disability during the early stages of the disease, patients generally progress to chronic pain which is refractory to medical treatments, leading them to seek surgical intervention. Joint replacement surgery is a highly effective treatment, but because of its inherent risks" and limitations on the durability of joint prostheses, it is medically desirable to delay joint replacement surgery in many patients. To detemine whether treatment of knee OA with hylan G-F20 (Synvisc®) was effective in late-stage patients who would otherwise be considered as candidates for knee replacement surgery, a prospective observational study was carried out independently by three orthopaedic surgeons.
392
Aspects of hyaluronan in joints
METHODS
A total of 989 consecutive patients in the practice of the investigators treated with hylan G-F20 (Synvisc®) for knee OA were prospectively followed for 6-24 months with a minimum follow up of 6 months. Both knees were treated in 377 patients. A total of 1366 knees were treated. Initially, patients received a single 3-injection course of hylan G-F20 over a 15-day period. Patients could receive treatment with a second 3-injection course ofhylan G-F20 as clinically required. The response to treatment was rated on a 5point scale (much worse, worse, same, better, much better) or the Hospital for Special Surgery Knee Rating Scale. Progression to knee replacement was determined from patient charts. The evaluation of knee replacement rates before and after the introduction of viscosupplementation was performed using data derived from practice coding records using ICD-9 (diagnostic) and CPT (treatment) codes. Adverse events in the injected knee, as well as systemic events, were recorded. RESULTS
The patients in this study were generally considered to be candidates for knee replacement surgery. Most had a long history of OA with significant cartilage erosion and bone changes evident by X-ray (Table I). The patients had failed multiple prior treatments and presented with chronic pain which limited their daily activities.
Table 1. Patient Population
#Patients # Knees Kelgren & Lawrence X-ray Grade % Grade VI % Grade III % Patients failed or Contraindicated to Other treatments: analgesics And/or NSAIDS
WEISS 233
WADDELL 648
MILLER 108
COMBINED 989
303
927
136
1366
61% 32%
83% 13%.
73% 23%
80% 16%
100% 100%
100% 100%
100% 100%
100% 100%
The majority of patients showed significant clinical improvement in both pain and function after the first course of treatment. This improvement lasted for 6 months or longer (Table 2).
393
Availability of highly clastoviscous hylan
.
. T a bl e 2 Effiectrveness AnallYSIS
% of knees significantly improved 6 months after 1st course Number of knees progressing to Knee replacement in patients who Received only one course % knees significantly improved 6 months after 2nd course Number of knees progressing to Knee replacement in patients who Received two courses
WEISS
WADDELL
MILLER
COMBINED
65%
63%
76%
64%
280f303
400f927
23 of 136
91 of 1366
54%
58%
92%
61%
001'57
140fl29
oof12
140f198
Fourteen percent of knees required retreatment with a second course ofhylan G-F20 at some point during the 18-month study period (Table 3). Patients receiving two courses of hylan G-F20 experienced comparable effectiveness to the first course of treatment. Table 3. Hylan G-F20 Courses" Administered Over a 2 Year Period
Single course
WEISS 303 knees
WADDELL 927 knees
MILLER 136 knees
COMBINED 1366
Two courses
57 knees
181 knees
12 knees
250
·A course of'hylan G-F20 treatment consists 00 intra-articular injections of2l11L,adminstered over a 15-day period.
Of the 1366 knees treated with Synvisc, 91 (6.7%) of knees progressed to knee replacement surgery after the first course of treatment and 105 progressed to knee replacement after 2 years, an overall rate of 7.7% (Table 2). To evaluate how the rate of knee replacement was affected by the availability of viscosupplementation, practice databases utilizing diagnostic (ICD-9) and treatment (CPT) codes were analyzed (Table 4). This analysis demonstrates a drop of 25 - 62% in the need for knee replacement in the same clinical practices before and after the use of viscosupplementation with hylan GF20. The large variation of this figure in the three practices is most likely due to differences in patient populations (Table 4). Table 5 summarizes the safety data collected in these 989 patients treated with hylan G-F20. No systemic adverse events related to hylan G-F20 treatment were noted in any of the patients. Local reactions in the injected knee occurred after approximately 1.9% of the injections. Occasionally, these reactions were acute, severely painful, and accompanied by significant effusion which sometimes contained a large number of white
394
Aspects of hyaluronan in joints
blood cells. None of these reactions were regarded as serious and all reactions resolved without sequelae. There were no incidents ofjoint infection in this study. Table 4. Analaysis of the Effect of Viscosupplementation on the Prevalence of Knee Replacement Surgery Based on Diagnostic and Treatment Coding Data
WEISS
% of Total OA Knee in practice which Progressed to knee replacement PRIOR to the AFTER the availability Availability of Of Viscosupplementation Viscosupplementation 4.7% 1.8%
0/0 Decrease
-62.0%
WADDELL
7.3%
4.8%
-34.0%
MILLER
9.1%
6.8%
-25.0%
*COMBINED
7.0%
4.5%
-36.0%
* average across all three practices
Table 5. Safety Analysis
Local adverse events (rate per injection) of all Courses
WEISS
WADDELL
MILLER
COMBINED
2.1%
1.8%
2.2%
1.9%
DISCUSSION These data demonstrate that viscosupplementation with hylan G-F20 is effective for managing pain and disability in patients with late stage OA of the knee. Considering that this patient group had failed multiple other treatments for knee OA and were referred to be considered for surgery, it is important that more than 92% of knees did not progress to knee replacement during the 2-year study period. This decreased need for knee replacement surgery is confirmed by the analysis of coding data which demonstrated a significant drop in total knee replacement surgery rates relative to a comparable patient pool taken from the period preceding the introduction of viscosupplementation. Data from a national database likewise indicates a nationwide drop in total knee replacement surgery since viscosupplementation products became available in the USA4• • The safety profile for hylan G-F20 observed in this trial compares favorably with other treatments for OA. The intra-articular injection of viscosupplements can be associated with post-injection flares of pain and swelling which, though sometimes appearing
Availability of highly clastoviscous hylan
395
significant, have had no sequelae other than being local, transient events. Viscosupplementation compares favorably with other alternatives for managing late stage OA patients it is less invasive with less morbidity and mortality than knee replacement. The treatment of OA with NSAIDS has been reported to be associated with 8,800 deaths and 52,000 hospitalizations in a single year in the USA5 • The safety profile observed in this group of patients treated with hylan G-F20 compares very favorably with these statistics. The success of viscosupplementation in managing patients with late stage OA provides a significant advance in orthopaedic therapeutics, with the major societal benefits being reduced disability and improved quality of life.
REFERENCES 1.
2. 3.
4. 5.
P. Tugwell, Economic evaluation of the mangement of pain in osteoarthritis, Drugs, 1996,52 (Suppl. 3),48-58. Impact of arthritis and other rheumatic conditions on the health-care system-United States, Morbidity and Mortality Weekly Report. 1997,48 (3), 349-353. J. H. Lonner and P. A. Lotke, Aseptic complications after total knee arthroplasty, Journal of the American Academy of Orthopaedic Surgeons, 1999, Vol. 7(5), Sept/Oct., 311-323. Hip and knee implant review, Orthopaedic Network News, 1999, Vol. 10 (3), July. G. Singh and R. Rosen, NSAID induced gastrointestinal complications: The ARAMIS perspective-I 997, 1. Rheumatol., 1998, (SuppI.51), 8-16.
CLINICAL EVALUATION OF HYALURONIC ACID COMBINED WITH TOLMETIN IN THE TREATMENT OF OSTEOARTHRITIS OF THE KNEE Sbaojin Wang l
,
Yanli Be2 ,
Peixue Ling 2,
Tianmin Zbang
3
l1heFirstAffiliatedHospital ofShandongMedica/ University, Jinan250012, China. 2Shandollg C.P.Frer;JaPharmaceutica1sCo..Ltd., Jinan250014, J ShandongBiopharmaceutica1s Institute,
China.
Jinan250014, China
ABSTRACf
67 out-patients with idiopathic osteoarthritis (OA) of the knee were enrolled in a six-week, double-blind trialof sodium hyaluronate (HA)plustolmetin sodium (HA-tolmetin) or HAinjected intra-articularly. 34 patients received 5 injections of 10mg of tolmetin sodium and 20 mg ofHA in 2ml of phosphate bufferat weekly intervals, the other 33 patients onlyreceived 20 mg ofHA in 2 ml of phosphate buffer in a similar regimen as control. Assessments of activities of daily living (ADL)and pain weremadebefore each uyection and 7 days after the final injection The results showedthat progressive reductions in themean scores of ADL and pain were significant in both groups, andthe meanscores of all parameters were lowerin HA-tolmetin group than in HAgroup. No evident side effects were observed in HA-tolmetin group. The results suggest that HA and tolmetinhavesynergism in thetreatment of OAof the knee. KEYWORDS
Hyaluronic acid,tolmetin, osteoarthritis, knee INTRODUCTION
The treatment of osteoarthritis (OA) of the knee is directed towards the reliefof symptoms. Nonsteroidal anti-inflammatory drugs (NSAIDs) playan important roledue to their dualanalgesic and anti-inflammatory action Sideeffects are the primary problems in many patients, leading to cessation ofthernpy. The local application ofNSAIDs can avoidthese effects by delivering low dosesdirectly to the affected site. High-moleeualr-weight sodium hyaluronate (HA)solution injected intra-articularly is proved to be effective in the treatment of OAof the kneeby a greatdeal of clinical reports. In this paper, we reportand evaluate the efficacy and safety of theformulation on volunteers withOAof the knee. MATERIALS & METHODS Patients
67 out-patients of either sex with idiopathic OA of the knee were enrolled in a six-week, double-blind trialofHA-tolmetin versus HAas control. Inclusion criteria: age 18-70; OA of the kneeverified by clinical examination, laboratory tests and radiographs according to Altman'; for patients taking NSAIDs or glucocorticoids, the wash-out time shouldbe no less than 2 weeks.
398
Aspects of hyaluronan in joints
Exclusion criteria: accompanying OAof the hip,which couldconfuse assessment; patients with malfunction ofliver and kidney. Drug
HA-tolmetin Injection: 2ml contains 20mg of HA (mean molecuar weight range 1.5x1062.0x106 Da) and lOmgoftolmetin sodium, provided by Shandong C'P FredaPhann.Co. HA Injection: SofastlM , 2ml con1ains 20mg of HA with a mean molecuar weight range 1.5xl 06_ 2.0x106 Da, provided by Shandong C.P. FredaPhann.Co. Treatment
On entering the trial, patients were divided randomly to either the HA-tolmetin or HA group, and underwent a series of pre-treatment assessments and necessary laboratory investigations. All iniections were given intra-articularly once a week for consecutive 5 weeks. The iniections of drugs and assessments wereperformed by different surgeons to eliminate any possible bias. Corticosteroids, otherNASIDs andanalgesics were not pennittedto be used in any form during thetreatment period. Evaluation of efficacy
The index assessed and scored according to Lequesne': (l)Activities of daily living (ADL) including 4 different activities, eachbeingscored on 4-point scale (0= no difficulty to 3= activity impossible). Theactivities were: getting up from sitting, climbing upand down stairs, squatting or bending on the knee, walking on uneven ground; (2) The grading of the severity of pain on movement, pain at rest and pain on press were assessed using 10 em visual analogue scales (VASl, All patients were assigned to one of the five severity grades according their scores beforeand after treatment respectively. Grade I: ~30; Grade II:2Q....29; Grade III: 11-19; Grade IV: 5-10; Grade V:(}-4. The efficacy was divided intofour grades. Excellent: scores for all indices weredownto 0, or the severity grade decreased no lessthan4 grades at the end of the treatment Good: the severity grade decreased no lessthan3 grades at the end of the treatment Fair: the severity grade decreased by 1-2 grades at the end of the treatment Poor: the decrease in severity grade did not surpassed original grade at the end of thetreatment Evaluation of safety
Before and afterthe treatment, urine and venous bloodsamples of each patient weretestedfor routine urinoscopy, hematological and blood biochemical screen investigation, and all suspected adverse reactions wererecorded. Statistical analysis Wilcoxen's Signed Rank Tests and T-testswere usedto analyze the significance of the difference between the groups before and aftertreatment Two-tailed statistical testswere usedand ~.05 wastaken as the threshold abovewhichdifferences or changesare considered as non-significant.
two
Hyaluronic acid combined with tolmetin
399
RESULTS &DISCUSSION The detailed information of patients in the two groups was listed in table 1. All patients completed the trial. Therewereno significant differences (P;::'0.05) in sex, age and the distribution of severity grades betweenthe two groupsbeforetreatment The information of the patientsof in the two treatmentgroupsbeforetreatment Sex Grade Groups Age(mean) No. Male Female I II III IV V HA-tolmetin 34 12 14-16(53.1) 6 13 8 7 0 21 22...{j2(50.5) HA 33 11 4 14 9 6 0 22 Tablel.
ADL There were progressive reductions in mean scores during the 5 week treatment in both groups (Table 2, Figure 1). After the treatment, there was significant difference between the two groups (P<0.01). Table 2.
The changes in the mean scores ofADL and pain dtuing the treatment period (mean±SD)
Grou ps HA-tolmetin HA
ADL Pre-treatment Post-treatment 7.91±1. 96· 1.68±1. SO" 7.30+1. 76 2.82+2.21
Pain Pre-treatment Post-treatment 15.03±4. OS· 1.18±2.34" 16.27+4. OS 4.00+3.00
*: P;::'0.05, **: P
Progressive reductions in mean pain scoreswere also foundin bothtreatment groupsduring the 5 week treatment(Table2 and Figure 2). Duringthe treatment period, therewere progressive reductions in both groups, the mean score reduction was greaterin HA-tolmetin group than in HA group, and the difference was significant (P
-1
-o-HA group - - HA-tolmetio group
-7 ' - - - - - - - - ' - - - - ' -
__'_
3
- ' -_ _----1
4
Time/weeks
Figure 1. The changesin the mean scoresofADLduringthe treatmentperiod
400
Aspects of hyaluronan in joints
-o-HAgroup - - - HA-tolmetin group
" - -_ _..J
-15 L -_ _......._ _--'-_ _- - ' 3
•
Time/weeks
Figure 2. Progressive reductions in themean scores of painduring the treatment period Efficacy evaluation
The distribution of severity of patients and the efficacy evaluated by doctors and patients at the end of the treatment was listed in thetable3.
Table3. The distribution of severity andthe efficacy at the endof thetreatment Groups
I
II
III
IV
HA-tohnetin HA
0 0
0 0
0 6
9 19
v
Efficacy"(by 00cta"s) Fair Good Poor
ExceIlwt 15 25 8 7
14 13
5 13
0 0
Efficacy"(by patiaJls) Fair Poor ExceIIaJt Good 18 9
12 13
4
11
0 0
*:P
Possibly drug-related events were reported by 3 patients in HA group afterthe third injection, but none in HA-tolmetin group. The joints of all the 3 patients swelled with aggravated pain several hours after injection, and the signs disappeared within 3 days since it occurred without treatment. Allof the laboratory screening investigations werenotchanged significantly. Tolmetin effectively suppresses inflammation and is used widely in the treatment ofOA. The oraladministration doseis 900-1200mg perday. Manypatients can'ttolerate itssideeffects, which limit its use in chronic disease such as OA. In this paper, the intra-articualr injection dose of tolmetin is 10mg oncea week, and noadverse effects wereobserved. The results suggest that the formulation may represent a valuable approach to the patients with OA of the knee with more efficacy and safety. REFERENCE
1. R. Altman, E. Asch, D. Bloch, G Bole, D. Borenstein, Development of criteria for the classification and reporting of osteoarthritis. ArthritisRheum., 1986, 29,1039-1049. 2. M Lequesne, Indices of severity and disease activity for osteoarthritis, Semin. Arthritis Rheum., 1991,20 (suppl 2),48-54. 3. K SriwatanakuI, W Kelvie, L. Lasagna, IF. Calimlim,0.F. Weis, et al, Studies withdifferent types of visual analog scales for measurement of pain. Clin. Pharmacol. Then, 1983, 34, 234-239.
IDENTIFICATION OF A NOVEL INTRACELLULAR BYALURONAN-BINDING PROTEIN, IHABP4 Lei Huang, Nicholas Grammatikakis, Masahiko Yoneda, Shib D. Banerjee, and Bryan P. Toole" Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111, USA
ABSTRACT The functions of hyaluronan in extracellular matrices and at the surface of cells have been extensively studied. However, it is now clear that significant pools ofhyaluronan are also present intracellularly and, in an attempt to define the function of intracellular hyaluronan, we have sought to identify intracellular hyaluronan-binding proteins. In previous studies we identified CDC37, a cell cycle regulatory protein, using a monoclonal antibody that recognizes a novel group ofhyaluronan-binding proteins. In this study, we have identified a second hyaluronan-binding protein with this antibody, and characterized its properties. This protein, which we term IHABP4, is an intracellular and specific hyaluronan-binding protein containing several hyaluronan-binding motifs, namely B(X7)B (where B denotes basic and X denotes non-acidic amino acids). Comparison of the deduced chick, mouse and human protein sequences indicates that the hyaluronan-binding motifs in these sequences are conserved. By biochemical fractionation and immunofluorescent localization of epitope-tagged IHABP4 we have shown that it is mainly present in the cytoplasm. We also confirmed the intracellularlocalization ofhyaluronan in actively cycling cells. These data support the possibility that intracellularhyaluronan and its binding proteins may play important roles in cell behavior.
INTRODUCTION Hyaluronan is a ubiquitous glycosaminoglycan (GAG) in extracellular and pericellular matrices. On the basis of its unique physical and chemical properties, it has been well documented that hyaluronan plays a critical role in dynamic structural changes within extracellular matrices during development and tissue remodelling, as well as in maintenance of mechanical properties and homeostasis ofmany tissues':'. Interactions ofhyaluronan with hyaluronan binding proteins (HABPs) are involved in establishing and modifying the structural properties of extracellular matrices. Furthermore, it is increasingly appreciated that hyaluronan is a crucial pericellular and cell surface component that actively participates in regulating cell behavior through its interactions with cell surface HABPs, such as CD44 and RHAM:M 1•4 Surprising new developments, however, have been the convincing demonstration of intracellular hyaluronan and its fluctuation during the cell cycle", as well as the discovery of several intracellular HABPS (IHABPs), including CDC37 6 , RHAMM/IHABP4, 7 and P32 8 . Although the interactions and functions of intracellular hyaluronan and IHABPs are not yet understood, their discovery raises an interesting question. Does intracellularhyaluronan, like extracellularhyaluronan, play an important role in regulating cellular behavior through interactions with IHABPs?
448
The action ofhyaluronan in cells
lDENTIFICATION OF A NOVEL INTRACELLULAR HABP, IHABP4
Usingthe monoclonal antibody (mAb), IVd4, which recognizes a novel group of chick HABPs9, we previously identified and characterized CDC37, an intracellular HABP which has now been shown to be an important cell cycle regulatory protein". In a more recent study, we have characterized a second novelHABP recognized by mAb IVd4, using similar strategiesto those previously described". This HABP, like CDC37, is also an intracellular protein containing multiple hyaluronan binding motifs, B(X7)B, in which B denotes arginine or lysine and X denotes any amino acid except glutamic or aspartic acid". Furthermore, we have cloned the chick, mouse and human homologs of this gene (GenBank/ EBI Data Bank accession numbers AF227683, AF227684, and AF241831), shown that the hyaluronan binding motifs are conserved among the deduced chick, mouse and human protein sequences, and directly demonstrated that the chick and mouse proteins bind hyaluronan specifically". IHABP4 contains multiple conserved hyaluronan-binding motifs throughout its sequence, notably one stretch with three overlapping motifs in the middle region ofthe protein sequence. These results implythat the hyaluronan-binding properties of this proteinhave functional importance. We have termedthis protein llIABP4 since three other IHABPshave so far been characterized: CDC376, RHAMM/ IHABp 4,7 and P32 8 and since the function of llIABP4 is not yet understood. Human IHABP4 appears to be the homolog of Ki-U57, an intracellular kinase-associated protein that was partially characterized previously". This is ofparticuiar interestsince two other intracellular HABPs, CDC37 and RHAMM are also associated with kinases. Ki-l/57, is expressed in activated, but not resting, human lymphocytes and is enriched in some tumor cells". Obviously, to understand the function of this novel HABP, further investigation will be needed including examination of the expression levels in various normal cells vs corresponding tumor cells, as well as its interaction with other regulatory or structural protein(s). DISCUSSION It has becomeincreasingly evidentthat hyaiuronan is present not only extracellularly but also in the cytoplasm, nucleusand other organelles within various types of tissues and cells" Recent studies provide evidence that rapid uptake of exogenous hyaluronan added to transformed lOTl/2 cellsleadsto accumulation in multiple subcellularcompartments, such as cellprocesses, perinuclear area and nucleus, and directly affectscell motility", Moreover, it has been shown that there is a dramatic increase in endogenous intracellular hyaluronan and re-distribution from nucleolus to cytoplasm (mainly the perinuclear area) and interchromosomal regions during mitosis of mitogen-stimulated smooth muscle cells and fibroblasts'. There is also an increase in pericellular hyaluronan during cell division":"; however, extracellular hyaluronan that becomes internalized after addition to dividing cells is distributed in a different pattern to that of endogenous intracellular hyaluronarr'. Similar changesin the amountsand distribution of intracellularhyaluronan-binding sites also occur in dividing cells'. Thus it is of interestthat Ki-l/57 is localized in the cytoplasm, nucleus and nucleolus of Hodgkin's and myeloma cell lines", indicating that hyaluronan and Ki-l/57, i.e. IHABP4, may have overlapping distributions. The data discussed above suggest a possible role for intracellular hyaluronan and IHABPsin cell division. However it is not clear the extent to which hyaluronan is directly targeted to variousintracellular sites after synthesis rather than being first secreted and then re-internalized. Synthesis and interactions of pericellular hyaluronan have been shown to promote cell detachment and rounding during mitosis'v", anchorage independent growth
Intracellular hyaluronan-binding protein. IHABP4
449
in culture":", and tumor growth and progression in vivo 17•20 . Yet hyaluronan internalization has also been implicated in some ofthese processes'r". Possibly a dynamic balance between pericellular hyaluronan and intracellular hyaluronan exists, and hyaluronan re-internalization might contribute to this balance. This dynamic balance could in tum be involved in regulating various cellular behaviors. In this regard it is noteworthy that internalization into endothelial cells of a complex ofheparan sulfate-proteoglycan and a heparin-binding protein targets the latter to mitochondria, consequently preventing apoptosis". Also, as mentioned above, exogenously added hyaluronan can be targeted to various intracellular compartments and results in increased cell motility", Interestingly, in the latter study, targeting of hyaluronan to the nucleus was blocked by a B(X7)B-containing peptide suggesting the possible involvement of an IHABP. Although re-internalization of hyaluronan, and other GAGs, is likely to contribute to intracellular pools, the question of how these polymers reach sites such as the cytosol, mitochondria or nucleus remains unclear. One possibility is that they are transported in a retrograde manner through the Golgi, endoplasmic reticulum and cytosol in a similar manner to some protein toxins, or by other less understood, "nonclassical" routes24,2S. Although re-intemalization subsequent to secretion is one likely pathway whereby GAGs become localized to intracellular sites, recent evidence' indicates that it is unlikely to be the sole pathway for distribution of intracellular hyaluronan. Another possible mechanism for intracellular targeting of hyaluronan arises from its unique mechanism of synthesis. Hyaluronan is synthesized at the cytoplasmic face of the plasma membrane and secretion takes place by extrusion during polymer elongation". It is thus conceivable that a subpopulation of hyaluronan polymer is directly deposited in the cytosol rather than crossing the plasma membrane; this in tum may be regulated by interaction with IHABPs. The most important, and puzzling, question arising from the various studies discussed above is the exact mechanism whereby intracellular hyaluronan might influence cellular events. It is possible that the high level of hydration associated with hyaluronarr' also plays a role in structural changes in the cytoskeleton or nuclear matrix during cell division or motility, e. g. in regulating cell shape or volume changes. Another possibility is that hyaluronan acts as an intracellular template for the assembly of interacting signaling factors, such as kinases, thus amplifying the efficiency with which they interact. Irrespective of the role of intracellular hyaluronan, it is to be expected that its functions will be regulated and/or mediated by IHABPs. However, direct evidence for intracellular interaction in situ and for the functional consequence of these interactions is needed to elucidate this possibility.
ACKNOWLEDGEMENTS This work was supported by National Institutes of Health Grants CA73839 and CA82867
REFERENCES I.
B.P.
Toole,
Glycoforum:
Science
of
hyaluronan
today.
b.tt.p.;.!/ww.w.,.g!):.l:.o.f.Q.ru.m.•grjp., 1999. 2. 3.
B.P. Toole, In: Proteoglycans: Structure, Biology and Molecular Interactions. R. Iozzo (ed.), Marcel Dekker, NY, 2000, pp. 61-92. W. Knudson, & C.B. Knudson, Glycoforum: Science of hyaluronan today. http:l(www.glycQfonnn.gt".jp. 1999.
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4. 5. 6.
I. Entwistle, C.L. Hall, & E.A. Turley, 1. Cell. Biochem., 1996,61,569-577. S.P. Evanko, & T.N. Wight, 1. Histochem. Cytochem., 1999,47, 1331-1342. N. Grammatikakis, A Grammatikakis, M. Yoneda, Q. Yu, S.D. Banerjee, & B.P. Toole, 1. Bioi. Chem., 1995,270, 16198-16205. V. Assmann, 1.F. Marshall, C. Fieber, M. Hofinann, & I.R. Hart, 1. Cell Sci., 1998, Ill, 1685-1694. TB. Deb, & K Datta, 1. Bioi. Chem., 1996,271,2206-2212. S.D. Banerjee, & B.P. Toole, Dev. Bioi., 1991, 146, 186-197. L. Huang, N. Grammatikakis, M. Yoneda, S.D. Banerjee, & B.P. Toole, 1. Bioi. Chem., 2000,275,29829-29839. B. Yang, B.L. Yang, R.C. Savani, & E.A. Turley, EMBO 1., 1994, 13,286-296. I. Kobarg, S. Schnittger, C. Fonatsch, H. Lemke, M.A Bowen, F. Buck, & H.P. Hansen, Exp. Clin. Immunogenet., 1997, 14, 273-280. L. Collis, C. Hall, L. Lange, M. Ziebell, R. Prestwich, & E.A. Turley, FEBS Letters, 1998,440,444-449. S.P. Evanko, LC. Angello, & TN. Wight, Arterioscler. Thromb. Vase. Biol., 1999, 19, 1004-1013. M. Brecht, U. Mayer, E. Schlosser, & P. Prehm, Biochem. 1., 1986,239,445-450. D. Rohde, H. Hansen, M. Hafuer, H. Lange, V. Mielke, M.L. Hansmann, & H. Lemke, Am. 1. Path., 1992, 140,473-482. R. Kosaki, K Watanabe, & Y. Yamaguchi, Cancer Res., 1999, 59, 1141-1145. R.M. Peterson, Q. Yu, I. Stamenkovic, & B.P. Toole, Am. 1. Path., 2000, 156, 2159-2167. N. Itano, T Sawai, O. Miyaishi, & K Kimata, Cancer Res., 1999,59,2499-2504. Q. Yu, B.P. Toole, & I. Stamenkovic,1. Exp. Med., 1997,186,1985-1996. M. Culty, M. Shizari, E.W. Thompson, & C.B. Underhill, 1. Cell Physio/., 1994, 160, 275-286. D. Liu, E. Pearlman, E. Diaconu, K Guo, H. Mori, T Haqqi, S. Markowitz, I. Willson, & M.S. Sy, Proc. Natl. Acad. Sci. U.S.A., 1996,93, 7832-7837. AM. Olofsson, M. Vestberg, H. Herwald, 1. Rygaard, G. David, KE. Arfors, V. Linde, H. Flodgaard, I. Dedio, W. Muller-Esterl, & E. Lundgren-Akerlund,1. Clin. Invest., 1999, 104, 885-894. J.M. Lord, & L.M. Roberts, 1. Cell BiD/., 1998, 140, 733-736. A. Cleves, Current Bioi., 1997,7, R318-R320. P.H. Weigel, v.e. Hascall, & M. Tammi,1. Bioi. Chem., 1997,272, 13997-14000.
7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25 26.
THE PRESENCE AND PROCESSING OF INTRACELLULAR HYALURONAN IN PROLIFERATING CELLS Stephen P. Evanko and Thomas N. Wight The Hope Heart Institute.I 124 Columbia Street, Seattle, WA 98104, USA
ABSTRACT Hyaluronan (HA) is found in the extracellular matrix of many tissues where it is thought to promote cell migration and proliferation. Recent data suggests that hyaluronan-dependent pericellular matrix formation is a rapid process which occurs as cells detach during mitosis and migration. Growing evidence for intracellular hyaluronan in tissues in vivo, together with evidence of intracellular hyaluronan-binding molecules, prompted us to examine hyaluronan distribution and uptake, and hyaluronan-binding sites within cells and their relationship to cell proliferation in vitro using a biotinylated hyaluronan-binding protein and fluorescein-labeled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyaluronan staining was seen in the cytoplasm in a diffuse, network-like pattern and within vesicles. Nuclear hyaluronan staining was observed and confirmed by confocal microscopy, and was often associated with nucleoli and nuclear clefts. After serum stimulation of 3T3 cells, there was a dramatic increase in cytoplasmic hyaluronan staining, especially during late prophase/early prometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan-binding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus following stimulation. Exogenous fluorescein-labeled hyaluronan was taken up more avidly into vesicles in growing cells, but appeared to be localized distinctly compared to endogenous hyaluronan. Processing of internalized fluorescein-HA was more rapid and the cytoplasmic distribution of labeled HA was more persistent in cells replated in the presence of PDGP or serum. These data suggest that HA synthesis and degradation may be closely coupled and that intracellular hyaluronan and/or its internalization and degradation may be important for cell proliferation.
KEYWORDS Hyaluronan, degradation, cell proliferation, mitosis, PDGP
INTRODUCTION The role of hyaluronan (HA) in cell proliferation is well documentedl rl, but the mechanisms by which it affects cell behavior are only beginning to be appreciated. One proposed role for HA is that it may facilitate cell detachment during mitosis and cellular Iocomotionl- 3, 5, Evidence is growing that HA is present in the cytoplasm and nuclei in a number of tissues in vivo6 - 8 , A number of intracellular HA binding molecules that may be important in regulating the cell cycle or in gene transcription have also been recently described 9- l l.
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We have recently found that HA is present the nucleus and cytoplasm of smooth muscle cells and fibroblasts and that intracellular HA may playa role during cell proliferation 4. This study further examines the presence and processing of intracellular HA in order to begin to address the possible role of intracellular HA in cell proliferation.
MATERIALS AND METHODS Various cell types were kindly provided by colleagues in the Department of Pathology, The University of Washington. Human SMC were provided by Dr. Russell Ross; Human skin fibroblasts were provided by Dr. Peter Byers; 3T3 01 cells were provided by Dr. Daniel Bowen Pope; and mammary epithelial and mammary tumor cells were provided by Dr. Karen Swisshelm.Staining for intracellular HA was done as previously described using a biotinylated hyaluronan binding region from cartilage proteoglycan, kindly provided by Dr. Charles Underhill, Department of Oncology, Georgetown University'[. Where appropriate, extracellular HA was removed prior to fixation by incubation of the cells with 2 Ulml Streptomyces hyaluronidase at 37°C for 1 h. Cells were fixed in 2.5% formalin in PBS for 10 min., 22°C and then permeabilized with 0.5% Triton X-100 for 10 min. The probe was used at a concentration of 2.5 l1g1ml in PBS containing 1% BSA, for 1 h at 22°C. The bound probe was localized with Texas Red-streptavidin. Controls for specificity of intracellular staining included digestion of the cells with Streptomyces hyaluronidase after permeabilization, and preincubation of the probe with an excess of HA. Fluorescein-labeled HA (fHA) was prepared as described previously12. Uptake and degradation of fHA was assessed in 3T3 cells, taking advantage of their rapid responsiveness to serum deprivation and the ease with which they can be synchronized. For uptake studies, cells seeded on coverslips were made quiescent in 0.5% serum for 48 h and then stimulated with 10 % PBS or 10 ng/ml PDGF in the presence of fHA (5 l1g1ml) and allowed to take up the labeled HA for 24 h. Cells were fixed and then stained for endogenous intracellular HA as described above. To examine the rate of degradation of internalized fHA, 3T3 cells were allowed to take up fHA (added at 5 l1g1ml) for 72 h under normal growth conditions (10% PBS), and then replated onto coverslips in DMEM containing either 0.5% PBS, 0.5% PBS plus 10 ng/ml PDGF, or 10% PBS. Cells were fixed at various times and examined by fluorescence microscopy for content of fHA. We found that it was necessary to preload cells with fHA and then replate them because in pilot experiments in which we tried to wash out the free, uninternalized fHA, the intracellular signal continued to increase after the washout. This was most likely due to continued endocytosis of fHA which had bound to the culture dish or extracellular matrix and was not effectively washed out. Localization of intracellular HA binding sites4 was done as described. Controls for specificity of binding of the fHA to permeabilized cells included blocking with excess unlabeled HA, digestion of fHA with hyaluronidase, digestion of the fHA with pronase (to eliminate the possibility that fluoresceinated protein contaminants could be binding), and incubation of the cells with free, uncoupled fluoresceinamine. The controls indicated that intracellular binding was specific (not shown). Visualization and morphometric analysis of HA-versican matrices were done on control or PDGF-treated SMC as previously descnbedl-'. Time-lapse photomicroscopy was used in conjunction with the particle exclusion assay to examine the kinetics of pericellular coat formation.
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RESULTS AND DISCUSSION Intracellular Hyaluronan Hyaluronan was localized in permeablilized cells using a highly specific biotinylated probe prepared from the cartilage protoeglycan, aggrecan. In non permeabilized cells, HA was seen in the extracellular matrix and on the cell surface as a tangled flocculent network (data not shown). Streptomyces hyaluronidase digestion removed the cell surface and pericellular matrix staining, leaving only residual punctate staining on the cell surface. In cells treated with hyaluronidase and then permeabilized, HA was seen within vesicles and also in a diffuse, network-like distribution throughout the cell, a distribution consistent with that of the endoplasmic reticulum (see Fig. IB, below). HA was also seen within the nucleus and was often prominently associated with nucleoli in human SMC and with nuclear clefts in human skin fibroblasts (data not shown). This was confirmed by colocalization of HA with nuclear staining using confocal microscopy. When cells were treated with Streptomyces hyaluronidase after permeabilization, all of the intracellular staining was abolished (data not shown). A variety of other cell types, such as bovine endothelial cells, mouse 3T3 cells, normal mammary epithelial cells and mammary tumor cells also contained abundant intracellular HA (data not shown). In endothelial cells, HA was more prominent intracellularly than extracellularly under these conditions. The distribution of HA within these cells was usually very similar to that described above, although in one of the tumor cell lines, there was a more punctate appearance to the network of intracellular HA staining. HA has been localized ultrastructurally within the nucleus in other cells, particularly in nucleoli and areas of heterochromatin around the nuclear periphery, consistent with our observations 8. HA was also found in the cisternal space of the rough endoplasmic reticulum. The cytoplasmic distribution observed in the present study is consistent with a distribution in the rough endoplasmic reticulum. HA was also clearly present within vesicles. These are most likely endosomes as fluorescein-labeled HA is intemalized into similar vesicles (see below). This raises the question of the source of the intracellular HA and whether endosomal vesicles containing HA may be transporting it back to the endoplasmic reticulum through a retrograde transport mechanism.
Intracellular and Extracellular HA is Increased in Proliferating Cells 3T3 cells were made quiescent by incubation in 0.5% serum for 48 h and then stimulated to proliferate by addition of serum to 10%. In the control, unstimulated cells, only small amounts of intracellular HA staining was detectable (See Fig. IA), whereas in the stimulated cells, abundant intracellular HA was detected (Fig. lB). The most prominent intracellular HA staining was seen in mitotic cells where the HA appeared to Figure 1. Intracellular hyaluronan staining in (A) control fill the entire cell in the area 3T3 cells and (B) cells stimulated with fetal bovine serum.
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surrounding the chromosomes during metaphase, and then filling the area between separating chromosomes during anaphase'[. Strong staining persisted through telophase. During late telophase, small amounts of HA could be seen within the thin, membrane "bridges" linking the two separating daughter cells, as if the organelle(s) in which the HA was located had been drawn out during partitioning. In many cases these HA-positive "bridges" between the cells remained long after mitosis and often appeared to connect the nucleolus of one cell to another. Permeabilized control and serum stimulated cells were incubated with fHA to localize intracellular HA binding sites. The fHA bound prominently to the nucleoli in the nucleus of control cells as well as to a faint, lace-like network in the cytoplasm (data not shown, see 4). In the stimulated cells, there was a notable increase in the amount of binding of fHA to the reticular network in the cytoplasm indicating an increase in the amount of available binding sites. In addition, in contrast to the control cells, the fHA bound extensively throughout the nucleus and nuclear periphery in the stimulated cells. It is well known that hyaluronan synthesis and secretion is increased during cell proliferation 1, 3. Therefore, we used the particle exclusion assay to determine if the hyaluronan was organized into the pericellular matrix in SMC stimulated with PDGF. Only 25% of the control cells displayed pericellular matrices. In cells stimulated with PDGF, the number of cells displaying hyaluronan coats increased to approximately 70%. Coats were thicker in the stimulated cells also. Hyaluronan coats were present around virtually all the mitotic cells (data not shown see ref. 3). Time lapse imaging showed that pericellular coat formation occured as cells were detaching from the substrate during mitotic cell rounding, and thus appeared to facilitate the release and sliding of the cell across the substrate. Thus, both intracellular and extracellular HA are coordinately increased during mitosis, and the amount and distribution of intracellular HA binding sites is altered. The source and function of the intracellular HA is not yet clear. When cells were allowed to internalize fluorescein-labeled HA (fHA) and then stained for endogenous intracellular HA, there was a distinct distribution of the exogenous and endogenous material'l, The exogenous fHA was largely confined to endosomal vesicles and did not appear to colocalize with the endogenous intracellular HA in the reticular cytoplasmic network. This would suggest that some of the intracellular HA may be derived from an intracellular source. On the other hand, if the intracellular cytoplasmic HA is derived from intemalization and translocation of pericellular material, it is possible that the cells translocate their own endogenous HA much more efficiently than the labeled HA. The idea that HA is translocated may be supported by the results of another study, which showed that IOTl/2 cells rapidly translocated Texas Red-labeled HA to the cytoplasm and nucleus in conjunction with migration l'l, However, it is difficult to be sure that the fluorescent label is still associated with the HA in these kinds of experiments. Incubation of living cells in the presence of relatively high concentrations of Streptomyces hyaluronidase (20 U/ml) for 18 h almost completely abolished the intracellular HA (data not shown) while short term incubation of cells with hyaluronidase had no effect (see Fig 1). This suggests that at least part of the intracellular HA is translocated from the extracellular matrix and the removal of pericellular HA by the hyaluronidase prevented its internalization. On the other hand, it is also possible that the hyaluronidase was somehow able to penetrate into the same compartment containing the intracellular HA. Incubation of cells with concentrations of chloroquine or ammonium chloride which have been shown to inhibit lysosomal degradation of HA had either no effect or slightly
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increased the amount of intracellular HA in the reticular network (data not shown). Thus, not surprisingly, HA appears to accumulate in endosomes and other compartments in the presence of the lysosomal inhibitors. This suggests that lysosomal degradation of HA is not necessarily a prerequirement for its apparent translocation. At this point, we can only speculate on the source of the intracellular HA. The observations by Kan that HA is present in the rough endoplasmic reticulumf suggest that hyaluronate synthase (HAS) may be active immediately after its synthesis and insertion into the ER membrane and can begin synthesizing HA en route to the cell surface. The dramatic upregulation of HA synthesis during proliferation and the increase in intracellular HA during mitosis makes this idea plausible. Therefore, some of the HA may indeed be derived from intracellular synthesis. Another possibility is that active HAS is internalized and transported to other compartments through a retrograde mechanism. As mentioned above, newly synthesized pericellular HA could be transported into the cell in this same way. Neither of these possibilities sufficiently explain how HA gets into the nucleus, however.
HA Degradation is Also Increased in Proliferation Normally cycling 3T3 cells were allowed to take up fHA for 72 h (Fig. 2A) and then replated in the presence of low serum or high serum, or stimulated with PDGF. In low serum, the cells rapidly become quiescent and assume a flattened morphology. In these cells, the fHA remained within distinct endosomal vesicles and was not degraded, even after 36 hours Figure 2. 3T3 cells were following replating (Fig. 2B). In contrast, the cells which were incubated with fHA for 72 h replated in high serum rapidly lost the fHA from the (A) and then viewed 36 h after replating in the presence endosomes (Fig. 2C). Identical results were seen if cells were of (B) 0.5 % bovine serum stimulated with PDGF. This indicates that processing and turnover of internalized HA occurs more rapidly in or (C) 10 % serum. proliferating cells. This suggests that the intracellular degradation of HA may have a regulatory role during cell proliferation, and that it is closely coupled to the increased synthesis which occurs at this time.
REFERENCES 1. M Brecht, U Mayer, E Schlosser, P Prehm. Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem. J. 1986;239:445-450. 2. E Deudon, E Berrou, M Breton, J Picard. Growth-related production of proteoglycans and hyaluronic acid in synchronous arterial smooth muscle cells. Int. J. Biochem. 1992;24:465-470.
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3. SP Evanko, IC Angello, TN Wight. Formation of hyaluronan and versican rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arter. Thromb. Vase. Bioi. 1999;19:1004-1013. 4. SP Evanko, TN Wight. Intracellular localization of hyaluronan in proliferating cells. J. Histochem. Cytochem. 1999;47:1331-1341. 5. M Yamagata, S Suzuki, S Akiyama, K Yamada, K Kimata. Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates. J Bioi Chem 1989;264:8012-8018. 6. RK Margolis, CP Crockett, W-L Kiang, RU Margolis. Glycosaminoglycans and glycoproteins associated with rat brain nuclei. Biochim. Biophys. Acta 1976;451:465-469. 7. K Furukawa, H Terayama. Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver. Biochim. Biophys. Acta 1977;499:278-289. 8. FW Kan, High-resolution localization of hyaluronic acid in the golden hamster oocytecumulus complex by use of a hyaluronidase-gold complex. Anat. Rec. 1990;228:370-382. 9. N Grammatikakis, A Grammatikakis, M Yoneda, Q Yu, SO Banerjee, BP Toole. A novel glycosaminoglycan-binding protein is the vertebrate momologue of the cell cycle control protein, Cdc37. J. Biol. Chem. 1995;270:16198-16205. 10. TB Deb, K Datta. Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein copurified with splicing factor SF2. J. Biol. Chem. 1996;271:2206-2212. 11. S Zhang, MCY Chang, 0 Zylka, S Turley, R Harrison, EA Turley. The hyaluronan receptor RHAMM regulates extracellular-regulated kinase. J. Bioi. Chem. 1998;273:1134211348. 12. AN de Belder, KO Wik. Preparation and properties of fluorescein-labelled hyaluronate. Carbo Res. 1975;44:251-257. 13. W Knudson, E Bartnik, CB Knudson. Assembly of pericellular matrices by COS-7 cell transfected with CD44 lymphocyte-homing receptor genes. Proc. Natl. Acad. Sci. USA 1993;90:4003-4007. 14. L Collis, C Hall, L Lange, M Ziebel, R Prestwich, E Turley. Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action. FEBS Letters 1998;440:444-449.
LOW MOLECULAR WEIGHT OLIGOSACCHARIDES OF HYALURONAN POTENTLY ACTIVATE DENDRITIC CELLS Christian C. Termeer!", Peter Prehm2 & Jan C. Simoni I
2
Department ofDermatology, University ofFreiburg, Hauptstr. 7, D-79/04 Freiburg, Germany Department ofBiochemistry, University ofMuenster, Wa/deyerstr. /5, D-48/49 Munster, Germany
ABSTRACT The ECM-component hyaluronate (HA) exists physiologically as a high molecular weight polymer (HMW-HA), but is cleaved at sites of inflammation, where it will be contacted by dendritic cells (DC). To determine the effects ofHA on DC, HA-fragments of different size were established. Only small HA-fragments of tetra- and hexasaccharide size (sHA), but not of intermediate size (INT-HA, MW 80-200 kDa) or HMW-HA (MW 1000600 kDa) induced immunophenotypic maturation of human monocyte-derived DC (upregulation ofHLA-DR. B7-l/2, CD83, downregulation ofCD115). Likewise, only sHA increased DC-production of the cytokines IL-l13, TNFa, IL-l2 as well as their allostimulatory capacity. These effects were highly specific for sHA, since they were not induced by other glycosaminoglycans (GAG's) such as chondroitinsulfate (CS) or heparansulfate (HS) or their fragmentation products. Interestingly, sHA-induced DC maturation does not involve the HA-receptors CD44 or RHAMM, since DC from CD44 deficient mice and wild type mice both responded similarly to sHA-stimulation, whereas RHAMM is not detectable in DC. These findings suggest that during inflammation interaction of DC with small HA-fragments induce DC-maturation. KEYWORDS Dendritic cells, T-cell stimulation, CD44, RHAMM INTRODUCTION Hyaluronan (HA) is a ubiquitous extracellular matrix (ECM) component, present at high concentrations in the skin, where it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes 1,2. In normal skin, HA exists as a high molecular weight (600-1000 kDa)(HMW-HA) nonsulfated glycosamino-glycan (GAG) composed of repeating units of D-glucuronic acid-N-acetyl-D-glucosamine. Functional properties of HMW-HA, are the maintenance and hydration of the cutaneous ECM, as well as the binding of various growth factors and smaller-sized GAG's. The physiological degradation of HMW-HA within the skin includes the uptake into keratinocytes, which is related to the high affinity HA-receptor CD443.4, and intracellular fragmentation to intermediate sized fragments (INT-HA 300-60 kDa). Fragmented HA is released by keratinocytes, passes the basement membrane and is liberated without
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significant further catabolism by dermal cells into lymphatic vessels". These fragments are degraded within skin draining lymph nodes", During inflammation platelet derived chemotactic factors like fibrin stimulate the influx and activation of fibroblastsv'', These cells directly degrade the surrounding ECMcomponents by the secretion of hyaluronidase resulting in increased tissue concentrations of small HA-fragments (sHA)7,8. Furthermore, cleavage of HA can be induced by reactive oxygen species released for example by granulocytes or in UV-irradiated skin, demonstrating that different proinflammatory stimuli can trigger unspecific degradation of HA 9,lO. During cutaneous immune responses antigen presenting cells (APC) like DC infiltrate skin, were they encounter antigen. This involves both the influx of DC progenitors from the blood stream, as well as the migration of antigen-laden DC residing within the epidermis, the Langerhans cell (LC), into the dermis ll ,l2 . Subsequently, DC emigrate from the dermis into lymphatic vessels and enter the regionallymphnodes to elicit a specific T-cell response by presentation of antigen in the context of MHC lor MHC II molecules!'. During this process DC become activated, which is associated with a distinct change in phenotype and function, termed DC-maturation 1l,IZ. These maturational events can be reproduced during in vitro culture of DC from progenitor cells in GM-CSF and IL-4 containing medium by the addition of "maturational stimuli", such as LPS, TNFa, Monocyte Conditioned Medium (MCM) or CD40-1igation 13,14. Mature human DC have a non-adherent dendritic phenotype, express selected surface markers like CDla and CD83 I S, but lack the CSF-l receptor CDI15 13,14. These phenotypic changes, which also include high expression ofMHC class I and class II as well as costimulatory molecules B7-1,B7-2 and CD40 result in an improved capacity to stimulate resting T_cells1I,IZ. Here, we became interested in the effects of HA-fragments of different size on DC and found low molecular weight HA-fragments, but not INT-HA or HMW-HA potently and specifically induce maturation of human and murine dendritic cells.
MATERIALS & METHODS Media and Reagents RPMI-1640 cell culture medium was supplemented with 1% Penicillin! Streptomycin, 1% L-Glutamine (All Seromed, Berlin, Germany) and 1% HSA (Bayer, Munich, Germany). All reagents, media and buffers used in our assays were checked by Limulus amebocyte lysate (LAL) assays, and were found to contain less than 0.06 EU/ml of endotoxin in accordance with the European Community standard value for aqua ad injectabilia. Polymyxin B, a cationic antibiotic that displaces Caz+ from anionic phospholipids, was obtained from Calbiochem, Bad Soden, Germany. Chondroitinsulfate C from sharks cartilage for clinical application was kindly provided by Sankyo Pharrna INC., Munich, Germany and was subjected to enzymatic digestion with IOU of Chondroitinase ABC from Proteus vulgaris (Sigma, Deisenhofen, Germany) overnight. Heparansulfate-Na Braun 10.000 I.U. (Braun, Melsungen, Germany) for clinical application was digested with 2 U Heparinase III from Flavobacterium heparinum (Sigma) at 37°C. Glyoxal and Glyceraldehyde were purchased from Sigma. The polyclonal rabbit-anti-mouse RHAMM-serum and the polyclonal rabbitanti-human RHAMM-serum have been described elsewhere'",
Low molecular weight oligosaccharides
459
Preparation of human DC's Monocytic cells were separated from buffy coats from healthy blood donors on a endotoxinfree Ficoll-paque plusTM gradient (Pharmacia, Freiburg, Germany) and incubated for 45 min. with a non-activating, anti-human-CD14 MACS™-Bead labelled mAb (Milteny Biotech, Bergisch Gladbach, Germany). CD14+ cells were purified by magnetic cell sorting using a MACS-system™ (Milteny). 5x106 monocytesl well were seeded into 6-well flat bottom plates (Costar, Bodenheim, Germany) and cultured for 4 days in RPMI 1640 (GffiCO BRL, Eggenstein, Germany) supplemented with 5% endotoxin tested human serum-albumin (Bayer Diagnostics, Munich, Germany), GCP/GMP quality 1000 Vlml GMCSF (LeukomaxTM, Novartis, Niirnberg, Germany) and 100 Vlml IL-4 (kindly provided by Schering-Plough, New York, VSA) at 37°C, 5% C02. Generation of murine bone-marrow derived DC DC were generated following the method of Inaba et a1. 17, with minor alterations. BM was harvested from the tibia and femur of C57BU6 mice (n=4). The cells were resuspended at 1x106 cellslml cRPMI-1640 (Gibco) with 40 ng/ml GM-CSF and 100 ng/ml IL-4 (both PromoCell, Heidelberg, Germany). Cells were fed on days 3 and 5 of culture, by replacing half the medium in each well with fresh cRPMI with GM-CSF and IL-4. On day three, nonadherent cells were aspirated, following gentle swirling of the plate. Loosely adherent cells, including DC were harvested by gentle pipetting, on day 6. DC were washed once and resuspended at approximately 5x lOS cells/ml in cRPMI. 8 ml of the cell suspension were underlayed with 2 ml 14.5% metrizarnide (Boehringer Ingelheim, Heidelberg, Germany) in a 14 ml conical bottomed tube (Becton Dickinson) and centrifuged at room temperature (22 °C) for 20 min at 600 x g. The low buoyant density cells were collected, washed twice and resuspended for use. Purity of lab positive cells was >70% as determined by flowcytometry. Preparation of the HA-fra!tments Hyaluronic acid (HEALON ) for clinical application (endotoxin content lower 0.1 ng/mg) was kindly provided by Pharmacia, Erlangen, Germany. Two types of HA-fragments from HEALONTM were generated: 1) !NT -HA was prepared for 2 min on ice using a Branson Sonifier with the output set at the microtip limit, as described I8. Samples were then separated by 0.5% agarose gel electrophoresis and visualized with the cationic dye Stains All (3,3' -dimethyl-9-methyl-4,5,4',5'-dibenzothiacarbocyanine, BioRad, Munich, Germany), as described!". 2) small HA-fragments (sHA) were generated by enzymatic digestion of INT-HA with bovine testis hyaluronidase (Sigma) for 12 h in 1 M Sodiumacetate buffer pH 5.0, 37°C. The fragments were separated on a Biogel PlO (BioRad) 3,5 x 115 cm column overnight. Samples were collected from the column with a for 12 h on 20 min/each. The HA-concentration of each sample was analyzed by addition of uronic-acid, as described 20 and was measured photometrically at 520 nm against destilled water. The detection of HA-fragment size in each sample was determined by ANTSlabelling technique and analyzed by 30% acrylamid gel-electrophoresis 21.
460
The action of hyaluronan in cells
T-cell isolation / T-cell proliferation assay Resting T-cells were obtained from the CDl4-depleted cell fraction ofPBMC's. Remaining antigen presenting cells were removed by positive selection with mAb's against HLA-DR (Clone .HB 145) and CDIlb (OKT-6)(both ATCC) for 45 min at 4°C, washed in PBS followed by incubation with a secondary goat-anti-mouse DynaI™-Bead-labelled mAb under the same conditions (Dynal, Hamburg, Germany). Flow-cytometry showed purity of CD3+ cells >85%, HLA-DR positive cells were less than 1%, and the cells did not respond to PHA-stimulation (IOng/ml). The unlabelled CD3+ cells were collected after magnetic sorting, washed and plated at Ixl06/well with 5x10 4/well mature allogeneic DC's in round bottom 96-well plates (Costar). On day 4, I ~Ci / well eH]-Thymidine (Amersham, Freiburg, Germany) was added for the final 18 h of culture. Finally, the radioactivity was determined by liquid scintillation spectroscopy (Canberra Packard, Dreieich, Germany). RT-peR Total RNA was isolated from dendritic cells and the RHAMM positive breast cancer cell line T47D 16 using the quick-prep kit (Pharmacia, Erlangen, Germany) according to the manufacturers instructions. eDNA was synthesized from 5J.1g of total RNA using the Superscript II reverse transcriptase (Gibco, Eggenstein, Germany). The product was subjected to 30 cycles at 94°C 1 min., 2 min., 55°C 3 min. using Taq-DNApolymerase (Gibco). 25J.1I-aliquots of the PCR reaction products were analysed by 1 % agarose gel electrophoresis. The primer were generated following the sequence published by Assmann et a1. 16 : Lower Primer: Pos.95I : 5' CAG GAA TAG AGA ACA CAA CG 3' Upper Primer: Pos.1719: 5' TCT TCC TTC TIC ATC TTC CAG C 3'
ire:
RESULTS HA-pre.garations of three different sizes were used; HWM-HA (endotoxin-free HEALON M), sonified HEALON™ yielding HA-fragments of intermediate size (INT-HA), small HA-fragments (sHA) generated by hyaluronidase-digestion of INT-HA. 0,5% agarose-gel-electrophoresis ofHMW-HA or INT-HA demonstrated a MW of 1000-200 kDa or 200-80 kDa, respectively (Fig.IA). The sHA-preparation was further separated on a polyacrylamid gel column and fractions eluted at 20 min intervals were collected. 30% acrylamid-gel-electrophoresis revealed that fractions collected at earlier timepoints contained larger oligosaccharides, e.g. fraction No.12 fragments of 4-14 oligosaccharide size (Fig.lB), whereas samples collected late contained only small fragments, e.g. fraction No.22 fragments of 4-6 oligosaccharide size (Fig.lB). For experimental use, three fractions covering a range of different sizes from 4-14 oligosaccharide length were adjusted to an HA-content of 1 mg/ml. Late fractions eluted from the column, which contained no detectable amounts ofHA, served as negative control. The effects of the different HA-preparations on the immunophenotype of human blood derived DC was determined by flow-cytometry. Day 4 immature DC's displayed a high surface expression ofCDla, CD44, rCAM-l, intermediate levels ofB7-1, B7-2, HLA-DR and CD1l5 while CD83 expression was low (data not shown). 48 h of stimulation with three different sHA fractions led to a dose-dependent phenotypic changes in DC including a marked upregulation of HLA-DR, B7-1, B7-2, rCAM-land CD83, as well as a
Low molecular weightoligosaccharides
461
downregulation of CDla and CDIl5 (data not shown). SHA concentrations as low as 10 ug/ml were found to match exactly the phenotypic maturation induced in the same DC by a 48 h treatment with 10 ug/ml LPS. Interestingly, sHA-fractions containing 4-16, 4-10, or 46 oligosaccharides all had similar effects (data not shown). Dose-titration experiments showed l Oug/ml of each sHA fraction to induce complete phenotypic DC-maturation (data not shown). This suggests that sHA-fragments of 4-6 oligosaccharide size which were present in all fractions (Fig.1B), are predominantly responsible for the DC-maturation. However, early fractions contain relatively less 4-6 oligosaccharides. We can therefore not exclude, that sHA-fragments of8-l6 oligosaccharides might also be effective. By contrast, HMW-HA or INT-HA obtained from the same stock of endotoxin free HEALON™ at concentrations as high as 100 ug/ml had no effect on DC. Additionally, DC were not affected by the column control, which did not contain detectable amounts of HA, excluding the possibility that components of the Biogel PIO gel induced DC maturation (data not shown). The maturation stimulated by sHA-fragments was long-lasting and irreversible, since sHA treated DC could be cultivated until day 14 without further addition of IL-4 and GMCSF and kept their mature phenotype (data not shown). ,----'--------,
Fig. 1 A
Fig.I Characterization of HApreparations A) 0.5% agarose gel electrophoresis shows the MW of HMW-HA is 1000-200 kOa, INT-HA fragments were of 20080 kDa size. B) Fractions containing sHA-oligosac-charides on a 30% poly-acrylamid gel electro-phoresis. Fractions collected at earlier timepoints contain larger oligosaccharides, whereas samples collected late contain only small fragments.
Since phenotypic DC-maturation is accompanied by the enhanced production of proinflammatory cytokines'v''', ELISA-assays were performed to determine the cytokine content in supernatants of sHA-stimulated DC's. The same three sHA-fractions, but not HMW-HA, INT-HA or the column control upregulated the IL-ll3 and TNFa-production in a dose-dependent fashion similar to the effect induced by LPS (Fig.2 and Table 1). We found l Oug/ml sHA effectively induced a significant TNFa-release, which was saturated at concentrations greater 50 ug/ml (Table I). By contrast, sHA treatment resulted only in a modest IL-12 secretion compared to LPS (Fig.2). Of note, IFNy' or IL-4 production by DC was not affected by either sHA or LPS (data not shown). The maturation of DC is also accompanied by their enhanced ability to stimulate T-cell mediated immune responses in vitro and in vivo 15,16. To test the effects of sHA on these functional properties, we first assessed the capacity of human DC to stimulate the proliferation of resting allogeneic T-cells in a standard MLR. sHA at concentrations as low as 10 ug/ml, like LPS, significantly enhanced the allostimulatory potential of DC (Fig.3). Again, this stimulatory activity was conferred by sHA-fragments of 4-16 oligosaccharide size (Fig. 3).
462
The action of hyaluronan in cells
DC-treatment
~-
2
*
sHA4-t4 size
=:'~*'
sHA4-IO size sHA4-6 size Ctrl.
INT-HA HMW-HA
*
LPg
o
50
1000 2000 3000 10 20 30 40 50
100 150
Fig.3 DC-treatment
H
3-Thymidine
incorporation CPM +/- SO xl 0
*
sHA 4-14 sHA 4-10 sHA 4-6
em, INT-HA HMW-HA LPS
o
20
40
60
80
100
120
Fig. 4 LPS A
B
140
Fig.2 sHA enhances cytokine production of DC Day 4 DC were incubated for 48 h with 20 ug/ml of sHApreparations (filled bars), 10 ug/ml LPS (textured bar), HMW-HA, INT-HA, left untreated (-) or the column control (CtrL)(open bars). ELISA-assays were performed with cell free supernatants for their content of IL-IJ3, TNFa, IL-12 and IFNy. Results are shown in pglml +/- SD of triplicate wells. * P>O.OO I comoared to untreated Dcr-),
~===::::::::::===~ Fig.3 sHA stimulated DC enhance T-cell proliferation DC were pre-treated with 20 ug/ml with sHA (filled bars), 10 Ilglml LPS (textured bar), HMW-HA, INT-HA, left untreated (-) or the column control (CtrL)(open bars)and co-incubated for 4 days with alloreactive T-cells, T-cell proliferation was determined on day 5 by 3[Hl-thymidine incorporation. Results are shown in counts per minute (CPM) +/- SD of triplicate wells. * P > 0.001 compared to untreated DC (-).
Fig.4 The sHA induced DCmaturation is LPS independent DC were incubated with sHA (A), or LPS (8), and analysed for HLA-DR expression by FACS. The thin solid line shows the isotype control, the dashed line HLA-DR expression on untreated DC, dotted line HLA-DR expression on sHA (A) or LPS (8) stimulated DC. Bold line: HLA-DR expression on DC pretreated with
Polvmvxin B.
Since LPS at concentrations as low as 50 pg/ml has been described to activate cells of the myelo-monocytic lineage 22, we wished to exclude that the sHA preparations used in this study contained trace amounts of LPS. All materials used during sHA generation as well as during DC cultures were endotoxin free as confirmed by LAL-assays (data not shown).
Low molecular weight oligosaccharides
463
Furthermore, addition of 10 ug/ml of the LPS-inhibitor Polymyxin B had no effect on the sHA induced upregulation ofMHC class II (Fig.4A), B7-1, B7-2 or CDla (data not shown), but in the same experiment inhibited all LPS effects (Fig.4B). These experiments rule out the possibility that the sHA effects on DC were due to LPS contamination. Fig. 5 ~-HEAIOh/
-tI+HE......hlIM7
11I47
B
D
,_0
T-cells + +
+
CD44 CD44 ICD44 ICD44 : CD44 ° 4
+i-i+i~
-i+i -i-
sHA ~~~-----~~~
o
JlUD
-1QUD
sno
CD44 has been shown to be the major cellular HA_receptor23,24. This raised the question whether CD44, which is expressed at high levels on DC, mediates sHA-induced DC maturation. Anti-CD44 mAb's either complete or as f(ab)-fragments, shown to block HA binding to human CD44 24 , revealed to be an inappropriate tool to address this issue, since they induced DC-clustering and partial activation data not shown and 24. As an alternative approach, we studied DC from CD44 deficient mice25 • First, we confirmed that sHA also induced maturation of murine DC prepared from the bone marrow of wildtype CS7BL/6 mice. This included upregulated expression of lab, B7-1, B7-2 as well as an enhanced allostimulatory capacity (Fig.S C,D,E). Incubation of the same murine DC with INT-HA or HMW-HA had no effect (data not shown). Importantly, sHA-induced maturation was also observed in DC generated from CD44-deficient CS7BL/6 mice (Fig. S A,B), again resulting in an significantly enhanced allostimulatory capacity comparable to that of sHA-stimulated wildtype DC (Fig. SE). This indicates, that for the effects of sHA on these DC CD44expression is not required. However, we can not exclude the possibility, that CD44 might
464
The action ofhyaluronan in cells
be involved in the sHA-mediated maturation of CD44 wildtype mice. We ould further exclude that s-HA exerted its effects via the second HA-receptor RHAMM, since we did not detect RHAMM m-RNA in mature or immature DC by RT-PCR (Fig. 6A) or RHAMM expression on the surface or cytoplasma of DC as determined by FACS (Fig. 6B). Fig. 6 A) Fig.6 RHAMM is not expressed by DC. A) Total mRNA was analysed by rt-PCR from LANE A- untreated DC, SLPS-stimulated DC, C- the RHAMM positive breast cancer cell line T47D, osHA-treated DC, E- HMWHA treated DC. B) DC were either analysed by FACS for their RHAMM-expression (the solid lines) The dotted lines show staining with a control serum.
surfaceexpression RHAMM. (800 bp)
cytoplasmicstaining 13-Aetin •
,ii'
FL'' '
,0'
10'
Table 1 The sHA-induced DC-maturation is specific for HA-oligosaccharides. Stimulus UMW-UA sUA
LPS (l 011g/mI) US sus CS-C sCS-C Glyceraldehyde Glyoxal
511l!/ml 5.3 +/- 0.6 5.7 +/- 1.6 1.7 +1- 0.3 2234 +1- 145 n.d. n.d. n.d, n.d. 4.7 +/- 1.6 8.4 +1- 3.2 6.6 +/- 0.7 5.5 +/- 0.8
1011e1ml ZO l1g/ml 5011g/ml 100l1c/ml 3.6 +/- 0.9 n.e, +/. 1.5 n.a. +/- 0.7 6.9 2.6 663.6 +/- 45.8 1875.3 +1- 201.5 2174.8 +1- 185.2 2076,2 +1- 104,8
--_.
-----
-
3.4 2.6 6.3 2.4 6.2 5.7
+1- 0.6 +1-1.5 +1- 2.6
cyt cvt n.d, 3.1 4.4 4.9
+1- 0.4 +1- 1.0
+/-1.3
---
._.
--
--
cyt cyt
cyt cyt
cyt cyt
n.a.
1.8
+1- 0.6
+1- 1.7 +1- 1.0
4.8 2.6 cvt
+1- 1.1 +1- 0.5
cvt CVt 3.8 8.3 0.5 cvt
+1- 0.6
---
cvt
---
--
-cyt cyt +1- 2.6 +1- 3.7 +1- 0.1
cvt
Human OC were incubated for 24 h with different concentrations of the indicated substances,1 0 IIg/ml LP5, or left untreated and the cell free supernatants were assayed for their TNFa-content. ReSUlts are shown in pg/ml TNFa +1- standard deviation (SO). cyt = cytotoxic. n.d. = not detectable.
To determine whether other glycosaminoglycans (GAG's) present in the ECM could also influence DC-maturation, DC were treated with purified chondroitinsulfate C (CS-C) or heparansulfate (HS) used either as a complete molecule or after enzymatic digestion to oligosaccharide size (sCS-C, sHS). Although CS and HS show high structural homology with HA being built of repeating disaccharide-units, they did not influence the TNFarelease by DC or the upregulation ofMHC class II and B7-1/ B7-2 molecules (Table I and data not shown). To exclude that high concentrations of reactive aldehyde-groups, present at the free N-acetyl-glucosamine end of each sHA molecule were responsible for DCmaturation, we tested the effects of the two small aldehydes glyoxal and glyceraldehyde. However these substances failed to induce DC-maturation when used in identical concentrations as sHA (5-50 ~glml)(Table I).
Low molecular weight oligosaccharides
465
DISCUSSION Here, we have shown that only small HA-fragments of 4-16 oligosaccharide size but not intermediate size fragments (INT-HA) or high molecular weight HA (HMW-HA) induce terminal and irreversible maturation of human and murine dendritic cells. This is in agreement with studies related to inflammation and wound healini 6•32 • SHA-fragments of 3-10 oligosaccharide size accelerated neoangiogenesis during wound repair within 4872h27. In agreement with our results, HMW-HA did not affect angiogenesis, even acting antiangiogenic at higher concentrations'". Additionally, the ability of sHA, but not HMWHA, to stimulate directly the growth and tube formation of endothelial cells was demonstrated in a number of models including chick chorioallantoic membranes, rat skin and cyroinjured murine skin grafts27.29. Furthermore, sHA-fragments of 10-16 oligosaccharide size had no effect on endothelial cell proliferation", thus parallelling our findings that only sHA fragments of 4-16, but not of 10-16 oligosaccharide size activate DC. On the other hand, both INT-HA fragments with a peak molecular size of 200 kDa as well as sHA of 6 oligosaccharide length have been described to activate murine alveolar macrophages via a NfkB/ IKB-dependent pathway". Moreover, they were shown to upregulate mRNA synthesis and protein secretion of the chemokines MIP-la., MIP-l~ and MCP_1 31 and to trigger nitric oxide synthase (iNOS) activiti 2. This is in partial contrast to our finding that INT-HA (MW 80-200 kDa) had no effect on DC. These discrepant results could be due to the different cell types studied, i.e. murine alveolar macrophages 30.32 versus human or murine dendritic cells (our study). On the other hand, they may be due to the method of INT-HA generation employed by McKee et al., which does not exclude the possibility that their preparations contain substantial amounts of sHA_fragments30-32 . Further, we wished to determine whether sHA-induced DC maturation involves the known HA-receptors CD44 or RHAMM 16,23.24. Importantly, the sHA-response of DC generated from CD44-deficient mice was identical to that of DC from CD44 expressing mice, demonstrating the sHA-effect to be independent of CD44. Similar conclusions were drawn, when the sHA-stimulated proliferation of vascular endothelial cells was studied 27· 29. Specifically, perinuclear CD44-staining of sHA-treated endothelial cells was not different from untreated cells 28, indicating that the sHA-uptake and specific Nf-KB dependent signalling was not mediated by CD44. The notion that sHA-fragments interact with cells independent of CD44 is supported by recent findings from Culty et a1. and Tammi et al. 23,33. Based on HMW-HA-competition studies these investigators concluded that only sHA-fragments of at least 6-10 oligosaccharide length, but not smaller fragments bind to CD44 on endothelial cells or keratinocytes, respectively. Further, we found no evidence that RHAMM is involved in sHA-mediated DC-maturation, since RHAMM mRNA or protein could not be detected in human DC's. CONCLUSION We have shown that only small HA-fragments of 4-16 oligosaccharide size, but not larger INT-HA or HMW-HA induce irreversible phenotypic and functional DC-maturation. This effect is highly specific for hyaluronan and was not observed after DC stimulatioon
466
The action ofhyaluronan in cells
with other ECM-glycosaminoglycans such as chondroitinsulfate C or heparan sulfate. These findings suggest that also in vivo sHA fragments generated at sites of inflammation activate DC migrating into or out of these tissues, thereby augmenting and perpetuating the immune-response.
ACKNOWLEDGEMENTS We thank Dr. R. Schmitts, University of Homburg, Homburg, Germany for CD 44 -/- mice.
REFERENCES 1. J.H. Poulsen, I. M. Jensen & U. Petersen. D-eH)glucosamine labelling of epidermal and dermal glycosaminoglycans in cultured human skin. J. Clin. Chem. Clin. Biochem. 1998, 26, 123-133. 2. R. Tammi & M. Tammi. Correlations between hyaluronan and epidermal proliferation as studied by 3H-glucosamine and 3H- thymidine incorporations and staining of hyaluronan on mitotic keratinocytes. Exp. Cell.Bioi. 1991, 195,524-527.
3. R. Tammi, A.M. Saamlinen, H.I. Maibach & M. Tammi. Degradation of newly synthesized high molecular mass hyaluronan in the epidermal compartments of human skin in organ culture. J. Invest. Dermatol. 1991,97, 126-130. 4. G. Gaya, I. Rodriguez, J.1. Jorcano, P. Vassalli & I. Stamenkovic. Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation. Genes & Develop. 1997, 11,996-1007. 5. R. Tammi, U.M. Agren, A.1. Tuhkanen & M. Tammi. Hyaluronan metabolism in skin. Progress in Histochemistry& Cytochemistry 1994, 29, 1-8. 6. R.I. Fraser, W.G. Kimpton, T.C. Laurent, R.N. Cahill & N. Vakakis. Uptake and degradation ofhyaluronan in lymphatic tissue. Biochem. J. 1988,256, 153-158. 7. P.H. Weigel, G.M. Fuller & R.D. LeBouef. A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing. J. Theor. Bioi. 1986, 119,219-234. 8. C.M. Taylor, J.M. Thompson & 1.B. Weiss. Matrix integrity and the control of angiogenesis. Int. J. Radiat. Bioi. 1991,60,61-64. 9. R. Moseley, R.I. Waddington & G. Embery. Degradation of glycosaminoglycans by reactive oxygen species derived from stimulated polymorphonuclear leukocytes. Biochim. Biophys. Acta 1997,1362,221-231.
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10. U.M. Agren, R.H. Tanuni & M. I. Tammi. Reactive oxygen species contribute to epidermal hyaluronan catabolism in human skin organ culture. Free Radic. Bioi. Med. 1997,23,996-1001. 11. D.N. Hart. Dendritic cells: Unique leukocyte populations which control the primary immune response. Blood 1997, 90, 3245-3287. 12. R.M. Steinman, L. Hoffman & M. Pope. Maturation and migration of cutaneous dendritic cells. J. Invest. Dermatol. 1995, 105, 2S. 13. N. Romani, D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser & G. Schuler. Generation of mature dendritic cells from human blood improved method with special regard to clinical applicability. J. Immunol. Meth. 1996, 196, 137-151. 14. A. Reddy, M. Sapp, M. Feldman, M. Subklewe & N. Bhardwaj. A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells. Blood 1997, 90, 3640-3646. 15. LJ. Zhou & T.F. Tedder. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J. Immunol. 1995. 154,3821-3829. 16. V. Assmann, J.F. Marshall, C. Fieber, M. Hofmann & I.R. Hart. The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells. J. Cell Sci. 1998, Ill, 1685-1694. 17. K. Inaba, M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara, S. Muramatsu & R.M. Steinman. Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class ll-negative progenitor in mouse bone marrow. Proc Natl Acad Sci USA 1993, 90, 3038-3042. 18. K. Kubo, T. Nakamura, K. Takagaki, Y. Yoshida & M. Endo. Depolyrnerisation of hyaluronan by sonication. Glycoconj. J. 1993, 10,435-439. 19. H.G. Lee & M. Cowman. An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal. Biochem. 1994,219,278-287. 20. T. Bitter & H. Muir. A modified uronic acid carbazol reaction. Anal. Biochem. 1960,4, 330-334. 21. P. Jackson. The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis. Mol. Biotech. 1996,5, 101-123. 22. S.L. Weinstein, C.H. June & A.L. DeFranco. Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CDI4. J. Immunol. 1993, 151,3829-383~
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The action ofhyaluronan in cells
23. M. Culty, H.A. Nguyen & c.a Underhill. The hyaluronan receptor (CD44) partipiciates in the uptake and degradation ofhyaluronan. J.Cell BioI. 1992,116, 1055-1063. 24. H. Haegel-Kronenberger, H. de la Salle, A. Bohbot, F. Oberling, J.-P. Cazenave & D. Hanau. Adhesive and/or signaling functions of CD44 isoforms in human dendritic cells. J. Immunol. 1998, 161,3902-3911. 25. R. Schmitts, J. Filmus, N. Gerwin, G. Senaldi, F. Kiefer, T. Kundig, A. Wakeham, A. Shahinian, C. Catzavelos, J. Rak, C. Furlonger, A. Zakarian, J. Simard, C. Paige, J.e. Gutierrez-Ramos & T.W. Mak. CD44 regulated hematopoietic progenitor distribution, granuloma formation and tumorigenicity. Blood 1997, 90, 2217-2233. 26. P. Schenck, S. Schneider, R. Miehlke & P. Prehm. Synthesis and degradation of hyaluronate by synovia from patients with rheumatoid arthritis. J. Rheumatol. 1995, 22, 400-405. 27. D.C. West, LN. Hampson, P.F. Schofield & S. Kumar. Angiogenesis induced by degradation products of hyaluronic acid. Science 1985,228,1324-1326. 28. A. Sattar, P. Rooney, S. Kumar, D. Pye, D.C. West, I. Scott & P. Ledger. Application of angiogenic oligosaccharides of hyaluronan increases blood vessel numbers in rat skin. J. Invest. Dermatol, 1994, 103,576-579. 29. V.C. Lees, T.-P. Fan & D.C. West. Angiogenesis in a delayed revascularization model is accelerated by angiogenic oligosaccharides ofhyaluronan. Lab. Invest. 1995, 73, 259266. 30. P.W. Noble, C.M. McKee, M. Cowman & H.S. Shin. Hyaluronan fragments activate an NF-KB/IKBa. autoregulatory loop in murine macrophages. J. Exp. Med. 1996, 183,23732378. 31. C.M. McKee, M.B. Penno, M. Cowman, M.D. Burdick, R.M. Strieter, C. Bao & P.W. Noble. Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. J.Clin.Invest. 1996,98,2403-2413. 32. C.M. McKee, C.J. Lowenstein, M.R. Horton, J. Wu, e. Bao, B.Y. Chin, A.M. Choi & P.W. Noble. Hyaluronan fragments induce nitric-oxide synthase in murine macrophages through a nuclear factor KB-dependent mechanism. J. Bioi. Chem. 1997, 272, 80138018. 33. R. Tammi, D. MacCallum, V.C. Hascall, J.P. Pienimaki, M. Hyttinen & M. Tammi. Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides. J Bioi Chem. 1998, 273, 28878-28888.
EFFECTS OF ELASTOVISCOUS SOLUTIONS OF HYLURONAN DERIVATIVES ON MECHANOTRANSDUCTION Elvira de la Pefia, Salvador Sala, Robert F. Schmidt & Carlos Belmonte" lnstituto de Neurociencias, Universidad Miguel Hernandez-Consejo Superior de Investigaciones Cientlficas. Campus de San Juan, Apdo. 18. San Juan de Alicante 03550. Alicante, Spain
ABSTRACT Sensory endings of nerve fibers signalling joint pain express stretch-activated ion channels which open in response to an increase in membrane tension. These stretchactivated channels have been well characterised in Xenopus laevis oocytes. We have investigated whether hylan solutions of different elastoviscosity used for intra-articular treatment of joint pain in humans, modify the response characteristics of native stretchactivated ion channels of oocytes. Patch-clamp recordings on intact oocytes and in isolated membrane patches (outside-out) were performed in Barth's solution (control condition) and after exposure to hylans of different elastoviscosity. For mechanical stimulation, monitored suction was applied through the microelectrode and the activity of stretch-activated channels was recorded. The activity of the channels was significantly reduced in the presence of high elastoviscous hylan A, 0.9 % polymer content, molecular weight 6 M and of a mixture of hylan A (90% by weight) and hylan B (10% by weight 0.8% total polymer content), labelled as Synvisc®, a clinically used hylan product. In contrast, when a non-elastoviscous solution of hylan (hylan A, molecular weight 96,000) was used, no effect was observed. In conclusion, stretch-activated channels have a decreased mechanical sensitivity in the presence of elastoviscous solutions of hylan, but not in the presence of non-elastoviscous solutions of hylan of the same concentration. These data suggest that the analgesic effect of intra-articular injection of elastoviscous solutions of hylans are due to a reduction of the sensitivity of joint mechano-nociceptors sensitivity in animals and in arthritic patients. KEYWORDS Mechanotransduction, stretch-activated channels, oocytes, elastoviscous solutions, hyaluronan, hylan, arthritic pain, nociceptors. INTRODUCTION Noxious stimuli are detected by the peripheral endings of a population of primary sensory neurones called nociceptive neurons'. In nociceptive endings, the stimulating energy (strong mechanical forces, irritant chemicals, heat or cold) is finally transformed into a train of electrical signals, the nerve impulses, whose firing frequency encodes some of the characteristics of the stimulus. Nerve impulses are transmitted to higher
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Aspectsof hyaluronan in joints
levels of the central nervous system where they elicit a sensation of pain. The mechanism by which external physical or chemical changes are transformed by nociceptive nerve endings into internal biochemical and/or electrical signals is incompletely understood'. The primary transducer element is located in the cell membrane and is formed by a molecular entity that is modified by the stimulus. In several types of sensory receptors, this transducing element is linked to perireceptor structures that may act to convey the stimulus to the primary transducer in the membrane or to modify some of the characteristics of the stimulus'. Nociceptor endings in the skin, joints, muscles and ligaments are devoid of encapsulated structures and thus appear as 'free' nerve endings". However, there is evidence that transmission of mechanical forces and availability of chemical substances acting on nociceptors may be modified by molecules present in the extracellular matrix. Hyaluronan, a polysaccharide present in all extracellular spaces and found at high concentrations in the capsule and synovial tissue of the joints 5.7 offers a prominent example of how the effect of the stimulus on sensory nerve endings can be modulated by molecular entities located in the extracellular matrix. Hyaluronan appears to act as an elastoviscous filter for the transmission of mechanical forces to nociceptor endings leading to an analgesic effect8 • The pain-reducing action of intraarticulary applied elastoviscous hyaluronan solutions was demonstrated in animal behavioural studies in the early 1970s. In arthritic racehorses and dogs, elastoviscous solutions of highly purified, high molecular weight hyaluronan produced analgesia'v", This effect was confirmed later in behavioural animal studies in rats!'. Based on these findings, elastoviscous hyaluronan or hylan (a hyaluronan derivative) solutions were introduced in veterinary and human medicine for the treatment of arthritic pain5.12.l4. The mechanism of this analgesic effect was further studied in neurophysiological experiments in healthy and arthritic joint models in cats and rats using hyaluronan and hylan. These studies demonstrated that elastoviscous fluid applied intraarticularly reduced movement-evoked nerve impulses signalling pain l5- 17 • In arthritis, the rheological properties of the synovial fluid (viscosity, elasticity, pseudoplasticity) are altered in parallel with the appearance of low molecular mass hyaluronan and it has been suggested that healthy, high molecular mass hyaluronan act as a shock absorber or mechanical stabiliser for nocieeptors of the joint". This hypothesis was supported by the observation that nociceptor activity in single nociceptor fibers of the normal and inflamed knee joint of the Cae·15 and rat" was reduced by intra-articular injection of high elastoviscous hylan solutions. This effect was not obtained by injection of degraded, low molecular weight hylan. The difference was explained as a reduction by the high elastoviscous hylan of the tension transmitted by joint movement to the transducing element, the mechanosensory channels present in the membrane of nociceptive nerve terminals. These mechanosensory channels are opened by membrane stretch, leading to depolarisation and finally to the production of nerve impulses 19. Nevertheless, there is no direct evidence that hyaluronan molecules modify the activity of mechanosensory channels. Moreover, it is unknown what type of interactions takes place between stretch-activated channels and high molecular weight hyaluronan or hylan molecules. We explored the influence ofhylans on the activity ofmechanosensory channels in Xenopus oocytes, a model of cell with well characterised native membrane channels activated by stretch 20.25.
Effects of clastoviscous solutions on mechanotransduction
409
MATERIALS & METHODS
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Figure 1. Recording methods .Mechanical stimulation was performed by applying a monitored suction (negative pressure) through the microelectrode. In the intact oocyte (A) the cellattached configuration was used and the oocyte was immersed either in Barth's solution or in a hylan solution. B. Diagram of the chamber employed to expose isolated membrane patches ( inside-out or out-side configuration) to different solutions. This chamber had two compartments (A and B) containing different solutions separated by a central smaller compartment (C) filled with mineral oil. The pipette with the attached membrane patch was moved from one compartment to the other through the oil compartment (arrow). Oocytes were obtained from adult Xenopus laevis. Oocytes at maturation stages V and VI were carefully dissected and stored in sterile Barth's medium (NaCI 88 mM, KCI ImM, MgS0 4 0.82 mM, Ca(N03)2 0.33 mM, CaCh 0.41 mM, NaHC03 2.4 mM, Tris/HCI 5mM, pH 7,4,200 mOsm). To remove the follicle cell layer, the oocytes were treated with O.5mg/ml collagenase in Barth's medium for 2h at 19°C and subsequently washed and kept at 19°C in Barth's medium with Penicillin/Streptomycin lOOD/ml until
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Aspects ofhyaluronan injoints
used for experiments. To remove the vitelline membrane, the defolliculated oocytes were shrunk in hypertonic "stripping" solution (aspartate-K 200 roM, KCl 20 mM, MgCb I rnlvl, EGTA 10 mM, HEPES 10 roM, pH 7,2) for about 5 min. Thereafter using two fine forceps the vitelline membrane was peeled off completely. The naked 00cytes were transferred to sterile Barth's medium without antibiotics, or to hylan solutions (see Results). Recordings were performed using the patch-clamp technique in 3 configurations, namely, cell-attached, inside-out and out-side out. We used glass micro electrodes pulled from borosilicate glass (outer diameter 1.5mm, inner diameter 0.86mm) on a Sutter P-87 puller and filled with different solutions depending of the patch configuration (electrode resistance 7-12 MO, tip size around 2.5 micrometers). All experiments were done at room temperature. Circumscribed membrane deformations were obtained by applying suction to the microelectrodes. The negative pressure was applied to the patch microelectrode via a polyethylene tube connected to a syringe. A pressure transducer was also connected to measure the pressure changes (Fig. IA). To expose an isolated membrane patch to different solutions, a special chamber was built (modified from Qin et aI., 198926) . As illustrated in Fig. lB, the chamber had two compartments (A and B) containing different solutions separated by a central smaller compartment C filled with mineral oil. During the experiment, the pipettes could be moved through the oil-containing slit from chamber A to chamber B, but the mixing of the solutions in these two compartments through the slit was prevented by the oil. The following hylan solutions were used: 1- hylan A, 0.9 % polymer content, molecular weight 6 M (HyA, 6M MW) 2- a mixture of hylan A (90% by weight) and hylan B (10% by weight 0.8% total polymer content), labelled as Synvisc®, the preparation used as therapeutics for the treatment of painful osteoarthritis in humans, 0.9% polymer content. 3- Hylan A, 0.9 % polymer content, molecular weight 96,000 (HyA 96,000 MW). These solutions were prepared in Barth's medium or in a calcium-free solution, as specified in each experiment. The composition of this solutions was (in mlvl): calcium-free solution, NaCI 130, EGTA 2, HEPES 10, 260 mOsm, and pH 7.4; high K+ frog solution (KFR), KCI 117.5, CaCh 1.8, HEPES 10, 240 mOsm, and pH 7.2. Hylan solutions were kindly provided by Biomatrix Inc. (Ridgefield, NJ, USA).
RESULTS & DISCUSSION
Intact oocytes After applying the pipette on the surface of the oocyte, stretch-activated channels were identified and characterised by application of a negative pressure to the pipette.
A. Electrophysiological properties ofthe stretch-activated channels The conductance and reversal potentials of the mechanosensitive ion channels were determined from records obtained with a given suction pressure and at various voltage clamp settings (Fig. 2A). They were calculated by plotting the sizes of the individual currents during a pressure application against the respective levels of voltage-clamps (i.e. the pre-selected voltage in the patch micro electrode, see also legend for details). The conductance was calculated from the slope of the linear fit of the data and the rever-
Effects of elastoviscous solutionson mcchanotransduction
411
sal potential [rom the intersection of this line with voltage axis (1=0). This was also made in KFR solution (a modified Barth's solution with a very high K'
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Figure 2. Current-voltage relationship of mechanosensitive channels. A. Sample recordings obtained by application of a negative pressure in the cell-attached configuration with different voltages in the pipette (Vp represented at the right side of the trace); the oocyte was immersed in Barth's solution. B. Effect of different external solutions. Squares: KFR solution (high K+ concentration); in this situation the membrane potential of the oocyte was OmV. Reversal potential, -3mV, conductance 28pS. Circles: Barth's solution. Reversal potential -31mV, conductance 30pS. Triangles: Synvisc®. Reversal potential -10 mY, conductance 21pS. Diamonds: HyA 96,000 MW. Reversal potential -54mV, conductance 31pS. Note that in all conditions conductances were similar.
412
Aspects ofhyaluronan in joints
The variability in the native resting membrane potential among individual oocytes (usually between -20 and -60 mY) explains the differences in reversal potential when a fixed voltage of + 40mV was applied through the microelectrode. Conductances and reversal potentials values of stretch-activated channels found in our experiments were similar to those described in the literature'". B. Activation threshold and intensity-response behaviour to negative pressure The opening probability of mechanosensitive ion channels in response to negative pressure has been shown to follow the Boltzmann distribution, where the mechanical free energy available for gating is linearly dependent on pressure".
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Figure 3. Activation threshold and intensity-response behaviour with negative pressures. A. Cell-attached recordings obtained in Barthssolution with increasing negative pressure values (indicated in the left side of the traces). B. Average currents «1» recorded in Barth's solution (dark circles, n= 9), and in the presence of HyA 6M MW (open squares, n=5); HyA 96,000 MW (open circles, n=6), and Synvisc® (open triangles, n=4). The pipette was always filled with Barth's solution. Average currents were normalised to the maximum control value. Each point plotted in the diagram is the average of at least 3 measurements in 3 different patches.
I
Effectsof elastoviscous solutions on meehanotransduetion
413
This was confirmed in our experiments (Fig. 3). We were looking for this presumed relationship by measuring the activation threshold and the intensity-response behaviour under control conditions (Barth's solution) and in the presence of external elastoviscous (HyA 6M MW and Synvisc®) and non-elastoviscous (HyA 96,000 MW) solutions (Fig. 3B). Suction pressure was applied in stepwise increments within the measuring range of the pressure transducer, i.e. from 0 to -60 mmHg. In those cases where no stretch activation could be achieved with suction pressures up to -60 mmHg, still higher but uncalibrated suctions were applied. In all cases included in Fig. 3B these high suction pressures led to channel activation, thus proving that channels with high activation thresholds were indeed present in the patched membrane area.
Isolated patches A. Comparative behaviour ofstretch-activated channels in the different recording configurations
A Cell-attached configuration Compartment A
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Figure 4. Comparative behaviour of stretch-activated channels in the different recording configurations. A. The micrelectrode was filled with Barth's solution and compartments A and B with a Ca2+-free solution to avoid the activation of Ca 2+_ gated cr channels in the excised patches. The voltage in the patch was fixed at Vp=+30mV in all recordings. B. The mieroelectrode was filled with a Ca2+-free solution, the chambers A and B were filled with Barth's solution. The voltage in the patch was fixed at +30mV in the first recording and -30mV in the others.
414
Aspects of hyaluronan in joints
The results shown in Fig. 3B strongly suggest that in the presence of high molecular weight hylan solutions (HyA 6M MW) stretch-activated channels needed more pressure, i.e. more stretching, to be activated than in presence of Barth's or non-elastoviscous solutions (HyA 96,000 MW). In Synvisc® the decrease in sensitivity to stretch was even more pronounced. In the cell-attached configuration, responses of stretch-activated channels under control and test solutions had to be explored in separate oocytes. Moreover, the amount of test solution on the surface of the patch was uncontrolled. Therefore, we attempted to record from isolated patches that could be exposed sequentially to control and to hylan solutions. We established first that the responses of stretch-activated channels obtained in the cell-attached patch configuration were also observed in isolated patches either in the inside-out or the outside-out configuration. For this purpose, we first recorded in the cell-attached mode to ascertain that stretch-activated channels were present in the patch, placing the intact oocyte in compartment A of the chamber represented in Fig. IB, filled with control solution. Thereafter, we isolated the patch from the oocyte and tested whether the stretch-activated channels remained operative after this procedure, which was indeed the case in the majority of our preparations. A further step in this initial set of experiments with this chamber was to test that the passage of the tip portion of the recording pipette with the attached patch (in either of the 2 configurations) through the oil-filled compartment C, did not have any influence on the stretch-evoked channel activity. FigA illustrates the response behaviour of the same stretch-activated channels in the cell-attached configuration versus the inside-out and outside-out patch configurations. The response of stretch-activated channels remained practically unchanged after the passage through the oil filled compartment C.
B. Effect ofelastoviscous and non-elastoviscous hylan solutions Fig. SA illustrates the results of one experiment performed to test the influence of elastoviscous solution of Synvisc® on the response of stretch-activated channels to a negative pressure applied to the outside-out patch. Compartment A was filled with Barth's medium a control solution while compartment B was occupied by Synvisc® solution (Fig. SA). A negative pressure failed to evoke any current flow through the patch even with much higher negative pressures than under control conditions. In the second series of tests, compartment B was filled with an non-elastoviscous solution (HyA 96,000 MW). A typical example of the results is illustrated in Fig. SB. The control response in compartment A to negative pressure is illustrated in the left trace of the figure. The patch was then moved to compartment B, which contained the the HyA 96,000 MW solution. Repeating the negative pressure to the patch in this solution gave practically the same responses than those under control and differences were not significant, indicating that this hylan was not able to change the properties of the channels.We conclude from these results that elastoviscous hylans caused a considerable decrease in the sensitivity of stretch-activated channels in these membranes, thus confirming the results obtained in the cell-attached configuration experiments.
Effects ofelastoviscous solutions on mechanotransduction
A
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415
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Compartment B HyA 96,000 MW
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Figure 5. Response to pressure of outside-out patches in the presence of non-elastoviscous and elastoviscous solutions. A. Stretch-activated channel opening induced by negative pressure when the patch was immersed in Barth's solution (left trace) and Synvisc® (right trace). No activity was observed in the presence Synvisc® in spite of the larger negative pressure applied. Voltage was fixed at -20mV in both recordings. Microelectrode was filled with Ca2+-free solution. B. Response to negative pressure in the presence of Barth's solution (left trace) and of HyA 96,000 MW (right trace). Voltage was fixed at -30mV in both recordings. CONCLUSIONS
The series of experiments reported above established unequivocally that both the cell-attached and the outside-out patch configurations are suitable to study the response behaviour of native stretch-activated channels from Xenopus laevis 2o •22,25. In the cellattached configuration the cell was immersed in the hylan solution and the pipette was applied on a portion of the cell surface that would be covered by a layer of the viscoelastic material of undetermined depth. The outside-out patch provided better controlled conditions since this situation mimics most closely the "real-life" conditions in intact cells and tissues in vivo and in vitro because the external surface of the membrane is fully exposed to the elastoviscous solution (or any other substance). In both types of experiments, the opening of stretch-activated channels by negative pressure was signifi-
416
Aspects of hyaluronan in joints
candy reduced in the presence of elastoviscous solutions but not when solutions of similar chemical composition but reduced viscoelasticity were applied. These results strongly suggest that hyaluronan networks interfere with the transmission of force to stretch-activated channels. Cells possess a pericellular matrix in which hyaluronan form with proteoglycans a tridimensional hyaluronan-proteoglycan complex (named 'pericellular molecular cage' by Balazs, 1998 8) that it is attached to the cell surface through specific hyaluronan receptors in a brush-like configuration. Mechanosensory channels, on the other hand are attached to both intracellular and extracellular linking proteins that convey mechanical force. These could be embedded in the 'pericellular molecular cage' which would be mechanically stabilised when exposed to an elastoviscous solution made up of molecules in a random coil configuration as high molecular weight hyaluronan and hylans", thus reducing tension transmitted to the different domains of the stretch-activated channel proteins.
ACKNOWLEDGEMENTS The authors wish to express their deep thanks to Prof. Endre A. Balazs for his inspiration, support and encouragement to perform this study and to Eva Quintero for technical assistance. The work was supported by a grant from Biomatrix Inc., Ridgefield, NJ, USA and by SAF- 99-066-C02-0l from Spanish Ministerio de Educaci6n y Cultura.
REFERENCES
1. C.S. Sherrington, 'The integrative action of the nervous system', Scribner, New York. 1906. 2. C. Belmonte, 'Signal transduction in nociceptors: general principles', In: Neurobiology ofnociceptors, C.Belmonte & F.Cervero (eds.), 1996, Oxford University Press, Oxford, pp.243-257. 3. S.M. Block, 'Biophysical principles of sensory transduction.' In: Sensory transduction, D.P. Corey & S.D. Roper (eds.), 1992, The Rockefeller University Press, New York, pp. 241-260. 4. K. Kruger & Z. Halata, 'Structure of nociceptor 'endings', In: Neurobiology of nociceptors, C.Belmonte & F.Cervero (eds.), 1996, Oxford University Press, Oxford, pp 3771. 5. E.A. Balazs, 'The physical properties of synovial fluid and the special role of hyaluronic acid', In: Disorders of the knee, A.J. Helfet (ed.), lB. Lippincott Co., Philadelphia, 1974, pp. 63-75. 6. E.A. Balazs & J.L. Denlinger, 'The synovial cell', In: Modern aging research 4. Comparative pathobiology of major age-related diseases: current status and research frontiers, D.G. Scarpelli & G. Migaki (eds.), 1984a, Alan R Liss Inc, New York, pp. 129-143. 7. E.A Balazs & J.L Denlinger, 'Sodium hyaluronate and joint function', Equine Vet. Sci. 1984b, 5, 217-227.
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8. E.A. Balazs, 'The viscoelastic intercellular matrix and control of cell function by hyaluronan', In: The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives, (Proceedings of the Wenner-Gren Foundation International Symposium held in honor of Endre A. Balazs, September 18-21, 1996, Stockholm, Sweden) T. Laurent (ed.), 1998, Portland Press, London, pp.l85-204. 9. N.W. Rydell, J'Butler & E.A. Balazs, 'Hyaluronic acid in synovial fluid.VI. Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of arthritis in track horses', Acta Vet. Scand. 1970, 11, 139-155. 10. N. Rydell & E.A. Balazs, 'Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of osteoarthritis and on granulation tissue formation', Clin. Orthop. 1971, 80, 25-32. 11. S. Gotah, J.Onaya, M. Abe, K. Miyazaki, A.Hamai, K. Horie, & K.Tokuyasa, 'Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats', Ann. Rheum. Dis. 1993,52, 817-822. 12. J.G. Peyron & E.A. Balazs, 'Preliminary clinical assessment ofNa hyaluronate injection into human arthritic joints', Path 01. Biol. 1974,22, 731-736. 13. C. Weiss, E.A. Balazs, R. St. Onge & J.L. Denlinger, 'Clinical studies of the intraarticular injection of Healon@ (sodium hyaluronate) in the treatment of osteoarthritis of human knees', In: Seminars in arthritis and rheumatism, vol II. J.H. Talbott (ed.), 1981, Grune & Stratton, New York, pp.143-144. 14. E.A Balazs & J.L. Denlinger, 'Sodium hyaluronate and joint function', Equine Vet. Sci. 1985,5,217-228 15. M.A. Pozo, E.A. Balazs & C. Belmonte, 'Reduction of sensory response to passive movements of inflamed knee joints by hylan, a hyaluronan derivative', Exp. Brain Res., 1997, 116,3-9. 16. C. Belmonte, M.A. Pozo & E.A. Balazs, 'Modulation by hylan and its derivatives (hylans) of sensory nerve activity signalling articular pain', In: The Chemistry, Biology and Medical Applications ofHyaluronan and its Derivatives, T. C. Laurent (ed.), 1998 London: Portland Press, pp. 205-217. 17. M. Pawlak , A. Gomis, S. Just, B. Heppelmann, C. Belmonte, & R. F. Schmidt, 'Mechanoprotective actions of elastoviscous hylans on articular pain receptors', This volume. 18. E.A. Balazs, D. Watson, I.F. Duff & S. Roseman, 'Hyaluronic acid in synovial fluid. 1. Molecular parameters of hyaluronic acid in normal and arthritis human fluids', Arthritis Rheum., 1967, 10,357-376. 19. F. Sachs, 'Stretch-sensitive ion channels: an update', In: Sensory Tranduction, 45 th Annual Symposium. Society of General Physiologists. D.P Corey & S.D.Roper (eds.), 1992, Chapter 15, pp. 241-270. 20. C. Methfessel, V. Witzemann, T. Takahashi, M. Mishina, S. Numa, & B. Sakmann, 'Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptors and sodium channels', Pliigers Arch., 1986,407,577-588. 21. V .Taglitti, & M. A. Toselli, 'Study of stretch-activated channels in the membrane of frog oocytes: interactions with ci+ ions', J. Physiol. 1988,407,311-328. 22. X.C. Yang.& F. Sachs, 'Block of stretch-activated ion channels in xenopus oocytes by gadolinium and calcium ions', Science, 1989,243,1068-1071.
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23. X.C. Yang, & F. Sachs, 'Characterization of stretch-activated ion channels in xenopus oocytes', J. Physiol., 1990,43, 103-122. 24. P. ROwen, & W, JR .MCbride, 'Rapid adaptation of single mechanosensitive channels in Xenopus oocytes, P.N.A.S., 1992,89, 7462-7466. 25. P. H. Owen, & W, JR MCbride,. 'The pharmacology of mechanogated membrane ion channels', Pharmacological Reviews, 1996,48,231-252. 26. D. Qin, M. Takano & A. Noma, 'Kinetics of ATP-sensitive K+ Channel revels with oilgate concentration jump method', Am. J. Physiol., 1989, 257, H1624-HI633. 27. B. Martinac, M.Buechner, A.H. Delcour, J. Adler, & C. Kung, 'Pressure-sensitive ion channel in Escherechia coli', P.N.A.S., 1987,84,2297-2301.
EFFECTS OF HYALURONAN ON EQUINE ARTICULAR TISSUE METABOLISM ·Stephen P Frean & Peter Lees Dept ofVeterinary Basic Sciences, Royal Veterinary College Hawkshead Lane, North Mymms, Hatfield, Hens AL9 7TA. United Kingdom
ABSTRACT
Polysaccharide preparations have been used extensively to treat osteoarthritis in the horse, despite little knowledge of these drugs' exact mechanisms of action. Investigation of proteoglycan synthesis, measured by 35S04 uptake, in response to hyaluronan by equine cultured chondrocytes and cartilage explants, was undertaken. Stimulation of proteoglycan synthesis was achieved at drug concentrations between 225 ug/ml, theoretically achievable at recommended clinical dose rates. A disease sub-entity important in equine osteoarthritis is acute sterile synovitis. Hyaluronan carries a specific label indication for treatment of synovitis in the horse. The potential anti-inflammatory action of hyaluronan was investigated using equine cultured synoviocytes, measuring prostaglandin E z synthesis by radioimmunoassay. Hyaluronan at a concentration of2000J.lg/mi significantly reduced prostaglandin Ez synthesis. KEYWORDS
Horse, osteoarthritis, cartilage, synovium, prostaglandin Ez, proteoglycan INTRODUCTION
A rationale in the pharmacological treatment of equine osteoarthritis is to limit the damage to, and stimulate repair of, articular cartilage. To this end hyaluronan is administered intra-articularly or intravenously. This study investigated the effects of hyaluronan on the synthesis of cartilage proteoglycans, a process critical to the maintenance ofnormal articular cartilage and the repair of damaged cartilage. Equine joint disease is frequently characterised by inflammation of the synovial membrane. Synoviocytes produce pro-inflammatory cytokines, metalloproteinases, and eicosanoids. Of the latter, prostaglandin E z (PGEz) has been suggested as an important mediator of articular inflammation and hyperalgesia through its enhancement of vascular permeability, its vasodilatory properties, and its sensitisation of joint nociceptors. Investigations of the effect ofhyaluronan on PGEz production by equine synoviocytes was undertaken. MATERIALS AND METHODS Source of tissue
420
Aspects ofhyaluronan in joints
Tissue was obtained from adult horses less than seven years old. Macroscopically normal cartilage was harvested under aseptic conditions from the distal metacarpi and synovium was harvested from the metacarpophalangeal joint capsule. Chondrocyte Culture
Following enzymatic digestion of cartilage with pronase and collagenase, chondrocytes were grown in monolayer culture in culture plates in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with heat-inactivated Foetal Calf Serum (FCS) (10% v:v). Cartilage Explant Culture
Explants were maintained in DMEM supplemented with FCS (10% v:v) in culture plates for 12h. At this point medium was changed with addition oftest drugs. Synoviocyte Culture
Synovial membrane was subjected to collagenase digestion to obtain synoviocytes; newly released cells were seeded in 25cm3 flasks. Flasks were maintained until cell replication was observed; at this stage synoviocytes were reseeded in 24-well plates and grown to confluence. Treatment of chondrocytes and cartilage explants with hyaluronan
Chondrocytes, maintained in FCS-supplemented DMEM, were treated with hyaluronan (0, 0.25, 2.5, 25, 250 and 2500 ug/ml) for 48h at 37°C. Cartilage explants were similarly treated with hyaluronan at concentrations of 0, 0.2,2,20 and 200 ug/ml. Assay of proteoglycan synthesis
Explants were subjected to treatment with 4M Guanidine HCI (Gu-HCl) / 2% CHAPS / 50mM sodium acetate containing proteinase inhibitors. Following Gu-HCl extraction, the explants were digested with papain. Chondrocyte cultures were subjected to the same procedure excluding the final digestion step with papain. Newly synthesised 35sulphated proteoglycans were separated from free 35S0 4 in medium fractions, explant or monolayer Gu-HCl extracts and papain digests by chromatography on Sephadex G-25M gel columns. Eluant was collected in O.5ml fractions directly into scintillation vials. Treatment of synoviocytes with hyaluronan
Synoviocytes were incubated under the following treatment conditions for 48h: unstimulated (medium only), LPS (l Oug/ml), LPS (l Oug/ml) and indomethacin (IO-6M), LPS (LOug/ml) plus hyaluronan (20, 200, 500, 1000 and 2000!lg/ml), hyaluronan only (1000 and 2000!lg/ml), and LPS [I Oug/ml) plus PBS equivalent in volume to hyaluronan (1000 and 2000!lg/ml).
Effects on equine articular tissue metabolism
421
Prostaglandin Ez assay
Radioimmunoassay for PGEz was conducted using tritiated PGEz tracer and commercially available antiserum raised against PGEz. RESULTS Prostaglandin E z production by synoviocytes
Synoviocytes incubated with LPS produced concentrations ofPGEz significantly higher than the basal levels of unstimulated cells (P<0.001 vs unstimulated). Unstimulated synoviocytes incubated with hyaluronan (1000 and 2000 ug/ml) showed decreases in PGE z synthesis of 21% and 61%, respectively, but these differences were not statistically significant. LPS-stimulated cells treated with hyaluronan (20, 200, 500 and 1000 ug/ml) showed no significant changes in the enhanced PGE z synthesis compared to LPSstimulated cells not treated with hyaluronan. The highest test concentration of hyaluronan preparation (2000~g/ml) yielded a significant reduction in PGE z production (p
422
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Effect of hyaluronan (HA) on prostaglandin E2 (PGE 2) synthesis by lipopolysaccharide (LPS}-stimulated synoviocytes (48 hour incubation; 37°C). Each treament group represents mean +/- SEM PGE 2 concentration of 4 replicate wells. Hyaluronan concentrations (in parentheses) are expressed in ug/ml. The LPS concentration is 10~.• P
(rather than, e.g., receptor-mediated) in nature i. A possible explanation of the bell-shaped dose-response curve of 35S04 incorporation might be that high concentrations ofhyaluronan physically coat cells or the cartilage explant surface and either block access to 35S04 precursor, or limit the secretion ofnewly synthesised sulphated proteoglycans into the culture medium. Experiments using methylcellulose (a chemically inert compound of similar viscosity to hyaluronan) have shown both this compound and hyaluronan to affect release of cellular components 2. Increased 35S04 incorporation by chondrocytes was noted in response to very low concentrations ofhyaluronan. It might be thoughtthat this stimulation lends credence to the recommended dose for intravenous administration (40mg per horse). This, however, appears less convincing in the light of measured physiological concentrations of hyaluronan; Tulamo et al reported endogenous serum hyaluronan concentrations of 0.19 to 0.76Jlglml, i.e., higher than drug concentrations found to stimulate proteoglycan synthesis 3. In addition, the physiological clearance ofhyaluronan is rapid. For example, in the rabbit, the estimated half-life was 2.5 to 4.5min, so that an intravenous dose would be eliminated rapidly 4. Further, there is a conceptual problem arising from the high molecular weight of commercially available hyaluronan preparations and their hence assumed inability to penetrate the synovial membrane to achieve concentrations in synovial fluid. Exposure ofmany cell types to LPS results in the induction ofcycloxygenase-2 (COX-2), with consequent synthesis of PGE2. The mechanisms of action of nonsteroidal antiinflammatory drugs (NSAIDs) and corticosteroids in inhibiting inflammatory eicosanoid synthesis are well established (inhibition of the actions of COX-2 and phopholipase A2, respectively, and, in the case of steroids inhibition of induction of COX-2 also). Given the dissimilarity in molecular structure ofhyaluronan to corticosteroids and NSAIDs, it might
Effects on equine articular tissue metabolism
423
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Figure 2
Proteoglycan synthesis by chondrocytes in response to hyaluronan. Synthesis is reflected in 35S04 incorporation over 48 hours at 37°C. Results represent mean data of two experiments with four replicates in each treatment group +/- SEM, hence N=8. *P
be considered unlikely that hyaluronan exerts its PGE z concentration-lowering effects through inhibition of these enzymes. The possibility ofhyaluronan inhibiting the release of PGE z into culture medium, rather than inhibiting the synthesis of PGE z, is worthy of consideration. Similarly, it has been sU1.gested that hyaluronan might prevent mobilisation of eicosanoid precursors: release of I C-Iabelled arachidonic acid from human synovial fibroblasts, in response to bradykinin and calcium ionophore, was inhibited by hyaluronan in a concentration-dependent manner 2. Phospholipids and their metabolites are found in appreciable amounts in synovial fluid and synovial tissues of arthritic joints. The capacity of hyaluronan to form complexes with phospholipids 5 suggests that the administration of hyaluronan into the joint space might provide a source of binding sites for these molecules. The observation of reduced PGEz synthesis in the presence of hyaluronan might be explained by this binding capacity of hyaluronan; the production of eicosanoids such as PGEz results from metabolism of cell membrane phospholipids. Hyaluronan might bind these molecules in stable complexes, rendering their breakdown more difficult. CONCLUSIONS
Hyaluronan (i) stimulates proteoglycan synthesis by equine cartilage in vitro, and (ii) inhibits PGE2 production by equine synoviocytes. These effects are observed at concentrations achievable by intra-articular injection, and may suggest reasons for the claimed efficacy ofhyaluronan as a treatment for osteoarthritis in the horse.
424
Aspects ofhyaluronan in joints
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Figure 3
Proteoglycan synthesis by cartilage explants in response to hyaluronan. Synthesis is reflected in 35S04 incorporation over 48 hours at 37°C. Results represent mean data of two experiments with four replicates in each treatment group +/- SEm, hence N=8. *P<0.05 vs untreated (Dunnett's test, 2-tailed). 0 extract; l1li medium; • digest; • total activity.
ACKNOWLEDGEMENTS This work was generously supported by the Home of Rest for Horses, Speen, Bucks, United Kingdom, the Horserace Betting Levy Board, and Bayer Animal Health, Bury St Edmunds, Suffolk, United Kingdom.
REFERENCES 1. T. C. Laurent, U. B. G. Laurant & J. R. E. Fraser, 'The structure and function of hyaluronan: an overview', Immunology and Cell Biology, 1996,74, AJ-A7. 2. K. Tobetto, T. Yasui, T. Ando, M. Hayaishi, N. Motohashi, M. Shinogi & I. Mori, 'Inhibitory effects of hyaluronan on [14C]arachidonic acid release from labeled human synovial fibroblasts', Japanese Journal ofPharmacology, 1992, 60, 79-84. 3. R. M. Tulamo, H. Saari & Y. T. Konttinen, 'Determination of concentration of hyaluronate in equine serum', American Journal ofVeterinary Research, 1990, 51,740742. 4. J. R. Fraser, T. C. Laurent, H. Pertoft & E. Baxter, 'Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit', Biochemical Journal, 1981, 200, 415-424. 5. P. Ghosh, N. Hutadilok & N. Adam, 'Interactions of hyaluronan (hyaluronic acid) with phospholipids as determined by gel permeation chromatography, multi-angle laser-light scattering photometry and I H-NMR spectroscopy', International Journal of Biological Macromolecules, 1994, 16,237-244.
SIGNAL TRANSDUCTION PATHWAYS IN HYALURONAN INDUCED PROLIFERATION OF ENDOTHELIAL CELLS Slevin M*\ Kumar Sl & Gaffney Jl JDepartment
ofBiological Sciences, Manchester Metropolitan University, Oxford Road, Manchester, UK.
2Department
ofPathology, Stopford Building, Manchester Victorian University, Manchester, UK.
ABSTRACT Knowledge of the signal transduction pathways involved in mediating the effects of oHA on target cells would be useful in defming potential selective targets for inhibitors of endothelial cell (EC) function in relevance to intervention in angiogenesis. We have previously shown that oHA induced mitogenesis involves activation of protein kinase C, MAP kinase and early response genes in bovine aortic EC (BAEC). Here we demonstrate the potential involvement of both G-protein and tyrosine kinase linked elements, suggesting the existence of cross-talk between separate signal transduction pathways. In the presence of oHA, both PLC r1 and PLC 131,132 and 133 were translocated to the plasma membrane. We also found that GI3 sub-units became strongly associated with PLC r1, and and immuno-neutralising antibodies loaded into cells using liposome mediated delivery, significantly reduced MAP kinase tyrosine phosphorylation. Furthermore, MAP kinase tyrosine phosphorylation as well as cell proliferation were significantly reduced in the presence of pertussis toxin. Ras was also activated in oHA treated cells, and the potent ras inhibitor Ftase 1 significantly inhibited cell proliferation. KEYWORDS Hyaluronic acid, signal transduction, endothelial INTRODUCTION Angiogenesis is essential for the growth and repair of normal tissues and diseases adversely affecting angiogenesis (eg. stroke, mycocardial infarction, diabetic retinotherapy, rheumatoid arthritis, neoplasia, AIDS) are the most common cause of human mortality and morbidity in the western world. A better understanding of the mechanisms and control of angiogenesis in these situations is fundamental in order to ameliorate morbidity and improve their cure rate. The target cell for neovascularisation is the blood-vessel endothelial cell and specific angiogenic molecules produced or induced by the tissues are believed to initiate this process. One of these angiogenic molecules, hyaluronan (HA), has differing roles in neovascularisation depending on its molecular mass. High molecular mass HA is anti-angiogenic, whereas oligosaccharides of HA, of specific size (oHA), stimulate endothelial cell proliferation and migration, two key events associated with neovascularisation, and induce angiogenesis in vivo. Liu et al. (1996) have reported that hyaluronidase (HAase) of tumour cells induces angiogenesis and that tumour cells use HAase as one of the "molecular saboteurs" to depolymerise HA in order to facilitate invasion. As a consequence, breakdown products of HA can further promote tumour establishment by inducing angiogenesis. A detailed study is required to characterise the signal transduction pathways involved in mediating
470
The action ofhyaluronan in cells
the effects of oHA on target cells. Recently, o-HA was shown to activate tyrosine phosphorylation of proteins, as well as ras in RSV-3YI cells (Serbulea et al 1999). CD44, the principal cell surface receptor for HA coupled to non-receptor protein tyrosine kinases including Lck, Fyn and Lyn in T lymphocytes (Ilangumaran et al 1999), and was linked to both ras and PKC activation in T-24 cells (Fitzgerald et al 2000). We have previously identified some of the cytoplasmic associated signalling intermediates in o-HA induced activation of EC. Here we describe activation of several membrane associated effectors which modulate proliferation in BAEC. MATERIALS AND METHODS BAEC were isolated, characterised. cultured and maintained as described previously (Slevin et al 1999). o-HA was used at a concentration of IOug/ml in all experiments.
CeU Proliferation Cells were loaded in triplicate onto 6 well plates at a concentration of 2x10 4and after attachment (4h), incubated for 72h in serum poor medium (SPM) containing 2% FCS together with the appropriate enzyme inhibitor, with or without 0HA. Statistically significant differences (p<0.05) were determined by analysis of variance using the student t test. Liposome mediated antibody loading Cells were made transiently permeable by treatment for 4 min with ice-cold L-a. lysophosphatidylcholine (1uM, 250ul, 4°C) in a mixture of glycerol (1.2M):PBS.(Piiper et al 1997) Antibodies were applied (1-5ug/ml) and after a further 30 sec, 2ml of preheated SPM. The cells were allowed to recover (3Omin, 3rC) before addition of o-HA. Efficiency of cell loading as well as recovery were monitored by trypan blue exclusion and FACS analysis (data not included). Western blotting Semi-confluent cells, were incubated in 6-well plates for 48h in SPM and following appropriate treatment, lysates were collected in RIPA buffer, protein concentrations determined using the Biorad reagent and proteins (20J,1g) separated by reducing SDS-PAGE using a 12% gel. Proteins from blotted gels were visualised using the appropriate monoclonal or polyclonal antibodies. Isolation of plasma membrane and cytoplasmic cell fractions These fractions were separated using a digitonin buffer based system as described in Slevin et al (1998). Immunoprecipitation/Coprecipitation Lysates from o-HA stimulated cells collected in RIPA buffer (1OOJ,1g) were incubated overnight with the appropriate antibody attached to protein A1G agarose beads. After washing, the complexes were boiled in sample buffer and protein separated by SDS-PAGE as described above. RESULTS AND DISCUSSION Expression and translocation of PLe isoforms HAEC expressed the y1, 131 and 132 isoforms of PLC, but not y2 or 3. Examination of the membrane fraction of o-HA activated cells, revealed translocation of PLCyl, as well as PLC13I, PLC132 and in particular PLC133 (Fig 1). Whilst PLCy isoforms are known to be activated by receptor tyrosine kinases, PLC13 isoforms are linked to G-protein coupled receptors, suggesting the possible existence of multiple signal transduction pathways activated by o-HA.
Signal transduction pathways
471
Coprecipitation studies Immunoprecipitates obtained after incubation of o-HA treated cells with antibodies to G~ sub-units, contained significant quantities of immunoreactive PLCy1 protein (Fig 2). A similar result was obtained when lysates were immunoprecipitated with anti-Gailo/t/z (data not shown). Only weak immunoreactivity to PLC~2 was found. Preincubation of cells with pertussis toxin (IOOng/ml, 30 min) reduced the association of PLCyl with G~ (Fig 3), suggesting that interaction between these two separate pathways may be important in regulation of o-HA function. Fig I) "5
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Expression of tyrosine phosphorylated MAP kinase in o-HA treated cells loaded with inhibitory G-protein antibodies
Effect of G proteins on MAP kinase tyrosine phosphorylation Map kinase (p42/p44) tyrosine phosphorylation was significantly reduced in BAEC loaded with antibodies directed towards Galilo/t/z and G~ sub-units (Fig 4), whilst antibodies directed to Gus/elf and Gq proteins had no effect (data not included). Pertussis toxin (IOOng/ml) was also able to block MAP kinase tyrosine phosphorylation. These results suggest a role for Go-protein linked elements in mediation of o-HA induced proliferation. Activation of ras Ras protein was activated within 2 min after addition of o-HA in BAEC (Fig 5), as measured following immunoprecipitation ofras-GTP bound to GSTRaf-I kinase fusion protein which recognised only the activated form (TCS Biologicals). Inhibition of ras activation using Ftase I (lOuM, ras farnesyltransfererase inhibitor, Calbiochem), reduced o-HA induced MAP kinase tyrosine phosphorylation (Fig 6). Proliferation studies Cell proliferation of BAEC was significantly reduced in the presence of both Ftase I (10uM) and pertussis toxin (lOOnM Ga protein inhibitor) as shown in Fig 7. As a control, bFGF treated cells also showed a reduction in proliferation in the presence ofFtase I but not pertussis toxin (data not included). GPantag 2A (a Gq protein inhibitor, Calbiochem), had no effect. Conclusion o-HA induced proliferation in vascular endothelial cells occurs by activation of signal transduction pathways which converge at MAP kinase. At least two separate pathways appear to be important. Tyrosine kinase linked elements including PLCy, she
472
The action ofhyaluronan in cells
(not shown here) and Raf-l kinase, together with G-protein linked elements including PLC13, GaJiJo/t/z and G13 sub-units are involved and possibly exhibit cross-talk before stimulation of ras and subsequently MAP kinase. We are in the process of establishing the importance of G13r sub-units in mediating o-HA responses using BAEC transfected with plasmid vectors containing 13-ARK lactive sequences (Koch et aI1994). Fig
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References Fitzgerald KA, Bowie AG, Skeffington BS, O'Neill LAJ Ras, protein kinase C zeta and I kappa B kinases I and 2 are downstream effectors ofCD44 during the activation ofNF-Kappa B by hyaluronic acid fragments in T-24 carcinoma cells. J Immunol (2000), 164;2053-2063. Ilangumaran S, Borisch B, Hoessli DC Signal transduction via CD44: Role of plasma membrane microdomains. Leukemia & Lymphoma., (1999) 35; 455-469. Koch WJ, Hawes BE, Inglese J, Luttrell LM and Lefkowitz RJ Cellular expresion of the carboxyl terminus of a G protein-couples receptor kinase attenuates Gpy-mediated signalling. J BioI Chem (1994),269,6193-6197. Liu D, Pearlman E, Guo EDK, Mori H, Haqqi S, Markowitz S, Willson J, Sy MS. (1996) Expression of hyaluronidase by tumour cells induces angiogenesis in vivo. Proc Natl Acad Sci, USA 93, 7832-37. Piiper A, StryjekKaminska D, Klengel R, Zeuzem S. (1997) Epidermal growth factor inhibits bombesin-induced activation of PLC-beta 1 in rat pancreatic acinar cells. Gastroenterology, 1I3; 1747-1755. Serbulea M, Kakuma S, Thant AA, Miyazaki K, Machida K, Senga T, Ohta S, Yoshioka K, Horra N, Hamaguchi MHyaluronan activates mitogen-activated protein kinase via Ras signalling pathway. Int J Oncology. (1999) 14,723-33. Slevin M, Krupinski J, Kumar S, Gaffney J. (1998) Angiogenic oligosaccharides ofhyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation. Lab Invest 78, 987-1003.
CONTROL OF BYALURONAN (HA) GENERATION IN RENAL PROXIMAL TUBULAR EPITHELIAL CELLS. Stuart. G. Jones, Suzanne. M. Jones and Aled. O. Phillips· Institute of Nephrology, University of Wales College of Medicine. Heath Park Cardiff, Wales. UK.
ABSTRACT Progression of renal disease is correlated to the degree of renal interstitial fibrosis. Proximal tubular cells (PTC) contribute to these pathological changes by the generation of cytokines and alterations in the composition of the extracellular matrix. Hyaluroanan (HA) is a ubiquitous connective tissue polysaccharide which regulates cell function and may therefore contribute to, and regulate, tissue remodelling. In the context of diabetic nephropathy, increased renal hyaluronan production, has been implicated in the glomerular hypei'cellularity in the streptozotocin model of diabetes. Recent studies have also implicated macrophage infiltration in the pathogenesis of diabetic nephropathy. In the current study we investigated PTC production of HA in response to alterations in glucose concentration and the macrophage derived cytokine IL-lB. The results demonstrate a time dependent increase in HA concentration in the culture supernatant in response to both 25mM D-glucose and IL-IB. Hyaluronan synthases (HAS) mRNA expression was examined by RT-PCR. HAS2 mRNA induction was seen following either 25mM D-glucose or IL-lB stimulation. HAS3 mRNA was constitutively expressed by PTC and was not influenced by the addition of either 25mM D-glucose or IL-l B. In contrast HAS 1 mRNA expression was not detected in either unstimulated or stimulated cells. Stimulation of HA generation by either IL-l or 25 roM D-glucose, was abrogated by inhibition of I-kappa B kinase (IKKB) using Sulindac, and by the use of the proteosome inhibitor PSI, thus implicating activation of NF-lCB in transcriptional activation of HAS2.
INTRODUCTION The aim of our work is to identify factors which in combination with hyperglycaemia, may contribute to both the initiation and progression of diabetic renal disease. It is now evident that progressive decline in renal function in diabetes is closely correlated with the degree of renal interstitial fibrosis 1.2. We have therefore, focused on those mechanisms which may induce changes in the interstitium, and in particular the mechanisms by which the proximal tubular epithelial cell (PTC) may be involved in their initiation. Recent studies have implicated macrophage infiltration in the pathogenesis of diabetic nephropathy 3-6. In the current study we have therefore examined the regulation of PTC HA generation in response to macrophage derived cytokines and elevated glucose concentration.
MATERIALS AND METHODS Cell cuLture: Experiments were performed utilising HK2 cells, (human renal proximal tubular epithelial cells immortalised by transformation with human papilloma virus [HPV] 16 E6lE7 genes 7) under standard culture conditions. Cells were grown to confluence and growth arrested in serum free medium for 48 hours, and all experiments where then performed under serum free conditions. Growth arrested cells were stimulated with recombinant IL-lB (0 to lOOng/ml) or 25 roM D-glucose. Supernatant samples were
474
The action of hyaluronan in cells
collected up to 96h post stimulation for quantification of HA. Control experiments were performed by the addition of 25 mM L-Glucose. or 5mM D-glucose. Alteration in HA synthesisr. In all experiments HA concentration in the cell culture supernatant was determined by an enzyme linked immunosorbant assay (HA "Chugai" quantitative test kit. Chugai Diagnostics). The effect of various stimuli on the expression of HAS 1. 2 and 3 mRNA expression was determined by Reverse Transcription and the Polymerase Chain Reaction (RT-PCR) as previously described 8 using specific oligoneucleotide primers. To confirm that HA generation was dependent on induction of HAS transcription, HK-2 cells were stimulated with IL-lB (lng/ml) or 25 mM D-glucose, in the presence of increasing (non-toxic) doses of either actinomycin-D (0-25Ong/ml) to inhibit transcription or cycloheximide (0-51lg/ml) to inhibit mRNA translation respectively. Involvement ofNF-kappa B: The dependence of HA generation of NF-kappa B activation was determined by inhibition of I-kappa B kinase (IKKB) using Sulindac, and by the use of the proteosome inhibitor PSI (Calbiochem)
RESULTS Alteration in HA production: Addition of either IL-lB or 25 mM D-glucose led to a time dependent increase in HA generation (Fig 1). The effect of IL-lS was significant at l2h. In contrast the effect of 25 mM D-glucose was delayed and only apparent 96h after its addition (Fig 1). The increase in HA synthesis following addition of 25mM D-glucose was unrelated to alterations in osmolarity since addition of 25 mM L-glucose did not affect the production of HA. Co-stimulation of cells with IL-lS and 25 mM D-glucose led to an additive rather than an augmented increase in HA generation. HAS2 mRNA induction was seen following IL-lB stimulation (Fig 2a). HAS3 mRNA was constitutively expressed by HK-2 cells and was not modified by the addition of IL-lB. In contrast HASI mRNA expression was not detected in either un-stimulated or stimulated cells. Inhibition of de novo gene transcription by actinomycin-D or mRNA translation by the addition of cycloheximide completely abrogated HA generation following addition of IL-lB.
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Addition of 25 mM D-glucose had similar effects on the expression of HA synthase, with an induction of HAS 2 mRNA expression, but no effect on constitutive HAS 3 mRNA expression (fig 2b). Similarly addition of 25 roM D-glucose did not induce the expression of HAS I mRNA which again remained undetectable by RT-PCR. The effect of glucose on HA generation, was also abrogated by addition of either actinomycin-D or cycloheximide
Mechanism of HA stimulation: Both IL-IB and 25 roM D-giucose induced alterations in HA generation by HK-2 cells were abrogated by the addition of Sulindac in a dose dependent manner (Fig 3). In contrast indomethacin had no effect on the generation of HA following addition of either stimulus. Inhibition of NF-kappa B activation by the addition of the proteosome inhibitor PSI also abrogated both the IL-IS and 25 roM D-glucose stimulated increase in HA (Fig 4).
DISCUSSION The data presented in the current manuscript demonstrate that HA generation by proximal tubular epithelial cells was stimulated by both 25roM D-glucose and the pro-inflammatory cytokine IL-IB. In addition the data demonstrates that stimulation of HA synthesis is dependent of transcriptional activation of HAS2. The different "kinetics" of HA stimulation following addition of either stimulus however suggests that the mechanisms by which they mediate these effects may differ. In addition to the inducible expression of HAS2 mRNA we have also demonstrated constitutive expression of HAS3 mRNA which was not influenced by the addition of either IL-iS or 25roM D-glucose. Previous studies have implicated alterations in HA synthesis in the pathogenesis of the glomerular abnormalities associated with diabetic nephropathy 6. In these studies alterations in HA were related to alteration in prostaglandin turnover. Although the effect of both IL-iS and 25 roM D-glucose were
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~ "'6 NO Figure 4: Inhibition of HA generation in response to addition of either IL-IB [A] or 25 mM D-glucose by the proteosome inhibitor PSI. Confluent monolayers of HK-2 cells were stimulated with either IL-IB [A] or 25 mM D-glucose in the presence of PSI. Control experiments were performed by stimulation with 5 mM D-glucose in the absence of PSI. Supematant samples were collected 24h after addition of IL-IB and 96h following addition of 25 mM D-glucose for determination of HA concentration. Data represents mean ± sd of 4 individual experiments, *p<0.OO5 for PSI vs either IL-IB [A] or 25 mM D-glucose [B] stimulated.
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inhibited by Sulindac, it is unlikely that this is related to alteration in prostaglandin turnover as neither IL-IB nor 25 roM D-glucose induced alterations in HA synthesis were inhibited by indomethacin. Recent studies suggest that Sulindac inhibits activation of NF-kappa B by inhibition of IKKB kinase activity 9. Our results demonstrate that induction of HA by either IL-IB or 25 mM D-glucose was associated with activation of NF-kappa B. Furthermore inhibition of NF-kappa B activation either by Sulindac or by the use of a proteosome inhibitor inhibited HA generation in response to either stimulus. The NF-kappa B pathway regulates the cellular response to a variety of stimuli including cytokines, bacterial and viral infection, and activation of cellular stress pathways. It is also critical for the control of cellular growth and has been demonstrated to be an important regulator of apoptosis. Its activation is known to be triggered by numerous pro-inflammatory cytokines such as IL-IB. This is the first demonstration however that the activation of NF-kappa B increases the synthesis of HA by transcriptional activation of HAS2, although transcriptional activation of HAS 1 by TNFa has recently been demonstrated in a myofibroblast cell line 10. Activation of NF-kappa B by elevated concentration of glucose has recently been demonstrated in vascular smooth muscle cells II. To our knowledge, however, our data is the first to demonstrate activation of NFkappa B following addition of 25 roM D-glucose in renal cells. Although it is known that increased synthesis of HA may be a feature of diabetic glomerulosclerosis, little to date is known regarding alterations in HA in the renal interstitium. It is interesting to speculate on the functional significance of increased HA production. Recent studies suggest that high molecular weight HA and low molecular weight HA oligosaccharides present different signals to cells. In general high molecular weight HA represents the normal homeostatic state whereas the generation of low molecular weight HA fragments, signals a disruption of the normal homeostatic environment. Several workers have reported that HA-oligosaccharides may stimulate gene expression and protein synthesis of chemokines 12 and interstitial collagens 13. In contrast high M w HA oligosaccharides inhibit the "bio-activity" of TGF-B and stimulate the secretion of tissue inhibitors of metalloproteinases 14, 15. These observations therefore suggest that high M w HA may be "anti-fibrotic" whereas, if unabated, the generation of low molecular weight HA fragments may disrupt the normal balance between cells and matrix and contribute to the pathophysiology of chronic tissue inflammation and fibrosis. The key to understanding the implication of the increased HA that we have described may therefore be the analysis of the molecular weight of the HA generated. Macrophage influx has previously been implicated in the pathogenesis of diabetic nephropathy, both in animal models 3, 4 and in human disease 5. This suggests that generation of macrophage derived cytokines such as IL-IB, in combination with the effect of elevated glucose concentrations may act synergistically to influence the pathogenesis of diabetic nephropathy. We have previously demonstrated that the combined effects of glucose and IL-IB may modulate the pro-fibrotic potential of the PTe in diabetic nephropathy, by an increase in the production of the pro-fibrotic cytokine TGF-BI 16. More recently augmented NF-kappa B activation by pro-inflammatory cytokines by elevated glucose concentrations has been demonstrated in vascular smooth muscle cells II. In the current manuscript we have demonstrated that stimulation with the combination of ILI-B and 25 mM D-glucose had an additive effect on the production of HA, which further supports the concept that the interaction of hyperglycaemia and macrophage derived pro-inflammatory cytokines may augment renal injury in diabetes. In summary we have demonstrated for the first time that increased HA synthesis in response to either IL-IB or elevated 25 roM D-glucose is dependent on NF-kappa B activated transcription of HAS 2. This may have implication for the pathogenesis of renal interstitial changes associated with diabetes mellitus. We have also demonstrated that HAS 3 is constitutively expressed and not induced in these cells, although the role of constitutive HA
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The action ofhyaluronan in cells
synthesis in these cells remains to be determined, Identification and contrasting the signalling pathways which mediate transcriptional activation ofHAS2 following stimulation by ll...-IB or 25 mM D-glucose therefore represent an important area for future research.
ACKNOWLEDGEMENTS SGJ is supported by the National Kidney Research Foundation and AOP is in receipt of a Wellcome Trust Advanced Training Fellowship.
REFERENCES l. A. Bohle, M. Wehrmann, O. Bogenschutz, C. Batz and G. A. Muller, The Pathogenesis of Chronic Renal Failure in Diabetic Nephropathy, Path. Res. Pract., 1991, 187,251-259.
2. P. H. Lane, M. W. Steffes, P. Fioretto and S. M. Mauer, Renal interstitial expansion in insulin-dependant diabetes mellitus. Kidney Int, 1993,43,661-667. 3. B. A. Young, R. J. Johnson, C. E. Alpers, C. E. Eng, K. Gordon, J. Floege and W. Couser, Cellular events in the evolution of experimental diabetic nephropathy, Kidney Int, 1995,47,935-944. 4. C. Sassy-Pringent, D. Heudes, C. Mandet, M. F. Belair, O. Michel, B. Perdereau, J. Bariety and P. Bruneval, Early glomerular macrophage recruitment in streptozotocin induced diabetic rats, Diabetes, 2000, 49, 466-475. 5. T. Furuta, T. Saito, T. Ootaka, J. Soma, K. Obara, A. Keishi and K. Yohinaga, The role of macrophages in diabetic glomerulosclerosis, Am. J. Kidney. Dis., 1993,21,5,480-485. 6. P. Mahadevan, R. G. Larkins, J. R. E. Fraser, A. J. Fosang and M. E. Dunlop, Increased hyaluronan production in the glomeruli from diabetic rats: link between glucose induced prostaglandin production and reduced sulphated proteoglycans, Diabetologia, 1995, 38, 298-305. 7. M. J. Ryan, G. Johnson, J. Kirk, S. M. Fuerstenberg, R. A. Zager and B. Torok-Storb, HK-2: An immortalized proximal tubule epithelial cell line from normal adult human kidney., Kidney Int, 1994,45,48-57. 8. A. O. Phillips, R. Steadman, N. Topley and J. D. Williams, Elevated D-glucose concentrations modulate TGF-Bl synthesis by human cultured renal proximal tubular cells: the permissive role of platelet derived growth factor., Am. J. Pathol., 1995, 147, 2, 362374. 9. Y. Yamasmoto, M. J. Yin, K. M. Lin and R. B. Gaynor, Sulindac inhibits activation of the NF-kB pathway, J. Biol. Chem., 1999,274,38,27307-27314. 10. T. Ohkawa, N. Ueki, T. Taguchi, Y. Shindo, M. Adachi, Y. Amuro, T. Hada and K. Higashino, Stimulation of hyaluronan synthesis by tumor necrosis factor alpha is mediated by the p50/p65 NF kapp B complex in MRC-5 myofibroblasts, Biochem. Biophys. Acta., 1999, 1448, 416-424.
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11. K. K. V. Yemeni, W. Bai, B. V. Khan, R. M. Medford and R. Natarajan, Hyperglycaemia induced activation of nuclear transcription factor kB in vascular smooth muscle cells, Diabetes, 1999,48,855-864. 12. C. M. McKee, M. B. Penno, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao and P. W. Noble, Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages, J. Clin. Invest., 1996,98,2403-2413. 13. P. Rooney, M. Wang, P. Kumar and S. Kumar, Angiogenic oligosacharides of hyaluronan enhance the production of collagens by endothelial cells, J Cell Sci, 1993, 105, 213-218. 14. P. Locci, L. Marinucci, C. Lilli, D. Martinese and E. Becchetti, Transforming growth factor 81 - hyaluronic acid interaction, Cell. Tissue. Res., 1995,281,317-324. 15. T. Yasui, M. Akatsuka, K. Tobetto, J. Umemoto, T. Ando, K. Yamashita and T. Hayakawa, Effects of hyaluronan on the production of stromolysin and tissue inhibitor of metalloproteinase-1 (TIMP-1) in bovine articular chondrocytes, Biomed Res, 1992, 13, 5, 343-348. 16. A. O. Phillips, N. Top1ey, R. Steadman, K. Morrisey and J. D. Williams, Induction of TGF-81 synthesis in D-glucose primed human proximal tubular cells: differential stimulation by the macrophage derived pro-inflammatory cytokines IL-18 and TNFa, Kidney Int, 1996, 50, 1546-1554.
PART?
CLINICAL APPLICATIONS OF HYALURONAN
MOLECULAR STRATEGIES FOR THE THERAPEUTIC UTILIZATION OF HYALURONAN Philip A. Band Biomatrix, Inc., 65 Railroad Avenue, Ridgefield, New Jersey 07657, USA
ABSTRACT All of the currently licensed medical indications for hyaluronan are based on viscoelastic preparations which take advantage of hyaluronan's unique physiochemical properties and biocompatibility. Well-established relationships exist between the molecular characteristics of different hyaluronan preparations and their applicability for specific indications. Despite the successful application of these principles in more than 30 million patients, considerable debate continues to surround the molecular mechanisms which underly hyaluronan's therapeutic utilization. Because hyaluronan can exert profound biological effects in experimental systems, biomechanical explanations of its mode of action are sometimes considered incomplete. A perspective is presented which explains the compatibility between these viewpoints based on well-documented relationships between the biological effects of hyaluronan and its concentration, molecular weight and rheological properties. Data supporting this perspective will be reviewed, and simple paradigms useful for product development will be suggested. KEYWORDS Hyaluronan, hylan, viscosurgery, viscosupplementation, viscoelastic. INTRODUCTION The use of hyaluronan in medicine is well established. More than 30 million patients have benefited from the clinical use of hyaluronan, making it one of the most successful products of biotechnology. The occasion of this symposium honoring Endre Balazs marks an appropriate time to review how the molecular and biological properties of hyaluronan relate to the different ways in which it is currently used in medicine, and to consider what general principles can be applied to the development of important new indications. The current therapeutic uses of hyaluronan derive directly from its unique physicochemical properties and their importance to the biology of the intercellular matrix. Endre A. Balazs is credited with realizing early on the broad potential for hyaluronan in medicine, and for devoting his considerable energies to bringing hyaluronan's benefits to patients in multiple ways 1. The first indications developed for hyaluronan were entirely new modalities, viscosurgery 2 and viscosupplementation 3, specifically made possible by hyaluronan's unique rheological properties and biocompatibility. Later products additionally took advantage of the ways in which hyaluronan's physiochemical and biological properties could address existing medical needs in novel ways, such as control of post-surgical adhesions, soft-tissue
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augmentation, tissue engineering, drug delivery and topical protection 4.5. The term "intercellular matrix therapy" was recently suggested to refer to this diverse group of indications for hyaluronan 5. Because the medical utility of hyaluronan's current indications is dependent on specific mechanical properties, they are generally regulated as medical devices, rather than as pharmaceutical agents. More recently, efforts have been increasing to develop pharmacologic indications for hyaluronan 6.7,8.9. Such approaches are typically based on an entirely different aspect of hyaluronan's biological activities, generally using hyaluronan solutions of low concentration and/or molecular weight with little or no viscoelasticity, and focusing on specific hyaluronan binding proteins (hyaladherins, see reference 10 for review). These efforts are not incompatible with the currently available medical devices based on viscoelastic hyaluronan preparations. Some general principles particularly important when developing new therapies with hyaluronan are evident from the successes and failures of the last thirty years, and some issues are still the subject of considerable debate and research effort. The regulatory status of hyaluronan-based products must be critically considered as it can vary between both indications and jurisdictions, and to some extent will dictate the logic and testing requirements for product licensing. This review will summarize the available information from the perspective of product development, and try to identify useful paradigms based on hyaluronan's molecular and biological properties. KEY PARAMETERS FOR HYALURONAN PRODUCTS
Several parameters related to the molecular properties of hyaluronan are important to consider when developing hyaluronan for any indication, particularly its molecular weight, its production from biological sources, and the diversity of its biological actions. Before considering the molecular strategies that have been used to customize hyaluronan for specific indications, these fundamental issues will be briefly addressed.
Source and Purity: Hyaluronan preparations of varying properties and quality are now widely available from many manufacturers. They are generally produced from one of two sources: avian tissue 11 (chicken combs, a byproduct of the food industry), or bacteria (streptococci, most commonly) 12.13. Both sources present specific manufacturing challenges because of hyaluronan's sensitivity to degradation (shear, thermal, free radical and oxidative) and its tendency to interact with and trap other molecules. The development of methods to reliably produce sterile, non-pyrogenic, noninflammatory, viscoelastic, hyaluronan preparations was critical to the extensive medical use of hyaluronan today II. Different production methods can yield widely varying products, particularly with respect to molecular weight distribution and impurities. It was particularly important to establish a methodology to routinely test and specify such medically useful preparations 14. The testing methods for hyaluronan are themselves technically challenging because of hyaluronan's high molecular weight, and because troublesome trace impurities are difficult to remove and hard to measure. There is still no universally accepted method to determine the molecular weight distribution of hyaluronan, and the available chromatographic columns do not effectively resolve polymers of very high molecular weight 14&. The first published standards for hyaluronan were for ophthalmic viscoelastic devices IS. In 1999, the European Pharmacofoeia provided general testing procedures and standards for hyaluronan products'". These include identification of the
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hyaluronan polysaccharide by infrared spectrometry, determination of intrinsic viscosity, and analyses for protein, nucleic acids, sulfate, trace metals and endotoxin. Though it is important that intrinsic viscosity is included as an estimate of molecular weight, it should be noted that this method provides no information on molecular weight distribution. Furthermore, there are no monographs available for chemically modified hyaluronans despite their widespread medical use. Nor does the pharmacopoeia address the suitability of a hyaluronan preparation for any specific indication, stating that the manufacturer must warrant this suitability. Developers of hyaluronan products must therefore take the responsibility to ensure that their hyaluronan source, testing methods and specifications are appropriate for their product's intended use. Rheology and Molecular Weight: The rheolorical properties of hyaluronan in aqueous solution have been extensively reviewed 1 • The two primary molecular parameters controlling its viscoelastic behavior are molecular weight and concentration. Both viscosity and elasticity increase exponentially as either concentration or molecular weight is increased. Because of this relationship, concentration and molecular weight must be jointly considered when developing product specifications to ensure consistency and the desired rheology range. Biological responses to hyaluronan are highly dependent on the molecular weight and concentration range evaluated (see section below on molecular weight and biological activity). Viscoelastic solutions typically have very different biological actions than non-viscoelastic solutions. The molecular weight and rheological profile required for a specific use can vary widely between indications, and often becomes the subject of competitive product debates. Understanding the principles governing hyaluronan's rheological behavior is critical to successful product development, and should likewise be considered when interpreting in vitro or laboratory studies. Residence Time: The turnover of hyaluronan varies widely between different tissues, but is generally rapid compared to other matrix polymers 18. Using methods developed to measure nanogram quantities of hyaluronan 19 and systemic turnover 20, its widespread distribution in body fluids and systemic clearance pathways became clearly established, adding a new dimension to the long-recognized distribution of hyaluronan in connective tissues. The rapid and voluminous daily turnover of hyaluronan is directly related to its water solubility because hialuronan is primarily catabolized via its movement as a component of tissue fluid 1. As such, in tissues subjected to different physical forces and therefore having differing rates of fluid flow, the half-life of hyaluronan can vary from 12 hours in the rabbit knee 22 to 7 days in the monkey vitreous 23. From the perspective of product development, residence time must be customized to the requirements for a specific indication. The desirable residence time can range from viscosurgical products that need only be present during the surgical procedure, to soft tissue augmentation implants which would ideally have a residence time of many years, and are therefore not feasible for unmodified hyaluronan. Hyaluronan products intended for systemic administration have an entirely different set of obstacles to overcome with respect to their pharmacodynamics, primarily their rapid plasma clearance and the difficulty of targeting to their intended site of action. The control of residence time is difficult to achieve without significant modification of hyaluronan's water solubility. For unmodified hyaluronan, residence time can only be increased by using concentrated high molecular weight solutions or
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"dry" solids 24, and the extent of prolongation is very limited. Effective control of residence time generalll requires chemical modification or formation of water insoluble complexesf" 2 (see below). Developing molecular strategies to prolong residence time has been important to creating new indications for hyaluronan.
MOLECULAR STRATEGIES FOR CUSTOMIZING HA PRODUCTS When developing therapeutic products based on hyaluronan, the requirements for a specific indication should be evaluated in terms of purity, rheology, molecular weight, physical form and residence time. These requirements are indication-specific and can be optimized by controlling the molecular properties of hyaluronan. Successful strategies for customizing hyaluronan include the use of well-defined fractions and physical forms, and more recently have resorted to complexation or covalent derivatization. Often it is necessary to combine strategies in order to achieve the desired properties. The molecular strategies which have proven useful are detailed here. Defined Hyaluronan Fractions: The idea of using a well-defined fraction of hyaluronan was first patented in 1979, which describes a preparation of ultra pure high molecular weight hyaluronan for medical purposes 11. This patent specifies a hyaluronan fraction with average molecular weight greater than 750,000, which is biologically inert as tested by vitreus exchange in primates 14. The exclusion of low molecular weight hyaluronan from the patent specification was especially insightful based on the accumulating evidence regarding biological activities of low molecular weight hyaluronan fractions and oligosaccharides (see section below, Molecular Weight and Biological Activity). Numerous publications and patents propose applications for lower molecular weight fractions of hyaluronan (below 750,000). A patent which led to the successful use of hyaluronan in skin care demonstrated that combining a high and low molecular weight fraction of hyaluronan was required to exert a "plumping" action on stratum corneum in the nude mouse 27, presumably related to the skin penetration of the low molecular weight fraction. Specific molecular weight fractions of hyaluronan have also been patented for wound healing and intra-articular treatment 28, Ophthalmic surgery provides a good example of how different molecular weight ranges of hyaluronan have been used to customize a range of viscosurgical fluids with widely varying properties and applications 29. Viscosurgical materials for the eye have been categorized as viscohesive, viscodispersive or viscoadaptive, and surgeons continue to define how specific rheological properties are best suited to specific surgical needs. Several indications under development are based on the biological activities of hyaluronan, sometimes using low molecular weight fractions or oligosaccharides. Hyaluronan oligosaccharides have been shown to be an.piogenic 9 and to inhibit tumor growth and metastasis in the B-16 melanoma model . Therapies based on specific biological actions of hyaluronan will probably require regulation as drugs, and will present a new series of technical challenges. Indications requiring systemic administration will need to overcome the high rate and capacity for systemic hyaluronan turnover 18. Alternative routes of administering hyaluronan have demonstrated the possibility of eliciting drug-like actions. A clinical trial of subcutaneous hyaluronan demonstrated a statistically significant benefit in patients with chronic bronchitis 30. Aerosolized hyaluronan has shown benefits in animal models of lung injury 31. These efforts to develop specific hyaluronan fractions and
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new routes of administration will require very different paradigms than those used to develop viscoelastic hyaluronan devices. Covalent Derivatization: The use of covalent derivatization to customize the properties of hyaluronan has been extensively developed in the last two decades, as recently reviewed 25. A variety of chemical reagents have been used, including aldehydes, carbodiimides, vinyl sulfone, and epoxides. The properties of hyaluronan can be modified by attaching pendant groups or by crosslin king hyaluronan molecules to each other. Efforts to derivatize hyaluronan are focused on creating materials more suitable than hyaluronan itself for a diversity of indications. A major goal in derivitizing hyaluronan is to control its water solubility. Crosslinking hyaluronan decreases solubility and increases residence time because the hyaluronan no longer readily dissolves in tissue fluid 25.32. Attachment of pendant groups can decrease water solubility by reducing or eliminating the negative charge on the carboxylate group or by increasing hydrophobic character via the pendant group itself 33. In some cases the carboxylate group has been converted into an ester with a positively charged side chain 34, allowing the formation of polyanionpolycation complexes with reduced water solubility. Covalent derivatization has been very successful from the perspective of developing licensed, hyaluronan-based medical products. The major successes have been for the treatment of osteoarthritis (Synvisc'", Biomatrix Inc., USA), for soft tissue augmentation (Hylaform'", Biomatrix Inc., USA; and Restylane'", Qmed AB Sweden) and for control of post-surgical adhesions (Seprafilm", Genzyme Inc., USA). Modified hyaluronan biomaterials have been tested as a matrix for cartilage 35 and skin 36 cells, and will likely prove useful to the burgeoning field of tissue engineering. Complexes: Because of its polyanionic character, higher order structure 37 and specific binding sites 10, hyaluronan can form tight complexes with many types of molecules. The properties of these complexes depend on many factors, including the molecular weight of the hyaluronan itself, the type of complexing molecule used (e.g. polymer vs small molecule, its degree of charge and hydrophobic character, etc.), and the ratio of complex components. The widely ranging properties of the resulting materials create new opportunities for using hyaluronan. Several good examples illustrate how specific hyaluronan complexes can be used to improve hyaluronan's utility. Complexes between hyaluronan and polycations can decrease solubility and mediate binding to anionic surfaces, such as to the skin 38. An ionic complex between Fe3+ and hyaluronan has decreased water solubility and increased residence time, making the complex more suitable for reducing postHyaluronan has been shown to form complexes with surgical adhesions'", phospholipids 40 and a wide range of specific proteins 41, creating many possibilities for therapeutic intervention. Mixtures: Numerous reports have described specific mixtures of hyaluronan with other polymers, adjuvants and active agents. It is not necessary for a tight association complex to form in order to provide improved utility. The goals are generally to modify rheological properties, physical form, delivery profile or residence time. A hyaluronan mixture with chondroitin sulfate has proven useful for ophthalmic phacoemulsification procedures because of its decreased cohesiveness and improved coating properties 42. Mixtures of hyaluronan with polyethylene oxide exhibit a rheological synergy that improves functionality and cost effectiveness 43.
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Ternary polymer mixtures containing hyaluronan, dextran sulfate and albumin form pH-sensitive films, which can modify the optical properties of the skin surface 44. Hylan G-F 20 (Synvisc'") is a hyaluronan-based mixture 45 which illustrates how multiple strategies must sometimes be combined when customizing hyaluronan for a specific indication (viscosupplementation). It contains a mixture of two hyaluronan derivatives: hylan A, a water-soluble derivative of molecular weight 6 million 46 and hylan B, a water insoluble derivative in the form of a hydrated elastic gel slurry 32. The mixture behaves as a uniform pseudoplastic fluid which can be easily administered into the joint via narrow gauge needles. The crosslinking conditions used for the hylan A and hylan B components, and their ratio in the mixture, were specifically optimized to create a synovial fluid replacement with viscosity and elasticity similar to those in a normal young adult, and to extend the product's intra-articular residence beyond that of hyaluronan 47.
Drug Delivery: Hyaluronan's combination of physiochemical functionality and biocompatibility enables the design of mechanically functional delivery systems. These can be placed in sensitive tissue compartments that are generally intolerant of foreign polymers. By providing biomechanical function and a favorable microenvironment, hyaluronan based delivery systems can augment drug activity. Obvious examples include the local delivery of agents to treat arthritis, vitreoretinal disease, wound healing, and post-surgical complications. Drug delivery systems using hyaluronan and its derivatives can be based on everything from simple mixtures to covalent adducts. The first published clinical study evaluating hyaluronan for the treatment of eauine arthritis compared cortisone alone to a mixture of hyaluronan plus cortisone 4 • A mixture of hyaluronan with thrombin and a radiocontrast agent provided a new material for percutaneous embolization 49. Electrostatic complexes between hyaluronan and cationic drugs like gentamycin or pilocarpine have been shown to provide sustained ocular release in both animal and human studies 50,51. A wide range of drugs has been covalentll attached to HA, including anti-inflammatory, cytotoxic and anti-microbial agents 52,5 • Efforts to use hyaluronan and its derivatives to improve the safety and effectiveness of drugs via controlled local delivery offer significant potential for development. MOLECULAR WEIGHT AND BIOLOGICAL ACTIVITY
Molecular weight, and its relationship to biological activity, is important to consider in detail. It was suggested two decades ago that hyaluronan could behave as either a mechanical body or a biochemical signal, depending on its concentration and molecular weight 54 • Recent work confirms that hyaluronan can exert very different biological effects depending on the circumstances. Generally, these differences in biological effects can be explained based on differences in the concentration, molecular weight and rheology range evaluated. The data presented below may allow some generalizations to be drawn about this distinction between hyaluronan as a rheological body and hyaluronan as a molecular signal. Elastoviscous solutions of high molecular weight hyaluronan are generally either inert or exert a "stabilizing" effect, whereas non-elastoviscous solutions of low molecular weight hyaluronan or oligosaccharides can be extremely biologically active and trigger specific cellular responses. Detailed examples of this paradigm are provided below. These two sides of the hyaluronan story are not incompatible, but their distinction is particularly important to consider from the perspective of product development.
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Rheological Effects on Tissues and Cells: Although viscoelastic medical devices based on high molecular weight hyaluronan are generally considered "inert" in the sense that they have no known pharmacologic action, this does not mean that they are inert in the biological sense. All implants exert biological effects in terms of the cellular response around them and the altered mechanical forces they create, both of which are often important to their therapeutic success. For this reason, it is important to keep in mind some well-known principles about the effects of viscoelastic media on cell behavior. Within connective tissue, the cells exist in an environment containing concentrated hyaluronan, which has been referred to as a "crowded" macro-molecular environment 55. Many of the chemical, enzymatic, diffusional and transport processes important to tissue physiology are sensitive to the macromolecular properties of this inter- and peri-cellular environment. Lymphomyleoid cells are rarticularly sensitive to changes in the rheological properties of their environment 5 , 56. Both their rate of migration and phagocytic activity can be inhibited in proportion to the viscosity of their media. These effects are not polymer-specific, being observed whether hyaluronan, DNA or gelatin are used to increase the media's bulk viscosity. Many aspects of lymphomyeloid cell biology are affected, including lymphoblast activation 56, production of reactive oxygen metabolites'" and prostanoid metabolism 58. The cell-protecting effects of hyaluronan afainst free radical damage have also been found to increase with molecular weight 5 • Despite the diversity of these effects, the data can be broadly generalized as showing that the presence of a viscoelastic medium surrounding lymphomyeloid cells has a "quieting" effect on the cells, in that it reduces activities associated with inflammation and attenuates the response to different types of stimuli. The biological effects of elastoviscous media have been demonstrated in numerous other in vitro and in vivo systems. Proliferation rates for synovial cells and fibroblasts were found to be inhibited by high concentrations of high molecular weight hyaluronan, and either unaffected or stimulated by hyaluronan of low concentration or low molecular weight 60. Animal studies have demonstrated that high molecular weight hyaluronan can provide an optimized environment for regeneration of bone 61 and dentin 62 • Intra-articular hyaluronan injections can delay the development of arthritic lesions 63,64,65, and in those cases where molecular weight was evaluated as a variable, the highest molecular weight, most viscoelastic preparations, were most effective. Though the mechanism of these effects on tissue damage and regeneration are unclear, they are in many ways reflective of hyaluronan's pleitropic effects during morphogenesis'". The effects of viscoelastic fluids on pain are particularly important when considering the use of hyaluronan to treat arthritis. Early studies of analgesia in animals demonstrated that hyaluronan could affect pain-driven behavior, such as limping in dogs and racing in horses 67. More recent animal studies have found that these local analgesic actions of hyaluronan depend on concentration and molecular weight in a way that demonstrates their relationship to rheological properties 68. Electrophysiologic studies have confirmed that the firing rate of nociceptors in an inflamed joint is sensitive to the viscoelasticity of fluids injected into the joint 69. This effect of hyaluronan on nociceptor sensitivity has now been reproduced in a second species 70, and the newly found influence of viscoelastic media on calcium channels 71 may provide a general mechanism for these phenomena. Biological Actions of Low Molecular Weight Hyaluronan: Although the biological actions of hyaluronan described above were all related to the use of high
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molecular weight viscoelastic preparations, an entirely different group of biological activities are only observed when low molecular weight hyaluronan is used. The first demonstration that significant biological activities could be generated by degrading hyaluronan was a series of experiments reported more than 50 years ago by Endre Balazs. The proliferation of embryonic fibroblasts was stimulated by hyaluronidase-degraded hyaluronan, but unaffected by the same non-digested preparation". Similar observations have now been reported in many different systems. During angiogenesis, concentrated solutions of high molecular weight hyaluronan are inhibitory 73, whereas degraded hyaluronan and oligosaccharides stimulate the proliferation of endotherial cells in vitro 74.75 and vasculogenesis in vivo 76. The chondrogenic differentiation of mesenchymal cells is likewise specifically stimulated by hyaluronan in the 200,000-400,000 molecular weight range, while unaffected by the identical hyaluronan preparation before degradation 77. Thus during both angiogenesis and chondrogenesis the molecular size of hyaluronan can affect the differentiation pathway expressed by the cells. The effects of hyaluronan on gene expression in macrophages 78 and epithelial cells 79 have likewise been shown to be highly dependent on molecular weight, with activation of gene expression only becoming evident when the hyaluronan is degraded to a molecular weight below about 0.5 million. These latter systems have been shown to include the control of the nuclear transcription factors NF-KB and AP-I, both involved in the control of inflammation. This relationship between the effects of hyaluronan on gene expression and its molecular weight is particularly interesting in light of recent evidence for an intra-cellular hyaluronan pathway 80, and in light of studies showing that cellular internalization of h{'aluronan is maximal for molecules in the molecular weight range ofO.I-O.S million 8. Numerous studies have identified cell-associated hyaluronan binding proteins (hyaladherins) which can mediate important biological activities of hyaluronan 10.41. 82. Although cell surface hyaladherins are commonly referred to as "hyaluronan receptors", it has been suggested that because of the crowded macromolecular environment around cells, there is a biomechanical aspect associated with hyaluronancell interactions that should always be considered 83. Because of the high molecular weight and polyvalent character of hyaluronan as a ligand, and its high concentration in tissue, hyaluronan receptors are probably sensitive to more than just the presence or absence of their ligand. Receptors may sense the state of the hyaluronan matrix surrounding the cell, and transmit this message to ion channels, mechanoreceptors and the cytoskeleton. A viscoelastic matrix of concentrated high molecular weight hyaluronan would transmit a different signal than a matrix whose viscoelasticity has been reduced by dilution (edema) or degradation of the hyaluronan.
Hyaluronan and Inflammation: The differences described above for the biological effects of high molecular weight hyaluronan at high concentration compared to low molecular weight or diluted hyaluronan solutions, may in some ways relate to the differences in the molecular state of hyaluronan in normal, inflamed and regenerating tissues. Inflammation can degrade hyaluronan via reactive oxygen metabolites 84.85 and can dilute the interstitium by increasing fluid flux from the microvasculature 86. Despite the degradation and dilution, inflamed tissues tend to contain increased levels of hyaluronan, probabli' due to the stimulation of hyaluronan synthesis by inflammatory cytokines 8 • This accumulation of hyaluronan in pathologic tissues may contribute to their clinical impairment, and its concentration in serum may serve as a useful clinical marker 88.89. It has been suggested that hyaluronan plays a dynamic role
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in connective tissue activation, and that increased hyaluronan turnover is a common component of inflammatory processes in a wide range of tissues and experimental systems 90. This altered state of the interstitium may be important for proliferative 91, morphologic 66 and reparative 92 responses. CONCLUDING REMARKS Looking back at the medical development of hyaluronan over the past three decades, it is clear that most of the paradigms that led to successful new therapies can be traced to Endre Balazs. His remarkable dedication and energy are known to all whom have worked with him. But more than anything, it is his vision from which we have all benefited. In the words of Albert Szent Gyorgyi: "Discovery consists of seeing what everybody has seen and thinking what nobody has thought." REFERENCES I.
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B. P. Toole, Hyaluronan in morphogenesis, J. Intern. Med., 1997, 242,35-40. N. Rydell & E.A. Balazs, Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of osteoarthritis and on granulation tissue formation, CUn. Orthop.• 1971, 80,25-32. S. Gotoh, J.1. Onaya, M. Abe, K. Miyazaki, A. Hamai, K.Horie, K. & K. Tokuyasu, Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats, Ann. Rheum. Dis., 1993,52,817-822. M. A. Pozo, E. A. Balazs & C. Belmonte, Reduction of sensory responses to passive movements of inflamed knee joints by hylan, a hyaluronan derivative, Exp. Brain Res.. 1997, 116,3-9.20. R.F. Schmidt, M. Pawlak, S. Just, B. Heppelmann, A. Gomez & C. Belmonte, Mechanoprotective actions of elastoviscous hylans on articular pain receptors, In: Hyaluronan 2000, J.F. Kennedy, G.O. Phillips, P.A. Williams & V.C. Hascall (eds.), Woodhead Publishing, Cambridge, UK, 2001 (in press). C. Belmonte, Effects of elastsoviscous hylans on mechanotransduction, In: Hyaluronan 2000, J.F. Kennedy, G.O. Phillips, P.A. Williams & V.C. Hascall (eds.), Woodhead Publishing, Cambridge, UK, 2001 (in press). E. A. Balazs, The influence of extracellular macromolecular polysaccharides on the development and growth of fibroblast cultures, In: Abstracts of the Vl Interruuionai Congress ofExperimental Cytology, Stockholm 1947. R. N. Feinberg & D.C. Beebe, Hyaluronate in vasculogenesis, Science, 1983, 220, 1177-1179. D. C. & S. Kumar, The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity, Exp. Cell Res., 1989, 183, 179-196. R. Deed, P. Rooney, P. Kumar, J. D. Norton, 1. Smith, A. J. Freemont & S. Kumar, Early-response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by non-angiogenic, high-molecularweight hyaluronan, Int. J. Cancer, 1997,71,251-256. V. Lees, T. Fan, T. & D. West, Angiogenesis in a delayed revascularization model is accelerated by angiogenic oligosaccharides of hyaluronan, Lab. lnvest., 1995, 73, 259-266. M. J. Kujawa, D. A. Carrino & A. I. Caplan, Substrate-bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture, Dev. Biol.• 1996, 114, 519-528. J. Hodge-Dufour, P. W. Noble, M. R. Horton, C. Bao, M. Wysoka, M. D. Burdick, R. M. Strieter, G. Trinchieri, E. Pure, Induction of IL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages, J. Immunol., 1997, 159,2492-2500. B. Oertli, B. Beck-Schimmer, X. Fan & R. P. WUthrich, Mechanisms of hyaluronan-induced up-regulation of ICAM-l and VCAM-l expression by murine kidney tubular epithelial cells: Hyaluronan triggers cell adhesion molecule expression through a mechanism involving activation for nuclear factor-kB and activating protein-I, J. Immunol., 1998, 161, 3431-3437. S.E. Evanko & T.N. Wight, The presence and processing of intracellular hyaluronan in proliferating cells, In: Hyaluronan 2000, J.F. Kennedy, G.O. Phillips, P.A. Williams & V.c. Hascall (eds.), Woodhead Publishing, Cambridge, UK, 2001 (in press). P. G. McGuire, J. J. Castellot Jr. & R. W. Orkin, Size-dependent hyaluronate degradation by cultured cells, J. Cell. Physiol., 1987, 133,267-276. J. Entwistle, C. L. Hall & E. A. Turley, HA receptors: Regulators of signalling to the cytoskeleton, J. Cell Biochem., 1996,61,569-577.
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THE QUESTION OF IMMUNOGENICITY OF HYLANS Nancy E. Larsen', Diane F. Mess\ Janet L. Denlmger/Biomatrix, lnc., 65 Railroad Avenue. Ridgefield. New Jersey 07657 USA Biology lnstitute., 65 Railroad Avenue, Ridgefield. New Jersey 07657 USA
2 Matrix
ABSTRACT In primates (Aotus species), chronic and repeated exposure to HA (hyaluronan), hylan A, and hylan B did not result in the formation of detectable antibody titer, nor did re-challenge with the HA or hylan test article elicit a humoral (antibody) or cellular (skin test) response. Nonclinical studies in primates (multiple intra-articular injections) demonstrated an absence of humoral or cellular immunity to hylan. In multiple, intra-articular-injection studies in rabbits the results were similar, with an absence of humoral or cellular immune-related events. Clinical studies reveal the presence of serum titers to avian protein and to endotoxin in control serum (from patients who had never been injected with hylan) and in serum from hylan-treated patients. In some cases, serum cross-reacted with hylan in the ELISA, producing a titer. There was no correlation between serum titer and clinical symptoms. The results are consistent with the nonimmunogenicity of the pure HA molecule, and the lack of meaningful immunogenicity associated with the presence of low concentrations of protein and endotoxin. INTRODUCTION Hyaluronans (HA) and hylans (HA derivatives) are used with increasing frequency in a variety of medical applications including ophthalmology, orthopedics, and dermatology". It is well established that the purified HA molecule is nonimmunogenic due to its conserved chemical structure, lack of species- and tissuespecificity, and nonforeignness 6.7. All hyaluronan preparations contain detectable and reproducible levels of protein and endotoxin, whether derived from avian comb tissue or from bacterial fermentation. Proteins and endotoxin in avian or bacterial HA products may theoretically behave as antigens, and therefore their presence must be considered in the assessment of the potential for immunogenic response to HA administration.
MATERIALS & METHODS Primate Studies Owl monkeys (Aotus species, approximately 1 kg body weight, 2-12 years of age, n=50) with previous multiple exposure to hyaluronan and hylans (hylan A and hylan B) via intra-vitreal, intra-articular, and/or intradermal injections were used in these studies. Forty-two (42) primates comprised the test group; eight (8) primates comprised the control, unexposed group. Serum was obtained at baseline and at 4 weeks after intradermal challenge with HA or hylan test article. Serum was assayed by ELISA for the presence of antibody titer. Cellular immunity was assessed at
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baseline and at 4 weeks post-challenge. In a second study, primates (n=20) were injected intra-articularly each week for an average of 6 months, via bilateral injections, with hylan G-F 20, a mixture of hylan A and hylan B. Serum was obtained at baseline (prior to treatment) and at monthly time points for 6 months, and assayed for the presence of antibody titer (ELISA). Cellular immunity was assessed at monthly time points via intradermal challenge with HA and/or hylan test article.
Clinical Studies Fifty-five human subjects with osteoarthritis of the knee joint were injected intra-articularly with Synvisc® (hylan G-F 20); up to 4 courses of Synvisc treatment were administered to each joint (up to 12 injections per joint) over a period of one year. Blood was taken from each subject before administration of Synvisc and at the end of the study. Serum was prepared from each blood sample and analyzed for the presence of antibodies using an ELISA procedure. Pre-injection serum was used as a negative control for the post-injection response. A 'normal range' of ELISA response was determined using ELISA data from pre-injection sera. The normal variance in ELISA result was used to identify serum samples with higher than normal responses.
RESULTS Primate Studies ELISA analysis - Immunogenicity study: Absorbance values of control sera (l: 16 dilution) were compared to absorbance values of test sera (l: 16 dilution) at
baseline and at 4 weeks. There was no statistically significant increase in the serum absorbance values (ELISA) from test primates as compared to control, untreated primates (Fig I). Primates that had been previously injected with hyaluronan, hylan A or hylan B did not have higher absorbance values at baseline or at 4 weeks postintradermal challenge with the HA or hylan test article, as compared to the control, unexposed primate group (Fig 2). Intra-articular study: Serum from 16 of 20 treated primates showed no increase in absorbance in the ELISA, while 4 primate sera showed mild increase in titer. Serum from these primates also cross-reacted strongly with endotoxin lipopolysaccharide (LPS) and/or avian protein. Skin challenge: Intradermal challenge with HA or hylan test articles produced negative results in all primates in all groups, at all time points.
Immunogenicity ofhylans
Figure 1 Evaluation of Humoral Immune Response to Hylans in Primates
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Clinical applications ofhyaluronan
Clinical Studies In pre-injection serum from unexposed subjects, the ELISA response to Synvisc ranged from absorbance of 0.039 to 0.201, corresponding to titers of < 8 to 512. In 54 of 55 patients, the post-injection titers were within the normal range. One patient had a postinjection serum response which was outside (higher than) the normal range. Pre-injection and post-injection serum from this patient produced a positive reaction toward avian protein in the ELISA test (elevated absorbance, elevated titer).One adverse event (swelling and pain) occurred during the second course of treatment with Synvisc (5th injection). This patient received 4 additional injections for a total of 9 of Synvisc in the affected joint without further local reaction.
CONCLUSIONS In the primate model, there was no increase in the nature or magnitude of the cellular (skin test) or humoral (antibody test) response following dermal challenge with hyaluronan or hylan test articles, indicating that a cellular or humoral immune response is not associated with repeated administration of hyaluronan or hylan preparations. The presence of preexisting titers to avian protein is associated with cross-reactivity to hylan in the ELISA. In intra-articular studies in the primate model, chronic injection of Synvisc (weekly for 6 months) is not associated with detectable local or systemic (immune) effects. Clinical study data indicate that pre-existing serum titer to avian protein and/or LPS may produce crossreactivity with hylan in the ELISA and that serum reactivity is rare and not causally related to adverse clinical response. This study confirms previous reports that hylan A and hylan B elicit immunological response in animals 8.
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6. 7. 8.
E.A. Balazs. Viscosurgery in the eye. Ocular Inflammation Ther., 1983, 1,91-92. E.A. Balazs. Viscosurgery. Transplantation/Implantation Today 1985, 2, 62-64. E.A. Balazs, E.A. Leshchiner, N.E. Larsen and P. Band. Applications of hyaluronan and its derivatives. In Biotechnological Polymers (Ed. Gebelein, C. G.), Technomic Publishing Co., Inc., Basel, 1993,41-65. K.W. Marshall. The current status of hylan therapy for the treatment of osteoarthritis. Today's Therapeut. Trends, 1997, 15,99-108. D.l. Piacquadio, N.E. Larsen, l.L Denlinger and E.A. Balazs. Hylan B gel (Hylaform) as a soft tissue augmentation material. In Tissue Augmentation in Clinical Practice: Procedures and Techniques (Ed. Klein, A. W.), Marcel Dekker, Inc., New York, 1998,269-291. W. Richter. Non-immunogenicity of purified hyaluronic acid preparations tested by passive cutaneous anaphylaxis. Int. Arch. Allergy Appl. ImmunoI., 1974,47,211-217. W. Richter, E. Rydel and E.O. Zetterstrom, Non- immunogenicity of purified sodium hyaluronate preparations in man. Int. Arch. Allergy Appl. Immunol. 1979,59,45-48. N.E. Larsen, C.T Pollak, K. Reiner, E. Leshchiner and E.A.Balazs. Hylan gel biomaterial: dermal and immunologic compatibility. J. Biomed. Mater. Res. 1993, 27, 1129-1134.
MECHANICAL INJURY OF HUMAN PERITONEAL MESOTHELIAL CELLS (HPMC) IS ACCOMPANIED BY AN INCREASE IN HYALURONAN SYNTHESIS. Susan Yung 1., Gareth J. Thomas' & Malcolm Davies' I
Department ofMedicine. Queen Mary Hospital. The University of Hong Kong. Pokfulam Road. Hong Kong, China. 'Department ofMedicine. University of Wales College ofMedicine. Heath Park. Cardiff, United Kingdom.
ABSTRACT Hyaluronan (HA) has been implicated in tissue repair but its role during injury to the peritoneum is poorly understood. To delineate its possible role we have investigated several aspects of HA metabolism in an in vitro model of peritoneal injury. Confluent, growth arrested HPMC were wounded and the synthesis of HA during the repair process determined by [3H]-glucosamine labelling. After an initial lag period of 12 h de novo synthesis of HA was up-regulated I.75±O.15-fold (n=3) above controls and remained so until the re-establishment of the monolayer. Cytochemical staining for UDP-glucose dehydrogenase was localised to the cells at the margin of the wound and also to those cells migrating into the wound. Using RTPCR, mRNA for HAS II was not detected in non-injured cells, but on wounding the monolayer, mRNA for HAS II was induced, maximal 12-24h after injury and remained elevated for up to 144h. In contrast mRNA for HAS III was constitutive before injury but its expression was reduced during the restitution of the wound. These findings suggest that HAS II and HAS III are differentially regulated in response to mesothelial damage. In a separate study, wounded cells were co-cultured with endotoxin-free HA (Mr 250,000 Da). The inclusion of HA (range 0-3.3 ug/ml) increased the rate of cell migration in a dose-dependent manner. This response to HA was abrogated by prior digestion of the HA to disaccharides with Streptomyces hyaluronidase. These findings suggest that HA plays a critical role in the response of the mesothelium to injury. KEYWORDS Hyaluronan, wound healing, hyaluronan synthase, uridine diphosphoglucose dehydrogenase INTRODUCTION Hyaluronan (HA) is a linear non-sulphated glycosarninoglycan synthesized by mesenchyma cells and is composed of the repeating disaccharide units N-acetyl-Dglucosamine and D-glucuronic acid [I]. HA synthesis occurs at the plasma membrane and involves the enzymes uridine diphosphoglucose dehydrogenase
482
The action ofhyaluronan in cells
(UDPGD) and hyaluronan synthase (HAS) I, II and III [2, 3J. UDPGD converts UDP-glucose to UDP-glucuronate which is subsequently transferred to nascent HA by HAS. HA is predominantly found in connective tissue where it can bind to cell surface receptors, for example CD44, the receptor for hyaluronic acid mediated motility (RHAMM) and to the proteoglycans aggrecan and versican [4, 5J. Despite its simple structure HA is involved in several physico-chemical and biological functions. Due to the presence of charged residues, it can accommodate many water molecules and thus may act as a space filler, lubricant and a hydrated matrix through which cells can migrate. HA has also been observed to play a pivotal role in cell migration, proliferation, embryogenesis, inflammation and injury [6-8J. Continuous ambulatory peritoneal dialysis (CAPD) is now commonly used as a treatment for end-stage renal failure. One consequence of CAPD is chronic inflammation of the peritoneum which leads to the denudation of the mesothelium, exposure of the interstitium to PD fluids and thus contributing to sub-mesothelial fibrosis [9J. Recent studies have demonstrated that HA plays a pivotal role in the response of the peritoneal membrane to chemical or bacterial injury during CAPD. Previously, we reported that HA levels are increased in the spent peritoneal fluids of patients under-going CAPD and that these levels are further increased during peritonitis and inflammation [10]. Furthermore, successful treatment of peritonitis results in the HA levels returning back to the resting levels. We have also reported that during inflammation of the peritoneum, elevated levels of HA is attributed in part by the increased synthesis of interleukin-Ibeta [IlJ. How HA is involved in the regeneration of the mesothelium remains to be determined. In this paper, we use an established in vitro model of human peritoneal mesothelial cells to evaluate the involvement of HA in peritoneal injury and healing. We report that cell damage results in the differential regulation of HAS II, an increased synthesis of HA during the initiation of mesothelial regeneration and that the addition of exogenous HA enhances the rate of monolayer regeneration. MATERIALS AND METHODS
Materials All chemicals and reagents were of the highest purity commercially available. Sodium hyaluronate (molecular weight 2.5 x 10') was a kind gift from Dr Ove Wik (Pharmacia Opthalmics Uppsala, Sweden) and was free of endotoxin and growth factors. Biotinylated hyaluronate-binding region protein (HABR) was a kind gift from Professor Michael Bayliss, Royal Veterinary School, London, U.K.
Cell culture Human peritoneal mesothelial cells (HPMC) were obtained from omental specimens using established methods [12]. De novo synthesis of hyaluronan
Prior to radiolabeling growth arrested HPMC were washed once with PBS and the monolayer wounded by multi-scratching using a sterile glass rod as previously described [13]. Two methods were used to follow the synthesis of ['H]-radiolabelled
Human peritoneal mesothelial cells
483
HA. In the first, cells were incubated with serum free medium containing ['H]glucosamine (25 J.lCilml) for varying time periods (0-144 h). At selected time points the culture medium (CM) was decanted and the cells released from the culture flasks by incubation with 0.001 %(w/v) EDT A and 0.125% (w/v) trypsin for 5 min at 37°C. In the second method cells were pulsed labelled for selective time periods and the cells released as above. In both approaches the detached cells were added to the CM and incubated with papain (3.5 U/ml) in the absence of EDTA and cysteine at 65°C for 24 h [10]. Aliquots of HA samples were quantified by high performance liquid chromatography, and their hydrodynamic size analysed under dissociative conditions on Sephacryl S-Iooo. To confirm the presence of HA, samples were incubated with 10 mU Streptomyces hyaluronidase [10]. Assessment of uridine diphosphoglucose dehydrogenase (UDPGD) activity HPMC grown in 35 mm dishes were wounded as described above and UDPGD activity of both control and wounded cells assessed according to the method of Mehdizadeh et at [14] until the re-establishment of the monolayer. Histochemical staining of free HA Wounded and control HPMC were cultured on glass coverslips and at selective time points, briefly washed with PBS and fixed with cold acetone for 2 min. Cells were incubated with HABR according to the method of Pitsillides et at [15]. RNA extraction and RT·PCR for HAS I, HAS II and HAS III Total RNA from control or wounded cells were extracted at selective time periods using RNA isolator (Genosys, Cambridge, UK) according to the manufacturers instructions. Equal amounts of total RNA (lug) from each sample were initially reversed transcribed into cDNA using Superscript RNase H" reverse transcriptase and random hexamers (hexadeoxyribonucleotides, pd[N]6' looJ,lM Pharmacia). Equal volumes (2 ul) of RT product were then subjected to PCR amplification. To confirm that equal amounts of cDNA was used and semi-quantify the PCR products, a-actin was used as the 'house-keeping' gene (25 cycles). The sequences of primers were designed as follows: Primer a-actin (sense) a-actin (antisense)
Sequence 5' -GGAGCAATGATCTTGATCTT-3' 5' -TCCTGAGGTACGGGTCCTTCC-3'
Size 204 bp
HAS I (sense) HAS I (antisense)
5' -AGCAGGACGCGCCCAAGCCCACTC-3' 5'-CTTCTTATTCTTGTTCTTTAGCGA-3'
562bp
HAS II (sense) HAS II (antisense)
5' -TCCCGGTGAGACAGATGAGT-3' 5' -ACCCGGTTCGTGAGATGC-3'
495bp
HAS III (sense) HAS III (antisense)
5' -AGTGCAGCTTCGGGGATGA-3' 5'-TGATGGTAGCAATGGCAAAGAT-3,
453 bp
484
The action of hyaluronan in cells
After amplification, 10 J.I1 of each PCR reaction mix was electrophoresed on a flatbed agarose gel (3% w/v) in IxTris-acetate-EDTA buffer containing ethidium bromide (0.5J.1g/ml) at 70 V for 2.5 h. Photographs of the gels were subsequently taken and the density of the bands obtained evaluated using a BioRad densitometer of the negatives and expressed as the ratio of specific primer to a-actin. Addition of exogenous HA to wounded HPMC HPMC were grown to confluence in 35 mm dishes, growth arrested for 72 h and wounded as described above. Cells were cultured with (a) serum free medium alone, (b) HA (50 - 3300 ng/ml) (c) HA (3300 ng/ml) predigested with Streptomyces hyaluronidase (10 mU) and the mixture boiled for 5 min and (d) boiled enzyme alone. 10% FCS was used as the positive control. The closure of the denuded area was monitored using an Axiovert 135 inverted microscope heated with an M heating stage. Data was analyzed using the software BioVisions (Improvisions Ltd, UK). Determination of Cell Growth and Viability Growth rates were determined using the MIT assay [16] and cell toxicity assessed by measuring lactate dehydrogenase released into cell-conditioned medium as previously described for mesangial cells by us [17]. RESULTS AND DISCUSSIONS In a previous study we demonstrated that when a mechanical wound was made in confluent quiescent HPMC monolayers, the cells at the wound edge migrated towards the centre of the wound with re-establishment of the monolayer achieved within 72 h [13]. Since a HA-rich matrix is known to have a profound effect on cellular movement, we determined whether HA was involved in the migratory behaviour of an injured monolayer of HPMC. To achieve this we determined mRNA expression of HAS I, HAS II and HAS ill using semi-quantitative RT-peR. The data demonstrated that when normalized to a-actin, there was an increase in the mRNA expression of HAS II 6h after wounding and that this was maximal between 12-24 h (I5-fold increase over control, see Figure 1). Thereafter its expression declined and by 48 h mRNA for HAS II returned to a level only slightly higher than that expressed in control quiescent cells. In comparison, whilst mRNA for HAS ill was constitutive in normal cells, a slight decline in its levels was observed 6 h after injury (Figure I). Maximal reduction of HAS ill was observed after 24 h (6-fold reduction over control cells). The level of mRNA for HAS ill returned to resting levels by 96 h. These findings were consistent in three different experiments with three different cell lines. Expression of mRNA for HAS I was not detected in either control or wounded cultures of HPMC (up to 40 cycles). The same primers for HAS I, however, successfully demonstrated HAS I gene products from RNA derived from human lymphocytes (data not shown). Throughout the experiments, we determined whether mechanical wounding was harmful to the cells. An aliquot of each supernatant during the course of the experiment was analysed for lactate dehydrogenase release. We demonstrated no change in lactate dehydrogenase release during control and wounded cells.
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485
Densitometric ratios of (a) HAS Il/o-actin and (b) HAS Ill/a-actin after RT-PCR of total RNA extracted from non-injured (0) and injured (II) cells (n= 3).
Temporal synthesis of HA in response to injury in cultured HPMC No change was observed in the de novo synthesis of HA in control and wounded cells during the initial 12 h, but thereafter, eH]-HA synthesis from wounded monolayers increased, with maximum synthesis observed between 18 to 24 h (Figure 2). 120
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486
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Gel filtration chromatography of [3H]-labeled HA purified from control and wounded monolayers showed no difference in their hydrodynamic size (Figure 3).
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HA is synthesized at the leading edge of the wound Localization of HA synthesis in wounded monolayers was achieved by UDPGD cytochemical staining. At the time of wounding (t=O) reaction product for UDPGD was evident throughout the culture, but after 24 h strong reaction product was observed at the leading edge and in cells entering the wound. This enhanced activity for UDPGD was also evident at 72 h in the area of contact between the two advancing edges. When probed with HABR a similar pattern was observed for the location of HA. This pattern of staining was not observed in cultures pre-incubated with Streptomyces hyaluronidase (data not shown). HPMC wound closure is enhanced in the presence of HA The concentration of HA has been shown to be significantly increased in spent effluents collected from CAPD patients during episodes of peritonitis suggesting a possible role for this glycosaminoglycan in the response to peritoneal inflammation and injury [10]. To investigate this we examined the effect of HA on the rate of cell migration in wounded cultures of confluent HPMC. Exogenous HA at doses in the range detected in the peritoneum of infected and non-infected fluid (50-3300 ng/ml) were added to the cells and the rate of cell migration monitored until re-establishment of the monolayer. We observed an increase in the rate of HPMC migrating into the wound in a dose-dependent manner (Figure 4). HA disaccharides obtained from digestion of HA with Streptomyces hyaluronidase did not increase migration of HPMC nor did the enzyme alone.
Human peritoneal mesothelial cells
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CONCLUSIONS In this study we have demonstrated that restitution of the peritoneal mesothelial monolayer is accompanied by an increase in HA synthesis. The increase in HA synthesis was localized to cells at the leading edge of the wound in addition to cells migrating into the denuded area. mRNA for HAS II was demonstrated to be involved during the recovery process whilst mRNA for HAS ill was only demonstrated to be present when the monolayer had fully recovered.
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Effect of exogenous HA on migration rate of HPMC after injury. Endotoxin- free HA (0 - 3.3 ug/ml), HA pre-digested with Streptomyces hyaluronidase and denatured hyaluronidase were added to wounded cultures of HPMC. The rate of migration of the cells were calculated according to the Materials and Methods. 10% FCS was also included as a positive control. Results are expressed as mean ± SO (n 3).
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ACKNOWLEDGMENTS Supported by a grant (044838/ZJ95/Z) from The Wellcome Foundation and the Kidney Research Foundation for Wales. All figures with kind permission from Kidney International.
REFERENCES I. T. C. Laurent, Structure of hyaluronic acid. In Chemistry and Molecular Biology
ofthe Intercellular Matrix. Balazs EA (ed) Academic, London pp 703-732. 2. P. Prehm. Identification and regulation of the eucaryotic hyaluronate synthase. In The Biology of Hyaluronan, Ciba Foundation Symposium 143, Wiley, Chichester, England, pp 21-40.
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The action of hyaluronan in cells
3. A. P. Spicer, L.A. Kaback, T. J. Smith, M. F. Seldin, Molecular cloning and characterization of the human and mouse UDP-glucose dehydrogenase genes. J Bioi Chem., 1998,273: 25117-25124. 4. B. P. Toole, Hyaluronan and its binding proteins, the hyaladherins. Curro Opin. Cell. Bioi.. 1990, 2, 839-844. 5. L Sherman, J Sleeman, P Herrlich, H Ponta, Hyaluronate receptors: key players in growth, differentiation, migration an tumor progression. Curro Opin. Cell Biol., 1994,6: 726-733. 6. T. C. Laurent, J. R. E. Fraser, The properties and turnover of hyaluronan. In Functions of Proteoglycans, Ciba Foundation Symposium 124, Wiley, Chichester, England, pp 9-24. 7. C. B. Underhill, H. A. Nguyyen, M. Shizari, M. Cutly, CD44 positive macrophages take up hyaluronan during lung development. Dev. Biol., 1993, 155,324-336. 8. O. Oksala, T. Salo, R. Tammi, L. Hakkinen, M. Jalkenen, P. loki, H. Larjava, Expression of proteoglycans and hyaluronan during wound healing. 1. Histochem. Cytochem., 1995,43,125-135. 9. J. W. Dobbie, Long-term effects of peritoneal dialysis on peritoneal morphology. Perit. Dial. Int., 1994, 14 [Suppl. 3],916-920. 10. S. Yung, G. A. Coles, J. D. Williams, M. Davies, The source and possible significance of hyaluronan in the peritoneal cavity. Kidney Int., 1994, 46, 527533. 11. S. Yung, G. A. Coles, M. Davies, IL-l beta, a major stimulator of hyaluronan synthesis in vitro of human peritoneal mesothelial cells: relevance to peritonitis in CAPD. Kidney Int .. 1996,50, 1337-1343. 12. E. Stylianou, L. A. Jenner, M. Davies, G. A. Coles, 1. D. Williams, Isolation, culture and characterization of human peritoneal mesothelial cells. Kidney Int., 1990,37,1563-1570. 13. S. Yung, M. Davies, Response of the human peritoneal mesothelial cell to injury: an in vitro model of peritoneal wound healing. Kidney Int., 1998,54,2160-2169. 14. S. Mehdizadeh, L. Bitensky, J. Chayen, The assay uridine diphosphoglucose dehydrogenase activity. Cell Biochemistry Function., 1991,9, 103-110. 15. Pitsillides AA, Archer CW, Prehm P, Bayliss MT, Edwards JC, Alterations in hyaluronan synthesis during developing joint cavitation. J Histochem Cytochem., 1995; 43: 263-273. 16. T. Mossmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. 1. Immunol. Methods., 1983, 65,55-63. 17. D. C. Wheeler, R. Chana, N. Topley, M. M. Petersen, M. Davies, 1. D. Williams, Oxidation of low density lipoprotein by mesangial cells may promote glomerular damage. Kidney Int., 1994,45, 1628-1636.
APOPTOSIS AND HYALURONAN-ENRICHED EXTRACELLULAR MATRIX DEGRADATION IN CUMULUS CELL-OOCYTE COMPLEX: IMPLICATION IN FERTILITY Monica Di Giacomo, AntoDella CamaioDi & AntoDietta Salustri" Histology and Embryology Section, Departmeru ofPublic Health andCell Biology, University ofRome Tor Vergata, via Orazio Raimondo 8.00173 Rome. Italy
ABSTRACT Oocyte fertilization rate progressively decreases with the increase of the interval time from the ovulation. This phenomenon follows and parallels the gradual dispersion of the cumulus expanded mass surrounding the ovulated oocytes. Mouse COCs, expanded in vitro with FSH in the presence of serum, undergo to dissociation similarly to in vivo gonadotropin-stimulated COCs. Metabolic labelling of the newly synthesized hyaluronan in this culture condition has allowed determining that this process depends on matrix disassembly and hyaluronan release. In the present paper we provide evidence that an increasing number of cumulus cells undergo to apoptosis after expansion and that hyaluronan release from the matrix closely correlates with this process. In addition, we show that when apoptosis is experimentally prevented, matrix is preserved and the fertilizability ofthe enclosed oocyte is prolonged. KEYWORDS Hyaluronan, apoptosis, fertilization, cumulus cell-oocyte complex. INTRODUCTION Following an endogenous LH-FSH surge or administration of an ovulatory dose of hCG, mural granulosa cells increase the synthesis of proteolytic enzymes and promote the formation of a rupture site in the follicle wall. Conversely, cells investing the oocyte, namely cumulus cells, synthesize an abundant muco-elastic extracellular matrix that produces a 20-40 fold increase in the volume of the cumulus cell-oocyte complex (COC), a process termed cumulus expansion or mucification. At ovulation, the expanded cae leaves the follicle through the ruptured wall, while mural granulosa cells remain in the follicle forming the corpus luteum. An essential component of the expanded cumulus matrix is hyaluronan (HA) (I). Combined action of gonadotropins and an oocyte soluble factor promotes the increase of the steady state level of HA synthase 2 mRNA and the synthesis of HA with more than 2 million Dalton molecular weight (2-5). Electron microscopy analysis shows that the long HA strands are organized into twisted fibrils that form a homogeneous mesh-like network anchored to cumulus cell surface and penetrating into the oocyte zona pellucida (6). Proteins are essential to organize and retain HA in such a highly structured matrix since protease treatment causes dissociation of the fibrils into individual hyaluronan filaments and dispersion of cumulus cell mass into single cells (7). In vitro and in vivo studies have identified the serum protein inter-atrypsin inhibitor (Iul) as an essential element in HA retention and cumulus expansion (8).
490
The action of hyaluronan in cells
However, it is not clear yet how this protein acts to promote such effect (9). Involvement of highly specific HA binding proteins synthesized by cumulus cells has been advanced (10). Indeed, an increase of the steady state level of TSG-6 mRNA occurs in COCs undergoing expansion (10). Noteworthy, the protein encoded by this mRNA has the property to bind both hyaluronan and IaI. Cumulus cells also synthesize the protein CD44 (11), a HA cell surfuce receptor implicated in the organization of pericellular coat in several cell types. When cumulus expansion is totally or partially inhibited in mice, a significantly lower number of COCs are ovulated and only a few of them can be fertilized (12-14). Therefore, cumulus expansion has an important role in the process of fertilization. Fertilization rate of the oocytes progressively declines with the increase of time from the ovulation and nearly falls to zero over 16 h of their permanence in the oviduct (for review see 15-17, 18). This decline follows and parallels the gradual loosening of the matrix and reduction in size of the cumulus mass. When COCs were examined at the electron microscopy during this period of time, some of the cumulus cells showed signs of degeneration (19). Mouse COCs, expanded in vitro with FSH in the presence of serum, undergo to reduction in size similarly to in vivo gonadotropin stimulated COCs. Metabolic labelling of the newly synthesized HA has allowed determining that this phenomenon depends on matrix disassemblyand HA release (20). In the present work we have studied the relationship between cell degeneration and hyaluronan matrix disassembly and whether oocyte fertilizability could be prolonged inhibitingthese phenomena. MATERIALS AND METHODS Isolation and culture of COCs
Mature 8-1O-week-old female Swiss CD-I mice were injected with 5 IV of pregnant mare serum gonadotropin (PMSG) in 0.1 ml of physiological saline. After 46-48 h, the animals were either sacrificed by cervical dislocation or injected with 5 IV of human chorionic gonadotropin (hCG) to induce ovulation. Ovaries were collected 46-48 after PMSG injection into the animals and transferred to Hepes buffered MEM containing I mg/ml BSA. Compact COCs were released into the medium by puncturing large antral follicles and collected by micropipette. For each culture 20 COCs were transferred to a 20 ul drop of culture medium MEM supplemented with 0.3 mM pyruvate, 50 ug/ml gentamicin, 3 mM L-glutamine, 5% FBS and 20 ng/ml FSH or 1 mM dbcAMP to induce cumulus expansion. The cultures were incubated at 37°C in a humidified atmosphere of 5% C02 in air for the times indicated in the text. For hyaluronan synthesis and organization studies, glycosaminoglycan labellingprecursors were added at the beginning of culture. For morphological studies ofCOCs expanded in vivo, oviducts were collected at 14 h from hCG iniection (ovulation time) and at following times as indicated in the text. COCs were released by puncturing the oviductal ampulla in MEM containing 1 mg/ml BSA. Qualitative and quantitative analysis of apoptosis
COCs were fixed for 30 min at 24°C with 4% paraformaldehyde in PBS (pH 7.4). Then, they were washed intensively with PBS, and treated for terminal deoxynucleotidyl
Extracellular matrix degradation
491
transferase-mediated dUTP nick end labelling (TUNEL) to reveal DNA breaks. Cells were permeabilized for 2 min with Triton X-I OO/PBS. After washes in PBS, COCs were incubated for 60 min at 37°C with terminal deoxynucleotyl transferase (TdT) enzyme in the presence of fluorescein isothiocyanate-corjugated dUTP (TUNEL reaction mixture). Negative controls, which were included in each staining replicate, consisted of incubating COCs in TUNEL reaction mixture without TdT enzyme. For positive controls, COCs were first treated with 1 mg/ml deoxyribonuclease I to induce DNA strand breaks followed by the TUNEL method. To visualize chromatin, COCs were counterstained with 5 ug/ml Hoechst 33258 for 5 min and washed extensively with PBS. COCs were individually transferred in a small volume on a slide and gently pressed under a coverslip till cwnulus cells, dispersed in the tridimensional structure of the matrix. lay on the same focal plane. Cells were inspected in an Olimpus Axioplan 2 microscope in fluorescent mode. Cells showing an homogeneous and moderate chromatin staining by Hoechst throughout the entire nucleus resulted TUNEL negative, and, therefore considered non apopotic cells. On the contrary, cells with nuclei containing condensed chromatin deeply stained with Hoechst that was either margined into masses aligned with the nuclear envelope or shrunken into a single mass or fragmented into multiple masses that occurred in clusters immediately adjacent to one another, resulted to be TUNEL positive, and therefore defined apoptotic cells. For quantitative analysis of apoptosis, six random fields of each COC were analyzed through a 400 X magnification in fluorescent mode. Total and apoptotic cells were counted and the percentage of apoptotic cells calculated for each COC. At least 10 COCs for each experimental sample have been analyzed. The data were expressed as means +/-SEM ofat least three independent experiments.
Quantitation of hyaluronan The amount of HA synthesized in the cultures were determined by metabolic labelling as described elsewhere (21). For each sample, 20 COCs were cultured in a 20 J..ll drop of medium in the presence of [35S] sulphate (60 J..lCilml) and [3H] glucosamine (100 J..l Ci/ml), At the end of each culture, the incubation medium was aspirated. Then. mediwn and cell-matrix fraction was treated with 20 J..lI of a papain solution (750 mIU final activity) for l h at 65°C. The extraction was completed by adding 1 vol of8 M guanidine HCl containing 4% (w/v) Triton X-lOO. Each extract was heated at lOO°C for 3 min to inactivate the papain and diluted to 500 J..lI by adding 0.1 M Tris, 0.1 M sodium acetate, pH 7.3, followed by elution on a column of Sephadex G50 (2 ml bed volume) equilibrated with 0.1 M Tris, 0.1 M sodiwn acetate and 0.5% Triton X-I00, pH 7.3. Aliquots from the excluded volume for each extract were digested with chondroitinase ABC (0.1 unit/ml) for 2 h at 37°C. An aliquot of each digest was chromatographed on a column of Sephadex G50 (4 ml bed volume) to determine the proportion of the radiolabeled macromolecules digested by the enzyme. The remaining portion of each sample was analyzed for its relative proportion of HA and DS disaccharides by high pressure liquid chromatography (HPLC) on Partisphere PAC equilibrated with acetonitrile:methanol:aqueous buffer in a ratio 52:12:36. The aqueous buffer contained 0.5 M Tris, 0.1 M boric acid, pH 8. The mass of HA synthesized during the labelling period was determined by calculating the specific activity of the UDP-Nacetylhexosamine pools from the ratio of 3H to 35S in the ADi-4S derived from the DS. Radioactivity was determined with a Beckman LS 3801 counter with spillover corrections determined for 35S standards.
492
The action ofhyaluronan in cells
In vitro fertilization In vitro fertilization was essentially performed as described elsewhere (22). Cumulus enclosed or denuded oocytes were transferred to 100 ~l drop of Whitten's medium supplemented with 15 mg/ml BSA. Subsequently, 10 ul ofa sperm suspension containing 5-10 x 106 spermatozoa/ml was added to oocyte cultures. Spermatozoa were obtained from the epididymis of CD l-proven breeders and were capacitated 2 h in the same medium used for insemination. After 6 h, the oocytes were observed by interference contrast microscope and those fertilized identified by the formation oftwo pronuclei. RESULTS AND DISCUSSION
Cumulus ceU apoptosis Expanded COCs are released from mouse ovarian follicles 14 h after an endogenous surge of gonadotropins or a hCG irYection into the animals. After ovulation, COCs progressively decrease in size and complete denudation of oocyte occurs at approximately 30 h from gonadotropin stimulus. When COCs were examined at the electron microscopy, some of the cumulus cells showed signs of degeneration (19). We have shown that these cells die through apoptotic process (23). At ovulation, 2 % of apoptotic cells/COC was found while this value increases approximately 8 and 15 fold at respectively 17 and 20 h from hCG injection. Similar temporal pattern of apoptosis occurred in COCs stimulated in vitro with FSH in the presence of FCS. In this culture condition apoptotic rate was 2% at 15 h, when full cumulus expansion was achieved, and 10% and 17% at respectively 20 and 24 h ofculture.
Correlation between cumulus ceU apoptosis and hyaluronan release from the matrix COCs stimulated in vitro with FSH in the presence of FCS begin to synthesize hyaluronan at 3 h and cease at 15 h of culture, when full expansion is achieved (Fig. 1). At this time almost all (80-900Al) the newly synthesized HA is retained in the matrix. A progressive HA release from the matrix to the culture medium occurs afterwards paralleling the apoptotic events described above. When COCs are cultured in the presence of dbcAMP, hyaluronan synthesis is stimulated and accumulated in the matrix with temporal pattern and extent similar to that observed with FSH. However, no significant amount of HA is released into the medium between 15 and 28 h of culture by dbcAMP stimulated COCs (Fig.2). Noteworthy, apoptosis does not occur in this culture condition (23). In addition, like in vivo, integrity of the cumulus is lost by ovulated COCs cultured in vitro for 14 h in control condition (28 h from hCG stimulation), while it is preserved by those COCs cultured for the same time period in the presence of dbcAMP (23).
Effect of maintenance of cumulus ceU-matrix on oocyte fertilizability In order to determine whether maintenance of cumulus cell-matrix integrity could prolong the fertilizability of oocytes, fertilization rate of ovulated COCs cultured for 14 h with and without dbcAMP was compared with that of COCs inseminated soon after ovulation (control). The results show that, when COCs were cultured in the absence of
Extracellular matrix degradation
493
dbcAMP, fertilization rate dropped to 20% of control. while, when they were cultured in the presence of dbcAMP, fertilization rate was about 70% of control.
14 12
0'
10
~
8
0
•
~total
~lT1Itrix
-.-rredium
:J:
'0
E
6
0< :J:
4
~
2 0
3
6
9
12
15
21
18
24
hours after FSHstlmulatlon
Figure 1.
Time course ofhyaluronan synthesis and its distribution between matrix and medium in COCs stimulated in vitro with FSH.
14 12
0'
10
~CII
8
'0
E
6
~
4
0
:J:
~
~total
~1T1Itrix
-.-rredium
2 0
3
6
9
12
15
18
21
24
27
hours after dbcAMPstlmulatlon
Figure 2.
Time course ofhyaluronan synthesis and its distribution between matrix and medium in COCs stimulated in vitro with dbcAMP.
494
The action of hyaluronan in cells
CONCLUSIONS In the present paper we provide evidence that an increasing number of cumulus cells undergo to apoptosis after expansion and that hyaluronan release from the matrix closely correlates with this phenomenon. In addition. we show that when apoptosis is experimentally prevented, matrix is preserved and the fertilizability of the enclosed oocyte is prolonged. These results and future studies on molecular mechanisms involved in COCs dispersion may allow to optimized culture conditions of oocytes utilized in assisted reproductive programs. ACKNOWLEDGEMENTS This work was supported by a MURST grant for National Project "Development and Differentiation of Germ Cells" and by CNR grant n.98.005I2.CT04.
REFERENCES 1. A. Salustri, M. Yanagishita, C. Underhill. T.C. Laurent & V.C. Hascall. Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicles, Dev. Bioi., 1992, 151, 541-51. 2. A Salustri, M. Yanagishita & V.C. Hascall, Mouse oocytes regulate hyaluronic acid synthesis and mucification by FSH-stimulated cumulus cells, Dev. Biol., 1990, 38, 26-32. 3. E. Tirone, C. D'Alessandris, V.C. Hascall, G. Siracusa & A. Salustri, Hyaluronan synthesis by mouse cumulus cells is regulated by interactions between folliclestimulating hormone (or epidermal growth factor) and a soluble oocyte factor (or transforming growth factor beta 1),1. Bioi. Chem., 1997,272,4787-94. 4. C. Fulop, A. Salustri & V.C. Hascall. Coding sequence of a hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex, Arch. Biochem. Biophys., 1997, 337, 261-66. 5. J.A Elvin, T.C. Amander, P. Wang, N.M. Wolfinan & M.M. Matzuk, Paracrine actions of growth differentiation factor-9 in the mammalian ovary, Mol. Endocrinol., 1999, 13, 1035-48. 6. AI. Yudin. G.N. Cheer & D.F. Katz, Structure of the cumulus matrix and zona pellucida in the golden hamster: a new view of sperm interaction with oocyteassociated extracellular matrix, Cell Tissue Res.. 1988,251,555-64. 7. G.N. Cherr, AI. Yudin & D.F. Katz, Organization of the hamster cumulus extracellular matrix: a hyaluronate-glycoprotein gel which modulates sperm access to the oocyte, Dev. Growth. Differ., 1990, 32, 353-65. 8. L.C. Chen, S.l.T. Mao & W.L. Larsen, Identification of a factor in fetal bovine serum that stabilizes the cumulus extracellular matrix, 1. BioI. Chem., 1992, 267, 12380-86. 9. L. Chen, H. Zhang, RW. Powers, P.T. Russell & W.l. Larsen. Covalent linkage between proteins of the inter-alpha-inhibitor family and hyaluronic acid is mediated by a factor produced by granulosa cells, 1. Bioi. Chem., 1996,271,19409-14 10. C. Fulop, R V. Kamath, Y. u, J.M. Otto, A. Salustri, B.R Olsen, T.T. Giant & V.C. Hascall. Coding sequence, exon-intron structure and chromosomal localization of murine TNF-stimulated gene 6 that is specifically expressed by expanding cumulus cell-oocyte complexes, Gene, 1997,202,95-102.
Extracellular matrix degradation
495
11. N. Ohta, H. Saito, T. Kuzumaki, T. Takahashi, M.M.lto, T. Saito, K. Nakahara, M. Hiroi, Expression ofCD44 in human cumulus and mural granulosa cells of individual patients in in-vitro fertilization programmes. Mol Hum Reprod, 1999, 5, 22-28. 12. L. Chen. P.T. Russell & W.J. Larsen. Functional significance of cwnulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cwnulus mass, Mol. Reprod. Dev., 1993,34,87-93. 13. K.A. Hess, L. Chen & W.J. Larsen. Inter-a-trypsin inhibitor binding to hyaluronan in the cwnulus extracellular matrix is required for optimal ovulation and development of mouse oocytes, Biol. Reprod., 1999,61,436-43. 14. H. Hizaki, E. Segi, Y. Sugimoto, M. Hirose, T. Saji, F. Ushikubi, T. Matsuoka, Y. Noda, T. Tanaka, N. Yoshida, S. Narumiya & A Ichikawa, Abortive expansion of cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2), P.NA.S., 1999, 31, 10501-06. 15. J.T. Lanman. Delays during reproduction and their effects on the embryo and fetus. 2. Aging of eggs, N Engl. J. Med, 1968,278, 1047-54. 16. AL. Smith & l.R. Lodge, Interactions of aged gametes: in vitro fertilization using in vitro-aged sperm and in vivo-aged ova in the mouse, Gamete Res., 1987, 16,47-56. 17. N.J. Winston. P.R. Braude & M.H. Johnson. Are failed-fertilized human oocytes useful? Hum. Reprod., 1993, 8, 503-07. 18. AJ. Wilcox, C.R. Weinberg & D.D. Baird, Post-ovulatory ageing of the human oocytes and embryo failure, Hum. Reprod., 1998, 13,394-97. 19. F.l. Longo, Ageing of mouse eggs in vivo and in vitro, Gamete Res., 1980, 3, 37993. 20. A Camaioni, V.C. Hascall, M. Yanagishita & A. Salustri, Effect of exogenous hyaluronic acid and serum on matrix organization and stability in the mouse cumulus cell-oocyte complex, J. Biol. Chem., 1993,268,20473-81. 21. A Salustri, M. Yanagishita & V.C. Hascall, Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus cell-oocyte complex during follicle stimulating hormone-induced mucification. J. Biol. Chem., 1989, 264, 13840-47. 22. B. Hogan, R. Beddington, F. Costantini & E. Lacy, in Manipulating The Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, New York, 1994, pp. 146-147. 23. M. Di Giacomo, A Camaioni, M. De Fetici, M. Piacentini & A Salustri, Inhibition of cumulus cell apoptosis by PKAII activators prolongs in vitro fertilizability of the oocyte, Dev. Biol., 2000, in preparation.
CLINICAL APPLICIATION OF SERUM HYALURONAN FOR LIVER DISEASES AND ITS SIGNIFICANCE *Takato Ueno", Osamu Hashimoto', Seisyu Tamaki!, Toshiro Ogata", Toru Nakamura', Ryuichiro Sakata', Kyuichi Tanikawa' & Michio Sata' 1
Research Centerfor Innovative Cancer Therapy and Second Department ofInternal Medicine, Kurume University School ofMedicine, 67 Asahi-machi, Kurume 830-0011, Japan. 2 Department ofSurgery Kurume University School ofMedicine, 67 Asahi-machi, Kurume 830-001 I, Japan. 3 International Institute for Liver Research, 2432-3, Aikawa-machi, Kurume, 839-0861, Japan
ABSTRACT
In this study, we demonstrated the clinical application of serum hyaluronan (HA) for liver diseases, which was obtained through our basic and clinical studies. We determined serum HA levels using a sandwich enzyme-binding assay kit (Chugai Diagnostics Science Co., LTD. Tokyo, Japan). Serum HA levels increased with progression of liver diseases, and serum HA determination was useful in the diagnosis of liver diseases. It is very important to decide the indication for interferon therapy in patients with chronic hepatitis, or to diagnose either cirrhosis or non-cirrhosis. Complications such as hepatocellular carcinoma and varices in digestive tracks tend to occur in cirrhosis. The efficiency of diagnosing cirrhosis in patients showing serum HA levels more than 130 n g/ml was approximately 90%, thus determining the serum HA level was useful for diagnosing liver cirrhosis. In addition, serum HA levels reflected the improvement of fibrosis in liver specimens of hepatitis C virus RNA negative patients after interferon- c¥ treatment. Moreover, the measurement of serum HA is useful as a predictor of hepatic regeneration after hepatectomy. In conclusion, measurement of serum HA is suggested to be useful in the diagnosis or comprehension of the progression of liver diseases. KEYWORDS
Hyaluronan, liver disease, hepatic fibrosis, hepatic sinusoidal capillarization, sinusoidal endothelial cell, interferon therapy, hepatectomy INTRODUCTION
In the liver, most HA is produced by hepatic stellate cells (HSCs), which are called Ito cells, fat-storing cells, and lipocytes. The cells are located in the Disse's spaces around hepatic sinusoids. HA in the tissue enters the blood stream through the lymph, the majority of HA in the blood is rapidly taken up via receptors into hepatic sinusoidal endothelial cells (SECs), where degradation follows 1.2 Generally, SECs have many fenestrae, presenting a sievelike appearance in the cytoplasm. In addition, the basement
446
Clinical applications of hyaluronan
membranes, which are visible in capillaries, are hardly seen along the basal side of SECs. In chronic liver diseases such as liver cirrhosis, however, hepatic sinusoidal capillarization frequently appears, and the SECs begin to show the morphological characteristics of capillary endothelial cells. That is, SECs which develop hepatic sinusoidal capillarization seem to decrease in HA degradation capacity. This is a review of reports as to the relationship between serum HA levels and liver disorders, as well as a summary of our own research regarding HA in liver diseases. We determined serum HA levels in our studies using a previously reported sandwich enzyme-binding assay (Chugai Diagnostics Science Co., LTD. Tokyo, Japan) 3.
BASIC APPROACH ON EVALUATIONOF SERUM HYALURONAN LEVEL IN CHRONIC LIVER DISEASES In previous reports, serum HA levels were elevated in patients with chronic liver diseases, especially liver cirrhosis 4-8. Turnover studies with labeled HA in patients with liver cirrhosis have disclosed that the clearance rate was slower in patients, but that the total amount of HA turnover was approximately the same as that in healthy persons 9. We clarified the cause of high blood HA concentration in liver cirrhosis using thioacetamide-induced liver cirrhosis model rats. In vivo observation of sinusoidal capillarization, and in vitro immunolocalization of factorVIII-related antigen and C4 C] HA binding in cultured SECs were determined. Basement membranes were observed
Figure 1.
Electron micrographs showing hepatic sinusoids of normal liver and cirrhotic liver in rats. Figure la: normal liver. Figure lb: cirrhotic liver. S: hepatic sinusoid, H: hepatocyte, E: SEC, I: HSC, arrows: fenestra, arrowheads: basement membrane.
Serum hyaluronan for liver diseases
Figure 2.
447
A fluorescent micrograph showing the localization of antifactorVlll-related antigen in cultured SECs. X 400
(dpm)
-
>'S:
~
2X 10 3
III Cl
P<0.001
c i5 c :is
« ::J:
1X103
U :::
14C-HA
HA+14C-HA
D: IE:
control rate trsated with physiological saline for 16 weeks
TAA-treated rat8
Figure 3.
Amount of C4C] HA binding to cultured SECs. The amount of C4C] HA binding to cultured SECs is significantly reduced in the thioacetamide -treated liver cirrhosis group compared with that in control rats. Levels of C4C] HA binding to cultured SEes obtained from control rats and thioacetamide-treated rats after HA preincubation are almost equivalent. 0: control rats, ~: thioacetarnide-treated rats
along the basal side of SECs and the fenestrae decreased with progression of hepatic fibrosis (Fig. I). Immunofluorescent reactive products of factorV1ll-related antigen were more abundant in cirrhotic rats compared with controls (Fig. 2). Amount of [14C] HA binding was significantly decreased in the cirrhotic rats compared with the controls (Fig. 3). Based on these results, one reason that the blood HA concentration increases markedly in liver cirrhosis is considered to be the reduction in the HA-binding ability of SECs 10. HA as well as other extracellular matrix components is produced primarily by
448
Clinical applications of hyaluronan
~ E il... ~
600
..
400
..E§ ::l
I'"~
800
o
....
°1
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e
..e
200
u:
Dl
mnn±SD '" cuntrols
Figure 4.
.....................n acute viral hepautls
ehronte heputltlll
8>
~
8
· ·········~· liver cirrhosis
ft,
8
alcoholic ratty liver
o.r. .;. · · ·..i alcoholic fibrosis
ulcohollc c1rrh05b
Serum HA levels in patients with various liver diseases.
HSCs in the liver, and HA production increases in the cirrhotic liver 11. Therefore, the HA produced may be released into the circulation and may partially account for the observed increase in blood HA concentration. In addition, the molecular weights of synthesized and depolymerized HA may differ from each other, and circulating HA seems to reflect both. In previous reports 4.8, marked elevation in serum HA is seen in patients with virusinduced cirrhosis, alcoholic cirrhosis, primary biliary cirrhosis, and primary sclerosing cholangitis. In our study, all disease groups showed mean serum HA levels higher than that of the normal subjects, and the level increased with progression of the disease (Fig. 4). Moreover, in patients with cirrhosis, the serum HA level was markedly elevated, and the measurement of serum HA is suggested to be useful in diagnosing the liver cirrhosis 12
USEFULNESS OF SERUM HYALURONAN DIAGNOSING LIVER CIRRHOSIS
MEASUREMENT
IN
To differentiate between liver cirrhosis and non-cirrhosis, cut-off levels of HA, type
N collagen, laminin and type III procollagen-N-peptide (P I1IP), indocianine green 15 min. retention rate (ICGRI5) and the platelet count in blood were established based on receiver operating characteristic curves according to various liver pathological diagnosis. The respective cut-off levels for HA, type Ncollagen, laminin and PIlIP, ICGR15 and the platelet count were each 130 7) glml, 250 7) glml, 2.5 V/ml, 1.0 V/ml, 18 % and 14 X 104 /mnr', Diagnostic efficiencies at these levels are 91 % for laminin, 90 % for HA, 83 % for type Ncollagen, 74 % for platelet count, 64 % for ICGR15 and 58 % forP I1IP. In addition, the diagnostic efficiency for liver cirrhosis was 96 % in patients showing HA level of more than 130 7) glml and type N collagen level exceeding 250 7) glml (Table I). The measurement of serum HA or serum HA and type N collagen concentrations was therefore useful for the diagnosis ofliver cirrhosis.
INTERFERON THERAPY IN PATIENTS WITH CHRONIC HEPATITIS C AND SERUM HYALURONAN Recently, some reports showed that interferon (IFN)- ~ treatment improved hepatic histology, including hepatic fibrosis, over a prolonged period in chronic hepatitis C
Serum hyaluronan for liver diseases
449
Diagnosis of liver cirrhosis using serum hepatic fibrosis Markers, ICGRI5, and platelet count
Table 1.
HAandLllminln
Type IV
HA
type
collll.gen
Cut-olfvlI.lue
Platelel
PIIlP
Lamlnln
leGR1S
count
IV
130nglml
250n!J"ml
collagen
2.5UJml
1,OU!ml
14X 10'/mm.l
18%
Senelllvny ("to)
87
77
88
58
60
67
64
Spaclllcity ('Yo)
90
86
95
98
57
72
63
Efficiency (%)
90
83
96
91
56
74
64
Comparison of histological scores before and after IFNtreatment between group 1 and group 2
Table 2.
G,oup2
Group 1 Histology
(~
Troatment
Before
After
Beforo
± 0.7
0.3
±
0.7 (P<0.02)
1.1
After
± 0.7
0.3
± 0.5 (p
CR
1.4
NR
1.1 ± 0.8
1.1 ± 0.7
1.3 ± 0.8 1.2 ± 0.4
CR
1.8 ± 0.9
1.5 ± 0.9
2.5 ± 1.0 1.4 ± 0.8 (p
NR
2.0 ± 0.7
1.8 ± 0.9
1.5
Grading
Staging
±
0.8
1.8
±
1.0
IFN- a ; Int.r1Mon-a I CAl camp"'. respond..... NA: norweaponde",
Table 3. Fibrosis Markers
Comparison of serum hepatic fibrosis markers before and after IFN- (~ treatment in group I and group 2 Group 1
Treatment
Before
Group 2
Ane,
CR
83
± 77
42 ± 32*
± 54
46
Before
Aner
100
± 88
45 ± 20*
55
± 60
36
HA
± 32
± 18
NR
81
CR
1.0 ± 0.4
0.8 ± 0.2·
1.1 ± 0.2 0.7 ± 0.2
NR
1.1 ± 0.3
1.0 ± 0.3
1.0 ± 0.2 0.8 ± 0.1
PiliP
CR
155 ± 97
102 ± 40·
103 ± 123
NR
144 ± 48
131 ± 43
165 ± 55 132 ± 45
99 ± 42
ly.N HA: hyaluronan, Pili P: type III procollagen N-peptlde, Ty.N: type W collag.n.IFN~a; Interteron·'l. CR; complete reapond ..... NR; non-relpondera p
*
(CH-C) 13-15. Serum fibrosis markers are considered to reflect hepatic fibrosis 12,16. We reported the clinical application of serum fibrosis markers in long-term follow-up patients with CH-C treated with IFN- ~ 17. Our study included 52 patients treated with IFN- (y (total 480 megaunits) for 6 months. All subjects underwent liver biopsy before and after treatment. Thirty-six patients who underwent liver biopsy less than 2 years after treatment were classified as group 1, and 16 patients who underwent liver biopsy more than 2 years after treatment as group 2. The two groups were subdivided into HCV RNA negative responders and positive non-responders. Liver specimens and serum fibrosis markers such as HA, Plll P, and typeLVcollagen were compared before and after treatment. In responders of groups 1 and 2, as shown in Table 2, grading score
450
Clinical applications ofhyaluronan Table 4.
Correlation between histological scores and serum hepatic fibrosis markers in group 1 and group 2 Group 1
Flbrosl8
Grading
Group 2
Markers
n
HA
70
0.116
0.341
29
0.147
0.449
PiliP
61
0.239
0.064
19
0.623
0.004
Ty.1V
81
0.352
0.005
19
0.399
0.05
p
---------------------------------------Staging
HA
70
0.291
0.815
29
0.458
0.013
PiliP
61
0.33
0.009
19
0.39
0.099
Ty.IV
61
0.351
0.006
19
0.259
0.284
HAi hyaluronan,pili Pj type III procollagen N-poplldo,Ty.N i type IV collagen, n; number"j correlation coofflclenl, Pi ~Y8Iu.
after treatment was significantly decreased compared with that before treatment. Staging score after treatment was significantly improved only in the responders of group 2. In the responders of group 2, serum HA level was significantly decreased compared with that before treatment (Table 3). In group 2, the grading score was significantly correlated with serum pIll P and typeNcollagen levels, and the staging score was significantly correlated with only serum HA level (Table 4). These findings indicate that the serum P IIIP and type N collagen levels reflect the activity, and serum HA reflects the degree of fibrosis in liver specimens of HCV RNA negative patients in a long-term follow-up of patients after IFN- (ll treatment.
SERUM HYALURONAN AS A PREDICTOR OF HEPATIC REGENERATION AFTER HEPATECTOMY IN HUMANS Curative resection for liver cancer is the most effective treatment, and well preserved liver function after hepatectomy results in improved quality of life. Therefore, the capacity for hepatic regeneration after hepatectomy is important for allowing surgeons to determine the appropriate extent of resection. It is known that cirrhotic livers regenerate slowly and incompletely. In addition, preoperative liver function tests inadequately estimate postoperative hepatic regeneration. We investigated the relationship between preoperative serum HA levels and hepatic regeneration rate using computerized tomographic volumetry, the hepatic fibrosis rate and immunolocalization of factorVllI-related antigen as a marker for hepatic sinusoidal capillarization. The serum HA levels was significantly correlated with the hepatic regeneration rate (Fig. 5). The hepatic regeneration rate of patients with serum HA levels above 200 7) g/ml and factor VlII-related antigen in the liver was low. From these results, the serum HA level is regarded as a useful predictor for hepatic regeneration after hepatectomy.
CONCLUSIONS The serum HA determination is useful in the diagnosis of liver disorders, especially, liver cirrhosis. The pathophysiological mechanisms of the increased serum level are not satisfactorily understood. Decreased clearance from the liver and increased HA production are both very important factors. Changes in SECs relative to the hepatic sinusoidal capillarization may also contribute to the decreased uptake or degradation of
Serum hyaluronan for liver diseases preoperutlon
Reml
a
Hepatic regeneration rate (HRR) =
451
postoperation
Rem 2
Rem 2 - Rem I
R-T
2
o o 0: 0:
:c
o
-1 - j - - - - , - - - , - - - , - - - - - , - - - - - , o 100 200 300 400 Serum HA Calculation of hepatic regeneration rate (HRR) (Figure 5a) and correlation between serum HA levels and HRR (Figure 5b). T: tumor, R: resected liver volume including tumor (ml), Rem 1: estimated remnant liver volume at surgery (ml), Rem 2: remnant liver volume after hepatectomy (ml)
b Figure 5.
HA in blood. This review, therefore, has focused on the role of measuring serum HA level in the diagnosis ofliver cirrhosis, on the evaluation of the improvement in hepatic fibrosis after IFN- 0: therapy, and on the usefulness as a predictor of hepatic regeneration after hepatectomy. Overall, it seems to be important to clarify the clinical value of monitoring the serum HA level in various liver disorders.
REFERENCES S. Eriksson, 1. R. E. Fraser, T. C. Laurent, H. Pertoft & B. Smedsrod, 'Endothelial cells are a site of uptake and degradation of hyalulonic acid in the liver' , Exp. Cell. Res., 1983,144,223-228. 2. N. Forsberg & S. Gustafson, • Characterization and purification of the hyaluronan-receptor on liver endothelial cells' , Biochem. Biophys. Acta, 1991, 1078,12-18. :-3. K. Chichibu, T. Matuura, S. Shichijo & M. Yokoyama, •Assay of serum hyaluronic acid in clinical application' , Clin. Chim. Acta, 1989, 181,317-324. 4- . A. Engstrom-Laurent, L. Loaf, A. Nyberg & T. Schroder, 'Increased serum 1.
452
Clinical applications ofhyaluronan
levels of hyaluronate in liver disease' , Hepatology, 1985,5,638-642. 5. T. Frebourg, B. Delpech, E. Bercoff, 1. Senant, P. Bertrand, Y. Deugnier & 1. Bourreille, 'Serum hyaluronate in liver diseases: study by enzymoimmunological assay' , Hepatology, 1986, 6, 392-395. A. Nyberg, A. Engstrom-Laurent, L. Loaf, 'Serum hyaluronate in primary 6. biliary cirrhosis-a biochemical marker for progressive liver damage' , Hepatology, 1988, 8, 142-146. 7. C. Babbs, N. Y. Haboubi, J. M. Mellor, A. Smith, B. P. Rowan & T. W. Warnes, 'Endothelial cell transformation in primary biliary cirrhosis: a morphological and biochemical study' , Hepatology, 1990, 11, 723-729. 8. G. Ramadori, G. Zohrens, M. Manns, H. Rieder, H. P. Dienes, G. Hess & K. H. Meyer Zum Buschenfelde, 'Serum hyaluronate and type III procollagen aminoterminal propeptide concentration in chronic liver disease. Relationship to cirrhosis and disease activity' , Eur. J Clin. Invest., 1991,21,323-330. 9. J. R. E. Fraser, A. Nyberg, A. Engstrom-Laurent & T. C. Laurent, 'Removal of hyaluronic acid from the circulation in rheumatoid disease and primary biliary cirrhosis' , J Lab. Clin. Med., 1986, 107, 79-85. 1 O. S. Tamaki, T. Ueno, T. Torimura, M. Sata & K. Tanikawa, 'Evaluation of hyaluronic acid binding ability of sinusoidal endothelial cells in rats with liver cirrhosis' ,Gastroenterology, 1996,111,1049-1057. 1 1. K. Murata, Y. Ochiai & K. Akashi, 'Polydispersity of acidic glycosaminoglycan components in human liver and the changes at different stages in liver cirrhosis' , Gastroenterology, 1985, 89, 1248-1257. 1 2. T. Deno, S. Inuzuka, T. Torimura, S. Tamaki, H. Koh, M. Kin, T. Minetoma, Y. Kimura, H. Ohira, M. Sata, H. Yoshida & K. Tanikawa, Serum hyaluronate reflects hepatic sinusoidal capillarization, Gastroenterology, 1993, 105, 475-481. 1 3. E. K. Manesis, C. Papaioannou, A. Gioustozi, G. Kafiri, J. Koshines & S. Hadziyannis, 'Biochemical and virological outcome of patients with chronic hepatitis C treated with interferon alfa-2b for 6 or 12 months: a 4-year follow-up of 211 patients' ,Hepatology, 1997,26, 734-739. 1 4. 1. F. Dufour, R. DeLellis, M. M. Kaplan, 'Regression of hepatic fibrosis in hepatitis C with long-term interferon treatment' , Dig. Dis. Sci., 1998, 43, 25732576. 1 5. D, T. Y. Lau, D. E. KleineR, M. G. Ghany, Y. Park, P. Schmid & J. H. Hoofnagle, "lu-year follow-up after interferon- (Y therapy for chronic hepatitis C' , Hepatology, 1998,28,1121-1127. 1 6. A. Castilla, J. Prieto & N. Fausto, 'Transforming growth factors {3 , and (Y in chronic liver disease: effects of interferon alfa therapy' ,N Engl. J Med., 1991, 324, 933-940, 1 7. T. Ueno, T. Ide, O. Hashimoto, Y. Uchimura, T. Torimura, R. Kumashiro, S, Inuzuka & M. Sata, 'Long-term follow-up of interferon-treated chronic hepatitis C and serum hepatic fibrosis markers' , Hepato-Gastroenterol., 2000, in press. 1 8. T. Ogata, K. Okuda, T. Dena, N. Sata & S. Aoyagi, 'Serum hyaluronn as a predictor of hepatic regeneration after hepatectomy in humans' , Eur. J Clin. Invest., 1999, 29, 780-785.
EFFECTS OF HYALURONAN USED AS A SUPPLEMENT IN PERITONEAL DIALYSIS SOLUTIONS Andrzej Breborowiczr", James B. Moberly", Krzysztof Pawlaczyk', Alicja Polubinska', Malgorzata Kuzlan-Pawlaczyk", Katarzyna WieczorowskaTobis', Kristen Ogle 2, Leo Martis', Dimitrios G. Oreopoulos'' /Department ofPathophysiology. Poznan Medical School III. Swiecickiego 6.60-781 Poznan. Poland 2 Baxter
Healthcare Corporation McGaw Park. Illinois. USA.
3 Division ofNephrology. University of Toronto. 399 Bathurst Street, M5T 288, Ontario, Canada
ABSTRACT Hyaluronan present in the peritoneal cavity is removed during the course of peritoneal dialysis. The addition of hyaluronan to dialysis f1uids has been proposed as a means to improve peritoneal transport functions and to protect the peritoneal membrane during peritoneal dialysis. Studies in rats have examined the effect of hyaluronan, added to dialysis solutions, on peritoneal permeability, inflammation and morphology. (0) Addition of 10 mg/dL high-molecular weight (hmw) hyaluronan (m.w. 1.8-2.4 x 10 to the dialysis solution caused significant improvements in fluid and solute transport. Net ultrafiltration of water across the peritoneum increased, whereas the membrane's permeability to protein declined. During an eight hour dwell in the peritoneal cavity, about 25% of the administered hyaluronan was absorbed, and concentrations in the peritoneal interstitium and in blood increased significantly. Absorbed hyaluronan was rapidly metabolised and, was not affected by the uremic status of the animal. Hyaluronan reduced the acute intraperitoneal inflammation induced by the initiation of peritoneal dialysis and the surgical procedure. In rats exposed to dialysis solution supplemented with hyaluronan (10 mg/dL) for one month, the interstitial connective tissue in the peritoneal membrane was less thickened than that of control rats, and the membrane had a lower permeability to protein and reduced hydraulic conductivity, potentially helping to promote the removal of water during dialysis. Thus, supplementation of peritoneal dialysis solutions with hyaluronan may improve peritoneal transport and fluid removal while helping to reduce peritoneal inflammation caused by the dialysis procedure. KEYWORDS
Hyaluronan, peritoneal dialysis, permeability, inflammation INTRODUCTION
Since its inception 20 years ago.continuous ambulatory peritoneal dialysis (CAPO) and automated peritoneal dialysis have become widely used as alternatives to hemodialysis for replacement of renal function in patients with end-stage renal disease
454
Clinical applications ofhyaluronan
(I). Worldwide, approximately 16% of patients with renal failure are managed using peritoneal dialysis, and in some countries, this percentage is much greater due to the potential cost benefits of the therapy. During a typical peritoneal dialysis exchange, ~2 liters of dialysis solution is infused into the peritoneal cavity, where it is left to dwell for a variable period and then drained out, removing uremic toxins that diffused from the peritoneal blood vessels into the solution. The instillation of dialysis solutions into the peritoneal cavity, however, is a non-physiological event which can induce an intraperitoneal inflammatory reaction (2). Furthermore, due to their acidity and high osmolality, dialysis solutions are bioincompatible and may cause injury to the peritoneal membrane with subsequent inflammatory response and healing. HY ALURONAN IN THE PERITONEAL CAVITY
Glycosaminoglycans, including hyaluronan, are produced locally within peritoneal cavity. These substances are lost from the peritoneal cavity during peritoneal dialysis due to the continuous infusion and removal of fluid. Staprans et al. found chondroitin sulfate and heparan sulfate in dialysate effluent from CAPD patients (3). Lipkin et al. (4) reported the presence of large quantities of hyaluronan in the effluent dialysate and found that hyaluronan concentration in dialysate exceeded the serum hyaluronan level, suggesting that hyaluronan is produced locally. Later it was shown that both peritoneal mesothelial cells and fibroblasts participate in hyaluronan synthesis (5,6), while peritoneal macrophages playa minor role (7). In stable CAPD patients, Yamagata et al. reported that the dialysate hyaluronan concentration gradually increased with time on dialysis and that this effect is more pronounced in patients with a hyperpermeable peritoneum (8). Increased permeability of the peritoneum reflects the damage done by bioincompatible dialysis fluids. In such situations increased intraperitoneal production ofhyaluronan may reflect an inflammatory response and subsequent healing (7,9). We hypothesized that in patients treated with chronic peritoneal dialysis, the healingpromoting effect of endogenous hyaluronan is not effective due to its dilution with high volumes of the infused dialysis fluid and repeated draining. Therefore, we proposed that supplementation of the dialysis fluid with exogenous hyaluronan may help to preserve the peritoneum as a living dialysis membrane. In preliminary experiments, it was demonstrated that the addition of hyaluronan or chondroitin sulfate to dialysis fluid over one week prevents functional deterioration of the peritoneum (10,11). This paper presents the results of additional experiments on the acute and chronic effect of highmolecular weight hyaluronan (m.w. 1.8-2.4 x 106 D) (Genzyme Pharmaceuticals, Cambridge, MA, USA) on the permeability, inllammatory response and morphology of the peritoneum in rats. EFFECT OF HY ALURONAN ON PERITONEAL PERMEABILITY
During acute peritoneal dialysis performed in awake, unrestrained male Wistar rats, addition of hyaluronan (10 mg/dL) to a standard dialysis solution (Dianeal 3.86% glucose, Baxter Europe, Northern Ireland) modified the permeability of the peritoneum to solutes and water. These changes were observed during long (4-8 hours) dialysis dwells. Thus, during a four hour dwell, transperitoneal removal of water (ultrafiltration) was increased by 25% and after eight hours by 24%, mainly due to inhibition of fluid reabsorption from the peritoneal cavity. Hyaluronan did not change the absorption of the osmotic solute (glucose) from the dialysate. The higher volume of drained dialysate
Supplement in peritoneal dialysis solutions
455
increased the small solute (creatinine) peritoneal clearance by 27% at eight hours; at the same time, peritoneal permeability to protein was reduced by 27%. HY ALURONAN ABSORPTION FROM THE PERITONEAL CAVITY
Despite its large molecular size, hyaluronan is readily absorbed from the peritoneal cavity. During peritoneal dialysis in rats performed with dialysis solution supplemented with hyaluronan at a concentration of 10 mg/dL (total amount of hyaluronan added to the peritoneal cavity was 3 mg), 12% ofthe administered hyaluronan was absorbed after four hours dwell and 26% after eight hours. After a four hour dwell, the hyaluronan concentration in the plasma increased by 147% compared to control animals exposed to Dianeal 3.86, and by 294% after an eight hour dwell. At the same time periods, hyaluronan concentration in the peritoneal interstitium increased by 127% and 115%, respectively. Absorbed hyaluronan was rapidly metabolised in the body; in rats, uremia induced by bilateral nephrectomy had no effect on this process. EFFECT OF HY ALURONAN ON PERITONEAL HEALING AND INFLAMMATION
The following experiments were performed using a rat model of peritoneal dialysis developed in our laboratory (12). Simultaneously with implantation of the peritoneal catheter in male Wistar rats, omentectomy was performed to prevent entrapment of the catheter by the omentum during peritoneal dialysis. This surgical procedure and the subsequent repeated intraperitoneal infusions of dialysis solution produced an intraperitoneal inflammatory reaction, reflected by the production of various inflammatory mediators and the influx of leukocytes into the peritoneal cavity. In rats implanted with peritoneal catheters and infused with peritoneal dialysis solution for eight days, the inflammatory response was at its maximum immediately after the procedure and gradually deelined during the following days. The inflammation was reflected by high dialysate cell count and increased dialysate level of the cytokines TNFa and Mep-I. The addition ofhmw hyaluronan (10 mg/dL) to the dialysis solution reduced the inflammatory response when compared to control animals dialyscd with standard dialysis solution (DianeaI3.86) (Table 1). Table I. Dialysate cell counts and inflammatory mediators following implantation of peritoneal catheters in rats. Rats were administered Dianeal 3.86% (D3.86) or D3.86 containing 10 mg/dL hyaluronan (D3.86+HA) for eight days following catheter insertion. Each group contained six animals (mean ± SD).
Parameter Cell count
1000
Group D 3.86 D 3.86+HA
lIml TNFa pg/mL
D 3.86 D 3.86+HA
x
MCP-l pg/mL
Days 1 5.89±2.23 2.l2±O.72 p
of dialysis after catheter implantation 2 4 8 6 3.69±O.35 1.76±O.21 1.49±O.16 1.48±O.18 2.02±O.21 1.19±O.O8 O.98±O.11 O.94±O.O7
7.89±2.0l 3.46±1.78 p
5.24±I.ll 1.11±O.88 p
456
Clinical applicationsofhyaluronan
In another experiment, we evaluated whether hyaluronan could reduce intraperitoneal inflammation in rats during chronic peritoneal dialysis. Peritoneal dialysis was performed for two weeks on alternating days using Dianeal 3.86 or Dianeal 3.86 supplemented with 10 mg/dL hyaluronan. Supplementation of the solution with hyaluronan reduced the inflammatory reaction, as shown by a reduction of inflammatory mediators in the dialysate (Table II). Table II. Dialysate cell counts and inflammatory mediators following alternating dialysis exchanges with Dianeal 3.86 or Dianeal 3.86 + 10 mg/dL hyaluronan for two weeks. Each group consisted of seven Wistar rats (mean ± SD).
Dianeal 3.86 Dianeal 3.86 +HA (lOmg/dL)
Dialysate cell count 1/JlL 2980 ± 225
TNFa mL 2.8 ±1.4
2145 ± 199 p<0.05
2.5±1.1
pg/mL 29.7± 10.5
IL-IO PglmL 43.6 ± 13.2
13.5 ± 6.4 p
35.7 ± 11.1 p
MCP-I
HYALURONAN AS AN ADDITIVE TO THE DIALYSIS FLUID DURING CHRONIC PERITONEAL DIALYSIS
In these experiments we set out to determine how the addition of hyaluronan to dialysis solution affects intraperitoneal inflammation and peritoneal structure and function during four weeks of peritoneal dialysis in rats. After implantation of the peritoneal catheter, rats were infused daily with the dialysis fluid (Dianeal 1.36% glucose). In earlier experiments, we found that one week is sufficient time for complete healing of peritoneum after omentectomy and implantation of the catheter (13). Therefore, the animals were divided into two groups at the end of one week, and during the following four weeks they were infused intraperitoneally with the following fluids: A. Dianeal 3.86 - control group (n=8) B. Dianeal 3.86 + hyaluronan 10 mg/dL (n=7) The peritoneal inflammatory response was determined at the beginning of dialysis and thereafter at one week intervals by measuring (in the dialysate effluent drained after a four hour dwell) the dialysate cell count and differential and concentration of cytokines TNFa, MCP-l and MIP-2. In all dialysed animals, the inflammatory response was the strongest at the start of the study and gradually declined, reaching minimal values around the second week of dialysis; thereafter it started to increase again. With the addition of hmw hyaluronan to the dialysis fluid, the inflammatory response was reduced. After four weeks of dialysis, the concentration of the studied cytokines in the dialysate was lower in rats treated with hyaluronan-supplemented dialysis fluid (Table Ill). There was also a tendency for a lower dialysate cell-count in the treated group: 1847±II01 in the Dianeal 3.86 group and 979±237 in the Dianeal 3.86 + Hyaluronan group. After four weeks of dialysis, the hyaluronan-treatcd animals tended to show a higher percentage of macrophages: 61.9±8.2% vs. 52.6±6.1% and a lower percentage of neutrophils: 13.7±5.6'Yo vs. 20.1± 11.4 in the Dianeal 3.86 group. We found that chronic intraperitoneal infusion of the dialysis fluid in rats causes an increase in the dialysate elastase activity. Addition of hyaluronan to the dialysis fluid reduced that effect, starting from the second week of dialysis (Fig. I). After four weeks
Supplement in peritoneal dialysis solutions
457
of dialysis with hyaluronan-supplemented dialysis fluid, the permeability of the rat peritoneum changed. Table III. Dialysate cytokine levels in rats dialysed for four weeks with Dianeal 3.86 (D) or Dianeal 3.86 supplemented with 1Omg/dL hmw hyaluronan (D+HA)
Time Start WeekI Week2 Wcck3 Wcek4
MIP-2 MCP-l TNFa pg/mL pg/mL pg/mL D+HA D+HA D D+HA D D 5.5±5.2 5.4±1.5 38.0±9.9 42.3±19.2 I08.2±79.7 102.4±24.5 8O.2±23.3 5.4±3.8 7.0±5.3 40.2±26.6 25.3±23.0 73.5±52.1 55.2±22.7 8.O±4.7 8.4±7.3 36.2±27.1 18.3±17.0 76.1±47.6 67.6±35.2 17.4±9.9 9.0±5.0 33.0±13.7 16.7±12.0 99.7±39.8 p
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Figure 1. Elastase activity in four-hour dialysate effluents during peritoneal dialysis in rats. Rats were treated for four weeks with Dianeal 3.86 (white bars) or Dianeal 3.86 supplemented with hmw hyaluronan (10 mg/dL) (black bars). Values are expressed as mean values ± SD. The character and magnitude of these changes was evaluated during four hour peritoneal equilibration tests using 30 ml of standard dialysis f1uid (Dianeal 3.86). At various time intervals during the equilibration test, bi-directional transperitoneal equilibration (blood - dialysate) of solutes and transport of fluid (resulting in changes in the dialysate volume) were examined.
458
Clinical applications ofhyaluronan
Starting from the end of the first hour of the dialysate dwell, fluid removal from the body, expressed as net ultrafiltration (difference between the infused and drained volumes of the dialysate) was higher in rats exposed for one month to Dianeal 3.86 supplemented with hyaluronan (Fig. 2). Transperitoneal equilibration of small solutes (urea, creatinine and glucose) was similar in all animals. However, after four hour dwell, transperitoneal equilibration of total protein, expressed as their ratio x 1000 of dialysate to blood concentrations was smaller in rats pretreated with hyaluronan: 40.0±7.9 vs. 59.1±17.9 (p<0.05) in control animals.
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Figure 2. Net ultrafiltration during a four hour dialysis exchange with Dianeal 3.86 in rats which were dialysed with Dianeal 3.86 (white bars) or Dianeal 3.86 supplemented with hmw hyaluronan 10 mg/dL (black bars) for four weeks. Values are expressed as mean values ± SD. At the end of four weeks of peritoneal dialysis, we took biopsies from the peritoneal membrane covering the liver surface in all the animals. In all cases, we found a thickening of the submesothelial interstitial tissue, with deposition of collagen. Rats exposed to hyaluronan tended to have less thickening of the peritoneal membrane: 16.8±11.7 urn vs. 23.7±9.7 urn in rats not exposed to exogenous hyaluronan. Also, rats dialysed with hyaluronan tended to show weaker staining for the presence of collagen (65.5±9.3 units vs. 74.7±23.7 units, respectively). DISCUSSION AND CONCLUSIONS
We have demonstrated that supplementation of dialysis solution with exogenous high molecular weight hyaluronan reduces the intensity of intraperitoneal inf1ammation and alters the kinetics of fluid and solute transport across the peritoneum in rats. In our studies, the concentration of hyaluronan used as additive to the dialysis fluid was over 50 times higher than its value in the overnight dialysate effluent from CAPD patients (4). However, hyaluronan levels in the peritoneal cavity may be much higher than that
Supplement in peritoneal dialysis solutions
459
deduced from effluent levels, and hyaluronan levels in the peritoneal fluid can be elevated up to WOO-fold in certain instances, such as during peritonitis (14). During peritoneal dialysis, it is probable that peritoneal hyaluronan levels are reduced due to dilution with infused fluid and repeated draining, and these levels may be biologically ineffective at suppression inflammation or maintaining peritoneal function. Wang et al. (15) also demonstrated increased fluid removal during acute peritoneal dialysis performed with hyaluronan supplemented fluid. Combined with our observations in both acute and chronic experiments, these data suggest that increased fluid removal during peritoneal dialysis with solutions containing hyaluronan or in rats pretreated with hyaluronan may be due to an accumulation of glycosaminoglycan in the peritoneal interstitium, which decreases hydraulic conductivity of the membrane and therefore reduces fluid reabsorption from the peritoneal cavity (16). It was surprising to note that significant amount of bmw hyaluronan is absorbed from the peritoneal cavity. However such absorption should not be a problem because of its rapid metabolism in the body, and the absence of kidneys does not impede this metabolic process (17). In rats, the addition of bmw hyaluronan to the dialysis f1uid reduced the intraperitoneal inflammatory response induced by peritoneal dialysis. Previously, others have demonstrated that hmw hyaluronan, in contrast to low molecular hyaluronan, has anti-inflammatory properties in vitro (18). Hyaluronan also suppresses the release of elastase from activated rat peritoneal leukocytes (19); indeed, in our experiments dialysate e1astasc activity was lower following administration of hyaluronan. This may explain why, during chronic dialysis in rats, the addition of hyaluronan to the dialysis f1uid helps to preserve the structural and functional integrity of peritoneum. We did not find that hyaluronan had a significant protective effect against dialysis-induced thickening of the peritoneal interstitium; however, there was a tendency for smaller overgrowth of the submesothelial tissue in the hyaluronan supplemented group. The increase in dialysate volume during dialysis with hyaluronan may provide higher peritoneal clearances, thereby increasing the adequacy of peritoneal dialysis. At the same time, due to reduced peritoneal permeability, protein is not lost from the body, which may help to prevent or reduce malnutrition. These results support the hypothesis that the addition of hyaluronan to peritoneal dialysis solutions may improve the efficiency of the peritoneal dialysis, decrease the inflammatory response and increase the longevity of peritoneum as the dialysis membrane. REFERENCES
I. R.P. Popovich, J.W. Moncrief, J.F. Decherd, J.B. Bomar, W.K. Pylc, The definition of a novel portable/wearable equilibrium dialysis technique. (Abstract) Trans.Am.Soc. Artif. Int. Organs 1976,5,64. 2. H..J. Bos , F. Meyer F, J.e. De Veld et al. Peritoneal dialysis fluid induces change of mononuclear phagocyte proportions. Kidney Int. 1989, 36, 20 - 26. 3. I. Staprans, C.F. Piel, J.M. Felts, Analysis ofselcctcd plasma constituents in continuous ambulatory peritoneal dialysis effluent. Am. .I. Kidney Dis. 1986, VII, 490-494. 4. G.W. Lipkin, M.A. Forbes, E.H. Cooper, J.H. Turney, Hyaluronic acid metabolism and its clinical significance in patients treated by continuous ambulatory peritoneal dialysis. Nephrol.Dial.Transplant. 1993,8,357-360 5. S. Yung, G.A. Coles, J.D. Williams, M. Davies, The source and possible significance of hyaluronan in the peritoneal cavity. Kidney lnt. 1994,46, 527-533
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Clinical applications of hyaluronan
6. A. Breborowicz, J. Wisniewska , A. Polubinska A, K. Wieczorowska-Tobis, L. Martis, D.G. Oreopoulos, Role of peritoneal mesothelial cells and fibroblasts in synthesis of hyaluronan during peritoneal dialysis. Perit. Dial. Int. 1998, 18, 382386 7. K.N. Lai, C.C. Szeto, K.B. Lai, C.W.K. Lam, D.T.M. Chan, J.C.K. Leung, Increased production ofhyaluronan by peritoneal cells and its significance in patients on CAPO. Am. J. Kidney Dis. 1999,33,318-324 8. K. Yamagata, C. Tomida, A. Koyama Intraperitoneal hyaluronan production in stable continuous ambulatory peritoneal dialysis patients. Perit. Dial. Int. 1999, 19, 131-137 9. P.H.Weigel, G.M.Fuller, R.D. LeBoeuf, A model for the role of hyaluronic acid and fibrin in early events during the inflammatory response and wound healing. J. Theor, BioI. 1986, 119,219-226 10. A. Breborowicz, K. Wieczorowska, 1. Martis, D.G. Oreopoulos, Glycosaminoglycan chondroitin sulphate prevents loss of ultrafiltration during peritoneal dialysis in rats. Nephron 1994,67,346-350 II. K. Wieczorowska, A. Breborowicz, 1. Martis, D.G. Oreopoulos, Protective effect of hyaluronic acid against peritoneal injury. Perit. Dial. Int. 1995, 15, 81-83 12. K. Wieczorowska-Tobis, K. Korybalska, A. Polubinska, MRadkowski, A. Breborowicz, D.G. Oreopoulos, In vivo model to study the biocompatibility of peritoneal dialysis solutions. InU.Artif.Organs. 1997,20, 673-677 13. K. Wieczorowska-Tobis, A. Polubinska, M. Kuzlan-Pawlaczyk, K. Pawlaczyk, A. Breborowicz, D.G. Oreopoulos, The characteristics of peritoneal healing after catheter insertion in a rat model of peritoneal dialysis. Advances in Peritoneal Dialysis 1998: 14,36-39 14. G.A.B. Edelstam, U.B.G. Laurent, O.E. Lundkvist, J.R.E. Fraser, Concentration and turnover of intraperitoneal hyaluronan during inflammation. Inflammation 1992, 16, 459-469 15. T. Wang, C. Chen, O. Heimburger, J. Waniewski, J. Bergstrom, B. Lindholm, Hyaluronan decreases peritoneal fluid absorption in peritoneal dialysis. J. Am. Soc. Nephrol. 1998,8,1915-1920 16. A. Polubinska, K. Pawlaczyk , M. Kuzlan-Pawlaczyk , K. Wieczorowska-Tobis, C. Chen, J.B. Moberly, L. Martis, A. Breborowicz, D.G. Oreopoulos, Dialysis solution containing hyaluronan: Effect on peritoneal permeability and inflammation in rats. Kidney Int. 2000, 57, 1182-1189 17. J.R. Fraser, T.C. Laurent, H. Pertoft, E. Baxter, Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit. Biochem. .J. 1981,200,415-424 18. B. Beck-Schimmer, B. Oertli, T. Pasch, R.P. Wuthrich, Hyaluronan induces Monocyte Chemoattractant Protein-I expression in renal tubular epithelial cells. J. Am. Soc. Nephrol. 1998, 9, 2283-2290 19. M. Akatsuka, Y. Yamamoto, K. Tobetto, T. Yasui, T. Ando, Suppressive effects of hyaluronic acid on elastase release from rat peritoneal leukocytes. J. Pharm. Pharmaeol. 1993,45, 110-114
PROTEOGLYCAN ENHANCES TIlE FORMATION OF mE SHAP·HYALURONAN COMPLEX AND ITS EFFECT IN HYALURONAN·RICH MATRIX Ming Zhaol, Masahiko Yoneda l, Usbeng Zbuo l, Lei Huang l, Hideto Watanabe2, Yoshibiko Yamada 2, Sbigeji Nagasawa 3, Hitoshi Nishimura 3 and Koji Kimata l 'Institute for Molecular Science ofMedicine, Aiehi Medical University, Nagakute, Aichi 480-1195, Japan 2Craniofacial Departmental Biology and Regeneration Branch, National Institute ofDental Research, National Institute ofHealth, Bethesda, MD 20892, USA -'Pharmaceutical Department, Hokkaido University, N15-W7, Kita-ku; Sapporo, Japan
ABSTRACf Hyaluronan (HA) was shown not only to be associated noncovalently with some extracellular molecules such as HA-binding proteoglycans but also to be covalently linked to SHAP (Serum-derived Hyaluronan-Associated Protein), the heavy chains of inter-alpha-trypsin inhibitor (IT!) through a unique ester linkage. Such non-covalent and covalent associations may play important roles in the formation and maintenance of HA-rich matrix. In the present study, we examined the binding activity of IT! to various extracellular matrix molecules by enzyme-linked immunosorbent assay, and found that the heavy chains but not the bikunin of ITI bound to PG-Mlversican, to link protein, and to the hyaluronan-binding region of aggrecan from various sources, which suggests specific interactions between the conserved regions of the heavy chains and the conserved HA-binding regions of these matrix molecules. In the further binding experiments using recombinant proteins for various portions of the heavy chains of human 111, we found that the polypeptide near the C-terminus of the heavy chain and the HA-binding region of aggrecan were involved in binding to each other. Such a noncovalent protein-protein interaction may be important as an initial step for the formation of the HA-SHAP complex as well as for the formation and stabilization of HA-rich matrix. KEYWORDS Inter-alpha-trypsin inhibitor, hyaluronan, extracellular matrix, proteoglycan INTRODUCfION Actively proliferating/migrating normal cells and some malignant tumor cells often have large, pericellular hyaluronan (HA)-eontaining matrices or "coats" which can be easily diminished by treatment with Streptomyces hyaluronidase. We therefore designated these matrices "HA-rich matrix". Recent evidence suggests important roles of the HA-rich matrix in normal and abnormal various cellular processes. Therefore, biochemical bases of the HA-rich matrix and the mechanism of the formation are important issues for understanding functions of the HA-rich matrix. In the HA-rich matrix, HA has been found to be noncovalently associated with CD44, a HA receptor on the cell surfaces and HA-binding proteoglycans such as PGMzversican or aggrecan in the extracellular matrix'. We showed previously that SHAP (Serum-derived HA-Associated Protein) is a member of the HA-rich matrix, which corresponds to the heavy chains of ITI and is covalently linked to HA through a unique ester linkage 2 • IT!, a plasma protease inhibitor family are widely distributed in human and other animal blood and comprises three genetically different peptides, two heavy chains (HCl
498
The action of hyaluronan in cells
and HC2) and a light chain (bikunin) 3. Bikunin is a proteoglycan and bears one chondroitin sulfate chain", Two heavy chains are covalently attached to this chondroitin sulfate chain by ester bonds s. The structure of ITI is exactly the same as those in the SHAP-HA complex except that HA was replaced by chondroitin sulfate. We found that the formation of the enlarged HA-rich matrices in cultured cells such as cancer cells needs ITI. However, it has remained to answer how ITI is involved in the formation of the HA-rich matrix and how the SHAP-HA complex is formed from ITI in the matrix.
METHODS
Quantitative assay Corthe area oC the HA-rich matrix Formalin-fixed red cells were added to cells cultured on dishes! and the rnatirx thus visualized as the exclusion area of the red cells under a microscope was measured by computer-assisted analysis.
Preparation and detection oC SHAP-HA Complex from the cultures The complex was extracted from the culture with 4 M guanidine HCl including 0.1 M Tris-HCl, pH 8.0 and protease inhibitors at 4°C for 24 h. For the formation in a test tube, the factor fraction (0.5ml) was mixed with 50 microgram of human ITI and 0.5 microgram of HA in 2.5 ml of Hanks SSS and then incubated at 37°C for 16 h in a test tube.The HA fraction was prepared with CsCI density gradient (twice) in 8 M guanidine HCI, under the dissociative condition. The samples were digested by the protease-free Streptomyces hyaluronidase. SHAP was detected with the immunoblotting using rabbit anti-human ITI antibody and HRP-conjugated protein A. The immune complexes were visualized by enhanced chemiluminescence assay. The density of bands on the X-ray film were measured by computer-assisted analysis",
RESULTS
Effect of ITI and SHAP-HA complex on Extracellular Matrix FM3A P-15A, a highly metastatic mouse mammary carcinoma cell line has been shown to form HA-rich extracellular matrices in serum-containing medium. The cells were able to form HA-rich matrices even in a serum-free medium although the sizes of the matrices were not so great. However, the average area of the HA-rich extracellular matrices of cells cultured in the presence of ITI is approximately twice as much as that of the cells cultured in absence of ITI (see Fig.1A). Subunits of ITI and three heavy chains (HC1, HC2 and HC3) showed no effects on the matrix formation (data not shown), neither do bikunin (see Fig. 1A). The addition of bovine serum induced the formation of HA-rich matirx with the largest size. We have observed the same effect when the serum factor was added to the culture media with ITI. The SHAP-HA complex formation requirs a serum factor. We prepared the factor fraction from bovine sera and confirmed that the fraction did not contain ITI. We showed that the mixture of HA, ITI and the factor fraction in a test tube was enough to form the SHAP-HA complex (see Fig.IS), but either ITI or the factor itself in mixing with HA was not (see Fig.IS). We expected that the SHAP-HA complex was formed in the HA-rich matrix when ITI and the serum factor were added to the culture media. We extracted and prepared the HA fraction from treated cells and then detected the SHAP bands on the membrane by immunoblotting (see Fig.IS). The results showed that the SHAP-HA complex made the volume of HA-rich matrix large(see Fig.l A).
m
binds to PG-Mlversican via the heavy chains Recently, the interactions of ITI with other matrix molecules were examined. ITI
Formation ofSHAP-hyaluronan complex
499
B
A
T
600
tJII12
400
200
o Non
Figurel
ITI
ITI Factor
Factor
Bikunin
ITI Factor
ITI
Factor
Effect of ITI on the area of HA-rich extracellular matrix. A: Mouse mammary carcinoma cells (FM3A P15A) were cultured in the chemically defined medium without serum. The dishes were added with 40 rnicogram protein of human ITI or the same amount of ITI and the factor fraction preparated from bovine sera. The area of extracellular matrix of the cells cultured for 2 days measured. B: The detection of the SHAP-HA complex prepared from the cultures.
showed binding activity with PG-M/versican, aggrecan, and link protein. The binding was through the medium of its heavy chain(s) but not bikunin. The binding region was defined to the C-terminal 48 amino acid residue (535-582) in HCl. This region has a hydrophobic sequence, LWAYLTI, conserved in HCl, HC2 and HC3. On the other hand, the binding regions in PGs were defined as the domain G1 7 • We have found that PG-M enhanced the SHAP-HAcomplex formation. The activity of PG-M was dose-dependent at the high concentrations of PG-M (see Fig. 2). This result suggested that PG-M gave the effect on the heavy chains of ITI, which approached to HA to form SHAP-HA complex. DISCUSSION AND CONCLUSION
The present results have shown that PG-M binds HA and IT!. The polypeptide near the C-terminal region of any of the heavy chains of ITI and the G1 domain of HAbinding proteoglycans such as PG-M bind each other. Chen et aI. observed the affinity binding of ITI to HAS, However, we could only observe the ionic interaction between m and HA (unpublished observation) which may be very weak under physiological conditions. It is likely, therefore, that the binding of PG-M to both HA and ITI may strengthen such a weak interaction beween HA and IT!. In addition, since PG-M have been shown to be able to bind to the heavy chains of ITI, it may also bind to the heavy chains (SHAP) covalently bound to HA and strengthen its own binding 10 HA We suggested previously thaI the formation of the SHAP-HA complex required serum factor(s). We have found that HA-binding proteoglycans greatly enhanced the formation of SHAP-HA complex, which, as discussed above, may be due to the enhanced interaction between ITI and HA by the binding of PG-M to both HA and IT!. Therefore, those proteoglycans may be one of factors involved in the formation of SHAP-HA complex in addition to serum factor(s). Taken into consideration together, the present results have let us assume that the binding of ITI 10 PG-M/versican may play an important role to regulate the formation of HA-rich matrix.
500
The action ofhyaluronan in cells
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REFERENCES 1 Knudson, W., Bartnik, E., and Knudson, C. 8. Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes. Proc Natl Acad Sci USA. 1993, 90(9):4003-4007 2. Zhao, M, Yoneda, M., Ohashi, Y., Kurono, S., Iwata, H., Ohnuki, Y., and Kimata, K. Evidence for the covalent binding of SHAP, heavy chains of inter-alpha-trypsin inhibitor, to hyaluronan. J. BiD/. Chern. 1995,270,26657-26663 3. Salier, J. P., Rouet, P., Raguenez, G., and Daveau, M The inter-alpha-inhibitor family: from structure to regulation. Biochem. J. , 1996,315,1-9 4. Hochstrasser, K., Schonberger, O. L., Rossmanith, I., Wachter, E. Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, V. Attachments of carbohydrates in the human urinary trypsin inhibitor isolated by affinity chromatography. Hoppe Seylers Z Physiol Chern 1981, 362(10), 1357-1362 5. Enghild, J. 1., Salvesen, G., Thogersen, I. 8., Valnickova, Z., Pizzo, S. V. and Hefta, S. A Presence of the protein-glycosaminoglycan-protein covalent cross-link in the inter-alpha-inhibitor-related proteinase inhibitor heavy chain 2/bikunin. J. BioL Chem., 1993, 268,8711-8716 6. Kida, D., Yoneda, M., Miyaura, S., Ishimaru, T., Yoshida, Y., Ito, T., Ishiguro, N., Iwata, H., and Kimata, K. The SHAP-HA complex in sera from patients with rheumatoid arthritis and osteoarthritis. J Rheumatoll999, 26(6):1230-1238 7. Yoneda, M., Zhao, M. et al Roles of inter-alpha-tyrpsin inhibiotor and hyaluronanbinding proteoglycans in hyaluronan-rich matrix formation. In: New frontiers in Medical Sciences: Redefining Hyaluronan. G. Abatangelo and P.H. Weigel, eds, 2_, Elsevier Science 8. V., pp21-30 8. Chen, L., Mao, S. J., Mclean, L R., Powers, R. W., and Larsen, W. J. Proteins of the inter-alpha-trypsin inhibitor family stabilize the cumulus extracellular matrix through their direct binding with hyaluronic acid. J. Bioi. Chem., 1994, 269(45):28282-28287
VISCOSURGERY: A HISTORICAL PERSPECTIVE Biomatrix Inc. 65 Railroad Avenue. Ridgefield. New Jersey, USA
ABSTRACT The therapeutic paradigm viscosurgery means the use of elastoviscous solutions or viscoelastic gels to protect sensitive tissues from mechanical damage, to create and maintain space for surgical procedures and to use them as "soft" tools to manipulate tissues during surgery. The first viscosurgical "tool" made of a purified hyaluronan called the non-inflammatory fraction of Na-hyaluronan (NIF-NaHA) developed in the late 1960s was tested in primates and later used in human eyes. These early clinical and nonclinical studies showed that the highly elastoviscous solution served to push back the retina during retinal detachment surgery and helped keep the retina in its reattached position after surgery. Hyaluronan solution was also shown to protect the corneal endothelium from mechanical damage during corneal transplantation. The protection of this single cell layer of endothelium is important because these cells do not regenerate in adults and their water pumping function is essential for maintaining the transparency of the cornea. Viscosurgery became widely used in the early 1980s when the first therapeutic product made of NIF-NaHA was made available worldwide for ophthalmic surgeons. Its major use today is in procedures when the cataractous lens is removed and replaced by a plastic lens, a procedure that was revolutionized by viscosurgery. Hylan B gel was used in retinal detachment surgery to push back the detached retina and keep it reattached in clinical studies. Hylan B gel was also used in primate studies and in pilot human studies after glaucoma surgery to maintain surgically formed outflow channels. Viscosurgical applications in orthopedics were tested clinically in arthroscopy using hylan solutions to protect the cartilage surface from mechanical damage from instruments. DEFINITION VISCOSURGERY: Elastoviscous fluids facilitate surgical procedures as surgical tools or implants to protect, manipulate and separate delicate tissues. HISTORICAL REVIEW 1958 - 1968: The concept of using an elastoviscous solution of high average molecular weight hyaluronan in the posterior segment as an internal retinal tamponade and vitreus replacement is introduced and tested in primatesI.2.3. 1968 - 1971: Biotrics, Inc. (Arlington, MA, USA) developed a high molecular weight (23 million) purified hyaluronan from human umbilical cord and rooster comb and defined it as the non-inflammatory fraction of Na-hyaluronan (NIF-NaHA) which is later patented.4,5.
462
Clinical applications ofhyaluronan
1970 - 1972: The first publications on pre-clinical and clinical studies using elastoviscous hyaluronan solution (NIF-NaHA). These publications established its use to facilitate reattachment of the retina and to protect the corneal endothelium and prevent postsurgical adhesions after corneal transplantation'v''". 1972 - 1976: Biotrics, Inc. trademarks the name Heaton" for their first highly purified hyaluronan (NIF-NaHA) and licensed it to Pharmacia AB (Uppsala, Sweden) for manufacturing and worldwide distribution rights for the treatment of arthritis in animals and humans and for ophthalmic surgery. 1979: The use of Healon® for protection of the corneal endothelium during intraocular lens implantation in patients followinfi cataract surgery is reported. The term "viscosurgery" is introduced (B.A. Balazs) 0. 1980: Healon" for viscosurgery is introduced by Pharmacia AB for general marketing at the American Intraocular Implant Society in Los Angeles, California. During the following years, Healon@ became available in most countries of the world. This is the first commercial use of hyaluronan as a therapeutic agent in humans. 1980 - 1982: The use of high molecular weight (>3 million) elastoviscous hyaluronan solution (Healon~ as a viscosurgical tool, to protect and manipulate tissues and to create tissue space in surgical procedures is established in animal studies and in clinical practice. The first international meeting on the use of Healon@ in eye surgery is held in Germanyll.1S. 1983: The first combination viscosurgical product made of 4% chondroitin sulfate (MW 25,000) and 3% hyaluronan (average MW 0.5 million) called Viscoat'" was mtroduced!" 1989: The first use of Healon@ to promote healing of chronic tympanic membrane perforation in ear surgeryt7·19. 1992: The use of Healon@ as a viscosurgical tool to facilitate the insertion of inner ear electrodes in cochlear implant surgery", 1992 - 1996: A great variety of viscoelastics mostly made of hyaluronan are available for ophthalmic viscosurgery. They represent a broad spectrum of rheological properties. The rapid development of new surgical procedures for lens extraction and intraocular plastic lens implantation often requires the use of two viscoelastics with different rheological properties during the surgery. The concepts of "cohesive" and "dispersive" viscoelastics are introduced21~2. Today, there are many hyaluronan solutions on the market for ophthalmic viscosurgery. They contain Na-hyaluronan of various average molecular weights (0.5 MW to > 4 million MW) and in various concentrations (1-2.3%). Consequently the rheological properties (elasticity and viscosity) of these fluids varies considerably. One preparation, in addition to low average molecular weight hyaluronan also contains chondroitin sulfate (Viscoat'").
Viscosurgery: a historical perspective
463
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RHEOLOGICAL PROPERTIES OF VISCOELASTICS The elastoviscosity of hyaluronan solution depends on the average molecular weight and the concentration of the molecule dissolved in the physiological buffer solutions. Greater concentration and higher molecular weight yields solution with greater viscosity and elasticity. Molecular interactions and the presence of low molecular weight glycosaminoglycans can also influence pseudoplasticity and elasticity of the solution. The elastoviscous behavior is also greatly dependent on the shear rate or frequency at 'which it is measured. (Fig. 1 & 2)24. Low molecular weight polysaccharides, like chondroitin sulfate (MW 25,000) in relatively large concentration (3% or more) have low viscosity and they also exhibit "stickiness". When these "sticky" solutions are mixed with viscous low molecular weight hyaluronan (average MW <0.5 million) a new property of the solution is observed. Such solutions will mix, disperse and eventually dissolve in water, but slower than their elastoviscous properties would predict. Therefore products made from such solutions are called "dispersivea?". Such a product is available for viscosurgery as a mixture of chrondroitin sulfate (4%) and hyaluronan (3%) under the trade name Viscoat'", In contrast, solutions made of high and very high molecular weight hyaluronan under high shear behave like elastic bodies and therefore they are called "cohesives", These products will not disperse easily under high shear I .
464
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REFERENCES 1. E. A. Balazs, Physiology of the vitreous body, In: Importance ofthe Vitreous Body in
2.
3.
4. 5. 6.
Retina Surgery with Special Emphasis on Reoperations, (C. L. Schepens, ed.), (Proceedings of meeting held in May 1958 in Ipswich, Mass.), Mosby, S1. Louis, 2948, 144-146. E. A. Balazs and D. B. Sweeney, The use of hyaluronic acid and collagen preparations in eye surgery, In: Controversial Aspects of the Management of Retinal Detachment, Vol. 3 (C. L. Schepens and C. D. J. Regen, eds.), Little, Brown and Company, Boston, 1965, 200-202. E. A. Balazs and D. B. Sweeney, The replacement of the vitreous body in the monkey by reconstituted vitreous and hyaluronic acid, In: Modem Problems in Ophthalmology, Vol. 4 (Surgery of Retinal Vascular Diseases, Amersfoort, 1963), (E. B. Streiff, ed.), Karger, Basel, 1966, 230-232. E. A. Balazs, Hyaluronic Acid and Matrix Implantation, Biotrics, Inc., Arlington, Mass., 1971. E. A. Balazs, Ultrapure Hyaluronic Acid and the Use Thereof, US Patent No.4 141 973, February 27,1979. E. A. Balazs, M. 1. Freeman, R. Kloti, G. Meyer-Schwickerath, F. Regnault and D. B. Sweeney, Hyaluronic acid and replacement of vitreous and aqueous humor, In: Modem Problems in Ophthalmology, Vol. 10, (Presented at the meeting: Secondary
Viscosurgcry: a historical perspective
465
Detachment of the Retina, Lausanne, 1970), (E. B. Streiff, ed.), Karger, Basel, 1972, 3-21. 7. P. Algvere, Intravitreal implantation of a high-molecular hyaluronic acid in surgery for retinal detachment, Acta Ophthalmol., 1971,49,975-976. 8. F. Regnault, Acide hyaluronique intravitreen et cryocoagulatin dans Ie traitement des formes graves de decollement de la retine, Bull. Soc. Ophthalmol. Fr., 1971. 9. R. Kloti, Hyaluronsaure als Glaskorpersubstituent, Schweiz: Ophthal. Ges., 1972, 165,351-359. 10. E. A. Balazs, D. Miller and R. Stegmann, Viscosurgery and the use of Na-hyaluronate in intraocular lens implantation, Paper presented at the International Congress and First Film Festival on Intraocular Implantation, Cannes, France, May, 1979. 11. E. L. Graue, F. M. Polack and E. A. Balazs, The protective effect of Na-hyaluronate to corneal endothelium, Exp. Eye Res., 1980, 31, 119-127. 12. D. Miller and R. Stegmann, Use of sodium hyaluronate in anterior segment eye surgery, J. Am. Intraocul. Implant Soc .1.,1980,6, 13-15. 13. L. G. Pape and E. A. Balazs, The use of sodium hyaluronate (Healon®) in human anterior segment surgery, Ophthalmology, 1980, 87, 699-705. 14. S. Stenkula, L. Ivert, I. Gislason, R. Tornquist and L. Weijdegard, The use of sodium hyaluronate (Healon~ in treatment of retinal detachment, Ophthal. Surgery, 1981, 12,435-437. 15. G. Meyer-Schwickerath, (Ed.), Viskochirurgie des Auges: Beitriige des ersten nationalen Healon@-Symposiums, October 15 & 16, 1982. 16. S. E. Harrison, D. B. Soil, M. Shaylgan and T. Clinch, Chondroitin sulfate: a new effective protective agent for intraocular lens insertion, Ophthalmology, 1982, 89, 1254-1260. 17. L. E. Stenfors, Repair of tympanic membrane perforations using hyaluronic acid: an alternative to myringoplaty, J. Laryngol Otol., 1989, 103, 39-40. 18. M. P. Rivas Lacarte, T. Casasin, F. Pumarola and A. Alonso, An alternative treatment for reduction of tympanic membrane perforations: Sodium hyaluronate, Acta Otolaryngol (Stockh), 1990,110,110-14. 19. C. Laurent, O. SOderberg, M. Anniko and S. Hartwig, Repair of chronic tympanic membrane perforations using application of hyaluronan or rice paper prosthesis, ORL, 1991, 53, 37-40. 20. F. Lehnhardt, Intralokluare elektrodenplazierung mittels Healon®, HNO, 1992, 40, 86-9. 21. S. Arshinoff, The physical properties of ophthalmic viscoelastics in cataract surgery, In: Proceeding of the National Ophthalmic Speakers Program, Medicopea International Inc., Montreal, 1992,7-12. 22. S. Arshinoff, Dispersive and cohesive viscoelastic materials in phacoemulsification, Ophthalmic Practice, 1995, 13:3,98-104. 23. H. B. Dick and O. Schwenn, Viscoelastics in Ophthalmic Surgery, Springer Verlang, Berlin, Heidelberg, New York, 2000. 24. H. Bothner Wik and O. Wik, Rheology of hyaluronan, In: The Chemistry, Biology and Medical Application of Hyaluronan and its Derivatives, (T. C. Laurent, ed.), Portland Press, London and Miami, 1998.
ENHANCED THROMBOXANE SYNTHESIS THROUGH THE INDUCTION OF CYCLO-OXYGENASE-2 BY BYALURONAN IN
RENAL CELLS Li Kang Sun Research Laboratory, Division of Nephrology, Cantonal Hospital, CH-9007. St. Gallen, Switzerland
INTRODUCTION
Eicosanoids, lipid inflammatory mediators. have potent effects on inflammation. and cycle-oxygenase (COX) initiates eicosanoids synthesis. Matrix degradation products such as fragmented hyaluronan acid (HA) display important proinflammatory effects on renal tubular epithelial cells (TECs) and macrophages (M€I>s). As HA accumulates considerably in renal injury. we therefore believe that HA could play a significant role in thromboxane-rnediated immune events in the kidney. Here in this review we discuss the consequences resulting from the HA induced COX-2 expression and subsequently the production of thromboxane A2 (TXA2) in TECs and M€I>s. HYALURONAN AND ITS PROINFLAMMATORY EFFECTS
Because of the interaction of hyaluronan (HA) with the immune system and other cells HA shows an anti-inflammatory effect. It was first reported in the mid 1980s that HA is often accumulated in tissues and fluids during disease [1-2]. HA has. therefore. to an increasing extent been utilised as a diagnostic marker. HA became an inflammatory factor. Previously we have reported that accumulation of HA in the cortical renal interstitium is linked to these classical proinflammatory events in the kidney [3]. We and others have shown by in vitro studies that the HA fragments with a molecular weight in the range of 60-600.000 Da have proinflammatory effects in mouse tubular epithelial cells (TECs) and macrophages (M€I>s). This includes the upregulation of chernokines, cytokines and adhesion molecules. Binding of HA to its specific cell surface receptor CD44 leads to a cascade of cellular activation events. which involves. among others. the nuclear translocation of NF-KB [4-6]. We have investigated the role of cyclo-oxygenase-2 (COX-2). the inducible isoform of COX. in formation of eicosanoids in kidney of inflammatory disease. We used an in vitro approach system to examine the expression of COX-I, COX-2 and the production of thromboxane A2 (TX~) in response to fragments of HA (I-lO00lJ.g/ml). The expression of COX-2 was then analysed on the mRNA and protein levels. the resulting TXB 2 production. a stable metabolite of TXA2• was calculated in cell culture supernatant in cell lines of TEC (MCT cell line) and M€I> (RAW 264.7 cell line) as indices of HA caused eicosanoids generation. COX-2 mRNA was also measured in vivo in MRL-Fas'P' mice. and in mice with anti-glomerular basement membrane (anti-GBM) nephritis. We observed, the expression of COX-2 was time (0.5-24h) and dose (11000IJ.g/ml) dependent on HA. whereas the mRNA level of COX-l remained unchanged under the same conditions. HA increased also the production of TXA2• and this HA-induced TXA2 production abolished completely by COX-2 selective inhibitor SC58125 (12.5 IJ.M) and celecoxib (O.25-51J.M), Additionally. the renal COX-2 mRNA levels were also enhanced 2.I-fold in MRL-Fas'P' mice compared with non-nephritic
502
The action ofhyaluronan in cells
MRL-++ mice. Similar results were found in mice with anti-GBM nephritis (5.5-fold upregulation of COX-2 mRNA). These observations indicate that (a) both COX isoforms, COX-I and COX-2, expressed in quiescent TEC and Met> cells; (b) HA induced the expression of COX-2, whereas the level of COX-I remained unchanged; (c) HA-induced TX~ production mediated primarily by COX-2 and not by COX-I; and (d) COX-2 expression was enhanced in kidneys of MRL-FaiP' mice and in kidneys of anti-GBM nephritis mice [7]. Because HA accumulates considerably in renal injury, we believe that HA could play a significant role in thromboxane (TX)-mediated immune events in the kidney. CD44 is the major cell surface receptor for HA. CD44 expressed constitutively by a wide range of cell types, including Met>s, TEC, lymphocytes, fibroblasts and proliferating mesangial cells [8-9]. CD44 has been attributed to a wide range of biological functions. By alternative splicing of its at least 10 variant exons CD44 created a large number of isoforms. The binding of HA to CD44 is also a highly regulated event, not all of the CD44 form constitutively bind HA [10]. In normal kidney, CD44 is only found on interstitial dendritic cells and passenger leukocytes [1112]. In contrast CD44 is considerably enhanced in inflammatory renal diseases, particularly on TECs and in glomerular crescents [13]. We have also reported the impressive up-regulation of CD44 on renal proximal TECs in MRL'P' mice with lupus nephritis and in CBA-kdkd mice with interstitial nephritis at sites where HA abundantly accumulated [14], which suggested the in vivo interaction between HA and CD44. To complement our observation as mentioned above, we have newly tested whether triggering via the CD44 by crosslinking is capable of activating the COX and TXA 2 pathway in RAW 264.7 cells. Crosslinking of CD44 IRAWB14.4, but not 1M 7.8.1, enhanced COX-2 mRNA expression, whereas COX-I mRNA did not change significantly in this cell line. Crosslinking of CD44 also increased the production of TM [15]. These findings complement the data from TXA 2 production enhanced by HA fragments in TEC and Met>, and suggest that CD44 must be one of the cell surface molecules, which mediate the HA-stimulated COX-2 expression and finally the enhanced TX~ production in these cell lines. Our findings expand the number of known proinflammatory effects by fragmented HA and suggest a link between the turnover of HA-CD44 interaction and prostanoid production in inflammatory tissue injury. THROMBOXANE AND CYCLO·OXYGENASE IN KIDNEY INFLAMMA· TORY DISEASE TXA 2, an eicosanoid metabolite from the sequential acnvines of COX and thromboxane synthase (TXS), has been originally discovered as a potent vasoconstrictor, and it may play an important role in proinflammatory events in the kidney [16]. An in vitro study of rat and human cells demonstrated the involvement of TM in angiogenesis and development of tumor metastasis [17]. In various forms of glomerular immune injury, TXA 2 production is enhanced in glomeruli [18-19]. In murine models of system lupus erythematosus (SLE). increased renal production of TXA 2 has been demonstrated both in the MRL-Fas'P' and in the NZBIW strains of lupus mice [20]. Similarly, the renal TX~ production is markedly elevated in the active phase of human SLE, and urinary levels correlate with disease severity [21]. The biological and pathological actions of TX~ occur following binding to its specific cell membrane receptor. Not only the production of TX~ was regulated during the inflammation process; marked increase of TX~ receptor protein in nephritic glomeruli was also
Enhanced thromboxane synthesis
503
demonstrated in anti-GBM rats. The enhanced intraglomerular TXA2 synthesis causes either a sequestration or desensitisation of its receptor [22]. The synthesis of TX is dependent on the activity of COX and TXS enzymes. COX is a key enzyme regulating formation of eicosanoids, and COX is therefore the major therapeutic target for nonsteroidal anti-inflammatory drugs (NSAIDs). COX initiates eicosanoids synthesis, the individual prostanoid species are generated then by special synthases. In the late 1980s two distinct COX genes were described which derive from different genes but share 60% amino acid identity [23-24]. The COX-I gene lacks a TATA box, which is typical of a "house-keeping" gene [25]. COX-2 contains a TATA box and several inducible enhancer elements including CIEBP, CRE and NF-lCB sites [26-27]. It has been suggested that the inducible isofonn COX-2 is responsible for the high prostanoid generation during inflammatory responses, although the participation of COX-I is constitutively expressed [28-29]. Enhanced expression of COX-2 has been found at inflammatory sites in animals and in patients with inflammatory diseases [16, 30]. Tomasoni and colleagues demonstrated that the expression of COX-2, but not COX-I, was enhanced parallel to the increased-TXfl, synthesis in peripheral blood mononuclear cells from patients with active lupus nephritis compared to patients in the inactive form of the disease and to healthy subjects [31]. Even though the COX-I gene is a typical "house-keeping" gene and the COX-2 gene contains several inducible enhancer elements, however, the "inflammatory COX-I" has been described in numerous studies [16-17, 32-33], and COX-2 has been suggested to adapt the physiological role of COX-I under physiological condition. COX-2 is expressed in macula densa, in the interstitial cells of medula, in mesangial cells, in neonates and in the spinal cord, and COX-2 should play an important role in the nephron development [34-41]. Considering the aspect of physiological COX-2 one should not ignore the pathological COX-2 in the COX-2 studies. The proinflammatory CQX-2 has been studied in detail. NF-lCB has been suggested as an important transcription factor for expression of the inducibel CQX-2 gene [42-44]. NF-lCB is one of the transcription factors, which HA activated during the HA fragments-induced proinflammatory process [5].
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TX~ is one of the eicosanoids, numerous studies have demonstrated that the other members of eicosanoids play an important role in inflammation, too. The different eicosanoids show a differential regulation mechanism. Penglis and colleagues [45] could demonstrate the differential regulation of prostaglandine E2 (PGE) and TXA 2, the effects of exogenous arachidinic acid (AA) and exogenous PGH2 have also been tested in this study. The distinct kinetics of PGE2 and TX~ synthesis have shown that the TXS was saturated at lower substrate concentrations than POE synthase. Eicosanoids serve as autocrine or paracrine mediators to signal changes within the immediate environment, 15_deoxy_A I2• , 4 POJ2 could regulate the expression of CQX-2 and thereby
504
The action ofhyaluronan in cells
the subsequent production of eicosanoids by activation of the peroxisome proliferatoractivated receptor-y (PPARy) or inhibition of the NF-1d3 [33, 46]. PGE2 potentiates COX-2 mRNA expression via an adenylyl cyclase/cAMP-dependent pathway [47]. The balance of different eicosanoids could probably be restored in part via feedback control [Fig. 1].
CONCLUSION HA activated the transcriptions factor NF-1d3, which is necessary for the expression of inducible COX-2 by numerous stimuli. In the kidney, synthesis and degradation of HA is mediated by specific enzymes, which are expressed in the kidney [48-49]. Impressive interstitial accumulation of HA occurs in various inflammatory renal diseases, which includes lupus nephritis, anti-GBM nephritis, tubulointerstitial nephritis and renal allograft rejection [3, 50-53]. Increased production of TX~ is a feature of several renal diseases, in particular in lupus nephritis [16]. It has been reported recently that COX-2 is upregulated in human kidneys with lupus nephritis, linking earlier observations of increased TXA2 production with the enhanced expression of this enzyme [21, 31]. HA, together with other proinflammatory stimuli, which are known to upregulate COX-2 in various cells types, including macrophages and tubular cells, could therefore cause disturbance of the local eicosanoids network and influence the immunological process [54-57]. System dependent observations give us an insight of the enhanced-TXA2 through the induction of COX-2 in inflammatory diseases. Further studies will be necessary to examine the in vivo relevance of these findings. Combined use of the selective inhibitors for COX-I, COX-2 and TXS, investigation of the balance between TXA2 and the other eicosanoids, and in vivo studies using a TX~ receptor antagonism pose the next challenge.
ACKNOWLEDGMENTS I wish to thank Dr. R. P. Wuthrich for his insightful discussion and reviewing this manuscript. I extend my thanks to Mrs. I. Schoenauer and Mr. P. Home for reviewing this manuscript.
REFERENCES I.
2.
3. 4. 5.
6.
L. Juhlin, A. Tengbald, J. P. Ortonne & 1. Ph. Lacour, 'Hyaluronate in suction blisters from patients with scleroderma and various skin disorders', Acta Dermatol. Venerol., 1986,66,409-413. Engstrorn-Laurent & R. Hallgren, 'Circulating hyaluronic acid levels vary with physical activity in healthy subjects and in rheumatoid arthritis patients. Relationship to synovitis mass and morning stiffness', Arthritis Rheum., 1987,30,1333-1338. V. Sibalic, X. Fan, 1. Lofting & R. P. Wuthrich, 'Upregulated renal tubular CD44, hyaluronan and osteopontin in kdkd mice with interstitial nephritis', Nephrol. Dial. Transplant., 1997, 12, 1344-1353. P. W. Noble, C. M. McKee, M, Cowman & H. S. Shin, 'Hyaluronan fragments activate an NF-KB/I!CBo. autoregulatory loop in murine macrophages', J. Exp. Med., 1996, 183, 2373-2378. B. Oertli, B. Beck-Schirnmer, X. Fan & R. P. Wuthrich, 'Mechanisms of hyaluronan-induced upregulation of rCAM-l and VCAM-l expression by murine kidney tubular epithelial cells: hyaluronan triggers cell adhesion molecule expression through a mechanism involving activation of nuclear factor-KB and activating protein-I', J. Irnmunol., 1998, 161,3431-3437. B. Beck-Schimmer, B. Oertli, T. Pasch & R. P. Wuthrich, 'Hyaluronan induces monocyte chemoattractant protein-I expression in renal tubular epithelial cells', J. Am. Soc. Nephrol.. 1998,9, 2283-2290.
Enhanced thromboxane synthesis 7. 8. 9.
10. 11. 12.
13.
14. 15. 16. 17.
18. 19.
20. 21.
22. 23. 24. 25.
26. 27,
28.
29.
30. 31.
505
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The action of hyaluronan in cells
32. Ch. 1. Smith, Y. Zhang, C. M. Kobildt, J. Muhammad, B. S. Zweifel, A. Shaffer, 1. 1. Talley, J. L. Masferrer, K. Seibert & P. C. Isakson, 'Pharmacological analysis of cyclooxygenase-I in inflammation', Proc. Natl. Acad. Sci., 1998,95,13313-13318. 33. D. W. Gilroy & P. R. Colville-Nash, 'New insights into the role of COX-2 in inflammation', J. Mol. Med., 2000, 78, 121-129. 34. R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois & M. D. Breyer, 'Cyclooxygenase-2 is associated with the macula densa of rat kidney and increased with salt restriction', 1. Clin. Invest., 1994,94,2504-2510. 35. Y. Guan, M. Chang, W. Chao, Y. Zhang, R. redha, L. Davis, S. Chang, R. N. Dubosi, C. M. Hao & M. Breyer, 'Cloning, expression, and regulation of rabbit cyclooxygenase-2 in renal medullary interstitial cells', Am. J. Physiol., 1997,273, FI8-F26. 36. V. L. Marcheselli & N. G. Bazan, 'Sustained induction of prostaglandin endoperoxide synthase-2 by seizures in hippocapus', J. Biol. Chern., 1996,271,24794-24799. 37. F. Beiche, S. Scheuerer, K. Brune, G. Geisslinger & M. Goppelt-Struebe, 'Up-regulation of cyc1ooxygenase-2 mRNA in the rat spinal cord following oeripheral inflammation', PEBS Letter, 1993,390,165-169. 38. M. Z. Zhang, J. L. Wang, H. F. Cheng, R. C. Harris & J. A. McKanna, 'Cyclooxygenase-2 in rat nephron development', Am. J. Physiol., 1997,273, F994-Floo2. 39. V. F. Norwood, S. G. Morham & O. Smithies, 'Postnatal development and progression of renal dysplasia in cyc1ooxygenase-2 null mice', Kidney Int., 2000, 58, 2291-2300. 40. T. Yang, J. B. Schnermann & J. P. Briggs, 'Regulation of cyclooxygenase-2 expression in renal medulla by tonicity in vivo and in vitro', Am. J. Physiol., 1999,277, FI-F9. 41. M. Klimhoff, 1. L. Wang, H. F. Cheng, R. Langenbach, J. A. McKanna, R. C. Harris & M. D. Breyer, 'Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development', Kidney Int., 2000, 57, 414-422. 42. F. D'Acquisto, T. Iuvone, L. Rombola, L. Sautebin, M. Di Rosa & R. Carnuccio, 'Involvement of NF-1CB in the regulation of cyclooxygenase-Z protein expression in LPS-stimulated 1774 macrophages', PEBS letter, 1997,418,175-178. 43. S. Nakao, Y. Ogata, E. Shimizu-Sasako, M. Yamazaki, S. Furuyama & H. Sugiya, 'Activation of NFkappaB is necessary for IL-lbeta-induced cyclooxygenase-2 (COX-2) expression in human gingival fibroblast', Mol. Cell Biochem., 2000, 209, 113- I 18. 44. Y. Chen, L. Yang & T. J. Lee, 'Oroxylin A inhibition of Iipopolysaccharede-induced iNOS and COX-2 gene expression via suppression of nuclear factor-kappaB activation', Biochem. Pharmacol. 2000,59,1445-1457. 45. P. S. Penglis, G. L. Cleland, M. Demasi, G. E. Caughey & M. J. James, 'Differential regulation of prostaglandin E, and thromboxane A, production in human monocytes: Implications for the use of cyclooxygenase inhibitors', J. Immunol., 2000,165,1605-1611. 46. H. Inoue, T. Tanabe & K. Umesono, 'Feedback control of cyclooxygenase-2 expression throgh PPARy', J. BioI. Chern., 2000, 275, 28028-28032. 47. B. Hinz, K. Brune & A. Pahl, 'Prostaglandin E, upregulates cyclooxygenase-2 expression in lipopolysaccharide-stimulated RAW 264.7 Macrophages', Biochem. Biophys. Res. Commun., 2000, 272,744·748. 48. E. Feusi, L. K. Sun, A. Sibalic, B. Beck-Schimmer, B. Oertli & R. P. Wuthrich, 'Enhanced hyaluronan synthesis in the MRL-Fas'P' kidney: Role of cytokines', Nephron, 1999,83, 66-73. 49. L. K. Sun, E. Feusi, A. Sibalic, B. Beck-Schimrner & R. P. Wuthrich, 'Expression profile of hyaluronidase mRNA transcripts in the kidney and in renal cells', Kidney Blood Press. Res., 1998, 21,413-418. 50. K. Nishikawa, G. Andres, A. K. Bhan, R. T. McCluskey, A. B. Collins, 1. L. Stow & I. Stamenkovic, 'Hyaluronate is a component of crescents in rat autoimmune glomerulonephritis', Lab. Invest., 1993, 68, 146-153. 51. R. P. Wuthrich, X. H. Fan, T. Ritthaler, V. Sibalic, D. J. Yu, J. Loffing & B. Kaiss1ing, 'Enhanced osteopontin expression and macrophage infiltration in MRL-Fas'P' mice with lupus nephritis', Autoimmunity, 1998,28,139-150. 52. J. Wu, G. Fan, K. Kitazawa & T. Sugisaki. 'The relationship of adhesion molecules and leukocyte infiltration in chronic tubulointerstitial nephritis induced by puromycin aminunucleoside in Wistar rats', Clin. Irnmunol, Immunopathol., 1996,79,229·235. 53. R. Hallgren, B. Gerdin & G. Tufveson, 'Hyaluronic acid accumulation and redistribution in rejecting rat kidney graft. Relationship to the transplantation edema', J. Exp. Med., 1990, 171,2063-2076. 54. S. Arias-Negrete, K. Keller & K. Chadee, 'Proinflammatory cytokines regulate cyclooxygenase-2 mRNA expression in human macrophages', Biochem. Biophys. Res. Commun., 1995,208,582-589.
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55. A. Schneider & R. A. Stahl, 'Cyclooxygenase-2 (COX-2) and the kidney: current status and potential perspectives', Nephrol. Dial. Transplant., 1998, 13, 10-12. 56. T. Yang, D. Sun, Y. G. Huang, A. Smart, J. P. Briggs & J. B. Schuermann, 'Differential regulation of COX-2 expression in the kidney by lipopolysaccharide: role of CDI4', Am. J. Physiol., 1999,277,
Flo-F16. 57. H. Kobayashi, G. W. Sun & T. Terao, 'Production of prostanoids via increased cyclo-oxygenase-2 expression in human amnion cells in response to low molecular weight hyaluronic acid fragment', Biochim. Biophys. Acta, 1998, 1425,369-376.
PART 8
KERATINOCYTES AND HYALURONAN
HYALURONAN METABOLISM AND DISTRIBUTION IN STRATIFIED, DIFFERENTIATED CULTURES OF EPIDERMAL KERATINOCYTES Sanna Pasonen-Seppinen 1*, Raija Tammi1, Markku Tammi 1, Michael HOIDf, Vincent C. HascaU2, Donald K. MacCaDum3 }Department ofAnatomy, University ofKuopio, P.O.Box J627, FIN-702 JJ Kuopio, Finland. ZDepartment ofBioengineering,
3Department ofAnatomy and Cell
Cleveland Clinic Res. Inst.• Cleveland, OH
Biology, University ofMichigan, Ann Arbor, MI.
ABSTRACT
Hyaluronan is a major intercellular matrix molecule in the vital cell layers of skin epidermis. An organotypic culture model was developed to study epidermal hyaluronan metabolism by using a rat epidermal keratinocyte line (REK) which exhibits histodifferentiation similar to that of the native epidermis when cultured at an air-liquid interface. Two different support matrices were used: reconstituted collagen fibrils with and without a covering basal lamina previously deposited by canine kidney cells. REKs formed a stratified squamous, keratinized epithelium on both support matrices. Hyaluronan and its receptor, CD44, colocalized in the basal and spinous layers similar to their distribution in the native epidermis. Most (~75%) of the newly synthesized hyaluronan was retained in the epithelium when a basal lamina was present while most (~80%) diffused out of the epithelium in its absence. While REKs on the two matrices synthesized hyaluronan at essentially the same rate, catabolism of the macromolecules was much higher in the epithelium on the basal lamina with a half-life of approximately one day, similar to its half-life in native human epidermis. The formation of a true epidermal compartment bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells provides a good model within which to study the regulation of synthesis and catabolism of epidermal hyaluronan, isolated from the influence of dermal cells, or supporting cells (feeder cells) included in a collagen matrix. KEYWORDS Hyaluronan, epidermis, keratinocyte, organ culture, collagen, basal lamina, basement membrane INTRODUCTION Hyaluronan is generally regarded as a component of connective tissue extracellular matrices, but it is also present in restricted locations within the nervous system I and in certain epithelial tissues 2 including its universal presence within stratified epithelia 3. In epidermis, hyaluronan is present in exceedingly high concentrations 4, in the small intercellular space between epidermal keratinocytes. Additionally, studies of human skin organ cultures have demonstrated that hyaluronan within the epidermis is rapidly
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Keratinocytes and hyaluronan
turned over, an observation that suggests that the epidermis possesses mechanisms to catabolize hyaluronan that are closely coordinated with its synthesis by keratinocytes 5. In skin, both epidermal keratinocytes and the cells present in the dermis (principally fibroblasts) synthesize hyaluronan 4. Because both epidermis and dermis form and contain hyaluronan, it is difficult to resolve the specific contribution of each component to the overall metabolism and content of this macromolecule in skin. To understand more completely the nature of hyaluronan synthesis and distribution in the epidermis, we have studied a rat epidermal cell line in culture, first in monolayer culture 6 and now we extend these studies to the metabolism of hyaluronan in a reconstituted epidermis formed by the rat epidermal keratinocytes when grown on collagen at the air-liquid interface. This organotypic culture model facilitates reformation of a stratified epithelium that closely recembles the native epidermis. In order to form a true epidermal compartment bounded superiorly by the stratum corneum and inferiorly by a basal lamina, we cultured the rat epidermal keratinocytes on a collagen matrix that was covered by a basal lamina previously deposited by Maden Darby canine kidney (MDCK) cells which were then removed from the matrix prior to adding the keratinocytes. The presence of the basal lamina on the collagen matrix altered the metabolism and distribution of hyaluronan when compared to the "open" collagen matrix alone. A comparison of hyaluronan metabolism on the two different culture substrates and the possible role of the basal lamina in altering the metabolism of hyaluronan are the subjects of this report. MATERIALS AND METHODS Keratinocyte culture REK stock cultures were grown in Dulbecco's modified Minimal Essential Medium (I g glucose / liter), 10 % fetal bovine serum, 50 Ilg / ml gentamicin S04 at 37°C in humidified 95% air /5% C02. Keratinocytes were subcultured twice a week in a usual manner as described in Tammi et al. (2000) 7. Collagen substrate Commercially available rat tail collagen (Sigma, St. Louis, MO) was dissolved at a concentration of 2.2 mg / ml in 0.034 N acetic acid at 4°C overnight. The dissolved collagen was dialyzed at 4°C against 2 x 500 volumes of 0.2 M NaCI in 50 mM trisHCl, pH 7.6, over 24 hours. The dissolved collagen, 800 Ill, was added to individual 24.5 mm diameter tissue culture inserts (Costar Transwell'", 3.0 urn pore size). Before use, collagen was polymerized by incubating the tissue culture inserts for 45 minutes at 37°C in a humidified atmosphere. Pre-formed basal lamina Maden Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (Bethesda, MD) and were subcultivated onto collagen gels in the tissue culture inserts (200,000 cells / 24.5 mm insert). The MDCK cells were fed 3 X per week for 18 - 22 days using the same medium as that used for the keratinocytes. MDCK cells were removed as described in Tammi et al. (2000) 7.
Metabolism and distribution
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Organotypic keratinocyte cultures Recently confluent cultures of REKs with little or no morphological evidence of stratification were subcultivated onto either: 1. collagen covered (designated BL -) or 2. basal lamina coated / collagen covered (designated BL +) Transwell'v culture inserts at a concentration of 150,000 - 200,000 REKs / 24.5 mm insert. The subcultivated REKs were grown for 2 days with culture medium present both in the well beneath the Transwell'" insert as well as on the surface of the cells. The culture medium was subsequently removed from the surface of the cells and the level of the medium beneath the Transwelll'' insert adjusted to the level of the REKs present on the BL- or BL+ collagen gels. The cultures for this study were grown for an additional seven days at 37°C in humidified 95% air /5% C02 with the medium being changed 3 X per week. Electron microscopy Culture substrates were processed for electron microscopy as described in Tammi et al, (2000) 7, and examined by scanning electron microscopy. The BL- and BL+ cultures were examined as either epoxy embedded 1 J.LDl thick light microscopic sections or as conventional electron microscopic thin sections. The cultures were fixed for 2 hours at 4°C in a 50:50 mixture of buffered 2.5% glutaraldehyde and 2% aqueous OS04 with the fmal mixture containing 4% sucrose and 2 mM CaCh. The samples were dehydrated in ethanol and propylene oxide and embedded in epoxy resin. Sections were stained with either toluidine blue (light microscope) or uranyl acetate and lead citrate (electron microscope.) Histochemical demonstration of hyaluronan and CD44 localization Hyaluronan and CD44 antibody (OX-50) stainings were done as described in Tammi et al. (2000) 7 and Wang et al. (1992) 8. Metabolic labeling of REK cultures Nine days after subcultivation the REK cultures were metabolically labeled with 20 /lCi/ml of eH]glucosamine and 100 /lCi/ml of 35S04 (both compounds were from Amersham, Little Chalfont, UK) in culture medium. Cultures were analyzed at 3, 6,9, 12, 18 and 24 hours after labeling. At 15 hours or 24 hours, the radiolabeled precursors were then added in a small aliquot from a concentrated stock solution to give fmal concentrations equivalent to the continuously labeled cultures. In each case, these cultures were incubated for an additional 3 hours giving labeling windows of 15 - 18 hours and 24 - 27 hours. Three "compartments" of the cultures (medium, epithelium, matrix) were analyzed. Values for two of the compartments (medium and matrix) were combined and expressed as a single value. At the end of each labeling interval, the medium (2 ml) was collected and the culture inserts were washed once with 0.5 ml Dulbecco's PBS which was added to the medium (= medium compartment). Thereafter the cultures were immersed in 1 ml 0.04% sodium EDTA in PBS for 5 min at 37°C to separate the epithelium from the matrix. The epithelial sheets (= epithelial compartment) were mechanically separated from the matrices using stereo microscope and fine needles. Purification of radiolabeled glycosaminoglycans and ion exhange cromatographyare described in Tammi et al. (2000) 7 and Tammi et al. (1998) 6.
514
Keratinocytes and hyaluronan
RESULTS AND DISCUSSION Morphology of the REK organotypic cultures To study hyaluronan metabolism in reconstituted epidermis, rat epidermal keratinocytes were cultivated on collagen substrate with or without basal lamina at airliquid interphase. REKs subcultured onto either substrate (BL- / BL+) stratified and exhibited the normal epidermal differentiation. There was no difference in general morphology between the two culture models. The major difference was evident at the junction of basal keratinocytes with the underlying matrix. Basal keratinocytes grown on collagen alone exhibited numerous, small cytoplasmic processes extending from the undersurface of the cell down into the meshwork of collagen fibrils. Basal cells apposed to the basal lamina exhibited a smooth undersurface, and ultrastuructural studies revealed numerous hemidesmisomes along basal cell plasma membrane. Electron microscopy of the REKs cultured on the two different substrata confirmed the light microscopic results. HA and hyaluronan reseptor (CD44) localization As in normal epidermis 8, REKs produced large amounts of hyaluronan, which was colocalized with the HA binding receptor, CD44. HA was present between keratinocytes in the spinous and basal cell layers, with the strongest signal present between adjacent keratinocytes comprising the spinous cell layer. In BL- cultures, but not in BL+ cultures, HA was demonstrable in the subjacent collagenous matrix. Biosyntheisis of g1ycosaminoglycans To study glycosaminoglycan biosynthesis and compartmentalization of hyaluronan in REK organotypic cultures, the REKs were metabolically labeled with eH]glucosamine and 35S04. The total incorporation of eH] into glycosaminoglycans is shown in Fig.l as a function of labeling time and for individual 3 hour labeling windows. In chondroitin sulfate and heparan sulfate biosynthesis, there was no difference between cultures with or without basal lamina (data not shown). However, in the biosynthesis of hyaluronan, the predominant glycosaminoglycan in REK cultures, there was a distinct difference between the two culture configurations with significantly less label in the BL+ configuration at times greater than 10 hours (Fig. 1). Because there is no significant difference between the rates of hyaluronan synthesis between the two culture configurations (Fig. I), this result indicates that cultures with a basal lamina are catabolizing significantly more of the hyaluronan that was synthesized during the labeling period. According to our results, we expect that the half life of a newly synthesized hyaluronan molecule in cultures with a basal lamina will be close to that observed for explants of human skin 5. Compartmentalization of hyaluronan In cultures lacking a basal lamina, abundant, large HA-molecules were found in the supporting collagenous matrix and underlying medium. In contrast, hyaluronan recovered from the basal lamina-covered matrix and underlaying medium was reduced in amount (Fig. 2) and of smaller molecular mass suggesting that macromolecular hyaluronan was retained within the epithelium. This indicates that the basal lamina
Metabolism and distribution
515
deposited by the MDCK cells formed an effective barrier to high molecular weight hyaluronan.
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Figure 2.
Newly synthesized hyaluronan (total, panel A), and amounts in the epithelium (panel B) and the subjacent matrix plus medium compartments of organotypic keratinocyte cultures (panel C). The curves show the data points fitted to logarithmic equations. The symbols are the same as in Fig. 1.
516
Keratinocytes and hyaluronan
Taken together, the key observations from these experiments were that: 1. the total amount of 3H-labeled hyaluronan accumulated in cultures without a basal lamina was greater than in cultures with a basal lamina, and 2. conversely, the total amount retained in the epithelium was greater in cultures with a basal lamina.
CONCLUSIONS Rat epidermal keratinocytes cultured on collagen or basal lamina-covered collagen matrices grow, stratify and differentiate in a manner similar to the native epidermal tissue. The distribution of hyaluronan and the hyaluronan receptor CD44 within the "epidermis" formed by the cultured keratinocytes is similar to that in native epidermis as is the exceptionally active synthesis and catabolism of hyaluronan by the keratinocytes. The addition of the basal lamina to the collagen matrix permits the formation of a true epidermal compartment bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells. The basal lamina forms a true barrier to the diffusion of macromolecular hyaluronan away from the keratinocytes and results in increased catabolism of the molecule within the epithelium. Thus, the rat epidermal keratinocytes cultured on a basal lamina-covered collagen gel provides a good model within which to study epidermal metabolism isolated from the influence of dermal cells or other supporting cells included in a collagen matrix.
REFERENCES 1. 2. 3.
4.
5.
6.
7.
8.
A. Bignami, M. Hosley & D. Dahl, Hyaluronic acid and hyaluronic acid-binding proteins in brain extracellular matrix. Anat. & Embryol., 1993, 188,419 - 33. R. Tammi, S. Ronkko, U. Agren & M.Tammi, Distribution ofhyaluronan in bull reproductive organs. 1. Histo. Cytochem., 1994a, 42, 1479 - 86. R. Tammi, M. Tammi, L. Hakkinen & H. Larjava, Histochemical localization of hyaluronate in human oral epithelium using a specific hyaluronate-binding probe. Archives ofOral Biol., 1990.35,219 - 224. R. Tammi, U. Agren, A.-L.Tuhkanen & M. Tammi, M, Hyaluronan metabolism in skin. IN: Progress in Histochemistry and Cytochemistry, (ed. W. Graumann) Stuttgart: Gustav Fischer Verlag,1994b pp 1 - 81. R. Tammi, A.-M. Siiiimiimen, H. I. Maibach, M & Tammi, Degradation of newly synthesized high molecular mass hyaluronan in the epidermal compartment and dermal compartments of human skin in organ culture. J. Invest. Dermatol. 1991,97, 126 - 130. R. Tammi, D. MacCallum, V. C. Hascall, J-P. Pienimaki, M. Hyttinen & M. Tammi, Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharidies but not hexasaccharides. 1. Bioi. Chem., 1998, 273, 28878 88. R. Tammi, M.Tammi, V. C. Hascall, M. Hogg, S. Pasonen & D. K. MacCallum, A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices, Histochem. Cell Bioi., 2000,113,265-277. C. Wang, M. Tammi & R. Tammi, Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages, Histochemistry, 1992, 98, 105-112.
HYALURONAN AND HYLAN IN THE TREATMENT OF OSTEOARTHRITIS Charles Weiss Chairman, Department ofOrthopaedics and Rehabilitation, Mount Sinai Medical Center Clinical Professor ofOrthopaedics University ofMiami School ofMedicine 6431 Pine Tree Drive Circle, Miami Beach, Florida 33141 USA
ABSTRACT
Endre Balazs' recogmtion that some forms of osteoarthritic pain are related to decreases in the elastoviscous properties of hyaluronan and his purification of a noninflammatory fraction of hyaluronan led him to develop the concept of viscosupplementation. The first hyaluronan viscosupplemcnt Healon® produced in his laboratory, had a mw of 2-3 million, and was safe and effective in the treatment of early to moderate human osteoarthritis. He subsequently developed hylans: cross-linked hyaluronans in which the carboxylate and acetamido groups are not chemically modified. Hylan G-F20 (Synvisc®) was specifically formulated to behave as a synovial fluid prosthesis. It has similar physicochemical properties to the synovial fluid found in young healthy adults, prolonged residence time, the same permeability, biocompatibility and lack of immunogenecity as umnodified hyaluronan. Clinical studies have demonstrated its safety and efficacy compared to saline, NSAIDs, and low molecular weight hyaluronans in all stages of osteoarthritis and over multiple courses. KEYWORDS
Hyaluronan, hylan, viscosupplementation, osteoarthritis INTRODUCTION
Osteoarthritis is the leading cause of disability in the United States in patients over 50. lts treatment consumes up to 1% of the gross national product, and the knee is the most commonly involved weight bearing joint.i" Recently viscosupplementation has been added to the therapeutic modalities available to treat this disease. This chapter will cover the history and mechanism of action of viscosupplements, clinical trials of hylan G-F20 in the treatment of osteoarthritis of the knee, its efficacy in advanced disease, comparison to NSAlDs, and to other clinically used hyaluronans. Its safety will be compared to other standard treatments. Finally on the basis of safety and efficacy the position of viscosupplementation with hylan G-F20 in the treatment algorithm for osteoarthritis of the knee will be defined. VISCOSUPPLEMENTATION: HISTORY AND MECHANISM OF ACTION.
More than thirty years ago Balazs proposed that some types of osteoarthritic pain are related to decreases in the elastoviscous properties of hyaluronan in osteoarthritic synovial fluid. s.6 In normal synovial fluid the concentration ofhyaluronan is 3 to 4 mg/ml
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Clinical applications ofhyaluronan
and its molecular weight averages 4-5 million. 7 It is a polyanion with regularly spaced mutually repellant negative charges composed of repeating units of N-acetyglucosamine and glucuronic acid. The hydrated molecule occupies 1000 times the volume of the unhydrated molecule.' At a concentration of 0.3 mg/ml these molecules completely fill their solvent; thus, these solutions and intercellular matrixes (concentration 3-4 mg/ml) consist of highly overlapping crowded entangled polyanionic molecular networks which influence every molecule, compound, fibril and cell that is bathed by them. A hyaluronan rich layer is concentrated on the articular cartilage surface corresponding to the lamina splendens and is imbibed into the tangential zone. 9,IO Levick found a similar layer on the surface of synovium and compared it to a filter cake formed as the synovial fluid hyaluronan is pumped through synovial tissue by joint movement. This acts as a molecular filter influencing the movement and distribution of large molecules such as fibrinogen and irnmuno- globulins into the joint. 11 The crowded molecular state and high molecular weight of this polyanion confers elastoviscous properties which are essential for its physiologic functions. In its undisturbed state synovial fluid has a high intrinsic viscosity (five hundred thousand times greater than of saline) and thus is able to sustain tissue spaces. 12 When subjected to slow movement, low strain frequency, low rates of deformation, the rate at which mechanical energy is transmitted to this concentrated network of molecules is slow enough to allow time for the molecules to configurationally adjust and line up parallel to the direction of flow (pseudoplasticity), and therefore dissipate energy through viscous flow and heat. This is called the loss modulus or dynamic viscous modulus of the material. When subjected to high strain frequencies or high rates of deformation, the molecules are unable to configurationally adjust and therefore become increasingly entangled and act as elastic solids storing energy. This is known as the storage modulus (dynamic elastic modulus) a measure of the material's elasticity. At rest or with slow joint movement normal synovial fluid behaves predominantly as a viscous fluid. On physiologic movement and when subject to stresses somewhat less than those occurring during slow walking the material begins to act predominantly (cross-over point) as an elastic solid. In pathologic synovial fluids (those with decreased molecular weight and/or concentration of hyaluronan) the fluids behave predominantly as viscous fluids without the tissue protection of an elastic solid when subject to deformation forces within the physiologic range.13- 1S Decreases in the molecular size and/or concentration of the hyaluronan molecule causes changes in the overlap and entanglement of this molecule which affects the rheological properties of the fluid. The behavior of cells known to be involved in the pathogenesis of osteoarthritis is affected by the rheological properties of the hyaluronan matrix which surrounds them. High molecular weight hyaluronan at concentrations present in normal young adult synovial fluid and connective tissues affects many activities of the lymphomyeloid system by decreasing cell migration, pinocytosis, prostaglandin and bradykinin release, mitosis, free radical production, lymphocyte activation, fibroblast migration, phagocytosis and stabilizes nociceptor activity.16-22 Hyaluronan solutions of low elastoviscous properties (low concentration and/or molecular weight) as are found in arthritic joints, inflammation, etc. have the reverse effect with increased: migration and division of leukocytes, macrophage activity, fibroblast migration, degradative and inflammatory enzyme release, free radical formation, increased nociceptor sensitization and activation. Synovial cells similarly are influenced by the hyaluronan in their environment. The addition of exogenous high molecular weight hyaluronan has been shown to stimulate synoviocytes to synthesize hyaluronan invitro while the addition of low molecular weight hyaluronan does not have that effect.23
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In 1968 Balazs purified the first non-inflammatory fraction of sodium hyaluronan (NifNaHa) which was suitable for medical use. 24 It had a molecular weight of 2-3 million. Studies in a canine model with surgically induced partial thickness cartilage injury demonstrated that the introduction of exogenous hyaluronan diminished pain (dogs used the hyaluronan treated knees in preference to the untreated knees) and increased healing. 25 The intra-articular injection of hyaluronan into the arthritic joints of race horses who were lame and had failed to respond to conventional treatment resulted in significant improvement in symptoms and return to racing. 25 This device was commercialized by Pharmacia for use in veterinary medicine as the first viscosupplement. The reduction in pain and limping in these horses persisted long after the exogenously introduced hyaluronan had cleared the joint.25 Subsequent studies demonstrated that the synovial fluid obtained from the arthritic joints of race horses had decreased elastoviscous properties and the synovial fluid obtained from those joints that responded to viscosupj,lementation treatment had a return of their elastoviscous properties to normal levels? 7 Those animals that failed to respond continued to have synovial fluid of reduced elastoviscous properties. The clinical improvement in race horses was dependent upon the molecular weight and rheological properties of the material injected into the joint i.e. the higher the molecular weight and concentration, the higher the elastoviscosity and the better the clinical result. 26 ,27 Several studies in Japan confirmed the relationship between pain response and the elastoviscosity of exogenously introduced hyaluronan. Gotoh, utilizing a bradykinin pain model in rats demonstrated that the hi~her the elastoviscosity of exogenously added hyaluronan the greater the pain reduction? Pain in arthritic synovitis produced in rats by injecting monosodium urate crystals intraarticularly was decreased by injecting high molecular weight hyaluronan one hour prior to urate injection. This study used a direct quantitative measure of the pain mediators bradykinin and prostaglandin E2 rather than a behavioral measure and demonstrated that only at the highest molecular weight hyaluronan did a significant decrease in pain mediator concentration occur.i" The first human studies of viscosupplementation in the treatment of osteoarthritis were conducted from 1969 to 1973: in Sweden by Rydell, in South Africa by Helfet,28 in France by Peyron,29,30 in the United States by Weiss and in Great Britain by St. Onge. 31 The hyaluronan Healon® used in these investigations had an average molecular weight of 2-3 million and was produced by Biotrics, Inc. In 1974 Helfet reported the results of a single injection in 20 hips and 36 knees with osteoarthritis: 95% of injections produced rapid relief of pain lasting an average of 6 weeks in hips and 10 weeks in knees. No adverse events were noted." In 1974 Peyron injected 23 osteoarthritic knees with 1,2,or 3 ml of Healon® (1% solution, l Orng/ml.) Two mls injected twice had the best results (74% positive responsej.i" A separate study demonstrated that symptomatic improvement after Healon® injection was accompanied by an increase in hyaluronan concentration and limiting viscousity number.'" This treatment was most effective in patients with a short duration of diseaser'" Weiss injected 2 ml of Healon® twice in two weeks in the osteoarthritic knees of 16 patients and 16 patients received sham injections. Evaluation from one to 12 weeks after the second injection showed a significant decrease in symptoms in the Healon® injected knees. Some difference though not statistically significant was present at 6 months in those patients with severe pain and mild to moderate disease." Isdel concluded in a dose range study with Healon® that 20 milligrams was the appropriate unit dose. 32 Overall Healon® was effective in relieving pain in patients who had high levels of pain, mild to moderate disease on x-ray and symptomatic arthritis of relatively recent onset. In patients with more advanced disease it
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Clinical applications ofhyaluronan
was less effective. Therefore Balazs set about to develop an ideal viscosupplement for arthritis having elastoviscous properties and molecular weight similar to that found in the synovial fluid of normal young adults, increased local tissue residence time, and the same bio-compatibility and permeability to metabolites and macromolecules as the native hyaluronan.P In 1987 two low molecular weight (500,000 - 800,000 mw) hyaluronans, Artz® manufactured by Seikegaku in Japan and Hyalgan® manufactured by Fidia in Italy, became available commercially to treat arthritic joints. For purposes of classification since more than 90% of hyaluronan molecules in normal synovial fluids are more than 2.0 M molecular weight products of average mw 2.0 M or more will be classified as high molecular weight and those below 2.0 M will be classified as low molecular weight. Clinical experience with Artz® indicated that, at the usual treatment dose (25 mg in 2.5 ml injected five times at one week intervalsj." patients with mild or moderate disease clinically and radiologically, had satisfactory results, while those with advanced radiologic damage or large effusions, did not." Hyalgan® is a I% solution of hyaluronan molecular weight 500,000 to 700,000: 2 ml contains 20 milligrams of hyaluronan. Five weekly injections are required for a positive effect as three weekly injections have shown no difference from saline controls. 30,35 A recent double-blind controlled study reported that 5 injections of Hyalgan were statistically significantly better than saline controls in patients with mild to moderate osteoarthritis of the knee and equivalent to NSAIDs. 36 In the 1980's Balazs and coworkers developed a hyaluronan derivative with the characteristics of an ideal viscosupplement. Hylan is the generic name for a family of a cross-linked hyaluronan polymers in which neither the carboxylate or the acetamido group of hyaluronan is chemically modified.37,38 Two hylan polymers are currently used in medicine: hylan A (average mw 6 million) which dissolves in aqueous solution to form a highly elastoviscous solution and hylan B which becomes hydrated in aqueous solution to form highly elastoviscous solids and gels. Hylan G-F20 (Synvisc®) is the specific hylan composition formulated to behave as a synovial prosthesis. It has similar physico-chemical properties to the synovial fluid found in a healthy young adult. It is highly elastic over a wide range of strain frequencies and its intra-articular residence time is extended compared to native hyaluronan and low molecular weight hyaluronans. 38,39 It has the same permeability, biocompatibility and lack of irnmunogenicity as unmodified hyaluronan. The comparisons of molecular weight distributions of hylan G-F20, normal synovial fluid, osteoarthritic synovial fluid and two low molecular weight products arc seen in Figure 1. In normal adult synovial fluid hyaluronan molecular weight distributions peak at 6-7 million and the majority of chains are distributed between 3 and 6 million. In osteoarthritic synovial fluid the molecular weight distribution is much broader, peaks at a lower molecular weight, and includes a significant proportion of chains below 3 million with very few below 1 million. The molecular weight distribution of hylan G-F20 is very similar to the distribution of molecular weights found in young healthy synovial fluid averaging 6 million and distributing mostly between 3-7 million. In contrast the molecular weight distribution of two other hyaluronan viscosupplementation products are lower than even the osteoarthritic synovial fluid. A comparison of the elastic and viscous behavior of normal and osteoarthritic synovial fluid compared with a hyaluronan and hylan viscosupplement is shown in Figure 2.
Treatment or osteoarthritis
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0,12 . , . - - - - - - - - - - - - - - - - - - - - - - - - ,
0,1
Hylan G-F 20 (MW6million)
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~ 0,08
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"
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Fig. I. The molecular weight distribution ofHA in normal and osteoarthritic synovial fluid and in viscosupplementation products." 100
0
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80
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,0
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, 60
, 30
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Fig.2 Elastic and viscous behavior of normal and osteoarthritic synovial fluid compared with low molecular weight HA and a Hylan viscosupplement.Y Hylan G-F20 was formulated to be similar to normal young adult synovial fluid which in the physiologic range of activity behaves predominantly as an elastic solid whereas other hyaluronan viscosupplements remain predominantly viscous fluids throughout all physiological ranges. When hylan G-F20 is injected into an osteoarthritic joint it raises the elastoviscousity of the endogenous synovial fluid into the normal range. This transformation can not occur with any other hyaluronan product on the market today.
472
Clinical applications ofhyaluronan
Recent neurophysiological studies in the cat knee confirm the observations of prior behavioral pain models and demonstrate the effects of elastoviscosity on nociceptor activity. The discharge frequencies of nociceptors of the medial articular nerve were measured after inflammatory and mechanical stimulation. Discharge frequency was significantly reduced after intra-articular injection of highly elastoviscous hylan G-F20 but was not when the same concentration of non-elastoviscous degraded hylan G-F20 was injected. The effect was most pronounced during movement of the inflamed joint. Thus elastoviscosity directly influences nociceptors responsible for pain perception. 21,22 Other factors of course play a role in the reduction of pain. The decrease in pain following the injection of low elastoviscous materials, is likely the result of repeated dilution of noxious stimuli, polyanionic levage, as well as repeated mechanical and possible physiochemical stimulation of synovial lining cells.4o RYLAN G-F20 CLINICAL EFFICACY Over the past several years the safety and efficacy of hylan G-F20 in the treatment of osteoarthritis of the knee has been evaluated in seven double-blind controlled studies: 3 vrs saline, I vrs arthrocentesis, 2 vrs NSAID and 1 vrs low molecular weight hyaluronan. Six additional studies have been reported: I multi-center retrospective study, I retrospective study comparing a low molecular weight hyaluronan to hylan G-F20, I open label study of hylan G-F20 vrs saline, and 3 prospective open label studies in advanced disease. The first two prospective multi-center randomized double-blind studies were designed to identify the optimal treatment regimen. Two 2 ml intra-articular injections of hylan GF20 administered two weeks apart were compared with three injections given one week apart and both were compared with a control treatment of intra-articular saline. Patients had predominantly Larsen grades II and III osteoarthritis. Two bi-weekly hylan G-F20 injections were significantly better than saline control by week four and remained so through week twelve (p< 0.05» for the outcome measurements of pain on weight bearing, overall pain and overall treatment evaluation. The three injection regimen was significantly better than two injection regimen by week 8 for the same parameters and remained so through week twelve.41 The third clinical trial was a multi-center prospective randomized, controlled (intra-articular saline) double-blind three injection regimen study. There were 57 knees in the hylan G-F20 population and 60 in the control. Clinical outcome measures included pain during weight bearing, night pain, pain during knee movement and overall assessment of pain.42 The mean age was 62 ±I years, 65% of patients were females, mean duration of osteoarthritis was 6 ±0.5 years, and 82% had radiographic Larsen grades II and III. Patients with effusions were excluded. In the hylan G-F20 group more than 50% of patients were symptom-free (VAS <20) at 12 weeks and significantly (p < 0.001) better than saline controls for patient evaluated weight bearing pain, night pain, pain on movement and overall pain. At 26 weeks these improvements continued (p<0.005) for weight bearing pain, night pain and decrease of activity. In the 26-week follow up period only 7% of patients in the hylan G-F20 group required NSAIDS and 4% required analgesics. In the control group, where 53% of patients required NSAIDS, corticosteroids or surgery these differences were statistically significant (p:'S0.05).42 A multi-center three injection double blind prospective, randomized, U.S. trial compared hylan G-F20 to arthrocentesis. 12% of patients had Kellgren Lawrence grade IV disease. 52 knees received hylan G-F20 and 52 arthrocentesis. After an initial 4 week
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washout period 31 patients, who worsened by more than 20 millimeters on a pain VAS scale after withdrawal of NSAlD, were considered a flare population. Patients with inflammatory disease were specifically included in this study. Hylan G-F20 was better than arthrocentesis in pain relief (p<0.0001) and reduction of effusion (p<0.005) in the intend to treat population as determined by an analysis of covariance. In patients who received two courses of treatment when followed up 26 to 36 weeks post treatment a categorical analysis showed that more than half had better than a 50% improvement in every outcome measure. This was significant for pain with motion (p<0.02), rest pain (p<0.05), and overall pain (p<0.05) compared to control. In the flare population there was significant (p<0.05) improvement in hylan G-F20 treated knees compared to control with >50% improvement in pain on motion, rest pain, night pain, walking and overall pain.43 Two double-blind multicenter, prospective randomized studies compared hylan G-F20 to NSAID. In the first there were three intend to treat populations: one received NSAID plus a control of 3 weekly arthrocentesis, the second received hylan G-F20 plus placebo pills; and, the third received NSAID plus hylan G-F20. Patients were studied for 26 weeks. 65% were females with a mean age of 61 ±1 and mean duration of disease of 6 ± 0.6 years. 73% had Kellgren-Lawrence grades II and III. All patients had baseline pain on motion of> 50 on a visual analog scale. At 12 weeks hylan G-F20 was at least equivalent to NSAID in improving pain parameters and overall assessment. By 26 weeks both hylan G-F20 groups were statistically superior to NSAID in percent of symptom free patients. Repeat measures analysis demonstrated statistically significant improvement of hylan G44 F20 versus NSAID alone (p<0.05) with regard to overall pain level. A second study compared hylan G-F20 to Diclofenac." There were three study groups: hylan G-F20 patients received arthrocentesis and 3 injections of hylan G-F20 plus a placebo pill Diclofenac patients received diclofenac plus three arthrocentesis and the double control patients received three arthrocentesis and a placebo pill. Primary outcome measures were the WOMAC pain, stiffuess and function osteoarthritis index, the Lequesne index and the overall assessment of response to treatment. At three months there was significant improvement (p<0.03) of hylan G-F20 over double control for all parameters of the WOMAC scores and no statistical significance between Diclofenac and control. All parameters of the Lequesne index demonstrated statistically significant improvement of hylan G-F20 over Diclofenac and over control and no difference between Diclofenac and control." This lack of difference between Diclofenac and control may be accounted for by the nature of the control, which consisted of 3 arthrocentesis thereby both diluting nerve stimulating or sensitizing agents such as bradykinins, prostaglandins etc. as well as stimulating the synovium mechanically.t" A multi-center retrospective study over a 2-1/2 years was carried out in 336 patients, (122 treated bilaterally), 458 knees, and 1537 injections.t" 56 knees in 41 patients had a second course, and mean patient age was 65 ± 0.7 years with a mean duration of disease of7.0 ± 6.2 years. 63% of the patients were female. 77% of the knees were rated better or much better in response to initial treatment, 21% were the same and 2% were worse or much worse. After a second course of treatment 87% of knees were rated better or much better, 9% the same and 4% worse. The mean time to retreatment was 8.2 months with a range of 2.4 to 18.6 months. Treatment was effective across the entire spectrum of disease with 58% of patients with Kellgren-Lawrence grade IV arthritis of the medial compartment of the knee rated better or much better. Up to 90% of patients with less severe disease were better and over 70% of patients were able to use less analgesics and NSAIDs. 46
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Clinicalapplications ofhyaluronan
An open label 12 week, multi-center study was conducted with hylan G-F20 in 222 patients, 256 knees, 56% had Larsen grades III and IV arthritis and had continued pain on activity despite treatment with analgesics and anti-inflammatory medications.f" After a two week wash out period patients received three arthrocentesis and 3 hylan G-F20 injections. All outcome measures: night pain, weight bearing pain, overall improvement were significantly improved (p=O.OOOI) one through 12 weeks post treatment. Improvement occurred in all grades of severity. A prospective randomized double-blind multi-center trial compared the clinical efficacy of a high molecular weight (hylan G-F20) to a low molecular weight hyaluronan (Artz® 750,000 mw) in the treatment of osteoarthritis of the knee.49 73 knees were studied in 70 patients, 38 received hylan G-F20 and 35 received Artz. A 3 injection regimen was utilized and evaluation was performed at 12 weeks. 51% of patients were female, mean age of 60 ±2 years, duration of osteoarthritis was 4.6 years, 76% had Larsen x-rays grades II and III. At 12 weeks patients receiving hylan G-F20 were significantly better (p<0.05) in all primary outcome measures (overall condition, most painful knee movement, weight bearing pain and overall pain) as well as symptom free patients. A retrospective study was reported of 200 patients who received either 5 weekly injections of low mw hyaluronan or 3 weekly injections of hylan G-F20. A 10 point Likert scale was used to rate pain and restriction of movement before and after treatment as well as severity of side effects. Both groups were significantly better than before treatment in weight bearing pain, mobility and pain at rest. Hylan G-F20 was significantly better than hyaluronan in relief of pain on weight bearing, improving mobility (p<0.03) and night time awakening pain (p<0.005).50 Three prospective open studies were carried out in late stage osteoarthritis of the knee. In Canada 60 patients on the que for total knee arthroplasty (TKA), all had grade IV Kellgren-Lawrence grade arthritis, chronic disabling pain, with or without effusion, mean age was 67± 1 years 65% were female. At one year there was a significant imErovement in the WOMAC A,B,& C scores (p<0.02) and 38% were able to delay surgery. 1 Miller reported a U.S. prospective study of 108 patients (52 men and 56 women) who were candidates for TKA. All patients had failed NSAIDs, corticosteroids and conservative treatment. 83% had Kellgren-Lawrence grade IV and 13% had grade III osteoarthritis. The Hospital for Special Surgery Rating Scale was utilized pretreatment and at 1, 3, 6, and12 months and every six months thereafter. 52% of eligible patients did not undergo TKA at 2.6years. 52 Weiss reported a study of 123 knees in 90 patients (65% female average age 74± years) 75% had Kellgren-Lawrencc grade IV arthritis and 19% grade III, all had failed conservative treatment, 85% failed NSAIDs, 2/3 failed inter-articular corticosteroids and 113 failed arthroscopic debridement. 65% of patients were better six months after treatment and 46% at 1 year; 64% of patients better at one year remained so at 2 years. 33 knees had a retreatment (mean time 15 mos.) and 78% remained better 12 mos. later. These results were not significantly altered by obesity, previous treatment failures including arthroscopic debridement, or intra-articular steroids. A practice analysis of the number of TKAs in the 2 years prior to and 2 years subsequent to me introduction of hylan G-F20 in this practice demonstrated a 52% decrease (62% by diagnostic code).53,s4 In summation these studies demonstrate that viscosupplementation with hylan G-F20 is effective over the broad spectrum of osteoarthritic disease of the knee. It is more effective than saline injections, arthrocentesis, NSAIDs and lower molecular weight hyaluronans.
Treatmentof osteoarthritis
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The results of a second course of treatment are better than the first course, and viscosupplementation has resulted in a decrease in the number ofTKAs. SAFETY In order to appropriately position viscosupplementation with hylan G-F20 in our armamentarium for the treatment of the osteoarthritic knee, it is important to consider both the efficacy and safety of this device compared to other standard treatments. A meta analysis of all of the prospective double-blind clinical trials with hylan G-F20 shows a local adverse reaction (pain and effusion) rate per injection of 2.1%.44 This was similar to the 2.7% per injection rate found in Lussier's retrospective analysis 55 and consistent with the open label safety trial (2.5%) conducted by Wobig.47 69% of patients who had a local adverse event were clinically improved and 66% had a subsequent event-free injection in the same knee.55 Lussier indicated that the number of reactions increased with extra articular injection of material.55 However, these effusions caused no permanent sequelae and did not jeopardize the success of treatment, nor was there any increase with subsequent courses and no evidence of an immune response.f Twelve systemic events were reported in 511 hylan G-F20 treated patients (559 knees in 7 clinical trials) each was a unique isolated event and there was no recurrence with repeat injections thus not apparently related to hylan itself. Prospective open studies in advanced disease had local events similar to those reported in the clinical trials, (4.4% Miller 52 and 2.1% Weiss 53,54 there were no permanent sequelae, patients often progressed to clinical improvement and there was no statistically significant increase in local events after a second course. Clinical trials comparing low molecular weight hyaluronan with hylan GF20 showed no significant difference in local reactions. 49,50 Safety data from post marketing medical surveillance through June of 2000 indicated that 3,279,593 hylan GF20 injections have been given worldwide. There were no systemic reactions attributed to hylan G-F20, as only 3,638 (0.11%) local reactions and 12 joint infections 1/273299 (0. 0004%) per injection were reported. NSAIDS are the most commonly used medication for osteoarthritic pain.58 Recent studies show that in the United States alone 16.5000 deaths per year occur, a rate of 0.22% for long term use of NSAIDs. GI ulcerations occur in 9-30% of patients on NSAIDS for more than 2 weeks. 10% of these peptic ulcers develop obstruction, perforation or hemorrhage.f" This risk is heightened and additional problems such as nephrotoxicity (4%) edema (4%) hyperkalemia (2%) hyponatremia (7%) occur in elderly patients who have the highest incidence of osteoarthritis of the knee.61 Significant hypertension (5mm increase in diastolic pressure) occurs in 1% of the patients taking NSAIDs) which has been demonstrated to increase the risk of stoke by 67% and coronary artery disease by 15%.60 All of these complications are increased by drug interactions i.e.: ACE inhibitors, Beta blockers, calcium channel blockers and diuretics commonly used for the treatment of mild hypertension arrhythmias, or congestive failure in elderly patients.61-65 Considering the lethal and debilitating systemic effects of these drugs particularly in patients with osteoarthritis of the knee over the age of 65, with ulcer disease, history of helicobacter pylori, arrhythmias, congestive heart failure, hypertension, use of diuretics, B-blockers, ACE inhibitors, bleeding disorders, allergies to aspirin, asthma, edema, and renal disease it is appropriate to use viscosupplemcntation treatment prior to NSAIDs in patients with these risk factors. Intra-articular corticosteroids have been used in osteoarthritic joints for almost 50 years despite the lack of evidence of long term efficacy. It decreases neutrophil migration,
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Clinical applications of hyaluronan
prostaglandin synthesis, interieukin secretion and acid hydrolase release from leukocytes.f" The use of intra-articular steroids has significant systemic and local side effects. A single injection of intra-articular steroids can suppress stress responses with suppression of endogenous cortisone production for 10 to 30 days.'" Rapid heart rate and elevated blood pressure are systemically described symptoms. Intra-articular injection of corticosteroids in diabetic patients often makes the diabetes more difficult to control. Steroid arthropathy after intra-articular injection of steroid was initially reported in 1958.68,69 Noyes et.al showed a significant reduction in the tensile strength of the monkey anterior cruciate ligament for 3 months following intra-articular injection." An increased incidence of avascular necrosis has been reported injoints following steroid injection.n,n An infection rate of one in 14,000 was reported by Hollanderr' this rate is almost 20 times more frequent than that reported after viscosupplementation with hylans. Post injection flares manifested by joint swelling and pain several hours after treatment and subsiding in 1 to 3 days, occurs in 2%, roughly the same percentage as occurs in response to hylan or hyaluronan injections.t" On occasion hypersensitivity to methylprednisolone or its carrier vehicle may occur and a case of anaphylaxis was reported in 1997.74 The use of intra-articular corticosteroids for osteoarthritis of the knee should be limited when one considers the systemic and local responses, to those cases where there is a clear inflammatory response i.e. effusion and inflammatory cells or acute crystalline arthropathy. Total knee arthroplasty is one of the most successful procedures in orthopaedics today, however it has significant morbidity and mortality. The death rate from pulmonary embolism is over 0.2%.75 Approximately 47% of patients will develop a deep vein thrombosis (DVT), despite prophylactic treatment. Five years later 24% of these patients will have a post-thrombosis syndrome consisting of pain, edema venous stasis, skin ulcers and discoloration, or restless leg syndrome, compared to 4% of patients who did not develop DVT after surgery." Over the life time of the prosthesis 1% of patients will develop a joint infection requiring removal of the prosthesis." This is 2500 times the infection rate after viscosupplementation injection. 10% of patients having TKA are likely to require revision surgery, and 5% of patients have unsatisfactory results due to permanent joint stiffuess. CONCLUSION
Two recent internationally accepted treatment algorithms have included viscosupplementation for treatment of the osteoarthritic knee'" and governmental statistics have demonstrated a decline in the number of TKA' s since the introduction of hylan G-F20 into the treatment algorithm for this diseasc.78,79 The treatment of osteoarthritis of the knee should start with patient education, weight loss, exercise, braces, canes, topical analgesics and simple analgesics such as acetomenophen. In patients especially those over 65, on medications (ACE inhibitors, diuretics, Beta blockers, etc.) who have hypertension, renal, cardio-pulmonary or gastrointestinal disease this should be followed by viscosupplementation. For the remainder short courses of NSAlDs may be appropriate. The use of intra-articular corticosteroids should be reserved for those patients who have an acute crystalline arthropaty or a recurrent inflammatory component of their arthritis. There is little evidence that corticosteroids provide long lasting clinical benefit and its use may be accompanied by significant systemic and local side effects. Certainly viscosupplementation should be considered prior to joint arthroplasty or surgical intervention.
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DEDICATION
This chapter is dedicated to Endre Balazs. His insight, ground breaking basic discoveries and the clinical application of hyaluronan in the treatment of osteoarthritis has relieved suffering in millions of patients. He exemplifies highest humanitarian ideals in the merger of science and medicine. REFERENCES
1. Maclan CH, Knight K, Paulus H, Brook RH, Shekelle PG. Cost attributable to osteoarthritis. J. Rheumatol1998; 2213-2218. 2. Tugwell P. Economic evaluation of the management of pain in osteoarthritis. Drugs 1996;52 Suppl. 3:48-58. 3. Felson DT. The epidemiology of knee arthritis: results from the Farmingham Osteoarthritis Study. SeminArthritis Rheum. 1990;20(3Suppll):42-50. 4. Impact of Arthritis and Other Rheumatic Conditions on the Health-Care SystemUnited States, 1997. Morbidity and Mortality Weekly Report 1997;48:349-353. 5. Balazs EA, editor. Hyaluronic acid and matrix implantation: a report on the biological activity and therapeutic use of hyaluronic acid. Arlington, MA:Biotrics, Inc. 1971. 6. Balazs EA, Watson D, Duff IF, Roseman S. Hyaluronic acid in synovial fluid. I. Molecular parameters of hyaluronic acid in normal and arthritic human fluids. Arthritis Rheum 1967; 10:357-76. 7. Balazs EA, Gibbs DA. The rheological properties and biological function of hyaluronic acid. In:Balazs EA, editor. Chemistry and molecular biology of the intercellular matrix. New York Academic Press; 1970:1241-53. 8. Laurent TC. Structure of hyaluronic acid. In:Balazs EA, editor. Chemistry and molecular biology of intercellular matrix . Vol. 2. New York: Academic Press; 1970:703-32. 9. Weiss C, Rosenberg L, Helfet AJ. An ultrastructural study of normal young adult human articular cartilage. J Bone Joint Surg 1968; 50A:663-74. 10. Balazs EA, Bloom GD, Swann DA. Fine structure and glycosarninoglycan content of the surface layer of articular cartilage. Fed Proc 1966; 25:1813-6. II. Levick JR, Synovial fluid. Determinants of volume turnover and material concentration. In:Kuettner KE, Peyron JG, Scheleyerbach R, Hascall VC, editors. Articular cartilage and osteoarthritis, New York: Raven Press; 1992. p.529-41. 12. Denlinger JL. Hyaluronan and its derivatives as viscoelastics in medicine. The chemistry, biology and medical applications of hyaluronan and its derivatives. (Proceedings of the Wenner-Gren Foundation International Symposium, Sep 18-21, 1996, Stockholm, Sweden. Vol. 72). London: Portland Press; 1997. p. 235-42. 13. Myers RR, Negami S, White RK. Dynamic mechanical properties of synovial fluid. Biorheology 1966; 3:197-209. 14. Balazs EA, Viscoelastic properties of hyaluronic acid and biological lubrication (Proceedings of the Symposium: Prognosis for Arthritis:Rheumatology Research Today and Prospects for Tomorrow, 1967). Univ Mich Med Ctr J (Suppl) 1968; 9:255-9.
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15. Balazs EA, The physical properties of synovial fluid and the special role of hyaluronic acid. In: Helfet AJ, editor. Disorders ofthe knee. r ed. Philadelphia: JB Lippincott; 1974. P. 63-75. 16. Darzynkiewicz Z, Balazs EA, Effect of connective tissue intercellular matrix on lymphocyte stimulation. I. Suppression of lymphocyte stimulation by hyaluronic acid. Exp Cell Res 1971; 66:113-23. 17. Balazs EA, Darzynkiewicz Z. The effect of hyaluronic acid on fibroblasts, monoculclear phagocytes and lymphocytes. In:Kulonen E, Pikkarainen J, editors. Biology of fibroblast (proceedings of the symposium in Turku, Finland, 1972). London: Academic Press, 1973. p. 237-52. 18. Forrester JV, Balazs Ea. Inhibition of phagocytosis by high molecular weight hyaluronate. Immunology 1980:40:435-46. 19. Aihara S, Murakami N, Ishii R, Kariya K, Azuma Y, Hamada K. Effects of sodium hyaluronate on the nociceptive response of rats with experimentally induced arthritis. Folia Pharmacol Japon 1992;100:359-65. 20. Gotoh S, Onaya J-I, Abe M, Miyazaki K, Hamai A, Horie K. Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats. Ann Rheum Dis 1993;52:817-22. 21. Belmonte C, Pozo MA, Balazs EA. Modulation by hyaluronan and its derivatives (hylans) of sensory nerve activity signalling articular pain. In: Laurent TC, editor. The chemistry, biology, and medical applications of hyaluronan and its derivatives (Proceedings of the Wenner-Gren Foundation International Symposium, Sept. 18-21, 1996, Stockholm, Sweden. Vol. 72). London: Portland Press, 1997. p. 205-17. 22. Pozo MA, Balazs EA, Belmonte C. Reduction of sensory responses to passive movements of inflamed knee joints by hylan, a hyaluronan derivative. Exp Brain Res 1997;116:3-9. 23. Smith MM, Ghosh P, The synthesis of hyaluronic acid by human synovial fibroblasts is influenced by the nature of the hyaluronate in the extracellular environment. Theumatol Int 1987:7:113-22. 24. Balazs EA, editor. Hyaluronic acid matrix implantation: a report on the biological activity and therapeutic use of hyaluronic acid. Arlington, MA: Biotrics, Inc.; 1971. 25. Rydell N, Balazs EA. Effect of the intra-articular injection of hyaluronic acid on the clinical symptoms of osteoarthritis and on granulation tissue formation. Clin Orthop 1971:25-32. 26. Butler J, Ryedell NW, Balazs EA. Hyaluronic acid in synovial fluid. VI. Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of arthritis in track horses. Acta Vet. Scand. 1970:11:139-155. 27. Balazs EA, Denlinger JL. Sodium hyaluronate and joint function. J Equine Vet Sci 1985;5:217-28. 28. Helfet, J: Management of osteoarthritis of the knee joint. In: Helfet AJ, ed. Disorders ofthe Knee. I" ed. Philadelphia: Lippincott, 1974: 179. 29. Peyron JG, Balazs EA: Preliminary clinical assessment of Na-hyaluronate injection into human arthritic joints. Pathol BioI 1976; 22:731-6. 30. Peyron JP: Intraarticular hyaluronan injections in the treatment of osteoarthritis: State-of-the art review. J. Rheum. Vol 20; Suppl39, Aug. 1993, pp.lO-15. 31. Weiss C, Balazs EA, St. Onge R, Denlinger JL: Clinical studies of the intra-articular injections of Healon® (sodium hyaluronate) in the treatment of osteoarthritis of human knees. Semin Arthritis Rheum 1981; 11: 143-4.
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32. Isdale AH, Hordon LD, Bird HA, Wright V: Intra-articular hyaluronate (Healon®); a dose ranging study in rheumatoid arthritis and osteoarthritis. J Drug Dev 1991: 2:939. 33. Balazs EA, Delingcr JL: Viscosupplementation: A new concept in the treatment of osteoarthritis. J Rheum. Vol 20; Suppl39. Aug. 1993; pp.3-9. 34. Shichikawa K, and the Drug Evaluation Committee of the Japanese Rheumatism Association: Evaluation of the effect of sodium hyaluronate to osteoarthritis of the knee. Ryumachi 1983: 23:280-90. 35. Russel JI, Michalek JB, Lawrence VA, Lessap OJA, Briggs BT, May GS: A randomized, placebo and non-intervention controlled trial of intra-articular 1% sodium hyaluronate in the treatment of knee osteoarthritis (abstr). Arthritis Rheum 1992: (suppl)) 35:B94. 36. Altman RD, Moskowitz R, Hyalgan Study Group. Intraarticular sodium hyaluronate (Hyalgan®) in the treatment of patients with osteoarthritis of the knee: a randomized clinical trial. 1. Rheumatol1998; 25:2203-12. 37. Balazs EA, Leshchiner E, Larsen NE, Band, P. Hyaluronan biomaterials: medical applications. In: Wise DL, Trantolo DT, Altobelli DE, Yaszemski MJ, Gresser JD, Schwartz ER, editors. Encyclopedic handbook of biomaterials and bioengineering. Vol. 2. New York: Marcel Dekker; 1995. P.1693-715. 38. Balazs EA, Leshchiner EA. Hyaluronan, its crosslinked derivative-hylan- and their medical applications. In: Inagaki H, Phillips GO, editors. Cellulosics untilization: research and rewards in cellulosics (Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future). New York: Elsevier Applied Science; 1989. P.233-41. 39. Band, P, Goldman A, Barbone A, ReinerK, Balazs EA. Intra-articular distribution and residence time ofhylan polymers (Abstract 433). Materials Research Society, Spring Meeting. San Francisco; 1995. 40. Denlinger JL: Metabolism of sodium hyaluronate in articular and ocular tissues (Thesis). Lille, France: Universite' des Sciences et Techniques de Lille, 1982:365. 41. Scale D, Wobig M, Wolpert W. Viscosupplementation of osteoarthritic knees with hylan: A treatment schedule study. Curr Ther Res 1994; 55:220-32. 42. Wobig, M, Dichut A, Maier R, Vetter G. Viscosupplementation with hylan G-F20: A 26-week controlled trial of efficacy and safety in the osteoarthritis of the knee. Clin Ther 1998; 20:410-23. 43. Moreland LW. New therapeutic options for treating knee osteoarthritis.Pharmacy and Therapeutics May, 1999: pp. 238-45. 44. Adams M. Viscosupplementation as an alternative to conventional treatment for the management of osteoarthritis ofthe knee. J Clin Rheumatol. Vol.5 No.6 (Suppl) Dec. 1999pp. SI8-23. 45. Dixon OJ, Hosie G, on behalf of the Primary Study Group. Double-blind double control comparison of viscosupplementation with hylan G-F20 (Synvisc®) against Diclofenac and control in knee osteoarthritis. Abstract No. 989. Poster presentation. AAOS Meeting, Anaheim, CA Feb. 4-8,1999. 46. Lussier A, Cividino AA, McFarlane CA, Olyszynski WP, Potasher WJ, de Medicis R. Viscosupplementation with hylan for the treatment of osteoarthritis: findings from clinical practice in Canada. J Rheumatol1996; 23:1579-85. 47. Wobig M, Beks P, Dichut A, Maier R, Vetter G. Open-label multicenter trial of the safety and efficacy of visco supplementation with hylan G-F20 (Synvisc) in primary
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oseoarthritis of the knee. J Clin Rheumatol Vol. 5, No.6 (Suppl) Dec. 1999 pp. S2431. 48. Wobig M. Hylan G-F20 (Synvisc) for the treatment of osteoarthritis of the knee: Clinical studies and practical considerations. J Clin Rheumatol Vol. 5 No.6 (Suppl) Dec. 1999pp. SI2-17. 49. Wobig M, Bach G, Beks P, Dickhut A, Runzheimer J, Schwieger G. The role of elastoviscosity in the efficacy of viscosupplementation for osteoarthritis of the knee: A comparison of hylan G-F20 and a lower-molecular-weight hyaluronan. Clin Ther 1999; 21: 1549-62. 50. Sripada P,Pritchard C, Banks PF. Comparison of efficacy ofhyaluronan and hylan GF20 in osteoarthritis. Presentation ACR Abstract No. 1353, Nov. 1999. 51. Bell M, Fallaha M, Lenczner E. et.al. Viscosupplementation with hylan G-F20 in total knee replacement candidates: An effective pain management therapy that may delay surgery (Abstract). Osteoarthritis Research Society International, Vienna, Austria, Sept. 16-19,1999. 52. Miller EH, Snyder MA, Heidt RS, Weich MC. Analysis of the results of viscosupplementation with hylan G-F20 in the treatment of osteoarthritis of the knee. A prospective study of 108 patients.(Abstract) American Academy of Orthopaedic Surgeons, Anaheim, CA, Feb. 4-8,1999. 53. Weiss C. The treatment of osteoarthritis of the knee with hylan G-F20 in orthopaedic practice. (Abstract FR062) 5 th World Congress OARSI, Barcelona, Spain October 46,2000 Osteoarthritis and Cartilage Vol.S Suppl B p.S86. 54. Weiss, C. Tillero, W, Balazs, L. Viscosupplementation with hylan G-F20 (Synvisc®) in orthpaedic practice: A prospective analysis. J. Southern Orthopaedic Association, Summer 1999, Vol.8 No.2: pp.129-30. 55. Lussier A, Cividino AA, McFarlane CA, Olysynzki WP, Potasher WJ, de Medicis R. Viscosupplementation with hylan for the treatment of osteoarthritis: findings from clinical practice in Canada. J Rheumatol1996; 23:1579-85. 56. Sanders PA, Grennan MD: Non-steroidal anti-inflammatory drugs versus simple analgesics in the treatment of arthritis. Baillieres Clin Rheumatol 1990;4:371-85. 57. Creamer P, Hochberg MC. Osteoarthritis. Lancet 1997;350:503-9. 58. Wagner PJ, Sculco T. NSAID Roundup: Focus on adverse effects. Orthopaedic Special Ed, Vol.6 No.1 of2 2000: pp 15-24. 59. Wolfe MM, Lichtenstein DR, Singh G. Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs. N Engl J Med. 1999;340:1888-99. 60. Collins R, Pete R, MacMahon S. Blood pressure, stroke, and coronary heart disease: part 2. Lancet. 1990;335: pp.827-38. 61. Whelton A, Maurath KM, Verburg, Geis GS. Renal safety and tolerability of celecoxib, a novel cyclooxygenase-2 inhibitor. Am. J Therap.(2000) 7(3): pp.159-75. 62. Pope JE,Anderson JJ, Felson DT. A meta-analysis of the effects of non-steroidal antiinflammatory drugs on blood pressure. Arch Intern Med 1993;153:477-484. 63. Johnson AG,Nguyen TV, Day RO. Do non-steroidal anti-inflammatory drugs affect blood pressure? A meta-analysis. Ann Intern Med 1994: 121:289-300. 64. Ruoff GE. The impact of non-steroidal anti-inflammatory drugs on hypertension; alternative analgesics for patients at risk. Clin Ther 1998:20:376-387. 65. Collins R, Peto R, MacMahon S. Blood pressure, stroke and coronary heart disease. Part 2, short-term reductions in blood pressure: overview of randomized drug trials in their epidemiological context. Lancet 1990;335:765-774.
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66. Rozental TD, Sculco TP. Intra-articular corticosteroids: An updated overview. The Am J. Ortho Jan 2000;pp.18-23. 67. Koehler ME, Urowitz MB, Killinger DW. The systemic effects of intra-articular corticosteroid. J. Rheumatol. 1974;1:171-125. 68. Chandler GN, Wright V. Deleterious effect of intra-articular hydrocortisone. Lancet. 1958;2:661-663. 69. Steinberg CL, Duthie RB, Pira AE. Charcot-like arthropathy following intra-articular hydrocortisone. JAMA. 1962;181:145-148. 70. Noyes FR, Grood ES, Nussbaum NS, Cooper SM. Effect of intra-articular corticosteroids on ligament properties. Clin Orthop. 1977;123:197-209. 71. McCarty DJ, McCarthy G, Carrera G. Intra-articular corticosteroids possibly leading to local osteonecrosis and marrow fat induced synovitis. J Rheumatol. 1991;18:10911094. 72. LaRoche D, Arlet J, Mazieres B. Osteonecrosis of the femoral and humeral heads after intraarticular corticosteroid injections. J Rheumatol. 1990;17:549-551. 73. Hollander JL. Intrasynovia1 corticosteroid therapy in arthritis. MD Med J. 1969;19:62-66. 74. Mace S, Vadas P, Pruzanski W. Anaphylactic shock induced by intra-articular injection of methylprednisolone acetate. J Rheumatol. 1997;24:1191-1194. 75. Clagett, GP, Anderson FA Jr., Geert SW. Prevention of venous thromboembolism. 5th ACCP Consensus Confon antithrombotic therapy. Chest. 1998 V 114 (5) supp!. 76. Hass S. Deep vein thrombosis: Beyond the operating table. Orthopedics. Vo!.23 No. 6 Supp!. June 2000 pp.629-632. 77. Wilson G, Kelley K, Thornhiss TS. Infection as a complication of total knee replacement arthroplasty. JBJS 72A 1990 pp. 878-883. 78. Orthopaedic Network News. 1999 Hip and knee update. Vo!. 10, No.4, Oct. 1999. 79. Orthopaedic Network News 2000 Hip and knee implant review. Vol. 11, No.3 July 2000. 80. Am College of Rheum. Reconunendation for medical management of osteoarthritis. Arth &Rheum. Vol. 43 No.9 Sept 2000 pp.1905-15. 81. Weiss, C. Why viscoelasticity is important for the medical uses of hyaluronan and hylans. New Frontiers in Medical Sciences: Redefining hyaluronan 2000 Elsevier pp.97. 82. Data on tile. Biomatrix, Inc. Ridgefield, NJ.
THE CONTROL OF ADHESIONS WITH HYLAN POLYMERS Charles Weiss Chairman, Department ofOrthopaedics and Rehabilitation, Mount Sinai Medical Center Clinical Professor ofOrthopaedics University ofMiami School ofMedicine 6431 Pine Tree Drive Circle, Miami Beach, Florida 33141 USA
ABSTRACT
Postsurgical adhesion formation is a significant medical and economic problem. Endre Balazs' purification of the first noninflammatory fraction of hyaluronan and his recognition of its role as a regulator of cell function, surface protector and as a barrier molecule led to its initial use as an antiadhesion device. Hylans (cross-linked hyaluronan molecules) were developed by Balazs and coworkers to enhance the elastoviscosity, molecular weight and local tissue residence time while retaining the biocompatibility and permeability of native hyaluronan. Hylan polymers have been specifically engineered as fluids, gels, membranes, and combinations to safely reduce adhesion formation after tendon, spinal, abdominal, endothelial and pelvic trauma. KEYWORDS
Hyaluronan, hylan, adhesion, tendon, spine, abdominal, pelvic, sinus, viscoseparation INTRODUCTION
Adhesions are pathologic connective tissue fibrous bands that join together adjacent connective tissues or organs as a consequence of physical, chemical, metabolic, inflammatory or infectious trauma or tumorous invasion. Adhesion formation constitutes a significant medical and economic burden to both the individual and to society due to pain, loss of function, complications and costs associated with attempted correction. Hyaluronan is a ubiquitous component of connective tissue present in the intercellular and interfibrillar matrix of skin, connective tissue, joint and tendon spaces, serosal and subserosal tissues. 1 It is concentrated on and within tissue and organ surfaces as a highly entangled polyanionic molecular network which influences every cell, fibril, compound, molecule and ion in the extracellular matrix.2•s It sustains tissue spaces i.e, between tendon sheath and tendon, dura and surrounding tissues, etc. having 500,000 times the viscosity of saline solution," It absorbs energy between adjacent cells and tissue surfaces dissipating it as heat under conditions of low shear stress bI viscous flow and under high strain frequencies absorbs energy as an elastic solid -9 and stabilizes the micro environment of normal tissues by inhibiting inflammatory mediators as well as the migration and mitosis of cells of the lymphomyloid system, phagocytes and fibroblasts.!" 12 It stabilizes nociceptor nerve fiber endings to allow for comfortable unnoticeable movement between adjacent tissue surfaces. IJ- 16 The molecular size and concentration of the hyaluronan layer on tissue surfaces acts as a molecular filter 4 keeping out large
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molecules such as fibrinogen which can act as a fibrinous scaffold between adjacent tissue surfaces, a precursor to adhesion formation. When this homeostatic state is interrupted by trauma the concentration, molecular size and molecular interaction of hyaluronan may be severely affected through bleeding, the release of serum, degredative enzymes, clot formation between adjacent tissue surfaces, reductions in plasminogen activator activity, and decreased fibrinolysis. A decrease in the elastoviscosity of hyaluronan at the trauma site stimulates the migration and mitosis of cells of the lymphomyloid system, the production of interleukin, prostaglandins, bradykinins, free radicals and the migration of phagocytes and fibroblasts into the wound area. IO-12 Decreased elastoviscosity sensitizes nociceptors 15,16 particularly to movement, causing pain, and encouraging immobilization of the local tissue environment thereby enhancing the ability of reparative cells to migrate into the wound and initiate the early phases of wound healing. These conditions also set the stage for adhesions to form as fibroblasts migrate along the fibrin pathways between adjacent tissue surfaces. For decades surgeons have attempted to inhibit adhesion formation by the use of gentle surgical technique, early mobilization and a variety of biologic and nonbiologic barrier materials. In the 1960s Endre Balazs recognized that hyaluronans could be an ideal biological barrier molecule and conceived the idea of viscoseparatiorr " the use of viscoelastic gels and fluids to separate tissues, prevent adhesions, decrease scar formation and facilitate healing. His development of a noninflammatory fraction of sodium hyaluronan (NIFNaHA) enabled this material to be used medically. This chapter will review his early work with hyaluronan and subsequent work with hylans in the prevention and control of adhesion formation. VISCOSEPARATION IN TENDON SURGERY.
Viscoseparation was first reported by Balazs and coworkers in several mammalian species using a variety of experimental models. 18,19 In a rabbit abrasion extensor hallucis longus (EHL) adhesion model 80% of tendons injected with NIFNaHA (mw 1-2 mil) prior to sheath repair had less adhesions than controls. A fascial adhesion model was studied in rabbits, guinea pigs, and monkeys: 82% of animals has less adhesions between abraded fascia and overlying subcutaneous tissue 3 to 6 weeks postsurgery. A third group of studies compared local tissue reaction (rejection) to an implanted foreign body (polyethylene tubes) and found no rejection on the hyaluronan treated side and 40% rejection on the saline side. The clinical relevance of this material was tested in a hand surgery model using excision of the superficialis tendon and division, crushing and repair of the profundus tendon in "no man's land" of the ring and long fingers in owl monkeys.i" Either saline or NIFNaHA (Healon®) was injected about the tendons which were then immobilized for 4 to 5 weeks. Subsequently a blinded evaluation of the range of motion of the fingers up to 12 weeks was performed. There was significant improvement (p
Control of adhesionswith hylan polymers
485
demonstrated significantly decreased adhesion formation and strength, in the hyaluronan treated compared to saline treated controls without interfering with healing or causing systemic reaction. The effect of hyaluronan increased with the elastoviscous properties of the hyaluronan used.22,23 It was felt however, that native hyaluronans lacked sufficient local tissue residence time and elastoviscous properties to be effective in clinical injuries to flexor tendons. This supposition was later borne out by Hagberg in a clinical study using exogenous hyaluronan after flexor tendon surgery following injury. However clinical and surgical variability, insufficient number of patients, len~th of follow up and patient cooperation restricted the evaluation of these clinical results? In the 1980s Balazs sought to overcome the shortcomings of decreased residence time and low molecular weight of native hyaluronans by producing cross linked hyaluronans called hylans. 25,26 Hylan A is a water soluble derivative average mw 6 million and hylan B a water insoluble hydrated elastic gel slurry. Hylans have increased molecular weight, increased local tissue residence time, the same biocompatibility and permeability as native hyaluronan, and do not interfere with normal clot (fibrin) formation 27 they do not cause granulation tissue, foreign body reaction or immunol0 reaction.28•32 This material was tested in a EHL tendon abrasion model in rabbits.' ,34 After abrading the EHL and closing the tendon sheath, the sheath was injected with saline, hylan gel or nonviscoelastic (degraded) hylan gel. Animals were sacrificed at 3 weeks and studied by pull-out force, histologic evaluation, and gross observation. Fifty-five percent of hylan treated tendons had no adhesions compared to only 5 % of controls and 7% of nonelastoviscous hylan gel (Table I) this was significant at the (p<0.0005) level. There was no difference in tensile strength of tendons repaired in the presence of either hylan gel or saline.
5ic
Table 1. Adhesion strength (g)* Rating None (NL) 183 ± 83 Mild <2XNL Moderate <3 XNL Severe >3 XNL
Tendon Adbesions**
n 12 4 2 4
Hylan B gel Nonviscous hylan % Strength (g) n % Strength (g) 55 179±47 I 7 129 18 300±10 I 7 390 9 480 ±60 4 29 499 ±63 18 1075 ±205 8 57 1182± 333
Treatment control n % Strength (g) 1 5 150 3 14 323±18 4 19 415 ±20 13 62 1064±244
* ±S.D. ** In a moderate traumamodel in the rabbit(pull-out force in g) Significance ofhylan B gel versus lowviscosity hylanor controlp
486
Clinical applications ofhyaluronan
VISCOSEPARATION IN SPINE SURGERY In the United States over 300,000 laminectomies for herniated discs or neural compression are performed annually. Up to 20% of patients will continue to have disability or pain 36,37 often due to perineural fibrosis or adhesion formation." The pathogenesis of these adhesions is associated with the migration of fibroblasts into the perineural space along the fibrin scaffolding formed as peridural veins bleed into the surgical site.J9-43 For more than 50 years a variety of biological and nonbiological materials have been utilized in an attempt to limit the extent of postsurgical adhesions to the dura. Hyaluronan has been shown to decrease perineural fibrosis in a number of animal models, 43,44 and was significantly more effective than Gel Foam 44 or free fat grafts." Native hyaluronan is, however, not an ideal barrier molecule as it has a relatively low molecular weight, rapidly diffuses away from the local tissue site prior to healing whereas the ability to decrease adhesions has been shown to be directly proportional to molecular weight.43 A specific hyaluronan derivative hylan G-F80 (Hylagel®) a mixture offour parts hylan B by volume and one part hylan A was engineered to prevent adhesions. It was studied in a controlled, double blind rabbit laminectomy model." After laminectomy saline or Hylagel® (hylan G-F80) was placed into the incision site prior to closure. Adhesion formation was graded by histology and dissection. Postlaminectomy adhesion formation to the dura was significantly diminished in the hylan treated rabbits (p<0.005 at 4, 8, 12 weeks post surgery) and adhesions that did form were softer and more pliable. Hylan was biologically inert when placed in the spinal fluid space, injected into the dura or into the extradural space. There was no evidence of acute inflammatory reaction and no systemic adverse events. Hylan G-F80 appears to be a safe and effective method of decreasing postlaminetomy adhesion formation. Currently an international study using Hylagel® is underway in Europe and in the United States, to determine its effectiveness in reducing post surgical perineural adhesion formation in patients undergoing intra-spinal lumbar surgical procedures. VISCO SEPARATION IN ABDOMINAL, PELVIC AND SINUS SURGERY Adhesion formation occurs in over 90% of patients after laparotomy,47-49 resulting in over 300,000 hospitalizations, more than 1.7 billion in costs and an overall 3% mortality.50, Hylafilm® is a hylan barrier membrane which adheres tightly to the wound surface, requires no sutures, has excellent biocompatibility and is completely resorbable. When studied in a rat cecal abrasion model it was 97% effective in preventing adhesions. One in 36 hylan treated animals had weak adhesions (separation force of 77 grams) whereas in the control animals 67% had strong adhesions (separation force >149 grams). There was no interference with wound healing as seen on histologic section and no signs of systemic toxicity.51 In a rat liver abrasion model studied at 7 days 11 of 12 Hylafilm® treated wounds had no adhesions compared to 11 of 12 control wounds which had significant adhesions. There were no local or systemic reactions and no change in healing." Postsurgica! or i~~a~o.ry a1hesion ~ormati~n is a si~~iticant problem in ~elv~c surgery, causmg pam, infertility 53~ 4 and high medical costs: Hylafilm® was studied m a rabbit uterine hom abrasion model 14 days post surgery. There were 15 Hylafilm® treated rabbits and 15 controls. Five Hylafilm® treated rabbits and only one control
Control or adhesions with hylan polymers
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rabbit were completely free of adhesions. The extent of adhesion formation (as a percentage of involvement of the length of the uterine hom) was evaluated by a blinded observer. Hylafilm® reduced the extent of adhesions from 46±5.9% to 19.8±4.3%. In the Hylafilm® treated animals there were no local tissue reactions, no inhibition of healing and no systemic reactions.V The development of postoperative synechiae with stenosis is a common problem for rhinologists after sinus surgery. Recently a highly hydrated hylan B gel (Hylasine®) has been used as a post-surgical dressing after endoscopic sinus surgery. In an open label study patients with chronic sinusitis requiring bilateral endoscopic ethmoidectomy were evaluated up to 5 weeks post surgery. On the treatment side the sinussectomy cavity was filled with Hylasine®. From week 2 to 5 there were consistent highly significant decreases in scar formation and postoperative stenosis on the Hylasine® treated side. 56 SUMMARY Endre Balazs' recognition of the importance of hyaluronan in the extracellular matrix as a regulator of cell function, tissue organization and protection, and his purification of the first NIFNaHA has played the defining role in the medical use of these molecules. His development of hylan polymers which have enhanced viscoelastic properties, and local tissue residence time while retaining the biocompatibility, permeability, carboxylate and acetamido groups of unaltered hyaluronan has permitted the engineering of these molecules to perform specific biologic functions, Since the mid 1980s hylan polymers have been shown in a number of tendon, fascial, spinal, endothelial, abdominal and pelvic post traumatic adhesion models to be effective in reducing adhesions, while not interfering with normal healing, nor producing local or systemic adverse reactions. DEDICATION This chapter is dedicated to Endre A. Balazs in recognition of his past accomplishments and in anticipation of his future contributions to the advancement of science and the betterment of mankind. REFERENCES I. Laurent TC, Fraser JRE. Hyaluronan. FASEB J 1992;6:2397-404. 2. Weiss C, Rosenberg L, Helfet AJ. An ultrastructural study of normal young adult human articular cartilage. J Bone Joint Surg 1968;50A:663-74. 3. Balazs EA, Bloom GD, Swann DA. Fine structure and glycosarninoglycan content of the surface layer of articular cartilage. Fed Proc 1966;25:1813-6. 4. Levick JR. Synovial fluid. Determinants of volume turnover and material concentration. In:Kuettner KE, Peyron JG, Scheleyerbach R, Hascall VC, editors. Articular cartilage and osteoarthritis, New York:Raven Press; 1992.p.529-4 I. 5. Balazs EA. The viscoelastic intercellular matrix and control of cell function by hyaluronan. In: Laurent TC, editor. The chemistry, biology, and medical applications of hyaluronan and its derivatives (Proceedings of the Wenner-Gren Foundation International Symposium, Sept. 18-21, 1996, Stockholm, Sweden. Vol. 72). London: Portland Press, 1997;21:185-203. 6. Denlinger JL. Hyaluronan and its derivatives as viscoelastics in medicine. The chemistry, biology and medical applications of hyaluronan and its derivatives.
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(Proceedings of the Wenner-Gren Foundation International Symposium, Sept 18-21, 1996, Stockholm, Sweden. Vol.72). London: Portland Press. 1997; p.235-42. 7. Myers RR, Negami S, White RK. Dynamic mechanical properties of synovial fluid. Biorheology 1966;3:197-209. 8. Balazs EA. Viscoelastic properties of hyaluronic acid and biological lubrication (Proceedings of the Symposium: Prognosis for Arthritis: Rheumatology Research Today and Prospects for Tomorrow, 1967). Univ Mich Med Ctr (Suppl) 1968;9:2559. 9. Gibbs DA, Merrill EW, Smith KA, Balazs EA. The rheology of hyaluronic acid. Biopolymers 1968;6:777-91. 10. Darzynkiewicz Z, Balazs EA. Effect of connective tissue intercellular matrix on lymphocyte stimulation. 1. Suppression of lymphocyte stimulationby hyaluronic acid. Exp Cell Res 1971;66:113-23. 11. Balazs EA, Darzynkiewicz Z. The effect of hyaluronic acid on fibroblasts, monoculclear phagocytes and lymphocytes. In: Kulonen E, Pikkarainen J, editors. Biology of fibroblast (proceedings of the symposium in Turku, Finland, 1972). London: Academic Press, 1973. P.237-52. 12. Forrester JV, Balazs EA. Inhibition of phagocytes by high molecular weight hyaluronate. Immunology 1980;40:435-46. 13. Aihara S, Murakami N, Ishii R, Kariya K, Azuma Y. Hamada K, et.al. Effects of sodium hyaluronate on the nociceptive response of rats with experimentally induced arthritis. Folia Pharmacol Japon 1992;100:359-65. 14. Gotoh S, Onaya JI, Abe M, Miyasaki K, Hamai A, Horie K, et.al. Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats. Ann Rheum Dis 1993;52:817-22. 15. Belmonte C, Pozo MA, Balazs EA. Modulation by hyaluronan and its derivatives (hylans) of sensory nerve activity signalling articular pain. In: Laurent TC, editor. The chemistry, biology and medical applications of hyaluronan and its derivatives (Proceedings of the Wenner-Gren Foundation International Symposium, Sept. 18-21, 1996, Stockholm, Sweden. Vol. 72). London:Portland Press, 1997, p.205-17. 16. Pozo MA, Balazs EA, Belmonte C. Reduction of sensory responses to passive movements of intIammed knee joints by hylan, a hyaluronan derivative. Exp Brain Res 1997;116:3-9. 17. Balazs EA. Hyaluronic acid and matrix implantation. A report on the biological activity and therapeutic use of hyaluronic acid. Arlington, MA' Biotrics, Inc.: 1971. 18. Rydell NW, Balazs EA. Effect of intra-articular injection of hyaluronic acid on the clinical symtoms of osteoarthritis and on granulation tissue formation. 1971. Clin. Orthop. No. 80, 25-32. 19. Balazs EA, Rydell NW, Freeman M1. Effect of hyaluronic acid on adhesion formation. 1971. In Hyaluronic Acid and Matrix Implantation, 2nd edition (Ed. Balazs EA.), Biotrix, Inc. Arlington, MA, Appendix 13. 20. St. Onge R, Weiss C, Denlinger JL, Balazs EA. A preliminary assessment of Nahyaluronate injection into "No-Man's Land" for primary flexor tendon repair. 1980. Clin. Orthop. 146,269-275. 21. Arnie! D, Ishizue K, Billings E, Wiig N, Vande Berg J, Akeson WHo Hyaluronan in flexor tendon repair. J Hand Surg. 1989, 14A, 837-843. 22. Thomas SC, Jones LC, Hungerford OS. Hyaluronic acid and its effect on postoperative adhesions in the rabit flexor tendon. 1986. Clin Orthop. 206, 281-289.
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23. Hagberg L. Gerdin B. Sodium hyaluronate as an adjunct in adhesion prevention after flexor tendon surgery in rabbits . .I Hand Surgery, 1992 17,935-941. 24. Hagberg L. Exogenous hyaluronate as an adjunct in the prevention of adhesions after flexor tendon surgery: A controlled clinical trial. .I Hand Surg 1992;17A:132-6. 25. Balazs EA, Leshchiner E, Larsen NE, Band P. Hyaluronan biomaterials:medical applications. In: Wise DL, Trantolo DT, Altobelli DE, Yaszemski MJ, Gresser JD, Schwartz ER, editors. Encyclopedic handbook of biomaterials and bioengineering. Vol. 2. New York:Marcel Dekker; 1995. P. 1693-715. 26. Balazs EA, Leshchiner E. Hyaluronan, its crosslinked derivative hylan and their medical applications. In:Inagaki H, Phillips GO, editors. Cellulosics utilization: research and rewards in cellulosics ( Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future). New York: Elsevier Applied Science; 1989. P.233-41. 27. Larsen, NE, Leshchiner E, Balazs EA. Belmonte C. Biocompatibility of hylan polymers in various tissue compartments. In Polymers in Medicine and Pharmacy (Proceedings of the Material Research Society, Spring Meeting, April 17-21, 1995, San Francisco, CA) (Eds. Mikos AG, Leong KW, Radomsky ML, Tamada JA, Yaszemski Ml.), Materials Research Society, Pittsburgh PA, 149-153. 28. Balazs EA, Denlinger, JL, Leshchiner E, Band P, Larsen N, Leshchiner A, Morales B. Hylan: hyaluronan derivatives for soft tissue repair and augmentation. 1988. In Biotech USA 1988 (Proceedings of the Fifth Intl, Congress on Biotechnology, Nov. 14-16, 1988, San Francisco, CA), Conference Management Corp., Norwalk, CT, 442451. 29. Balazs EA, Band PA, Denlinger JL, Goldman AI, Larsen NE, Leshchiner EA, Leschiner A, Morales B. 1991. Matrix engineering. Blood Coagul, Fibrinolysis 2, 173-178. 30. Biomatrix Premarketing Approval Application for Synvisc® hylan G-F20. 1994. Volume 1, section III, Summary of data: nonclinical summary. 31. Larsen NE, Pollack CT, Reiner K, Leshchiner E, Balazs EA. Hylan gel biomaterial: dermal and immunologic compatability. 1993. J. Biomed. Mater. Res. 27, 1129-1134. 32. Larsen NE, Bosniak SL, Miller K, Pollack CT, Leshchiner EA, Balazs EA. Evaluation (in vivo) of hylan B (hylan gel) soft tissue implants. 1995. Society for Biomaterials, 21 sl Ann. Meeting, March 18-22, 1995, San Francisco, CA, 312. 33. Weiss C, Levy HJ, Denlinger JL, Suros JM, Weiss HE. The role of Na-hylan in reducing post surgical tendon adhesions. 1986. Bulletin of the Hospital for Joint Diseases, Orthopaedic Institute 46:9-15. 34. Weiss C, Suros JM, Michalow A, Denlinger JL, Moore M, Tejeiro W. The role of Na-hylan in reducing post surgical tendon adhesions: Part 2. 1987. Bulletin of the Hospital for Joint Diseases. Orthopaedic Institute 47:31-39. 35. Amadio PC, Hunter JM, Jaeger SH, Wehbe MA, Schneider LH. The effect of vincular injury on the results of flexor tendon surgery in zone 2..1 Hand Surgery Vol. 10, No.5, Sept. 1985. pp.626-32. 36. Cook SD, Prewett AB, Dalton JE, Whitecloud III TS. Reduction in peridural scar formation after laminectomy with Polyactive® membrane sheets. 1994. Spine 19, 1815-1825. 37. Vital Health Statistic Data from National Center for Health Statistics, ICD-9 Code: 80.51. 38. Burton CV, Kirkaldy-Willis WH, Yong-Hing K, Heithoff KB. Causes of failure of surgery on lumbar spine. 1981. Clin. Orthop. 157,191-199.
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39. North RB, Campbell IN, James CS, Conover-Walker MK, Wang H, Piandatosi S, Rybock JD, Long DM. Failed back surgery syndrome: 5year follow-up in 102 patients undergoing repeated operation. 1991. Nuerosurgery 28, 685-691. 40. Ross JS, Robertson JT, Frederickson RCA, Petrie JL, Obuchowski N, Modic MT, deTribolet N. Association between peridural scar and recurrent radicular pain after lumbar discectomy: Magnetic resonance evaluation. Neurosurgery 38, 855-863. 41. LaRocca H, Macnab I. The laminectomy membrane. Studies in its evaluation, characteristics, effects and prophylaxis in dogs. 1974. J Bone Joint Surg. [Br] 56, 545-550. 42. Cauchoix J, Ficat C, Girgard B. Repeat surgery after disc excision. 1978. Spine 3, 256-259. 43. Songer, MN, Rauschning W, Carson EW, Pandit SM. Analysis of peridural scar formation and its prevention after lumbar laminotomy and discectomy in dogs. 1995. Spine 20, 571-580. 44. Songer MN, Ghosh L, Spencer DL. Effects of sodium hyaluronate on peridural fibrosis after laminotomy and discectomy. 1990. Spine 15,550-554. 45. Abitbol 11, Lincoln TL, Lind Bl, Amiel D, Akeson WH, Gartin SR. Preventing postiaminectomy adhesion. A new experimental mode1.1994. Spine 19, 1809-1814. 46. Weiss C, Dennis J, Suros JM, Denlinger J, Badia A, Montane I. Sodium hyaluronate for the prevention of postiaminectomy scar formation. 1989 Trans. Orthop. Res. Soc. 13,44. 47. Menzies D, Ellis H. Intestinal obstruction from adhesions - how big is this problem? Ann R Coil Surg Eng11990; 72:60-63. 48. Menzies D. Peritoneal adhesions: incidence, cause and prevention. Surg. Annual 1992; 24:27-45. 49. Holmahl RB. Adhesions: prevention and complications in general surgery. Eur J Surg. 1997:163:169-174. 50. Ray NF, Denton WG, Thamer M, Henderson SC, Perry S. Abdominal adhesiolysis:inpatient care and expenditures in the United States in 1994. J Am Coil Surg. Jan. 1998.186;1:1-9. 51. Harris ES, Foresman PA, Rodeheaver GT, Larsen NE, Balazs EA. Efficacy of a resorbable hylan barrier membrane in the prevention of adhesions in a rat cecal abrasion model. 1996. Fifth World Biomaterials Congress, May 29-June 2, 1996, Toronto, Canada, 371 (abstract). 52. Balazs EA, Larsen NE, Pollak CT, Miller BA. The effects pf Hylafim® film on adhesion formation in vivo in a rabbit uterine hom model and rat liver adhesion model. (Abstract) 4 th International Conference on PostOperative Healings & Adhesions, October 22-24, 1999, sponsored by Wayne State University, Ft. Lauderdale, Florida. Pg. 74. 53. Diamond MP, DeCherney AH. Pathogenesis of adhesion formation/reformation: application to reproduce pelvic surgery. Microsurgery 1987; 8:103-107. 54. Steege JF, Stout AL. Resolution of chronic pelvic pain after laparoscopic lysis of adhesions. Am J Obstet Gyneco11991; 165:278-283. 55. Di Zerega GS. Contemporary adhesion prevention. Fertil Sterill994; 61:219-235. 56. Kimmelman C, Edelstein D, Goldman A, Domareki W, Romasz R. Hyalasine® (Hylan B) as a postsurgical dressing for endoscopic sinus surgery. (Abstract) Annual Meeting of Otolaryngology Head and Neck Surgery, Washington DC. Sept. 25,2000.
INTRACELLULAR HYALURONAN IN EPIDERMAL KERATINOCYTES Raija Tammlt, Kirsi Rillat, Juha-Pekka Pienlmakit, Michael Hogg2, Donald K. MacCallum3 , Vincent C. Hascalls and Markku Tammil IDepartment of Anatomy. University of Kuopio, P.O.B. 1627. FlN·702Il Kuopio, Finland. 2Department of Biomedical EngineeringlND20. Lerner Research Institute. The Cleveland Clinic Foundation. 9500 Euclid Avenue. Cleveland. OH 44195 3Department ofAnatomy and Cell Biology. University of Michigan. Ann Arbor. Ml
ABSTRACT Rat epidermal keratinocyte monolayer cultures (REKs) actively synthesize hyaluronan (HA) most of which is retained on the cell surface or released into culture medium. However, a small proportion ofHA also resides in an intracellular compartment (IC-HA). We characterized IC-HA localization and processing in REKs using specific staining with HA-binding probe, and its size by gel filtration of metabolically labeled HA. About 3% of REKs exhibited abundant IC-HA, while half of the cells lacked any microscopically demonstrable IC-HA. IC-HA was localized in 200-600 nm cytoplasmic vesicles. Dual staining of IC-HA with markers for lysosomes showed no colocalization. When REKs were treated with HAlO (decasaccharide), all IC-HA disappeared, while lfA<J, ~, and sulfated glycosaminoglycans had no effect. All IC-HA was cleared 10 min after addition of HA 10, and a 50%-reduction was reached in 5 min. An anti-CD44 mab, that increases HA on the cell surface, also increased IC-HA. Inhibiton of endocytosis via coated pits and caveolae did not reduce the amount of IC-HA. Perturbation of lysosomal activity caused accumulation of IC-HA. Most of the IC-HA had low MW « 40 kDa) compared to the HA in the medium and on cell surface (> 2000 kDa). The data demonstrate a rapid uptake and lysosomal degradation of IC-HA in REKs via a receptor dependent route separate from coated pits and caveolae, that involves a receptor with HA 10 specificity, identified as CD44 or functionally dependent on CD44. The IC-HA consists of small HA fragments which may have a specific role in cellular homeostasis.
KEYWORDS Hyaluronan, epidermis, keratinocyte, endocytosis, CD44, hyaluronan oligosaccharides, coated pits, caveolae
INTRODUCTION Hyaluronan is the major extracellular matrix component in epidermis I. The estimated concentration of hyaluronan between keratinocytes exceeds one rng/rnl, while its half life is only about one day 2, 3. Obviously, there must be an efficient catabolic mechanism which balances the rapid synthesis of HA, and maintains the hyaluronan concentration in a range that supports the normal cell-cell interactions in stratification and differentiation. Partial degradation of epidermal hyaluronan in organ cultures is also indicated by the decrease in the molecular weight of newly synthesized hyaluronan molecules (> 4x10 6 Da) to Ix106 and 0.5x106 Da after 24 and 48 h chases, respectively 3. Experiments in rat epidermal keratinocytes in organotypic cultures also demonstrated hyaluronan catabolism by keratinocytes 4. Keratinocytes express a high level of CD44 5, a receptor
518
Keratinocytes and hyaluronan
responsible for hyaluronan uptake in other cells 6, 7 and mice with blocked epidermal CD44 expression accumulate hyaluronan in skin 8, These results collectively suggest that hyaluronan uptake involves the CD44 receptor on keratinocytes. We previously found that rat epidermal keratinocytes (REKs) contain a pool of hyaluronan resistant to trypsin and Streptomyces hyaluronidase treatments 9, suggesting an intracellular location. The present study characterizes this pool in more detail, and shows that even in monolayer cultures, REKs actively internalize and metabolize hyaluronan in a receptor-mediated fashion via an endocytic route that is not dependent on coated pits or caveolae, but involves CD44. Increased amounts of intracellular HA are seen in proliferating and migrating keratinocytes. This is particularly interesting because it has been recently suggested that intracellular hyaluronan is involved in novel cell functions beyond those involved in extracellular matrix organization and regulation 10. 11. MATERIALS & METHODS
Cell culture A newborn rat epidermal keratinocyte line (REK) was developed by MacCallum and Lillie 12. REKs were cultured in Dulbecco's MEM (low glucose, Life Technologies, Paisley, UK) with 10% fetal bovine serum (HyClone, Logan, UT). For biochemical assays and radiolabeling, the cells were seeded at 100.000/ml and grown close to confluency in 6-well plates (Costar Corp., Cambridge, MA). For microscopic studies, the cells were plated in 8-well chamber slides (Lab-Tek, Nalge Nunc Int., Naperville, IL).
Modification of hyaluronan uptake and degradation To study the receptor mediated uptake of hyaluronan, we treated recently confluent REK cultures with hyaluronan oligosaccharides 9 or with chondroitin, chondroitin sulfate A, chondroitin sulfate C or heparan sulfate (Seikagaku Kogyo Co., Tokyo, Japan). Other cultures were incubated with anti-CD44 mabs; Ox 50 (Biosource Int., Camarillo, CA) or Hermes 3 (a gift from Dr. Sirpa Jalkanen, Turku) or with non-immune mouse IgG (Sigma). To block endocytosis through clathrin coated pits, we treated cells with chlorpromazine (Sigma), or hypertonic serum free medium with 0.4 M sucrose as described 13. To block uptake via caveolae, we treated cells with filipin (Sigma) or nystatin (Sigma) 14, To inhibit macropinocytosis, we treated cells with arniloride IS, The function of lysosomes was perturbed by using either ammonium chloride or chloroquine (Sigma) 16. Receptor recycling was inhibited by adding monensin (Sigma) to the culture medium.
Isolation of intracellular hyaluronan and assay of hyaluronan and chondroitin sulfate disaccharides The REK cultures were radio labeled with 20 and 100 MCilml of [3H]glucosamine and [35S]sulfate (Amersham, Little Chalfont, UK), Cell surface associated hyaluronan was removed by incubating the cells with trypsin (0.2 % trypsin-a. I % EDT A, Sigma) either alone or followed by incubation with Streptomyces hyaluronidase (15 TRU/ rnl in HBSS) for 4-12 h at 4°C. Hyaluronan remaining in the cell fraction after trypsin and extracellular hyaluronidase treatment was designated "intracellular". The samples were digested with proteinase K or papain, and hyaluronan and other glycosaminoglycans were precipitated with cold ethanol, followed by precipitation with CPC 9. Samples were subjected to the analysis of specific disaccharides as described previously 9. 4. Samples were digested with Streptococcus hyaluronidase and chondroitinase ABC (Seikagaku) and injected onto a Superdex Peptide column. The eluent was monitored at 232 nrn, and the fractions were counted for 3H and 35S activities. The carrier hyaluronan produced a disaccharide peak quatitated by absorbance at 232 nm,
Epidermal keratinocytes
519
which was used to monitor the recovery (about 80%). The chemical content of newly synthesized hyaluronan was calculated from the dual labeling data as described in detail previously 4.
Hyaluronan size assay The samples were digested with proteinase K, ethanol precipitated and chromatographed on Sephacryl S-1OOO (Pharmacia, Uppsala, Sweden). Healon (4Ilg) was added as a carrier to each fraction, followed by precipitation with ethanol at -20°C. The precipitates were analyzed for labeled hyaluronan using Superdex chromatography of specific disaccharides as described above.
Staining for endogenous intracellular hyaluronan and markers of cellular compartments The staining protocol was essentially as decribed before 9. The pericellular hyaluronan was removed by treating paraformaldehyde fixed cells with Streptomyces hyaluronidase (10 TRU/rnl for 20 min). Thereafter, the cells were permeabilized in 0.1 % Triton-X 100, and incubated with a biotinylated complex of hyaluronan binding region of cartilage proteoglycan and link protein (bHABC) 17. The bHABC specifically bound to HA was visualized either using avidin-biotin peroxidase (Vector Laboratories, Inc., Burlingame, CA) and DAB (Sigma), or by Texas Red streptavidin or by FITC-avidin D (Vector) . Optical densities of DAB-stained cells were measured as described 9. In dual staining protocols, monoclonal antibodies (against transferrin receptor, cathepsin D, caveolin I, and CD44) were mixed with bHABC. Texas Red-labeled antimouse antibody combined with FITC-avidin was used to visualize the mabs. For electron microscopy REKs were stained for HA as described above and postfixed with reduced osmium tetroxide. For dual staining of HA and CD44, antimouse secondary antibody, conjugated to 5 nm gold particles (Amersham, Little Chalfont, UK) was applied. Fluid phase and coated pit uptake were visualized by incubating REKs in the presence of lysine fixable, Texas Red-labeled dextran (MW 10,000, Molecular Probes, Oregon, USA) and FITC-Iabeled transferrin (Molecular Probes). Fluorescein-labeled hyaluronan preparations were gifts from Drs. Ronald Midura and Jayne Lesley.
RESULTS Hyaluronan in cytoplasmic vesicles bHABC localized most of the HA on plasma membranes of REKs. However, some was also located in vesicle-like structures close to the nucleus, suggesting an intracellular location 9. Treatment of living REK cultures or fixed cells with Streptomyces hyaluronidase before permeabilization and staining for hyaluronan did not remove this perinuclear staining. Confocal microscopy of cells pretreated with hyaluronidase exhibited a vesicle-like hyaluronan signal mostly in a perinuclear position. Transmission electron microsopy showed that the HA was localized in membrane bound vesicles.
Content of intracellular hyaluronan REKs contain a pool of hyaluronan that is resistant to trypsin digestion, a treatment that removes virtually all hyaluronan from the cell surfaces. The size of this trypsinresistant hyaluronan pool was larger in low cell density cultures, as demonstrated by analysis of parallel cultures seeded at different densities and metabolically labeled on the next day. Cultures with fewer REKs (14 x 104 cells/cm 2) exhibited a tenfold higher intracellular hyaluronan content than REKs seeded at 88 x 104 cells/cmz (see Fig. 1).
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The influence of cell density on intracellular hyaluronan, REKs were plated at two different densities and metabolicaly labeled 24 h later for 20 h. The HA in the cell layer was recovered after removal of the pericellular HA with trypsin as described in Materials and Methods. The amount was measured with Superdex gel filtration of specific disaccharides after enzyme digestion.The number of cells per cm 2 (x 10-3 ) at the end of labeling is given on the x-axis,
Increased intracellular hyaluronan accumulation following lysosome inhibition Histochemical staining of REK cultures treated with the inhibitors of lysosomal functions, ammonium chloride and chloroquine, showed increased accumulation of intracellular HA-positive vesicles (Table 1). Optical density (OD) measurements of the intracellular hyaluronan showed an increase with both agents used. Also apigenin an inhibitor of hyaluronidase activity, caused and increase in intracellular HA (see Table 1). Most of the lysosomes visualized with anti-cathepsin D staining were hyaluronan negative, and most of the hyaluronan positive structures were negative for cathepsin D, although some occasional colocalization was present. So little if any of the visualized HA was in the lysosomes. These findings suggest that the hyaluronan positive vesicles represent prelysosomal endosomes, and that the final degradation in the acidified compartment is too rapid to allow HA detection.
Ha-oligosaccherlde-lnduced reduction of intracellular hyaluronan staining
REK cultures were incubated with HA-decasaccharides (HAlO's) at concentrations between 0.1-1 mg/ml for 4 h were totally negative. OD measurements of the hyaluronan stainings confirmed the finding and showed that HA-oligosaccharides HAg or smaller, and the related glycosaminoglycans chondroitin sulfate and heparan sulfate had no effect on the intracellular HA accumulation (see Table 1). The optical density of the DAB was reduced to 60% of the starting value in 5 min after adding HA 12, and reached a stable basal level in 10 min.
Epidermal keratinocytes
521
Table 1. Influences of various treatments on the intracellular HA staining Effect
Treatment
Concentration
Ammonium chloride Chloroquine Monensin Apigenin
1-10 mM 0.5 -2 mM l-12J.IM 0.02-0.25 ug/ml
+ + + +
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0.2 mg/ml 0.2mg/ml 0.3 mg/ml
= =
Chlorpromazin Sucrose (hyperosmolarity) Filipin Nystatin Amiloride
0.5 - 2 ug/ml 0.4 M 0.25 -15 ug/ml
+
I-IO~M
2-3mM
+ + + + =
- decrease = no change + increase
Uptake via coated pits, caveolae and pinocytosis The formation of coated pits is inhibited by chlorpromazine and hyperosmotic medium. Neither of these treatments decreased the amount of intracellular hyaluronan in REK cultures (see Table 1). Transferrin receptor, which uses coated pit pathway, was present in REKs but did not colocalize with HA to a large extent. Filipin and nystatin, reported to inhibit the formation of caveolae, failed to cause any reduction in the intracellular hyaluronan staining (Table 1). REKs do not exhibit extensive caveolae, and the immunostaining with a mab against caveolin I gave a diffuse signal with occasional cells exhibiting a more intense punctate staining. The distribution patterns of caveolin I and hyaluronan were clearly different. REKs showed active pinocytosis of Texas red labeled dextran, a commonly used fluid phase endocytosis marker. Cells showing active dextran uptake often also showed intense intracellular HA staining. However, most of the hyaluronan positive vesicles were dextran negative. The large size of the HA positive vesicles suggested that they might be formed via macropinocytosis. However, arniloride, an inhibitor of macropinocytosis, was unable to influence the content of intracellular hyaluronan in REKs (see Table I).
Molecular mass distribution of intracellular hyaluronan Samples of REK culture media, and intracellular material were eluted on Sephacryl S 1000, and the fractions were analyzed for hyaluronan using Superdex Peptide gel chromatography of specific disaccharides. Most of the intracellular hyaluronan eluted after Kay = 0.5, indicating a molecular mass below 100 kDa (Fig. 2), with the most abundant size eluting at Kav 0.67, a molecular weight of =30 kDa. In a contrast, medium hyaluronan eluted in the excluded volume, indicating a molecular mass greater than 2000 kDa (see Fig. 2). Also most of the hyaluronan released from the cell surface by trypsin treatment eluted close to the excluded volume (data not shown). Although hyaJuronan of <400 kDa size formed a minor proportion of the total hyaluronan in the extracellular compartments, the total amount in this size range was somewhat higher than that within the cell (Fig. 2).
522
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Molecular weight distribution of HA synthesized by REKs on Sephacryl S1000 chromatography. HA was metabolically labeled with 3H-glucosamine and recovered as described in Materials and Methods. Fractions were analyzed for HA using Superdex gel filtration of the specific disaccharides after enzyme digestion. The arrows indicate the elution positions of HA of size 400 kDa and 90 kDa as estimated from the calibration curve supplied by the manufacturer, and the elution position of HA oligosaccharides of 24- 26 units, MW about 5 kDa.
Intracellular hyaluronan and CD44 in REKs REKs stained with anti-CD44 antibodies showed a strong plasma membrane signal. Most of the intracellular, hyaluronan positive vesicles were CD44 negative. However, both confocal microscopy and immunoelectron microscopy showed occasional colocalization in small diameter vesicles close to the plasma membrane. Monensin, which is an inhibitor of receptor recycling, caused accumulation of intracellular CD44 and hyaluronan. Large circular structures with CD44 on the perimeter surface and hyaluronan inside were present in confocal analysis of the cytoplasm. QX50, a monoclonal anti-CD44 antibody which increases pericellular hyaluronan staining in REKs 9, caused a consistent, dose-dependent increase in the intracellular hyaluronan positive area (see Table 1). A normal, isotypically matched mouse IgG and Hermes3, an anti-CD44 antibody against human CD44 which does not recognize rat CD44, only slightly increased the level of intracellular hyaluronan. Endocytosis of exogenous hyaluronan Following an incubation at 4 °C for 30 min and a chase at 37°C FITC-labeled hyaluronan was observed in cytoplasmic vesicles. The presence of an excess of HAoligosaccharides inhibited most of the binding and internalization, when the FITC-HA concentration was 10 ug/ml or lower. At 20 ug/ml FITC-HA concentration, HAoligosaccharides (I mg/ml) caused approximately a 50% reduction in the amount of internalized HA. At higher concentrations the oligosaccharides failed to compete with FITC-HA uptake. The FITC-HA positive vesicles in oligosaccharide-treated specimens also contained TR-dextran. Although most of the exogenous FITC-Iabeled HA and endogenous HA were in separate vesicles, a few vesicles showed colocalization.
Epidermal keratinocytes
523
CONCLUSIONS Rat epidermal keratinocytes contain a pool of endogenous intracellular hyaluronan. The mean molecular mass of endogenous intracellular hyaluronan is smaller than that of extracellular hyaluronan. REKs also internalize exogenous high-molecular weight hyaluronan. Hyaluronan at lower concentrations is internalized by receptor mediated uptake, but at higher concentrations in the incubation medium, fluid phase uptake also takes place. The receptor mediated uptake does not utilize coated pits or caveolae. The hyaluronan uptake rate in REKs is modulated by CD44 antibodies. However, the receptor specificity requires HA decasaccharides which suggests that other molecules in addition to CD44 may take part in the binding and internalization. Once internalized, the half life of HA before degradation to a level undetectable by the bHABC probe is short, less than 10 min. Mitotic and migrating cells show elevated levels of intracellular hyaluronan suggesting that rapid hyaluronan metabolism plays role in these processes.
ACKNOWLEDGEMENTS We thank Ms. Arja Venalainen, and Ms. Riikka Tiihonen for technical help in the chromatographic assays and for preparing the light and electron microscopic specimens. The financial support from the Academy of Finland, Finish Cancer Foundation and EVO Funding of Kuopio University Hospital is acknowledged.
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R. Tamrni, 1. Ripellino, R. U. Margolis, H.I. Maibach & M. Tamrni, 'Hyaluronate accumulation between upper spinous cells in human epidermis treated with retinoic acid in skin organ culture', J. Invest. Dermatol., 1989,92,326-332.
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U. M. Agren, M. Tamrni & R. Tamrni, 'Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture' , J. Cell. Physiol. , 1995 ,164, 240-248.
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R. H. Tamrni, M. I. Tamrni, V. C. Hascall, M. Hogg, S. Pasonen & D. K. MacCallum, 'Collagen substrates surfaced with a pre-formed basal lamina alter the metabolism and distribution of hyaluronan in epidermal keratinocyte *organotypic' culture', Histochem. Cell. BioI., 2000, 113,265-277.
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A-L. Tuhkanen, M. Tamrni, A. Pelttari, U. M. Agren & R. Tamrni, 'Ultrastructural analysis of human epidermal CD44 reveals preferential distribution on plasma membrane domains facing the hyaluronan-rich matrix pouches', J. Histochem. Cytochem., 1998,46,241-248.
6.
D. J. Aguiar, W. Knudson & C. B. Knudson, 'Internalization of the hyaluronan receptor CD44 by chondrocytes', Exp. Cell Res., 1999,252,292-302.
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M. Culty, T. E. O'Mara, C. B. Underhill, H. Yeager Jr. & R. P. Swartz, 'Hyaluronan receptor (CD44) expression and function in human peripheral blood monocytes and alveolar macrophages', J. Leukoc. Biol., 1994,56,605-611.
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G. Kaya, I. Rodriguez, 1. L. Jorcano, P. Vassalli & I. Stamenkovic, 'Selective
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Keratinocytes and hyaluronan suppression of CD44 in keratinocytes of mice bearing an antisense CD44 trans gene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation', Genes Develop., 1997, 112,996-1007.
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R. Tammi, D. MacCallum, V. C. Hascall, I-P. Pienimaki, M. Hyttinen & M. Tammi'Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides', J. Biol. Chem., 1998,273,28878-28888.
10. S. P. Evanko & T. N. Wight, , Intracellular localization ofhyaluronan in proliferating cells', 1. Histochem. Cytochem., 1999,47,1331-1342. 11. L. Collis, C. Hall, L. Lange, M. Ziebell, R. Prestwich & E. A. Turley, , Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action', FEBS Lett., 1998,440,444-449. 12. D. K. MacCallum & J. H. Lillie, 'Evidence for autoregulation of cell division and cell transit in keratinocytes frown on collagen at an air-liquid interface', Skin. Pharmaco!' 1990, 3, 86 - 96. 13. J. Yannariello-Brown , C. T. McGary & P. H. Weigel, 'The endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells is Ca( +2)-independent and distinct from a Ca(+2)-dependent hyaluronan binding activity', 1. Cell. Biochem., 1992,48, 73-80. 14. J. E. Schnitzer, P. Oh , E. Pinney & J. Allard, 'Filipin-sensitive caveolae-mediated transport in endothelium: Reduced transcytosis, scavanger endocytosis, and capillary permeability of select macromolecules', J. Cell Biol., 1994, 127,1217-1232. 15. M. A. West, M. S. Brestscher & C. Watts, 'Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells', 1. Cell Biol., 1989, 109,2731-2739. 16. 1. Mellman, R. Fuchs & A. Helenius, , Acidification of the endocytotic and exocytotic pathwats', Annu. Rev. Biochem., 1986, 55,663-700 17. R. Tammi, U. M. Agren, A-L. Tuhkanen & M. Tammi, 'Hyaluronan metabolism in skin', Progr. Histochem., 1994, 29/2.
EVALUATION OF THE INFLUENCE OF HYALURONAN AND HYALURONAN FRAGMENTS ON HUMAN KERA TINOCYTES DURING UV IRRADIATION Doreen Gerlach ", Christoph Huschka, Wolfgang Wohlrab Department ofDermatology, Martin-Luther-University ofHalle-Wittenberg, Emst-Kromayer-Str. 5-8. D-06097 Halle. Germany. [email protected]
ABSTRACT
In response to the attack of reactive oxygen species (ROS) caused by UV irradiation, skin has developed a complex antioxidant defence system. We investigated the influence of hyaluronan (HA) or hyaluronan fragments (HAP) on UV-irradiated human keratinocytes. We studied the vitality of the cells by the determination of the cell number and the DNA synthesis efficiency using BrdU incorporation. The intracellular changes ofthe ROS content were determined by a dihydrorhodamine 123 test (DHR). Our results indicate that both HA and HAP protect keratinocytes against the consequences ofUVA and UVB irradiation. As a result ofUV irradiation an increase in the content of intracellular peroxides was observed. In the presence of HA or HAF, a reduction in the content of ROS could be achieved. With rising doses of UVB the number of viable keratinocytes was decreased, while the additional treatment with HA or HAP led to a significant improvement of viability. The present results might be explained by the anti-inflammatory and radical capturing qualities ofHA or HAP. KEYWORDS
Hyaluronan fragments, keratinocytes, ultraviolet irradiation, reactive oxygen species INTRODUCTION
The enormous increase in skin cancer is related to the exposure to UV light and the UV-induced photo dynamic reactions. The most important mechanism of the UVinduced damage of biomolecules is based on the activation of oxygen, the formation of peroxides and the induction of radical chain reactions. The long, linear, non-branched chain of HA is very sensitive to cleavage by reactive oxygen species (ROS) 1. The objective of this work was to investigate in vitro reactions of HA or HAP on UV-irradiated human keratinocytes. MATERIALS & METHODS Chemicals
Hyaluronan fragments (HAP) were kindly provided from Dr. 1. H. Ozegowski (Department of Microbiology, Friedrich-Schiller-University, lena, Germany). Keratinocyte serum free medium (SFM) was obtained from Gibco Life Technologies GmbH (Eggstein, Germany). Gentianaviolet was purchased from Riedel-de Haen
526
Keratinocytes and hyaluronan
(Seelze, Germany). Cell Proliferation ELISA, BrdU (colorimetric), was from Boehringer (Mannheim, Germany). Methanol, ethanol and PBS were from Merck (Darmstadt, Germany). DHR was purchased from Sigma (Deisenhofen, Germany). Cell culture HaCaT-ceIIs were kindly provided from Prof. Dr. Norbert E. Fusenig (Cancer Research Centre, Heidelberg, Germany). Cells of passages 24th to 33th were used. Cells of these passages ensure a good reproducibility 2-3. HaCaT cells are suitable for radiation studies with ultraviolet light s. The cells were cultured in 96 well plates in SFM supplemented with 0.1-0.2 ng/ml epidermal growth factor (EGF) and 25 ug/ml bovine pituitary extract at 37°C in a humidified atmosphere containing 5 % C02. Preincubation of confluent keratinocytes with HA or HAF means that the respective substance was added to SFM at defined intervals prior to UV irradiation. After the incubation period, the medium including the not bound or not incorporated HA or HAF was rejected and replaced by PBS only. HA or HAF treatment without preincubation means that the respective substance was added in PBS to the cells immediately prior to UV irradiation and leaved on the cells during the irradiation. Ultraviolet irradiation The keratinocytes were irradiated using an UV-irradiation chamber (Dr. Grobel Elektronik, Germany), whereby the non-irradiated control cells were covered by an aluminium foil. The UVR sources were an F15T8 lamp (Sankyo Denki) with a peak at 309 nm for UVB and a TL-D 15W/05 lamp (Philips) with a peak at 370 nm for UVA. Viability and DNA synthesis After irradiation, PBS was replaced by SFM followed by an incubation period of 24 h. To evaluate the viability of HA or HAF treated keratinocytes, the crystal violet assay was carried out 4. SFM was removed and viable cells were fixed with methanol for 10 min and rinsed with water four times. The keratinocytes were then stained for 10 min with an 0.1 % crystal violet solution. After rinsing with water four times, the dye was solved by 0.1 mol/l trisodium citrate in 50 % ethanol. The extinction (1..=540 nm) was measured using a microplate reader iEMS (Labsystems). DNA synthesis efficiency was assessed by BrdU incorporation into newly synthesised DNA. The ELISA reaction product was quantified by measuring the absorbance at 370 nm and 490 nm. Content of peroxides For the examination of the UV-induced formation of intracellular peroxides, the oxidation of DHR to the red fluorescent rhodamine 123 was measured according to Peus et al. 6. DHR was added to the medium (containing HA or HAF in the case of preincubation) at a final concentration of 5 11M and the cells were incubated for 45 min. Then the cells were washed twice with PBS. According to whether a preincubation or no preincubation was intended, PBS or PBS containing HA or HAF was added to the cells, and then the irradiation was carried out. The plates were measured 10 min after the end of irradiation at wavelengths of 485 nm and 538 nm using a fluorescence reader Fluoroskan Ascent (Labsystems; Finland).
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Statistics Each bar represents the mean ± S.D. Differences between the means were tested using the Student-Newman-Keuls method (t-test). Differences were considered to be statistically significant ifp < 0.05. RESULTS & DISCUSSION With increasing doses of UVB, the content of viable keratinocytes and the DNA synthesis decreased (Fig. 1). The decrease in DNA synthesis was more intensive than the decrease in the content of viable cells but there was a similar dose dependence. Reasons for the UV-induced decrease in DNA synthesis are the reaction of DNA with peroxides and the formation of pyrimidine dimers. As a result of UV-irradiation, the content of peroxides was significantly increased. In the range of comparable physiological doses, the generation of peroxides were higher by UVA irradiation than by UVB irradiation (Fig. 2 and Fig. 3). ~
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Effect ofUVB irradiation on the content of peroxides in human keratinocytes. * p<0.05; n = 8 (mean ± S.D.) untreated vs. UVB.
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In the presence of HA or HAP without irradiation, viability and proliferation behaviour of confluent HaCaT-cells were not altered (concentration range 0.01-100 ~M; incubation for 24 h, 48 h and 72 h - data not shown). HA or HAP do not absorb UVA or UVB light (Fig. 4 and Fig. 5). The antiproliferative and cytotoxic effects of UVB irradiation (120 ml/crn") could be partly abolished by preincubation with HA or HAF (Fig. 6). A short preincubation for I h is no less effective than a long preincubation for 24h. The UV induced increase in the content of peroxides could also be partly prevented by preincubation with HA or HAP (Fig. 7 and 8). A short preincubation time is more effective than a long preincubation time (Fig. 7). This is probably caused by a rapid metabolism of HA or HAF by the keratinocytes or a degradation of HA or HAF by ROS 7. This process is concentration-dependent as well (Fig. 8). In the case of the addition of HA or HAF only during the irradiation procedure, the content of peroxides was decreased too (Fig. 9). UV irradiation led to high amounts of peroxides in keratinocytes which are lower in the case of HAF or HA preincubation and in the presence of HAF or HA only during the UV irradiation. We suppose that for the tests with preincubation of HA or HAF a receptor linkage is necessary for the protective effect.
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530
Keratinocytes and hyaluronan
The results of the viability indicate a time and concentration dependent influence of HA or HAF on UVB-irradiated cells. The scavenger qualities alone could not be responsible for the protective effect of HA and HAP because ascorbic acid was more effective in scavenging ROS (Fig. 7) but without protecting from cell death (data not shown). An absorbance effect like in the case of sunscreens could be excluded (Figures 4 and 5). A short preincubation was as effective as a long preincubation - pro radical scavenger theory - contra protein biosynthesis theory. A CD44 receptor intervention could be possible. A positive influence of HA or HAF on the inflammation process as supposed by the use in orthopaedics or wound healing is also possible. The penetration behaviour of HA or HAF is not well established, and, to our knowledge, an effective transdermal transport of HA or HAF has not been described so far. Due to the lower molecular weight, however, it should be assumed that topical applied HAP can better penetrate than HA. Consequently, exogenous applied HAF may protect epidermal human keratinocytes from damage caused by exposure to sunlight. CONCLUSIONS HA or HAF are able to scavenge ROS which are generated during UV irradiation and can protect keratinocytes from UV damages. Thus, it would be conceivable that HAF could be used for sunscreens or other topical applications. REFERENCES I. C. L. Hawkins, M. J. Davies, Direct detection and identification of radicals generated during the hydroxyl radical-induced degradation of hyaluronic acid and related materials, Free Radic. Bioi. Med, 1996, 21 (3), 275-290. 2. B. Bonnekoh, J.Roeding, G. R. F. Krueger, M. Ghyczy, G. Mahrle, Increase of lipid fluidity and suppression of proliferation resulting from liposomal uptake by human keratinocytes in vitro, Brit. J Dermatol., 1991, 124,333-340. 3. R. Laub, B. Bonnekoh, G. Mahrle, Alteration der KI67-positiven Wachstumsfaktoren in vitro kultivierter Keratinozyten, Dermatol. Monatsschr. , 1991, 177, 1-9. 4. P. Boukamp, R. T. Petrussevska, D. Breitkreutz, J. Hornung, A. Markham, N. E. Fusenig, Division of Differentiation and Carcinogenesis in Vitro, 1. Cell Biology, 1988, 106,761-771. 5. J. Lehmann, D. Pollet, S. Peker, V. Steinkraus, U. Hoppe, Kinetics of DNA strand breaks and protection by antioxidants in UVA-or UVB-irradiated HaCaT keratinocytes using the single cell gel electrophoresis assay, Mutation Research, 1998,407,97-108. 6. D. Peus, R. A. Vasa, A. Meves, M. Pott, A. Beyerle, K. Squillace, M. R. Pittelkow, H202 Is an Important Mediator of UVB-Induced EGF-Receptor Phosporylation in Cultured Keratinocytes, 1. Invest. Dermatol., 1998, 110,966-971. 7. U. M. Agren, R. H.Tammi, M. I. Tammi, Reactive oxygen species contribute to epidermal hyaluronan catabolism in human skin organ culture, Free Radic. Bioi. Med, 1997,23,996-1001.
HY ALURONAN DERIVATIVES IN POSTSURGICAL ADHESION PREVENTION Daniele Pressato', Emilio Bigonl, Massimo Donal, A1essandra Paveslo', Davide Renier', Luciana Bonaflni', Pier Andrea De Iaco' & Mario Lise 3 I Fidia Advanced Biopolymers via Ponte della Fabbrica 3/A, 35031 Abano Terme, Padova, Italy. 2 Unita Complessa di Istituti di Ostetricia e Glnecologia, Ospedale S. Orsolal, Universita di Bologna. via Massarentl 13,40138 Bologna, Italy. 3 Dipartimento di Scienze Oncologiche e Chirurgiche. Sezione di Clinica Chirurgica Generale II. Universita di Padova, via Giustiniani 2, 35128 Padova, Italy.
ABSTRACT Adhesion formation after gynecological and general surgery is a major cause of morbidity and complications which include infertility, small bowel obstruction, pelvic pain and lengthy re-operations. Moreover, adhesion formation after abdominal and pelvic surgery causes a substantial economical burden due to increased medical care costs and loss of patient productivity. Several approaches have been suggested to prevent or reduce adhesions: mini-invasive surgery, pharmacologic therapy and intraperitoneal instillation of solutions or polymers. Current methods of reducing adhesions often involve "barriers", both resorbable and not, or combinations of both, aimed at separating adjacent abdominal organs and promoting peritoneal wound healing. Hyaluronan (HA) and its derivatives have recently been identified as promising biodegradable barriers for adhesion prevention. A new, absorbable, crosslinked hyaluronan derivative, (HYALOBARRIER'" gel), with improved viscosity and prolonged residence time, and a benzylic ester derivative HYAFF™ II have shown good biocompatibility and efficacy in reducing adhesions in abdominal, gynecological and E.N.T. surgery. Moreover, combinations of HA with synthetic, non-absorbable meshes used in large abdominal defects, have proved effective in reducing adhesions between intestinal and prosthetic material. A review of the preclinical and clinical data on Fab Hyaluronan derivatives in the prevention of postsurgical adhesions will be presented. KEYWORDS Adhesion prevention, cross-linked hyaluronan, HYAFfT M 11, surgical mesh, plasmacoating. INTRODUCTION Postsurgical adhesions represent one of the most common complications following surgical procedures and are an inevitable outcome of the wound healing process in the peritoneum and other anatomical structures. The adhesions that occur in response to injury in various kinds of surgery, i.e. abdominal, gynecological, E.N.T. and orthopedic surgery, are fibrotic scars that connect internal organs or parts of the body that normally are separated. They occur at the site of surgical procedure
492
Clinical applications ofhyaluronan
although they may also occur elsewhere due to indirect causes such as inflammatory diseases, infection, foreign body reactions or therapy. The physiological mechanism of adhesion formation still remains unclear. However, it is now generally agreed that, for the abdomino-pelvic area, the loss of fibrinolytic activity of the peritoneum due to a reduced activity of the plasminogen activators with persistence of fibrinous exudate, is a major cause of permanent fibrous adhesions I. In most patients, adhesions have severe clinical consequences such as bowel obstruction, chronic pelvic pain (but not always) 2-3 and infertility 4.5. The development of adhesions may interfere with intraperitoneal therapies and can make a surgical reintervention more difficult, because of the risk of injury to the bowel. The rate of adhesions is very high: authors have reported that adhesions occur in 55% to 100% of patients after surgery. The incidence of adhesions in the female reproductive tract and in different components of the abdominal wall range from 71% to 94% 6. Menzies et al. found that 93 % of patients who had undergone at least one previous abdominal operation had adhesions, compared with only lOA % of patients who had never had any previous abdominal operations 7. Moreover, adhesions represent a significant economical burden: a 1998 study indicated that the costs of hospitalization for the lysis of adhesions in 1994 was approximately $ 1.3 billion in the United States 8. Between 1990 and 1994 the number of surgical abdominal operations for adhesiolysis increased from 91, I 07 to 107,290 and the costs increased from 2,080 to 3,220 million dollars 9. Various methods have been suggested to reduce or prevent adhesions, and several experimental and clinical studies have shown improvement in pain after adhesiolysis 9-10. However adhesions almost always reform and so the surgical procedure is sometimes useless II. The reduction of incisional areas by means of laparoscopy reduces, but does not eliminate the problem 12. Pharmacological therapies such as the use of antiinflammatory 13, antioxidant, anticoagulant and fibrinolytic agents'':" are effective in reducing adhesion formation, but the results of the relative clinical trials have often been conflicting 16. The use of surgical barriers both resorbable and not has also been experimented, nevertheless their antiadhesive properties are often compromised in critical conditions such as inadequate hemostasis and bleeding", In particular kinds of surgery i.e, incisional hemia repair or acquired abdominal wall defects where synthetic mesh implantation is required, a reduction of adhesions was seen when these non-absorbable devices were coupled with biodegradable polymers 18-19. Recently, experimental preclinical trials have shown that hyaluronan (HA), a natural component of the extracellular matrix, when applied intraperitoneally or used as a "coating" on the injured surfaces, reduces postoperative adhesions after abdomino-pelvic surgery 20. The efficacy of HA depends on the molecular weight as well as the viscosity of the preparation used. However, the results were conflicting, the rapid degradation and dispersion of unmodified HA from the site of administration" reduced its antiadhesiogenic properties. These findings indicated a need to modify HA chemically in order to increase its viscosity and consequently its residence time. Bums 22 et a1. showed that a gel form of modified HA reduced the incidence and severity of de novo adhesions. New hyaluronan derivatives including auto-crosslinked gels (HY ALOBARRIER gel) 23, benzylic ester derivatives (HY AFF iIIl II) in the form of non-woven fabrics and technologies to apply HA to synthetic meshes by means of a plasma coating process 24 have been developed by Fidia Advanced Biopolymers (Fab). The auto-crosslinking reaction of HA produces a significant increase in the gel's viscosity and a prolonged residence time compared to the starting hyaluronan solution, with an improvement in its antiadhesive effect. The esterification ofHA with benzylic alcohol permits the processability of the polymer and a non-woven pad can be obtained. The hyaluronan coating on synthetic surgical mesh for abdominal surgery improves the biocompatibility of the device 25, thus leading to a reduction in the risk of bowel adhesions.
Postsurgical adhesion prevention
493
Different animal models and surgical protocols involving pelvic surgery in laparotomy and laparoscopy, abdominal surgery and E.N.T. surgery were extensively utilized during the preclinical and clinical development ofFab's hyaluronan derivatives in adhesion prevention. In experimental trials the products were tested in comparison to antiadhesive barriers currently available in clinical practice. In the clinical trials the effectiveness of the devices was compared vs. control patient groups that received conventional surgical procedures for abdomino-pelvic and E.N.T. surgery, but no antiadhesive treatments were applied. MATERIALS & METHODS
Preclinical experiments Different Hyaluronan derivatives were purchased from Fidia Advanced Biopolyrners (Abano T. Italy) and selected for the preclinical animal experiments in different kinds of surgery. The crosslinked derivative gel (RYALOBARRlERlI> gel) was tested in abdominal and gynecological surgery both in laparoscopy and laparotomy. The synthetic HA plasmacoated devices were tested in a standardized model oflarge abdominal wall defect repair. In all experiments, the animals were provided with standard chow and water ad libitum. Animal care and surgery were performed under the National Guidelines of the Ministry of Health # 116/92. Abdominal surgery Forty New Zealand female white rabbits weighing 3-4 Kgs were randomly allocated into two treatment groups (n=20): no treatment (control group) and RYALOBARRlERlI> gel (treated group). At the time of laparotomy for adhesion evaluation, observers were blinded to the treatment applied. The animals were kept fasting for at least 24 hours before surgery, and anesthetized by intramuscular injection with SO mg/kg of Ketamine hydrochloride and 1.6 mg/kg Xylazine hydrochloride. They were then placed in supine position, shaved on the abdomen and prepped. After a 10 ern mid line laparotomy the parietal peritoneum of the right side was exposed and a 10 em" defect including both parietal peritoneum and the muscular fascia was created by sharp dissection. The cecum was then exposed, and abraded starting from the Sth to the io- haustrum distally from the ileocecal junction until bleeding appeared. The cecum was then returned to its normal anatomical position. The abdominal cavity was washed with Ringer's lactate solution and accurate hemostasis was performed. Ten weeks after surgery, the animals were euthanized and the grade and severity of adhesions were blindly evaluated applying a 0-11 adhesion score", The incidence of adhesion-free animals was also evaluated. Gynecological surgery Two different experimental standardized protocols were followed in this type of surgery: HYALOBARRIER iIll gel was tested in 64 female rabbits subjected to laparoscopy and 69 animals which received laparotomic surgery. In laparoscopy, adhesions (protocol 1) were induced by means of two different surgical trauma: abrading the uterine horns and denuding a delimited area of the adjacent peritoneum. Following surgery accurate hemostasis was achieved by means of an electrocoagulator and the animals were randomly assigned to HYALOBARRIER iIll gel treatment (n=22), TC7 Interceed'", a commercially available antiadhesive barrier (n=20), or no treatment (n=22). Treatments were applied uniformly over the raw surfaces. Six weeks after surgery, adhesions were blindly evaluated applying a
494
Clinical applications ofhyaluronan
scoring system (0-4 scale)" and determining the percentage of animals with severe adhesions. In laparotomic surgery (protocol 2) the four major blood vessels of each uterine hom ligament were excised until abundant bleeding appeared. No hemostasis or cautery was performed. Following surgery, the animals were randomly assigned to HYALOBARRIER gel (n=20) treatment, Intergelf" (n=12), Seprafilm" (n=17) (two hyaluronan based devices), or no treatment. The materials were applied in order to cover completely all injured anatomical sites. Two weeks after surgery, the animals were euthanized and the adhesions were graded using a 0-4 score" and comparing the percent of adhesion-free animals in each treatment group. Abdominal surgery (large defect). Two different surgical meshes based on ePTFE and polyester mesh used in clinical practice for incisional hernia repair, were surface modified by hyaluronan plasma treatment. The biomedical devices were steam-sterilized. Seventy-two rabbits were anesthetized with ketamine/xylazine i.m. injection and subjected to the following surgical procedures: a 12 em' full-thickness parietal defect was created and the omentum was removed. The animals were randomly divided into four groups and the surgical meshes for abdominal repair were fixed to the abdomen after isolation of the muscular plane by means of non-absorbable sutures. Two groups received either ePTFE or polyester unmodified meshes (controls), the remaining two groups received the same devices subjected to HA-plasma coating treatment (treatments). Two months after surgery, a secondlook laparotomy was performed, the adhesions were blindly assessed applying a site-specific adhesion score", and the incidence of adhesion-free animals was calculated. The stability of the implanted meshes was evaluated, as was the presence of inflammatory reaction by means of histological observations. Clinical trials. Various clinical trials have been designed to demonstrate the safety and efficacy of hyaluronan derivatives: the crosslinked hyaluronan derivative HYALOBARRIER gel was tested in abdominal and gynecological surgery, whereas the benzylic ester derivative HYAFFTM 11 in non-woven form (commercial name Merogel") was tested in E.N.T. surgery. In abdominal surgery, 20 patients with ulcerative colitis and familial polyposis who were scheduled for colectomy and ileal pouch-anal anastomosis with diverting-loop ileostomy were enrolled in a pilot trial. Before abdominal closure, the patients were randomly assigned to treatment or control (untreated). The patients in the treated group received the gel uniformly distributed on the intestinal loops. second look laparoscopy for adhesion evaluation was programmed when patients returned for ileostomy closure. In gynecological surgery, three different trials were designed involving the following surgical procedures: laparoscopic surgery (study 1), hysteroscopic surgery (study 2) and laparotomy (study 3). In the first multicenter controlled clinical trial, eighty patients subjected to myomectomy, ovarian cysts removal, adhesiolysis, conservative treatment of ectopic pregnancy and endometriosis removal, randomly received HYALOBARRlER gel (n=40) or no treatment (n=40). In the second trial, sixty patients subjected to hysteroscopic surgery for septatae uterus, intrauterine adhesions, endometrial ablation, endometrial polyps and submucosal myoma randomly received the gel (n=30) or no treatment (n=30). Patients subjected to laparotomic surgery for myomectomy were enrolled in the third multicenter study and randomly assigned to a HYALOBARRIER treatment group or untreated control group. All the study protocols were approved by ethical review and all patients gave written informed consent. For ethical considerations, in the gynecological
Postsurgical adhesion prevention
495
surgery studies, the second look laparoscopy required for adhesion assessment (6-12 months after surgery) was envisaged in the protocol at the discretion of the principal investigators. In the E.N.T. trial, inclusion criteria involved patients undergoing functional endoscopic sinus surgery (FESS) for bilateral or unilateral chronic sinusitis. Patients randomly received Merogel" in the treated group and FESS with standard postoperative treatment without application of the product in the control group. The safety of the hyaluronan-based device was evaluated recording the occurrence of adverse events at each control visit 1, 14, 30, 60 and 90 days after surgery. Its effectiveness was evaluated at 14, 30 and 90 days following initial surgery recording the presence of adhesions in the cavities by video sinus endoscopy. RESULTS
Preclinical Experiments Abdominal surgery At the time of second look laparotomy the gel had been completely absorbed. No abdominal hernias, infection, or other adverse events were recorded during postsurgical monitoring. The mean adhesion scores were respectively 0.65±0.45 and 7.73±0.83 for the HY ALOBARRIER gel and untreated control groups. In this case, significant differences were seen between the two groups (P = 0.0001 non parametric Kruskal-Wallis test). The percentage of adhesion-free animals was significantly greater in the group treated with ACP gel than in the untreated control group: 90% HY ALOBARRIER gel vs. 15% control (P = 0.0001 Chi-square test).
Gynecological surgery In the laparoscopic protocol, six weeks after surgery at the second look observation when Blauer's scoring system of adhesions (0-4 scale) was applied, the HYALOBARRIER gel treatment showed the lowest grade of postsurgical adhesions (1.25±0.28) compared to TC7 Interceed" (2.45±0.22) and the untreated group (2.24±O.26). Significant differences were found between the HYALOBARRIER gel vs. TC7 Interceed''" and untreated control groups (P < 0.05 Kruskall-Wallis non-parametric test). The HY ALOBARRIER gel treatment group showed the highest percentage of animals with severe adhesions (35%) compared to the TC7 Interceed" (85%) and to the untreated group (66%). In the laparotomic protocol (protocol 2) the group of animals treated with HY ALOBARRIER gel presented a low adhesion grade (1.15±0.32) and a percentage of adhesion-free animals corresponding to 55%. The other treatments also gave a low adhesion grade (Intergel" 1.17±O.46, Seprafilm" 2.29±O.44) and a percentage of adhesion-free animals corresponding to 58% in the Intergel" and 35% in the Seprafilm" groups. Statistically significant differences were found in all treatments vs. untreated control (P < 0.05 Kruskal-Wallis non-parametric test and Chi-square tests) but no differences were found between the treatment groups.
Abdominal surgery (large defect) At the time of second-look laparotomy, the new-grown mesothelial layer in the surgical defect was incorporated into all prosthesis, no evidence of seroma or intra-abdominal abscess was present. The HA-coated synthetic membranes gave lower adhesion score meanS±sem than the unmodified surgical devices. The HA-treated ePTFE group developed less severe adhesions
496
Clinical applications ofhyaluronan
(filmy and avascular adhesions) between the biomaterials and the intestinal loops or liver then the other treatments. The resulting adhesion grade was 0.44:1:0.30 in the HA-plasmacoated ePTFE group and 2.72±0.97 in the corresponding uncoated controls. The scores of HA-coated polyester meshes and the uncoated biomaterial were respectively 3.44±1.06 and 6.56±l.lS, a statistically significant difference was seen by applying the Kruskal-Wallis nonparametric test (P < 0.05). The same trend was seen by evaluating the incidence of adhesionfree animals in each treatment group: 66.6% of animals in the HA-ePTFE treated group showed absence of adhesions vs. S9.9% of the uncoated ePTFE group. A decrease in the percentage of adhesion-free animals was seen in the group treated with polyester-based devices: the percentages of animals with no adhesions were respectively 50% in the polyester-coated mesh group vs. 22% in the uncoated control group (P < 0.05, Chi-square test) that showed strong and widespread adhesions between mesh and bowel and liver. On histological observation, the thickness of the muscle-mesh interface did not differ between HA treated membrane groups and uncoated controls. Similarly, no inflammatory reaction or fibrosis at the muscle-mesh interface was seen.
Clinical Trials To date, 20120 patients have been enrolled in the pilot abdominal surgery study. Postoperatively, no complications or adverse events in the short or long term occurred in treated and untreated groups. Adhesion assessment by second look laparoscopy is currently ongoing. SO/SO patients in the gynecological laparoscopy and 60160 in hysteroscopy were enrolled and they received an average of20 and 10 ml of gel respectively. Clinical data recorded during the postoperative course indicate the absence of any serious adverse events. No statistically significant differences were observed in the incidence of the most frequent adverse events, fever and pelvic pain, which normally occurred in the postoperative period. This data confirmed the good tolerability of HYALOBARRIERiI> gel. Hospitalization time was no longer for the treated patients than for the untreated control group. Adhesion evaluation by second look laparoscopy is still ongoing. The gynecological trial in laparotomy is currently ongoing, to date 50/100 patients have been enrolled and no complications or serious adverse events have been observed. 66 patients were enrolled in the E.N.T. trial for a total of 117 nasal cavities subjected to surgery. The two study groups were statistically comparable for demographic characteristics. No serious adverse events occurred in the treated (Merogel") and control groups. During the control visit, clinical evaluation by means of video-endoscopy demonstrated a significant reduction (P < 0.05) in the percentage of sinuses with adhesions 30 and 90 days after surgery in the Merogel" group in comparison to the control (1.9% vs. 14.9% and 3.6% vs. 23.7% respectively). DISCUSSION Various methods of adhesion prophylaxis have been used during experimental and clinical trials. Practicing an appropriate surgical technique is the most effective strategy for reducing the risk of adhesion formation. The reduction of incisional areas by means of miniinvasive surgery, limiting trauma, avoiding introducing foreign bodies into the peritoneal cavity and achieving an adequate hemostasis, are surgical procedures that every surgeon should adopt. However, improvements in surgical technique alone have not satisfactorily reduced the incidence of adhesion formation. The barrier methods based on hyaluronan and its derivatives seem to be promising because of their efficacy and absence of serious side effects. The devices are used as a barrier to
Postsurgical adhesion prevention
497
separate adjacent surfaces while the wound healing process occurs. This method was demonstrated to be effective because it produces an intraperitoneal "flotation" during peritoneal healing, avoiding fibrin bridge formation between adjacent organs. However, in many cases the solution's low viscosity and consequent rapid clearance represent a critical point, reducing the antiadhesive efficacy of the device. The hyaluronic acid derivatives HYALOBARRIER@ gel and HY AFFTM 11 (Merogel" are new, absorbable biomaterials, highly biocompatible as previously demonstrated in a series of toxicological studies, and effective barriers which can stay in place for an adequate time period prior to degradation. The results of our experimental studies showed that HYALOBARRlERII!> gel used intraperitoneally in an amount sufficient to cover the injured surfaces inhibits adhesion formation in different surgical procedures even in presence of critical conditions such as bleeding or inadequate hemostasis. Moreover, in clinical trials both in abdominal and gynecological surgery HYALOBARRIERII!> gel showed good tolerability without any adverse events attributable to the use of the device. Clinical results in the E.N.T. trial show that a hyaluronan benzylic ester derivative in nonwoven form (Merogel" can limit the incidence of postoperative adhesions compared to the standard surgical procedure. This clinical study suggested that hyaluronan-based devices may act as a barrier between the damaged surfaces interacting favorably in the postoperative healing process of sinus mucosa. In abdominal surgery, particularly in the large defects, the best preventive measure is peritoneum interpositioning. However, the peritoneal layer is not always present, consequently the use of a synthetic mesh is often required. The direct contact of the surgical mesh with underlying viscera can increase the risk of adhesions and fistula formation. In an experimental trial we demonstrated that HA applied to a synthetic device using a plasma coating process, can improve the biocompatibility properties reducing significantly the risk of adhesions without compromising the tensile strength characteristics of the devices. In conclusion, the Hyaluronan plasma-coating process appears promising, and should become a valuable tool in enhancing the tolerability of a synthetic material. Further clinical trials will be designed to confirm the experimental data.
CONCLUSIONS To date, preclinical studies, clinical safety and efficacy data, have indicated that Fab. hyaluronan derivative-based devices are effective in reducing adhesions in different kinds of surgery. The devices are non-toxic, non-antigenic, biocompatible and well tolerated and therefore represent promising devices for the prevention of postoperative adhesions.
REFERENCES 1. G.S. diZerega. The cause and prevention of postsurgical adhesions: a contemporary update. In: Gynecological Surgery and Adhesion Prevention. M.P. Diamond, G. diZerega, c.B. Linsky & R.L. Reid (eds.), Wiley-Liss Inc., New York, 1995, pp. 27-37. 2. H. Ellis, 'The Clinical Significance of Adhesions: Focus on Intestinal Obstruction', Eur. J. Surg. 1997; Suppl. 577:5-9. 3. J.F. Steege & A.L. Stout, 'Resolution of Chronic Pelvic Pain After Laparoscopic Lysis of Adhesions', Am J Obstet Gynecol 1991, 165, 278-283. 4. M.G.R. Hull, C.M.A. Glazener, N.J. Kelly, D.l. Conway, P.A. Foster et aI, 'Population study of causes, treatment and outcome of infertility' ,Br. Med. J., 1985,291, 1693-1697.
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Clinical applications ofhyaluronan
5. M.P. Diamond & A.H. De Cherney, 'Pathogenesis of Adhesion Formation/Reformation: Application to Reproductive Pelvic Surgery', Microsurgery, 1987,8,103-107. 6. M.P. Diamond, 'Incidence of postsurgical adhesions, In: Peritoneal Surgery. G.S. diZerega (ed.), Springer-Verlag, New York:, 1999:217-220. 7. D. Menzies & H. Ellis, 'Intestinal obstruction from adhesions - how big is the problem?', Ann. Royal ColI. Surg. Engl., 1990,72,60-63. 8. N.F. Ray, W.G. Danton, M. Thamer, S.C. Henderson & S. Perry, 'Abdominal adhesiolysis: impatient care and expenditures in the United States in 1994', 1. Am. CoIl. Surg., 1998, 186, 1-9. 9. S.D. Wexner, 'Prevention and treatment of adhesive small bowel obstruction', In: Proceedings of the 90th Congress of the Italian Society of Surgery, L. Pozzi (ed.), Padua October 19th_ 22th, 1997, pp. 248-252. 10. J.F.Steege & a.L.Stout 'Resolution of chronic pelvic pain after laparoscopic lysis of adhesions' Am. J. Obstet. Gynecol., 1991, 165,278-283. 11. A Hershlag, M.P. Diamond & A.H. DeCherney, 'Adhesiolysis. Clin. Obstet. Gynecol., 1991, 34, 395-402. 12. Operative Laparoscopy Study Group, 'Postoperative adhesion development after operative laparoscopy: evaluation at early second-look procedures', Fertil. SteriI., 1991, 55,700-704. 13. R. Marana, A.A Luciano, L. Muzii, V.E. Marendino & S. Mancuso, 'Laparoscopy versus laparotomy for ovarian conservative surgery: a randomized trial in the rabbit model', Am. J. Obstet. Gynecol., 1994, 171,861-864. 14. A.M. Kappas, G.H. Barsoum, 1.B. Ortiz, M.R.B. Keighley, 'Prevention of peritoneal adhesions in rats with Verapamil, Hydrocortisone Sodium Succinate, and Phosphatidylcholine', Eur J Surg., 1992, 158,33-35. 15. AG. Turcapar, C. Ozarslan, E. Erdem, C. Bumin, N. Erverdi et aI., 'The effectiveness of low molecular weight heparin on adhesion formation in experimental rat model', Int. Surg. 1995,80,92-94. 16. M.P. Diamond, C.B. Linsky, T. Cunningham, L. Kamp, E. Pines, A.H. DeCherney & G.S. diZerega, 'Adhesion Reformation: Reduction by the Use of Interceed (TC7) Plus Heparin. 1. Gynecol. Surg., 1991,7,1-6. 17. R.L. Reid, Ha P.M. Hn, J.E.H Spence, T. Tulandi, AA. Yuzpe, D.M. Wiseman, 'A randomized clinical trial of oxidized regenerated cellulose adhesion barrier (lnterceed, TC7) alone or in combination with heparin', Fertil. Steril. 1997,67,23-29. 18. P.K. Amid, AG. Shulman, I.L. Lichtenstein, S. Sostrin, J. Young, M. Hakakha, 'Experimental evaluation of a new composite mesh with selective property of incorporation to the abdominal wall without adhering to the intestines', J. Biomed. Mater.
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Res., 1994,28,373-375. 19. U.K. Dasika, W.D. Widmann, 'Does lining Polypropylene with Polyglactin mesh reduce intraperitoneal adhesions?', Am. Surg. 1998,64,817-820. 20. J.W. Bums, K. Skinner, J. Colt et al., 'Prevention of Tissue Injury and Postsurgical Adhesions by Precoating Tissues with Hyaluronic Acid Solutions', 1. Surg. Res., 1995, 59, 644-652. 21. D.A Grainger, W.R. Meyer, AH. De Cherney et a!., 'The Use of Hyaluronic Acid Polymers to Reduce Postoperative Adhesions. J. Gynecol. Surg., 1991,7,97-101. 22. J.W. Bums, K. Skinner, J. Colt, L. Burgess, R. Rose & M.P. Diamond, 'A hyaluronate based gel for the prevention of postsurgical adhesions: evaluation in two animal species', Fertil, Steril., 1996,66,814-821. 23. M. Mensitieri, L. Ambrosio, L. Nicolais, D. Bellini & M. O'Regan, 'Viscoelastic properties modulation of a novel autocrosslinked hyaluronic acid polymer', 1. Mater. Sci. Mater. Med., 1996,7,695-698. 24. A Pavesio, D. Renier, C. Cassinelli & M. Morra, 'Anti-adhesive surface through Hyaluronan coatings', Medical Device Technology, 1997,8,20-27. 25. P. Narayanan, 'Surface functionalization by RF plasma treatment of polymers for immobilization of bioactive-molecules', J. Biomater. Sci. Polymer ed., 1994,6, (2), 181193. 26. S.P. Boyers, M.P. Diamond & AH. De Cherney, 'Reduction of postoperative pelvic adhesions in the rabbit with Gore-Tex surgical membrane', Fertil. Steril., 1988,49, 10661070. 27. K.L Blauer & RL. Collins, 'The effect of intraperitoneal progesterone on postoperative adhesion formation in rabbits', Fertil. Steril. 1988,49,144-149. 28. L. Hagberg, O. Wik & B. Gerdin, 'Determination of biomechanical characteristics of restrictive adhesions and of functional impairment after flexor tendon surgery: a methodological study of rabbits', J. Biomechanics, 1991,24,935-942.
EFFECT OF HYALURONAN ON MATRIX METALLOPROTEASE EXPRESSION IN FIBROBLASTS AND KERA TOCYTES. Isnard N., Legeais J·M., Renard G., Robert L. Laboratoire de Recherche en Ophtalmologie, Esc B3 6eme etage, Hotel Dieu, I place du Parvis Notre-Dame. 75004 Paris. France.
[email protected]
ABSTRACT Both hyaluronan and matrix metalloproteases (MMPs) are thought to be involved in tissue remodelling in a variety of physiological and pathological processes such as embryonic development, morphogenesis, wound healing or tumor progression. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMP-s. In order to explore the possible relationship between these processes we studied the effect of hyaluronan on MMP-s expression (biosynthesis and activation) in culture of human skin fibroblasts and corneal keratocytes (explant cultures and cell cultures). These cells were shown to exhibit distinct phenotypes as far as matrix biosynthesis is concerned. Using a synthetic substrate: N-Suc(ala)3pNA we measured elastase-type endopeptidase activity produced by fibroblasts and keratocytes and characterised the MMPs by zymography. Hyaluronan added to fibroblast as well as keratocytes cultures stimulated the membrane bound elastase type endopeptidase activity in a dose dependant fashion. In presence of 1 mglml of hyaluronan there was an increase of MMPs expression and also an activation of these MMPs both by fibroblasts and keratocytes. KEYWORDS Hyaluronan, MMP-s, Fibroblasts, Keratocytes. INTRODUCTION Matrix metalloproteinases (MMP-s) are a family of extracellular matrix degrading enzymes that are thought to play a crucial role in tissue remodelling. MMP-s are involved in a variety of physiological and pathological processes such as embryonic development, morphogenesis, wound healing or tumor progression (1). Several MMP-s are expressed at low levels in normal tissues and are upregulated during pathological processes and tissue remodelling. Hyaluronan an extracellular polysaccharide plays an important role in a variety of physiological and pathological processes (2, 3,4). In wound healing and/or inflammation MMP-s and hyaluronan are both upregulated. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan (5) and also of matrix metalloproteases (6). We wanted to study the
532
Keratinocytes and hyaluronan
correlation between these processes both in human skin fibroblasts and corneal keratocytes. MATERIALS & METHODS Explant cultures : Cornea and skin were obtained from healthy donors and came from surgery departments of our hospital. Corneal rims remaining after grafting were cut in fragments of 1 mrrr' and cultivated in DMEM with or without hyaluronan (1 mg/ml) for 48 hours at 37°C with shaking. Dermal fragments were cultivated using the same protocol. The culture media were kept and the tissue extracts were obtained after incubation by homogenising, skin or corneal fragments in 0.1 M Tris-HCI pH8 buffer containing 1 roM cacr, 1 roM MgClz, 1 roM zinc acetate, 0.01 % Hrij 35 and 0.01 % NaN3, using an Ultraturax. Cell cultures : Corneal keratocytes and skin fibroblasts were cultured for 24 hours in DMEM without fetal calf serumwith or without hyaluronan (l mg/ml). Culture media were kept and the cells were sonicated in 0.1 M Tris-HCI pH8 buffer containing 1 roM CaClz, I roM MgCl z, 1 roM zinc acetate, 0.01 % Hrij 35 and 0.01 % NaN3. The cell extracts correspond to the intra and pericellular fractions. Elastase type endopeptidase activity: In order to determine elastase type endopeptidase activity we used a synthetic substrat : N-Suc(ala)3pNA as described (7). Gelatin zymography : Explant and cell culture media and cell or tissue extracts were studied by gelatin zymography as described (8). They were electrophoresed at 150 Volts on 10 % acrylamid gels containing 1 mg/ml gelatin. The gels were first incubated in a 2.5 % triton X-IOO in order to eliminate SDS and then in 0.1 M Tris buffer containing 0.1 M CaCl z and I roM Zinc acetate. After 24 hours of incubation at 37°C the gels were stained with Coomassie blue and destained in water. The activities of both active or inactive forms of MMP-s were quantified by densitometry using a morphometric software (Visiolab©). RESULTS & DISCUSSION Keratocyte and fibroblast cell cultures : As shown on the zymograms of Fig.I, MMP-2 is expressed, in its inactive pro-form by both cell types but MMP-9 in its inactive pro-form only by fibroblasts. In presence of hyaluronan, MMP-2 was increased by both cell types as shown by densitometric scanning of zymograms by about 20 %.
Effect on matrix metalloprotease expression
533
B MMP·9~
MMP·2
Figure 1 : Gelatin zymography of culture MMP·2~ media of keratocyte (A) and skin fibroblast (B) Lane I : culture medium, control Lane 2 : culture medium, with I mg/ml hyaluronan
Cornea explant culture :
MMp·9
MMP·2
45kDA
Figure 2 : Gelatin zymography of tissue extracts (lanes 1 and 2) and culture media (lanes 3 and 4) of corneas cultured with (lanes 2 and 4) or without (lanes I and 3) hyaluronan. As shown on Fig.2, in cornea explant culture both MMP-2 and MMP-9 are expressed in their active and inactive forms. Both activities were higher in the culture medium than in the tissue homogenates showing that both enzymes are excreted by the cells during incubation. The activity of MMP-2 was somewhat more important than that of MMP-9. In presence of hyaluronan MMP-activity was slightly increased, by about 15 %. The major effect of hyaluronan was the activation of latent MMP-9 to its active form. The ratio of active to inactive form of MMP-9 increased in presence of hyaluronan from 0.75 to 3.4 in the corneal extract. The same increase of the ratio of active to inactive form of MMP-9 was even higher in the culture medium where it increased from 0.9 without hyaluronan to 4.6 in presence of hyaluronan.
534
Keratinocytesand hyaluronan
Skin explant cultures :
MMP·9
MMP-2
Figure 3 : Gelatin zymography of extracts (lanes 1 and 2) and extracellular media (lanes 3 and 4) of skin explant cultures, with (lanes 2 and 4) or without (lanes I and 3) hyaluronan. Fig.3 shows similar experiment carried out on skin explant cultures. Both MMP-2 and MMP-9 are expressed both in the culture medium and in the tissue homogenates. In the extracellular medium, the activity of MMP-9 was somewhat higher than the activity of MMP-2. The addition of hyaluronan increased here also the conversion of the inactive form of these enzymes to their active form. This ratio increased from 0.85 to 1.2 for MMP-2 and from 5.9 to 6.7 for MMP-9 in the culture media. Elastase type activity : The titration of elastase type endopeptidase activity with the synthetic substrate showed a dose dependant increase for skin fibroblasts at 1 mg/ml (+ 30 %, P < 0.05) and at 2 mg/ml (+ 65 %, P < 0,001) hyaluronan (7). In keratocytes, the increase was of the same order as in fibroblasts at 1 mg/ml hyaluronan (+ 52 %, P < 0.01) and no further increase was seen at 2 mg/ml hyaluronan. These results confirm and extend previous studies carried out with the same synthetic substrate which showed also an increase of the elastase-type endopeptidase activity of human skin fibroblasts in presence of 1 mg/ml hyaluronan (9). CONCLUSIONS We compared the expression of the latent and active forms of MMP-s in human skin fibroblasts and corneal keratocytes as well in skin and corneal explant cultures as well as the action of hyaluronan on these activities. The comparison of cell cultures and explant culture show that the presence of the extracellular matrix influences the expression of MMP-2 and MMP-9 as well as the conversion of the inactive pro-form to the active form. The ratio of active to the inactive form was different in cornea and skin explant cultures. For both cell-types in explant cultures a large part of both MMP-2 and MMP-9 activities was excreted in the culture medium.
Effect on matrix metalloprotease expression
535
The addition of hyaluronan increased the conversion of the inactive to the active fOlTIlS, in the medium, for both tissues. For cornea explant cultures this ratio increased in presence of I mg/ml hyaluronan from 0.9 to 4.6 for MMP-9. For skin explant cultures the increase was from 0.85 to 1.2 for MMP-2 and from 5.9 to 6.7 for MMP-9. Both fibroblast and keratocyte cell cultures, at the 3rd passage expressed MMP-2 in its inactive form, but only small amounts of MMP-9 could be detected in the extracellular media of skin fibroblasts. MMP-2 and MMP-9 activities taken together were lower in cell cultures than in explant cultures. Hyaluronan at 1 and 2 mg/ml produced a dose dependent increase of elastase-type endopeptidase activity in fibroblasts. With keratocytes, an increase of 52 % was seen at 1 mg/mI hyaluronan and no further increase at 2 mg/mI. These results confirm and extend our previous observations concerning the increase of elastase type endopeptidase activity of hyaluronan (9). The main action of hyaluronan appears to be an increase of the conversion of latent forms of these enzymes to their active form. It appears that this activation concerns both MMP-2 and MMP-9. The differences noticed between corneal and skin cells confirm our previous conclusions based on the study of glycosaminoglycan biosynthesis, concerning the difference between the phenotypes of keratocytes and fibroblasts. (10, 11)
REFERENCES 1. Shapiro S. Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Current Opinion in cell Biology, 1998, 10 (5) : 602-608. 2. Kielty CM., Whittaker SP., Grant ME., Shuttleworth CA. Type VI collagen microfibrils : evidence for a structural association with hyaluronan. J Cell BioI., 1992, 118 (4) : 979-990. 3. Toole B. Hyaluronan in morphogenesis. J. of Internal Medicine, 1997, 242 : 35-40. 4. Inoue M., Katami C. The effect of hyaluronic acid on corneal epithelial cell proliferation. Invest. Ophthalmol. Vis. Sci., 1993, 34 (7) : 2313-23-15. 5. Heldin P., Asplund T., Ytterberg D., Thelin S., Laurent T. Characterization of the molecular mechanism involved in the activation of hyaluronan synthetase by plateletderived growth factor in human mesothelial cells. Biochem. J., 1992,283 : 165-170. 6. Kossakowska A., Edward D., Prusinkiewicz C., Zhang M., Guo D., Urbanski S., Grogan T., Marquez L., Janowska-Wieczorek A. Interleukin-6 regulation of matrix metalloproteinase (MMP-2, MMP-9) and tissue inhibitor of metalloproteinase (TIMP-l) expression in malignant non-Hodgkin's lymphoma. Blood, 1999,94 (6) : 2080-2089. 7. Isnard N., Legeais J-M., Renard G., Robert L. Effect of hyaluronan on MMPexpression and activation. Cell Bioi Int , 2001, in press. 8. Kleiner D., Stetler-Stevenson W. Quantitative zymography : detection of picogram quantities of gelatinases. Analytical Biochemestry., 1994,218 : 325-329. 9. Bernard E., Hornebeck W., Robert L. Effect of hyaluronan on the elastase-type activity of human skin fibroblasts. Cell BioI. Int., 1994, 18 (10) : 967-971. 10. Fodil-Bourahla I., Drubaix I. and Robert L. Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastin-larninin receptor. Mechanisms of Ageing and Development, 1999, 106: 241-260. 11. Dupuy F, Peterszegi G., Legeais J-M., Robert A-M., Robert L. and Renard G. Stabiblityof glycosaminoglycans during in vitro aging of human keratocytes. In preparation.
AGING AND REGULATION OF HYALURONAN BIOSYNTHESIS. COMPARATIVE STUDIES ON HUMAN SKIN FIBROBLASTS AND CORNEAL KERA TOCYTES. Robert L.*, Fodil I., Isnard N., Dupuy F., Robert A.M., Renard G. Laboratoire de Recherche en Ophtalmologie, Esc B3 6eme etage, Hotel Dieu, 1 place du Parvis Notre-Dame. 75004 Paris, France.
[email protected]
ABSTRACT Human skin fibroblasts and corneal keratocytes were compared for their GAGbiosynthetic activity as a function of passage number (in vitro replicative senescence) by comparing the fraction of total label eH-glucosamine) incorporated in total glycoconjugates and individual GAG-s. It appeared that keratocytes synthesised about as much non-GAG glycoconjugates (glycoproteins) as fibroblasts. Incorporation in GAG-s increased with passage number for fibroblasts and remained constant for keratocytes. Hyaluronan as % of total GAG-s was somewhat higher in fibroblasts than in keratocytes. A strong increase of incorporation was seen at the latest passage (15th) close to replicative senescence of fibroblasts but titration with hyaluronectin showed a decrease of hyaluronan concentration, interpreted as a sign of post-synthetic degradation of hyaluronan in pre-senescent fibroblasts. Regulation of GAG-biosynthesis as a function of passage number permitted thus to differentiate fibroblast and keratocyte phenotypes.
KEYWORDS Hyaluronan, GAG-s, fibroblasts, keratocytes, replicative senescence.
INTRODUCTION Regulation of glycosaminoglycan (GAG) biosynthesis is part of the phenotypic expression of extracellular matrix (ECM) biosynthesis by mesenchymal cells. Their phenotype can best be characterised by their "program" of matrix biosynthesis 1-3. This "program" comprises the "choice" of genes coding for matrix components to be expressed (qualitative aspect) and for the quantitative regulation (up or down) of the expression of the chosen genes. This "program" of regulated matrix biosynthesis starts during embryonal development and continues during maturation and aging. The structure and function of tissues, and their susceptibility to pathological modifications are largely dependent on the correct execution of this program and also on its modifications during aging. Regulation of GAG-biosynthesis represents an important part of the above outlined "program". The biological properties and role of individual GAG-s in both tissues studied are sufficiently well known to render useless any repetition in a short article. With these premises in mind we shall describe our studies on the expression of GAG-s
538
Keratinocytes and hyaluronan
in skin fibroblasts and corneal keratocytes, both of human origin, in explant and cell culture and as a function of passage number (in vitro aging) 4.
MATERIALS & METHODS Cells Fibroblasts were cultured from skin samples obtained from plastic surgery, using standardised procedures 5, Corneas were obtained from the French Eye Bank after preservation in explant culture conditions as described 6, Cell cultures were derived from the corneas as described 7 and passaged in Dulbecco modified Eagle's minimal medium (DMEM) with 10 % foetal bovine serum and antibiotics. Biosynthesis of GAG-s was studied by adding 3H-glucosamine to the cultures (1 /lCi/ml) for a further incubation for 24 to 48 hours. Cells and medium were separated and individual GAG-s determined after selective degradation by specific enzymes, enabling the determination of incorporated radioactivity. For hyaluronan the Streptomyces hyaluronidase was used, for chondroitin 4 and 6 sulfate chondroitinase AC or ABC of Proteus vulgaris, for dermatan sulfate the chondroitin sulfate B-Iyase of Flavobacterium heparinum, for keratan sulfate, keratanase I from Pseudomonas, all from Sigma (St. Louis, MO.). For heparan sulfate, nitrous acid degradation was used 8. Digested GAG-s were separated from undigested macromolecules by gel filtration after precipitation of the undigested fraction by absolute ethanol containing 1 % potassium acetate. Hyaluronan concentration was determined by the titration method described by Delpech et al. 9 using hyaluronectin purified from sheep brain as described by Delpech and Halevent 10, Statistical significance was calculated using the non-parametric method of Mann and Whitney,
RESULTS & DISCUSSION Fibroblasts As shown on table I, skin fibroblasts incorporated actively 3H-glucosamine in total glycoconjugates. Incorporation in total glycoconjugates increased from the 5th passage (relatively "young" cells) to the 15th passage (cells near the end of their replicative lifespan) by about 45 %. Part of this incorporation concerned non-GAG glycoconjugates, essentially glycoproteins. This fraction was of the order of 28 % of total incorporation at the 5th passage and decreased to 14 % of total incorporation at the 15th passage. The relative amount of GAG-s synthesised appears thus to increase with passage number. Hyaluronan represented about 40 % of total GAG biosynthesis at the 5 th passage and somewhat less, about 35 % at the 15th passage. The relative amount of the other GAG-s, chondroitin sulfates and heparan sulfate changed less. A large proportion of the neosynthesised GAG-s was secreted in the culture medium (figures in parenthesis in table I), This is surprising for heparan sulfate, supposed to be localised mainly on cell membranes. Fig. I shows the increase of incorporation of the tracer with passage number for chondroitin 4 and 6 sulfates and dermatan-sulfate as well as for heparan sulfate. Little change was noticed between the 5th and 10t h passages, but a strong (and significant) increase is seen at the 15th passage, mainly for dermatan sulfate and heparan sulfate. No significant change was seen for chondroitin 4 and 6 sulfates.
539
Aging and regulation of biosynthesis. Table I.
Incorporation of 3H-glucosamine in total glycoconjugates and GAG-s by human skin fibroblasts in cell culture at the 5th and 15' h passages. The figures in parenthesis represent the % of total incorporated radioactivity recovered in the culture medium. (Recalculated from Table I of FodilBourahla et al., 1999). Hyal : hyaluronan, CSABC : total chondroitin-sulfates, HS : heparan sulfate. JH-GlcN Incorporation in non GAGPassage incorporated in glycoconjugates number total glycoconjugates cpmll06 cells 6 cpmll0 cells. 5
91278 ± 13123
25450
15
132871 ± 6 951
18636
% of total incorporation
in individual GAG-s
Hyal. 40 (92) 35 (88)
CSABC 29 (77)
28 (86)
HS 31 (70) 37 (76)
Fig. 2 shows the incorporation of the tracer in hyaluronan (upper graph) with a quite important increase between passage 10 and 15, as for the sulfated GAG-s (Fig. 1). When however the titrable concentration of hyaluronan was determined using hyaluronectin as the reagent there was a progressive decrease with passages (lower graph of Fig. 2). This discrepancy between the two methods might be due to a rapid post-synthetic degradation of newly synthesised hyaluronan.
50000 [J
o
Chondrctttn-sulfates A,C Dermatan sulfate
[J Heparan sulfate
40000 ~
1l ~ =
30000
~
i
20000
13' 10000
PDS Population doubling level
Figure 1.
3H-glucosamine incorporation in chondroitin sulfates A and C, in dermatan sulfate and in heparan sulfate. The values are expressed as cpm per 106 cells.
540
Keratinocytes and hyaluronan
A 50000
.....'"
40000
.l!l
e 30000
= ~
~=.. 20000 '"
10000 0
PD5
PD 10
PD 15
Population doubling level
B 80000 .i!l ~
...e
60000
t
40000
'-'
c .S c
20000 0
PD5
PD 10
PD 15
Populationdoublinglevel
Figure 2.
A : Incorporation of 3H-glucosamine in hyaluronan by fibroblasts at increasing passages. B : Titration of the concentration of hyaluronan present in the culture media with hyaluronectin.
Aging and regulation of biosynthesis.
541
Cornea and keratocytes Table II shows the results of 3H-glucosamine incorporation in cornea explant cultures. The fraction incorporated in stromal glycoproteins was comparable to the figures found for fibroblasts, the major fraction of the label was found in secreted or stroma - bound GAG-s. Hyaluronan represented 32 % (tissue bound) to 38 % (secreted) of total GAG synthesis.
Table II.
Incorporation of 3H-glucosamine in GAGs in human cornea during a 48 hours explant culture. Results expressed as % of total GAGs incorporation. Average values ± S.D. 6 parallel experiments.
% of total GAG-s
Retained in corneal tissue
Secreted in the medium
Hyaluronan
32.1 ± 3.0
38.1 ± 4.4
Keratan sulfate
18.6 ± 1.9
13.8 ± 1.8
Dermatan sulfate
20.9 ± 1.3
12.9 ± 2.0
Chondroitin sulfates (A+B +C)
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9.4 ± 1.3
When keratocytes were cultured through 10 passages total incorporation in GAG-s remained relatively stable. Incorporation in total GAG-s did not show any significant variation with increasing passage number. About 75 % of the neosynthesised GAG-s were secreted in the culture medium. As shown on Fig. 3 this remarkable stability of GAG-biosynthesis was seen for every individual GAG-s synthesised between the 4 th and 10th passages. The presented results clearly show that the fibroblast and keratocyte phenotype are quite different as far as GAG-biosynthesis is concerned. Keratocytes synthesised about as much non-GAG glycoconjugates as fibroblasts. They also exhibited an increase of incorporation of 3H-glucosamine with increasing passage number in hyaluronan as well as in sulfated GAG-s (Figs. I and 2). Keratocytes synthesised comparable proprtions of hyaluronan (32 to 38 % of total GAG-s) as fibroblasts (35 to 40 % of total GAG-s). GAG-synthesis by keratocytes did not show the passage dependent shift seen with fibroblasts. Keratocyte phenotype characterised by GAG-biosynthesis appears therefore more stable than fibroblast phenotype at least as far as shown by the study of replicative senescence.
542
Keratinocytes and hyaluronan
A
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Incorporation of 3H-glucosamine in individual GAG-s secreted (A) and retained in cells (B) at increasing passages of keratocytes expressed as % of incorporation in total GAG-s. Average of results from 5 different donors ± SD. Dermatan sulfate was counted in the sum of chondroitin sulfate, resulting in a sum of sulfated GAG-s above 100 %.
CONCLUSIONS
These experiments clearly establish a phenotype difference between human skin fibroblasts and corneal keratocytes judged by the relative amount of GAG-s synthetised during successive passages (in vitro aging).
REFERENCES 1. Robert L., Le fibroblaste, definition de son phenotype par son "programme" de biosynthese de la matrice extracellulaire. Pathol. Biol., 1992, 40, 851-858. 2. Robert L., Mechanisms of aging of the extracellular matrix. Role of the elastinlaminin receptor, Novartis Price Lecture, Gerontology, 1998,44,307-317. 3. Robert L., Le vieillissement, , Belin-CNRS Ed., Paris,1994.
Aging and regulation of biosynthesis.
543
4. Hayflick L., The cellular basis for biological aging. In • Handbook of the biology of aging.: Finch C.E., Hayflick L. (eds.), Van Nostrand Reinhold Company, New York, 1977, pp 159-186. 5. Fodil-Bourahla 1., Drubaix 1., Robert L., Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastinlaminin receptor, Mech. Ageing. Develop .. 1999, 106,241-260. 6. Isnard N., Legeais J.M., Renard G., Robert L., Effect of hyaluronan on MMPexpression and activation. Cell BioL. lnt, 2001, 25 (8), 735-739. 7. Dupuy F., Savoldelli M., Tixier J.M., Robert A.M., Robert L., Legeais J.M., Renard G., Chemotactic penetration of keratocytes in ePTFE polymer in vitro, J. Biomedical Materials Research, 2000, 56,487-493. 8. Shively J.E., Conrad H.E., Formation of Anhydrosugars in the chemical depolyperisation of heparin. Biochemistry, 1976, 15, 3932-3942. 9. Delpech B., Bertrand P., Maingonnat C., Girard N., Chauzy C., Enzyme-linked hyaluronectin : A unique reagent for hyaluronan assay and tissue location and for hyaluronidase activity detection. AnaL. Biochem.,1995, 229, 35-41. 10. Delpech B., Halevent c., Characterization and purification from human brain of a hyaluronic acid-binding glycoprotein, hyaluronectin, , J. Neurochem., 1981,36(3), 855-859.
EFFECTS OF KGF AND TGF-8 ON HYALURONAN SYNTHESIS AND DISTRIBUTION IN EXTRA-, PERI·, AND INTRACELLULAR COMPARTMENTS OF EPIDERMAL KERATINOCYTES Karvinen S.,* Tammi M., Tammi R. Dept. ofAnatomy, University ofKuopio r.o.so» 1627, 70211 Kuopio, Finland
ABSTRACT The aim of this study was to examine the effects ofKGF and TGF-13 on HA synthesis and proliferation of rat epidermal keratinocytes. KGF stimulated proliferation at small doses (I and 10 ng/ml). TGF-13 inhibited keratinocyte proliferation at 10 ng/ml. Both growth factors increased the migration of the cells. HA localization was studied with a histochemical staining assay using biotinylated aggrecan Gl domain link-protein complex. In a part of the samples extracellular HA was enzymatically digested prior to permeabilization of the membranes to specifically examine intracellular HA. A five to sixfold increase of intracellular HA staining intensity as compared to control was observed with both growth factors at 24 hours. A twofold increase in pericellular HA was also found with KGF, but no marked effect was seen with TGF-I3. Metabolic incorporation of eH] glucosamine and 35S04 into labelled hyaluronan and chondroitin disaccharides from medium, trypsinate and cell samples revealed a sixfold increase of intracellular HA with KGF at 100 ng/ml. A twofold increase of HA in medium samples was also seen with KGF, but it had no effect on trypsinate samples. TGF-13 at 10 ng/ml had no significant effect on extracellular HA, but induced a twofold increase of intraand pericellular HA. KEYWORDS Hyaluronan, keratinocytes, KGF, TGF-I3, proliferation, migration INTRODUCTION Hyaluronan (HA) is a large, linear polysaccharide that forms the main component of the extracellular matrix in the epidermis of skin7.8. It forms a loose, gel-like matrix, which enhances the exchange of molecules between cells and the bloodstream", and creates an environment that favors the migration of cells". Keratinocyte growth factor (KGF), is a powerful mitogenic agent. secreted by stromal fibroblasts, that influences only epithelial cells'. In epidermis. KGF receptors are primarily expressed in the spinous cell layer in which the differentiation of keratinocytes takes place. KGFR can also be found in the basal cell layer, but not in the granular- or clear cell layers". No previous results have been published linking KGF with hyaluronan. Transforming growth factor beta (TGF-I3) is encoded by three different genes as three isoforms (TGF-J3 1,2 and 3). It controls cell proliferation and differentation in several cell types and also balances the effects of other growth factors either by enhancing their
546
Keratinocytes and hyaluronan
effects or inhibiting them". TGF-13 signaling requires the binding of TGF-13 to type II receptor and the complex binding to type I receptor!'. TGF -13 has been found to stimulate HA synthesis in fibroblasts". MATERIALS AND METHODS Cell proliferation
Newborn rat epidermal keratinocytes were seeded into 24 well plates at 60.000 cells /well. The following day, KGF (0,1,10 and 100ng/ml, Sigma, USA) or TGF -13 (0,1,5 and 10ng/ml, Gibco, Scotland) was added. The cells were incubated at 37°C, and the cell number counted on the three following days (at 24, 48 and 72). The growth medium (MEM, Gibco) was removed, the cells washed with HBSS (EuroClone) and incubated with 0.05%trypsin-0.02% EDTA until detached. The cells were harvested and wells washed with HBSS. The cells were counted using a hemacytometer on Olympus CK2 inverted phase contrast microscope. Migration
The cells were seeded at 600.000 cells/well on 6 well plates and grown for 24 h. A cell-free area was introduced by scraping the monolayer crosswise with a sterile I000111 pipette tip, washed with HBSS and fresh medium was added. The width of the cell-free area was approximately 1000 urn, TGF-B at 5 and 10 ng/ml or KGF at 10 and 100 ng/ml was added to two wells each, 2 wells on each 6-well plate remained as contro!. The area covered by the cells in 8 crossing areas in duplicate wells of each growth factor concentration was measured immediately after scraping and 24 h later using a phase conrast microscope, videocamera and NIH-image® software. The distance the cells had migrated was calculated as follows: (-.Jb--.Ja)/2, where a= area covered by the cells at Oh and b= area covered by the cells after 24 h. The results (pixels) were converted to micrometers. Staining of HA
The cells were seeded into 8-well chamber slides at 20.000 cells/well, and cultured for 48 h at 37°C. Fresh medium was added with KGF at 100 ng/ml, or TGF-13 at 10 ng/ml for 4, 6, and 24 h. The cells were fixed with 2% paraformaldehyde 0.05% glutaraldehyde in sodium phosphate buffer, pH 7.4 (PB) for 20 min, washed with PB, permeabilized with Triton X-IOO, and incubated with a specific hyaluronan binding probe" overnight at 4°C, followed by avidin-biotin-complex (Vector, CA) and diaminobenzidine with 0.005% hydrogen peroxide. Optical densities were analysed by a light microscope and digital camera. In part of the slides, extracellular hyaluronan was digested prior to the permeabilisation of the membranes with Streptomyces hyaluronidase (Seikagaku). Metabolic labeling assay
The cells were seeded into 6 well plates at 200.000 cells/well and cultured until subconfluent. Fresh medium containing CH] glucosamine (2011Ci/ml) and 35 S04 (10 llCi/ml) was added for 24 h. The medium was harvested and wells washed with precooled HBSS which was collected with the medium. The cells were trypsinized, and 50
Effects of KOF and TOF-13 on synthesis
547
III of the cell suspension was taken aside to count the cell number. The wells were washed twice with HBSS, the cells sentrifuged in the cold (5 min at 4000 rpm) and washed once with 250111 cool HBSS containing 10% fetal bovine serum. All washes were combined with the trypsin solution and the cells digested overnight at 4°C with HBSS containing 41lg HA (Healon, Pharmacia) and 20 TRU Streptomyces hyaluronidase, and washed with cool HBSS. All samples were kept in -20°C prior to analysis. The analysis was done as described in Tammi et ai, 1998, except that the CPC and ethanol precipitations were replaced by dialysis with a 10 kDa cut off. RESULTS AND DISCUSSION The effects ofKGF and TGF-8 on keratinocyte proliferation
The cells were cultured in the presence of the growth factors, and counted. Neither KGF nor TGF-13 influenced cell numbers at 24 h. The number of cells in wells containing TGF-13 was slightly lower than in control cells at 48 h (Fig.1). At 72 h TGF-13 at 10 ng/ml had decreased proliferation while the lesser amounts had no significant effect. At 48 and 72 h, KGF at 10 and 1 ng/ml caused an increase in proliferation, but 100 ng/ml showed no effect (Fig.I). It has been previously reported that KGF does not increase keratinocyte proliferation until the cells are subconfluenr'. The cell density at 24 h culture may therefore have been too low to bring about the full stimulatory effect of KGF. TGF-13 has been reported to inhibit keratinocyte proliferation as revieved by Sporn et a1 5 . The reason why the lesser amounts did the opposite is not known. 5 10 0
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TGF -8 and KGF both increase the migration of rat epidermal keratinocytes
To investigate the effects on migration we cultured cells with different concentrations of the growth factors for 24 hours. The keratinocytes with no added growth factors, migrated approximately 150 11m in 24 hours. TGF-13 increased migration at both doses with slightly higher response at higher concentration. The increase was 30-40 %. KGF
548
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also caused an increase in migration, approximately 25-30% to control. As with TGF-B, the higher concentration showed a more marked response.
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Effects ofKGF and TGF-13 on synthesis
549
Histochemical staining of HA Stainings with a specific probe suggested that TGF-B does not have a significant effect on the amount of total HA. However, TGF-B showed a time dependent increase in intracellular hyaluronan, reaching 5 times the control level at 24h (Fig 3). KGF showed no effect on total HA at 4 and 6 h, but induced a threefold increase at 24 h. KGF, like TGF-B increased intracellular hyaluronan. The increase was two- and fourfold at 4 and 6 h, respectively, and sixfold at 24 h (Fig 3).
The effect of KGF and TGF-8 on newly synthesized HA in extra-, peri-, and intracellular compartments Biochemical experiments were done to determine HA secretion into growth medium, and to confirm the OD results on total and intracellular HA. TGF-B did not significantly effect the amount of HA secreted into medium during the 24h labeling time (Fig. 4). TGF -13 increased the amount of HA in the pericellular compartment at higher doses, but the lower doses had no effect. In the intracellular compartment, however, TGF-B increased the amount of hyaluronan approximately twofold at 10 ng/ml. KGF showed a twofold increase in the amount ofhyaluronan secreted into medium (Fig. 4). The effect was dose-dependent, and was strongest at 100 ng/ml KGF. KGF had no marked effect on trypsinate (pericellular) samples, but did increase the amount of intracellular hyaluronan up to six times that of the control cells. 120 ,
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550
Keratinocytes and hyaluronan
CONCLUSIONS In conclusion, KGF and TGF-B differ on their effect on keratinocyte proliferation and the amount of hyaluronan in extra-, and pericellular compartments. However, they both significantly increase the amount of intracellular HA and cell migration as compared to control cells.
REFERENCES 1. P.W.Finch, J.S.Rubin, T.Miki, D.Ron, S.A. Aaronson, Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth, Science, 1989, 245, 752-5. 2. M.D. Hines, B.L. Allen-Hoffmann, Keratinocyte growth factor inhibits cross-linked envelope formation and nucleosomal fragmentation in cultured human keratinocytes,1. BioI Chern, 1996,271,6245-51. 3. C.B. Knudson and W. Knudson, Similar epithelial-stromal interactions in the regulation of hyaluronate productioon during limb morphogenesis and tumor invasion, Cancer Lett, 1990, 52, 113-22. 4. W.J. LaRochelle, O.R. Dirsch, P.W. Finch, H.G. Cheon, M. May, C. Marchese, J.H. Pierce and S.A. Aaronson, Specific receptor detection by a functional keratinocyte growth factor- immunoglobulin chimera, J. Cell BioI, 1995, 129,357-66. 5. M.B. Sporn, A.B. Roberts, L.M.Wakefield and R.K. Assoian, Transforming growth factor-beta: biological function and chemical structure, Science, 1986,233,532-4. 6. M. Suzuki, T. Asplund, H. Yamashita, C.H. Heldin and P. Heldin, Stimulation of hyaluronan biosynthesis by platelet-derived growth factor-BB and transforming growth factor-beta I involves activation of protein kinase C, Biochem J, 1995, 307, 817-21. 7. R. Tammi, J.A. Ripellino, R.U. Margolis and M.Tammi, Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a spesific probe, J Invest Dermatol, 1988, 90, 412-4. 8. R. Tammi, U.M. Agren, A.L. Tuhkanen and M. Tammi, Hyaluronan metabolism in skin, Prog Histochem Cytochem, 1994,29, 1-81. 9. E.A. Turley, L. Austen, K. Vandeligt and C. Clary, Hyaluronan and cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells, J Cell Bioi, 1991,112,1041-7. 10.R. Tammi, D. MacCallum, V.C. Hascall, J-P. Pienimaki, M. Hyttinen and M. Tammi, Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides, J BioI Chern, 1998, 273, 28878-88. 11. 1.L.Wrana, L. Attisano, J Carcamo, A. Zentella, 1. Doody, M.Laiho, X.F. Wang and J. Massague, TGF beta signals through a heteromeric protein kinase receptor complex, Cell, 1992, 71, 1003-14.
HYALURONAN STIMULATES KERATINOCYTE MIGRATION AND ACTIVATES THE TRANSCRIPTION FACTOR AP-l IN KERA TINOCYTES THROUGH THE JNK PATHWAY Kari Torrbnen, Monica Yabal, Kirsi Rilla, Kai Kaarniranta, Raija Tammi, Mikko J. Lammi & Markku Tammi Department of Anatomy. University of Kuopio, P.D.S 1627. Fin-70211 Kuopio, Finland
ABSTRACT
Hyaluronan (HA) has been considered a passive extracellular matrix (ECM) polysaccharide, but recent studies have shown its importance in controlling many cell functions including motility, proliferation and adhesion, which imply signaling from ECM to cytosol. Hyaluronan is a major ECM component in stratified epithelia such as skin epidermis. We found that hyaluronan added to the growth medium of newly plated human skin keratinocytes increased cell migration in an in vitro wound-healing assay. Hyaluronan also increased the transcription factor AP-I, as determined by gel shift assays. The kinase signals that apperently led to the increased AP-I level were associated with the activation of c-Jun, mainly via the JNK pathway as early as 10 min after the addition of hyaluronan, and with the minimum concentration of 10 ng/ml. ERKI was also slightly activated, while p38 MAPkinase was not affected. KEYWORDS
Hyaluronan, AP-I, keratinocyte, MAPK INTRODUCTION
Hyaluronan is a major matrix macromolecule in the extracellular space of human epidermis I. One function of hyaluronan in epidermis is the maintenance of some extracellular space between keratinocytes and thus facilite diffusion of nutrients to cells and waste from cells, but it is obvious that hyaluronan is also a signal molecule and mediator of ECM information. Mammalian cells hav several mitogen activated protein kinase (MAPK) cascades linked to different signal transduction pathways. These cascades activate either one of the extracellular regulated kinases (ERK) 2, c-Jun NH2-terrninal kinases (JNK) , or stress activated protein kinase (SAPK) 3 or p38 kinases 4. The ERK pathway is closely related to activation of growth factor receptors while JNK and p38 pathways are often associated with cellular stress and cytokine receptors. MAPK activation often induces activating protein-I (AP-I), the transcription factor that contributes to the expression of specific sets of genes 5. The AP-I family of transcription factors is composed of homodimers or heterodimers of the protein families Jun (v-Jun, c-Jun, JunB, JunD), Fos (v-Fos.c-Fos, FosB, Fral, Fra2) and activating
552
Keratinocytes and hyaluronan
transcription factor (ATF2, ATF3ILRFl, B-ATF) 6. A central component of most AP-l complexes is c-Jun, which is expressed at low levels in many different cell types and greatly induced by external stimuli like growth factors, cytokines, and UV irradiation 5. It has been shown that c-Jun is essential for ras-transformation 7. We found that AP-l was specifically activated by exogenous HA in epidermal keratinocytes and studied the intracellular MAPK signaling routes involved in the activation.
MATERIALS & METHODS Cell culture The continuous human keratinocyte cell line (HaCat, kindly provided by Dr. Neubert Fusenig, German Cancer Research Center, Heidelberg, Germany) was cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 3 mM glutamine and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37°C. The medium was changed every second day.
Migration The HaCat cells were trypsinized, diluted I:5, plated to 24-well plates and grown until almost confluent. A cell-free area was introduced by scraping the monolayer crosswise with a sterile 1000 III pipette tip, washed with Hank's balanced salt solution (Euroclone, U.K.) and a fresh medium with different concentrations of HA was added. The area covered by the cells in 8 crossing areas in 8 wells of every concentration was measured immediately after scraping and 24 h later with Olympus CK 2 inverted phase contrast microscope, Panasonic Wv-CD 130-Lvideocamera and NIH image software. The average distance the cells had migrated was calculated as follows: db - ~a) / 2, where a= area covered by the cells initially and b= area covered by the cells after 24 h.
Incubation with hyaluronan The cells were incubated with purified high MW hyaluronan (Provisc, Alcon Laboratories Inc., Fort Worth, TX, USA), or hyaluronan-oligosaccharidesat different time points after trypsinization. The incubation time with hyaluronan varied between 10 min and 24 h. The cells were harvested on ice bath with a rubber policeman, washed with PBS and collected by centrifugation at 2000 x g.
Nuclear protein extraction and gel mobility shift assay of AP·l Nuclear proteins were extracted by the hypotonic lysis method of Goldstone and Laving. Equal quantities of protein extracted from the cells 9 were incubated with the 32P-Iabeled oligonucleotide at +4°C for 20 min and subjected to the gel mobility shift assay. The c-Jun oligonucleotide 5'-CGCTTGATGAGTCAGCCGGAA-3' was labeled with T4 polynucleotide kinase (Promega, Madison, WI, USA) according to the manufacturer's protocol. The labeled oligonucleotides were purified by NAP-columns (Pharmacia Biotech, Uppsala, Sweden).The oligonucleotide and cell extract mixtures were
Stimulates keratinocyte migration
553
loaded onto 5% non-denaturing PAGE and electrophoresed for 2h at 4°C with Bio-Rads Mini-Protean II apparatus (Bio-Rad, Hercules, CA, USA) and using a voltage of 150V. The gels were dried with Bio-Rads gel drier and analyzed by autoradiography on Kodak MR-film (Eastman Kodak Laboratories Ltd, Rochester, NY, USA). Antibodies Specific MAPK antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) that only recognize the phosphorylated forms of these kinases were used to determine activation in the different MAPK pathways. An antibody against c-Jun (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used to determine the total amount of c-Jun protein. Phosphorylation of c-Jun was determined using phospho-c-Jun antibody (New England Biolabs, Beverly, MA, USA). Total protein extraction The culture dishes were put on ice bath, washed with ice-cold PBS, and the cells were isolated as described above. The cells were quickly fozen in liquid nitrogen, lysed in ice cold extraction buffer (10 roM Tris, pH 7.4,100 roM NaCl, I roM EDTA, 1 roM EGTA, I roM NaF, 2 mM Na3V04, 1% Triton X-IOO, 10% glycerol, 0.1% SDS, 0.5% deoxycholate), and incubated on ice for 10 min. The total solubilized proteins from the supernatant of a 4000 x g centrifugation for 15 min at 4°C were quantified by the Bradford assay 9. Immunoblotting Equal amounts of total protein extracts were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred onto Immobilon-NC membranes (Millipore, Bedford, MA, USA) by 2.5 mNcm2 constant current with a Sammy'P' semidry blotter (Schleicher & Schuell, Dassel, Germany). Nonspecific binding was blocked by 2% BSA in 10 roM Tris, 150 roM NaCl, pH 7.4 (Tris-saline buffer) overnight at 4°C. Membranes were immunoblotted with the phospho-specific p-JNK, p-Erk and p-p38 antibodies, diluted to 0.2 ug/ml with 2% BSA and 0.2% Tween 20 in the Tris-saline buffer for one hour. The membranes were washed four times with the Tris-saline buffer with 0.2 % Tween 20, incubated for one hour with 1:10000 dilution of horseradish peroxidase conjugated secondary antibody (anti-rabbit IgG for c-Jun and anti-mouse IgG for MAPkinases) (Zymed Laboratories, Inc., CA, USA), and washed four times with 0.2% Tween 20 in Tris-saline buffer. Immune complexes were visualized using the NENTM chemiluminescence detection kit according to manufacturer's instructions (Life Science Products, Inc., Boston, MA, USA).
RESULTS AND DISCUSSION Hyaluronan induces HaCat cell migration HaCat cells were cultured in 24-well plates as described in materials and methods. In vitro wounding assay was done and different hyaluronan concentrations were added for
554
Keratinocytes and hyaluronan
24h. Hyaluronan induced cell migration in a dose dependent way as shown in figure 1.
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Stimulateskeratinocyte migration
555
Hyaluronan induces the activation protein-l (AP-l) in HaCaT cells The HaCat cells were treated with high molecular weight hyaluronan or a 18-24 mer oligosaccharide (HA18-24) (10 ug/ml) for different periods. The nuclear proteins were collected and analyzed with a gel shift assay. As shown in figure 2A the amount of AP-I capable of binding DNA increased after hyaluronan stimulation. Similar addition of PBS only had no effect. The maximum response was gained in 1-2 hours. The HA18-24 produced a response similar to that of high molecular weight hyaluronan. Figure 2B shows that the total level of c-Jun also increases, with the strongest response at 60 min, while no change was detected in c-Fos (figure 20), the other component of AP-l. Increased phosphorylation of c-Jun occured earlier than total c-Jun, with the maximum responce in 30 min (figure 2C).
JNK is phosphorylated immediately after hyaluronan stimulation In order to know which of the MAP kinases were involved in AP-l activation we treated the HaCat cells with hyaluronan for different time periods and analyzed the total proteins by Western blotting. Figure 2E shows the phosphorylation of JNK already 10 min after hyaluronan addition. The highest levels of phosphorylated JNK were achieved in 20 min and the active form of the enzyme returned to control level in two hours. Extracellularly regulated kinase-l (Erk-I) was also phosphorylated after hyaluronan stimulation as shown in figure 2F. The amount of the phosphorylated enzyme peaked at 30 minutes after hyaluronan stimulation, however the activation was not as strong as that of JNK. Erk2 level was not significally affected by hyaluronan (Fig. 2F), and p38 remained at control level, too (Fig. 20). This suggests that the level of AP-I increased mainly via stimulation of JNK activity.
Hyaluronan stimulates c-Jun and pJNK dose dependently The HaCat cells were treated with different concentrations of high molecular weight hyaluronan for 20 min, the cells were harvested and the total proteins were analysed by immunoblotting. Increased cell content of c-Jun protein was observed with the minimum of lOng/ml hyaluronan, as shown in figure 3. The phosphorylation of JNK by hyaluronan occurs with lOnglmJ concentration, too.
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556
Keratinocytes and hyaluronan
CONCLUSIONS High molecular weight hyaluronan stimulates HaCat cell migration, triggers JNK signal transduction cascade, elevates c-Jun and increases AP-l transcription factor by time and dose dependent way.
REFERENCES 1. 2. 3.
4.
5. 6. 7. 8.
9.
R. Tammi, U. M. Agren, A. L. Tuhkanen and M. Tammi, Hyaluronan metabolism in skin, Prog Histochem Cytochem, 1994,29,2,1-81. E. Cano and L. C. Mahadevan, Parallel signal processing among mammalian MAPKs, Treds Biochem Sci, 1995,20, 117-122. D. Lallemand, 1. Ham, S. Garbay, L. Bakiri, F. Traincard, O. Jeannequin and C. M. Pfarr, Stress-activated protein kinases are negatively regulated by cell density, Embo 1,1998,17,19,5615-5626. G. J. Fisher, H. S. Ta1war, J. Lin, P. Lin, F. McPhilips, Z. Wang, X. u, Y. Wan, S. Kang and J. 1. Voorhees, Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo, 1. Clin. Invest, 1998, 101,6, 1432-1440. M. Karin, The regulation of AP-1 activity by mitogen-activated protein kinases, J Bioi Chem, 1995,270, 16483-16486. M. Karin, Z. Liu and E. Zandi, AP-I fuction and regulation, Curr Op Cell Bioi, 1997, 9,240-246. R. Johnson, B. Spiegelman, D. Hanahan and R. Wisdom, Cellular transformation and malignancy induced by ras require c-Jun, Mol Cell Bioi, 19%, 16,4504-4511. S. D. Goldstone and M. F. Lavin, Prolonged expression of c-jun and associated activity of the transcription factor AP-l, during apoptosis in a human leukemic cell line, Oncogene, 1994,9,2305-2311. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem, 1976, 72, 248-54.
HYALURONAN SYNTHASE 2 (HAS2) REGULATES MIGRATION OF EPIDERMAL KERATINOCYTES Kirsi Rlllat, Mikko Lammit, Reijo Slronent, Vincent C. Hascall2, Ronald Mlduraz, Markku Tammlt and Raija Tamml! / Department of Anatomy, University of Kuopio, SF-702ll Kuopio, Finland. 2Department of Biomedical Engineering, Connective Tissue Biology Section, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195.
ABSTRACT Hyaluronan (HA) is a linear polysaccharide abundant in the extracellular space between epidermal keratinocytes. It is synthesized at the inner face of the plasma membrane by hyaluronan synthases (Has). We probed the importance of hyaluronan in keratinocytes by establishing cell lines carrying exogenous hyaluronan synthase 2 (Has2) gene(s) in sense and antisense orientations in order to increase and decrease their hyaluronan synthesis, respectively. The cell lines with the sense Has2 cDNA showed increased HA synthesis, while most cell lines with Has2 antisense cDNA contained less HA. Has2 antisense cells differed from control cell lines; they spread at a slower rate and retained a rounded morphology for a longer time. Further, during the first 24 hours after plating, proliferation was delayed in the antisense cell lines. In an in vitro wounding assay the antisense cells showed a significantly decreased migration rate as compared to controls. Cell lines with the Has2 sense cDNA were similar to the control cell lines in spreading and proliferation rates. However, they migrated faster than control cell lines. KEYWORDS Hyaluronan, keratinocyte,migration, spreading INTRODUCTION Hyaluronan is an ubiquitous high molecular weight glycosarninoglycan tissue matrix space filling, angiogenesis, cell migration, differentiation, tumor invasion and metastasis, and inflammation 1.2. Hyaluronan has been suggested to facilitate metabolic exchange between circulation and the tissue in the extracellular space of human epidermis 3. The metabolic turnover of hyaluronan in the small extracellular space between adjacent keratinocytes is markedly fast, with a half life of about one day in human skin organ culture 4 as well as in organotypic rat keratinocyte cultures 5. This suggests that hyaluronan may also contribute to some dynamic cellular processes such as the migratory activity in moving upwards from the basal layer and the shape change from columnar to squamous 3. Hyaluronan is synthesized at the inner face of the plasma membrane by hyaluronan synthase (Has). They act by alternative addition of glucuronic acid and N-acetylglucosamine to the growing hyaluronan chain, which is simultaneously extruded through the membrane into the extracellular space. Three isoenzymes have been identified in vertebrates, designated as Hasl, Has2 and Has3 (see review 6 and references therein), each with distinct kinetic properties and product size 7,8. Hyaluronan has been suggested to contribute to cell migration as a structural component of the extracellular space 2. In addition, hyaluronan controls cell locomotion by interacting with and signaling through specific cell surface receptors. Although a positive correlation between tissue hyaluronan content and enhanced migration has been noted
558
Keratinocytes and hyaluronan
repeatedly 9,10, direct evidence for the contribution of hyaluronan synthesis or tissue concentration has been missing. The aim of this study was to modulate endogenous hyaluronan synthesis by upregulation and downregulation of Has2 in keratinocytes, and to examine the consequences in terms of their proliferative and migratory activities, and their morphology.
MATERIALS & METHODS Transfections A rat Has2 full-length eDNA (4172 bp, Gen Bank #AFOO8201) was ligated into the multiple cloning site of the eukaryotic expression vector pCI-neo (5474 bp, Promega, Madison, WI) in sense and antisense orientation. A newborn rat epidermal keratinocyte cell line (REK) II was transfected according to the manufacturer's instructions with FuGENE™6 transfection reagent (Boehringer-Mannheim, Mannheim, Germany) combined with I !-Ig of plasmid DNA.
Migration Analysis The transfected and control cells were seeded at 500,000 cells/35 mm plates and grown until confluence. A cell-free area was introduced by scraping the monolayer crosswise with a sterile I ml pipette tip which cleared cells from =1000 urn wide lanes. The areas covered by the cells in 8 crossing areas in duplicate wells of every cell line were measured immediately after scraping and 24 h later.
Spreading Analysis of individual cells The spreading rates of cells in the different cell lines were determined by measuring the areas occupied by individual cells at 3, 6, 9, and 24 h after plating. Keratinocytes were seeded on 8-well chamber slides and 5 images of each cell line were captured at every time point using a CoolSNAP CCD camera (Photometries) mounted on a Nikon Eclipse TE300 inverted microscope with a 20 x DIC objective. The areas of individual cells were measured using NIH Irnage®software.
RESULTS & DISCUSSION Cell lines Most of the Has2 sense and antisense cell lines showed increased and decreased: a) hyaluronan secretion into medium, and b) in situ hyaluronan staining of the cell layers, respectively. The presence of the gene constructs and their copy numbers were estimated by Southern blotting. The transfected Has2 gene constructs were present in all selected cell lines in copy numbers ranging from I to 10, as estimated by the band intensities.
Spreading The average area occupied by the sense cell lines was similar or slighty larger than that of the mock-transfected and parental controls. In contrast, spreading was significantly slower in antisense cells, and after 24 h they only covered about half of the area of the control and sense cell lines (Fig. 1). There was a delay in the initiation of the proliferation cycle in antisense cells (data not shown) which was probably associated with incomplete spreading, since most normal cells cannot divide without a proper state of matrix adhesion.
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Migration Has2 overexpression consistently enhanced the migration of REK cells while migration of cells expressing Has2 antisense gene was always reduced (Fig. 2). The rnock-transfected keratinocyte migration front moved an average of 7 um/h, while the antisense cells migrated 4 um/h and sense cells 9 urn/h. The reduced migration of antisense cells was not completely restored by exogenous hyaluronan indicating the importance of endogenous hyaluronan synthesis in these cells.
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560
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CONCLUSIONS This study indicates that Has2 directed hyaluronan synthesis represents an important regulatory control of the migration of epidermal keratinocytes. The expression levels of Has2 also affected their phenotype in other aspects, including general cell spreading, f?rmation of lamellipodia, and the onset of proliferation. These findings are completely in line with the abundance of hyaluronan in the epidermis and with its rapid turnover during the migration and spreading of differentiating keratinocytes, and in their role in wound healing.
ACKNOWLEDGEMENTS This work was supported by Academy of Finland, grant # 40807, Finnish Cancer Foundation, EVO funds of Kuopio University Hospital and Kuopio University Biotechnology Funds.
REFERENCES T. C. Laurent and J. R. E. Fraser, Hyaluronan, Faseb J. , 1992, 6, 2397-2404. W. Y. Chen and G. Abatangelo, Functions of hyaluronan in wound repair, Wound Repair Regen ., 1999,7, 2, 79-89. 3. R. Tammi, 1. A. Ripellino, R. U. Margolis and M. Tammi, Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe, J. Invest. Dermatol., 1988,90,412-414. 4. R. Tammi, A.-M. Saamanen, H. 1. Maibach and M. Tammi, Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organ culture, J. Invest. Dermatol., 1991,97, 126130. 5. R. Tammi, M. Tammi, V. C. Hascall, M. Hogg, S. Pasonen and D. K. MacCallum, Collagen substrates surfaced with a pre-formed basal lamina alter the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures, Histochern. Cell. Biol., 2000, in press. 6. P. H. Weigel, V. C. Hascall and M. Tammi, Hyaluronan synthases, J. BioI. Chem., 1997,272,13997-14000. 7. J. Brinck and P. Heldin, Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44, Exp. Cell Res. , 1999,252,2,342-351. 8. N.ltano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, T. Shinomura, M . Hamaguchi, Y. Yoshida, Y. Ohnuki, S. Miyauchi, A. P. Spicer, J. A. McDonald and K. Kimata, Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties, J. Biol. Chern. ,1999,274,35,25085-25092. 9. S. L. Schor, A. M. Schor, A. M. Grey, J. Chen, G. Rushton, M. E. Grant and 1. Ellis, Mechanism of action of the migration stimulating factor produced by fetal and cancer patient fibroblasts: effect on hyaluronic and synthesis, In Vitro Cell Dev. B io I., 1989,25,8,737-746. 10. 1. R. Ellis and S. L. Schor, Differential effects ofTGF-betal on hyaluronan synthesis by fetal and adult skin fibroblasts: implications for cell migration and wound healing, Exp. Cell Res., 1996,228,2,326-333. 11. H. P. Baden and J. Kubilus, The growth and differentiation of cultured newborn rat keratinocytes, J.lnvest. Dermatol., 1983, 80, 2,124-130. 1. 2.
EGF REGULATES HAS2 EXPRESSION, CONTROLS EPIDERMAL THICKNESS AND STIMULATES KERATINOCYTE MIGRATION Markku I. Tammi l *, Juha-Pekka Pienimakil , Kirsi RiUal , Csaba Fiilop2, Mikko J. Lammi), Reijo Slronent, Ronald Midura 2, Vincent C. Hasca1l2, Merja Luukkonent, Kari Torronen l , TUna Lehtot, & Raija Tammi! I
Department of Anatomy. University of Kuopio, P.O.B. 1627. 70211 Kuopio, Finland
2Department ofBiomedical Engineering. Lerner Research Institute. Cleveland Clinic Foundation. Cleveland. OH 44195. USA.
ABSTRACT High concentrations of hyaluronan reside in the small space between the vital kertinocyte layers of human and animal epidermis and influence keratinocyte interactions, including growth, mobility and differentiation. We have previously found that the content of epidermal hyaluronan in human skin organ cultures is decreased and increased by cortisol and retinoic acid, and associated with enhanced and retarded terminal differentiation, respectively. To further substantiate this idea, we incubated epidermal keratinocytes with epidermal growth factor (EGF), and found a marked increase in hyaluronan synthesis which correlated with faster migration in an in vitro wounding assay of keratinocyte monolayers. EGF increased hyaluronan also in stratified, differentiated organotypic cultures, and increased the height of vital epidermis and reduced the thickness of the cornified layers, findings in line with an inhibition of terminal differentiation of keratinocytes. The stimulation of hyaluronan synthesis by EGF was due to upregulation of hyaluronan synthase 2 (HAS2) but not HAS 1 or HAS3. A part of the EGF influence on the structure of epidermis, and on skin wound healing, is thus mediated through its control of HAS2 expression.
KEYWORDS Hyaluronan, keratinocytes, epidermis, migration, wound healing, epidermal growth factor.
INTRODUCTION Skin epidermis and other stratifying epithelia contain abundant hyaluronan in a narrow extracellular space surrounding the epithelial cells 1. The half life of labelled epidermal hyaluronan in human skin organ culture is -1 day 2, indicating fast local turnover by keratinocytes. The importance of hyaluronan in the multilayered squamous epithelia is not completely understood, but we have hypothesized that the high concentration of hyaluronan is necessary to maintain an extracellular space for the nutritional needs of the more superficial cell layers, while the fast turnover allows the modulation of cell shape that occurs during differentiation, and the migration of kertinocytes in wound healing 3. In the epidermis of human skin organ cultures, hyaluronan synthesis and concentration is increased by retinoic acid 1, a widely used drug in skin diseases. At the same time,
562
Keratinocytes and hyaluronan
retinoic acid retards the differentiation of keratinocytes in epidermis. Glucocorticoids have also extensive use as a drug in skin diseases, and inhibit hyaluronan synthesis when applied in pharmacological doses 4. Glucocorticoids promote normal differentiation and decrease the thickness of the vital layers of epidermis. It thus appears that enhanced hyaluronan metabolism is associated with faster growth and remodelling of the vital part of epidermis, while decreased hyaluronan synthesis allows an earlier terminal differentiation of the upper epidermal cells. Whether the above mentioned correlation between hyaluronan metabolism and keratinocyte differentiation can be extended to other factors that modulate epidermal differentiation was examined in the present studies on the effect of epidermal growth factor (EGF). EGF is one of the most powerful agents that influences the behavior of keratinocytes. It transmits its information through the EGF-receptor (EGF-R), which belongs to the erbB-receptor tyrosine kinase family 5.6. In other cells, signaling through EGF-R regulates many cellular processes, including cell adhesion, expression of matrix degrading proteinases, and cell locomotion; these phenomena are all important e.g. in skin wound healing. Keratinocyte EGF-R expression is transiently elevated after wounding, and is important to keratinocyte proliferation and migration during reepithelization (for review, see 7). Exogenously added EGF and overexpression of EGF-R result in enhanced ligand-mediated migration ofkeratinocytes and faster reepithelialization 8.
MATERIALS AND METHODS
Cell culture A newborn rat epidermal keratinocyte (REK) cell line 9 was cultured in Dulbecco' s MEM (Gibco, Grand Island, NY) with 10 % fetal bovine serum (PBS) (HyClone, Logan, UT), streptomycin (50 J.Ig1ml), penicillin (50 Ulml) and 1-2 mM L-glutamine. Cells were trypsinized when they reached confluency using 0.02 % EDTA (w/v)/0.025 % trypsin (w/v) (Sigma, St. Louis, MO). For biochemical assays and radiolabeling, the cells were grown close to confluency in 6-well plates and incubated in the presence of 20 J.ICi/ml of [3H]glucosamine, and 100 to 200 J.ICi/ml [35S]sulfate (Amersham, Little Chalfont, UK). Organotypic cultures at air-liquid interface were performed as described 10.
Isolation of radiolabeled glycosaminoglycans After labeling in 6-well plates (9.6 cm 2/well), medium was removed and cell layer washed with 400 J.Il of Hank's solution (HyClone). For each culture, the medium and wash were combined and designated as "medium". Each cell layer was trypsinized, the trypsin solution and washes of the well and centrifuged cell pellet with MEM were combined and designated as "trypsinate". The resulting cell pellet was designated as the "intracellular" fraction. Carrier (6 J.Ig hyaluronan, Healonts, Pharmacia Uppsala, Sweden) was added to each sample to improve the recovery of radiolabeled glycosaminoglycans during the purification procedures and analysis. Medium, trypsinate and intracellular fraction were each digested for 1.5 h in 50 mM sodium-acetate, containing 200 J.Ig1ml papain (Sigma), roM EDTA and 5 roM cysteine, pH 6. Papain was inactivated in a boiling water bath and glycosaminoglycans precipitated with 1 % cetylpyridinium chloride, centrifuged, supernatant removed by aspiration and discarded. The precipitates were washed with water, dissolved in 4 M guanidine-HCl, and precipitated with ethanol. Details of the
EGF regulates HAS2 expression
isolation and analysis have been published previously
563
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Hyaluronan assay with double labeling Each purified sample was digested in 50 mM ammonium acetate, pH 7.0, with 25 mU chondroitinase ABC and 1 mU of Streptococcus hyaluronidase for 3 h at 37°C (both undigested material (heparan sulfate) and digestion products were analyzed on a Superdex Peptide column (Pharmacia) monitored for 3H and 35$ radioactivity, and for carrier hyaluronan at 232 nm 11. Incorporation of 35S04 provides a measure of the amount of the chondroitinldermatan sulfate synthesized during the labeling period. The [3H]galactosamine, derived from [3H]glucosamine, incorporated into the same chondroitinldermatan sulfate disaccharides provides an estimate of the effective specific activity of the UDP-N-acetylhexosamine precursor pool, and hence can be used to determine the chemical content of newly synthesized 3H-labelled hyaluronan 10. RT -rca with Hast, Has2, Has3 and GAPDH primers Keratinocytes were cultured in -28 cm 2 dishes until confluency and scraped into TRlzol®-reagent (Gibco) for total RNA isolation according to the instructions of the manufacturer. The RT-PCR reactions of Dnase treated samples were done with the RNA PCR Core Kit (Perkin Elmer, Branchburg, NJ). To obtain rat Hasl and Has3 specific primers, cDNA sequences were amplified from rat keratinocyte RNA with mouse Hasl and Has3 specific primers using RT-PCR. The PCR products were cloned into a pSportl (Gibco) plasmid and sequenced. Primers for Hasl and Has3 were 5'-GCT CTA TGG GGC GTT CCT C-3' and 5'-CAC ACA TAA GTG GCA GOO TCC-3', 5'-ACT CTG CAT CGC TGC CTA CC-3' and 5'-ACA TGA CIT CAC GCT TGC CC-3', respectively. Rat Has2 and GAPDH specific primers, 5'-TCG GAA CCA CAC TGT TTG GAG TG-3' and 5'-CCA GAT GTA AGT GAC TGA TIT GTC CC-3', and 5'TGA TGC TGG TGC TGA GTA TG-3' AND 5'-GGT GGA AGA ATG GGA GIT GC-3', were designed from GeneBank sequenced AFOO820I and MI770I, respectively. For quantitation of Has2 mRNA, a shortened (internal standard) Has2 cDNA containing the primer binding sites identical to those in the wild type Has2 cDNA was prepared by PCR. After in vitro transcription, the shortened Has2 RNA strand was purified and quantitated at 260 nm. RT-PCR was performed with constant amounts of the wild type and different concentrations of the shortened Has2 RNAs. The resulting products were run on an agarose-gel and quantitated by EtBr fluorescence by digital image analysis. Assay of keratinocyte migration Keratinocyte cultures approaching confluency were wounded by two perpendicular lines(-1 mm wide) scraped with a disposable pipette tip. The cleared areas were measured by an inverted microscope again 18 h after the treatment. The change in the area was measured and converted to mean migration distance (1JIll) of the cell front. Hyaluronan microscopy Cell layers were washed with Hank's balanced salt solution and fixed at room temperature for 20 min in PBS with 2 % paraformaldehyde with or without 0.5 %
564
Keratinocytes and hyaluronan
glutaraldehyde. The fixed cells were washed with 0.1 M sodium phosphate buffer, pH 7.4 (PB), and then blocked in 1 % BSA (w/v) containing 0.1 % Triton-Xl00 (v/v) in PB. Hyaluronan staining was done with biotinylated hyaluronan binding complex (bHABC), purified from a 4 M guanidine-HCl extract of bovine articular cartilage after dialysis and trypsin digestion, as described previously 11. The probe is a purified mixture of biotinylated G 1 domain of aggrecan and link protein. The bHABC probe, diluted to 5 lJg/ml in 3 % BSA (w/v), was added to the fixed cells, and incubated overnight at 4°C. After washing, avidin-biotin peroxidase (ABC-standard kit, Vector Laboratories, Inc., Burlingame, CA) was added for I h. The color was developed using 3,3'-diaminobenzidine (DAB) (0.05 %) and H202 (0.03 %). Counterstaining was done with hematoxylin for 2 min before mounting in Aquamount (BDH Laboratory Supplies, Poole, England). For confocal analysys Texas Red-labelled streptavidin (dilution 1:1000) was used instead of ABC. The cells were mounted with Vectashield (Vector), and viewed with a Perkin Elmer UltraVIEW confocal microscope (Wallac-LSR, Oxford, UK). Optical densities of the DAB stained cultures were measured as described previously 11. The specificity of the staining was confirmed by the lack of signal after pre-digesting fixed cultures with Streptomyces hyaluronidase or preincubating the bHABC probe with HA oligosaccharides. RESULTS Specific stimulation of keratinocyte hyaluronan synthesis by EGF The amounts of newly synthesized 3H-hyaluronan, 3H135S-chondroitin sulfate, and chondroitinase resistant glycosaminoglycans (mainly heparan sulfate) were determined using the double label method. Hyaluronan synthesis rate was highest with 20 ng/ml EGF, a concentration routinely used in subsequent experiments. The synthesis of other glycosarninoglycans (heparan sulfate and chondroitin sulfate) was not altered appreciably by EGF treatment. Since serum also stimulates hyaluronan synthesis, we examined the interactive effects of EGF and serum. Approximately similar increase of hyaluronan synthesis was seen whether the basal medium contained 0.5 % or 10 % serum, suggesting that factors in serum. such as IGF-l and PDGF that might contribute to increased synthesis, are additive with EGF. Since the vitality of cells may suffer in extended cultures with low serum concentration, 10 % serum was used in later experiments. Time course of EGF-induced hyaluronan synthesis Small aliquots of the radiolabeled precursors were added into the medium at different times following introduction of EGF, to monitor the time dependence of hyaluronan synthesis in 6 h labeling windows (Fig. 1). The medium change alone caused a marked increase of total hyaluronan synthesis. Nevertheless, EGF-treatment showed an additional -2-5 fold increase of newly synthesized total hyaluronan above the control at all time windows. The majority of hyaluronan synthesized in extended labeling periods was found in the culture medium, but a significant fraction remained associated with the cell layer in the 6 h labeling windows, either with the "trypsinate" or the "intracellular" fractions (Fig. 1). The newly synthesized hyaluronan in these fractions was substantially increased by EGF, peaking at the 0-6 and 6-12 h labeling windows. The "trypsinate" hyaluronan
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represents molecules bound to the receptors, mainly CD44, while most of the "intracellular" hyaluronan probably represents endocytosed material destined for lysosomal degradation. The EGF-induced increase of hyaluronan associated with the cell layer was also seen by staining the keratinocyte cultures with the hyaluronan specific probe (bHABC) (Fig. 2) and confirmed by determination of the total optical densities. The EGF-induced increase of intracellular hyaluronan was specifically measured in cultures digested with Streptomyces hyaluronidase to remove cell surface hyaluronan, then permeabilized and stained with bHABC. Morphological changes of keratinocytes and organotypic keratinocyte cultures
Shortly after introduction of EGF, the subconfluent, flattened keratinocytes began to round up and increase their membrane ruffles and microspikes, which was followed by cell elongation and the appearance of lamellipodia. These changes in morphology were apparent in a few of the cells even after 1 h, and most cells showed the altered morphology
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Keratinocyte morphology and hyaluronan distribution in the presence of EGF. A control culture is shown in (A) and one treated with EGF for 18 h in (B). Hyaluronan is present on membrane patches and ruffles of the wellspread, non-polar control cells, while in the elongated, apparently migrating EGF-treated cells hyaluronan signal is generally enhanced and most abundant in the pericellular region.
at 6 h and thereafter (Fig. 2). REKs cultured at the air-liquid interphase form 3-4 vital cell layers on top of which there are several fully differentiated comeocytes. Hyaluronan is histologically visible between all the vital cell layers and treatment with EGF dramatically increases its staining intensity. At the same time, the height and number of the vital cell layers increases, suggesting delayed terminal differentiation of the keratinocytes. Localization of the cell-associated hyaluronan Most of the hyaluronan in control cultures resided in plasma membrane patches II. In EGF-treated cells, the hyaluronan signal intensity was generally increased. also covering the membrane ruffles and microspikes in cells undergoing rounding. In the cells elongated by EGF, the mid-portion (around the nucleus) and the trailing edge showed an intense hyaluronan signal (Fig. 2). The amount of hyaluronan that accumulated in response to EGF treatment was sufficient to exclude sedimenting red blood corpuscles on keratinocyte surfaces, a frequently used test of hyaluronan coat formation and hyaluronan synthesis. Confocal images showed more hyaluronan also at the underside of the EGF-treated cells than in control cells. The intracellular hyaluronan signal was present in structures conforming to cytoplasmic vesicles which were generally larger and markedly more abundant in EGFtreated cultures. No nuclear hyaluronan signal was found. The accumulation of intracellular hyaluronan in response to EGF was most conspicuous in rounding cells, while cells retaining a more flattened morphology contained less. Stimulation of migration by EGF treatment The cells became elongated in EGF-treated cultures, a common finding in cells with enhanced motility. A dose dependent stimulation in the migration of the keratinocytes by EGF was confmned by artificial wounding of the cell layer, and quantitation of the speed at which the cells migrated into the cleared area (Fig. 3). The migratory activity peaked at the same EGF concentration as for the synthesis of hyaluronan. The cell number almost
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Effect of EGF on keratinocyte migration and proliferation. (A) Confluent keratinocyte cultures were artificially wounded, and the migration distance of keratinocytes at the wound edge after 24 h was measured as described in Methods. (B) The numbers of keratinocytes after 24-48 h growth in 2-200 ng/ml of EGF were counted in a hemocytometer. The bars show the range of duplicate cultures.
doubled by 24 h, independent of the presence of EGF (2-200 ng/ml) and further increases at 48 h were also independent of EGF except with some inhibition at the 200 ng/mllevel. Thus, EGF had no significant effect on the proliferation rate of the keratinocytes in conditions that showed a strong stimulation in hyaluronan synthesis and migration.
EGF-induced increase in hyaluronan synthase 2 (8as2) mRNA In order to reveal changes in the expression of the different hyaluronan synthases responsible for the enhanced hyaluronan synthesis by EGF, we estimated the mRNA levels of rat Hasl, HasZ and Has3 using RT-PCR. This analysis suggested that Has I and Has3 mRNA levels were not augmented by EGF treatment (Fig. 4A). In contrast, Has2 level was increased by EGF. However, the basal level ofHasZ mRNA in the keratinocytes was so low that the level of increase in its two transcripts was difficult to estimate by Northern blot. Therefore, we used quantitative RT-PCR with an internal, truncated cRNA standard, and compared the sample band densities with a set of standards. A low average copy number was noted before the change to fresh medium (-6/cell) (Fig 4B). While the level of HAS2 mRNA also increased in control cultures following change into fresh medium, the number of HasZ mRNA copies was 1.5-8 times higher in the EGF treated cultures at all time points examined, peaking at 6 h with -54 copies per cell.
DISCUSSION AND CONCLUSIONS The present data demonstrate the wide fluctuations in HasZ mRNA copy numbers that occur during regular keratinocyte cultures, and the strong influence of EGF. The changes in Has2 mRNA and hyaluronan synthesis levels closely corresponded with each other in time and quantity, strongly suggesting a tight transcriptional regulation of hyaluronan synthesis.
568
Keratinocytes and hyaluronan
B A
a>
Co
Hasl Has2 Has3 GAPDH
C'
EGF
~
"a o
60 40
~
~
... 02 6 12 18 24 Time after medium change (h)
Figure 4.
Hyaluronan synthase mRNA expression in EGF-treated cultures. (A) Total RNAs isolated from equal numbers of keratinocytes 3 h after change into fresh medium with or without EGF were reverse transcribed and amplified with 35 peR cycles for the different Has types and GAPDH. (B) Assay of Has2 mRNA at different time points following change into EGF containing (open circles) and control medium (closed circles), utilizing the mRNA internal standards.
While it is obvious that the synthesis rate of hyaluronan in various cell types is controlled by cytokines and growth factors, including EGF, the contribution of the three known Has genes in this regulation remains uncertain. In particular, the interactions of different hormonal and growth factor effectors need further characterization, as indicated by an earlier report showing upregulation of Hasl mRNA in keratinocytes treated by TGF~ in the presence of hydrocortisone 12, a strong suppressor of Has2 13. The intracellular accumulation of endogenous hyaluronan in perinuclear vesicles by EGF treatment was quite striking and unexpected, likely a result of enhanced receptormediated uptake, since our unpublished data show that the intracellular hyaluronan staining enhanced by EGF is reduced by incubation with hyaluronan decasaccharides, known to displace hyaluronan from keratinocyte surface receptors II. Our preliminary data in EGF-treated cultures also indicate less than expected accumulation of hyaluronan in 24 h labelings, as compared to values integrated from shorter labeling windows. Rapid catabolism has not been demonstrated in other cell cultures, but hyaluronan is known to have a very short half life in the epidermis of human skin organ cultures 2 and in organotypic keratinocyte cultures 10. While the biological importance of the hyaluronan turnover in keratinocytes is not currently known, its association with the enhanced motility of the cells seems most plausible. The data in this study establish that hyaluronan, a major extracellular matrix molecule in stratified epithelia like epidermis, is specifically increased in keratinocytes by EGF upregulation of Has2, and that Has2 regulation can induce rapid and dramatic changes in their cellular environment. considering the small extracellular space into which hyaluronan is synthesized. The biological consequences include enhanced keratinocyte migration during wound healing and thickened vital epidermis likely due to delayed differentiation. The direct influence of Has2 expression on keratinocyte migration was demonstrated by transfection of Has2 genes in sense and antisense orientations (see Rilla et al., in this volume). The ability of epidermal keratinocytes to rapidly cover an open wound is a biologically
EGF regulates HAS2 expression
569
crucial motility response of keratinocytes. Healing of skin wounds involves transient upregulation of EGF-receptors 14, and is aided by EGF-like growth factors 8. These studies suggest that stimulated EGF-receptor signaling and enhanced hyaluronan metabolism are intimately connected with each other and with the epithelial wound healing process.
ACKNOWLEDGEMENTS Technical help of MS Arja Venalainen, MS Riikka Tiihonen and MS Paivi Perttula is gratefully acknowledged. Financial support was received from the Academy of Finland, Finnish Cancer Foundation and EVa Funds of Kuopio University Hospital.
REFERENCES R. Tammi, J. A. Ripellino, R. U. Margolis, H.I. Maibach & M. Tammi, 'Hyaluronate accumulation in human epidermis treated with retinoic acid in skin organ culture', J. Invest. Dermatol., 1989, 92, 326-332. 2. R. Tammi, A. M. Saamanen, H. I. Maibach & M. Tammi, 'Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organ culture', J. Invest. Dermatol., 1991, 97, 126130. 3. M. Tammi & R. Tammi, Hyaluronan in the epidermis. in Science ofHyaluronan Today, V. Hascall and M. Yanagishita, Editors. 1999. Http://www.glycoforum.gIjp/science/hyaluronan/hyaluronanE.html 4. U. M. Agren, M. Tammi & R. Tammi,'Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture', J. Cell Physiol., 1995, 164,240-248. 5. E. Tzahar & Y. Yarden, 'The ErbB-2IHER2 oncogenic receptor fo adenocarcinomas: from orphanhood to multiple stromal ligands' , Biochim. Biophys. Acta, 1998,1377, M25-37. 6. K. L. Carraway 3rd & L. C. Cantley, 'A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling', Cell, 1994, 78, 5-8. 7. L. G. Hudson & L. J. McCawley, 'Contributions of the epidermal growth factor receptor to keratinocyte motility, Microsc. Res. Tech., 1998,43,444-455. 8. L. J. McCawley, P. O'Brien & L. G. Hudson, 'Overexpression of the epidermal growth factor receptor contributes to enhanced ligand-mediated motility in keratinocyte cell lines' ,Endocrinology, 1997, 138, 121-127. 9. D. K. MacCallum & J. H. Lillie, 'Evidence for autoregulation of cell division and cell transit in keratinocytes grown on collagen at an air-liquid interface', Skin Pharmacol., 1990, 3, 86-96. 10. R. H. Tammi, M. I. Tammi, V. C. Hascall, M. Hogg, S. Pasonen & D. K. MacCallum, 'A pre-formed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices', Histochem. Cell Bioi., 2000, 113,265-277. 11. R. Tammi, D. MacCallum, V. C. Hascall, J. P. Pienimaki, M. Hyttinen & M. Tammi, 'Hyaluronan bound to CD44 on keratinocytes in displaced by hyaluronan decasaccharides and not hexasaccharides', J. Bioi. Chem., 1998,273,28878-28888. 12. Y. Sugiyama, A. Shimada, T. Sayo, S. Sakai & S. Inoue, 'Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF- beta upregulates their expression in cultured human skin cells' , J. Invest. Dermatoi., 1998, 110, 116-121. 1.
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Keratinocytes and hyaluronan
13. W. Zhang, C. E. Watson, C. Liu, K. J. Williams & W. P. Werth, 'Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy', Biochem. J., 2000,349,91-97. 14. C. M. Stoscheck, L. B. Nanney & L. E. King, Jr., 'Quantitative determination of EGF-R during epidermal wound healing', J. Invest. Dermatol., 1992, 99, 645-649.
INDEX activation energy 185 adhesion assays 444 adhesive anti- 67-68 adhesive bio- 68 adhesive force 72 aggrecan 332, 358, 497 albumin adsorption 293-304 alginate fibre 280 amide derivatives of hyaluronan 261 262 ' amidyl radical 157 anchorage-independent growth 349353 angiogenesis 469 annealing 206 antibodies 437 anti-cancer activity 419-426 anti-inflammatory 501 anti-proliferative activity 419-426 antisense probes 241 apoptosis 489-494 Arrhenius plots 120,121 articular chondrocytes 334 atomic force microscopy 67-74, 109-116,137-139 bacterial hyaluronan 37 basal lamina 511-516 Benoit-Doty relation 39 binding activity 342-347 binding affinity 368 binding assays 356 binding crystal 411- 416 bio compatibility 269 bio-adhesive 68 biocompatible gel 285-292 biodegradability 269 biological activity 306 biomaterials 261-266, 269-275 biostability 277-284 biosynthesis 228-236 biosynthesis glycosaminoglycan 537-542 birefringence 214,215,216 bladder cancer cells 419, 424 bone marrow 459 bowel disease 381
breast cancer cell lines 443-445 brevican 358 bromine free radical 143-150 calcium oxalate 411-416 cancer cells 419-426, 443-445 cancer, skin 525 cartilage 332 casein kinase II assay 367 caverolae 517 CD44 331-339,341-347,350-353, 381-388,396-398,408,419-426, 457-466,517-524 CD44 expression 443-445 CD44 hyaluronan receptor 355-364 CD44 overexpression of 350 cell activation 342 cell adhesion 341, 443 cell biological role of hyaluronan cell culture 332,390-398,412,473, 482,518,522,562 cell death 438-441 cell growth 484 cell hyaluronan interactions 349-354 cell migration 444 cell proliferation 451-455, 470 545550 ' cell proliferation assay 445 cell surface 374 cell surface CD 44 341-347 cell surface expression 415 cell surface receptors 408 cell-associated hyaluronan 566 cellular proliferation 421 cellular signalling 301-304,361 chain-chain association 123-134 characteristic ratio 76 chimeric glycosaminoglycan 233 chloramides 156 chlorine free radical 143-150 chondrocyte 331-339 chondroitin 233 chondroitin sulphate 233,518 Chrohn's disease 382 clinical applications of hyaluronan cloning 356 coated pits 521 cold crystallisation 324 collagen 511-516 colon 382 compliance 197
572
Index
confocal micrograph 385-388 confocal miscroscopy 412,438 confocal-FRAP 123-134 conformational properties 89-97, 99102 corneal keratinocytes 532-535, 537542 creep experiments 195-200 critical overlap parameter 181-188 critical shear rate 95, 187 crosslinked derivatives 261, 264268, 269-275 crosslinked hyaluronan 182-191, 201-204 crosslinking process 277-284 crystal binding 411-416 cummulus cell-oocyte complex 489494 cyclo-oxygenase 501-504 cystoskeleton 331-339 cystosplasmic vesicles 519 cytokines 305, 429-434, 473-478 cytoplasmic cell fractions 470 DEAC ion exchange resin 99-102 deacetylated derivative of hyaluronan 261, 263 degradation 455 degradation of hyaluronan 141-149, 401 dendritic cells 457-466 derivatisation of hyaluronan 278 diabetic renal disease 473 differential scanning calorimetry 205, 208,316-321,324-327 diffusion coefficients 270 dihydroxyalkyl radical 153 DNA 429-434 DNA bioconjugate 305-310 DNA synthesis 526-530 Dorfman, Albert 29-33 drug delivery 265 dynamic light scattering 43 dynamic measurements 188 dynamic viscosity 203 eicosanoids 501 elastase 532 elastic modulus 202, 203 elastic modulus of hyaluronan gels 296 elasticity 201-204
electron microscopy 513 electron microscopy of combined hyaluronan 269-275 electrophoresis 254 electrophoretic mobility 305-308 embryogenesis 245-248 endocytosis 401-409, 522 endometrial tissue 238 endometrium 237-244 endopeptidase activity 532 endothelial cells 302,303,401-409, 419-426,469-472 endothelial hyaluronan receptors 353-364 enthalpy of crystallisation 316-321 , 324-327 enthalpy of melting 206-208,316321, 324-327 enzymatic hydrolysis 249-252 enzyme assays 229 enzyme treatment 412 epichlorohydrin 279 epidermal growth factor 561-569 epidermal keratinocytes 545-550, 557-560, 561-569 epidermis 511-516,517-524 epithelial cells 473-478,561 EPR experiments 152-159 erk kinase 373-377 erythrocytes 390, 391 esterified hyaluronan 278 esters of hyaluronan 261, 264 ethidium bromide 307 eukaryotic plasma membrane 245248 excluded volume 77 exclusion, 27 expression of hyaluronan 414, 415 extensional flow 209-218 extracellular hyaluronan 447 extracellular matrix 332, 498-500, 511 fertility 489-495 fibrinogen, adsorption 293-304 fibroblasts 531-535 field flow fractionation of hyaluronan 55-64 first order rate constant 146,147 flow cytometric analysis 440 flow cytometry 333, 342, 444
Index fluorescein labelled hyaluronan 452455 fluorescent-labelled HA 436 force measurements 67-74 force-separation curves 213, 217 formaldelyde 182 fragmentation, polysaccharide 151159 free radical 141-150 free radicals, resistance to 281-283 galactosidase gene 305, 306 gamma ray crosslinked hyaluronan 266,267 Gaussian cloud 80 gel biocompatible 285-292 gel filtration chromatography - see gel permeation chromatography gel mobility shift assay 552 gel permeation chromatography, of hyaluronan 47-54,55-64,100102,127,230,286 gel slurry 195-200 gelatin zymography 532-535 gelation of hyaluronan 205-208 gels derivatised hyaluronan for 293304 gel-sol transition temperature 206 gene targeting 246-248 gene transfer 305-310 glass transition 316-321,324-327 glucosamine (3 H labelled) 538-542 glutaraldehyde 261,265 glycoconjugates 538-542 glycosaminoglycan biosynthesis 514, 537-542 glycosaminoglycans 231-256 glycosaminoglycans, radiolabelled 562 glycosyl transferase 227-236 glycosylation sites 253-258 grafting, polymer 233 HABPI 365-371 HABPS447 haptotaxis 444 HARE proteins 401-409 Healon 181-192,212-218 heat shock protein 72, 435-441 heparin 369 histochemical demonstration 513 histochemical staining 483
573
Huggin's constant 181,184 Huggin's equation 85, 92 hyaladherins 7, 162,365,373,407 hyaluronan anti-cancer activity 419426 hyaluronan behaviour in solution 3745 hyaluronan binding 341-347,360, 365-371, 373-377 hyaluronan binding sites 452 hyaluronan biological properties of 117-122 hyaluronan biosynthesis 288 hyaluronan biosynthesis of 8,31 hyaluronan cell biological role 7 hyaluronan chain characterisation 38,39 hyaluronan characterisation, 4, 5 25,26 hyaluronan chemical structure 41 hyaluronan clinical applications 9 hyaluronan confocal-FRAP 123-134 hyaluronan conformational properties 89-97, 99-012 hyaluronan covalently linked 67-74, 278-284 hyaluronan critical shear rate 95 hyaluronan crosslinked 99-102, 181192,201-204,261-268,269-275, 277-280 hyaluronan crystal binding 411-416 hyaluronan degradation 8,9, 141149,401,455,518 hyaluronan derivatives 261-266 hyaluronan DNA matrix 305-310 hyaluronan dynamic light scattering of 43 hyaluronan expression 414,415 hyaluronan fluorescein labelled 123134,341-347,451-455 hyaluronan force measurements 6774,109-116,137-139 hyaluronan from umbilical cords 1, 2 hyaluronan gel 285-292, 293-304 hyaluronan gel permeation chromatography of 47-54, 55-64, 91-97, 99-102, 127, 143-150 hyaluronan history of 1-10, 17-23 hyaluronan hydration of 45 hyaluronan hydrogelation 205-208,
574
Index
314 hyaluronan hydrolysis 249-252 hyaluronan in joint fluids 3 hyaluronan in rooster comb 3 hyaluronan in Streptocci 3 hyaluronan intracellular 447-449, 451-455 hyaluronan intrinsic persistence length 39 hyaluronan intrinsic viscosity 75-78, 82, 91-93, 105, 184 hyaluronan ion exchange of 99-102 hyaluronan isothermal titration calorimetry 161-171 hyaluronan light scattering on 91-97, 143-150 hyaluronan Mark-Houwink parameters 91, 184 hyaluronan metabolism 511-516 hyaluronan molecular mass 46-54, 55-64,91-97, 143-150,184,286, 431,521 hyaluronan molecular modelling of 39-44 hyaluronan networks of 6, 7 hyaluronan NMR 3 C) studies 117122,281 hyaluronan NMR 161-171 hyaluronan oligosaccharides 436441,457-466 hyaluronan pathology of 9 hyaluronan phase transition 323-327 hyaluronan physiological functions 26,27 hyaluronan production of 221-226 hyaluronan radius of gyration 25, 39, 4950,80,89,90, 127 hyaluronan receptors 355, 359, 396, 401-409 hyaluronan rheological properties 5, 6, 89-97, 103-108, 175-180, 195200, 201-204 hyaluronan secondary structure 118 hyaluronan self association 99, 123134,137-139 hyaluronan self diffusion 125-134 hyaluronan shear rate dependence 93 hyaluronan staining 412, 546 hyaluronan stimulation 555
e
hyaluronan structure 3, 4 hyaluronan sulphated derivative 293304 hyaluronan synthases 227-235,237244,245-248,473,481,557 hyaluronan synthesis 474,481-487 hyaluronan synthesis rate 565 hyaluronan tertiary structures 117122 hyaluronan turnover 401 hyaluronan viscoelasticity 89-97 hyaluronan viscometry of 47-54 hyaluronan x-ray diffraction of 44 hyaluronan zero shear viscosity 94, 95, 96 hyaluronan-binding proteins 7, 407, 447-449 hyaluronan-binding proteoglycans 497-500 hyaluronan-cell interactions 349-354 hyaluronan-protein interactions 161171, 293-304 hyaluronic acid-based polyurethane derivatives 313-322 hyaluronidase 230, 249-252, 253258,436,445 hyaluronidase Streptomyces 332, 390-398,497,538 hyaluronidase, resistance to 281-283 hybridisation 239,246-248 hydration of hyaluronan 46 hydrodynamic diameter 75-78 hydrogel hyaluronan 314 hydrogelation 205-208 hydrogels 293, 261, 265 hydrolysis 249-252 hydroxy radicals 141-150,151-159 Hylon 181-192, 195-200,209-218 hyperglycemia 473 hypochlorite 151-159 I CAM 1408 IHABP4447-449 immunohistochemistry 239 immunoblotting 553 immunocytochemistry 403-408 immunofluorescent antibody staining 357 immunohistochemical staining 361, 384 immunoperoxidase 357
Index immunoprecipitation 470 immunostaining 437-441 inflammation 381, 531 inflammatory disease 502 inflammatory disorders 429-434 integral-membrane proteins 357 intracellular HA 447-449,451-455 intrinsic viscosity 75-78,82,83,9193, 105, 106, 184 isocyanate 313 isothermal titration calorimetry 161171 joint protection 209-218 keratinocyte migration 563 keratinocytes 511-516, 517-524, 525-530,531-535,545-550,551555,557-560,561-568 kidney 412 kidney inflammatory disease 502 kinases 367, 471, 551 knee joint 210 Kratky plot 40 leucocyte adhesion 381-388 light scattering, of hyaluronan 47-54, 55-64,91-97, 143-150,286 link module 161-171, 341 link superfamily 357-362 liver cells 401-409 liver kinase assay 367 lyase 230 lymph node 408 lymphatic endothelium 359-363 lymphocytes 390-398 mammary cells 351-353 Mark-Houwink parameters 91,184 Martin equation 85, 86 MDCK cells 411-416 menstrual cycle 237-244 mesothelial cells 481-487 metabolic labelling 412-415,513 metabolic labelling assay 546 metal ion complex 300-304 metalloprotease expression 531-535 Meyer, Karl Alexander 3, 17-23 mitogen activated 551-555 mitosis 451-455 molecular mass of hyaluronan 4754, 55-64, 76, 91-97, 184 molecular motion 316 monoclonal antibodies 401-408
575
monocyte adhesion assay 383 mutagenesis 231 mycobacterial DNA 423 myeloperoxidase 151, 152 nanomechanically patterned surface 293-304 nephrolithiasis 411-416 neurocan 358 nitroso spin traps 154 NMR 161-171, 281, 283 NMR of crosslinked hyaluronan 269-275 Northern blotting analysis 437-441 nuclear protein extractor 552, 553 oligomers, hyaluronan 350 oligosaccharides of hyaluronan 436, 457-466,517-524 opposed jets 214 organ culture 332,511-516 oxidised hyaluronan 264 Pasteurella 227 pathology of hyaluronan PDGF 451-455 pericellular matrix 331-339,389-398 peristence length 39, 76, 77, 83 peritoneal mesothelial cells 481-487 peroxides 525-530 peroxynitrite 142-150 persistency test 287 phase diagram 318 phase transition 323-327 phase transition temperatures 316 phenotypic expression 537 phosphorylation 368-371 photoimmobilisation 295, 301 pinocytosis 521 plasma membrane 470 plasmid DNA 306 polymerase chain reaction 229 polymerisation of hyaluronan 229 polymorphic structure 317 polymorphism 253-258 polyurethane derivatives 313-322, 323-327 polyvinyl alcohol 279 porcine excisional wound model 307 progesterone 368 proliferating cells 497 prostate cancer 419, 424 protein extraction 553
576
Index
protein heat shock 435-441 protein hyaluronan binding 365-371, 407,447-449 protein-hyaluronan interactions 161171 proteins HARE 401-409 proteins integral-membrane 357 proteoglycans 231-256, 497-500 proximal tubular cells 473-478 radius of gyration 29, 39, 49, 50, 80, 89,90,127 receptors, hyaluronan 396, 401-409 REK cultures 517-524 relaxation times 188, 270 renal cells 473-478,501-504 renal tubule epithelium 412 reticulation 266 RHAMM 373-377,408,457-466 rheological analysis 294, 296 rheological behaviour 181-192, 201204 rheological creep experiments 195200 rheological flow profiles 175-180 rheological properties 89-97, 103108 rheumatoid synovial fluid 152 rhodamine 526 RNA extraction 483 rooster combs 99, 110 rubber elasticity theory 190 SANS 40 SAXS, of crosslinked hyaluronan 269-275 scanning electron microscopy 307 SEC - see gel permeation chromatography secondary structure 118 self association 99, 123-134, 137139 self diffusion 125-134 semi-dilute regime 185 semi-dilute solution 84 sense probes 241 serum electrophoretic pattern 254 shear flow measurements 185 shear flow of synovial fluid 209-218 shear rate dependence 93 shift factors 189 sialidase treatment 253, 255
signal transduction 469-472 signalling molecules 554 skin cancer 525 skin epidermis 561 skin fibroblasts 532-535, 537-542 specific viscosity 79, 81, 105 sperm 365-371 spermadine 369 spin trapping 154-159 spreading analysis 558 squeeze flow 209-218 stagnation point 215 staining of hyaluronan 412, 546 stratified epithelia 511, 561 Streptococcus bacteria 182, 221226,227 Streptococcus equi 90, 138 Streptomyces hyaluronidase 332, 390-398,497,538 subconfluent cultures 414 sulindac 476-478 sulphated derivative of HA 293-304 superoxide radical 141-150 sustained release 308 synovial cells 389-398 synovial fluid 117 synovial fluid 209-218 synthases, hyaluronan 217-235, 237-244, 245-248 T-cell stimulation 457-466 TEMPO - mediated oxidation 264, 265 tertiary structures 117-122 testicular hyaluronidase 230 thermal analysis 294, 297 thermal properties of hyaluronan derivatives 313-322 thromboxane 501-504 tissue augmentation 201, 202 tissue collection 238 tissue distribution 406 tissue engineering 293 tissue extraction 254 transcription factor 551-555 transfectants 350-353 transfected cells 356 transfections 306-310, 558-560 transmembrane 408 trypsin inhibitor 497 TSG-6161-171
Index
TSG-protein 358 tumour growth, inhibition of 349-353 TXA2502 Ugi's reaction product 265 umbilical cord 1, 2, 402 uniaxial compression 214 uridine diphosphoglucose dehydrogenase 483 urine 411 UV irradiation 525-530 versican 358,381-388,497-498 vesicles 519 vinyl sulfone 182 viscoelastic measurements 91-97 viscometric activation energy 185 viscometry of hyaluronan 47-54 viscosity of polymer solutions 79-87 viscosupplementation 195 viscous modulus 202, 203 water absorption capacity 281 water holding capability 323 water uptake 294 Western blotting 333, 439, 470 Wharton's jelly 117 wound assay 444 wound healing 481-486, 531, 561 wound repair 411-416 wounded cells 483 wounding-induced migration 559 Yamakawa and Fuji model 41 zero shear viscosity 94, 95, 96, 104, 197,204 zymogram technique 254
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