Pathophysiology and Clinical Applications of Nitric Oxide

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Pathophysiology and Clinical Applications of Nitric Oxide

Part B

The Endothelial Cell Research Series A series of significant reviews of basic and clinical research related to the endothelium. Edited by Gabor M.Rubanyi, Berlex Biosciences, Richmond, California. Volume One Endothelium-Derived Hyperpolarizing Factor edited by Paul M.Vanhoutte Volume Two Endothelial Modulation of Cardiac Function edited by Malcolm J.Lewis and Ajay M.Shah Volume Three Estrogen and the Vessel Wall edited by Gabor M.Rubanyi and Raymond Kauffman Volume Four Modern Visualisation of the Endothelium edited by J.M.Polak Volume Five Pathophysiology and Clinical Applications of Nitric Oxide edited by Gabor M.Rubanyi Volumes in Preparation Mechanical Forces and the Endothelium P.I.Lelkes Vascular Endothelium in Human Physiology and Pathophysiology P.Vallance and D.Webb Morphogenesis of Endothelium W.Risau This book is part of a series. The publisher will accept continuation orders which may be cancelled at any time and which provide for automatic billing and shipping of each title in the series upon publication. Please write for details.

Pathophysiology and Clinical Applications of Nitric Oxide Part B Edited by

Gabor M.Rubanyi Berlex Biosciences Richmond, California USA

harwood academic publishers Australia • Canada • China • France • Germany • India Japan • Luxembourg • Malaysia • The Netherlands Russia • Singapore • Switzerland

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Printed in Singapore. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-203-30372-5 Master e-book ISBN

ISBN 0-203-34370-0 (Adobe eReader Format) ISBN: 90-5702-415-2 (Print Edition) ISSN: 1384-1270

DEDICATION

During the printing of this book we received the news of the award of the 1998 Nobel Prize in Medicine to Dr Robert Furchgott, Dr Louis Ignarro and Dr Ferid Murad for their pioneering work in the discovery of nitric oxide as a novel mediator of biological functions. In the name of all contributors to this book and numerous other scientists in the field we congratulate Bob, Lou and Ferid on this great personal accomplishment and we would like to dedicate this volume to them. The Nobel Prize also reflects a well-deserved appreciation of the entire field, some important recent progress of which is presented here.

CONTENTS

Preface

ix

Contributors

xi

Part B II. Nitric Oxide and Pathophysiology of Diseases 13

Nitric Oxide and Its Role in Hemostasis E.M.Battinelli, M.R.Trolliet and J.Loscalzo

228

14

Hypertension E.Nava and T.F.Luscher

248

15

Pulmonary Hypertension J.Dupuis, D.Langleben and D.J.Stewart

263

16

Atherosclerosis: Role of NO J.P.Cooke and P.S.Tsao

279

17

Nitric Oxide in Myocardial and Splanchnic Ischaemia/Reperfusion A.M.Lefer and R.Scalia

296

18

Nitric Oxide and Circulatory Shock C.Thiemermann

317

19

Kidney Diseases P.Gross, H.Dorniok, D.Reimann and M.Plug

328

20

The Role of Nitric Oxide in Preterm Labour and Pre-eclampsia R.E.Garfield, I.Buhimschi, G.R.Saade and K.Chwalisz

342

21

Multiple Sclerosis J.E.Merrill

367

22

Regulation of Nitric Oxide and Inflammatory Mediators in Human Osteoarthritis-Affected Cartilage: Implication for Pharmacological Intervention A.R.Amin, M.G.Attur and S.B.Abramson

389

23

The Role of Nitric Oxide in Rheumatoid Arthritis M.Stefanovic-Racic and C.H.Evans

404

24

Gastrointestinal Function in Shock A.L.Salzman

415

vii

III. Therapeutic Applications 25

Nitric Oxide Donors J.A.Hrabie and L.K.Keefer

442

26

Nitric Oxide Inhalation AM.Atz and D.L.Wessel

458

27

Nitric Oxide Synthase Inhibitors J.F.Parkinson, J.J.Devlin and G.B.Phillips

491

28

The Pharmacology of Peroxynitrite-Dependent Neurotoxicity Blockade J.S.Althaus, G.J.Fici and P.F.Von Voigtlander

508

29

Hemoglobin: Its Role as a Nitric Oxide Scavenger S.R.Fischer and D.L.Traber

524

30

Therapeutic Implications of Recombinant Endothelial Nitric Oxide Synthase Gene Expression in Cerebral and Peripheral Arteries A.F.Y.Chen, T.O'Brien and Z.S.Katusic

540

31

Gene Therapy Approaches with iNOS E.Tzeng and T.R.Billiar

554

Index

572

Part A 1

The Beginnings of Research on Nitric Oxide in Biology: A Historical Perspective R.F.Furchgott

1

I.

Generation and Biological Actions of Nitric Oxide

2

Structural Variations on a Theme of Nitric Oxide Production by Three Isoforms of Nitric Oxide Synthase B.S.Masters, P.Martásek and L.J Roman

17

3

Nitric Oxide Synthase Gene Regulation K.K.Wu

39

4

Localization and Subcellular Targeting of Nitric Oxide Synthases G.García-Cardena and W.C.Sessa

51

5

Molecular Mechanisms of Peroxynitrite Reactivity A.J.Gow and H.Ischiropoulos

59

6

Nitric Oxide, Peroxynitrite and Poly (ADP-Ribose) Synthetase: Biochemistry and Pathophysiological Implications C.Szabó

69

7

Nitric Oxide and Gene Transcription J.K.Liao and P.Libby

99

viii

8

Nitric Oxide and the Regulation of Vasoactive Genes D.V.Faller

121

9

Cyclooxygenase: An Important Transduction System for the Multifaceted Roles of Nitric Oxide D.Salvemini

155

10

Regulation of Nitric Oxide Synthase Expression and Activity by Hemodynamic Forces B.E.Sumpio, B.O.OluwoleX.Wang and M.A.Awolesi

171

11

Nitric Oxide Synthase Regulation by 17p-Estradiol K.Kauser and G.M.Rubanyi

195

12

Mice Deficient in eNOS and nNOS Isoforms P.L.Huang

209

Index

I/1

PREFACE

The identification that nitric oxide (NO) mediates endothelium-dependent vasodilation and that NO is synthesized by nitric oxide synthase (NOS) from L-arginine represented key discoveries of the 1980s. These observations opened the way to unprecedented research activity in the early 1990s which led to new insights into the physiology and pathophysiology of numerous biological and disease processes. Research on NO has continued at a high pace in the past 5 years and it is by now clear that in addition to the cardiovascular system, NO is implicated in the function and disease of several other organs and systems as well (e.g., inflammatory diseases in the brain, joints, lung and gut; immunological disorders, cancer, diseases of the reproductive organs, etc.). It is also evident that we are entering into a new phase of NO research: several of the basic research observations are turning into novel therapeutic principles, and these are being pursued in clinical trials. Selective inhibitors of nitric oxide synthase isoforms are being discovered, novel NO-donors are being developed and the first reports on gene therapy approaches have been published—just to name a few examples which illustrate the point. Athough some excellent books have been published in the past about NO research, this work is the first that summarizes the quantum leap from basic sciences to clinical applications of novel therapeutic principles emerging from this decade-long research activity. The book is divided into two parts. Part A starts with a historical perspective written by Dr Robert Furchgott. His cornerstone observation in 1980 about the essential role of the endothelium in acetylcholineinduced vasorelaxation undoubtedly represented the start of this whole field. The rest of this volume is dedicated to the theme Generation and Biological Actions of Nitric Oxide. Leading experts give state-of-theart overviews of various aspects of this theme, including the description of NOS function and regulation, the biological actions of NO, and the functional consequences of NOS gene knock-out in mice.

x

Part B has two sections. The first of these describes the known role of NO (its deficiency and/or excess) in the pathophysiology of diseases, such as those of the cardiovascular, kidney, reproductive, nervous and skeletal systems. The second section of Part B summarizes the recent progress achieved with therapeutic applications of NO. This section describes, among other topics, the discovery and therapeutic application of new NO donors, the therapeutic use of NO inhalation, selective inhibitors of NOS isoforms, and gene therapy approaches with both the constitutive and inducible forms of NOS. Based on the scope, the excellent contributions by the leading experts, and the very efficient and professional work of the publisher, I believe this book on the pathophysiology and clinical application of nitric oxide is a one-of-a-kind work which will be of interest and benefit to both the experts working in the field and interested professionals of a wide variety of disciplines.

CONTRIBUTORS

Abramson, Steven B Department of Rheumatology and Medicine Hospital for Joint Diseases Rm. 1401, 301 East 17th Street New York, NY 10003 USA Althaus, John S CNS Diseases Research Unit 7251 Pharmacia and Upjohn Inc. 301 Henrietta Street Kalamazoo, MI 49001 USA Amin, Ashok R Department of Rheumatology and Medicine Hospital for Joint Diseases Rm. 1600, 301 East 17th Street New York, NY 10003 USA Attur, Mukundan G Department of Rheumatology and Medicine Hospital for Joint Diseases

Rm. 1600, 301 East 17th Street New York, NY 10003 USA Atz, Andrew M Cardiac Intensive Care Unit Children’s Hospital, Farley 653 300 Longwood Avenue Boston, MA 02115 USA Awolesi, Mark A Department of Surgery (Vascular) Yale University School of Medicine 333 Cedar Street, FMB 137 New Haven, CT 06510 USA Battinelli, Elisabeth M Department of Medicine and Biochemistry Whitaker Cardiovascular Institute Boston University School of Medicine 80 E Concord Street, W-507 Boston, MA 02118–2394

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USA Billiar, Timothy R Department of Surgery University of Pittsburgh 479 Scaife Hall Pittsburgh, PA 15261 USA Buhimschi, Irina Department of Obstetrics and Gynecology University of Texas Medical Branch Medical Research Building, Rm. 11.104 301 University Boulevard Galveston, TX 77555–1062 USA Chen, Alex FY Department of Anesthesiology and Pharmacology Mayo Clinic St Mary’s Hospital 200 First Street SW Rochester, MN 55905 USA Chwalisz, Kristof Research Laboratories Schering AG Berlin Germany Cooke, John P Division of Cardiovascular Medicine Stanford University School of Medicine 300 Pasteur Drive, CVRC Stanford, CA 94305–5406 USA Devlin, James J Department of Immunology and Pharmaceuticals Discovery Berlex Biosciences 15049 San Pablo Avenue Richmond, CA 94804–0099 USA Dorniok, Heike Department of Medicine Universitätsklinikum CG Carus Fetscherstrae 76

D-01307 Dresden Germany Dupuis, Jocelyn Terrence Donnelly Heart Centre St Michael’s Hospital 30 Bond Street, Rm. 712-B Toronto, Ontario Canada M5B 1W8 Evans, Christopher H Room C-313 Presbyterian University Hospital 200 Lothrop Street Pittsburgh, PA 15261 USA Faller, Douglas V Cancer Research Center, Rm. K701 Boston University Medical Campus 80 East Concord Street Boston, MA 02118 USA Fici, Gregory J CNS Diseases Research Unit 7251 Pharmacia and Upjohn Inc. 301 Henrietta Street Kalamazoo, MI 49001 USA Fischer, Stefanie R Investigational Intensive Care Unit The University of Texas Medical Branch 610 Texas Avenue Galveston, TX 77555–0833 USA Furchgott, Robert F Department of Pharmacology SUNY Health Science Center at Brooklyn 450 Clarkson Avenue, PO Box 29 Brooklyn, NY 11203 USA García-Cardeña, Guillermo Department of Pharmacology Yale University School of Medicine Boyer Center for Molecular Medicine 295 Congress Avenue

xiii

New Haven, CT 06536–0812 USA Garfield, Robert E Department of Obstetrics and Gynecology University of Texas Medical Branch Medical Research Building, Rm. 11.104 301 University Boulevard Galveston, TX 77555–1062 USA Gow, Andrew J University of Pennsylvania Medical Center 1 John Morgan Building 3620 Hamilton Walk Philadelphia, PA 19104–6068 USA Gross, Peter Department of Medicine Universitätsklinikum CG Carus FetscherstraBe 76 D-01307 Dresden Germany Hrabie, Joseph A SAIC Frederick National Cancer Institute—Frederick Cancer Research and Development Center Bldg. 469–2, PO Box B Frederick, MD 21702 USA Huang, Paul L Cardiovascular Research Center Massachusetts General Hospital—East 149 East 13th Street, 4th Floor Charlestown, MA 02129–2060 USA Ischiropoulos, Harry University of Pennsylvania Medical Center 1 John Morgan Building 3620 Hamilton Walk Philadelphia, PA 19104–6068 USA Katusic, Zvonimir S Departments of Anesthesiology and Pharmacology Mayo Clinic

St Mary’s Hospital 200 First Street SW Rochester, MN 55905 USA Kauser, Katalin Department of Cardiovascular Research Berlex Biosciences 15049 San Pablo Avenue Richmond, CA 94804–0099 USA Keefer, Larry K Chemistry Section Laboratory of Comparative Carcinogenesis National Cancer Institute—Federick Cancer Research and Development Center Building 538 Frederick, MD 21702–1202 USA Langleben, David Jewish General Hospital 3755 Cote St Catherines Rd Montreal, Quebec Canada H3T 1E2 Lefer, Allan M Department of Physiology Jefferson Medical College Thomas Jefferson University 1020 Locust Street Philadelphia, PA 19107–6799 USA Liao, James K. Atherosclerosis and Vascular Medicine Unit Brigham and Womens Hospital and Harvard Medical School 221 Longwood Avenue, LMRC-316 Boston, MA 02115–5817 USA Libby, Peter Atherosclerosis and Vascular Medicine Unit Brigham and Womens Hospital and Harvard Medical School 221 Longwood Avenue, LMRC-316 Boston, MA 02115–5817

xiv

USA Loscalzo, Joseph Department of Medicine and Biochemistry Whitaker Cardiovascular Institute Boston University School of Medicine 80 E Concord Street, W-507 Boston, MA 02118–2394 USA Lüscher, Thomas F Division of Cardiology University Hospital Bern Römistrasse 100, CH 8091 Zürich Switzerland Martasek, Pavel The Robert A Welch Foundation Department of Biochemistry The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, TX 78284–7760 USA Masters, Bettie Sue The Robert A Welch Foundation Department of Biochemistry The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, TX 78284–7760 USA Merrill, Jean E Hoechs Marion Roussel Inc. Route 202–206 PO Box 6800 Bridgewater NJ 08807–0800 USA Nava, Eduardo Department of Physiology Univeristy of Murcia School of Medicine Murcia Spain O’Brien, Timothy Divisions of Endocrinology and Metabolism

Mayo Clinic St Mary’s Hospital 299 First Street SW Rochester, MN 55905 USA Oluwole, Babalola O Department of Surgery (Vascular) Yale University School of Medicine 333 Cedar Street, FMB 137 New Haven, CT 06510 USA Parkinson, John F Department of Immunology and Pharmaceuticals Discovery Berlex Biosciences 15049 San Pablo Avenue Richmond, CA 94804–0099 USA Phillips, Gary B Department of Immunology and Pharmaceuticals Discovery Berlex Biosciences 15049 San Pablo Avenue Richmond, CA 94804–0099 USA Plug, Maria Department of Medicine Universitätsklinikum CG Carus Fetscherstrae 76 D-01307 Dresden Germany Reimann, Doreen Department of Medicine Universitätsklinikum CG Cams Fetscherstrae 76 D-01307 Dresden Germany Roman, Linda J The Robert A Welch Foundation Department of Biochemistry The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive

xv

San Antonio, TX 78284–7760 USA Scalia, Rosario Department of Physiology Jefferson Medical College Thomas Jefferson University 1020 Locust Street Philadelphia, PA 19107–6799 USA Sessa, William C Department of Pharmacology Yale University School of Medicine Boyer Center for Molecular Medicine 295 Congress Avenue New Haven, CT 06536–0812 USA Rubanyi, Gabor M Berlex Biosciences 15049 San Pablo Avenue Richmond, CA 94804–0099 USA Saade, George R Department of Obstetrics and Gynecology University of Texas Medical Branch Medical Research Building, Rm. 11.104 301 University Boulevard Galveston, TX 77555–1062 USA Stefanovic-Racic, Maja Room C-313 Presbyterian University Hospital 200 Lothrop Street Pittsburgh, PA 15261 USA Stewart, Duncan J Terrence Donnelly Heart Centre St Michael’s Hospital 30 Bond Street, Rm. 712-B Toronto, Ontario Canada M5B 1W8 Salvemini Daniela Searle Research and Development c/o Monsanto Company

800 N.Lindbergh Boulevard St Louis, MO 63167 USA Sumpio, Bauer E Department of Surgery (Vascular) Yale University School of Medicine 333 Cedar Street, FMB 137 New Haven, CT 06510 USA Salzman, Andrew L Division of Critical Care Medicine Children’s Hospital Medical Center 3333 Burnet Avenue Cincinatti, OH 45229–3039 USA Szabó, Csaba Division of Critical Care Medicine Children’s Hospital Medical Center 3333 Burnet Avenue Cincinatti, OH 45229–3039 USA Thiemermann, Christoph The William Harvey Research Institute St Bartholomew’s Hospital and the Royal London School of Me dicine and Dentistry Charterhouse Square London, EC1M 6BQ UK Traber, Daniel L Investigational Intensive Care Unit The University of Texas Medical Branch 610 Texas Avenue Galveston, TX 77555–0833 USA Trolliet, Maria R Department of Medicine and Biochemistry Whitaker Cardiovascular Institute Boston University School of Medicine 80 E Concord Street, W-507 Boston, MA 02118–2394 USA Tsao, Philip S Division of Cardiovascular Medicine

xvi

Stanford University School of Medicine 300 Pasteur Drive, CVRC Stanford, CA 94305–5406 USA Tzeng, Edith Department of Surgery University of Pittsburgh 497 Scaife Hall Pittsburgh, PA 15261 USA Von Voigtlander, Philip F CNS Diseases Research Unit 7251 Pharmacia and Upjohn Inc. 301 Henrietta Street Kalamazoo, MI 49001 USA Wang, Xiujie Department of Surgery (Vascular) Yale University School of Medicine 333 Cedar Street, FMB 137 New Haven, CT 06510 USA Wessel, David L Cardiac Intensive Care Unit Children’s Hospital, Farley 653 300 Long wood Avenue Boston, MA 02115 USA Wu, Kenneth K Division of Hematology Department of Internal Medicine University of Texas at Houston Medical School 6431 Fannin, MSB 5.016 Houston, TX 77030 USA

Nitric Oxide and Pathophysiology of Diseases

13 Nitric Oxide and Its Role in Hemostasis Elisabeth M.Battinelli, Maria R.Trolliet and Joseph Loscalzo Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Masssachusetts 02118, USA

Nitric oxide (NO) is a short-lived, free radical gas that is found in a variety of cell types and organ systems. In vascular hemostasis, NO is important for preventing platelet activation, adhesion, and aggregation. NO may play a role in the development of hemostatic disorders that occur when NO availability is altered. In this chapter, we discuss the importance of NO in the hemostatic response with particular emphasis on the pathogenesis of thrombotic and hemorrhagic diatheses. Key words: Fibrinolysis, hemostasis, nitric oxide, thrombosis INTRODUCTION Hemostasis depends, in part, on the ability to form a protective platelet plug in the event of vascular injury and thereby prevent excessive blood loss. A poorly controlled or excessive hemostatic response, however, can be manifested as hemorrhage or thrombosis, respectively. The primary hemostatic response is dependent upon platelets and products of the endothelium that modulate platelet function. One mediator that has been shown to have antiplatelet activity is nitric oxide (NO). Under resting conditions the endothelium is stimulated by flow to produce NO, which can regulate platelet adhesion and aggregation (Pohl and Busse, 1989; Cooke et al., 1991; de Graaf et al., 1992). The production of NO leads to activation of soluble guanylyl cyclase with a concomitant increase in cGMP, which is a principal mediator of the effects of NO. NO can inhibit platelet adhesion and aggregation, and can also induce disaggregation of previously aggregated platelets (Radomski and Moncada, 1991a,b; Radomski et al., 1987a,b,c). Thus, in different

NITRIC OXIDE AND ITS ROLE IN HEMOSTASIS

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disease states in which less or more nitric oxide is being produced by the vasculature, platelet function may be adversely altered. The platelet response may also be modulated in patients with cardiovascular disease who use organic nitrates, such as nitroglycerin, which act as NO donors. While lack of NO may lead to thrombosis, an excess of NO may be detrimental such that platelet aggregation is impaired and a bleeding diathesis results. In this chapter we will provide evidence for the importance of NO in regulating platelet function and hemostasis, and illustrate hemostatic disorders that arise when NO production or bioactivity is altered. PATHOGENESIS OF THROMBOSIS The process of thrombus formation involves the interaction of elements in the vessel wall with platelets and activated coagulation factors (Figure 13–1). In arteries, a thrombus may occlude the vessel resulting in decreased or absent blood flow to tissues, potentially leading to ischemia or infarction. One of the most important rheologie properties of flowing blood that regulates hemostasis is the blood’s tendency to move in a pattern of concentric, cylindrical laminae, termed laminar flow (Goldsmith et al., 1986). These laminae are arranged such that a gradient exists within the vessel, with the greatest velocities found in the laminae closest to the center of the vessel and the lowest velocities found near the vessel wall. Because of cell size and charge properties, the laminae in the center of the vessel are enriched in red blood cells while the laminae closest to the vessel wall are enriched in platelets. Since the blood velocity is relatively low in the vicinity of the vessel wall, platelets have a long residence time in that domain, thereby allowing for rapid activation should the vessel become damaged. Another hemodynamic factor that is important for understanding the hemostatic function of platelets is shear rate (defined as the product of blood viscosity and shear stress), which is determined by the velocity gradient in the vessel. According to Poiseuille’s Law, velocity is inversely proportional to the crossectional area of a vessel or a vascular bed. Thus, blood velocity is highest in those vessels that have the smallest crossectional area, such as the aorta. Capillaries have the largest crossectional area, and, therefore, the lowest velocities. Large vessels with high velocities have higher rates of shear than do small vessels with low velocities. Increases in the shear rate lead to an increase in platelet deposition, while low shear rates are associated with preferential fibrin deposition (Loscalzo, 1994). Under normal circumstances, platelet activation is suppressed. There are three known endothelial products that inhibit platelet activation: cyclooxygenase and lipooxygenase metabolites, such as prostacyclin and 13-hydroxyoctadecadienoic acid (13-HODE) respectively; ecto-nucleotidase ADP diphosphohydrolase (ecto-ADPase); and nitric oxide (Radomski et al., 1987b; Moncada, 1982; Buchanan et al., 1991). Prostacyclin has been shown to inhibit platelet aggregation (Radomski et al., 1987a), while 13HODE regulates the adhesiveness of the vessel wall, modulating integrin receptor expression (Buchanan et al., 1991). The ecto-nucleotidases metabolize ADP to AMP and adenosine, resulting in a decrease in platelet recruitment and activity by the dinucleotide agonist (Marcus et al., 1993); this molecule has recently been identified as CD39 (Marcus et al., 1997). The role of NO as an inhibitor of platelet aggregation will be elucidated next.

Correspondence: Joseph Loscalzo, Whitaker Cardiovascular Institute, Boston University School of Medicine, 80E. Concord Street, Room W507, Boston, MA 02118–2394, USA. Tel: (617) 638–4890; Fax: (617) 638–4066

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Figure 13-1. A simplified scheme of the hemostatic response.

Platelet Adhesion Adhesion of platelets to the subendothelial matrix following vascular injury (or contact with foreign surfaces in the blood) is the initial event in the process of thrombosis. Binding interactions occur between glycoproteins found on the platelet surface and the connective tissue of the subendothelium. Some of these glycoproteins include: GPIb/IX/V, GPIa/IIa, and GPIIb/GPIIIa. GPIb, which noncovalently complexes with GPIX and GPV, is the main surface receptor for the adhesion of platelets to the subendothelium. Adhesion is mediated by von Willebrand factor (vWF), which links the platelet integrin to collagen in the subendothelium (Vaughan, 1996). After these components come into contact, vWF undergoes a conformational change that is essential for the platelet and subendothelium to remain in contact. Direct binding of the platelet to collagen is mediated by GPIa/IIa.

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Figure 13-2. Representation of the normal pathways of coagulation.

Platelet Recruitment and Aggregation After adhesion to the subendothelial matrix, the platelet initiates a series of reactions that include release of adenosine 5'-diphosphate (ADP) and serotonin (5-HT) from densegranules; and release of a-granule constituents, including fibrinogen, vWF, vitronectin, fibronectin, thrombospondin, platelet factor 4, and platelet-derived growth factor (PDGF) (Vaughan, 1996). At the same time, the eicosanoid thromboxane A2 (TxA2) is formed from arachidonic acid via membrane-bound phospholipase C. Through the release of ADP, 5-HT, and TXA2, more platelets are recruited to the area. Platelet recruitment occurs through the GPIIb/GPIIIa complex, also known as the fibrinogen receptor, which undergoes a calcium-dependent conformational change to become active (George et al., 1984; Ginsberg et al., 1988). Its ligand, fibrinogen, has two different sequences that can directly interact with platelets, including two RGD sequences in the a chain and a dodecapeptide sequence near the carboxyterminus of the chain (Hawiger et al., 1982). Plateletto-platelet linkages thereby form with fibrinogen as the bridge between GPIIb/IIIa molecules on adjacent platelets. Simultaneously, the platelet aggregate activates the coagulation cascade via the assembly of prothrombinase on the platelet surface. This series of reactions leads to further platelet aggregation by the production of yet another platelet agonist, thrombin (Loscalzo, 1994). Thrombin is produced by cleavage of its inactive precursor prothrombin in the common pathway of the coagulation cascade (Figure 13–2). This reaction occurs through the action of activated factor X (Xa) and activated factor V (Va) along with calcium and phospholipid cofactors. Factor X activation can occur through either the intrinsic or extrinsic pathways. The intrinsic pathway is activated by contact of the blood with subendothelial structures like collagen or basement membrane components in areas of vessel damage. Factor VII undergoes a conformational change to expose its active site, which converts the zymogen prekallikrein to kallikrein and converts factor XI to activated factor XI (XIa). Next, factor XIa converts the zymogen factor IX into activated factor IX (IXa) that, in turn, activates factor X (Colman et al., 1984). The extrinsic pathway is activated when the

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membrane proteolipid, tissue factor, is either exposed in the subendothelium to flowing blood or induced to form on the surface of endothelial cells or vascular smooth muscle cells by inflammatory mediators such as cytokines and endotoxin. Tissue factor activates factor VII directly, which then activates factor X of the common pathway as described above (Wilcox et al., 1989; Marmur et al., 1991). In recent years, the exclusivity of the intrinsic and extrinsic coagulation pathways has been questioned. Crosstalk between early events in these pathways has been identified that both complicates our understanding of clotting mechanisms and adds yet another level of redundancy to hemostatic defense. The platelet plug that forms in response to vascular injury is not stable. The fibrin meshwork formed by the action of thrombin on fibrinogen is required in order to add stability. Fibrin is produced when the serine protease thrombin cleaves the A and B chains of fibrinogen. Fibrin can then be further stabilized through transamidation reactions by factor XIIIa, which itself is generated by the action of thrombin on factor XIII (Loscalzo, 1994). Endothelial Defense Mechanisms If the hemostatic response were to occur inappropriately or without proper regulation, pathological thrombosis could result. The endothelium and the platelet itself release mediators that attenuate the hemostatic response, providing a mechanism of defense against unbridled hemostasis and thrombus formation. Two such mediators are prostacyclin and nitric oxide. Prostacyclin has been shown to inhibit platelet activation through cAMP-dependent mechanisms, while NO impairs platelet adhesion and activation, in part, by an increase in cGMP (Moncada et al., 1982; Radomski et al., 1990). The endothelium itself is also able to regulate thrombosis by degrading any prothrombotic vasoactive amines present in the blood, inactivating thrombin, and inducing expression of thrombomodulin, a thrombinbinding surface protein that facilitates thrombin-dependent activation of protein C, a naturally occurring anticoagulant that degrades factors Va and VIIIa (Esmon et al., 1993). The fluidity of the blood is also maintained by endothelial surface glycosaminoglycans that catalyze the binding of the anticoagulant serine protease inhibitors (serpins), antithrombin III and heparin cofactor II, to specific coagulation proteins, such as thrombin, thereby attenuating coagulation and platelet activation (Rosenberg et al., 1994). The endothelium also contributes to thrombus dissolution by producing molecules essential for fibrinolysis, including plasminogen activators. Fibrinolysis Plasmin, the fibrinolytic counterpart to thrombin, is the principal mediator of fibrinolysis. This enzyme is converted from its plasma zymogen plasminogen by plasminogen activators, including tissue-type plasminogen activator (t-PA) and the urokinase-type plasminogen activators, all of which are serine proteases. Plasmin, once formed, cleaves fibrin leading to the dissolution of the fibrin clot or the thrombus. The mechanism by which platelets regulate fibrinolysis has not been established. Platelets can directly bind plasminogen and t-PA, leading to enhanced activity of plasminogen (Adelman et al., 1988). Platelet granules contain a myriad of substances that can regulate the fibrinolytic response, including plasminogen activator inhibitor-1 (PAI-1), 2-antiplasmin, Cl esterase inhibitor, and 2-macroglobulin, all of which are inhibitors of fibrinolysis (Ouimet and Loscalzo, 1994) (Figure 13–3). PAI-1 is one of the most important modulators of fibrinolysis, representing 60% of the plasminogen activator inhibitory activity in plasma (Ouimet and Loscalzo, 1994). Other roles for platelets in fibrinolysis include facilitation of clot retraction,

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Figure 13-3. Molecules involved in plasminogen activation and its subsequent conversion to plasmin. Solid lines indicate activation; dashed lines indicate inhibition.

potentiation of plasminogen activation, modulation of fibrinolysis in the microenvironment by secretion of a-granule contents, and protection from factor XIIIa inactivation. Plasmin has been shown to have the ability both to activate and inhibit platelets (Niewiarowski et al., 1973; Guccione et al., 1985). High concentrations of plasmin can act as a platelet agonist, yet low concentrations of plasmin lead to inhibition of platelet aggregation. At very high concentrations, plasmin modifies the platelet GPIIIa fibrinogenbinding domain leading to impaired fibrinogen binding and platelet aggregation, thus providing an explanation for the dichotomous actions of plasmin on platelet functional responses (Pasche et al., 1991). In addition to the endothelial and fibrinolytic systems discussed above, other systems also exist to limit the extent of thrombosis. Anithrombin III is a serpin that inhibits the formation of fibrin by inhibiting thrombin as well as other coagulation factors, including factors IXa, Xa, XIa, and XIIa (Rosenberg et al., 1973). Heparin and endothelial heparan sulfate potentiate the anticoagulant actions of antithrombin III by catalyzing the binding of this serpin to these serine proteases (Bjork et al., 1976). Proteins C and S provide another mechanism of anticoagulation. Protein C is activated by the thrombomodulin-thrombin complex, and in its active form is able to inactivate factors Va and VIIIa (Clouse et al., 1986). Protein S is a cofactor for activated protein C, which has the added ability to potentiate fibrinolysis by complexing to plasminogen activator inhibitor -1 and enhancing plasminogen activation by t-PA. NITRIC OXIDE SYNTHESIS Nitric oxide synthesis occurs in both the vascular endothelium and the platelet. NO is produced when the terminal guanidino nitrogen of L-arginine undergoes a 5-electron oxidation to form L-citrulline and NO (Palmer et al., 1988; Nathan, 1992) (Figure 13–4). This reaction is catalyzed by the nitric oxide synthase (NOS) family of enzymes. There are two main isoform classes of NOS, a constitutive NOS (cNOS) and inducible NOS (iNOS, Nos2 gene product), although the distinction between these isoform classes has been recently blurred by the finding that the “constitutive” endothelial isoform (eNOS or Nos3 gene product) can be upregulated by exercise and the “inducible” isoform is constitutively expressed in the kidney (Green et al., 1996; Markewitz et al., 1993). The differences between these isoforms are shown in Table 13–1. Constititutive NOS is found in vascular endothelial cells (eNOS) as well as in neuronal cells (nNOS, No si gene product) and is regulated by Ca2+ and calmodulin. Ca2+ is released from intracellular stores when acetylcholine or bradykinin stimulates inositol 1,4,5-triphosphate (IP3) binding to endoplasmic reticulum receptors with the subsequent release of intracellular Ca2+. This increase in Ca2+ leads to its binding to calmodulin, which then binds to and activates cNOS (Dinerman et al., 1993; Bredt et al., 1990; Busse et al.,

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Table 13±1. Characterization of inducible a nd constitutive nitric acid synthases.

Figure 13±4. Diagram of nitric oxide synthesis by nitric oxide synthase (NOS).

1990; Mayer et al., 1989). The production of NO by cNOS is, therefore, rapid, transient, and continuous until the Ca2+ level returns to baseline. Inducible NOS has been found in many cell types including the macrophage, and the neutrophil (Marietta et al., 1988; Yui et al., 1991). Inducible NOS is stimulated by exposure to bacterial endotoxin or cytokines (Drapier et al., 1988; Stuehr et al., 1987; Ding et al., 1988), and its activity is not dependent upon Ca2+ concentration, but, rather, is regulated at the transcriptional level (Xie et al., 1992). The production of NO by iNOS is delayed in comparison to production by cNOS, but the amounts of NO generated and the duration of its production by this isoform far exceed those of cNOS. The mechanism of activation of NO synthase in the endothelial cell has not been elucidated. Since the constitutive form of NO synthase is Ca2+-dependent, it has been postulated that this cation is responsible for the activation of NO synthase in vivo (Xie et al., 1992). The process of endothelial activation may lead to activation of Ca2+ channels leading to an increase in intracellular calcium, thereby regulating expression of NO synthase and NO production.

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NITRIC OXIDE AND GUANYLYL CYCLASE The enzyme guanylyl cyclase has been shown to mediate, in part, the platelet inhibitory actions of NO. NO binds to the heme moiety of guanyly cyclase and induces a conformational change that displaces the iron out of the plane of the porphyrin ring (Ignarro, 1986). This effect results in the enzymatic conversion of guanosine 5'-triphosphate to cyclic guanosine 3',5'-monophosphate (cGMP) with concomitant stimulation of cGMP-dependent protein kinase. Cyclic GMP-dependent protein kinase phosphorylates intracellular enzyme targets that are responsible for regulation of intracellular calcium levels. Regulation of calcium level in platelets has been suggested as one mechanism of action of guanylyl cyclase. The mechanisms of action of cGMP-dependent protein kinase are many, including inhibition of platelet fibrinogen binding to the GPIIb/IIIa receptor, inhibition of phosphorylation of myosin light chains and of protein kinase C, and stimulation of phospholipase A2- and C-mediated responses (Radomski et al., 1991a,b). Cyclic GMP itself inhibits receptor-mediated calcium influx in platelets. An increase in soluble guanylyl cyclase leads to a reduction of intracellular Ca2+ in platelets through cGMP-mediated inhibition of Ca2+ release from intracellular stores, an increased rate of Ca2+ extrusion, and decreased Ca2+ entry from extracellular stores (Nakashima et al., 1986; Matsuoki et al., 1989; Morgan and Newby, 1989; Geiger et al., 1992; Johansson and Hayness, 1992). An inhibitor of guanylyl cyclase, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin1-one, blocks these cGMP-dependent effects resulting in an increase in platelet aggregation (Moro et al., 1996). Inhibition of cGMP-mediated platelet responses can also occur through cyclic nucleotide phosphodiesterases, which can degrade cGMP. Radomski and colleagues showed that M+B 22984, a cGMP phosphodiesterase inhibitor, can potentiate L-arginine mediated anti-platelet effects (Radomski et al., 1990). PLATELETS AND NITRIC OXIDE Nitric oxide synthase isoforms exist in human platelets (Muruganandam et al., 1994). An eNOS-like isoform has been isolated from platelets and appears to require similar co-factors as does eNOS itself. The molecular size of this constitutively expressed NOS was found to be 80 kDa in contrast to the 130-kDa protein found in endothelial cells, which could represent either differential splicing of the Nos3-like transcript or be the result of post-translational processing. An isoform of NOS homologous with iNOS has also been characterized in platelets. Since the platelet is anucleate with a minimal pool of mRNA (Djaffar et al., 1991), and thus able to synthesize only modest amounts of protein, most platelet proteins are derived from its precursor cell, the megakaryocyte. The megakaryocyte expresses both constitutive and inducible NOSs (Lelchuk et al., 1992). Release of NO from the platelet has recently been established (Zhou et al., 1995). Stimulation of NO synthase, dependent upon L-arginine, leads to activation of soluble guanylyl cyclase and, thereby, increases cGMP, with concomitant inhibition of platelet aggregation (Radomski et al., 1990). Freedman and colleagues have recently demonstrated that NO can also inhibit platelet recruitment to a growing platelet thrombus (Freedman et al., 1997). L-arginine has been shown to inhibit platelet aggregation in vivo and in vitro. A single, saturable, sodium-independent transporter system for movement of L-arginine into human platelets has recently been postulated (Vasta et al., 1995). Radomski (1990) showed that L-arginine inhibited platelet aggregation induced by ADP and arachidonic acid. Yet, inhibition of thrombin-induced aggregation occurred only when other platelet antagonists were present, including prostacyclin and cGMP phosphodiesterase inhibitors. The mechanism underlying NO’s ability to regulate platelet aggregation is currently under investigation. Platelet agonists induce aggregation by increasing intracellular calcium levels via stimulation of the inositol

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3-phosphate pathway. NO’s ability to regulate intracellular calcium levels through cGMP and cGMPdependent protein kinase has, therefore, been postulated as its mechanism of action, as described above. Recently, a molecular mechanism of how NO may modulate platelet aggregability via cGMP has been proposed. Reep and colleagues have suggested that phosphorylation of the signaling molecule rap 1 in platelets is induced by NO stimulation of guanylyl cyclase and activation of cGMP-dependent protein kinase (Reep et al., 1996). They showed that incubation with S-nitroso serum albumin leads to inhibition of collagen-stimulated platelet aggregation that was correlated with phosphorylation of rap 1 in a dosedependent manner. These data suggest that cGMP leads to activation of a signaling cascade within the platelet that results in modulation of platelet aggregation. It appears that NO released from both endothelial cells and platelets regulates the platelet response and prevents thrombus formation. NO from endothelial cells suppresses platelet adhesion and aggregation. When stimulated with bradykinin, endothelial cells release NO in amounts sufficient to inhibit platelet adhesion, as is the case when NO is released under constant flow conditions in coronary and pulmonary vessels (Venturini et al., 1989; Pohl et al., 1989). Endothelial NO decreases aggregation of platelets immediately downstream from where it is released, suggesting that the endothelium is responsible for regulating platelets in its vicinity as opposed to platelets at a distance from the endothelial source of NO (De Graaf et al., 1992). Release of endothelial NO in vivo by cholinergic stimulation and by substance P leads to inhibition of platelet aggregation induced by collagen or ADP (Humphries et al., 1990). NO from the endothelial cell has also been shown to have the ability to disaggregate preformed platelet aggregates (Radomski et al., 1987a). One of the mechanisms by which endothelial NO can regulate platelet function has recently been elucidated by Murohara and colleagues (1995). P selectin is normally present in the Weibel-Palade bodies found in endothelial cells and also in the -granules of platelets. Once the endothelial cell is activated, the granules fuse with the plasma membrane and P selectin is rapidly translocated to the extracellular surface. NO synthase inhibitors, such as L-NAME, induce endothelial P-selectin expression on the endothelial cell surface in the rat mesenteric circulation in vivo. This suggests that NO regulates adhesion of platelets to the endothelium by decreasing expression of the P selectin needed for initial attachment of the platelet to the endothelium. Michelson and colleagues have shown that S-nitroso-N-acetylcysteine can markedly inhibit upregulation of P-selectin on the platelet surface (Michelson et al., 1996). Murohara and colleagues elucidated the mechanism of P-selectin expression by suggesting that thrombin stimulation leads to an increase in P-selectin on the surface of platelets through the activation of phosphoinositol-specific phospholipase C (PLC), which leads to an increase in intracellular Ca2+ and activation of phosphorylation cascades within the platelet. They have shown that phorbol myristylacetate (PMA), a PKC activator, stimulates rapid P-selectin expression. Also, N,N,N-trimethylsphingosine, an inhibitor of PKC, has been shown to inhibit P-selectin expression on thrombin-stimulated cells. Since it has also been shown that NO and cGMP inhibit PKC activity, the authors postulated that NO may regulate PKC through phosphorylation by a cGMP-dependent kinase and, therefore, leads to down regulation of P-selectin expression on the surface of both platelets and endothelial cells. Other blood-borne cell types have also been found to release NO, possibly augmenting the vascular NO pool provided by the platelet and the endothelial cell. The contribution of NO released from neutrophils in the regulation of platelet function in vivo has not been established; however, it has been shown that human neutrophils and mononuclear cells release a factor that has a similar pharmacological profile as NO with the ability to inhibit platelet aggregation (Salvemini et al., 1989). The neutrophil itself can be regulated by NO, with NO inhibiting adhesion and chemotaxis of stimulated cells. (Kubes, 1991; Moilanen, 1993).

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Effect of S-Nitrosothiols on Platelet Aggregation NO reacts with thiols in the presence of oxygen to produce S-nitrosothiols. In plasma, S-nitrosothiols are the primary redox state of NO (Stamler et al., 1992). S-nitrosothiols have been suggested to be a more stable source of NO in plasma, in contrast to free NO, and, therefore, S-nitrosothiols have been postulated to be a storage pool of NO. S-nitrosothiols activate platelet soluble guanylyl cyclase and elevate cGMP levels, leading to inhibition of platelet aggregation (Mellion et al., 1985). Loscalzo and colleagues showed that nitroglycerin and nitroprusside can inhibit platelet aggregation at pharmacologically achievable concentrations in vivo and this effect is potentiated by thiols (Loscalzo and Welch, 1995). Mendelsohn and colleagues showed that S-nitroso-N-acetylcysteine, an S-nitrosothiol compound, decreased the intracellular calcium flux in response to ADP stimulation leading to inhibition of fibrinogen binding in activated platelets, an effect accompanied by a rise in intracellular cGMP (Mendelsohn et al., 1990). Keaney and colleagues showed that endogenous NO can react with serum albumin to form S-nitroso-albumin, which was shown to have antiplatelet properties (Keaney et al., 1993). Simon and colleagues bolstered the importance of S-nitrosothiols in platelet function by showing that the platelet surface is able to facilitate the release of NO from S-nitrosothiols (Simon et al., 1993). Synergy of NO with other Platelet Inhibitors Other factors produced by the endothelial cell and the platelet work in concert with NO to potentiate inhibition of platelet activation and aggregation. Some of these factors include PGI2, PGD2, and t-PA (Radomski and Moncada, 1991a,b; Loscalzo, and Vaughn, 1987). It appears, however, that no single factor is able to limit the activated-platelet response; rather the synergy of several factors is essential for limiting platelet aggregation optimally. Prostacyclin synthesis by endothelial cells is stimulated by mechanical injury, PDGF, and bradykinin (Gerrard and White, 1982). Prostacyclin, a potent inhibitor of aggregation, is a very weak inhibitor of platelet adhesion, and NO does not synergize with prostacyclin as an inhibitor of adhesion. Prostacyclin’s ability to limit platelet adhesion appears to be regulated by cGMP-dependent responses as opposed to its usual mechanism of action through cAMP-dependent responses. Radomski and colleagues have shown that NO and PGI2 do not act synergistically to inhibit adhesion since there is no antiadhesive actions of PGI2 (Radomski et al., 1987a). In the clinical realm, inhibition of NO production in vivo decreases the ability of aspirin to attenuate platelet aggregation (Golino et al., 1992). NO and prostacyclin appear to synergize with each other through an NO-induced increase in cGMP with subsequent inhibition of cGMP-inhibited cAMP phosphodiesterase (PDE), resulting in potentiation of prostacyclin’s cAMPmediated antiplatelet effects (Maurice and Haslam, 1990). The actions of NO can also be potentiated by protective mechanisms within the platelet. Superoxide anions produced by damaged endothelial cells, leukocytes, and smooth muscle cells can inactivate NO through the formation of peroxynitrite, which is further metabolized to nitrite and nitrate. Peroxynitrite is very highly reactive and able to cause cell toxicity through lipid peroxidation and nitrosation of protein tyrosyl residues. Moro has demonstrated that peroxynitrite stimulates platelet aggregation and limits NO and prostacyclin’s ability to impair aggregation (1994). Superoxide dismutase (SOD), an enzyme which catalyzes the dismutation of Superoxide into water and hydrogen peroxide, can protect NO from oxidative inactivation by superoxide. SOD potentiates the ability of NO to prevent adhesion and aggregation of platelets induced by thrombin in vivo. Administration of superoxide dismutase to damaged and stenotic carotid arteries leads to a decrease in thrombus formation at the site of vascular damage (Meng et al, 1995).

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THROMBOSIS AND NO DEFICIENCY Decreased production of NO can lead to thrombosis. Clinical evidence substantiates the antiplatelet role of NO. Endothelial damage induced by laser injury leads to inhibition of NO release and, consequently, platelet aggregation (Rosenblum et al., 1987). Nishimura and colleagues showed that nitric oxide synthase blockade in vivo enhances the accumulation of activated platelets in areas of endothelial damage within brain pial arterioles (Nishimura et al., 1991). L-Nitroarginine methyl ester (L-NAME), an inhibitor of NO synthesis, potentiates the pulmonary accumulation and prolongs the disaggregation of [111In]-labeled platelets induced by ADP, PAF, and thrombin in vivo (Radomski and Moncada, 1991a,b). In experimental coronary stenosis in rabbits, massive platelet adhesion and formation of aggregates on the surface of the damaged endothelium was observed in those animals treated with NG-monomethyl-L-arginine (L-NMMA), another inhibitor of nitric oxide synthesis, prior to the production of the stenosis (Herbaczynska-Cedro et al., 1991). Folts and colleagues used a canine model of coronary artery stenosis to demonstrate that intravenous nitroglycerin, an organic nitrate, can improve ischemia associated with acute coronary artery disease by inhibition of platelet thrombus formation (Folts et al., 1991). In addition, the antithrombotic effects of nitrates are potentiated by pretreatment of the animals with reduced thiols such as N-acetylcysteine. Impaired NO synthesis has been demonstrated in hypertension (Cadwgan and Benjamin, 1991) and in diabetes mellitus (Calver et al., 1992), and this deficiency could be involved in the thrombotic complications that occur in these disease processes. In addition, studies with inhibitors of NOS have shown that decreased generation of NO in vivo can lead to platelet and polymorphonuclear leukocyte activation and thrombosis (May et al., 1991; Herbaczynska-Cedro et al., 1991; Golino et al., 1992; Yao et al., 1992). When L-NMMA was given to healthy subjects and platelet function monitored in vivo, hemostasis was disturbed and platelets became activated as measured by the increase in plasma levels of markers of platelet activation, including -thromboglobulin and platelet factor 4 (Bodzenta-Lukaszyk,1994). We recently studied two brothers with a cerebral thrombotic disorder and found that their platelets could not be inhibited by NO. The activity of the antioxidant enzyme, plasma glutathione peroxidase, was found to be decreased in the patients’ plasma, accounting for their insensitivity to NO. Addition of exogenous glutathione peroxidase led to restoration of platelet inhibition by NO. The mechanism of action of glutathione peroxidase involves reduction of lipid hydroperoxides to their corresponding alcohols. Granule secretion following platelet activation results in release of eicosanoids, including lipid hydroperoxides, which are then reduced by glutathione peroxidase (Freedman et al., 1995). Impaired metabolism of lipid hydroperoxides, as occurs when glutathione peroxidase activity is decreased, can lead to an increase in hydroperoxyl radical concentration which can, in turn, react with and reduce bioactive NO. These reactions thus impair the ability of NO to inhibit platelet activation, leading to a thrombotic propensity (Freedman et al., 1996). Previously, Freedman and colleagues had shown that glutathione peroxidase can potentiate the ability of the naturally occurring S-nitrosothiol, S-nitroso-glutathione, to inhibit platelets (Freedman et al., 1995). NO AND ATHEROTHROMBOSIS Thrombosis is a major determinant of atherosclerosis. In humans and animals with atherosclerosis, the ability of the endothelium to synthesize bioactive NO is greatly reduced (Chester et al., 1990). Impaired NO production may promote atherosclerosis by several mechanisms. Mitogens, like PDGF, have been postulated to be responsible for the proliferation of smooth muscle cells within atherosclerotic lesions (Radomski and Moncada, 1991a,b). Mitogen activity can be reduced by suppressing their release from

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stimulated platelets, an effect supported by NO. Administration of L-arginine, the substrate for NOS, was found to restore impaired production of NO, decrease activation of platelets and leukocytes, and limit the extent of the vascular lesions associated with this disease in an animal model of hypercholesterolemia (Dexter et al., 1991; Cooke and Tsao, 1992). Vascular injury occurs when balloon angioplasty is performed to treat occlusive arterial lesions. Angioplasty results in endothelial denudation and varying degrees of smooth muscle cell injury. Normally, endothelial injury exposes the procoagulant molecules located in the subendothelium, leading to platelet adhesion, aggregation, and thrombosis. Marks and colleagues found that local administration of a relatively stable S-nitrosothiol, polyS-nitroso-bovine serum albumin, can inhibit intimai proliferation and platelet deposition after denuding arterial injury (Marks et al., 1995). Another group used balloon injury to denude the endothelium of rat carotid arteries, thereby limiting the ability of the endothe lium to produce NO (von der Leyen et al., 1995). They restored NO production by using gene transfer techniques to deliver the Nos3 gene to the site of vascular injury, leading to inhibition of neointimal lesion formation. In coronary bypass grafting, either an artery or a vein may be used as a new conduit. It has been suggested that more effective release of NO by arterial vessels may be responsible for greater patency of arterial grafts than venous bypass grafts (Luscher et al., 1989). Production of NO is stimulated by ADP and other agents in the internal mammary artery but not in the saphenous vein. Thus, the lower NO synthesis rate in the saphenous vein coronary bypass grafts may explain the patency differences. Atherosclerotic plaques have been demonstrated to contain the cytokines, IL-1 and TNF- (Arbustini et al., 1991). These cytokines can induce iNOS in cells within the plaque, so that they may be partly involved in the balance between prothrombotic and antithrombotic processes by this mechanism. In injured arteries, vascular smooth muscle cell iNOS is rapidly induced within one day of injury. This leads to inhibition of platelet adhesion and maintenance of blood flow in the injured vessel (Yan et al., 1996). Yet, production of NO via iNOS has also been shown to produce cellular toxicity via peroxynitrite generation. Peroxynitrite, which decreases the bioavailability of NO, has been observed to produce nitrostyrosine at sites of endothelial injury in atherosclerotic lesions (Kooy et al., 1994). Lipoproteins that may be present in atherosclerotic lesions can also regulate NO activity. Low-density lipoprotein (LDL) can decrease the bioactivity of NO by an unknown mechanism (Flavahan et al., 1992). Possible explanations for this decreased activity include inhibition of NOS activity by LDL, inactivation of NO itself, or changes in NO metabolism. In addition, LDL may decrease the uptake of L-arginine into platelets, and this may lead to a decrease in the NOS activity, thereby promoting thrombosis (Chen et al., 1994). The presence of high-density lipoproteins (HDL), however, is beneficial since HDL decreases platelet activitity by increasing NOS activity in platelets. NO AND BLEEDING DIATHESES Lipopolysaccharide (LPS or endotoxin) is responsible for the development of septic shock associated with bacterial infection. Severe hypotension and disturbances in the hemostatic balance are among the major clinical symptoms of septicemic reactions. The bleeding diathesis in sepsis is believed to be caused by disseminated intravascular coagulation with consumption coagulopathy, fibrinolysis, and thrombocytopenia (Radomski and Moncada, 1991a,b). Stimulation of endothelial cells with LPS and interferon- , a cytokine produced during inflammatory reactions, results in expression of iNOS. This enzyme is responsible for producing large amounts of NO which are sufficient to inhibit platelet aggregation. Shultz and Raij (1992) reported that LPS can induce production of endogenous NO, which is critical for preventing LPS-induced platelet-dependent renal thrombosis. Another consequence of septic shock that is due to upregulation of

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NOS enzymes is hypotension. Keaney and colleagues used methylene blue—an inhibitor of guanylyl cyclase and of NOS, and also a direct NO oxidant—to reduce NO production and restore mean arterial pressure in a rabbit model of LPS-induced hypotension (Keaney et al., 1994). Bleeding complications that accompany endotoxemia may, thus, result from upregulated expression of iNOS in vascular cells and leukocytes. Hemostatic defects, including prolonged bleeding times and decreased platelet adhesion and aggregation, also occur in uremia. In uremia, metabolites of the urea cycle accumulate, including L-arginine, which may account for the increased NO production in these patients (Radomski and Salas, 1995). Bode-Boger and colleagues showed that L-arginine increases the excretion of NO byproducts, including nitrate. They demonstrated that the addition of L-arginine to the diets of hypercholesterolemic rabbits increases the ratio of L-arginine to the endogenous NO synthase inhibitor assymetric dimethylarginine (ADMA) thereby restoring urinary nitrate excretion (Bode-Boger et al., 1996). It has been shown that the prolonged bleeding times in uremie rats can be normalized by systemic administration of L-NMMA. NO donors activate the plasma fibrinolytic system in peripheral vascular disease patients by inhibiting the release of PAI-1 (Gryglewski, 1993). Gryglewski reported that PGI2 synergizes with the NO donor molsidomine to increase the activity of the fibrinolytic system in patients with peripheral vascular disease. This cooperative effect is attributed to PGl2’s ability to release t-PA from cells coupled with the NO donor’s ability to inhibit release of the antifibrinolytic molecule, PAI-1. This work has been substantiated by the results of Korbut and colleagues, who showed that SIN-1, an NO donor, can also inhibit release of PAI-1 from platelets (Korbut et al., 1993). Inhalation of NO results in an increase in the template bleeding time (Hogman et al., 1993). Simon and colleagues (Simon et al., 1995) found that inhibition of NO production by L-NMMA shortens the bleeding time modestly. In vitro, the effects of NO were significantly different, resulting in a significant decrease in platelet activation or aggregation. PHARMACOLOGICAL ROLE OF NO DONORS IN VASCULAR THROMBOSIS Organic nitrates have been used for over a century in the treatment of cardiovascular disease. The potential benefit of nitroglycerin as an inhibitor of platelet aggregation was first observed in 1967 by Hampton (Loscalzo and Welch, 1995). Organic nitrates by themselves are weak inhibitors of aggregation in vitro; however, following ADP stimulation, nitroglycerin has been shown to be a potent inhibitor of aggregation ex vivo (Chirkov et al., 1993). Intravenous administration of nitroglycerin and isosorbide mononitrates can lead to inhibition of the platelet activation (Wallen et al., 1994); these agents have also been shown to inhibit thrombosis in animals (Werns et al., 1994; Plotkine et al., 1991). Patients suffering from myocardial infarction and treated with nitroglycerin show a decrease in platelet adhesion and aggregation in comparison to patients not given this agent (Diodati et al., 1990). Kuritzky showed that nitroglycerin can disaggregate platelet-rich thrombi in the retinal circulation (Kuritzky et al., 1984). The effectiveness of the organic nitrates correlates with the extent of vascular injury. An animal model in which arterial injury is induced in pigs by balloon angioplasty showed increased inhibition of platelet deposition in the injured area after treatment with nitroglycerin in comparison to pigs with milder injury treated with nitroglycerin (Lam et al., 1988). Loscalzo showed that nitroglycerin requires intracellular reduced thiols in its mechanism of platelet inhibition (Loscalzo, 1985). The requirement of thiols as potentiators of organic nitrates may also be beneficial for limiting or targetting the effects of NO. It would be useful to inhibit platelet aggregation using organic nitrates without evoking the deleterious effects that NO has on blood pressure. Radomski and

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colleagues have shown that S-nitrosoglutathione is able to inhibit platelet aggregation without altering blood pressure in rats (Radomski et al., 1992). Similar results have been obtained by intraarterial injections of Snitrosoglutathione (de Belder et al., 1994). Organic nitrites may also affect preformed platelet aggregates. Stamler showed that nitrogylcerin can disaggregate platelet aggregates (Stamler et al., 1989). Others have shown that nitroglycerin can reduce platelet adhesion to injured vessels (Lam et al., 1988). NO donors that do not require metabolic breakdown include sodium nitroprusside, SIN1, molsidomine, and S-nitroso-N-acetylpenicillamine (SNAP). Nitroprusside has been shown to inhibit platelet aggregation in vivo and in vitro (Levin et al., 1982). Molsidomine and SIN-1 lead to inhibition of thrombosis in jugular and mesenteric venous systems in patients suffering from myocardial infarction and controls (Wautier et al., 1989). Molsidomine and isosorbide dinitrate synergize with prostacyclin and prostaglandin E1 to inhibit platelet activation (Sinzinger et al., 1992). SNAP has been shown to inhibit prostaglandin synthesis by inhibiting lipooxygenase, a key enzyme for arachidonic acid metabolism to oxidized eicosanoid derivatives in human platelets (Maccarrone et al., 1996) The beneficial properties of aspirin in limiting platelet aggregation and thrombosis have been well established (Patrono, 1994). In an attempt to improve upon the antiplatelet actions of aspirin, while limiting the negative gastric mucosal effects that occur with prolonged treatment, Wallace and colleagues studied the activity of a nitric oxide-releasing, gastricsparing aspirin derivative. The nitroxybutylester derivative of aspirin (NCX 4215) was seven times more potent than aspirin in limiting platelet aggregation in vitro without inducing gastric mucosal injury. NCX 4215 was found to release NO in the presence of platelets and to increase platelet levels of cGMP (Wallace et al., 1995). CONCLUSIONS In this chapter we have reviewed the importance of nitric oxide in regulating hemostasis. Our understanding of the role of NO in the hemostatic system has dramatically increased in this past decade. Advances in knowledge of the molecular mechanisms by which NO modulates the hemostatic response will lead to the development of novel therapeutic strategies to prevent the pathological processes of NO-deficient thrombotic disorders and NO-dependent bleeding diatheses. ACKNOWLEDGMENTS The authors wish to thank Stephanie Tribuna for her help in the preparation of this manuscript. REFERENCES Adelman, B., Rizk, A. and Manners, E. (1988) Plasminogen interactions with platelets in plasma. Blood, 72, 1530–1535. Arbustini, E., Grasso, M, Diegli, M. and Pucci, A. (1991) Coronary atherosclerotic plaques with and without thrombus in ischaemic heart syndromes: a morphologic, immunohistochemical and biochemical study. American Journal of Cardiology, 68, 36B–149B. Bjork, I. and Nordenman, B. (1976) Acceleration of the reaction between thrombin and antithrombin III by nonstoichiometric amounts of heparin. European Journal of Biochemistry, 68, 507–511. Bode-Boger, S.M., Boger, R.H., Krenke, S., Junher, W. and Frolich, J.C. (1996) Elevated L-arginine/ dimethylarginine ratio contributes to enhanced NO production by dietary L-arginine in hypercholesterolemic rabbits. Biochemical and Biophysical Research Communications, 219, 598–603.

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14 Hypertension Eduardo Nava1 and Thomas F.Lüscher2 1

Department of Physiology, University of Murcia School of Medicine, Murcia, Spain. 2Division

of Cardiology, University Hospital, Zürich, Switzerland

The endogenous release of nitric oxide (NO) contributes to the maintenance of blood pressure and this, in turn, is a stimulus for NO production. The role of NO in hypertension is very controversial. Recent research work is showing the existence of profound differences in the role of NO depending on the model of hypertension. In genetic and renovascular hypertension, the production of NO is increased probably as a compensatory mechanism against the overproduction of different vasoconstrictors. In genetic hypertension, however, the bioactivity of NO is diminished. In salt-sensitive hypertension, NO production is impaired probably due to a deficiency of the substrate for NO synthase. In human essential hypertension, pharmacological experiments reveal an impaired NO dilator mechanism. NITRIC OXIDE AND THE REGULATION OF BLOOD PRESSURE Since its discovery, an increasing body of evidence shows that NO is a widespread biological mediator implicated in many physiological and pathophysiological processes (Moncada and Higgs, 1993). Nitric oxide is synthesized from L-arginine by a family of enzymes called NO synthases (Figure 14–1). One isoform of these is a Ca++-dependent enzyme (endothelial NO synthase, eNOS) which is constitutively expressed in endothelial cells (Busse and Mülsch, 1990) and plays a crucial role in the regulation of blood pressure and vascular tone (Rees et al., 1989; Vallance et al., 1989). Definite proof of the role of NO in the maintenance of blood pressure is the fact that mice lacking the gene for eNOS develop hypertension (Huang et al., 1995). Inhibitors of NO production cause endothelium-dependent contractions of isolated arteries, decrease blood flow and induce pronounced and sustained hypertension when infused intravenously or

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Figure 14±1. The L-arginine/NO pathway in the blood vessel wall. sGC=soluble guanylate cyclase; cGMP =cyclic 3',5'guanosine monophosphate; LPS=lipopolysaccharide; TNF=tumor necrosis factor; IL-1= interleukin Ib; cNOS/ iNOS=constitutive/inducible NO synthase; ADMA=asymmetrical dimethylarginine; L-NMMA=monomethyl-Larginine; L-NAME=nitro-L-arginine methylester.

given orally in vivo (Rees et al, 1989, 1991; Vallance et al., 1989; Takase et al., 1994). Thus, the cardiovascular system is exposed to a continuous NO-dependent vasodilator tone and withdrawal of it mimics many features of human hypertension including target organ damage (Palmer et al., 1992; Blot et al., 1994). In situations of NO deprivation, which can be simulated by treating animals chronically with NO synthesis inhibitors, the endothelium can replace the functional abilities of NO provided antihypertensive treatment is supplied (Takase et al., 1996). This is probably because the endothelium increases the release of alternative relaxing factors which in normal conditions are not present. The activity of eNOS is modulated by shear stress on the endothelial surface and by a variety of receptoractivated agonists as well as hormones (Sessa et al., 1994; Schmidt et al., 1990; Weiner et al., 1994). It is not totally clear, however, whether variations in blood pressure per se can affect the release of endotheliumderived NO. Kelm and colleagues have suggested that only fluctuations in blood flow, but not in blood pressure can stimulate eNOS (Kelm et al., 1991). Nevertheless, studies performed in our lab demonstrate that acute, pharmacologically-induced, drops in blood pressure cause NO release to decrease (Figure 14–2,

Correspondence: Thomas F.Lüscher, M.D., Professor of Medicine, Cardiology University Hospital, CH-8091 Zürich, Switzerland. Tel: +41 1 255 2121; Fax: +41 1 255 4251.

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Figure 14±2. Effects of lowering blood pressure with a continuous infusion of prostacyclin or adenosine on the plasma concentration of nitrate. The decrease in plasma nitrate suggests a downregulation of NO production when blood pressure diminishes, (from Nava et al. (1996c), by permission).

Nava et al., 1996c) and, on the contrary, elevations in pressure are followed by an increased production (Figure 14–3, Nava et al., 1994), suggesting that high blood pressure upregulates NO production and vice versa. The kidney plays a pivotal role in the long-term regulation of blood pressure. A large number of studies evidence the capacity of the kidney to produce NO and its relevant role in renal function (Salazar et al., 1993; Cowley et al., 1995; Biondi et al., 1992; Fenoy et al., 1995). It has been demonstrated that the kidney is very sensitive to the reduction of NO as low doses of NOS inhibitors reduce sodium and water excretion without affecting renal hemodynamics or systemic arterial pressure (Salazar et al., 1993). Within the kidney, the medulla, which is importantly involved in pressure-induced natriuresis (Cowley et al., 1992), produces most of the NO-dependent cyclic GMP (Biondi et al., 1992). There are increasing grounds for believing that NO participates in the control of renal medullary blood flow. Firstly, NOS inhibitors decrease medullary, but not cortical blood flow, when selec lively applied in the medullary interstitial space and this is associated with sodium retention and development of hypertension (Cowley et al., 1995). Also, treatment with these inhibitors blunts the pressure-natriuresis response by impairing the renal medullary vasodilation produced by increasing arterial pressure (Fenoy et al., 1995). We have recently presented evidence showing that Ca++-dependent NOS activity is considerably higher in the renal medulla than in the renal cortex and that the activity in the renal medulla is also much higher than in the heart and aorta (Figure 14–4, Nava et al., 1996a). These results indicate that the renal medulla has a greater potential to produce NO compared to the renal cortex or cardiovascular tissues involved in blood pressure regulation. Because NO appears to be highly relevant for renal function it is conceivable that small alterations in the renal NO production, which do not affect systemic vascular tone, lead to hypertension because of an impaired regulation of sodium and water excretion (see part 4).

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Figure 14±3. Effects of angiotensin II (3 ug kg−1min-1, solid lines),, substance P (3 ug kg−1min−1, dotted lines),, and prostacyclin (0.3–0.6 mg kg−1min−11, dashed lines) on blood pressure (closed symbols) and plasma nitrate (open symbols). Plasma nitrate concentration follows the changes in blood pressure caused by angiotensin II and prostacyclin. For substance P, however, there is an inverse relation between blood pressure and plasma nitrate. * statistically significant (p <0.05) compared to basal levels (from Vava et al. (1994), by permission).

NITRIC OXIDE IN SPONTANEOUS HYPERTENSION The SHR is a widely used model of genetic hypertension (Kurtz et al., 1994). The capacity of SHR vessels to relax has been a matter of controversy long before endothelium-derived relaxing factor (EDRF) was known. Spector et al. (1996) showed that the SHR vessels relax better than their normotensive counterparts from WKY rats. In contrast, Cohen and Berkowitz (1976) found relaxations to the same agonists diminished in the SHR. The role of the endothelium in the SHR has shown to be quite peculiar from the very initial studies after EDRF was described. Endothelium-dependent responses to pharmacological (Konishi et al., 1983; Lüscher et al., 1986; Tesfamariam et al., 1998; Diederich et al., 1990; Li and Joshua 1993) or mechanical stimuli (Koller and Huang, 1994; 1995) are often, but not always (Angus et al., 1992; Li and Bukoski, 1993) found defective. It has been suggested that this impairment could be due to a diminished smooth muscle ability to relax (Cohen and Berkowitz, 1976; Konishi and Su, 1983), to an excessive production of prostanoidcontractile factor(s) rather than a depressed release of relaxing factors (Lüscher and Vanhoutte, 1986; Diederich et al., 1990) or to a decrease in endothelium-derived hyper-polarizing factor (Hayakawa et al., 1993). Therefore, the role of NO in this form of hypertension is unclear and the numerous existing reports are often contradictory. For example, in vitro studies using NOS inhibitors to test the ability of the endothelium to generate NO have shown that the SHR produces less NO (Li and Joshua, 1993; GilLongo et al., 1996). However, in vivo studies display a variety of results: less NO production (Schleiffer et al., 1991), equal production (Yamazaki et al., 1991) and more production (Gil-Longo et al., 1996; Lacolley et al., 1991). Measurements of the stimulated release of NO by assessment of the depressor responses to endothelium-dependent vasodilators also confront the in vitro results (Schleiffer et al., 1991; Yamazaki et al., 1991). Direct measurements of NO or NO-related substances has led to no definitive conclusion. Research works done in the heart suggest an increased production of NO in the SHR (Kelm et al., 1995;

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Figure 14±4. Ca2+-dependent NO synthase (NOS) activity in the kidney (a) and the heart and aorta (b) in normotensive Wistar-Kyoto rats (WKY, white bars) and spontaneously hypertensive rats (SHR, black bars). In both strains of rats the activity was much higher in the renal medulla. In the medulla and heart of the SHR the activity was significantly higher than in the same tissues of the WKY. *p<0.05 compared to the same kind of tissue of the WKY. (From Nava et al. (1996a), by permission).

Nava et al., 1995b). The kidney of these rats may release normal (Hayakawa et al., 1993) or increased amounts of NO (Nava et al., 1996a). Cultured endothelial cells from SHR appear to generate less NO (Malinski et al., 1993) and the aortic endothelium shows a rarefied staining of eNOS antibodies that worsens with ageing (Cuevas et al., 1996). On the contrary, Sawada et al. find changes in serum concentration of NO metabolites upon stimulation with acetylcholine unaltered in the SHR (Sawada et al., 1994). Smooth muscle cyclic GMP has been reported to be increased (Papapetropoulos et al., 1994), normal (Arnal et al., 1993), diminished (Amer et al., 1988) or both depending on the vessel studied (Amer et al., 1974). Table 14–1 resumes the different findings on the biology of NO in the SHR. The biology of the endothelium in the SHR is indeed very intriguing and our research has been devoted to this model of hypertension for many years. In the late eighties we demonstrated that the impaired relaxations of aortic segments from SHR are not caused by a deterred production of EDRF but instead, by an increased release of prostanoid contractile factors (Figure 14–5, Lüscher and Vanhoutte, 1986, 1988). With the advent of the NO age and using new methodologies we have confirmed the initial observations on the release of EDRF in this form of hypertension. NOS is indeed upregulated in the heart (Nava et al., 1995b), kidney (Nava et al., 1996a) and mesenteric resistance arteries (Nava et al., 1995a) obtained from SHR. Moreover, the concentration of the oxidative product of NO, nitrate, is higher in hypertensive rats as compared to normotensive controls (Nava et al., 1995a). Thus, the basal release of NO is increased in rats with spontaneous hypertension. Hayakawa et al., (1993) have confirmed our findings: they report that, although the vasoactive effects of acetylcholine in the kidney are actually abnormal, the measured release of NO from renal vessels is not diminished but slightly higher in the SHR. Treatment with inhibitors of the endothelium-

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Table 14±1. Status of the endothelial function in a wide variety of studies and experimental approaches found over the years. P = pharmacological study; A = analytical study of NO or NO-related substances; vt = in vitro study; vv = in vivo study; A = augmented; N = normal; D = diminished.

derived hyperpolarizing factor (EDHF) bring the vasoactive effects of acetylcholine to a normal status, suggesting that, in the SHR, an increased EDHF production, but not a decreased NO, occurs (Hayakawa et al., 1993). Further studies carried out in our lab have demonstrated that the accumulation of cyclic GMP in the heart and mesenteric resistance arteries is similar in WKY and SHR (Nava et al., 1995a). Moreover, the NOdependent vasodilator tone, assessed by the blood pressure effects of L-NAME, is not higher in hypertensive rats as would be expected in a situation in which the production of NO is increased. Thus, endogenously-produced NO, otherwise increased in SHR, is inefficacious in this condition. Probably an additional event takes place that prevents NO to accomplish its normal hemodynamic functions. The hypertrophied and fibrotic intimal layer of hypertensive vessels (Lindop et al., 1994) may represent a physical barrier for NO accounting for the blunted hemodynamic actions of NO. Also the chemical environment that NO encounters, such as oxidative stress can determine its fate as the results from Nakazono et al. (1991) and Tschudi et al. (1996) suggest. The mechanism by which high blood pressure leads to an increased production of NO is not clear yet. It is known that the release of NO by endothelial cells can be altered by changes in shear stress (Buga et al., 1991) and that mRNA and protein for cNOS can be induced by mechanical forces different from shear forces (Awolesi et al., 1995). It is plausible that not only shear stress, but also other mechanical factors such as blood pressure and pulsatile stretch contribute to this phenomenon. As mentioned before, we found that acute increases in blood pressure do enhance the release of NO, suggesting that pressure per se is a stimulus for eNOS (Figures 14–2, 14–3, Nava et al., 1994).

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Figure 14±5. Different mechanisms of endothelial dysfunction in genetic (i.e. spontaneous; left) and salt-induced hypertension of the rat (right). ACh=acetylcholine; ADP=adenosine diphosphate; cGMP=cyclic 3',5'-guanosine monophosphate; NE=noradrenaline; 5-HT=5-hydroxytryptamine (serotonin); PGH2=prostaglandin H2.;EDCF=endothelium-derived contracting factor. (Modified from Lüscher and Vanhoutte (1980), by permission).

isolated coronary vessels is augmented in the SHR (Kelm et al., 1995). We have recently found that the activity of constitutive NOS is higher in endothelium of hearts from adult SHR (Nava et al., 1995b). Very young prehypertensive SHRs have, in contrast, lower cNOS activity than the normotensive, indicating that the increased activity of cNOS in these cells is indeed related to hypertension (Figure 14–6, Nava et al., 1995b). Later studies showed that the augmented activity of cNOS in the hypertensive heart happens at the expense of the left ventricle where the highest differential pressure in the cardiovascular system is found (Nava et al., 1995b). These observations demonstrate that high blood pressure upregulates NOS in the heart. It appears that cardiac NOS activity remains unchanged within the normotensive blood pressure range, and that there is a pressure threshold above which upregulation of the enzyme takes place. An enhanced production of NO in the hypertensive heart probably acts as a compensatory mechanism by decreasing myocardial contractility and causing vascular dilatation. Nitric oxide in the hypertensive heart may also protect from hypertrophy. High blood pressure causes cardiac hypertrophy and fibrosis which often leads to left ventricular failure (Anversa et al., 1993). Nitric oxide, which is also a potent inhibitor of smooth muscle cell growth and migration (Dubey and Lüscher, 1993), might protect the heart from these deleterious effects of hypertension. Studies performed in kidneys from SHR show that the activity of Ca2+-dependent NOS in the medulla, where the long-term control of blood pressure takes place, is higher than in the normotensive counterparts (Figure 14–4, Nava et al., 1996a). This observation discards the possibility that hypertension in the SHR is related to a NO-dependent inability of the renal medulla to handle water and sodium (Cowley et al., 1995). The mechanism responsible for the enhancement of the NOS activity in the renal medulla of SHR is not known. It could be a compensatory response to high arterial pressure or to an increased release of certain vasoactive substances. Indeed, the altered renal function of the SHR could be secondary to an increased production of these factors (Lu et al., 1994; Kato et al., 1995). Very recent discoveries from our lab

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Figure 14±6. Blood pressure (a) and activity of constitutive NO synthase (cNOS; b) in hearts of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY) at age 3 weeks (young) and 18 weeks (adult). The increased activity of cNOS appears to be related to the hypertensive status of of the animals and not to age or strain differences. *,**p<0.05 and p<0.01 versus normotensives, p<0.01 versus young animals, (from Nava et al. (1995b), by permission).

demonstrate that the increased activity of NOS in the medulla of SHR occurs at the expense of neuronal NOS (nNOS) rather than eNOS as would have been expected (Nava et al., 1996b). Using immunocytochemistry we have seen that nNOS stains collecting tubular cells suggesting that the SHR kidney may compensate high blood pressure by enhancing the transport mechanism of ions between the interstitium and the tubules. NITRIC OXIDE IN RENOVASCULAR HYPERTENSION Renovascular hypertension is as controversial as genetic hypertension. We and others have observed that endothelium-dependent vascular relaxation is impaired suggesting a diminished production of EDRF (Dohi et al., 1991 ; Lockette et al., 1986). Nakamura and Prewitt found that stimulated and also basal release of NO are deficient in 1K1C hypertensive rats (Nakamura and Prewitt, 1992). However, Sigmon and Beierwaltes have demonstrated that treatment with an inhibitor of NO synthesis affects similarly clipped

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and non clipped kidneys as well as normotensive controls (Sigmon and Beierwaltes, 1994). They concluded that in this model of hypertension, the endothelium is not dysfunctional but is a critical component in the adaptation to increased blood pressure. Interestingly, Pucci et al., (1994) have proven that vasodilatation in aortic coarctation-induced hypertension is mediated by NO while in salt-induced hypertension, it is not. Recent studies show higher amounts of cyclic GMP and NO metabolites in 1K1C hypertensive rats compared to sham operated rats (Figure 14–7, Dubey et al., 1996). Thus, the latest trend is towards the believing that the production of NO is increased in renovascular hypertension to compensate high blood pressure as occurs in genetic hypertension. NITRIC OXIDE IN SALT-SENSITIVE HYPERTENSION We reported ten years ago that Dahl salt-sensitive rats have impaired endothelium-depend-ent relaxations, however no release of vasoconstrictor prostanoids could be demonstrated as in the SHR (Figure 14–4, Lüscher and Vanhoutte, 1988; Lüscher et al., 1987). This suggested that a decreased EDRF production contributed to the pathogenesis of this form of hypertension. Chen and Sanders have reported that inhibition of NO synthesis causes a higher increase in blood pressure in normotensive rats than in Dahl salt-sensitive rats, suggesting that the synthesis of NO is lower in these animals (Chen and Sanders, 1991, 1993). They also demonstrate that NO production, as assessed by the pharmacological effects of NO inhibitors, improves by increasingly feeding NaCl to salt-resistant Dahl rats and administration of L-arginine is effective in lowering blood pressure in the salt-sensitive strain, but not the salt-resistant or the SHR (Chen and Sanders, 1991). In this line Ikeda et al., (1995) have demonstrated that neuronal NOS activity is lower in kidneys from saltsensitive Dahl rats as compared to salt-resistant. Hayakawa et al., (1993) have performed interesting experiments demonstrating that renal blood vessels from Dahl salt-sensitive rats display a reduced response to acetylcholine which is accompanied by a decreased release of NO. These findings contrast with those in the SHR, were the impaired responses to acetylcholine are accompanied by a normal or even increased production of NO (see part 2). Studies on the biology of NO in other models of saltsensitive hypertension have provided results which point to similar directions. Blood vessels from DOCA salt-sensitive hypertensive rats elicit impaired endothelium-dependent relaxations (Voorde and Leusen, 1986) and cyclic GMP accumulation is diminished (Otsuka et al., 1988) suggesting that a decreased production of NO may be involved. Hayakawa and coworkers (1993) have demonstrated that these impaired relaxations are paralleled by a diminished release of NO from perfused kidney vessels. Beneficial effects of treatment with L-arginine on endothelial function have also been shown (Hayakawa et al., 1994; Laurant et al., 1995) in this model of hypertension. Rees and colleagues have recently reported that another model of saltsensitive hypertension, the Sabra hypertensive-prone rat, shows a decreased activity of cNOS in aortic endothelial cells and the concentration of nitrate in plasma is lower than in the Sabra hypertensive-resistant rat (Figure 14–8, Rees et al., 1996). They conclude that a decrease in NO generation plays a role in the susceptibility of Sabra hypertensive-prone rat to develop hypertension and that the resistance to hypertension of the Sabra hypertensive-resistant strain is related to increased NO generation. Thus, saltsensitive hypertension may be partly caused by a diminished production or activity of NO, which is an important factor in the kidney’s ability to excrete water and sodium (Salazar et al., 1993). Studies in humans reveal that the ability of L-arginine to produce NO is diminished in salt-sensitive patients when being salt-loaded (Higashi et al., 1996). This finding discourages the possible plans for treating saltsensitive hypertension with L-arginine, as studies in animals had suggested (Chen and Sanders, 1991; Hayakawa et al., 1994; Laurant et al., 1995).

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Figure 14±7. Graph showing circulating NO metabolites in platelet-poor serum obtained from paired groups of normotensive 1K rats and hypertensive 1K1C rats 2,3,4, and 5 wekks after clipping. *p<0.05 compared to corresponding value for 1K. (From Dubey et al (1996), by permission).

Figure 14±8. (a). Blood pressure response to L-NMMA and; (b) plasma concentrations of NO metabolites in the Sabra hypertension-resistant (open symbols) and hypertension-prone (closed symbols) rat. The smaller response of the hypertension-prone rats to L-NMMA indicates a diminished NO-dependent vasodilatory tone. *p<0.05 (Modified from Rees et al., (1996), by permission).

NITRIC OXIDE IN HUMAN HYPERTENSION Human experimentation has shown so far a diminished production of NO, both basal (Calver et al., 1992; Panza et al., 1993b) or stimulated (Figure 14–9, Panza et al., 1990; Linder et al., 1990). However, most of the studies have been performed measuring the changes in forearm blood flow in response to NO inhibitors or vasodilators and few direct measurements of NO release have been done. Benjamin and colleagues have reported an altered NO synthesis in platelets in essential hypertension (Cadwgan and Benjamin, 1993) and a diminished production of NO as assessed by urinary nitrate (Benjamin et al., 1995). Studies carried out by

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Figure 14±9. Changes in forearm vascular resistance caused by acetylcholine and sodium nitroprusside in normotensives and hypertensives. The response to acetylcholine, but not that to nitroprusside was impaired in hypertensive patients. * p<0.05. (From Linder et al (1990), by permission).

Taddei et al. (1993) have demonstrated that essential hypertension may be associated to an increased production of a cyclooxygenase-dependent endothelium-derived vasoconstrictor substance. In contrast, renovascular and hyperaldosteronism hypertensive patients reveal no role for this substance (Taddi et al., 1993). This group has also shown that acetylcholine-mediated forarm vasodilatation is reduced in normotensive offsprings of essential hypertensive patients, thus demonstrating that endothelial disfunction can precede the appearance of hypertension (Taddi et al., 1992). Vallance et al. (1994) have demonstrated the existence of an inverse relationship between blood pressure and the drop in blood flow in the brachial artery in response to a NO inhibitor (in relation to the responses to norepinephrine) (Calver et al., 1994). Thus, the higher the blood pressure, the lower the production of NO is. Smooth muscle relaxation appears to be maintained in hypertensive subjects (Panza et al., 1993b; Taddi et al., 1993), but not every study agrees with this assumption (Preik et al., 1994). Although some animal studies show beneficial effects of Larginine administration in lowering blood pressure (Chen and Sanders, 1991), in humans hypertensives no decreased availability of substrate for NO has been proven (Panza et al., 1993a). There has been no success either in finding a role for oxidative stress in essential hypertension in humans (García et al., 1995). Unfortunately, no study has been carried out so far focusing of analysis of NO release in specific types of hypertension. It would be interesting to find out whether the concepts on the role of NO that are being established in animal models of hypertension, are valid as well in the different types of hypertension in humans.

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Lüscher, T.F. and Vanhoutte, P.M. (1986) Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension, 8, 344–348. Lüscher, T.F. and Vanhoutte, P.M. (1988) Mechanisms of altered endothelium-dependent responses in hypertensive blood vessels. In: Relaxing and Contracting Factors, ed., Vanhoutte, P.M., pp. 495–509. Clifton, N.J.: Humana Press. Lüscher, T.F, Raij, L. and Vanhoutte. P.M. (1987) Endothelium-dependent vascular responses in normotensive and hypertensive Dahl rats. Hypertension., 9, 157–163. Malinski, T., Kapturczak, M., Dayharsh, J. and Bohr, D. (1993) Nitric oxide synthase activity in genetic hypertension. Biochem. Biophys. Res. Commun., 194, 654–658. Moncada, S. and Higgs A. (1993) Mechanisms of disease. The L-arginine-nitric oxide pathway. N. Eng. J. Med., 329, 2002–2012. Nakamura, T. and Prewitt, R.L. (1992) Alteration of endothelial function in arterioles of renal hypertensive rats at two levels of vascular tone. J. Hypertens., 10, 621–627. Nakazono, K., Watanabe, N., Matsuno, K., Sasaki, J. and Sato, T. (1991) Does superoxide underlie the pathogenesis of hypertension?. Proc. Natl. Acad. Sci. USA, 88, 10045–10048. Nava, E., Leone, A.M., Wiklund, N.P. and Moncada, S. (1994) Detection of release of nitric oxide by vasoactive substances in the anaesthesized rat. In, The Biology of Nitric Oxide, Eds: M.Feelisch, R.Busse and S. Moncada, pp. 179–181, London: Portland Press. Nava, E., Llinás, M.T., González, J.D. and Salazar, F.J. (1996a) Nitric oxide synthase activity in renal cortex and medulla of normotensive and spontaneously hypertensive rats. Am. J. Hypertension, In press. Nava, E., Moreau, P. and Lüscher, T.F. (1995a) Basal production of nitric oxide is increased, but inefficacious in spontaneous hypertension. Circulation, 92, 1–347. Nava, E., Noll, G. and Lüscher, T.F. (1995b) Increased activity of constitutive nitric oxide synthase in hearts from spontaneously hypertensive rats . Circulation, 91, 2310–2313. Nava, E., Salazar, J., Martímez de Velasco, J., Martínez-Murillo, R., Fernández, A.P., Serrano, J. and Rodrigo, J. (1996b) Distribution of neuronal nitric oxide synthase immunoreactivity in normal and hypertensive rat kidneys. J. Am. Soc. Nephrol, 1, 1540. Nava, E., Wiklund, N.P. and Salazar, F.J. (1996c) Changes in nitric oxide release in vivo in response to vasoactive substances. Br J Pharmacol. 119, 1211–1216. Otsuka, Y, Dipiero, A., Hirt, E., Brennaman, B. and Lockette, W. (1988) Vascular relaxation and cGMP in hypertension. Am. J.Physiol, 254, H163-H169. Palmer, R.M.J., Bridge, L., Foxwell, N.A. and Moncada, S. (1992) The role of nitric oxide in endothelial damage and its inhibition by glucocorticoids. Br. J.Pharmacol, 105, 11–12. Panza, J.A., Casino, PR., Badar, D.M. and Quyyumi, A.A. (1993a) Effect of increased availability of endotheliumderived nitric oxide precursor on endothelium-dependent vascular relaxation in normal subjects and in patients with essential hypertension. Circulation, 87, 1475–1481. Panza, J.A., Casino, PR., Kilcoyne, C.M. and Quyyumi, A.A. (1993b) Role of endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation, 87, 1468–1474. Panza, J.A., Quyyumi, A.A., Brush, I.E. and Epstein, S.E. (1990) Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N. Eng. J. Med., 323, 22–27. Papapetropoulos, A., Marczin, N., Snead, M.D., Cheng, C., Milici, A. and Catravas, J.D. (1994) Smooth muscle cell responsiveness to nitrovasodilators in hypertensive and normotensive rats. Hypertension, 23, 476– 484. Preik, M., Kelm, M., Köster, A., Heinzelmann, Notz, W. and Strauer, B.E. (1994) Reduced nitric oxidedependent vasodilation following application of organic nitrates in patients with essential hypertension. In: The Biology of Nitric Oxide, eds., M.Feelisch, R.Busse and S.Moncada, pp. 179–181. London: Portland Press. Pucci, M.L., Miller, K.B., Dick, L.B., Guan, H., Lin, L. and Nasjletti, A. (1994) Vascular responsiveness to nitric oxide synthesis inhibition in hypertensive rats. Hypertension, 23, 744–751. Rees, D., Ben-Ishay, D. and Moncada, S. (1996) Nitric oxide and the regulation of blood pressure in the hypertensionprone and hypertension-resistant Sabra rat. Hypertension, 28, 367–371.

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Rees, D.D., Palmer, R.M.J. and Moncada, S. (1989) Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc. Natl. Acad. Sci. USA, 86, 3375–3378. Rees, D.D., Palmer, R.M.J., Schulz, R., Hodson, H.F. and Moncada, S. (1991) Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br. J.Pharmacol., 101, 746–752. Salazar, F.J., Alberola, A., Pinilla, J.M., Romero, J.C. and Quesada, T. (1993) Salt-induced increase in arterial pressure during nitric oxide synthesis inhibition. Hypertension, 22, 49–55. Sawada, Y, Sakamaki, T., Nakamura, T., Sato, K., Ono, Z. and Murata, K. (1994) Release of nitric oxide in response to acetylcholine is unaltered in spontaneously hypertensive rats.J. Hypertens., 12, 745–750. Schleiffer, R., Pernot, F, Van Overloop, B. and Gairard, A. (1991) In vivo involvement of endothelium-derived nitric oxide in spontaneously hypertensive rats: effects of NG-nitro-L-arginine methyl ester. J. Hypertension, 9, S192–S193. Schmidt, H.H.H.W., Zernikow, B., Baeblich, S. and Böhme, E. (1990) Basal and stimulated formation and release of Larginine-derived nitrogen oxides from cultured endothelial cells. J. PharmacoL Exp. Ther., 254, 591–597. Sessa, W.C., Pritchard, K., Seyedi, N., Wang, J. and Hintze, T.H. (1994) Chronic exercise in dogs increses coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ. Res., 74, 349–353. Shirasaki, Y, Kolm, P., Nickols, G.A. and Lee, T.J.-F. (1988) Endothelial regulation of cyclic GMP and vascular responses in hypertension. J.PharmacoL Exp. Ther., 245, 53–58. Sigmon, D.H. and Beierwaltes, W.H. (1994) Nitric oxide influences blood flow distribution in renovascular hypertension. Hypertension, 23, 134–139. Spector, S., Fleisch, J.H., Maling, H.M. and Brodie, B.B. (1996) Vascular smooth muscle reactivity in normotensive and hypertensive rats. Science, 166, 1300–1301. Taddei, S., Virdis, A., Mattei, P. and Salvetti, A. (1993) Vasodilation to acetylcholine in primary and secondary forms of human hypertension. Hypertension, 21, 929–933. Taddei, S., Virdis, A., Mattei, P., Arzilli, F. and Salvetti, A. (1992) Endothelium-dependent forearm vasodilation is reduced in normotensive subjects with familial history of hypertension. J. Cardiovasc. Pharmacol., 20, S193–S195. Takase, H., Moreau, P., Küng, C.F., Nava, E. and Lüscher, T.F. (1996) Antihypertensive therapy improves endotheliumdependent relaxation of resistance arteries in nitric oxide deficient hypertension: Effect of verapamil and trandolapril. Hypertension, 27, 25–31. Tesfamariam, B. and Halpern, W. (1988) Endothelium-dependent and endothelium-independent vasodilation in resistance arteries from hypertensive rats. Hypertension, 11, 440–44. Tschudi, M.R., Mesaros, S., Lüscher, T.F. and Malinski, T. (1996) Direct in situ measurement of nitric oxide in mesenteric resistance vessels. Increased decomposition by superoxide in hypertension. Hypertension, 27, 32–35. Vallance, P., Collier, J. and Moncada, S. (1989) Effects of endothelium derived nitric oxide on peripheral arteriolar tone in man. Lancet, ii, 997–1000. Voorde, J.V. and Leusen, I. (1986) Endothelium dependent and independent relaxation of aortic rings from hypertensive rats. Am. J.Physiol., 250, H711-H717 Weiner, C.R, Lizasoain, I., Baylis, S.A., Knowles, R.G., Charles, I.C. and Moncada, S. (1994) Induction of calciumdependent nitric oxide synthase by sex hormones. Proc. Natl. Acad. Sci. USA, 91, 5212–5216. Yamazaki, J., Fujita, N. and Nagao, T. (1991) NG-monomethyl-L-arginine-induced pressore response at developmental and established stages in spontaneouly hypertensive rats. J.Pharmacol Exp. Then, 259, 52–57.

15 Pulmonary Hypertension Jocelyn Dupuis1, David Langleben2 and Duncan J.Stewart3 1Montreal

Heart Institute, 2Sir Mortimer B. Davis Jewish General Hospital, McGill University, 3Terrence Donnelly Heart Centre, St.Michael's Hospital, University of Toronto, Canada

Unlike systemic vascular beds, the pulmonary circulation must accommodate the entire cardiac output, and yet maintain a much lower arterial pressure. This is accomplished by virtue of a very low normal pulmonary vascular resistance (PVR) in physiological conditions. However, elevation of PVR occurs as a consequence of a wide variety of pulmonary, cardiac and systemic disorders (secondary pulmonary hypertension (SPH)) or as a primary disease (primary pulmonary hypertension (PPH)). The role of abnormalities of the L-arginine/NO pathway in the pathogenesis of PH is uncertain. The evidence both supporting and refuting an important contribution is reviewed. In animal models of PH, the evidence suggests a significant impairment of endothelium-dependent dilation, particularly in the larger conduit pulmonary arteries. The evidence is less compelling that reduced NO synthesis or activity is responsible for vasoconstriction and remodeling of smaller resistance vessels. Similarly, the human data is controversial. Although clearly more studies are needed, it might be reasonable to conclude that the clinical experience to date is generally compatible with the more extensive literature from animal studies. Regardless of whether decreased NO activity plays a pathogenic role in PH, there is a good rationale supporting therapeutic strategies to increase pulmonary vascular NO in the treatment of this disorder, and various therapeutic approaches are reviewed. Keywords: Nitric oxide, pulmonary hypertension, endothelial dysfunction, hypoxia, monocrotaline, nitric oxide synthase.

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INTRODUCTION The human pulmonary circulation is in a series circuit with the systemic circulation, so that the entire cardiac output (approximately 5 L/min in resting humans) also passes through the lungs. Given the large cross-sectional area of the lung capillary bed, and that the pulmonary venous drainage system into the left atrium of the heart also normally operates at a low pressure (0–12 mm Hg), the lung circulation has a low vascular resistance and is able to accommodate blood flow at a low driving (pulmonary arterial) pressure, as compared to the systemic circulation. Moreover, in the resting state, some of the capillary bed is minimally recruited, allowing the pulmonary blood flow to rise dramatically during exercise, with only slight rises in pulmonary arterial pressure. A precapillary arteriolar segment regulates the pulmonary vascular resistance with exquisite homeostatic control, and responds to a variety of factors, including oxygen tension, pH, flow, and circulating and neurohormonal mediators. Pulmonary hypertension remains an important clinical problem. It is defined as an elevated pressure in the pulmonary circulation, often with decreased pulmonary blood flow. The right ventricle, designed to pump at a low pressure, must generate the increased pressure to force blood through the lungs, and it may ultimately fail, resulting in the demise of the patient. Depending on the etiology, the increased pulmonary vascular resistance may be due to vasoconstriction, thickening or structural remodeling of blood vessel walls by cellular proliferation or alterations in extracellular matrix, thrombosis and obliteration of vessels, or a combination of these events. The end result is a reduction in the cross-sectional area of the vessel lumen. The causes of pulmonary hypertension may be broadly divided into “precapillary” and “postcapillary” (Reeves and Groves, 1984). Furthermore, after a careful search to exclude secondary diseases, primary pulmonary hypertension may be diagnosed. Diseases which result in increased pulmonary venous pressure, including left heart failure and mitral valve disease, are the commonest “postcapillary” causes, and represent commonest cause of pulmonary hypertension in humans. The other four major “secondary” causes of pulmonary hypertension include lung disease (obstructive, restrictive, interstitial andhypoxic), chronic thromboemboli, autoimmune or collagen-vascular diseases (such as scleroderma, the CREST syndrome, polymyositis, and mixed connective tissue disease variants), and congenital heart disease with left-to-right shunts. Each of these etiologies offers paradigms for understanding some of the common or differing aspects of the pathophysiologic mechanisms active in pulmonary hypertension. Physical entrapment or compression of vessels may be combined with vasoconstriction from hypoxia (COPD or interstitial disease). Plugging of large, midsized or small arteries is seen in thromboembolic pulmonary hypertension. The collagen vascular diseases cause intimai sclerosis of pulmonary microvessels, as well as causing pulmonary fibrosis. They may also predispose to increased pulmonary vasoconstriction (akin to Raynaud’s phenomenon of the extremities). Longstanding left-to-right intracardiac shunts, with persistent high flow and high shear stress in the pulmonary bed, induce endothelial injury and vascular remodeling that narrows the bed, ultimately resulting in an Eisenmenger physiology. Primary pulmonary hypertension is still an enigma (Rubin, 1997). It presents mainly between the ages of 20 to 40 years, and has a femaleimale ratio of approximately 2:1, with an incidence is 1–2 per million. The most frequent histologie abnormality of the lung circulation is termed “plexogenic pulmonary arteriopathy”. Structural remodeling of pulmonary microvessels, by medial thickening, intimai proliferation and formation of plexiform lesions, progressively increases pulmonary vascular resistance (Voelkel et al., 1997). Vasospasm may be present to varying degrees. Until recently, the clinical course was relentlessly downhill, leading to death. A minority (15–25%) of patients respond well to high-dose calcium channel blockers (Rich et al., 1992), and recent studies have shown dramatic survival benefit from chronic intravenous infusions of prostacycline for patients who are in functional classes III and IV (Barst et al., 1996). Addition

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of warfarin anti-coagulation also improves survival, presumably by reducing the occurrence of thrombosisin-situ in pulmonary microvessels (Fuster et al., 1984; Rich et al., 1992). Primary pulmonary hypertension has been reported in families and a gene has been localized to chromosome 2 (Nichols et al., 1997). Other risk factors for primary pulmonary hypertension include HIV infection, cirrhosis of the liver, and intake of anorexic agents (Abenhaim et al., 1996). Endothelial dysfunction is a feature common to most types of pulmonary hypertension, and it contributes to the vascular abnormalities seen in these disorders. The pulmonary endothelium has important metabolic activity for many vasoactive and cytokine mediators circulating in the blood, including angiotensin I, bradykinins, 5-hydroxytryptamine (serotonin), catecholamines and other neurohormones. This activity is altered in primary pulmonary hypertension and many types of secondary pulmonary hypertension, and may contribute to the vasoconstriction and cellular proliferation seen in these disorders (Catravas et al., 1992). Reduced production of prostacycline and increased production of endothelin–1 have been described in primary pulmonary hypertension (Giaid et al., 1993; Stewart et al., 1991; Christman et al., 1992). As is discussed below, reduced endothelial-mediated vasorelaxation, implying decreased nitric oxide production, has been reported in primary and secondary pulmonary hypertension. Anorexic agents may alter pulmonary endothelial handling of 5hydroxytryptamine and catecholamines, resulting in high local levels of these vasoconstrictors and smooth-muscle mitogens. The endothelium is an essential element in the homeostasis of thrombosis and fibrinolysis. It normally maintains the balance in favor of anticoagulation and fibrinolysis, thereby promoting vascular patency. Increased thrombotic and decreased fibrinolytic activity has been reported in primary pulmonary hypertension (Voelkel et al., 1997). This thrombus formation and fibrin deposition will itself narrow the vascular lumen, and it may also act as a nidus for cellular ingrowth. The endothelium has a crucial role in the control of inflammation and in regulating growth of other cells (smooth muscle, fibroblast, pericyte) in the vessel wall. It produces cell adhesion molecules which mediate the attachment of inflammatory cells and platelets and, when activated, it produces cytokines, including interleukins and platelet activating factor, which amplify inflammation and alter cell growth. The endothelium also introduces molecules into the extracellular matrix of the vessel wall (proteoglycans, collagens, elastins), which contribute to the control of cell growth and to vascular elastance and compliance. There is at present no good animal model of primary pulmonary hypertension or plexogenic pulmonary arteriopathy. Our understanding of the formation of plexiform lesions is limited. Animal models of secondary pulmonary hypertension have been developed, and two are discussed here. Monocrotaline, a pyrrolizidine alkaloid, will induce pulmonary hypertension when it is converted to active pyrroles in species with the necessary hepatic microsomal enzymes. The pyrroles then travel through the venous system to the next downstream organ, the lung. Endothelial permeability and dysfunction are early findings, with subsequent activation of many secondary mediator cascades that ultimately lead to vascular remodeling and thrombosis. The monocrotaline model may most appropriately be considered a model of the human Acute Respiratory Distress Syndrome (ARDS), which has a variety of triggers, including trauma, burns and sepsis, and which involves early important endothelial injury and dysfunction. Pulmonary vascular remodeling and pulmonary hypertension develop with progressive ARDS. Hypoxia-induced pulmonary hypertension can be reproduced in many species, but sensitivity to hypoxia varies between species and strains (Weir and Archer, 1957). Acute hypoxia is a potent vasoconstrictor of the pulmonary circulation. This effect may be mediated by closure of potassium channels on smooth muscle cells. In vivo, acute hypoxic vasoconstriction is at least in part moderated by release of nitric oxide from the endothelium. Chronic hypoxia causes ongoing vasoconstriction, but it also induces muscularization of large and small pulmonary arteries, and polycythemia (Jones et al., 1991). With return to normoxia, the vascular remodeling regresses more slowly than the constriction and polycythemia resolve. Use of these animal models allows

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the study of various aspects of hypoxia, and they are relevant to chronic lung diseases and to high-altitude exposure. NITRIC OXIDE PRODUCTION BY THE PULMONARY CIRCULATION: ROLE IN CONTROL OF PULMONARY VASCULAR RESISTANCE Endothelium-dependent dilation of pulmonary arteries, like that of systemic vessels, was recognized in the seminal report of Furchgott and Zawadzski (Furchgott and Zawadzski, 1980), and has since been confirmed by many other groups (Gruetter and Lemke, 1986; Cherry et al., 1982; Chand and Altura, 1981). In a manner analogous to that of the systemic vasculature, the pulmonary vascular endothelium produces nitric oxide (NO) by converting L-arginine to L-citrulline through the action of the endothelial constitutive NOsynthase (NOS III). NO and NO breakdown products (NOX) can be measured in the effluent of isolated saline-perfused lungs, demonstrating that there is substantial basal production of NO by the pulmonary vasculature (Wang et al., 1995; Spriestersbach et al., 1995; Nelin et al., 1996). This production is increased by the receptor-independent stimulator of endothelial NO production, A-23187 (Spriestersbach et al., 1995), while it is reduced by inhibitors of NO-synthase, L-NMMA and L-NAME (Wang et al., 1995; Spriestersbach et al., 1995; Nelin et al., 1996). Again, as in systemic vessels, the effect of NO on pulmonary smooth muscle cells is mediated by the stimulation of soluble guanylate cyclase (sGC) and the increased production of cyclic 3',5'-guanosine monophosphate (cGMP) (Ignarro et al., 1987a; Ignarro et al., 1987b). Cyclic GMP levels are two-fold higher in the presence versus the absence of the pulmonary endothelium and an inhibitor of sGC, methylene blue, reduces these levels while increasing pulmonary vascular tone (Ignarro et al., 1987b). Inhibition of phosphodiesterases increases the levels of cGMP in the pulmonary effluent and potentiates the response to NO-induced pulmonary vasodilation (Thusu et al., 1995; Ichinose et al., 1995; Cohen et al., 1996). NO is also present in the exhaled air (Gustafsson et al., 1991; Cremona et al., 1995), although the source of exhaled NO gas is largely naso-pharyngeal in origin (Phillips et al., 1996; Kimberly et al, 1996; Dillon et al., 1996; Lundberg et al., 1995). However, experimental evidence suggests that pulmonary vascular production also contributes to NO in exhaled air in isolated porcine lungs (Nelin et al, 1996; Cremona et al., 1995), and this is increased by acetylcholine and reduced by L-NAME (Nelin et al., 1996; Cremona et al., 1995). As well, exhaled NO is reduced by perfusion of isolated lungs by blood (Cremona et al., 1995), presumably due to the binding of vascular-derived NO to hemoglobin. In contrast to the systemic vasculature, endothelial NO release is not diminished by aging in the rat pulmonary artery (2). This difference between the aorta and the pulmonary artery may at least in part reflect the lower pressures to which the latter is exposed throughout life. The site of action of NO in the pulmonary circulation has been studied using the double occlusion method in isolated perfused lung preparations (Roos et al., 1994; Rimar and Gillis, 1995; Lindeborg et al., 1995; Hakim, 1994; Ferrario et al., 1996). Both in response to the endogenous release of endothelial NO by acetylcholine as well the pharmacological generation of NO by sodium nitroprusside, preferential dilation occurs at the level of small precapillary arteries, and to a lesser degree in small veins and larger pulmonary arteries (Roos et al., 1994; Ferrario et al., 1996). Pulsatile flow during hypoxia is associated with an increase in EDRF production by precapillary pulmonary arteries (Hakim, 1994), suggesting a shear-stress induced increase in NO release at this level of the circulation, again analogous to systemic vessels (Griffith et al., 1987). Inhaled NO also causes dilation of small pulmonary arteries and veins (Roos et al., 1994; Rimar and Gillis, 1995; Lindeborg et al., 1995), whereas, as expected, it does not affect the tone of the larger upstream capacitance vessels (Roos et al., 1994). This preferential effect of NO on small resistence pulmonary vessels needs to be kept in mind in the interpretation of in vitro studies of pulmonary vascular

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reactivity which often use larger capacitance vessels. Thus, some of the apparent discrepancies in data derived from models of experimental pulmonary hyper tension examining vascular reactivity of isolated pulmonary conduit vessels versus studies in isolated intact lungs or in vivo may be explained by the greater contribution of the small precapillary arteries to changes in pulmonary vascular resistance and therefore pulmonary pressures. Role of NO in Maintaining Normal Pulmonary Pressures The pulmonary circulation is characterized by a very low vascular resistance and it is attractive to speculate that a high basal release of NO by the endothelium of pulmonary resistance vessels contributes significantly to low baseline pulmonary vascular tone. Many studies have addressed this issue by measuring pulmonary vascular tone and blood flow after the inhibition of endogenous NO production using various NOS antagonists, however, often with conflicting results. Some investigators have reported little or no increase in pulmonary vascular tone after NOS inhibition (Wanstall et al., 1995; Russ and Walker, 1993; Lindeborg et al., 1995; Hasunuma et al., 1991; Hakim, 1994; Ferrario et al., 1996; Barnard et al., 1993), whereas others have shown more substantial effects (Rodman et al., 1990; Nelin et al., 1996; Madden et al., 1995; Isaacson et al., 1994; Cremona et al., 1995; Cooper et al., 1996; Blitzer et al., 1996). In general, the modulation of basal vascular tone by NO is accentuated in situations of increased pulmonary vascular tone induced by hypoxia or by pharmacological stimulation with vasoconstrictor substances, such as angiotensin II, endothelin-1 or the thrombaxne A2 mimetic U-46619 (Wanstall et al., 1995; Russ and Walker, 1993; Roos et al., 1994; Rimar and Gillis, 1995; Persson et al., 1994; Liu et al., 1991a,b; Hasunuma et al., 1991; Ferrario et al., 1996; Adnot et al., 1991). In isolated rat lungs, increases in flow associated with the simultaneous elevation of arterial and venous pressures (so that the transpulmonary gradient is maintained constant) were inhibited by L-NAME (Barnard et al., 1993), consistent with a role of NO in buffering increases in pulmonary perfusion pressure. However, against such a role is the observation that chronic inhibition of NO production by L-NAME for three weeks in normal rats reduced cardiac output and increased systemic arterial pressure, without affecting pulmonary pressure (Hampl et al., 1996; Hampl et al., 1993). In the same experiments, the perfusion pressure-flow relationship of isolated lungs was only mildly shifted upwards by long-term L-NAME, much less than that of animals subjected to chronic hypoxia. Moreover, chronic hypoxia, but not L-NAME administration, caused hypertensive pulmonary remodeling whereas long-term supplementation with the NO precursor, L-arginine did not prevent these changes in the hypoxic animals (Hampl et al., 1996). The modest role of NO in the maintenance of normal pulmonary hemodynamics was further underscored by recent studies of targeted disruption of the endogenous NOS III gene in mice (Steudel et al., 1997). These mice exhibited only mild increases in pulmonary arterial pressure, associated with increased pulmonary vascular resistance measured in vivo, but no noticeable morphological abnormalities of the pulmonary vasculature. In conclusion, despite the evidence that there is substantial basal NO production by the normal pulmonary circulation, the current weight of evidence would support only a modest contribution in the maintenance of the normally low basal pulmonary vascular resistance. Rather, it appears that NO may play a more significant role in attenuating increases in pulmonary vascular tone in response to pharmacological of pathophysiological stimuli that result in pulmonary hypertension.

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Role Of NO In Acute Hypoxic Pulmonary Vasoconstriction Acute hypoxia causes pulmonary vasoconstriction resulting in increased pulmonary arterial pressures and is often used as a constrictor stimulus in the evaluation of pulmonary vascular reactivity. Acute hypoxic pulmonary vasoconstriction is also of considerable clinical importance because of the numerous pathological conditions which produce hypoxia, including respiratory distress syndromes in newborns and adults, pulmonary edema, pulmonary embolism, and pneumonia, just to name a few. A number of studies have suggested that hypoxic vasoconstriction is dependent on the presence of an intact endothelium (Rubanyi and Vanhoutte, 1988; Rodman et al., 1997; Hoshino et al., 1988; Holden and McCall, 1984), consistent with a contribution of reduced NO production (or activity) to hypoxic pulmonary hypertension. In isolated rat pulmonary arteries, the inhibition of EDRF production mimics effect of hypoxia (Rodman et al., 1990), and hypoxic vessels contain lower levels of cGMR Moreover, these hypoxic vessels exhibited reduced responsiveness to acetylcholine, whereas relaxation in response to authentic NO or 8-bromo-cGMP was preserved. In contradistinction to these findings in isolated vessels, there is general consensus that in intact lungs in vitro or in vivo, withdrawal of NO does not contribute vasoconstriction in response to acute hypoxia, but rather its release may attenuate the increase in pulmonary vascular tone (Sprague et al., 1992; Robertson et al., 1990; Persson et al., 1990; Nelin et al., 1996; Nelin and Dawson, 1993; Blitzer et al., 1996; Archer et al., 1989). Indeed, acute hypoxia is associated with an increase, not a decrease, in NO release into the perfusate of isolated rat lungs (Wang et al., 1995). The mechanism of increased NO production is unknown but could be related to the increase in shear stress caused by the hypoxic constriction (Hakim, 1994; Barnard et al., 1993). Moreover, the pharmacological stimulation of NO release by acetylcholine, bradykinin or A-23187 (McMurtry, 1985; Archer et al., 1990), as well as the administration of authentic NO itself (Frostell et al., 1993; Archer et al., 1990), reduces hypoxic vasoconstriction, pointing to a potential therapeutic role for NO in the treatment of pulmonary hypertension in this condition. The L-arginine-NO Pathway In Experimental Models Of Pulmonary Hypertension Rats exposed to hypoxia for a period of three weeks or more develop pulmonary hypertension with structural remodeling of the pulmonary arteries in the form of concentric hypertrophy. This model is particularly relevant to pulmonary hypertension which develops in patients with chronic obstructive pulmonary disease (normobaric hypoxia) and to the less frequent condition of high altitude pulmonary hypertension (hypobaric hypoxia). The model of hypoxic pulmonary hypertension in rats has, therefore, been extensively utilized to study the potential pathophysiological role of NO in pulmonary hypertension. While there is general agreement that the L-arginine-NO pathway plays an important role in the modulation of pulmonary vascular tone in response to chronic hypoxia, there is more controversy surrounding nature of this modulation. Some investigators have reported reduced NO production or activity (Rodman et al., 1990; Maruyama and Maruyama, 1994; Eddahibi et al., 1992; Carville et al., 1993; Adnot et al., 1991), whereas others have demonstrated preserved and even enhanced NO activity (Wanstall et al., 1995; Russ and Walker, 1993; Resta and Walker, 1994; Oka et al., 1993; Isaacson et al., 1994; Hampl et al., 1993; Barer et al., 1993). Several hypotheses have been advanced in an effort to reconcile these apparent discrepancies, including differences in species, experimental conditions, duration of hypoxia, and data from intact lungs versus isolated pulmonary vessels. For example, many of the studies demonstrating reduced NO activity were performed using isolated pulmonary conduit arteries (Rodman et al., 1990; Maruyama and Maruyama, 1994; Eddahibi et al., 1992; Carville et al., 1993), and thus may not faithfully predict the behaviour of the small arteries that determine the resistance of the intact pulmonary vascular bed. In isolated lungs, a reduction in NO activity was described in one report (Adnot et al., 1991), however, this has not been

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reproduced in the majority of studies (Resta and Walker, 1996; Russ and Walker, 1993; Resta and Walker, 1994; Oka et al, 1993; Hampl et al., 1993; Barer et al., 1993) in which NO activity has been found to be preserved or increased during long-term exposure to hypoxia, serving to attenuate increases in pulmonary vascular tone. As well, increased levels of NOX (NO, NO2, and NO3) have been recovered from the pulmonary effluent of chronically hypoxic rats (Isaacson et al., 1994). This was associated with increased NOS activity as determined by conversion of 3H-Larginine to 3H-L-citrulline (Shaul et al., 1995) in the lung homogenates, and increased expression of NOS III mRNA and protein (Xue and Johns, 1996; Shaul et al., 1995). Others have confirmed the upregulation of NOS III expression in the rat chronic hypoxia model of pulmonary hypertension (Le Cras et al., 1996; Xue et al., 1994), and increased expression of NOS III protein has been shown by immunostaining to be tightly correlated with the development of vascular hypertrohy in hypoxic pulmonary hypertension (Xue and Johns, 1996). Monocrotaline (MCT) is an alkaloid derivative which in rats is metabolized by the liver to dehydromonocrotaline, a substance highly toxic to pulmonary vascular endothelium. Rats injected with MCT develop pulmonary hypertension due predominantly to increased precapillary vascular resistance, right ventricular hypertrophy and medial hypertrophy of pulmonary arteries that are well established after three weeks. Large hilar pulmonary arteries from MCT-treated animals demonstrate impaired endotheliumdependent dilation to ADP, A23187 and acetylcholine (Mathew et al., 1995), which was not reversed by Larginine or superoxide dismutase suggesting that neither a deficiency of the substrate for NOS, nor inactivation of NO by free radicals, contributed to the apparent reduction in NO activity. Isolated pulmonary intralobar arteries (~700 µm in diameter) exhibited preserved but reduced relaxation in response to acetylcholine (Madden et al., 1994). This decrease in endothelium-dependent dilation was interpreted as being due to reduced arterial distensibility secondary to vascular remodeling rather than a decrease in the release or activity of NO (Madden et al., 1994). This is consistent with the observation in the same model, that the inhibition of NO synthesis by L-NAME caused a greater decrease in pulmonary artery diameter in vessels from MCT-treated compared with those from normal rats (Madden et al., 1995). Moreover, in intact isolated lungs from MCT-treated rats perfused at constant flow and preconstricted by hypoxia, there was an increase in responsiveness to acetlycholine (Archer et al., 1990) suggesting an increase in receptor-mediated stimulation of NO production in this model of pulmonary hypertension. Recently, it has also been shown that immunoreactive NOS III was increased in pulmonary arterial endothelium of lungs from MCT-treated animals compared to control rats (Resta et al., 1997), whereas there was no change in NOS III expression in pulmonary veins. Therefore, it is hard on the face of the evidence reviewed here to conclude that a decrease in endothelial NO production or activity underlies pulmonary hypertension in the animal models most thoroughly investigated. Rather, it is possible that in these animal models of pulmonary hypertension, the NO-Larginine pathway may in fact be upregulated perhaps as a mechanism of compensation, serving to buffer increases in pulmonary vascular resistance and attenuate the degree of elevation in pulmonary arterial pressures. Thus, although a defect in the NO system may not be a primary cause of pulmonary hypertension in these models, it could be argued that in more advanced states of pulmonary hypertension there is a relative deficit of NO activity since clearly there is failure of this compensatory mechanism. It follows that various means of increasing NO production or activity could represent potentially useful therapeutic strategies for the treatment of pulmonary hypertension.

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The L-arginine-NO Pathway in Human Pulmonary Hypertension The first report of endothelium-dependent dilation of human pulmonary arteries appeared in 1987 (Greenberg et al., 1987). Relatively normal segments of pulmonary arteries obtained from patients undergoing surgery for removal of malignant tumors exhibited relaxation to acetylcholine that was abolished by removal of the endothelium (Greenberg et al., 1987). In normal humans, the systemic administration of the inhibitor of NO synthesis, L-NMMA (300 mg i.v.) produced increases in systemic and pulmonary vascular resistance, resulting in a modest increase in pulmonary arterial pressures (Stamler et al., 1994; Blitzer et al., 1996). Although, in part, the increases in pulmonary vascular resistance might have occurred passively as a consequence of the decrease in cardiac output caused by the increased afterload, the increase in pulmonary artery pressure stongly supports a contribution of NO to basal pulmonary vascular tone. Another recent study was designed to exclude the passive influence of changes in systemic hemodynamics on the pulmonary circulation by direct infusion of L-NMMA and acetylcholine into a branch of the pulmonary artery, measuring changes in pulmonary blood flow using a doppler flow wire (Cooper et al., 1996). Infusion of acetylcholine produced a dose-dependent increase in pulmonary blood flow while LNMMA caused a reduction, demonstrating that normal basal pulmonary vascular tone is influenced by a continuous local release of NO by the pulmonary vasculature (Cooper et al., 1996). The acute hypoxic increase in pulmonary arterial pressures in humans, as in animals, is also attenuated by endothelium-derived NO, again consistent with role for this vasodilator mediator as a counter-regulatory mechanism (Blitzer et al., 1996). In healthy subjects, infusion of the NO substrate, L-arginine, did not significantly modify pulmonary hemodynamics (Mehta et al., 1995). However, in a small group of patients with various forms of pulmonary hypertension, L-arginine infusion for 30 minutes caused a 15.8±3.6% reduction in pulmonary arterial pressure and a 27.6±5.8% reduction in pulmonary vascular resistance (Mehta et al., 1995). In subgroup analysis it was noted that patients with secondary causes of pulmonary hypertension (nearly all of which had heart failure), were particularly responsive to L-arginine infusion while those with primary or scleroderma pulmonary hypertension showed little response. The same authors demonstrated that the infusion of L-arginine was accompanied by an increase in circulating levels of L-citrulline, the immediate product of L-arginine metabolism by NOS (Mehta et al., 1995), and by an increase in exhaled NO (Mehta et al., 1996). Peak levels of L-citrulline, but not L-ornithine (the direct metabolite of L-arginine by the hepatic urea cycle) correlated with the reduction in pulmonary pressures, however, somewhat counterintuitively the basal L-arginine levels correlated inversely rather than directly with pulmonary hemodynamic responsiveness (Mehta et al., 1995). None-the-less, it appears that L-arginine has potent short-term vasodilating effects in some patients with pulmonary hypertension that is mediated at least in part through increased endogenous production of NO. Although pulmonary hypertension in humans does not appear to be related to a systemic decrease in L-arginine availability, reduced cellular transport of Larginine (Durante et al., 1996; Bogle et al., 1996) or endogenous inhibitors of NO synthesis (Vallance et al., 1992) may be contributing factors. As well, it is possible that, as a result of an upregulation of NOS activity as part of a compensatory mechanism, any limitation in NO availability is aggravated by increased L-arginine utilization. Isolated pulmonary vascular rings taken from patients with chronic obstructive pulmonary disease exhibited reduced basal and receptor mediated endothelium-dependent dilation (Dinh-Xuan et al., 1991), but preserved responses to endothelium-independent, NO donor compound, sodium nitroprusside. The magnitude of impairment in endothelium-dependent dilation correlated with the severity of intimai hypertrophy and the degree of hypoxia, based on measurements of oxygen partial pressure in arterial blood (Dinh-Xuan et al., 1991), suggesting a link between endothelial dysfunction and disease severity. Similarly, the same group has shown that pulmonary arteries from patients with Eisenmenger’s syndrome also have

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reduced response to acetylcholine, even exhibiting paradoxical vasoconstriction at higher concentrations (Dinh Xuan et al., 1990). Consistent with these functional observations in larger pulmonary arteries, it has been reported that there is reduced expression of NOS III protein by immunostaining and mRNA by in situ hybridization in lungs from patients with pulmonary hypertension of various etiologies (Giaid and Saleh, 1995) in proportion to the severity of hypertensive morphological vascular abnormalities. In particular, the most severe cases, i.e. those associated with plexiform lesions, exhibited very little NOS III expression (Giaid and Saleh, 1995). However, as with animal models of pulmonary hypertension, there is not yet general agreement concerning the pathophysiological role of reduced NO activity in human pulmonary hypertension. Another study in a similar group of patients with pulmonary hypertension (Xue and Johns, 1995), reported an increase in NOS III immunoreactivity in pulmonary vascular endothelium, rather than a decrease, even in patients with severe primary pulmonary hypertension. Clearly, more studies in humans with pulmonary hypertension are required to resolve this controversy. It is perhaps worthwhile to note, as reviewed in the previous sections, that in animals studies a pattern is emerging supporting increases in NO synthesis in pulmonary hypertension in the smaller precapillary resistance vessels likely as a mechanism of compensation, whereas endothelium-dependent dilation in larger conduit pulmonary arteries appears to be reduced, possibly secondary to the effects of increased pulmonary arterial pressure and pulsatility. The limited data available to date from clinical studies is not necessarily incompatible with a similar interpretation. THERAPEUTIC CONSIDERATIONS From the above discussion we can conclude that the L-arginine-NO pathway likely plays a critical role in pulmonary hypertension; either in the case of a primary impairment in endothelial NO production or activity, or a relative deficiency, in which case the NO system acts as a failed mechanism of compensation. In any event, strategies to increase pulmonary vascular NO activity should be beneficial in the treatment of this condition. The simplest approach is the systemic administration of NO-donor compounds such as sodium nitroprusside or nitroglycerin. The obvious limitation in the therapeutic utility of these compounds is their profound effects on systemic arterial pressures. Patients with severe pulmonary hypertension are often in a delicate balance of hemodynamic compensation, which can be easily upset by systemic vasodilation, sometimes with fatal consequences. Alternate pharmacological approaches have been tested in experimental models, including the delivery of inhibitors of phospodiesterase via the tracheobronchial tree to elevate pulmonary cGMP (Ichinose et al., 1995), or the direct inhalation of analogues of cGMP (Lawson et al., 1995). In addition, the infusion of L-arginine, as discussed above, has been applied therapeutically to a limited number of clinical situations, including persistent pulmonary hypertension of the new born (McCaffrey et al., 1995) and pulmonary hypertension complicating heart failure. Newer NO donor compounds have also been tested in experimental models of pulmonary hypertension (Vanderford et al., 1994), but their potential clinical utility remains to be explored. The ideal pharmacological agent will deliver NO only to the pulmonary circulation. Being a gas, it is highly feasible to deliver NO by inhalation. It is beyond the scope of this manuscript to review the extensive literature which has developed recently on the therapeutic utility of inhaled NO. A more detailed analysis can be found in several recent reviews of the topic (Adnot and Raffestin, 1996). This treatment modality is now being commonly used in the management of pulmonary hypertension in several clinical situations including, persistent pulmonary hypertension of the newborn (Kinsella and Abman, 1993; Kinsella et al., 1992; Roberts et al., 1992; Abman, 1995; Lönnqvist et al., 1994), pulmonary hypertension following cardiac surgery (Shah et al., 1995; Ivy et al., 1996; Breuer et al., 1995; Beghetti et al., 1995; Journois et al.,

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1994; Rich et al., 1993; Haydar et al., 1992), respiratory distress syndrome (Pappert et al., 1995; Gerlach et al., 1993), among others (Channick et al., 1994; Weitzberg et al., 1993; Scherrer et al., 1996; Williamson et al., 1996). This approach has also been applied to the screening of pulmonary hypertension (Adatia et al., 1995; Sitbon et al., 1995) to establish vasodilator reserve either in evaluation of patients with primary disease (Sitbon et al., 1995), or in patients awaiting heart transplantation for heart failure (Adatia et al., 1995). Its therapeutic utility in patients with pulmonary hypertension of nonreversible causes, such as in primary pulmonary hypertension, is in question since the withdrawal of inhaled NO can precipitate a rebound increase in pulmonary vascular resistance (Lavoie et al., 1996; Miller et al., 1995), leading to destabilization of the delicate hemodynamic balance and fatalities have been reported (Partanen and Nieminen, 1995). However, inhaled NO has been successfully used as a bridge to transplantation in such cases (Snell et al., 1995). In the case of primary pulmonary hypertension in particular, the therapeutic challenge is to provide increases in NO activity selectively in the pulmonary circulation for prolonged periods, possibly for life. Strategies have been developed for delivery of long-term inhaled NO to patients with primary pulmonary hypertension in an ambulatory setting (Channick et al., 1996), but role in the treatment of the devastating condition remains to be established. In future, a gene therapy approach might offer the advantage of continuous overproduction of NO in the lung, at least for the period of transgene expression. Adenoviralmediated transfer of endothelial NOS through the tracheobronchial tree has been shown to reduce hypoxic vasoconstriction in rats (Janssens et al., 1996), without affecting systemic arterial pressure. However, adenoviral-mediated gene transfer may have limited therapeutic utility clinically due to the inflammatory response induced by viral proteins and relatively short duration of transgene expression (Lemarchand et al., 1993). Newer adenoviral constructs may overcome these limitations, or nonviral approaches to gene transfer in the lung may provide alternate approaches in the development of more clinically relevant gene transfer strategies for the treatment of pulmonary hypertension. REFERENCES Abenhaim, L., Moride, Y, Brenot, F. et al. (1996) Appetite suppressant drugs and the risk of primary pulmonary hypertension. N. Engl. J. Med., 335, 609–616. Abman, S.H. (1995) Inhaled nitric oxide therapy of severe neonatal pulmonary hypertension. Acta Anaesthesiol. Scand., 39 (Suppl. 105), 65–68. Adatia, L, Perry, S., Landzberg, M., Moore, P., Thompson, J.E. and Wessel, D.L. (1995) Inhaled nitric oxide and hemodynamic evaluation of patients with pulmonary hypertension before transplantation. J. Am. Coll. Cardiol, 25, 1656–1664. Adnot, S., Raffestin, B., Eddahibi, S., Braquet, P. and Chabrier, P.-E. (1991) Loss of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J.Clin. Invest., 87, 155– 162. Adnot, S. and Raffestin, B. (1996) Pulmonary hypertension: NO therapy? Thorax, 51, 762–764. Archer, S.L., Tolins, J.P., Raij, L. and Weir, E.K. (1989) Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium derived relaxing factor. Biochem. Biophys. Res. Commun., 164, 1198–1205. Archer, S.L., Rist, K., Nelson, O.P., DeMaster, E.G., Cowan, N.J. and Weir, E.K. (1990) Comparison of the hemodynamic effects of nitric oxide and endothelium-dependent vasodilators in intact lungs. J. Appl. Physiol, 68, 735–747. Barer, G., Emery, C., Stewart, A., Bee, D. and Howard, P. (1993) Endothelial control of the pulmonary circulation in normal and hypoxic rats. J. Physiol, 463, 1–16. Barnard, J.W., Wilson, P.S., Moore, T.M., Thompson, W.J. and Taylor, A.E. (1993) Effect of nitric oxide and cyclooxygenase products on vascular resistance in dog and rat lungs. J. Appl Physiol, 74, 2940–2948.

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Roos, C.M., Rich, G.F., Uncles, D.R., Daugherty, M.O. and Frank, D.U. (1994) Sites of vasodilation by inhaled nitric oxide vs. sodium nitroprusside in endothelin-constricted isolated rat lungs. J.AppL Physiol., 77, 51–57. Rubanyi, G.M. and Vanhoutte, P.M. (1988) Calcium and activation of the release of endothelium-derived relaxing factor. Ann. NY Acad. Sci, 522, 226–233. Rubin, LJ. (1997) Primary Pulmonary Hypertension. N.Engl. J.Med., 336, 111–117. Russ, R.D. and Walker, B.R. (1993) Maintained endothelium-dependent pulmonary vasodilation following chronic hypoxia in the rat. J. Appl Physiol., 74, 339–344. Scherrer, U., Vollenweider, L., Delabays, A., Savcic, M., Eichenberger, U., Kleger, G.R., Fikrle, A., Ballmer, P.E., Nicod, P. and Bärtsch, P. (1996) Inhaled nitric oxide for high-altitude pulmonary edema. N.Engl. J.Med., 334, 624–629. Shah, A.S., Smerling, A.J., Quaegebeur, J.M. and Michler, R.E. (1995) Nitric oxide treatment for pulmonary hypertension after neonatal cardiac operation. Ann. Thorac. Surg., 60, 1791–1793. Shaul, P.W., North, A.J., Brannon, T.S., Ujiie, K., Wells, L.B., Nisen, P.A., Lowenstein, C.J., Snyder, S.H. and Star, R.A. (1995) Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. Am.J.Respir. Cell Mol. Biol, 13, 167–174. Sitbon, O., Brenot, F, Denjean, A., Bergeron, A., Parent, F, Azarian, R., Herve, P., Raffestin, B. and Simonneau, G. (1995) Inhaled nitric oxide as a screening vasodilator agent in primary pulmonary hypertension: A dose-response study and comparison with prostacyclin. Am.J.Respir. Crit. Care Med., 151, 384–389. Snell, G.I., Salamonsen, R.F, Bergin, P., Esmore, D.S., Khan, S. and Williams, T.J. (1995) Inhaled nitric oxide used as a bridge to heart-lung transplantation in a patient with end-stage pulmonary hypertension. Am. J.Respir. Crit. Care Med., .151, 1263–1266. Sprague, R.S., Thiemermann, C. and Vane, J.R. (1992) Endogenous endothelium-derived relaxing factor opposes hypoxic pulmonary vasoconstriction and supports blood flow to hypoxic alveoli in anesthetized rabbits. Proc. Natl. Acad. Sci USA, 89, 8711–8715. Spriestersbach, R., Grimminger, F, Weissmann, N., Walmrath, D. and Seeger, W. (1995) On-line measurement of nitric oxide generation in buffer-perfused rabbit lungs. J.Appl Physiol., 78, 1502–1508. Stamler, J.S., Loh, E., Roddy, M.A., Currie, K.E. and Creager, M.A. (1994) Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans. Circulation, 89, 2035–2040. Steudel, W., Ichinose, F, Huang, PL., Hurford, WE., Jones, R.C., Bevan, J.A., Fishman, M.C. and Zapol, W.M. (1997) Pulmonary vasoconstriction and hypertension in mice with targeted disruption of endothelial nitric oxide synthase (NOS 3) gene. Circ. Res., 81, 34–1. Stewart, D.J., Levy, R.D., Cernacek, P. and Langleben, D. (1991) Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease? Ann. Intern. Med., 114, 464–469. Thusu, K.G., Morin, F.C., III, Russell, J.A. and Steinhorn, R.H. (1995) The cGMP phosphodiesterase inhibitor zaprinast enhances the effect of nitre oxide. Am.J.Respir. Crit. Care Med., 152, 1605–1610. Vallance, P., Leone, A., Calver, A., Collier, J. and Moncada, S. (1992) Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. J.Cardiovasc. Pharmacol., 20 Suppl. 12, S60-S62. Vanderford, P.A., Wong, J., Chang, R., Keefer, L.K., Soifer, S.J. and Fineman, J.R. (1994) Diethylamine/nitric oxide (NO) adduct, an NO donor, produces potent pulmonary and systemic vasodilation in intact newborn lambs.J.Cardiovasc. Pharmacol., 23, 113–119. Voelkel, N.F, Tuder, R.M. and Weir, E.K. (1997) Pathophysiology of primary pulmonary hypertension:from physiology to molecular mechanisms. In: Primary pulmonary hypertension, edited by Rubin, L.J. and Rich, S., pp. 83–133. New York: Marcel Dekker. Wang, D., Hsu, K., Hwang, C.-P. and Chen, H.I. (1995) Measurement of nitric oxide release in the isolated perfused rat lung. Biochem. Biophys. Res. Commun., 208, 1016–1020. Wanstall, J.C., Hughes, I.E. and O’Donnell, S.R. (1995) Evidence that nitric oxide from the endothelium attenuates inherent tone in isolated pulmonary arteries from rats with hypoxic pulmonary hypertension. Br.J.Pharmacol, 114, 109–114. Weir, E.K. and Archer, S.L. (1957) The mechanism of acute hypoxic vasoconstriction. FASEB J., 9, 183–189.

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16 Atherosclerosis: Role of NO John P.Cooke and Philip S.Tsao Division of Cardiovascular Medicine, Stanford University, Stanford, CA, USA

INTRODUCTION The most common cause of morbidity and mortality in Europe and the United States is atherosclerosis, a clinical entity that most physicians will deal with frequently in their practice. Atherosclerosis is a complex process that is thought to be initiated by a “response to injury” of the endothelium (Ross, 1997). The specific injuries that precipitate atherogenesis are not known, although risk factors have been identified. These include hypercholesterolemia, hypertension, diabetes mellitus, and tobacco use. Another important risk factor is a family history of premature atherosclerosis (i.e., first-degree relatives who have incurred myocardial infarction or stroke under the age of 60). This risk factor likely represents predisposing genetic factors that have not yet been elucidated. In addition to these traditional risk factors, there is accumulating evidence that elevated plasma levels of lipoprotein(a) and homocysteine also accelerate the process of atherosclerosis. Finally, obesity, type A personality, and sedentary lifestyle predispose to adverse vascular events (e.g., myocardial infarction). Elevated levels of serum cholesterol seem to be necessary for the progression of atherosclerosis. In countries where dietary intake of cholesterol and serum cholesterol levels are low, myocardial infarctions are much less common than in countries where individuals consume a Western diet and have higher cholesterol levels. It is the low-density lipoprotein (LDL) fraction of serum cholesterol that is positively correlated with adverse vascular events. Elevated levels of LDL cholesterol (particularly oxidized LDL) perturb the cell membrane, alter permeability and secretion, and are associated with the expression of intercellular adhesion molecules, cytokines, and growth factors. Recent evidence suggests that specific glycoprotein adhesion molecules

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(e.g., vascular cell adhesion molecule-1, or VCAM-1) and chemokines (e.g., monocyte chemotactic protein-1, or MCP-1) elaborated by endothelial cells may participate in monocyte adhesion and infiltration in vessels exposed to high levels of serum cholesterol (Cybusky and Gimbrone, 1991; Berliner et al., 1995). The expression of these adhesion molecules and chemokines may explain the observation that within several days of a high cholesterol diet, monocytes adhere to the endothelium, particularly at intercellular junctions (Ross, 1997). The monocytes migrate into the subendothelium, where they begin to accumulate lipid and become foam cells. This is the earliest event in the formation of the fatty streak. These activated monocytes (macrophages) release mitogens and chemoattractants that recruit additional macrophages as well as vascular smooth muscle cells into the lesion. As foam cells accumulate in the subendothelial space they distort the overlying endothelium, and eventually may even rupture through the endothelial surface. In these areas of endothelial ulceration, platelets adhere to the vessel wall, releasing epidermal growth factor, platelet-derived growth factor, and other mitogens and cytokines that contribute to smooth muscle migration and proliferation. These factors induce smooth muscle cells in the vessel wall to proliferate and migrate into the area of the lesion. These vascular smooth muscle cells undergo a change in phenotype from a “contractile” cell to a “secretory” cell. Secretory vascular smooth muscle cells elaborate extracellular matrix (i.e., elastin), which transforms the lesion into a fibrous plaque. The smooth muscle cells may also become engorged with lipid to form foam cells. The lesion grows with the recruitment of more cells, the elaboration of extracellular matrix, and the accumulation of lipid until it is transformed from a fibrous plaque to a complex plaque. The complex plaque typically is characterized by a fibrous cap which overlies a necrotic core. The necrotic core is composed of cell debris and cholesterol, and contains a high concentration of the thrombogenic tissue factor, secreted by macrophages. In later stage lesions, calcification may occur. Calcifying vascular cells in the vessel wall can transform into osteoblast-like cells and secrete bone proteins such as osteopontin (Parhami et al., 1997). Microscopic examination of these areas reveals histology very similar to bone tissue. Oxidized lipoprotein stimulates the elaboration of bone protein by these vascular cells. Intriguingly, oxidized lipoprotein reduces bone formation by osteoblasts (Parhami et al., 1997). This recent finding may account for the clinical observation that some patients with atherosclerosis (typically elderly women) appear (by X-ray) to have nearly as much calcium in their aorta as in their spine—one speculates as to which structure is holding them upright! By virtue of its bulk the complex plaque may limit blood flow. With moderate-sized lesions (i.e., occupying 50% of the cross-sectional area of the lumen), ischemia occurs only when the tissue supplied by the vessel requires more blood (e.g., exercise-induced myocardial ischemia, manifested by exertional angina). As the lesion becomes larger (i.e., 80–90% of the cross-sectional area), it may limit basal blood flow, causing ischemia at rest (e.g., rest angina). The complicated plaque is the major cause of acute cardiovascular events (e.g., unstable angina, myocardial infarction, embolie stroke, acute arterial occlusion). Hemorrhage into the plaque (secondary to spontaneous rupture of vasa vasorum supplying the lesion) can cause rapid expansion of the plaque and even luminal obstruction. Ulceration or rupture of the fibrous plaque (i.e., due to hemodynamic force or balloon angioplasty) exposes the highly thrombogenic necrotic core, leading to local thrombosis and/or distal embolization.

Correspondence: John P.Cooke, MD, PhD, Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305–5246, USA

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Plaque rupture is the most common cause of myocardial infarctions and stroke. Rupture of the complex plaque exposes the flowing blood to the highly thrombogenic constituents of the plaque (the foam cells, which elaborate tissue factor). Microscopic examination of the ruptured plaque generally reveals that the rupture has occurred at the shoulder of the lesion. In this area the fibrous cap can be seen to be thinned. Immunohistochemical studies reveal an intense concentration of macrophages in the area, which are elaborating copious amounts of metalloproteinases (Knox et al., 1997). These macrophages appear to be undermining the fibrous cap, weakening it and predisposing to its rupture under the stress of hemodynamic forces. It is not known what may be the trigger for this inflammation and destruction of the fibrous cap. However, an infectious etiology must be considered. A viral role in atherogenesis was proposed over two decades ago (Fabricant et al., 1973, 1978), and more recently there is accumulating evidence, albeit circumstantial, that infectious agents may trigger inflammatory processes that contribute to the pathophysiology of atherosclerosis. Seroepidemiological links have been made between cytomegalovirus infections and atherosclerosis in patients undergoing vascular surgery or in those with transplant coronary artery disease (Adam et al., 1987; Gratton et al., 1989; MacDonald et al., 1989). There is also histopathological evidence of the presence of cytomegalovirus or herpes simplex virus in atherosclerotic lesions in human vessels (Gyorkey et al., 1984; Benditt et al., 1983; Hajjar et al., 1987) Moreover, an avian herpes virus (Marek’s disease virus) can induce atherosclerosis in chickens, which can be prevented by immunization (Minick et al., 1079). Trials of antibiotics in patients with atherosclerosis are under way, in an attempt to test the hypothesis that infection plays a role in the progression of atherogenesis. The fibrous cap, weakened by the degradative action of the macrophages, ruptures under the stress of hemodynamic forces. With rupture of the plaque, thrombus forms in the fissures of the lesion. The thrombus often extends into, and may occlude, the lumen. Plaque rupture and thrombus formation is the most common cause of heart attack and stroke. Furthermore, as the thrombus organizes it can contribute to growth of the lesion and increase the symptoms of the patient. CLINICAL PRESENTATIONS OF ATHEROSCLEROSIS Atherosclerosis causes symptoms as the lesion impinges on the lumen of the vessel. Usually this reduction of lumen diameter takes decades to develop, and symptoms gradually increase over time as the lesion becomes flow-limiting (when it occupies about 50% of lumen diameter). Coronary atherosclerosis causes angina, typically described as a retrosternal pressure with exertion. Symptoms of coronary disease may appear at rest when the lesion becomes greater than 70% of lumen diameter. Carotid artery disease may cause transient ischemie attacks (amaurosis fugax; transient paresthesis, paraplegia, or dysphasia) or stroke. Disease in the leg arteries causes intermittent claudication (cramping pain with walking, relieved by standing still). Renal artery stenosis may cause hypertension and renal insufficiency. Mesenteric artery stenosis causes post-prandial pain and weight loss. When lesions in the conduit vessel become subocclusive (e.g., occupying greater than 80% of the crosssectional area), basal blood flow may be limited. Under these conditions, alterations may also occur at the level of the microvasculature (European Working Group on Critical Leg Ischemia, 1991). Because of reduced blood flow (and reduced shear stress), platelets and leukocytes may be more likely to adhere to the endothelium (as adherence is inversely dependent upon the level of shear stress). Neutrophils become activated and begin to generate superoxide anion. In this milieu of oxidative stress the endothelium begins to express adhesion molecules and chemokines, which further promotes interaction of circulating blood elements with the endothelium. Platelets release serotonin and thromboxane All, which induce vasoconstriction and further stimulate platelet adherence and aggregation. The beneficial effects of some

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pharmaceuticals (such as prostaglandin El analogues, naftidrofuryl, and pentoxifylline) may be due in part to their ability to disrupt this cycle of platelet and leukocyte interaction with the vessel wall. When atherosclerotic arterial occlusive disease of a vessel progresses to the point that symptoms cannot be medically controlled, interventional techniques, such as catheter balloon angioplasty or atherectomy, endarterectomy, or venous bypass grafting, are indi cated. Acute failure of these procedures is due generally to thrombosis precipitated by gross endothelial denudation and exposure of the thrombogenic constituents of the plaque. Hemorrhage into the vessel wall due to plaque rupture or dissection also contributes to early closure. Long-term success of these catheter-based procedures is limited by a different pathophysiologic process known as myointimal hyperplasia (Cooke and Candipan, 1994). This process is characterized by migration of vascular smooth cells from the media to the intima where they proliferate and transform into secretory cells, synthesizing extracellular matrix that contributes to the lesion. This process is precipitated by endothelial denudation, platelet-vessel wall interactions, and the release of growth factors such as plateletderived growth factor and fibroblast growth factor from platelets and injured vascular smooth muscle cells. Inflammatory cells also contribute, releasing cytokines (such as interleukin1) and other paracrine substances that stimulate migration and proliferation. In addition to myointimal hyperplasia, which causes intimai thickening and narrowing of the lumen, another process called vascular remodeling participates in renarrowing of the vessel. Vascular remodeling is not well understood, but can be observed as a reduction of the outer diameter of the vessel, possibly secondary to adventitial fibrosis. The combination of myointimal hyperplasia and negative remodeling may cause symptomatic restenosis in over 30% of patients within 6 months of balloon angioplasty or atherectomy, and contributes to most late failures of bypass grafts. ROLE OF THE ENDOTHELIUM Atherosclerosis begins with an alteration in the adhesiveness of the endothelium for circulating monocytes. In our laboratory, the focus is on this role of the endothelium in the initiation of atherogenesis. Specifically, we have directed our attention to an endogenous anti-atherogenic molecule that is derived from the endothelium. Most recently, we have found that the production of this factor can be modulated; a reduction in its synthesis accelerates atherosclerosis, whereas an increase in its synthesis suppresses and can even reverse atherosclerosis. This endothelium-derived relaxing factor is now known to be nitric oxide (NO). NO is derived from the metabolism of L-arginine to L-citrulline and NO by the enzyme NO synthase (Moncada and Higgs, 1995). NO is the most potent endogenous vasodilator known, and it exerts its actions in the same way as do exogenous nitrovasodilators such as nitroglycerine. NO released from the endothelium diffuses to the subjacent vascular smooth muscle and activates soluble guanylate cyclase within the vascular smooth muscle, leading to the production of cyclic guanosine monophosphate (cGMP). This cyclic nucleotide is the second messenger for the action of endothelium-derived NO as well as exogenous nitrovasodilators, and it activates cGMP-dependent kinases and phosphatases that mediate vascular smooth muscle relaxation. NO is not only a potent vasodilator but also has important effects on circulating blood elements. NO inhibits platelet adherence and aggregation (Cohen, 1995). Together, the endothelial products NO and prostacyclin confer a resistance to platelet-vessel wall interaction. NO exerts its effect on platelet reactivity in part by stimulating intra-platelet production of cGMP which subsequently phosphorylates proteins which regulate platelet activation and adherence. Platelets themselves contain small amounts of NO synthase and are capable of generating NO which may act as a brake on their activation. NO also inhibits adherence of leukocytes to the endothelium (Kubes et al., 1991). This salutary effect of NO was first discovered using models of ischemia-reperfusion. When the coronary artery of an experimental

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animal is ligated, this induces ischemia of the myocardium subserved by that vessel. When the ligature is released, the ensuing reperfusion is associated with further injury to the myocardium which is in part due to the adherence and infiltration of neutrophils, and the concomitant release of oxygen-derived free radicals. The adherence of leukocytes and subsequent reperfusion injury can be markedly inhibited by the simultaneous perfusion of the coronary artery by exogenous NO donors (Johnson et al., 1990). NO AND MODULATION OF MONOCYTE ADHERENCE Other investigators have looked at the interaction of monocytes with endothelial cells. The ability of monocytes to bind to endothelial cells in vitro is inhibited by exogenous NO in a dose-dependent manner (Bath et al., 1991). These investigations suggest that NO is a potent modulator of leukocyte-vessel wall interactions. The mechanism by which NO inhibits monocyte adherence and infiltration is under investigation in our laboratory and others. NO has both acute and chronic effects on monocyte adhesion. Within minutes of exposure to exogenous NO, endothelial cells become more resistant to monocyte adherence (Tsao et al., 1995). Because of the rapid time course, this effect of NO must be due to inhibition of signalling pathways involved in adhesion— perhaps by a cGMP-dependent mechanism. More chronic exposure to NO suppresses gene expression of adhesion molecules (such as VCAM-1), and chemokines (such as MCP-1) involved in monocyte adhesion and infiltration (Zeiher et al., 1995; Tsao et al., 1996, 1997). By contrast, inhibition of NO synthesis increases the expression of endothelial proteins required for monocyte adhesion (Tsao et al., 1996). NO appears to exert its effects on gene expression by blocking the activation of specific transcriptional proteins (such as nuclear factor KB) (Figure 16–1) (Zeiher et al., 1995; Tsao et al., 1996, 1997). Accumulating evidence supports the hypothesis that NO exerts its effect on monocyte adherence and chemotaxis in part by modulating the activity of redox-responsive transcriptional pathways (Tsao et al., 1997; Marui et al., 1993; DeCaterina et al., 1995). We have observed that vascular smooth muscle cells exposed to oxidized lipoprotein or cytokines begin to elaborate superoxide anion. This generation of reactive oxygen species is associated with transcription of MCP-1 mRNA and increased chemotactic activity for monocytoid cells (Tsao et al., 1997). These effects are all suppressed by the NO-donor, DETANO. These observations indicate that NO reduces transcription of MCP-1; it is also possible that NO may reduce the half-life of MCP-1 mRNA although this was not directly examined in this study. NO may act by reducing intracellular oxidative stress. There are several possible mechanisms by which NO may reduce oxidative stress. NO can scavenge superoxide anion, although the product of this reaction, peroxynitrate anion, is itself a highly reactive free radical (Ischiropoulos, 1992; Lin, 1995). However, it is possible that peroxynitrate anion could subsequently nitrosylate sulfhydryl groups to form S-nitrosothiols (Radi et al., 1991). This class of molecules is known to induce vasodilation, inhibit platelet aggregation, and interfere with leukocyte adherence to the vessel wall (Stamler et al., 1992, 1989). Another mechanism by which NO may ameliorate oxidative stress is by terminating the autocatalytic chain of lipid peroxidation that is initiated by oxidized LDL or intracellular generation of oxygen-derived free radicals. Indeed, exogenous NO inhibits copper-induced oxidation of LDL cholesterol, causing a lag in the formation of conjugated dienes (Hogg et al., 1993). Finally, NO may directly suppress the generation of oxygen-derived free radicals by nitrosylating, and thereby inactivating oxidative enzymes. This hypothesis is supported by the observation that the generation of superoxide anion by stimulated neutrophils is reduced by their exposure to exogenous NO (Clancy et al., 1992). This is due to the inactivation of NADPH oxygenase, a multimeric enzyme, with cytosolic and particulate components. The particulate component is vulnerable to nitrosylation by NO (either at its heme moiety or sulfhydryl group), which prevents its

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Figure 16±1. Atherosclerotic risk factors such as hypercholesterolemia, hypertension, tobacco, and diabetes mellitus lead to increased free radical production and decreased nitric oxide activity in endothelial cells. This endothelial dysfunction not only has acute effects on vascular tone, but also chronic effects on vessel structure. Increased superoxide anion leads to activation of NFicB via phosphorylation and degradation of the inhibitor protein IKB a. NFicB is then free to translocate into the nucleus to initiate transcription of proatherogenic genes such as VCAM-1 and MCP-1. Nitric oxide can inhibit these processes by inhibiting superoxide production, directly scavenging superoxide anions, as well as increasing the transcription and activity of iKBoc. Moreover, since NO is a paracrine factor, it can have important inhibitory effects on circulating leukocytes and underlying smooth muscle cells.

association with the cytosolic component, and reconstitution of the active enzyme. A similar phenomenon may occur in endothelial cells. This would explain the observation of Niu and colleagues (1994), who reported that antagonism of endogenous NO production increases oxidative stress in HUVECs, as demonstrated using redox-sensitive fluorophores. Furthermore, Pagano and colleagues (1993) have shown that exogenous NO donors inhibit the generation of superoxide anion by the endothelium of rabbit thoracic aortae treated ex vivo with antagonists of superoxide dismutase. It is well established that hypercholesterolemia reduces the activity of endothelium-derived NO (Heistad et al., 1984; McLenahan et al., 1991). In parallel, the endothelium begins to generate superoxide anion (McLenahan et al., 1991). This alteration in endothelial redox state triggers the oxidant-sensitive transcriptional cascade that results in the activation of genes encoding molecules that regulate endothelial adhesiveness (Ohara et al., 1991). The cytokine-induced activation of VCAM-1 and MCSF in cultured endothelial cells is suppressed by antioxidants or NO donors (Tsao et al., 1997; Marui et al., 1993). This effect of NO appears to be due in

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part to stabilization and/or increased expression of IicBa, which complexes with NFicB to inhibit its transcriptional activity (Marui et al., 1993). Our data are consistent with this model of an oxidant-responsive NFKB-mediated pathway that is modulated by NO. Recently, Zeiher and colleagues (1995) have also provided evidence that NO inhibits MCP-1 expression in cytokine-stimulated HUVECs, in a cGMP-independent fashion. To summarize, there is accumulating in vitro and in vivo evidence that NO suppresses the expression of monocyte adhesion molecules and chemokines. NO appears to exert its effects by reducing intracellular oxidative stress, thereby defusing oxidant-triggered transcription. Therapeutic strategies to enhance vascular NO activity may therefore inhibit the progression of atherosclerosis as well as restore normal vasoreactivity. EFFECTS ON VASCULAR GROWTH NO also regulates the growth of vascular smooth muscle cells. In vitro, NO-donors inhibit the proliferation of vascular smooth muscle cells; this effect is mimicked by exogenous administration of 8-bromo-cGMP, a stable analog of the second messenger of NO action (Garg and Hassid, 1990). Other agents such as atrial natriuretic peptide which increase the intracellular levels of cGMP inhibit proliferation of vascular smooth muscle cells in culture. Does NO inhibit the proliferation of vascular smooth muscle cells in vivo? Some initial studies indicate that NO does indeed play an important role in controlling vascular growth. In a number of disease states where the release of NO is reduced or abolished, such as restenosis, hypercholesterolemia, and hypertension, there is an increase in the proliferation of vascular smooth muscle cells within the media and the intima. In experimental animal models, augmentation of endogenous NO synthesis (as with administration of the NO precursor L-arginine), inhibits “restenosis” (myointimal hyperplasia) after balloon angioplasty (Candipan, 1996). In one study, after subjecting the carotid artery to balloon angioplasty, we successfully transfected the vessel with a plasmid construct containing the gene encoding NO synthase. This gene transfer had the effect of enhancing NO synthesis locally and inhibiting myointimal hyperplasia (von der Ley en et al., 1995). More recently, we have shown that the intramural administration of a single dose of L-arginine, at the time of balloon angioplasty, can markedly inhibit myointimal hyperplasia 2–4 weeks later (Schwarzacher et al., 1997). This effect of L-arginine is associated with increased local production of NO, probably due to utilization of L-arginine by induced NO synthase in vascular smooth muscle cells in the injured area. ENDOTHELIAL DYSFUNCTION: ROLE IN VASCULAR DISEASE Proliferation of vascular smooth muscle cells, as well as monocyte adherence and infiltration, platelet adherence, and aggregation, are key processes involved in atherogenesis. Because endothelium-derived NO inhibits each of these processes, we have proposed that NO is an endogenous anti-atherogenic molecule (Cooke and Tsao, 1993). Therefore an endothelial injury or alteration which results in a reduction in NO activity could promote atherogenesis. Indeed, a number of disorders that are associated with atherosclerosis, are also associated with an impairment in the ability of the endothelium to elaborate NO (Cooke and Dzau, 1993). In animal models, and in humans, hypercholesterolemia, hypertension, homocysteinemia, diabetes mellitus, and exposure to tobacco are each associated with impaired NO-dependent vasodilation. This endothelial impairment occurs well before any structural changes of atherogenesis are detected. Indeed when otherwise normal vessels are exposed in vitro to oxidized lipoproteins, an impairment in NO-

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dependent vasodilation can be detected within minutes. This reduction in NO activity is due to a reduced synthesis and/or an increased degradation of the molecule. If a reduction in NO activity promotes atherogenesis, a restoration of NO activity might be expected to retard progression of the disease. In support of this hypothesis, a wide variety of interventions that inhibit the progression of atherosclerosis, are also associated with improvements in NO-dependent vasodilation, including antilipid agents, antioxidants, estrogen, angiotensin-converting enzyme inhibitors, and L-arginine (Cooke et al., 1992). This of course does not imply causality, and the improvement in endothelial function could be a secondary phenomenon. However, we have accumulated evidence that indicates a direct role for endothelium-derived NO as an anti-atherogenic molecule. We reasoned that if we could increase the synthesis of NO by the vessel wall, a number of key processes in atherosclerosis would be inhibited and progression of the disease halted. To test this hypothesis NZW rabbits were placed on normal or high cholesterol diet; some of the animals on the high cholesterol diet also received supplemental dietary arginine or methionine to increase the intake of these amino acids six-fold (Cooke et al., 1992; Wang et al., 1994). After 10 weeks the thoracic aortae and coronary arteries were harvested for studies of vascular reactivity and histomorphometry. The supplemental dietary arginine did not alter the lipid profile nor any hemodynamic parameters in the hypercholesterolemic animals; the only difference between the hypercholesterolemic groups was that plasma arginine was doubled in the hypercholesterolemic animals receiving dietary arginine supplementation. At 10 weeks, the thoracic aortae and coronary arteries were harvested for studies of vascular reactivity and histomorphometry. As expected, NO-dependent vasodilation to acetylcholine was inhibited in the hypercholesterolemic animals receiving vehicle. By contrast hypercholesterolemic animals receiving L-arginine had an improvement in NOdependent vasodilation. The improvement of NO-dependent vasodilation in hypercholesterolemic animals receiving L-arginine was associated with a striking effect on vascular structure. A significant reduction was observed in the surface area of the thoracic aorta involved by lesions in the hypercholesterolemic animals receiving arginine (Figure 16–2) (Cooke et al., 1992). In the left main coronary artery the differences were even more striking; no lesions at all were observed in the hypercholesterolemic animals receiving L-arginine (Wang et al., 1994). The mechanism by which dietary arginine inhibits atherogenesis is being elucidated in our laboratory. It appears to be due in part to an inhibition of monocyte-endothelial cell interaction in the hypercholesterolemic animals. After only 2 weeks of a high cholesterol diet, we find that the thoracic aorta of hypercholesterolemic animals has increased adhesiveness for monocytes (Tsao et al., 1994). Compared with the thoracic aortae from normal animals, those from hypercholesterolemic rabbits manifest a three-fold increase in the number of adherent cells. The increase in cell binding is attenuated in thoracic aortae from hypercholesterolemic animals receiving dietary arginine supplements (Tsao et al., 1994). This is associated with an increase in the release of NO from these tissues (Tsao et al., 1994), as well as a reduced vascular expression of MCP-1 (Tsao et al., 1997). To provide further evidence that NO regulates monocyteendothelial cell interaction, some animals were exposed to nitro-arginine, an antagonist of NO synthase. The number of adherent cells was dramatically increased in normocholesterolemic animals receiving nitroarginine (Tsao et al., 1994). The increased adhesiveness of the endothelium in the nitro-arginine treated animals was associated with an increased vascular expression of MCP-1 (Tsao et al., 1997). Thus, reductions in NO activity by hypercholesterolemia, or inhibition of NO synthesis, is associated with increased monocyte-endothelial cell binding, possibly due to the increased expression of specific chemokines and adhesion molecules.

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Figure 16±2. Photomicrographs of thoracic aortae from New Zealand White rabbits fed a high cholesterol diet in the absence (top panel) or the addition (bottom panel) of dietary arginine.

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NO AND APOPTOSIS More recently we have found that administration of arginine can also restore NO activity in hypercholesterolemic rabbits with established lesions. This improvement in NO activity is associated with regression of pre-existing lesions (Candipan et al., 1996). Preliminary studies indicate that the enhancement of vascular NO activity causes regression by inducing apoptosis of macrophages within the lesion. Apoptosis has been reported to occur in vascular cells of human atherosclerotic plaque (Isner et al., 1995; Geng and Libby, 1995). Factors involved in the initiation and regulation of apoptosis in atherosclerosis have not been fully elucidated, but immunohistochemical studies provide evidence for several proteins known to participate in apoptosis, including p53 (Bennett et al, 1995). Among the myriad pathways that may be involved, there is accumulating evidence to implicate L-arginine/NO synthase. Cytokine-mediated activation of iNOS induces apoptosis of macrophages in vitro (Messmer et al., 1995). The effect of iNOS activation is augmented by additional L-arginine, and attenuated by antagonists of NO synthase. To determine if modulation of vascular NO activity could induce apoptosis of macrophages in the intimai lesion, we performed the following study. Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 weeks and subsequently placed on 2.5% L-arginine HC1 in the drinking water, and the cholesterol diet continued for two weeks, at which time the aortae were harvested for histological studies. We observed by Hoechst staining that L-arginine treatment increased the number of apoptotic cells (largely macrophages) in the intimai lesions by three-fold. In subsequent studies, aortae were harvested for ex vivo studies. Aortic segments were incubated in cell culture medium for 4 to 24 hours with modulators of NO synthase pathway. The tissues were then collected for histological studies, and the conditioned medium collected for measurement of nitrogen oxides by chemiluminescence. Addition of sodium nitroprusside to the medium caused a time-dependent increase in apoptosis of vascular cells (largely macrophages) in the intimai lesion. L-arginine (10−3 M) had an identical effect on apoptosis, which was associated with an increase in NOX released into the medium. These effects were not mimicked by D-arginine, and they were antagonized by the NO synthase inhibitor, L-nitroarginine (10−4 M). The effect of L-arginine was not influenced by an antagonist of cGMP-dependent protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8-Br-cyclic GMR These results indicated that supplemental L-arginine induces apoptosis of macrophages in intimai lesions by its metabolism to nitric oxide, which acts via a cyclic-GMP independent pathway. Furthermore, these studies explain our previous observation that supplementation of dietary arginine induces regression of atheroma in this animal model. It is likely that iNOS expressed by cells within the lesion is responsible for the effect of L-arginine. Indeed, previous immunohistochemical studies have detected iNOS in the intimai macrophages and vascular smooth muscle cells of human atherosclerotic plaque (Ohara et al., 1993). These are activated cells which also produce superoxide anion. In this milieu, the product of iNOS is quickly transformed into peroxynitrite anion, a highly reactive free radical (Ischiropoulos et al., 1992). Peroxynitrite anion is cytotoxic and induces apoptosis (Lin et al., 1995). Peroxynitrite anion can also affect cell function by nitrosylating tyrosine residues that are involved in the signal transduction of transmembrane receptors. Using monoclonal antibodies directed against nitrotyrosine, evidence of peroxynitrite formation has been observed in human atherosclerotic plaque (Beckman et al, 1993). This is relevant to the present study, since peroxynitrite anion is likely the NO species mediating the effects observed in this study. Previous studies have suggested that apoptosis induced by iNOS activity may be mediated in part by mechanisms independent of cGMP (Pollman et al., 1996). Consistent with this observation is our finding that the various manipulations of the cGMP pathway did not influence apoptosis in the lesions of this animal model.

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The activation of iNOS may have complex effects on the evolution of atherosclerotic plaque. By inducing cell death, iNOS activation may contribute to the development of the “necrotic core” of complex lesions. One might also speculate that iNOS may be involved in the characteristic atrophy of the media beneath atheroma or the dissolution of the fibrous cap by activated macrophages. Peroxynitrite anion produced by these activated macrophages could induce apoptosis of vascular smooth muscle. Furthermore, peroxynitrite anion may reduce collagen formation by vascular cells, and activate metalloproteinases which degrade extracellular matrix (Rajagopalan et al., 1996). These actions of peroxynitrite anion would contribute to plaque instability and have led some to explore antagonism of iNOS as a potential therapeutic avenue. However, it is likely that such a strategy would have unintended consequences. Antagonism of iNOS activity could promote platelet aggregation, leukocyte adherence, vasoconstriction, and proliferation of vascular smooth muscle cells and macrophages. We speculate that iNOS may be in fact a countervailing force in the accretion of atherosclerotic plaque. Furthermore, by reducing proliferation and by promoting apoptosis of macrophages in the lesion, iNOS activation may lead to plaque stabilization and even regression, as suggested by our observations. It is worthy of emphasis that both macrophages and vascular smooth muscle cells contribute to the intimai lesion in the balloon-injured hypercholesterolemic rabbits, but we observed that it was largely the macrophages that were undergoing apoptosis. ADMA: A NEW RISK FACTOR FOR ATHEROSCLEROSIS? The mechanism by which hypercholesterolemia impairs the L-arginine/NO pathway is probably multifactorial, and dependent upon the stage of atherosclerosis. Most studies have reported that the defect in hypercholesterolemia and therosclerosis is reversed by exogenous L-arginine (Cooke et al., 1991; Drexler et al., 1991; Creager et al., 1992). Recently, asymmetric dimethylarginine (ADMA) has been characterized to be an endogenous, competitive inhibitor of NO synthase (Vallance et al., 1992). ADMA has been shown to be synthesized by human endothelial cells (Pickling et al., 1993). The plasma level of ADMA is elevated in hypercholesterolemic rabbits (Bode-Böger et al., 1996; Böger et al., 1997a) as well as in hypercholesterolemic (Böger et al., unpublished observation) and atherosclerotic humans (Böger et al., 1997b) concomitantly with impaired endothelial NO elaboration. We have examined the effect of ADMA upon cultured endothelial cells. Incubation of endothelial cells with ADMA (at concentrations that are observed in hypercholesterolemic humans) inhibited NO production. This effect was associated with increased endothelial superoxide radical elaboration and NFkBB activation, resulting in enhanced MCP-1 expression and endothelial adhesiveness for monocytes. These effects of ADMA were reversed by L-arginine. Vascular NO activity is decreased in hypercholesterolemia and atherosclerosis, leading to impaired endothelium-dependent vasodilation (Creager et al., 1990), increased platelet aggregability (Wolf et al., 1997), and monocyte adhesiveness for the endothelium (Theilmeier et al., 1997). This impaired NO activity may contribute to the development and progression of atherosclerosis. Indeed, in animal models of hypercholesterolemia, pharmacological inhibition of NO synthase promotes atherosclerosis (Tsao et al., 1994b; Cayatte et al., 1994; Naruse et al., 1994). On the other hand, as previously mentioned, enhancement of endogenous NO formation with L-arginine supplementation improves endothelial function, inhibits platelet activation and monocyte adhesiveness for the endothelium, and slows progression of lesions or even induces regression (Cooke et al., 1992; Böger et al., 1995; Candipan et al., 1996; Tsao et al., 1994b; Creager et al., 1990; Tsao et al., 1994a). The mechanism(s) leading to the impaired NO activity in hypercholesterolemia and atherosclerosis has remained unclear. Increased degradation and/or reduced synthesis of NO may be involved. The mechanism

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Figure 16±3. Asymmetric dimethylarginine (ADMA) can competitvely inhibit the production of NO from NOS. ADMA is normally converted by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline, which is then recycled back to L-arginine. ADMA can also decrease the uptake of L-arginine via the cationic transporters.

by which L-arginine exerts its beneficial effects is likely through enhanced production of endotheliumderived NO. However, it is not yet completely understood how exogenous L-arginine increase NO elaboration in hypercholesterolemia, given the low Km of the purified endothelial NO synthase (2.9 µM (Pollock et al., 1991)) and the relatively high physiological plasma concentrations of L-arginine (60–100 µmol/L), which are unchanged in hypercholesterolemia (Laghi-Pasini et al., 1992). We suggest that this “L-arginine paradox” is likely due to the presence of endogenous competitive NOS inhibitors like ADMA (Figure 16–3) (Böger et al., 1997a). In 1992, Vallance et al., (1992) observed that ADMA, but not SDMA, competitively inhibits NO synthesis. ADMA plasma levels, which are 1.0±.0.1 mmol/L in healthy humans, are elevated to 2.2±.0.2 mmol/L in hypercholesterolemic individuals (Cooke et al., unpublished observation). In elderly patients with peripheral arterial disease and generalized atherosclerosis, ADMA levels range from 2.5 to 3.5 mmol/L, corresponding to the severity of the vascular disease (Böger et al., 1997b). Increased ADMA levels are associated with reduced NO elaboration in hypercholesterolemic subjects (Laghi-Pasini et al., 1992) and in atherosclerotic patients (Böger et al., 1997b), as judged by reduced urinary nitrate excretion and impaired endothelium-dependent, NO-mediated forearm vasodilation. We have found that intracellular ADMA levels within endothelial cells are considerably higher than those in conditioned media from human endothelial cells. The same may be true for ADMA levels in endothelial cells in vivo in respect to human plasma. Our observation that endothelial cells are a source of ADMA corroborates the previous finding by Fickling et al (1993) that human endothelial cells release ADMA and

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SDMA. ADMA competitively inhibits endothelial NO synthesis in an autocrine manner. This observation is in line with previous findings that exogenous ADMA concentrations between 1 and 10 µM affect the activity of the NO synthase in rat mesentery tissue (Kurose et al., 1995) in rat brain (Faraci et al., 1995), and in cultured macrophages (Fickling et al., 1993). Faraci et al., (1995) calculated an IC50 value for the inhibition of NO production in rat cerebellar homogenate by ADMA of 1.8 ± .1 µM, and Fickling et al., (1993) reported that 2 and 10 µM ADMA inhibited nitrite production in LPS-simulated J774 macrophages by 17 and 33% respectively. Taken together with our recent observations, these data suggest that ADMA may be a potential autocrine regulator of endothelial NO synthase. The source of ADMA in endothelial cells is currently unclear. Dimethylarginines are likely the result of degradation of methylated proteins (McDermott, 1976; MacAllister et al., 1996). The specific enzyme Sadenosylmethionine: protein arginine N-methyl-transferase (protein methylase I) has been shown to methylate internal arginine residues in a variety of polypeptides, yielding NG-monomethyl-L-arginine, NG,NG-dimethyl-L-arginine, and NG, NG-dimethyl-L-arginine upon proteolysis (Rawal et al., 1995). The metabolism of ADMA, but not SDMA, occurs via hydrolytic degradation to citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) (MacAllister et al., 1994). Inhibition of DDAH causes a gradual vasoconstriction of vascular segments, which is reversed by L-arginine (MacAllister et al., 1996). This latter finding also suggests that regulation of intracellular ADMA levels affects NO synthase activity. Intriguingly, recent data from our laboratory indicate that low-density lipoprotein increases endothelial cell ADMA elaboration. Although the mechanism by which LDL may affect ADMA formation or metabolism is currently unknown, this finding suggests that elevated ADMA levels may mediate some of the effects of LDL on the endothelium in an autocrine manner. Most importantly, within the concentration range we found in cultured endothelial cells (5–0 µM), ADMA can induce some of the pathophysiological changes of the endothelium that occur in hypercholesterolemia. Inhibition of endothelial NO synthase activity is associated with a concentration-dependent increase of endothelial adhesiveness for human THP-1 monocytoid cells. This effect of ADMA and L-NMMA mimics the increase in adhesiveness observed when endothelial cells are pre-incubated with nLDL; in both cases, adhesion is diminished by co-incubating endothelial cells with L-arginine. Supplementation of cholesterol-fed rabbits with L-arginine has previously been shown to reduce aortic endothelial asgesiveness for monocytes (Tsao et al., 1994bb). The cumulative data strongly suggest that NO is an endogenous anti-atherogenic molecule. Therefore impairment of NO activity (by hypercholesterolemia, diabetes mellitus, hypertension, homocysteinemia, or tobacco use), likely plays a role in the initiation of atherosclerosis. Preliminary studies indicate that many disorders associated with endothelial dysfunction and/or atherogenesis are also associated with elevated levels of ADMA. SUMMARY Vascular disease begins with an alteration in the endothelium which is characterized by an increase in intracellular oxidative stress, and the activation of oxidant-response genes regulating the expression of adhesion molecules and chemokines. These changes promote interaction of the endothelium with circulating blood elements. Monocyte infiltration and foam cell formation ensue, followed by further endothelial dysfunction and damage which precipitates platelet adherence and proliferation of vascular smooth muscle. These key processes in atherogenesis are opposed by nitric oxide. NO suppresses the expression and signaling of adhesion molecules involved in monocyte adhesion to the vessel wall, and inhibits platelet adherence and vascular smooth muscle cell proliferation. The NO synthase pathway is perturbed by hypercholesterolemia and other metabolic disorders that predispose to atherosclerosis. It is likely that basic

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Stamler, J.S., Mendelsohn, M.E., Amarante, P., Smick, D., Andon, N., Davies, P.P., Cooke, J.R and Loscalzo, J. (1989) N-acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor. Circ. Res., 65, 789–95. Stamler, J.S., Simon, D.I., Osborne, J.A., Mullins, M.E., Jaraki, O., Michel, T., Singel, D.J. and Loscalzo, J. (1992) Snitrosylation of proteins with nitric oxide: Synthesis and characterization of biologically active compounds. Proc. Natl. Acad. Sci. USA, 89, 444–48. Theilmeier, G., Chan, J., Zalpour, C. et al. (1997) Adhesiveness of mononuclear cells in hypercholesterolemic humans is normalized by dietary L-arginine. Arterioscler. Thromb. Vase. Biol., (in press). Tsao, P., McEvoy, L.M., Drexler, H., Butcher, E.G. and Cooke, J.P. (1994b) Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine. Circulation, 89, 2176–2182. Tsao, PS., Buitrago, R., Chan, J.R. and Cooke, J.P. (1996) Fluid flow inhibits endothelial adhesiveness: Nitric oxide and transcriptional regulation of VCAM-1. Circulation, 94, 1682–1689. Tsao, P.S., Lewis, N.R, Alpert, S. and Cooke, J.R (1995) Exposure to shear stress alters endothelial adhesiveness: Role of nitric oxide. Circulation, 92, 3513–3519. Tsao, P.S., Theilmeier, G., Singer, A.H. et al. (1994a) L-arginine attentuates platelet reactivity in Hypercholesterolemic rabbits. Arterioscler. Thromb., 14, 1529–1533. Tsao, P.S., Wang, B.Y., Buitrago, R. et al (1997) Nitric oxide regulates monocyte chemotactic protein-1. Circulation, 96, 934–940. Vallance, R, Leone, A., Calver, A., Collier, J. and Moncada, S. (1992) Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. J.Cardiovasc. PharmacoL, 20(Suppl. 12), S60–S62. von der Ley en, H., Gibbons, G.H. et al. (1995) Gene therapy inhibiting neointimal vascular lesion: In vivo transfer of endothelial cell nitric oxide synthase gene. Proc. Natl Acad. Scl USA, 92, 1137–1141. Wang, B., Singer, A., Tsao, R et al. (1994) Dietary arginine prevents atherogenesis in the coronary artery of the hypercholesterolemic rabbit. J.Am. Coll. Cardiol, 23, 452–58. Wolf, A., Zalpour, C., Theilmeier, G. et al (1997) Dietary L-arginine supplementation normalizes platelet aggregation in hypercholesterolemic humans. J.Am. Coll Cardiol, 29, 479–185. Zeiher, A.M., Fisslthaler, B. et al. (1995) Nitric oxide modulates expression of monocyte chemoattractant protein-a in cultured human endothelial cells. Circ. Res., 76, 980–986.

17 Nitric Oxide in Myocardial and Splanchnic Ischemia/ Reperfusion Allan M.Lefer and Rosario Scalia Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA

INTRODUCTION Abrupt shutting off blood flow (i.e., ischemia) to a vascular bed followed by reperfusion (i.e., reestablishment of blood flow) results in a severe form of injury to the tissue perfused by that vascular bed. This phenomenon is known as “reperfusion injury”. Reperfusion injury occurs in a variety of vascular beds, including the coronary (Van Benthuysen et al., 1987), mesenteric (Lefer et al., 1991), renal (Lieberthal et al., 1989), cerebral (Rosenblum et al., 1992), and skeletal muscle (Korthuis et al., 1988) circulations. One of the earliest important events after reperfusion of an ischemic bed is a significant degree of endothelial dysfunction characterized by the loss of endothelium-derived relaxing factor (EDRF) (Tsao et al., 1990), now known to be nitric oxide (NO) (Moncada et al., 1990). This reduction in NO formation and release is observed both in response to challenge with endothelium-dependent vasodilators (e.g., acetylcholine, A23187), so-called agonist-mediated NO release, as well as in response to NO synthase inhibitors [e.g., NGnitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA)], which unmask the basal release of NO (Moncada et al., 1990). A significant and sustained endothelial dysfunction has been observed in myocardial ischemiareperfusion (Tsao et al., 1990) and in splanchnic ischemia-reperfusion (Carey et al., 1992). This may contribute significantly to the reperfusion injury of the underlying tissue, because NO is known to induce vasorelaxation (Furchgott and Zawadzki, 1980), inhibit platelet aggregation (Radomski et al., 1991), quench superoxide radicals (Rubanyi et al., 1986; Gryglewski et al., 1986), and attenuate adherence of polymorphonuclear (PMN) leukocytes to the endothelium (McCall et al., 1988; Kubes et al., 1991). The endothelial dysfunction occurring in ischemia/reperfusion, is characterized by an abrupt decrease in activity

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of nitric oxide released from the affected endothelium. This marked decrease in NO activity is an important event signalling early pathophysiologic changes and serves as the hallmark of the first phase of reperfusion injury, termed the “endothelial trigger”. This endothelial trigger sets up the cascade of pathophysiological events manifest in reperfusion injury leading to a second major event resulting in tissue injury (Lefer and Lefer, 1993). This second major event, directly dependent on the endothelial dysfunction is known as the “neutrophil amplification” step. This relationship was originally coined by Bulkley (1989) in reference to splanchnic ischemia/reperfusion but clearly pertains to myocardial ischemia/reperfusion and to that of other regional vascular beds (e.g., renal, cerebral) (Lieberthal et al., 1989). It also occurs in total body ischemia/ reperfusion such as that occurring in hemorrhage/reinfusion (Wang, 1993) and in endotoxemia/sepsis (Siegfried et al., 1992; Wang et al., 1995). This chapter will focus on the role of nitric oxide in myocardial ischemia-reperfusion (MI/R) and splanchnic ar tery occlusion reperfusion (SAO/ R) injury, since most of the NO related data have been obtained in these two forms of ischemia/reperfusion (I/R). NO AND THE ENDOTHELIUM IN ISCHEMIA/REPERFUSION A. Time Course of NO Changes in Ischemia/Reperfusion A variety of well designed experiments have been conducted in order to characterize the time course of loss of endothelial derived nitric oxide (EDNO) in ischemia/reperfusion. The first complete study of this type was reported by Tsao et al. (1990) who studied MI/ R in cats. Cats were subjected to 90 minutes of complete occlusion of the left anterior descending (LAD) coronary artery followed by either no reperfusion (i.e., 0 min) or reperfusion of 2.5, 5, 10, 20, 60, 180 and 270 minutes. LAD coronary artery rings were challenged with two endothelium-dependent vasodilators: acetylcholine (ACh), a receptor mediated endothelium-dependent dilator, and A23187, a non-receptor mediated endothelium-dependent dilator. Responses to these agents were compared to an endothelium-independent dilator, acidified sodium nitrite (NaNO2). These responses were also compared to vasorelaxant responses obtained with the same three vasodilators in the non-ischemic control left circumflex coronary artery. Figure 17–1 illustrates the vasorelaxant responses of these LAD coronary artery rings to ACh and NaNO2 at key time points. Surprisingly, total occlusion of the LAD for 90 minutes without reperfusion did not diminish the vasorelaxant response to either ACh or NaNO2. However, 90 minutes of ischemia followed by only 2.5 minutes of reperfusion resulted in a marked attenuation of the vasorelaxation response of ACh (also to A23187) but not to NaNO2. This pattern is characteristic of endothelial dysfunction and is due to reduced NO release by the affected endothelium. Over the ensuing post-reperfusion time (i.e., 5 to 270 minutes) the vasorelaxation response to ACh diminished even more, but at no time was there an attenuation in the vasorelaxant response to NaNO2. Although not shown in Figure 17–1, the responses to A23187 paralleled those to ACh at all times. Thus, endothelial dysfunction originated in the first 2.5 minutes of reperfusion, and was exacerbated and sustained over the entire 4.5 hour post-reperfusion observation period. Moreover, intravenous infusion of recombinant human superoxide dismutase (hSOD) just prior to reperfusion prevented most of the endothelial dysfunction occurring after reperfusion (Tsao et al., 1990). Thus, endothelial dysfunction was manifest in terms of agonist mediated NO release by the endothelium (i.e.,

1Supported

N.I.H.

in part by Research Grant No. GM-45434 from the National Institute of General Medical Science of the

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pulsed, release). However, the endothelium usually releases NO continuously in low concentrations (i.e., basal release). Basal release is more difficult to quantify, but can be done by measuring the degree of vasocontraction of vascular rings to a nitric oxide synthase (NOS) inhibitor (e.g., L-NMMA, L-NAME). Using this technique, Ma et al. (1993) showed that the vasocontraction of cat LAD coronary arteries to LNAME was markedly diminished 5 min post-reperfusion following 90 min of ischemia. The response was exacerbated at all post-reperfusion times over the 270 min observation period just as in the case of reduced ACh and A23187 responses. Thus, the endothelial dysfunction occurring following reperfusion of an ischemic coronary vasculature is manifest both in agonist-mediated (i.e., pulsed release of NO) as well as in basal release of NO (i.e., low level continuous release of NO). Additional important information on the mechanism of the endothelial dysfunction was provided by Tsao and Lefer (1990). This study was performed in isolated rat hearts perfused with Krebs-Henseleit buffer rather than with blood. Hearts were subjected to global ischemia (i.e., total cessation of coronary flow for 20 min) followed by reperfusion for periods of 2.5 to 45 minutes with Krebs-Henseleit solution gassed with either 95%O2+5%CO2 or with 95%N2+5%CO2. In addition, superoxide radical release was determined by chemiluminescent methods. As expected, there was a profound endothelial dysfunction 2.5 minutes following reperfusion characterized by markedly attenuated vasorelaxation of the coronary microcirculation (i.e., a smaller decrease in coronary perfusion pressure) to ACh but not to nitroglycerin (NTG). This endothelial dysfunction gradually worsened over the remainder of the 45 min post-reperfusion period. Moreover, there was a large pulse of superoxide radical released by the ischemic-reperfused vasculature within the first minute following reperfusion which lasted for about 1 to 2 minutes. The burst of superoxide radical production preceded the endothelial dysfunction, and in the absence of blood cells in the perfusate could be traced to the endothelium itself. Moreover, infusion of hSOD, but not the hydroxyl radical scavenger MPG, given at the time of reperfusion, abolished both the pulse of superoxide release, and the endothelial dysfunction. However, the most surprising finding in this study is shown in Figure 17–2. The interesting result in this study was that reperfusion with 95%N2+5%CO2 abolished the superoxide release and the endothelial dysfunction clearly pointing to reoxygenation (i.e., re-introduction of oxygen) as the cause of the endothelial dysfunction. In additional studies, it was determined that myocardial ischemia/ reperfusion in the intact rat results in much the same degree of coronary microvascular endothelial dysfunction (Tsao and Lefer, 1991). Moreover, studies on the recovery of normal endothelial function revealed that the endothelial dysfunction persisted for six to seven weeks following ischemia/reperfusion (Tsao and Lefer, 1991) probably as a function of the slow generation of new endothelial cells. This time course of the onset of endothelial dysfunction observed in MI/R in cats and rats is not unique to the coronary circulation, but is very similar to that occurring in the mesenteric vasculature. Thus, splanchnic artery occlusion/reperfusion (SAO/R) also results in a profound endothelial dysfunction in the superior mesenteric artery (SMA) of the rat (Lefer and Ma, 1991, 1993). Moreover, the endothelial dysfunction occurs as early as 2.5 min post-reperfusion and progresses to its maximal state by 60 min post-reperfusion (Lefer and Ma, 1993). Endothelial dysfunction has been observed in cats subjected to SAO/R (Carey et al., 1992; Karasawa et al., 1991) and thus is not species specific. In summary, a severe form of endothelial dysfunction occurs within 2.5 min of reperfusion of the ischemic coronary or mesenteric circulation. The endothelial dysfunction is characterized by a markedly diminished release of NO assessed either by agonist stimulation or by basal release. There is some question of whether the dysfunction occurs in all segments of the ischemic-reperfused vasculature (i.e., large vessels, microvessels) (Quillen et al., 1990). The antecedent event most likely to bring about the endothelial dysfunction is rapid reoxygenation of the previously ischemic vasculature resulting in a burst of superoxide radicals which inactivate NO and may actually injure the NO biosynthetic process. Recent evidence

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Figure 17±1. Representative force recording of coronary artery rings isolated from cats subjected to either ischemia alone or ischemia followed by reperfusion. At the arrow, 20 nM U–4619 was given to add tone to the rings. Before reperfusion (time 0) the response to the endothelium-dependent vasodilator ACh (100 pM to 100 nM) was normal and comparable to that induced by the endothelium-independent vasodilator NaNO2 (100 nM to 100 µM). However, after reperfusion, the ACh-induced relaxation was significantly attenuated with maximal decrement observed after 270 min of reperfusion. All NaNO2 responses showed normal relaxation. This pattern of response is indicative of endothelial dysfunction.

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Figure 17±2. Effects of oxygenation on ischemia and reperfusion-induced endothelial dysfunction of the rat coronary circulation. In isolated rat hearts perfused with Krebs-Henseleit solution, reperfusion with 95% N2+5% CO2 prevents the endothelial dysfunction typical of reperfusion injury when reperfused with 95% O2+5% CO2. In these conditions, reoxygenation of ischemic tissues is a critical step for the production of superoxide radicals which are responsible of endothelial damage. Control hearts were not subjected to ischemia/reperfusion, but were time matched controls perfused at 20 ml/min for 65 minutes. All values are means ± SEM for 6–12 rings.

suggests that a critical deficit in tetrahydrobiopterin (TB4) may be a key factor in the endothelial dysfunction (Tiefenbacher et al., 1996). B. Role of NO in Endothelial Adhesion Molecules In recent years, considerable evidence has been gathered which points toward the conclusion that NO attenuates leukocyte-endothelium interaction (i.e., inhibits adherence of leukocytes to the endothelium). One of the earliest findings in this regard is that NO inhibits formyl-methionyl-leucyl-phenylalanine (fMLP) induced PMN aggregation (McCall et al., 1988). Three years later, Kubes et al. (1991) showed that superfusion with L-NMMA, a NOS inhibitor, over the cat mesenteric microvasculature resulted in a profound increase in adherence of leukocytes to the venular endothelium. This enhanced adhesiveness could be overcome by addition of a monoclonal antibody directed against the leukocyte 2-integrin CD18, the major neutrophil adhesion molecule responsible for firm cellular adhesion. Additional evidence has been obtained in the coronary vasculature. In a series of studies employing nitric oxide donors, Siegfried and coworkers (Siegfried et al., 1992a, 1992b; Johnson et al., 1990) showed that a variety of different types of NO donors (e.g., sydnonimine, cysteine, nitrate) attenuated release of superoxide radical by cat neutrophils resulting in a NO-sparing effect on the coronary endothelium and on the mesenteric vascular endothelium (Carey et al., 1992). This has been subsequently confirmed in human neutrophils for superoxide (Clancy et al., 1992; Moilanen et al., 1993) and for hydrogen peroxide (Forslund

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and Sundqvist, 1995). Although these data strongly pointed toward a marked anti-neu trophil effect of NO, they fell short of associating the leukocyte-endothelial interaction with specific cell adhesion molecules. More recently, efforts were made to ascertain which cell adhesion molecules were down-regulated by nitric oxide. A recent study (Lefer et al., 1993) employing human aortic endothelial cells (HAECs) in culture found that NO donors or the NO precursor, L-arginine decreased basal ICAM-1 surface expression. This has been followed up by DeCaterinia et al. (1995) who showed that NO donors can also down-regulate the mRNA and protein for ICAM-1 and VCAM-1 in cultured human endothelial cells. Down-regulation of these important endothelial cell adhesion molecules was related to suppression of the transcription factor NF B and can also be initiated by oxidized low density lipoproteins (oxo-LDL) (Liao et al., 1995). More recently, Armstead et al. (1997) reported that NO donors or L-arginine down-regulated the mRNA and protein expression of P-selectin in human cultured iliac artery and vein endothelial cells. Moreover, LNAME upregulated P-selectin expression in these same cell types. These studies carried out in cultured human endothelial cells under carefully controlled conditions are important in understanding the cell signalling mechanisms responsible for the regulatory effects of NO on cell adhesion molecules. However, they do not yield information on whether these relationships pertain to the intact animal under physiologically relevant conditions. To answer these questions, studies have been conducted in vivo employing intravital microscopy (See the section on microcirculatory changes in NO and leukocyteendothelium interaction). NEUTROPHILS AND NO IN ENDOTHELIAL DYSFUNCTION AND TISSUE NECROSIS As mentioned above, endothelial dysfunction is the hallmark of the early events following reperfusion of a previously ischemic vasculature. This applies to both the coronary and the mesenteric circulation. The key event in this endothelial dysfunction is a loss of endothelial derived nitric oxide (EDNO). One of the major triggers of endothelial dysfunction is a large early burst of superoxide radicals. This early superoxide burst is thought to originate from the hypoxic-reoxygenated endothelium itself (Zweier et al., 1987). This “endothelial trigger” sets up the microvasculature for the consequences of endothelial dysfunction, since superoxide is known to quench NO and thus inactivate EDNO (Rubanyi and Vanhoutte, 1986; Gryglewski et al., 1986). One of the probable sources of the endothelial superoxide is the xanthine-oxidase system (Bulkley, 1989). This endothelial production of superoxide is an important event in the early endothelial dysfunction, occurring in the first minute of reperfusion (Tsao et al., 1990; Tsao and Lefer, 1990; Lefer et al., 1990). However, this early loss of NO is not sufficient by itself to produce marked tissue injury. In the isolated perfused cat heart, rendered globally ischemic and then reperfused with and without neutrophils, about 25% of the endothelial dysfunction occurs in the absence of neutrophils and 75% of the dysfunction occurs upon perfusion with activated PMNs (Tsao et al., 1992). These results are summarized in Figure 17–3 in which coronary artery rings were isolated from isolated cat hearts, perfused either with buffer (no PMNs) or with PMNs activated by fMLP. These hearts were made ischemic (i.e., perfused at 15% of control coronary flow for 90 minutes) followed by reperfusion at control flows for 20 minutes. These data are consistent with the findings of Tsao and Lefer (1990) showing that PMNs play a key role in propagating the endothelial dysfunction and in contributing to the cell injury of the underlying parenchymal cells (i.e., cardiac myocytes in the case of myocardial ischemia/reperfusion, and liver and intestinal cells in the case of splanchnic ischemia/reperfusion). Another aspect of the interaction of PMNs with the NO generated by the endothelium in ischemia/ reperfusion is the interesting effect the oxidative burst of neutrophils has on residual NO from the

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Figure 17±3. Role of neutrophils in cat coronary endothelial dysfunction. Cat hearts were perfused under control (nonischemic) conditions or reduced ischemia for 90 min at 15% of control flows, and reperfused for 20 minutes. Coronary artery rings were then isolated and studied. Addition of inactivated neutrophils to isolated cat coronary artery rings did not interfere with acetylcholine (ACh)-induced relaxation. Concomitant incubation of cat coronary rings with fMLPactivated neutrophils significantly impaired the endothelial-mediated dilator response to ACh, but not to NaNO2. All values are means ± SEM, numbers of experiments are indicated at the bottom of the bars.

dysfunctional endothelium. This large secondary burst of superoxide from activated PMNs blocks any further release of NO from the endothelium, and results in a vasospasm-like contraction of coronary arteries (Ma et al., 1991; Ohlstein and Nichols, 1989). Results obtained in both cat (Ma et al., 1991) and rabbit (Ohlstein and Nichols, 1989) coronary arteries clearly show that activated PMNs induce a marked and sustained vasoconstriction in isolated arterial segments. The basis for this constriction, somewhat analogous to a coronary vasospasm, is that the PMNs activated by fMLP or LTB4, release an oxidative burst of superoxide radicals which antagonize the basal release of NO by the endothelium. This vasospasm doesn’t occur in an artery denuded of endothelium or when the PMNs are not activated, indicating that the vasocontraction is clearly due to the superoxide released by activated PMNs acting on endothelium derived NO. This vasocon striction can be washed out, and the arteries tested for their ability to relax to endothelium-dependent dilators (e.g., acetylcholine). Figure 17–4 shows responses to key agents delineating the key principles involved in this phenomenon. Addition of inactivated PMNs (Control) does not interfere with the acetylcholine (ACh) induced vasorelaxation (i.e., NO-dependent relaxation). However activation of the PMNs with fMLP markedly reduced the ACh induced relaxation (i.e., from 93% to 18%). If human superoxide dismutase (hSOD) is added to the PMNs which scavengers superoxide anions released from the activated PMNs, then the NO is spared, and a marked vasorelaxation to ACh occurs. This protection does

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not occur in the presence of the hydroxyl radical scavenger, mercaptopropionyl glycine (MPG). Not only are activated PMNs necessary for this reaction, but the activated PMNs must adhere to the endothelium in order to attenuate the endothelium-dependent relaxation. If one adds a monoclonal antibody (i.e., R15.7) directed against the common -chain of the leukocyte 2-integrins (i.e., CD18), one can also protect the ACh-induced vasorelaxation, whereas a control isotype antibody (R3.1) is ineffective in this regard (Figure 17–4). This anti-CD 18 antibody also worked effectively in attenuating cardiac necrosis and preserving coronary endothelial function when administered in vivo just before reperfusion of the ischemic myocardium (Ma et al., 1991). Indeed, if one blocks adherence of PMNs to the coronary endothelium with a neutralizing monoclonal antibody directed against either ICAM-1 (Ma et al., 1992) or P-selectin (Weyrich et al., 1993) or an anti-selectin oligosaccharide (Buerke et al., 1994) one preserves vascular endothelial function as well as attenuates myocardial reperfusion. Moreover, critical reductions in basal NO release to the coronary vascular endothelium were shown to promote PMN adhesion to the ischemicreperfused endothelium (Ma et al., 1993). Thus, preservation of endogenous NO in the coronary vascular endothelium is a salient feature of the protection against reperfusion injury in myocardial ischemia/reperfusion. MICROCIRCULATORY CHANGES IN NO AND LEUKOCYTEENDOTHELIUM INTERACTION Due to its strategic anatomic position, between the large conductance vessels and the parenchymal tissue of body organs, the microcirculation is a primary candidate as a target and mediator of cardiovascular diseases and inflammatory states. Recently, particular emphasis has been attributed to the physiology and physiopathology of the microvascular endothelium as a major factor in the regulation of circulatory function. The vascular endothelium contributes to the control of the cardiovascular system by synthesis and release of endothelium-derived nitric oxide (NO) under normal as well as pathophysiologic conditions (Moncada et al., 1990). NO is synthesized by vascular endothelial cells via the action of constitutive NO synthase in the L-arginine and citrulline pathway (Moncada et al., 1990; Ignarro, 1989). Beyond its role as a vasodilator substance, NO exerts a variety of other functions in the vascular endothelial environment. In particular, NO is known to inhibit platelet aggregation, inhibit leukocyte endothelial interaction and maintain capillary vascular integrity (Lefer and Lefer, 1993; Ignarro, 1989). Thus, the occurrence of endothelial dysfunction or decreased NO release during pathophysiological conditions (e.g., ischemia-reperfusion) not only affects vascular tone, but also disrupts many other facets of the normal microcirculatory environment. In this regard, it is well established that impaired endothelial production or release of NO occurs in a variety of models of ischemia-reperfusion of different organs, such as heart (Tsao et al., 1990) and splanchnic viscera (Karasawa et al., 1991). Despite the fact that restoration of blood flow to ischemic areas is essential for tissue salvage, reperfusion of the ischemic tissue also triggers a series of events which enhance and extend ischemic tissue injury (Tsao et al., 1990; Lefer and Lefer, 1993; Parks and Granger, 1986). At the present time, one of the most important factors believed to play a central role in this reperfusion-induced tissue injury is circulating leukocytes (i.e., polymorphonuclear leukocytes). Evidence indicates that leukocyte accumulation in the post-ischemic tissue is in part dependent on early endothelial dysfunction (Tsao et al., 1990). Several investigators have demonstrated that basal release of NO is significantly decreased in the microcirculation very early following ischemia-reperfusion, and this decrease in basal NO release results in a significant increase in PMN adherence to the vascular endothelium (Ma et al., 1993 ; Horgan et al., 1990; Kurtel et al., 1992). These data taken together indicate that ischemia followed by reperfusion results in a

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Figure 17±4. Cat coronary artery rings isolated from control cats and incubated with autologous PMNs (1×106). Neutrophil-induced endothelial dysfunction can be attenuated by two different strategies. The addition of hSOD but not the hydroxyl radical scavenger MPG, to co-incubated cat coronary rings with activated neutrophils, restores the response of the endothelium to ACh. Inhibition of activated PMN adherence to the vascular endothelium by a monoclonal antibody against CD18 but not an irrelevant control MAb (R3.1) can also protect the vascular endothelium from neutrophil mediated injury, as confirmed by the preservation of ACh-induced vasorelaxation in isolated coronary rings. All values are means ± SEM; numbers in bars are numbers of rings studied.

rapid endothelial dysfunction associated with a reduced NO release, which promotes leukocyte-endothelial interaction and leukocyte accumulation in the post-ischemic tissue. This interrelationship has been studied also by several investigators (Kubes et al., 1991; Arndt et al., 1993; Kubes et al., 1992) who have been able to mimic increases in leukocyte-endothelium interaction observed following ischemia-reperfusion through the use of nitric oxide synthase inhibitors such as NG monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME). Both L-NMMA and L-NAME have been shown to increase leukocyte-endothelium interaction and in particular leukocyte adherence to the microvascular endothelium and leukocyte emigration from the microcirculation (Kubes and Granger, 1992). These effects have been attributed to the loss of endothelial NO, as evidenced by the fact that administration of exogenous NO or L-arginine, the substrate for NO synthase, can override the effects of both L-NMMA and L-NAME (Gauthier et al., 1994; Davenpeck et al., 1994). This inhibition of endothelial NO synthesis, even in the absence of ischemia and reperfusion, results in enhanced leukocyteendothelial interaction. Another important factor involved in leukocyte-endothelial interaction during the inflammatory process initiated by ischemia-reperfusion is the expression of cell adhesion molecules. Identification and characterization of the leukocyte and endothelial cell adhesion molecules that mediate leukocyteendothelial interaction have been crucial in determining the mechanism controlling the trafficking of

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leukocytes in the vasculature and the movement of leukocytes out of the circulation into areas of tissue injury. In fact, an important aspect of enhanced leukocyte-endothelial interaction during ischemiareperfusion is the expression of specific endothelial cell adhesion molecules. P-selectin, a member of the selectin family of adhesion glycoproteins is one of the earliest endothelial adhesion molecules involved in leukocyte recruitment to the site of inflammation. Davenpeck and coworkers (Guathier et al., 1994; Davenpeck et al., 1994a, 1994b) extensively studied the interrelationship between NO and cell adhesion molecules and their relationship to ischemia-reperfusion. These investigators employed intravital microscopy of the rat mesenteric microvasculature to further investigate the interaction between NO and cell adhesion molecules. In one of their first studies, Davenpeck et al. (1994b), demonstrated that ischemiareperfusion of the mesenteric circulation, a condition which has been shown to dramatically reduce endothelial NO, resulted in a rapid increase in leukocyte rolling and adherence to the venular endothelium during the first 30 min following reperfusion. At the same time, a marked increase of P-selectin expression in the venular endothelial surface was found 30 min after reperfusion. These results clearly indicate that the critical reduction in NO release 30 min after reperfusion is correlated with an increased upregulation of Pselectin on the endothelial surface (Davenpeck et al., 1994b). In a follow-up study, Gauthier et al. (1994) showed in the same model of ischemia-reperfusion of the rat mesenteric circulation, infusion of an NO donor markedly attenuated post-reperfusion rolling and adherence of leukocyte to the venular endothelium. This clearly demonstrates that restoration of physiological levels of NO in the systemic circulation during microcirculatory perturbations results in a reduced leukocyte-endothelial cell interaction and amelioration of the associated circulatory shock. In order to further investigate this interrelationship between NO and cell adhesion molecules, Davenpeck et al. (1994a), also showed that exposure of the rat mesenteric microvasculature to NOS inhibitors mimicks the effect of ischemia-reperfusion. The key results from this study are depicted in Figure 17–5. The number of adherent leukocytes in the microvasculature is very low during normal physiological conditions. However, exposure of the rat mesentery to increasing concentrations of L-NAME from 25 to 100 μM resulted in a progressive concentration-dependent increase in leukocyte adherence. Equimolar concentration of Larginine, but not of its stereoisomer D-arginine, was able to overcome the L-NAME-induced leukocyte adherence, thus suggesting the specific NO-mediated mechanism of L-NAME-induced leukocyte adherence. Moreover leukocyte adherence could be markedly reduced by administration of a monoclonal antibody directed against P-selectin. Similar results were obtained with leukocyte rolling. Immunohistochemical localization of P-selectin showed upregulation of this adhesion molecule in the rat mesenteric microvasculature following direct inhibition of nitric oxide synthase by L-NAME (Davenpeck et al., 1994a). These findings, taken together, clearly point to the fact that a dynamic negative feedback relationship exists between nitric oxide release and leukocyte-endothelial cell interaction. Reduced nitric oxide release from the microvascular endothelium, during pathophysiologic conditions, leads to an increased adhesion molecule expression on the endothelial cell surface and enhancing NO exogenously can markedly diminish these processes. This phenomenon plays an important role in the margination and emigration of leukocytes from the blood stream and their accumulation in injured tissues. MOLECULAR MECHANISMS OF NO IN REGULATION OF CELL ADHESION MOLECULE EXPRESSION A critical factor involved in leukocyte-endothelial interaction is the expression of cell adhesion molecules on the microvascular endothelium (Carlos and Harlan, 1994). To date, three major families of cell adhesion

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Figure 17±5. L-NAME-induced inhibition of nitric oxide synthase increases leukocyte adherence along the venular endothelium of the rat mesenteric microcirculation utilizing intravital microscopy. This phenomenon was inhibited by equimolar concentration of L-arginine and was also blocked by the systemic administration of an antibody against Pselectin (PB 1.3). Therefore, loss of endogenous nitric oxide release upregulates leukocyte rolling, the prelude to leukocyte adherence. All values are means ± SEM for 6 rats in each group.

molecules which regulate leukocyte-endothelial cell interaction are known to play a role in ischemiareperfusion: a) selectins (i.e., P-selectin, L-selectin, E-selectin (Bevilacqua and Nelson, 1993; Lefer et al., 1994); b) the 2 integrins (e.g., CD11/18) (Springer, 1990), and c) the immunoglobulin superfamily (e.g., ICAM-1, VCAM-1, PECAM-1) (Springer, 1990). The expression of selectins (i.e., P-selectin) and immunoglobulin superfamily members (e.g., ICAM-1 and VCAM-1) on the microvascular endothelium has been recently correlated to impairment of nitric oxide synthesis and release from endothelial cells, during pathological perturbations of the microcirculation (e.g., ischemia-reperfusion and hyperlipidemia). In particular, rapid (i.e., 10–15 min) P-selectin endothelial cell surface expression has been linked to leukocyte accumulation and tissue injury in several in vivo models of acute inflammation generally associated with a reduced release of nitric oxide (Weyrich et al., 1993; Gauthier et al., 1994; Davenpeck et al., 1994a). Furthermore, other investigators have shown, employing intravital microscopy, that the NO synthase inhibitor L-NAME directly induces P-selectin expression, thus facilitating neutrophil rolling and adherence in the rat mesenteric microcirculation in vivo (Davenpecket al., 1994a). P-selectin and ICAM-1 expression on endothelial cells were also found to be upregulated during induction of hypercholesterolemia in cholesterol fed animals (Gauthier et al., 1995). More recently, nitric oxide was also found to be able to reduce cytokine-induced expression of an other physiologically relevant effector molecule characteristic of endothelial activation during atherogenesis (i.e., VCAM-1) (DeCaterina et al., 1995). Despite these numerous data regarding nitric oxide-induced modulation of adhesion molecules on the endothelial cell surface, the precise molecular mechanisms of this cell signalling pathway are still poorly

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understood. Nevertheless, at the present time, only a few mechanisms have been proposed for explaining NO-mediated regulation of adhesion molecules expression. The first mechanism is related to the fact that both endothelial dysfunction (i.e., loss of endothelial NO) and P-selectin expression are believed to be mediated by the generation of oxygen-derived radicals at the time of reperfusion. Virtually all cells generate oxygen derived free radicals through normal physiological pathways such as the mitochondrial electron transport pathway and the NADPH pathway (Grisham and Granger, 1989). Normally, adequate mechanisms exist within the cell to scavenge these radicals and thus prevent injury to the cell. In this connection, endogenous NO is a known scavenger of oxygen derived free radicals and in particular the superoxide radical (Rubanyi et al., 1991). Accordingly, Gaboury et al. (1993) recently demonstrated that superoxide radicals introduced directly into the rat mesenteric microcirculation following generation by hypoxanthine-xanthine oxidase results in P-selectin mediated leukocyte rolling and adherence. Thus reduced endogenous NO synthesis by endothelial cells as a result of superfusion with LNAME could lead to an excess of oxygen-derived free radicals within these cells, stimulating expression of endothelial cell adhesion molecules (Davenpeck et al., 1994a). A similar mechanism may occur outside the endothelial cell as well, since NO also scavenges radicals at the endothelial cell surface (Rubanyi et al., 1991). Also consistent with this hypothesis is the observation that both L-arginine and 8-bromo-cGMP can significantly attenuate the L-NAME induced increase in P-selectin expression (Davenpeck et al., 1994a). More recently, a second line of in vitro studies has focused attention on the potential role of protein kinase C (PKC) as a signalling molecule in these processes. Murohara et al. (1995), examined the hypothesis that L-NAME stimulates P-selectin expression on platelets via PKC activation utilizing flow cytometry. These workers showed it is possible to induce P-selectin surface expression in cat platelets, as well as PKC activity on platelet membranes, following a 10 min incubation with either phorbol-12myristate-13 acetate (i.e., a well known PKC stimulator), thrombin, or L-NAME. Conversely, the specific PKC inhibitor UCN-01 (i.e., 7-hydroxystauroporine) was able to attenuate L-NAME-induced P-selectin expression on isolated platelets (Figure 17–6), thus confirming the role of PKC activation in NO-mediated P-selectin cell surface expression (Murohara et al., 1995). These two purported mechanisms of cell signalling between nitric oxide and cell adhesion molecules could explain some of the molecular mechanisms by which nitric oxide can regulate cell adhesion molecule expression on endothelial cells. Actually, P-selectin, which is constitutively stored in Weibel-Palade bodies of endothelial cells (McEver et al., 1995), is rapidly translocated (i.e., in 10 min) to the endothelial surface upon stimulation with histamine, thrombin, hydrogen peroxide or hypoxia-reoxygenation (Lorant et al., 1991). These temporal relationships are consistent with signalling times for both cyclic GMP and protein kinase C. More complex molecular mechanisms have been recently reported in the case of other endothelial cell adhesion molecules (i.e., E-selectin, ICAM-1 and VCAM-1) in which upregulation of endothelial cells requires de novo synthesis of proteins and therefore requires 2 to 4 hours to induce changes. DeCaterina et al. (1995) have recently shown that, in cytokine-stimulated human saphenous vein endothelial cells, several NO donors inhibit VCAM-1 expression by 35–55%, and also reduce to a lesser extent E-selectin and ICAM-1 level in similar conditions. In the case of VCAM-1, these investigators also showed that nitric oxide inhibits VCAM-1 gene transcription, in part by inhibiting NFKB (DeCaterina et al., 1995). These observations offer new insights into the biology of nitric oxide and the physiological pathways by which, NO can modulate the expression of cell adhesion molecules, thus affecting the pathology of the inflammatory and immune response.

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NO, NO DONORS AND PEROXYNITRITE IN ISCHEMIA/ REPERFUSION One of the major endogenous agents which is important in preserving vascular homeostasis is nitric oxide. Nitric oxide is produced in low nanomolar concentrations by the vascular endothelium (Kelm and Schrader, 1990). NO can diffuse to the basilar surface of the endothelial cells and then to vascular smooth muscle cells where it can induce vasorelaxation of the vascular smooth muscle cells. NO can also diffuse to the luminal surface where it interfaces with the blood and exerts some very important physiologic actions including inhibition of platelet aggregation (Radomski et al., 1991), quenching of superoxide radicals (Rubanyi and Vanhoutte, 1986; Gryglewski et al., 1986; Stewart et al., 1988) and attenuation of leukocyte adherence to the endothelium (McCall et al., 1988; Kubes et al., 1991; Gauthier et al., 1994; Davenpecketf et al., 1994a, 1994b; Gaboury et al., 1993). These effects occur at low nanomolar concentrations, below concentrations which are necessary to produce vasodilation (Gauthier et al., 1994; Davenpeck et al., 1994a, 1994b; Gaboury et al., 1986). In ischemia/reperfusion, authentic NO gas dissolved in physiological solutions (e.g., 0.9% NaCl) yielding local NO concentrations of 2 to 10 nM, has been shown to exert protective effects in both myocardial ischemia/reperfusion (Johnson et al., 1991) and in splanchnic ischemia/reperfusion (Aoki et al., 1990). The major effect was an attenuation of tissue injury and improved biochemical sequelae. Figure 17–7 illustrates the effect of authentic NO on myocardial necrosis in cats subjected to MI/R. In the case of myocardial ischemia/reperfusion this was related to reduced neutrophil infiltration into the reperfused myocardium (Johnson et al., 1991). These results were confirmed by the administration of the inorganic NO releasing agent sodium nitrite (NaNO2) at pH 2.0 (Johnson et al., 1990). These were the earliest reports that addition of physiological amounts of NO could exert beneficial effects in severe life-threatening forms of ischemia/ reperfusion. However, these agents require significant improvement in order to be clinically useful. The next advance occurred with the synthesis of third generation organic NO donors. These are organic nitrates that release NO in solution, and are much more potent than classical NO donors (e.g., nitroglycerin, nitroprusside) in addition to inducing much less tolerance than these agents. Also, these newer agents are more stable compounds, and do not release superoxide radicals or cyanide, as do some of the earlier NO donors (e.g., SIN-1). Several of these newer NO donors have been studied in myocardial ischemia/ reperfusion. Thus, C87–3754, a sydnonimine NO donor, but not its non-NO releasing control compound C88–3934, markedly protected the ischemic cat myocardium from necrosis, and preserved the integrity of the coronary vascular endothelium (Siegfried et al., 1992a). These results were followed by a study employing another NO donor, a cysteine analog SPM-5185, and its non-NO donating control compound SPM-5267 in the same cat model of myocardial ischemia/reperfusion (Siegfried et al., 1992b). In this second study, the major mechanism of the cardioprotection was identified as inhibition of neutrophilendothelium interaction and subsequent release of superoxide by activated PMNs (Siegfried et al., 1992b). This was subsequently confirmed using the same NO donor in dogs (Lefer et al., 1993), and another NO donor in isolated rat hearts perfused with PMNs (Pabla et al., 1996). The same principles apply to splanchnic ischemia-reperfusion since the sydnonimine, C87–3754 also protected cats subjected to splanchnic artery occlusion/reperfusion (Carey et al., 1992). This NO donor increased survival and attenuated the formation of a myocardial depressant factor (MDF) (Lefer, 1987) in this lethal form of shock. Moreover, the NO donor preserved the integrity and function of the superior mesenteric arterial endothelium (Carey et al., 1992). In all cases, infusion of the NO donor occurred at a rate which did not exert any detectable effect on arterial blood pressure (i.e., was sub-vasodilator). More recently, the precursor of NO, the amino acid L-arginine has been studied in myocardial ischemiareperfusion (Weyrich et al., 1992; Nakanishi et al., 1992). In the same cat model of myocardial ischemia/ reperfusion as that employed in the NO donor studies, intravenous infusion of L-arginine was also effective

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Figure 17±6. Representative fluorescence histograms of P-selectin cell surface expression on feline platelets, as assessed by flow cytometry. Inhibition of nitric oxide synthase by 1 mM L-NAME significantly increaseds P-selectin expression. This L-NAME mechanism selectively operates on nitric oxide synthase as confirmed by the observation that co-incubation with equimolar concentration of L-arginine, but not D-arginine, can override L-NAME effects. The PKC inhibitor (UCN-01) also inhibited L-NAME-induced P-selectin expression, thus confirming the role of PKC in nitric oxide cell signaling.

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Figure 17±7. Effect of authentic NO on myocardial necrosis in cats subjected to MI/R. Nitric oxide significantly reduced the amount of necrotic myocardial tissue, following ischemia-reperfusion of cat coronary circulation for 90 minutes of ischemia and 270 minutes of reperfusion. NO infusion was administered lose to the heart just before reperfusion, and continued for the entire reperfusion period. All values are means ± SEM for 6 cats in each group.

in reducing infarct size as well as attenuating neutrophil infiltration into the ischemic reperfused myocardium and preserving coronary vascular endothelial function (Weyrich et al., 1992). Similar effects were observed in a dog model of myocardial ischemia-reperfusion injury (Nakanishi et al., 1992). Thus, availability of arginine may be important in maintaining endothelial NO in ischemia/reperfusion. Recently, Tiefenbacher et al. (1996) showed that there is also a deficit in tetrahydrobiopterin, (TB4) a critical cofactor in the biosynthesis of NO. Where TB4 is replaced, much of the post-reperfusion endothelial dysfunction is prevented. It should be emphasized that there are many published reports showing that either low doses of NO gas or NO donors or higher doses of L-arginine or TB4 are protective in ischemia/reperfusion (Tsao et al., 1990; Siegfried et al., 1992a, 1992b; Gauthier et al., 1994; Lefer et al., 1993; Pabla et al., 1996). However, there are some dissenting studies in which NO synthase inhibitors (e.g., L-NAME) also have been shown to be protective in myocardial hypoxia-reoxygenation models (Mathies et al., 1992; Schulz and Wambolt, 1995) and in either hypoxic-reoxygenated pigs or isolated perfused rat hearts, suggesting that NO may not be good under all conditions. It is difficult to distinguish among several possible explanations from these findings; including the possibility that (a) L-NAME may be cardiotoxic at high doses due to coronary constrictor or other effects, (b) NO may exert different effects in ischemia/reperfusion from hypoxia/reoxygenation, or (c) there is cardiotoxicity in these models due to formation of peroxynitrite. However, the last potential

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Figure 17±8. Effects of the nitric oxide metabolite peroxynitrite on ischemic-reperfused rat hearts. Infusion of peroxynitrite to (800 nM) ischemic hearts markedly attenuated the neutrophils-induced myocardial injury, as confirmed by improved postischemic LVDP values throughout reperfusion. All values are means ± SEM for 6–7 hearts in each group.

explanation is unlikely, due to recent findings showing physiologically relevant concentrations of peroxynitrite to be cardioprotective (Lefer et al., 1997a). Recently, authentic peroxynitrite (ONOO−) was added to isolated perfused rat hearts to yield a local concentration of 800 nM, a concentration calculated to be comparable to biologically achievable concentrations (Lefer et al., 1997a). In rat hearts subjected to ischemia for 30 minutes and reperfusion for 45 minutes, ONOO− had no detectable effect on left ventricular developed pressure (LVDP), dP/dt max, or coronary flow. When rat hearts were subjected to ischemia/reperfusion and perfused with rat neutrophils (i.e., PMNs), a severe reduction in LVDP, dP/dt max, and coronary flow occurred. However, perfusion with 800 nM ONOO− in ischemic/reperfused rat hearts perfused with PMNs resulted in a marked amelioration of the cardiodepression observed with the pH matched vehicle for ONOO− (Lefer et al., 1997a). Figure 17–8 illustrates the major cardioprotective effect of ONOO− in the isolated perfused rat hearts. Physiological concentrations of ONOO− clearly protected the heart against post-ischemia/reperfusion induced cardiac stunning. These results also correlated closely with reduced MPO activity and fewer PMNs in the ischemic rat hearts reperfused with PMNs (Lefer et al., 1997a). Thus, ONOO− markedly attenuated accumulation of PMNs in the ischemia/reperfused rat heart, and preserved left ventricular contractility in the face of ischemia-reperfusion with neutrophils.

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Recently, these studies in isolated perfused hearts have been confirmed in vivo in cats subjected to myocardial ischemia/reperfusion. In these experiments, ONOO− was infused directly into the heart to achieve a local concentration of 400 nM. Under these conditions, ONOO− significantly preserved the reperfused ischemic myocardium by 55% (Lefer et al., 1997b). These ONOO− treated cats also exhibited a significant preservation of coronary endothelial function (i.e., maintained NO release) as well as attenuated PMN infiltration into the ischemic/reperfused myocardium (Lefer et al., 1997b). Thus, physiologically relevant concentrations of ONOO− (i.e., 400–800 nM) act as a cardioprotective agent in ischemia/ reperfusion. Earlier studies demonstrating cytotoxic effects of ONOO− employed concentrations of 500 μM to 1.5 mM ONOO− to produce significant cell death (Szabo et al., 1996). These extremely high concentrations are unlikely to ever occur in vivo, since ONOO− is formed from the equimolar combination of NO and superoxide radical (Pryor and Squadritto, 1995; Beckman et al., 1990) and NO is present in blood at 1–10 nM (Kelm and Schrader, 1990). Even if NO levels increase by a factor of 100 to 1,000, this could only yield maximal ONOO− concentrations of 10 µM. With a half-life of < 1 second, ONOO− would probably never achieve these cytotoxic levels in vivo. In summary, physiological levels of NO are important in preserving endothelial cell integrity and preventing increased leukocyte-endothelium interaction as well as platelet adherence to the endothelium. Physiological concentrations of NO therefore prevent leukocyte infiltration into inflamed areas following reperfusion of an ischemic circulation (e.g., myocardium, splanchnic region). This results in markedly limiting tissue necrosis and protects the function of these ischemic-reperfused organs. These effects of physiologic concentration of NO are very important and lead to cytoprotective effects. These cytoprotective effects need to be studied further to fully elucidate their molecular mechanisms. REFERENCES Aoki, N., Johnson, G. III and Lefer, A.M. (1990) Beneficial effects of two forms of NO administration in feline splanchnic artery occlusion shock. Am.J.Physiol., 258, G275–G281. Armstead, V.E., Minchenko, A.G., Schuhl, R.A., Hayward, R., Nossuli, T.O. and Lefer, A.M. (1997) Regulation of Pselectin expression in human endothelial cells by nitric oxide. Am. J.Physiol, 273, H740–746. Arndt, H., Russell, J.B., Kurose, I., Kubes, P. and Granger, D.N. (1993) Mediators of leukocyte adhesion in rat mesenteric venules elicited by inhibition of nitric oxide synthesis. Gastroenterology, 105, 675–680. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. and Freeman, B.A. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA, 87, 1620–1624. Bevilacqua, M.P. and Nelson, R.M. (1993) Selectins. J. Clin. Invest., 91, 379–387. Buerke, M., Weyrich, A.S., Zheng, Z., Gaeta, F.C.A., Forrest, M.J. and Lefer, A.M. (1994) Sialyl Lewisxcontaining oligosaccharide attenuates myocardial reperfusion injury in cats. J.Clin. Invest., 93, 1140– 1148. Bulkley, G.B. (1989) Mediators of splanchnic organ injury: overview and perspective. In: Splanchnic Ischemia And Multiple Organ Failure, edited by A.Marston, G.B.Bulkley, R.G.Fiddian-Green and U.H.Haglund, pp. 191–193. London: Edward Arnold. Carey, C, Siegfried, M.R., Ma, X-L, Weyrich, A.S. and Lefer, A.M. (1992) Antishock and endothelial protective actions of a NO donor in mesenteric ischemia and reperfusion. Cire. Shock., 38, 209–216. Carlos, T.M. and Harlan, J.M. (1994) Leukocyte-endothelial adhesion molecules. Blood, 84, 2068–2101. Clancy, R.M., Leszczynska-Piziak, J. and Abramson, S.B. (1992) Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action in the NADPH oxidase. J. Clin. Invest., 90, 1116–1121. Davenpeck, K.L., Gauthier, T.W. and Lefer, A.M. (1994) Inhibition of endothelial-derived nitric oxide promotes Pselectin expression and actions in the rat microcirculation. Gastroenterology, 107, 1050–1058.

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18 Nitric Oxide and Circulatory Shock Christoph Thiemermann The William Harvey Research Institute, St.Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom Tel: +44 171 982 6119; Fax: +44 171 251 1685; E-Mail: [email protected]

Nitric oxide (NO) is generated by three different isoforms of NO synthase, two of which are expressed constitutively (in endothelium: eNOS, brain: nNOS), while one is induced by endotoxin or cytokines (iNOS). Expression of iNOS in many organs/tissues in septic shock results in an enhanced formation of NO which contributes to circulatory collapse and possibly organ injury/dysfunction and host defence. Inhibition of NOS activity in shock has beneficial and adverse effects, which have tentatively been linked to inhibition of iNOS and eNOS activity, respectively. Although inhibition of NOS activity attenuates the circulatory failure in endotoxin (or septic) shock in many species, it is less clear whether an enhanced formation of NO by iNOS contributes to the organ injury or mortality associated with shock. Studies subjecting mice in which the iNOS gene has been inactivated by gene-targeting to endotoxemia support the notion that NO from iNOS contributes to hypotension and host defence, but provide controversial results regarding the role of iNOS in organ injury and mortality. The role of NO in the pathophysiology of humans with shock is less well investigated. Clearly, degree of iNOS induction in patients with septic shock is lower that in rodents with endotoxemia. Nevertheless, in patients with septic shock (placebo-controlled multi-center study, 156 patients per group with matched SAPS II scores), inhibition of NOS activity with NG-methyl-L-arginine (L-NMMA) improved haemodynamics and reduced the time to resolution of shock, without causing any major side effects. Key words: circulatory shock, multiple organ failure, selective iNOS inhibitors, aminoguanidine, aminoethyl-isothiourea.

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INTRODUCTION Septic shock, regardless of its aetiology, is defined as sepsis (systemic response to infection) with hypotension resulting in impaired tissue perfusion and oxygen extraction (Parrillo, 1990). The definition of septic shock is independent of the presence or absence of a multiple organ dysfunction syndrome (MODS), which is defined as impaired organ function such that homeostasis cannot be maintained without intervention (Baue, 1993). MODS may either occur as is a direct result of a well-defined insult to a specific organ (e.g. primary MODS) or as a consequence of an exaggerated host response (secondary MODS). This enhanced host response termed systemic inflammatory response syndrome (SIRS) may occur in response to infection, multiple trauma, haemorrhage, ischaemia and immune-mediated organ injury (Baue, 1993). Current therapeutic approaches for septic shock include antimicrobial chemotherapy, volume replacement, inotropic and vasopressor support, oxygen therapy and mechanical ventilation as well as haemodialysis and haemofiltration. These have, however, failed to make a substantial impact on the high mortality associated with septic shock (Nathanson et al., 1994) and, hence, septic shock remains the major cause of death in noncoronary intensive care units with an estimated mortality ranging between 50 and 80%. As shock is also by far the most common cause of prolonged admission to an intensive care unit, the clinical and socioeconomic importance of this illness is substantial. Since the discovery in 1990 that an enhanced formation of endogenous nitric oxide (NO) contributes to (i) the hypotension caused by endotoxin and TNF (Vane and Thiemermann, 1990; Kilbourn et al., 1990a,b), (ii) the vascular hyporesponsiveness to vasoconstrictor agents (Julou-Schaeffer et al., 1990; Rees et al., 1990), and (iii) the protection of liver integrity in rodents with sepsis (Billiar et al., 1990), there has been an ever increasing interest in the role of NO in the pathophysiology of animals and man with septic shock. The overproduction of NO in animal models of circulatory shock is due to an early activation of eNOS (which is transient, e.g. after bolus administration of endotoxin) and (more importantly) the delayed induction of iNOS activity in macrophages (host defence), vascular smooth muscle (hypotension, vascular hyporeactivity, maldistribution of blood flow) and parenchymal cells (contributing to organ dysfunction or organ integrity) (Thiemermann, 1994, 1995; Szabo, 1996; Rees, 1995). The formation of NO by eNOS (and potentially also by iNOS) also exerts beneficial effects in shock including vasodilatation, prevention of platelet and leukocyte adhesion, improvement of microcirculatory blood flow and augmentation of host defence. Thus, it is not surprising that basic and clinical scientists have advocated the use of contrasting therapeutic approaches including (i) inhibition of NOS activity, (ii) enhancement of the availability of NO (NO-donors, NO-inhalation) or (iii) a combination of both approaches (Thiemermann, 1994, 1995; Szabo, 1996; Rees, 1995). NITRIC OXIDE AND THE PATHOPHYSIOLOGY OF ENDOTOXIN SHOCK The circulatory failure associated with shock of various aetiologies is characterised by severe hypotension (peripheral vasodilatation), hyporeactivity of the vasculature to vaso-constrictor agents, myocardial dysfunction, maldistribution of organ blood flow and reduced tissue oxygen extraction. There is now good evidence that an enhanced formation of NO contributes to several of these pathophysiological features of septic shock. For instance, an enhanced formation of NO due to activation of eNOS (acute phase of shock) and particularly following the induction of iNOS in the vascular wall (late phase of shock) importantly contributes to the hypotension in animals (rat, dog, pig, sheep, baboons) and man with septic shock. Interestingly, endotoxin does not cause hypotension in mice in which the gene for iNOS has been inactivated (“iNOS knock-out” mice) (MacMicking et al., 1995). Thus, the hypothesis (Thiemermann and Vane, 1990) that an enhanced formation of endogenous NO importantly contributes to the hypotension

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associated with endotoxic shock, is now supported by numerous studies (in various different species from rodents to man) using different pharmacological (e.g. prevention of iNOS expression, inhibition of iNOS activity with non-selective or iNOS-selective inhibitors, use of agents which scavenge NO etc.) or molecular biological approaches (e.g. gene-targeting of the iNOS gene). The peripheral vascular failure in animals and man with septic shock also results in a progressive attenuation of the presser effects afforded by norepinephrine and other vasoconstrictor agents (epinephrine, vasopressin, angiotensin II, serotonin, histamine, calcium, potassium). This vascular hyporeactivity contributes to the therapy-refractory hypotension in septic shock. Clearly, the hyporeactivity of blood vessels obtained from animals exposed to endotoxic or hemorrhagic shock (for several hours) to catecholamines is largely—but not exclusively—due to an enhanced formation of NO secondary to the induction of iNOS. In endotoxemia, an NO-mediated vascular hyporeactivity occurs in conductance, resistance as well as venous vessels (see Parratt and Stoclet, 1995 for review). Prolonged periods of septic shock also result in the development of an endothelial dysfunction, which is characterised by the impairment of “endothelium-dependent vasodilatation” and therefore presumably eNOS activity. The mechanism(s) of this endothelial dysfunction may include the downregulation of the expression of the eNOS gene by pro-inflammatory cytokines such as TNF, endothelial cell damage due to cytotoxic effects of NO, peroxynitrite or oxygen-derived radicals, and (to a lesser extent) the inactivation of NO by oxygen radicals (see Thiemermann, 1994, 1995). The role of NO in the development of the organ injury/dysfunction (MODS) associated with shock is still controversial. There is evidence that shock is associated with a maldistribution of organ blood flow which may well lead to a reduction in oxygen delivery to specific areas within an organ (Baue, 1993). Regardless of any local reduction in oxygen delivery (e.g due to hypotension and hence a reduction in perfusion pressure), shock also leads to the development of a marked defect in tissue oxygen extraction which is likely to contribute importantly to the development of MODS. Although the role of NO from iNOS in this defect in tissue oxygen extraction is not very well investigated, there is evidence (primarily from in vitro studies) that the excessive generation of NO by iNOS causes direct cytotoxic effects. For instance, large amounts of NO cause an autoinhibition of mitochondrial respiration by inhibiting several key enzymes in the mitochondrial respiratory chain (NADH-ubiquinone reductase, succinate-ubiquinone oxidoreductase) or in the Krebs’ cycle (e.g. cis-acconitase) resulting in a shift in glucose metabolism from aerobic to anaerobic pathways (Morris and Billiar, 1994; Thiemermann, 1995). NO and superoxide anion generate peroxynitrite anions (Beckmann et al., 1990), which causes strand breaks in DNA (Zingarelli et al., 1996). The occurrence of DNA strand breaks triggers a futile, energy-consuming repair cycle by activating the nuclear enzyme poly (ADP)ribosyltransferase (PARS). Activation of PARS results in the rapid depletion of the intracellular concentration of NAD+ (its substrate) slowing the rate of glycolysis, electron transfer and ATP formation which ultimately results in cell death (“PARS suicide hypothesis”) (Schraufstetter et al., 1986). Inhibitors of PARS activity (e.g. 3-aminobenzamide, nicotinamide) attenuate the inhibition of cellular respiration caused by peroxynitrite (Zingarelli et al., 1996). The generation of mice deficient in iNOS (iNOS mutant or “iNOS knock-out” mice) has helped to shed a further light into the physiological and/or pathophysiological importance of the generation of NO by iNOS (Wei et al., 1995; MacMicking et al., 1995 ; Laubach et al, 1995). Most importantly, these studies provide strong evidence that the enhanced formation of NO by iNOS (e.g. in macrophages) importantly contributes to host defence against infection. For instance, MacMicking and colleagues demonstrate that iNOSdeficient mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Moreover, iNOS-deficient mice (but not wild-type or heterozygous mice) were highly susceptible to infection with the protozoa parasite Leishmania major and exhibited reduced non-specific inflammatory responses to carageenin (Wei et al., 1995). The importance of NO as a mediator of host defence is also

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highlighted by studies demonstrating that non-selective inhibitors of NOS activity increase mortality in mice challenged with live bacteria (infection) rather than endotoxin (endotoxaemia). In contrast, the hypotension (see above) and the early (but not the late) mortality caused by endotoxin is reduced in iNOS deficient mice, while the degree of liver injury is unaltered (MacMicking et al., 1995; Laubach et al., 1995). Taken together, these studies support the view that an enhanced formation of NO by iNOS defends the host against infectious agents and tumour cells, while an excessive induction of iNOS in other tissues (e.g. vasculature) may cause shock and possibly tissue injury. PREVENTION OF THE EXPRESSION OF INOS PROTEIN In 1990, dexamethasone was the first drug shown to prevent the expression of iNOS protein and activity caused by endotoxin in cells and tissues (Radomski et al., 1990; Rees et al., 1990). Since then, the list of xenobiotics which prevent the induction of iNOS activity and protein is ever growing and now includes antibodies to TNF , soluble TNF receptors, the endogenous IL-1 receptor antagonist, IL-4, IL-10, IL-11, IL-13, PAF-receptor antagonists, dihydropyridine-type calcium channel antagonists, glibenclamide, Nacetylserotonin (an inhibitor of the salvage pathway for the generation of BH4), 2,4-diamino-6hydroxypyrimidine (DAHP, an inhibitor of the activity of GTP cyclohydrolase and, hence, BH4 biosynthesis), tyrosine kinase inhibitors (genistein, tyrphostins, erbstatin), inhibitors of the activation of the nuclear transcription factor NF B (rotenone, PDTC, butyrolated hydroxyanisole) or inhibitors of I Bprotease (TPCK); to name but a few (see Szabo and Thiemermann, 1995). It should, however, be pointed out that many agents which prevent the expression of iNOS protein have to be administered prior to (or shortly after) endotoxin to prevent induction of iNOS, circulatory failure or MODS. In contrast, once hypotension (and presumably iNOS induction) has occurred, the administration of these agents show no or only minor beneficial effects (depending on their mechanism of action) on haemodynamics and organ function. Moreover, most of the above agents exert many effects, other than the prevention of the expression of iNOS protein and, hence, it is difficult to delineate which of their beneficial effects in animal models of shock are due to the inhibition of iNOS activity or secondary to other effects. For instance, calpain inhibitor I (inhibitor of the proteolysis of I B and, hence, of the activation of NF B) and dexamethasone, but not the serine and cysteine protease inhibitor chymostatin, attenuate (i) the circulatory failure (hypotension and vascular hyporeactivity to norepinephrine), (ii) the multiple organ dysfunction (liver and pancreatic injury/dysfunction, increase in lactate, hypoglycemia), and (iii) the induction of iNOS and cyclooxygenasc-2 (COX-2) protein and activity (in lung and liver) of rats with endotoxic shock (Ruetten and Thiemermann, 1987). Although the inhibition of the activation of NF B by calpain inhibitor I (and possibly dexamethasone) prevented (i) the circulatory failure as well as the development of organ injury/dysfunction in this study, and (ii) the expression of iNOS protein, the causal link between these two events remains unclear, for NF B plays a role in the transcription of many other genes (e.g. COX-2, adhesion molecules etc.). Agents, such as inhibitors of the activation of NF B or tyrosine kinase, which interfere with the expression of one (iNOS) or more (e.g. adhesion molecules) proteins which play a role in the pathophysiology of shock may well lead to novel therapeutic approaches for diseases associated with local or systemic inflammation, they are less suitable to delineate the role of iNOS in the pathophysiology of shock.

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INHIBITION OF NOS ACTIVITY In contrast to therapeutic approaches aimed at preventing the expression of iNOS protein, agents which inhibit the activity of NOS offer the opportunity for a late intervention in animals and patients which have developed hypotension and early signs of the onset of organ dysfunction. The discovery of an L-arginineanalogues which inhibits NOS activity, NG-methyl-L-arginine (L-NMMA), provided the first tool to explore beneficial or side effects of NOS inhibitors in shock. The subsequent discovery of the NOS inhibitors NGnitro-L-arginine (L-NA) and its methyl ester (L-NAME; Moore et al, 1990), which in contrast to L-NMMA were cheap and readily available, stimulated numerous studies aimed at evaluating the role of NO in septic shock by using (high doses of) L-NAME. This was somewhat unfortunate, as L-NAME is a more potent inhibitor of eNOS than iNOS activity and, hence, caused many adverse effects resulting from the inhibition of eNOS activity including excessive vasoconstriction (e.g. fall in cardiac output, pulmonary hypertension etc) and enhanced adhesion of platelets and neutrophils to the endothelium (Thiemermann, 1995). Thus, LNAME reduces oxygen delivery and exacerbates organ injury in (many, but not all) animal models of endotoxic or septic shock. These results are not necessarily solely due to the use of very large amounts of LNAME, but rather a reflection of the fact that L-NAME is a more selective inhibitor of eNOS than iNOS activity. In rats with endotoxemia, even infusion of very low doses of L-NAME (e.g. 0.03 to 0.3 mg/kg/h) result in a dose-related increase in blood pressure (due to inhibition of eNOS activity) without reducing the rise in the plasma levels of nitrite/nitrate (an indicator of iNOS activity) or the organ injury caused by endotoxin (Wu et al., 1996). The hypothesis that the basal release of NO by eNOS has an important role in the regulation of regional blood flow and adhesion of blood-borne cells to the endothelium (beneficial effects of NO), while the excessive generation of NO by iNOS “in the wrong place at the wrong time” contributes to some aspects of the pathophysiology of shock (harmful effects of NO), has stimulated the search for selective inhibitors of iNOS activity. The following paragraphs highlight some aspects of the chemistry and pharmacology of NOS inhibitors which are more potent inhibitors of iNOS than eNOS activity. For a more detailed and complete account of the chemistry and iso-enzyme selectivity of NOS inhibitors, the interested reader is referred to a recent, excellent review of this topic (Southan and Szabo, 1996). L-NMMA is a competitive inhibitor of the binding of L-arginine to NOS and, hence, excess of L-arginine reverses the inhibition of NOS activity by L-NMMA. Although L-NMMA inhibits all isoform of NOS to a variable degree, it is a more potent inhibitor of iNOS than eNOS activity in cultured cells (Gross et al., 1990). As L-NMMA is only a moderately selective inhibitor of iNOS activity, it is not entirely surprising that L-NMMA exerts both beneficial as well as adverse effects in models of shock, which have been tentatively linked to its ability to inhibit iNOS and eNOS activity, respectively. Thus, the effects of LNMMA in any given model of shock depend on (i) the selectivity of iNOS inhibition achieved with a given dose of L-NMMA, (ii) the degree of iNOS activity in a given model of shock and (iii) the time point of the administration of L-NMMA. As the degree of iNOS induction caused by endotoxin varies between species, L-NMMA is more likely to exert a beneficial effect in shock when used in low doses (greater selectivity) in a species (e.g rat) with a relatively substantial degree of iNOS induction, while adverse effects of L-NMMA are more common when large doses of this L-arginine analogue are used in models/species which do exhibit only a minor degree of iNOS induction. Moreover, when L-NMMA is given prior to endotoxin (e.g. at a time point when there is no induction of iNOS activity), effects due to inhibition of eNOS activity (adverse effects) may predominate. In contrast, when given after (injection of endotoxin) the onset of hypotension, infusions of relatively low doses of L-NMMA (3 to 10 mg/kg/h) have been convincingly demonstrated to exert beneficial hemodynamic effects in rodents (mice, rat), sheep and baboon models of endotoxemia and sepsis. In the baboon, administration of live E.coli bacteria (2 109 colony forming units)

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resulted (after 4 h) in a significant increase in the serum levels of biopterin, neopterin and nitrate, suggesting that administration of live bacteria results in the induction of GTP cyclohydrolase I and iNOS within 4 h (Strohmeier et al., 1995). In this models, infusion of L-NMMA (5 mg/kg/h) attenuated (i) the rise in the serum levels of nitrate and creatinine (an indicator of renal dysfunction), (ii) the hypotension and fall in peripheral vascular resistance and (iii) the substantial 7-day mortality caused by severe sepsis in this species (Daryl Rees and Heinz Redl, personal communication). These findings clearly document that the circulatory failure caused by septic shock in baboons is largely mediated by an enhanced formation of NO by iNOS and that inhibition of iNOS with L-NMMA (or other more selective inhibitors of iNOS activity) improves outcome in this model. Aminoguanidine was the first relatively selective inhibitor of iNOS activity discovered (Corbett et al., 1992). Aminoguanidine is a more potent inhibitor of iNOS than eNOS activity in vitro and in vivo (Misko et al., 1993; Griffith et al., 1993; Joly et al., 1994; Wu et al., 1995). Aminoguanidine attenuates the delayed hypotension in rats (Wu et al., 1995) and rabbits (Seo et al, 1996) with endotoxin shock and improved survival in mice challenged with endotoxin (Wu et al., 1995). In rats subjected to endotoxemia, aminoguanidine also decreased the (i) degree of bacterial translocation presumably by preventing the injury to the gut mucosal barrier (Sorrell et al., 1996), (ii) the disruption of the blood brain barrier (Boje, 1996), the increase in pulmonary transvascular flux (Arkovitz et al., 1996), (iv) the liver injury/dysfunction (Wu et al., 1996), (v) as well as the decrease in total cytochrome P450 content (liver) and the subsequent reduction of the metabolism of ethylmorphin and metazolam (Muller et al., 1996). In contrast, aminoguanidine did neither attenuate the impairment in gluconeogenesis (Ou et al., 1996), nor the cardiac depresssion caused by endotoxin in the rat (Klabunde and Coston, 1995). Unfortunately, there is little information regarding the effects (and side effects) of aminoguanidine in models of shock or sepsis in larger animals. Moreover, the interpretation of the mechanism(s) by which aminoguanidine exerts its beneficial effects in shock is, however, difficult, as aminoguanidine is not a very specific inhibitor of iNOS activity, as this agent elicits many effects, which appear to be unrelated to the inhibition of iNOS activity (non-specific effects) (see Thiemermann, 1997 for review). S-substituted isothioureas (ITUs) are non-amino acid analogues of L-arginine and also potent inhibitors of iNOS activity with variable isoform selectivity (Garvey et al., 1994; Szabo et al., 1994; Southan et al., 1995). The most potent isothioureas are those with only short alkyl chains on the sulphur atom and no substituents on the nitrogen atoms. For instance, S-ethyl-ITU is a potent competitive inhibitor of all isoforms of NOS, while aminoethyl-ITU and S-methyl-ITU are more selective inhibitors of iNOS than of eNOS activity (Southan et al., 1995). Although S-substituted isothioureas also inhibit nNOS activity, they do not cross the blood brain barrier and, hence, may well be useful tools to elucidate the role of an enhanced formation of NO from iNOS in disease states including circulatory shock and inflammation. In rodent models of endotoxin shock, S-methyl-ITU or Aminoethyl-ITU reduce the circulatory failure (hypotension and vascular hyporeactivity to norepinephrine) and the liver injury/dysfunction (Szabo et al., 1994; Thiemermann et al., 1995). In blood vessels obtained from rats with endotoxemia, S-methyl-ITU also attenuated the vasodilator effect and the rise in vascular cGMP caused by L-arginine (Martinez et al., 1996). Moreover, S-methyl-ITU also reduced the vascular leak in the pulmonary vasculature of rats with endotoxic shock (Arkovitz et al., 1996). In a porcine model of endotoxemia, injection of aminoethyl-ITU (10 mg/kg i.v. at 3 h after endotoxin) restored hepatic arterial blood flow (from reduced to normal levels) and increased hepatic oxygen consumption, without affecting cardiac output (Saetre et al., 1997). Like aminoguanidine, isothioureas are also likely to elicit effects which are unrelated to their ability to inhibit NOS activity. For instance, aminoethyl-ITU is a radical scavenger and exerts beneficial effects in models of disease/pathology known to be mediated by oxygen-derived free radicals. Moreover, aminoethyl-ITU also

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prevents the expression of iNOS protein caused by endotoxin in cultured macrophages and in the rat in vivo (Ruetten and Thiemermann, 1996). The number of novel NOS inhibitors which differ in chemistry and selectivity towards certain isoenzymes of NOS is ever increasing (see Southan and Szabo, 1996). In the context of this brief review the NOS inhibitor, L-NG-(l-iminoethyl)lysine, deserves to be mentioned, for this agent exhibits a 30-fold selectivity for iNOS over bNOS. Moreover, L-NG-(1-iminoethyl)lysine exerts potent anti-inflammatory effects in a rat model of adjuvant arthritis (Connor et al., 1995). Moreover, certain amidines including 2iminopiperidine, butyramine, 2-aminopyridine, propioamidine and acetamidine inhibit NOS activity. Interestingly, both 2-iminopiperidine and butyramidine are more potent inhibitors of iNOS activity than LNMMA in murine macrophages (Southan et al., 1996). Recently, an analogue of acetamidine termed 1400W [N-(3-(aminomethyl)benzyl)acetamidine] has been reported to be a slow, tight binding inhibitor of human iNOS. The inhibition by 1400W of the activity of human iNOS was (i) extremely potent (Kd value ~7nM), (ii) dependent on the co-factor NADPH and (iii) either irreversible or extremely slowly reversible. Most notably, 1400W was approximately 5000-fold more potent as an inhibitor of iNOS activity than of eNOS activity. Moreover, the inhibition by this agent of the activity of eNOS or nNOS activity was reversible by L-arginine, while iNOS inhibition was not. In a rat model of vascular injury caused by endotoxin, 1400W was 50-fold more potent as an inhibitor of iNOS than eNOS activity (Garvey et al., 1997). Thus, 1400W appears to be the most potent and selective inhibitor of iNOS activity known to date and, hence, will be an ideal tool to elucidate the role of NO from iNOS in shock and other diseases associated with the induction of iNOS. NITRIC OXIDE SYNTHASE INHIBITION IN HUMANS WITH SEPTIC SHOCK Although our understanding of the role of NO in animal models of circulatory shock has improved substantially over the past years, our knowledge regarding the biosynthesis and importance of NO in the pathophysiology of patients with shock (of various aetiologies) is still very limited. Indeed, a Medline search covering the time period from 1987 to November 1995 revealed that only 8 to 14% of all of the publications which included the key word ‘nitric oxide’ also included the key word ‘human’ (Preiser and Vincent, 1996). Endotoxin and cytokines (when given in combination) causes the expression of iNOS as well as the formation of NO in various human cells (primary or cell lines) including hepatocytes, mesangial cells, retinal pigmented epithelial cells and lung epithelial cells (Morris and Billiar, 1994; Preiser and Vincent, 1996). Elevated plasma levels of nitrite/nitrate occur in patients receiving IL-2 chemotherapy. In contrast, there is also evidence that the plasma levels of nitrite/nitrate are lower in patients after trauma, surgery and in patients with HIV infections. Interestingly, the increase in iNOS activity in leukocytes obtained from patients with sepsis appears to correlate with the number of failing organs, but not with blood pressure. Taken together, these studies support the conclusion that septic shock in man is associated with an enhanced formation of NO. It should, however, be stressed that the increase in the plasma levels of nitrite/ nitrate elicited by endotoxin, cytokines or bacteria in rodents (10-fold) is substantially higher than the observed increases in the plasma levels of these metabolites of NO in other animal species (pig, sheep etc) or humans. Early reports of beneficial haemodynamic effects of L-NMMA in humans with septic shock (Petros et al., 1994) stimulated a placebo-controlled multi-center study involving 312 patients with septic shock, which was designed to evaluate the effects of L-NMMA on the resolution of shock at 72 h (primary endpoint). The severity of illness according to the SAPS II score was similar between placebo and the LNMMA group. Infusion of L-NMMA enhanced mean arterial blood pressure and systemic vascular resistance index and decreased cardiac output (from elevated towards normal levels). L-NMMA had no

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effect on left ventricular systolic work index indicating that the fall in cardiac output was not due to an impairment in cardiac contractility. In patients which had an elevation in mean pulmonary artery pressure, infusion of L-NMMA resulted in a further transient increase in mean pulmonary artery pressure. Interestingly, L-NMMA did not affect the thrombocytopenia or the renal dysfunction caused by sepsis. Most notably, 41% of patients treated with L-NMMA, but only 21% of patients treated with placebo, recovered from shock within 72 h. There was also a strong trend for a reduction in mortality (at day 14) in patients treated with L-NMMA (Robert Grover, personal communication). CONCLUDING REMARKS In the last years numerous agents have been reported to be more potent inhibitors of iNOS than eNOS activity, and some of these have been evaluated in animal models of endotoxemia. There is good evidence that inhibition of iNOS activity attenuates the circulatory failure in endotoxin (or septic) shock in many species. Although some inhibitors of iNOS activity reduce the organ injury/dysfunction associated with endotoxic or septic shock, it is still unclear whether these effects are directly related to the ability of these agents to inhibit iNOS activity, or secondary to improvements in haemodynamics (e.g. L-NMMA) or (at least in part) due to non-specific effects (e.g aminoguanidine, aminoethyl-isothiourea). Studies using mice in which the iNOS gene has been inactivated by gene-targeting to elucidate the role of iNOS in endotoxemia support the notion that NO from iNOS contributes to hypotension and host defence, but provide controversial results regarding the role of iNOS in organ injury and mortality (Wei et al., 1995; Laubach et al., 1996, MacMicking et al., 1995). Our knowledge about the physiological role of NO from iNOS in sepsis (e.g. host defence) is relatively limited, and hence it remains difficult to predict the adverse effects of (even very selective) inhibitors of iNOS activity. Many of the reported adverse effects of NOS inhibition in shock appear to be secondary to the inhibition of eNOS activity (e.g. with large doses of L-NAME), while the infusion of lower doses of L-NMMA (3 to 10 mg/kg/h given after the onset of shock) results in beneficial haemodynamic effects in animals and man with shock, without causing any (apparent) side effects. Any potential adverse effects of non-selective NOS inhibitors may be overcome by combining these agents with NO-donors (e.g. SNAP), which may improve regional haemodynamics and inhibit the adhesion of platelets and PMNs to the endothelium in the absence of NO-synthesis by endothelial cells (due to eNOS inhibition) (Wright et al, 1992). Moreover, the rise in pulmonary vascular resistance caused by L-NMMA (e.g in patients with sepsis and pre-existing pulmonary hypertension) can be overcome by combining the systemic administration of this NOS-inhibitors with NO gas inhalation therapy. Indeed, in anaesthetised pigs with endotoxaemia, NO-gas inhalation alone was sufficient to improve pulmonary haemodynamics, gas exchange and arterial blood gases, while L-NMMA attenuated the circulatory failure. The combination of both therapies, however, was superior to either alone in improving survival (Klemm et al., 1995). The recent discovery of an analogue of acetamidine (1400W), which is a very selective inhibitors of iNOS activity, may enable future studies aimed at using this (or similar pharmacological tools) to elucidate the role of NO from iNOS in the multiple organ failure and mortality associated with septic shock. ACKNOWLEDGEMENTS The author is a Senior Fellow of the British Heart Foundation (FS/96018).

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REFERENCES Arkovitz, M.S., Wispe, J.R., Garcia, V.F. and Szabo, C. (1986) Selective inhibition of the inducible isoform of nitric oxide synthase prevents pulmonary transvascular flux during acute endotoxemia. J.Pediatr. Surg., 31, 1009–15. Baue, A.E. (1993) The multiple organ or systems failure syndrome. In Pathophysiology of shock, sepsis and organ failure, edited by G.Schlag & H.Redl, pp. 1004–18. Berlin: Springer. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. and Freeman, B. (1990) Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA, 87, 1620–24. Billiar, T.R., Curran, R.D., Harbrecht, B.G., Stuehr, D.J., Demetris, A.J. and Simmons, R.L. (1990) Modulation of nitrogen oxide synthesis in vivo: NG-monomethyl-L-arginine inhibits endotoxin-induced nitrite/nitrate biosynthesis while promoting hepatic damage. J.Leukoc. Biol, 48, 565–569. Boje, K.M. (1996) Inhibition of nitric oxide synthase attenuates blood-brain barrier disruption during experimental meningitis. Brain Res., 720, 75–83. Conner, J.R., Manning, P.T., Settle, S.L., Moore, W.M., Jerome, G.M., Webber, R.K. et al. (1995) Suppression of adjuvant-induced arthritis by selective inhibition of inducible-nitric oxide synthase. Eur. J.Pharmacol., 273, 15–24. Corbett, J.A., Tilton, R.G., Chang, K., Hasan, K.S., Ido Y, Wang J.L. et al. (1992) Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes, 41, 552–558. Garvey, E.P., Oplinger, J.A., Furfine, E.S., Kiff, R.J., Laszlo, F, Whittle, B.J.R. and Knowles, R.G. (1997) 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric oxide synthase in vitro and in vivo. J.Biol Chem., 272, 4959–63. Garvey, P.E., Oplinger. J.A., Tanoury, G.J., Sherman, P.A., Fowler, M., Marshall, S. et al. (1994) Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas. J.Biol. Chem., 269, 26669–76. Griffith, M.J., Messent, M., MacAllister, R.J. and Evans, T.W. (1993) Aminoguanidine selectively inhibits inducible nitric oxide synthase. Br. J.Pharmacol., 110, 963–8. Gross, S.S., Stuehr, D.J., Aisaka, K., Jaffe, E.A., Levi. R. and Griffith, O.W. Macrophage and endothelial nitric oxide synthesis: cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methyl-arginine. Biochem Biophys Res Commun, 170, 96–103. Joly, G.A., Ayres, M., Chelly, F. and Kilbourn, R.G. (1994) Effects of NG-methyl-L-arginine, NG-nitro-L-arginine and aminoguanidine on constitutive and inducible nitric oxide synthase in rat aorta. Biochem. Biophys, Res. Commun., 199, 147–54. Julou-Schaeffer, G., Gray, G.A., Fleming, I., Schott, C., Parratt, J.R. and Stoclet, J.C. (1990) Loss of vascular responsiveness induced by endotoxin involves the L-arginine pathway. Am.J.Physiol., 259, H1038–43. Kilbourn, R.G., Gross, S.S., Jubran, A., Adams, J., Griffith, O.W., Levi, R. et al. (1990) NG-methyl-L-arginine inhibits tumour necrosis factor-induced hypotension: implications for the involvement of nitric oxide. Proc. Natl Acad. Sci. USA , 87, 3629–32. Kilbourn, R.G., Juburan, A., Gross, S.S., Griffith, O.W., Levi, R., Adams, J. et al. (1990) Reversal of endotoxinmediated shock by NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis. Biochem. Biophys. Res. Commun., 172, 1132–8. Klabunde, R.E. and Coston, A.F. (1995) Nitric oxide synthase inhibition does not prevent cardiac depression in endotoxic shock. Shock, 3, 73–8. Klemm, P., Thiemermann, C., Winklmaier, G., Martorana, PA. and Henning, R. (1995) Effects of nitric oxide synthase inhibition combined with nitric oxide inhalation in a porcine model of endotoxic shock. Br. J. Pharmacol., 114, 363–368 Laubach, V.E., Sheseley, E.G., Smithies, O. and Sherman, PA. (1995) Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc. Natl. Acad. Sci. USA, 92, 10668– 92. MacMicking, J.D., Nathan, C., Horn, G. et al. (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell, 82, 641–650.

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Martinez, M.C., Muller, B., Stoclet, J.C. and Andriantsitohaina, R. (1996) Alteration by lipopolysaccharide of the relationship between intracellular calcium levels and contraction in rat mesenteric artery. Br. J. Pharmacol., 118, 1218–22. Misko, T.P, Moore, W.M., Kasten, T.P, Nickols, D.A., Corbett, J.A., Tilton, R.G. et al. (1993) Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur. J.Pharmacol., 233, 119–125. Morris, S.M. and Billiar, T.R. (1994) New insights into the regulation of inducible nitric oxide synthase. Am. J.Physiol., 266, E829–39. Muller, C.M., Scierka, A., Stiller, R.L., Kim, Y.M., Cook, D.R., Lancaster, J.R. et al. (1996) Nitric oxide mediates hepatic cytochrome P450 dysfunction induced by endotoxin. Anesthesiology, 84, 1435–42. Nathanson, C., Hoffmann, W.D., Suffredini, E.F., Eichacker, PQ. and Banner, R.L. (1994) Selected treatment strategies for septic shock based on proposed mechanism of pathogenesis. Ann. Intern. Med., 120, 771– 83 Ou, J., Molina, L., Kim, Y.M. and Billiar, T.R. (1996) Excessive NO production does not account for the inhibition of hepatic gluconeogenesis in endotoxemia. Am.J.Physiol., 271, 621–8. Parillo, J.E. (1990) Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction and therapy. Ann. Intern. Med., 113, 227–242. Petros, A., Lamb, G., Leone, A., Moncada, S., Bennett, D. and Vallance, P. (1994) Effects of a nitric oxide synthase inhibitor in humans with septic shock. Cardiovasc. Res., 28, 34–39. Preiser, J.C. and Vincent, J.L. (1996) Nitric oxide involvement in septic shock: Do human beings behave like rodents? In Yearbook of Intensive Care and Emergency Medicine, edited by J.L.Vincent, pp. 358–365, Berlin: Springer. Radomski, M.W., Palmer, R.M.J. and Moncada, S. (1990) Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc. Natl. Acad. Sci. USA., 87, 10043–10047. Rees, D.D., Cellek, S., Palmer, R.M.J. and Moncada, S. (1990) Dexamethasone prevents the induction of nitric oxide synthase and the associated effects on the vascular tone: an insight into endotoxic shock. Biochem. Biophys. Res. Commun., 173, 541–47. Rees, D.D., Cellek, S., Palmer, R.M.J. and Moncada, S. (1990) Dexamethasone prevents the induction of nitric oxide synthase and the associated effects on the vascular tone: an insight into endotoxic shock. Biochem. Biophys. Res. Commun., 173, 541–47. Rees, D.D. (1995) Role of nitric oxide in the vascular dysfunction in septic shock. Biochem. Soc. Trans., 23, 1025–9. Ruetten, H. and Thiemermann, C. (1996) Prevention of the expression of inducible nitric oxide synthase by aminoguanidine or aminoethyl-isothiourea in macrophages and in the rat. Biochem. Biophys. Res. Commun., 225, 525–530. Saetre, T., Gundersen, Y., Thiemermann, C., Lilleansen, P. and Aasen, A.O. (1997) Aminoethyl-isothiourea, a selective inhibitor of inducible nitric oxide synthase activity, improves liver circulation and oxygen metabolism in a porcine model of endotoxaemia. Shock; in press. Schraufstatter, I., Hinshaw, D., Hyslop, P., Spragg, R. and Cochrane, C. (1986) Oxidant injury of cells. DNA strand breaks activate polyadenosine diphosphate ribose polymerase and lend to depletion of nicotinamide adenine dinucleotide. J. Clin. Invest. 77, 1312–19. Seo H.G., Fujiwara, N., Kaneto, H., Asashi, M., Fujii, J. and Taniguchi, N. (1996) Effect of the nitric oxide synthase inhibitor, S-ethyl-isothiourea, on cultured cells and cardiovascular functions of normal and lipopolysaccharide-treated rabbits. J.Biochem., 119, 553–8. Sorrells, D.L., Friend, C., Koltuksuk, U., Courcoulas, A., Boyle, P., Garrett, M. et al. (1996) Inhibition of nitric oxide with aminoguanidine reduces bacterial translocation after endotoxin challenge in vivo. Arch. Surg., 131, 1155–63. Southan, G.J., Szabo, C. and Thiemermann, C. (1995) Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity. Br.J.Pharmacol., 114, 510–16. Southan, G.J., Szabo, C., O’Conner, M.P., Salzman, A.C. and Thiemermann, C. (1995) Amidines are potent inhibitors of nitric oxide synthases: Preferential inhibition of the inducible isoform. Eur. J. Pharmacol., 291, 311–318. Southan, G.J. and Szabo, C. (1996) Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem. Pharmacol., 51, 383–394.

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Strohmeier, W., Werner, E.R., Redl, H., Wachter, H. and Schlag, G. (1995) Plasma nitrate and pteridine levels in experimental bacteremia in baboons. Pteridines, 6, 8–11. Szabo, C., Southan, G.J. and Thiemermann, C. (1994) Beneficial effects and improved survival in rodent models of septic shock with S-methyl-isothiourea sulfate, a novel, potent and selective inhibitor of inducible nitric oxide synthase. Proc. Natl. Acad. Sci. USA, 91, 12472–76. Szabo, C. and Thiemermann, C. (1995) Regulation of the expression of the inducible isoform of nitric oxide synthase. Adv. Pharmacol., 34, 113–54. Szabo, C. (1995) Alterations in nitric oxide production in various forms of circulatory shock. Horizon, 1, 2–32. Thiemermann, C., Ruetten, H., Wu, C.C. and Vane, J.R. (1995) The multiple organ dysfunction syndrome caused by endotoxin in the rat: Attenuation of liver dysfunction by inhibitors of nitric oxide synthase. Br. J. Pharmacol., 116, 2845–2851. Thiemermann, C. and Vane, J.R. (1990) Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharide in the rat. Eur.J.Pharmacol., 182, 591–5. Thiemermann C. (1994) The role of L-arginine:nitric oxide pathway in circulatory shock. Adv. Pharmacol., 28, 45–79. Thiemermann, C., Ruetten, H., Wu, C.C. and Vane, J.R. (1995). The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase. Br.J.Pharmacol., 116, 2845–2851. Thiemermann, C. (1998). The use of selective inhibitors of inducible nitric oxide synthase. Sepsis, 1, 123–129. Wei, X., Charles, I.G., Smith, A. et al. (1995) Altered immune responses in mice lacking inducible nitric oxide synthase. Nature, 375, 408–411. Wright, C.E., Rees, D.D. and Moncada, S. (1992) Protective and pathological roles of nitric oxide in endotoxic shock. Cardiovasc. Res., 26, 48–57. Wu, C.C., Chen, S.J., Szabo, C., Thiemermann, C. and Vane, J.R. (1995) Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock. Br.J.Pharmacol., 114, 1666–1672. Wu, C.C., Ruetten, H. and Thiemermann, C. (1996) Comparison of the effects of aminoguanidine and NG-nitro-L-arginine methyl ester on the multiple organ dysfunction caused by endotoxemia in the rat. Eur.J.Pharmacol., 300, 99–104. Zingarelli, B., O’Conner, M., Wong, H., Salzman, A.L. and Szabo, C. (1996). Peroxynitrite-mediated DNA breakage activates polyadenosine diphosphate ribosyl sythetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide. J.Immunol., 156, 350–358.

19 Kidney Diseases Peter Gross, Heike Dorniok, Doreen Reimann and Maria Plug Department of Medicine, Universitatsklinikum C.G.Carus, Fetscherstaβe 76 LD-01307 Dresden, FRG

The occurrence of constitutive (NOS III) or inducible (NOS II) isoforms of NO-synthase has been described in virtually all vascular and most tubular segments of the normal kidney, including intrarenal arteries and arterioles involved in the regulation of renal perfusion. Stimulation of nitric oxide by intrarenal infusion of acetylcholine decreases renal vascular resistance and increases renal blood flow. NOS I is present in the macula densa; it is instrumental in blunting the tubulo-glomerular feedback in the condition of salt loading. In postischemic acute renal failure, nitric oxide reportedly reaches high concentrations during the incipient hypoxic phase. These concentrations may cause damage to cell membranes. Conversely during the reperfusion phase endogenous nitric oxide may be beneficial to the postischemic kidney. In uremia and dialysis nitric oxide is increased explaining the phenomenon of uremic bleeding. However in some uremic patients endogenous NOS inhibitors may accumulate, reducing NO generation and possibly contributing to hypertension. IgAnephropathy in patients and glomerulonephritis in experimental models have been found to be conditions of increased nitric oxide, which usually has beneficial rather than detrimental effects upon the kidney. Incipient diabetes mellitus induces a preferential increase of NO-generation at sites of diabetic nephropathy, preventable by aminoguanidine. The nephrotoxicity of cyclosporine A is associated with defective renal vasodilatation in response to acetylcholine, implying a lack of nitric oxide. In humans L-arginine fails to correct this defect however. In summary, several kidney diseases will be amenable to nitric oxide based therapies in the future. Key words:Acute renal failure, uremia, glomerulonephritis, IgA-nephropathy, diabetes mellitus, cyclosporine A.

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INTRODUCTION The kidney is one of the most densely vascularized organs of the body. Its function depends on the maintenance of perfusion pressures within narrow limits. However, these limits vary substantially from segment to segment of the renal vasculature. Accordingly, the kidney utilizes an array of vascular control mechanisms to regulate segmentai perfusion as needed. One major regulatory mechanism is related to renal nitric oxide. In this chapter we shall discuss published evidence on the role of nitric oxide in renal disease. As an introduction we will briefly outline the normal functions of NO in the kidney. Recent histochemical work by Bachmann et al. (1995) has delineated the localization of NO-synthases in the normal kidney of the rat, the mouse and the human. The authors observed a strong signal of neuronal NO-synthase (NOS I) in the macula densa. They described an intimate spatial relation between NOS I positive cells of the macula densa and their adjacent renin containing cells in the endothelium of afferent arterioles. In the renal vasculature the constitutive NO synthase (NOS III) was located in the endothelium of cortical and medullary blood vessels. Glomerular arterioles showed strong labelling in the endothelia of both afferent and efferent arterioles. At the capillary level the glomerular tuft showed endothelia positive for NOS III. In addition to the prominent findings of NO synthase in renal vascular structures a more subtle presence of NOS I and NOS III has been described in virtually all renal tubular epithelia (summarized in: Hill-Kapturczak et al., 1995). Studies in normal kidneys have shown that the renal response to calcium mobilizing hormones such as bradykinin or acetylcholine is a release of NO that leads to vasodilatation (Welch et al., 1991). These findings probably reflect the widespread distribution of NOS III in renal blood vessels that was mentioned just previously. Recent interest has centered on the role of NOS I in the macula densa. Wilcox and Welch (1996) showed a blunting of the tubuloglomerular feedback response in the salt loaded rat and this was mediated by nitric oxide. In other words, the salt loaded state enhanced nitric oxide of macula densa origin and this permitted an improved afferent arteriolar blood flow, an enhanced single nephron glomerular filtration rate and enhanced NaCl excretion. As far as a potential role for NOS in renal tubular epithelia is concerned, observations in isolated tubular cells (Neuringer et al., 1991) as well as in isolated perfused kidneys (Gabbai et al., 1992) have been obtained. They suggest that NO may have direct tubular effects to increase reabsorptive transport. Nonetheless a general stimulation of renal NOS III by infusion of bradykinin or acetylcholine will be followed by natriuresis and diuresis. Thus the vascular effects of nitric oxide in the kidney will override any tubular effects. POST-ISCHEMIC ACUTE RENAL FAILURE The role of nitric oxide in the pathophysiology of acute renal failure is ambiguous. Acute renal failure is a condition of renal vasoconstriction, decreased glomerular filtration rate, tubular obstruction and tubular epithelial cell damage. Most available studies of models of post-ischemic acute renal failure have suggested that NOS activity is increased in this condition (Robinette and Conger, 1993; Waz et al., 1994). Yaqoob et al., (1996b) analyzed the effects of hypoxia—which is held to be the initiating event in post-ischemic acute renal failure—on tubular epithelial cells in vitro. They induced hypoxia in freshly isolated proximal tubules of the rat and they directly monitored NO generation with an amperometric NO sensor. Within 7 min. of the induction of hypoxia, NO release started to rise dramatically and thereafter this increase was maintained Correspondence: Peter Gross, M.D., Nephrologie, Klinik für Innere Medizin III, Universitatsklinikum C.G. Carus, Fetscherstr. 76, D-01307 Dresden, F.R.G., Tel:0049 351 458 2645; Fax:0049 351 458 533.

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during a stable phase of elevation (Figure 19–1). Cell membrane damage as assessed by measurements of lactate dehydrogenase (LDH) was first detectable about 8 min. after the start of NO release. The NOS inhibitor L-NAME prevented the hypoxia induced increase in NO in a dose dependent manner and this occurred in parallel to incremental cytoprotection. Noiri et al., (1996) used bilateral crossclamping of the renal pedicle for 45 min. to induce ischemia and they studied the kidneys 24 hrs after reperfusion. Eight hours before the crossclamping the authors injected the rats with antisense oligonucleotides targeting the inducible NOS (NOS II). As a result of the injection a selective knock-down of NOS II in proximal tubules prevailed at the time of ischemia. This was followed by complete functional protection of the kidney from renal failure, an effect that could not be imitated by giving the nonspecific NOS inhibitor L-NAME. The observed effects were attributed to specific rescue of tubular epithelium from lethal injury; as demonstrated in the study, application of antisense constructs did not affect endothelial NOS (NOS III). In a different study in vivo, Conger et al., (1996) examined the role of NO in a rat model of norepinephrine induced acute renal failure one week after its induction by infusing norepinephrine. They concluded that NOS activity was maximally stimulated in their model. The NOS isoform involved in the stimulation was NOS III, not NOS II. It was suggested that the increased NOS activity was a response to the ischemia induced renal vasoconstrictor tone. Administration of the NO inhibitor L-NAME precipitated further profound vasoconstriction in an already malperfused kidney. Considering the discrepant results of Yaqoob et al. (1996b) and Conger et al. (1996) we would like to point to the difference in time of observation in the two studies: Whereas Yaqoob et al (1996b) performed measurements within minutes of induction of hypoxia, Conger et al. (1996) studied nitric oxide one week after the initiation of hypoxia by norepinephrine. Together these experimental models indicate a sequence of NO functions in the evolution of postischemic acute renal failure. In this chain of events different isoforms of NOS in different tissues appear to be involved in succession. Furthermore the importance of the balance between nitric oxide and superoxide for hypoxia-reoxigenation injury merits consideration as pointed out by Yaqoob et al (1996a). In summary the role of nitric oxide in post-ischemic acute renal failure is dependent on time after injury and tissue compartment involved. More specific antagonists of the isoforms of NOS will be required in the future before any therapeutic strategies in patients with acute renal failure can be considered. Radiocontrast nephropathy is a specific kind of post-ischemic acute renal failure. Diabetes mellitus and arterio-sclerosis are known conditions that put patients at risk for radiocontrast nephropathy and these underlying disturbances have been characterized for their defective endothelium-dependent vasorelaxation. In a recently described animal model in the rat the radiocontrast agent iothalamate when injected alone increased outer medullary blood flow by approximately 100% (Agmon et al., 1994). Pretreatment by the NO inhibitor L-NAME reduced basal medullary blood flow and transformed the medullary vasodilator response to radiocontrast into a prolonged vasoconstriction. Acute renal failure developed consistently. It was associated with selective necrosis of medullary thick ascending limbs. A comparable set of experiments in radiocontrast nephropathy was also reported by Andrade and Seguro (1996). It is therefore conceivable that endothelial NO-synthase (NOS III) is impaired in patients at risk for radiocontrast nephropathy. This impairment may be a crucial element in the pathogenesis of the subsequent acute renal failure. Future studies with L-arginine or with intrarenally effective NO donors in patients at risk will be helpful in clarifying the therapeutic potential of these agents. UREMIA AND DIALYSIS It is still an unresolved question whether nitric oxide generation is increased in uremia leading to pathologic changes, or whether the opposite applies because of endogenous inhibitors of NO formation.

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Figure 19±1. (A) Recording of NO concentration in the incubation medium of freshly harvested isolated rat proximal tubules during normoxia (lower curve) and hypoxia (upper curve). (B) Effect of 7.5 min and 15 min of normoxia (C) and hypoxia (H) on LDH release. (Reprinted by permission of the publisher from Yaqoob et al., 1996).

In 1993 Noris et al. (1993) found that L-NMMA normalized the prolongued bleeding time of uremic rats. Extending their study to include patients in chronic renal failure they observed higher NO generation in uremic platelets than in control. They also observed higher cGMP concentrations in uremic platelets and higher L-arginine concentrations in uremic serum versus control. Moreover uremic plasma potently induced NO synthesis by cultured endothelial cells. On the basis of these findings, Noris et al. (1993) proposed a role for enhanced nitric oxide synthesis in uremia to contribute to platelet dysfunction, uremic bleeding and dialysis hypotension. Recent measurements of intradialytic blood levels of nitric oxide in patients

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undergoing chronic hemodialysis (Roccatello et al (1996a) showed higher baseline levels of NO in dialysis patients than in controls and levels increased further during the dialysis procedure. In addition exposure of blood cells to dialysis membrane material has been shown to initiate NO release from neutrophils (Roccatello et al., 1996b) and from vascular endothelial cells that had been in contact with such blood cells (Amore et al., 1995a). However, Vallance et al. (1992) found that uremic patients cannot eliminate endogenous dimethylarginines (DMA), normally excreted in urine. One of the DMA’s, NG,NG-dimethylarginine (ADMA) inhibits NO synthesis in a manner similar to L-NMMA. Uremic patients had higher plasma concentrations of ADMA than control; plasma concentrations of ADMA changed in parallel to the plasma creatinine concentration. Synthetic ADMA as well as urine extracts containing ADMA inhibited NOS II isolated from induced macrophages in a dose dependent manner and similar to L-NMMA. The authors proposed that uremia is associated with an accumulation of endogenous ADMA, inhibiting NO generation and thereby eventually contributing to hypertension and immundysfunction of renal failure. Some of the discrepancies between the studies mentioned previously may be explained by work of Arese et al. (1995). They tested the effects of uremic plasma from patients on NO generation by several different cell lines in culture expressing NOS II and NOS III. They found that 79% of uremic plasma samples inhibited NOS II and NOS III, while 20% stimulated the latter. Plasma taken after a hemodialysis session usually showed a reduced inhibitory capacity; in several cases of “inhibitory plasma” postdialysis plasma samples even stimulated NOS activity. These results indicate that uremia may be a condition in which activating and inhibiting stimuli of NOsynthases compete with each other. This and the possibility of differences in ADMA formation or access to compartimentalized NOS are likely explanations for the discrepancies of results in different studies. GLOMERULONEPHRITIS Glomerulonephritites are a group of renal disorders in which immune glomerular inflammation—usually mediated by cytokines—predominates. Therefore NO-synthases would be expected to have a role in at least some forms of glomerulonephritis. The available evidence indeed shows that nephritic glomeruli often generate increased amounts of NO which seems to be primarily derived from infiltrating macrophages. Thus Kashem et al. (1995) studying the renal biopsies of 28 patients with IgA glomerulonephritis were able to demonstrate the presence of NOS II in most, a finding that was completely absent in renal tissue from control. Observations suggested a macrophage related origin of the NOS II. Of note the authors were able to correlate the expression of NOS II with the degree of adjacent tubulo-interstitial damage in IgA glomerulonephritis. In fact a recent study by Tetsuka et al. (1996) indicated that nitric oxide may enhance the degree of inflammation in the kidney by co-inducing cyclooxigenase-2 expression causing formation of PGE2, an inflammatory mediator. Cattell et al. (1990), Jansen et al. (1994) and Sever et al. (1992) determined nitrite production in models of an accelerated nephrotoxic glomerulonephritis in rats. This was done by measuring the urinary nitrite excretion rate in vivo and by assessing the nitrite generation rate in vitro in cultured glomeruli. The latter were isolated from experimental animals. It was observed that nitrite generation by isolated nephritic glomeruli increased approximately 100-fold during the 24 h induction phase of this glomerulonephritis. Over the following 7 days, nitrite generation returned to baseline. Parallel findings to those in isolated glomeruli were obtained in measurements of urinary nitrite. In these particular models the induction phase of glomerulonephritis is characterized by an influx of macrophages into the glomeruli. Isolated macrophages from the glomeruli of nephritic kidneys were indeed found to show stimulated nitric oxide production and this was attributable to NOS II (Cattell et al., 1991). In a subsequent

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study from the same laboratory Waddington et al. (1996) used a single i.v. injection of arginase to test the role of stimulated NO generation in the pathophysiology of their nephritic model. Arginase is an enzyme that depletes intracellular stores of L-arginine. In the study of Waddington et al. (1996) arginase reduced NO generation by 50%. Surprisingly NO suppression by arginase worsened the manifestations of glomerulonephritis. Thus there was increased proteinuria and intraglomerular thrombus formation in response to arginase. Waddington et al. (1996) concluded that the stimulation of nitric oxide in their model was protective rather than detrimental. A comparable set of experimental observations has also led Munger et al. (1994) to propose a beneficial role of nitric oxide in their animal model of nephrotoxic glomerulonephritis. They reduced endogenous nitric oxide formation 90% by giving the nonspecific NOS inhibitor L-NAME during the phase of granulocyte infiltration of the glomeruli. In response to the treatment with L-NAME the measured parameters of glomerulonephritis worsened:the glomerular filtration rate and the renal blood flow were strongly reduced whereas proteinuria and leucocytic infiltration of the kidney increased. However the results of this study may not simply reflect the direct effects of NO inhibition on the nephritic glomerulus, since the model was simultaneously characterized by systemic hypertension (153±6 mmHg versus 118±7), which lasted several days. Systemic hypertension is usually associated with intrarenal hypertension and the latter may worsen glomerulonephritis in itself. A more extended experiment than that of Munger et al. (1994) was reported by Tikkanen et al. (1994). They studied a model of Heymann-nephritis in the rat to which they gave treatment with L-NAME during 12 weeks of observation. The L-NAME treated rats became hypertensive, too, as was observed in the model of Munger et al. (1994). 12 weeks of L-NAME failed to improve the glomerulonephritis in the model of Tikkanen et al. (1994); instead proteinuria, peritubular infiltrates of monocytes and histologie damage of the kidneys all worsened in response to L-NAME. Tikkanen et al. (1994) concluded that nitric oxide was antiproliferative and antiinflammatory in their model and therefore protective to the kidney. Recently, Gabbai et al. (1996) studied experimental tubulointerstitial nephritis by giving L-NAME and L-NIL—two different NOS inhibitors—to test the role of nitric oxide. L-NAME worsened kidney function and renal pathologic morphology and it augmented antigenspecific IgG, as well as cytokines induced such as interferon y, IL-4 and IL-2. According to their data interstitial injury and functional deterioration are prevented by NO in experimental interstitial nephritis. There is only one experimental model of glomerulonephritis in which nitric oxide appeared to be detrimental while NO inhibition was therapeutic. Ketteler et al. (1993b) performed experiments in Thy-I glomerulonephritis in rats. This model is believed to resemble IgA-nephropathy in humans. Thy-I glomerulonephritis is induced by an injection of anti-thymocyte antibodies. The latter bind to an antigen on renal glomerular mesangial cells. As a consequence of the binding complement is activated and lysis of mesangial cells ensues causing a glomerulonephritis. Ketteler et al. (1993a) gave L-NMMA 60 min before the injection of anti-thymocyte antibodies. L-NMMA prevented lysis of mesangial cells even though the binding of antibodies to mesangial cells and the influx of macrophages were unaltered. L-NMMA also improved proteinuria, expression of TGF-, fibronectin and tenascin. When the experiments were repeated in rats on a protein-restricted diet—presumably causing a degree of depravation of L-arginine—similar improvements of the pathologic changes of the model were observed even when no L-NMMA was given. Taken together the role of nitric oxide in different models of glomerulonephritis is not uniform. In addition to the classical pathways of NO-stimulation and effect additional mechanisms such as autoinduction of NOS-II by NO (Pfeilschifter, 1995) or induction of cyclooxigenase-2 expression by NO (Tetsuka et al., 1996) may be involved. In the majority of the reported models the evidence suggests a protective role of endogenous nitric oxide to the kidney. It is surprising therefore that few if any attempts

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have been made to test the potential therapeutic benefit from NO precursors such as L-arginine, or NO donors supplemented to models of glomerulonephritis. DIABETES MELLITUS According to recent evidence nitric oxide overproduction is involved in the genesis of early diabetic nephropathy, whereas subsequent NO deficiency may contribute to the late changes of glomerulosclerosis. Diabetic nephropathy is closely correlated with the magnitude and duration of antecedent hyperglycemia. One of several mechanisms involved in this process is related to NOS stimulation by glycated protein. Nonenzymatic glycation of long-lived proteins occurs during hyperglycemia. Amore et al. (1997) investigated the effects of glycated albumin on the production of nitric oxide by endothelial cells in vitro. Glycated albumin induced a dose response related increase of NO-synthase activity that was as much as 16fold higher than baseline (Figure 19–2). The effect was still detectable with low concentration of glycated albumin, resembling the concentrations occurring in vivo in diabetic patients. The observed effects were attributable to induction of NOS IL In another set of experiments Sabbatini et al. (1992) infused glycated serum proteins into normal rats. This was found to cause glomerular hyperfiltration, one of the early signs of incipient diabetic nephropathy. In the rat model of streptozotocin induced diabetes early renal changes consisted of significantly higher renal blood flow and glomerular hyperfiltration. In this early stage of diabetes there were also an increased urinary cGMP-excretion rate as well as increased concentrations of nitrite in plasma and urine (Bank and Aynedjian, 1993; Cooper et al., 1993; Dai et al., 1993). Treatment with the NOS inhibitors L-NA or L-NAME diminished glomerular filtration rate and renal blood flow of the diabetic rats to significantly greater extent than in normal control (Bank and Aynedjian 1993; Cooper et al., 1993; Mattar et al., 1994; Tolins et al., 1992). Exogenous NO in the form of nitroglycerin did not confer an additional effect on hemodynamics of diabetic animals (Cooper et al., 1993). The results were interpreted to indicate that NO generation was submaximal in the early stages of streptozotocin-induced diabetes in the rat. In measurements of renal hemodynamics made 6 weeks after induction of streptozotocin diabetes, Tolins et al. (1992) observed an upward shift of the renal curve of blood flow autoregulation. In this way diabetic rats had a higher renal blood flow than controls at low perfusion pressures. Because these changes were inhibitable by L-NAME the results were again taken as evidence of an NO-mediated diabetic effect (Tolins et al., 1992). Corbett et al. (1992) described aminoguanidine as a preferential inhibitor of NOS II Testing the effects of aminoguanidine in diabetic rats they observed the changes induced by aminoguanidine on blood flow or vascular leakage of albumin to be restricted to tissues manifesting vascular dysfunction of diabetes. The results were taken to show that diabetes induces a preferential increase of NO-generation in tissues and vascular beds that are the sites of diabetic vasculopathy. In the later stages of diabetes it is likely that nitric oxide release from the renal vasculature is reduced, similar to observations made in systemic arteries in patients with diabetes (Johnstone et al., 1993). In the same vein, Wang et al. (1993) studying isolated glomeruli in culture obtained from rats 2 to 3 weeks after induction of diabetes found basal cGMP as well as stimulated cGMP to be no larger in diabetic glomeruli than in control. Craven et al. (1994) also showed a progressive impairment of NO-dependent cGMP-generation by glomeruli of diabetic rats depending on the duration of the diabetes. The impaired NO-dependent cGMP-formation and/or decreased NO-synthases of advanced diabetes may contribute to diabetic glomerular capillary hypertension, in this way participating in the pathogenesis of diabetic glomerular injury (Zatz et al., 1986). Taken together the experimental data strongly indicate an overproduction of nitric oxide contributing to the incipient stage of diabetic nephropathy. Accordingly it will be interesting to find out from ongoing clinical trials whether aminoguanidine in patients with diabetes proves to be therapeutic or not.

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Figure 19±2. Dose response curve of NOS activity of murine endothelial cells after incubation with graded concentrations of glycated albumin. (Reprinted by permission of the publisher from Amore et al., 1997).

NEPHROTOXICITY OF CYCLOSPORINE A Systemic hypertension and vasoconstriction of the renal microcirculation are well known side-effects of cyclosporine A (Mason, 1990). Several studies provide evidence that cyclosporine A impairs formation of nitric oxide or its vasodilatory effects (Stephan et al., 1995), directly or by enhanced endothelial superoxide anion-generation, which causes inactivation of nitric oxide (Rubanyi and Vanhoutte, 1986; Diederich et al., 1994). Several authors have shown that NOS-inhibition worsens the nephrotoxicity of cyclosporine A (Amore et al., 1995; Pollock and Polakowski, 1996; Gardner et al., 1996). Gardner et al. (1996) treated rats with cyclosporine A plus the NOS inhibitor L-NAME for 3 weeks. Cyclosporine A dosage was chosen in such a way that cyclosporine A blood levels were within the therapeutic range for human renal transplant patients. Rats treated with cyclosporine A alone showed proximal tubular collapse and vacuolization. However rats receiving the combination of cyclosporine A plus L-NAME had vascular abnormalities consisting of endothelial and arteriolar medial hyperplasia. The relation between cyclosporine A and nitric oxide has also been studied by supplementing L-arginine. Gallego et al. (1993) administered cyclosporine A without and with L-arginine to rats. After 15 days of cyclosporine A the rats showed inhibitions of endothelium-dependent vasodilatation, diuresis, natriuresis and urinary cGMP-excretion rate. The response of the cyclosporine A treated rats to endothelium-independent agents such as sodium nitropresside and atrial natriuretic peptide was unaltered. The toxic effects of cyclosporine A were acutely overcome by administration of the amino acid L-arginine. The coadministration of L-arginine for 15 days prevented the functional effects of cyclosporine A toxicity. L-arginine normalized endotheliumdependent vasorelaxation impaired by cyclosporine A in isolated femoral arteries (Gallego et al., 1994). Bobadilla et al. (1994) also gave cyclosporine A together with L-arginine to rats. L-arginine caused vasodilatation and improved the glomerular filtration rate by approximately 115%, both in comparison to measurements made under cyclosporine A alone.

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Much of the evidence of an impairment of the NO-pathway in endothelial cells in response to cyclosporine A rests on the demonstration of defective acetylcholine induced vasorelaxation (Gallego et al., 1993; Gallego et al., 1994; Diederich et al., 1994; Stephan et al., 1995). No direct measurements of NO or of one of its stable metabolites have been reported. It is therefore remarkable that recent reports have suggested an effect of cyclosporine A to increase the expression of nitric oxide synthase. Amore et al. (1995) observed an increased expression of mRNA for NOS III and NOS II in cyclosporine A toxicity in the kidney, as did Nakayama et al. (1996) but not Pichier et al. (1996). It is possible therefore that cyclosporine A may have direct effects to inhibit NO activity or that increased superoxide-generation and subsequent NO-inactivation (Diederich et al., 1994) are involved in the demonstrated impairment of the NOpathway. With respect to the beneficial effects of L-arginine in experimental cyclosporine A induced nephrotoxicity it is disappointing that such effects have not been reproducable in humans. Gaston et al., (1995) tested 12 renal transplant recipients, 6 of whom were treated with cyclosporine A. They infused 30 g of L-arginine over 120 min and they compared the results obtained with those in 6 transplant recipients on azathioprine, not receiving cyclosporine A. The latter group responded to L-arginine infusion with a decrease of the mean arterial blood pressure, a decrease of renal vascular resistance, an increased renal blood flow and an improved glomerular filtration rate. In contrast, L-arginine—while decreasing mean arterial blood pressure—failed to improve renal blood flow or glomerular filtration rate in the transplant recipients receiving cyclosporine A. The observed effects were specifically related to L-arginine, because branched-chain amino acids did not change any of the parameters measured. A recent study in 11 adolescent kidney transplant recipients receiving cyclosporine A who were given L-arginine for 6 weeks also failed to show a benefit in terms of renal blood flow, glomerular filtration rate or proteinuria (Zhang et al., 1995). Even though L-arginine-related effects may involve insulin stimulation and its subsequent effects on nitric oxide (Giuliano et al., 1997) it is obvious that L-arginine is insufficient in humans to overcome the effects of cyclosporine A on the nitric oxide pathway. MISCELLANEOUS Glomerulosclerosis The endogenous progression of renal insufficiency to endstage renal failure, a process of progressive renal glomerulosclerosis, has been of substantial interest to nephrologists. Because glomerulosclerosis is associated with hyperperfusion of residual renal tissue the question has been raised whether nitric oxide contributed to glomerulosclerosis. An accepted animal model to study glomerulosclerosis is 5/6 nephrectomy in rats. Aiello et al. (1994) studying homogenized renal tissue from 5/6 nephrectomy found basal release of NO to be significantly lower than control. Gehlen et al. (1994) assessed mRNA of NOS II, NOS III and soluble guanylate cyclase A and B by 3 weeks after the induction of 5/6 nephrectomy in the rat. No significant changes from control were found. Functional studies of the effects of NO supplementation have been reported by Ingram et al. (1994), Corner et al. (1996) and Cianciaruso et al. (1996). Ingram et al. (1994) concluded that the reduction of proliferating cell nuclear antigen as well as that of endothelin-1 mRNA in renal cortical tissue from 5/6 nephrectomized rats receiving 2 weeks of supplementation with Larginine indicated a beneficial effect counteracting glomerular hypertrophy of residual tissue. Corner et al., (1996) treated 5/6 nephrectomized rats for 5 months with molsidomine, a donor of nitric oxide. Molsidomine prevented the hypertension of 5/6 nephrectomy and prolongued survival. Rats receiving molsidomine had less proteinuria and higher creatinine clearance than vehicle treated controls. However in the absence of any control treatment with an NO-independent antihypertensive it is not certain that the

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observed benefit was primarily related to NO rather than to its antihypertensive effect. Cianciaruso et al., (1996) gave oral L-arginine to 11 patients with renal insufficiency for 6 months in a controled manner. In the group treated with L-arginine urinary excretion rate of nitrate rose significantly thereby demonstrating increased formation of nitric oxide. However the systemic blood pressure, the glomerular filtration rate and the proteinuria were similar in L-arginine treated and control patients. The study therefore failed to show a protective effect of L-arginine supplementation on the outcome of renal function in patients with glomerular disease. Taken together published data favour the view that the nitric oxide pathway is not of major importance in glomerulosclerosis or in the endogenous progression of renal insufficiency. Pregnancy and Preeclampsia The question has been raised whether deficient nitric oxide production might contribute to the vasoconstriction of the kidney and other vascular beds in preeclampsia (Danielson and Conrad, 1995; Umans and Lindheimer, 1995). Normal gestation in humans and rats is charcterized by a fall in peripheral vascular resistance and arterial blood pressure. Renal vascular resistance normally shows an even larger decline and glomerular filtration rate and renal blood flow peak during mid-gestation at 20 to 40% above pre-conception values. Danielson and Conrad (1995) infused the NOS inhibitors NAME and L-NMMA into virgin control rats and gravid rats on day 14 of gestation. The gravid rats showed significantly increased baseline glomerular filtration rate and renal blood flow while the renal vascular resistance was decreased 30 to 40%. During infusion of NAME or L-NMMA, renal vascular resistance, glomerular filtration rate and renal blood flow were equalized in the pregnant and virgin rats. The gravid animals were more responsive to nitric oxide synthesis inhibition than the virgin ones. These experiments demonstrated a critical role for nitric oxide in the maternal vasodilatory response of normal pregnancy. The findings increase the probability that deficient nitric oxide production might contribute to vasoconstriction of preeclampsia. Ureteral Obstruction Unilateral ureteral obstruction is associated with intense renal vasoconstriction. Baseline NO production was increased in acute unilateral ureteral obstruction (Salvemini et al., 1994). Acute NOS inhibition worsened the vasoconstriction of acute unilateral ureteral obstruction further (Chen et al., 1994). These observations are compatible with the hypothesis that increased nitric oxide may represent a compensatory response to unilateral ureteral obstruction. REFERENCES Agmon, Y., Peleg, H., Greenfeld, Z., Rosen, S. and Brezis, M. (1994) Nitric oxide and prostanoids protect the renal outer medulla from radiocontrast toxicity in the rat. Journal of Clinical Investigation, 94, 1069–1075. Aiello, S., Noris, M., Corna, D., Benigni, A., Zoja, C., Todeschini, M. et al. (1994) Defective renal synthesis of nitric oxide in rats with reduced renal mass. Journal of the American Society of Nephrology, 5, 936. Amore, A., Bonaudo, R., Ghigo, D., Arese, M., Costamagna, C., Cirini, P. et al (1995a) Enhanced production of nitric oxide by blood-dialysis membrane interaction. Journal of the American Society of Nephrology, 6, 1278–1283. Amore, A., Cirina, P., Mitola, S., Peruzzi, L., Gianoglio, B., Rabbone, I. et al. (1997) Nonenzymatically glycated albumin enhances nitric oxide synthase activity and gene expression in endothelial cells. Kidney International, 51, 27–35. Amore, A., Gianoglio, B., Ghigo, D., Peruzzi, L., Porcellini, M.G., Bussolino, F. et al. (1995b) A possible role for nitric oxide in modulating the functional cyclosporine toxicity by arginine. Kidney International, 47, 1507–1514.

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Andrade, L. and Seguro, A.C. (1996) Hypercholesterolemia aggravates radiocontrast nephrotoxicity: protective role of nitric oxide. Journal of the American Society of Nephrology, 7, 1837. Arese, M., Strasly, M., Ruva, C., Costamagna, C., Ghigo, D., MacAllister, R. et al. (1995) Regulation of nitric oxide synthesis in uremia. Nephrology Dialysis Transplantation, 10, 1386–1397. Bachmann, S., Bosse, H.M. and Mundel, P. (1995) Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. American Journal of Physiology, 268, F885–898. . Bank, N. and Aynedjian, H.S. (1993) Role of EDRF in diabetic renal hyperfiltration. Kidney International, 43, 1306–1312. Bobadilla, N.A., Tapia, E., Franco, M., Lopez, P., Mendoza, S., Garcia-Torres, R. et al. (1994) Role of nitric oxide in renal hemodynamic abnormalities of cyclosporine nephrotoxicity. Kidney International, 46, 773– 779. Cattell, V., Cook, T. and Moncada, S. (1990) Glomeruli synthesize nitrite in experimental nephrotoxic nephritis. Kidney International, 38, 1056–1060. Cattell, V., Largen, P., De Heer, E. and Cook, T. (1991) Glomeruli synthesize nitrite in active Heymann nephritis; the source is infiltrating macrophages. Kidney International, 40, 847–851. Chen, R.N., Inman, S.R., Stowe, N.T. and Novick, A.C. (1994) Endothelium derived relaxing factor and renal blood flow during acute ureteral obstruction. Journal of the American Society of Nephrology, 5, 600. Cianciaruso, B., Bellizzi, V., Minutolo, R., Andreucci, M., Russo, R., Conte, G. et al. (1996) Chronic supplementation of L-arginine in patients with moderate renal failure secondary to chronic glomerulonephritis. Journal of the American Society of Nephrology, 7, 1316. Conger, J., Robinette, J., Villar, A., Raij, L. and Shultz, P. (1996) Increased nitric oxide synthase activity despite lack of response to endothelium-dependent vasodilators in postischemic acute renal failure in rats. Journal of Clinical Investigation, 96, 631–638. Cooper, M.E., Komers, R. and Allen, T.J. (1993) Diabetic hyperfiltration:role of nitric oxide. Journal of the American Society of Nephrology, 4, 546. Corbett, J.A., Tilton, R.G., Chang, K., Hasan, K.S., Ido, Y., Wang, J.L. et al. (1992) Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes, 41, 552–556. Corna, D., Noris, M., Luzzana, E., Benigni, A., Zoja, C., Todeschini, M. et al. (1996) A nitric oxide donor either as prophylaxis or therapy slows renal disease progression and prolongs survival in remnant kidney. Journal of the American Society of Nephrology, 7, 1852. Craven, P.A., Studer, R.H. and De Rubertis, F.R. (1994) Impaired nitric oxide-dependent cyclic guanosin monophosphate generation in glomeruli from diabetic rats. Journal of Clinical Investigation, 93, 311– 320. Dai, F.X., Skopec, J., Diederich, A. and Diederich, D. (1993) Effects of aminoguanidine, an inhibitor of inducible nitric oxide synthase in the diabetic rat. Journal of the American Society of Nephrology, 4, 791. Danielson, L.A. and Conrad, K.P. (1995) Acute blockade of nitric oxide synthase inhibits renal vasodilation and hyperfiltration during pregnancy in chronically instrumented conscious rats. Journal of Clinical Investigation, 96, 482–490. Diederich, D., Skopec, J., Diederich, A. and Dai, EX. (1994) Cyclosporine produces endothelial dysfunction by increased production of superoxide. Hypertension, 23, 957–961. Gabbai, F.B., Khang, S., Gold, D. and Kelly, C.J. (1996) Inhibition of inducible nitric oxide synthase intensifies interstitial injury and functional deterioration in experimental interstitial nephritis. Journal of the American Society of Nephrology, 7, 1699. Gabbai, F.B., Peterson, O.W., Khang, S. and Blantz, R.C. (1992) Glomerular hemodynamics in the isolated perfused rat kidney in control and during administration of L-NMMA. Journal of the American Society of Nephrology, 3, 561. Gallego, M.J., Farre, A.L., Riesco, A., Monton, M., Grandes, S., Barat, A. et al. (1993) Blockade of endotheliumdependent responses in conscious rats by cyclosporine A: Effect of L-arginine. American Journal of Physiology, 264, H708–H714. Gallego, M.P., Garcia-Villalon, A.C., Lopez-Farre, A.J., Garcia, J.L., Garron, M.P., Casado, S. et al. (1994) Mechanisms of the endothelial toxicity of cyclosporine A. Role of nitric oxide, cGMP, and Ca2+. Circulation Research, 74, 477–484.

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Gardner, M.P., Houghton, D.C., Andoh, T.E, Lindsley, J. and Bennett, W.M. (1996) Clinically relevant doses and blood levels produce experimental cyclosporine nephrotoxicity when combined with nitric oxide inhibition. Transplantation, 61, 1506–1512. Gaston, R.S., Schlessinger, S.D., Sanders, P.W., Barker, C., Curtis, J.J. and Warnock, D.G. (1995) Cyclosporine inhibits a renal response to L-arginine in human kidney transplant recipients. Journal of the American Society of Nephrology, 5, 1426–1433. Gehlen, E, Wagner, J., Lippoldt, A., Ciechanowicz, A. and Ritz, E. (1994) Gene regulation of the components of the renal nitric oxide system in subtotally nephrectomized rats. Journal of the American Society of Nephrology, 5, 330. Giugliano, D., Marfella, R., Verrazzo, G., Acampora, R., Coppola, L., Cozzolino, D. et al. (1997) The vascular effects of L-arginine in humans. Journal of Clinical Investigation, 99, 433–438. Hill-Kapturczak, N., Kapturczak, M.H., Malinski, T. and Gross, P. (1995) Nitric oxide and nitric oxide synthase in the kidney: potential roles in normal renal function and in renal dysfunction. Endothelium, 3, 253– 299.. Ingram, A., Parbtani, A., Ly, H., Shankland, S.J. and Scholey, J.W. (1994) The effect of dietary supplementation with Larginine on cytokine expression in remnant glomeruli. Journal of the American Society of Nephrology, 5, 581. Jansen, A., Cook, T., Taylor, G.M., Largen, P., Riveros-Moreno, V., Moncada, S. et al. (1994) Induction of nitric oxide synthase in rat immune complex glomerulonephritis. Kidney International, 42, 1107–1112. Johnstone, M.T., Creager, S.J., Scales, K.M., Cusco, J.A., Lee, B.K. and Creager, M.A. (1993) Impaired endotheliumdependent vasodilation in patients with insulin-dependent diabetes mellitus. Circulation, 88, 2510–2516. Kashem, A., Endoh, M., Yamauchi, E, Yano, N., Nomoto, Y. and Sakai, H. (1995) Inducible NOS in human glomerulonephritis; the source is infiltrating monocytes/macrophages. Journal of the American Society of Nephrology, 6, 923. Ketteler, M., Narita, I., Border, WA. and Noble, N.A. (1993a) Nitric oxide-inhibition prevents mesangial cell lysis in experimental glomerulonephritis. Journal of the American Society of Nephrology, 4, 610. Ketteler, M, Narita, I., Bress, D.H., Noble, N.A. and Border, W.A. (1993b) Aminoguanidine inhibits NOsynthesis in nephritic glomeruli. Journal of the American Society of Nephrology, 4, 610. Mason, J. (1990) The pathophysiology of Sandimmune (cyclosporine) in man and animals. Pediatrie Nephrology, 4, 554–574. Mattar, A.L., Ribeiro, M.O., Fujihara, C.K., Padiha, R.M., De Nucci, G. and Zatz, R. (1993) Effects of acute and chronic nitric oxide blockade on renal function of diabetic rats. Journal of the American Society of Nephrology, 4, 799. Munger, K.A., Fogo, A., Nassar, G. and Badr, K.F. (1994) A protective role for nitric oxide in the acute neutrophil dependent phase of nephrotoxic nephritis in rats. Journal of the American Society of Nephrology, 5, 588. Nakayama, Y., Nonoguchi, H., Kitamura, K., Kiyama, S. and Tomita, K. (1996) Induction of nitric oxide synthase II in cyclosporine A induced acute renal failure. Journal of the American Society of Nephrology, 7, 1830. Neuringer, J., Zeidel, M., Troy, J.L., Zayas, M., Otuchere, G. and Brenner, B.M. (1991) NW-nitro-L-arginine methyl ester inhibits renal sodium transport in vivo and in vitro. Journal of the American Society of Nephrology, 2, 510. Noiri, E., Peresleni, T., Miller, F and Goligorsky, M.S. (1996) in vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia. Journal of Clinical Investigation, 97, 2377– 2383. Noris, M., Benigni, A., Boccardo, P., Aiello, S., Todeschini, M. et al. (1993) Enhanced nitric oxide synthesis in uremia: Implications for platelet dysfunction and dialysis hypotension. Kidney International, 44, 445– 450. Pichler, R.H., Andoh, T.F., Hugo, C, Shankland, S.J., Nangaku, M., Ophascharoensuk, V. et al. (1996) Differential expression of nitric oxide synthase isoforms in CSA nephropathy: potential regulation by osteopontin. Journal of the American Society of Nephrology, 7, 1763. Pfeilschifter, J. (1995) Does nitric oxide, an inflammatory mediator of glomerular mesangial cells, have a role in diabetic nephropathy? Kidney International, 48, S 50-S 60. Pollock, D.M. and Polakowski, J.S. (1996) Inhibition of nitric oxide production potentiates the functional response to cyclosporine. Journal of the American Society of Nephrology, 7, 1571. Robinette, J.B. and Conger, J.D. (1993) Nitric oxide synthase activity is increased—not decreased—in vasculature of acute renal failure kidneys. Journal of the American Society of Nephrology, 4, 743.

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Roccatello, D., Mengozzi, G., Alfieri, V., Martina, G., Paradisi, L., Sena, L.M. et al. (1996a) Nitric oxide release in blood from hemodialysis patients: a cross-over study. . Journal of the American Society of Nephrology, 7, 1418. Roccatello, D., Mengozzi, G., Menegatti, E., Paradisi, L., Sena, L.M. and Piccoli, G. (1996b) Early release of nitric oxide by neutrophils interacting with hemodialysis membranes. Journal of the American Society of Nephrology, 7, 1496. Rubanyi, G.M. and Vanhoutte, P.M. (1986) Superoxide anions and hyperoxia inactivate endothelium derived relaxing factor. American Journal of Physiology, 250, H822–H827. Sabbatini, M., Sansone, G., Uccello, F, Giliberti, A., Conte, G. and Andreucci, V.E. (1992) Early glycosylation products induce glomerular hyperfiltration in normal rats. Kidney International, 52, 875–881. Salvemini, D., Seibert, K., Masferrer, J.L., Misko, T.P., Currie, M.G. and Needleman, P. (1994) Endogenous nitric oxide enhances prostaglandin production in a model of renal inflammation. Journal of Clinical Investigation, 93, 1940–1947. Sever, R., Cook, T. and Cattell, V. (1992) Urinary excretion of nitrite and nitrate in experimental glomerulonephritis reflects systemic immune activation and not glomerular synthesis. Clinical Experimental Immunology, 80, 326–329. Stephan, D., Billing, A., Krieger, J.P., Grima, M., Fabre, M., Hofner, M. et al. (1995) Endothelium-dependent relaxation in the isolated rat kidney: impairment by cyclosporine A. Journal of Cardiovascular Pharmacology, 26, 859–868. Tetsuka, T., Daphna-Iken, D., Miller, B.W., Guan, Z., Baier, L.D. and Morrison, A.R. (1996) Nitric oxide amplifies interleukin 1-induced cyclooxigenase-2 expression in rat mesangial cells. Journal of Clinical Investigation, 97, 2051–2056. Tikkanen, L, Uhlenius, N., Tikkanen, T., Holthöfer, H., Törnroth, T., Fyhrquist, F. et al. (1994) Renoprotective role of nitric oxide in Heymann nephritis. Journal of the American Society of Nephrology, 5, 594. Tolins, J.P., Brown, B.M., Raij, L. and Mauer, S.M. (1992) Nitric oxide modulates renal hemodynamics and autoregulation in diabetic rats. Journal of the American Society of Nephrology, 3, 555. Umans, J.G. and Lindheimer, M.D. (1995) The renal adaptation to pregnancy is now “NOS”-talgic. Journal of Clinical Investigation, 96, 4–5. Vallance, P., Leone, A., Calver, A., Collier, J. and Moncada, S. (1992) Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. The Lancet, 28, 997–1000. Waddington, S., Cook, H.T., Reaveley, D., Jansen, A. and Cattell, V. (1996) L-arginine depletion inhibits glomerular nitric oxide synthesis and exacerbates rat nephrotoxic nephritis. Kidney International, 49, 1090–1096. Wang, Y.X., Brooks, D.P. and Edwards, R. (1993) Attenuated glomerular cGMP production and renal vasodilatation in streptozotocin-induced diabetic rats. American Journal of Physiology, 246, R952–R956. Waz, W., Dandona, R., Love, J., Bofinger, D., Van Liew, J. and Feld, L. (1994) Nitric oxide production increases in post-ischemic rat kidneys. Journal of the American Society of Nephrology, 5, 913. Welch, W.J., Wilcox, C.S., Aisaka, K., Gross, S.S., Griffith, O.W., Fontoura, T. etal (1991) Nitric oxide synthesis from L-arginine modulates renal vascular resistance in isolated perfused and intact rat kidneys. Journal of Cardiovascular Pharmacology, 17, S165–S168. Wilcox, C.S. and Welch, W.J. (1996) TGF and nitric oxide: effects of salt intake and salt-sensitive hypertension. Kidney International, 49, (Suppl. 55), S9–S13. Yaqoob, M., Edelstein, C.L. and Schrier, R.W. (1996a) Role of nitric oxide and superoxide balance in hypoxiareoxigenation proximal tubular injury. Nephrology Dialysis Transplantation, 11, 1743–1746. Yaqoob, M., Edelstein, C.L., Wieder, E.D., Alkhunaizi, A.M., Gengaro, P.E., Nemenoff, R.A. et al. (1996b) Nitric oxide kinetics during hypoxia in proximal tubules: effects of acidosis and glycine. Kidney International, 49, 1314–1319. Zatz, R., Dunn, B.R., Myer, T.W., Anderson, S., Rennke, H.G. and Brenner, B.M. (1986) Prevention of diabetic glomerulopathy by pharmacologie amelioration of glomerular capillary hypertension. Journal of Clinical Investigation, 77, 1925–1930.

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20 The Role of Nitric Oxide in Preterai Labour and Preeclampsia Robert E.Garfield1, Irina Buhimschi1, George R.Saade1 and Kristof Chwalisz2 1

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Texas, USA 2

Research Laboratories of Schering AG, Berlin, Germany

INTRODUCTION Female reproduction is generally governed by changes in circulating levels of hormones, particularly the sex steroids that occur during the reproductive cycle. Evidence reported by us and others indicate that nitric oxide (NO) may control many physiological processes in females and that an absence of sufficient NO may lead to several pathological conditions. NO synthesis can be altered in various tissues by sex steroid hormones and cytokines. Therefore, the sex steroids, in particular oestrogens and progesterone, may be responsible for many normal and abnormal states through modulation of NO synthesis or action. In this brief review, we will outline how NO controls some of the important physiological functions during normal pregnancy and how derangement of these mechanism leads to pathological states such as pre-eclampsia and preterm labour. Preterai Labour and Preterm Birth Preterm birth (i.e. birth before 37 completed weeks of gestation) is one of the major causes of maternal and neonatal morbidity and mortality in the world. Preterm birth occurs in about 10% of pregnancies in most European and North American countries and in over 20% of pregnancies in less developed countries. With a world-wide birth rate of about 90 million babies per year, preterm labour is a major health issue and it is the leading cause of infant mortality. It is estimated that approximately 13 million infants are born prematurely worldwide each year (Berkovitz and Papiernik, 1993). Prematurity is responsible for 75% of infant mortality and 50% of long-term neurological handicaps, including blindness, deafness, developmental delay,

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cerebral palsy, and chronic lung disease (Berkovitz and Papiernik, 1993; Creasy, 1993). Thus, any treatment which prolongs the length of gestation could have a profound effect on neonatal mortality and morbidity. The survival rate improves approximately by 2% per day from the 23rd to the 26th week of pregnancy (i.e. from 16% at 23 weeks to 57% at 26 weeks) reaching 80% at 28 weeks and over 90% after 30 weeks of gestation (Haywood et al., 1994). The health care costs incurred as a result of prematurity are enormous. In the US, for example, it was estimated that the total cost per survivor weighing 900 g at birth (approximately 26 weeks of gestation) is in excess of the total average life-time earnings per survivor. Over 4 billion US$ (35% of health care costs for infants) were spent for the treatment of low-birth-weight infants (Iams, 1995). Evidence points toward a multifactorial origin of preterm labour as a syndrome. Several factors including genital or systemic infection, maternal or foetal stress, and low socio-economic status, are associated with preterm labour and preterm birth. However, work conducted by Romero and colleagues (1988), and confirmed by many other investigators, suggests that only 25–30% of all preterm deliveries are associated with an acute inflammatory process, often a result of intrauterine infection. The aetiology of the vast majority of preterm labour is largely unknown. Both intrauterine or systemic infections have been proposed as an important cause of preterm labour (Romero et al., 1988). Systemic maternal infections such as pyelonephritis, pneumonia, and malaria, for example, are associated with preterm labour and preterm birth. On the other hand, colonisation of the lower genital tract with a variety of micro-organisms may lead to ascending intrauterine infection which in turn may cause preterm labour. Infection-related preterm labour most likely is the result of host defence mechanisms involving the release of inflammatory cytokines in response to bacterial products (i.e. LPS, endotoxin). It is believed that the pro inflammatory cytokines (IL-1, TNF , IL-8, etc.) stimulate the production of uterotonins, such as prostaglandins, leukotrienes and oxytocin, in the decidua and foetal membranes, eventually leading to the onset of labour (Kelly, 1994). The cytokines may also trigger local mechanisms of cervical ripening and maturation of foetal membranes. This involves the recruitment of white blood cells, the release of metalloproteinases and finally the degradation of the extracellular matrix, leading to effacement and dilatation of the uterine cervix or to the rupture of foetal membranes. Both maternal and foetal stress can lead to an increased secretion of the corticotropin-releasing hormone (CRH) and other stress hormones. CRH, which increases in the maternal plasma, prior to the onset of labour (McLean et al., 1995), can stimulate myometrial contractility indirectly through the release of uterine prostaglandins (Benedetto et al., 1994). It is also known that CRH is expressed in the placenta (Riley et al., 1991). The increased foeto-placental secretion of CRH, due to foetal stress (e.g. during placental insufficiency), has been proposed as one of the mechanisms underlying preterm labour (McLean et al., 1995). Currently, none of the available treatments for preterm labour have been shown to decrease perinatal mortality. A comprehensive review of 328 randomised, placebo-controlled trials clearly demonstrated that current therapy is unsatisfactory (Higby et al., 1993). This analysis showed that:(a) magnesium sulphate is not superior to placebo, (b) -adrenergic receptor agonists (betamimetics) effectively stop premature labour for only 24–8 hours, (c) the only tocolytic drugs that may be effective are the cyclooxygenase-inhibitors (indomethacin). Nevertheless, betamimetics and magnesium sulphate are still the most widely used

Correspondence: R.E.Garfield, Ph.D., University of Texas Medical Branch, Department of Obstetrics and Gynaecology, Division of Reproductive Sciences, 301 University Blvd., Galveston, Texas 77555–1062, USA. Tel:(409) 772–7590; Fax:(409) 772–2261; E-mail:[email protected]

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tocolytics. Therefore, new tocolytic agents with improved efficacy and reduced side-effects are urgently needed. Both the early diagnosis and prediction of preterm labour remain a major stumbling block in the fight against this major health problem. In the studies described above (Higby et al., 1993), up to 80% of patients diagnosed clinically as being in preterm labour who were given placebo actually deliver more than one week later. On the other hand, delayed diagnosis of preterm labour may also significantly contribute to the failure of tocolytic therapy. Moreover, objective assessment methods of the preparatory (conditioning) changes occurring in the uterus and cervix, long before the onset of active labour, are still not available. Pre-eclampsia Pre-eclampsia affects about 7–10% of pregnant women and is another major cause of maternal and perinatal mortality and morbidity as well as a significant contributor to increased health care costs. The pathophysiology of this condition remains unclear. At the present time, the only therapy for pre-eclampsia is delivery along with acute management or prevention of the related complications. Pre-eclampsia usually does not become clinically manifest until the late third trimester, but the microvascular changes occur weeks before. Classically, pre-eclampsia is defined as the triad of hypertension, proteinuria and pathological oedema associated with foetal growth retardation. Hemodynamically, preeclampsia is characterised by diminished blood volume with haemoconcentration and consequent rheological changes (Arias, 1975; Klee et al., 1993) and by vasoconstriction with increased peripheral resistance (Lin and Walters, 1979; Mabie et al., 1989). It is also associated with endothelial damage and dysfunction leading to increased vascular resistance and loss of refractoriness to vasopressor agents along with increased capillary permeability (Roberts and Redman, 1993). ROLE OF NITRIC OXIDE IN THE CONTROL OF UTERINE AND CERVICAL FUNCTION NO as an Inhibitor of Uterine Contractility There are numerous mechanisms involved in the regulation of uterine contractility. The conversion of the pregnant myometrium from a quiescent to an active and reactive state during labour, a condition dominated by rhythmic, synchronous and forceful contractility, is the result of complex interactions between an array of systems and events. Normal, as well as abnormal labour are believed to be preceded by a series of hormonal changes, including changes in tissue levels or response to steroid hormones (mainly progesterone), prostaglandins and cytokines. These events activate a number of smooth muscle cell processes transforming the myometrium to a higher level of excitability and conductivity. At the same time a down-regulation of mechanisms responsible for uterine relaxation occurs. We refer to this state as the “preparatory” or “conditioning” step a requirement for achieving which is required to achieve effective labour and delivery (Garfield and Yallampalli, 1993; Garfield, 1994). The discovery in the past decade that NO plays a pivotal role in uterine relaxation and pregnancy maintenance should be viewed as a major breakthrough. These findings have changed our understanding of the mechanisms leading to the initiation of parturition. Prior to this time research was mainly focused on the release of stimulatory mediators or uterotonins (i.e. oxytocin, prostaglandins, endothelin) with little attention being paid to inhibitory or relaxing mechanisms. The experimental studies reviewed below

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strongly suggest that the NO system plays a significant role in the maintenance of uterine quiescence during pregnancy and abnormalities in this system lead to preterm delivery. An L-arginine-NO-cyclic guanosine monophosphate (cGMP) pathway has been shown to exist in the rat (Izumi et al., 1993; Yallampalli et al., 1994), guinea pig (Chwalisz et al., 1994), rabbit (Sladek et al., 1993), and human uterus (Buhimschi et al., 1995a). In all these species, the NO system is upregulated during pregnancy (Izumi et al., 1993; Conrad et al., 1993; Natuzzi et al., 1993; Yallampalli et al., 1994, Sladek and Roberts, 1996) and inhibits uterine contractility until term but not during delivery, suggesting that this intrinsic, autocrine, NO generating activity of the uterus during gestation may contribute to the maintenance of uterine quiescence during pregnancy and its withdrawal prior to term may trigger parturition (Izumi et al., 1993; Yallampalli et al., 1994). We and other authors have identified several rate limiting steps in the NO-cGMP pathway that seem to be gestationally regulated. Measurement of total NO generating activity from tissue explants showed that the rat uterus is capable of generating NO in much higher amounts during pregnancy than in the nonpregnant state. Tissues from labouring and postpartum tissues exhibited a low NO generating activity, similar to those from nonpregnant animals (Buhimschi et al., 1996) (Figure 20–1 A). This intrinsic property of the rat uterus to generate NO seems to be located at the level of the myometrial cells since removal of the decidua resulted in only a minor decrease in NO production (Figure 20–1B). The literature is discrepant as to which isoforms of NOS are present in the uterus. However, different species have been examined. The fact that these studies have been performed in different species may have contributed to the confusion. The sheep (Figueroa and Massmann, 1995) and guinea-pig uterus (Weiner et al., 1994a) exhibit primarily constitutive NOS activity regulated by oestrogen and not progesterone. These studies suggest that the increase in NOS activity during pregnancy or after oestrogen treatment is due to increased expression of e and bNOS but not iNOS (Weiner et al., 1994a). Other authors identified only a calmodulin dependent-155 kDa product which they attributed to bNOS in the rat uterus (Jaing et al., 1996). Immunohistochemically, this enzyme was not confined to nerve cells but was widely distributed in several non-neural cells (Schmidt et al., 1992). In contrast, another study identified iNOS mRNA in the rat uterine smooth muscle and increased expression after exposure to LPS (Nakaya et al., 1996). We investigated the presence and gestational differences in NOS isoforms expressed in the rat uterus and concluded that only iNOS and eNOS were present during pregnancy (Buhimschi et al., 1996) (Figure 20–2A to F). We identified bNOS in the nonpregnant rat uterus (unpublished results). However, during pregnancy bNOS levels were virtually absent (Buhimschi et al., 1996) (Figure 20–2C and D). We did not observe a definite correlation between NOS expression measured using RT-PCR, Western blots and total NO generating capacity. Although we detected an absolute increase in iNOS mRNA in the uterus, iNOS protein measured using monoclonal antibodies did not increase significantly during pregnancy. However, a difference in banding pattern was observed between late gestation and during labour (Buhimschi et al., 1996). Similarly, a previous study reported a discrepancy between gene transcription and iNOS mRNA levels in cultured macrophages and detected both transcriptional and post-transcriptional regulation of the enzyme (Weisz et al., 1994). We speculate that although iNOS gene transcription ceases at term, the protein remains present but its activity is abruptly attenuated by post-translational modifications. Such mechanism for the regulation of iNOS has been already described in cultured mouse macrophages (Förstermann and Kleiert, 1995). Furthermore in these cells LPS but not -interferon prolonged the iNOS mRNA half-life from 1 to 4–6 hours (Weisz et al., 1994). Most likely iNOS expression is regulated at several levels:transcriptional, posttranscriptional and post-translational. We also believe that in addition to the relative abundance of the NOS isoforms, quantitative differences in NO production (i.e. picomols for the constitutive and nanomols for the inducible NOS) (Berdeaux, 1993) may also be important in the gestational modulation of the total in vivo

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Figure 20±1. Nitrite production by the nonpregnant (NP) and pregnant rat uterus (A) and myometrium (B) during gestation (19 to 21), day 22 morning of the delivery date (22NL), term delivery (22L) and one day postpartum (ppl). Pieces of whole uterine wall (A) or after removal of the endometrium or decidua (B) were incubated in minimum essential medium for 24 hours. Nitrates in the medium were reduced to nitrites by the acid treated cadmium method. Total nitrites were measured using the Greiss reaction. Statistical analysis: ANOVA followed by post-hoc tests using Fisher’s least significance criteria. Means, shown with ± SEM, with at least one common superscript are not different at a value of p < 0.05. N=5–6 animals in each group.

NO generating activity. This may account for the discrepancies observed between the presence of the enzymes (i.e. increased expression of eNOS during labour: Figure 20–2F) and the NO generating capacity of these tissues (i.e. decreased during labour: Figure 20–1A). NOS activity, both calcium dependant and calcium-independent, (Buhimschi et al., 1995a; Ramsay et al., 1996) and NOS isoforms have also been identified in the human uterus (Telfer et al., 1995). However, the NO synthetic activity of the human myometrium is generally low compared to the rat myometrium and is even lower during pregnancy compared to nonpregnant or labouring patients (Buhimschi et al., 1995a; Ramsay et al., 1996). In addition, the human myometrium is not relaxed by L-arginine (Buhimschi unpublished observation; Jones and Poston, 1997). The human uterus, however has a much higher and sensitivity to NO or NO donors and this sensitivity is gestational dependent (Buhimschi et al., 1995a). It is therefore plausible that the human myometrium is mainly a target for NO but not a source whereas the murine uterus is both a target and a source of NO. Potential sources of NO include gestational tissues adjacent to the myometrium such as placenta or amnion. Both iNOS (Figure 20–3A to C) and eNOS (Figure 20–3D to F) isoforms are present in rat placenta. iNOS exhibits a major down-regulation which starts prior to term (Purcell, et al., 1997) while expression of the endothelial isoform is maintained relatively constant throughout gestation and labour. Using immunohistochemistry iNOS was noted to be confined to the peripheral placental layer (trophospongial cell layer), mainly within the glycogen cells and trophospongial giant cells, in close proximity to the decidua and myometrium (Figure 20–4). NO is usually viewed as a very short acting substance synthesised within or in close proximity of its target tissue. Its half-life of only few seconds may not be consistent with a “paracrine” function for

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Figure 20±2. Western blots of NOS isoforms of the rat uterus (myometrium and decidua) on day 19 of gestation (d19) and during term labor (d22L) from two representative animals at each time are illustrated in panels A (iNOS) C (nNOS) and E (eNOS). Positive controls: A: mouse macrophage lysate (M+); C: cytosol from rat cerebellum (B+); E: lysate from human endothelial cells (E). Densitometric quantification of the bands from 4–7 animals in each group are shown for each isoform below the blot (B: iNOS; D: nNOS; F: eNOS). The densities of bands were normalized against the positive control band within the same gel. Statistical analysis: ANOVA followed by post-hoc tests using Fisher’s least significance criteria. The mean (± SEM) marked with asterisk is statistically different (eNOS on d22L) from the value obtained on d19 (eNOS) at a value of p < 0.05.

placental NO. However, recent data suggest that NO may be carried to remote sites by haemoglobin as a cysteine nitrosylated, thus prolonging its half life and increasing its bioavailability to distant target tissues (Stamler and Simon, 1991). In addition, NO rapidly diffuses to distances of 200–400 mm within tissues (Lancaster, 1994) a distance which is well within the range of placental NO to act on the uterus. The changes in placental iNOS expression suggest a “paracrine” role for NO in regulating uterine contractility, uteroplacental blood flow and immunomodulation required for pregnancy maintenance. In addition, the withdrawal of both placental and uterine NO at term may be involved in the initiation of labour. There is also some controversy regarding which NOS isoforms are present in human placental tissue and whether they are gestationally regulated. Some studies revealed the presence of both calcium-dependent and calcium independent NOS activities (Morris et al., 1995), the latter being significantly higher during the first trimester as compared to term (Sooranna et al., 1995; Ramsay et al., 1996). Other studies, either failed to identify the presence of iNOS mRNA (Garvey et al., 1994) or described calcium independent activity using functional assays but could not identify the iNOS protein on Western blots (Myatt et al. 1993). Both studies (Garvey et al., 1994; Myatt et al., 1993) concluded that the NO synthase enzyme in human placenta appears to correspond to the calcium-calmodulin dependent endothelial isoform. Moreover, other authors could not demonstrate differences in total placental NOS activity at between labouring and nonlabouring (Di Iulio et al., 1996; Thomson et al., 1997). The major drawback of the studies on human placental tissue is

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Figure 20±3. Western blots of the inducible (Panels A and B) and endothelial (Panels C and D) isoforms of the rat placenta on days 16 (d16), 18 (d18), 20 (d20) of gestation, on day 22 morning of the delivery date (22NL) and during term delivery (22L) from 2–3 representative animals at each time are illustrated. Positive controls: A, B: mouse macrophage lysate (M+); D, E: lysate from human endothelial cells (E). Densitometric quantification of the bands are shown for each isoform below the blots (C: iNOS; F: eNOS). The densities of bands were normalized against the positive control band within the same gel. Statistical analysis: ANOVA followed by post-hoc tests using Fisher’s least significance criteria. Means (± SEM) with at least one common superscript are not different at a value of p <0.05. No significant differences were observed in eNOS levels at the times noted above.

that the samples were collected at term from labouring and nonlabouring patients delivered by elective caesarean section. It is possible that at the time caesarean section was performed (which is usually dictated by foetal maturity) iNOS expression may have already been downregulated to the point of non-detection as we have observed in our studies in rats. Based on the above studies, we believe that there is at least a dual (uterine and placental) source of NO during gestation acting in a paracrine (placental) and autocrine (uterine) manner to regulate uterine contractility (Purcell et al., 1997). The relative contribution of each of these is species dependent. Figure 20–5 is a hypothetical representation of the NO sources in the human as compared to the rat. In the human uterus, the placental NO production (paracrine) by far exceeds the intrinsic myometrial NO synthetic activity (Ramsay et al., 1996). This would explain why L-arginine produces a dose-dependant relaxation of rat (Yallampalli et al., 1993) but not the human uterus (Buhimschi, unpublished observation; Jones and Poston, 1997). NO concentration in the tissue decreases exponentially as the distance from the source of NO production increases (Lancaster, 1994). Since the human myometrium relies on placental production of NO and therefore lower local concentration, its sensitivity to NO should be higher than that of the rat myometrium. This is supported by our findings (Buhimschi et al., 1995a). This model also explains the

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Figure 20±4. Localisation of iNOS on day 16 (A) versus day 22 of gestation (B) in lightly counterstained sections. Immunohystochemistry was performed using polyclonal antibodies: L: labyrinth; TCL: trophoblastic cell layer; My: myometrium. Bars bracket the TCL. Magnification: 70×.

poor correlation observed by us (Buhimschi et al., 1995a) and other authors (Weiner et al., 1994b) between the NO and cGMP production from the pregnant human and guinea pig uterine tissue, respectively. In both these species, a high cGMP-generating activity was described with NO synthesis almost absent. It is probable that the myometrium in these species is designed to be a target rather than a source of NO. The in vitro findings of decreased effectiveness of NO donors in vitro in tissues from labouring animals (Izumi et al., 1993; Yallampalli et al. 1993; Buhimschi et al., 1996) raises doubt as to the clinical usefulness of NO donors as tocolytics since the responsiveness of the uterus to NO and cGMP is downregulated at this time. However, we have shown that the uterus responded differently to spontaneously releasing NO adducts, such as diethylenetriamine/NO (DETA/NO) when the compound is given to the whole animal versus when used in isolated tissue in vitro. Namely, uterine tissue isolated from animals during labour (either term or preterm) was relatively nonresponsive to NO while that collected from nonlabouring rats demonstrated a significant reduction in contractility when exposed to NO in organ chambers (Figure 20–6B). This was in contrast to our in vivo studies which indicated that the NO donor had a more pronounced inhibitory effect on intrauterine pressure during term labour as compared to its effect in nonlabouring preterm animals (Buhimschi et al., 1997) (Figure 20–6A). It is possible that the difference in effects between the in vitro and in vivo functions is related to a pharmacological difference or to an effect on alternate pathways present only in vivo. There are also clinical reports documenting the uterine relaxant effect of nitroglycerin (GTN), in pregnant women. GTN has been successfully used to relax the human uterus for a variety of clinical indications including external cephalic version, inverted uterus, hypertonic uterus and breech extractions (Peng et al., 1989; DeSimone et al., 1990; Altabef et al., 1992; Roblin et al., 1991; Greenspoon and Kovacic, 1991). Although these reports are preliminary in nature, they support the role of NO in uterine relaxation. In a pilot observational clinical study, transdermal nitroglycerin (Deponit 10, Schwarz Pharma)

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Figure 20±5. Hypothetical distribution of the of the NO sources and target organs in the rat versus human uteroplacental interface.

inhibited preterm contractions in 13 women (Lees et al., 1994). The study has since included 30 women treated with GTN patches and 30 retrospective control patients treated either with placebo or ritodrine (Black et al., 1996). The average prolongation of gestation was 46 days in the GTN group compared to 27 days in historical controls. Based on these data, a multicenter (15 centres), randomised, controlled trial was initiated to compare GTN patch treatment to intravenous ritodrine and different doses of GTN to placebo. Although, the final results are not yet available, the interim analysis indicates that transdermal GTN is at least as effective as ritodrine (Black et al., 1996; C. Lees, personal communication). The side-effect profile, however, was substantially better for GTN. Although prolongation of gestation is an important target, the ultimate aim of any tocolytic therapy must be to decrease perinatal mortality and morbidity. Such results are not yet available. The Role of NO in Cervical Ripening and Dilatation The ripening of the cervix is another key event in the preparatory changes preceding the onset of labour. By the end of gestation, the cervix, which consists mainly of connective tissue, undergoes considerable softening, shortening and dilatation. The underlying biochemical events for this process are still poorly understood. However, a complex cascade of degradative enzymes (matrix metalloproteinases, MMPs) accompanied by a rearrangement of extracellular matrix (EM) proteins and glycoproteins (Leppert, 1992) appear to be involved. Liggins (1981) was the first to suggest that cervical ripening is similar to an inflammatory process. Infiltration of white blood cells into the cervix occurs in women (Junqueira et al., 1980) and guinea pigs (Hegele-Hartung et al., 1989) at term. This influx of cells is accompanied by rearrangement of the extracellular matrix and increase in activated and degranulated polymorphonuclear granulocytes and macrophages, the source for collagenases and matrix metalloproteinases (MMP) which are involved in dissolution of the connective tissue matrix (Chwalisz et al., 1991; Romero et al., 1990). There is also evidence that other mediators like prostaglandins (Hollingsworth et al., 1980), relaxin (Downing and

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Figure 20±6. (A) Response curves (% decrease in pressure versus time) after administration of DETA/NO (5 mg i.p.: solid symbols) and DETA (3.1 mg i.p.: open symblos) in anesthetized rats during pregnancy (circles) and during spontaneous labour (squares). Relaxation responses as % of control activity were significantly different in both labouring and pregnant animals injected with DETA/NO compared to the groups injected with DETA only (Two-way repeated measures ANOVA). (B) DETA/NO dose relaxation-response curves for longitudinal uterine strips from rats during pregnancy on d19 (solid triangles) and during spontaneous labour (open triangles). Each point represents the mean and SEM from 5 animals in each group. The value for each animal was obtained by averaging the values from 3–6 strips. DETA/NO significantly decreased spontaneous contractility only on day 19.

Sherwood, 1985) and cytokines (Chwalisz et al., 1994) are involved in the remodelling of the connective tissue associated with parturition. Progesterone seems to exert an overall control of cervical ripening, since the antiprogestins are effective agents in inducing cervical ripening in all species investigated to date including humans (Chwalisz and Garfield, 1994). The effect of antiprogestins does not appear to be dependent on prostaglandins, which have been thought for a long time to be the key mediators of cervical ripening (Kelley, 1994). Neither indomethacin (Chwalisz, 1994) nor the specific COX-II inhibitor flosulide (Shi et al., 1996a) inhibited the physiological and antiprogestin-induced cervical ripening in animals or humans. NO plays a complex role in inflammation. It is well established that both LPS and the pro-inflammatory cytokines are the most potent inducers of iNOS (high-output isoform) expression in the macrophages. NO and its oxidation products accumulate at the site of inflamation and NOS inhibitors can suppress inflammation (Evans et al., 1995). The induction of iNOS in various tissues (e.g. in arthritic joints) can lead to the sustained production of high concentrations of NO which may induce pro-inflammatory effects including vasodilatation, oedema, cytotoxicity, tissue remodelling, and the mediation of cytokine-dependent processes. Such might be the case in the cervix during the ripening process. Studies from our group have shown that tissue explants from cervices of rats collected during labour, either at term or preterm after antiprogesterone treatment, have increased NO synthetic activity. During the

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Figure 20±7. (A) Nitrite production by the nonpregnant (NP) and pregnant rat cervix during gestation (19 to 21), day 22 morning of the delivery date (22NL), term delivery (22L) and one day postpartum (ppl). The cervix was defined as the less vascular tissue with parallel lumina between the uterine horns and the vagina and was incubated in minimum essential medium for 24 hours. Nitrates in the medium were reduced to nitrites by the acid treated cadmium method. Total nitrites were measured using the Greiss reaction. Statistical analysis: ANOVA followed by post-hoc tests using Fisher’s least significance criteria. Means, shown with ± SEM, with at least one common superscript are not different at a value of p<0.05. N=5–6 animals in each group. (B) Nitrite production in nonpregnant (NPLPS) and pregnant rat uterus and cervix on day 20 of gestation (d20LPS) after i.p. administration of 400 (μg/kg LPS or vehicle (NP and d20, respectively). The animals were sacrificed 6 hours after injection. Statistical analysis: ANOVA followed by post-hoc tests using Fisher’s least significance criteria. Means (± SEM) with different superscripts are different at a value of p<0. 05. N=4 animals in each group.

rest of the gestational period, NO generation in the cervix remained low similar to that seen in nonpregnant animals (Figure 20–7A). Interestingly, administration of LPS resulted in a marked increase in NO production in the cervix in both the pregnant and nonpregnant states while in the uterus the increase was only noted in the nonpregnant rat (Figure 20–7B). Therefore the uterine iNOS during pregnancy and cervical iNOS during labour are already upregulated and cannot be induced any further by LPS (Buhimschi et al., 1996). All three NOS isoforms are present in the rat cervix and, in contrast to the uterus, the gestational changes in mRNA production are in agreement with the changes in NO synthetic activity. There is an absolute increase in iNOS and bNOS expression in the ripened cervix. eNOS is present but at constant levels throughout pregnancy, labour and postpartum (Figure 20–8). In vitro functional studies on the tensile properties of isolated rat cervices using incremental stretch have shown that the cervical extensibility increases gradually from day 19 to day 22 morning of the delivery day and further on day 22. Cervices collected during labour show a further increase in extensibility compared to cervices collected on the morning of the delivery day (Figure 20–9A). These findings are similar to those from previous studies (Harkness and Harkness, 1959). We observed that although the duration of gestation remains unchanged, in vivo treatment of rats with L-NAME (50 mg/day) significantly prolongs the duration of active foetal expulsion. In addition, overnight incubation of cervices from rats on day 20 of gestation with L-NAME significantly decreases their compliance as they become stiffer behaving more like cervices from rats at earlier gestation (Figure 20–9B). Since the change in cervical extensibility during rat pregnancy

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Figure 20±8. Western blots of NOS isoforms of the rat cervix on day 19 of gestation (d19) and during term labor (d22L) from two representative animals at each time are illustrated in panels A (iNOS) C (nNOS) and E (eNOS). Positive controls: A: mouse macrophage lysate (M+); C: cytosol from rat cerebellum (B+); E: lysate from human endothelial cells (E) Densitometric quantification of the bands from 4–7 animals in each group are shown for each isoform below the blot (B: iNOS; D: nNOS; F: eNOS). The densities of bands were normalized against the positive control band within the same gel. Statistical analysis: ANOVA followed by posthoc tests using Fisher’s least significance criteria. The means (± SEM) on day 22L marked with asterisks are statistically different from the value obtained on d19 for the respective isoform at a value of p<0.05 (*) or <0.01 (**).

is gradual (Figure 20–9A) while the change in NO production is more abrupt (Figure 20–7A), it is probable that NO is involved in the dilatation of the cervix as a final step of the ripening process, rather than earlier. We were also able to enhance ripening by applying a gel formulation of sodium nitroprusside on the cervix of pregnant guinea pigs. This treatment led to a significant increase in cervical extensibility accompanied by the rearrangement of collagen fibres, stromal oedema, arterial dilatation, and infiltration of macrophages, lymphocytes and granulocytes (Shi et al., 1996b; Chwalisz et al., 1997). These studies suggest that NO exerts a powerful physiological function during cervical ripening, acting probably at the end of the inflammatory cascade. NO may act in concert with PGE2 to induce local vasodilatation, increase vascular permeability and promote leukocyte infiltration. In addition, NO may directly regulate the activity of metalloproteinases (MMPs). These enzymes are Zn++ and Ca++-dependent, and may be controlled by NO in a manner similar to guanylate cyclase. Indeed, NO stimulates gelatinase activity in rat mesangial cells (Trachtman et al., 1996). NO action at various locations (uterus, cervix, implantation sites, ovary, vasculature, etc.) may be mediated in part by MMPs. In support of this concept is the finding that cytokines, which are known to regulate NO synthesis, also modulate MMPs (Goetzl et al., 1996). Recent studies show that there is cross-talk between NO and cyclooxygenase (COX) another critical enzyme in many inflammatory responses, including cervical ripening. The prostaglandin pathway shares a

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Figure 20±9. (A) Cervical extensibility (measured as the reciprocal of the slope of a straight line fitted through the linear part of the maximum force versus displacement curve) during gestation (days 15, 16, 19 to 21), day 22 the morning of the delivery day (22NL) and during term labor (22L). Statistical analysis: ANOVA followed by post-hoc tests using Fisher’s least significance criteria. Means (± SEM) with at least one common superscript are not different at a value of p<0.05. N=4 on each day of gestation. (B) Extensibility of cervices incubated overnight with 3 mM L-NAME and their matched controls. N=6 in each group. The mean (± SEM) marked with asterisk is significantly lower at a value of p<0.005 (paired t test).

number of striking similarities with the NO pathway. COX exists in at least two isoforms: COX-1 which is constitutively present in most tissues and is involved in the physiological production of prostaglandins, and COX-2 which, like iNOS is cytokine-inducible and is expressed in inflammatory cells (Xie et al., 1991; Sirois and Richards, 1992). In response to endotoxin, IL-1, and TNF- , COX-2 produces large amounts of pro-inflammatory prostaglandins. Recent in vitro and in vivo studies demonstrate that NO directly stimulates COX-2 activity, thus increasing prostaglandin production during inflammation (Salvemini et al., 1995). Conversely, prostaglandins modulate NO production (Di Rosa et al., 1995). Therefore, the interactions between NO and prostaglandins represent a new powerful mechanism that may alter the course of an inflammatory process. It appears likely that this mechanism also operates in the cervix during the ripening process. Our studies, indicate that the NO system may be a new target for novel therapeutic agents capable of both stimulating (NO donors) and inhibiting (NOS inhibitors) cervical ripening. Local cervical application of agents which do not stimulate uterine contractions may have certain advantages over prostaglandins which are known to cause tetanic uterine contrac tions and foetal compromise. Preliminary clinical studies support the concept that NO donors can be used to ripen the human cervix (Thomson et al., 1997). NITRIC OXIDE AND PRE-ECLAMPSIA While the aetiology of pre-eclampsia remains unclear, during the past decade several theories on the origin of pre-eclampsia have been proposed (Table 20–1). However, as long as the cause of pre-eclampsia remains

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Table 20±1. Major ethio-pathogenic theories of preeclampsia.

unclear it is extremely difficult to assess whether the described changes have a causal relationship with preeclampsia or are just a response to the clinical syndrome. In 1973, Gant et al. reported the results of a study in which they followed normal pregnant women longitudinally and infused them with increasing concentrations of angiotensin II. The concentration of angiotensin that was required to raise the diastolic blood pressure by 20 mmHg was determined and referred to as the Effective Presser Dose (EPD). At the end of the study, the patients were separated into those who had a normal pregnancy and those who developed pre-eclampsia in the third trimester. They found that those patients who ultimately completed a normal pregnancy had a higher EPD than nonpregnant patients. The EPD in normal pregnancy increased progressively throughout gestation. In those that ultimately developed pre-eclampsia, the EPD was initially the same as those with normal pregnancy outcome but progressively decreased, reaching below non-pregnant levels long before the disease became clinically manifest. This study indicated that normal pregnancy is associated with an early and progressive refractoriness to pressor agents and that preeclampsia is preceded by an increased sensitivity to pressor agents. Few years later, the same investigators reported the same findings in patients with pre-existing chronic hypertension who became pregnant (Gant et al., 1977). Those who completed a normal pregnancy had a progressively increasing resistance to angiotensin while those who ultimately developed superimposed preeclampsia lost that resistance to angiotensin early in their gestation. This indicated that hypertension in preeclampsia was just a delayed clinical manifestation of an abnormality at the microvascular level that was unique to pregnancy and reversible with delivery. It became apparent that to understand the aetiology of preeclampsia, we must first determine the pathways responsible for the changes in vascular reactivity in a normal pregnancy. Initial efforts concentrated on the renin-angiotensin system, the cyclooxygenase products and endothelin. One by one however, these theories fell into disfavour (Ito et al., 1992; Mitchell, 1991; Brown et al., 1990). Following the report by Furchgott and Zawadski (1980), attention was directed to EDRF or NO as ideal candidates to play a central role in pre-eclampsia, a condition known to be associated with endothelial dysfunction (Roberts and Redman, 1993). Several in vitro studies using a variety of isolated arteries from different animals have shown that EDRF and NO are important in the regulation of vascular reactivity in pregnancy. The in vitro release of EDRF in response to acetylcholine and the calcium ionophore, A23187, is increased in pregnancy (Matsumoto et al., 1992; Weiner et al., 1989). Increased EDRF production also alters the arterial contractile response to a variety of agents during pregnancy (Weiner et al., 1991, 1992; Hull et al., 1992; Grigs et al., 1993). These effects have been found to depend on the animal and the vessel used. Furthermore, regional variation in the stimulated release of endothelium-derived NO of the thoracic was also observed (Gregg et al., 1995). It was noted that at any resting tension, rings from pregnant rabbits

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contracted significantly less than those from non-pregnant rabbits (Belfort et al., 1993). In a separate experiment using the same model, it was showed that pregnancy is associated with a decrease in the response of isolated aortic rings to serotonin, endothelin and the thromboxane analogue U46619, but not to phenylephrine. Depending on the agent used, this refractoriness was partially or totally reversed by removal of the endothelium or inhibition of nitric oxide synthesis, respectively (Saade et al., 1994a). Studies in the intact animal have also confirmed the importance of the endothelium in vascular homeostasis in pregnancy. The vasoconstrictor responses to a variety of agonists are decreased in the in situ blood-perfused mesenteric vessels of pregnant rats, an effect partially reversed by nitric oxide synthase blockade (Chu and Beilin et al., 1993). Animal Models of Pre-eclampsia Many attempts have been made to mimic or induce pre-eclampsia in different animal species, including nonhuman primates. Several animal models have been described, none of which have a complete resemblance to human pre-eclampsia and some of the findings could not be reproduced by other investigators. A preeclampsia-like condition can be produced in 50–83% of pregnant ewes by a 72-hour food deprivation. This condition is characterised by maternal hypertension, foetal depression (hypoxia), premature delivery and maternal death due to seizures (Thatcher and Keith, 1986). A similar condition occurs in late pregnant guinea-pigs either spontaneously or induced by ketosis (Wagner, 1976). Whether these conditions are similar to human pre-eclampsia remains unclear since systematic studies on blood pressure and other symptoms of pre-eclampsia are not available. Pre-eclampsia-like conditions can occur naturally in nonhuman primates (Stout and Lemmon, 1969) and there are reports of experimentally-induced pre-eclampsia in baboons and Rhesus monkeys by partial occlusion of the aorta (Cavanagh et al., 1985; Combs et al., 1993). However, none of these models have convincingly reproduced the clinical syndrome associated with pre-eclampsia. Effects of NOS Inhibition During Pregnancy in Animals Numerous studies performed in nonpregnant animals have indicated that inhibition of NO synthesis, usually by competitive inhibition of the NOS enzymes, resulted in a dramatic increase in blood pressure. We and other authors have reported that chronic inhibition of NO synthesis in pregnant rats with the L-arginine analogue NG-nitro-L-arginine methyl ester (L-NAME) causes hypertension, proteinuria (Baylis et al., 1992) and foetal growth retardation without affecting gestational length (Yallampalli and Garfield, 1993; Molnár et al., 1994). Glomerular damage (Baylis et al., 1992) and histopathological changes in the placental bed (Osawa, 1996) similar to human pre-eclampsia are also present. The structural alterations of the glomeruli seem present only in gravid animals and not in virgin animals that have undergone the same treatment (Molnár et al., 1994). The increased blood pressure and foetal growth retardation can be reversed by simultaneous infusion of L-arginine but not D-arginine (Buhimschi et al., 1995b). Further studies in our laboratory using the Sprague-Dawley rats with chronic NO inhibition as a model identified that there is an initial rise in pressure on the day following the initiation of L-NAME treatment (usually on day 17 or 18 of pregnancy) which persists for one day only and then rapidly returns to near the range for the untreated control pregnant rats (Figure 20–10). The day before the onset of labour, the blood pressure starts to increase again and remains elevated throughout the postpartum period. This was in contrast to the sustained blood pressure elevation noted by other investigators in nonpregnant rats treated with L-NAME (Molnár and Hertelendy, 1992; Liao et al., 1996). We also performed studies that compared

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Figure 20±10. Effect of NG-nitro-L-arginine methyl ester (L-NAME) on systolic blood pressure of pregnant versus nonpregnant females or normal male rats. Blood pressure levels were measured on day 17 before the osmotic minipumps were implanted S.C., and then daily up to the tenth day postpartum (pp10). Animals received 150 mg/kg/day L-NAME in saline solution or saline only (controls). Each point in each of the final groups represents mean ± SEM from 5 male rats, 8 nonpregnant or 13 pregnant female rats.

the systolic blood pressure of nonpregnant rats both males and females with the systolic blood pressure of pregnant rats after L-NAME infusion and identified the existence of a blood pressure lowering mechanism during the pregnant state. This was confirmed by the observation that the blood pressure in the L-NAME treated pregnant rats becomes similar to the L-NAME treated nonpregnant rats shortly after pregnancy termination. Moreover, the response to L-NAME is independent of the sex of the animal (Figure 20–10). The compensatory mechanism responsible for this refractoriness is unknown, but it probably represents a foetal and/or placental protective mechanism specific for pregnancy. The refractoriness of the blood pressure to the effects of nitric oxide synthase inhibition seen only in pregnancy resembles that described with other pressor agents (i.e. vasopressin, angiotensin, epinephrine, norepinephrine) in vivo and in vitro using human or animal models (Gant et al., 1987). Moreover, the initiation of L-NAME treatment early in pregnancy (day 11) resulted in sustained hypertension throughout mid gestation that gradually decreased close to term. Postpartum the blood pressure rose again to levels higher than those seen during midgestation (Shi et al., 1997). In conclusion, the decrease in blood pressure which occurs in pregnancy despite continuous infusion of L-NAME is dependant on a mechanism operative only during late gestation in rats. The late gestational period in rats (i.e. day 17–22) is marked by important changes in steroid hormones such as progesterone withdrawal and rise in oestrogen prior to labour (Puri and Garfield, 1982). We tested

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Figure 20±11. (A) Systolic blood pressure in rats in rats chronically infused with 150 mg/kg/day L-NAME or saline alone during pregnancy (with or without L-NAME infusions) and postpartum after injections of progesterone (A: P4, 6 mg/kg/day), promegestone (B: R5020, 1.5 mg/kg/day); levonorgestrel (C: 1-NOR, 1.5 mg/kg/day) or mifepristone (D: RU486, 30 mg/kg/day). The L-NAME or saline osmotic mini-pumps were inserted on day 17 (d17) after the first blood pressure measurement. Progestins, antiprogestins or vehicle (sesame oil) were administrated S.C. daily from postpartum day 1 (pp1) up to postpartum day 10 (pp10). Each point in each of the final groups of rats represents mean ± SEM for 5 rats. Means with at least one common superscript are not different at a value of p<0.05.

the hypothesis that steroid hormones (namely oestrogen, progesterone or androgens) modulate the refractoriness to L-NAME. We observed that pregnane derivatives such as progesterone (Figure 20–11 A) and promegestone (R5020: a “pure” progesterone receptor agonist devoid of any antimineralocorticoidic activity, Figure 20–11B) and not the 19-nortestosterone derivatives such as levonorgestrel have hypotensive effects after L-NAME inhibition (Figure 20–11C). Antiprogestin administration resulted in a further increase of blood pressure levels (Figure 20–1 ID). Androgens (Figure 20–12B), but not oestrogens (Figure 20–12A), also decreased blood pressure after NO blockade. The hypotensive effect of progestins in rats continuously infused with L-NAME is apparent not only during pregnancy and postpartum (Buhimschi et al., 1995b) but also in nonpregnant female and male rats (Liao et al., 1996). Promegestone was also able to lower blood pressure in spontaneously hypertensive rats. Our conclusion was that progesterone and androgens, but not oestrogens, may be important in regulating the vascular adaptation during pregnancy. However, progesterone may not be the main factor responsible for the refractoriness to L-NAME during late gestation since our more recent data show that blood pressure falls concomitantly with progesterone withdrawal (Shi et al., 1997). Another possibility is that chronic NO blockade produced experimentally in rats induces, through feedback regulation, increased expression of NOS that may compensate at least partially for the lack of NO.

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Figure 20±12. (A) Systolic blood pressure in rats in rats chronically infused with 150 mg/kg/day L-NAME or saline alone during pregnancy (with or without L-NAME infusions) and postpartum after injections of 17 -oestradiol (A: 17 E2 30 μg/kg/day) or 5 -dihydrotestosterone (B: 5 -DHT, 0.3 mg/kg/day). The L-NAME or saline osmotic minipumps were inserted on day 17 (d17) after the first blood pressure measurement. 17 -Oestradiol and 5 dihydrotestosterone or vehicle were administrated S.C. daily from postpartum day 1 (pp1) or 2 up to postpartum day 10 (pp10). Each point in each of the final groups of rats represents mean ± SEM for 5 rats. Means with at least one common superscript are not different at a value of p<0.05.

however, had no effect (Buhimschi et al., 1997d) indicating the importance of the balance between vasoconstrictor and vasodilator prostaglandins. A recent study performed by Sibai and colleagues (Lubarski et al., 1997), using non-pregnant versus pregnant Wistar-Kyoto rats with chronic NO inhibition, and which confirmed our findings with L-NAME, also showed that in contrast to L-NAME (which inhibits eNOS to a greater extent than iNOS), aminoguanidine (a selective iNOS inhibitor) administration does not result in increased blood pressure in either pregnant or nonpregnant rats. This study (Lubarski et al., 1997) also confirms our previous hypothesis based on experiments on the rat uterus that the different NOS isoforms may have different roles during pregnancy. While iNOS is the isoform involved in regulating uterine contractility, the vascular adap tation to pregnancy is dependent on the activity of the endothelial isoform. We also believe that, to some extent, these isoforms may complement each other and act to balance NO production in case of a deficiency of one of the isoforms. In vitro and In vivo Studies on Human Pre-eclampsia Studies on isolated vessels from different animal species demonstrate that the physiological vascular adaptation to pregnancy (increased blood volume, increased cardiac output and decreased vascular resistance) is accompanied by an increase in endogenous NO production (Weiner et al., 1994; Nathan et al., 1995) and enhanced responsiveness of the vascular smooth muscle to NO (Izumi et al., 1994). The available data from human pregnancy, however, is not as conclusive. Steele et al. (1993) reported no difference between isolated uterine arteries from pregnant and non-pregnant patients in response to norepinephrine. Removal of the endothelium, however, increased the response of the vessels from pregnant women but the inhibition of nitric oxide synthase had no effect. Using a system similar to that reported described by Angus and colleagues (He et al., 1988) it was demonstrated that at any internal circumference, omental arteries from pre-eclamptic patients had a higher wall tension than those from normotensive patients. This

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difference was present in the activated, passive and relaxed states denoting that these vessels had different mechanical and structural differences (Saade et al., 1994b). Using isolated human omental artery in organ chambers, it has also been shown that pregnancy reduces the contractile effect of the thromboxane analogue U46619. This refractoriness was dependent on the presence of an intact endothelium but was not affected by nitric oxide synthase inhibition (Saade et al., 1995). In a separate experiment, it was showed that the response of isolated omental artery to norepinephrine is decreased during normal pregnancy. This refractoriness to norepinephrine was absent in omental arteries from preeclamptic patients which had similar responses to those from non-pregnant women (Belfort et al., 1995). Finally, other investigators have shown that the endothelium-dependent relaxation of isolated subcutaneous and umbilical arteries is impaired in pre-eclampsia (Pinto et al., 1991; McCarthy et al., 1993). Recently, a few in vivo studies evaluating the role of nitric oxide in vascular reactivity in pregnancy or pre-eclampsia have been published. The stable isotope [15N]L-arginine was infused into normal pregnant volunteers and used the rate of production of [15N] labelled nitrite/nitrate to estimate nitric oxide production at different stages of pregnancy and postpartum. We found that arginine flux and nitric oxide production are increased in early, but not late pregnancy as compared to postpartum (Goodrum et al., 1996). Other investigators have measured the response of local blood flow to various agents as a method to evaluate the role of nitric oxide in human pregnancy in vivo. lontophoretic administration of acetylcholine and sodium nitroprusside into the volar aspect of the forearm resulted in marked increase in the skin blood flow, as measured with laser Doppler. The degree of vasodilatation was not significantly different between non-pregnant, normotensive pregnant and pre-eclamptic women (Eneroth-Grimfors et al., 1993). Ford et al. (1996) measured the change in venous diameter in response to L-NMMA infusion. L-NMMA resulted in venoconstriction in women immediately postpartum but not in the same women 12–16 weeks postpartum or in nonpregnant controls. Measuring the forearm blood flow response to brachial artery infusion of L-NMMA, Anumba et al. (1997) found that normal pregnant women showed an enhanced constrictor response when compared to nonpregnant volunteers, indicating increased basal nitric oxide activity in pregnancy. The effect of L-NAME, however, was not different between normal pregnant and pre-eclamptic patients, arguing against a decrease in nitric oxide activity in pre-eclampsia. However, these are acute studies that may not duplicate conditions of chronic impairment of the NO system. Two recently published studies used Doppler ultrasound to determine the effects of NO donors on uterine and umbilical vessels in preeclamptic women. Both studies showed that NO dilated the umbilical vasculature but had no effect on the uterine artery Doppler indices or on the systemic blood pressure (Grunewald et al., 1995; Makino et al., 1997). These, along with the data from our laboratory summarised in the previous section, indicate that the endothelium is an important modulator of the refractoriness seen in pregnancy. Whether this endothelial effect is totally or partially secondary to increased nitric oxide production in the human pregnancy remains to be determined. REFERENCES Altabef, K.M., Spencer, J.T. and Zinberg, S. (1992) Intravenous nitroglycerin for uterine relaxation of an inverted uterus. Am. J. Obstet. Gynecol., 166, 1237–1238. Anumba, D.O.C., Ford G.A., Boys R.J. and Robson, S.G. (1996) The role of nitric oxide in the modulation of vascular tone in normal pregnancy. Br. J. Obstet. Gynecol., 103, 1165–1170. Arias, F. (1975) Expansion of intravascular volume and foetal outcome in patients with chronic hypertension and pregnancy. Am. J.Obstet. Gynecol., 123, 610–616.

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Baylis, C., Mitruka, B. and Deng, A. (1992) Chronic blockade of nitric oxde synthesis in the rat produces systemic hypertention and glomerular damage. J. Clin. Invest., 20, 278–281. Belfort, M., Saade, G., Kramer, W., Suresh, M., Moise, K. and Vedernikov, Y. (1995) Effects of selected vasoconstrictor agonists on isolated omental artery from premenopausal nonpregnant women, and from normotensive and preeclamptic pregnant women. Am. J.Obstet. Gynecol., 172, 360 [Abstract #366]. Belfort, M., Saade, G., Van Den Veyver, I., Valdez-Torres, A., Hsu, H., Longmire, S. et al. (1993) Pregnancy reduces the maximal tension developed in rabbit thoracic aorta. In: Proceedings of the 40th annual meeting of the Society for Gynecologic Investigation, pp. 306 [Abstract # P247]. Benedetto, C., Petraglia, F., Marozio, L., Chiarolini, L., Florio, P., Genazzani, A.R. et al. (1994) Corticotropin-releasing hormone increases prostaglandin F2 alpha activity on human myometrium in vitro. Am. J. Obstet. Gynecol., 171, 126–131. Berdeaux, A. (1993) Nitric oxide: an ubiquitous messenger. [Review] Fundam. Clin. Pharmacol. 7, 401–411. Berkovitz, G.S. and Papiernik, E. (1993) Epidemiology of preterm birth. Epidemiol. Rev., 15, 414±443. Black, R.S., Flint, S., Lees, C. and Campbell, S. (1996) Preterm labour and delivery. Eur. J.Pediatr., 155, 2–7. Brown, C.E., Gant, N.F., Cox, K., Spitz, B., Rosenfeld, C.R. and Magnes, RR. (1990) Low-dose aspirin II. Relationship of angiotensin II pressor responses, circulating eicosanoids, and pregnancy outcome. Am. J. Obstet. Gynecol. 163, 1853–61. Buhimschi, C., Buhimschi, I., Yallampalli, C., Ckwalisz, K. and Garfield RE. (1997a) Contrasting effects of DETA/ NO, a spontaneously releasing nitric oxide donor, on pregnant rat uterine contractility in vitro versus in vivo. Am. J. Obstet. Gynecol., 177, 690–701. Buhimschi, L, Chwalisz, K., Saade, G. and Garfield, R.E. (1997b) The effect of an endothelin antagonist on blood pressure in a rat model of preeclampsia. Am. J. Obstet. Gynecol., 176, S102, [Abstract #335]. Buhimschi, I., Chwalisz, K., Saade, G. and Garfield, R.E. (1997c) The effect of prostacyclin agonists on blood pressure in a rat model of preeclamspia. Am. J. Obstet. Gynecol., 176, S102, [Abstract #336]. Buhimschi, L, Chwalisz, K., Saade, G. and Garfield, R.E. (1997d) The effect of indomethacin on blood pressure in a rat model of preeclamspia. Am. J.Obstet. Gynecol., 176, S106, [Abstract #336]. Buhimschi, L, Ali, M., Jain, V., Chwalisz, K. and Garfield, R.E. (1996) Differential regulation of nitric oxide in the rat uterus and cervix during pregnancy and labour. Hum. Reprod., 11, 1755–1766. Buhimschi, I., Yallampalli, C., Dong, Y.L. and Garfield, R.E. (1995a) Involvement of a nitric oxide-cGMP pathway in control of human uterine contractility during pregnancy. Am. J. Obstet. Gynecol., 172, 1577– 1584. Buhimschi, L, Yallampalli, C, Chwalisz, K. and Garfield, R.E. (1995b) Preeclampsia-like conditions induced by nitric oxide inhibition: effects of L-arginine, D-arginine and steroid hormones. Hum. Reprod., 10, 2723– 2730. Cavanagh, D., Rao, P.S., Knuppel, R.A., Desai, U. and Balis, J.U. (1985) Pregnancy-induced hypertension: development of a model in the pregnant primate (Papio anubis). Am.J.Obstet. Gynecol, 151, 987–999. Chu, Z.M. and Beilin, L.J. (1993) Mechanisms of vasodilation in pregnancy:studies of the role of prostaglandins and nitric-oxide in changes of vascular reactivity in the in situ blood perfused mesentery of pregnant rats. Br.J.Pharmacol, 109, 322–29. Chwalisz, K., Ciesla, I. and Garfield, R.E. (1994) Inhibition of nitric oxide (NO) synthesis induces preterai parturition and preeclampsia-like conditions in guinea pigs. In Proceedings of the 41th annual meeting of the Society for Gynecologic Investigation, pp. 26, [Abstract #O36]. Chwalisz, K., Benson, M., Scholz, P., Daum, J., Beier, H.M. and Helge-Hartung, C. (1994) Cervical ripening with cytokines interleukin 8, interleukin 1b and tumor necrosis factor a in guinea-pigs. Hum. Reprod., 2, 2173–2181. Chwalisz, K. (1994) The use of progesterone antagonists for cervical ripening and as an adjunct to labour and delivery. Hum. Reprod., 9, 131–161. Chwalisz, K. and Garfield, R.E. (1994) Antiprogestins in the induction of labour. Ann. New York Acad. Sci, 734, 387–413. Chwalisz, K., Hegele-Hartung, C., Schulz, R., Shi Shao, Q., Louton, P.T. and Elger, W. (1991) Progesterone control of cervical ripening—experimental studies with the progesterone antagonists onapristone, liloprostone and mifepristone.

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21 Multiple Sclerosis Jean E.Merrill* Department of Immunology, Berlex Biosciences, 15409 San Pablo Avenue, Richmond, CA 94804, USA

Nitric oxide (NO) has been shown to be capable of modulating the induction of the immune response, the permeability of the blood brain barrier (BBB), trafficking of cells to the central nervous system (CNS), and local responses in the inflammatory milieu. Therefore, it might be predicted that NO could be both beneficial and harmful in an autoimmune disease such as MS. This chapter will deal with the various aspects of NO as it pertains to MS by exploring in vivo and in vitro models and evidence from MS patients’ tissue. Key words: Cytokines, multiple sclerosis, nitric oxide, experimental allergic encephalomyelitis, free radicals. INTRODUCTION Multiple sclerosis (MS) is thought to be an inflammatory, autoimmune demyelinating disease in which myelin and the myelin-producing cells, called oligodendrocytes, are destroyed. Axonal insulation, called myelin, is a multilamellar membrane required for efficient nerve conduction. When myelin is disrupted, conduction slows and clinical signs become evident. In MS plaque tissue, oligodendrocytes die by necrosis; this is evidenced by their hypertrophied cell bodies and disrupted plasma and mitochondrial membranes. The prevalence of actively phagocytosing cells in the plaques has implicated activated macrophages as myelin and oligodendrocyte destroyers. Their presence infers that, among other toxins, free radicals of oxygen and nitrogen are at work in this pathology.

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CYTOKINES IN MS AND EAE Proinflammatory cytokines such as interferon gamma (IFNγ), interleukin-1 beta (IL-1 ), and tumor necrosis factor alpha (TNF ), or ligands for the Fc receptor (FcR) and complement receptor (C3biR) can activate macrophages and glia to produce free radicals of oxygen and nitrogen in the rodent (Murphy et al., 1995). While proinflammatory cytokines do not directly activate adult human macrophages and glia to produce NO, they most likely amplify NO production incurred by additional membrane perturbations or the crosslinking of cell surface molecules other than cytokine receptors (DeMaria et al., 1994; Bukrinsky et al., 1995; Vouldoukis et al., 1995). In that regard, proinflammatory cytokines could play a role in NO production in the peripheral immune system as well as in the CNS. The studies examining proinflammatory cytokines at the protein and/or mRNA levels in MS patients’ blood plasma, cerebrospinal fluid (CSF), brain tissue, and cultured blood leukocytes that among other proinflammatory cytokines, IFNγ IL-1, and TNFα are elevated (Hofman et al., 1986, 1989; Merrill et al., 1989; Hauser et al., 1990; Merrill et al., 1992; Huberman et al., 1993; Link et al., 1994; Merrill and Benveniste, 1996). An elevation in TNFα and IFNγ predicts a relapse in MS and the number of circulating IFNγ-positive blood cells correlates with moderate to severe disability (Beck et al., 1988; Link et al., 1994). IL-1 is constitutively produced by MS patients’ freshly isolated lymphocytes in vitro in the absence of stimulation, suggesting in vivo activation (Merrill et al., 1989). In clinical trials, treatment of MS patients with IFNγ exacerbated the disease (The IFN MS Study group, 1993), while treatment with interferon- (IFNβ) improved patients’ clinical scores. It is therefore believed that IFNβ may benefit MS patients by inhibiting IFNγ-inducible major histocompatibility antigen class II (MHCII) genes, among others (Ransohoff et al., 1991). Interestingly, the mRNAs for transforming growth factor beta (TGFβ) and interleukin-4 (IL-4), cytokines which can be both pro- and antiinflammatory, are also elevated in MS patients’ blood cells as determined by in situ hybridization (Link et al., 1994). These studies provide evidence for an association between activated macrophages and microglia and their cytokine products with the inflammatory loci in MS CNS tissue. In the case of experimental allergic encephalomyelitis (EAE), it is also clear that the clinical and histological findings are dependent on the macrophage as an effector cell. EAE is thought to be a CD4+ T cell-dependent, Thl-mediated disease in which MHCII is elevated on macrophages and microglia. In preclinical EAE, IL-2, IL-2R, and IFNγ are all elevated, while IL-4 and TNFα appear at the height of clinical disease in the brains and spinal cords of these animals (Merrill et al., 1992b). Interleukin-12 (IL-12), which induces the Thl response, unmasks latent EAE in an otherwise resistant strain of mouse (Segal and Shevach, 1996). The presence of interleukin-10 (IL-10) in EAE brain tissue correlates with recovery from disease in one study (Kennedy et al., 1992), but fails to abrogate disease in another study (Cannella et al., 1996). The ability of retinoid to reduce EAE correlates with increased IL-4 and decreased IL-2, TNF , and IFN (Racke et al., 1995). Interference with TNF or IL-1 function in vivo in EAE animals by use of specific antibodies to either the ligand or receptor, soluble receptors, or receptor antagonists ameliorates the disease (Ruddle et al., 1990). Physical or functional elimination of macrophages from EAE-susceptible animals prevents disease induction (Huitinga et al., 1990). Pretreatment of EAE animals before disease induction with antiinflammatory cytokines such as interleukin-13 (IL-13) or IL-10 inhibits the disease, while IL-4 enhances the disease in one model (Cash et al., 1994; Willenborg et al., 1995; Rott et al., 1994). IL-6 suppresses demyelination in the Theiler’s virusinduced model (Rodriguez et al., 1994). IL-13 inhibits both clinical and histological signs in EAE and may Correspondence: Hoechst Marion Roussel Inc., Route 202–206, P.O. Box 6800, Bridgewater, NJ 08807– 0800, USA.

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do so through its capacity to down-regulate IL-1, TNFα, and NO production by macrophages and microglia (Cash et al., 1994). In a different way of inhibiting EAE, Khoury et al. (1992) have demonstrated that myelin basic protein-induced oral tolerance is associated with the upregulation of TNFβ, IL-4, and prostaglandin E (PGE). Both MHCII and TNF production, elevated before the oral tolerance, are downregulated in myelin-fed animals (Khoury et al., 1992). All these data point to a role for IL-12, TNF , IL-1 , IFN , and IL-2 in disease induction and perpetuation, while IL-10, IL-4, IL-6, IL-13, IFN , and PGE negatively regulate the proinflammatory events and thereby ameliorate disease in the animal model. EVIDENCE FOR THE INVOLVEMENT OF NO IN MS AND DEMYELINATING MODELS NO in Animal Models Only recently has significant progress been made in studying the role of NO in the pathogenesis of experimental animal models of MS and other neuroimmunologic disorders. MacMicking et al. (1992) first reported elevated spontaneous NO and superoxide anion (O2−) release ex vivo by both peripheral and CNSderived neutrophils and mononuclear cells isolated from Lewis rats with EAE. The release of both free radicals was augmented by incubation of these cells with encephalitogenic T cells, probably via the release of the proinflammatory cytokines TNFα, IL-1β and IL-2. The observation that both peripheral and CNSderived cells produced NO, suggesting that the inflammatory cells responsible for mediating EAE in this model were likely to be activated prior to entering the CNS. Koprowski et al. (1993) used iNOS-specific oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR) to evaluate iNOS induction in the brains of rodents with both encephalitic viral diseases and EAE. Intraocular injection of herpes simplex virus type-1 (HSV-1) was associated with iNOS mRNA expression in all mice with clinical signs of encephalitis. In rats infected with borna disease virus (BDV), iNOS mRNA induction was observed to be highest at a time when animals had severe neurological symptoms associated with perivascular necrosis. In guinea pig MBP-induced acute Lewis rat EAE, iNOS mRNA induction was evident prior to, during, and after clinical symptoms. Okuda et al (1995) showed iNOS-positive macrophages in spinal cords of mice with actively induced EAE, correlating with the peak of disease. The reactivity of NO with heme and non-heme iron (Fe) centres to form electron paramagnetic resonance (EPR)-detectable Fe-NO complexes was utilized in a study by Lin et al. (1993) to demonstrate endogenous NO formation in an adoptive transfer EAE model. Definitive EPR spectra of Fe-NO complexes of ironsulfur proteins were observed in all 10 spinal cord samples from female SJL/J mice with EAE. No evidence for the formation of EPR-detectable Fe-NO complexes was detected in peripheral tissues such as spleen, liver and blood of the affected animals. The SJL/J EAE model in this study exhibited a chronic relapsingremitting disease course with animals exhibiting clinical symptoms for prolonged periods post T cell transfer. The observation that EPR-detectable NO adducts could be detected in spinal cords of animals with EAE at 14–75 days post transfer strongly supports the contention that NO may be an important mediator of chronic inflammation. The results of the above studies show that iNOS mRNA and protein (and presumably enzyme activity) are induced in rodent EAE models of MS and rodent models of virally-mediated inflammatory encephalitis. A determination of how much NO is actually produced in these models and whether it is sufficient to mediate cytotoxicity was addressed by Hooper et al. (1995) using a novel method for spin trapping NO in vivo. In adoptive transfer EAE in Lewis rats, 20–30 VI NO were observed in the spinal cord on days 4 and 5

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post T cell transfer, correlating with hind limb paralysis on day 4 and general paralysis on day 5. Although elevated NO levels were detected in brain, they were substantially less than in spinal cord, consistent with the ascending course of this disease with the spinal cord as the primary site of lesion development. In the same study, BDV and rabies models were also studied to direct inflammation to the brain. In both viral diseases NO production in the brain was highest at the time of onset of neurological symptoms. The importance of these observations with NO spin traps in vivo is that (1) they establish that CNS tissues in both viral and T-cell mediated encephalitis are exposed for prolonged periods to very high levels of NO; (2) they establish that high output NO synthesis localizes to the site of inflammatory disease; (3) they confirm and extend the utility of a new technique to directly determine the efficacy of therapeutic agents directed at inhibiting NO production in the CNS. Such therapeutic agents as nitric oxide synthase (NOS) inhibitors or inhibitors of iNOS induction have been tested in animal models. Inhibitors of iNOS in EAE There is obviously a strong rationale for testing the potential efficacy of iNOS inhibitors in treating EAE. The first reported study of an iNOS inhibitor in EAE was by Cross et al. (1994) in an adoptive transfer EAE model in SJL/J mice. Aminoguanidine, a fairly selective but weak inhibitor of iNOS, was used for the study at high doses: 100 and 200 mg/kg/day s.c. or 400 mg/kg/day i.p., with treatment starting on the day of T cell transfer. A substantial decrease in mean maximum clinical score was observed at 400 and 200 but not at 100 mg/kg/day in aminoguanidine treated animals compared to placebo. A delay in disease onset was only observed for 400 mg/kg/day. Histological analysis of spinal cords from EAE mice revealed a reduction in inflammation, demyelination and axonal damage, which reached significance at the highest dose only. There was no non-specific effects of aminoguanidine on the immune system, since the compound did not decrease proliferation of MBP-specific T cells in vitro. Inducible NOS inhibitors have also been studied in Lewis rat EAE and experimental allergic neuritis (BAN) (Zielasek et al., 1995; Ruuls et al., 1996; Zhao et al., 1996). Four L-arginine analog NOS inhibitors were tested: N-monomethyl-L-arginine (100 mg/kg/day given p.o.), N-nitro-L-arginine (87.5 mg/kg/day given p.o.), aminoguanidine (50 and 200 mg/kg/day given i.p.) and N-nitro-L-arginine methyl ester (NAME; 150 mg/kg/day given i.p). Ammonium acetate was used as a control. In EAN, a modest protective effect on disease score, compound muscle action potential, demyelination and inflammation was seen for Nmonomethyl-L-arginine only. NAME had a modest effect on clinical score only and neither aminoguanidine nor N-nitro-L-arginine had any effect on any parameter studied. In one study in acute EAE, no beneficial effect of any compound was observed. In the other two EAE studies in Lewis rats, N-nitro-L-arginine (125 mg/kg twice a day i.p.) and N-monomethyl-L-arginine (225 μg/kg once a day i.v.) showed a modest exacerbation of clinical score (Ruuls et al., 1996), while aminoguanidine (100 mg/kg/twice a day s.c.) profoundly decreased the incidence, severity, and disease duration in the treated animals (Zhao et al., 1996). Some of the disparity between the aminoguanidine results obtained in Lewis rat EAE and in SJL/J mouse EAE is reconcilable. The one Lewis rat study did use lower doses and a different route of administration than the SJL/J study and this may account in part for the differences. Unlike the Zhao study, where iNOS was examined and found to be absent in the aminoguanidine treated animals’ inflammatory foci, in neither of the other two studies was it shown that the administered NOS inhibitor actually inhibited iNOS in situ or that NO production in the affected tissue was actually blocked. Bioavailability of aminoguanidine and other standard L-arginine analog NOS inhibitors and their subsequent access to the spinal cords and brains is likely to be a key issue in designing future therapeutics and testing them in animal models.

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Evidence for Involvement of NO in the Pathology of MS Within the demyelinated lesions in MS brain, activation of both astrocytes and microglia in the form of gliosis has been reported. In addition, the accumulation of inflammatory macrophages and the presence of NO-inducing proinflammatory cytokines like IFNγ, IL1 , and TNF at the lesion edge have suggested NO may contribute to the disease process (reviewed in Merrill and Murphy, 1996). Evidence to support this hypothesis includes the presence of astrocyte-associated NADPH diaphorase staining in chronic and acute MS brain lesions. iNOS mRNA has been detected in these plaques by RT-PCR (Bô et al., 1995); iNOS protein, nitrotyrosine staining, and in situ RT-PCR for iNOS colocalizes with Ricinus communis agglutinin-1 staining, a marker for macrophages/microglia. iNOS and NO were found in MS but not control brain tissue (Bagasra et al., 1995). Recently, DeGroot et al., (1997) have also found strong iNOS positivity in cells in MS lesions; the cells were identified as parenchymal and perivascular macrophgages and were only iNOS positive in active demyelinating areas. Our own studies suggest that astrocytes, microglia, inflammatory macrophages, and even endothelial cells make iNOS in MS tissue (Figure 21–1). Increased NO production probably occurs in MS given that there are elevated levels of nitrite and nitrate levels in CSF of patients (Johnson et al., 1995). In the same study, increased levels of neopterin, a precursor of the iNOS enzyme cofactor tetrahydrobiopterin, was also reported in CSF from MS patients. The reaction of NO with O2− forms peroxynitrite (ONOO−), a strong trans-nitrosating agent capable of nitrosating susceptible protein thiols, such as cysteine. This chemical reaction may result in formation of nitroso-amino acids, such as nitrosocysteine, potentially making them immunogenic. Thus it is of interest that MS patients have elevated circulating antibodies to conjugated S-nitrosocysteine epitopes suggesting nitrosation of proteins in vivo (Boullerne et al., 1995). Evidence for NO-Induced Damage of Oligodendrocytes In vitro As discussed above, activated monocytes, macrophages, and microglia secrete various cytokines, including IL1 , TNF , and NO. Microglia are cytotoxic mediators of oligodendrocyte cell death in vitro. Microglial cytotoxicity and NO production appear to be mediated partially and indirectly by TNF . That is, TNF does not induce NO by itself, nor can soluble TNF directly kill oligoden drocytes; TNF associated with the cell surface of microglia may play a role in the cytotoxicity (Zajicek et al., 1992; Chao et al., 1993; Merrill et al., 1993; Merrill and Benveniste, 1996). TNF secretion, NO production, and/ or cytotoxicity by these cells can be regulated by several types of agents, including antiinflammatory cytokines, antioxidants, and compounds which raise intracellular cAMP (see discussion below). We and others have previously shown that nitric oxide can impair mitochondrial function in oligodendrocytes and astrocytes (Bolaños et al., 1994; Mitrovic et al., 1994) and that it induces a necrotic like cell death in oligodendrocytes in vitro (Merrill et al., 1993; Mitrovic et al., 1995). Nevertheless, not all oligodendrocytes from primary brain cultures are killed by NO. Indeed, we have discovered NO-resistant oligodendrocyte cell lines suggesting that there are differential sensitivities among maturational subsets of oligodendrocytes to the toxic effects of this molecule (Mackenzie-Graham et al., 1994; Mitrovic et al., 1994). It has been proposed that a cell which makes NO may be able to protect itself from such toxic effects on intracellular proteins. This has been suggested for neurons and seems to be the case for microglial cells (Mitrovic et al., 1994; Nussler and Billiar, 1993;Simmons and Murphy, 1992) and astrocy tes (Bolaños et al., 1994). Inculture, the myelin producing cell, the oligodendrocyte, is vulnerable to toxicities mediated by complement, antibodies, cytokines, oxygen free radicals and nitric oxide produced by macrophages (Merrill et al., 1993; Merrill and Benveniste, 1996). Studies performed in this laboratory have demonstrated an IFNγinduced, NO-dependent microglia cell-mediated cytotoxicity of oligodendrocytes. Ameboid rodent

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Figure 21±1. Immunohistochemical analysis by double staining of markers for macrophages/microglia and iNOS in a chronic active lesion in MS. A. Staining for the macrophage/microglial cell marker CD68 (DAKO, Carpinteria, CA used at 1:100). The primary antibody was followed by a secondary affinity purified, Texas Red-conjugated goat F(ab’)2 anti-mouse IgG (Molecular Probes, Eugene, OR used at 1:50). B. The same section was stained with a rabbit IgG affinity purified antibody to an NH2 terminal peptide of human iNOS which does not cross react with bNOS or ecNOS (see Ding et al., J. Biol. Chem.); this antibody was generated at Berlex Laboratories and used at 1:500. This was followed by a secondary affinity purified, fluoresceinconjugated goat F(ab’)2 anti rabbit IgG at 1:50 (Molecular Probes). The section was viewed using a Zeiss Axioplan microscope with 35mm camera attachment and fluorescein and rhodamine filters used either separately or together (C). Double staining shows that CD68-positive inflammatory macrophages and peri vascular microglia are iNOS positive; single staining of iNOS at the blood brain barrier also suggests that endothelial cells are positive.

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microglial cells, after treatment with PMA, LPS, and/or IFNγ, produce micromolar concentrations of NO within 24h. NO production, as well as oligodendrocyte lysis, is inhibited by NOS inhibitors and anti-TNFα antibodies, thus suggesting that NO may be the mechanism of macrophage/microglial cell killing of oligodendrocytes. (Merrill et al., 1993) These in vitro results suggest a direct role for NO in oligodendrocyte cell loss and primary demyelination in EAE and MS in vivo. Primary glial cells in vitro are differentially affected by nitric oxide. Cultures enriched for microglia, astrocytes or oligodendrocytes were treated with S-nitroso-N-acetyl D, L-penicillamine (SNAP), an NOreleasing chemical. There was a significant decrease in the function of the ferrosulfur-containing mitochondrial enzyme, succinate dehydrogenase, in SNAP-treated oligodendrocytes and astrocytes, whereas microglia were unaffected. In addition, morphological changes and single stranded DNA breaks occurred in oligodendrocytes, but not in astrocytes and microglia. Oligodendrocytes were also less easily rescued from the toxic effects of NO by oxyhemoglobin than were astrocytes. A subpopulation of oligodendrocytes were killed by NO via a necrotic, non-apoptotic mechanism (Mitrovic et al., 1994,1995). These findings strongly suggest that the myelin producing cell is more sensitive to NO than the other two glial cell types. CYTOKINE-NO REGULATORY PATHWAYS Cytokine Regulation of NO Much of the regulation of NO production in macrophages and glia has been studied in the rodent. In humans, such cells are clearly turned on to produce the type II or inducible nitric oxide synthase (iNOS) and NO when certain cell surface receptors are crosslinked; these include CD4, a non-CD4 binding site for HIV-1, CD23, and CD69 (DeMaria et al., 1994; Reiling et al., 1994; Bukrinsky et al., 1995; Vouldoukis et al., 1995; Koka et al., 1995; Nicholson et al., 1996; Vodovotz et al., 1996). The role of cytokines in augmenting NO induction in human glia and macrophages in vitro may be very different from that in rodent cells in vitro and even different from effects in vivo in human disease . Inducers of NO: proinflammatory cytokines IFNγ is a strong inducer of iNOS and NO in rat glia and macrophages and this production is synergistically increased in the presence of TNFα or lipopolysaccharide (LPS) (Merrill et al., 1993; Reiling et al., 1994; Murphy et al., 1995; Dileepan et al., 1995). About half of the LPS-induced, but not IFNγ-induced, NO is mediated through TNFα (Merrill et al., 1993). IFNγ and IL-1β, but not LPS, are inducers of iNOS and NO in human glial cells derived from fetal brain tissue (Lee et al., 1993; Koka et al., 1995; Ding et al., 1997), but not from neonatal or adult brain-derived glial cells (Murphy et al., 1995; this laboratory’s unpublished observations). Stevenson et al. (1995) have shown that interleukin-12 (IL-12), probably through the induction of TNFα and IL-1β, induces NO. IL-2 in mice and humans in vivo induces NO (Yim et al., 1995). Interleukin-6 (IL-6) has no effect on NO induction (Rockett et al., 1992; Chiang et al., 1994; Tanaka et al., 1995) nor does IFNβ alone (Fast et al., 1993; Fujihara et al., 1994; MacMicking et al., 1995). Nevertheless, mouse macrophages primed by suboptimal doses of LPS will be triggered by IFNβ to produce NO (Fujihara et al., 1994; Zhang et al., 1994).

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Modulators of NO: antiinflammatory cytokines, antioxidants, and elevators of cAMP Since it is clear that certain cytokines block EAE and that NO is involved in EAE (see above), it is of interest to know whether blockade of NO by these same cytokines might account for their inhibition of EAE. Doyle et al. (1994) report that IL-13 suppresses NO production but not respiratory burst and oxygen radicals in murine macrophages; this cytokine also suppresses TNFα, though to a lesser extent. Such a finding suggests a mechanism for the inhibition of development of EAE by IL-13 (Cash et al., 1994). It also demonstrates the independence of oxygen and nitrogen free radical production in these cells. This is an important observation given that ONOO− may cause greater damage in cells and tissues than NO, but that both O2− and NO need not be inhibited at the same time for an effect by an antiinflammatory cytokine. In cultures of rodent macrophages and microglia, it is almost universally accepted that TGFβ inhibits iNOS and NO through several mechanisms (Ding et al., 1990; Merrill et al., 1993; Vodovotz et al., 1993, 1994; Alleva et al., 1994; Boulard et al., 1995). These include inhibition of transcription and translation of iNOS, decreased stability of iNOS mRNA, increased degradation of iNOS protein, and stimulation of arginase, thereby depleting the enzyme substrate L-arginine (Kanno et al., inhibition of NO; interestingly, this inhibition is not through the activation of IL-10, IL-1994; Chesrown et al., 1994). IL-11 attenuates an inflammatory response in mice through 6, or TGF (Trepicchio et al., 1996). In the rodent, the capacity of IL-4 and IL-10 to inhibit iNOS and NO appears to depend on pretreatment of the cells being stimulated with IFNγ; activated mouse cells are barely inhibited by IL-4 and IL-10 (Gazzinelli et al., 1992b; Oswald et al., 1992; Cunha et al., 1992; Schneemann et al., 1993; Romani et al., 1994; Bogdan et al., 1994; Appelberg, 1995). Multiple mechanisms for this inhibition have been suggested: (1) through the reduction of TNFα (Fiorintino et al., 1991; Gazzinelli et al., 1992a; Chao et al., 1993); (2) inhibition of the activation of protein kinase C (Sands et al., 1993); or (3) induction of arginase (Corraliza et al., 1995). These cytokines may synergize with each other or with TGFβ (Oswald et al., 1992; Alleva et al., 1995). Still other studies have found modest or no inhibition of NO (Murphy et al., 1995; Strassmann et al., 1994) or an increase in NO by IL-10 (Chesrown et al., 1994). As with the rodent, depending on the state of activation of human monocytes/ macrophages, IL-4 may up- or downregulate NO. Nevertheless, IL-4 regulation of NO in the human works in an apparently opposite fashion from that in the rodent. IL-4 can directly induce iNOS and NO production in resting monocytes from normal healthy human donors (reviewed in Merrill and Benveniste, 1996). This activation can be amplified by pretreatment with IFNγ (Pierre-Kolb et al., 1994). In spontaneously high NO producer monocytes, especially those from allergy patients, IL-4 abrogates NO (Mautino et al., 1994). Nevertheless, in one study IL-4 has also been shown to induce NO in murine splenocytes (Tian and Lawrence, 1995). The production of elevated NO in allergy patients and the ability of IL-4 to induce NO indirectly in peripheral blood mononuclear cells is probably related to the fact that crosslinking of the molecule CD23, the low affinity IgE Fc receptor [Fc RII], by IgE or anti-Fc R antibody leads to NO (Becherel et al., 1994; Paul-Eugene et al., 1995; Vouldoukis et al., 1995). IL-4 induces IgE production as well as an increase in soluble CD23. Interestingly, high levels of IL-4 and NO could feedback negatively, possibly through the elevation of cAMP, on both IgE production and NO (Becherel et al., 1994; Paul-Eugene et al., 1995), which may explain the IL-4 inhibition of “spontaneously” produced NO from allergy patients (Mautino et al., 1994). A brief rise in intracellular cAMP in unstimulated cells leads to a small but significant direct induction of iNOS, as well as leading to the amplification of iNOS and NO by subsequent crosslinking of CD23 (Alonso et al., 1995). However, in cells stimulated by TNFα, IL-1β, or LPS, prolonged elevation of cAMP, via adenyl cyclase activators (e.g. PGE2), phosphodiesterase (PDE) inhibitors or -adrenergic agonists, inhibits NO production (Bulut et al., 1993; Feinstein et al., 1993; Murphy et al., 1995). PDE inhibitors such as pentoxifylline (PTX), isobutyl-methylxanthine (IBMX) and iloprost variously inhibit TNF production,

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cytotoxicity and O2− production in addition to NO production (Marotta et al., 1992; Kozaki et al., 1993; Takahashi et al., 1994), possibly through the inhibition of NFκB which is involved in NO and TNFα induction (Biswas et al., 1993). Data supporting a role for both TNFα and NO in EAE are therefore complemented by studies demonstrating that the PDE inhibitors PTX and rolipram inhibit EAE in rodents and primates most likely through the inhibition of cytokines, inflammation, demyelination and NO (Rott et al., 1993; Genain et al., 1995; Sommer et al., 1995). In studies in our own laboratory, IL-4 inhibited human glial cell production of TNF at ng/ml and NO production at mg/ml concentrations. While inhibiting rat TNF at ng/ ml concentrations, IL-4 had no effect on rat NO production. The induction of the iNOS gene in rodents and humans is different, suggesting that the regulation of the gene may also be species-specific. In our studies, rat microglia are in an activated state in vivo at birth; they therefore may have been refractory to inhibition by IL-4 and IL-10 in our in vitro studies (St. Pierre et al., 1997). In our hands, IL-13 inhibited NO in rodent glia and human glial cell cultures. The inhibition of TNFα and microglial cell cytotoxicity by IL-13 may be unrelated events: IL-13 downregulation of IL1 may account for the depression in TNFα production while cytotoxicity may be inhibited partially because of the NO inhibition (Cash et al., 1994; Doyle et al., 1994). St. Pierre et al. (1997) have shown that IFNγ/LPS induction of rat microglial cell TNFα was strongly inhibited by IFNβ, while NO was only weakly inhibited. This suggests that there are even differences in NO regulation by cytokines between mice and rats. Furthermore, the significant inhibition of human glial cells’ NO and TNFα point out the complex mechanisms of actions and species-specific differences in IFNβ regulatory pathways. Antioxidants will also inhibit mediators of inflammation. Nicotinamide, an oxygen free radical scavenger (Andersen et al., 1995) and poly ADP ribose synthase (PARS) inhibitor (Zingarelli et al., 1996) has been reported to inhibit macrophage mediated cytotoxicity (Kröncke et al., 1991) and nitric oxide production through transcriptional (Hauschildt et al., 1991; Pellat-Deceunynck et al., 1994) and posttranscriptional effects (Andersen et al., 1995; Cetkovic-Cvrlje et al., 1993). N-acetyl cysteine (NAC), a precursor of glutathione, is another oxygen radical scavenger (Althaus et al., 1994) and inhibits TNFα production by blocking oxidation of the IkB-NFkB complex and thereby preventing NFκB from translocating to the nucleus (Dröge et al., 1994; Gaiter et al., 1994; Hayashi et al., 1993; Schreck et al., 1992). Mayer and Noble (1994) demonstrated that NAC protected oligodendrocytes from programmed cell death. In studies on rodent glia in vitro, both antioxidants inhibited cytotoxicity and TNF but not NO production. This suggests that neither NFκB nor significant ADP ribosylation are required for NO induction in rat microglia, unlike what has been described for normal macrophages and macrophage cell lines in the mouse (Hauschildt et al., 1991; Pellat-Deceunynck et al., 1994). On the other hand, TNFα gene expression and microglial mediated cytotoxicity probably require oxygen radicals, which should then be scavenged by NAC or nicotinamide (Schreck et al., 1992; Hayashi et al., 1993; Dröge et al., 1994). Additionally, NO mediates DNA strand breaks in oligodendrocytes (Parkison et al., 1996), thus preserving NAD and ATP pools in these by nicotinamide (Zingarelli et al., 1996), thus preserving NAD and ATP pools in these cells (Kröncke et al., 1991; Althaus et al., 1994). Since rolipram and PTX have been shown to inhibit EAE in rodents and primates (see above), and because the elevation of cAMP has been demonstrated to inhibit both TNF and NO induction, cytotoxicity, and superoxide anion production variously in human and mouse glia and leukocytes (Bulut et al., 1993; Feinstein et al., 1993; Kozaki et al., 1993; Marotta et al., 1992; Takahashi et al., 1994), our laboratory was interested in assessing cAMP elevation in the function of human and rodent glia following the treatment with Iloprost, a protacyclin analogue, and the two phosphodiesterase inhibitors IBMX and PTX. It has been proposed that the mechanism of inhibition of TNFα and NO by the elevation of cAMP is through the inhibition of NFκB-mediated transcription (Biswas et al., 1993; Chao et al., 1992; Han et al.,

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1990; Strieter et al., 1988) or through elevation of cAMP (Kambashi et al., 1995; Strassmann et al., 1994). We found that the elevation of cAMP by Iloprost was much more effective than the inhibition of cAMP degradation by IB MX or PTX. Why TNF production was more senstitive to elevation of cAMP than iNOS induction was in both rat and human glial cultures is not known. Several reports have suggested that chronic elevation of high levels of cAMP is required for NO inhibition (Marotta et al., 1992; Feinstein et al., 1993; Bulut et al., 1993); we may have not achieved such levels in our cultures with these agents. In the assessments described here, with all three classes of inhibitors, we wish to reemphasize that when TNF was completely inhibited, NO and cytotoxicity were only partially or not at all inhibited. This supports our contentions 1) that microglial cell killing of oligodendrocytes is not mediated by soluble TNF ; 2) that TNF does not include NO; and 3) that microglial cell-mediated killing is partly NO-dependent (Merrill et al., 1993). Nevertheless, all three microglical cell-mediated events may be involved at some point or other in lesion formation in MS, and therefore interference with these three processes might be of benefit in treating MS. (St.Pierre et al, 1997) NO Regulation of Cytokines In some autoimmune diseases, including MS, a pernicious proinflammatory cycle may account for the clinical and histopathological chronicity. In this regard, it is quite noteworthy that NO and/or ONNO directly upregulate production of IL-1β, TNFα, interleukins-8 and -12 (IL-8, IL-12) and hydrogen peroxide (H2O2) in macrophages. Exposure of a mouse macrophage cell line to SNAP induces IL-12 p40 mRNA (Rothe et al., 1996). Nitrogen radicals also indirectly enhance cytokine induction of TNFα (Fülle et al., 1991; Magrinat et al., 1992; Lander et al., 1995a, b; Bigler et al., 1993; Andrew et al., 1995; Marcinkiewicz et al., 1995; Rothe et al., 1996). This induction is mediated at the transcriptional level possibly through the induction of NFκB (Lander et al., 1995a,b; Andrew et al., 1995). Lander (1995b) suggests that NO, through enhancement of GTPase activity and G-protein-mediated events, stimulates the translocation of NFκB to the nucleus. Nevertheless, in some cases, NO inhibits LPS-induced IL-1β and TNFα in macrophages (Fülle et al., 1991; Bigler et al., 1993). In endothelial cells, NO inhibits NFκB translocation by stabilizing the NFκB:IκB complex by preventing 1κB degradation (Peng et al., 1995; DeCaterina et al., 1995). These cases illustrate the complexity of the effects of free radicals in signal transducing events in the macrophage at different stages of activation and point to the danger in generalizing NO effects on NFκB in all cells. Other Effects of NO on the Immune System While it is clearly a proinflammatory molecule locally at sites of tissue damage, NO may also protect against autoimmune disease when acting early and systemically. Mutant mice, in which the iNOS gene is defective, as well as IFNγ receptor knock out mice (with impaired NO production), have a significantly stronger anti-CD3 response and Thl response to infectious agents than wild type mice (Matthys et al., 1995; Wei et al., 1995). This suggests that NO may inhibit T cell responses leading to delayed type hypersensitivity by inhibiting T cell proliferation (Stewart et al., 1994; Matthys et al., 1995), either through suppression of IFNγ or induction of PGE2 (Blanco et al. 1995). NO has also been shown to inhibit leukocyte adhesion and migration by its interference with GDI 11/CD 18 (leukocyte functional antigen-1, LFA-1) expression (Kubes et al., 1991). NO also down-regulates MHCII expression in macrophages, thereby inhibiting antigen presentation (Sicher et al., 1994). In other words, early in the disease process in MS or EAE, NO might actually protect against autoimmune events initiated in the peripheral blood.

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NO Regulation of Gene Transcription in Inflammation and CNS Repair The role of oxygen and nitrogen radicals in nervous system pathology has been extensively reviewed elsewhere. Free radicals have been substantially implicated in autoimmune and infectious inflammatory conditions, neurodegenerative diseases, and trauma of the brain and spinal cord; at pathological levels in these conditions, radicals cause DNA damage, mitochondrial dysfunction, and apototic or necrotic cell death in neurons, macroglia, or even (in some cases) in microglia (Dawson and Dawson, 1994; Murphy and Grzybicki, 1996; Merrill and Murphy, 1996; Mitrovic et al., 1996; Parkinson et al., 1996). Because NFκB’s movement to the nucleus and subsequent binding to DNA is regulated by the redox state in the cytoplasm and nucleus, a substantial number of cytokines and adhesion molecules, as well as T cell and B cell functions will be affected (Kaltschmidt et al., 1993; Raes et al., 1995; Sun and Oberley, 1996). Thus free radicals can have a profound influence on inflammatory immune mediators derived from glia or immune cells during a pathological condition. The effects of free radicals can be demonstrated on upstream signal transduction pathways leading to gene transcription as in the case of their effects on phosphatases, tyrosine kinases (p21ras, p56lck, and p59fyn) (Raes et al., 1995; Sun and Oberley, 1996), serine/ threonine kinases (Raf-l) (Seko et al., 1996), and the heme-regulated eIF-2 kinase (Raes et al., 1995). The list of redox-regulated transcription factors/activators is fairly long and includes p53, AP-1, NF-κB, Ets, Sp-1, glucocorticoid receptor, and Egr-1 among others (Remacle et al., 1995; Sun and Oberley, 1996). Many of these signal transduction pathways are utilized by neurons and glia during development of the nervous system as well as in the adult brain and spinal cord in maintaining homeostasis. Because of their c-fos-dependence, trophic and survival factors for both glia and neurons such basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), and nerve growth factor (NGF) are all upregulated by hydrogen peroxide (H2O2) (Pechan et al., 1992; Sundaresan et al., 1995). By ejecting the metal from the protein, nitroso compounds could potentially destabilize zinc fingers in enzymes and transcription factors, rendering them less functional (Rice et al., 1993); this has implications for the transcription of Type III nitric oxide synthase (NOS) in endothelial cells, the neurofilament gene in neurons, and myelin genes in oligodendrocytes, all of which are regulated by zinc-finger containing transcription factors (Elder et al., 1992; Wariishi et al., 1995; Armstrong et al., 1995). In a dose-dependent manner, the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) can either stimulate or inhibit protein production and proliferation in primary oligodendrocyte precursors; NO inhibits myelin basic protein mRNA levels in mature oligoden-drocytes (Mackenzie-Graham et al., 1994; Mitrovic et al., 1996). It is hypothesized that this regulation may occur via effects on c-fos (Remacle et al., 1995) or p59 fyn (Umemori et al. (1994). With respect to inflammation and demyelination in MS, the impact of this form of regulation would be greatest in the repair of myelin during remyelination. REGULATION OF NO PRODUCTION IN GLIAL CELLS NO Production by Oligodendrocytes The production of NO by oligodendrocyte cultures is of interest, given that within the whole primary oligodendrocyte population, which is composed of bipotential precursors, committed oligoden drocytes, and mature myelin basic protein (MBP)-producing oligodendrocytes, there is a subset of NO-sensitive cells (Merrill et al., 1993; Mitrovic et al., 1994, 1995). We have recently demonstrated that primary rat oligodendrocytes produce nitric oxide as a consequence of the induction of the iNOS gene. Inhibition of transcription or translation results in the absence of iNOS protein in oligodendrocytes and the lack of

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detectable NO in the culture supernatants. The protein is identical in size to that induced in other human and rodent glial cells (Merrill et al., 1997; reviewed in Merrill and Murphy, 1996). The staining for iNOS enzyme in oligodendrocytes suggests that, if the iNOS is functional, at least half of the GalC+/CNPase+ oligodendrocytes in these cultures are able to produce NO. Levels of iNOS mRNA in vitro and function of iNOS enzyme (as determined by L-citrulline production) indicate that microglia are better producers than oligodendrocytes and astrocytes. Alveolar macrophages appear to be better producers than brain microglia under the conditions tested. Higher concentrations of LPS are required to induce oligodendrocytes than astrocytes (Simmons and Murphy, 1992; Galea et al., 1994) or microglia (Zielasek et al., 1992; Merrill et al., 1993). These data suggest that cell-specific factors such as ability to respond to certain stimuli and regulatory events at the transcriptional and translational levels may be responsible for such differences. Nevertheless, oligodendrocytes respond just as well to the cytokines IL1 and IFNγ in iNOS/ NO production; both of these cytokines have been implicated in MS (Merrill and Murphy, 1996; Murphy et al., 1995). Fundamentally, the fact that oligodendrocytes can produce NO may not be surprising, given that Gold et al. (1996) have demonstrated iNOS activation in the myelinating cell in the peripheral nervous system, the Schwann cell. In this study, Schwann cells produced 15 μM nitrite after 48 hours induction with IFNγ and TNFα, which proved to be the peak in both the amount and time of NO production. The amount of NO produced by Schwann cells was able to inhibit T cell proliferation thereby leading to T cell apoptosis, indicating that Schwann cell-derived NO could be immunoregulator. Some NO-producing cells seem to be refractory to NO-mediated toxicity as in the case of microglia and NOS-positive neurons (Dawson and Dawson, 1994; Mitrovic et al., 1994, 1995; Murphy et al., 1995). Cells like astrocytes, macrophages, and hepatocytes, while insensitive to self injury from endogenously produced NO, may be more sensitive to exogenous NO from a neighboring cell. Nevertheless, these cells seem to be able to repair the NO-mediated damage, possibly by converting to glycolysis during the repair phase (Drapier and Hibbs, 1989; Mitrovic et al., 1994; Stadler et al., 1991). In the case of oligodendrocytes, it will be important to determine whether the NO-producing oligodendrocytes are sensitive to their own chronically produced endogenous nitric oxide. Given the variety of toxic effects that exogenous NO has on oligodendrocytes, from mitochondrial dysfunction to DNA breaks, it will also be interesting to relate the role of endogenously-induced NO to specific toxicities which NO produces in cells (Mitrovic et al., 1994, 1995). Using immortalized, cloned oligodendrocyte cell lines, it appears to us that a more mature line is less sensitive to exogenous NOinduced cell death than one of the less mature lines (Mackenzie-Graham et al., 1994). In spite of this, NO still affects mature cells since it inhibits the expression of myelin basic protein mRNA in these cells (Mackenzie-Graham et al., 1994). We are currently assessing the putatively differential capacity of these cell lines to produce NO. Human Glial Cell NO Production Since the production of iNOS and NO in the central nervous system has such potentially harmful effects on both neurons and oligodendrocytes and may play a role in such diseases as CNS AIDS and MS, the cells of the nervous system may have evolved a protective mechanism to prevent spurious levels of NO formation even in the presence of iNOS gene induction (Xie et al., 1992). We have shown that human fetal astrocytes and microglia respond to IFNγ and IL1 by producing iNOS mRNA within hrs and iNOS protein within a day. However in our experience, these cells do not make NO for a period of 24–48 hrs after the appearance of the enzyme. Another study on human glia also reported this delay in the appearance of NO, but the kinetics of iNOS mRNA and protein were not investigated (Lee et al., 1993). This is in contrast to the

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appearance of iNOS and NO production by rodent glia in vitro within 8–24 hrs of stimulation (Simmons and Murphy, 1992). Many human cells such as heptacytes, chondrocytes, endothelial cells and some glioblastoma cells have been shown to express iNOS and release large amount of NO after stimulation with bacterial products and/or cytokines, showing similar kinetic patterns to that in rodent glial cells (Chakravarthy et al., 1995; Grabowski et al., 1996; Blanco et al., 1995). While human glial cells display transcription and translation of iNOS similar to other cell types, they have different kinetics of NO production compared to these other human cell types (Ding et al., 1997). Recent work from the laboratory of Dennis Stuehr has identified the fact that iNOS is not a functional enzyme until it is in a conformationally active state created by its dimerization (Baek et al., 1993). The binding of heme and tetrahydrobiopterin (BH4) to the enzyme play a significant role in forming and stabilizing active dimeric iNOS (Tzeng et al., 1995). To understand if this temporal lag between iNOS enzyme synthesis and NO production was due to insufficient intracellular levels of the iNOS cofactor BH4 we added BH4 to the cultures. We were able to show a significant increase in NO production in a dose- and time-dependent manner, suggesting that the rate limiting step in NO production is cofactor induction and activation of iNOS. The issue of whether human macrophages make NO is being resolved through data generated from in vivo assessments. While NO production in murine macrophages accumulates in a linear fashion after a lag of 6 hours after stimulation of cytokines (Evans et al., 1994; Assreuy et al., 1994), human mononuclear phagocytes express iNOS mRNA and protein but produce no or little NO in vitro (Murray et al., 1992; Schneemann et al., 1993; Padgett et al., 1992; Sakai et al., 1993; Denis, 1994; Weinberg et al., 1995; Albina, 1995; Perez-Perez et al., 1995). Although post translational regulation has been speculated, some investigators have excluded the possibility of the regulation by BH4 because increasing BH4 levels does not enable them to produce high levels of NO (Weinberg et al., 1995). This suggests that NO production in human glia and macrophages may be regulated differently. Clearly, glial cells and macrophages could respond differently to cytokine stimulation, thereby initiating different mechanisms for regulating iNOS catalytic activity. Studies examining tissues from humans with a variety of diseases have clearly shown that human macrophages do make iNOS and NO in vivo (Bagasra et al., 1995; Nicholson et al., 1996; Vodovotz et al., 1996). The inability to induce NO in vitro by crosslinking of cytokine receptors clearly points to the complexity of the induction of NO. It confirms that our inability to mimic the in vivo event in vitro may explain the lack of NO induction in macrophages. A better understanding of mechanisms of disease induction in patients may point the way to better in vitro modeling of iNOS/NO production in human cells. REFERENCES Albina, J.E. (1995) On the expression of nitric oxide synthase by human macrophages. Why no NO? J. Leuk. Biol., 58, 643–649. Alleva, D.G., Burger, C.J. and Elgert, K.D. (1994) Tumor-induced regulation of suppressor macrophage nitric oxide and TNFα production. Role of tumor-derived IL-10, TGFβ and prostaglandin E2. J. Immunol., 153, 1674–1686. Alonso, A., Carvalho, J., Alonso-Torre, S.R., Nunez, L., Bosca, L. and Crespo, M.S. (1995) Nitric oxide synthesis in rat peritoneal macrophages is induced by IgE/DNP complexes and cyclic AMP analogues. Evidence in favor of a common signaling mechanism. J. Immunol., 154, 6475–6483. Althaus, J.S., Oien, T.T., Fici, G.J., Scherch, H.M., Sethy, V.H. and vonVoigtlander, P.P. (1994) Structure activity relationships of peroxynitrite scavengers: an approach to nitric oxide neurotoxicitry. Res. Comm. Chem. Pathol. Pharmacol., 83, 243–252. Andersen, H.U., Larsen, P.M., Fey, S.J., Karlsen, A.E., Mandrup-Poulsen, T. and Nerup, J. (1995) Two dimensional gel electrophoresis of rat islet proteins. Interleukinlb-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide. Diabetes, 44, 400–407.

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22 Regulation of Nitric Oxide and Inflammatory Mediators in Human Osteoarthritis-Affected Cartilage: Implication for Pharmacological Intervention Ashok R.Amin1,2,*, Mukundan G.Attur1 and Steven B.Abramson1,2 1

Department of Rheumatology and Medicine, Hospital for Joint Diseases, 301 East 17th Street, Room 1600, New York, NY 10003, USA 2Departments

of Pathology, Medicine and Kaplan Cancer Research Center, New York University Medical Center, New York, NY 10016, USA

Human OA-affected cartilage demonstrates spontaneous superinduction of NO and PGE2 production in ex vivo conditions which is sensitive to inhibitors of RNA transcription and protein translation. Human OA-affected cartilage expresses NOS that has properties similar to ncNOS and iNOS. The spontaneous release of NO is regulated by autocrine IL-1 or paracrine IL-17. Intra-articular NO downregulates PGE2 production in OAaffected cartilage, whereas increases in intracellular cAMP or PKC activation downregulates the spontaneous (or cytokine induced) NO production. The OA-affected cartilage also releases several other inflammatory mediators which include TNF , IL-6 and IL-8. Various anti-inflammatory drugs used in the treatment of arthritis including aspirin, sodium salicylate, tetracyclines, cyclosporine and rapamycin inhibit NO production in OAaffected cartilage. Thus, OA cartilage is a rich source of inflammatory mediators, a site of activated cytokine production and of prodigious amounts of both NO and PGE2. So conceived, OA cartilage is a tissue “inflamed”, brimming with phologistic products that can serve as targets of future pharmacological intervention. Key words: Osteoarthritis, nitric oxide, prostraglandins, inflammation. OSTEOARTHRITIS AND CARTILAGE Osteoarthritis or “osteoarthrosis”: Which term is more appropriate? Classically, osteoarthritis (OA), unlike rheumatoid arthritis (RA), is defined as an inherently non-inflammatory disorder of movable joints characterized by deterioration of articular cartilage and the formation of new bone at the joint surfaces and margins (Hough, 1993). In contrast to RA, the synovial fluid in OA typically contains few neutrophils (< 3,

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Figure 22±1. The various effects of intracellular NO on chondrocyte dysfunction. Condensed from Blanco et al., 1995b; Frenkel et al., 1996, Murrel et al., 1995; Taskiran et al., 1994, Pelletier et al., 1996., Amin et al., 1997c).

000/mm3), and, except for advanced disease, the synovium does not exhibit significant cellular proliferation or infiltration by inflammatory leukocytes. The molecular pathogenesis of OA is increasingly understood by the elucidation of events within the articular cartilage. For example, altered dynamic equilibrium between matrix synthesis and degradation by human chondrocytes has recently been implicated as having a primary role in the degeneration of articular cartilage resulting in OA (Goldring, 1993; Pelletier et al., 1991, 1996). This includes upregulation of catabolic activities, such as secretion of degradative proteases, and/or downregulation of anabolic activities such as collagen and proteoglycan synthesis. ROLE OF NITRIC OXIDE ON CHONDROCYTE FUNCTION Among the various human cell types, chondrocytes are among the most prodigious sources of NO, upon stimulation with cytokine and endotoxins. NO can be induced in human, bovine, rabbit and equine chondrocytes. (Amin et al., 1995a; Palmer et al., 1992, 1993; Taskiran et al., 1994). NO has profound effect on chondrocyte functions. As shown in Figure 22–1, it is reported to stimulate metalloproteases, downregulate synthesis of proteoglycans and collagen type II, inhibit actin polymerization, induce apoptosis, downregulate IL-IRA expression and augment aggregan release from cartilage. These effects together would not only augment cartilage degradation, but also inhibit repair mechanisms. NOS IN OA-AFFECTED CARTILAGE Normal or OA-affected human chondrocytes when grown in monolayers and stimulated with cytokines ± endotoxins show the presence of a 133 kD iNOS in western blot analysis (Sakurai et al., 1995; Maier et al., 1994; Geng et al., 1995; Blanco and Lotz, 1995). Analysis of NOS directly in osteoarthritis-affected cartilage shows the presence of at least two forms of NOS: a) a neuronal-like NOS based on western blot

Correspondence: Ashok R.Amin, Tel: 212–598–6537; Fax: 212–598–7604.

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analysis, size of the protein and antibody reactivity (Amin et al., 1995a); b) an iNOS based on in situ hybridization (Evans et al., 1996). In view of the complex functional expression of more than one isoform of NOS in various cell types (Togashi et al., 1997; Belmont et al., 1997), the expression and regulation of NOS(s) in OA-affected cartilage also seems to be more complex than previously anticipated, especially in view of the recent observation that ncNOS regulates iNOS and NFκB expression in the same cells (Togashi et al., 1997). Even if we were to assume that the OA-NOS is a single isoform, it certainly harbors unique properties. For example, (I) the spontaneously released NO (in OA-affected cartilage expiant assays) is sensitive to cyclohexamide and NF- B inhibitor (PDTC), but not to hydrocortisone at concentrations sufficient to block cyclooxygenase-2 (COX-2) activity in the same OA-affected cartilage (Amin et al., 1997a); (II) OA-NOS shows similar and distinct susceptibility (to iNOS) to pharmacological active drugs used for arthritis therapy as described below; (III) unlike iNOS, OA-NOS expression is insensitive to TGF (Amin et al., 1995a,b; Blanco et al., 1995a; Nathan and Xie, 1994); (IV) we have also observed that unlike normal human chondrocytes grown in monolayers (stimulated with IL-1 or LPS) to induce iNOS and NO production (Geng et al., 1995), the spontaneous (or IL-1 induced) production of NO by OA-affected cartilage is sensitive to PMA (PKA activation) and elevated intracellular cAMP levels. In view of these results, it is tempting to speculate that the cartilage, like the brain (both which are avascular and alymphatic), demonstrates “uncharacteristic” regulation of NOS and NO production (involving differentially expressed NOS s and NO production) during different physiological, developmental and pathophysiological conditions (Togashi et al., 1997; Peunova and Enlkolopov, 1995). These studies together indicate that the characterization and regulation of OA-NOS(s) require further study. NITRIC OXIDE PRODUCTION IN OA-AFFECTED CARTILAGE Human OA-affected cartilage when incubated in F-12 medium (without supplement), such as serum growth factors or cytokines, spontaneously releases substantial amounts of nitric oxide (> 1 μM Nitrite/100 mg cartilage) within 48 h in ex vivo conditions (Amin et al., 1995a and Figure 22–2). Similar observations in OA/RA-affected cartilage has also been made independently by Sakurai et al. (1995). Incubation of normal human cartilage under similar conditions does not show detectable (< 0.1 μM) amount of nitrite in the medium (Figure 22–2) This spontaneous superinduction of NO by OA-affected cartilage is sensitive to cycloheximide and NF B inhibitors (PDTC) and insensitive to TGF and hydrocortisone in sufficient quantities to inhibit iNOS and COX-2 in murine macrophages (Amin et al., 1995a, 1997a; Blanco et al., 1995). The NO production in OA-affected cartilage can be upregulated by cytokines (IL-l and TNF ) +endotoxins. SECONDARY SIGNALS INVOLVED IN MODULATION OF OA-NOS To evaluate the role of secondary signals in the regulation of the spontaneously released NO in OA-affected cartilage, experiments with OA cartilage organ cultures were set up as described in Figure 22–3. Addition of TNF or IL-1 to these OA-cartilage expiant cultures augmented the release of NO by 100% and 500% respectively. However, PMA or increase in intracellular cAMP induced by forskolin, or PMA+cAMP (synergistically) blocked (~50–90%) the spontaneous release of NO in these same organ cultures. These experiments show that the spontaneous release of NO (and IL-1 mediated NO release) can be downregulated by PMA, cAMP and PMA+cAMP in a synergistic manner. Therefore, PMA and cAMP (activators of PKC and PKA respectively) are negative modulators of IL-1 dependent effects in OA-

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affected cartilage with respect to NO release. The action of these negative modulators in OA-explants are quite distinct from those seen in normal chondrocytes grown in monolayers and stimulated with IL-1 or LPS as described above (Geng et al., 1995). REGULATION OF COX-2 AND PGE2 PRODUCTION IN OA-AFFECTED CARTILAGE Prostaglandins are produced at elevated levels in inflamed tissues including rheumatoid synovium (Bombardier et al., 1991; Davies et al., 1984). PGEi and PGE2 contribute to synovial inflammation by increasing local blood flow and potentiating the effects of mediators such as bradykinin and IL-I that induce vasopermeability (Davies and Mclntrye, 1992). These inflammatory mediators also trigger osteoclastic bone reabsorption (Robinson et al., 1975). Previous studies have shown that stimulation of articular cartilage leads to synthesis of (in order of decreasing quantity) PGE2, PGF2, PGI2α and TXA2 (Bandara and Evans, 1992). Cartilage specimens from OA-affected patients spontaneously released PGE2 at 48 h in ex vivo culture at levels at least 50-fold higher than that seen in normal cartilage and 18-fold higher than that seen in normal cartilage+cytokines+endotoxin. The average amount of PGE2 released (in ex vivo conditions) by 1 g of OAaffected cartilage is ~280 ng/ml in 48 h. The superinduction of PGE2 production coincides with the upregulation of COX-2 in OA-affected cartilage based on mRNA analysis and western blot analysis of cartilage extracts (Amin et al, 1997a). The spontaneous production of both NO and PGE2 by OA cartilage explants is regulated at the level transcription and translation, in that both are sensitive to inhibition by cycloheximide and PDTC. The cross regulation of NO/PGE2 in OA-affected cartilage was examined. The NOS inhibitor L-NMA inhibited intracellular NO production in OA-affected cartilage by >90%, but augmented significantly (twofold) the spontaneous production of PGE2 in the same explants. Similarly, addition of exogenous NO donors to OA-affected cartilage significantly inhibited PGE2 production by more than 75%. Cytokine (IL-lβ and TNFα)+endotoxin stimulation of OA cartilage explants in ex vivo conditions augmented PGE2 and NO production. Addition of L-NMA (which inhibited NO production) further augmented cytokine-induced PGE2 production by at least fourfold. Inhibition of PGE2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE2 did not significantly affect the spontaneous NO production in OA-affected cartilage. These data indicate that human OA-affected cartilage in ex vivo conditions shows (a) superinduction of PGE2 due to upregulation of COX-2, which may inhibit chondrocyte proliferation (Blanco and Lotz, 1995) and (b) spontaneous release of NO that acts as an autacoid to attenuate the production of COX-2 products such as PGE2. The scheme shown in Figure 22–4 demonstrates the regulation of NO and PGE2 in OA-affected cartilage. REGULATION OF NO AND PGE2 PRODUCTION BY IL-lβ IN OA-AFFECTED CARTILAGE Interleukin (IL) 1β plays a central role in the pathophysiology and cartilage damage/ degradation in arthritis. In non-inflammatory arthropathies such as osteoarthritis the synovial-derived IL-1β has been implicated in the disease process. We have recently observed that human OA-affected cartilage expresses upregulated IL-1β mRNA not seen in normal cartilage (Attur et al, 1997a). The OA-affected cartilage (but not normal cartilage) in ex vivo conditions spontaneously releases (together with NO and PGE2) detectable amounts of autocrine IL-1β, which is known to be involved in cartilage damage and inflammation. The

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Figure 22±2. Spontaneous release and cytokines+endotoxin induced nitric oxide (A) and PGE2 (B) from normal and OA-affected cartilage explants in ex vivo conditions. (A) Knee articular cartilage from three OA patients and three normal individuals was cut into 3 mm discs; 4–6 discs were placed in organ culture in 2 ml medium in the presence and absence of 500 μM L-NMA in triplicate (n=3) The release of nitrite in ex vivo conditions after 48 h in the presence or absence of IL-1 (1 ng/ml)+TNF (100 u/ml)+LPS (100 μg/ml) was monitored (Gilliam et al., 1993), (B) OA affected and normal cartilage (from 3 individuals) was incubated in F-12 medium ± IL-l +TNF +LPS ± cycloheximide (1 μg/ml) in triplicate (n=3) as described above. The release of PGE2 was monitored at 48 h by RIA. In these experiments the levels of PGE2 were normalized and represented as ng/ml of PGE2/gm wet wt. of cartilage.

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Figure 22±3. Modulation of NO production by secondary signaling molecules in OA-affected cartilage. OAaffected cartilage explants were set up as described in Figure 22–2 from three different patient material (A, B, and C) in triplicate (n=3) Control designates the spontaneous release of NO by the OA-affected cartilage. The explants were incubated with IL-1β (1 ng/ml), TNFα (100U/ml) and LPS (100 μg/ml) in the presence and absence of PMA (10 ng/ml) and /or forskolin (30 μM) designated as cAMP. The nitrite was estimated at the end of 48 h.

autocrine IL-1β released by the OA-affected cartilage (for at least 72 h in ex vivo conditions) is in sufficient quantities to modulate at least two mediators, NO and PGE2. Addition of recombinant soluble IL-l receptor (but not soluble TNFα receptor) significantly attenuates the spontaneous release of both NO and PGE2 in these OA-affected cartilage in ex vivo conditions (Figure 22–5). These experiments demonstrate that human OA-affected cartilage itself releases sufficient amounts of functionally active autocrine IL-1P that may facilitate or augment cartilage degradation and inhibit cartilage repair, and therefore lead the OA-affected cartilage into an autodestructive pathway (Attur et al., 1997 a). ROLE OF TNF AND CARTILAGE SNAKE VENOM LIKE PROTEASE'S (C-SVP) / TNFα CONVERTASE IN OA-CARTILAGE We have recently described a snake venom-like protease isolated from a differential display screen between normal and osteoarthritis (OA)-affected cartilage (and designated c-SVP). This protein shows sequence identity to TNFα convertase recently reported in macrophages and a variety of other non-chondrocyte cells. (Patel et al., 1998; Black et al., 1997; Moss et al., 1997). The mRNA for c-SVP is broadly distributed and is differentially expressed in a variety of human tissues. The c-SVP mRNA, like stromelysin and collagenase-1, is upregulated in arthritis-affected cartilage but not in normal cartilage. OA-affected cartilage explants also expressed low levels of TNFα mRNA that could not be detected in normal cartilage. The OA-

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Figure 22±4. Regulation of PGE2 production by intracellular/extracellular nitric oxide in OA-affected cartilage. The OA-affected cartilage spontaneously releases NO/PGE2 in substantial quantities (Amin et al. 1995a, 1997a) One of the endogenious/intraarticular stimulus is IL-lβ. Intra-articular and/or exogenous NO suppresses the spontaneous/cytokine +endotoxin induced PGE2 production by COX 2. Inhibition of endogenous (spontaneous or cytokine+endotoxin induced) NO in the cartilage by NOS inhibitors (L-NMA) causes a derepression of COX-2 mediated PGE2 production and thereby significantly augmenting PGE2 production in OA-affected cartilage.

affected cartilage spontaneously released detectable levels (> 1 pg/ml) of IL-1α, IL-6, IL-8 and TNFα in ex vivo conditions. Addition of soluble TNFα receptor: Fc in the presence or absence of soluble interleukin-1β receptor (sIL-lR) (with which it acted synergistically) significantly attenuated the spontaneous release of IL-8 in the OA-cartilage explant assay. These data indicate a functional paracine and/or autocrine role of TNFα in OA-affected cartilage that may depend, in part, on upregulated levels of cartilage derived c-SVP (TNFα convertase). These experiments further emphasize the fact that at least two proinflammatory cytokines (IL-β and TNFα) produced in an autocrine fashion by OA-affected cartilage regulate other inflammatory (NO and PGE2) and chemotactic (IL-8) mediators, that may contribute to the autodestructive pathway of the cartilage in OA. REGULATION OF NO BY IL-17 IN OA-AFFECTED CARTILAGE Interleukin-17 (IL-17), also designated (CTLA-8), produced by activated T cells shows ~57 homology to the open reading frame 13 of a lymphotropic virus, Herpesvirus saimiri (Broxmeyer, 1996). IL-17 has been

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Figure 22±5. The autocrine and paracrine effect of IL-1 and IL-17 on NO/PGE2 production in OA-affected cartilage. OA-affected cartilage in ex vivo conditions shows upregulation of IL-1 mRNA and spontaneous release of IL-1 which augments NO/PGE2 production within the cartilage. Addition of soluble IL- R (but not soluble TNF R:Fc) downregulates the spontaneous production of NO/PGE2 in OA-affected cartilage (Attur et al., 1998a) Normal or OAaffected human cartilage which does not show detectable amounts of IL-17 is sensitive to exogenous IL-17. IL-17 upregulates NO/PGE2 production in OA-affected cartilage independent of IL-1 signaling (Attur et al., 1997).

shown to activate transcription factor NF-κB, to induce expression of G-CSF, IL-6, IL-8, PGE2 and surface ICAM-1 in fibroblasts, and also to enhance proliferation of T cells induced by suboptimal co-stimulation with PHA (Broxmeyer, 1996). Our recent studies indicate that NO and PGEa production in OA-affected cartilage can be augmented by IL-17 in ex vivo conditions. IL-17 dependent production of both NO and PGE2 was sensitive to cycloheximide and PDTC, but not to soluble IL-1 receptor. These data indicate that IL-17 activation pathway in chondrocytes requires NFkB activation, but is independent from IL-1 signaling (Figure 22–5). Thus indicating that the IL-17 dependent NO production in OA-affected cartilage is independent of IL-1 signaling (Attur et al., 1997). IL-17 also upregulates cytokines such as IL-6, G-CSF and IL-8 in synovial cells (Fossiez et al, 1996). These cytokines have been detected in OA and RA synovial fluids and are reported to be released also by chondrocytes (Schlaak et al, 1996; Recklies et al, 1992; Venn et al., 1993). While it is likely that IL-17 may play a more important role in RA due to the greater prominance of infiltrating T cells, it is tempting to speculate that this recently described cytokine, as has been suggested for IL-1 , also contributes to the pathogenesis of OA. Indeed, the initial studies of IL-17 may constitute the proverbial tip of the iceberg. The fact that IL-17 regulates other cytokines as well as pleotropic secondary signaling messenger molecules, such as NO (Schmidt and Walter, 1994) and PGE2 (Tsujii and DuBois, 1995) expands its potential array of catabolic effects, particularly in chondrocytes where the overexpression of these mediators has been

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implicated in progressive cartilage destruction (Schmidt and Walter, 1994; Amin et al, 1995a, 1997a; Blanco et al, 1995; Frenkel et al., 1996, Murrel et al, 1995, Taskiran et al., 1994). PHARMACOLOGICAL INTERVENTION OF NITRIC OXIDE IN OSTEOARTHRITIS AFFECTED CARTILAGE Several known pharmacological agents used in the treatment of arthritis have been shown to inhibit nitric oxide production in various cell types. Among these agents, we have shown that NSAIDs (Amin et al., 1995b)), tetracyclines (Amin et al., 1996), Cytokine Suppressive Anti-Inflammation Drugs (CSAIDs) (Attur et al, 1998a) and immunosuppressive drugs (Attur et al., 1998b) inhibit nitric oxide production in osteoarthritis-affected cartilage and/ or murine macrophages stimulated with LPS (Figure 22–6). EFFECT OF NSAIDs Although nonsterodial anti-inflammatory drugs (NSAIDs) clearly inhibit cyclooxygenase responsible for the synthesis and release of prostaglandins (Vane, 1994; Furst, 1994), these actions are not sufficient to explain all the anti-inflammatory effects of NSAIDs. NSAIDs act independently of cyclooxygenase to inhibit activation of neutrophils (Abramson et al, 1985, 1994), promote apoptosis (Lu et al, 1995) and inhibit NFκB activation (Kopp and Ghosh, 1994). The role of NFKB in the action of NSAIDs is controversial (Frantz and O’Neill, 1995). It should be noted that Farivar and Brechers (1996) have recently shown that salicylates inhibit iNOS transcription in cardiac fibroblasts in a NFκB independent manner. Since nitric oxide has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases, we examine the action of NSAIDs on NO/PGE2 production in two systems. We have reported that exposure of LPS-stimulated murine macrophages to therapeutic concentration of aspirin (ICso=2 mM) and hydrocortisone (ICso=1–5 μM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1–2 mM), indomethacin (5–20 μM), and acetaminophen (60–120 juM) has no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50=20 mM) significantly inhibited nitrite production. Immunoblot analysis of iNOS in the presence of aspirin showed inhibition of iNOS expression (IC50=2mM). Sodium salicylate variably inhibited iNOS expression (0–35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal anti-inflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of COX-2 because both aspirin and indomethacin inhibited PGE2 synthesis by >85%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translation/ postranslational modification and directly on the catalytic activity of iNOS. We also examined the effects of selected NSAIDs on the spontaneous production of PGE2 and NO in rat chondrosarcoma cells and cultured human cartilage explants obtained after knee replacement surgery from patients with OA. OA-affected cartilage constitutively expressed both PGE2/NO (Attur et al, 1998c). Based on these observations we evaluated the effect of anti-inflammatory drugs on COX-2 and OA-NOS expression. Aspirin (IC50=2 μM), tenidap (IC50=30µM) and sodium salicylate (20–30% inhibition at 3 mM), but not indomethacin (5–20 μM) or hydrocortisone (1 µM), inhibited the spontaneous release of NO from OA-affected cartilage in ex vivo conditions within 48 h and sustained inhibition for at least 72 h. Rat

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Figure 22±6. Pharmacological intervention of NO/PGE2 in murine macrophages stimulated with LPS and OA-affected cartilage spontaneously releasing NO and PGE2. Glucocorticoids inhibit iNOS in murine macrophages but not in OAaffected cartilage (Amin et al., 1995a, 1997a) Tetracyclines (TETRA), minocyclines, doxycycline and chemically modified tetracycline: (CMTs) decreases NO production in both macrophages and OA-affected cartilage. (Amin et al., 1996, 1997b) The action of TETRA in macrophages is at the level of mRNA stability. (Amin et al., 1996; Trachman et al., 1996) Cyclosporine inhibits NOS/COX-2 expression in macrophages and down-regulates NO production in OAaffected cartilage (Attur et al., 1998b) CSAID do not inhibit COX-2 and iNOS in murine macrophages stimulated with LPS but inhibit PGE2 and NO production in OA-affected cartilage (Attur et al., 1998a) Aspirin down-regulated iNOS protein production and also inactivates iNOS in cell free extracts to inhibit NO production (Amin et al., 1995b) Rapamycin (RAPA) inhibits both PGE2 and NO production with significant effect on their respective mRNA expression and protein expression in macrophages stimulated with LPS (Attur et al., 1998b) NSAIDS such as indomethacin and salicylate (NaSAL) do not inhibit significantly the NO production n LPS stimulated macrophages or OA-affected cartilage (Amin et al., 1995b), whereas salicylates and indomethacin (INDO) inhibits PGE2 production in murine macrophages stimulated with LPS.

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chondrosarcoma cells maintained in vivo spontaneously release NO in ex vivo conditions (DiCesare et al, 1998). The 133 kD iNOS expressed by a rodent chondrosarcoma cell line, like the OA-NOS was susceptible to both aspirin (IC50=2–3 mM) and sodium salicylate (IC50=2–3 mM), but not indomethacin and hydrocortisone (IC50=1 uM). It should be noted that aspirin (1 mM), indomethacin (5–20 μM), tenidap (30 joM) and hydrocortisone (1 juM) inhibited COX-2 dependent PGE2 production in OA-affected cartilage (Attur et al, 1998c). Together the above results indicate that: (1) aspirin > tenidap > sodium salicylate but not indomethacin and hydrocortisone (at pharmacological concentrations) inhibit NO production in human OA-affected cartilage via a mechanism(s) independent of COX-2 expression, (2) rodent chondrosarcoma 133 kD iNOS like the human OA-cartilage OA-NOS is susceptible to inhibition by NSAIDs such as aspirin and sodium salicytate, but differs from macrophage iNOS with respect to susceptibility to hydrocortisone and (3) COX-2 present in OA cartilage is sensitive to pharmacological concentrations of aspirin, indomethacin, tenidap and hydrocortisone. Thus, these data further illustrate that some NSAIDS exert PGE2 independent actions (e.g., NOS inhibition) which may contribute to their effectiveness as anti-inflammatory agents. Moreover, the data demonstrate that both COX-2 and NOS isoform exhibit differential tissue and sensitivity to pharmacological agents which needs to be accounted for an anti-inflammatory drug strategies. A NOVEL MECHANISM OF ACTION OF TETRACYCLINES Doxycycline and minocycline are members of the tetracycline family of broad-spectrum antibiotics. During recent years, it has been established that tetracyclines, which are rapidly absorbed and have a prolonged half-life, exert biological effects independent of their antimicrobial activity (Golub et al., 1991, 1992; Uitto et al., 1994). Such effects include inhibition of matrix metalloprotease (MMPs) [including collagenase (MMP-1), gelatinase (MMP-2), and stromelysin (MMP-3) activity] and prevention of pathogenic tissue destruction (Golub et al., 1991). Furthermore, several studies have also suggested that tetracyclines and inhibitors of metalloproteases inhibit tumor progression (De Clerk et al., 1994), bone reabsorption (Rifkin et al., 1994), and angiogenesis (Maragoudakis et al., 1994) and may have anti-inflammatory properties (Ramamurthy et al., 1994). Tetracyclines have recently been shown to have “chondroprotective” effects in inflammatory arthritides in animal models (Yu et al., 1992). In view of these effects of tetracyclines on cellular functions and the provocial role of NO in the above pathophysiological conditions, we investigated the effect of tetracyclines on NOS expression and NO production. We evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent iNOS. Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1β-induced OA-NOS activity in ex vivo conditions. Similarly, minocycline > doxycycline inhibited both LPS and IFNγ-stimulated iNOS in RAW 264.7 cells in vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors of L-arginine that directly inhibit the specific activity of iNOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS mRNA, protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for β-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of β-actin expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inflammatory diseases, these

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observations suggest that tetracycline’s should be evaluated as potential therapeutic modulators of NO for various pathological conditions (Amin et al., 1996). We have also investigated the ability of chemically modified tetracyclines (CMT), [which retain their anti-MMP activity but lack antimicrobial activity], to inhibit nitric oxide production in murine macrophages stimulated with LPS. These CMTs [CMT-3 (IC50~2.5 μg/ml) and CMT-8 (IC50~10 fig/ml), but no CMT-1, -2 or -5] inhibited nitrite production in LPS-stimulated macrophages (Amin et al., 1997b). Unlike competitive inhibitors of L-arganine which inhibited the specific activity of iNOS in cell-free extracts, CMTs exerted no such direct effect on the enzyme. CMTs could, however, be shown to inhibit both iNOS mRNA and protein expression in LPS-stimulated cells, which would account for the inhibition of nitrite accumulation in the medium. CMTs had no significant effects on the levels of mRNA for glyceraldehyde-3phosphate dehydrogenase (GAPDH). These studies indicate that a novel mechanism of CMTs action is the inhibition of iNOS expression and nitrite production, which may have beneficial effects in the treatment of chronic inflammatory disease. In summary, we have entered a new era in the conceptualization of the pathogenesis of osteoarthritis. The debate, “osteoarthritis versus osteoarthrosis”, must be reframed: if we require of inflammatory processes all of the classical signs of inflammation (e.g., rubor, color, dolor, etc.), then the more narrow conception, that OA is a biomechanically driven process, interrupted by brief and episodic inflammation, will prevail. However, in the modern molecular era we can alternatively define “inflammation” as a process characterized by the release and activation of toxic cellular mediators which promote tissue injury, resulting in some, but not all of the classical signs of inflammation, including “functia laesa”, loss of function. By this definition OA cartilage is a rich source of such inflammatory mediators, a site of activated cytokine production and of prodigious amounts of both NO and PGE2. So conceived, OA cartilage is a tissue “inflamed”, brimming with phologistic products that can serve as targets of future pharmacological intervention. Which conceptual framework shall we choose, osteoarthritis or osteoarthrosis? The implications are clear: exciting interventional “anti-inflammatory” strategies for the former, pharmacological nihilism for the latter. REFERENCES Abramson, S., Korchak, H., Ludewig, R., Edelson, H., Haines, K. et al. (1985) Modes action of aspirin-like drugs. Nat. Acad. Sci. USA., 82, 7227–31. Abramson, S. Leszynska-Piziak, J., Clancy, R.M. and Weissman, G. (1994) Inhibition of neutrophil functions function by aspirin-like drugs (NSAIDS): requirement for assembly of heterotrimeric G proteins in bilayer phospholipid. Biochem. Pharmac., 47, 563–72. Amin, A.R., DiCesare, P., Vyns, P., Attur, M., Tzeng, E., Billar, T.R. et al. (1995a) The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: Evidence for upregulated neuronal nitric oxide synthase. J. Exp. Med., 183, 2097–2102. Amin, A.R., Vyas, P., Attur, M., Leszczynska-Pizak, J., Patel, I.R., Weissman, G. et al. (1995b) The mode of action of aspirin-like drugs: Effect on inducible nitric oxide synthase. Proc. Natl. Acad. Sci. USA, 92, 1926–1930. Amin, A.R., Attur, M., Vyas, P., Thakker, G., Patel, I., Patel, P. et al. (1996) A novel mechanism of action of tetracyclines: Effect on nitric oxide synthase. Proc. Natl. Acad. Sci. USA., 93, 14014–14019. Amin, A.R., Attur, M., Patel, R.N., Thakker, G.D., Marshall, P.J. Rediske, J. et al. (1997a) Superinduction of cyclooxygenase-2 in human osteoarthritis-affected cartilage: Influence of nitric oxide. J. Clin. Invest., 99, 1231–1237 Amin, A.R., Patel, R.N., Thakker, G.D., Lowenstein C, Attur, M. and Abramson, S.B. (1997b) Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and cehmically modified tetracyclines. FEBS Letters, 410, 59–264.

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23 The Role of Nitric Oxide in Rheumatoid Arthritis Maja Stefanovic-Racic and Christopher H.Evans Room C-313 Presbyterian University Hospital, 200 Lothrop Street, Pittsburgh, PA 15261, USA Tel: (412) 648±1090; Fax: (412) 648±8412; E-mail:[email protected]

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic joint inflammation and loss of articular cartilage. Accumulated evidence suggests that nitric oxide (NO) synthesis is elevated in both animal models of RA and human RA. The articular chondrocytes appear to be the dominant intraarticular source of NO, which is produced in large amounts following the exposure of these cells to interleukin-1 (IL-1). NO could influence RA at the three points—as an immunomodulator, as a modulator of inflammation and as a modulator of cartilage loss. The literature concerning the first two is confusing. However, NO has been repeatedly found to inhibit matrix synthesis, and thus impair cartilage repair. Interestingly, NO also prevents matrix degradation, so it could be chondroprotective. Further research is required in order to better define the overall effect of NO on cartilage metabolism. Inhibitors of NOS are good prophylactic agents in some, but not all, animal models of RA, but they are weak therapeutic agents. Although the future treatment of human RA might include therapeutics that would specifically target NO production, they are unlikely to offer any advantage overcurrently used immunosuppressive or antiinflammatory agents. However, their chondroprotective potential could be of most significance, as no presently used antiarthritic drug has ever shown such effects. Key words: rheumatoid arthritis, articular cartilage, chondrocytes, interleukin-1, prostaglandin E2, proteoglycans.

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INTRODUCTION Rheumatoid arthritis (RA) is an autoimmune systemic condition that primarily affects synovial joints. Diseased joints are characterized by persistent inflammation and loss of articular cartilage. Although symptoms of inflammation can be relieved by nonsteroidal antiinflammatory drugs or steroids, and diseasemodifying agents might slow down the course of disease, cartilage destruction can be neither prevented nor stopped, leading ultimately to irreversible loss of joint function. Thus, development of novel, chondroprotective therapeutics would be particularly welcome. Such an improvement is hindered by our poor understanding of the biology of arthritis. Genetic, biochemical and environmental factors are thought to be involved in the aetiopathogenesis of RA. Among the biochemical mediators, nitric oxide (NO) has been increasingly recognized as an important player in the pathophysiology of this disease. As such, NO might become the specific target of future pharmacological manipulation, leading to improved treatment of RA. NITRIC OXIDE SYNTHESIS IS INCREASED IN ARTHRITIS In patients with RA, elevated systemic NO production could be expected as a result of immune response and widespread inflammation. In addition, such a short-lived molecule would have to be synthesized locally within the joint to exert its actions on arthritic tissues. Indeed, elevated levels of breakdown products of NO, nitrite and nitrate, have been detected in the serum, synovial fluid and urine of patients with RA. Using chemiluminescence, Farrell et al. (1992) detected higher serum and synovial fluid levels of nitrite in patients with RA than in those with osteoarthritis (OA) or healthy controls. In addition, synovial fluid nitrite was higher than serum nitrite in both forms of arthritis, implying nitric oxide synthesis within the joint. Similar results for serum nitrite levels were reported by Ueki et al. (1996). They also found significant correlation between serum nitrite concentrations and several indices of the inflammatory activity, including early morning stiffness, the number of inflamed joints, and the serum levels of C reactive protein, tumour necrosis fator-alpha (TNF-α) and interleukin-6 (IL-6). Jacob et al. (1992) reported that concentrations of nitrite and nitrate were also higher in synovial fluid samples from arthritic joints than those with traumatic tears. Stichtenoth et al. (1995) measured the urinary nitrate excretion in RA patients with high disease activity. Again, the rate of nitrate excretion was much higher in urine samples from RA patients than in those from healthy controls. The treatment with prednisolone (a steroid drug) decreased profoundly the indices of disease activity but the effect on the urinary nitrate excretion was much weaker. This suggests that NO is not just a by-product of systemic inflammation and is still being produced following the therapy, perhaps in remaining arthritic joints. Finally, Grabowski et al. (1996) analysed the serum concentrations of nitrite and nitrate as well as the nitrite/ nitrateicreatinine ratio in morning urine samples, following an overnight fast in order to avoid the influence of dietary nitrate intake. They found that urinary nitrite/ nitrate:creatinine ratios were significantly elevated in RA patients comparing to healthy volunteers. Patients with RA also had the elevated serum nitrite/nitrate levels, but the difference was not significant. This suggests that urinary nitrite/nitratexreatinine ratio could be used as the simple, sensitive assay to follow endogenous nitric oxide production in chronic diseases such as RA. Nitric oxide production is also elevated in several animal models of RA. Rats with adjuvant-induced arthritis have increased levels of nitrite/nitrate in both plasma (Connor et al, 1995) and urine (StefanovicRacic et al, 1994). Weinberg et al. (1994) have also reported elevated urinary nitrite/nitrate excretion in MRL/lpr mice, which develop a spectrum of spontaneous autoimmune manifestations, including arthritis. However, the increased NO production does not simply reflect the intensity of either local or systemic inflammation in RA. When Stefanovic-Racic et al. (1994) used antiinflammatory drug indomethacin to

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treat rats with adjuvant-induced arthritis, they were able to suppress inflammation but not urinary nitrite/ nitrate excretion. Moreover, Cannon et al (1996) measured urinary nitrite/nitrate excretion and expression of iNOS mRNA in the joint, liver and spleen tissues and compared them with severity of disease in rats with two different models of RA. Despite similar arthritic scores, NO production was significantly higher in adjuvant-than in collagen-induced arthritis. Furthermore, a prior injection with Mycobacterium bo vis heatshock protein suppressed both NO synthesis and disease severity in adjuvant-induced arthritis, but not in collagen-induced arthritis, suggesting an NO-dependent and NO-independent mechanism of disease, respectively. CELLULAR SOURCES OF NO IN THE ARTHRITIC JOINT In order to target pharmaceutically NO synthesis within the joint, we have to determine which cells are major local producers of NO. Several types of cells are normally present within joint connective tissues. They include chondrocytes, present in articular cartilage; cells within the synovium, such as type A synoviocytes, which resemble macrophages, type B synoviocytes, which are fibroblastic, and endothelial cells lining the synovial capillaries; and, in certain joints, meniscal fibrochondrocytes and fibroblasts of intraarticular ligaments. During inflammation they are joined by infiltrating cells, such as T and B lymphocytes, macrophages, neutrophils and mast cells. Both resident mesenchymal cells and inflammatory cells represent a potential source of NO in arthritic joints. Rabbit articular chondrocytes were the first intraarticular cells found to produce substantial quantities of NO in vitro, in response to a single stimulus, interleukin-1 (IL-1) (Stadler et al, 1991; Palmer et al, 1992). Interestingly, IL-1 also plays a significant role in the pathophysiology of RA. Subsequently, IL-1-stimulated NO synthesis has been documented in chondrocytes derived from rat, bovine, porcine, equine and human cartilage. Inducible NO synthase (iNOS) has been cloned from human chondrocytes and found to be almost identical to iNOS detected in human hepatocytes (Charles et al, 1993; Maier et al, 1994). The capacity of chondrocytes to produce NO appears to be dependent on their position within cartilage matrix. Fukuda et al (1995) reported that superficial cells produce more NO in response to lower amounts of IL-1 than do cells from the deeper zones of cartilage. The higher rate of NO synthesis by IL-1 stimulated superficial chondrocytes might be the result of the more abundant iNOS mRNA levels detected in these cells by Hayashi et al (1997). Presumably, this would lead to more iNOS protein and increased NO output. Rabbit synovial fibroblasts can also synthesise NO upon stimulation by IL-1 (StefanovicRacic et al, 1994), and so can meniscal fibrochondrocytes (Cao et al, 1994) and ligament fibroblasts (Stefanovic-Racic, 1996). Human synovial fibroblasts, unlike chondrocytes, seem to require more complex stimulation. Grabowski et al (1996) found that various combinations of IL-1, TNF-α and interferon-gamma (IFN-γ) can stimulate these cells to produce significant quantities of NO while individual cytokines failed to do so. Other human joint cells might also be able to synthesize NO under appropriate conditions. Recent ex vivo studies provided the evidence of increased iNOS expression and NO production in cells derived from RA joints. Sakurai et al (1995) were first to report the presence of iNOS in rheumatoid synovium and cartilage. Although the rate of spontaneous NO synthesis by both types of tissues was relatively low, it increased significantly upon stimulation by a combination of IL-1, TNF- and lipopolysaccharide (LPS). The inducible NOS was predominantly expressed in chondrocytes, type A, macrophage-like synoviocytes and endothelial cells, as shown by immunohistochemical analysis and in situ hybridization. The spontaneous NO production by synovial cultures prepared from RA patients was also detected by Mclnnes et al (1996). Interestingly, they identified type B, fibroblasts-like synoviocytes as the major cell type expressing iNOS whereas synovial macrophages and CD3+ T lymphocytes were mostly

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iNOS-negative. St. Clair et al (1996) documented the increased level and activity of iNOS in mononuclear blood cells from patients with active RA, as compared to healthy controls. Moreover, McCartney-Francis et al (1993) found elevated NO synthesis in peripheral blood mononuclear cells obtained from rats with streptococcal cell wall fragments-induced arthritis. Since blood mononuclear cells are recruited to RA joints, it would be interesting to see whether these infiltrating cells have the potential to express iNOS. Although Grabowski et al. (1996) could not detect NO production by leucocytes derived from synovial fluid, Sakurai et al. (1995) did find the iNOS mRNA and protein in some mononuclear cells that infiltrate the rheumatoid synovium. Similarly, Connor et al. (1995) reported that within the synovium derived from rats with adjuvantinduced arthritis the iNOS immunoreactivity was localized primarily to infiltrating macrophages. Thus, inflammatory white blood cells could produce NO, but articular chondrocytes appear to be the dominant source of NO in the joint (Evans and StefanovicRacic, 1995). NITRIC OXIDE AS AN IMMUNOMODULATOR Since RA is thought of as an autoimmune disease, its manifestations could be influenced by immunomodulatory actions of NO. Nitric oxide could affect immune cells either directly, through metabolic preturbations, or indirectly, by regulating the production of various molecules which, in turn, modulate the activity of these cells. As T lymphocytes are considered to play a major role in both the initiation and maintenance of synovial inflammation in RA, effects of NO on these cells could be particularly important. Nitric oxide and its derivative S-nitrosoglutathione can suppress proliferation of human T-lymphocytes, as shown by Merryman et al. (1993) and Bauer et al. (1997). By inhibiting the expression of la by macrophages (Sicher et al, 1994), NO could also affect the presentation of potential (auto)antigents to synovial T-cells. Such effects of NO could be beneficial especially during early stages of RA, at the time of Tlymphocytes’ migration into the joint, local activation and proliferation. However, lalenti et al (1993) found that inhibition of NO biosynthesis by rats with adjuvant-induced arthritis led to reduced antigen-stimulated proliferation of T-cells. Furthermore, NO could inhibit the secretion of T-lymphocyte-associated cytokines. In mice, Taylor-Robinson et al (1994) reported that NO suppresses the release of cytokines specific for T helper type 1 (Th1) cells, such as interleukin-2 (IL-2) and IFN-γ. In agreement with these data, Wei et al. (1995) noticed that mice deficient in iNOS developed a stronger Thl type of immune response. Such a selective inhibitory action of NO might be important in rheumatoid joints, where Thl cells predominate and are considered pathogenic. Unfortunately, these results could not be repeated in human T-lymphocytes. Bauer et al (1997) found that NO inhibits the secretion of cytokines by both Thl and Th2 cells indiscriminately. On the other hand, Merryman et al. (1993) could not detect any effect of NO on IL-2 production. It would be interesting to find out the reason for such discrepancies and, even more importantly, whether NO modulates the activity of human synovial Tlymphocytes. In later stages of RA, these cells become dysfunctional and respond poorly to stimulation. Although such profile is typical of mature T-cells, chronic exposure to NO might contribute to this unresponsiveness. B lymphocytes represent another potential target of NO actions within joints. By inhibiting the secretion of some T cell-specific cytokines such as interleukin-4 (IL-4) and IL-2 (Bauer et al., 1997), NO could indirectly suppress the production of autoantibodies by B cells, including rheumatoid factors. Interleukin-6 is another B-cell activator, and NO has been shown to inhibit the secretion of IL-6 by several cell types, such as macrophages (Stadler et al, 1993; Deakin et al, 1995) and articular chondrocytes (Hauselmann et al, 1995). This, in turn, might lower the quantities of immune deposits present in articular cartilage of patients with RA. Cartilage-bound immune complexes are believed to exacerbate joint inflammation by activating

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complement and inflammatory cells. Moreover, Hayashi and Jasin (1995) reported that NO produced by chondrocytes may suppress crosslinking of immune deposits at the articular surface. Thus, by inhibiting the formation of immune complexes NO could have antiinflammatory effect in RA joints. On the other hand, NO might promote long-term survival of B cells within rheumatoid joints by maintaining the expression of the Bcl-2 protooncogene (Genaro et al., 1995). Further studies on the imrmmomodulatory effects of NO in RA are clearly required. NITRIC OXIDE AS A MODULATOR OF INFLAMMATION Inflammation represents complex series of reactions in response to a noxious stimulus. The first signs of acute inflammation are vasodilation and increased vascular permeability, leading to oedema. Polymorphonuclear leucocytes are recruited from blood, followed by mononuclear cells. Activated infiltrating and resident cells release a large number of mediators, including NO. The combined actions of white blood cells and proinflammatory mediators leads to tissue destruction, pain and, sometimes, loss of function, while antiinflammatory agents try to control the damage. Evidence suggests that, depending upon the circumstances, NO may either promote or suppress inflammation. In animal models of acute local inflammation, NO has been shown to promote both vasodilation (Lippe et al., 1993) and vascular permeability (lalenti et al., 1992). Accordingly, mice lacking the iNOS gene developed less carrageenininduced oedema (Wei et al., 1995). Similar effects of NO has been detected in models of acute systemic inflammation. Furthermore, NO inhibits adhesion and emigration of polymorphonuclear leucocytes, presumably by suppressing the expression of CDI I/CD 18 and P-selectin adhesion molecules (Kubes et al., 1991), release of chemoattractant interleukin-8 (IL-8) (De Caterina et al., 1995; Hauselmann et al., 1995) and actin polymerisation at the neutrophil plasma membrane (Clancy et al., 1995). Although polymorphonuclear cells are present in the rheumatoid joint, activated macrophages represent the dominant cell type in inflammatory synovitis. However, data regarding the effect of NO on monocyte migration are contradictory. De Caterina et al. (1995) reported that the decreased expression of vascular cell adhesion molecule1 (VCAM-1) and to a lesser extent intercellular adhesion molecule-1 (ICAM-1) by NO was followed by reduced monocyte adhesion to endothelial cells in vitro. However, results of in vivo experiments done by Mulligan et al. (1992) suggested that NO might actually stimulate recruitment of mononuclear cells. Nitric oxide could also promote activation of mononuclear cells (Lander et al., 1993) and release of certain proinflammatory cytokines, such as IL-1 (Hill et al., 1996) and TNF-α (Eigler et al., 1993; Deakin et al., 1995; Wang et al., 1997), while suppressing the synthesis of interleukin-1 receptor antagonist (IL-1Ra) (Pelletier et al., 1996). This way NO may contribute to a cytokine environment typical of rheumatoid joints, which is characterized by substantial amounts of IL-1 and TNF-a and the relatively low level of IL-1Ra. The release of prostaglandin E2 (PGE2), which is found at elevated levels in the rheumatoid joint, can be either stimulated (Salvemini et al., 1993) or inhibited by NO (Stadler et al., 1993; Hauselmann et al., 1995), depending on the cell type and experimental conditions. The relative concentration of NO seems to be especially important, as large amounts of NO inhibit the synthesis of PGE2 whereas lesser amounts of NO might potentiate it (Stadler et al., 1991; Swierkosz et al., 1995). Prostaglandin E2 is a powerful proinflammatory mediator, which promotes local vasodilation and vascular permeability and sensitizes pain receptors. Furthermore, in animal models of local inflammation (Fujii et al., 1996) and RA (StefanovicRacic et al., 1994) inhibition of either PGE2 or NO synthesis had similar antiinflammatory effects. Thus, regulation of PGE2 synthesis by NO might help define the overall NO activity as either pro- or antiinflammatory. Moreover, PGE2 seems to upregulate IL-6 release in inflammation (Hinson et al., 1996) and

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adjuvant-induced arthritis (Anderson et al., 1996). In some cells, such as macrophages and chondrocytes, where NO inhibits PGE2 production, the synthesis of IL-6 is also suppressed (Stadler et al., 1993; Hauselmann et al., 1995). In addition to being B-cell stimulator, IL-6 induces acute phase proteins and inhibits insulin-like growth factor-I synthesis by the liver (De Benedetti et al., 1997). Therefore, by modulating PGE2 and, consequently, IL-6 release, NO could affect several indices of systemic inflammation in RA, and the growth defect in children with systemic juvenile RA. Local acute inflammation can be resolved completely or it can lead to chronic inflammation, which is characterized by persistent mononuclear cell infiltration, proliferation of connective tissue cells and the laying down of various matrix macromolecules, particularly collagen. In an animal model of non-immune chronic inflammation Vane et al. (1995) demonstrated maximal iNOS activity in the granulomatous tissue at the time of intense macrophage infiltration. This activity decreased as granuloma became more vascularized and fibrotic. Since NO inhibits the expression of VCAM-1 and ICAM-1 (De Caterina et al., 1995), which are elevated in immune-mediated chronic inflammation (Abe et al., 1996), one would expect NO to reduce the intensity of mononuclear cell infiltration. However, luvone et al. (1994) found that NO seems to promote recruitment of macrophages and granuloma formation. In addition to chronic cellular infiltration the rheumatoid synovium is characterised by the hyperplastic growth and neoangiogenesis, leading to development of a pannus. Nitric oxide has been reported to either stimulate (Morbidelli et al., 1996) or inhibit (PipiliSynetos et al., 1993) proliferation of endothelial cells. Similarly, collagen synthesis, which is involved in the fibrotic process, could be inhibited by NO, as shown by Trachtman et al. (1995), or promoted, as found by Schaffer et al. (1997). Therefore, further research is required to elucidate a role of NO in rheumatoid synovitis. NITRIC OXIDE AND CARTILAGE METABOLISM The mobility of our synovial joints is dependent on the presence of normal cartilage, the metabolism of which is regulated by articular chondrocytes. In RA there is a pronounced imbalance in matrix turnover, characterized by impaired matrix synthesis and increased degradation, leading eventually to total loss of cartilage. Stimulation of articular chondrocytes with IL-1 can mimick many of metabolic perturbations seen in rheumatoid cartilage, including the expression of iNOS. Such endogenously produced NO mediates the IL-1 induced suppression of proteoglycan biosynthesis by rabbit (Taskiran et al., 1994; Tamura et al, 1996), human (Hauselmann et al, 1994) and rat chondrocytes (Jarvinen et al, 1995). For some unknown reason, NO has no effect on proteoglycan production by bovine chondrocytes (Stefanovic-Racic et al., 1996). Furthermore, NO seems to protect bovine (Stefanovic-Racic et al., 1996; Hanglow et al., 1995) and rabbit (Stefanovic-Racic et al., 1997) cartilage proteoglycans in organ culture from IL-1 stimulated degradation, presumably by decreasing the activity of stromelysin and other matrix metalloproteinases (MMPs). These IL-1-inducible proteolytic enzymes are believed to play a role in cartilage destruction. Protective effects of NO can be seen even after its synthesis declines, suggesting long-term influence on chondrocyte metabolism. Surprisingly, Tamura et al. (1996) found that NO mediates the effects of IL-1 on both MMPs’ activity and proteoglycan release by monolayer cultures of rabbit chondrocytes. Thus, certain effects of NO might be dependent on the experimental conditions. This is a particular concern with chondrocytes, because they rapidly de-differentiate when removed from their surrounding matrix. In such cases, organ cultures are preferable over monolayers of chondrocytes, as they more closely resemble the natural environment of these cells. One important question is whether NO modulates proteoglycan breakdown in human cartilage, thus contributing to the observed resistance of this tissue to catabolic actions of IL-1. If both anabolic and catabolic

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pathways of cartilage proteoglycans are modulated by NO, then the overall effect of these actions has to be determined in order to define NO as either chondroprotective or chondrodestructive agent. The synthesis of collagen, another major macromolecule of cartilage matrix, is also suppressed by NO in cultures of rabbit chondrocytes (Cao et al., 1997). Although the role of NO in collagen breakdown has not been established, IL-1-induced production of MMPs, such as collagenase and gelatinase, is limited by NO (Stefanovic-Racic et al., 1997). Since these enzymes have been implicated in collagen degradation, NO could be also collagenprotective. In addition to its suppressive actions on the biosynthesis of proteoglycans and collagen, NO inhibits chondrocyte proliferation (Blanco and Lotz, 1995; Tamura et al., 1996), migration and attachment to fibronectin (Frenkel et al., 1996). Thus, NO could contribute to the limited ability of chondrocytes to mount a repair response following an injury or disease. Finally, Blanco et al. (1995) reported that high levels of NO induce apoptosis in human chondrocytes. This could explain why superficial articular chondrocytes, which produce most NO (Fukuda et al., 1995; Hayashi et al., 1997), are also the first cells to die in rheumatoid cartilage (Mitchell and Shepard, 1970). NITRIC OXIDE AND THE TREATMENT OF RHEUMATOID ARTHRITIS There is thus considerable evidence that NO, produced at elevated amounts in RA, could play multiple roles in this disease. Since some of these effects could be beneficial while others are clearly damaging, it is of most importance to determine the overall impact of NO actions on the development of RA. Several groups of investigators have used animal models of RA to study just that. McCartney-Francis et al. (1993) found that inhibition of NO synthesis by NG-monomethyl-L-arginine (L-NMA) prevented the development of streptococcal cell wall-induced arthritis. These findings agree with those by lalenti et al. (1993), StefanovicRacic et al. (1994) and Connor et al. (1995) using adjuvant-induced arthritis as a model and NG-nitro-Larginine methyl ester (L-NAME), L-NMA and Niminoethyl-L-lysine (L-NIL), respectively. Even the spontaneous development of arthritis in autoimmune MRL/lpr mice was suppressed in similar way by LNMA, as shown by Weinberg et al. (1994). Although these data implay that inhibition of NO synthesis could be beneficial in RA, the situation is not so simple. Firstly, L-NMA, L-NAME and other non-selective inhibitors of NOS are not suitable for the treatment of RA since suppression of constitutive NOS activities by these agents could cause serious sideeffects, such as increased peripheral vascular resistance. This could further impair blood flow and nutritional supply in rheumatoid joints, and, hence, promote the tissue destruction. Therefore, iNOS-specific inhibitors, such as L-NIL, would need to be used. Secondly, NOS inhibitors in above mentioned studies were applied prophylactically, whereas in human disease anti-rheumatic drugs are used therapeutically. Unfortunately, LNMA did not show therapeutic properties in adjuvantinduced arthritis (Stefanovic-Racic et al., 1995). Thirdly, NO might not be such an important mediator after all. Thus, mice defficient in iNOS are still susceptible to collagen-induced arthritis (Mudgett, J.S., personal communication). Nitric oxide seems to play a less significant role in this model of RA than in adjuvant-induced arthritis (Cannon et al., 1996). Finally, these studies do not provide a mechanism of NO action. Inhibitors of NO synthesis could simply inhibit an immune response to antigens used in animal models. The suppression of T-cell proliferation in LNAME treated animals and lack of therapeutic effects of L-NMA support this hypothesis. In such case, NO inhibitors might not have any advantage over presently used immunosuppressive drugs, such as methotrexate. If the action of NO inhibitors is based mainly on the prevention of synovitis, then they might not be as beneficial in human disease as they are in animal models of RA. This has already been shown for indomethacin and other non-steroidal antiinflammatory drugs. Direct chondroprotective effect of NO

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manipulation represents the most intriguing and promising option, since no other treatment can prevent cartilage loss in RA. In this case novel, chondrocyte specific NO modulators would have to be developed and preferably used within the first two years of disease onset as this is the period of time when joint erosions, which are closely linked to long-term disability, develop (Brooks, 1993). REFERENCES Abe, Y., Sugisaki, K. and Dannenberg, A.M. Jr. (1996) Rabbit vascular endothelial adhesion molecules: ELAM1 is most elevated in acute inflammation, whereas VCAM-1 and ICAM-1 predominate in chronic inflammation. Journal of Leukocyte Biology, 60, 692–703. Anderson, G.D., Hauser, S.D., McGarity, K.L., Bremer, M.E., Isakson, P.C. and Gregory, S.A. (1996) Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin6 in rat adjuvant arthritis. Journal of Clinical Investigation, 97, 2672–2679. Bauer, H., Jung, T., Tsikas, D., Stichtenoth, D.O., Frolich, J.C. and Neumann, C. (1997) Nitric oxide inhibits secretion of T-helper 1- and T-helper 2-associated cytokines in activated human T cells. Immunology, 90, 205–211. Blanco, F.J. and Lotz, M. (1995) IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE2. Experimental Cell Research, 218, 319–325. Blanco, F.J., Ochs, R.L., Schwarz, H. and Lotz, M. (1995) Chondrocyte apoptosis induced by nitric oxide. American Journal of Pathology, 146, 75–85. Brooks, P.M. (1993) Clinical management of rheumatoid arthritis. The Lancet, 341, 286–290. Cao, M., Stefanovic-Racic, M., Georgescu, H.I., Miller, L.A. and Evans, C.H. (1998) Generation of nitric oxide by differentiated lapine meniscal cells and its effect on collagen biosynthesis: stimulation of collagen production by arginine. Journal of Orthopaedic Research, 16, 104–111. Cao, M., Westerhausen-Larson, A., Niyibizi, C., Kavalkovich, K., Georgescu, H.I., Rizzo, C.F. et al. (1997) Nitric oxide inhibits the synthesis of type II collagen without altering Col2Al mRNA abundance: prolyl hydroxylase as a possible target. Biochemical Journal, 324, 305–310. Cannon, G.W., Openshaw, S.J., Hibbs, J.B.Jr., Hoidal, J.R., Huecksteadt, T.P. and Griffiths, M.M. (1996) Nitric oxide production during adjuvant-induced and collagen-induced arthritis. Arthritis and Rheumatism, 39, 1677–1684. Charles, I.G., Palmer, R.M.J., Hickery, M.S., Bayliss, M.T., Chubb, A.P., Hall, V.S. et al. (1993) Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte. Proceedings of the National Academy of Sciences of the USA, 90, 11419–11423. Clancy, R., Leszczynska, J., Amin, A., Levartovsky, D. and Abramson, S.B. (1995) Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymeriztion in human neutrophils. Journal of Leukocyte Biology, 58, 196–202. Connor, J.R. Manning, P.T., Settle, S.L., Moore, W.M., Jerome, G.M., Webber, R.K. et al. (1995) Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. European Journal of Pharmacology, 273, 15–24. Deakin, A.M., Payne, A.N., Whittle, B.J.R. and Moncada, S. (1995) The modulation of IL-6 and TNF- release by nitric oxide following stimulation of J774 cells with LPS and IFN-γ. Cytokine, 7, 408–416. De Caterina, R., Libby, P., Peng, H.-B., Thannickal, V.J., Rajavashisth, T.B., Gimbrone, M.A. et al. (1995) Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. Journal of Clinical Investigation, 96, 60–68. De Benedetti, R, Alonzi, T., Moretta, A., Lazzaro, D., Costa, P., Poli, V. et al. (1997) Interleukin 6 causes growth impairment in transgenic mice through a decrease in insulin-like growth factor-I. A model for stunted growth in children with chronic inflammation. Journal of Clinical Investigation, 99, 643–650. Eigler, A., Sinha, B. and Endres, S. (1993) Nitric oxide releasing agents enhance cytokine-induced tumor necrosis factor synthesis in human mononuclear cells. Biochemical and Biophysical Research Communications, 196, 494–501.

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Evans, C.H. and Stefanovic-Racic, M. (1995) Possible role of inducible nitric oxide in articular chondrocytes in the pathogenesis of arthritic swelling. Reply letter. Arthritis and Rheumatism, 38, 1530–1531. Farrell, A.J., Blake, D.R., Palmer, R.M. and Moncada, S. (1992) Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Annals of Rheumatic Diseases, 51, 1219–1222. Frenkel, S.R., Clancy, R.M., Ricci, J.L., Di Cesare, P.E., Rediske, J.J. and Abramson, S.B. (1996) Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly. Arthritis and Rheumatism, 39, 1905–1912. Fujii, E., Irie, K., Ogawa, A., Ohba, K. and Muraki, T. (1996) Role of nitric oxide and prostaglandins in lipopolysaccharide-induced increase in vascular permeability in mouse skin. European Journal of Pharmacology, 297, 257–263. Fukuda, K., Kumano, R, Takayama, M., Saito, M., Otani, K. and Tanaka, S. (1995) Zonal differences in nitric oxide synthesis by bovine chondrocytes exposed to interleukin-1. Inflammation Research, 44, 434–437. Genaro, A.M., Hortelano, S., Alvarez, A., Martinez-A., C. and Bosca, L. (1995) Splenic B lymphocytes programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels. Journal of Clinical Investigation, 95, 1884–1890. Grabowski, P.S., England, A.J., Dykhuizen, R., Copland, M., Benjamin, N., Reid, D.M. et al (1996) Elevated nitric oxide production in rheumatoid arthritis. Arthritis and Rheumatism, 39, 643–647. Grabowski, P.S., Macpherson, H. and Ralston, S.H. (1996) Nitric oxide production in cells derived from the human joint. British Journal of Rheumatology, 35, 207–212. Hanglow, A.C., Rowan, K., Lusch, L. and Coffey, J.W. (1995) Degradation of bovine cartilage proteoglycan in vitro is enhanced by inhibition of nitric oxide synthase. Inflammation Research, 44 (suppl. 2), S151– S152. Hauselmann, H.J., Oppliger, L., Michel, B.A., Cao, M., Larkin, L.A., Stefanovic-Racic, M. et al. (1995) Nitric oxide and the synthesis of prostaglandin E2 and cytokines by human articular chondrocytes. Transactions of the Orthopaedic Research Society, 20, 355. Hauselmann, H.J., Oppliger, L., Michel, B.A., Stefanovic-Racic, M. and Evans, C.H. (1994) Nitric oxide and proteoglycan synthesis by human articular chondrocytes in alginate culture. FEBS Letters, 352, 361–364. Hayashi, T., Abe, E., Yamate, T., Tagushi, Y. and Jasin, H.E. (1997) Nitric oxide production by superficial and deep articular chondrocytes. Arthritis and Rheumatism, 40, 261–269. Hayashi, T. and Jasin, H.E. (1995) Covalent cross-linking of immune complexes on the articular cartilage surface. Role of nitric oxide. Arthritis and Rheumatism, 38(suppl.), S163. Hill, J.R., Corbett, J.A., Kwon, G., Marshall, C.A. and McDaniel, M.L. (1996) Nitric oxide regulates interleukin 1 bioactivity released from murine macrophages. The Journal of Biological Chemistry, 271, 22672–22678. Hinson, R.M., Williams, J.A. and Shacter, E. (1996) Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation; Possible role of cyclooxygenase-2. Proceedings of the National Academy of Sciences of the USA, 93, 4885–4890. lalenti, A., lanaro, A., Moncada, S. and Di Rosa, M. (1992) Modulation of acute inflammation by endogenous nitric oxide. European Journal of Pharmacology, 211, 177–182. Ialenti, A., Moncada, S. and DiRosa, M. (1993) Modulation of adjuvant arthritis by endogenous nitric oxide. British Journal of Pharmacology, 110, 701–706. Iuvone, T., Carnuccio, R. and Di Rosa, M. (1994) Modulation of granuloma formation by endogenous nitric oxide. European Journal of Pharmacology, 265, 89–92. Jacob, T., Morrell, M., Manzi, S., Verdile, V., Simmons, R.L. and Peitzman, A. (1992) Elevated nitrates in inflammatory joint disease: nitric oxide in arthritides. Arthritis and Rheumatism, 35(suppl. 5), R9. Jarvinen, T.A.H., Moilanen, T., Jarvinen, T.L.N. and Moilanen, E. (1995) Nitric oxide mediates interleukin1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage. Mediators of Inflammation, 4, 107–111. Kubes, P., Suzuki, M. and Granger, D.N. (1991) Nitric oxide: an endogenous modulator of leukocyte adhesion. Proceedings of the National Academy of Sciences of the USA, 88, 4651–4655. Lander, H.M., Sehajpal, P., Levine, D.M. and Novogrodsky, A. (1993) Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds. The Journal of Immunology, 150, 1509–1516.

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Lippe, I.T., Stabentheiner, A. and Holzer, P. (1993) Participation of nitric oxide in the mustard oil-induced neurogenic inflammation of the rat paw skin. European Journal of Pharmacology, 232, 113–120. Maier, R., Bilbe, G., Rediske, J. and Lotz, M. (1994) Inducible nitric oxide synthase from human articular chondrocytes: cDNA cloning and analysis of mRNA expression. Biochimica and Biophysica Acta, 1208, 145–150. McCartney-Francis, N., Alien, J.B., Mizel, D.E., Albina, I.E., Xie, Q,-W., Nathan, C.F. et al. (1993) Suppression of arthritis by an inhibitor of nitric oxide synthase. The Journal of Experimental Medicine, 178, 749– 754. Mclnnes, I.E., Leung, B.P., Field, M., Wei, X.Q., Huang, F.-P, Sturrock, R.D. et al. (1996) Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients. The Journal of Experimental Medicine, 184, 1519–1524. Merryman, P.P., Clancy, R.M., He, X.Y. and Abramson, S.B. (1993) Modulation of human T cell responses by nitric oxide and its derivative, S-nitrosoglutathione. Arthritis and Rheumatism, 36, 1414–1422. Mitchell, N. and Shepard, N. (1970) The ultrastructure of articular cartilage in rheumatoid arthritis: a preliminary report. American Journal of Bone and Joint Surgery, 52, 1405–1423. Morbidelli, L., Chang, C.H., Douglas, J.G., Granger, H.J., Ledda, F. and Ziche, M. (1996) Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. American Journal of Physiology, 270, H411– H415. Mulligan, M.S., Warren, J.S., Smith, C.W., Anderson, D.C., Grace, C, Rudolph, A.R. et al. (1992) Lung injury after deposition of IgA immune complexes. The Journal of Immunology, 148, 3086–3092. Palmer, R.M.J., Andrews, T., Foxwell, N.A. and Moncada, S. (1992) Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes. Biochemical and Biophysical Research Communications, 188, 209–215. Pelletier, J.-P, Mineau, E, Ranger, P., Tardif, G. and Martel-Pelletier, J. (1996) The increased synthesis of inducible nitric oxide inhibits IL-Ira synthesis by human articular chondrocytes: possible role in osteoarmritic cartilage degradation. Osteoarthritis and Cartilage, 4, 77–84. Pipili-Synetos, E., Sakkoula, E. and Maragoudakis, M.E. (1993) Nitric oxide is involved in the regulation of angiogenesis. British Journal of Pharmacology, 108, 855–857. Sakurai, H., Kohsaka, H., Liu, M.-E, Higashiyama, H., Hirata, Y., Kanno, K. et al. (1995) Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides. Journal of Clinical Investigation, 96, 2357–2363. Salvemini, D., Misko, T.P., Masferrer, J.L., Seibert, K., Currie, M.G. and Needleman, P. (1993) Nitric oxide activates cyclooxygenase enzymes. Proceedings of the National Academy of Sciences of the USA, 90, 7240–7244. Schaffer, M.R., Efron, P.A., Thornton, F.J., Klingel, K., Gross, S.S. and Barbul, A. (1997) Nitric oxide, an autocrine regulator of wound fibroblast synthetic function. The Journal of Immunology, 158, 2375–2381. Sicher, S.C., Vazquez, M.A. and Lu, C.Y. (1994) Inhibition of macrophage la expression by nitric oxide. The Journal of Immunology, 153, 1293–1300. Stadler, J., Harbrecht, E.G., Di Silvio, M., Curran, R.D., Jordan, M.L., Simmons, R.L. et al. (1993) Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells. Journal of Leukocyte Biology, 53, 165–172. Stadler, J., Stefanovic-Racic, M., Billiar, T.R., Curran, R.D., Mclntyre, L.A., Georgescu, H.I. et al. (1991) Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharides. The Journal of Immunology, 147, 3915–3920. St. Clair, E.W., Wilkinson, W.E., Lang, T., Sanders, L., Misukonis, M.A., Gilkeson, O.S. et al. (1996) Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoid arthritis patients. The Journal of Experimental Medicine, 184, 1173–1178. Stefanovic-Racic, M. (1996) Synthesis of nitric oxide by articular tissues, and its role in cartilage matrix turnover. Ph.D. Thesis, School of Medicine, University of Pittsburgh, Pittsburgh, USA. Stefanovic-Racic, M., Meyers, K., Meschter, C., Coffey, J.W., Hofffman, R.A. and Evans, C.H. (1994) Nmonomethyl arginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats. Arthritis and Rheumatism, 37, 1062–1069.

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Stefanovic-Racic, M., Meyers, K., Meschter, C., Coffey, J.W., Hofffman, R.A. and Evans, C.H. (1995) Comparison of the nitric oxide synthase inhibitors methylarginine and aminoguanidine as prophylactic and therapeutic agents in rat adjuvant arthritis. The Journal of Rheumatology, 22, 1922–1928. Stefanovic-Racic, M., Mollers, M.O., Miller, L.A. and Evans, C.H. (1997) Nitric oxide and proteoglycan turnover in rabbit articular cartilage. Journal of Orthopaedic Research, 15, 422–429. Stefanovic-Racic, M., Morales, T.I., Taskiran, D., Mclntyre, L.A. and Evans, C.H. (1996) The role of nitric oxide in proteoglycan turnover by bovine articular cartilage organ cultures. The Journal of Immunology, 156, 1213–1220. Stefanovic-Racic, M., Stadler, J., Georgescu, H.I. and Evans, C.H. (1994) Nitric oxide synthesis and its regulation by rabbit synoviocytes. The Journal of Rheumatology, 21, 1892–1898. Stichtenoth, D.O., Fauler, J., Zeidler, H. and Frolich, J.C. (1995) Urinary nitrite excretion is increased in patients with rheumatoid arthritis and reduced by prednisolone. Annals of Rheumatic Diseases, 54, 820–824. Swierkosz, T.A., Mitchell, J.A., Warner, T.D., Bottting, R.M. and Vane, J.R. (1995) Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids. British Journal of Pharmacology, 114, 1335–1342. Tamura, T., Nakanishi, T., Kimura, Y., Hattori, T., Sasaki, K., Norimatsu. H. et al. (1996) Nitric oxide mediates interleukin-1 induced matrix degradation and basic fibroblast growth factor release in cultured rabbit articular chondrocytes: a possible mechanism of pathological neovascularization in arthritis. Endocrinology, 137, 3729–3737. Taskiran, D., Stefanovic-Racic, M., Georgescu, H.I. and Evans, C.H. (1994) Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1. Biochemical and Biophysical Research Communications, 200, 142–148. Taylor-Robinson, A.W., Liew, F.Y., Severn, A., Xu, D., McSorley, S.J., Garside, P. et al. (1994) Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells. European Journal of Immunology, 24, 980–984. Trachtman, H., Futterweit, S. and Singhal, P. (1995) Nitric oxide modulates the synthesis of extracellular matrix proteins in cultured rat mesangial cells. Biochemical and Biophysical Research Communications, 207, 120–125. Ueki, Y., Miyake, S., Tominaga, Y and Eguchi, K. (1996) Increased nitric oxide levels in patients with rheumatoid arthritis. The Journal of Rheumatology, 23, 230–236. Vane, J.R., Mitchell, J.A., Appleton, L, Tomlinson, A., Bishop-Bailey, D., Croxtall, J. et al. (1994) Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proceedings of the National Academy of Sciences of the USA, 91, 2046–2050. Wang, S., Yan, L., Wesley, R.A. and Danner, R.L. (1997) Nitric oxide increases tumor necrosis factor production in differentiated U937 cells by decreasing cyclic AMP. The Journal of Biological Chemistry, 272, 5959– 5965. Wei, X.-Q., Charles, I.G., Smith, A., Ure, J., Feng, G.-J., Huang, F.-P. et al (1995) Altered immune responses in mice lacking inducible nitric oxide synthase. Nature, 375, 408–411. Weinberg, J.B., Granger, D.L., Pisetsky, D.S., Seldin, M.F., Misukonis, M.A., Mason, S.N. et al. (1994) The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease expression in MRL1pr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine. The Journal of Experimental Medicine, 179, 651–660.

24 Gastrointestinal Function in Shock Andrew L.Salzman Critical Care Medicine, Children's Hospital Medic al Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA Pediatrics and Cellular and Molecular Physiology, University of Cincinnati College of Medicine, USA

Nitric oxide (NO) plays a major role in the response of the gastrointestinal tract to circulatory shock. NO serves a variety of diverse functions in the gut and has been characterized as a neurotransmitter in the myenteric plexus (Miller et al., 1993b), a paracrine hormone (Falcone and Bohlen, 1990; Stark and Szurszewski, 1992), a second messenger (Änggård, 1994; Adler et al., 1995), and a cytotoxin (Kurose et al., 1993; Änggård, 1994). NO deficiency and excess have been associated with pathophysiologic states (Szabóô and Thiemermann, 1994a), manifested by inflammation (Berisha et al., 1994; Yan et al., 1995; Wizemann et al., 1994), altered mesenteric perfusion (Holm, 1993; McCall et al., 1989), macromolecular epithelial hyperpermeability (Salzman et al., 1994a), and ischemia/reperfusion injury (Eppinger et al., 1995). It is anticipated that clinical interventions to selectively decrease or augment intestinal NO production may have utility in the therapeutic management of various shock states. The optimal application of these approaches, as well as an understanding of their anticipated toxicities, must consider the physiologic and pathologic actions of NO in the bowel (Änggård, 1994; Salzman, 1995). This review focuses on the role of NO in the gut during various shock states and discusses the potential therapeutic options in the management of NO-related disease. NITRIC OXIDE: BIOSYNTHESIS AND SOURCES IN THE GASTROINTESTINAL TRACT Gastrointestinal NO synthesis has been detected at rest and in shock. NO is produced by most cell types and tissues in the gastrointestinal tract, including the submucosa, muscle, nerves, and endothelium (Gao et al.,

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1995a, 1995b; Lovchik et al., 1995; Dollberg et al., 1995; Warner et al., 1995). Three NOS isoforms have been unequivocally identified in the bowel: the constitutive isoforms ecNOS and bNOS, which produce low levels of allosterically regulated NO (Abu-Soud and Stuehr, 1993; Dawson et al., 1993), and the inducible isoform iNOS, which produces higher levels of NO and is synthesized de novo in response to proinflammatory stimuli (Miller et al., 1993a, 1993; Miller et al., 1995). In neuronal elements of the bowel, bNOS-derived NO acts as a non-adrenergic noncholinergic neurotransmitter (Miller et al., 1993b; Mourelle et al., 1993a). By staining for NADPH diaphorase activity, NOS activity has been inferred to reside in myenteric neuronal bodies and efferents to the circular muscle (Barry et al., 1994; Nichols et al., 1993). Ultrastructural immunochistochemical techniques have localized bNOS to the enteric neural myenteric plexus and circular muscle layer of canine ileum and colon. bNOS appears to be present exclusively in neurons containing vasoactive intestinal peptide (VIP), but does not share the same organelles (Berezin et al., 1994). The distribution of bNOS varies along the axis of the gastrointestinal tract; the colon is especially rich in bNOS-containing neurons (Nichols et al., 1993). The physiologic role of bNOS is apparent from the gross anatomic abnormalities in bNOS deficient mice (pyloric stenosis) (Huang et al., 1993) and in clinical syndromes characterized by localized bNOS deficiency in the gastroesophageal junction (achalasia) (Mearin et al., 1993). The constitutive NOS isoform ecNOS, which serves an essential function in maintaining microvascular patency, has been identified histologically in the endothelium of submucosal arterioles, independent of their perivascular innervation (Nichols et al., 1993). ecNOS may also be present in enterocytes (Blachier et al., 1991; M’Rabet-Touil et al., 1993), although the NOS activity does not demonstrate the typical calcium dependence associated with the constitutive isoforms. The level of expression of both bNOS and eNOS is generally constant, unrelated to the presence of circulatory shock. The inducible NOS isoform, iNOS, is rarely detected under physiologic conditions (Cook et al., 1994). In circulatory shock, iNOS expression is upregulated (Kostka and Daniel, 1993), as demonstrated by increased levels of iNOS mRNA (Wilson et al., 1994), immunoreactivity (Mercer et al., 1996), and enzymatic activity in gastric (Rachmilewitz et al., 1994) and intestinal homogenates (Tepperman et al., 1994; Boughton-Smith et al., 1993b; Boughton-Smith et al., 1993a). iNOS expression has been identified in many cell types which populate the bowel, including the endothelium (Gross et al., 1991), smooth muscle (Mourelle et al., 1992; Stark and Szurszewski, 1992), and monocyte/macrophages (Drapier and Hibbs, 1994). NO production has also been measured from rat enterocytes stimulated in vitro by y-interferon, TNF-α, IL-1β, and endotoxin (Grisham, 1993), and in vivo by systemic administration of endotoxin (Tepperman et al, 1994). Similar results have been obtained in cultured transformed human enterocytes, such as DLD-1 and Caco-2BBe, in response to treatment combinations of TNF-α, IL-1β, IFN-γ, and endotoxin (Sherman et al., 1993). Gastrointestinal iNOS expression is greatly increased in experimental models of endotoxic shock, peaking at 4 hours and virtually resolving by 24 hours. Regional differences in expression are marked, with most iNOS protein localized to the jejunum and ileum (Mercer et al., 1996). NO is generated in the bowel to a limited extent by granulocytes and monocytes which infiltrate the mucosa and submucosa (McCall et al., 1989). In sepsis and circulatory shock, leukocytes enter the mucosa in response to chemotaxins, such as IL-8, which are produced by immunostimulated enterocytes (Eckmann et al., 1993). The relative contribution to bowel iNOS activity from resident cells (enterocytes) and infiltrating cells (leukocytes) has not been rigorously established, but by immunohistochemical analysis it is evident that most iNOS expression is localized to the enterocyte (unpublished observation). Both crypt and apical enterocytes express iNOS in endotoxic rodent models. We have observed in an in vitro system utilizing Caco-2BBe cell monolayers, a reductionist model which replicates enterocyte differentiation, that cytokine-mediated iNOS expression increases more than 10 fold as enterocytes mature into polarized differentiated monolayers

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(Salzman et al., 1997). The in vivo correlate of the crypt-villus developmental regulation of iNOS expression is currently under investigation. NO may also be generated in mammals by inorganic means. Nitrate is concentrated in the saliva and may be reduced by oral bacteria to nitrite. Nitrite, in turn, may be further reduced by gastric acid to NO (Änggård, 1994). Administration of Ha antagonists or antacids for stress ulcer prophylaxis may interfere with this pathway, by alkalinizing the gastric fluid. Evacuation of gastric contents, which is a common practice in the early management of severe shock, would be expected to remove the source of nitrite, and thereby also contribute to decreased gastric lumenal NO production. The increased association of nosocomial pneumonia and gastric alkalinization has previously been ascribed to the loss of the the antibacterial effect of low gastric pH (Hoyt, 1993). An alternative speculation is that the loss of inorganically derived gastric NO may impair bacterial clearance from the GI tract and predispose to bacterial aspiration and pulmonary infection. NO is highly lipophilic and readily traverses cell membranes to create a sphere of activity, acting as paracrine hormone or a cytotoxic effector species (Änggård, 1994; Ignarro, 1989). The limit to NO diffusion is not intrinsic but rather is a function of its interaction with nucleophiles, such as oxygen and iron-sulfur centers, which serve as a molecular sink (Henry et al., 1993). Because of the multiple sources of NO within the bowel, it is often difficult to associate a particular action of NO with its precise cellular origin. Similarly, pharmacologic inhibition of NO synthases in the bowel may produce highly complex effects, depending on the cells producing and interacting with NO (Rachmilewitz et al., 1995; Boughton-Smith et al., 1993c; Thompson et al., 1994; Arndt et al., 1993; Grisham et al., 1994; Kubes and Granger, 1992; Miller et al., 1993a; Kubes, 1994; Boughton-Smith et al., 1993b). Signal Transduction of iNOS Expression in the Gastrointestinal Tract Circulatory shock induces a generalized inflammatory process in the gut, mediated by multiple arms of the humoral and cellular immune response. Pro-inflammatory cytokines initiate and coordinate this complex response, some produced systemically but many generated de novo in the bowel (Fiocchi, 1996), including IL-12, IFN-γ, IL-4, IL-6, IFNγ, and IL-10. The specific combinations of cytokines which regulate iNOS expression in vivo have not been defined. Microbial pathogens may also interact with the apical surface of the gut mucosa, potentially stimulating iNOS expression in the underlying epithelium. In vitro studies in cultured enterocytes suggest that that there may be a complex interplay between microbes and mucosal cytokines in the regulation of epithelial NO production in the bowel (Salzman et al., 1997). The in vivo signal transduction of iNOS expression in the gut is incompletely understood, due to the complex diversity of intestinal cell types and the potential interactions of microorganisms and associated toxins located in the bowel lumen. Data from reductionist in vitro models of the epithelium, however, have revealed many of the pro-inflammatory signals which may contribute to upregulated gastrointestinal iNOS expression in enterocytes. iNOS induction varies greatly across species and cell type, due to differences in signal transduction pathways and in the cis-acting transcriptional control elements of the 5' flanking region of the iNOS gene (Xie et al., 1993; Spink et al., 1995). Of the cell types resident in the bowel, or which infiltrate into the gut mucosa during shock, monocytes are best characterized in terms of their capacity to upregulate iNOS expression. The predominant cell type in the bowel, however, is the enterocyte; it is significant therefore that these cells have the capacity to upregulate iNOS expression and produce abundant NO (Linn et al., 1997; Salzman et al., 1995, 1997). Microbial pathogens which colonize the intestinal lumen are capable of inducing iNOS expression in enterocytes primed by pro-inflammatory cytokines (Salzman et al., 1997). Relatively few microorganisms

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are required to induce iNOS expression. In sepsis and shock there is typically an increased colonization of the bowel, due to impaired peristalsis and the resultant bacterial overgrowth, thus increasing the probability of enteric iNOS expression (Wirthlin et al., 1996). The ability of gram negative organisms to induce iNOS expression, as well as other pro-inflammatory species such as IL-8 and ICAM, is optimized by bacterial invasion into the enterocyte (Huang et al., 1996; Eckmann et al., 1993; Jung et al., 1995). Non-invasive mutants strains weakly induce iNOS expression and NO production, compared to invasion-competent parental strains (Salzman et al., 1997). Induction of iNOS expression by intact organisms is not mediated by cell wall associated lipopolysaccharide (LPS). Micromolar concentrations of LPS, a purified constituent of the gram negative bacterial cell wall, can independently induce iNOS expression in some, but not all, transformed human intestinal epithelial cell lines. Although this concentration of LPS exceeds reported serum levels during sepsis (Strohmaier et al., 1996), it is within the range measured in the bowel lumen, which may be extremely high. LPS- and microbialmediated induction of in vitro iNOS expression in cultured enterocytes requires co-incubation with IFN-γ (Salzman et al., 1997), a pro-inflammatory cytokine produced by mononuclear cells in the lamina propria (Fiocchi, 1996). The mechanism by which IFN-γ primes human enterocytes for iNOS expression is unknown (Salzman et al., 1997). It is clear, however, that it operates at the pre-translational level. Nuclear run-on analysis demonstrates that IFNγ acts synergistically with LPS, IL-1β, and intact microorganisms to activate human iNOS transcription (Stamler et al., 1992; Linn et al., 1997). Indeed, treatment with either LPS or IL-1β alone does not induce iNOS transcription above basal levels of expression in DLD1 cells (Linn et al., 1997; Wilson et al., 1997). IFN-γ, in contrast, is capable of independently inducing iNOS expression in some cultured enterocyte cell lines, such as Caco-2BBe, but not others, such as DLD-1. In monocytes and macrophages, LPS and intact microorganisms induce the expression of multiple proinflammatory genes (Xie et al., 1994; Keller et al., 1995). LPS has similarly been demonstrated to induce the expression of pro-inflammatory genes, such as IL-8, in some intestinal epithelial cell lines (SW620 and HT29) but the signal transduction events underlying this process are poorly understood (Eckmann et al., 1993). Surface bound CD14 (Tobias et al., 1993), the classic receptor for LPS, is not expressed in intestinal epithelial cells (Pugin et al., 1993). Non-CD14 mediated signal transduction elicited by LPS and intact bacteria has been demonstrated under strict serum-free conditions in leukocytes via the CD11/CD18 receptor (Ingalls and Golenbock, 1995) and in transfected epithelial cells transiently expressing CD lie/CD 18, but the relevance of these mechanisms to intestinal epithelial iNOS expression is unclear (Ingalls and Golenbock, 1995). Future studies will be required to establish the identity of the LPS receptor in intestinal epithelial cells which mediates iNOS expression. Differing patterns of iNOS induction by intact bacteria and purified LPS suggest that gram negative bacteria stimulate iNOS signal transduction in enterocytes by a pathway not involving cell wall associated LPS (Degroote et al, 1995). IFN-γ has been shown to induce iNOS expression in conjunction with gram negative organisms, independent of their LPS content (Salzman et al, 1997). Thermal pre-treatment of Salmonella, but not LPS, inhibited NO production in DLD-1 cells, suggesting that bacterial-mediated induction of iNOS in IFN-y primed intestinal epithelial cells is mediated chiefly by a heat-labile moiety. The identity of the heat-labile component is unknown, but may represent a bacterial cell wall protein which is important in bacterial-enterocyte interaction and eukaryotic signal transduction (Salzman et al, 1997). Expression of human iNOS mRNA in intestinal epithelial cells induced by cytokines or microbes is efficiently inhibited by agents which interrupt the nuclear translocation of NF-κB. NO production and iNOS mRNA expression by DLD-1 cells are blocked by the anti-oxidant pyrrolidine dithiocarbamate (PDTC), which acts presumably at a point proximal to the phosphorylation of the inhibitory protein IκBα (Xie et al., 1994). iNOS expression in enterocytes is also inhibited by various serine protease inhibitors, such as N-α-

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-p-tosyl-lysine chloromethyl ketone, which inhibit the proteolytic degradation of IkB and the resultant nuclear translocation of NF-κB (Salzman et al., 1995). Similarly, in vitro administration of peptide aldehyde proteosomal inhibitors inhibits the nuclear translocation of NF-κB and the production of NO by cultured human enterocytes (Wilson et al., 1997). Tyrosine kinase activation has been implicated in iNOS signal transduction in rodent vascular smooth cells (Marczin et al., 1993) and in cultured human enterocytes (Salzman et al., 1995). Mucosal growth factors, such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EOF), inhibit tyrosine kinase activation, but have diverse effects depending on the cell type and species on iNOS iNOS mRNA expression and on iNOS activity at the post-transcriptional level (Gazzinelli et al., 1992; Schini et al., 1992; Dong et al., 1993; Goureau et al., 1994; Asano et al., 1994). In cultured human enterocytes, however, bFGF or EGF do not effect iNOS mRNA expression or activity (Salzman et al., 1995). The specific role of tyrosine kinase in iNOS expression is unclear and may be related to IFN-γ stimulated transcription factors, such as IRF-1, which have been implicated in the regulation of the murine iNOS promoter (Martin et al., 1994) and might interact with an IRE sequence in the 5' enhancer of the human iNOS gene (Linn et al., 1997). Alternatively, tyrosine kinase activity may be induced by bacterial interaction with the enterocyte. Invasion of human epithelial cells by Salmonella typhimurium, for example, induces tyrosine phosphorylation of the epidermal growth factor receptor (Galán et al., 1992). Hypoxia-reoxygenation, which has been demonstrated in pulmonary epithelial cells to generate oxygen free radicals and induce a variety of pro-inflammatory cytokines, has been shown not to induce iNOS expression in cultured human enterocytes, when given alone or in combination with cytokines (Salzman et al., 1995). Incubation under a hypoxic atmosphere for 24 hours, without reoxygenation, also has not been shown to upregulate iNOS expression, unlike a variety of other pro-inflammatory species, including NF-KB, which are hypoxia-responsive in certain cell types (Salzman et al., 1995). Similarly, oxidant stress with H2O2, a known activator of NF-κB in lymphocytes, does not induce NO production in cultured enterocytes nor effect the formation of NO in response to cytokines (Salzman et al., 1997b). Molecular Regulation of iNOS Expression in the Gastrointestinal Tract The human iNOS gene is expressed in cultured enterocytes at very low levels under resting conditions (Salzman et al., 1995). Indeed, iNOS activity and the production of NO are barely detectable (Salzman et al., 1995; Sherman et al., 1993). Nonetheless, using more sensitive assays, such as polymerase chain reaction and nuclear run-on analysis, it is evident that human iNOS is expressed at a low constitutive level (Chu et al., 1995; Linn et al., 1997). Consistent with these observations, reporter assays in which the 5' flanking region of the human iNOS gene is placed upstream of a luciferase reporter construct region exhibit low levels of basal stimulation in transfected enterocytes (Linn et al., 1997). The inability to detect iNOS mRNA expression at the level of Northern analysis implies that the half-life of the primary transcript is short, due to nuclear processing or cytoplasmic degradation. Exposure of cultured human enterocytes to cytokine combinations, such as IL-1β and IFN-γ, induces a 2– 4 fold increase in RNA synthesis at the nuclear level, whereas steadystate iNOS mRNA expression is upregulated by more than 30-fold (Salzman et al., 1995). It would appear thus that the cytokine-mediated elevation in steady-state iNOS mRNA levels is mediated in part by transcription, but that posttranscriptional regulation is the dominant factor governing the overall balance of iNOS expression (Linn et al., 1997). We have also observed that the increase in primary transcripts of human iNOS mRNA is fully inhibited by cycloheximde (Linn et al., 1997), an inhibitor of protein synthesis, indicating that de novo

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synthesis of protein factors is required for cytokine-mediated induction of human iNOS transcription in enterocytes. Utilizing transient transfection of human iNOS reporter constructs, the cytokine-responsive sequence elements in DLD-1 cells have been localized between 10.9 and 9.6 kb upstream of the transcription initiation site (Linn et al., 1997). No further induction has been identified using promoter segments terminating at -13.1 kb. We have observed that the cytokine-responsive region between 10.9 and 9.5 kB functions independent of distance and orientation relative to the reporter gene, demonstrating the behavior of a classical enhancer (Linn et al., 1997). The specific cytokine which induces expression of the human iNOS enhancer in enterocytes is currently unknown. Singular addition of cytokines does not stimulate iNOS transcription after a short-term exposure (Linn et al., 1997), whereas after prolonged exposure, IFN-γ is able to independently upregulate expression in cultured enterocytes (Salzman et al., 1995). The molecular mechanism by which a single cytokine may stimulate iNOS expression is not understood currently. Several potential ds-regulatory elements in the proximal human iNOS promoter and distal enhancer regions have been identified (Chartrain et al., 1994; Linn et al., 1997). In the proximal human iNOS promoter, a consensus sequence for the pro-inflammatory transcription factor NF-κB binding exists at position −115 bp (Chartrain et al., 1994; Nunokawa et al., 1994), in addition to two additional potential binding sites between—8.7 to −10.7 kb (Linn et al., 1997). Evidence favoring a role for NF-κB in the induction of the human iNOS gene in enterocytes has been provided only indirectly, by pharmacologic studies utilizing serine protease inhibitors and the anti-oxidant pyrollidine dithiocarbamate (Salzman et al., 1995). More definitive proof awaits site-specific mutagenesis of the NF-KB regions in reporter constructs. Such a demonstration will have practical implications in the near future as novel anti-inflammatory agents which inhibit IκB kinase activity are available for clinical application. Potential binding sites for additional transcription factors induced by IL-1β or IFNγ have also been identified (Linn et al., 1997). The region of the human iNOS enhancer between positions −10.3 and −10.5 kb, for example, contains two recognition sites for interferon regulatory factor 1 (IRF-1), a transcriptional activator that is required for IFN-γ—dependent activation of the murine iNOS promoter (Kamijo et al., 1994; Martin et al., 1994). Sites have also been described for STAT1, a pro-inflammatory transcription factor activated by Janus kinase (JAK)-dependent phosphorylation in response to IFN-γ stimulation (Darnell, 1996). The downstream IRF-1 site is also bounded by a recognition site for the AP-1 (jun/fos) family of transcription factors, components of which are transcriptionally induced by interleukin-1β (Angel and Karin, 1991). REGULATION OF GASTROINTESTINAL FUNCTION BY ENDOGENOUS NITRIC OXIDE It has been popular to classify the various NO synthase isoforms in the gut into a beneficial and deleterious grouping, but this is oversimplified and somewhat misleading. The so-called “beneficial” isoforms (bNOS and cNOS) have been implicated in neurotransmission (Miller et al., 1993b), vascular perfusion (Vallance et al., 1989), microvascular patency (Geiger et al., 1992), and inhibit leukocyte activation and adhesion (Ney et al., 1990). In contrast, the “deleterious” isoform (iNOS) contributes to mutagenesis (Nguyen et al., 1992), inhibits ATP formation (Stadler et al., 1994; Corbett et al., 1994; Drapier and Hibbs, 1994; Kurose et al., 1993; Welsh et al., 1991), and may trigger apoptosis (Sarih et al., 1993). Given the diverse group of cell populations that form the mammalian gut, it is not surprising that all three isoforms of NOS play a role in the response of the bowel to shock.

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Mesenteric Perfusion NO modulates the extent and distribution of blood flow within the layers of the wall of the gut (Holm, 1993; McCall et al., 1989) via its effect on the vascular tone of mesenteric resistance vessels (Diederich et al., 1990) and submucosal arterioles (Bohlen and Lash, 1992). NO mediates mesenteric vascular relaxation via two independent mechanisms: 1) stimulation of guanylyl cyclase, which then increases the concentration of intracellular cGMP, and 2) attentuation of adrenergic constriction in the intestinal microvasculature (Nase and Boegehold, 1996). Intravital microscopic studies demonstrate that NOS inhibition augments sympathetic neurogenic vasoconstriction in mesenteric small feed arteries, first-order arterioles, and secondorder arterioles (Nase and Boegehold, 1996). Under physiologic conditions, the endothelium regulates the underlying intestinal vascular smooth muscle in response to endocrine, neural, metabolic, and physical stimuli, in order to match the demand for perfusion required for digestion, tissue oxygenation, and mucosal integrity (Boughton-Smith et al., 1993c; Pawlik et al., 1993; Kiraly et al., 1993; Piqué et al., 1992). Inhibition of basal production of NO by inhibitors of NOS, e.g., LN-monomethylarginine, results in a substantial decrease in the perfusion of the gastrointestinal tract (Kubes and Granger, 1992). Increased perfusion, on the other hand, results from stimulation of NO stimulation by pentagastrin (Pique et al., 1992) and calcitonin gene related-peptide (Holzer et al., 1993; Lambrecht et al., 1993), in addition to shear stress and the other signalling pepides. Ingestion of food results in the release of NO by activation of ecNOS in the gastric mucosa via the release of pentagastrin (Holm, 1993; Pique et al., 1992) and/or central vagal activity (Kiraly et al., 1993). Gastrointestinal hormones, such as gastrin (Pique et al., 1992) and cholecystokinin (Lambrecht et al., 1993), stimulate the synthesis of NO by the constitutive NOS isoform, via binding to receptors on the luminal surface of the mesenteric endothelium and inducing the opening of calcium channels. Under pathophysiologic conditions, iNOS-derived NO in the gut wall may diffuse to nearby vascular smooth muscle (Yamada et al., 1993; Piqué et al., 1992; Stark and Szurszewski, 1992) and induce gastrointestinal hyperemia (Grisham and Yamada, 1992; Stark and Szurszewski, 1992). In experimental models of well-compensated sepsis, ileal blood flow is typically markedly increased (Fink et al., 1987; Lang et al., 1984). In unresuscitated models of acute septic shock, prior to the induction of iNOS, mesenteric perfusion is reduced. In rats, acute normovolemic anemia induced by rapid blood loss has been shown to lead to a rapid rise in NOS activity and gastric hyperemia, thereby mitigating the fall in oxygen carrying capacity (Piqué et al., 1992; Panes et al., 1992). In ischemiareperfusion states, NO production by the mesenteric vascular endothelium is altered, but in an age-dependent fashion: endothelial cell production of NO in mesenteric artery is attenuated in 3 day old swine but amplified in 35 day old swine (Nowicki, 1996). These results suggest that ischemia-reperfusion in immature subjects may induce a regional vasculopathy, manifested by a loss of endothelial NO production, local vasoconstriction, and gut necrosis (Nowicki, 1996). In contrast, mature subjects compensate for the loss of blood flow by an upregulation of ecNOS activity in the endothelium (Nowicki, 1996). Accordingly, subsequent exposure to ischemia-reperfusion is well tolerated. This adaptation, known as intestinal preconditioning, is mediated by a transient increase in NO production and is eliminated by NOS inhibition (Hotter et al., 1996). Electrolyte and Fluid Regulation Both pro-absorptive and pro-secretory effects of NO have been demonstrated in intestinal in vivo and ex vivo preparations (Barry et al., 1994; Wilson et al., 1994; Takeuchi et al., 1994; Hällllgren et al., 1993). NO has been found to act synergistically with prostaglandins to support alkaline secretion by the gastroduodenal mucosa in response to acid and food (Konturek et al., 1993). In contrast, other reports have described a

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secretory effect in the gastroduodenal mucosa of the rat in response to NOS inhibition (Takeuchi et al., 1994). In the ileum, NOS inhibitors promote secretion of water and ions (Barry et al., 1994), whereas in the colon supplemental NO induces secretion and inhibits sodium absorption (Wilson et al., 1994). NO also contributes to the decrease in gastric acid secretion characteristic of sepsis: the acid secretory response to pentagastrin is ablated by treatment of rats with endotoxin (Martinez-Cuesta et al., 1992). Co-administration of the isoform nonselective NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), partially restores the response. A total restoration of pentagastrin-induced acid secretory response in endotoxic rats requires inhibition of both NOS and platelet activating factor (PAF) activity (MartinezCuesta et al., 1992). The overall effect of NO on salt and water homeostastis in the bowel during shock states is unknown. Gastrointestinal Motility Circulatory shock disrupts bowel motility, as manifested by gastroparesis and intestinal ileus (Wirthlin et al., 1996; Salzman, 1995). The loss of normal peristalsis may interfere with the provision of enterai nutrition, enhance enteric bacterial proliferation, and necessitate gastric decompression and parenteral hyperalimentation (Wirthlin et al., 1996). Although many mediators no doubt influence gut motility in sepsis and shock, several lines of investigation point to a major role for NO: First, exogenous addition of NO to the gut decreases enteric motility (Slivka et al., 1993). Second, NO is a non-adrenergic non-cholinergic (NANC) neurotransmitter in the enteric nervous system. In concert with VIP, which is co-localized with bNOS in the gut, NO coordinates propogation of gut contents and sphincter relaxation (Miller et al., 1993b; Mourelle et al., 1993a). Third, excessive NO, produced by iNOS during shock, inhibits smooth muscle activity generally (Mourelle et al., 1992; Stark and Szurszewski, 1992). Exposure of isolated gallbladder strips to endotoxin, for example, induces a marked elevation of iNOS and a loss of muscle tone, suggesting that excessive NO may be responsible for distension of the gallbladder commonly observed in sepsis (Mourelle et al., 1992).Whereas inhibition of NOS may be therapeutic in certain inflammatory conditions, there is the potential risk of blocking muscular relaxation, as has been observed in resting canine colonie smooth muscle (Huizinga et al., 1992). In addition to its effect on intestinal motility, NO also regulates sphincteric tone via NANC neural pathways (Slivka et al., 1993; Kaufman et al., 1994). NO has been shown to have a profound inhibitory effect on the tone of the sphincter of Oddi in an ex vivo rabbit preparation (Slivka et al., 1993; Mourelle et al., 1993b) and in in vivo studies in prairie dogs (Kaufman et al., 1994). NO acts directly to inhibit smooth muscle contraction by inhibiting the release of excitatory mediators. Indeed, NO is the primary inhibitory determinant of the contractile activity of smooth muscle in the wall of the intestine (Daniel et al., 1994). NO also acts indirectly to cause smooth muscle relaxation via its effect on VIP release (Allescher et al., 1996). Studies of rat enteric synaptosomes demonstrate that VIP can be released by calcium dependent mechanisms by NO agonists or NO-dependent mechanisms. It is thought that the release of VIP is induced by a presynaptic stimulatory mechanism of NO and that this effect may enhance or contribute to the action of NO (Allescher et al., 1996). The production of NO depends on nerves with N-type Ca++ channels. There is evidence of a tonic Ca++-dependent NO output in isolated, perfused, canine ileal segments. Distension of the stomach after ingestion of food is similarly due to activation of NANC nerves and is NO mediated (Desai et al., 1991).

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Lumenal Antibiosis The production of iNOS-derived NO by the intestinal epithelium may serve an important physiologic function as an endogenous antibacterial effector species. Under normal conditions, the intestinal lumen contains an enormous burden of bacteria, in number exceeding the quantity of eukaryotic cells of the host! The ability of the enteric mucosa to prevent transepithlial passage of prokaryotes into the splanchnic viscera is a testament to the adequacy of the mucosal barrier. In part, the barrier function of the mucosa is structural, dependent upon the fusion of adjacent cells to form a nearly impenetrable intercellular connection, termed the zonula occludens (Salzman et al., 1994a). An immunologic dynamic barrier also exists, represented by the numerous antibacterial molecular species released by enterocytes into the intestinal lumen. A range of antibacterial products are synthesized by enterocytes, including the cryptdin family, complement components, superoxide anion, and NO (Harwig et al., 1995; Moon et al., 1997). NO is a potent inhibitor of bacterial energetics, as evidenced by the in vivo inhibition of aconitase activity in E. coli subjected to exogenous authentic NO (Gardner et al., 1997). The susceptibility of E. coli to the effects of NO is highly dependent upon the ambient conditions. Low oxygen tension greatly potentiates the loss of bacterial aconitase activity, such that nanamolar concentrations of NO are effective in reducing cellular respiration (Gardner et al., 1997). It is quite possible, but as yet unproven, that iNOS expression in the gut mucosa plays a critical role in host defense. Therapeutic regimens which target iNOS activity might be expected, accordingly, to decrease the efficiency of the intestinal mucosal barrier and increase the risk of systemic infection. INTERACTION OF NITRIC OXIDE WITH OTHER MEDIATORS OF INFLAMMATION IN THE GASTROINTESTINAL TRACT Nitric Oxide and Oxygen Centered Free Radicals In vitro and in vivo data demonstrate that NO per se may not be the only relevant cytotoxic species in circulatory shock (Szabô and Salzman, 1995; Zingarelli et al., 1996; Szabó et al., 1995; Szabo et al., 1996). Accumulating evidence now implies that peroxynitrite, a reaction product of NO and superoxide anion, is a final common effector of cytotoxicity and tissue injury (Szabó et al., 1996a). Simultaneous generation of nitric oxide and superoxide favors the production of a toxic reaction product, peroxynitrite anion (ONOO-) (Beckman et al., 1990; Pry or and Squadrito, 1995). In in vitro systems, the ratio of superoxide and NO determines the reactivity of peroxynitrite: excess NO reduces the oxidations elicited by peroxynitrite (Rubbo et al., 1994; Villa et al., 1994; Szabó et al., 1995). Under in vivo conditions, peroxynitrite is formed, as the end-products of specific oxidative processes triggered by peroxynitrite can be detected (see below). The oxidant reactivity of peroxynitrite is mediated by an intermediate with a biological activity of hydroxyl radical, which is not hydroxyl radical per se, but rather, peroxynitrous acid or its activated isomer (Rubbo et al., 1994). Immunohistochemical evidence implies that peroxynitrite is produced in endotoxic shock (Wizemann et al., 1994) and ischemia-reperfusion injury (Ischiropoulos et al., 1995). Although there is no available agent to specifically scavenge or neutralize peroxynitrite, indirect evidence suggests that much of the toxicity of NO is, in fact, mediated by peroxynitrite. Peroxynitrite is more cytotoxic than NO or superoxide in a variety of experimental systems (Hausladen and Fridovich, 1994; Castro et al., 1994; Brunelli et al., 1995; Szabó and Salzman, 1995; Bolanos et al., 1995; Denicola et al., 1995). For example, peroxynitrite, and not NO, is a potent initiator of DNA strand breakage (Szabó et al., 1996a). Scavenging oxygen radicals or

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peroxynitrite limits the toxicity of NO generating drugs (Szabó et al., 1996; Burkart et al., 1995), suggesting that when large amounts of NO are generated in biological systems, there are sufficient amounts of basal superoxide (produced by the mitochondria, for example), to form peroxynitrite and cause consequent cytotoxicity. Nitric Oxide and Cyclooxygenase Metabolites NO can activate or inactivate a variety of enzymes in a cGMP-independent fashion. NOmediated activation of cyclooxygenase (COX) has been described in various cells including macrophages (Stadler et al., 1993; Salvemini et al., 1994; Corbett et al., 1993). This action is related to the reaction of NO with the iron-heme center in the active site of the enzyme. On the other hand, NO at high concentrations, inhibits COX activity (Stadler et al., 1993). Activation of COX by NO may have pro-inflammatory effects in the gastrointestinal tract. Cyclooxygenases are similar to nitric oxide synthases in the sense that they exist in two distinct isoform, a constitutive and a cytokine-inducible. The expression of the inducible isoform (COX-2) has been demonstrated in the gut, in response to pro-inflammatory stimuli. It is therefore conceivable that large amounts of NO, produced by iNOS, activate COX-2 during intestinal inflammation, and inhibition of NOS may reduce the production of NO as well as the production of prostaglandins (Salvemini et al., 1995; Sautebin and DiRosa, 1994). EFFECT OF NO ON INTESTINAL ENERGETICS Overproduction of NO in the gut during sepsis and other forms of circulatory shock is suspected to have a deleterious action on the capacity of enterocytes to maintain optimal levels of oxidative phosphorylation and ATP formation. Several mechanisms have been proposed to account for the toxicity of NO towards cellular respiration: NO has been shown in vitro to attack iron-sulfur clusters in the active catalytic centers of key enzymes participating in ATP formation. NADH-ubiquinone-oxidoreductase and succinate-ubiquinone oxidoreductase, the first two enyzmes of the electron transport chain, are inhibited by exogenous NO, thereby interfering with cellular ATP formation (Drapier and Hibbs, 1994; Stadler et al., 1994; Kurose et al., 1993; Welsh et al., 1991). NO also has been shown to disrupt the Krebs cycle via its inactivation of the catalytic center of c/s-aconitase (Stadler et al., 1994; Corbett et al., 1994). Several groups have questioned whether NO is independently toxic to aconitase, suggesting instead that the reaction product peroxynitrite, and not NO per se, is the active toxic species (Hausladen and Fridovich, 1994; Castro et al., 1994). A recent study has clarified the question of NO-mediated toxicity by showing that the enzyme is highly susceptible to NO attack under conditions of substrate occupancy (Gardner et al., 1997). Moreover, the same study demonstrated that the susceptibility of aconitase is potentiated by acidosis and hypoxia. Indeed, at oxygen tensions of 20–30 torr NO was found to inactivate aconitase at concentrations in the low micromolar range (Gardner et al., 1997). Hypoxia to this degree is present under resting conditions in the apical villi, on account of a unique vascular supply that provides inhomogeneous oxygen delivery (Shepard and Kiel, 1992). The crypt-villus oxygen gradient arises secondary to two anatomic features of the villus: first, a countercurrent arteriovenous plexus in the villus core, and second, a single terminal arteriole in the villus, which ensures that crypt cells are well oxygenated at the expense of apical villi, which are last in line to obtain oxygen (Shepard and Kiel, 1992). Moreover, in the setting of circulatory shock, mucosal hypoxia,

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acidosis, and NO production are simultaneously increased, creating a powerful combination favoring NOmediated inactivation of aconitase. NO has also been shown to attack cytochrome oxidase, the terminal enzyme in the electron transport chain which provides a reducing equivalent to molecular oxygen. The rate constant for this reaction is large, implying its potential relevance under conditions of high output NO production, such as septic shock. The relative contribution of this reaction to the total NO-mediated impairment of oxidative phosphorylation is unknown. Given that the reaction rate of NO with cytochrome oxidase exceeds that of cyanide, an extremely potent respiratory toxin, it is probable that NO-mediated inactivation of cytochrome oxidase is of fundamental importance to the loss of cellular energetics in circulatory shock. Finally, inflammatory induction of NO production has been shown in vitro (Dimmeler et al., 1993; Zhang and Snyder, 1992) and in vivo in rodents to lead to the auto-monoADP ribosylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the regulation of glycolysis (Dimmeler et al., 1992). Clinical evidence in support of this process has been found in inflammatory bowel disease. Colonic mucosal biopsies from patients with active disease have elevated intestinal NO production and absent GAPDH activity (Boughton-Smith et al., 1993a; Molinay Vedia et al., 1992; Zhang and Snyder, 1992). In concert with the oxygen-centered free radical superoxide, NO forms the potent oxidant peroxynitrite in a rapid diffusion-limited reaction (Pryor and Squadrito, 1995). PeroxynitritQ is rapidly protonated to peroxynitrous acid, an unstable intermediate, in a reaction which is accelerated by acidosis and thus, presumably by shock. Peroxynitrous acid has a vibrationally active structure with hydroxyl-radical like activity which results in the oxidation of multiple cellular targets, including membrane lipids, DNA, and tyrosine residues. The effect of peroxynitrite on DNA is of particular importance, as evidenced by DNA cleavage in solutions of end-labelled DNA restriction fragments (King et al., 1992) and DNA nicking of the supercoiled plasmid pBR322 (Salgo et al., 1995). Peroxynitritemediated DNA single strand breakage potently induces the activation of poly-ADP ribosyl synthetase (PARS) activation (Szabo et al., 1996a; Zingarelli et al., 1996), an abundant nuclear enzyme previously thought to participate in DNA repair. Peroxynitrite-mediated DNA strand breakage activates the C-terminal zinc-finger domain of PARS, which in turn activates an N-terminal ribosylase. PARS activity catalyzes the covalent modification of nuclear proteins, such as histones and an auto-modification domain of PARS itself, adding polymers of ADPribose, derived from NADH (Berger, 1991). The consequent consumption of the cellular substrate pool of NADH produces generalized cellular failure resulting from energetic depletion (Berger, 1991). Such a mechanism of injury has been proposed to underly the common response to such diverse genotoxic agents as free radicals and oxidants (Yamamoto et al., 1993; Schraufstatter et al., 1986; Akerfeldt, 1959; Zingarelli et al., 1996; Szabó et al., 1996a). As predicted by this mechanism, the inhibition of PARS activity restores NAD and ATP levels in cultured enterocytes exposed to exogenous peroxynitrite (Kennedy et al., 1998). The physiologic role of PARS is unknown; indeed, the phenotype of the PARS knockout mouse is essentially normal (Wang et al., 1995). ROLE OF NITRIC OXIDE IN GASTROINTESTINAL MUCOSAL INJURY Excessive NO production has been proposed as a final common effector of inflammatory injury to the intestinal mucosal barrier (Salzman et al., 1994b; Unno et al., 1995; Salzman, 1995). Experimental models of circulatory shock demonstrate that endotoxicosis induces high levels of NOS activity in villus absorptive cells and colonocytes in rats, reducing ex vivo cellular viability from 95% to 80% and contributing to the damage associated with colonie mucosal inflammation (Tepperman et al., 1993; Tepperman et al., 1994).

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Pretreatment with a NOS inhibitor preserves viability and prevents inflammation (Tepperman et al., 1993; Tepperman et al., 1994). Ex vivo studies of enterocytes harvested from endotoxic rats also show that induction of epithelial iNOS activity is associated with a decrease in cellular viability (Tepperman et al., 1994). Abrogation of in vivo NOS activity by pharmacologic means prevents the loss of mucosal barrier function in an endotoxic rodent model, if the inhibitor is administered in a manner that preserves the beneficial actions of the constitutive NOS isoform (Laszlo et al., 1994). The utility of NOS inhibition is highly dependent upon the timing of administration, however. In the early phase of endotoxic shock, NOS inhibition is deleterious (Boughton-Smith et al., 1994; Laszlo et al., 1994; Boughton-Smith et al., 1993b), in agreement with reports that exogenous provision of NO during early endotoxic shock attenuates LPSinduced macroscopic jejunal damage. Later administration of isoform non-selective NOS inhibitors, at a time when iNOS upregulation has occurred, is salutary, preventing hyperpermeability in endotoxic rats (Boughton-Smith et al., 1993c). The concept that selective inhibition of the inducible isoform is beneficial has gained further support from endotoxic shock studies correlating iNOS-selective inhibition with reductions in intestinal mucosal hyperpermeability (Mitchell Fink; personal communication). In vitro studies using Caco-2BBe monolayers, a model which avoids confounding effects of NO on mesenteric perfusion and neutrophil adhesion, confirm that exogenous administration of authentic NO or NO donors diminishes ATP, dilates tight junctions, and induces hyperpermeability to hydrophilic macromolecules in a cGMP-independent manner (Salzman et al., 1994a). Endogenous NO production, stimulated by exposure to IFN-γ, has also been shown to play a pivotal role in the increase in permeability of Caco-2BBe monolayers (Unno et al., 1995). NO-mediated hyperpermeability is associated with a disruption of cytoskeletal architecture, including a reduction in fluorescein-phalloidin staining of junctional actin (Salzman et al., 1994a). The mechanism of cytoskeletal alteration is likely to be secondary to an NOmediated reduction in cellular ATP (Salzman et al., 1994a). In addition to its independent toxicity, NO also contributes to peroxynite formation and the consequent activation of the PARS pathway. Exposure of cultured Caco-2BBe cell monolayers to SIN-1, which generates peroxynitrite in situ, induces DNA strand breaks, energetic depletion, and paracellular hyperpermeability (Kennedy et al., 1998). Inhibition of PARS activity partially restores NAD+, ATP, and paracellular permeability, demonstrating that peroxynitrite triggered barrier function is mediated, in part, by the creation of DNA strand breaks and the activation of the PARS pathway. In vivo demonstration that the PARS pathway mediates tissue and organ dysfunction has been provided in various experimental models of inflammatory injury, in which PARS inhibitors are protective (Szabó et al., 1996b; Zingarelli et al., 1996). Intestinal structure and mucosal barrier function are preserved by in vivo inhibition of PARS in various experimental models of intestinal shock and inflammation, including splanchnic occlusion and reperfusion (Cuzzocrea et al., 1997). Since it is well-established that impaired intracellular energetics are associated with a loss of intestinal epithelial barrier function (Salzman et al., 1994a), it is probable that PARS inhibitors block the development of hyperpermeability by preventing ATP depletion. In contrast, NOmediated in vitro barrier failure, although associated with a reduction in ATP concentration, does not result in a reduction in NAD+, nor in an increase in DNA strand breaks and PARS activity (Kennedy et al., 1998). Inhibition of PARS activity, as expected, does not reverse NO-mediated barrier dysfuction in in vitro systems (Kennedy et al., 1998). Since shock of all etiologies is associated with tissue acidosis and hypoxia, altered pH and oxygen tension may be key clinically relevant determinants of the toxicity of gut free radical and oxidant production. As mentioned above, in vitro aconitase inactivation by NO is greatly potentiated by low oxygen tension and reduced pH. In addition, lowered pH favors the conversion of peroxynitrite to the more toxic species peroxynitrous acid. Hyperpermeability of cultured intestinal epithelial cell monolayers is

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exacerbated by incubation with NO and peroxynitrite donors under mildly acidotic conditions (Kennedy et al., 1998). No studies have been reported examining the effect of hypoxia on NO or peroxynitritemediated barrier injury. NO deficiency states, as occur in the early phase of partial and total splanchnic ischemia, have also been associated with the loss of intestinal barrier function at the level of the mesenteric microvasculature and epithelial mucosa (Salzman et al., 1994b, 1993; Kubes and Granger, 1992; Kubes, 1993; Kubes, 1992; Kanwar et al., 1994a), although some studies have failed to confirm this association (Laszlo et al., 1994). Exogenous repletion of NO ameliorates splanchnic ischemia induced hyperpermeability (Kubes, 1994), independent of vascular perfusion (Payne and Kubes, 1993). Conversely, NOS inhibitors exacerbate barrier dysfunction induced by splanchnic ischemia-reperfusion injury (Kubes, 1994). Supplemental NO reduces injury in several models of gut injury, including 1) caustic injury from ethanol (Kiraly et al., 1993; Lambrecht et al., 1993), mineral acid (Matsueda et al., 1993), or bile-acid (Lifrak et al., 1993), 2) ischemia (Caplan et al., 1994), 3) ischemia/reperfusion injury (Kubes, 1994), and 4) early endotoxin-induced damage (Boughton-Smith et al., 1994). The beneficial action of NO in these conditions may be related to its augmentation of mesenteric perfusion, resulting from NO-mediated guanylyl cyclase activation, but increased blood flow does not completely account for all of the protective actions cited above (Payne and Kubes, 1993). A likely mechanism for the salutary effect of exogenous NO in these conditions may be related to its inhibitory effect on leukocyte adhesion (Kubes, 1992), platelet activation (Geiger et al., 1992), and mast cell activation (Kanwar et al., 1994b). For example, NO downregulates the surface expression of CD11/CD18 (Kubes et al., 1991; Miller et al., 1994) and non-specific NOS inhibition augments leukocyte adhesion and tissue infiltration in the postcapillary venules of the rat mesentery and mucosa (Arndt et al., 1993; Kurose et al., 1994; Miller et al., 1994). Indeed, long-term inhibition of the endothelial NOS isoform, cNOS, via the oral administration of the substituted arginine derivative L-NAME, produces ileitis characterized by a marked mucosal infiltration by granulocytes (Miller et al., 1994). Similarly, endogenous NO deficiency, which occurs during the early phase of splanchnic ischemiareperfusion (Kanwar et al., 1994a), is associated with an increase in neutrophil infiltration into the intestinal mucosa. Exogenous supplementation of NO prevents mucosal leukocyte infiltration in this setting (Andrews et al., 1994). Thus, insufficient NO production may have pro-inflammatory effects via the consequent activation of mast cells, platelets, and neutrophils. Excessive NO may also be injurious via its direct actions as a cytotoxic effector species or in combination with oxygen centered free radicals to produce the highly toxic reaction product peroxynitrite. Similarly, the pharmacologic manipulation of tissue NO levels in the gut during shock is subject to toxicity from either excessive supplementation of NO or excessive blockade of NO synthesis. Given that the degree of NO production is related to the phase of shock and other proinflammatory signals and that the toxicity of NO is highly dependent upon ambient conditions of pH and oxygen tension, it may be clinically difficult to guage the requirement for NOS inhibition or NO supplementation during the rapid evolution of circulatory shock. PHARMACOLOGICAL MODULATION OF GASTROINTESTINAL FUNCTION VIA INFLUENCING NITRIC OXIDE HOMEOSTASIS Enteral Nitric Oxide Replacement Therapy Application of exogenous NO allows for regional alleviation of pathologic vasoconstriction, when delivered directly into a hollow viscus, such as the lung (Nelin et al., 1994; Etches et al., 1994). Therapeutic use of eraterally-administered soluble NO donors has not yet been described but may be expected to induce a

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local mucosal hyperemia in the gut, without systemic hypotension. This approach could be useful in the treatment of various forms of mesenteric hypoperfusion by inducing a gastrointestinal steal phenomenon. NO has been positively implicated in wound healing in colonie lesions; thus, it would be not unexpected to find that local admininstration of enteral NO donors might be an effective therapeutic for gastrointestinal ulcers. During hypodynamic forms of circulatory shock, mesenteric perfusion is significantly compromised. In this setting, enteral NO donors could promote better mesenteric perfusion. Ideally, such agents could be added to enteral formulas used in circulatory shock in order to pharmacologically manipulate post-prandial hyperemia and facilitate successful enteral feeding. The use of soluble NO donors for regional vasodilitation has been complicated by systemic uptake and hypotension. Recently, we developed a series of novel substituted NO donors of the NONOATE class which are restricted from transepithelial flux on the basis of a charged amino side-group (Brilli et al., 1997). These compounds relieve pulmonary hypertension when given by an inhaled route, without an effect on systemic blood pressure or systemic vascular resistance. Current studies are underway to establish the role of these compounds as enteral supplements in the preservation of gut mucosal blood flow in shock states. The potential use of soluble NO donors raises important issues as to the toxicity of NO, alone and in combination with superoxide anion (Robbins et al., 1995). The basis for NOmediated toxicity is multifactorial and it is presently unclear which mechanisms may be clinically relevant: First, NO may interact with the iron center in hemoglobin, altering its oxidation state to form methemoglobin, a species which is unable to transport oxygen (Lonqvist et al., 1994). In the presence of adequate levels of erythrocytic methemoglobin reductase, exogenous administration of NO rarely results in a level of methemoglobin in excess of several percent and is well tolerated (Robbins et al., 1995). Second, NO may participate in deaminative reactions with DNA, potentially acting as a mutagen (Robbins et al., 1995). There have been no long-term follow-up in vivo studies of chronic NO exposure to exclude this possibility. Clearly, before enterally administered NO can be considered as a chronic therapy there would need to be great attention given to issues of genetic injury. Third, NO is a known inhibitor of platelet activation, via its elevation of intracellular cGMP concentration (Venturini et al., 1992); thus, hemorrhage is a potentially important toxicity. Fourth, NO in combination with superoxide anion NO forms a potent oxidant, peroxynitrite. In light of the many potential toxicities of exogenous NO delivery, several approaches are now under development to improve the therapeutic ratio of this approach (Ichinose et al., 1995). Ichinose et al., have observed that Zaprinast, an inhibitor of the phosphodiesterase which breaks down cGMP, prolongs the action of NO, suggesting that lower concentrations of NO may be equally efficacious as regional vasodilators (Ichinose et al, 1995). Adjunctive therapies which bolster endogenous anti-oxidant defenses in the gut may also have a future role as prophylaxis for enteral NO therapy. Selective Inhibition of Nitric Oxide Synthase Isoforms The use of iNOS-selective inhibitory compounds for the prevention of gastrointestinal dysfunction in circulatory shock may have clinical utility, as suggested by the salutary effects of isoform-selective agents in rodent models of endotoxic shock (Southan and Szabo, 1996). A variety of novel potent iNOS selective compounds are currently under development and moving toward clinical trials for the treatment of various inflammatory conditions, including septic shock. A non-selective NOS inhibitor, L-NMA, is currently in a Phase 3 pivotal trial for septic shock; its clinical impact on gastrointestinal function in shock is uknown. Concerns with a non-selective approach are warranted, however, given the importance of constitutive NOS activity to the maintenance of mesenteric perfusion, microvascular patency, peristalsis, electrolyte secretion, and mucosal production of mucus (Szabó and Thiemermann, 1994b). Generalized inhibition of

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all of these salutary functions of constitutively derived NO may have clinically relevant side-effects and bears close examination. The generation of peroxynitrite in the early phase of circulatory shock, prior to the upregulation of iNOS, illustrates the potential importance of constitutively-derived NO to the pathophyisology of shock and complicates therapeutic approaches based on iNOSselective inhibition. Indeed, the relative pathologic roles of cNOS and iNOS derived NO in shock are currently unclear. If cNOS-derived NO does play a critical pathologic role in organ injury in circulatory shock, and is not merely a pathologically irrelevant epiphenomenon, then approaches which rely upon NOS inhibition may be of limited benefit. iNOSselective inhibitors would be expected, in this instance, to provide only a partial protection and nonselective inhibitors might be unacceptably toxic, based upon their abrogation of all the myriad housekeeping functions of cNOS-derived NO. A more promising approach may be to combine iNOS-selective inhibition with a scavenger of peroxynitrite, thereby neutralizing the toxic effects of peroxynitrite generated by either NOS isoform. We have recently reported the discovery of a novel agent, mercaptoethylguanidine (MEG), which acts in both these capacities (Szabo et al., 1997), functioning as a potent and selective iNOS inhibitor and a potent scavenger of peroxynitrite. We have obtained preliminary data demonstrating that MEG protects intestinal mucosal barrier function in splanchnic oclusion-reperfusion models in rodents; its effect on gut permeability in experimental models of endotoxic shock has not yet been evaluated. Inhibition Of iNOS Expression Enzymatic inhibition of iNOS activity may be advantageous in circulatory shock, since the signal transduction cascade resulting in free radical formation is generally far advanced at the time of clinical presentation and not amenable to cytokine-inhibitor based therapies. Nonetheless, there are clinical situations in which a persistent stimulation of cytokine synthesis is present, usually in the setting of an undrained infection or non-viable tissue. In these instances it may be useful to consider a therapeutic approach which blocks the intracellular signal transduction of iNOS expression. Several targets have now been identified which lend themselves to pharmacologic manipulation. Steroid pre-treatment inhibits iNOS induction potently in certain cell types, such as rodent macrophages (Szabó, 1995), rat aortic smooth muscle (Rees et al., 1990), human megakaryocytes (Lelchuk et al., 1992), and human mesangial cells (Nicolson et al., 1993), but weakly in others, such as human chondrocytes (Palmer et al., 1993). In contrast, in intestinal epithelial cells dexamethasone only slightly reduces the level of iNOS mRNA in the early phase of cytokine induction and has no effect on total NO production or iNOS activity over a longer period of cytokine stimulation (Salzman et al., 1995). These celltype specific features highlight the importance of evaluating the regulation of NO synthesis at multiple levels, including signal transduction, transcription, enzyme synthesis, and enzymatic activity. Additional therapeutic choke-points at the transciptional level which may be considered include the blockade of NF-κB nuclear translocation, tyrosine kinase activation, and IRF-1 upregulation. As with steroids, each of these other non-specific strategies is likely to interfere with the regulation of unrelated genes and produce unacceptable toxicity. FUTURE TRENDS The future introduction of novel NOS inhibitors, NO scavengers, regionally-specific NO donors, isoformselective inhibitors of iNOS expression, and specific inhibitors of iNOS transcription will allow for a more

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precise manipulation of tissue NO levels in the gut during shock. The selection of agents will depend in part on the relative toxicities of targeting a particular isoform and will be influenced to a large degree by issues of timing and the status of the underlying inflammatory process. The importance of peroxynitrite, as a toxic byproduct of NO, is increasingly appreciated and will lead to the generation of novel scavenging agents to protect the intestinal mucosa. Singular inhibition of iNOS is unlikely to be of significant benefit, given the production of peroxynitrite from cNOS-derived isoforms. The ideal agent(s) of the future for the treatment of gut injury in shock will need to simultaneously 1) selectively inhibit iNOS-derived NO, thereby decreasing the direct toxicity of NO and reducing the formation of peroxyntrite, 2) provide a low level of supplemental NO in conditions of NO deficiency, and 3) scavenge residual peroxynitrite. A diagnostic means of non-invasively monitoring gut NO production in real time would be a considerable advantage in rationally treating NO-mediated enteric disease during shock. CONCLUSIONS NO has an established role as a regulator of gastrointestinal physiology. Alone and in combination with oxygen-centered free radicals, NO also represents an important pathophysiological mediator, contributing to inflammation in various forms of circulatory shock. Until recently, the interest in the biology of NO has been confined to the realm of basic investigation. With the advent of novel delivery systems to provide exogenous NO and the discovery of isoform-selective NOS inhibitors to reduce NO formation, there are now new therapeutic means to modulate mesenteric vascular resistance and intestinal mucosal inflammation. Recent interest in the interaction of NO and oxygen-centered free radicals has resulted in greater appreciation of the important pathologic role played by peroxynitrite. The centrality of peroxynitritemediated injury, and in particular its activation of poly (ADP-ribose) synthetase via the induction of DNA strand breaks, has broad implications for novel therapeutic approaches to reduce bowel injury in shock. Application of peroxynitrite scavengers and PARS inhibitors may be salutary in various forms of circulatory shock and is currently the subject of intense investigation in experimental models of splanchnic ischemiareperfusion injury and endotoxic shock. REFERENCES Abu-Soud, H. and Stuehr, D.J. (1993) Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer. Proceedings of the National Academy of Science, 90, 10769–10772. Adler, K., Fischer, B., Li, H., Choe, N. and Wright, D. (1995) Hypersecretion of mucin in response to inflammatory mediatros by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase. American Journal of Respiratory Cellular Molecular Biology, 13, 526–530. Akerfeldt, S. (1959) Acta Chemica Scandanavia, 13, 1479–1480. Allescher, H.D., Kurjak, M., Huber, A., Trudrung, P. and Schusdziarra, V. (1996) Regulation of VIP release from rat enteric nerve terminals: evidence for a stimulatory effect of NO. American Journal of Physiology, 271, G568–G574. Andrews, F.J., Malcontenti-Wilson, C. and O’Brien, P.E. (1994) Protection against gastric ischemia-reperfusion injury by nitric oxide generation. Digestive Diseases Science, 39, 366–373. Angel, P. and Karin, M. (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochimica et Biophysica Acta, 1072, 129–157. Arndt, H., Russell, J.B., Kurose, L, Kubes, P. and Granger, D.N. (1993) Mediators of leukocyte adhesion in rat mesenteric venules elicited by inhibition of nitric oxide synthesis. Gastroenterology, 105, 675–680.

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Salzman, A.L., Eaves-Pyles, T, Linn, S.C., Denenberg, A.G., Morelli, P.J. and Szabó, C. (1997) Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells. Gastroenterology, 114, 93–102. Salzman, A.L., Bernstein, T. and Szabó, C. (1997a) Maturation of intestinal epithelial cells regulates the inducibility of human iNOS expression. Japanese Journal of Pharmacology, 75(5), 99p. Salzman, A.L., Denenberg, A. and Szabó, C. (1997b) Degradation of IκB isoforms in IL-1 stimulated human intestinal epithelial cells is not redox sensitive. Critical Care Medicine, 25, A69. Sarih, M., Souvannavong, V. and Adam, A. (1993) Nitric oxide synthase induces macrophage death by apoptosis. Biochemical and Biophysical Research Communications, 191, 503–508. Sautebin, L. and DiRosa, M. (1994) Nitric oxide modulates prostacyclin biosynthesis in the lung ofendotoxintreated rats. European Journal of Pharmacology, 262, 193–196. Schini, V.B., Durante, W., Elizondo, E., Scott-Burden, T., Junquero, D.C., Schafer, A.I. et al. (1992) The induction of nitric oxide synthase activity is inhibited by TGF-β, PDGFAB and PDGBB in vascular smooth muscle cells. European Journal of Pharmacology, 216, 379–383. Schraufstatter, I.U., Hinshaw, D.B., Hyslop, P.A., Spragg, R.G. and Cochrane, C.G. (1986) Oxidant injury of cells: DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide. Journal of Clinical Investigation, 77, 1312–1320. Shepard, A. and Kiel, J. (1992) A model of countercurrent shunting in the intestinal villus. American Journal of Physiology, 262, H1136–ZH1142. Sherman, P.A., Laubach, V.E., Reep, B.R. and Wood, E.R. (1993) Purification and cDNA sequency of an inducible nitric oxide synthase from a human tumor cell line. Biochemistry, 32, 11600–11605. Slivka, A., Stamler, J., Chuttani, R., Carr-Locke, D. and Loscalzo, J. (1993) Inhibition of sphincter of Oddi function by the NO carrier S-nitroso-N-acetlycysteine in rabbits ex vivo and in humans during ECRP . Gastroenterology, 104, A378 (Abstract). Southan, G.J. and Szabó, C.S. (1996) Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochemical Pharmacology, 51, 383–394. Spink, J., Cohen, J. and Evans, T. (1995) The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase. Journal of Biological Chemistry, 270, 29541–29547. Stadler, J., Harbrecht, B.C., DiSilvio, M., Curran, R.D., Jordan, M.L., Simmons, R.L. et al. (1993) Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells. Journal of Leukocyte Biology, 53, 165–172. Stadler, J., Curran, R.D., Ochoa, J.B., Harbrecht, E.G., Hoffman, R.A., Simmons, R.L. et al. (1994) Effect of endogenous nitric oxide on mitochondrial respiration of rat hepatcytes in vitro and in vivo. Archives of Surgery, 126, 186–191. Stamler, J.S., Simon, D.I., Osborne, J.A., Mullins, M.E., Jaraki, O., Michel, T. et al. (1992) S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc. Natl Acad. Sci., 89, 444–448. Stark, M.E. and Szurszewski, J.H. (1992) Role of nitric oxide in gastrointestinal and hepatic function and disease. Gastroenterology, 103, 1928–1949. Strohmaier, W., Werner, E.R., Wachter, H., Redl, H. and Schlag, G. (1996) Pteridine and nitrite/nitrate formation in experimental septic and traumatic shock. Shock, 6, 254–258. Szabó, C. and Thiemermann, C. (1994) Invited opinion: role of nitric oxide in hemorrhagic, traumatic, and anaphylactic shock and in thermal injury. Shock, 2, 145–155. Szabó, C. (1995) Alterations in the production of nitric oxide in various forms of circulatory shock. New Horizons, 3, 3–32. Szabó, C. and Salzman, A.L. (1995) Endogenous peroxynitrite is involved in the inhibition of cellular respiration in immuno-stimulated J774.2 macrophages. Biochemical and Biophysical Research Communications, 209, 739–743. Szabó, C., Salzman, A.L. and Ischiropoulos, H. (1995) Peroxynitrite-mediated oxidation of dihydrorhodamine 123 occurs in early stages of endotoxic and hemorrhagtic shock and ischemia-reperfusion injury. Federation of European Biochemical Societies Letters, 372, 229–232.

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Szabó, C., Day, B.J. and Salzman, A.L. (1996) Evaluation of the relative contribution of nitric oxide and peroxynitrite to the suppression of mitochondrial respiration in immunostimulated macrophages, using a novel mesoporphyrin superoxide dismutase analog and peroxynitrite scanvenger. Federation of European Biochemical Societies Letters, 381, 82–86. Szabó, C., Zingarelli, B., O’Connor, M. and Salzman, A.L. (1996a) DNA strand breakage, activation of polyADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite. Proceedings of the National Academy of Science, 93, 1753– 1758. Szabó, C., Zingarelli, B. and Salzman, A.L. (1996b) Role of poly-ADP ribosyltransferase activation in the nitric oxideand peroxynitrite-induced vascular failure. Circulation Research, 78, 1051–1063. Szabó, C., Ferrer-Sueta, G., Zingarelli, B., Southan, G.J., Salzman, A.L. and Radi, R. (1997) Mercaptoethylguanidine and related guanidine nitric oxide synthase inhibitors react with peroxynitrite and protect against peroxynitriteinduced oxidative damage. Journal of Biological Chemistry, 272, 9030–9036. Takeuchi, K., Ohuchi, T., Miyake, H. and Okabe, S. (1994) Stimulation by nitric oxide synthase inhibitors of gastric and duodenal HCO3− secretion in rats. Journal of Pharmacology and Experimental Therapeutics, 266. 1512–1519. Tepperman, B.L., Brown, J.F. and Whittle, B.J. (1993) Nitric oxide synthase induction and intestinal epithelial cell viability in rats. American Journal of Physiology, 265, G214–G218. Tepperman, B.L., Brown, J.F., Korolkiewicz, R. and Whittle, B.R. (1994) Endotoxin challenge promotes neutrophilindependent nitric oxide synthase induction, injury, and cGMP formation in rat colonic epithelial cells. Gastroenterology, 106, A782 (Abstract). Thompson, J.A., Sadowska-Krowicka, H., Rossi, J., Clark, D.A. and Miller, M.J. (1994) Inducible nitric oxide synthase gene expression in guinea pig ileitis: a model of IBD prevented by aminoguanidine. Gastroenterology, 106, A782 (Abstract). Tobias, P.S., Soldau, K., Kline, L., Lee, J.D., Kato, K., Martin, T.P. et al. (1993) Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein. Journal of Immunology, 150, 3011–3021. Unno, N., Menconi, M.J., Smith, M. and Fink, M.P. (1995) Nitric oxide mediates interferon-γ-induced hyperpermeabiilty in cultured human intestinal epithelial monolayers. Critical Care Medicine, 23, 1170– 1176. Vallance, P., Collier, J. and Moncada, S. (1989) Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet, 997–999. Venturini, C.M., Weston, L.K. and Kaplan, J.E. (1992) Platelet cGMP, but not cAMP, inhibits thrombin-induced platelet adhesion to pulmonary vascular endothelium. American Journal of Physiology, 263 (2 pt 2), H606-H612. Villa, L.M., Salas, E., Darley-Usmar, M., Radomski, M.W. and Moncada, S. (1994) Peroxynitrite induces both vasodilation and impaired vascular relaxation in the isolated perfused rat heart. Proceedings of the National Academy of Science, 91, 12383–12387. Wang, Z.Q., Auer, B., Sting, L., Berghammer, H., Haidacher, D., Schweiger, M. et al (1995) Mice lacking ADPRT and poly (ADP-ribosylation) develop normally but are susceptible to skin disease. Genes and Development, 9, 510–520. Warner, R.L., Paine, R., Christensen, P.J., Marletta, M.A., Richards, M.K., Wilcoxen, S.E. et al. (1995) Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase. American Journal of Respiratory Cellular and Molecular Biology , 12, 649–661. Welsh, N., Eizirik, D.L., Bendtzen, K. and Sandler, S. (1991) Interleukin-1β-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase. Endocrinology, 129, 3167–3173. Wilson, K.T., Ciancio, M.J. and Chang, E.B. (1994) Inducible nitric oxide synthase mRNA expression is increased in intestinal mucosa of endotoxemic rats and is inhibited by dexamethasone. Gastroenterology, 106, A793 (Abstract). Wilson, K.T., Xie, Y., Musch, M.W. and Chang, B.C. (1994) Sodium nitroprusside stimulates anion secretion and inhibits sodium chloride absorption in rat colon. Journal of Pharmacology and Experimental Therapeutics, 266, 224–230. Wilson, L., Denenberg, A., Xue, V., Szabó, C. and Salzman, A.L. (1997) Proteolysis of I-kappa B is not required for the nuclear translocation of NF-kappa B: discovery of a novel pathway of p50/p65 activation. Shock, 7, 50.

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Wirthlin, D.J., Cullen, J.J., Spates, S.T., Conklin, J.L., Murray, J., Caropreso, D.K. et al. (1996) Gastrointestinal transit during endotoxemia: the role of nitric oxide. Journal of Surgical Research, 60, 307–311. Wizemann, T.M., Gardner, C.R., Laskin, J.D., Quinones, S., Durham, S.K., Goller, N.L. et al. (1994) Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia. Journal of Leukocyte Biology, 56(6), 759–768. Xie, Q., Whisnant, R. and Nathan, C. (1993) Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers indubibility by interferon gamma and bacterial lipopolysaccharide. J. Experimental Medicine, 177, 1779–1784. Xie, Q., Kashiwabara, Y. and Nathan, C. (1994) Role of transcription factor NF-κB/Rel in induction of nitric oxide synthase. Journal of Biological Chemistry, 269, 4705–4708. Yamada, T., Abell, C. and Grisham, M.B. (1993) Colonic hyperemia induced by bacterial cell wall polymers: potential role of nitric oxide. Gastroenterology, 104, A291 (Abstract) Yamamoto, K., Tsukidate, K. and Farber, J.L. (1993) Differing effects of the inhibition of poly(ADP-ribose) polymerase on the course of exidative cell injury in hepatocytes and fibroblasts. Biochemical Pharmacology, 46, 483–491. Yan, Z.Q., Hansson, G.K., Skoogh, B.E. and Lotvall, J.O. (1995) Induction of nitric oxide synthase in a model of allergic occupational asthma. Allergy, 50, 760–764. Zhang, J. and Snyder, S.H. (1992) Nitric oxide stimulates auto-ADP-ribosylation of glyceraldehyde-3-phsophate dehydrogenase. Proceedings of the National Academy of Science, 89, 9382–9385. Zingarelli, B., O’Connor, M., Wong, H., Salzman, A.L. and Szabó, C. (1996) Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolyusaccharide. Journal of Immunology, 156, 350–358. Zingarelli, B., Salzman, A.L. and Szabó, C. (1996) Protective effects of nicotinamide against nitric oxide mediated vascular failure in endotoxic shock: potential involvement of poly ADP ribosyl synthetase. Shock, 5, 258–264. Änggård, E. (1994) Nitric oxide: mediator, murderer, and medicine. Lancet, 343, 1199–1206.

Therapeutic Applications

25 Nitric Oxide Donors Joseph A.Hrabie1 and Larry K.Keefer2 1Chemical

Synthesis and Analysis Laboratory, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 USA

2

Chemistry Section, Laboratory of Comparative Carcinogenesis, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 USA

The historical basis for the study of NO donors is described followed by a presentation of the chemistry of the principal classes of NO-releasing compounds. Current uses as pharmaceuticals as well as selected pharmacological studies are presented for each group along with potential advantages and disadvantages. An analysis of the potential for future drug development based on NO release technology is presented with the conclusion that the S-nitrosothiols and the diazeniumdiolates offer excellent opportunities for developing future pharmaceutical agents. Key words: nitric oxide, prodrugs, NO donor molecules. HISTORICAL ROLES OF NITRIC OXIDE Any discussion of modern nitric oxide chemistry deserves to be preceded by a brief discussion of the scientific relevance of nitric oxide in the years before the physiological relevance of the molecule finally blossomed. Much of the recent work directed towards the development of medically useful nitric oxide donors relies on studies conducted for other purposes before such a tremendous biological role became evident and often with the mind-set that NO was not compatible with life.

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Nitric Oxide in Chemistry The most nearly ubiquitous role of NO in chemistry, learned by all in freshman chemistry but soon forgotten, is as an intermediate in the oxidation of ammonia to nitric acid and thence to fertilizer. This chemistry has not proven particularly useful in the design of NO donors. Much more pertinent chemistry has resulted from the second major traditional role of NO as the first step in the air pollution cascade frequently referred to as NOx chemistry. Considerable research into the reactions of metal nitrosyls has resulted from the study of the air pollution problem (Pandey, 1983). Although not as widely known, considerable knowledge of NO chemistry has resulted from its use as a chemical reagent. The synthesis of caprolactam by photolytic reaction of NO with cyclohexene is the basis of commercial production of nylon in some countries (McCleverty, 1979). The fact that NO could be developed as an alternative to sodium nitrite in diazonium salt-based chemistry was also an early reason to study the molecule (Brackman and Smit, 1966). Nitric Oxide in Biology: Villain and Friendly Killer Traditional studies of NO in biological systems centered on the negative effects of exposure, particularly from air pollution and tobacco smoke. Exposure to NO was considered to be a potential cause of cancer via the resulting formation of nitrosating agents (Williams, 1988). Metal complexes of NO were shown to nitrosate amines (Brackman and Smit, 1965). Ironically, concerns over the toxicity of NO by-products such as nitrite and nitrate encouraged the study of biological systems exhibiting elevated levels of these potential signals of increased cancer risk and thus led to the discovery that NO can be a friendly killer when employed by activated macrophages as part of the body’s immune response (Stuehr and Marletta, 1987). Nitric Oxide Donor Leads Provided by Established and Investigational Clinical Agents Long before the revolution in NO physiology many compounds now known and, at times in the past, speculated to act as pharmaceuticals via formation of NO had come into favor. While the details of the release of NO from each will be described later, their structures are presented in Figure 25–1 as typifying the leads that history provided in the search for new NO donors. Foremost among these were the clinical nitro vasodilator s nitroglycerin (GTN), amyl nitrite, isosorbide dinitrate and sodium nitroprusside (SNP). All are now known to act via NO formation. Additionally, the antibiotics alanosine (an investigational antineoplastic) and dopastin (an antihypertensive) as well as their chemical cousin cupferron had been shown (Alston et al., 1985) to release NO via enzymatic oxidation. Nitric Oxide Donor Leads Provided by Established Chemistry In addition to the biological roots discussed above, many of the new classes of NO donors to be discussed below have their basis in previously known chemistry. Thus, our own development of the diazeniumdiolates was initially linked to the first reports of a reaction between anaerobic NO and secondary amines (Drago and Paulik, 1960). Since we still find it instructive to turn back to these reports occasionally it is worth noting several potential chemical connections of the other NO donors.

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Similarities in the behavior of the organic nitrites and the S-nitrosothiols (thionitrites) were noted long ago (Oae et al., 1978) and both are used in organic synthesis to effect various types of nitrosation chemistry (Doyle et al., 1977, Oae and Shinhama, 1983). In like fashion, much of the reactivity that is involved in the case of oximes, furoxans and related compounds has been reviewed (Freeman, 1973). In fact, the possible interconversion of one of the newer proposed classes of NO donor, the 3,4-dihydrodiazete 1,2-dioxides, with the isomeric furoxans has been considered (Greene and Gilbert, 1975). Yet another interesting debate that has been ongoing for a long time in the chemical community is the role of nitroxyl (HNO as opposed to NO) in the reactions of Piloty’s acid (Smith and Hein, 1960), one of the sulfonated hydroxylamines described below. CLASSES OF NITRIC OXIDE DONOR COMPOUNDS The existence of a wide variety of NO donors is useful because each releases NO under a unique set of conditions depending upon the chemistry involved and each is thus applicable to a different pharmacological problem. Nitric oxide donors are available which require activation by reduction or oxidation and these two processes may be enzymatic or metal-mediated. Many NO donors which spontaneously decompose without the need for either form of activation are also available and are among the most promising candidates for drug development. Consideration of materials which produce potential NO derivatives such as nitroxyl or peroxynitrite is also required in exploring the full range of possible NO-based pharmaceuticals. Equally important is the range of by-products formed by all of the NO-releasing materials. In the ensuing sections an attempt is made to provide a discussion of each of these issues as they relate to each of the important classes of NO donors. Nitrate and Nitrite Esters Two of the most widely known nitrovasodilators fall into this classification. The use of nitroglycerin as a treatment for angina and the blood pressure effects of amyl nitrite, a modern-day drug of abuse, are probably among the most widely known tidbits of medical knowledge possessed by the general public. Nitroglycerin must be activated by a three electron reduction during its metabolic conversion to NO (Murad, 1990) as shown in equation 1. It is not possible to specify an exact sequence of physiological events leading to NO generation because multiple pathways are known both enzymatic and nonenzymatic. Among the systems studied in detail, various thiols appear to be the source of the reducing power (being oxidized to disulfides, RSSR, in the process) (Keen et al, 1976). Irradiation of nitrite esters with UV light can also lead to the release of some nitric oxide (Barton et al., 1979).

The major disadvantage to the use of nitrate esters such as nitroglycerin is that patients develop a tolerance to the drug requiring increasingly larger doses to achieve the desired pharmacological effect. Various mechanisms have been proposed to explain nitrate tolerance including enhanced vascular production of

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Figure 25±1. Historical (pre-1987) potential NO donors

superoxide ion (O2–) (Münzel et al., 1995), which is known to react instantly with NO thus destroying it (Koppenol et al., 1992), and exhaustion of the enzyme-cofactor (thiol) system which performs the reduction shown in equation 1 (Chung and Fung, 1990). Although neither of these explanations is likely to represent a complete picture of this complex physiology, consideration of the thiol depletion problem has led to the development of a new type of nitrate ester which exhibits reduced tolerance development by incorporating the necessary thiol groups in the same molecule as the nitrate (Kojda et al., 1995).

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Amyl nitrite is an example of an NO-generating pharmaceutical administered by inhalation. Release of NO occurs via reaction with thiols (Ignarro et al., 1981) to form S-nitrosothiols (see later section). It is considered unlikely that many major new advances in pharmacology will arise from the nitrate/nitrite class of NO donors. Although their supply of NO is rapidly bioavailable, they are easily deactivated by hydrolysis to the parent alcohol and nitrate/nitrite ion. Additionally, it is known from organic chemistry that these compounds can produce complex mixtures of potentially harmful products such as carcinogenic nitrosamines in vivo via nitrosation and transnitrosation (Doyle et al., 1983). Such chemistry can render even the most carefully planned pharmaceutical useless as a site-specific agent and produce undesirable systemic consequences. Sodium Nitroprusside and Other Metal Nitrosyls Sodium nitroprusside (SNP, Figure 25–1) is often used in crisis situations to stabilize patients with acute systemic hypertension. Intravenous administration provides such an immediate infusion of NO that the blood pressure can be reliably controlled by adjusting the infusion rate. This is presumably a result of the fact that a simple one-electron reduction can produce NO release and hemoglobin is an excellent oneelectron reducing agent. There is evidence that the loss of one cyanide ligand precedes NO release (Bates et al., 1991) and that a membrane-based enzyme may be involved in biological tissues (Kowaluk et al., 1992). SNP can also release NO photochemically (Arnold et al., 1984). Similar nitrosyl complexes of other transition metals form easily. The bound NO in these complexes may be formally NO– or nitrosonium (NO+) as well as neutral. A good review of these reactions exists (Fontecave and Pierre, 1994). Of particular interest among these are the ruthenium nitrosyl chlorides referred to as “caged nitric oxide” because they release NO on photolysis (Bettache et al., 1996). The cited review also provides references to interesting research suggesting a role for formation of iron nitrosyls in hemoproteins in their activation. One interesting naturally occurring analog of these compounds is a salivary hemoprotein nitrosyl used by a bloodsucking insect to promote optimal blood flow while feeding (Ribeiro et al., 1993). Related polynitrosyl iron-sulfur clusters containing the anions [Fe2S2(NO)4]2– and [Fe2S3(NO)7]–, referred to as Roussin’s red and black salts, have been used in research. The extent of NO release from these complexes has been reported to vary with the light intensity in the room (Matthews et al., 1994) and so these are of limited value although their photochemistry may be exploited to induce radiation sensitization of hypoxic tumors (Bourassa et al., 1997). Sydnonimines and Related Compounds Molsidomine (Figure 25–2) is in clinical use as an antianginal drug and is the best known of the sydnonimines whose NO release is attributed to the “N-NO” element grouping. Unfortunately, the oxidative activation which produces NO release also produces a stoichiometric amount of superoxide (O2–) which can react with the NO to form peroxynitrite (ONOO–), a strong oxidant capable of doing extensive damage (Koppenol et al., 1992). A series of analogs which some claim release less superoxide are compounds containing an additional nitrogen in the heterocyclic ring, the oxatriazoles (Figure 25–2). The compounds where R is an aryl ring have been shown to be many times more potent as antiplatelet agents than molsidomine (Kankaanranta et al., 1996). Production of nitrous oxide upon photolysis has been reported (Bjerre et al., 1979) which would tend to indicate the formation of some nitroxyl (NO–) ions.

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Figure 25±2. NO-releasing heterocyclic rings.

The intermediacy of SIN-1A in the NO release of molsidomine would suggest that other N-nitroso compounds may be NO donors. Unfortunately, many of these materials (such as most nitrosamines and Nnitrosoureas) are powerful carcinogens but it should be noted that they have been shown to elicit some NOlike activity. The effect of streptozotocin (a derivative of N-methyl-N-nitrosourea) on vasodilation (Thomas and Ramwell, 1989a) is most likely attributable to NO (Kröncke and Kolb-Bachofen, 1996). Certain bis(Nnitrosoamino)benzenes have been shown to release NO more efficiently on photolysis than the metal nitrosyls mentioned above (Namiki et al., 1997).

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Inorganic Salts Sodium nitrite (NaNOa) is a classical nitrosating agent used to form diazonium salts in organic synthesis. Nitrite is also the chief decomposition product of NO in oxygenated solutions (Wink et al., 1993). However, acidification of nitrite-containing solutions produces NO gas via disproportionation of nitrous acid (HNO2, Schwarz-Bergkampf, 1972). This process is also accompanied by the formation of nitrogen dioxide (NO2) and nitrosating agents and is thus not useful medicinally. NO is also generated on the skin surface via bacterial reduction of sweat nitrate, possibly as a way to combat infection (Weller et al., 1996). Irradiation of nitrite anions with UV light can form NO in the absence of oxygen or acid (Reszka et al., 1996). Similarly, peroxynitrite (ONOO–), the product of NO deactivation by superoxide (O2–), can behave as a secondary source of NO via nitrosation of alcohols, amines or thiols present in vivo. One interesting recent discovery is that the hydroxyl groups of sugars such as glucose react with peroxynitrite to form NOreleasing compounds (Moro et al., 1995). Among the other inorganic compounds that have been shown to form some NO are azide ion (N3–) and hydroxylamine (H2NOH). Both of these are oxidatively metabolized by catalase/H2O2 or various peroxidases (Markert et al., 1994). These reactions are not sufficiently well defined (and, in fact, probably involve the formation of nitroxyl as well as NO) to recommend these materials as specific NO donors although hydroxylamine has been used as a vasorelaxing agent in a pharmacological research setting (Mian and Martin, 1995). Furoxans and Related Compounds Furoxans (Figure 25–3) are nitrovasodilators known to evolve NO in the presence of thiols such as glutathione or cysteine in proteins (Feelisch et al., 1992). Note that nitroxyl (NO–) is also formed in this reaction. Furoxans can also be made to fragment chemically in the gas phase via ring opening (Hwang et al., 1995). The bisoximes formed in furoxan deactivation are actually possible precursors to the cyclic azo dioxides (Singh, 1975), the diazete 1,2-dioxide analogs of which are known to undergo thermal reversion to NO as shown in Figure 25–3 (Severina et al., 1993). Most of these materials, as well as related ones prepared by nitrosation of certain α,β-unsaturated oximes at the beta carbon (Freeman and Gannon, 1969), have not been investigated as NO donors. Oximes A few simple oximes (R2C=NOH) exhibit weak vasoactivity (Thomas and Ramwell, 1989b) but the useful NO-donating oximes are more complex. Derivatives of FK-409 (Figure 25–4), first isolated from a fermentation broth (Hino et al., 1989), decompose spontaneously to NO at pH 7.4 at rates which vary with the substitution pattern. These compounds have been commercialized under the trademark “NORs.” Deprotonation to generate a conjugated system is probably the first step toward NO release (Kita et al., 1995) which explains the stability in acidic solutions. Unfortunately, one full equivalent of NO is not released and formation of some nitroxyl is believed to be occurring (Décout et al., 1995). Recently, a number of quinuclidin-3-one oximes (Figure 25–4) have been reported to release NO. These incorporate a different type of conjugated system than FK-409 and require oxidation for activation (Koikov et al., 1996).

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Figure 25±3. Additional NO-releasing heterocyclic rings.

Since N-hydroxy-L-arginine (Figure 25–4) is an intermediate in the enzymatic conversion of L-arginine to NO, one might expect such N-hydroxyguanidines to produce NO on oxidation but peracid oxidation of a model hydroxyguanidine has been reported to produce nitroxyl rather than NO (Fukuto et al., 1993b). NADPH-dependent oxidation of hydroxyarginine to NO as carried out by the nitric oxide synthases (NOSs) can, of course, be used as a source of NO (Pufahl and Marletta, 1993). Sulfonated Hydroxylamines and Other NO± Donors As mentioned previously, nitroxyl (NO–) can mimic the pharmacological effects of NO. In fact, NO is the one-electron oxidation product of NO– and can be formed from it in biological systems (Fukuto et al., 1993a). Thus, while NO– can be rapidly inactivated by dimerization followed by dehydration to produce nitrous oxide, if formed in low concentrations it can be a useful source of NO. Piloty’s acid (Figure 25–5) and its N,O-diacylated derivatives can release NO– by simple hydrolysis (Fukuto et al., 1992) but there is some indication that they can also release NO oxidatively (Zamora et al., 1995). Cyanamide (H2NCN) is known to form NO– by catalase/hydrogen peroxide oxidation (Nagasawa et al., 1990). Angeli’s salt (Na2N2O3) is known to decompose to NO– anaerobically but also forms NO in acidic solution (Bazylinski and Hollocher, 1985) and at high dilution in neutral buffer (Doyle and Mahapatro, 1984; Maragos et al., 1991). Reaction with one-electron oxidants such as ferricyanide has been shown to result in increased NO production (Zamora et al., 1995).

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Figure 25±4. Oxime NO donors.

Figure 25±5. Sulfonated hydroxylamines.

S-Nitrosothiols (Thionitrites) The S-nitrosothiols are sulfur analogs of the alkyl nitrites. Due to the widespread occurrence of the amino acid cysteine in proteins and small bioactive peptides this is a very promising group of compounds and has already proven to be fertile ground for research in NO physiology. Much of the interesting nature of this functional group originates in the duality of both the process for forming the S-NO bond and the process by which it is broken. Nitrosothiols may be formed by the traditional nitrosation reactions of organic chemistry ( ) but, more interestingly from a physiological point of view, they form directly from the reaction of NO with thiols in the presence of oneelectron oxidants (Butler and Williams, 1993). Conversely, NO can be released from these compounds by spontaneous dissociation (2RSNO→ 2RS+2NO; 2RS-→ RSSR) or by the action of one electron reductants (to regenerate the original thiol). In this case, both methods of NO regeneration are of potential significance to medicinal chemists since cleavage to

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NO has been shown to be effected by both iron and copper ions (for a review see Williams, 1996) and heterolytic cleavage with transfer of both NO+ and NO– has been demonstrated (Arnelle and Stamler, 1995). The interconversion of nitrosothiols and NO occurs so readily that it is believed to play a key role in the in vivo storage, transport and activity of what would otherwise be a far too reactive free radical to serve such diverse roles in bioregulation as described in Volume 1 (for a review see Stamler, 1995). In fact, several nitrosothiols have been identified as normal constituents of human physiological fluids (Gaston et al., 1993), including S-nitrosocysteine, S-nitrosoglutathione and S-nitrosoalbumin. As mentioned above in the relevant sections, nitrosothiols have been shown to be involved as active intermediates in the NO donation of several of the other classes of compounds covered in this chapter (Ignarro et al., 1981). Intensive drug development efforts are underway to capitalize on the many favorable attributes of the Snitrosothiols. These efforts have resulted in the preparation of S-nitrosoalbumin derivatives (Stamler et al., 1992b) and S,S’-dinitroso penicillamine dipeptides (Moynihan and Roberts, 1994). Several potential pharmaceutical in which the potential benefits of NO have been added to materials with other mechanisms of action have also been prepared. Examples of these are S-nitrosylated tissue plasminogen activator (Stamler et al., 1992a) and S-nitrosocaptopril (an angiotensin converting enzyme inhibitor; Nakae et al., 1995). Many advances in drug design can be expected from work with these and other S-nitrosothiols. Diazeniumdiolates (N2O2– Compounds) Our own work with NO donors has centered upon compounds containing the anionic N2O2− functional group and derivatives thereof (Figure 25–6). The possible pharmaceutical uses of these materials were first reported in 1991 (Vanin et al., 1991, Maragos et al., 1991) and many have now become commercially available. These materials have shown numerous advantages in pharmacological research and drug development (see below) but a single simple name for the compounds is not one of those advantages. The diazeniumdiolates are structurally related to the N-nitrosohydroxylamines, are often designated by the trivial (as organic chemists use the word) name “NONOates” and are sold by certain laboratory supply houses under the trade name “NOCs.” The correct full chemical designation is as 1-substituted diazen-1ium-1,2-diolates. Yet another designation for these materials is to describe their mode of synthesis as nucleophile/NO adducts. By far the most important examples to date have been those where the nucleophile residue is a secondary amine. They are easily prepared merely by exposing the free amine to NO gas ( )and the process is spontaneously reversible when they are dissolved in water or other protonating media ( ). This requirement for a proton source to regenerate NO means that the diazeniumdiolates are stable for prolonged periods in alkaline environments and this fact has been used to considerable advantage in the research setting (Keefer et al., 1996a; Hanson et al., 1995). The simplicity of this system led us to an equally simple method for naming the resulting NO donors for convenience in communication. As can be seen in Figure 25–6, the compounds are named by interposing a virgule between a suitable abbreviation for the amine precursor and the letters NO. The reaction of polyamines with NO has been used to produce a large number of zwitterionic NO donors which all produce NO with first-order kinetics and half lives at pH 7.4 and 37°C ranging from one minute to one day (Hrabie et al., 1993). Thus, by varying the amine structure, diazeniumdiolates with time-release profiles suitable for most research studies can be prepared. When a virtually instantaneous release of NO is required, the amino acid L-proline can be reacted with NO in methanolic sodium methoxide to yield

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Figure 25±6. Selected diazeniumdiolates.

PROLI/ NO (half-life 2 sec; Figure 25–6) (Saavedra et al., 1996). Since these synthesis reactions are conducted in organic solvents, materials that are not water soluble may also be derivatized with the N2O2– group and this may be extended to the preparation of NO-releasing polymers (Smith et al., 1996). As with the S-nitrosothiols above, the fact that these compounds are in reality derivatives of other molecules presents the interesting possibility of converting known pharmaceuticals into NO donors to achieve a double-barrel action.

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One of the greatest advantages of the diazeniumdiolates over all the other NO donors is that they lend themselves readily to prodrug development via alkylation at the terminal oxygen to produce stable Oalkylated derivatives (Figure 25–6, Saavedra et al., 1992). These prodrugs are stable even under acidic conditions and may thus be useful as oral dosage forms. Selective activation of these compounds by localized enzymatic activity may provide an excellent method for targeting NO release to a specific site in vivo (Saavedra et al., 1997). Similarly, derivatives of these compounds may be prepared by coordination of the bidentate N2O2– ligand with a transition metal (Christodoulou et al., 1993). The ability to “protect” the N2O2– functional group also makes it possible to conjugate a potential nitric oxide releasing molecule to a small peptide or protein (Keefer et al., 1996b) and adds great versatility to the organic synthetic chemistry of these molecules. Extensive pharmacological testing of the diazeniumdiolates is underway. The shortacting compounds have been found to have a number of beneficial cardiovascular effects (Zhang et al., 1996) including the inhibition of acute stent thrombosis (Kaul et al., 1996). Longer-acting materials have been shown to inhibit vascular smooth muscle cell proliferation (and thus perhaps restenosis; Mooradian et al., 1995). FUTURE COURSES OF DRUG DEVELOPMENT WITH NO DONORS The compounds which have the most potential for future development are in our view those which are based on straightforward derivatization applicable to a broad range of organic molecules and compatible with a large number of organic functional groups. While the serendipitous discovery of a single pharmaceutical in any of the NO donor classes described in this chapter is possible, modern drug development frowns on such approaches. We believe that the chemical versatility of the S-nitrosothiols provides an excellent opportunity for control of the NO donation process. Additionally, since these are naturallyoccurring substances, research data collected in the normal course of studies in biology will be available to aid the effort. The diazeniumdiolates also appear to offer excellent possibilities for rational drug design. Although (for the most part) these are not natural materials, the procedures used in their formation are gentle and their mode of action is uncomplicated by direct metabolism of the anionic diazeniumdiolate functional group. The exploitation of both the S-nitrosothiols and the diazeniumdiolates as pharmaceuticals will undoubtedly rely on a systematic manipulation of their structures to achieve both specificity of action and potency. For the S-nitrosothiols this of necessity involves variation of the backbone of the structure to which they are attached although if this carrier were then to be linked to another pharmacophore via chemistry similar to that now employed in protein modification this could provide two points of control. The diazeniumdiolates appear to offer the opportunity to exercise control over at least three independent factors: the reactivity of a covalently bound substituent on the N2O2− oxygen or the total absence of such a group; the influence of the primary carrier molecule to which it is attached; and the pharmacology of any secondary carrier to which the entire NO donor may be conjugated. This would suggest that future application of the methods of rational drug design, possibly assisted by a combinatorial chemistry approach, will lead to the development of many new pharmaceuticals and treatment protocols based upon diazeniumdiolate derivatives. REFERENCES Alston, T.A., Porter, D.J.T. and Bright, HJ. (1985) Generation of nitric oxide by enzymatic oxidation of Nhydroxy-Nnitrosamines. J. Biol. Chem., 260, 4069–4074.

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Maragos, C.M., Morley, D., Wink, D.A., Dunams, T.M., Saavedra, J.E., Hoffman, A. et al. (1991) Complexes of -NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. J. Med. Chem., 34, 3242–3247. Markert, M., Carnal, B. and Mauël, J. (1994) Nitric oxide production by activated human neutrophils exposed to sodium azide and hydroxylamine: the role of oxygen radicals. Biochem. Biophys. Res. Commun., 199, 1245–1249. Matthews, E.K., Seaton, E.D., Forsyth, M.J. and Humphrey, P.P.A. (1994) Photon pharmacology of an ironsulphur cluster nitrosyl compound acting on smooth muscle. Br. J. PharmacoL, 113, 87–94. McCleverty, J.A. (1979) Reactions of nitric oxide coordinated to transition metals. Chem. Rev., 79, 53–76. Mian, K.B. and Martin, W. (1995) The inhibitory effect of 3-amino-l,2,4-triazole on relaxation induced by hydroxylamine and sodium azide but not hydrogen peroxide or glyceryl trinitrate in rat aorta. Br. J. PharmacoL, 116, 3302–3308. Mooradian, D.L., Hutsell, T.C. and Keefer, L.K. (1995) Nitric oxide (NO) donor molecules: effect of NO release rate on vascular smooth muscle cell proliferation in vitro. J. Cardiovasc. Pharmacol., 25, 674–678. Moro, M.A., Darley-Usmar, V.M., Lizasoain, L, Su, Y., Knowles, R.G., Radomski, M.W. et al. (1995) The formation of nitric oxide donors from peroxynitrite. Br. J. Pharmacol., 116,, 1999–2004. Moynihan, H.A. and Roberts, S.M. (1994) Preparation of some novel S-nitroso compounds as potential slowrelease agents of nitric oxide in vivo. J. Chem. Soc. Perkin Trans. I, 1994, 797–805. Münzel, T., Sayegh, H., Freeman, B.A., Tarpey, M.M. and Harrison, D.G. (1995) Evidence for enhanced vascular superoxide anion production in nitrate tolerance: a novel mechanism underlying tolerance and crosstolerance. J. Clin. Invest., 95, 187–194. Murad, F. (1990) Drugs used for the treatment of angina: organic nitrates, calcium-channel blockers, and -adrenergic antagonists. In Goodman and Oilman's The Pharmaceutical Basis of Therapeutics, 8th ed., edited by A.G. Oilman, T.W. Rail, A.S. Nies and P. Taylor, pp. 764–783. New York: Pergamon Press. Nagasawa, H.T., DeMaster, E.G., Redfern, B., Shirota, F.N. and Goon, D.J.W. (1990) Evidence for nitroxyl in the catalase-mediated bioactivation of the alcohol deterrent agent cyanamide. J. Med. Chem., 33, 3120– 3122. Nakae, L, Takahashi, M., Kinoshita, T., Matsumoto, T. and Kinoshita, M. (1995) The effects of S-nitrosocaptopril on canine coronary circulation. J. PharmacoL Exp. Ther., 274, 40–6. Namiki, S., Arai, T. and Fujimori, K. (1997) High-performance caged nitric oxide: a new molecular design, synthesis, and photochemical reaction. J. Am. Chem. Soc., 119, 3840–3841. Oae, S., Kim, Y.H., Fukushima, D. and Shinhama, K. (1978) New syntheses of thionitrites and their chemical reactivities. J. Chem. Soc. Perkin Trans. I, 1978, 913–917. Oae, S. and Shinhama, K. (1983) Organic thionitrites and related substances. A review. Org. Prep. Proced. Int., 15, 165–198. Pandey, K.K. (1983) Transition metal nitrosyls in organic synthesis and in pollution control. Coord. Chem. Rev., 51, 69–98. Pufahl, R.A. and Marletta, M.A. (1993) Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis. Biochem. Biophys. Res. Commun., 193, 963–970. Reszka, K.J., Bilski, P. and Chignell, C.F. (1996) EPR and spin trapping investigations of nitric oxide (-NO) from UV irradiated nitrite anions in alkaline aqueous solutions. J. Am. Chem. Soc., 118, 8719–8720. Ribeiro, J.M.C., Hazzard, J.M.H., Nussenzveig, R.H., Champagne, D.E. and Walker, F.A. (1993) Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science, 260, 539–541. Saavedra, J.E., Billiar, T.R., Williams, D.L., Kim, Y.-M., Watkins, S.C. and Keefer, L.K. (1997) Targeting nitric oxide (NO) delivery in vivo. Design of a liver-selective NO donor prodrug that blocks tumor necrosis factor-induced apoptosis and toxicity in the liver. J. Med. Chem., 40, 1947–1954. Saavedra, J.E., Dunams, T.M., Flippen-Anderson, J.L. and Keefer, L.K. (1992) Secondary amine/nitric oxide complex ions, R2N[N(O)NO]−. O-Functionalization chemistry. J. Org. Chem., 57, 6134–6138. Saavedra, I.E., Southan, G.J., Davies, K.M., Lundell, A., Markou, C., Hanson, S.R. et al. (1996) Localizing antithrombotic and vasodilatory activity with a novel, ultrafast nitric oxide donor. J. Med. Chem., 39, 4361– 4365. Schwarz-Bergkampf, E. (1972) Analytical behavior of nitrous acid. Fresenius' Z. Anal. Chem., 259, 343–345.

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Severina, I.S., Ryaposova, I.K., Volodarsky, L.B., Mozhuchin, D.C., Tichonov, A.Y, Schwartz, G.Y. et al. (1993) Derivatives of l,2-diazetidine-l,2-di-N-oxides—a new class of soluble guanylate cyclase activators with vasodilatory properties. Biochem. Mol. Biol. Int., 30, 357–366. Singh, P. (1975) Convenient synthesis of bicyclic and polycyclic cis azoN,N'- dioxides. J. Org. Chem., 40, 1405– 1408. Smith, D.J., Chakravarthy, D., Pulfer, S., Simmons, M.L., Hrabie, J.A., Citro, M.L. et al. (1996) Nitric oxidereleasing polymers containing the [N(O)NO]– group. J. Med. Chem., 39, 1148–1156. Smith, P.A.S. and Hein, G.E. (1960) The alleged role of nitroxyl in certain reactions of aldehydes and alkyl halides. J. Am. Chem. Soc., 82, 5731–5740. Stamler, J.S. (1995) S-Nitrosothiols and the bioregulatory actions of nitrogen oxides through reactions with thiol groups. Curr. Topics Microbiol. Immunol., 196, 19–36. Stamler, J.S., Simon, D.I., Jaraki, O., Osborne, J.A., Francis, S., Mullins, M. et al. (1992a) S-nitrosylation of tissue-type plasminogen activator confers vasodilatory and antiplatelet properties on the enzyme. Proc. Natl. Acad. Sci. USA, 89, 8087–8091. Stamler, J.S., Simon, D.I., Osborne, J.A., Mullins, M.E., Jaraki, O., Michel, T. et al. (1992b) S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc. Natl. Acad. Sci. USA, 89, 444–448. Stuehr, D.J. and Marletta, M.A. (1987) Synthesis of nitrite and nitrate in murine macrophage cell lines. Cancer Res., 47, 5590–5594. Thomas, G. and Ram well, P.W. (1989a) Streptozotocin: a nitric oxide carrying molecule and its effect on vasodilation. Eur. J. Pharmacol., 161, 279–280. Thomas, G. and Ramwell, P.W. (1989b) Vascular relaxation mediated by hydroxylamines and oximes: their conversion to nitrites and mechanism of endothelium dependent vascular relaxation. Biochem. Biophys. Res. Commun., 164, 889–893. Vanin, A.F., Vedernikov, Y.I., Galagan, M.E., Kubrina, L.N., Kuzmanis, Y.A., Kalvin’sh, I.Y. et al. (1991) Angeli salt as a producer of nitrogen oxide in the animal organism. Biochemistry (NY), 55, 1048–1052. Weller, R., Pattullo, S., Smith, L., Golden, M., Ormerod, A. and Benjamin, N. (1996) Nitric oxide is generated on the skin surface by reduction of sweat nitrate. J. Invest. Dermaiol, 107, 327–331. Williams, D.L.H. (1988) Nitrosation. Cambridge: Cambridge Univ. Press. Williams, D.L.H. (1996) Literature highlights—38. nitric oxide release from S-nitrosothiols (RSNO)—the role of copper. Transition Met. Chem. (London), 21, 189–191. Wink, D.A., Darbyshire, J.F., Nims, R.W., Saavedra, J.E. and Ford, P.C. (1993) Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol., 6, 23–27. Zamora, R., Grzesiok, A., Weber, H. and Feelisch, M. (1995) Oxidative release of nitric oxide accounts for guanylyl cyclase stimulating, vasodilator and anti-platelet activity of Piloty’s acid: a comparison with Angeli’s salt. Biochem. J., 312, 333–339. Zhang, P., Ohara, A., Mashimo, T., Sun, J., Shibuta, S., Takada, K. et al. (1996) Cardiovascular effects of an ultra-shortacting nitric oxide-releasing compound, zwitterionic diamine/NO adduct, in dogs. Circulation, 94. 2235–2240.

26 Nitric Oxide Inhalation Andrew M.Atz and David L.Wessel Cardiac Intensive Care Unit, Children's Hospital, Farley 653, 300 Longwood Avenue , Boston, MA 02115, USA

In the lung, as elsewhere in the body, nitric oxide (NO), formed from L-arginine and molecular oxygen in a reaction catalyzed by NO synthase induces vasodilation through a cyclic guanosine monophosphate dependent pathway. Since NO exists as a gas it can be delivered by inhalation to the alveoli and then to the blood vessels which lie in close proximity to ventilated lung. Because of its rapid inactivation by hemoglobin, inhaled NO may achieve selective pulmonary vasodilation when pulmonary vasoconstriction exists. It has advantages over intravenously administered vasodilators which cause systemic hypotension and increase intrapulmonary shunting. Inhaled NO lowers pulmonary artery pressure in a number of diseases without the unwanted effect of systemic hypotension. This is especially dramatic in children with cardiovascular disorders and postoperative patients with pulmonary hypertensive crises. When pulmonary hypertension coexists with severe pulmonary parenchymal disease and hypoxemia, inhaled NO reduces intrapulmonary shunt fraction and improves oxygenation. Prospective randomized trials in persistent pulmonary hypertension of the newborn demonstrated improved oxygenation and decreased morbidity with inhaled NO. Similar studies are awaited in adults with acute respiratory distress syndrome, where pulmonary hypertension is a less prominent feature of the disease and any reversibility of the pathologic process must be demonstrated in carefully designed trials. Other actions of inhaled NO may further increase its therapeutic potential: inhaled NO attenuates proliferation of vascular smooth muscle, inhibits platelet aggregation, provides cytoprotection of donor organs, ameliorates harmful aspects of ischemiareperfusion injury, may promote angiogenesis in the immature lung, and improves the oxygen carrying capacity of sickle hemoglobin.

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Key words: Inhaled nitric oxide, pulmonary hypertension, cardiac surgery, acute respiratory distress syndrome, lung transplantation, persistent pulmonary hypertension of the newborn. MECHANISM AND SITE OF ACTION Nitric oxide (NO) has achieved prominence as a ubiquitous, endogenous molecule with a myriad of functions. The discovery of its biologic role has aided our understanding of disease mechanisms, and suggested new treatments for a variety of illnesses. The obligatory role of the endothelium in acetylcholineinduced vasodilation suggested the existence of an endothelium derived relaxing factor (Furchgott and Zawadzki, 1980), now accepted to be NO (Palmer et al., 1987; Ignarro et al., 1987). Nitric oxide formed from L-arginine and molecular oxygen in a reaction catalyzed by NO synthase induces vasodilation through acyclic guanosine monophosphate (cGMP) dependent pathway (Palmer et al., 1988). Since NO exists as a gas it can be delivered by inhalation to the alveoli and then to blood vessels which lie in close proximity to ventilated lung (Figure 26–1). Because of its short half-life and rapid inactivation by hemoglobin (Rimar and Gillis, 1993), inhaled NO may achieve selective pulmonary vasodilation when pulmonary vaso-constriction exists (Frostell et al., 1991; Wessel et al., 1993; Roberts et al., 1993) whether secondary to endothelial dysfunction or as a result of an abundance of vasoconstricting influences. Inhaled NO may achieve better oxygenation when administered in the presence of ventilation perfusion mismatch (Rossaint et al., 1993; Roberts et al., 1992; Kinsella et al, 1993a; Finer et al, 1994). Therefore, it has advantages over intravenously administered vasodilators which cause systemic hypotension and increase intrapulmonary shunting. Endothelium derived NO exerts important modulating influences on systemic and pulmonary vascular tone in health and disease (Frostell et al, 1993a; Celermajer et al, 1993; Stamler et al, 1994; Celermajer et al, 1994; Lipsitz et al, 1996; Van Camp et al, 1994). The effectiveness of inhaled NO as a pulmonary vasodilator, together with the diminished response to the endothelium dependent vasodilator acetylcholine in patients in whom endothelial injury is associated with the disease state, (Wessel et al., 1993; Adatia et al, 1994; Wessel et al, 1993) raises the question whether a deficiency of endogenously released NO is responsible for the elevation in pulmonary vascular tone. Nitric oxide is thought to be released continuously under basal conditions and inhibition of basal release may lead to an increase in systemic vascular resistance (Vallance et al, 1989). The perfusion of isolated human lungs with methylene blue, an inhibitor of NO mediated vessel relaxation, leads to an increase in pulmonary vascular resistance (Cremona et al, 1991). Similarly, competitive inhibitors of NO synthase administered in fetal animals produced sustained pulmonary hypertension in the newborn (Abman et al., 1990). Thus, endothelial damage with a reduction in endogenous NO could account for pulmonary vasoconstriction, whether imposed by a disease process (e.g., acute lung injury, long standing ventricular septal defect) or a transient side effect of treatment (e.g., cardiopulmonary bypass). Relaxation at both pre and postcapillary levels by inhaled NO has been demonstrated in animal models of pulmonary hypertension (Gao et al., 1995; Mikiyasu et al., 1996). In the perfused human lung inhaled NO affected primarily arterial vessels but during extreme venous constriction could dilate at the postcapillary level as well (Rimar and Gillis, 1995). In adults with acute lung injury, NO has a predominant vasodilating effect on the pulmonary venous vasculature (Benzing and Geiger, 1994). The increased responsiveness seen in pediatrie patients with pulmonary venous hypertension to NO may result from pulmonary vasorelaxation at a combination of pre and postcapillary vessels (Adatia et al., 1995; Atz et al., 1996a). It appears that inhaled NO, unlike intravenous NO donors, are limited in action to small resistance arteries and veins, and unable to dilate larger capacity vessels (Roos et al., 1994).

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Figure 26±1. Nitric oxide (NO) is endogenously formed from L-arginine after acetylcholine (ACh) binds to muscarinic receptors (M) on the intact endothelium and stimulates endothelial nitric oxide synthase (NOS). NO exists as a gas and can be delivered to alveoli. It diffuses across alveolar membrane to closely adjacent vessels. Constricted pulmonary vessels dilate as result of increased intracellular cGMP production in smooth muscle cells. Nitric oxide is immediately inactivated by hemoglobin with the formation of methemoglobin (met Hb), nitrosylhemoglobin (NO Hb), nitrates (NO3-) and nitrites (NO2-) limiting its activity to the pulmonary circulation. PA=pulmonary artery; LA=left atrium.

Nitrovasodilator therapy is not new. After the first sublingual application of nitroglycerin to treat the symptoms of angina pectoris (Murrell, 1879), over a century passed before the biologically active ingredient of nitroglycerin (i.e., NO) was revealed. Prior to this discovery, NO was discussed relevant to its role as a by-product of high temperature combustion (smokestacks, cigarettes), its role in the generation of nitrogen dioxide (acid rain), its presence as a contaminant in medical and industrial gases, and its curiously high affinity for hemoglobin (Oda et al., 1980). Coincident with the recognition of NO as an important endogenous mediator of vascular relaxation, researchers were exploiting the unique characteristics of this molecule to assess pulmonary diffusion capacity in humans (Meyer et al., 1990); its great affinity for hemoglobin offered alternatives to the use of carbon monoxide for this test. These events culminated in the initial description of the use of inhaled NO in pulmonary hypertension (Higenbottam et al., 1988) and the elegant studies by Zapol and collaborators showing selectivity and clinical efficacy of this molecule when inhaled. (Frostell et al., 1991). Since then, inhaled NO has emerged rapidly as a potential therapy for human pulmonary hypertensive disease. It represents the clinical application of advances in vascular biology which have occurred over the last twenty years.

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PULMONARY VASCULAR DISEASE Inhaled NO has emerged as an important diagnostic and therapeutic agent in patients with acute or chronic pulmonary vascular disease. Its hemodynamic effect has been demonstrated in patients with primary pulmonary hypertension and secondary pulmonary hypertension associated with pulmonary thromboembolism, collagen vascular disease, cardiac disease (congenital or acquired), heart or lung transplantation, and other disorders. Primary Pulmonary Hypertension The prognosis for patients with primary pulmonary hypertension is poor (Oakley, 1985). Response to acute vasodilator testing in patients with primary pulmonary hypertension is an important marker for survival (Houde et al., 1993) and may identify patients who would benefit from chronic medical therapy (Rich et al., 1992; Barst et al., 1994). The use of systemic vasodilators is not without risk in these patients (Buch and Wennevold, 1981; Packer et al., 1982, 1984). Therefore, most would recommend a trial of a short acting vasodilator, such as prostacyclin, to predict response. The effects of prostacyclin and inhaled NO have been compared in primary pulmonary hypertension. A comparable change in the pulmonary hemodynamics to both agents was found but inhaled NO was more specific and selective (Pepke-Zaba et al., 1991). Others have demonstrated an acute response to inhaled NO in children (Kinsella et al., 1993b) and adults (Sitbon et al., 1995) with primary pulmonary hypertension. It has been suggested that the improved survival in some patients on long-term intravenous prostacyclin therapy is unrelated to its acute effects and may be related either to antiproliferative effects on smooth muscle or antiregulatory effects on platelets (Higenbottam et al., 1993). Nitric oxide is present in the exhaled breath of humans (Gustafsson et al., 1991) and may also have antiproliferative effects on smooth muscle and inhibit platelet adhesion (Garg and Hassid, 1989; Moncada et al., 1991). It is possible that in suitable patients a low dose inhalation of NO might become a useful agent in the treatment of chronic pulmonary hypertension both by reversing the vasoconstrictor component when present and encouraging the regression of smooth muscle which obliterates the pulmonary vascular bed (Roberts et al., 1995; Kolpakov et al., 1995). However, potential long-term risks must be considered seriously. Demonstration of pulmonary vasoreactivity in patients with end stage pulmonary disease may differentiate patients who would benefit from long term medical therapy from those with fixed resistance who may require more urgent consideration of lung transplantation (Houde et al., 1993). Microscopic changes in the lungs of patients with primary pulmonary hypertension are histologically indistinguishable from the pulmonary vascular changes of the Eisenmenger complex. Typically in this disease, the lungs have been exposed to years of high pressure and flow from an unrepaired ventricular septal defect. Pulmonary vascular resistance rises inexorably, ultimately with reversal of blood flow through the defect. Resultant cyanosis will be exacerbated by any vasodilator not specific for the pulmonary circulation. Although inhaled NO offers only mild salutary effects in this disease, it has prompted reevaluation of therapeutic options. Pulmonary Thromboembolism Acute massive pulmonary embolism abruptly increases pulmonary artery pressure, pulmonary vascular resistance, and without treatment may lead to right heart failure, hypoxemia, and death. The prognosis of chronic thromboembolism is also generally poor. While inhaled NO is not a curative option its selective pulmonary vasodilatory actions have been successfully used in both animals and humans to decrease

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pulmonary artery pressure (Bottiger et al., 1996) and to improve oxygenation, by reversing right to left flow at an atrial level shunt (Estagnasie et al., 1994) and redistributing pulmonary blood flow to better ventilated alveoli. It has been effectively used as an adjunct to surgical thromboendarterectomy by improving both right heart function and gas exchange (Gardeback et al., 1995; Pinelli et al., 1996). It may also be of therapeutic value by inhibiting platelet aggregation following pulmonary embolism (Gries et al., 1997). CARDIOVASCULAR DISEASE Testing Vasoreactivity Pulmonary hypertension complicates the evaluation, clinical course and outcome of many patients with heart disease. It is often a crucial factor in determining the timing or type of intervention, and has been invoked as the primary determinant of mortality in many lesions, especially among children (Haworth, 1984; Hoffman et al., 1981). Although considerable uncertainty exists about parameters which will insure safe operability, an increased or fixed elevation in pulmonary vascular resistance may deny patients a chance for corrective surgery. Subsequently they may develop progressive obliterative pulmonary vascular disease (Eisenmenger complex) and severely reduced life expectancy. The demonstration of maximal pulmonary vasodilation in patients with a reactive pulmonary bed using a specific and safe agent is therefore an important objective. Oxygen has been used during catheterization for decades and remains part of standard vasodilator testing. However, a negative response to acute treatment with oxygen has been seen in some patients who did indeed have a reactive pulmonary bed (Bush et al., 1987; Houde et al., 1993). Inhaled NO has been demonstrated to vasodilate the pulmonary vascular bed with minimal systemic effects without increasing intrapulmonary shunting. Changes in cardiac output during vasodilator testing can confound the interpretation of calculated resistance but generally cardiac output is unaffected by inhaled NO. Nitric oxide can be accurately and quickly administered by either ventilator or mask, and with modern delivery techniques can be easily delivered to patients undergoing cardiac catheterization. Demonstration of a reactive pulmonary bed in patients being evaluated for heart-lung transplantation has enabled patients to be offered a single organ (heart) instead of heart-lung block with successful results (Adatia et al., 1995; Kieler-Jensen et al., 1994). Identification of patients with elevated but reactive pulmonary vasculature will identify those patients to may need more intensive postoperative care (Fyfe et al., 1991; Behrendt et al, 1986). Those patients demonstrating pulmonary reactivity to NO preoperatively presumably would be excellent candidates for NO therapy in the postoperative period if pulmonary hypertension is problematic. Studies of vasoreactivity in children during cardiac catheterization using NO found variable responsiveness that seemed to parallel the progression of established vascular disease, and suggested that vasodilator testing with NO may be helpful in selecting patients for operation (Berner et al., 1996). Congenital Heart Disease Although preoperative testing of pulmonary vasoreactivity is useful for diagnostic purposes, postoperative treatment of pulmonary hypertensive crises in children with congenital heart disease is therapeutically important (Tournois et al., 1994). Reactivity of the pulmonary vascular bed is related to the presence and degree of preoperative pulmonary hypertension, magnitude of preoperative left to right shunts, and to duration of cardiopulmonary bypass. On cardiopulmonary bypass pulmonary blood flow is supplied only by

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Figure 26±2. Patients with pulmonary hypertension were studied both pre and postoperatively with acetylcholine (ACH) and NO. Baseline mean pulmonary artery pressure decreased 27±4% preoperatively but only 9±2% postoperatively with the endothelium dependent vasodilator acetylcholine. However, after the attenuated response to ACH was demonstrated, postoperative inhalation of NO immediately lowered mean pulmonary artery pressure by 26±3%. The functional integrity of the smooth muscle was intact in the presence of endothelial dysfunction resulting from cardiopulmonary bypass (Wessel et al., 1993).

the vasovasorum via the bronchial circulation which may be inadequate to prevent ischemic damage to the endothelium and subsequently compromise endogenous production of NO. Transient pulmonary vascular endothelial cell dysfunction could be demonstrated in neonates and older children by documenting the loss of endothelium dependent vasodilation during the immediate postoperative period (Wessel et al., 1993) (Figure 26–2). Pulmonary vasodilation to acetylcholine was present preoperatively but attenuated postoperatively while response to inhaled NO was present both pre and postoperatively. This suggested that the functional integrity of the smooth muscle was intact in the presence of endothelial dysfunction resulting from cardiopulmonary bypass. Elevated pulmonary vascular resistance from atelectasis, microemboli, platelet plugging of vessels or other fixed obstructive processes could not be invoked as the cause of the blunted response to acetylcholine since resistance decreased so dramatically with NO. Plasma levels of cGMP in postoperative patients were unchanged after acetylcholine but rose more than three fold during pulmonary vasodilation with NO. This finding was consistent with the purported role of cGMP as the second messenger effecting smooth muscle relaxation. Attention focused on the endothelium as an important organ to address in the management of pulmonary hypertension. It also highlighted the potential importance of maintaining at least some antegrade flow from right ventricle into pulmonary arteries (and endothelium) during extracorporeal membrane oxygenation

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(ECMO). The heart should be permitted to eject some flow into the pulmonary arteries rather than allowing ECMO to provide total cardiopulmonary bypass for several hours or days. Surgical correction of congenital heart disease, in contrast to palliation with shunts or pulmonary artery bands, has been extended to the neonate where it is emerging as the preferred approach to many defects in most major centers (Castaneda et al., 1989; Wernovsky et al., 1995a). However, perioperative care of the newborn and infant requires an appreciation of the relative intolerance of the immature myocardium to increased afterload. The right ventricle must face the potential challenges of the transitional pulmonary circulation rendered ischemic and reactive by cardiopulmonary bypass simultaneously with transiently impaired ventricular function caused by the adverse effects of cardiopulmonary bypass and in many instances a right ventriculotomy. Thus, it is imperative that one minimize right ventricular afterload during the early postoperative hours as the ischemic-reperfusion injury transiently depletes myocardial reserve and cardiac output declines (Wernovsky et al., 1995b). Pulmonary hypertensive crises are dramatic events which threaten the life of an infant despite a good surgical repair (Wheller et al., 1979; Del Nido et al., 1987). In such situations the pulmonary artery pressure rises to systemic or suprasystemic levels, the systemic blood pressure falls and the transcutaneously monitored arterial oxygen saturation decreases. In a report of a series from one large center, half of the postoperative cardiac children who had pulmonary hypertensive crises died during their hospitalization (Hopkins et al., 1991). Inhaled NO can therefore be used to selectively manipulate the pulmonary vasculature after endothelial injury and represents an important advance in pharmacologic strategies aimed at treatment of the diseased or abnormally reactive pulmonary circulation. It has become a valuable tool in the treatment of postoperative pulmonary hypertension in congenital heart disease. Infants with total anomalous pulmonary venous connection (TAPVC) frequently have obstruction of the pulmonary venous pathway as it connects anomalously to the systemic venous circulation. When pulmonary venous return is obstructed preoperatively, pulmonary hypertension is severe and demands urgent surgical relief. Increased neonatal pulmonary vasoreactivity, endothelial injury induced by cardiopulmonary bypass, and intrauterine anatomic changes in the pulmonary vascular bed in this disease (Haworth, 1982) contribute to postoperative pulmonary hypertension. In one study 20 infants presenting with isolated TAPVC over a three year period were monitored for pulmonary hypertension. Nine patients had postoperative pulmonary hypertension and were treated with a 15 minute trial of inhaled NO at 80 ppm. Five patients received prolonged treatment with NO at 20 ppm or less. A mean percentage decrease of 42% in pulmonary vascular resistance and 32% in mean pulmonary artery pressure was demonstrated. There was no significant change in heart rate, systemic blood pressure or vascular resistance (Atz et al., 1996b) (Figure 26–3). Patients with TAPVC, congenital mitral stenosis, and other pulmonary venous hypertensive disorders appear to be the most responsive to NO. These infants are born with significantly increased amounts of smooth muscle in their pulmonary veins (Newfeld et al., 1980; Haworth and Reid, 1977). Histologic evidence of muscularized pulmonary veins as well as pulmonary arteries (Ferencz and Dammann, 1957) suggest the presence of vascular tone and capacity for change in resistance at both the arterial and venous sites. The increased responsiveness seen in younger patients with pulmonary venous hypertension to NO may result from pulmonary vasorelaxation at a combination of pre and postcapillary vessels. Pulmonary vasoconstriction in the postoperative newborn is exquisitely responsive to inhaled NO. However, reactive pulmonary vasoconstriction may not be the only cause of elevated pulmonary artery and right ventricular pressures. Differentiation between pulmo nary vasoconstriction and anatomic obstruction to pulmonary blood flow may be difficult, especially in neonates. Branch pulmonary artery stenosis, hypoplastic distal pulmonary arteries, or iatrogenic causes of obstruction to pulmonary blood flow, may be

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Figure 26±3. Percentage change in hemodynamic variables from baseline after 15 minutes of NO at 80 ppm in 9 neonates with pulmonary hypertension following repair of total anomalous pulmonary venous connection. There is marked specificity for the pulmonary circulation with a decrease of 42% in pulmonary vascular resistance (PVR) and 32% in mean pulmonary artery pressure (mPAP). There was no significant change in heart rate (HR), systemic blood pressure (BP) or vascular resistance (SVR). Although not statistically significant cardiac index (CI) increased by 10% (Atz and Wessel, 1997a).

reflected by elevated pressure in the main pulmonary artery. A definitive diagnosis may require invasive and potentially dangerous investigation of the circulation. Inhaled NO was used diagnostically in neonates with pulmonary hypertension after cardiac surgery to discern those with reversible vasoconstriction. Nine of 15 patients responded to a 15 minute trial with a reduction in mean pulmonary artery pressure from 35±4 to 26±4 mmHg and pulmonary vascular resistance from 17±6 to 10±4 U-m2. There were insignificant changes in systemic hemodynamics. Six patients did not respond to inhaled NO with either a decrease in proximal pulmonary artery pressure or an increase in systemic oxygen saturation. In each of these patients subsequent investigation, prompted by the failed response to inhaled NO, revealed anatomical obstruction to pulmonary blood flow. Thus, failure of the postoperative newborn with pulmonary hypertension to respond to NO successfully discriminated anatomical obstruction to pulmonary blood flow from pulmonary vasoconstriction (Adatia et al., 1996). Judicious use of a trial of inhaled NO may be of value to rule out pulmonary vasoconstriction and redirect investigation toward reassessment of the surgical result. Failure of the patient to show response to NO should be regarded as strong evidence of anatomic and possibly surgically remediable obstruction. Successful use of inhaled NO in a variety of congenital heart defects following cardiac surgery has been reported by several groups (Roberts et al., 1993; Curran et al., 1995; Journois et al., 1994; Luciani et al., 1996). Descriptions of use after Fontan procedures (Yahagi et al., 1994; Miller et al., 1993), total anomalous pulmonary venous connection (Tibballs, 1993; Girard et al., 1993b; Morris et al., 1995), and following VSD repair (Berner et al., 1993) have been described. Some studies suggest that there is a correlation between the response to NO and the extent of preoperative pulmonary hypertension (Beghetti et al., 1995; Miller et al., 1994b).

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ECMO support for severe cardiopulmonary failure after cardiac surgery in newborns and children has been advocated in many centers (Kulik et al., 1996; Ziomek et al., 1992). Since postoperative pulmonary hypertension following reparative cardiac operations is believed to be life threatening, yet reversible, NO treatment in this condition may diminish the need for ECMO. At one institution 6 of 10 patients who met ECMO criteria were managed with NO instead and survived to hospital discharge (Goldman et al., 1996a). This compares favorably with published survival rates in postcardiotomy patients supported by ECMO (Kulik et al., 1996). Although there are no randomized trials examining the benefit of NO among cardiac patients, this information suggests that a trial of inhaled NO should be considered in these patients prior to cannulation for ECMO. Adult Cardiac Disease Adults undergoing cardiac operations with postoperative pulmonary hypertension and right ventricular dysfunction have also been effectively treated with inhaled NO. Effective use following mitral valve surgery and coronary artery bypass grafting has been reported. Inhaled NO has been demonstrated to selectively vasodilate adults with pulmonary hypertension due to acquired mitral stenosis following mitral valve replacement with an approximate 20% change in pulmonary vascular resistance and a 10% change in pulmonary artery pressure (Snow et al., 1994; Rich et al., 1993a; Girard et al., 1992). More recently variability of response to NO in adult postoperative patients with valvar disease has been shown (Fullerton et al., 1997b). Responsiveness to NO in congenital mitral stenosis has been reported with a mean percentage decrease of 48 ± 4% in pulmonary vascular resistance and 28 ± 3% in mean pulmonary artery pressure (Atz et al., 1996a). Children with pulmonary venous hypertensive disorders existing since birth (e.g., congenital mitral stenosis or obstructed anomalous pulmonary venous drainage) may have increased vasoconstriction and thus increased response to inhaled NO when compared to adults with acquired mitral stenosis or patients with other congenital heart lesions without pulmonary vein muscularization. Cardiac Transplantation Elevated pulmonary vascular resistance is a risk factor prior to cardiac transplantation (Addonizio et al., 1987; Kirklin et al., 1988) which is manifested by an increased risk of right ventricular failure postoperatively (Bando et al., 1993; Murali et al., 1993). Vasodilator therapy is therefore often required and inhaled NO with its selectivity to the pulmonary vasculature would appear to be an ideal treatment. Comparisons with sodium nitroprusside, prostacyclin, and prostaglandin El demonstrated that NO provided similar pulmonary vasodilation with prostacyclin but importantly was the only vasodilator that was selective following transplantation (Kieler-Jensen et al., 1995; Williams et al., 1995; Girard et al., 1993a; Auler et al., 1996). Comparative Effects Numerous studies have compared the individual or combined effect of inhaled NO with other intravenous or inhaled vasodilators. In 6 adults with primary pulmonary hypertension, oxygen and inhaled NO appeared to have less potent selective pulmonary vasodilation compared to aerosolized prostacyclin and iloprost (Olschewski et al., 1996). However, in animal models inhaled NO and inhaled prostacyclin have similar selective pulmonary vasodilation; intravenous prostacyclin had less selectivity than inhaled NO in adults after cardiac surgery (Snow et al., 1994; Zobzl et al., 1995). In an experimental model of hypoxic vasoconstriction inhaled prostacyclin achieved only 65% of the reduction in pulmonary vascular resistance

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produced by inhaled NO and only with NO did right ventricular ejection fraction increase (Zwissler et al., 1995). A prospective randomized cross-over trial involving 13 children with pulmonary hypertension following cardiac repair showed both more effective and selective pulmonary vasodilation with NO at 20 ppm compared to intravenous prostacyclin at 20 ng/kg/min (Goldman et al., 1995a). Synergistic use of NO with aerosolized or intravenous prostacyclin (Parker et al., 1997; Schranz et al., 1993), atrial natriuretic peptide (Ivy et al., 1996), dipyridamole (Kinsella et al., 1995b; Fullerton et al., 1997a), or specific type V phosphodiesterase inhibitors (Cohen et al., 1996) holds considerable promise for more effective control of pulmonary hypertension. As a result of its vasodilator effect, inhaled NO has been demonstrated to improve right ventricular failure (Gatecel et al., 1995). It has been shown to assist the management of patients with right heart failure in those already on left ventricular assist device, thereby obviating the need for ECMO (Yahagi et al., 1995). PULMONARY DISEASE Neonatal and Infant Respiratory Failure Persistent pulmonary hypertension of the newborn Persistent pulmonary hypertension of the newborn (PPHN) is a syndrome characterized by increased pulmonary vascular resistance, right to left shunting of blood and severe hypoxemia. PPHN is frequently associated with pulmonary parenchymal abnormalities, including meconium aspiration, pneumonia, sepsis, lung hypoplasia, and dysplastic alveolar capillary structure. In some instances there is no evidence of pulmonary parenchymal disease and the etiology is unknown. Treatment strategies, including alkalinization, hyperventilation and use of intravenous vasodilators are aimed at lowering pulmonary vascular resistance but are associated with adverse effects and are not always successful (Roberts and Shaul, 1993). Extracorporeal membrane oxygenation (ECMO) has improved survival for neonates with refractory hypoxemia but may be associated with hemorrhagic, neurologic and other complications (Bartlett et al., 1985; O’Rourke et al., 1989; UK collaborative ECMO Trial Group, 1996). Although survival for PPHN has improved, better treatment would further reduce mortality rates and morbid outcomes. Impaired endogenous NO production has been suggested by reports of decreased concentrations of cGMP in infants with PPHN (Christou et al., 1997). Animal models of PPHN showed selective pulmonary vasodilation and improved outcomes with inhaled NO (Zayek et al., 1993). Thus, there is sound physiologic basis for use of inhaled NO as a selective pulmonary vasodilator in this disease. Early investigations suggested that this drug improved oxygenation in patients with PPHN who were administered 6–80 ppm of NO with oxygen. Although promising, these initial studies were small case series with physiologic rather than clinical outcomes and lacked a control group. Subsequent trials were informative but until recently were still limited by lack of controls, extensive treatment crossover designs, or inherent limitations of multi-center trials with varying definitions of standard clinical practice. Several studies have shown sustained improvement in oxygenation with NO (Roberts et al., 1992; Kinsella et al., 1992; Finer et al., 1994) (Figure 26–4). Efficacy of NO in the treatment of PPHN has been recently affirmed in single and multicenter randomized trials (The Neonatal Inhaled Nitric Oxide Study Group. 1997; Roberts et al., 1997; Wessel et al., 1997). Severe hypoxemia is usually the main indication for ECMO. Inhaled NO not only improves oxygenation but reduces the need for ECMO. Lung recruitment strategies facilitated by high frequency ventilation may enhance the efficacy of NO (Abman and Kinsella, 1995;

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Figure 26±4. Median percentage change in oxygenation index (OI) during the first 24 hours of study among patients with persistent pulmonary hypertension of the newborn who were randomized to control or NO treatment. The significant reduction in oxygenation index (OI) during the first 15 minutes was sustained during subsequent times compared to baseline (*) or to control patients (†) (p<0.05) (Wessel et al., 1997).

Kinsella et al., 1997). The data generated from these randomized trials along with followup studies designed to examine long term outcomes in these patients (Rosenberg et al., 1997) will likely provide the clinical basis for regulatory approval of inhaled NO as a noninvestigational drug. Respiratory distress syndrome After demonstrating the utility of inhaled NO in the term infant during the period of transitional circulation, investigators turned their attention to respiratory distress syndrome of prematurity. Initial animal models of prematurity showed marked improvement in pulmonary hemodynamics and gas exchange in severe experimental hyaline membrane disease (Kinsella et al., 1994). Safe ranges for NO, nitrogen dioxide, methemoglobin, and metabolites such as peroxynitrite are not clearly known in the premature infant. However, limited clinical trials in premature infants with gestational ages as young as 24 weeks have shown significant improvement in oxygenation and a decrease in mean airway pressure with inhaled NO (Peliowski et al., 1995; Ahluwalia et al., 1994). Randomized trials are now underway. Concerns persist regarding inhaled NO action on platelets, especially in premature infants at significant risk for intracranial hemorrhage. Adverse effects of NO on the premature lung are unknown. Pneumonia Reports have shown dramatic improvement in oxygenation in children with pneumonia caused by respiratory syncytial virus, group B streptococcus, and other organisms (Thompson et al., 1995; Leclerc et

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al., 1994). This is presumably attributable to improved ventilation perfusion matching. In animal models following endotoxemia inhaled NO improved pulmonary hypertension, pulmonary edema, and oxygenation (Ogura et al., 1994b, 1994a). Overwhelming pneumonia is a devastating complication that may be exacerbated by cardiopulmonary bypass. Mild infectious pneumonitis or bronchiolitis in the young preoperative infant can turn to life threatening respiratory failure during postoperative recovery. As an inhaled vasodilator, NO therapy addresses both aspects of the disease: pulmonary hypertension and hypoxia. Inhaled NO by virtue of its antioxidant effects, inhibition of unwanted platelet aggregation, and suppression of deleterious inflammatory responses during reperfusion injury may even have a role in routine prophylactic use for all patients at risk of post bypass respiratory complications. Congenital diaphragmatic hernia The pathophysiology of congenital diaphragmatic hernia results from a combination of pulmonary hypoplasia, pulmonary hypertension, and perhaps surfactant deficiency. Initial case reports demonstrated the successful use of inhaled NO following surgical repair (Frostell et al., 1993b). Follow up studies have shown variable results and are less encouraging (Shah et al., 1994; Kinsella et al., 1997). Some studies suggest that NO may be more effective if administered after exogenous surfactant treatment (Karamanoukian et al., 1995), along with perfluorocarbon-associated gas exchange (Wilcox et al., 1995), or after ECMO support (Karamanoukian et al., 1994). It is likely the variability in responsiveness is due to the degree of pulmonary hypoplasia vs. reversible pulmonary vasoconstriction in individual patients. Acute Lung Injury Acute lung injury is associated with significant morbidity and mortality related to pulmonary hypertension, right ventricular failure, and ventilation-perfusion mismatch (Ring and Stidham, 1994). Treatment of pulmonary hypertension with intravenous vasodilators opposes the desired action on ventilation perfusion relationships. In animal models of acute lung injury, inhaled NO significantly reduced pulmonary artery pressure and vascular resistance, improved transpulmonary vascular efficiency and ventilation-perfusion matching resulting in better oxygenation (Hillman et al., 1995). Acute Respiratory Distress Syndrome Inhaled NO has shown variable improvements in oxygenation and pulmonary hypertension in patients with acute respiratory distress syndrome (ARDS) (Rossaint et al., 1993). Selective pulmonary vasodilation may be most pronounced in patients with the greatest degree of pulmonary vasoconstriction (Bigatello et al., 1994; Puybasset et al., 1995) and baseline pulmonary vascular resistance may be the best marker predicting beneficial response to NO (Lowson et al., 1996). Inhibition of platelet aggregation has been suggested as an additional beneficial effect of NO in respiratory failure (Samama et al., 1995). Inhaled NO dilates vessels near ventilated units of lung, redistributing blood flow preferentially away from poorly ventilated segments. As a result ventilation-perfusion matching improves (Figure 26–5). Use of multiple inert gas techniques has supported this contention. This reasoning is further strengthened by studies showing enhancement of hypoxic pulmonary vasoconstriction by almitrene with improvement in ventilation perfusion matching. Additional improvement when inhaled NO was administered together with almitrene suggested redistribution by predominant local vasodilation in response to inhaled NO near

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Figure 26±5. Inhaled NO in lung disease is preferentially delivered to areas where alveolar ventilation (VA) is high. Blood vessels in proximity to poorly ventilated alveoli are affected by hypoxic vasoconstriction. By redirecting pulmonary blood flow to dilated vessels near well ventilated lung, intrapulmonary shunt declines and oxygenation improves.

well ventilated alveoli. Vasoconstriction by almitrene dominated local control in the poorly ventilated segments (Wysocki et al., 1994; Lu et al., 1995). Eleven patients with ARDS treated with ECMO underwent alteration of mixed venous oxygen tension with and without inhaled NO. Hypoxic vasoconstriction at the lowest mixed venous saturations increased as manifested by higher pulmonary vascular resistance. Inhaled NO had its most pronounced vasodilatory effect during the most hypoxic vasoconstriction. In contrast the NO induced improvement in ventilationperfusion matching was independent of mixed venous saturation (Benzing et al., 1997). By decreasing right ventricular afterload often right ventricular ejection fraction will improve (Rossaint et al., 1995; Offher et al., 1995). In a swine model of ARDS right ventricular total power decreased in relation to decreased RV afterload, not due to a change in intrinsic right ventricular contractility (Cheifetz et al., 1996). Pulmonary edema is one pathologic consequence of increased microvascular permeability in acute lung injury. Inhaled NO substantially decreases the pulmonary vascular permeability from oxidant injury (Poss et al., 1995; Kavanagh et al., 1994) and neutrophil mediated capillary leak (Guidot et al., 1995). It is speculated that endothelial damage by oxidative injury causes increased capillary leak and that NO may improve this not only by local vasodilation decreasing pulmonary capillary pressure (Benzing et al., 1995) but via some anti-inflammatory mechanism (Guidot et al., 1995). Inhaled NO has resulted in reduced neutrophil accumulation in endotoxin induced lung injury (Friese et al., 1996) as well as reduced capacity for superoxide production by neutrophils in infants with pulmonary hypertension during NO delivery and for

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Figure 26±6. In six patients following lung transplantation, inhaled NO reduced pulmonary artery pressure (PAp) by a mean percentage change of 24% and pulmonary vascular resistance (PVR) by a mean percentage change of 31% (*p<0. 05 for both). The arterial oxygen tension tended to increase from 98±21 to 147±21 (p=0.08) with an decrease in intrapulmonary shunt fraction from 28% to 21% (*p<0.05) (Adatia and Wessel, 1994).

up to 4 days later (Gessler et al., 1996). As in the treatment of pulmonary vasoconstriction, inhaled prostacyclin has similar efficacy to inhaled NO in ARDS (Zwissler et al., 1996; Pappert et al., 1995; Walmrath et al., 1996). In a recent prospective randomized multicenter study, NO was well tolerated and associated with a significant improvement in oxygenation over the first 4 hours of treatment and improved oxygenation index over the first 4 days compared to placebo. (Dellinger et al, 1998). Further studies are necessary however to determine if NO can actually improve clinical outcome in ARDS. Improvement in mortality may be demonstrable in only a very select subpopulation of patients with ARDS. Lung Transplantation For children and adults with end stage lung disease, either primary, or secondary to congenital heart disease, lung transplantation offers the only hope of active long-term survival with 2-year survival rates of 70–80% (Calhoon et al., 1991; Armitag et al., 1993). Management of postoperative pulmonary complications remains challenging. Transient graft dysfunction occurs in up to 20% of patients (Haydock et al., 1992) and is associated with pulmonary hypertension, edema, right ventricular failure and respiratory failure (Starnes et al., 1992; Pasque et al., 1992). Supportive treatment which assumes that the injury is reversible is directed towards positive-pressure ventilation, vasodilator therapy, inotropic drugs and at times mechanical support of the circulation. Supportive therapy carries its own risks of barotrauma, hemodynamic instability, infection, bleeding and neurologic injury (Haydock et al., 1992; Starnes et al., 1992). Again in this clinical scenario the injured lung vasculature is unresponsive to the endotheliurn dependent vasodilators but highly responsive to inhaled NO with significant decreases in pulmonary artery pressure and dramatic increases in PaO2 (Adatia et al., 1994; Macdonald et al., 1995; Myles and Venema, 1995)

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(Figure 26–6). Response to NO is proportional to the degree of endothelial dysfunction (Lindberg et al., 1996). The lack of a large donor pool of pediatric sized lungs dictate that a wide size range be accepted. However, size disparity in pediatric lung transplantation is a significant mortality risk factor (Lillehei et al., 1994). Inhaled NO in animal models significantly reduced pulmonary vascular resistance when pulmonary hypertension was induced by airway hyperinflation and supraphysiologic lung volumes (Ibla et al., 1996). These data suggests that inhaled NO may be particularly effective in children who receive organs from undersized donors. Evidence now exists that NO can be administered to the donor lung to enhance preservation during storage and transport to the recipient (Date et al., 1996; Bacha et al., 1996; Okabayashi et al., 1996). Some centers now routinely ventilate donor lungs with inhaled NO and continue into the postoperative period to blunt the reperfusion injury phase after transplantation. Other Pulmonary Diseases Variable and sometimes dramatic response to inhaled NO has been shown in other pulmonary processes such as high altitude pulmonary edema (Scherrer et al., 1996), chronic obstructive pulmonary disease (Adatia et al., 1993; Roger et al., 1996; Moinard et al., 1994), and in restrictive and fibrotic lung diseases (Channick et al., 1994; Jolliet et al., 1995; Williamson et al., 1996). The use of inhaled NO in testing for reversible elements of pulmonary vasoconstriction in these diseases could provide an important diagnostic tool for chronic pulmonary diseases. SICKLE CELL DISEASE Recent work shows that therapeutic concentrations of inhaled NO (80 ppm) increase the oxygen affinity of sickle hemoglobin in vitro and in vivo. (Head et al, 1997) An increase in oxygen affinity will decrease polymerization of deoxygenated sickle hemoglobin and therefore reduce sickling of erythrocytes. There was no change in oxygen affinity with NO in normal hemoglobin A erythrocytes. This is a newly defined mechanism of action of NO and suggests novel treatments for complications due to sickle cell disease. Inhaled NO may ameliorate complications associated with sickle cell disease by dilating the pulmonary vascular bed, reducing afterload on the right ventricle, redistributing pulmonary blood flow to better ventilated areas of lung, and by reducing sickling in the lung by alteration of hemoglobin S oxygen affinity. Dramatic clinical improvements in children with sickle cell disease suffering from acute chest syndrome have been reported during inhaled NO treatment (Atz and Wessel, 1997b). Proper large prospective trials need to be undertaken to further elucidate the importance of this novel therapy in acute chest syndrome. Early use of NO in this group of patients may decrease the incidence of painful crises and acute chest syndrome or reduce the need for more aggressive therapies such as exchange transfusion or mechanical ventilation. BRONCHODILATION Nitrosovasodilators can relax airway smooth muscle cells in vitro, a result of increased cGMP at the smooth muscle. Inhaled NO at 80–300 ppm has shown to reduce the bronchoconstrictor effect of methacholine in rabbits (Hogman et al, 1994b), in guinea pigs (Dupuy et al., 1992), and dogs (Gwyn et al., 1996). Similar studies in methacholine challenged human volunteers has been much less than that reported in animals or in humans treated with more traditional beta-sympathomimetic agents (Sanna et al., 1994). Asthmatic patients have significantly higher concentrations of exhaled NO compared to controls which may reflect induction

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of NO synthase in the lower airways (Kharitonov et al., 1994; Alving et al, 1993). Thirteen mild asthmatics were challenged with methacholine induced bronchospasm and then treated with inhaled NO. A minor but significant relaxation of airway tone was found (Kacmarek et al., 1996). A study of 12 pediatric asthmatics showed no improvement in pulmonary function tests after inhaling 10 or 40 ppm of NO compared to baseline (Pfeffer et al., 1996). It appears that measurement of exhaled NO may give insight into airway inflammatory diseases, or serve as a monitor of illness severity. Further studies are necessary to determine whether inhaled NO will have any significant therapeutic role in reactive airway disease. EXTENDED USE Although outpatient use of inhaled NO has been reported in a small number of adults (Channick et al., 1996), its use in younger patients with heart disease or as a therapeutic bridge to lung or heart lung transplantation is largely unstudied. NO inhibits smooth muscle growth (Garg and Hassid, 1989) and matrix protein synthesis in the extracellular matrix (Kolpakov et al., 1995). It also reduces hypoxic remodeling in the rat lung (Kouyoumdijian et al., 1994; Roberts et al., 1995), suggesting that it might have a salutary effect on scarring or pathologic remodeling in the human lung. The antioxidant and antiproliferative effects of NO combined with its antihypertensive action might provide a theoretical basis for prolonged treatment of idiopathic pulmonary hypertension. This might be particularly applicable to infants, who by virtue of their young age, have substantial capacity for smooth muscle regression, alveolar growth and angiogenesis. Four infants less than 4 months old with severe unexplained pulmonary hypertension (biopsy proven and presumed to be fatal) were treated with a 25 day regimen of inhaled nitric oxide and the angiogenesis factor heparin. At the end of the treatment period they had significantly lower (nearly normal) pulmonary artery pressures without recurrence of pulmonary hypertension in 3 of 4 during 2 years of follow-up. (Atz and Wessel, 1998) While no conclusion can be drawn from such limited experience, it has prompted reevaluation of our notion about presumed irreversibility of “primary” pulmonary hypertension early in life. SAFETY ISSUES Delivery of Inhaled NO Although there is little doubt that NO reduces pulmonary artery pressure and improves oxygenation in many patients with reversible components to their pulmonary vascular and lung disease, improper or inadequate delivery and monitoring can carry considerable patient risk (Body et al., 1995; Young and Dyar, 1996; Atz and Wessel, 1997c). The first described use of inhaled NO in humans employed a primitive delivery system utilizing a Douglas bag (Pepke-Zaba et al., 1991). Several different systems subsequently were adapted by investigators and varied with the clinical circumstance, patient characteristics, and duration of treatment (Miller et al., 1994a; Wessel et al., 1994). Modifications have allowed delivery of NO into continuous flow ventilators (Betit et al., 1995), high frequency ventilators and continuous positive airway pressure systems (Kinsella and Abman, 1993), a ventilator nebulizer to deliver NO during only inspiration (Rossaint et al., 1993), and pulsed delivery in spontaneously ventilating patients (Charmick et al., 1996). Systems specifically designed for transportation of patients dependent on NO have also been described (Kinsella et al., 1995a; Goldman et al., 1995b; Dhillon et al., 1996). An ideal delivery system uses medical grade quality gas manufactured by a Food and Drug Administration approved process. It minimizes the duration of gas in the delivery circuit, can deliver a wide range of precise NO doses with uniform mixing despite variable flow rates. It should have on-line analysis

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Figure 26±7. Microprocessor controlled, mass flow regulated delivery and monitoring system. Nitric oxide (NO) is delivered through a pressure stabilizing tank to a NO gas module. The desired concentration of NO is set by a knob on the front of the ventilator. The flow in the NO gas module is microprocessor controlled along with air and oxygen gas modules (Lindberg et al., 1997).

of NO, nitrogen dioxide and oxygen; incorporate stringent controls for exhaled gases; and have alarms to protect against excessive dosing or inadvertent discontinuation. Because rebound pulmonary hypertension (Atz et al., 1996b; Miller et al., 1995) or respiratory collapse (Lavoie et al., 1996; Goldman et al., 1996b) after prolonged inhalation of NO in some patients represents an additional hazard of abrupt interruption of NO delivery, an appropriate back up supply of NO must be in place. The system should be adaptable to different clinical situations, oxygen and NO concentrations should be independently controlled, and when used in conjunction with mechanical ventilation should not interfere with ventilator functions. Commercial products are now available that utilize mass flow controller technology capable of rapid and precise regulation and mixing of NO, oxygen, and air gas flows (Lindberg et al., 1997) (Figure 26–7). When integrated into a microprocessor-governed, flow-sensing circuit these devices promise to markedly improve the variability and precision of “homemade” systems enabling the set NO concentration to remain

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constant during the dynamic flow of a single breath regardless of flow or ventilatory mode. They may be contained within standard ventilator housing with two separate control panels (oxygen and NO) directing output for the three relevant modules (air, oxygen, NO). Alternatively more flexible systems, similarly controlled, are available to function in series with the most common mechanical ventilators. Dosage Measurements of NO and nitrogen dioxide currently are made just proximal to the airway. The alveolar concentration of these gases following therapeutic inhalation is unknown. Nitric oxide uptake by bronchial epithelium and lung fluid may affect final alveolar concentrations and may vary with disease. Because these effects are still not clarified, establishment of lowest effective dosage therapy is important to minimize possible toxicities. Animal work using experimental models of pulmonary hypertension and preliminary adult human investigations yield conflicting results with greatly varying conclusions regarding the dose of NO required to produced either a threshold response or 50 percent of maximal reduction in pulmonary artery pressure (Rossaint et al., 1993; Sitbon et al., 1995; Gerlach et al., 1993; Puybasset et al., 1994; Finer et al., 1994; Day et al., 1995; Lonnqvist et al., 1995). Both animal and human studies have suggested that there is a dose response relationship with regards to maximal pulmonary vasodilation up to doses of 80 or more ppm (DeMarco et al., 1996; Rich et al., 1993b; Berger et al., 1993; Dyar et al., 1993; Roberts et al., 1993). Optimal dosing of inhaled NO which will maximize pulmonary vascular relaxation without incurring toxic side effects, systemic hypotension, or deleterious effects on venous admixture is unclear. Maximal pulmonary vasodilator response to inhaled NO may occur at higher doses than that which produce optimal ventilation perfusion matching in patients with elevated pulmonary artery pressure and severe pulmonary parenchymal disease (Maruyama et al., 1995). By redistributing pulmonary blood flow away from under-ventilated alveoli toward better ventilated areas of lung, inhaled NO in very low concentrations (< 1 ppm) may improve intrapulmonary shunt fraction and raise PaO2. This effect may be optimized at doses of inhaled NO that are low (1–10 ppm) even though maximal pulmonary vasodilation occurred in the same patients at higher NO doses (10–100 ppm) (Gerlach et al., 1993). Improved oxygenation was lost at the higher NO doses although pulmonary vasodilation was maximized. Presumably this occurred from a “spillover” effect of NO into poorly ventilated lung with loss of preferential delivery to and vasodilation of better ventilated areas. No data are available for childhood diseases comparing the NOinduced dose response changes in oxygenation simultaneously with changes in pulmonary vascular resistance. This is especially important in the critically ill population of children with acute severe pulmonary parenchymal disease that complicates their pulmonary hypertensive congenital heart disease where dose response may be quite variable (Buhrer et al., 1995; Lonnqvist et al., 1995). In 17 neonatal and pediatric patients with ARDS treated with NO the best effective dose was 10 ppm in pediatric patients and 20 ppm in neonatal patients with ARDS plus PPHN (Demirakca et al., 1996). Thus, the desirable dose may depend in part on the degree of the pulmonary artery hypertension versus the severity of intrapulmonary shunting from lung disease. Monitoring Broad guidelines for the clinical use of inhaled NO and the monitoring of nitrogen dioxide toxicity have emerged from a National Heart Blood and Lung Institute conference (Zapol et al., 1994). Chemiluminescent and electrochemical analyzers are the two methods of NO and nitrogen dioxide analysis

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that are most widely used in experimental protocols (Fajardo et al., 1995; Betit et al., 1996; Strauss et al., 1996). Electrochemical analyzers are simpler to operate, inexpensive and portable, and are increasingly reliable for clinical monitoring. Nitric oxide and nitrogen dioxide analysis can also be obtained from more complicated infrared and mass spectrometry methods (Stenqvist et al., 1993; Etches et al., 1995). Toxicity At the relatively low levels of NO used therapeutically (1–80 ppm), the metabolic fate of inhaled NO is an accumulation of nitrate and nitrite in plasma, a small increase in methemoglobin but little detectable nitrosylhemoglobin (Jacob et al., 1994; Young et al., 1996). Possible toxicities of inhaled NO include methemoglobinemia due to the intravascular binding to hemoglobin (Rimar and Gillis, 1993), cytotoxic effects in the lung due to either free radical formation, development of excess nitrogen dioxide, peroxynitirite production (Radi et al., 1991), or injury to the pulmonary surfactant system (Matalon et al., 1996; Hallman et al., 1996). Carcinogenic (Nguyen et al., 1992) and teratogenic potential of inhaled NO exist as well as effects on glutathione metabolism, unknown effects on immature or immunocompromised lung, potential interaction with other heme containing proteins, and effects on platelet function and hemostasis. Methemoglobin Approximately 80–90% of inhaled NO is absorbed into the bloodstream and reacts with hemoglobin within the erythrocyte to form nitrosylhemoglobin and methemoglobin from which nitrites are generated by oxidation (Yoshida and Kasama, 1987). In a study of healthy volunteers, NO was inhaled at 32 to 512 ppm for 3 hours. There appeared to be a dose dependent increase in methemoglobin, but doses of 128 ppm or less never resulted in methemoglobin levels > 5%. Assuming a model of first order pharmacokinetics it was predicted that at a dose of 128 ppm methemoglobin concentrations will plateau at 3.5–4.0% (Young et al., 1994) This would represent a clinically insignificant reduction in oxygen carrying capacity of the blood. This data cannot be extrapolated to ill patients or to neonates because newborn humans, unlike many other mammals, have a reduced NADHmethemoglobin reductase activity compared with adults (Chun-Lap Lo and Agar, 1986; Choury et al., 1983). Preliminary data would indicate that methemoglobinemia may be a rare but important consequence of NO treatment. Methemoglobin levels > 5% were seen in 4 of 123 patients in one study. One patient who received continuous NO at 80 ppm responded to a vitamin C injection and blood transfusion, all others responded to a decrease in the NO dose alone (Wessel et al., 1994). Elevated methemoglobin levels in patients with PPHN were seen only among patients randomised to receive the highest concentration of NO (80 ppm). (Davidson, 1998) Nitrogen dioxide Recognition of the environmental hazards posed by the higher oxides of nitrogen both in the atmosphere and the workplace has prompted numerous laboratory, clinical, and epidemiologic studies. Although inhalation of nitrogen dioxide at doses as low as 2 ppm has been associated with terminal bronchial epithelium hypertrophy and alveolar cell hyperplasia in animals (Shiel, 1967), studies in humans have not generally demonstrated significant alterations in pulmonary mechanics when inhaled for brief periods of time (Frampton et al., 1991). Nitrogen dioxide causes histologie changes to the lung at doses of 25 ppm and in animals doses of 5000 ppm have produced pulmonary edema, hemorrhage, and death (Greenbaum et al.,

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1967). The rate of conversion of NO into nitrogen dioxide is determined by the concentrations of NO and oxygen and their contact or resident time; conversion is accelerated as higher concentrations are used and influenced by temperature and humidity (Foubert et al., 1992; Miyamoto et al., 1994; Nishimura et al., 1995). Absorption of nitrogen dioxide by soda lime has been useful but complicates delivery of precise concentrations of NO (Ishibe et al., 1996; Pickett et al., 1994). Safety limits of 5 ppm of nitrogen dioxide have been set by the Occupational Safety and Health Administration (CDC, 1988). Fifty to sixty percent of nitrogen dioxide is retained within the lung. Once absorbed, it remains within the lung for prolonged periods and reacts with water to form nitric and nitrous acids which are thought to be responsible for the pulmonary toxicity of nitrogen dioxide (Goldstein et al., 1977). Severe left ventricular dysfunction Caution should be exercised when administering NO to patients with severe left ventricular dysfunction and pulmonary hypertension. In adults with ischemic cardiomyopathy, sudden pulmonary vasodilation may occasionally unload the right ventricle sufficiently to increase pulmonary blood flow and harmfully augment preload in a compromised left ventricle (Hayward et al., 1996; Semigran et al., 1994; Loh et al., 1994). The attendant rise in left atrial pressure may produce pulmonary edema (Bocchi et al., 1994). This is not likely to arise from any negative inotropic effect of NO (Hare et al., 1997) and may be ameliorated with vasodilators or diuretics. A different but related phenomenon may be operative in the newborn with severe left ventricular dysfunction and pulmonary hypertension. In these patients the systemic circulation may depend in part on the ability of the right ventricle to sustain cardiac output through a right to left shunt across the patent ductus arteriosus. Selective pulmonary vasodilation may redirect the right ventricular output to the lungs and away from the systemic circulation. Therefore in newborns with severe left ventricular dysfunction, predominantly left to right shunting at the foramen ovale and exclusively right to left shunting at the ductus arteriosus, NO should be used with extreme caution, if at all (Henrichsen et al., 1996; Wessel et al., 1997; Beghetti et al., 1997). Rebound pulmonary hypertension Appreciation of rebound pulmonary hypertension and its transient characteristic (Figure 26–8) may facilitate weaning from NO and has important implications for patients with persistent pulmonary hypertensive disorders when interruption of NO is necessary (Atz et al., 1996b; Francoise et al., 1996). If the underlying pulmonary hypertensive process has not resolved, then the tendency for an abrupt increase in pulmonary artery pressure may be hazardous if NO therapy must be withdrawn or interrupted (Grover et al., 1992; Miller et al., 1995; Lavoie et al., 1996). If withdrawal of NO is necessary before resolution of the pathologic process, hemodynamic instability may be expected. If a labile patient with pulmonary hypertension is stabilized with NO prior to transfer to a specialized center for further management, NO should be available during patient transport. The reason for this rebound phenomenon is unclear but may involve suppression of endogenous production of NO from the pulmonary endothelium (Goldman et al., 1996b). Negative feedback inhibition by NO has been demonstrated for inducible (Assreuy et al., 1993) and endothelial (Ravichandran et al., 1995) nitric oxide synthase. Alternatively inhaled NO may play an unknown role in the modulation of endogenous pulmonary vasoconstrictors or alter membrane receptor conformation in vascular smooth muscle which could account for withdrawal effects.

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Figure 26±8. A bedside recording of pulmonary artery (PA) pressure, with systolic, diastolic and mean values in mmHg. After prolonged therapy with NO at 10 ppm, withdrawal results in a transient rebound pulmonary hypertension which resolves without intervention within 15 minutes.

Coagulopathy Initial animal reports suggesting an increased bleeding time during NO inhalation (Hogman et al, 1994a), have not proven clinically important in humans. Vasodilation caused by increased circulating cGMP may also result in increased cGMP levels in platelets resulting in reduced platelet adhesion and aggregability (Malmros et al., 1996). Studies in healthy volunteers show mild if any attenuation of platelet function with NO inhalation (Albert et al, 1996). Inhibition of platelet aggregation has in fact been suggested as an additional beneficial effect of NO in respiratory failure (Samama et al., 1995) or pulmonary embolism (Gries et al, 1997). REFERENCES Abman, S.H., Chatfield, B.A., Hall, S.L. and McMurtry, I.F. (1990) Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am. J. Physiol., 259, H1921-HI927. Abman, S.H. and Kinsella, J.P. (1995) Inhaled nitric oxide for persistent pulmonary hypertension of the newborn. The physiology matters! Pediatrics, 1153–1155. Adatia, I., Thompson, J., Landzberg, M. and Wessel, D.L. (1993) Inhaled nitric oxide in chronic obstructive lung disease. Lancet, 341, 307–308. Adatia, I., Lillehei, C., Arnold, J.H., Thompson, J.E., Palazzo, R., Fackler, J.C. and Wessel, D.L. (1994) Inhaled nitric oxide in the treatment of postoperative graft dysfunction after lung transplantation. Ann. Thorac. Surg., 57, 1311–1318. Adatia, I., Perry, S., Landzberg, M., Moore, P., Thompson, J.E. and Wessel, D.L. (1995) Inhaled nitric oxide and hemodynamic evaluation of patients with pulmonary hypertension before transplantation. J. Am. Coll. Cardiol. 25, 1656–1664. Adatia, I., Atz, A.M., Jonas, R.A. and Wessel, D.L. (1996) Diagnostic use of inhaled nitric oxide after neonatal cardiac operations. J. Thorac. Cardiovasc. Surg., 112, 1403–1405. Adatia, I. and Wessel, D.L. (1994) Therapeutic use of inhaled nitric oxide. Current Opinion in Pediatrics, 6, 583–590. Addonizio, L.J., Gersony, W.M., Robbins, R.C., Drusin, R.E., Smith, C.R., Reison, D.S., Reemtsma, K. and Rose, E.A. (1987) Elevated pulmonary vascular resistance and cardiac transplantation. Circulation, 76 (suppl. V), V-52–V-55.

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Ahluwalia, J.S., Kelsall, A.W.R., Raine, J., Rennie, J.M., Mahmood, M., Oduro, A., Latimer, R., Pickett, J. and Higenbottam, T.W. (1994) Safety of inhaled nitric oxide in premature neonates (letter). Acta Paediatr., 83, 347–348. Albert, J., Wallen, N.H., Broijersen, A., Frostell, C. and Hjemdahl, P. (1996) Effects of inhaled nitric oxide compared with aspirin on platelet function in vivo in healthy subjects. Clinical Science, 91, 225–231. Alving, K., Weitzberg, E. and Lundger, J.M. (1993) Increased amount of nitric oxide in exhaled air of asthmatics. Eur. Respir. J., 6, 1368–1370. Armitage, J.M., Fricker, F.J., Kurland, G., Hardesty, R.L., Michaels, M., Morita, S., Starzl, T.E., Yousem, S.A., Jaffe, R. and Griffith, B.P. (1993) Pediatric lung transplantation the years 1985 to 1992 and the clinical trial of FK506. J.Thorac. Cardiovasc. Surg., 105, 337–346. Assreuy, J., Cunha, F.Q., Liew, F.Y. and Moncada, S. (1993) Feedback inhibition of nitric oxide synthase activity by nitric oxide. Eur. J. Pharmacol, 108, 833–837. Atz, A.M., Adatia, I., Jonas, R.A. and Wessel, D.L. (1996a) Inhaled nitric oxide in children with pulmonary hypertension and congenital mitral stenosis. Am. J. Cardiol, 11, 316–319. Atz, A.M., Adatia, I. and Wessel, D.L. (1996b) Rebound pulmonary hypertension after inhalation of nitric oxide. Ann. Thorac. Surg., 62, 1759–1764. Atz, A.M. and Wessel, D.L. (1997a) Inhaled nitric oxide in the neonate with congenital heart disease. Seminars in Perinatology, 21, 441–455. Atz, A.M. and Wessel, D.L. (1997b) Inhaled nitric oxide in sickle cell disease with acute chest syndrome. Anesth., 87, 988–990. Atz, A.M. and Wessel, D.L. (1997c) Delivery and monitoring of inhaled nitric oxide. Current Opinion in Critical Care., 3, 243–249. Atz, A.M. and Wessel, D.L. (1998) Inhaled nitric oxide and heparin for infantile primary pulmonary hypertension. Lancet, 351, 1701. Auler, J.J., Carmona, M., Bocchi, E., Bacal, F., Fiorelli, A., Stolf, N. and Jatene, A. (1996) Low doses of inhaled nitric oxide in heart transplant recipients. J. Heart Lung Transplant., 15, 443–450. Bacha, E., Herve, P., Murakami, S., Chapelier, A., Mazmanian, G., de Montpreville, V., Detruit, H., Libert, J. and Dartevelle, P. (1996) Lasting beneficial effects of short-term inhaled nitric oxide on graft function after lung transplantation. J. Thorac. Cardiovasc. Surg., 112, 590–598. Bando, K., Konishi, H., Komatsu, K., Fricker, F.J., Del Nido, P.J., Francalancia, N.A., Hardesty, R.L., Griffith, B.P. and Armitage, J.M. (1993) Improved survival following pediatric cardiac transplantation in high-risk patients. Circulation, 88(part 2), 218–223. Barst, R.J., Rubin, L.J., McGoon, M.D., Caldwell, E.J., Long, W.A. and Levy, P.S. (1994) Survival in primary pulmonary hypertension with long-term continuous intravenous prostacyclin. Ann. Intern. Med., 121, 409– 415. Bartlett, R., Roloff, D., Cornell, R., Andrews, A., Dillon, P. and Zwischenberger, J. (1985) Extracorporeal circulation in neonatal respiratory failure: a prospective randomized study. Pediatrics, 76, 479–487. Beghetti, M., Habre, W., Friedli, B. and Berner, M. (1995) Continuous low dose inhaled nitric oxide for treatment of severe pulmonary hypertension after cardiac surgery in paediatric patients. Br. Heart J., 73, 65–68. Beghetti, M., Berner, M. and Rimensberger, P.C. (1997) Inhaled nitric oxide can cause severe systemic hypotension. J. Pediatr., 844. Behrendt, D.M., Crowley, D. and Uzark, K. (1986) Potential for reversibility of pulmonary vascular obstructive disease in children after cardaic transplantation. Am. J. Cardiol., 58, 1124–1126. Benzing, A., Brautigam, P., Geiger, K., Loop, T., Beyer, U. and Moser, E. (1995) Inhaled nitric oxide reduces pulmonary transvascular albumin flux in patients with acute lung injury. Anesth., 83, 1153–1161. Benzing, A., Mois, G., Brieschal, T. and Geiger, K. (1997) Hypoxic pulmonary vasoconstriction in nonventilated lung areas contributes to differences in hemodynamic and gas exchange responses to inhalation of nitric oxide. Anesth., 86, 1254–1261. Benzing, A. and Geiger, K. (1994) Inhaled nitric oxide lowers pulmonary capillary pressure and changes longitudinal distribution of pulmonary vascular resistance in patients with acute lung injury. Acta Anaesthesiol. Scand.. 38, 640–645.

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Berger, J.I., Gibson, R.L., Redding, G.J., Standaert, T.A., Clarke, W.R. and Truog, W.E. (1993) Effect of inhaled nitric oxide during group B streptococcal sepsis in piglets. Am. Rev. Respir. Dis., 147, 1080–1086. Berner, M., Beghetti, M., Ricou, B., Rouge, J.C., Prêtre, R. and Friedli, B. (1993) Relief of severe pulmonary hypertension after closure of a large ventricular septal defect using low dose inhaled nitric oxide. Intensive Care Med., 19, 75–77. Berner, M., Beghetti, M., Spahr-Schopfer, I., Oberhansli, I. and Friedli, B. (1996) Inhaled nitric oxide to test the vasodilator capacity of the pulmonary vascular bed in children with long-standing pulmonary hypertension and congenital heart disease. Am. J. Cardiol., 77, 532–535. Betit, P., Adatia, I., Benjamin, P., Thompson, J.E. and Wessel, D.L. (1995) Inhaled nitric oxide: evaluation of a continuous titration delivery technique developed for infant mechanical ventilation and manual ventilation. Resp. Care, 40, 706–715. Betit, P., Grenier, B., Thompson, J. and Wessel, D. (1996) Evaluation of four analyzers used to monitor nitric oxide and nitrogen dioxide concentrations during inhaled nitric oxide administration. Resp. Care, 41, 817– 825. Bigatello, L.M., Hurford, W.E., Kacmarek, R.M., Roberts, J.D.J. and Zapol, W.M. (1994) Prolonged inhalation of low concentrations of nitric oxide in patients with severe adult respiratory distress syndrome. Anesth., 80, 761–770. Bocchi, E.A., Bacal, F., Auler, J.O.C., de Carvalho Carmone, M.J., Bellotti, G. and Pileggi, F. (1994) Inhaled nitric oxide leading to pulmonary edema in stable severe heart failure. Am. J. Cardiol., 74, 70–74. Body, S.C., Hartigan, P.M., Shernan, S.K., Formanek, V. and Hurford, W.E. (1995) Nitric oxide: delivery, measurement, and clinical application. J. Cardiothorac. Vasc. Anesth., 9, 748–763. Bottiger, B.W., Motsch, J., Dorsam, J., Mieck, U., Gries, A., Weimann, J. and Martin, E. (1996) Inhaled nitric oxide selectively decreases pulmonary vascular resistance following acute massive pulmonary microembolism in piglets. Chest, 110, 1041–1047. Buch, J. and Wennevold, A. (1981) Hazards of diazoxide in pulmonary hypertension. Br. Heart J., 46, 401– 403. Buhrer, C., Merker, G., Falke, K., Versmold, H. and Obladen, M. (1995) Dose-response to inhaled nitric oxide in acute hypoxemic respiratory failure of newborn infants: a preliminary report. Pediatr. Pulmonol., 19, 291–298. Bush, A., Busst, C., Knight, W.B. and Shinebourne, E.A. (1987) Modification of pulmonary hypertension secondary to congenital heart disease by prostacyclin therapy. Am. Rev. Respir. Dis., 136, 767–769. Calhoon, J.H., Grover, F.L., Gibbons, W.J., Bryan, C.L., Levine, S.M., Bailey, S.R., Nichols, L., Lum, C. and Trinkle, J.K. (1991) Single lung transplantation——alternative indications and techniques. J. Thorac. Cardiovasc. Surg., 101, 816–825. Castaneda, A.R., Mayer, J.E., Jonas, R.A., Lock, J.E., Wessel, D.L. and Hickey, P.R. (1989) The neonate with critical congenital heart disease: repair-a surgical challenge. J. Thorac. Cardiovasc. Surg., 98, 869–875. CDC (1988) NIOSH recommendations for occupational safety and health standards. MMWR, 37, 21. Celermajer, D.S., Cullen, S. and Deanfield, J.E. (1993) Impairment of endothelium-dependent pulmonary artery relaxation in children with congenital heart disease and abnormal pulmonary hemodynamics. Circulation, 87, 440–46. Celermajer, D.S., Dollery, C., Burch, M. and Deanfield, J.E. (1994) Role of endothelium in the maintenance of low pulmonary vascular tone in normal children. Circulation, 89, 2041–2044. Channick, R.N., Hoch, R.C., Newhart, J.W., Johnson, F.W. and Smith, C.M. (1994) Improvement in pulmonary hypertension and hypoxemia during nitric oxide inhalation in a patient with end-stage pulmonary fibrosis. Am. J. Respir Crit. Care Med., 149, 811–814. Channick, R.N., Newhart, J.W., Johnson, F.W., Williams, P.J., Auger, W.R., Fedullo, P.E and Moser, K.M. (1996) Pulsed delivery of inhaled nitric oxide to patients with primary pulmonary hypertension. Chest, 109, 1545– 1549. Cheifetz, I.M., Craig, D.M., Kern, F.H., Black, D.R., Hillman, N.D., Greeley, W.J., Ungerleider, R.M., Smith, P.K. and Meliones, J.N. (1996) Nitric oxide improves transpulmonary vascular mechanics but does not change intrinsic right ventricular contractility in an acute respiratory distress syndrome model with permissive hypercapnia. Crit. Care Med., 24, 1554–1561.

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27 Nitric Oxide Synthase Inhibitors John F.Parkinson1, James J.Devlin2 and Gary B.Phillips2 1

Departments of Immunology and 2Pharmaceuticals Discovery, Berlex Biosciences Inc., 15049 San Pablo Ave., Richmond, CA 94804±0099, USA

INTRODUCTION Research on the L-arginine / nitric oxide (NO·) pathway has dramatically increased our understanding of fundamental physiological processes involved in regulating vascular tone, neurotransmission and immune function (Nathan, 1992; Feldman et al., 1993; Bredt and Snyder, 1994). NO- itself is a very simple molecule, yet understanding its signaling functions and its use by the immune / host defense system as a destructive tool continues to confront us with new challenges. This complexity derives from the exquisite chemical reactivity of NO- with a plethora of biological targets, including molecular oxygen, superoxide, transition metal centers and sulfhydryl groups (Stamler et al., 1994). Given the complexity of NO· chemistry and biology, it comes as no surprise that cells and tissues have developed equally complex genetic and enzymatic mechanisms for the biosynthesis of NO- in order to maintain tight control on its functions. Three major isoforms of nitric oxide synthase (NOS) are expressed in mammalian systems and they are derived from distinct genes: neuronal NOS (NOS-1), cytokineinducible NOS (NOS-2) and endothelial NOS (NOS-3). The enzymology and the molecular biology of the major NOS isoforms are reviewed in detail by other contributors to this book and have previously been reviewed by the authors (Parkinson and Phillips, 1997). The initial development of NO· research made tremendous strides through the use of non-selective NOS inhibitors, principally the substrate-based L-arginine analogs, N-methylL-arginine and N-nitro-L-arginine. The need, however, for better pharmacological tools with which to study the NO· pathway both in vitro and in vivo and also the tremendous potential therapeutic value of NOS inhibitors in an extraordinarily broad array of clinical settings, has provided the impetus both in academia and in industry for the discovery of

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potent and isoform-specific NOS inhibitors. The purpose of this Chapter is to review the most recent advances in the development of these inhibitors. The reader is referred to additional chapters in this book which extensively review the pharmacological and potential therapeutic applications of some of these inhibitors. APPROACHES TO NOS INHIBITORS Detailed enzymological studies and structure-function analyses of the major NOS isoforms have provided a number of mechanistic approaches to achieving NOS inhibition, which are summarised in Table 27–1. The purpose of the current Chapter is not to review all possible approaches to NOS inhibition since some of them provide non-selective compounds of little pharmacological value e.g. non-specific calmodulin antagonists and iodonium-based flavoprotein reductase inhibitors (reviewed by Nathan, 1992). The focus of this article will be on mechanism-based inhibitors of NOS since these provide the most likely basis for achieving a high degree of target enzyme specificity and thus a greater degree of pharmacological specificity and utility. All NOS isoforms catalyze the conversion of L-arginine to L-citrulline and NO- through N-hydroxy-Larginine (N-OH-L-arginine), as shown in Scheme 27–1 below:

Scheme 27–1. Electron transfer in NOS. The NOS isoforms are dimeric, self-contained electron transfer enzymes in which reducing equivalents are provided by NADPH and transferred via the FAD and FMN-containing flavoprotein reductase domain to a heme-containing oxygenase (“catalytic”) domain, as shown in Scheme 27–2 below: Scheme 27–2. Stoichiometry of the NOS Reaction. Electron transfer from the reductase domain to the catalytic domain of NOS dimers is reversibly regulated by Ca2+/calmodulin in NOS-1 and NOS-3 whereas in NOS-2 electron transfer is “continuous” due to the presence of tightly bound calmodulin as an enzyme “subunit” (reviewed in Parkinson and Phillips, 1997). The role of the NOS catalytic domain and the heme catalytic center in both steps of the overall reaction described in Scheme 27–1 is now well accepted (White and Marletta, 1992; Stuehr and Ikeda-Saito, 1992; McMillan et al., 1992) For example, both steps in Scheme 27–1 are inhibitable by carbon monoxide, a wellknown heme poison (Pufahl and Marietta, 1993). These seminal observations regarding the NOS mechanism of catalysis form the basis for two principal approaches to drug discovery of mechanism-based NOS inhibitors, namely: (i) direct enzyme inhibitors based on the substrate L-arginine e.g. guanidino- and isothiourea-containing compounds and (ii) direct enzyme inhibitors based on ligation of the catalytic heme center e.g. imidazole-containing compounds. The latter approach stems directly from the similarity between NOS and cytochrome P450 active site chemistry and mechanism (see Parkinson and Phillips, 1997). Although NOS isoforms were identified as high affinity BH4-binding and BRrdependent enzymes quite some time ago, a clear catalytic role for BH4 in either of the steps described in Scheme 27–1 has remained

Correspondence: Dr. J.F. Parkinson, Department of Immunology, Berlex Biosciences Inc., 15049 San Pablo Ave., Richmond, CA 94804–0099, USA.

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Table 27±1. Approaches to NOS inhibition.

elusive (reviewed in Parkinson and Phillips, 1997). The prevailing evidence suggests that BH4 behaves primarily as an allosteric regulator of NOS activity via promoting L-arginine binding (Klatt et al., 1994), kinetic enhancement and stabilisation of NOS dimer formation (Baek et al., 1993), preventing NOS autoinactivation (Griscavage et al., 1994) and coupling NADPH oxidation to NO· biosynthesis

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(thereby avoiding uncoupled O2–formation). Despite this lack of direct evidence for BH4 involvement in NOS catalysis, it cannot be excluded that BH4 is required for some hitherto unanticipated role in catalysis, such as stabilisation of the enzyme-bound reaction intermediate N-OH-L-arginine at the active site during enzyme turnover. The allosteric role of BH4 as a NOS regulator has provided sufficient stimulus to pursue BH4 antagonists as potential NOS inhibitors via either (i) direct BH4-binding antagonists to NOS or (ii) indirect inhibitors of BH4 via inhibition of the de novo or salvage pathways for BH4 synthesis in cells. An example of the first approach is 4-amino-BH4, a recently described potent antagonist of BH4 binding to NOS (Werner et al., 1996). An example of the second approach is 2,4-diamino-6-hydroxypyrimidine phosphate (DAHP) which has been used to demonstrate the dependence of NOS-2 formation and NOproduction in smooth muscle cells on co-induction of GTP cyclohydrolase-1 and de novo BH4 synthesis (Gross and Levi, 1992) The development of isoform-specific BH4-based NOS antagonists is not as advanced as the direct enzyme inhibitor approach. This likely stems from three major impediments to drug discovery efforts in this area: (i) the very high binding affinity (low nM) of NOS for BH4 compared to the relatively weak binding affinity (low μM) for substrate, (ii) the complexities of pterin chemistry and (iii) significant questions regarding the specificity of this approach vis-à-vis other BH4-dependent enzymes e.g. the amino acid hydroxylases whose deficiency is known to cause clinical problems and dihydropteridine reductase which is important for BH4 re-cycling (see Werner et al., 1996). This area remains promising for future research and may yet lead to novel approaches to NOS antagonism. The following sections will focus on the direct enzyme inhibitor approach for which there has been considerably more progress and a greater source of published literature and patents. MECHANISM-BASED NOS INHIBITORS The active site of NOS is unique in biological systems as far as we know. It is reasonable to assume that the safety of a therapeutic will be related to the degree to which it is specific for its target versus other systems. For this reason, drug discovery efforts in the field of NOS inhibitors have focused on mechanism-based enzyme inhibitors directed at the active site of NOS. Recently, significant progress has been made on the discovery of potent NOS inhibitors with reasonable selectivity between isoforms. This section will focus on a review of these developments (for other recent reviews see Parkinson and Phillips, 1997; Southan and Czabo, 1996; Macdonald, 1996a). Arginine Analogs The most widely used inhibitors of NOS are structurally related to the substrate L-arginine. Many compounds in this category have been published, but three stand out as useful tools. N-Methyl-L-arginine (NMA: compound 1) is a non-selective inhibitor with which most other compounds are compared (IC50, human NOS-1/NOS-2/NOS-3:7.1/3.3/5.3 μM) (Faraci et al., 1996). N-Nitro-L-arginine (NNA, compound 2) is selective for NOS-1 inhibition (IC50, human NOS-1/NOS-2/NOS-3: 0.50/7.6/0.50 μM) (Moore et al., 1996). Aminoguanidine (AG; compound 3) is selective for NOS-2 inhibition (IC50, human NOS-1/NOS-2/ NOS-3: 30.3/6.40/22.0 μM) (Faraci et al., 1996). N-Nitro-L-arginine methyl ester (NAME) is an inactive pro-drug that generates NNA (Southan et al., 1995). While these are the most commonly cited NOS inhibitors, they have been previously reviewed (Kerwin et al., 1995; Griffith and Kilbourn, 1996) and will not be discussed further except for comparison purposes.

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Two interesting arginine analogs are L-thiocitrulline (Narayanan and Griffith, 1994; Frey et al., 1994; compound 4) and S-methyl-L-thiocitrulline (Narayanan and Griffith, 1994; Furfine et al., 1994; compound 5). Although similar in potency to NMA for rat NOS-1 and NOS-2 (Narayanan and Griffith, 1994), thiocitrulline binds to NOS to give a type II difference spectrum that is characteristic of a compound coordinating directly to the sixth coordination site of the heme Fe (Frey et al., 1994). The thiourea is not basic and can not form a salt bridge with an acidic residue as can the guanidine of arginine. Thiourea can, however, form hydrogen bonds similar to those made by guanidine. The inactivity of the isosteric product Lcitrulline indicates that the sulfur is critical for binding. The more potent and more basic S-methyl-Lthiocitrulline (compound 5) is also competitive with L-arginine but, unlike L-thiocitrulline, binds NOS to give a type I difference spectrum (as does arginine), indicating it does not directly ligate the heme at the NOS catalytic center. SMethyl-L-thiocitrulline is isosteric with and less basic than NMA, but significantly more potent: Ki, human NOS-1/OS-2/NOS-3:0.0012/0.040/0.011 μM (Furfine et al., 1994). An unspecified sulfur binding may thus be invoked to explain the increased potency of S-methyl-L-thiocitrulline over NMA (see Figure 27–2C). Other S-alkyl thiocitrullines have also been reported (Makino and Nagafuji, 1996). Two compounds, N-(l-iminoethyl)-L-ornithine (McCall et al., 1991; Moore, et al., 1994; compound 6) and compound 7 (Furfine et al., 1994) illustrate the importance of basic nitrogens for NOS inhibitor binding. N-(l-Iminoethyl)-L-ornithine is an inhibitor of NOS with similar potency to NMA: IC50 rat NOS-1 / mouse NOS-2:3.9/2.2 μM. Replacement of the chain nitrogen with sulfur (Furfine et al., 1994) or methylene (Kerwin et al., 1995) results in inactive compounds, indicating that the chain nitrogen is important for binding, presumably through either a hydrogen bond or binding to an acidic residue (see Figure 27–2 A). Replacement of portions of the saturated carbon chain with unsaturation, sulfoxides, or sulf ones give compounds with similar potencies (Shearer et al., 1995; Hodson et al., 1995). Replacement of the acid moiety with a diol (compound 8) results in a more selective compound with similar potency: IC50 human NOS-1/ NOS-2/OS-3:150/12/8,420 μM (Hallinan et al., 1995a; Hallinan et al., 1995b). Interestingly, lengthening the chain of compound 6 by one carbon (compound 9) gives a NOS-2 selective compound: IC50 rat NOS-1/ mouse NOS-2:92/3.3 μM (Moore, et al., 1994). The results from these two compounds indicate that there are at least minor differences in the active sites of these two enzymes. NOS contact sites for the primary amine and carboxyl group of L-arginine have been explored using analogs of the mechanism-based inhibitor N-allyl-L-arginine (compound 10; Olken and Marletta, 1992; Robertson et al., 1995). Against bovine NOS-1, the ester and parent compound have similar potencies, but the acetamide formed by acetylation of the amino acid nitrogen is significantly less potent, indicating that the amine moiety is more important for binding than the acid function. While this conclusion may be true, interpretation of the results of the ester relies on the assumption that the ester is not hydrolyzing to the acid, which was not examined. Alcohols have also been used as substitutes for the acid moiety. Murad et al., have prepared analogs of NNA with the acid replaced with an alcohol and unsaturation in the chain that have NOS-1 selectivity (compound 11): IC50 rat NOS-1/mouse NOS-2:3.0/75 μM (Murad et al., 1995a, 1995b). Alternatively, the -phosphinic and phosphonic acid derivative of NNA and NMA were found to be much less potent than the corresponding amino acid derivatives (Cowart et al., 1996). Amidine-containing Inhibitors This series of compounds is grouped together since they all contain an amidine moiety (C(=NH)NH2), which is also contained within the guanidine moiety of arginine. Experimental evidence suggests some of these compounds are competitive with L-arginine, but evidence has not been reported for all of them. Some

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of these inhibitors may thus have different mechanisms or binding site(s) on NOS. The grouping is simply to illustrate the structural similarity of the inhibitors. One group of compounds reported in several studies is the isothioureas (ITUs; Garvey et al., 1994a. 1994b: Nakane et al., 1995: Bashaw et at., 1995: Macdonald 1996b: Stratman et al., 1996; Honda et al., 1996). It has been proposed that these compounds bind with the SR group pointing towards the heme, the R group in a small hydrophobia pocket and the nitrogens binding to the same site as the non-reacting guanidine nitrogens present in the substrate L-arginine (Garvey et al., 1994a). An upper limit on the bulk accommodated by the small hydrophobic binding pocket can be inferred from compound 12. A two order of magnitude difference in potency exists when comparing R=isopropyl versus isobutyl; for human enzymes the Ki for NOS-1/NOS-2/NOS-3 is 0.037/0.010/0.022 and 6.4/1.3/8.3 μM, respectively. Some bulk seems to be beneficial, however, since R=isopropyl is more potent than R=Me (Ki NOS-1/NOS-2/NOS-3:0.16/0.12/0. 20 μM). Binding studies of compound 12 (R=Et) with mouse NOS-2 indicate a type I difference spectrum. Although these compounds have been reported to be selective: R=Et, rat NOS-1/mouse NOS-2/bovine NOS-3:0.25/0.013/0.37 μM (Nakane et al., 1995) only hints of selectively have been seen using human

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isoforms, illustrating an important caveat when comparing results obtained with NOS inhibitors using enzymes from different species. The Glaxo-Wellcome group has also reported inhibitory bis-ITUs (Garvey et al., 1994a). Compound 13 has selectivity for human NOS-2 (Ki NOS-1/NOS-2/NOS-3:0.25/0.047 / 9.0 μM). In the same series a very potent and partially selective NOS-1 inhibitor was reported (Compound 14: KI NOS-1/NOS-2/NOS-3:0. 0013/0.0087/0.090 μM). The symmetrical nature of these bis-ITUs suggests that this inhibitor series may be binding to both active sites of the dimeric enzyme, but this hypothesis remains unproven. Binding of one

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isothiourea moiety has been proposed to be similar to the binding of the ITU of Compound 12, but the binding of the rest of the bis-ITU may be more interesting, since it is imparting selectivity to the inhibitor. Some cyclic ITUs are also NOS inhibitors (Moore et al, 1996, Garvey et al., 1994a; Basha et al., 1995; Strobel et al., 1996a; Yata et al., 1996; Strobel et al., 1996b; Calaycay et al, 1996). Examples are 2aminothiazole (Compound 15, R=H) and 2-aminothiazoline (Compound 16) which have Ki values for NOS-1/NOS-2/NOS-3 of 12/48/10 and 0.41 / 0.26 / 0.35 μM, respectively. Neither compound has selectivity, but they are competitive with L-arginine. The potency of the thiazole increases significantly by placement of a methyl group in the 5 position (Compound 15, R=Me). Similar cyclic ITUs, with modest selectivity towards NOS-2, have been reported by another group (Nakane et al., 1995; Basha et al., 1995). The most potent and selective thiazine is shown (Compound 17, R=Me, rat NOS-1 / mouse NOS-2/bovine NOS-3:0.034/0.0036/0.15 μM). The methyl group imparted a 100-fold potency enhancement for NOS-2, but the increase against both constitutive isoforms was less (Compound 17, R=H, NOS-1/NOS-2/NOS-3:0.54/0. 30/1.2 µM). The mechanism of 2-amino-5,6-dihydro-4H-1,3-thiazine (compound 17, R=H) was explored and binding proposed to be similar to L-arginine, with possible binding to the dioxygen binding site (Calaycay et al., 1996). The corresponding oxygen heterocycles were significantly less active against NOS-2, as in the case of thiocitrulline, indicating the importance of the sulfur for binding (Basha et al., 1995). A series of acylated cyclic ITUs are also NOS inhibitors (Shah et al., 1996). Cyclic amidines can also inhibit NOS (Hansen et al., 1995; Guthikonda et al., 1996; Hansen et al., 1996a, 1996b; Katsura et al., 1996). The most NOS-2 selective compound reported contained a sevenmembered ring, compound 18 (rat NOS-1/mouse NOS-2:7.8/0.081 μM). Changing the position of the alky1 group had large effects on potency as illustrated in Figure 27–1. Listed is a group of six-membered ring cyclic amidines that differ only in the positioning of the methyl group. As with the ITUs, small steric modifications by differential placement of a methyl group were found to impart large potency changes. 2-Aminopyridines (aromatized cyclic amidines) have been reported to be selective NOS2 inhibitors (Basha et al, 1995, 89; Faraci et al., 1996) An example of a selective compound in this series is 2aminopicoline (Compound 19, NOS-1/NOS-2/NOS-3:0.25/0.025/0.32 μM). While the methyl group enhanced potency by a factor of thirty over 2aminopyridine, further substitution at the 4-position caused a decrease in potency (Basha et al., 1995). Compound 19 was found to be competitive with L-arginine (Faraci et al., 1996). Acylaminopyridines have also been reported to be potent inhibitors of human NOS-2 (Guthikonda et al., 1996). Acyclic amidines have also been reported (Tjoeng et al., 1995; Oplinger et al., 1996; Garvey et al., 1997). An amidine (Compound 20) designed originally from a bis-isothiourea has very high NOS-2 selectivity in purified human enzymes, with selectivities greater than 200 and 5000-fold reported (versus NOS-1 and NOS-3 respectively). This compound shows interesting kinetics in that it has a slow on rate but is a tight binding inhibitor (Garvey et al., 1997). A group of bis-amidines that are very potent and selective inhibitors of NOS-1 has been reported (Macdonald, 1995; Macdonald et al., 1994; Gentile et al., 1995; Macdonald et al., 1996, 1996e). Starting with a weak, non-selective lead, this group has been able to produce very potent and selective compounds (Compound 21 ; Ki NOS-1/NOS-2/NOS-3:0.002/14/0.37 μM) (Macdonald, 1995). While preparing analogs of aminoguanidine and looking for NOS-2 selective compounds, the diaminobenzimidazole (Compound 22) was found to be NOS-1 selective: R=Me, rat NOS-1/human NOS-2/ human NOS-3:5.8/>100/>100 μM (Henley and Tinker, 1995). Substituting an isopropyl group (Compound 22, R=CH(CH3)2) for the methyl “destroyed” activity. A related unsubstituted compound was less active (Compound 22, R=H, IC50 rat NOS-1/human NOS-2/human NOS-3:77/25/>100 μM) and was shown to be competitive with arginine. .

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2-Iminobiotin, compound 23 (R=NH), was investigated as an L-arginine analog and found to weakly inhibit NOS: Ki, rat NOS-1 / mouse NOS-2:38/22 μM (Sup et al., 1994). Biotin (R=O) and thiobiotin (R=S), analogous compounds to L-citrulline and Lthiocitrulline from L-arginine, were both inactive. The acid moiety was found not to be important for binding. Heterocyclic Inhibitors Imidazole (Compound 24) and imidazole-containing compounds are well known for binding hemecontaining enzymes (Cole and Robinson, 1990) and have been shown to inhibit NOS(Wolff et al., 1993a, 1993b, 1994;Mayer et al., 1994; Wolff and Gribin, 1994;Handy et al., 1995; Babbedge et al., 1993; Moore et al., 1993; Chabin et al., 1996). Imidazole is a reversible, arginine-competitive inhibitor of bovine NOS-1 and recombinant human (rH) NOS-2 with modest selectivity: IC50, rH NOS-1/NOS-2/NOS-3:175/59/189 μM (Moore et al, 1993; Chabin et al., 1996). Imidazole also competes with BH4 for binding to rH NOS-2

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Figure 27±1. Activities of six-membered ring analogues. The figure shows the structures of six related analogues of a six-membered ring NOS inhibitor. Inhibitor potencies indicated (nM) are against mouse NOS-2. The figure illustrates that small structural changes, i.e. incorporation and location of a methyl group, cause large differences in inhibitor potency.

and difference spectra indicate that it acts as the sixth ligand of the heme in NOS (Chabin et al., 1996). These results provide indirect evidence that the heme catalytic center, L-arginine and BH4 are oriented close to each other in the NOS active site. Imidazole-based antifungal agents can inhibit NOS-1, with their predominant mode of action arising from a hydrophobic interaction with the calmodulin binding site (Wolff et al., 1993). Not surprisingly, with this mechanism, these compounds do not inhibit mouse NOS-2, which has the calmodulin much more tightly bound (Wolff and Gribin, 1994b). l-(2-Aminoethyl)imidazole shows a three-fold increase in activity for rH NOS-2 over imidazole: 22 μM (Chabin et al., 1996). Another group of imidazoles that have received attention are 1-phenylimidazoles (Wolff and Gribin, 1994; Garvey et al., 1994; Moore et al., 1993; Hoelscher et al., 1997). 1-Phenylimidazole (Compound 25) is a relatively selective inhibitor of NOS-2 over the constitutive isoforms: Ki, GH3 NOS-1/mouse NOS-2/bovine NOS-3:40/0.7/50 μM (Wolff and Gribin, 1994). The selectivity, but not the potency, is retained with rH NOS: 429 / 33 / 611 μM (Garvey et al., 1994). Unlike the unsubstituted compound, l-(trifluoromethylphenyl)-lH-imidazole (Compound 26) is reported to be selective only against NOS-3: IC50, rat NOS-1 /mouse NOS-2/bovine NOS-3:27 / 28 / 1,000 μM (Moore et al., 1993). A limited group of compounds were tested and electron withdrawing groups on the 4 position of the phenyl ring seem to decrease potency against rHNOS-2 signifi-cantly. This is proposed to be due to decreased electron density around the imidazole nitrogen which is interacting with the heme. 1Phenylimidazole has been found to be competitive with BH4 and L-arginine in both rHNOS-2 and a type II difference spectra indicates that it interacts with the heme in a manner analogous to imidazole (Garvey et al., 1994). However, against other isoforms and the same isoform, but different species, the compounds have not been found to be competitive with both arginine and BH4 (Garvey et al., 1994a; Wolff et al., 1994). Chabin et al., (1996) speculate that the isoform and species differences may be due to alternate orientations of the arginine and BH4 binding sites in the active enzyme. Various indazoles are NOS inhibitors and 7-nitroindazole (Compound 27) was reported to be a potent and relatively selective inhibitor of NOS-1: Ki, GHs NOS-1/mouse NOS-2/bovine NOS-3:0.16/1.6/0.8 μM (Babbedge et al., 1993; Moore et al., 1993; Chabin et al., 1996: Moore et al., 1993: Wolff and Gribin. 1994: Bland-Ward and Moore. 1995). The compound did not detectably inhibit NOS-3 activity in vivo while inhibitng the NOS-1 activity. The differences in in vivo versus in vitro selectivity may be due to uptake differences in the corresponding cells (Southan and Szabo, 1996). Interestingly, 7nitroindazole inhibits NOS-1 competitively with respect to arginine and BH4 (Wolff and Gribin, 1994), whereas NOS-2 and NOS-3 were found to be inhibited competitively only with respect to BH4 (Wolff et al., 1994). Wolff and colleagues proposed that the indazole binds to the heme Fe based mainly on the structural similarities to benzimidazole and imidazole, but no spectroscopic evidence was provided to support this hypothesis (Wolff

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et al., 1994). Subsequent studies have found a more potent analog with less selectivity, 3-bromo-7nitroindazole (Compound 28): IC50, rat NOS-1/rat NOS-2/bovine NOS-3: 0.17/0.86/0.62 μM (Bland-Ward and Moore, 1995). Regardless of whether the heterocycle is interacting with the heme, a proximal relationship of the arginine and BH4 binding sites is implicated, and a potential difference of location of these two sites in the isoforms may be utilized for subsequent design of isoform-selective compounds. Active Site Models Currently there is no data about the NOS active site from X-ray crystallographic analysis that can provide clear direction for rational design of NOS inhibitors. What is known from enzymology studies is that the arginine binds very close to the heme. This can be inferred from the numerous enzymological studies which demonstrate perturbation of the heme spectrum by the substrate L-arginine and by L-arginine-based NOS inhibitors, many of which cause a type I substrate-like difference spectrum (see above). In addition, it has also been demonstrated that compounds combining both a heme-binding and a substrate-like moiety can be identified e.g. thiocitrulline (see also above). At present, the only way to build a model of the active site is by using data from the inhibitors that are known to interact there. Arginine-based inhibitors are an excellent place to start, since small modifications have been made to inhibitors that may be extrapolated to small modifications in the developing active site

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(Figure 27–2). Basicity of the guanidine moiety does not seem to be critical, since NNA and S-methyl-Lthiocitrulline, less basic compounds, both bind NOS with high affinity. The chain NH does, however, seem to be important for some hydrogen bonding interaction. The non-reacting nitrogen does not seem to be important for binding since N-(l-Iminoethyl)-L-ornithine is a potent inhibitor. Analysis of data from the ITUs suggests that the reactive nitrogen is located in a hydrophobic pocket that can only tolerate small alkyl groups. The binding of thiocitrulline is slightly different in that the sulfur is ligating the heme directly (Figure 27–2B). Discovery of the ITUs and related compounds suggests the presence of a strong sulfur binding site. Sulfur-containing compounds, such as S-methyl-L-thiocitrulline and the isothioureas (compounds 10–12) bind tightly (Figure 27–2C). The acyclic ITUs may also hydrogen bond to the same site mentioned above for the chain N-H. A common feature of the amidines is the dramatic activity difference noted with minor modifications in the inhibitor structure. The ITUs (Compound 12) show a potency increase from R=methyl to ethyl and then a decrease going to R=propyl. Similar effects of minor modifications have been noted in the aminopyridines and the cyclic amidines, indicating a restricted binding site, at least in the dimensions explored thus far. Interestingly, in overlapping the structures of the aminopyridines and ITUs at the two nitrogens, the 4-position of the aminopyridines can overlap with the R group of the ITUs. 1-Phenylimidazole and related compounds (Figure 27–2D) and 7-nitroindazole present different binding schemes due to their different structures. Both heterocycles are postulated to interact with the heme, but supporting evidence is only available for 1-phenylimidazole. They also seem to present different binding schemes depending on the isoform being investigated. This is implicated by their apparent competition with BH4. Whether binding to the BH4 site imparts competitive binding to the arginine site via an allosteric interaction or direct occupation of the arginine site is not known. Future Directions for NOS Inhibitor Discovery Considerable progress has been made in understanding the structure and function of the family of NOS isoforms and in developing first generation isoform-selective NOS inhibi tors. What is most lacking in this field is the availability of a three-dimensional structure for the active site of NOS. Availability of this information, preferably for all three NOS isoforms, with bound inhibitors at the active site would greatly assist in the rational design of more potent and much more selective mechanism-based NOS inhibitors. Significant progress has been made in developing E. coli and insect cell expression systems for obtaining large quantities of recombinant NOS isoforms and NOS domains suitable for crystallographic studies (McMillan and Masters, 1993; Roman et al., 1995; Gachhui et al., 1997; Chen et al., 1997). These systems have already been used to identify specific glutamate residues involved in the binding of the substrate Larginine and have been used to propose molecular models of the NOS active site (Gachhui et al., 1997; Chen et al., 1997). The elucidation of the three dimensional structures of the isoforms of NOS will undoubtedly result in significant advances in inhibitor design. REFERENCES Babbedge, R.C., Bland-Ward, P.A., Hart, S.L. and Moore, P.K. (1993) Inhibition of rat cerebellar nitric oxide synthase by 7-nitro indazole and related substituted indazoles. Br. J.Pharmacol., 108, 225–228. Bland-Ward, P.A. and Moore, P.K. (1995) 7-Nitro indazole derivatives are potent inhibitors of brain, endothelium and inducible isoforms of nitric oxide synthase. Life Sciences, 57, 131–135. Bredt, O.S. and Snyder, S.H. (1994) Nitric oxide: a physiologic messenger molecule. Ann. Rev. Biochem., 63, 175–195

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Figure 27±2. Models for active site binding interactions of NOS inhibitors. This figure illustrates the proposed binding site of some inhibitors. A) The proposed binding site of arginine and its analogues. The basicity of the guanidine moiety does not seem to be critical for binding, but the chain NH does seem to be important for some hydrogen binding interaction. The non-reacting nitrogen does not seem to be important for binding. B) The proposed binding site of thiocitrulline. The binding of thiocitrulline is slightly different than arginine in that the sulfur is bonded directly to the heme. C) Proposed binding of isothioureas (ITUs). The acyclic ITUs, like arginine, may also hydrogen bond to the same site mentioned above for the chain NH. D) Proposed binding of phenylimidazole. Phenylimidazole has been postulated to interact with the heme. It has also been found to be competitive with BH4 and arginine in some isoforms indicating possible interaction with both of these binding sites. Basha, F.Z., et al, (1995) Presented at the 210th National Meeting of the American Chemical Society, Chicago, IL; Aug., 1995paper MEDI 244. Calaycay, J.R., Kelly, T.M., MacNaul, K.L., McCauley, E.D., Qi, H., Grant, S.K., Griffin, P.R., Klatt, T., Raju, S.M., Nussler, A.K., Shah, S., Weidner, J.R., Williams, H.R., Wolfe, G.C., Geller, D.A., Billiar, T.R., MacCoss, M., Mumford, R.A., Tocci, M.J., Schmidt, J.A., Wong, K.K. and Hutchinson, N.I. (1996) Expression and immunoaffinity purification of human inducible nitric oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-l,3-thiazine. J. Biol Chem., 271, 28212–28219. Chabin, R.M.; McCauley, E., Calaycay, J.R., Kelly, T.M., MacNaul, K.L., Wolfe, G.C., Hutchinson, N.I., Madhusudanaraju, S., Schmidt, J.A., Kozarich, J.W. and Wong, K.K. (1996) Acive-site Structure Analysis of Recombinant Human Inducible Nitric Oxide Synthase Using Imidazole. Biochemistry, 35, 9567–9575. Chen, P.P., Tsai, A.L., Berka, V. and Wu, K.K. (1997) Mutation of Glu-361 in human endothelial nitric-oxide synthase selectively abolishes L-arginine binding without perturbing the behavior of heme and other redox centers. J. Biol. Chem., 272(10), 6114–6118

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Cole, P.A. and Robinson, C.H. (1990) Mechanism and inhibition of cytochrome P-450 aromatase. J. Med. Chem., 33, 2933–2942. Cowart, M., Kowaluk, E.A., Kolhaas, K.L., Alexander, K.M., and Kerwin, J.F., Jr. (1996), Synthesis of Phosphoruscontaining Amino Acid Analogs as Inhibtors of Nitric Oxide Synthase. Biorg. Med. Chem. Lett., 6, 999–1002. Esser, C.K., Hagmann, W.K., Hoffman, W.F., Shah, S.K., Wong, K.K., Chabin, R.M., Guthikonda, R.K., Maccoss, M., Caldwell, C.G., and Durette, P.L. (1996) Substituted 2-Aminopyridines as Inhibitors of Nitric Oxide Synthase. WO patent 96/18616. Faraci, W.S., Nagel, A.A., Verdries, K.A., Vincent, L.A., Xu, H., Nichols, L.E., Labasi, J.M., Salter, E.D. and Pettipher, E.R. (1996) 2-Amino–-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo. Br. J. Pharmacol, 119, 1101–1108. Feldman, P.L., Griffith, O.W. and Stuehr, D.J. (1993) The Surprising Life of Nitric Oxide. Chem. Eng. News, 71, 26–38 Frey, C, Krishnaswamy, N., McMillan, K., Spack, L., Gross, S.S., Masters, B.S., and Griffith, O.W. (1994) LThiocitrulline, J. Biol. Chem., 269, 26083–26091. Furfine, E.S., Harmon, M.F., Paith, J.E., Knowles, R.G., Salter, M., Kiff, R.J., Duffy, C., Hazelwood, R., Oplinger, J.A. and Garvey, E.P (1994) Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline. J. Biol. Chem., 269, 26677–26683. Gachhui, R., Ghosh, O.K., Wu, C., Parkinson, J.F., Crane, B.R. and Stuehr, D.J. (1997) Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding. Biochemistry, 36, 5097–5103 Garvey, E.R, Oplinger, J.A., Tanoury, G.J., Sherman, RA., Fowler, M., Marshall, S., Harmon, M.F., Paith, J.E. and Furfine, E.S. (1994a) Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas. J. Biol Chem., 269, 26669–26676. Garvey, E.R, Tanoury, G.J., Oplinger, J.A. and Furfine, E.S. (1994b), Enzyme Inhibitors. WO patent 94/12165. Garvey, E.R, Oplinger, J.A., Furfine, E.S., Kiff, R.J., Laszlo, F, Whittle, B.J. and Knowles, R.G. (1997) 1400W is a Slow, Tight Binding, and Highly Selective Inhibitor of Inducible Nitric-oxide Synthase in vitro and in vivo. J.Biol Chem., 272, 4959–963. Gentile, R.J., Murray, R.J., MacDonald, J.E. and Shakespeare, W.C. (1995) Amidine derivatives with nitric oxide synthetase activities. WO patent 95/05363. Griffith, O.W. and Kilbourn, R.G., (1996) Nitric Oxide Synthase Inhibitors: Amino Acids. Methods in Enzymology, 268, 375–392. Gross, S.S. and Levi, R. (1992) Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J. Biol Chem., 267, 25722–25729 Guthikonda, R.N., Hagmann, W.K., Maccoss, M., Shah, S.K. and Burette, P.L. (1996a) Substituted 2Acylaminopyridines as Inhibitors of Nitric Oxide Synthase. WO patent 96/18617. Guthikonda, R.N., Grant, S.K., Maccoss, M., Shah, S.K., Shankaran, K., Caldwell, C.G. and Durette, P.L. (1996b) Cyclic Amidine Analogs as Inhibitors of Nitric Oxide Synthase. WO patent 96/14844. Hallinan, E.A., Tsymbalov, S., Moore, W.M., Currie, M.G. and Pitzele, B.S. (1995a) Arginine Based Inhibitors of Induced Nitric Oxide Synthase. Biology of Nitric Oxide, part 5, 10, 94. Hallinan, E.A., Tjoeng, F.S., Fok, K.F., Hagen, T.J., Toth, M.V., Tsymbalov, S. and Pitzele, B.S. (1995b) LN6-(lIminoethyl)lysine Derivatives Useful as Nitric Oxide Synthase Inhibitors. WO patent 95/24382. Handy, R.L. C., Wallace, P., Gaffen, Z.A., Whitehead, K.J. and Moore, P.K. (1995) The antinociceptive effect of l-(2trifluoromethylphenyl)-imidazole, a potent inhibitor of neuronal nitric oxide synthase in vitro, in the mouse. Br. J. Pharm., 116, 2349–2350. Hansen, D.W., Currie, M.G., Hallinan, E.A. Fok, K.F., Hagen, T.J., Bergmanis, A.A., Kramer, S.W., Lee, L.F., Metz, S., Moore, W.M., Peterson, K.B., Pitzele, B.S., Spangler, D.R, Webber, R.K., Toth, M.V., Trivedi, M. and Tjoeng, F.S. (1995) Amidino derivatives useful as nitric oxide synthase inhibitors. WO patent 95/11231.

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Hansen, D.W., Jr., Hagen, T.J., Kramer, S.W., Metz, S., Peterson, K.B., Spangler, D.R, Toth, M.V., Fok, K.F., Webber, R.K., Tjoeng, F.S., Pitzele, B.S. and Hallinan, E.A. (1996a) Nitric Oxide Synthase Inhibitors Derived from Cyclic Amidines. WO patent 96/35677. Hansen, D.W., Jr., Hallinan, E.A., Hagen, T.J., Kramer, S.W., Metz, S., Peterson, K.B., Spangler, D.R, Toth, M.V., Fok, K.F., Bergmanis, A.A., Webber, R.K., Trivedi, M., Tjoeng, F.S. and Pitzele, B.S. (1996b) Cyclic Amidino Agents useful as nitric oxide synthase inhibitors. WO patent 96/33175. Henley, P. and Tinker, A.C., (1995) 1,2-Diaminobenzimidazoles: Selective inhibitors of nitric oxide synthase derived from aminoguanidine. Bioorg. Med. Chem. Lett., 5, 1573–1576. Hoelscher, P., Rehwinkel, H., Burton, G., Phillips, G. and Parkinson, J.F. (1997) New imidazole derivatives are nitric synthase inhibitors. WO patent 9715555-A2. Honda, T., Makino, T. and Nagafuji, T. (1996) Aniline Derivative Having the Effect of Inhibiting Nitrogen Monoxide Synthase. WO patent 96/18607. Hodson, H.F., Palmer, R.M.J., Sawyer, D.A., Knowles, R.G., Franzmann, K.F., Drysdale, M.J., Smith, S., Davies, P.L, Clark, H.A.R. and Shearer, B.C. (1995) Enzyme Inhibitors. WO patent 95/34534. Katsura, Y., Nishino, S. and Tomishi, T. (1996) Amidine Derivatives, WO patent 96/30350. Kerwin, J.F., Lancaster, J.R. and Feldman, P.L. (1995) Nitric oxide: a new paradigm for second messengers. J. Med. Chem., 38, 4343–4362 Macdonald, J.E. (1995) Brain selective antagonists of nitric oxide synthase. Presentation at the 4th Annual International Business Communications Conference on Nitric Oxide; Philadelphia, P.A; March, 1995. Macdonald, J.E., Gentile, R.J. and Murray, R.J. (1994) Guanidine derivatives useful in therapy. WO patent 94/ 21621. Macdonald, J.E., Shakespeare, W.C., Murray, R.J. and Matz, J.R. (1996) Bicyclic Amidine Derivatives as Inhibitors of Nitric Oxide Synthetase. WO patent 96/01817. Macdonald, J.E. (1996a) Nitric Oxide Synthase Inhibitors. In Annual Reports of Medicinal Chemistry, edited by J.A.Bristol, pp. 221–230. Macdonald, J.E. (1996b) Bicyclic Isothiourea Derivatives Useful in Therapy. WO patent 96/24588. Macdonald, I.E. (1996c) Isothiourea Derivatives as NO Synthase Inhibitors. WO patent 96/09286. Makino, T. and Nagafuji, T. (1996), Amino Acid Derivative Having Nitrogen Monoxide Synthetase Inhibitor Activity. WO patent 96/06076. Mayer, B., Klatt, P., Werner, E.R. and Schmidt, K. (1994), Identification of imidazole as L-arginine-competitive inhibitor of porcine brain nitric oxide synthase. FEBS Letters, 350, 199–202. McCall, T.B., Feelisch, M., Palmer, R.M. and Moncada, S. (1991), Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. Br. J. Pharmacol., 102, 234–238. McMillan, K. and Masters, B.S. (1993) Optical difference spectrophotometry as a probe of rat brain nitric oxide synthase heme-substrate interaction. Biochemistry, 32, 9875–9880 McMillan, K., Bredt, D.S., Hirsch, D.J. Snyder, S.H., Clark, J.E. and Masters, B.S. (1992) Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide. Proc. Natl. Acad. Sci, 89, 11141–11145 Moore, W.M., Webber, R.K., Fok, K.F., Jerome, G.M., Connor, J.R., Manning, P.T., Wyatt, P.S., Misko, T.P., Tjoeng, F.S. and Currie, M.G. (1996) 2-Iminopiperidine and other 2-Iminoazaheterocycles as Potent Inhibitors of Human Nitric Oxide Synthase Isoforms. J. Med. Chem., 39, 669–672. Moore, P.K., Wallace, P., Gaffen, Z., Hart, S.L. and Babbedge, R.C. (1993a) Characterization of the novel nitric oxide synthase inhibitor 7-nitroindazole and related indazoles: antinociceptive and cardiovascular effects. Br. J. Pharmacol., 108, 219–224. Moore, P.K., Babbedge, R.C., Wallace, P. Gaffen, Z.A. and Hart, S. (1993b) 7-Nitroindazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure. Br. J. Pharmacol., 108, 296–297. Moore, W.M., Webber, R.K., Jerome, G.M., Tjoeng, F.S., Misko, T.P. and Currie, M.G. (1994) L-N6-(1iminoethyl) lysine: a selective inhibitor of inducible nitric oxide synthase. J. Med. Chem., 37, 3886–3888.

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Murad, F., Kerwin, J.F. and Gorsky, L.D. (1995a) Guanidino Compounds as Regulators of Nitric Oxide Synthase. US patent 5380945. Murad, F, Kerwin, J.F. and Gorsky, L.D. (1995b) Guanidino Compounds as Regulators of Nitric Oxide Synthase. US patent 5478946. Nakane, M., Klinghofer, V., Kuk, J.E. Donnelly, J.L., Budzik, G.P., Pollock, J.S., Basha, F. and Carter, G.W. (1995) Novel potent and selective inhibitors of inducible nitric oxide synthase. Mol. Pharm., 47, 831– 834 . Narayanan, K. and Griffith, O.W. (1994) Synthesis of L-thiocitrulline, L-homothiocitrulline, and S-methylLthiocitrulline: a new class of potent nitric oxide synthase inhibitors. J. Med. Chem., 37, 885–887. Nathan, C. (1992) Nitric oxide as a secretory product of mammalian cells. FASEB J., 6, 3051–3064 Olken, N.M. and Marletta, M.A. (1992) NG-allyl- and NG-cyclopropyl-L-arginine: two novel inhibitors of macrophage nitric oxide synthase. J. Med. Chem., 35, 1137–1144. Oplinger, J.A., Garvey, E.P., Furfine, E.S., Shearer, B.G. and Collins, J.L. (1996), Acetamidine Derivatives and their use as Inhibitors for the Nitric Oxide Synthase. WO patent 96/19440. Parkinson, J.F. and Phillips, G.B. (1997) Nitric Oxide Synthases: Enzymology and Mechanism-Based Inhibitors. In The Endothelium in Clinical Practice: Source and Target of Novel Therapies, edited by G.M. Rubanyi and V.J.Dzau, pp. 95–124. New York: Marcel Dekker Inc. Pufahl, R.A. and Marletta, M.A. (1993) Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis. Biochem. Biophys. Res. Commun., 193, 963–970 Robertson, J.G., Bernatowicz, M.S., Dhalla, A.M., Muhoberac, B.B., Yanchunas, J., Matsueda, G.R. and Villafranca, J.J. (1995) Inhibition of bovine brain nitric oxide synthase by a-amino and a-carboxyl derivatives of NG-allyl-Larginine. Bioorg. Chem., 23, 144–151. Roman, L.J., Sheta, E.A., Martasek, P., Gross, S.S., Liu, Q. and Masters, B.S. (1995), High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli. Proc. Natl. Acad. Sci., 92, 8428–8432. Shah, S.K., Grant, S.K., Maccoss, M., Shankaran, K., and Guthikonda, R.N. (1996) Substituted Heterocycles as Inhibitors of Nitric Oxide Synthase. WO patent 96/14842. Shearer, B.G., Franzmann, K.W. and Hodson, H.F. (1995) Aminoacid Derivatives as NO Synthase Inhibitors. WO patent 95/00505. Southan, G.J., Gross, S.S. and Vane, J.R., (1995) Esters and amides of NG-nitro-L-arginine act as prodrugs in their inhibition of inducible nitric oxide synthase. Enzymology and Biochemistry, 4–7. Southan, G.J. and Szabo, C. (1996) Selective Pharmacological inhibition of Distinct Nitric Oxide Synthase Isoforms. Biochem. Pharmacol., 51. 383–394. Stamler, J.S., Singel, D.J. and Loscalzo, J. (1992) Biochemistry of nitric oxide and its redox-activated forms. Science, 258, 1898–1902. Stratman, N.C., Fiei, G.J. and Sethy, V.H. (1996)U-19451A: A Selective Inducible Nitric Oxide Synthase Inhibitor. Life Sciences, 59, 945–951. Strobel, H., Bohn, H., Klingler, O., Schindler, U., Schonafinger, K. and Zoller, G. (1996a) 2-Amino-l,3-thiaepine und deren Verwendung als Hemmstoffe der Stickstoffmonoxid-Synthase. EP patent 0718294. Strobel, H., Bohn, H., Klemm, P., Klingler, O., Schindler, U., Schonafinger, K., and Zoller, G. (1996b) 2-Amino1,3thiazine als Hemmstoffe der Stickstoffmonoxid-Synthase. EP patent 0713704 Stuehr, D.J. and Ikeda-Saito, M. (1992) Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical. J.Biol Chem., 267, 20547–20550 Sup, S.J., Green, B.G. and Grant, S.K. (1994) 2-Iminobiotin is an inhibitor of nitric oxide synthases. Biochem. Biophys. Res. Comm., 204, 962–968. Tjoeng, F.S., Fok, K.F. and Webber, R.K. (1995) Amidino derivatives useful as nitric oxide synthase inhibitors. WO patent 95/11014. Werner E.R., Pitters E., Schmidt, K., Wachter, H., Werner-Felmayer, G. and Mayer, B. (1996) Identification of the 4amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase. Biochem. J., 320, 193–196.

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28 The Pharmacology of Peroxynitrite-Dependent Neurotoxicity Blockade John S.Althaus*, Gregory J.Fici and Philip F.Von Voigtlander CNS Diseases Research, Pharmacia & Upjohn, Inc., Kalamazoo, MI 49001, USA

Our laboratory has approached the development of peroxynitrite scavengers from two different perspectives. First, we were compelled by reports of scavenging activity of cysteine and sought to investigate this structural template in order to discover optimization based on structure activity relationships. Second, based on expertise in the area of inhibitors or lipid peroxidation, we were interested in discovering direct or indirect scavenging activity of our “lazaroid” class of synthetic antioxidants. With respect to the cysteine template, alkylation of the beta carbon and restricting conformational mobility were the most effective structural changes that lead to enhanced scavenging activity. Emerging as an important lead, penicillamine methyl ester was found to be protective in both in vitro and in vivo models of brain injury. Alternatively, peroxynitrite has the capacity of initiating lipid peroxidation. Lazaroid drugs which inhibit lipid peroxidation blocked the toxicity of peroxynitrite in cell culture. Mechanistic studies indicated that toxicity blockade was based on an indirect inhibition of lipid peroxidation rather than on a direct scavenging of peroxynitrite. In subarachnoid hemorrhage, the clinical candidate tirilazad, a lazaroid, was found to decrease mortality and increase functional outcome in human patients. We speculate that the possible role of peroxynitrite toxicity in SAH may be attenuated by tirilazad. Knowing that the toxicity of peroxynitrite proceeds through different pathways, the most effective therapy for the blockade of peroxynitrite toxicity was one which addressed these multiple mechanisms. A combination treatment which targeted the chemistry of peroxynitrite reactivity regarding sulfhydryl oxidation and initiation of lipid peroxidation was used to test the hypothesis. The concept was supported based on an observed synergism in activity with combination treatment. Finally, use of scavengers at millimolar concentrations to block high

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concentrations of oxidants implies a low potency for their mechanism. However, because scavenger reactivity is stoicheometrically based, it may be incorrect to conclude that because a scavenger exhibits millimolar potency in vitro that it will therefore lack potency therapeutically. Within the context of a given experiment, potency or activity at a given concentration should be used primarily to evaluate the relative effectiveness of a scavengers within a group. From such data, second-order rate constants can be reasonably calculated if for example a secondorder rate constant of a probe (e.g. cysteine at K2=5.9×103 M−1 s−1) that is run in the same assay is known. Assuming adequate pharmacokinetics, it will be the rate constant that will ultimately determine the potential therapeutic utility of a scavenger. Within this context, the organoselenium mimics of glutathione reductase appear to be the most promising leads. Key words: Peroxynitrite, scavengers, selenium, kinetics, sulfhydry, lazaroids. INTRODUCTION The apparent toxicity of nitric oxide observed in biological systems is controversial (Lipton et al., 1993). Many believe that nitric oxide itself possesses little toxicity but instead is a precursor to a fairly potent oxidant called peroxynitrite (Beckman et al., 1990). Peroxynitrite can be formed in a test tube by mixing superoxide and nitric oxide (Bough and Zafiriou, 1985). The rate of this reaction is 6.7×109 M−1 s−1 (Thomson et al., 1995) which is even faster than the enzymatic rate between superoxide and superoxide dismustase (SOD) (Riley et al., 1991). This is an extremely fast reaction and is in fact close to the diffusion limited rate (1010 M–1 s–1) (Visscher et al., 1988). This means that when superoxide and nitric oxide collide there is basically no other option but to reactively condense and form peroxynitrite. In vivo, there is ample opportunity for superoxide and nitric oxide to collide. The isoenzymatic systems of nitric oxide sythase in all of its forms and the inflammatory cell systems as well as mitochondrial electron leakages are ubiquitous sources for nitric oxide and superoxide respectively. As such, these sources virtually guarantee that peroxynitrite will be formed especially during critical events of pathophysiology. Unfortunately, no one has directly measured peroxynitrite in vivo. Primarily because in an aqueous environment at physiological pH, peroxynitrite decays very rapidly (~t1/2 < 1 s) (Denicola et al., 1993). Add physiological concentrations of buffers, exchangeable protons, sulfhydryls and antioxidants to name a few, and the decay of peroxynitrite is even more dramatic (Padmaja and Huie, 1993). The evidence that peroxynitrite exists in vivo is based primarily on kinetic arguments and on observations of biochemical fingerprints. That is, peroxynitrite will react with tyrosine/tyrosyl to give nitro-tyrosine/tyrosyl adducts (Ischiropoulos et al., 1992). Studies showed that 15N labeled L-arginine fed to macrophage resulted in 15N labeled nitro-tyrosine upon macrophage stimulate (Shigenaga et al., 1994). Antibodies have been raised to nitro-tyrosine/tyrosyl and provide specific staining of this pathological marker (Haddad et al., 1994). Colocalization of nitro-tyrosyl, NOS and SOD staining associated with pathophysiological lesions provide compelling arguments implicating peroxynitrite as an important cytotoxin (Chou et al., 1996). There are several different pathways that describe the nature of peroxynitrite reactivity (Liu et al., 1994). The most direct way is a benign intramolecular rearrangement that yields the nitrate anion (Zhang et al., 1994). This reaction proceeds through a high energy intermediate exhibiting the character of both hydroxyl radical and nitrogen dioxide radical (Koppenol et al., 1992). However, if along the way toward the

Correspondence: John S. Althaus, Parke-Davis, 2800 Plymouth Road, Ann Arbor, Michigan 48105, Lab #20–302N, USA.

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Table 1. Comparisons of the scavenging rates of various biologically relevant species.

formation of this high energy intermediate, peroxynitrite encounters a sulfydryl group; a proton; or chelated metal then the chemical fate will be different (Liu et al., 1994). Sulfhydryl groups will be oxidized; aromatics will be hydroxylated and lipids peroxidized; and aromatics will be nitrated respectively (Liu et al., 1994). Each of these later pathways may have pathological consequences. Of interest to the biochemical pharmacologist is the issue of whether or not these toxic reactions can be controlled therapeutically. To many it would seem that the endogenous antioxidants of cellular systems would normally be sufficient to handle oxidative stress provided by peroxynitrite. Or stated differently, given the fact that the cellular concentration of glutathione in the cell is as high as 5 mM as one example, can any therapeutic delivered non-toxically be expected to perform better that the natural antioxidant systems already in place? In response to these concerns, there is evidence to suggest that it may be possible to control peroxynitrite toxicity therapeutically. First, it is apparent that the endogenous cellular antioxidant systems are not sufficient to guard against peroxynitrite toxicity based on the staining of nitro-tyrosine residues associated with CNS trauma or neurodegeneration (Good et al., 1996). Second, the effective antioxidant concentration of glutathione may be much less than 5 mM based the possible activity of nitric oxide sequestration via thiol nitrosylation (Stamler and Loscalzo, 1992). The criterion for discovering a therapeutically effective scavenger will ultimately depend on the magnitude of the second order reaction rate between peroxynitrite and that scavenger. Perhaps the reason that cellular antioxidant systems fail to adequately defend against oxidation caused by peroxynitrite is because these systems react slower than the chemistry inherent in the structural/functional systems of the cell. An examination of the reported reaction rates between peroxynitrite and several biologically relevant species appears to confirm this later statement. In Table 28–1 we see that the scavenging rates of several antioxidants are orders of magnitude less than those of three functional enzymatic systems. Ultimately, the activity of any scavenging species will be a product of the reaction rate times the concentration (while requiring adequate pharmacokinetic properties). Nevertheless, it seems reasonable as a goal to develop scavengers that react as fast or faster than the most sensitive biological systems of peroxynitrite toxicity. One such scavenger is the drug ebselen which is reported to have a second-order rate constant with respect to peroxynitrite of 1×106 M–1 s–1 (Masumoto and Sies, 1996). However, specific blockade by ebselen of peroxynitritedependent toxicity in cases of neurotrauma and neurodegeneration remains to be established. OBJECTIVE Our laboratory has approached the development of peroxynitrite scavengers from two different perspectives. First, we were compelled by reports of the scavenging activity of cysteine and sought to investigate this

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structural template in order to discover optimization based on structure activity relationships. Second, based on expertise in the area of inhibitors of lipid peroxidation, we were interested in discovering direct or indirect scavenging activity of our “lazaroid” class of synthetic antioxidants. SULFHYDRYL SCAVENGERS Mechanism We envision the mechanism of peroxynitrite scavenging by aminosulfhydryl containing molecules (i.e. the cysteine template) as at least a three step process (Figure 28–1). First, the reaction between superoxide and nitric oxide yields peroxynitrite that initially exits in a relatively stable and non-reactive cis conformation. Peroxynitrite as an anion is attracted to the positive charge of the amino group of cysteine. A proton is transferred from this amino group to peroxynitrite yielding peroxynitrous acid. Peroxynitrous acid then adopts a more stable yet more reactive trans conformation. At physiological pH, the amino group of cysteine is reprotonated by a hydronium ion. Second, peroxynitrous acid is now posi tioned to react with cysteine covalently. A nucleophilic attach takes place between the d-orbital electrons of the cysteine sulfur and the partially positive charge of the peroxynitrous acid nitrogen. A proton exchange between sulfur and double bonded oxygen occurs concertively. Third, a rearrangement occurs with the transfer of a proton to the terminal oxygen and the weakening of the oxygen-oxygen peroxyl bond. This bond breaks and water is eventually liberated. The resultant structure formed is nitro-cysteine. Recently the kinetics of the reaction between glutathione and peroxynitrite were described (Zhang et al., 1997). The product of this second-order reaction is nitro-glutathione which is analogous to the product nitro-cysteine in the mechanism described above. Assays Structure activity relationships of analogs related to the cysteine template were examined as scavengers of peroxynitrite (Althaus et al., 1994). Briefly, peroxynitrite in a dose dependent manner inhibited the binding of I125 cAMP to a polyclonal antibody used in the radioimmunoassay of cAMP. Analogs of cysteine were tested for blockade of the toxic inhibition of antibody binding caused by peroxynitrite at 10 mM. Of the effective peroxynitrite scavengers; cysteine esters, penicillamine esters and cysteamine; penicillamine itself was most effective. The study was extended to include capillary electrophoresis as a method for analyzing the structural integrity of polyclonal and monoclonal antibodies exposed to peroxynitrite (Althaus et al., 1995b). Changes in peak definition and migration time of the antibody preparation were clearly associated with peroxynitrite exposure. When penicillamine was added to the antibody solution prior to exposure, the characteristic changes in the electrophoretic migration of antibody with peroxynitrite were not observed. Instead an additional peak was found that was tentatively identified as S-nitro-penicillamine. This is in agreement with the product S-nitro-glutathione which forms when peroxynitrite and glutathione react as reported recently (Zhang et al., 1997). The dose-dependent effects of peroxynitrite on the electrophoretic migration of the monoclonal antibody were further studied. Results showed that the kinetics of migrational change with peroxynitrite could be described mathematically (Althaus et al., 1997). The data fit a model that described first order reactivity at one site, and non-specific linear reactivity at multiple sites. Amino acid analysis of the antibody protein exposed to increasing concentration of peroxynitrite confirmed the correctness of this model. That is, tyrosine residues were lost at the lowest concentration of peroxynitrite and at higher concentrations several other primarily polar amino acids were lost at a more or less linear rate.

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Figure 28±1. Chemical mechanism regarding the reactivity between peroxynitrite and cysteine, the aminosulfhydryl template.

Further kinetic analysis showed that the formation of nitro-tyrosine with peroxynitrite roughly paralleled the loss of tyrosine. We developed an in vitro assay based on the formation of nitro-tyrosine from peroxynitrite and tyrosine. For enhancement of assay, nitro-tyrosine was converted to aminotyrosine using the reducing agent lithium aluminum hydride. Aminotyrosine is electrochemically active so that a sensitive measurement was made using HPLC with electrochemical detection. Based on this approach tyrosine at 10 mM was then exposed to peroxynitrite at 1 mM, and the amount of aminotyrosine was measured (N0). The reaction was repeated in the presence of 10 or 1 mM scavenger, and the amount of aminotyrosine was again measured (N’). We defined the relative effectiveness (f) of a scavenger as the ratio N’/ N0. In the absence of scavenger, “f” was defined as 1.0. Figure 28–2 shows the results of several effective scavengers. Consistent with data from the antibody assay, penicillamine was the most effective scavenger. Penicillamine has consistently proven to be more effective than cysteine as a scavenger even though structurally they differ by only two additional beta methyl groups in the case of the former. We wanted to know if the methyl groups of penicillamine increased

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Figure 28±2. Relative effectiveness (f) determined by the nitration of tyrosine (TYR) with various scavengers of peroxynitrite. Comparisons are made between hydrogen sulfide (HSH), cysteine (CYS) and penicillamine (PEN). Bars represent means plus S.E.M. for two determinations. The reaction conditions were as follows: Peroxy nitrite, 1 mM; tyrosine, 10 mM; scavengers, 1 mM; and ammonium acetate buffer, 50 mM at pH 7.

effectiveness by increasing the nucleophilicity of sulfur (enthalpic considerations) or by restricting rotational flexibility about the alpha-beta carbon bond based on molecular orbital calculations (entropie considerations). Similar analysis has been applied to the effectiveness of nipecotic acid as an inhibitor of GABA uptake (Althaus and Martin, 1989). Two model systems of the two scenarios above were tested and the results are found in Figure 28–3. A comparison of the “f” values for ethyl thiol vs. t-butyl thiol indicated that effectiveness was not enhanced by the electron induction properties of methyls. However a comparison of aminoethane thiol vs. aminocyclohexane thiol suggested that decreasing conformational entropy by using a restricted ring system resulted in a more effective scavenger. We tested the toxicity of peroxynitrite in several cell culture systems using the uptake of the amino acid isobutyric acid as a marker of cell viability (Buxser and Bonventre, 1981; Decker et al., 1993; Vroegop et al., 1995). Figure 28–4 shows that consistent with previous results, penicillamine was more effective and potent than cysteine as a neuroprotectant of murine spinal cord neurons exposed to peroxynitrite. In this same model, penicillamine or penicillamine methyl ester were also found to attenuate the loss of vitamin E and the formation of oxidized glutathione in cells exposed to peroxynitrite (Scherch et al., 1993). In cerebellar granule cells that were treated with the drug buthionine sulfoxamine (BSO) which causes a 90% depletion of glutathione, the potency of peroxynitrite toxicity was increased by a factor of 10 (Fici et al., 1996). Figure 28–5 shows a comparison of L-cysteine and D-penicillamine as neuroprotectants in this cellular model of glutathione depletion. Here the relative potency and effectiveness for these two drugs is reversed. One possible explanation for this reversal in activity is that under conditions of glutathione depletion, peroxynitrite-dependent intracellular oxidative stress may predominate and may be more readily addressed by scavengers that are transported into the cell. The in vitro studies above suggests that penicillamine may provide protection from the oxidative damage that results when nitric oxide and superoxide react to form peroxynitrite. We used the salicylate trapping

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Figure 28±3. Relative effectiveness (f) determined by the nitration of tyrosine with various scavengers of peroxynitrite. Comparisons of scavengers are made between (from left to right) ethane thiol (10 mM), t-butane thiol (10 mM), aminoethane thiol (1 mM), and aminocyclohexane thiol (1 mM). Bars represent means plus S.E.M. for two determinations. The reaction conditions were as follows: Peroxynitrite, 1 mM; tyrosine, 10 mM; scavengers, 1 mM or 10 mM; and ammonium acetate buffer, 50 mM at pH 7.

method (Althaus et al., 1995a) to evaluate the effectiveness of penicillamine to possibly lower nonenzymatic hydroxylating reactions in normal mice. The results showed that when the product of nonenzymatic (Zhang and Piantadosi, 1994) hydroxylation (2,3-dihydroxybenzoic acid) is expressed as a ratio to salicylate (2,3-DHBA/ Salicylate) both D-penicillamine and its ester reduced this measurement in plasma but only the ester was effective in brain (Figure 28–6). In vivo, the identity of the hydroxylating species is unknown. Conventional wisdom would suggest that the source is hydroxyl radical via the Fenton reaction (Floyd et al., 1986). We suggest that peroxynitrite should be considered as an alternative source. For many years it has been known that peroxynitrite can hydroxylate phenols (Halfpenny and Robinson, 1952). Recently this observation has been confirmed and studied more extensively (Van Der Vliet et al., 1994; Ramezanian et al., 1996). The chemistry of hydroxylation in brain and the physiological behavior of head injured mice is described (Hall et al., 1993). We used this model to evaluate D-penicillamine and its ester as a therapy (Figure 28–7). The results show that both drugs were effective in partially preventing the decline in “Grip Score” (time spent clinging to a taut string), however, the ester appeared to be more potent. This enhanced potency may stem from blood-brain-barrier penetration or enhanced tissue uptake of the ester although the fact that D-penicillamine exhibited any activity would suggest that vascular sources of peroxynitrite are important as well. Additionally, based on the copper chelating activity of penicillamine (Bredesen et al., 1996), we cannot rule out the possibility that penicillamine can reduce oxidative stress from traumatic head injury by preventing the formation of Fenton-directed hydroxyl radical.

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Figure 28±4. Effect of penicillamine methyl ester (Pen Ester), D-penicillamine (D-Pen), L-penicillamine (L-Pen) and Lcysteine (L-cys) against peroxynitrite toxicity (1 mM) in murine spinal cord neurons. Control indicates no exposure to either peroxynitrite or scavengers. We define maximum viability as 100% of control. *p<0.05 as indicated versus 0 mM drug for each scavenger. Results are means plus S.E.M. with n=4.

Conclusion Cysteine was used as a template to develop scavengers of peroxynitrite. Alkylation of the beta carbon and restricting conformational mobility were the most effective structural changes that lead to enhanced scavenging activity. Emerging as an important lead, penicillamine methyl ester was found protective in both in vitro and in vivo models of brain injury. LIPID PEROXIDATION INHIBITORS Mechanism Peroxynitrite can initiate lipid peroxidation (Radi et al., 1991). In Figure 28–8, the high energy intermediate possessing hydroxyl and nitrogen dioxide radical character extracts a hydrogen atom from a lipid which initiates a chain reaction involving the formation of oxygen-dependent lipid peroxyl radicals. Inhibitors of lipid peroxidation such as tirilazad act as chain terminators by reacting with peroxyl radicals to form an adduct which fragments into a lipid alcohol and a stable 5-alkoxy tirilazad radical (Church et al., 1994). As such these drugs act as indirect scavengers of peroxynitrite oxidation. Assays Inhibitors of lipid peroxidation from a chemical class called “lazaroids” (Althaus et al., 1993; Hall, 1997) have been tested against peroxynitrite toxicity in murine spinal cord neurons and cerebellar granule cells

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Figure 28±5. Effect of L-cysteine (L-Cys) and D-penicillamine (D-Pen) against peroxynitrite toxicity (100 μM) in cerebellar granule cells. Cerebellar granule cells were first treated for 24 hr. with buthionine sulfoximine (BSO). The BSO treatments resulted in 90% depletion of cellular reduced glutathione. Exposure of cells to 100 (iM peroxynitrite resulted in a 50% reduction in AIB uptake. We define this point as 0% protection. Results are means ± S.E.M. for n= 3. The % protection by L-cysteine was significantly greater than that for D-penicillamine at 100 μM and 300 uM.

described above. In Figure 28–9, the 2-methyaminochroman PNU-78517F and the pyrrolopyrimidine PNU-91736B partially blocked the toxicity of 1 mM peroxynitrite. In the case of PNU-91736B, nearly complete protection was observed at 100 μM. The stochiometry between PNU-91736B and peroxynitrite for near protection is about 0.1. This illustrates that PNU-91736B is presumably not acting directly as a scavenger but instead is acting as a chain terminator of lipid peroxidation. In this case far less drug may be required on a molar basis in order to provide protection. Lazaroids were also effective in blocking peroxynitrite toxicity in cerebellar granular cells depleted of glutathione by BSO (Fici et al., 1997, 1996). In this paradigm, pyrrolopyrimidines exhibited an EC50 value for protection of 1 μM vs. 100 uM for the 21-aminosteroid PNU-74006F (tirilazad). The increased potency presumably resulted from a lower oxidation potential (Hall et al., 1997). Mechanistically we believe that of significance is the fact that during the course of protection, tirilazad blocked the peroxynitrite-dependent increase in cellular lipid hydroperoxides but failed to block the formation of nitro-tyrosine residues. This suggests that blockade of protein nitration may not be required to achieve protection from peroxynitritemediated cell death (Fici et al., 1996). In human patients afflicted with subarachnoid hemorrhage (SAH), tirilazad administration was associated with decreased mortality and improved functional outcome (Hall, 1997). It is interesting to speculate on the role of peroxynitrite in this condition and the associated protective mechanisms of tirilazad. Studies of subarachnoid hemorrhage in humans shows that biochemically there is an associated increase in energy metabolism (resulting in depleted energy stores) and an increase in the release of the excitotoxin glutamate (Enblad et al., 1996). Model studies suggest that these are conditions capable of producing peroxynitrite based on the identification of protein nitration as an associated consequence (Schulz et al., 1996, 1995; Beal et al., 1995).

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Figure 28±6. Ratio of 2,3-dihydroxybenzoic acid (DHBA) to salicylate in mouse plasma or forebrain. Penicillamine or its ester were administered s.c. 30 min before sacrifice. Salicylate (300 mg/kg) was administered i.p. 15 min before sacrifice. Values equal mean plus S.E.M. for six animals. Effects were significant as indicated at *p<0.05 vs. saline by ANOVA.

Conclusion Peroxynitrite has the capacity of initiating lipid peroxidation. Lazaroid drugs which inhibit lipid peroxidation blocked the toxicity of peroxynitrite in cell culture. Mechanistic studies indicated that the toxicity blockade was based on an indirect inhibition of lipid peroxidation rather than a direct scavenging of peroxynitrite. In subarachnoid hemorrhage, the clinical candidate tirilazad, a lazaroid, was found to decrease mortality and increase functional outcome in human patients. We speculate that the presumed role of peroxynitrite toxicity in SAH may be attenuated by tirilazad. COMBINED THERAPY Assay It is clear that aminothiols and inhibitors of lipid peroxidation prevent the toxicity of peroxynitrite by two different mechanisms. We wanted to know if a combination treatment of scavenger and lipid peroxidation inhibitor would yield a more effective blockade of peroxynitrite toxicity. In cerebellar granule cells that were deplete of glutathione, we tested for additive protection using penicillamine as a concurrent treatment with peroxynitrite exposure and lazaroids as a post-treatment (Fici et al., 1997). At 100 μM or 300 uM peroxynitrite, the effects of the lazaroids PNU-74006F or PNU-101033E were at least additive with penicillamine. In the case of penicillamine (1 mM) with PNU-101033E (1 μM), peroxynitrite toxicity (300 μM) was completely blocked and a synergistic affect of the combined therapy was observed.

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Figure 28±7. Dose-response relationship for the effects of penicillamine (Pen) and penicillamine methyl ester (Pen Ester) administered by i.v. bolus at 3–5 min post-injury on early (1 hr) neurological recovery (i.e., grip score) in mice subjected to a severe concussive head injury. All values are mean plus S.E.M. for n= 15. Significant effects were found as indicated for *p<0.05.

Conclusion Knowing that the toxicity of peroxynitrite proceeds through different pathways, the most effective therapy for the blockade of peroxynitrite toxicity would be one which addresses these multiple mechanisms. A combination treatment which targeted the chemistry of peroxynitrite reactivity regarding sulfhydryl oxidation and initiation of lipid peroxidation was an attempt to test the hypothesis. The concept was supported based on results which showed a potential for synergism with combined treatment. FUTURE DIRECTION Assay Recently, a series of seleno-organic compounds were tested as scavengers of peroxynitrite (Briviba et al., 1996). These compounds were found to be at least 100 times more reactive than the sulfur counterparts. The compound that emerged as the most effective scavenger of peroxynitrite was ebselen, a glutathione peroxidase mimic (Ochi et al., 1992). A secondorder rate constant for ebselen was reported although the specifics of the experiment were not given (Masumoto and Sies, 1996). We report here the results of a selenodithiole-uracil analog exhibiting an apparent second-order rate constant with peroxynitrite of 1.2×106 M−1 s−1. Details and the results of the stopped flow experiment are shown in Figure 28–10.

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Figure 28±8. Chemical mechanism regarding the indirect reactivity between peroxynitrite and inhibitors of lipid (LH) peroxidation (e.g. Tirilazad, a “lazaroid”).

Conclusion Finally, use of scavengers at millimolar concentrations to block high concentrations of oxidants implies a low potency for their mechanism. However, because scavenger reactivity is stoicheometrically based, it may be incorrect to conclude that because a scavenger exhibits millimolar potency in vitro it will therefore lack potency therapeutically. Within the context of a given experiment, potency or activity at a given concentration can be used to evaluate the relative effectiveness of a scavenger. That is because from such data, a second-order rate constant can be reasonably calculated if for example a second-order rate constant of a probe (e.g. cysteine at K2=5.9×103 M−1 s−1) that is run in the same assay is known (Radi et al., 1991b). Assuming adequate pharmacokinetics, it will be the rate constant that will ultimately determine the potential therapeutic utility of a scavenger. Within this context, the organoselenium mimics of glutathione peroxidase appear to be the most promising leads. ACKNOWLEDGEMENT The authors would like to acknowledge the experimental contributions made to this work by Edward D.Hall, Heidi M.Scherch and Paula K.Andrus. REFERENCES Althaus, J.S., Fici, G.F., Plaisted S.M., Kezdy, F.J., Campbell, C.M., Hoogerheide, J.G. and VonVoigtlander, P.P. (1997) Protein nitration by peroxynitrite: A method for monitoring nitric oxide neurotoxicity. Microchemical Journal, 56, 155–164.

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Figure 28±9. Effect of lazaroid compounds, PNU-78517F and PNU-91736B against peroxynitrite toxicity (1 mM) in murine spinal cord neurons. Control indicates no exposure to either peroxynitrite or scavengers. We define maximum viability as 100% of control. *p<0.05 as indicated versus 0 mM drug for each lazaroid compound. Results are means plus S.E.M. with n=4. Althaus, J.S., Andrus, P.K., Hall, E.D. and VonVoigtlander, P.F. (1995a) Improvements in the salicylate trapping method for measurement of hydroxyl radical levels in the brain. In Central Nervous System Trauma Research Techniques, edited by S.T. Ohnishi and T. Ohnishi, pp. 437–444. New York: CRC Press. Althaus, J.S., Fici, G.J. and VonVoigtlander, P.F. (1995b) Antibody transformation by peroxynitrite as determined using capillary electrophoresis: a feasibility study. Res. Commun. Mol. Pathol. Pharmacol, 87(3), 359– 366. Althaus, J.S., Oien, T.T., Fici, G.J., Scherch, H.M., Sethy, V.H. and VonVoigtlander, P.F. (1994) Structure activity relationships of peroxynitrite scavengers an approach to nitric oxide neurotoxicity Res. Commun. Chem. Pathol. Pharmacol , 83(3), 243–254. Althaus, J.S., Hall, E.D. and VonVoigtlander P.F. (1993) Lazaroids—potent agents in medical brain protection? In Spektrum der N'eurorehabilitati on, edited by K. Von Vild, pp. 42–49. New York: W. Zuckschwerdt Verlag. Althaus, J.S. and Martin, D.L. (1989) Entropy as a factor in the binding of gamma-aminobutyric acid and nipecotic acid to the gamma-aminobutyric acid transport system. Neurochem. Res., 14(4), 311–316. Beal, M.F., Ferrante, R.J., Henshaw, R., Matthews, R.T., Chan, PH., Kowall, N.W., Epstein, C.J, and Schulz, J.B. (1995) 3-Nitropropionic acid neurotoxicity is attenuated in copper/zinc superoxide dismutase transgenic mice. J. Neurochem., 65(2), 919–922. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. and Freeman, B.A. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U.S.A., 87(4), 1620–16244. Blough, N. V and Zafiriou, O.C. (1985) Reaction of superoxide with nitric oxide to form peroxonitrite in alkaline aqueous solution . Inorg. Chem., 24, 3502–3504. Bredesen, D.E., Wiedau-Pazos, M., Goto, J.J., Rabizadeh, S., Roe, J.A., Gralla, E.B., Ellerby, L.M. and Valentine, J.S. (1996) Cell death mechanisms in ALS. Neurology, 47(4 Suppl 2), S36-S38.

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Figure 28±10. Stopped flow kinetics of the decay of a selenodithiole uracil analog, PNU-130164E, upon mixing with peroxynitrite. Peroxynitrite in 100 μM sodium hydroxide was mixed with PNU-130164E in ammonium acetate buffer. The initial concentrations of PNU-130164E and peroxynitrite at mixing were both 10 jtiM and the final pH of the solution was 7. The decay of PNU-130164E was monitored at 430 nm. Once peroxynitrite and PNU-130164E were mixed, the solution traveled 0.3 sees in time before reaching the cuvette for measurement. During this time interval, approximately 77% of PNU-130164E decayed and a steady-state concentration of 2.3 μM was maintained until flow was stopped. The inset is a replot of the inverse in concentration measured over the time interval between 10.8 and 11.6 sec. Because the reactants were mixed in stochiometrically equivalent concentrations, a simple linear relationship between inverse concentration and time applies and gives an apparent second-order rate constant (k2=1.2×106 M−1 s−1) as the slope of the line. The actual second-order rate constant would be greater if corrected for the spontaneous decay of peroxynitrite. Buxser, S. and Bonventre, P.F. (1981) Strphylococcal enterotoxins fail to disrupt membrane interity or synthetic function of Henle 407 intestinal cells. Infect. Immun., 31, 929–934. Briviba, K., Roussyn I., Sharov, VS. and Sies,H. (1996) Attenuation of oxidation and nitration reactions of peroxynitrite by selenomethionine, selenocystine and ebselen. Biochemical Journal, 319, 13–15. Castro, L., Rodriguez, M. and Radi, R. (1994) Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J. Biol Chem, 269(47), 29409–29415. Chou, S.M., Wang. H.S. and Komai, K. (1996) Colocalization of NOS and SOD1 in neurofilament accumulation within motor neurons of amyotrophic lateral sclerosis: an immunohistochemical study. J. Chem. Neuroanat., 10(3–4), 249–258. Church, D.F., Althaus, J.S., Koeplinger, K.A., Dugus, T.D., VonVoigtlander, P.P. and McCall, J.M. (1994) Tirilazad mesylate (TM) is oxidized to a stable radical intermediate. J. Free Rad. Biol. Med. Abstr., 2(3), D6. Crow, J.R, Beckman, J.S. and McCord, J.M. (1995) Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite. Biochemistry, 34(11), 3544–3552.

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29 Hemoglobin: Its Role as a Nitric Oxide Scavenger S.R.Fischer and D.L.Traber Investigational Intensive Care Unit, The University of Texas Medical Branch, 610 Texas Avenue, Galveston, TX-77555±0833, USA

Hemoglobin is able to bind nitric oxide (NO) at the iron ion of the heme and at amino termini, ultimately leading to the formation of nitrate/nitrite and methemoglobin. Apart from binding hemoglobin it has been proposed that hemoglobin may also act as a NO donor by release of nitrosothiols. NO is an important mediator for regulation of vascular tone, vascular reactivity and distribution of regional blood flow, sepsis-induced vasodilation, and myocardial depression. Several modified hemoglobin solutions have been developed to increase plasma half-life and decrease oxygen affinity. Scavenging of NO by hemoglobin increased systemic blood pressure in healthy and septic animals and induced pulmonary hypertension. In sepsis, decreased vascular reactivity for catecholamines was reversed and there was a tendency towards improvement of myocardial depression. Hemoglobin administration in healthy animals has been associated with reduced renal and pancreatic perfusion, in septic animals hemoglobin had little effect on regional blood flow. In sepsis, glomerular filtration rate and urine output decreased and after hemoglobin returned to presepsis levels. Hemoglobin had no effect on hypoxic pulmonary vasoconstriction which was blunted during sepsis. Hemoglobin infusion increased endothelin-1 levels and blockade of endothelin-1 production prior to hemoglobin administration resulted in attenuation of the increase in systemic blood pressure indicating an interaction between both substances. Although hemoglobin enhanced activity of lipopolysaccharide in vitro, so far no increased toxicity or impaired host-defense has been documented in vivo. More studies are needed to evaluate long-term effects. Keywords: Hemoglobin, nitric oxide, sepsis, scavenger.

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STRUCTURE OF HEMOGLOBIN The hemoglobin molecule consists of four polypeptide chains held together by noncovalent attractions. Each contains a heme group and a single oxygen binding site. Hemoglobin A consists of two and two (3chains. The iron ion of the heme can be in the ferrous (Fe2+) or the ferric (Fe3+) oxidation states; the corresponding forms of hemoglobin are called ferrohemoglobin and ferrihemoglobin. Ferrihemoglobin is also called methemoglobin. Only ferrohemoglobin can bind oxygen. The interactions of hemoglobin with O2, CO2 and nitric oxide (NO) take place at the heme iron and the amino termini. Although mostly recognized for its ability to transport O2 and CO2, hemoglobin’s ability to bind NO has recently received increased attention. That it can bind NO has long been known, indeed, the first publication dates from more than 100 years ago (Hermann, 1865). NO PHYSIOLOGY AND PATHOPHYSIOLOGY The biological effects of an endothelium-derived relaxing factor (EDRF) were first described by Furchgott and Zawadzki in 1980. Today, EDRF is believed to be identical with NO. NO is a small, uncharged, highly reactive radical molecule that diffuses freely across membranes and has a biological half-life of 2–30 seconds. It is produced by at least three isoenzymes of NO synthase, including two constitutive forms: cNOS is found in endothelial cells and participates in regulation of basal vascular tone; and bNOS in neuronal tissue. Both are always present and are dependent on the presence of Ca2+ ions. The third, calciumindependent form, the inducible NO synthase (iNOS), is found in macrophages and, after exposure to endotoxin, cytokines and other inflammatory mediators, it produces NO in large quantities. It is thought to be the source of the excess of NO that is involved, among other mediators, in causing hypotension and vascular hyporeactivity, in immunmodulation and host defense, and in cell aggregation. NO is produced from L-arginine, resulting in NO and L-citrulline. It quickly diffuses to nearby target cells through cell membranes and tissues. As a radical, it binds to metals, one of them iron. The enzyme guanylate cyclase in endothelial cells contains a heme group and has a high affinity for NO. Upon binding with NO it is activated, producing cyclic 3’5 -guanosine monophosphate which, in turn, mediates smooth muscle relaxation or other effects (Craven and DeRubertis, 1978). NO is quickly bound by hemoglobin-forming nitrosylhemoglobin with an affinity 1500 times higher than that of CO2. The reaction with ferrohemoglobin A is fast and the binding of NO strong. Both oxy- and deoxyhemoglobin are able to bind NO. Deoxyhemoglobin binds to NO, forming relatively stable deoxyhemoglobin-NO complexes; upon contact with oxygen it releases nitrate and is oxy dated to methemoglobin. Oxyhemoglobin and NO combine, but quickly transform to yield methemoglobin and nitrate (Fronticelli and Karavitis, 1994). The reaction of NO and methemoglobin, on the other hand, is slow, unstable, and easily reversible. Methemoglobin in unable to carry oxygen; it is reduced to form ferrohemoglobin by the methemoglobin reductase system in erythrocytes. Nitrates and nitrites are elevated in diseases such as sepsis with increased NO production (Evans et al., 1993; Barthlen et al., 1994). Recently, it has been recognized that the formation of S-nitrosothiols from NO may also play a role in mediating the effects of NO (Jia et al., 1996). S-nitrosothiols have NO-like vasorelaxant activity but do not react with the hemes of either oxy- or deoxyhemoglobin. S-nitrosohemoglobin (SNOHb) is formed between S-nitrosothiols and highly reactive SH groups of the -chains of hemoglobin. In their experiments Jia et al. detected that arterial but not venous blood from rats contained significant levels of SNOHb. A reverse relationship was found for nitrosylhemoglobin, which was detected in much higher concentrations in deoxygenated erythrocytes. The authors postulated that in the lung either elimination of NO or intramolecular transfer from heme to the SH groups of the -chains takes place, S-nitrosylation. This

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reaction was faster in the oxyconformation than the deoxyconformation. Dissociation of this complex yielded S-nitrosothiols. SNOHb may act as a NO donor in the systemic circulation and may thus serve in regulation of vascular tone. In summary, hemoglobin can interact with NO in several ways. The best known reaction is the conversion of NO to nitrate and nitrite. This results in the inactivation of NO. The reaction is mediated more by oxy- than deoxyhemoglobin. Hemoglobin can also combine with NO to form a nitrosothiol. Thus, in the presence of oxyhemoglobin NO is inactivated and NO-mediated vasodilation is ablated. On the other hand, if oxyhemoglobin has combined with NO to form nitrosothiol, deoxygenation will result in its release and, consequently, vasodilation. HEMOGLOBIN SOLUTIONS In the search for a blood substitute, research regarding hemoglobin solutions has exclusively focused on its properties as an oxygen carrier. Hemoglobin solutions free of cell membranes (stroma-free hemoglobins) were developed. However, cell-free hemoglobin is a poor choice as an oxygen carrier owing to its high oxygen affinity in the absence of 2,3-diphosphoglycerate, which does not allow adequate oxygen unloading in the peripheral tissues. In addition, cell-free hemoglobin quickly dissociates intoo -dimers, which are rapidly cleared by the kidney, shortening plasma half-life. Further, these dimers, precipitating in the proximal tubulus, can cause renal failure. For these reasons, bovine hemoglobin, human recombinant (Petros et al., 1991) and chemically modified hemoglobin solutions have been developed, with decreased oxygen affinity, prolonged plasma half-life from 8–36 hrs (Dietz et al., 1996), and without renal toxicity. Owing to the many functional groups on the surface of the hemoglobin molecule, many modifications are possible, including pyridoxalation, polymerization, conjugation, and intramolecular cross-linking. Other solutions include polyhemoglobin, a polymer of hemoglobin molecules and a copolymer of hemoglobin and albumin or encapsulation of hemoglobin molecules in synthetic membranes, forming so-called hemosomes. Examples of modified hemoglobins are diaspirin cross-linked hemoglobin (DCLHb) (Malcolm et al., 1994) and pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) (Malchesky et al., 1990). DCLHb is a hemoglobin whose subunits are cross-linked with diaspirin. PHP is prepared from outdated human red cells by modification of the deoxygenated stroma-free hemoglobin. It is pyridoxalated with pyridoxal 5'-phosphate to lower oxygen affinity and conjugated with activated polyoxyethylene to increase molecular weight and plasma half-life. The molecules then can be cross-linked, producing polymeric species of PHP (Yabuki et al., 1990; Malchesky et al., 1990). The development and characterization of different hemoglobin solutions have been comprehensively reviewed by DeVenuto (DeVenuto, 1983). EFFECTS OF HEMOGLOBIN Isolated Vessel Preparations Even before NO was recognized as a major mediator of vascular smooth vessel relaxation it was noted that hemoglobin was a rapidly acting inhibitor of endothelium-dependent relaxation (Martin et al., 1985). In isolated vessel preparations incubated with hemoglobin solutions, hemoglobin was shown to cause a decrease in acetylcholine or interleukin l induced relaxation, whereas endothelium-independent relaxation was not affected (Martin et al., 1985). Soluble hemoglobin was at least 10 times more potent than hemoglobin in erythrocytes (Rioux et al., 1994). Removal of the endothelium or NOS inhibition blocked the

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response to hemoglobin (Petros et al., 1994).Vascular contraction after incubation with hemoglobin was demonstrated in arteries as well as in veins of mesenteric, renal, femoral and pulmonary vessels. The magnitude of contraction varied considerably between the vessels. It was greater in the arteries than the corresponding veins, with the exception of the pulmonary vein, which exhibited a stronger contraction than the pulmonary artery. Since the vessels in these experiments were not pretreated with vasorelaxants, the authors concluded that the vasoconstriction elicited by hemoglobin was caused by inhibition of the basally released NO, which contributes to vasomotor tone (Petros et al., 1994). There is vast evidence that endotoxemia or sepsis causes vascular hyporeponsiveness to catecholamines (Julou Schaeffer et al., 1990; Weigert et al., 1995; Grissom et al., 1996; Yaghi et al., 1995; Nelson et al., 1996) although endogenous catecholamine levels are elevated in sepsis (Hahn et al., 1995). Expression of iNOS has been documented in aortas of septic rats, and incubation with a selective iNOS inhibitor reversed the depressed phenylephrineevoked contractions of denuded aortic rings (Weigert et al., 1995). Nonspecific NOS inhibitors showed similar results (Grissom et al., 1996), pointing to NO as a mediator for vascular hyporesponsiveness. Cellfree hemoglobin significantly potentiated the contractions to phenylephrine in control aortic rings from healthy rats and also reversed the hyporeactivity to phenylephrine of aortic rings incubated with endotoxin (Kilbourn et al., 1994). Whole red blood cells had no effect, indicating that only cell-free hemoglobin is able to scavenge NO produced in endotoxin-treated vascular tissue. Animal Models Cardiovascular effects Several investigators have studied the effects of hemoglobin in intact animal models during healthy conditions and during endotoxemia or sepsis. Hemoglobin solutions were found to cause an increase in mean arterial pressure (MAP) (Sharma and Gulati, 1994; Katsuyama et al., 1994; Schultz et al., 1993; Malcolm et al., 1994; Yamakawa et al., 1990). DCLHbinfusion in healthy rats increased MAP in a dosedependent manner, accompanied by a decrease in heart rate (Schultz et al., 1993; Malcolm et al., 1994). A similar increase in MAP as with DCLHb was achieved by NOS inhibition with L-NAME; the combination of both agents, however, yielded no further increase in MAP. Administration of L-arginine and nitroglycerine, a NO donor, significantly blunted the increase in MAP (Katsuyama et al., 1994; Schultz et al., 1993). Exchange transfusions with PHP in healthy rats while hemodynamic stability was maintained resulted in an increase in MAP even above baseline levels (Yamakawa et al., 1990). Exchange transfusion after induction of hemorrhagic shock in swine with hemoglobin and (3,5-dibromosalicyl) fumarate cross-linked hemoglobin (aaHb) also caused a prompt increase in MAP and systemic vascular resistance, accompanied by a decrease in cardiac output (Hess et al., 1993). In contrast to the healthy state, where NO production is thought to predominantly originate from cNOS, inflammatory states induced by cytokines, endotoxin or bacterial infusion cause induction of iNOS with production of large amounts of NO (Freas et al., 1995). In experiments from our laboratory, endotoxin- and bacterial infusion in sheep induced a hyperdynamic circulation, with increased cardiac output, systemic arterial hypotension due to decreased systemic vascular resistance, tachycardia, and increased pulmonary artery pressure (Meyer et al., 1994; Lingnau et al., 1996). Pulmonary shunt fraction was elevated as a result of blunted hypoxic pulmonary vasoconstriction and increased pulmonary vascular permeability, leading to interstitial pulmonary edema (Theissen et al., 1991; Meyer et al., 1994). NOS inhibition reversed the hyperdynamic circulation, resulting in a return of MAP and cardiac output to levels existing before sepsis (Meyer et al., 1994; Lingnau et al., 1996). Administration of different amounts of PHP (50, 100 and 200

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Figure 29±1. Mean arterial blood pressure (MAP) and systemic vascular resistance (SVRI) (mean ± SEM) in four groups of septic sheep. All sheep received a 48-hr infusion of Pseudomonas aeruginosa (106 colony-forming units/kg/ hr). After 24 hrs 50 mg, 100 mg, or 200 mg PHP, or the carrier solution was infused over 30 min. Sepsis led to hypotension and a decrease in SVRI. PHP caused a dose-dependent increase in MAP and SVRI.*p<0.05 vs. Ohrs, †p<0. 05 vs. 24 hrs, $p<0.05 vs. control group. Data from Bone et al. (1996a).

mg/kg) as a one time bolus in septic sheep led to an immediate increase in MAP. The magnitude of this increase and its duration were dose-dependent. MAP did not rise above the value it had before induction of sepsis (Bone et al., 1996a) (Figure 29–1). A continuous infusion of PHP at 20 mg/kg/hr was also sufficient to return MAP to presepsis levels (Bone et al., 1996b). A steady state was reached approximately 4 hours after begin of the infusion. Aranow et al. also reported an increase in MAP but not in systemic vascular resistance index after infusion of cross-linked hemoglobin into endotoxemic swine (Aranow et al., 1996).

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Figure 29±2. Mean pulmonary artery pressure (PAP) and pulmonary vascular resistance index (PVRI) in four groups of septic sheep (mean± SEM). All sheep received a 48-hr infusion of Pseudomonas aeruginosa (106 colony-forming units/ kg/hr). After 24 hrs 50 mg, 100 mg, or 200 mg PHP, or the carrier solution was infused over 30 min. PAP increased during sepsis, but PVRI remained unchanged. PHP administration caused a further increase in increase in PAP and increased PVRI. *p<0.05 vs. 0 hrs, †p<0.05 vs. 24 hrs, $p<0.05 vs. control group. Data from Bone et al. (1996a).

The vasopressor effect of hemoglobin was also exhibited in the pulmonary vasculature of healthy animals, leading to pulmonary hypertension; in septic animals it caused the already elevated pulmonary artery pressure to rise even further (Aranow et al., 1996; Hess et al., 1993; Poli de Figueiredo et al., 1996). In our experiments, both administration of PHP as a bolus or an continuous infusion were accompanied by an increase in pulmonary arterial pressure and pulmonary vascular resistance, although high peaks could be avoided with the continuous infusion (Figure 29–2). Pulmonary vascular resistance could be lowered by infusion of dobutamine and prostacycline (unpublished data).

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Hypoxic pulmonary vasoconstriction (HPV), a mechanism of the pulmonary vasculature that diverts blood flow away from poorly oxygenated alveoli to better ventilated lung areas, is blunted by NO inhalation (Pison et al., 1993; Romand et al., 1994; Welte et al., 1993; Channick et al., 1994; Booke et al., 1996a) and augmented by NOS inhibitors (Liu et al., 1991; Sprague et al., 1992). Within one hour of endotoxin administration HPV was abolished (Theissen et al., 1991). HPV can be induced in sheep by separate lung ventilation with one lung hypoxia while the other lung was ventilated with oxygen to avoid global hypoxemia. Under normal, healthy conditions over 50% of the blood flow to the hypoxic lung is diverted to the oxygenated lung. After 24 h of sepsis during bacterial infusion with Pseudomonas aeruginosa, this fraction decreased to 23%. Administration of a NOS inhibitor improved but did not restore HPV (Fischer et al., 1996). PHP, on the other hand, at 20 mg/kg/min, showed no effect on HPV (Fischer et al., 1997b). A possible explanation is that PHP may have formed nitrosothiols during these experimental conditions. Since the pulmonary arterial blood entering the hypoxic lung remained deoxygenated, it may have released nitrosothiol. However, whether this is indeed the case remains to be shown. Vascular hyporesponsiveness to catecholamines was demonstrated in the intact animal model by Booke et al., who showed in septic sheep that the norepinephrine dose necessary to maintain MAP at presepsis levels had to be progressively increased over the 24-hr period of its administration (Booke et al., 1996e). In another series of experiments we found that the dose of norepinephrine required to increase MAP by 10 mmHg in septic sheep was significantly reduced during PHP infusion, suggesting that PHP sensitized the vasculature to catecholamines (Fischer et al., 1997a). The effects of hemoglobin solutions on myocardial function have been studied in isolated perfused hearts from healthy animals; however the results are not uniform (Biessels et al., 1992; Looker et al., 1992; Chatterjee et al., 1986). While some studies found that modified hemoglobin caused an increase in coronary pressure and coronary vascular resistance (Biessels et al., 1992; Looker et al., 1992), other investigators could not demonstrate an influence on coronary flow resistance (Chatterjee et al., 1986). Ventricular contractility, assessed by left ventricular pressure and pres sure-volume relation, was well maintained during perfusion with the hemoglobin solution. In contrast to the modified hemoglobin PHP, stroma-free hemoglobin caused sinus bradycardia (Chatterjee et al., 1986). Myocardial depression during endotoxemia and sepsis has been extensively studied, but its cause is still not completely understood (Kumar and Parrillo, 1995). NO has been implicated as one of the mediators. Production of iNOS and NO in the myocardium during endotoxemia with a decrease in contractility of excised myocardial tissue has been demonstrated within several hours of endotoxin exposure (Schulz et al., 1992; Brady et al., 1992; Balligand et al., 1993). Cardiomyocyte depression induced by serum from septic patients could be reversed in vitro by NOS inhibitors (Kumar et al., 1993). Little information exists on the influence of hemoglobin on cardiac contractility in sepsis. We assessed myocardial contractility in septic sheep using the left ventricular endsystolic pressurediameter ratio (Emax) (Harada et al., 1997). The left ventricular diameter was measured with ultrasonic crystals positioned on the left ventricle, and a catheter tip manometer placed into the left ventricle. The preload of the left ventricle was varied with occlusion of the inferior vena cava. After infusion of Pseudomonas aeruginosa Emax decreased and had dropped by 53% after 12 hrs. Infusion of PHP tended to improve Emax, although this improvement was not statistically significant. Infusion of 5 μg/ kg^min dobutamine increased Emax, an effect, that lasted for at least 4 hours after discontinuation of the dobutamine infusion. Figure 29–3 shows the effect of PHP alone versus PHP with dobutamine on Emax during sepsis. Prostacycline infusion at 0.1 μg/kg/min had a similar effect, but its use was limited by systemic hypotension. Indirect measures of myocardial contractility, such as stroke volume index and left and right ventricular stroke work index, showed few changes after PHP.

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Figure 29±3. Left Ventricular end-systolic maximum elastance (Emax) in septic sheep (mean±SEM). All sheep received a 24-hr infusion of Pseudomonas aeruginosa (106 colony-forming units/kg/hr). After 12 hrs they received an 8hr infusion of PHP at 20 mg/kg/hr. Dobutamine was infused at 5 μg/kg-min in one group 4 hrs after beginning PHP for one hour. During sepsis Emax decreased by 53% at 12 hrs. PHP tended to increase Emax. Dobutamine increased Emax and its effect lasted on even after discontinuation of dobutamine. Data from Harada et al. (1997).

Effect on oxygenation Infusion of hemoglobin exacerbated hypoxemia and ventilation-perfusion abnormalities in endotoxemic swine (Aranow et al., 1996) and in bacteremic dogs (Crowley et al., 1993). In contrast to these findings, Bone et al. did not observe worsening of PaO2 or oxygen saturation after PHP administration (Bone et al., 1996a). Oxygen delivery increased during sepsis while oxygen uptake was unchanged. After PHP administration, oxygen delivery decreased but stayed well above baseline (presepsis) values. Oxygen extraction and uptake were unaffected by PHP. At these concentrations there also was no significant increase in methemoglobin levels. Effects on regional blood flow The effects of hemoglobin solutions on regional blood flow have been studied in healthy animals, animals after exchange transfusion, and septic animals. Blood flow to different organs was assessed either by microsphere technique or ultrasonic flow probes. Bolus infusion of 400 mg/kg of DCLHb in healthy, anesthetized rats had little effect on blood flow to brain, muscle skin, and most splanchnic organs, with the exception of stomach and spleen, which showed a transient increase in blood flow (Sharma and Gulati, 1994). Ulatowski et al. (1996) found after infusion of bovine fumaryl -crosslinked hemoglobin as exchange transfusion in healthy cats, a 40–70% decrease in blood flow to the kidneys, intestines and adrenal glands. In hyperdynamic sepsis, the distribution of blood flow between organs is altered (Bersten and Sibbald, 1989) owing to impaired autoregulatory function of the vasculature by endothelial damage and vascular hyporesponsiveness. Administration of a NO scavenger that increased systemic vascular resistance and vascular responsiveness to vasoconstrictors might further worsen blood flow to vascular beds with already compromised blood flow. Several investigators, including our own laboratory, have addressed this topic. The results are reported as changes in absolute blood flow to the organs as well as changes in

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distribution of blood flow expressed as a fraction of cardiac output, so-called redistribution. Most investigators have found either no redistribution or some redistribution during sepsis towards various organs in different patterns. However, almost all studies reported redistribution away from the pancreas (Bersten et al., 1990; Lang et al., 1984; Raper et al., 1991; Fish et al., 1986; Breslow et al., 1987; Ferguson et al., 1978; Booke et al., 1996b), and some report redistribution away from the kidney (Bersten et al., 1990; Raper et al., 1991; Breslow et al., 1987). Administration of DCLHb to septic rats caused no immediate changes in regional blood flow, but 24 hrs later there was an increase in perfusion to the splanchnic organs, the brain and the coronaries (Mourelatos et al., 1996). Aranow and colleagues found no change in renal and mesenteric blood flow after infusion of cross-linked human hemoglobin in hypodynamic, endotoxemic swine (Aranow et al., 1996). In contrast, ileal mucosal perfusion, measured by a laser Doppler flow probe, decreased during endotoxemia and further after hemoglobin administration. Studies from our laboratory with PHP infusion in hyperdynamic septic sheep showed no change in regional perfusion after hemoglobin (Bone et al., 1996c). In fact, perfusion to the pancreas tended to be increased during PHP infusion. Further, lactate, an indicator of tissue ischemia, was not elevated just before or after PHP administration compared to baseline. Effects on renal function Sepsis is frequently associated with acute renal failure despite adequate volume resuscitation and maintained systemic arterial pressure. Renal failure is characterized by a fall in glomerular filtration rate, oliguria, low fractional sodium excretion, proteinuria and increased plasma renin activity, findings consistent with hypoperfusion or volume contraction (Walker et al., 1986). In addition, levels of atrial natriuretic peptide (ANP) are elevated (Redl et al., 1992; Hinder et al., 1996). In preliminary studies in our laboratory, infusion of PHP into septic sheep normalized decreased glomerular filtration pressure, measured as ureteral occlusion pressure, and increased urinary output (Jourdain et al., 1997) (Figure 29–4). Previous studies with NOS inhibition using L-NMMA showed similar results (Hinder et al., 1996). The increase in glomerular filtration pressure could be mediated by the increase in systemic blood pressure. In addition, mesangial cells within the glomerulus as well as glomerular endothelial and epithelial are able to produce NO, and NOS inhibitors and PHP may have local effects on these cells within the glomerulus (Nicolson et al., 1993; Shultz et al., 1991). We saw no evidence of nephrotoxicity in our animal models of sepsis when hemoglobin was administered for up to 48 hrs. However, these results from previously healthy animals without underlying renal disease may not be extrapolated to organs affected by chronic disease. The effects of hemoglobin and whether hemoglobin exhibits any nephrotoxicity on diseased kidneys are unknown. Effect on laboratory values Few reports exist on the effect of modified hemoglobin solutions on laboratory values, markers of organ function. Also, any hemoglobin solution intended for clinical use should not interfere with common laboratory tests used for patient monitoring. In our septic sheep, PHP infusion at the given concentrations had little effect on laboratory values. Sepsis by itself caused small increases in g-glutamyl transferase, aspartate-amino-transferase, and bilirubin, but not in alanine-amino-transferase and alkaline phosphatase levels. None of these parameters were influenced by PHP infusion, indicating no adverse effect of PHP on liver function (Bone et al., 1996c). These findings are supported by the work of other investigators, who reported no change in liver function tests or liver histology after PHP infusion compared to a control group (Eldridge et al., 1996). Serum creatinine and blood urea nitrogen rose during sepsis but serum creatinine

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Figure 29±4. Left ureteral occlusion pressure (LUOP) as a measure of glomerular filtration pressure and urine output (mean ± SEM) in septic sheep. All sheep received a 48-hr infusion of Pseudomonas aeruginosa (106 colony-forming units/kg/hr). The treatment group received a 12-hr infusion of PHP at 20 mg/kg/hr after 24 hrs of sepsis; the control group received only the carrier. Urine output is shown as the cumulative urine output at a certain time point. LUOP fell during sepsis and was restored to baseline levels after PHP administration. This correlated with an increased urine output compared with the control group. *p<0.05 vs. 0 hrs, †p<0.05 vs control group. Data from Jourdain et al. (1997).

and creatinine clearance were not influenced by PHP. Serum lipase, osmolality, oncotic pressure, or serum electrolytes did not change with sepsis alone or with PHP infusion. In contrast to others who reported marked elevation in methemoglobin levels with modified hemoglobin solutions (Lee et al., 1995), methemoglobin was not significantly elevated in our animals, and neither were free iron levels. In vitro interference of low concentrations of PHP with laboratory tests were noted with lactate dehydrogenase assays and bilirubin determination (Bone et al., 1996c). These results indicate that PHP causes little interference for routine monitoring of laboratory values, and had no detectable adverse effect on organ function. Effect on endotoxin and bacterial growth Modified hemoglobin is cleared from the circulation by the reticulo endothelial (RE) system (Dietz et al., 1996). The long-term effects on the RE system have not been determined. Concern has been expressed as to whether this effect on the RE system could interfere with its the host-defense functions. Further, Roth et al. have demonstrated that lipopolysaccharide (LPS) binds to hemoglobin, resulting in increased methemoglobin formation, increased intravascular retention of LPS, and increased biological activity of LPS as manifested by enhancement of LPS activation of Limulus amebocyte lysate, increased release of human mononuclear cell tissue factor and enhanced production of human endothelial cell tissue factor (Yoshida et al., 1995; Roth et al., 1994). Our in vivo data do not support these findings, however. Pulmonary bacterial clearance of Pseudomonas aeruginosa, white blood cell counts or differential blood counts were not different between PHP-treated and untreated control animals (Bone et al., 1996c). Our findings are supported by those of Crowley et al. (1993), who observed no change in white blood cell count and clearance of Escherichia coli bacteria after infusion of stroma-free cross-linked hemoglobin in dogs. Further, should hemoglobin interfere with the ability of the RE system to kill bacteria, one would expect higher bacterial tissue counts. Bacterial cultures from heart, lung, liver, spleen, kidney and mesenteric lymph node from PHP-treated as well as untreated sheep surprisingly showed significantly lower bacterial counts in liver, spleen and kidney in the PHP group. In the other organs there was no difference in tissue bacterial counts. A possible explanation is the formation of oxygen radicals, which are responsible for killing organisms. Scavenging of NO by hemoglobin may make more oxygen radicals available for killing

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the bacteria. On the other hand, oxygen radical formation results in increased lipid peroxidation and other forms of cell damage (Motterlini et al., 1995). Further studies on the interactions of hemoglobin, bacterial killing and inflammatory mediators are needed. Interaction with endothelin-1 Apart from hemoglobin’s ability to bind NO there is increasing evidence of an interaction between hemoglobin and endothelin-1. Endothelin-1 is the most potent vasoconstrictor described to date and participates in the regulation of basal vascular tone. At the level of the endothelial cell, proendothelin (bigET) is cleaved to endothelin, the biologically active product. Infusion of DLCHb in healthy rats induced a rise in plasma endothelin-1 levels and tissue endothelin-1 levels in thoracic aorta and renal medulla (Gulati et al., 1995). We found endothelin-1 plasma levels in healthy sheep below the detection limit. After 24 hrs of sepsis they were significantly elevated, and within the first hour of administration of a PHP bolus a 30-fold increase in plasma endothelin-1 levels occurred, which diminished in parallel with the decreasing cardiovascular response to PHP (Bone et al., 1996c). Endothelin-1 production in cultured endothelial cells is stimulated by hemoglobin in vitro (Cocks et al., 1991). It has been proposed that hemoglobin may facilitate the conversion of proendothelin to endothelin-1. After blocking the conversion of proendothelin to endothelin-1 or pretreatment with an endothelin-1 receptor antagonist, administration of a modified hemoglobin in rats resulted in an attenuated increase in MAP compared to control conditions (Schultz et al., 1993; Gulati et al., 1995). These findings suggest that the effects of hemoglobin may not be exclusively mediated by NO scavenging. HEMOGLOBIN AND CLINICAL STUDIES Previous reports hemoglobin administration caused flu-like symptoms, headache, muscle aches, abdominal pain, hypertension, bradycardia, chest pain and laboratory abnormalities (Hess, 1996). However, several modified hemoglobin solutions are currently undergoing clinical investigation for different indications. Hemoglobin has been given in small doses to healthy volunteers and healthy anesthetized patients with no reported adverse effects (Przybelski et al., 1996; Jacobs and Hughes, 1996). One study reported an increase in mean pulmonary artery pressure from 20 to 27 mmHg after administration of 50 mg/kg cell-free hemoglobin (Garrioch et al., 1996). In a recent study hemoglobin use for intraoperative transfusion therapy was associated with increased plasma amylase and lipase levels without clinical evidence, however, of damage to the pancreas (Lessen et al., 1996). Administration of sequential boluses of 100 mg/kg DCLHb to septic patients receiving vasopressor therapy led to a rapid rise in MAP with significant reduction in catecholamine requirements (Rhea et al., 1996). SUMMARY Hemoglobin is able to bind NO and through this mechanism causes vasoconstriction in healthy and septic persons, resulting in increased systemic vascular resistance and MAP. Pulmonary hypertension, however, is an undesirable effect. Hemoglobin restores impaired vascular reactivity to catecholamines in sepsis and tends to improve cardiac dysfunction. The impaired glomerular filtration rate is improved by hemoglobin. While hemoglobin caused a decrease in renal and pancreatic blood flow in healthy animals, blood flow in septic animals was largely unaffected by hemoglobin administration. Although no increased bacterial growth has been found in septic animals receiving hemoglobin, the interactions of hemoglobin with host-

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defense mechanisms require more investigations, as does hemoglobin’s fate in the RE system in which it is taken up. After administration of large amounts of hemoglobin, methemoglobinemia may occur, impairing oxygenation. Among the clinical applications for hemoglobin are its use as an oxygen carrier during blood replacement therapy and as an NO scavenger in sepsis. The results of these studies are encouraging, but more trials confirming hemoglobin’s short- and long-term safety are warranted. REFERENCES Aranow, J.S., Wang, H., Zhuang, J. and Fink, M.R (1996) Effect of human hemoglobin on systemic and regional hemodynamics in a porcine model of endotoxemic shock. Crit. Care Med., 24, 807–814. Balligand, J.L., Ungureanu, D., Kelly, R.A., Kobzik, L., Pimentai, D., Michel, T. and Smith, T.W. (1993) Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J. Clin. Invest., 91, 2314–2319. Barthlen, W., Stadler, J., Lehn, N.L., Miethke, T., Bartels, H. and Siewert, J.R. (1994) Serum levels of end products of nitric oxide synthesis correlate positively with tumor necrosis factor alpha and negatively with body temperature in patients with postoperative abdominal sepsis. Shock, 2, 398–401. Bersten, A. and Sibbald, W.J. (1989) Circulatory disturbances in multiple systems organ failure. Crit. Care Clin., 5, 233–254. Bersten, A.D., Gnidec, A.A., Rutledge, F.S. and Sibbald, W.J. (1990) Hyperdynamic sepsis modifies a PEEPmediated redistribution in organ blood flows. Am. Rev. Respir. Dis., 141, 1198–1208. Biessels, P.T., Hak, J.B., Bleeker, W.K., van Beek, J.H. and Bakker, J.C. (1992) Effects of modified hemoglobin solutions on the isolated rabbit heart. Biomat. Artif. Cells Immobil. Biotechnol., 20, 693–696. Bone, H.G., Schenarts, P.J., Booke, M, Me Guire, R., Harper, D., Traber, L.D. and Traber, D.L. (1996a) Pyridoxalated hemoglobin polyozyethylene conjugate normalizes the hyperdynamic circulation in septic sheep. Crit. Care Med. (in press) Bone, H.G., Schenarts, P.J., Fischer, S., Traber, L.D. and Traber, D.L. (1996b) Continuous infusion of modified hemoglobin normalizes hemodynamics in septic sheep. Anesthesiology, 85, A227. Bone, H.G., Schenarts, P.J., Fischer, S.R., Me Guire, R., Harper, D., Traber, L.D. and Traber, D.L. (1996c) Modified hemoglobin reverses hyperdynamic circulation in septic sheep without effecting organ function. J. Appl. Physiol (submitted). Booke, M, Bradford, D.W., Hinder, R, Harper, D., Brauchle, R.W., Traber, L.D. and Traber, D.L. (1996a) Effects of inhaled nitric oxide and nebulized prostacyclin on hypoxic pulmonary vasoconstriction in anesthetized sheep. Crit. Care Med., 24, 1841–1848. Booke, M., Hinder, R, McGuire, R., Traber, L.D. and Traber, D.L. (1996b) Nitric oxide synthase inhibition versus norepinephrine in ovine sepsis—effects on regional blood flow. Shock, 5, 362–370. Booke, M., Hinder, R, McGuire, R., Traber, L.D. and Traber, D.L. (1996c) Nitric oxide synthase inhibition versus norepinephrine for the treatment of hyperdynamic sepsis in sheep. Crit. Care Med., 24, 835–844. Brady, A.J., Poole Wilson, P.A., Harding, S.E. and Warren, J.B. (1992) Nitric oxide production within cardiac myocytes reduces their contractility in endotoxemia. Am. J. Physiol, 263, H1963-H1966. Breslow, M.J., Miller, C.R, Parker, S.D., Walman, A.T. and Traystman, R.J. (1987) Effect of vasopressors on organ blood flow during endotoxin shock in pigs. Am. J. Physiol, 252, H291-H300. Channick, R.N., Newhart, J.W., Johnson, F.W. and Moser, K.M. (1994) Inhaled nitric oxide reverses hypoxic pulmonary vasoconstriction in dogs. A practical nitric oxide delivery and monitoring system. Chest, 105, 1842–1847. Chatterjee, R., Welty, E.V., Walder, R.Y., Pruitt, S.L., Rogers, RH. and Arnone, A. (1986) Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1-lysine 99 alpha 2). J. Biol Chem., 261, 9929–9937. Cocks, T.M., Malta, E., King, S.J., Woods, R.L. and Angus, J.A. (1991) Oxyhaemoglobin increases the production of endothelin-1 by endothelial cells in culture. Eur. J. Pharmacol, 196, 177–182.

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30 Therapeutic Implications of Recombinant Endothelial Nitric Oxide Synthase Gene Expression in Cerebral and Peripheral Arteries Alex F.Y. Chen1, Timothy O'Brien 2 and Zvonimir S. Katusic1,* 1

Departments of Anesthesiology and Pharmacology,

2Divisions of Endocrinology and Metabolism, Mayo Clinic, 200 First Street SW, Rochester,

Minnesota 55905, USA

Vascular gene therapy involves the transfer of functional genes to vascular tissues in order to correct the malfunction of a specific gene. For gene therapy to be feasible, a safe and efficient means of introducing genes into cells in a living individual is required. In vivo gene transfer can be accomplished using viral and nonviral vectors. For gene transfer to the cerebral and peripheral vascular systems, adenoviral vectors are the most efficient. However, the use of currently available adenoviral vectors is limited due to the transient nature of transgene expression, cytotoxicity and the development of an immune response to virally transduced cells. Transfer and functional expression of recombinant endothelial nitric oxide synthase gene (eNOS) to different vascular beds have been demonstrated both in vivo and ex vivo. The most recent studies of vascular eNOS gene transfer are reviewed in this article, and the potential use of eNOS gene therapy for a number of vascular diseases is also discussed. Although the feasibility of this approach has been demonstrated in animal models, currently available vectors have a number of technical and safety limitations. Therefore, these problems remain to be solved before eNOS gene therapy for vascular diseases can be applied in clinical settings. Key words: Adenoviral vector, adventitia, cerebral and peripheral arteries, endothelium, fibroblasts, gene therapy, nitric oxide synthase. INTRODUCTION Nitric oxide (NO) plays an essential role in the regulation of vascular tone of both cerebral and peripheral beds (Katusic and Cosentino, 1994; Brian et al., 1996; Cooke and Dzau, 1997). A number of vascular

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diseases have been shown to be associated with an impaired vascular NO production and/or activity, including subarachnoid hemorrhage-induced cerebral vasospasm, arteriosclerosis, thrombosis, and hypertension (Dusting, 1995; Cooke and Dzau, 1997). The field of vascular gene transfer has been rapidly developing in the past decade, and important advances have been made in vector technology, transgene expression efficacy, and site-specific gene targeting (Nabel, 1995; Walther and Stein, 1996; Wilson, 1996). Recent results from studies of eNOS gene transfer and expression in the cerebral and peripheral vasculature are of considerable importance in advancing our understanding of transgene expression and function, and represent potential therapeutic strategies for the vascular disorders associated with a deficiency of NO production and/or activity. In this review, the most recent advances in eNOS gene transfer studies are presented and their significance in the study of vascular biology and vascular diseases are discussed. NITRIC OXIDE SYNTHASE AND GENE THERAPY OF VASCULAR DISEASES Nitric Oxide Synthases in the Regulation of Vascular Tone Nitric oxide is a potent endogenous vasodilator. It is synthesized from the guanidino nitrogens of L-arginine through a process that consumes five electrons and results in the formation of co-product L-citrulline by a family of nitric oxide synthases (NOS). The process involves the transfer of electrons between five cofactors including flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiopterin (BH4), heme, and calmodulin (CaM), and requires three co-substrates including L-arginine, nicotinamide adenine dinucleotide phosphate (NADPH), and molecular oxygen (O2) (Knowles and Moncada, 1994). Three isoforms of NOS, encoded by three distinct genes on different chromosomes, have been isolated and purified. Both the neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively activated and expressed upon calcium-calmodulin binding following an increase in intracellular calcium. The inducible isoform (iNOS) is activated upon immunological stimulation independent of calcium. All three NOS isoforms are present in cerebral and peripheral vascular beds (Katusic and Cosentino, 1994; Chen and Lee, 1993, 1995), and the activation of the soluble guanylate cyclase with the production of cyclic 3',5'guanosine monophosphate (cGMP) have been identified as one of the primary mechanisms for NO-induced vasodilation (Moncada et al., 1991). Vascular Gene Transfer Vascular gene transfer refers to the introduction of genes into relevant cells of the blood vessel wall. It holds significant promise as a tool in the study of gene expression and regulation in vascular biology, and as a therapeutic means of controlling local vascular function under diseased conditions (O’Brien, 1997). Two methods have been used to transfer genes to the vessel wall: cell-mediated and direct gene transfer. In cellmediated gene transfer, autologus cells are harvested, grown in culture and the gene is delivered to these cells in vitro. At a later time, the genetically-modified cells are returned to the host animal (Nabel et al., 1989). In direct gene transfer, the gene is delivered directly to the tissue in vivo (Nabel et al., 1990). The direct method has many advantages for in vivo gene transfer in the clinical setting. However, direct delivery of genes to the vessel wall in vivo has a number of technical difficulties. In most animal studies to date, the “Correspondence: Tel: 507–255–5156; Fax: 507–255–7300; E-mail: [email protected].

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Table 30±1. Characteristics of vectors used in vascular gene transfer (Advantages, left; Disadvantages, right)

vector has been delivered to a surgically isolated segment of artery in which the side branches have been ligated. Catheter-based delivery to the porcine coronary arteries in vivo was recently described and was reported to be inefficient (French et al., 1994). Thus, operative delivery to peripheral arteries is substantially more efficient than catheter-based delivery to the coronary arteries. In recent years, both viral and non-viral vectors have been widely used for vascular gene transfer studies. Viral vectors currently used for vascular gene transfer include adenovirus, adeno-associated virus, retrovirus, and most recently, lentivirus (Nabel, 1995; Reifers and Kreuzer, 1995; Heistad and Faraci, 1996; O’Brien, 1997). These recombinant viruses are genetically modified to be replication-incompetent, and they contain an inserted cDNA sequence of interest (i.e. LacZ, eNOS, etc.) and an appropriate promoter (i.e. CMV, RSV, etc.). Non-viral vectors that have been studied in vascular gene transfer include liposomes and molecular conjugates for receptor-mediated gene delivery. Viral vectors generally have higher efficiency in transgene expression but are also more immunogenic than non-viral vectors. A comparison of these vectors with major advantages and disadvantages for vascular gene transfer is summarized in Table 30–1 (Smith, 1995; Wilson, 1996; Gunzburg and Salmon, 1996; O’Brien, 1997).

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Recombinant adenovirus has recently become the best choice among viral vectors for vascular gene transfer studies (Schneider and French, 1993). The human adenovirus is a non-enveloped linear doublestranded DNA virus with a 36 kb viral genome (Wilson, 1996). The niche of adenoviral vectors for vascular gene transfer includes its ability to transduce both dividing and non-dividing cells with high efficiency ex vivo and in vivo, and the ability to generate high titer stock vector (i.e. up to 1012 pfu/ml). The viruses enter the host cells via an endocytosis process through the interaction between viral penton complex and their cell surface receptors (Wickham et al., 1993; Bergelson et al., 1997). Upon entry into the cell, the viruses are taken up into endosomes, which are then disrupted by the virus, resulting in viral DNA release into the cytoplasm. The viral DNA then enters the nucleus, where it is not incorporated into the host chromosome but remains episomal (O’Brien, 1997). The major drawbacks of first generation adenoviral vectors include the capsid protein-induced, cell-mediated (i.e. CD8+ T-cell) immune response which may limit the duration of transgene expression and prevent repeated administration of the vector (Wilson, 1996; Wood et al., 1996). Currently available third generation adenoviral vectors deleted of all viral DNA and cell type specific promoters for vascular cell targeting may eventually overcome these problems (Walther and Stein, 1996; Chen et al., 1997; Ilan et al., 1997). Many studies have examined the use of adenoviral vectors to transduce the vessel wall. Much of the recent work concerning adenoviral-mediated gene transfer to the arterial wall in vivo has utilized a marker vector, encoding -galactosidase, to demonstrate the feasibility of adenoviral-mediated gene transfer to the arterial wall and to optimize the system. The feasibility of this approach was demonstrated when adenoviral vectors were used to transfer the genes for -galactosidase and the cystic fibrosis transmembrane conductance regulator to the carotid artery and jugular vein of sheep in vivo (Lemarchand et al., 1993). In vivo gene transfer to injured rat carotid arteries was demonstrated and a therapeutic window between viral doses of 1010 and 1011 pfu/ml described (Schulick et al., 1995a). This group also demonstrated in a non-injured rat carotid artery that vascular wall gene transfer was localized to the endothelium and adventitia (Schulick et al., 1995b). This may be due to the fact that the internal elastic lamina forms a barrier to the transport of vector particles to the media (Rome et al., 1994). Construction of Recombinant Adenoviral Vector Encoding Endothelial Nitric Oxide Synthase Gene We generated a recombinant adenoviral vector encoding an eNOS gene, driven by the cytomegalovirus immediate early promoter, through homologous recombination techniques (Figure 30–1). A full length human serotype 5 wild adenovirus was rendered replicationincompetent by the deletion of the early 1 (E1) region of the virus, with the replacement of a cDNA sequence of the bovine aortic endothelial cell eNOS gene construct. The vector was propagated at high titers on 293 cells, a human embryonic kidney carcinoma helper cell line that expresses the El region in trans thus enabling the virus to replicate (Spector and Samaniego, 1995; Chen et al., 1997a). Another adenoviral vector encoding an Escherichia coli galactosidase (LacZ) reporter gene (AdCMVLacZ) was used as a control vector for both ex vivo and in vivo vascular gene transfer studies (Katusic et al., 1996; Cable et al., 1997; Chen et al., 1997a, 1997b, 1997e, 1997d; Kullo et al., 1997a, 1997b, 1997c).

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IMPLICATIONS OF RECOMBINANT NITRIC OXIDE SYNTHASE GENE TRANSFER FOR CEREBRAL AND PERIPHERAL VASCULAR DISEASES Gene Transfer to the Cerebral Vasculature eNOS gene transfer to cerebral arteries In contrast to gene transfer to the peripheral vasculature, gene delivery to the cerebral vasculature has unique problems. A segment of cerebral artery cannot be occluded in order to allow localized gene delivery due to problems with ischemia. One potential approach to transduction of the cerebral vasculature would be delivery of genes to the vascular adventitia via the cerebrospinal fluid (CSF). Adenoviral-mediated reporter gene transfer (i.e. -galactosidase) to the cerebral vessels has been described by administration of vector to the CSF, either under normal conditions (Ooboshi et al., 1995) or in the presence of cisternal blood (Muhonen et al., 1997). This is an attractive approach to transduction of cerebral blood vessels, because of significant problems associated with the interruption of cerebral blood flow which is required for luminal administration of recombinant DNA. We have recently demonstrated that adenoviral vector-mediated eNOS gene transfer to canine cerebral arteries ex vivo resulted in functional transgene expression in the adventitia and endothelium, leading to increased basal production of cGMP with a subsequent reduction in UTP-induced vasoconstriction and enhancement in endothelium-dependent vasorelaxation (Katusic et al., 1996; Chen et al., 1997a). These findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall. More recently, we have successfully delivered adenoviral vectors encoding eNOS gene and galactosidase reporter gene into canine cerebral blood vessels in vivo via CSF, by means of vector injection into the cisterna magna (Chen et al., 1997b). Transgene expression are localized in the adventitial fibroblasts of major cerebral arteries, as shown by electron microscopy immunogold labeling (Chen et al., 1997b, 1997c). In eNOS gene- but not galactosidase reporter gene-transduced cerebral arteries, bradykinin-induced relaxations were significantly augmented. The change in vasoreactivity was also accompanied by increased cGMP production (Chen et al., 1997b, 1997c, 1997d). These results suggest that perivascular eNOS gene delivery via CSF and functional expression is a feasible approach that does not require interruption of cerebral blood flow. eNOS gene transfer and cerebral vasospasm In vivo functional expression of recombinant eNOS gene in cerebral blood vessels with increased local nitric oxide production may have important clinical implications. Subarachnoid hemorrhage-induced cerebral vasospasm, for instance, has been shown to be associated with an impaired L-arginine-nitric oxidecGMP pathway (Katusic et al., 1993; Cosentino et al., 1993; Kim et al., 1988, 1992), including a decrease in eNOS mRNA level (Hino et al., 1996) and loss of NOS immunoreactivity (Pluta et al., 1996). Experimental vasospasm could be alleviated by intravenous administration of glycerol trinitrate (Frazee et al., 1981), a well-known nitrovasodilator that releases nitric oxide intracellularly (Moncada and Higgs, 1995), intracarotid infusion of nitric oxide (Afshar et al., 1995), or restoration of endogenous nitric oxide production in arterial wall by administration of L-arginine and superoxide dismutase (Kajita et al., 1993, 1994). The administration of an adenoviral vector via CSF with functional expression of recombinant eNOS in cerebral arteries raises the possibility of providing continuous NO supply to the underlying smooth muscle

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Figure 30±1. Generation of recombinant adenoviral vectors encoding cDNA for eNOS: (Top), The shuttle vector contains Ad5 sequences (1–454 and 3334–6231) flanking the CMV promoter, a cloning polylinker and polyadenylation signal, Np, nucleotide position; (Bottom), After inserting cDNA sequence of eNOS into pACCMVpLpA to generate pACCMVNOS, the recombinant adenovirus was constructed through homologous recombination between plasmid pACCMVNOS and the Ad5 genome in 293 cells. (Original figure from Chen et al., 1997a; Reprinted with permission.)

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cells, and may become a potentially feasible therapeutic strategy in alleviating this devastating complication of subarachnoid hemorrhage. Cerebral vasospasm following subarachnoid hemorrhage occurs between 4 and 12 days after subarachnoid hemorrhage (Weaver, 1995). It is a transient phenomenon, probably due to stimulation of the cerebral blood vessels by blood present in the CSF, which is associated with high morbidity and mortality. Adenoviral vector-mediated transfer of eNOS gene may be useful in this setting as transient transgene expression would be an advantage. Intracranial delivery and functional expression of recombinant eNOS gene in the cerebral vasculature, therefore, may provide a novel and feasible approach for the treatment of certain cerebrovascular diseases such as vasospasm. Gene Transfer to the Peripheral Vasculature eNOS gene transfer and restenosis Angioplasty is one of the most commonly used forms of therapy in the treatment of occlusive vascular disease. A common complication of this procedure is restenosis which occurs 3–6 months after therapy. Systemic therapies have not been successful in the prevention of this complication. Smooth muscle cells form the principal cellular component of arteries and play a key role in formation of neointima in various vascular disease states including restenosis (Ross, 1993). One approach to the prevention of postangioplasty restenosis would be to inhibit smooth muscle cell proliferation at the time of the procedure. As the formation of neointima is a multifactorial process, pleiotropic therapeutic approaches are more likely to be successful than those which act on a single mechanism (Lafont et al., 1995). Nitric oxide is a simple diatomic molecule which has many putative effects in vascular biology including inhibition of monocyte adhesion, platelet aggregation and smooth muscle cell proliferation (Figure 30–2). We have recently demonstrated that adenoviral mediated gene transfer of eNOS to porcine coronary artery smooth muscle cells in vitro results in functional recombinant enzyme expression and inhibition of smooth muscle proliferation (Kullo et al., 1997a). This observation suggests that overexpression of eNOS in smooth muscle cells results in generation of biologically active NO. We also demonstrated that the activity of recombinant eNOS could be stimulated by the addition of sepiapterin, a precursor of tetrahydrobiopterin, to the tissue culture medium. These observations suggest that increasing local nitric oxide production via eNOS gene transfer may have a unique role in inhibiting neointima formation in vivo. Local delivery of NO is difficult due to its short half-life and limited solubility in aqueous solutions. Gene transfer of nitric oxide synthase, the enzyme responsible for the generation of NO from L-arginine, may overcome these difficulties. Recently, the feasibility of this approach was demonstrated when gene transfer of endothelial nitric oxide synthase to the injured rat carotid artery was shown to inhibit restenosis (von der Leyen et al., 1995). Direct delivery of eNOS gene to the endothelial cells of the rabbit carotid artery results in increased local production of nitric oxide (Kullo et al., 1997b). It should be noted, however, that the vector was delivered to an isolated segment of the rabbit carotid artery in vivo by surgical means. Occlusion of blood flow was therefore necessary. We also demonstrated that adventitial delivery of eNOS to the rabbit carotid artery in vivo resulted in enhanced vasorelaxation (Kullo et al., 1997c). In agreement with our data, similar results were also observed in a recent ex vivo study by Ooboshi et al. (1997). In contrast to intraluminal delivery, occlusion of blood supply was not necessary with adventitial delivery of vector in vivo. Furthermore, as restenosis may involve adventitial remodeling, in addition to smooth muscle cell proliferation (Scott et al., 1996; Shi et al., 1996; Zalewski and Shi, 1997), it will be interesting to study whether adventitial administration of eNOS will result in inhibition of myointimal hyperplasia after balloon injury. As restenosis of the coronary vessels after angioplasty is the most significant clinical problem, obtaining

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efficient gene transfer to the coronary vasculature will be critical. Delivery of adeno viral vectors to the pericardium has recently been described (Lamping et al., 1997) and this may be a potential route to deliver vectors to the adventitia of the coronary blood vessels. eNOS gene transfer and atherosclerosis Local or systemic gene delivery could be used in the treatment of atherosclerosis. In animal models, most progress has been made with the latter approach (Kozarsky and Wilson, 1995). Hyperlipidemia is a significant risk factor for cardiovascular disease (Cooke and Dzau, 1997). Gene therapy approaches to inherited dyslipidemia have been explored (for a recent review, see O’Brien, 1996). A local vascular wall approach to the treatment of atherosclerosis using gene delivery will be more difficult. Recently, topical application of adenoviral vector to the periarterial sheath of both normal and atherosclerotic arteries in cynomolgus monkeys and rabbits was demonstrated (Rios et al., 1995; Heistad and Faraci, 1996). This approach led to marker gene expression in about 20% of the adventitial cells in the femoral and carotid arteries, with no expression of the transgene in the intima or media (Heistad and Faraci, 1996). Since atherosclerosis affects the vasculature in a diffuse manner and local gene delivery may not suffice, gene delivery to the vessel wall as a therapeutic approach to atherosclerosis will need the development of improved vectors capable of widespread vascular transduction. As reduced NO bioavailability has been implicated in cholesterol-induced vascular dysfunction (Cooke and Dzau, 1997), we recently examined the effect of eNOS gene transfer to atherosclerotic vessels on vascular reactivity (Mozes et al., 1997). In this study, we demonstrated that overexpression of eNOS in atherosclerotic rabbit aorta resulted in improvement of endothelium-dependent vasorelaxation. Thus, gene therapy approaches resulting in overexpression of eNOS in the vascular wall may be of some utility in the future therapy of cholesterol-induced vascular dysfunction and atherosclerosis. eNOS gene transfer and vein bypass graft failure Although the internal mammary artery is the conduit of choice for myocardial revascularization (Loop et al., 1986), the greater saphenous vein is the most commonly utilized bypass conduit due to its availability and versatility in reaching distal coronary branches which cannot be bypassed with the IMA. However, 12 to 20% of saphenous vein coronary artery bypass grafts occlude during the first post-operative year (Grondin et al., 1974) and subsequent risk of graft occlusion is approximately 4% per year (Grondin et al., 1979). Early graft attrition, exclusive of technical failure, is associated with luminal thrombosis whereas subsequent failure of saphenous vein grafts is due to fibrointimal hyperplasia and atherosclerosis (Fitzgibbon et al., 1986). Two potential molecular approaches to reducing vein graft occlusion would be to enhance NO generation or decrease smooth muscle cell proliferation in the saphenous vein graft. In the former, nitric oxide synthase gene would be transferred to the vein graft at the time of harvest. As nitric oxide is known to inhibit many of the putative processes involved in graft failure, nitric oxide synthase overexpression may decrease the rate of occurrence of this problem. We have recently demonstrated that ex vivo adenoviral mediated eNOS gene transfer to the human saphenous vein is feasible and results in increased production of nitric oxide in the vascular wall (Cable et al., 1997).

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Figure 30±2. Schematic diagram of vascular effects of recombinant eNOS gene transfer. Viral or non-viral vector encoding eNOS gene can be transferred and expressed in either adventitial fibroblasts or endothelial cells of blood vessel wall, resulting in increased recombinant eNOS protein expression and subsequently increased nitric oxide production in the underlying smooth muscle cells. The possible biological effects of recombinant nitric oxide formation in vascular wall include augmentation of vasodilatation, inhibition of platelet aggregation, leukocyte adhesion, and smooth muscle mitogenesis.

eNOS gene transfer and pulmonary hypertension Diseases of the pulmonary vasculature are significant causes of morbidity and mortality (Collins-Nakai and Rabinovitch, 1993). Treatment options for these disorders are currently limited. Specific diseases in this category include primary pulmonary hypertension and hypoxic chronic pulmonary hypertension. Transfer of genes encoding vasodilators or inhibitors of smooth muscle cell proliferation may therefore provide a local therapeutic approach to pulmonary hypertension. Adenoviral-mediated gene transfer to the pulmonary vasculature has been described (Lemarchand et al., 1994). However, the extent and distribution of expression has been variable. In one report, cathetermediated gene transfer to the pulmonary vasculature of the rat was unsuccessful (Schachtner et al., 1995).

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In contrast, gene transfer to the pulmonary vasculature of pigs has been reported to result in transgene expression in the pulmonary vessels and alveolar septa (Muller et al., 1995). The different results obtained in these reports may be due to the species studied. Genes have also been delivered to the lungs via the airways (Rosenfeld et al., 1992). Indeed, this is the route of choice for gene therapy approaches to the treatment of cystic fibrosis (Wilson et al., 1994). More recently, airway administration of the gene for endothelial nitric oxide synthase has been shown to reduce acute hypoxic pulmonary vasoconstriction in rats (Janssens et al., 1996). Thus, gene transfer to the airways via aerosolization may be a potential therapeutic approach to pulmonary hypertension. SUMMARY AND FUTURE DIRECTIONS The feasibility of transferring eNOS genes to the vasculature has now been demonstrated in a number of animal models both in vivo and ex vivo as reviewed in this chapter. However, many problems need to be overcome before this technology enters the clinical arena. The main difficulty is the limitations of currently available gene transfer vectors. Improvements in vector design are currently under intensive investigation. While, improved liposomes and expression plasmids hold the most promise, viral vector development is also being pursued and newer generation vectors are already being tested (Wilson, 1996). Biosafety issues will remain a concern with viral vectors. Another formidable problem is the technical issue of how to deliver the vectors to the vessel wall in clinical practice. Most successful attempts at gene transfer have used open surgical administration of vector with ligation of vessel side branches. Catheter-mediated gene delivery would be more practical but results using this system have not been encouraging. Therefore, in addition to improvements in vector design, enhanced methods of in vivo gene delivery to the vessel wall will be required prior to widespread clinical application of this technology. Taken together, vascular gene transfer is a powerful experimental approach to study recombinant transgene expression and function in vascular biology and attempt to manipulate the arterial wall for therapeutic purposes. Future studies in this field will shed more light in determining its application for human vascular gene therapy. ACKNOWLEDGMENTS The studies were supported in part by National Institutes of Health grants HL-44116, HL-53524 (Z.S.K.), NIH Institutional Training Grant GM08288 (A.F.Y.C.), American Heart Association/Minnesota Affiliate grantin-aid and Mayo Clinic intramural research grant (A.F.Y.C. and T.O.), and funds from the Mayo Clinic and Foundation. REFERENCES Afshar, J.K.B., Pluta, R.M., Boock, R.J., Thompson, B.C. and Oldfield, E.H. (1995) Effect of intracarotid nitric oxide on primate cerebral vasospasm after subarachnoid hemorrhage. Journal of Neuro surgery, 83, 118– 122. Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S. et al. (1997) Isolation of a common receptor for coxsackie B virus and adenoviruses 2 and 5. Science, 275, 1320–1323. Brian, J.E., Jr., Faraci, F.M. and Heistad, D.D. (1996) Recent insights into the regulation of cerebral circulation. Clinical and Experimental Pharmacology and Physiology, 23, 449–457. Cable, D.G., O’Brien, T., Schaff, H.V. and Pompili, V.J. (1997) Recombinant eNOS-transduced human saphenous veins: gene therapy to augment nitric oxide production in bypass conduits. Circulation, 96(Suppl. II), 173–178.

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Chen, A.F.Y., O’Brien, T., Tsutsui, M., Kinoshita, H., Pompili, V.J., Crotty, T.B., et al (1997a) Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery. Circulation Research, 80, 327–335. Chen, A.F.Y., Smith, L.A., Crotty, T.B., O’Brien, T. and Katusic, Z.S. (1997b) In vivo eNOS gene transfer in cerebral arteries via cerebrospinal fluid. FASEB Journal, 11, A246 (Abstract). Chen, A.F.Y., Smith, L.A., Grotty, T.B., O’Brien, T. and Katusic, Z.S. (1997c) In vivo adenoviral-mediated expression of recombinant endothelial nitric oxide synthase gene cerebral arteries. Journal of Cerebral Blood Flow and Metabolism, (Abstract) 17(Suppl.), 5452. Chen, A.F.Y., Smith, L.A., Crotty, T.B., O’Brien, T. and Katusic, Z.S. (1997d) Effects of in vivo recombinant endothelial nitric oxide gene transfer on vascular reactivity of canine cerebral arteries. Stroke, 28, 260 (Abstract). Chen, F.Y. and Lee, T.J.F. (1993) Role of nitric oxide in neurogenic vasodilation of porcine cerebral artery. Journal of Pharmacology and Experimental Therapeutics, 265, 339–345. Chen, F.Y. and Lee, T.J.F. (1995) Arginine synthesis from citrulline in perivascular nerves of cerebral artery. Journal of Pharmacology and Experimental Therapeutics, 273, 895–901. Chen, H.-H., Mack, L.M., Kelly, R., Ontell, M., Kochanek, S. and Clemens, P.R. (1997) Persistence in muscle of an adenoviral vector that lacks all viral genes. Proceedings of the National Academy of Science USA, 94, 1645–1650. Collins-Nakai, R.L. and Rabinovitch, M. (1993) Pulmonary vascular obstructive disease. Cardiology Clinician, 11, 675–687. Cooke, J.P. and Dzau, V.J. (1997) Nitric oxide synthase: Role in the genesis of vascular disease. Annual Review of Medicine, 48, 489–509. Cosentino, F., Sill, J.C. and Katusic, Z.S. (1993) Endothelial L-arginine pathway and relaxations to vasopressin in canine basilar artery. American Journal of Physiology, 264, H413-H418. Dusting, G.J. (1995) Nitric oxide in cardiovascular disorders. Journal of Vascular Research, 32, 143–161. Fitzgibbon, G.M., Leach, A.J., Leon, W.J., Burton, J.R. and Kafka, H.P. (1986) Coronary bypass graft fate: angiographie study of 1179 vein grafts early, one year and 5 years after operation. Journal of Thoracic Cardiovascular Surgery, 91, 773–778. Frazee, J.G., Giannotta, S.L. and Stern, W.E. (1981) Intravenous nitroglycerin for the treatment of chronic cerebral vasoconstriction in the primate. Journal of Neurosurgery, 55, 865–868. French, B.A., Mazur, W., Ali, N.M., Geske, R.S., Finnigan, J.P, Rodgers G.P et al. (1994) Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries, atherosclerotic arteries and two models of coronary restenosis. Circulation, 90, 2402–2413. Grondin, C.M., Lesperance, J., Bourassa, M.G., Pasternac, A., Campneau, L. and Grondin, P. (1974) Serial angiographie evaluation in 60 consecutive patients with aortocoronary artery vein grafts 2 weeks, 1 year and 3 years after operation. Journal of Thoracic Cardiovascular Surgery, 67, 1–6. Grondin, C.M., Campeau, L., Lesperance, J., Solymoss, B.C., Vouhe, P., Castonguay, Y.R. et al. (1979) Atherosclerotic changes in coronary grafts six years after operation. Journal of Thoracic Cardiovascular Surgery, 77, 24–31. Gunzburg, W.H. and Salmons, B. (1996) Development of retro viral vectors as safe, targeted gene delivery systems. Journal of Molecular Medicine, 74, 171–182. Heistad, D.D. and Faraci, P.M. (1996) Gene therapy for cerebral vascular disease. Stroke, 27, 1688–1693. Hino, A., Tokuyama, Y., Weir, B., Takeda, J., Yano, H., Bell, G.I., et al. (1996) Changes in endothelial nitric oxide synthase mRNA during vasospasm after subarachnoid hemorrhage in monkeys. Neurosurgery, 39, 562–568. Ilan, Y., Droguett, G., Chowdhury, N.R., Li, Y, Sengupta, K., Thummala, N.R., et al. (1997) Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits longterm gene expression. Proceedings of the National Academy of Science USA, 94, 2587–2592. Janssens, S.P., Bloch, K.D., Nong, Z., Gerard, R.D., Zoldhelyi, P. and Collen, D. (1996) Adenoviral-mediated transfer of the human endothelial nitric oxide synthase gene reduces acute hypoxic pulmonary vasoconstriction in rats. Journal of Clinical Investigation, 98, 317–324. Kajita, Y., Suzuki, Y., Oyama, H., Tanazawa, T., Takayasu, M., Shibuya, M., et al. (1993) Combined effect of Larginine and superoxide dismutase on the spastic basilar artery after subarachnoid hemorrhage in dogs . In Cerebral Vasospasm, edited by J.M. Findlay, pp. 239–242. Tokyo: Elsevier Science Publishers.

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Kajita, Y., Suzuki, Y., Oyama, H., Tanazawa, T., Takayasu, M., Shibuya, M., et al. (1994) Combined effect of Larginine and superoxide dismutase on the spastic basilar artery after subarachnoid hemorrhage. Journal of Neurosurgery, 80, 476–483. Katusic, Z.S. and Cosentino, F. (1994) Nitric oxide synthase: From molecular biology to cerebrovascular 1.Transduces non-dividing cells physiology. News in Physiological Sciences, 9, 64–67. Katusic, Z.S., Milde, J.H., Cosentino, F. and Mitrovic, B.S. (1993) Subarachnoid hemorrhage and endothelial Larginine pathway in small brain stem arteries in dogs. Stroke, 24, 392–399. Katusic, Z.S., Tsutsui, M., Chen, A.F.Y., O’Brien, T. and Crotty, T.B. (1996) Effects of recombinant endothelial nitric oxide synthase gene expression on endothelium-dependent relaxations in canine arteries. Journal of Vascular Research, 33 (Supplement 1, Abstract), 188. Kim, P., Sundt, T.M. Jr. and Vanhoutte, P.M. (1988) Alterations in endothelium-dependent responsiveness of the canine basilar artery after subarachnoid hemorrhage. Journal of Neurosurgery, 69, 239–246. Kim, P., Sundt, T.M. Jr. and Vanhoutte, P.M. (1992) Reduced production of cGMP underlies the loss of endotheliumdependent relaxations in the canine basilar artery after subarachnoid hemorrhage. Circulation Research, 70, 248–256. Knowles, R.G. and Moncada, S. (1994) Nitric oxide synthase in mammals. Biochemical Journal, 298, 249– 258. Kozarsky, K.F. and Wilson, J.M. (1995) Gene therapy of hypercholesterolemic disorders. Trends in Cardiovascular Medicine, 5, 205–209. Kullo, I.J., Schwartz, R.S., Pompili, V.J., Tsutsui, M., Milstein, S., Fitzpatrick, L.A., et al. (1997a) Expression and function of recombinant endothelial nitric oxide synthase in coronary artery smooth muscle cells. Arteriosclerosis Thrombosis and Vascular Biology, 17, 2405–2412. Kullo, I.J., Mozes, G., Schwartz, R.S., Gloviczki, P., Tsutsui, M., Katusic, Z.S., et al. (1997b) Enhanced endotheliumdependent relaxations after gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries. Hypertension, 30, 314–320. Kullo, I.J., Mozes, G., Schwartz, R.S., Gloviczki, P., Crotty, T.B., Katusic, Z.S., et al. (1997c) Adventitial gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity. Circulation, 96, 2254–2261. Lafont, A., Guerot, C. and Lemarchand, P. (1995) Which gene for restenosis? Lancet, 346, 1442. Lamping, K.G., Rios, C.D., Chun, J.A., Ooboshi, H., Davidson, B.L. and Heistad, D.D. (1997) Intrapericardial administration of adenovirus for gene transfer. American Journal of Physiology, 272, H310-H317. Lemarchand, P., Jones, M., Yamada, I. and Crystal, R.G. (1993) In vivo gene transfer and expression in normal uninjured blood vessels using replication defective recombinant adenovirus vectors. Circulation Research, 72, 1132–1138. Lemarchand, P., Jones, M., Danel, C., Yamada, I., Mastrangeli, A. and Crystal, R.G. (1994) In vivo adenovirusmediated gene transfer to lungs via pulmonary artery. Journal of Applied Physiology, 76, 2840–2845. Loop, ED., Lytle, B.W., Cosgrove, D.M., Stewart, R.W., Goormastic, M., Williams, G.W. et al. (1986) Influence of the internal mammary artery graft on 10-year survival and other cardiac events. New England Journal of Medicine, 314, 1–16. Moncada, S. and Higgs, E.A. (1995) Molecular mechanisms and therapeutic strategies related to nitric oxide. FASEB Journal, 9, 1319–1330. Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacology Review, 43, 109–142. Mozes, G., Kullo, I., Cable, D., Crotty, T.B., Gloviczki, P., Katusic, Z.S. et al. (1997) Nitric oxide synthase gene transfer improves cholesterol-induced vasomotor dysfunction in the rabbit aorta. Journal of Investigative Medicine, 45, 256A (Abstract). Muhonen, M.G., Ooboshi, H., Welsh, M.J., Davidson, B.L. and Heistad, D.D. (1997) Gene transfer to cerebral blood vessels after subarachnoid hemorrhage. Stroke, 28, 822–829. Muller, D.W.M., Gordon, D., San, H., Yang, Z., Pompili, V.J., Nabel, G.J. et al. (1995) Catheter-mediated pulmonary vascular gene transfer and expression. Circulation Research, 75, 1039–1049. Nabel, E.G. (1995) Gene therapy for cardiovascular disease. Circulation, 91, 541–548.

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Nabel, E.G., Plautz, G., Boyce, P.M., Stanley, J.C. and Nabel G. (1989) Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science, 244, 1342–1344. Nabel, E.G., Plautz, G. and Nabel, G.J. (1990) Site-specific gene expression in vivo by direct gene transfer into the arterial wall. Science, 249, 1285–1288. O’Brien, T. (1996) Gene therapy and inherited dyslipidemia. Endocrinology Practice, 2, 37–43. O’Brien, T. (1998) Gene transfer and vascular disease. The Irish College of Physicians and Surgeons, in press. Ooboshi, H., Chu, Y, Rios, C.D., Faraci, P.M., Davidson, B.L. and Heistad, D.D. (1997) Altered vascular function following adenovirus-mediated overexpression of endothelial nitric oxide synthase. American Journal of Physiology, 273, H265-H270. Ooboshi, H., Welsh, M.J., Rios, C.D., Davidson, B.L. and Heistad, D.D. (1995) Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue. Circulation Research, 77, 7–13. Pluta, R.M., Thompson, E.G., Dawson, T.M., Snyder, S.H., Boock, R.J. and Oldfield, E.H. (1996) Loss of nitric oxide synthase immunoreactivity in cerebral vasospasm. Journal of Neurosurgery, 84, 648–654. Reifers, F. and Kreuzer, J. (1995) Current aspects of gene therapy: Implications for vascular interventions. Journal of Molecular Medicine, 73, 595–602. Rios, C.D., Ooboshi, H., Piegors, D., Davidson, B.L. and Heistad, D.D. (1995) Adenovirus-mediated gene transfer to normal and atherosclerotic arteries—a novel approach. Arteriosclerosis, Thrombosis, and Vascular Biology, 15, 2241–2245. Rome, J.J., Shayani, V., Flugelman, M.Y., Newman, K.D., Farb, A., Virmani, R., et al. (1994) Anatomic barriers influence the distribution of in vivo gene transfer into the arterial wall. Modeling with microscopic tracer particles and verification with a recombinant adenoviral vector. Arteriosclerosis and Thrombosis, 14, 148– 161. Rosenfeld, M.A., Yoshimura, K., Trapnell, B.C., Yoneyama, K., Rosenthal, E.R. and Dalemans, W., et al. (1992) In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell, 68, 143–155. Ross, R. (1993) The pathogenesis of atherosclerosis: a perspective for the 1990’s. Nature, 362, 801–809. Schachtner, S., Rome, J.J., Hoyt, R.F., Newman, K.D., Virmani, R. and Dichek, D.A. (1995). In vivo adenovirusmediated gene transfer via the pulmonary artery of rats. Circulation Research, 76, 701–709. Schachtner, S., Rome, J.J., Hoyt, R.F., Newman, K.D., Virmani, R. and Dichek, D.A. (1995) In vivo adenovirusmediated gene transfer via the pulmonary artery of rats. Circulation Research, 76, 701–709. Schneider, M.D. and French, B.A. (1993) The advent of adenovirus: gene therapy for cardiovascular disease. Circulation, 88, 1937–1942. Schulick, A.H., Newman, K.D., Virmani, R. and Dichek, D.A. (1995a) In vivo gene transfer into injured carotid arteries. Optimization and evaluation of acute toxicity. Circulation, 91, 2407–2414. Schulick, A.H., Dong, G., Newman, K.D., Virmani, R. and Dichek, D.A. (1995b) Endothelium-specific in vivo gene transfer. Circulation Research, 77, 475–85. Scott, N.A., Cipolla, G.D., Ross, C.E., Dunn, B., Martin, F.H., Simonet, L., et al. (1996) Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation, 93, 2178–2187. Shi, Y, O’Brien, Jr., J.E., Fard, A., Mannion, J.D., Wang, D. and Zalewski. (1996) Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries. Circulation, 94, 1655–1664. Smith, A.E. (1995) Viral vectors in gene therapy. Annual Review of Microbiology, 49, 807–838. Spector, D.J. and Samaniego, L.A. (1995) Construction and isolation of recombinant adenovirus with gene replacements. Methods in Molecular Genetics, 7, 31–44. von der Leyen, H.E., Gibbons, G.H., Morishita, R., Lewis, N.P., Zhang, L., Nakajima, M., et al. (1995) Gene therapy inhibiting neointimal vascular lesion: In vivo transfer of endothelial cell nitric oxide synthase gene. Proceedings of the National Academy of Science USA, 92, 1137–1141. Walther, W. and Stein, U. (1996) Cell type specific and inducible promoters for vectors in gene therapy as an approach for cell targeting. Journal of Molecular Medicine, 74, 379–392.

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31 Gene Therapy Approaches with iNOS Edith Tzeng and Timothy R.Billiar Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Atherosclerosis contributes to 50% of all mortality in the United States and Europe. The pathogenesis of atherosclerosis has been intensely studied but no effective means of preventing this disease is currently available. Therapies for atherosclerosis are directed at disrupting occlusive atherosclerotic plaques or bypassing diseased arterial segments with conduits. While these therapies have had considerable impact, they are still limited by the very nature of vascular healing which promotes an exuberant proliferative process and results in restenosis at sites of intervention. Restenosis secondary to intimal hyperplasia is the predominant etiology of the therapeutic failures of conventional interventions such as balloon angioplasty and surgical bypass grafting (Ross, 1993). There is tremendous need to elucidate the processes initiated by vascular injury that are responsible for intimal hyperplasia in hopes of developing more effective, preventative therapies. PATHOGENESIS OF INTIMAL HYPERPLASIA The arterial wall is composed of the intima, media, and adventitia. The intima consists of the endothelium and, in some species, 1–2 layers of SMCs as well (Schwartz et al., 1995a). These cells are clearly demarcated from the SMCs of the media by the internal elastic lamina. Vascular injury stimulates the development of a “neointima” at the site of injury. While the etiology of intimal hyperplasia is multifactorial, the most common initiating event is the dirsuption of the endothelium (Gibbons and Dzau, 1996). The endothelium is the normal source of regulatory molecules that modulate its own physiologic responses and the growth of the underlying SMCs while minimizing the accessibility of inflammatory cells

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to the vascular wall (Gibbons and Dzau, 1996). Injury characteristically results in the loss of the endothelium and is quickly followed by platelet adhesion with thrombus formation (Schwartz et al., 1995a). An inflammatory response is initiated, marked by macrophage and lymphocyte infiltration. Vascular healing has been well characterized in rodents (Schwartz et al., 1995b; Clowes et al., 1983). About 24 hours after the insult, medial SMC proliferation can be detected (Clowes et al., 1983). This “first wave” replication is mediated by basic fibroblast growth factor (bFGF) released locally by the injured SMCs (Olsen et al., 1992; Lindner and Reidy, 1991). Basic FGF is a potent mitogen for both SMCs and endothelial cells. Inhibition of this first wave response using antisense oligonucleotides directed against cell cycle genes (Simmons et al., 1994; Biro et al., 1993) diminishes intimal hyperplasia. The “second wave”, occurring between post-injury days 3–14 (Clowes et al., 1983), consists of SMC migration across the internal elastic lamina to form the neointima. The cellular signals that mediate SMC migration include platelet-derived growth factor (PDGF) (Ferns et al., 1991; Jackson et al., 1993), transforming growth factor (TGF ) (Schwartz et al., 1995), and angiotensin II (Prescott et al., 1991). Following SMC migration, neointimal SMCs undergo extensive proliferation and deposit extracellular matrix (Clowes et al., 1986). This “third wave” response accounts for the most significant increase in neointima thickness. The signals regulating this third phase of vascular healing are still under investigation. NITRIC OXIDE IN VASCULAR PHYSIOLOGY AND PATHOPHYSIOLOGY Normal human vascular physiology is orchestrated by an array of counter-regulatory mediators. One such mediator is endothelium-derived relaxing factor, originally described in 1980 by Furchgott and Zawadzki (1980) and eventually identified as nitric oxide (NO) (Palmer et al., 1987; Ignarro et al., 1987). This NO, originating from a constitutively expressed NO synthase (ecNOS) in the endothelium, is responsible for modulating vasomotor tone, acting through a cGMP intermediate (Palmer et al., 1987; Ignarro et al., 1987). NO also possesses a number of other vasoprotective properties. NO inhibits platelet adhesion and aggregation (Padomski et al., 1987), also through a cGMP-mediated pathway. in vitro data indicate that NO inhibits vascular SMC proliferation (Garg and Hassid, 1989) while promoting endothelial cell growth (Guo et al., 1995; Ziche et al., 1994). Finally, NO has been shown to reduce leukocyte infiltration of the endothelial barrier (Kubes et al., 1991). Endothelial cNOS is localized to the caveolae of the endothelial plasma membrane (Garcia-Cardena et al., 1996), and it is likely that both the lumenal surface and the endothelial-SMC interface are constantly “bathed” in NO at concentrations adequate to exert the above physiologic actions. In light of these vascular actions of NO, reduced NO availability or, conversely, providing a therapeutic source of NO may have profound influences on the vascular wall. The precise role of NO in the pathogenesis of atherosclerosis and restenosis is still under investigation. However, several studies implicate NO deficiency in the establishment and progression of these maladaptive states. Early events in the formation of atherosclerotic plaques include the accumulation of lipid-ladened macrophages in the vascular intima and disturbance of the endothelium (Ross, 1993). Even though the endothelium overlying a plaque may appear morphologically intact, endothelial NO synthesis capacity may in fact be functionally impaired. Similarly, NO consumption may be increased. Oxidized lipoproteins, a common component of atherosclerotic plaques, can bind and inactivate NO (Chin et al., 1992), thereby creating an environment depleted of NO. Diabetic patients have a predilection for

Correspondence: Edith Tzeng, MD, Department of Surgery, University of Pittsburgh, 497 Scaife Hall, Pittsburgh, PA 15261, USA. Tel: (412) 648–1935; Fax: (412) 648–9551; E-mail: [email protected].

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Figure 31±1. Events at sites of vascular injury. At sites of vascular injury resulting from angioplasty, surgical bypass, or endarterectomy, the endothelium is frequently denuded. This leaves behind an exposed internal elastic lamina that attracts platelets and leukocytes to adhere. These activated cells release a variety of cytokines and growth factors which can promote the migration and proliferation of the medial smooth muscle cells. NO, which is normally produced by the endothelial NOS, is capable of combatting the invasion of platelets and leukocytes as well as inhibiting smooth muscle proliferation. However, loss of the endothelium at sites of injury eliminates this source of NO and, as a consequence, the protection from thrombosis and intimal hyperplasia afforded by NO.

atherosclerosis which may stem from the progressive deposition of glycosylation products in the vasculature that may also consume NO (Bucala et al., 1991). In support of this, atherosclerotic arteries have been shown to be less vasoresponsive to NO-dependent agonists such as acetylcholine but are still responsive to “authentic” NO (Chester et al., 1990), suggesting a reduction in functional endogenous NO levels. Similarly, patients suffering from hypercholesterolemia demonstrate defective NO bioactivity (Casino et al., 1993). The process of intimal hyperplasia that occcurs following therapeutic manipulation may also stem from inadequate NO availability (Figure 31–1). Endothelial denudation with damage to the underlying internal elastic lamina and SMCs is evidenced at sites of arterial manipulation (Schwartz et al., 1995a; Gibbons and Dzau, 1996). Exposed collagen and SMCs are prothrombogenic and invite leukocyte and platelet adhesion, events which normally would have been minimized by endothelial NO synthesis was intact. Leukocytes, platelets, and SMCs release many chemotactic and mitogenic factors that then facilitate cellular migration and proliferation (Schwartz et al., 1995a; Gibbons and Dzau, 1996) and culminates in the creation of a neointimal lesion. It has been reported that iNOS is transiently expressed by vascular SMCs at sites of balloon injury in rat carotid arteries (Hansson et al., 1994). This iNOS expression in response to vascular injury may represent an inherent mechanism to provide an alternate source of NO until endothelial integrity is reestablished. Nonetheless, neointima formation still occurs despite this endogenous iNOS expression. The provision of exogenous supplemental NO, however, can attenuate intimai hyperplasia (McNamara et al., 1993; Davies et al., 1994; Guo et al., 1994; Marks et al., 1995; Lee et al., 1996), indicating that the induced source of NO is not sufficient in itself to completely inhibit intimal hyerplasia.

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Table 31±1. NO reduces intimai hyperplasia.

NITRIC OXIDE IN THE TREATMENT OF INTIMAL HYPERPLASIA While the role of NO in the establishment of intimal hyperplasia is still unclear, the benefit of NO in preventing this healing response is much more evident (Table 31–1). McNamara et al. (1993) reduced intimal hyperplasia by 39% in a rabbit model of vascular injury by supplementing dietary L-arginine. The benefit of dietary arginine was reversed with the concurrent administration of NG-nitro-L-arginine, an inhibitor of NOS activity, suggesting the benefit of dietary L-arginine is mediated through NO. Similarly, Davies et al. (1994) attenuated neointima formation by 47% in jugular vein grafts interposed into the carotid circulation in rabbits as well as preserved graft vasoreactivity with L-arginine alimentation. Guo et al. (1994) administered the NO donor, SPM-5185, systemically to rats subjected to carotid artery injury. This donor reduced neointimal dimensions by 82% and also accelerated reendothelialization. Alternatively, a single dose of S-nitroso-serum albumin (Marks et al., 1995), a naturally occurring NO adduct with a prolonged biologic half-life, locally administered to denuded rabbit femoral arteries reduced intimal/media ratios by 77% and reduced platelet deposition. Finally, inhalational NO (80 parts per million) delivered for 14 days inhibited intimal hyperplasia in a rodent model of vascular injury (Lee et al., 1996). The above studies strongly suggest that supplemental NO delivered to injured blood vessels may have therapeutic utility. Each of these methods of NO delivery has potential serious shortcomings if clinical use is comtemplated. For example, systemic administration of NO donors in a continuous fashion may cause profound hypotension or may lead to tolerance over a period of time. Inhaled NO also required a prolonged administration for effectiveness. More importantly, the complexity of NO biology and all its purported cytotoxic effects (Stamler, 1995) are incentives to limit NO delivery to a site-specific fashion to avoid systemic exposure. To achieve this goal, the best method available today may be the transfer of a NOS gene to regions of therapeutic intervention which would result in NOS expression restricted to the site of vascular injury (Figure 31–2). Excess NO would be minimally disseminated because it would be neutralized by hemoglobin in flowing blood such that toxicity, if any, would be obviated.

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Figure 31±2. iNOS gene therapy. One method to restore or augment NO production at sites of vascular injury is to transfer the iNOS gene directly to regions of endothelial loss. In this fashion, only cells (namely the exposed medial smooth muscle cells) at the injury site will express the iNOS enzyme and produce NO. This NO, however, will be able to reach a greater population of cells, including adherent platelets and leukocytes, because of its highly diffusable nature. Systemic toxicity, nonetheless, would be avoided due to the ability of hemoglobin to inactive any NO that escapes into the circulation.

VASCULAR GENE THERAPY Gene therapy for the prevention of intimal hyperplasia has been an area of great interest over the past few years, stemming from the failure of conventional systemic therapies, including antiplatelet agents and anticoagulants, to control neointima formation as well as to prevent the progression of atherosclerosis. These conventional interventions have had limited success in offsetting early thrombotic complications after surgical procedures but have not proven to be efficacious in preventing restenosis secondary to intimal hyperplasia. With these limited treatments, the interest in local modalities, especially gene therapy, was heightened. A number of candidate genes have been assessed with a wide range of success in different animal models of vascular injury. In order to discuss cardiovascular gene therapy, a review of gene delivery methods is necessary. For gene therapy to be successful, a way to encapsulate the gene and guide it into a target cell is essential. Optimization of the binding of the vector (a vehicle for gene delivery) to the cell, internalization of the vector, transport of the genetic material into the cell nucleus while preventing its degradation, and finally, expression of the transferred gene is also essential (Wilson, 1996). The two main types of gene delivery vehicles are the non viral and viral vectors. Nonviral methods involve placing the gene of interest into a plasmid containing a mammalian promoter and polyadenylation signal that will permit transcription and translation of the gene product. Such “expression” plasmids are internalized into cells by a number of

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Table 31±2. Characteristics of adenoviral and retroviral vectors.

* CFU, colony forming units; PFU, plaque forming units

techniques: microinjection into the nucleus of cells (Capecchi, 1980), calcium phosphate coprecipitation (Kato et al., 1986), liposome-mediated uptake (Stewart et al., 1992), electroporation (Potter et al., 1984), and particle bombardment (Klein et al., 1987). These transfection techniques are limited by low gene transfer efficiency as well as signficant tissue toxicity. Viral methods of gene delivery utilize viral mechanisms to introduce foreign DNA into cells. Two of the most frequently employed viral vectors are the retrovirus and the adenovirus (Table 31–2). The retrovirus, an RNA virus, is extremely attractive for gene therapy because an the retroviral life cycle requires genomic integration of viral sequences, allowing for “permanent” incorporation of retroviral genes into cells (Varmus et al., 1983; Miller, 1992; Friedmann, 1989). The simplicity of the retroviral genome permitted the creation of recombinant vectors in which structural sequences have been replaced with foreign genes. Infectious virus is then generated by introducing the recombinant retroviral genome into packaging cells that express the deleted structural proteins (Mann et al., 1983; Danos and Mulligan, 1988). Recombinant virus is infectious but is unable to self-replicate. These vectors are very useful for in vitro manipulation of cells. Because of the obligate integration step, however, retroviruses can only target actively proliferating cells. This requirement represents a major obstacle for in vivo applications. Adenovirus is a large DNA virus responsible for a number of common human ailments. The life cycle of this virus proceeds from infection, replication of the viral genome and synthesis of viral proteins, viral packaging, and release of progeny virus by cell lysis (Stratford-Perricaudet and Perricaudet, 1994). Unlike the retrovirus, the adenoviral genome has numerous overlapping coding sequences that make it difficult to generate deletions that do not impair viral packaging and infectivity. Nonetheless, recombinant adenoviral vectors have been successfully engineered (Berkner, 1992; Graham et al., 1977). These adenoviral vectors are deleted of their early genes that regulate late, structural gene expression (Babiss and Ginsberg, 1984; Babiss et al., 1985). However, low levels of viral proteins are still expressed, and these proteins may initiate the host inflammatory response following recombinant adenovirus exposure (Yang et al., 1995). Adenoviruses do not undergo genomic integration, and therefore, gene expression is transient, ranging from days to weeks (Wilson, 1996; Stratford-Perricaudet and Perricaudet, 1994; Berkner, 1992). The length of time of gene expression is a function of host immunity (Wison, 1996). Adenoviral proteins in the original infecting virus or proteins expressed post-infection stimulate both humoral and cellular immune responses (Yang et al., 1995) that culminate in the destruction of infected cells. This host response is a potential disadvantage of adeno viruses. None theless, adenoviruses are extremely useful for gene therapy because

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Table 31±3. Results of vascular gene therapies.

*PFU, plaque forming units; †I/M, intima/media ratio

virus can be prepared in large quantities and at high titers, both of which facilitate in vivo gene delivery (Schneider and French, 1993; Tzeng et al., 1996a). Delivering genes to blood vessels is not a new concept. In 1990, Nabel et al. reseeded denuded arterial surfaces and vein grafts with endothelial cells engineered with retroviral vectors (Nabel et al., 1989; Wisob et al., 1989). This, however, was a tedious method of gene delivery that required the collection, propagation, and in vitro transfection of endothelial cells prior to returning the cells to the patient. With the development of adenoviral vectors, direct in vivo gene delivery became feasible but the questions of what gene should be employed and will gene transfer have the desired therapeutic effect still have to be addressed. Ohno et al. (1994) transferred the herpes simplex thymidine kinase gene (tk) to balloon injured pih ilio-femoral arteries using an adenoviral vector. Thymidine kinase converts the prodrug ganciclovir to a cytotoxic metabolite. Treatment of pigs that received tk with ganciclovir led to a dramatic reduction in injury-induced neointima formation (Table 31–3). In another study, a modified retinoblastoma gene (Rb) was transferred into both rats and pigs undergoing arterial injury (Chang et al., 1995a). Rb is a tumor suppressor that blocks cell cycle progression in its unphosphorylated state. Animals treated with a modified, constitutively active Rb gene demonstrated a marked inhibition of intimal hyperplasia (Table 31–3). Both of these studies strongly support the utility of vascular gene delivery to treat restenosis. Another cell cycle inhibitor, p21, was shown to reduce neointimal lesion formation (Chang et al., 1995b) in a rodent model of vascular injury. More recently, a gene therapy strategy utilized the gene for hirudin (Rade et al., 1996), an anticoagulant that also possesses antiproliferative properties. Hiruden gene transfer into a rabbit model of vascular injury also reduced neointima formation. While all these therapies were partially effective in inhibiting thrombosis and intimal hyperplasia, each only addresses a single aspect of the vascular healing process. The singularity of the therapeutic effect of each gene may limit the utility of these therapies because the etiology of intimal hyperplasia is multifactorial. NOS gene transfer has the theoretic advantage in that NO can target a number of the events involved in intimal hyperplasia (Schwartz et al., 1995b; Clover et al., 1983; Plson et al., 1992; Lindner and Reidy, 1991; Simons et al., 1994).

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ecNOS vs. iNOS Given that a deficiency of endothelium-derived NO may be involved in the pathogenesis of vascular occlusive diseases, it was logical to consider replacing ecNOS activity at sites of vascular injury to restore NO synthesis capacity. Such an approach was tested by von der Leyen et al. (1995) in a rat carotid artery injury model (Table 31–3). Using liposomes complexed with the protein coat of the inactivated hemagglutinating virus of Japan (HVJ) (Kato et al., 1991), they were able to achieve levels of ecNOS activity at sites of vascular injury comparable to that measured in vessels with an intact endothelium. ecNOS-treated carotid arteries exhibited ~70% reduction in neointimal thickness as compared to control injured vessels. While the rat model has limitations in predicting therapeutic outcomes in humans (Ross, 1993; Schwartz et al., 1995a), these results suggest that NOS gene transfer as a method of locally delivering NO may prevent injury-induced intimal hyperplasia. What is the role of iNOS in cardiovascular gene therapy strategies? We commonly associate iNOS with disease pathophysiology such as in refractory systemic vasodilation seen with profound sepsis (Kilbourn et al., 1990) as well as in the pathogenesis of autoimmune diseases (Weinberg et al., 1994; Schmidt et al., 1992; McCartney-Francis et al., 1993; Corbett et al., 1991). Nonetheless, there are several theoretical advantages of using iNOS over ecNOS for cardiovascular gene therapies. For clinical applications, vascular gene delivery to the targeted vessel wall must be accomplished with minimal flow occlusion time, especially in the coronary or cerebral circulation. With even the most efficient delivery system available (adenoviral vectors), gene transfer efficiency may be low under these conditions. The attractiveness of iNOS gene transfer is that iNOS synthesizes much larger quantities of NO (Nathan and xie, 1994) which can diffuse to target a wide field of cells (Lancaster, 1994). Theoretically, only a small number of cells would have to express iNOS for the NO effect to be realized. In comparison, many more cells would have to express ecNOS to synthesize a similar concentration of NO. In addition, iNOS enzymatic activity does not require intracellular calcium fluxes to be activated (Nathan and Xie, 1994), in contrast to ecNOS, and will be maximally activated in the absence of agonist stimulation. EFFICACY OF iNOS GENE TRANSFER In developing iNOS gene transfer for vascular applications, it was initially necessary to evaluate various vascular target cells for their ability to sustain iNOS activity and tolerate high levels of NO synthesis. Employing a retro viral vector (Tzeng et al., 1996b) carrying the human hepatocyte iNOS cDNA (Geller et al., 1993), vascular endothelial cells and SMCs were engineered to express iNOS constitutively. Endothelial cells possess ecNOS activity. Therefore, these cells should support maximal iNOS activity as well as tolerate the higher levels of NO synthesis. This was indeed the case (Tzeng et al., 1996b). Endothelial cells that expressed iNOS proliferated and adhered as well as cells that were engineered with a control retroviral vector. In sharp contrast, SMCs which do not normally express NOS in the resting state were unable to maximally support recombinant iNOS enzymatic activity despite the expression of high levels of iNOS protein (Tzeng et al., 1996b). This finding was a consequence of the absence of tetrahydrobiopterin (BH4) synthesis in resting SMCs (Nakayamaet al., 1994; Tzeng et al., 1996c). BH4 is an essential cofactor that maintains NOS in an active dimeric configuration (Baek et al., 1993; Tzeng et al., 1995), and the rate-limiting biosynthetic step is regulated by GTP cyclohydrolase I (Werner et al., 1993) (Figure 31–3). GTP cyclohydrolase I is not expressed in resting SMCs but is induced by many of the same stimuli that induce iNOS expression in these cells (Nakayama et al., 1994). The addition of BH4 to cultured SMCs engineered to express iNOS resulted in the production of high levels of NO (Tzeng et al., 1996b).

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Figure 31±3. Diagramatic depiction of the BH4 biosynthetic pathway.

These pilot studies indicated that endothelial cells and SMCs responded differently to genetic manipulations to express iNOS and that optimal NO synthesis in SMCs may require the provision of supplemental BH4. As described earlier, retroviruses are limited in their ability to transduce nondividing cells (Varmus, 1988; Miller, 1992; Friedman, 1989; Tzeng et al., 1996a), and therefore, are not very useful for direct in vivo gene transfer. However, they are still useful for in vitro applications. Organ culture systems have been described in which balloon catheterinjured arteries were examined for proliferative activity (Takeshita et al., 1994). Maximal cellular proliferation in these cultured vessel segments was detected by 5–7 days postinjury. Using this model, arterial segments isolated from pig femoral arteries were injured, placed into organ culture (Tzeng et al., 1996b), and were infected with the iNOS retro viral vector or a vector carrying a control gene 5 days post-injury. The vessels were cultured for an additional 9 days at which time they were examined for myointimal hyperplasia. Retroviral transduction in this delayed fashion improved gene transfer efficiency from undetectable, if performed immediately after arterial injury, to 2–5% (Tzeng et al., 1996b). This still low transfer efficiency, nonetheless, yielded a significant increase in both NO synthesis and cGMP release when the blood vessels were cultured in the presence of BH4. Without supplementing this cofactor, both NO and cGMP remained at control levels. This low gene transfer efficiency also completely inhibited injuryinduced myointimal hyperplasia (Tzeng et al., 1996b). This beneficial effect of iNOS gene transfer was completely reversed with a NOS inhibitor, indicating the protective effect was mediated by NO. Despite the in vitro nature of this experiment, the results did indicate that low level iNOS gene transfer to injured porcine arteries could generate sufficient levels of NO to inhibit intimal hyperplasia and suggested that concurrent BH4 supplementation may also be necessary. In order to test the efficacy of iNOS gene transfer in vivo, a more efficient vector was needed. The ideal vector for this application is an adenoviral vector (Schneidner and French, 1993; Tzeng et al., 1996a). The generation of an iNOS adenoviral vector was complicated by the cytotoxicity of high level NO synthesis on the 293 cells used to produce recombinant adenovirus. These cells were sensitive to the high concentrations of NO produced during recombinant virus propagation. In addition, virus production is also impaired by the arginine analogs that are typically used to inhibit NO synthesis. Despite these obstacles, an adenoviral vector

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carrying the human iNOS cDNA was successfully constructed and infectious virus was collected (Shears, 1998). A very popular and frequently employed animal model of intimal hyperplasia is the rat carotid artery injury model (Ross, 1993; Schwartz et al., 1995a). While rats have limitations in predicting outcomes of therapies in humans (Ross, 1993; Vesselinovitch, 1988, Anderson, 1992), it was still an excellent initial model in which to study iNOS gene transfer. Balloon catheter injury of rat carotid arteries reproducibly creates thick neointimal lesions by 2 weeks post-injury (McNamara et al., 1993; Guo et al., 1994; Chang et al., 1995a,b; von der Leyen et al., 1995). The lesions typically measure ~1.5 to 2 fold that of the underlying media. Adenovirus mediated iNOS gene transfer to these arteries immediately following balloon injury using low concentrations of virus (~106 plaque forming units/rat, 60 minute incubation period) consistently reduced neointima formation by over 95% when examined 2 weeks post-injury (Table 31–3) (Shears, 1998). Similar treatment with a control adenoviral vector at the same low concentration did not reduce intimal hyperplasia. These results indicated that iNOS gene transfer successfully inhibited intimal hyperplasia in a rodent model using very low concentrations of adeno virus. They also suggested that the in vivo level of the cofactor BH4 was sufficient to support iNOS activity. Adenoviral vectors allow transient transgene expression, typically lasting for 1–2 weeks in an immunocompetent host (Wilson, 1996; Tzeng et al., 1996a). The question that arises is whether transient gene expression is adequate to correct the problem being targeted? Will iNOS expression that persists for 1– 2 weeks be sufficient to block intimal hyperplasia or will it merely delay the process by 1–2 weeks? When rats treated with the iNOS adeno virus were examined 6 weeks post-injury, there was still minimal intimal hyperplasia evident. This is in sharp contrast to control animals in which the neointimal lesions had progressed to near occlusion. These findings indicate that transient iNOS gene transfer can prevent this vascular healing process. These data also suggest that NO may work by attenuating the infiltration of inflammatory cells and preventing the release of growth factors that incite SMC migration and proliferation. If these early events are inhibited, the cascade of events involved in intimal hyperplasia may no longer occur or may occur at a much reduced level. With the limitations of the rodent model (Ross, 1993; Vesselinovitch, 1988; Anderson, 1992), a number of investigators studying intimal hyperplasia have employed the porcine model (Ohno et al., 1994; Chang et al., 1995a). The porcine vascular healing response has been well characterized and is very similar to the process observed in humans (Sims, 1989). In addition, these animals can be induced to develop systemic atherosclerosis using dietary manipulations (Reitman et al., 1982). This atherosclerosis also resembles the process observed in humans. Finally, pig blood vessels more closely approximate human proportions (peripheral as well as coronary arteries). In pigs, iliac artery injury with a balloon catheter reproducibly created a neointimal lesion, although not as extensive as that observed in rats. Adenovirus iNOS gene transfer (108 plaque forming units/pig) reduced injury-induced intimal hyperplasia by 70% at 3 weeks post-injury (Shears, 1998) (Table 31–3). The gene transfer efficiency was approximately 5–10% of surface cells in the lumen of the treated vessel. Histologic examination of the treated arteries revealed that the cells targeted by the adenoviral vector were the outer most layers of subintimal SMCs. These results indicate that the efficacy of iNOS gene transfer is not species specific and is still very effective in preventing injury-induced intimal hyperplasia in the more relevant pig model. While higher concentrations of adenovirus are required to achieve the therapeutic effect in pigs than in rats, the adenovirus dose is still 10–100 fold less than used in other gene therapies (Table 31–3). This suggests that low levels of iNOS gene transfer within the injured vessel wall can produce sufficient quantities of NO to elicit the desired therapeutic effect.

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TETRAHYDROBIOPTERIN: NECESSARY OR UNNECESSARY? The cell population most likely to be targeted during vascular gene therapy directed at injury-induced intimal hyperplasia are the SMCs because the endothelium is often denuded during the injury process. Unstimulated SMCs lack the biosynthetic machinery for BH4 (Nakayama et al., 1994; Tzeng et al., 1996c). It would seem intuitive that iNOS gene transfer targeted at SMCs would be doomed to failure due to this lack of cofactor essential for iNOS activation (Tzeng et al., 1996b; 1996c). However, results in both rodents and pigs have indicated that this is not the case. Instead, there appears to be sufficient BH4 available in vivo to at least support some level of iNOS activity (Shears, 1998). What is the source of this BH4? There are several possibilities. First, the injury process may induce GTP cyclohydrolase I expression in vascular SMCs, thereby providing endogenous BH4 biosynthesis in the blood vessel walls. This is very plausible since it has been shown that balloon injury of rat carotid arteries induces iNOS expression (Hansson et al., 1994) and many of the stimuli that induce iNOS also induce GTP cyclohydrolase I (Nakayama et al., 1994). Another potential explanation may be that circulating biopterin derivatives may serve as substrates for alternate BH4 biosynthetic pathways within SMCs (Figure 31–3). This is also plausible. We have observed that fetal calf serum, which contains biopterins, can support some iNOS activity in cultured SMCs engineered to express iNOS (Shears, 1998). Finally, it has been shown that BH4 or its derivatives can be transported directly between cells (Tzeng et al., 1996c). BH4 synthesized by adjacent cells such as regenerating endothelial cells may be accessible to SMCs. Even though iNOS gene transfer in both rats and pigs has therapeutic efficacy, it is not known whether iNOS activity following gene transfer is optimized. The provision of additional BH4 to the targeted arterial beds may further increase the amount of NO synthesized, and therapeutic levels of NO may be achieved with even lower viral load and a further reduction in gene transfer efficiency. An attractive method of supplementing BH4 may be the simultaneous transfer of the GTP cyclohydrolase I and iNOS genes. We have shown that GTP cyclohydrolase I and iNOS activities can exist in separate cell populations but BH4 can still be utilized by the cells expressing iNOS (Tzeng et al., 1996c). MECHANISMS BY WHICH NO INHIBITS INTIMAL HYPERPLASIA The mechanisms by which iNOS gene transfer inhibits intimal hyperplasia are still under investigation but they most likely involve the properties that we typically associate with NO, namely its anti-proliferative and anti-inflammatory actions. Even low concentrations of NO are antiproliferative to SMCs. The early studies of NO effects on SMC growth were performed with NO donors (Garg and Hassid, 1989; Guo et al., 1995). However, similar anti-proliferative effects were elicited in SMCs after iNOS gene transfer (Tzeng et al., 1996d). These effects were evident even in the presence of very low concentrations of NO. How NO attenuates SMC growth is not fully understood but it does, in part, rely upon cGMP (Garg and Hassid, 1989; Comwell et al., 1994; Assender et al., 1992; Moro et al., 1996). Very low NO levels can still stimulate soluble guanylate cyclase to generate high levels of cGMP (Tzeng et al., 1997a). However, inhibiting cGMP production with a selective soluble guanylate cyclase inhibitor (Moro et al., 1996; Tzeng et al., 1997a) only partially restores SMC proliferative activity. This raises the likelihood that another pathway may also be involved in NO-mediated antiproliferation. As for the anti-inflammatory effects of NO, it can be speculated that iNOS gene transfer may alter the up-regulation of leukocyte adhesion in response to vascular injury (Kubes et al., 1994). NO has been shown to transcriptionally inhibit intercellullar adhesion molecule-1 (ICAM-1) (Niu et al., 1994; De Caterina et al., 1995) and vascular cell adheison molecule-1 (VCAM-1) (De Caterina et al., 1995) in vitro by preventing the activation of the transcriptional factor NFB (Collins et al., 1995). Whether this occurs in vivo has yet to be demonstrated.

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NO-MEDIATED CYTOTOXICITY A great body of literature has been devoted to the cytotoxic actions of NO (Morris and Billier, 1996; Schmidt and Walter, 1994; Zhang et al., 1994). If NO generated during iNOS gene transfer is indeed cytotoxic to the cells expressing iNOS as well as the cells exposed to NO diffusion (Lancaster, 1994), iNOS gene therapy would have limited applicability. However, a number of NO’s toxic actions have recently been determined to be the consequence of using NO donors that release magnitudes greater amounts of NO than could ever be encountered in vivo. Some cells (Fehsel et al., 1995; Xie et al., 1995; Li et al., 1991), are exquisitely sensitive to NO even in modest concentrations. However, many more cell types are resistant to NO-mediated cytotoxicity (Kin et al., 1991a; Tzeng et al., 1997; Mannick et al., 1994). NO’s cytotoxicity has now been linked to its metabolic products and its reaction with other radicals such as superoxide (Stamler, 1994; Hausladen and Fridovich, 1994). Instead, there is an abundance of evidence that suggest NO is cytoprotective. Kim et al. (1997b) reported that hepatocytes treated with a modest dose of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 μM) are rendered résistent to both TNFoc- and Fas-mediated apoptosis through the inactivation of caspase-3-like proteases. Similarly, vascular endothelial cells engineered to express iNOS as a source of NO acquired resistance to endotoxin-induced apoptosis (Tzeng et al., 1997b). This effect could be reproduced by treating the cells with 100 μM SNAP. This protective effect of NO in endothelial cells is mediated, at least in part, through the inactivation of caspase-3-like proteases (Kim et al., 1997b; Enari et al., 1997). These observations, together with the anti-proliferative effect of NO on SMC growth, indicate that iNOS gene transfer may be ideal for the treatment of vascular diseases. NO may prevent the overgrowth of intimal SMCs as well as participate in the re-endothelialization process by protecting endothelial cells from stimuli that promote apoptosis (Tzeng et al., 1997; Dimmeler et al., 1997) while accelerating their regeneration (Guo et al., 1995; Ziche et al., 1994). OTHER APPLICATIONS OF iNOS GENE TRANSFER Atherosclerosis is the predominant disease process that underlies the formation of occlusive lesions. The pathogenesis of atherosclerosis may include a deficiency of NO production or availability (Chin et al., 1992; Chester et al., 1990). At the same time, both ecNOS and iNOS expression have been demonstrated to be up-regulated at sites of atherosclerotic plaques (Kanazawa et al., 1996; Buttery et al., 1996), suggesting that NO may be involved in the pathogenesis of atherosclerosis. The applicability of iNOS gene transfer for the prevention of atherosclerosis still remains to be proven but there is a great deal of theoretic promise for this therapy. While NO generated by iNOS gene transfer may not prevent atherosclerosis, it may serve to attenuate the vasoconstriction (Furchgott and Zawadzki, 1980; Palmer et al., 1987; Ignarro et al., 1987) and reduce the incidence of thrombotic events (Radimski et al., 1987) at sites of pre-existing lesions, and thus, reduce occlusive complications. Because atherosclerosis is a chronic disease process, successful treatment of this disease process may require gene therapy vectors that permit prolonged iNOS expression and NO synthesis. Vectors that allow for long-term gene expression are currently under development and are based on adeno-associated viruses (Flotte and Carter, 1995) and herpes viruses (Glorioso et al., 1995). Other forms of vascular complications may also be amenable to iNOS gene therapy. One such complication is associated with cardiac transplantation. Following transplantation, chronic rejection, which afflicts all grafts to some level, results in a progressive arteriosclerosis characterized by concentric medial and intimal hypertrophy (Ardehali, 1995; Gibbons, 1995; Mehra et al., 1995). Ultimately, this arteriosclerosis culminates in the occlusion of end-arteries within transplanted cardiac allografts (Ardehali, 1995; Ventura et al., 1995). The pathogenesis of transplantation arteriosclerosis is believed to be related to the inflammation associated with chronic allograft rejection (Russell et al., 1994a,b; Russell, 1995). The extent

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of the arteriosclerosis correlates with increasing episodes of acute rejection and the requirement for higher doses of immunosuppressants, an indicator of the severity of the ongoing rejection. The cytokines released by invading lymphocytes provide the stimuli for SMC migration and proliferation. iNOS expression is present in these rejecting allografts (Tanaka et al., 1995; Devlin et al., 1994) and inhibiting NO synthesis by this induced enzyme results in accelerated and enhanced arterial disease (Shears et al., 1997), suggesting that iNOS is a normal defense response that may be useful to battle allograft rejection. Once again, however, the iNOS response did not evolve to combat transplantation arteriosclerosis, a byproduct of human intervention, and it does not appear to be sufficient to control the disease. Nonetheless, using iNOS gene therapy, allografts may be engineered to produce higher levels of NO sufficient to control this process. In a rodent model of chronic rejection using aortic transplantation, iNOS gene transfer resulted in a dramatic reduction in allograft arteriosclerosis 6 weeks post-transplantation (Shears et al., 1997). What remains to be elucidated is whether the generated NO functions merely to inhibit SMC proliferation or whether it may also serve some role in reducing the incidence or severity of allograft rejection itself. REFERENCES Andersen, P.G. (1992) Restenosis: animal models and morphometric techniques in studies of the vascular response to injury. Cardiovasc. Pathol, 1, 263–278. Ardehali, A. (1995) Heart transplantation: accelerated graft atherosclerosis. Adv. Card. Surg., 6, 5–205. Assender, J.W., Southgate, K.M., Hallett, M.B. and Newby, A.C. (1992) Inhibition of proliferation, but not of Ca2+ mobilization, by cyclic AMP and GMP in rabbit aortic smooth-muscle cells. Biochem. J., 288, 527– 32. Babiss, L.E. and Ginsberg, H.S. (1984) Adenovirus type 5 early region 1 gene product is required for efficient shutoff of host protein synthesis. J. Virol., 50, 202–212. Babiss, L.E., Ginsberg, H.S. and Darnell, J.E. Jr. (1985) Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport. Mol. Cell. Biol., 5, 2552– 2558. Back, K.J., Thiel, B.A., Lucas, S. and Stuehr, D.J. (1993) Macrophage nitric oxide synthase subunits: purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. J. Biol. Chem., 268, 21120–21129. Berkner, K.L. (1992) Expression of heterologous sequences in adenoviral vectors. Curr. Top. Microbiol. Immunol., 58, 38–66. Biro, S., Fu, Y.M., Yu, Z.X. and Epstein, S.E. (1993) Inhibitory effects of antisense oligonucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration. Proc. Natl. Acad. Sci. USA, 90, 654– 658. Bucala, R., Tracey, K.J. and Cerami, A. (1991) Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilation in experimental diabetes. J. Clin. Invest., 87, 432–438. Buttery, L.D., Springall, D.R., Chester, A.H., Evans, T.J., Standfield, E.N., Parums, D.V., Yacoub, M.H. and Polak, J.M. (1996) Inducible nitric oxide synthase is present within human atherosclerotic lesions and promotes the formation and activity of peroxynitrite. Lab. Invest., 75, 77–85. Capecchi, M. (1980) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell, 22, 479–488. Casino, P.R., Kilcoyne, C.M., Quyyumi, A.A., Hoeg, J. and Panza, J.A. (1993) The role of nitric oxide in endotheliumdependent vasodilation of hypercholesterolemic patients. Circulation, 88, 2541–2547. Chang, M.W., Barr, E., Lu, M.M., Barton, K. and Leiden, J.M. (1995) Adenovirus-mediated over-expression of the cyclin-cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J. Clin. Invest., 96, 2260–2268. Chang, M.W., Barr, E., Seltzer, J., Jiang, Y.-Q., Nabel, G.J., Nabel, E.G, Parmacek, M.S. and Leiden, J.M. (1995) Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science, 267, 518–522.

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Index

1-syntrophin, 52 -granules, 237 2 integrins, 308, 312 -thromboglobulin, 240 -VLDL, 64 -IRE, 44 ·O2-, 101, 160 ·OH, 160 l-(2-aminoethyl)imidazole, 514 13-hydroxyoctadecadienoic acid (13-hode), 231 1400W, 329 15N-L-arginine, 7, 21 17 -estradiol: anti-atherosclerotic effect of, 199 physiological substitution doses of, 202 protection of NO by, 198 regulation of NOS-III enzyme activity by, 198 regulation of NOS-III gene expression by, 196 role of increased endothelial NO, 199 upregulation of endothelial NO production by, 196 vasculoprotective effects of, 195 1-phenylimidazoles, 514

2-amino-5,6-dihydro-4H-1,3-thiazine, 512 2-aminopicoline, 512 2-aminopyridine, 512 2-aminopyridines, 512 2-aminothiazole, 512 2-aminothiazoline, 512 2-deoxy glucose, 133 2-iminobiotin, 514 3-aminobenzamide, 88 3-bromo-7-nitroindazole, 515 4-amino-BH4, 506 7-nitroindazole, 515 A23187, 2 Acetaminophen, 11 Acetylcholine, 1 Acid-activatable inhibitory factor (BRPIF), 9 Acidosis, 435 Aconitase, 433, 435 Activated endothelium, 100 Activated factor V (Va), 232 Activated factor X (Xa), 232 Activated macrophages, 8 Activated PMNs, 306, 316 Acute arterial occlusion, 284 572

INDEX

Acute bronchopneumonia, 53 Acute cardiovascular events, 284 Acute exercise, 174 Acute lung injury, 482 Acute rejection, 581 Acute renal failure, 336 Acute respiratory distress syndrome (ARDS), 269, 483 Acyclic amidines, 513 Adeno-associated virus, 556 Adenosine, 456, 457 Adenovirus, 556, 574, 579 Adhesion molecule: expression, 311 Adhesion molecules, 287, 310, 312 Adjuvant-induced arthritis, 329, 414 ADP ribosylation, 436 ADP, 2 Adrenal glands, 51 Adult cardiac disease, 479 Adventitia, 558, 562 aFGF, 131 Air pollution: role of NO, 455 Alveolar macrophages, 60 Amidine-containing inhibitors, 510 Amidines, 506 Amino acid hydroxylases, 508 Aminogaunidine, 159, 328, 378, 506 Amyl nitrite, 456, 457, 458 Androgens, 366 Angeli’s salt, 463 Angina, 285 Angiogenesis, 13, 471 Angiogenesis: the role of NOS and hemodynamic forces, 188 Angioplasty, 560 Angiotensin II, 271, 362, 370 Angiotensin-converting enzyme inhibitors, 290 Animal models, 290 Anorexie agents, 269 Anoxia, 3 Antibiotics, 456 Antihypertensives, 456, 459 Anti-IFN antibodies, 43 Anti-inflammatory effects, 106 Antioxidant defense pathways, 62 Antioxidants, 70, 108, 113, 290, 383 Antiprogestins, 359 Antisense, 43

Antithrombin III, 234 Antithrombotic processes, 241 AP endonucleases, 72 AP-1, 45, 100, 104, 134, 136, 188, 287, 430 Apoptosis, 75, 155, 292, 398, 405, 419, 431 Arachidonic acid, 3, 156 ARDS, 484 Arginase, 340, 382 Arteriosclerosis, 555 Arteriovenous anastomosis, 173 Arteriovenous fistula, 174 Arthritis, 14, 397, 413 Arylimidazoles, 506 Aspirin, 243 Assy metric dimethylarginine (ADMA), 242 Astrocytes, 379, 381, 387 Asymmetric dimethylarginine (ADMA), 294, 295 Atherectomy, 285 Atherogenesis, 100, 286, 290, 312 Atherosclerosis, 121, 173, 240, 294, 569, 578, 581 Atherosclerosis: progression, 290 Atherosclerotic lesions, 53 Atherosclerotic plaques, 199 Atherothrombosis, 240 ATP, 2, 69 Atrial natriuretic peptide, 289 Autoimmune diseases, 384, 405, 413 Autooxidation, 60 Azide, 133, 461 B lymphocyte, 43 Balloon angioplasty, 240, 285, 289, 569 Basic fibroblast growth factor (bFGF), 124, 129, 131, 429, 569 Bcl-2, 417 BH4, 18, 28, 198, 507, 577 BH4 binding antagonists, 506 BH4 synthesis inhibitors, 506 Bioavailable NO, 199 Biopterin, 328 Bis-amidines, 513 Bleeding diathesis, 229 Bleeding time, 338 Blood pressure, 251, 252, 257 Blood pressure regulation, 254 Blood velocity, 230, 231 Bone reabsorption, 13 Bovine hemoglobin, 541

573

574

INDEX

Bowel injury, 442 BQ123, 367 Bradykinin, 2 Brain constitutive NOS (bNOS), 195, 352 Brain injury, 523 Brain, 21 Bronchodilation, 486 BRPIF, 9 C reactive protein, 414 C57BL/6 mice, 212 Ca2+, 9 Caesarean section, 356 Caged nitric oxide, 459 Calcification, 173 Calcitonin gene related-peptide (CGRP), 431 Calcium channel blockers, 268 Calcium ionophore (A23187), 2 Calcium phosphate coprecipitation, 573 Calcium pumps, 70 Calmodulin, 7, 25, 28, 198, 235 Calmodulin antagonists, 506 Calpain inhibitor, 326 cAMP, 161, 165, 187, 382, 399 cAMP phosphodiesterase, 239 cAMP responsive element, 187 Capillary migration and proliferation, 188 Capillary permeability, 351 Carbon monoxide, 134 Cardiac hypertrophy, 258 Cardiac myocytes, 54 Cardiac necrosis, 308 Cardiac output, 330 Cardiac transplantation, 479, 581 Cardioprotection, 316, 317 Cardiopulmonary bypass, 475 Cardiovascular disease, 173 Carotid artery disease, 285 Cartilage, 397, 413, 415 Cartilage metabolism, 418 CAT1, 26 Catalytic domain, 507 Cationic amino acid transporter 1 (CAT1), 26 Cationic liposomes, 557 Caveolae, 20 Caveolin, 25 Caveolin-1, 54 Caveolin-3, 54 CCAAT-enhancer-binding protein , 131

CD 11/CD 18, 101, 428, 438 CD 12, 428 CD 36, 144 CD 39, 231 CD4+T cells, 376 Cell death, 318 Cell proliferation: inhibition by diazeniumdiolates, 466 Cellular antioxidant systems, 524 Cellular thiols, 62 Central nervous system, 10, 375 Ceramide, 134 Cerebral ischemia: differing roles of nNOS and eNOS, 218 Cerebrospinal fluid, 376 Cerebrovascular blood flow, 219 Cerebrovasculature, 218 Cervical ripening, 350, 358 c-fos, 386 cGMP pathways, 293 cGMP, 3, 101, 112, 578 Chemically modified tetracycline (CMT), 14 Chemoattractant, 283 Chemokines, 283, 287, 426, 571 Chemotaxis, 238 Cholecystokinin, 431 Chondrocyte, 398 Chondrocytes, 399, 413, 415, 441 Chondrosarcoma, 12 Chorioallantoic membrane (CAM), 189 Chronic allograft rejection, 581 Chronic exercise, 175 Chronic obstructive pulmonary disease, 485 Chronic pulmonary disease, 140 Chronic rejection, 581 Chronic thromboembolic, 268 Cicaprost, 367 Circulatory failure, 323, 324, 326 Circulatory shock, 78, 310, 324, 328, 329, 432, 440 Circulatory shock: hypodynamic, 439 Cirrhotic rats, 175 Citrulline, 8 c-jun, 134 Clot retraction, 233 cNOS mRNA, 134 CNS trauma, 524 CNS, 375 Coagulation cascade, 232

INDEX

Coagulation factors, 229 Coagulopathy, 492 Collagen synthesis, 418 Collagenase IV (MMP-9), 121 Collagen-vascular disease, 268 Complex lesions, 293 Cone-plate viscometer, 176 Congenital diaphragmatic hernia, 482 Congenital heart disease, 268, 475 Consumption coagulopathy, 241 Copper chelating activity, 531 Coronary arteries, 196 Coronary artery bypass grafting, 479 Coronary artery, 292 Coronary microcirculation, 304 Coronary vasospasm, 307 Corticotropin-releasing hormone (CRH), 350 COX, 155 COX-1 activation: NO-mediated, 160 COX-1, 155, 156, 361 COX-2 activation: NO-mediated, 162 COX-2, 11, 155, 156, 162, 182, 361, 399, 401, 435 CRE, 187, 188 c-Rel, 103 c-Src, 128, 134 Cu,Zn superoxide dismutase, 60 Cultured hepatocytes, 204 Cupferron, 456, 457 Cyanamide, 463 Cyanide, 133 Cyclic amidines, 512 Cyclic AMP, 44 Cyclic guanosine monophosphate (cGMP), 3, 189, 256, 286 Cyclic nucleotide phosphodiesterases, 236 Cyclic strain, 171, 179 Cyclic strain: endothelin, 181 iNOS gene expression, 184 PDGF, 181 proliferation and migration, 179 morphological changes, 179 tPA, 181 Cycloheximide, 430 Cyclooxygenase, 155, 231, 405, 434 Cyclosporine A, 342 Cysteine, 523

575

Cysteine: enzyme cofactor in NO production, 461 Cystic fibrosis, 558 Cytochrome oxidase, 435 Cytochrome P480, 132 Cytokines, 39, 241, 269, 283, 286, 287, 323, 329, 349, 375 Cytomegalovirus, 285 Cytotoxicity due to PARS activation, 73 DEA/NO, 465 Decidua, 350, 352, 354 Delayed type hyper sensitivity, 385 Demyelination, 378, 383 Deoxy-hemoglobin-NO complexes, 539 DETA/NO, 465 Dexamethasone, 43, 202, 326 Diabetes mellitus, 74, 84, 240, 283, 341 Diaminobenzimidazole, 514 Diaminohyroxypyrimidine, 506 Diapirin cross-linked hemoglobin, 541 Diazeniumdiolates, 456, 464, 466 Diazonium salts, 455, 460 Dietary arginine, 290, 292, 293 Dihydrodiazete dioxides, 456, 461 Dihydropteridine reductase, 508 Dimethylargininedimethylaminohydrolase (DDAH), 296 Dimethylarginines, 339 Diphenyleneiodonium, 506 Direct enzyme inhibitors: based on the substrate, 507 based on ligation of the catalytic heme center, 507 Disseminated intravascular coagulation, 241 Ditutyryl-cAMP, 165 DLD-1 cells, 428 Dlg, 52 DNA, 70 D-NMMA, 7 DOC A salt-sensitive hypertensive rats, 260 DOPA synthesis, 60 Dopastin, 456, 457 Duchenne muscular dystrophy, 52 Dynamic exercise, 173 Dystrophin-glycoprotein complex, 20 Ebselen, 525 ecto-ADPase, 231 Edema, 163 EDHF, 7

576

INDEX

EDRF, 1, 99, 216, 255 EGF, 43, 131 EGF-receptor, 131 Egr-1, 100, 104 Eisenmenger complex, 475 Eisenmenger physiology, 268 Eisenmenger’s syndrome, 275 Elastic membrane, 176 Electron nuclear double resonance (ENDOR) spectroscopic techniques, 21 Electron paramagnetic resonance (EPR), 377 Electroporation, 572 Embolie stroke, 284 Embryonic stem (ES) cells, 210 Encephalitis, 377 Endothelial cell injury, 199 Endothelial cells, 54, 60, 74 Endothelial damage, 351 Endothelial denudation, 286 Endothelial dysfunction, 75, 100, 199, 290, 301, 325, 363 Endothelial dysfunction: in circulatory shock, 79 Endothelial heparan sulfate, 234 Endothelial nitric oxide synthase, 22, 125 Endothelial NOS knockout mice, 209 Endothelial NOS mutant mice: cardiac contractility and relaxation, 217 hypertension and blood pressure response, 216 response to vascular injury and atherosclerosis, 218 Endothelial ulceration, 284 Endothelial-derived NO, 112 Endothelial-leukocyte interaction, 101 Endothelin, 268, 367 Endothelin-1 receptor subtypes, 125 Endothelin-1, 121, 122, 124, 271 Endothelium, 558 Endothelium-dependent modulation of vascular tone, 195 Endothelium-dependent relaxation, 173, 259 Endothelium-derived hyperpolarizing factor, 7, 255 Endothelium-derived relaxing factor, 1 Endotoxemia, 323, 328 Endotoxic shock, 324, 329 Endotoxin, 43, 165, 323, 329, 425, 544 Energy metabolism, 132 eNOS activating protein (ENAP), 26 eNOS expression: upregulation of, 41 eNOS gene: expression and regulation of, 41

eNOS gene transfer, 555 eNOS gene transfer: and atherosclerosis, 561 and cerebral vasospasm, 560 and pulmonary hypertension, 563 and restenosis, 560 and vein bypass graft failure, 563 eNOS mRNA, 41 eNOS promoter function, 41 eNOS, 22, 40, 121, 182, 187, 209, 352, 556 eNOS: oligomeric status, 23 protein-protein interactions, 25 tissue distribution, 53 subcellular targeting, 54 mice deficient in, 209 Enterocytes, 425 Epidermal growth factor (EGF), 429, 284 Epithelial cells, 74 EPR spin-trapping, 21, 22 ER , 199 ER , 199 ERKO mice, 199 Erythrocytes, 486 Erythropoietin, 134 ES cells, 210 E-selectin, 100, 312 Essential hypertension, 251, 261 Estrogen, 290 Estrogen receptors, 199 Estrogen: vasculoprotective effect, 200 ET-1, 130 Ethinyl estradiol, 198 Excision repair, 72 Exercise, 173 Exhaled NO, 270 Experimental allergic encephalomyelitis (EAE), 376 Extracellular matrix proteins, 129 Extracellular matrix, 269, 284, 293, 359 Extracorporeal membrane oxygenation, 476 Extrinsic pathways, 232 FAD, 18 Female reproduction, 349 Female sexual steroid hormones: cardioprotection by, 195 Fenton-reaction, 531 FGF, 43

INDEX

Fibrinogen, 231 Fibrin deposition, 231, 269 Fibrinogen receptor, 231 Fibrinolysis, 229, 233, 241, 269 Fibroblast mitogenesis, 122 Fibroblasts, 112 Fibrochondrocytes, 415 Fibronectin, 129, 231, 419 Fibrotic lung diseases, 485 Fibrous cap, 284 Fibrous plaque, 284 Fissures, 285 Five-electron oxidation, 17 FK-409, 462 Flavoprotein oxidoreductases, 132 Flavoprotein reductase inhibitors, 506 Flexercell strain unit, 179 Flk-1, 124 Flt-1, 124 fMLP, 308 FMN, 18 Foam cells, 100, 283 Foetal depression, 364 Foetal stress, 350 Formyl-methionyl-leucyl-phenylalanine (fMLP), 305 Free radical, 3 Fuoxans, 456, 461 G 13, 44 GAF, 113 Gallbladder, 433 GAS, 44 Gastrin, 431 Gastrointestinal dysfunction, 440 Gastrointestinal function, 431 Gastrointestinal steal phenomenon, 439 Gastrointestinal tract, 425 Gastroparesis, 432 G-cyclase, 3 Gelatinase, 13 Gender difference, 196 Gene delivery methods, 573 Gene knockout approaches: developmental effects, 211 genetic background, 212 limitations to, 211 tissue specificity, 211 Gene knockout technology, 209 Gene therapy, 276

Gene transfer: to the cerebral vasculature, 558 Genetic factors, 283 Genetic hypertension, 251 Genetic injury, 439 Gestation, 349, 354 Gliosis, 379 Global ischemia, 304 Glomerulonephritis, 339 Glucocorticoids, 106 Glutathione peroxidase, 240 Glutathione: enzyme cofactor in NO production, 461 S-nitroso, 464 Glycolysis, 132, 436 Glycoproteins, 231 Gp 120 glycoprotein, 164 Growth factors, 283 Growth factors: regulation by oxygen tension and nitric oxide, 131 GTP cyclohydrolase I, 198, 328, 577, 508 GTP/GDP exchange, 109 Guanosine 3',5'-monophosphate (cGMP), 236 Guanosine 5'-triphosphate, 236 Guanylate cyclase, 3, 155, 236, 431 Gut necrosis, 432 HDL oxidation, 199 Heart failure, 276 Heart-lung transplantation, 475 Heme, 539 Heme binders, 506 Heme domain dimer, 28 Heme proteins, 132, 159 Heme-binding protein, 133 Heme-containing enzymes, 514 Heme-containing sensor, 121 Hemodynamic factor, 230 Hemodynamic forces, 173, 285 Hemoglobin, 4 Hemoglobin: clinical studies, 549 its role as a nitric oxide scavenger, 539 NO donor, 539 release of nitrosothiols, 539 Hemoglobin (effects on): endotoxin and bacterial growth, 548 isolated vessel preparations, 541 oxygenation, 545

577

578

INDEX

regional blood flow, 546 renal function, 547 Hemoglobin solutions, 539 Hemorrhage, 229, 286 Hemorrhagic diathesis, 229 Hemostasis, 229 Hemostatic defects, 242 Hemostatic disorders, 229 Hemostatic response, 229 Heparan sulfates, 129 Heparin, 234 Hepatocytes, 415 Herpes simplex virus, 43, 285 Herpes simplex virus type-1(HSV-l), 377 Heterocyclic inhibitors, 514 HIF-1, 134 HIF-1 binding site, 128 High altitude pulmonary edema, 485 High cholesterol diet, 283, 290 High-density lipoproteins (HDL), 241 Histamine, 2 Histocompatibility antigen class II (MHCII), 376 Homocysteine, 283 Hormone replacement therapy, 196 Hormones, 349 Host-defense, 323, 325, 434 Human atherosclerotic plaques, 64 Human cartilage, 12 Human hypertension, 260 Human neutrophils, 60 Human pulmonary hypertension, 274 Human recombinant hemoglobin, 541 Human saphenous vein endothelial cells, 313 Human umbilical vein EC, 41, 182 Hydrocortisone, 12 Hydrogen peroxide (H2O2), 59, 104, 384 Hydronephrotic kidney, 162 Hydroxyguanidines, 462 Hydroxyl radical, 434, 531 Hydroxylamines: formation of NO from, 461 Hydroxy-L-arginine: intermediate in arginine metabolism, 462 as NO donor, 463 Hypercholesterolemia, 64, 240, 283, 289, 290, 294, 296 Hypercholesterolemic animals, 292 Hypercholesterolemic patients, 198 Hyperpermeability, 438 Hypertension, 173, 240, 251, 283, 285, 289, 351, 555

Hypertension: genetic, 251 renovascular, 251 Hypertrophy, 257 Hypotension, 241, 323, 324 Hypoxanthine, 312 Hypoxia, 121, 268, 272, 273, 336, 435, 437 Hypoxia: vascular response to, 123 regulation of vasoactive genes by, 123 transcription factors regulated by, 135 acute responses to, 139 chronic responses to, 140 Hypoxia-inducible transcription factor- 1 (HIF-1), 135 Hypoxia-reoxygenation, 316, 429 Hypoxic pulmonary vasoconstriction, 272, 544 Hypoxic vasoconstriction, 269, 272 ICAM-1, 41, 100, 306, 312 ICI 182780, 199 IFN -, 113, 140, 376, 428 IGF, 129 IKB kinase, 104, 430 IKB , 104, 289 induction of, 110 IkB-protease, 326 Il-l , 113 IL-l , 140, 158, 163, 204 IL-13, 382 IL- 17, 404 IL-2, 416 IL-4, 43, 383 IL-6, 101, 131 IL-8, 101, 134, 426 Ileus, 432 Iloprost, 367 Imidazole, 506, 514 Immune response, 456 Immunoglobulin superfamily, 312 Immunomodulator, 413, 416 Immunosuppressive, 413 Impaired vascular NO production, 555 In situ hybridization, 415 Inactivated hemagglutinating virus of Japan, 576 Indazoles, 506, 515 Indomethacin, 165, 350, 414 Inducible nitric oxide synthase, 26, 113, 195 Inducible nitric oxide synthase: tissue distribution, 51

INDEX

regulation by oxygen tension, 126 Infant mortality, 349 Infarct size, 59 Infarction, 230 Inflammation, 328 Inhaled NO, 276, 478, 572 Inhaled NO: delivery, 487 dosage, 487 monitoring, 487 toxicity, 487 Inherited dyslipidemia, 561 Inhibitors of phospodiesterase, 276 iNOS, 26, 40, 69, 107, 140, 162, 165, 209, 241, 292, 352 iNOS: catalytic activities, 30 gene therapy approaches with, 569 NO production, 31 oligomeric status, 28 subcellular targeting, 53 iNOS cDNA, 576 iNOS enhancer, 430 iNOS gene: transcriptional activation of, 44 promoter structure, 44 iNOS gene expression: induction of, 43 iNOS gene transfer: efficacy of, 576 other applications of, 581 iNOS holo-enzyme, 28 iNOS inhibitor, 378 iNOS knock-out mice, 324, 325 iNOS protein, 576 Insulin-like growth factor-I, 418 Integrin receptor: expression, 231 Intercellular adhesion molecules, 283 Interferon , 40 Interleukin (IL) l , 403 Interleukin-1, 40, 415 Interleukin-4 (IL-4), 376, 416 Interleukin-6, 414 Interleukin-8, 384, 417 Interleukin-10, 376 Interleukin-12, 376 Interleukin-13, 376 Intestinal hyperpermeability, 75 Intima, 562

579

Intimal hyperplasia, 173 Intimal hyperplasia: pathogenesis of, 569 Intimal thickening, 286 Intracellular signal molecules, 44 Intrapulmonary shunt fraction, 471 Intrauterine infection, 350 Intrinsic pathways, 232 IRE, 429 IRF-1, 45, 113, 429 Iron chelators, 134 Iron ion, 539 Ischemia, 230, 284 Ischemia/reperfusion, 287, 301, 302, 307 309, 310, 312, 313, 434, Ischemic heart disease, 173 Ischemic myocardium, 308 Isoamyl nitrite, 457 Isobutyl-methylxanthine (IBMX), 383 Isolated rat hearts, 304 Isosorbide dinitrate, 456, 457 Isothioureas, 328, 506, 510 ISRE, 45 Janus kinase, 430 JNK, 100 Kallikrein, 232 Ketosis, 364 Kidney, 51 Krebs cycle, 435 Kupffer cells, 165 Labour, 351, 354 LAD coronary artery, 302 Laminar flow, 176, 230 Laminar shear stress, 181 Laminin, 129 L-arginine analogs, 506 L-arginine binding site, 40 L-arginine paradox, 294 L-arginine transport, 275 L-arginine, 7, 30, 198, 507 L-arginine: NO pathway, 10 supplementing dietary, 571 Lazaroids, 532 Lazaroid class of synthetic antioxidants, 523 L-citrulline, 17, 507

580

INDEX

LDL cholesterol, 283 LDL oxidation, 41 LDL, 64 Left ventricular failure, 257, 258 Leishmania major, 325 Lentivirus, 556 Leukocyte adherence, 287, 305, 311 Leukocyte adhesion molecules, 134 Leukocyte infiltration, 318 Leukocyte-endothelial interaction, 310 Leukocytes, 285, 571 Lewis rat, 378 LHRH, 161 Lipid peroxidation inhibitors: mechanism, 532 assays, 532 Lipid peroxidation, 288, 523 Lipopolysaccharide (LPS), 39, 241, 428 Lipoprotein (a), 283 Liposome-mediated uptake, 573 Liposomes, 576 Lipoxygenase, 231 L-NAME, 102, 128, 186, 188, 327, 364, 419, 542 L-NIL, 159, 162, 419 L-NMMA, 7, 159, 188, 327, 403, 419 L-NNA, 20 Long term potentiation, 54, 221 Low-density lipoprotein (LDL), 241, 296 LPS, 39, 53, 140, 156, 202 L-selectin, 312 L-thiocitrulline, 509 Lung capillary bed, 267 Lung circulation, 267 Lung disease, 268 Lung, 51, 471 Lung transplantation, 484 Lysophosphatidylcholine, 40, 41 Macrophages, 74, 113, 164, 339, 456, 465 MAHMA/NO, 465 Malaria, 350 MAP kinases, 44, 100, 108 Mass spectrometry, 7 Mast cells, 438 Maternal hypertension, 364 Matrix metalloprotease (MMPs), 13, 358, 418 MCP-1 expression, 292, 294 MCP-1, 287 M-CSF, 101

MDF, 44 Mdx mice, 52 Mechanism-based NOS inhibitors, 507 Media, 562 Medullary interstitial space, 254 Megakaryocyte, 237 MEKK1, 108 Mercaptoethylguanidine (MEG), 440 Mercaptopropionyl glycine (MPG), 308 Mesenteric circulation, 305 Mesenteric microcirculation, 312 Mesenteric microvasculature, 305 Mesenteric perfusion, 440 Metal complexes of NO, 456, 459 Metalloproteinases, 284, 293, 350, 359, 398 Methemoglobin, 490, 539 Methotrexate, 420, 506 Methylated proteins, 296 Methylene Blue, 4 MHC class II antigens, 113 Microcirculation, 308, 310 Microglia, 376, 379, 384, 387 Microvascular endothelial dysfunction, 305 Microvascular endothelium, 311, 312 Microvasculature, 285 Mitochondria, 62, 73 Mitochondrial dysfunction, 387 Mitochondrial respiration, 325 Mitogenic factors, 571 Mitral valve surgery, 479 Molecular conjugates, 557 Molsidomine, 242, 459 Monocrotaline, 273 Monocyte: adherence, 287, 290 infiltration, 290 Monocytes, 189 MRL/lpr mice, 419 MSP, 43 Mucosa, 426, 438 Mucosal barrier, 433 Multiple organ dysfunction syndrome (MODS), 323, 326 Multiple sclerosis, 375 Mutagenesis, 431 Myelin, 375, 381 Myelin basic protein, 376 Myocardial contractility, 257 Myocardial damage, 257 Myocardial depressant factor (MDF), 316

INDEX

Myocardial function, 545 Myocardial infarction, 242, 283, 284 Myocardial ischemia/reperfusion, 59, 308, 315, 316 Myointimal hyperplasia, 286, 289 Myometrial contractility, 350 Myometrium, 351, 354 Myristoylation, 54 nNOS: spectral characterization, 20 N-(l-iminoethyl)-L-ornithine, 509 Na+/K+ ATP-ase, 70 N-acetylcysteine(NAC), 100, 105, 113, 240, 384 NAD, 69 NADH oxidase, 101 NADPH oxidase, 132 NADPH, 7 NADPH-cytochrome P-450 reductase, 18 N-allyl-L-arginine, 509 NANC nerves, 9 Necrotic core, 284, 293 Neointimal lesion formation, 241 Neointimal SMC, 570 Neonatal and infant respiratory failure, 480 Neonatal mortality, 349 Neopterin, 328, 379 Neovascularization, 188 Neurodegeneration, 524 Neuronal injury, 60, 74 Neuronal nitric oxide synthase: bidomain structure, 18 flavoprotein domain, 18 heme domain, 18 oligomeric status of, 19 Neuronal NOS, 258 Neuronal NOS knockout mice, 209 Neuronal NOS mutant mice: pyloric stenosis, 213 airway responsiveness, 214 role in renin release, 215 aggressive behavior, 215 Neuronal postsynaptic density protein, 21 Neuronal tyrosine hydroxylase, 43 Neurotoxicity, 523 Neutrophil amplification, 301 Neutrophil infiltration, 315, 316 Neutrophils, 53, 238, 285, 287, 306, 405, 438 NF-KB activation, 107, 294

581

NF-KB, 44, 100, 103, 138, 289, 306, 326, 383, 384, 385, 398, 405, 429, 430 NF-KB: enhanced nuclear translocation of, 109 NG-monomethyl-L-arginine, 7 NG-nitro-L-arginine, 20 N-hydroxy-L-arginine, 507 Nicotinamide, 384 Nitrate/nitrite, 539 Nitrates: NO formation from, 458, 461 Nitration of tyrosine residues, 62 Nitric oxide, 3, 17, 69, 130, 155, 229 Nitric oxide: activation of the COX enzymes by, 158 and the regulation of vasoactive genes, 121 cyclic strain upregulation of, 182 gene transcription, 99 inhibition of PG synthesis, 164 in the treatment of intimal hyperplasia, 571 toxicity of, 523 Nitric oxide and tissue injury, 59 Nitric oxide chemistry: conversion to nitrite, 460 diazonium salt formation, 456 nitrosating agents, 456 nylong form, 455 reaction with secondary amines, 456, 464 reaction with superoxide, 459, 461 Nitric oxide donors, 305, 455 Nitric oxide sensor, 132 Nitric oxide synthase expression: regulation by hemodynamic forces, 171 Nitric oxide synthase inhibitors, 505 Nitric oxide synthase, 129, 234 Nitric oxide synthase: isoforms of, 17 gene regulation, 39 modulation by PGs, 165 regulation by 17 -estradiol, 195 oxidation of hydroxyarginine by, 463 gene therapy of vascular diseases, 556 genetic approach to, 209 Nitric oxide-derived oxidants, 59 Nitrites: formation from NO, 460 inorganic, 460 NO formatin from, 458 Nitro-cysteine, 526

582

INDEX

Nitro-glutathione, 526 Nitrogen dioxide, 490 Nitrogen oxides, 17 Nitroglycerin, 242, 456, 457, 458 Nitrosamines: formation from nitrites/nitrates, 458 potential for NO-donation, 459 Nitrosation, 455, 456, 460, 461, 463 Nitrosonium ion, 59 Nitrosothiols, 100, 109 Nitrosylation, 288 Nitrotyrosine, 64, 241, 293, 379 Nitro-tyrosine residues, 524 Nitrous acid, 460 Nitrous oxide: formation from nitroxy, 459, 463 Nitrovasodilators, 3 Nitrovasodilators : amyl nitrite, 456, 457, 458 isosorbide dinitrate, 456, 457 nitroglycerin, 456, 457, 458 sodium nitroprusside, 456, 457, 459 Nitroxyl: formation from Angeli’s salt, 463 formation from cyanarmide, 463 formation from furoxans, 461 formation from hydroxylamines, 456, 463 formation from oximes, 462 nitrous oxide from, 459 oxidation to NO, 463 NMDA, 10, 164 NMDA receptor, 52, 60 N-methyl-L-arginine, 506 N-nitro-L-arginine, 506 nNOS, 40, 209, 556 nNOS: alternatively spliced, truncated form of, 22 mice deficient in, 209 subcellular targeting, 52 superoxide production, 22 tissue localization, 21 NO, 3, 140, 187, 195, 479, 577 NO: as an anti-inflammatory mediator, 113 comparative toxicity of, 71 delivered by inhalation, 471 role in the regulation of vascular tone, 555 NO bioavailability, 562

NO deficiency, 239 NO donor, 72, 101, 158, 188, 242, 243, 276, 287, 306, 310, 313, 315, 316, 357, 441, 572 NO gas, 139 NO synthase, 7 NO–2, 8 NO−3, 9 NO2Arg, 159 NOCs, see diazeniumdiolates NO-mediated cytotoxicity, 580 Non-cGMP-dependent effects, 112 Nonenzymatic glycation, 341 NONOATE, 439 NONOates, see diazeniumdiolates Non-selective NOS inhibitors, 505 Nonsterodial anti-inflammatory drugs (NSAIDs), 405 Non-viral methods, 573 Non-viral vectors, 557 Norepinephrine, 161 Normocholesterolemic animais, 292 NOS, 173 NOS: active site models, 516 electron transfer, 507 expression of, 40 shear stress induced expression of, 181 transcriptional regulation of, 186 NOS activity: autoregulation, 127 NOS autoinactivation, 507 NOS catalytic domain, 507 NOS dimers, 507 NOS inhibitor, 327 NOS inhibitors: approaches to, 505 NOS-I (nNOS), 17, 195, 505 NOS-II (iNOS), 17, 195, 202, 505 NOS-II: protein structure, 26 inhibited by 17-estradiol, 201 NOS-III (eNOS), 17, 22, 195, 505 NOS III: mRNA, 273 protein, 273 NOS III-deficient mouse, 251, 271 NSAID, 159, 163 Obstructive pulmonary disease, 272

INDEX

Occlusion/reperfusion, 305 Oct-2, 43 Oestrogen, 349 Oligodendrocyte, 375, 379, 386, 387 Organ injury, 323 Organic nitrates, 242 Organic nitrites, 456, 458 Osteoarthritis, 397, 414 Osteoarthrosis, 397 Osteopontin, 284 Ovariectomized rats, 202 Ovariectomy, 196 Oxatriazoles, 459 Oxidant injury, 484 Oxidant stress, 429 Oxidant-sensitive transcription factors, 100 Oxidative burst, 307 Oxidative modification of LDL, 199 Oxidative processes, 70 Oxidative stress, 59, 256, 261, 285, 287, 289 Oxidized forms of nitric oxide, 59 Oxidized lipoprotein, 284, 287, 290 Oxidized low density lipoproteins, 306 Oximes: NO release from, 462 review of chemistry, 456 vasoactivity, 462 Oxygen, 121, 486 Oxygen delivery, 325 Oxygen free radical, 429 Oxygen sensor, 132 Oxygenation, 471 Oxyradicals, 73 P selectin, 237 p21 ras, 100, 109, 134 p50 (NF-KB1) 103 p52 (NF-KB2) 103 p53, 292 p65 (Rel A), 103 PAF, 134, 432 PAI-1, 233 Pancreatic islet cells, 51 Parallel-plate flow chamber, 176 Parkinson’s disease, 60 PARS, 69 PARS: potential regulation of iNOS expression, 85 PARS activation, 74

583

PARS cleavage, 78 PARS knockout mouse, 88, 436 Partial reduction of oxygen, 59 Parturition, 352 Paw inflammation, 162 PDGF, 569 PDGF-B, 121, 124 PDTC, 100, 105, 113, 399 PDZ, 21 PDZ motif, 52 PECAM-1, 312 Penicillamine methyl ester, 523 Pentagastrin, 431 Pentoxifylline, 383 Perfusion-bioassay, 5 Peripheral nitregic nerves, 51 Peripheral vascular disease, 173, 242 Peristalsis, 440 Permeability, 283 Peroxidase, 156 Peroxynitrate anion, 287, 293 Peroxynitrite Reactivity: molecular mechanisms, 54 Peroxynitrite scavengers, 523 Peroxynitrite, 22, 59, 69, 104, 155, 160, 239, 241, 313, 316, 325, 379, 434, 523 Peroxynitrite: DNA injury, 72 single strand breakage, 72 formation by superoxide, 459, 461 reaction with sugars, 461 oxidative biochemistry of, 60 Peroxynitrite-mediated nitration of tyrosine, 63 Peroxynitrous acid, 61, 436, 438, 525 Persistent pulmonary hypertension of the newborn, 480 PGEl, 189 PGE2, 156, 359, 401 PGF2, 156 PGG2, 156 PGH2, 156 PGI2, 134 Phenylimidazoles, 506 Phosphatases, 386 Phosphodiesterase, 439 Phosphoinositol-specific phospholipase C (PLC), 238 Physical forces, 171 Pial vessel, 220 Piloty’s acid, 456, 463 PIN, 22

584

INDEX

PKC activation, 313 PKC inhibitors, 134 Placenta, 350, 355 Plaque instability, 293 Plaque rupture, 284, 286 Plasmin, 233 Plasminogen activator inhibitor, 233 Plasminogen activator: tissue-type, 233 urokinase-type, 233 Plasminogen, 233 Platelet aggregation, 122 Platelet deposition, 231 Platelet factor 4, 231, 240 Platelet inhibitors, 238 Platelet recruitment, 237 Platelet: activation, 229 adhesion, 229, 231, 285 aggregation, 229, 231, 285 Platelet-derived growth factor, 231, 284 Platelets, 8, 112, 284, 438 PLGF, 131 PMA, 43 PMNs, 308 Pneumonia, 350, 482 Poiseuille’s law, 231 Poly (ADP) ribosyltransferase (PARS), 325, 383 Poly (ADP-ribose) synthetase, 69 Poly amines: NO donors from, 465 Polymorphonuclear (PMN) leukocytes, 240, 301, 309, 417 Postmenopausal women, 196 Postoperative pulmonary hypertension, 479 Post-prandial hyperemia, 439 Post-translational processing, 236 Pre-eclampsia, 344, 349, 362 Pregnancy, 344, 349, 352, 363, 368 Prekallikrein, 232 Premature atherosclerosis, 283 Pressure-induced natriuresis, 252, 254 Preterm labour, 349 Primary pulmonary hypertension: animal model, 269 Progesterone, 349, 359 Progressive obliterative pulmonary vascular disease, 475 Pro-inflammatory genes, 102 PROLI/NO, 465 Prostacyclin, 129, 231, 239, 268, 479

Prostaglandin El, 479 Prostaglandin, 417 Prostaglandins biosynthesis, 156 Prostaglandins, 155, 359 Prostanoid contractile factors, 255 Prostanoids, 129 Protein C, 233 Protein kinase C, 382 Protein tyrosine phosphatases, 100 Proteinuria, 351 Proteoglycan, 398, 419 Prothrombin, 232 Prothrombinase, 232 PSD-93, 21 PSD-95, 21, 52 P-selectin, 306, 310, 312 Pulmonary arterial pressure, 267, 471 Pulmonary arteries, 274 Pulmonary circulation, 267 Pulmonary conduit arteries, 273 Pulmonary disease, 480 Pulmonary fibrosis, 268 Pulmonary hemodynamics, 271, 274 Pulmonary hypertension, 121, 267, 471, 474, 539, 543 Pulmonary hypertension: experimental, 272 postcapillary, 268 precapillary, 268 Pulmonary hypertensive crises, 471 Pulmonary microvessels, 268 Pulmonary parenchymal disease, 471 Pulmonary remodeling, 271 Pulmonary transvascular flux, 328 Pulmonary vascular disease: primary pulmonary hypertension, 473 pulmonary thromboembolism, 474 Pulmonary vascular resistance (PVR), 267, 268, 274 Pulmonary vascular tone, 270, 274 Pulmonary vasoconstriction, 477 Pulmonary vasodilation: selective, 471 Pulmonary vessel, 128 Pulsatile flow, 172 Pulsatile stretch 257 Pyelonephritis, 350 Pyloric stenosis, 425 Pyrrolidine dithiocarbamate (PDTC), 105, 429, 430 Quantitative reverse transcriptase PCR, 182

INDEX

Radiation sensitizers, 459 Radical chain propagation, 70 Radiocontrast nephropathy, 338 Raf, 44, 128 Rap 1 , 237 Ras, 44 Rat islet cells, 163 RAW 264.7 cells, 14 Reactive oxygen intermediate, 133 Reactive oxygen species, 104 Rebound pulmonary hypertension, 491 Recombinant endothelial nitric oxide synthase gene expression: therapeutic implications, 555 Recombinant human superoxide dismutase (hSOD), 302 Recombinant viruses, 556 Redox state, 238 Redox-regulated transcription factors, 386 Redox-responsive transcriptional pathways, 287 Reduced thiols, 240 Reductase domain, 507 Refractoriness, 365 Regional blood flow, 173 Regression, 292 Rel B, 103 Relaxin, 359 Rel-related proteins, 103 Remyelination, 386 Renal function, 252 Renovascular hypertension, 258 Reoxygenation, 305 Reperfusion injury, 60, 81, 301 Reproductive cycle, 349 Respiratory distress syndrome, 481 Restenosis, 289, 569 Restenosis: inhibition by diazeniumdiolates, 466 Retinal circulation, 242 Retinoid, 376 Retrovirus, 556, 574 Retroviruses, 577 Rheologie properties, 230 Rheumatoid arthritis, 397, 413 Rheumatoid synovium, 401 Right ventricular dysfunction, 479 Rolipram, 383 Roussin’s salts, 459 Rupture, 284

Sabra hypertensive-prone rat, 260 Saliva, 426 Salmonella, 428 Salt-sensitive hypertension, 251, 259 Sandwich mount procedure, 2 Scavengers of peroxynitrite, 70 Schwann cell, 387 Scleroderma, 141 Secondary pulmonary hypertension, 269 Selectins, 312 Sepsis, 323, 328, 426, 539 Septic shock, 140, 162, 241, 323, 324 Serine protease inhibitors, 429 Serine protease, 233 Serine-threonine protein kinase, 133 Serotonin, 2, 285 Serum cholesterol, 283 Severe left ventricular dysfunction, 490 Sexual steroid hormone, 195, 349 Shear rate, 230 Shear stress, 5, 122, 171, 257, 272 Shear stress: morphological changes, 179 Shock, 425 Sickle cell disease, 121, 141, 485 Sickle hemoglobin, 471, 485 SIN-1, 113, 460, 459 Site-specific gene targeting, 555 Skeletal muscle, 51 SMC migration, 284, 570 SMC proliferation, 569 Smooth muscle cells, 74 Snake venom-like protease, 403 SNAP, see S-nitroso-N-acetyl-penicillamine S-nitrosoalbumin, 464 S-nitrosocaptopril, 464 S-nitrosoglutathione, 113, 416 S-nitrosohemoglobin, 539 S-nitroso-N-acetylcysteine, 238 S-nitroso-N-acetyl-penicillamine, 138, 527 S-nitroso-N-acetyl-penicillamine: peptide derivatives, 464 structure, 457 S-nitrosothiols, 238, 456, 463, 466, 539 S-nitrosylation, 100, 109, 134 SNP, see sodium nitroprusside SOD, 5, 524 Sodium channels, 70 Sodium nitrite, 460

585

586

INDEX

Sodium nitroprusside, 138, 293, 456, 457, 459, 479 Sodium retention, 254 Sodium salicylate, 11 Sp-1, 188 SPER/NO, 465 Sphincter of Oddi, 433 Sphincteric tone, 433 Sphingomyelin, 134 Spinal cord, 51 Splanchnic ischemia, 307 Splanchnic ischemia/reperfusion, 301, 315, 316 SPM-5185, 572 Spontaneous hypertension, 254 Spontaneously hypertensive (SH) rats, 175 S-substituted isothioureas, 328 Stabilization of NF-KB, 107 Stable homodimer, 20 STAT, 100, 113, 430 Steroid, 441 Stomach, 51 Streptozotocin, 459 Stress ulcer, 426 Stroke, 285 Stromelysin, 13 Subarachnoid hemorrhage, 523, 533, 555 Subendothelial matrix, 231 Subendothelial space, 284 Substance P, 2, 188, 237 Sulfated proteoglycans, 144 Sulfhydryl groups, 70 Sulfhydryl oxidation, 523 Sulfhydryl scavengers: mechanism, 525 Sulfonated, 456 Superoxide anion, 5, 239, 285, 287, 289, 293, 325, 384, 434, 439 Superoxide dismustase, 5, 70, 239, 308, 524 Superoxide production, 30 Superoxide radical, 59, 199, 294, 301, 306, 307, 312, 313, 318, 343, 523 Superoxide radical release, 304 Surgical bypass grafting, 569 SV129 mice, 212 Sympathetic ganglia, 51 Synovial fibroblasts, 415 Synovial fluid, 414 Synoviocytes, 415 Synoviocytes: type A, 415

type B, 415 Synovitis, 417 Synovium, 397, 415 Systemic blood pressure, 539 Systemic inflammatory response syndrome (SIRS), 323 Systemic sclerosis (scleroderma), 121, 141 Systemic vascular tone, 254 Tetracyclines: doxycycline, 13 minocycline, 13 Tetrahydrobioproterin, 316, 379, 579 TGF , 44, 122, 131, 399, 569 TGF- , 131, 382 Th1, 416 Th2, 416 Thiols: amyl nitrite and, 458 furoxans and, 461 nitroglycerin metabolism and, 458 Thionitrites, see S-nitrosothiols THP-1, 296 Thrombin, 2, 232 Thrombocytopenia, 241 Thrombomodulin, 233 Thrombosis, 229, 269, 555 Thrombosis: inhibition by diazeniumdiolates, 466 Thrombospondin, 121, 129, 231 Thrombospondin-1 : regulation by oxygen tension and nitric oxide, 128 Thromboxane A2, 285 Thrombus, 229, 340 Tirilazad, 523, 533 Tissue acidosis, 437 Tissue factor, 232 Tissue factor, 284 Tissue injury, 312, 434 Tissue necrosis, 306, 318 Tissue plasminogen activator: S-nitrosyl, 464 T-lymphocytes, 106 TNF- , 113, 118, 325, 403 TNF , 382 TNF-RE, 45 Tocolytics, 357 Tolerance: to nitrates, 458

INDEX

Topoisomerase-mediated repair, 72 Total anomalous pulmonary venous connection, 477 Transfection techniques, 574 Transforming growth factor beta (TGF), 376 Transgene expression efficacy, 555 Transient ischemic attacks, 285 Transplant coronary artery disease, 285 Transplantation arteriosclerosis, 581 Transporter system, 237 Trifluoroperazine, 506 Tumor necrosis factor-alpha, 40, 414 TxA2, 156 Tyrosine hydroxylase, 70 Tyrosine kinase, 429 Tyrosine kinases, 134, 386 U-46619, 271 Ubiquitin-proteasome pathway, 105 Ulceration, 284 Unstable angina, 284 Uremia, 242, 338 Urinary nitrate excretion, 295 Urinary nitrate, 261 Urinary tract infections, 53 Uterine arteries, 368 Uterine contractility, 351 Uterine smooth muscle, 352 Uterotonins, 350 Uterus, 51 Vascular cell targeting: cell type, 557 specific promoters, 557 Vascular contractile failure: in circulatory shock, 79 Vascular dysfunction, 562 Vascular gene therapy, 555, 572 Vascular gene transfer, 555, 556 Vascular hyporeactivity, 325 Vascular hyporesponsiveness, 324, 545 Vascular inflammation, 100 Vascular injury, 229, 231 Vascular O2 sensing, 132 Vascular remodeling, 140, 268, 269, 286 Vascular smooth muscle cells, 112, 140, 162 Vascular smooth cells: migration, 286 proliferation, 290 Vascular tone, 216

Vascular tone: local regulation of, 122 Vasculoprotection, 200 Vasoactive intestinal peptide (VIP), 425 Vasoconstrictor substance, 261 Vasodilators, 471 Vasodilators: furoxans as, 461 oximes as, 462 streptozotocin as, 459 Vasomotor tone, 541 Vasopressin, 2 VCAM-1, 41, 100, 105, 306, 312 Vector technology, 555 VEGF receptor, 124, 128 VEGF, 121, 128, 129 VEGF: regulation by oxygen tension and nitric oxide, 127 Venular endothelium, 305, 310 Vessel bifurcation, 172 Viral DNA, 557 Viral methods: of gene delivery, 574 Viral vectors, 556 Viscosity, 176 Vitronectin, 231 Voltage dependent K+ channel, 52 vWF, 231 W-7, 506 Weibel-Palade bodies, 237, 313 Wound healing, 439 Xanthine oxidase, 312 Xanthine-oxidase system, 306 ZO-1, 52

587