Bronchial Vascular Remodeling in Asthma and COPD

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Bronchial Vascular Remodeling in Asthma and COPD

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LUNG BIOLOGY IN HEALTH AND DISEASE

Executive Editor Claude Lenfant Former Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland

1. Immunologic and Infectious Reactions in the Lung, edited by C. H. Kirkpatrick and H. Y. Reynolds 2. The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal 3. Bioengineering Aspects of the Lung, edited by J. B. West 4. Metabolic Functions of the Lung, edited by Y. S. Bakhle and J. R. Vane 5. Respiratory Defense Mechanisms (in two parts), edited by J. D. Brain, D. F. Proctor, and L. M. Reid 6. Development of the Lung, edited by W. A. Hodson 7. Lung Water and Solute Exchange, edited by N. C. Staub 8. Extrapulmonary Manifestations of Respiratory Disease, edited by E. D. Robin 9. Chronic Obstructive Pulmonary Disease, edited by T. L. Petty 10. Pathogenesis and Therapy of Lung Cancer, edited by C. C. Harris 11. Genetic Determinants of Pulmonary Disease, edited by S. D. Litwin 12. The Lung in the Transition Between Health and Disease, edited by P. T. Macklem and S. Permutt 13. Evolution of Respiratory Processes: A Comparative Approach, edited by S. C. Wood and C. Lenfant 14. Pulmonary Vascular Diseases, edited by K. M. Moser 15. Physiology and Pharmacology of the Airways, edited by J. A. Nadel 16. Diagnostic Techniques in Pulmonary Disease (in two parts), edited by M. A. Sackner 17. Regulation of Breathing (in two parts), edited by T. F. Hornbein 18. Occupational Lung Diseases: Research Approaches and Methods, edited by H. Weill and M. Turner-Warwick 19. Immunopharmacology of the Lung, edited by H. H. Newball

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20. Sarcoidosis and Other Granulomatous Diseases of the Lung, edited by B. L. Fanburg 21. Sleep and Breathing, edited by N. A. Saunders and C. E. Sullivan 22. Pneumocystis carinii Pneumonia: Pathogenesis, Diagnosis, and Treatment, edited by L. S. Young 23. Pulmonary Nuclear Medicine: Techniques in Diagnosis of Lung Disease, edited by H. L. Atkins 24. Acute Respiratory Failure, edited by W. M. Zapol and K. J. Falke 25. Gas Mixing and Distribution in the Lung, edited by L. A. Engel and M. Paiva 26. High-Frequency Ventilation in Intensive Care and During Surgery, edited by G. Carlon and W. S. Howland 27. Pulmonary Development: Transition from Intrauterine to Extrauterine Life, edited by G. H. Nelson 28. Chronic Obstructive Pulmonary Disease: Second Edition, edited by T. L. Petty 29. The Thorax (in two parts), edited by C. Roussos and P. T. Macklem 30. The Pleura in Health and Disease, edited by J. Chrétien, J. Bignon, and A. Hirsch 31. Drug Therapy for Asthma: Research and Clinical Practice, edited by J. W. Jenne and S. Murphy 32. Pulmonary Endothelium in Health and Disease, edited by U. S. Ryan 33. The Airways: Neural Control in Health and Disease, edited by M. A. Kaliner and P. J. Barnes 34. Pathophysiology and Treatment of Inhalation Injuries, edited by J. Loke 35. Respiratory Function of the Upper Airway, edited by O. P. Mathew and G. Sant’Ambrogio 36. Chronic Obstructive Pulmonary Disease: A Behavioral Perspective, edited by A. J. McSweeny and I. Grant 37. Biology of Lung Cancer: Diagnosis and Treatment, edited by S. T. Rosen, J. L. Mulshine, F. Cuttitta, and P. G. Abrams 38. Pulmonary Vascular Physiology and Pathophysiology, edited by E. K. Weir and J. T. Reeves 39. Comparative Pulmonary Physiology: Current Concepts, edited by S. C. Wood 40. Respiratory Physiology: An Analytical Approach, edited by H. K. Chang and M. Paiva 41. Lung Cell Biology, edited by D. Massaro 42. Heart–Lung Interactions in Health and Disease, edited by S. M. Scharf and S. S. Cassidy 43. Clinical Epidemiology of Chronic Obstructive Pulmonary Disease, edited by M. J. Hensley and N. A. Saunders

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44. Surgical Pathology of Lung Neoplasms, edited by A. M. Marchevsky 45. The Lung in Rheumatic Diseases, edited by G. W. Cannon and G. A. Zimmerman 46. Diagnostic Imaging of the Lung, edited by C. E. Putman 47. Models of Lung Disease: Microscopy and Structural Methods, edited by J. Gil 48. Electron Microscopy of the Lung, edited by D. E. Schraufnagel 49. Asthma: Its Pathology and Treatment, edited by M. A. Kaliner, P. J. Barnes, and C. G. A. Persson 50. Acute Respiratory Failure: Second Edition, edited by W. M. Zapol and F. Lemaire 51. Lung Disease in the Tropics, edited by O. P. Sharma 52. Exercise: Pulmonary Physiology and Pathophysiology, edited by B. J. Whipp and K. Wasserman 53. Developmental Neurobiology of Breathing, edited by G. G. Haddad and J. P. Farber 54. Mediators of Pulmonary Inflammation, edited by M. A. Bray and W. H. Anderson 55. The Airway Epithelium, edited by S. G. Farmer and D. Hay 56. Physiological Adaptations in Vertebrates: Respiration, Circulation, and Metabolism, edited by S. C. Wood, R. E. Weber, A. R. Hargens, and R. W. Millard 57. The Bronchial Circulation, edited by J. Butler 58. Lung Cancer Differentiation: Implications for Diagnosis and Treatment, edited by S. D. Bernal and P. J. Hesketh 59. Pulmonary Complications of Systemic Disease, edited by J. F. Murray 60. Lung Vascular Injury: Molecular and Cellular Response, edited by A. Johnson and T. J. Ferro 61. Cytokines of the Lung, edited by J. Kelley 62. The Mast Cell in Health and Disease, edited by M. A. Kaliner and D. D. Metcalfe 63. Pulmonary Disease in the Elderly Patient, edited by D. A. Mahler 64. Cystic Fibrosis, edited by P. B. Davis 65. Signal Transduction in Lung Cells, edited by J. S. Brody, D. M. Center, and V. A. Tkachuk 66. Tuberculosis: A Comprehensive International Approach, edited by L. B. Reichman and E. S. Hershfield 67. Pharmacology of the Respiratory Tract: Experimental and Clinical Research, edited by K. F. Chung and P. J. Barnes 68. Prevention of Respiratory Diseases, edited by A. Hirsch, M. Goldberg, J.-P. Martin, and R. Masse 69. Pneumocystis carinii Pneumonia: Second Edition, edited by P. D. Walzer

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70. Fluid and Solute Transport in the Airspaces of the Lungs, edited by R. M. Effros and H. K. Chang 71. Sleep and Breathing: Second Edition, edited by N. A. Saunders and C. E. Sullivan 72. Airway Secretion: Physiological Bases for the Control of Mucous Hypersecretion, edited by T. Takishima and S. Shimura 73. Sarcoidosis and Other Granulomatous Disorders, edited by D. G. James 74. Epidemiology of Lung Cancer, edited by J. M. Samet 75. Pulmonary Embolism, edited by M. Morpurgo 76. Sports and Exercise Medicine, edited by S. C. Wood and R. C. Roach 77. Endotoxin and the Lungs, edited by K. L. Brigham 78. The Mesothelial Cell and Mesothelioma, edited by M.-C. Jaurand and J. Bignon 79. Regulation of Breathing: Second Edition, edited by J. A. Dempsey and A. I. Pack 80. Pulmonary Fibrosis, edited by S. Hin. Phan and R. S. Thrall 81. Long-Term Oxygen Therapy: Scientific Basis and Clinical Application, edited by W. J. O’Donohue, Jr. 82. Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure, edited by C. O. Trouth, R. M. Millis, H. F. Kiwull-Schöne, and M. E. Schläfke 83. A History of Breathing Physiology, edited by D. F. Proctor 84. Surfactant Therapy for Lung Disease, edited by B. Robertson and H. W. Taeusch 85. The Thorax: Second Edition, Revised and Expanded (in three parts), edited by C. Roussos 86. Severe Asthma: Pathogenesis and Clinical Management, edited by S. J. Szefler and D. Y. M. Leung 87. Mycobacterium avium–Complex Infection: Progress in Research and Treatment, edited by J. A. Korvick and C. A. Benson 88. Alpha 1–Antitrypsin Deficiency: Biology • Pathogenesis • Clinical Manifestations • Therapy, edited by R. G. Crystal 89. Adhesion Molecules and the Lung, edited by P. A. Ward and J. C. Fantone 90. Respiratory Sensation, edited by L. Adams and A. Guz 91. Pulmonary Rehabilitation, edited by A. P. Fishman 92. Acute Respiratory Failure in Chronic Obstructive Pulmonary Disease, edited by J.-P. Derenne, W. A. Whitelaw, and T. Similowski 93. Environmental Impact on the Airways: From Injury to Repair, edited by J. Chrétien and D. Dusser 94. Inhalation Aerosols: Physical and Biological Basis for Therapy, edited by A. J. Hickey

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95. Tissue Oxygen Deprivation: From Molecular to Integrated Function, edited by G. G. Haddad and G. Lister 96. The Genetics of Asthma, edited by S. B. Liggett and D. A. Meyers 97. Inhaled Glucocorticoids in Asthma: Mechanisms and Clinical Actions, edited by R. P. Schleimer, W. W. Busse, and P. M. O’Byrne 98. Nitric Oxide and the Lung, edited by W. M. Zapol and K. D. Bloch 99. Primary Pulmonary Hypertension, edited by L. J. Rubin and S. Rich 100. Lung Growth and Development, edited by J. A. McDonald 101. Parasitic Lung Diseases, edited by A. A. F. Mahmoud 102. Lung Macrophages and Dendritic Cells in Health and Disease, edited by M. F. Lipscomb and S. W. Russell 103. Pulmonary and Cardiac Imaging, edited by C. Chiles and C. E. Putman 104. Gene Therapy for Diseases of the Lung, edited by K. L. Brigham 105. Oxygen, Gene Expression, and Cellular Function, edited by L. Biadasz Clerch and D. J. Massaro 106. Beta2-Agonists in Asthma Treatment, edited by R. Pauwels and P. M. O’Byrne 107. Inhalation Delivery of Therapeutic Peptides and Proteins, edited by A. L. Adjei and P. K. Gupta 108. Asthma in the Elderly, edited by R. A. Barbee and J. W. Bloom 109. Treatment of the Hospitalized Cystic Fibrosis Patient, edited by D. M. Orenstein and R. C. Stern 110. Asthma and Immunological Diseases in Pregnancy and Early Infancy, edited by M. Schatz, R. S. Zeiger, and H. N. Claman 111. Dyspnea, edited by D. A. Mahler 112. Proinflammatory and Antiinflammatory Peptides, edited by S. I. Said 113. Self-Management of Asthma, edited by H. Kotses and A. Harver 114. Eicosanoids, Aspirin, and Asthma, edited by A. Szczeklik, R. J. Gryglewski, and J. R. Vane 115. Fatal Asthma, edited by A. L. Sheffer 116. Pulmonary Edema, edited by M. A. Matthay and D. H. Ingbar 117. Inflammatory Mechanisms in Asthma, edited by S. T. Holgate and W. W. Busse 118. Physiological Basis of Ventilatory Support, edited by J. J. Marini and A. S. Slutsky 119. Human Immunodeficiency Virus and the Lung, edited by M. J. Rosen and J. M. Beck

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120. Five-Lipoxygenase Products in Asthma, edited by J. M. Drazen, S.-E. Dahlén, and T. H. Lee 121. Complexity in Structure and Function of the Lung, edited by M. P. Hlastala and H. T. Robertson 122. Biology of Lung Cancer, edited by M. A. Kane and P. A. Bunn, Jr. 123. Rhinitis: Mechanisms and Management, edited by R. M. Naclerio, S. R. Durham, and N. Mygind 124. Lung Tumors: Fundamental Biology and Clinical Management, edited by C. Brambilla and E. Brambilla 125. Interleukin-5: From Molecule to Drug Target for Asthma, edited by C. J. Sanderson 126. Pediatric Asthma, edited by S. Murphy and H. W. Kelly 127. Viral Infections of the Respiratory Tract, edited by R. Dolin and P. F. Wright 128. Air Pollutants and the Respiratory Tract, edited by D. L. Swift and W. M. Foster 129. Gastroesophageal Reflux Disease and Airway Disease, edited by M. R. Stein 130. Exercise-Induced Asthma, edited by E. R. McFadden, Jr. 131. LAM and Other Diseases Characterized by Smooth Muscle Proliferation, edited by J. Moss 132. The Lung at Depth, edited by C. E. G. Lundgren and J. N. Miller 133. Regulation of Sleep and Circadian Rhythms, edited by F. W. Turek and P. C. Zee 134. Anticholinergic Agents in the Upper and Lower Airways, edited by S. L. Spector 135. Control of Breathing in Health and Disease, edited by M. D. Altose and Y. Kawakami 136. Immunotherapy in Asthma, edited by J. Bousquet and H. Yssel 137. Chronic Lung Disease in Early Infancy, edited by R. D. Bland and J. J. Coalson 138. Asthma’s Impact on Society: The Social and Economic Burden, edited by K. B. Weiss, A. S. Buist, and S. D. Sullivan 139. New and Exploratory Therapeutic Agents for Asthma, edited by M. Yeadon and Z. Diamant 140. Multimodality Treatment of Lung Cancer, edited by A. T. Skarin 141. Cytokines in Pulmonary Disease: Infection and Inflammation, edited by S. Nelson and T. R. Martin 142. Diagnostic Pulmonary Pathology, edited by P. T. Cagle 143. Particle–Lung Interactions, edited by P. Gehr and J. Heyder 144. Tuberculosis: A Comprehensive International Approach, Second Edition, Revised and Expanded, edited by L. B. Reichman and E. S. Hershfield

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145. Combination Therapy for Asthma and Chronic Obstructive Pulmonary Disease, edited by R. J. Martin and M. Kraft 146. Sleep Apnea: Implications in Cardiovascular and Cerebrovascular Disease, edited by T. D. Bradley and J. S. Floras 147. Sleep and Breathing in Children: A Developmental Approach, edited by G. M. Loughlin, J. L. Carroll, and C. L. Marcus 148. Pulmonary and Peripheral Gas Exchange in Health and Disease, edited by J. Roca, R. Rodriguez-Roisen, and P. D. Wagner 149. Lung Surfactants: Basic Science and Clinical Applications, R. H. Notter 150. Nosocomial Pneumonia, edited by W. R. Jarvis 151. Fetal Origins of Cardiovascular and Lung Disease, edited by David J. P. Barker 152. Long-Term Mechanical Ventilation, edited by N. S. Hill 153. Environmental Asthma, edited by R. K. Bush 154. Asthma and Respiratory Infections, edited by D. P. Skoner 155. Airway Remodeling, edited by P. H. Howarth, J. W. Wilson, J. Bousquet, S. Rak, and R. A. Pauwels 156. Genetic Models in Cardiorespiratory Biology, edited by G. G. Haddad and T. Xu 157. Respiratory-Circulatory Interactions in Health and Disease, edited by S. M. Scharf, M. R. Pinsky, and S. Magder 158. Ventilator Management Strategies for Critical Care, edited by N. S. Hill and M. M. Levy 159. Severe Asthma: Pathogenesis and Clinical Management, Second Edition, Revised and Expanded, edited by S. J. Szefler and D. Y. M. Leung 160. Gravity and the Lung: Lessons from Microgravity, edited by G. K. Prisk, M. Paiva, and J. B. West 161. High Altitude: An Exploration of Human Adaptation, edited by T. F. Hornbein and R. B. Schoene 162. Drug Delivery to the Lung, edited by H. Bisgaard, C. O’Callaghan, and G. C. Smaldone 163. Inhaled Steroids in Asthma: Optimizing Effects in the Airways, edited by R. P. Schleimer, P. M. O’Byrne, S. J. Szefler, and R. Brattsand 164. IgE and Anti-IgE Therapy in Asthma and Allergic Disease, edited by R. B. Fick, Jr., and P. M. Jardieu 165. Clinical Management of Chronic Obstructive Pulmonary Disease, edited by T. Similowski, W. A. Whitelaw, and J.-P. Derenne 166. Sleep Apnea: Pathogenesis, Diagnosis, and Treatment, edited by A. I. Pack 167. Biotherapeutic Approaches to Asthma, edited by J. Agosti and A. L. Sheffer

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168. Proteoglycans in Lung Disease, edited by H. G. Garg, P. J. Roughley, and C. A. Hales 169. Gene Therapy in Lung Disease, edited by S. M. Albelda 170. Disease Markers in Exhaled Breath, edited by N. Marczin, S. A. Kharitonov, M. H. Yacoub, and P. J. Barnes 171. Sleep-Related Breathing Disorders: Experimental Models and Therapeutic Potential, edited by D. W. Carley and M. Radulovacki 172. Chemokines in the Lung, edited by R. M. Strieter, S. L. Kunkel, and T. J. Standiford 173. Respiratory Control and Disorders in the Newborn, edited by O. P. Mathew 174. The Immunological Basis of Asthma, edited by B. N. Lambrecht, H. C. Hoogsteden, and Z. Diamant 175. Oxygen Sensing: Responses and Adaptation to Hypoxia, edited by S. Lahiri, G. L. Semenza, and N. R. Prabhakar 176. Non-Neoplastic Advanced Lung Disease, edited by J. R. Maurer 177. Therapeutic Targets in Airway Inflammation, edited by N. T. Eissa and D. P. Huston 178. Respiratory Infections in Allergy and Asthma, edited by S. L. Johnston and N. G. Papadopoulos 179. Acute Respiratory Distress Syndrome, edited by M. A. Matthay 180. Venous Thromboembolism, edited by J. E. Dalen 181. Upper and Lower Respiratory Disease, edited by J. Corren, A. Togias, and J. Bousquet 182. Pharmacotherapy in Chronic Obstructive Pulmonary Disease, edited by B. R. Celli 183. Acute Exacerbations of Chronic Obstructive Pulmonary Disease, edited by N. M. Siafakas, N. R. Anthonisen, and D. Georgopoulos 184. Lung Volume Reduction Surgery for Emphysema, edited by H. E. Fessler, J. J. Reilly, Jr., and D. J. Sugarbaker 185. Idiopathic Pulmonary Fibrosis, edited by J. P. Lynch III 186. Pleural Disease, edited by D. Bouros 187. Oxygen/Nitrogen Radicals: Lung Injury and Disease, edited by V. Vallyathan, V. Castranova, and X. Shi 188. Therapy for Mucus-Clearance Disorders, edited by B. K. Rubin and C. P. van der Schans 189. Interventional Pulmonary Medicine, edited by J. F. Beamis, Jr., P. N. Mathur, and A. C. Mehta 190. Lung Development and Regeneration, edited by D. J. Massaro, G. Massaro, and P. Chambon 191. Long-Term Intervention in Chronic Obstructive Pulmonary Disease, edited by R. Pauwels, D. S. Postma, and S. T. Weiss

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192. Sleep Deprivation: Basic Science, Physiology, and Behavior, edited by Clete A. Kushida 193. Sleep Deprivation: Clinical Issues, Pharmacology, and Sleep Loss Effects, edited by Clete A. Kushida 194. Pneumocystis Pneumonia: Third Edition, Revised and Expanded, edited by P. D. Walzer and M. Cushion 195. Asthma Prevention, edited by William W. Busse and Robert F. Lemanske, Jr. 196. Lung Injury: Mechanisms, Pathophysiology, and Therapy, edited by Robert H. Notter, Jacob Finkelstein, and Bruce Holm 197. Ion Channels in the Pulmonary Vasculature, edited by Jason X.-J. Yuan 198. Chronic Obstuctive Pulmonary Disease: Cellular and Molecular Mechanisms, edited by Peter J. Barnes 199. Pediatric Nasal and Sinus Disorders, edited by Tania Sih and Peter A. R. Clement 200. Functional Lung Imaging, edited by David Lipson and Edwin van Beek 201. Lung Surfactant Function and Disorder, edited by Kaushik Nag 202. Pharmacology and Pathophysiology of the Control of Breathing, edited by Denham S. Ward, Albert Dahan and Luc J. Teppema 203. Molecular Imaging of the Lungs, edited by Daniel Schuster and Timothy Blackwell 204. Air Pollutants and the Respiratory Tract: Second Edition, edited by W. Michael Foster and Daniel L. Costa 205. Acute and Chronic Cough, edited by Anthony E. Redington and Alyn H. Morice 206. Severe Pneumonia, edited by Michael S. Niederman 207. Monitoring Asthma, edited by Peter G. Gibson 208. Dyspnea: Mechanisms, Measurement, and Management, Second Edition, edited by Donald A. Mahler and Denis E. O'Donnell 209. Childhood Asthma, edited by Stanley J. Szefler and Søren Pedersen 210. Sarcoidosis, edited by Robert Baughman 211. Tropical Lung Disease, Second Edition, edited by Om Sharma 212. Pharmacotherapy of Asthma, edited by James T. Li 213. Practical Pulmonary and Critical Care Medicine: Respiratory Failure, edited by Zab Mosenifar and Guy W. Soo Hoo 214. Practical Pulmonary and Critical Care Medicine: Disease Management, edited by Zab Mosenifar and Guy W. Soo Hoo

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215. Ventilator-Induced Lung Injury, edited by Didier Dreyfuss, Georges Saumon, and Rolf D. Hubmayr 216. Bronchial Vascular Remodeling in Asthma and COPD, edited by Aili Lazaar 217. Lung and Heart–Lung Transplantation, edited by Joseph P. Lynch, III and David J. Ross The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.

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Bronchial Vascular Remodeling in Asthma and COPD

Edited by

Aili Lazaar University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A.

New York London

Informa Healthcare USA, Inc. 270 Madison Avenue New York, NY 10016 © 2006 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2981-1 (Hardcover) International Standard Book Number-13: 978-0-8247-2981-3 (Hardcover) Library of Congress Card Number 2006040438 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Bronchial vascular remodeling in asthma and COPD / edited by Aili Lazaar. p. ; cm. -- (Lung biology in health and disease ; v. 216) Includes bibliographical references and index. ISBN-13: 978-0-8247-2981-3 (hardcover : alk. paper) ISBN-10: 0-8247-2981-1 (hardcover : alk. paper) 1. Asthma--Chemotherapy. 2. Lungs--Diseases, Obstructive--Chemotherapy. I. Lazaar, Aili. II. Series. [DNLM: 1. Asthma--drug therapy. 2. Angiogenesis Modulating Agents--therapeutic use. 3. Lung--blood supply. 4. Muscle, Skeletal--drug effects. 5. Pulmonary Circulation--drug effects. 6. Pulmonary Disease, Chronic Obstructive--drug therapy. WF 553 B8695 2006] RC591.B76 2006 616.2’380061--dc22

2006040438

Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

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To my parents.

Introduction

“Remodeling” is a simple and promising word. Dictionaries define it as “to model anew; to mend.” For most, the implication is that when completed, remodeling will result in better function as well as structure. Does the reality substantiate this implication, not to say expectation, in biology and disease? Remodeling in diseases of the lung, especially of the airways, has been the subject of considerable interest for decades. Indeed, in the early 1960s, it was reported that long-term asthmatic patients showed persistent, irreversible airway obstruction (1). This was a surprising observation almost contradicting the fundamental definition of asthma as being a disease characterized by reversible airway obstruction. From then, it did not take long to establish that the "drift" from reversible to irreversible was due to the airway structure modification, that is, remodeling. Then, when asthma became further defined as an inflammatory disease, it was shown that the trigger for remodeling was inflammation. Chronic obstructive pulmonary disease also evidences a remodeling process, triggered as well by inflammation. However, in chronic obstructive pulmonary disease the inflammatory milieu is very different from that of asthma—chronic obstructive pulmonary disease inflammation being dominated by macrophages and neutrophils instead of eosinophils that dominate in asthma. It has been demonstrated that if the airway wall undergoes a change in response to inflammation, then the airway vessels will as well. Of course, it has been known for a long time that the hypoxia resulting from chronic obstructive plumonary disease leads to vascular reaction and remodeling essentially v

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Introduction

manifested by pulmonary hypertension. However, only recently has the role of endothelial cell apoptosis and that of the potency, or lack of potency, of the vascular endothelial growth factor started to receive attention and dominate the field. Increased vascularity and altered vascular permeability in the wall of airways of chronic obstructive pulmonary disease patients are now well established, especially in small airways. Asthma also leads to vascular remodeling; however it seems to be in larger airways than in chronic obstructive pulmonary disease. There is some evidence that the vascular proliferation seen in asthma may actually contribute to airway wall thickening, and thus airway obstruction. The field of vascular remodeling is complex and very active. Evidence indicates that it also has great significance because what is learned about vascular remodeling can lead to new therapeutic approaches and interventions. The mere fact that this book is possible attests to the importance and promise of the field. Dr. Aili Lazaar, editor of Bronchial Vascular Remodeling in Asthma and COPD, has assembled a cadre of distinguished and well-known contributors from many countries, indicating a huge international interest in this area of research. The collective experience reported in this book is unique and an asset to the series of monographs Lung Biology in Health and Disease. I am grateful to the editor and the authors for this contribution. Claude Lenfant, MD Gaithersburg, Maryland, U.S.A.

Reference 1.

American thoracic society definitions and classifications of chronic bronchitis, asthma and emphysema. Am Rev Respir Dis 1962; 85:762–768.

Preface

Asthma, a common chronic disease affecting millions of people worldwide, causes substantial social and economic impact. Although current therapies effectively target airway inflammation and bronchoconstriction, there is less evidence to suggest that these treatments alter the progression of chronic structural changes within the airways of patients with severe asthma, including subepithelial deposition of collagen, increases in airway smooth muscle cell mass, mucus gland hyperplasia, and mucosal neovascularization. Collectively, these histologic findings define airway remodeling, the subject of another edition in this series. Considerable research effort and clinical interest focuses on mechanisms of airway inflammation and hyperresponsiveness in both asthma and chronic obstructive pulmonary disease; however, our knowledge of bronchial vascular remodeling remains limited. In addition, the process of bronchial vascular remodeling may be vastly different that that seen in the pulmonary or coronary vessels, due to tissue-specific responses to hypoxia and inflammation. Mucosal neovascularization, along with bronchial vascular dilatation and leakage, may contribute to airway wall remodeling by altering wall thickness and compliance. Less well understood is the potential for angiogenic factors derived from lung stromal cells to contribute to other aspects of airway remodeling, such as expression of adhesion molecules, deposition of extracellular matrix, or promotion of airway hyperresponsiveness. Angiogenic factors may also be important for survival of alveolar cells. vii

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This book brings together investigators with expertise in basic science, physiology, pathology, pharmacology, and medicine in an attempt to elucidate the mechanisms that regulate the development of bronchial vascular remodeling in asthma and chronic obstructive pulmonary disease. The efficacy of current pharmacotherapy, as well as the potential impact of novel approaches, are also discussed. Aili Lazaar

Contributors

Vijay K. T. Alagappan Cardiopulmonary and Molecular Biology Lab, Department of Pharmacology, Erasmus Medical Center, University Medical Center, Rotterdam, The Netherlands Saskia Appelmans The Center for Transgene Technology and Gene Therapy, University of Leuven, and Flanders Interuniversity Institute for Biotechnology (VIB), Leuven, Belgium Tiffany Bamford Department of Medicine, Monash Medical School, and The Alfred Hospital, Prahran, Australia Ellen C. Breen Division of Physiology, Department of Medicine, University of California—San Diego, La Jolla, California, U.S.A. Peter Carmeliet The Center for Transgene Technology and Gene Therapy, University of Leuven, and Flanders Interuniversity Institute for Biotechnology (VIB), Leuven, Belgium Alfredo Chetta Section of Respiratory Diseases, Department of Clinical Sciences, University of Parma, Parma, Italy Edward M. Conway The Center for Transgene Technology and Gene Therapy, University of Leuven, and Flanders Interuniversity Institute for Biotechnology (VIB), Leuven, Belgium ix

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Contributors

Horace M. DeLisser Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. Daphne E. deMello Saint Louis University Health Sciences Center and Cardinal Glennon Children’s Hospital, St. Louis, Missouri, U.S.A. Andres Hurtado Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, and Department of Biomedical Engineering, University of Miami College of Engineering, Miami, Florida, U.S.A. Nele Kindt The Center for Transgene Technology and Gene Therapy, University of Leuven, and Flanders Interuniversity Institute for Biotechnology (VIB), Leuven, Belgium Andor R. Kranenburg Cardiopulmonary and Molecular Biology Lab, Department of Pharmacology, Erasmus Medical Center, University Medical Center, Rotterdam, The Netherlands Nicholas W. Morrell Division of Respiratory Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, U.K. Dario Olivieri Section of Respiratory Diseases, Department of Clinical Sciences, University of Parma, Parma, Italy Thomas B. Pulimood Division of Respiratory Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, U.K. Hari S. Sharma Cardiopulmonary and Molecular Biology Lab, Department of Pharmacology, Erasmus Medical Center, University Medical Center, Rotterdam, The Netherlands Sorachai Srisuma Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. and Division of Respiratory Physiology, Department of Physiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand Elizabeth M. Wagner Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland, U.S.A.

Contributors

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Peter D. Wagner Division of Physiology, Department of Medicine, University of California—San Diego, La Jolla, California, U.S.A. Adam Wanner Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, Miami, Florida, U.S.A. John W. Wilson Department of Respiratory Medicine, Monash Medical School, and The Alfred Hospital, Prahran, Australia

Contents

Introduction Claude Lenfant . . . . v Preface . . . . vii Contributors . . . . ix

1. Developmental Origins of the Bronchial Vasculature: Experimental Approaches to Study the Structure and Function of Bronchial Vasculature . . . . . . . . . . . . . . . . . 1 Daphne E. deMello I. Anatomy . . . . 1 II. Angiogenic Processes and Their Control . . . . 4 III. Experimental Approaches to Study the Bronchial Circulation . . . . 10 IV. Summary . . . . 15 References . . . . 16

2. Noninvasive Measurement of Airway Blood Flow. . . . . . 25 Andres Hurtado and Adam Wanner I. Introduction . . . . 25 II. Structural and Functional Basis of Noninvasive Techniques . . . . 26 III. Noninvasive Techniques for Airway Blood Flow Measurement . . . . 30 IV. Conclusions . . . . 38 References . . . . 39 xiii

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3. Molecular Mechanisms of Angiogenesis. . . . . . . . . . . . . . . . 45 Edward M. Conway, Saskia Appelmans, Nele Kindt, and Peter Carmeliet I. II. III. IV. V. VI.

Introduction . . . . 45 Vessel Growth—In the Embryo and Adult . . . . 46 Vascular Endothelial Cell Growth Factor . . . . 48 Vasculogenesis . . . . 51 Angiogenesis . . . . 53 Summary . . . . 65 References . . . . 66

4. Chemokine Regulation of Angiogenesis . . . . . . . . . . . . . . . . 81 Sorachai Srisuma and Elizabeth M. Wagner I. II. III. IV. V.

Introduction . . . . 81 The Chemokines . . . . 82 Role of Chemokines in Angiogenesis . . . . 90 Role of Chemokines in Other Lung Pathologies . . . . 93 Summary . . . . 97 References . . . . 97

5. The Role of the Extracellular Matrix in Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Horace M. DeLisser I. II. III. IV. V. VI. VII. VIII. IX.

Introduction . . . . 105 The ECM of the Quiescent Endothelium . . . . 105 The Angiogenic Matrix . . . . 108 Integrins and the ECM . . . . 112 ECM and Proliferation . . . . 114 ECM and EC Apoptosis . . . . 114 ECM and Endothelial Cell Migration . . . . 115 ECM and Capillary Morphogenesis . . . . 116 Summary and Conclusions . . . . 117 References . . . . 117

6. Angiogenesis in the Asthmatic Airway . . . . . . . . . . . . . . . . 127 John W. Wilson and Tiffany Bamford I. Introduction . . . . 127 II. Summary . . . . 136 References . . . . 137

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7. Bronchial Vascular Remodeling in Emphysema/ Chronic Bronchitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Hari S. Sharma, Andor R. Kranenburg, and Vijay K. T. Alagappan I. Introduction . . . . 147 II. Airway Remodeling . . . . 148 III. Vascular Remodeling in Chronic Obstructive Pulmonary Disease . . . . 149 IV. Growth Factors Involved in Vascular Remodeling . . . . 154 V. Vascular Remodeling in Emphysema and Chronic Bronchitis . . . . 157 VI. Conclusion . . . . 160 References . . . . 162

8. Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease . . . . . . . . . . . . . . . . . . . . . . . 169 Nicholas W. Morrell and Thomas B. Pulimood I. II. III. IV. V. VI. VII. VIII.

Introduction . . . . 169 The Normal Pulmonary Circulation . . . . 169 Effects of COPD on Pulmonary Hemodynamics . . . . 172 Effects of COPD on Pulmonary Vasculature . . . . 173 Morphological Changes . . . . 174 Cellular Changes . . . . 176 Mechanisms of Vascular Remodeling in COPD . . . . 178 Genetic Influences on Pulmonary Vascular Remodeling and Pulmonary Hypertension in COPD . . . . 185 IX. Therapeutic Approaches . . . . 186 References . . . . 188

9. Angiogenesis: Lessons Learned from Skeletal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Peter D. Wagner and Ellen C. Breen I. Angiogenesis: Overview of a Rapidly Changing Field . . . . 197 II. Initiation and Regulation by Pro- and Antiangiogenic Growth Factors . . . . 199 III. Scope—Focus on Skeletal Muscles: Which Genes Are Turned on, Which Are Important, What the Stimuli Are . . . . 200 IV. Relevance to the Lungs: Airways and Parenchyma, Focus on VEGF . . . . 206 V. Summary . . . . 207 References . . . . 207

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10. Pharmacologic Modulation of Bronchial Vascular Remodeling: Current Therapies and Novel Approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Alfredo Chetta and Dario Olivieri I. II. III. IV.

Introduction . . . . 213 Current Therapies . . . . 214 Novel Therapeutic Approaches . . . . 219 Conclusions . . . . 221 References . . . . 222

Index . . . . 227

1 Developmental Origins of the Bronchial Vasculature: Experimental Approaches to Study the Structure and Function of Bronchial Vasculature

DAPHNE E. DEMELLO Saint Louis University Health Sciences Center and Cardinal Glennon Children’s Hospital, St. Louis, Missouri, U.S.A.

I. Anatomy A. Gross Anatomy

The bronchial circulation is one of the double arterial and venous systems of the lung. The other “pulmonary” circulation arises from the pulmonary artery, supplies the alveolar capillary surface, and is the main functional artery of the lung. The “bronchial” circulation should correctly be called the airway circulation as it provides nutrient supply to the trachea, the bronchi, and peripheral airways up to the level of the terminal bronchiole. The origin of the airway circulation is from systemic arteries, and there is considerable interspecies and intraspecies variability (1–5). Here, the description is restricted to the human. In the fourth week of gestation, primitive bronchial arteries arise from the dorsal aorta in the neck and are distributed to the airways (6). By the sixth week of gestation, airway branching is at the segmental or lobar level, and at this time, the primitive bronchial arteries disappear. Definitive bronchial arteries arise from the thoracic aorta between the T3 and T7 vertebrae during the ninth to twelfth weeks of gestation. In about 40% of individuals a single bronchial artery is present for each lung. The right bronchial artery may arise directly from the aorta or from an intercostal, subclavian, or internal thoracic artery. On the left, about 70% of 1

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people have two or more bronchial arteries, generally arising directly from the aorta. The bronchial arteries supply the distal trachea and carina, and they ramify within the walls of the bronchi and intrapulmonary airways. The branches form a plexus in the peribronchial space by anastomosing with each other, and small arterioles penetrate the muscular wall of the airway to form a submucosal plexus. The two plexuses (submucosal and peribronchial) travel along the entire airway up to the level of the terminal bronchiole, where the bronchial arteries give off capillaries that communicate with pulmonary capillaries in airway walls (Fig. 1). The normal diameter of the bronchial artery at the hilum is less than 2 mm (8), and in its intrapulmonary portion, it is much smaller than the diameter of the accompanying pulmonary artery. Venous drainage of intrapulmonary airways (70% of the total) is via pulmonary veins into the left atrium, resulting a small degree of venous admixture (Fig. 2) (9). Thus, elevation of left heart pressure causes congestion not only of the alveolar region but of the intrapulmonary airways as well. True bronchial veins drain blood from large hilar structures and extrapulmonary airways to the right atrium. B. Microscopic Anatomy

In sections of the lung, the bronchial vessels are to be found within the bronchovascular sheath in the bronchial wall. Normally these vessels are readily recognized and distinguished from the pulmonary artery that is also present within the bronchovascular sheath by their relatively small diameter compared Submucosal venules

Bronchial artery

Bronchial artery

Bronchial muscle

Adventitial venules

Bronchial vein

Bronchial artery

Bronchial vein

Figure 1 Schematic depiction of the bronchial vasculature within the airway wall. A network of arborizing vessels is present in the submucosa as well as in the peribronchial region external to the bronchial smooth muscle. Source: From Ref. 7.

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Trachea Bronchial Arteries

Bronchus Bronchopulmonary Anastomoses

Bronchial Vein Vena Azygos

Alveoli Bronchial Veins Lymph Gland

Pulmonary Vein Pleura

Neuro-vascular Bundle

Figure 2 Diagrammatic representation of the bronchial circulation. Bronchial veins from the extrapulmonary airways, and hilar pleura drain to the right side of the heart via the Azygos veins; intrapulmonary bronchial veins anastomose with the pulmonary veins at the level of the alveoli, and drain to the left atrium. Source: From Ref. 10.

to the accompanying pulmonary artery. However, when the bronchial vessels are dilated secondary to disease, the distinction between the two systems— bronchial and pulmonary—may be difficult. In such instances the vessel structure may help in the identification because the bronchial artery has only an internal elastic lamina, whereas the pulmonary artery has both an internal and external elastic lamina. Bronchial-to-pulmonary arterial anastomoses can occur, the best example being the communication between bronchial arteries and pulmonary capillaries and veins (11). Larger, coiled anastomoses, some short (1 to 2 mm) and narrow (50 to 100 mm in diameter) and others longer (10 to 40 mm) and wider (300 to 400 mm in diameter), have been described (12). Presumably the coiled structure regulates the pressure gradient from bronchial to pulmonary arteries. Under normal conditions the bronchial to pulmonary connections are closed, but they have the capacity to shunt up to 10% of the cardiac output when the pulmonary arterial circulation is acutely obstructed, as in pulmonary embolism. In chronic disease states, such as bronchiectasis or congenital heart disease, these bronchopulmonary shunts can become rather prominent.

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deMello II.

Angiogenic Processes and Their Control

A brief review of the processes involved in blood vessel formation is in order. From studies of the assembly of blood vessels in the yolk sac of the chick embryo, two processes have been identified. Angiogenesis is the branching of new vessels from preexisting ones, and vasculogenesis is the development of vessels from blood lakes (13). We have recently demonstrated in the mouse that the same processes participate in the formation of the early lung’s vasculature (14). In mouse embryos from 9 to 20 days gestation, a correlation was made between light and transmission electron microscopy of the developing lungs and scanning electron microscopy (SEM) of mercox lung vascular casts, to demonstrate the process of early lung vascular development. At nine days, lakes appear within the primitive mesenchyme surrounding the epithelial-lined lung bud, presumably initiated by angioblast precursors in the mesenchyme, and later, hematopoietic cells appear within the lakes or spaces. Between 10 and 12 days, the density of these lakes within the peripheral lung mesenchyme increases, and at this time, the pulmonary artery has four generations of central branches near the hilum. In the peripheral mesenchyme surrounding the lung, vessels develop by the process of vasculogenesis and vascular casting reveals, at first, no communication between the peripheral vessels and the central vessels that develop by angiogenesis or sprouting from the main pulmonary vessels. Between 12 and 14 days, five to seven generations of central vessels are evident, and both conventional and supernumerary arteries are present. By this time, a connection is established between the central and peripheral systems that permits filling by mercox of the peripheral vessels and visualization of their casts. The complexity of the peripheral casts increases progressively to term, reflecting both increased peripheral connections and growth of additional peripheral vessels, which now occurs by angiogenesis (Figs. 3–7). In summary, early lung vascular development in the mouse occurs by two concurrent but separate systems: vasculogenesis in the periphery and angiogenesis centrally. A third process of fusion between the two systems establishes the pulmonary circulation. Further growth of peripheral vessels then proceeds by angiogenesis. One could speculate regarding the necessity for the two processes of vascular development in the lung. Perhaps the hemangioblast precursors that migrate with the primitive lung mesenchyme carry the information that dictates the eventual overall topography of the pulmonary vascular tree, i.e., the branching pattern for the conventional and supernumerary arteries. The developing airway and its accompanying pulmonary artery could then be “drawn” to the sites of vasculogenesis by a chemoattractant mechanism completing the vascular circuit and permitting formation of the air-blood barrier within the airspace wall. The air-blood barrier is an important structure essential for all independent air-breathing and gas exchange and results from fusion of the basement membranes of the alveolar epithelial cell and the underlying capillary

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Figure 3 Transmission electron micrograph of fetal mouse thorax. At 10 days, the mesenchymal cells around the lung bud become thin and endothelial-like around a space or “lake” within which hematopoietic precursors are seen. (Mag !1458). Source: From Ref. 14.

endothelial cell. It is located at the distal end of the pulmonary arterial tree and is immediately proximal to the pulmonary venous segment. Perhaps the process of vasculogenesis plays a role in determining the site for the formation of air-blood barriers. In the human, a lethal disorder, alveolar capillary dysplasia, results from perturbation of this process of air-blood barrier formation. When it affects all

Figure 4 Photomicrographs of mercox casts of fetal mouse pulmonary vasculature. At 12 days (left), only four generations of arterial central branches are present, and no vascular connection is seen to the peripheral lung. At 13 days (right), mercox filling of some peripheral vessels is present. Abbreviation: PA, pulmonary artery. Source: From Ref. 14.

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Figure 5 Mercox cast of the fetal mouse pulmonary vascular tree. At 15 days (left), there is an increase in the density of small peripheral vessels, which increases more dramatically at 16 days (right). Source: From Ref. 14.

lobes of the lung, gas exchange cannot occur, and the infant succumbs to hypoxemia (15). The genes, factors, receptors, and mechanisms that orchestrate this seminal event in lung vascular development remain to be identified. The developmental processes contributing to human lung vascular development were evaluated using the Carnegie collection of human embryos and fetuses. The collection is housed in the Human Developmental Anatomy Center at the National Museum of Health and Medicine of the Armed Forces

Figure 6 A high magnification scanning electron micrograph of casts of some of the peripheral vessels from a 13-day fetus reveals bulbous profiles with slender projections extending at right angles between them (arrows). (Mag !320).

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Figure 7 Scanning electron micrograph of mercox lung vascular cast in a 15-day fetus: extensive connections between the central, and peripheral systems are present revealing a complex peripheral network. Blind ending precapillary branches approach the future capillary network at right angles, a stage in coalescence. Residual constrictions suggest these mark the sites of fusion. (Mag !320).

Institute of Pathology in Washington, D.C. It consists of paraffin embedded and serially sectioned and stained human embryos and fetuses of different developmental stages, all staged according to Streeter’s criteria (16). To determine the temporal relationship of the appearance of veins, arteries, and airways and their topographic distribution in the growing lobes, serial sections of human embryos from Streeter stages 10 to 23 and of human fetuses were examined. Because of the serial nature of the sections it was possible to identify for both arteries and veins the origin, path, and appearance of a lumen and, particularly in the early stage of arterial development, to identify a blind ending. As new features appeared, the size, location, and distribution of various structures could be followed in the serial sections, permitting a virtual threedimensional reconstruction of the vascular systems as well as of the airways, and connective tissue septa. In the early glandular stage (Streeter stage 15–23, 5 to 8 weeks), the pulmonary arterial tree that accompanies the airways outside the lung is represented by two thick-walled vessels with a small lumen. The right and left pulmonary arteries and the pulmonary artery within the lung are represented only by a cord of cells. The peripheral mesenchyme contains only lakes or thin-walled vascular channels. In the late glandular phase (8–16 weeks), the pulmonary artery reaches upto the last several airway generations but is small and has a narrow lumen. Side branches that would represent supernumerary arteries are not numerous, suggesting that at this time only conventional branches have developed.

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By 10–11 weeks, a network of thin-walled peripheral vessels lies close to the most peripheral airway branch but still outside the epithelial basement membrane. Between 11 and 16 weeks, the vascular network increases in complexity. During the canalicular phase (16 to 24 weeks), when airway branching is complete, a pulmonary artery is present at the very end of the airway as the ultimate few branches represent the future alveolar region. Between 20 and 21 weeks, small vessels bulge into the airway lumen, representing the air-blood barrier. In the saccular phase (24 weeks), an extensive microcirculation is formed by transformation of the mesenchyme so that blood flows from the pulmonary artery through the capillary bed in the alveolar wall to pulmonary veins. This study indicated that in the formation of the human pulmonary vasculature as well, two processes, angiogenesis and vasculogenesis, occur that contribute to the central and peripheral vessels, respectively (Figs. 8 and 9) (17). The assembly of blood vessels in an organ is a complex process requiring the interaction of a number of genes and factors. Experiments involving overexpression or knockout growth factors or genes provide an insight into the candidate genes and factors involved in these complex regulatory mechanisms (18,19). For example, vascular endothelial growth factor (VEGF) and its receptor proteins (VRP) are expressed in developing lung epithelial and endothelial cells, respectively, suggesting a role in the processes of assembly of blood vessel wall and in the overall pattern of vessel branching within an organ (20–23). Knockout of the VEGF gene, or even its reduced expression in heterozygosity, results in lethal defects in vessel formation in the mouse embryo (18,24,25). Knockout of the Flt-1

Figure 8 Saggital section of a human embryo thorax at 50 12 days. The bronchial artery accompanies only the first few generations of bronchi, whereas in the peripheral subpleural region (arrows), an extensive network of sinusoids resulting from coalescence of blood lakes has formed. No connection is seen between the central and peripheral systems. (Mag !200, H&E).

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Figure 9 Photomicrograph of human fetal lung between 22 and 23 weeks. The distal end of the pulmonary artery (short arrows) encircles airspaces at the end of the airway. Airblood barriers (long arrows) have formed. (Mag !200, H&E).

(VEGF receptor) gene produces a lethal defect in angiogenesis (26), and knockout of the Flk-1 (VEGF receptor) gene produces a lethal failure of vasculogenesis (27). A number of other genes and factors have been shown to play a role in lung vascular development (28–37), and at least two, VEGF, and Angiopoietin-1, play a role in vessel survival and plasticity, even in adult life (38). In the murine airway, Baluk et al. (39) in a recent study showed that turning on VEGF expression in the airway epithelium [using a tet-on inducible transgenic system driven by the Clara cell (CC10) promoter] induced angiogenesis in the form of endothelial sprouts from venules, which grew towards the epithelium, significantly increasing airway mucosal vessel density. Although the newly formed vessels acquired “mature” characteristics, they began to regress within three days after the withdrawal of VEGF, and vessel density returned to normal after 28 days. In addition to demonstrating the control of airway angiogenesis by VEGF, this study illustrates reversibility of the angiogenic process and regression of newly formed vessels, a feature of clinical relevance in disease states resulting from altered airway vascularity. In a mouse model of hind limb ischemia, neovascularization developed in response to upregulation of VEGF messenger RNA (mRNA), protein expression in skeletal myocytes, and endothelial cells in the ischemic limb (40). Administration of a neutralizing VEGF antibody produced impairment of the neovascularization. Bronchial angiogenesis appears to have different triggering mechanisms from pulmonary angiogenesis, because when bronchial angiogenesis is induced, as with monocrotaline, pulmonary angiogenesis is not present in regions of bronchial angiogenesis (41).

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deMello III.

Experimental Approaches to Study the Bronchial Circulation

Several experimental approaches have been used to study the bronchial vasculature. These studies have been extensively reviewed previously (1,2,40) and will be discussed briefly here under the sections, “In Health,” and “In Disease.” A. In Health Structure

The normal bronchial circulation has been studied in different species, including dogs, sheep, rabbits, pigs, and fowl (40,41). Generally, dye or casting material has been injected into the bronchial circulation to demonstrate the anatomy. Species differences exist in the number of main bronchial arteries and veins, their distribution, and anastomoses with the pulmonary circulation (40). In the rat as early as fetal day 15.5, a vessel corresponding to the right bronchial artery was identified along the right lateral surface of the bronchus (42). In the human, the mucosa of the small airways average 28 vessels/mm2 of tissue and represent less than 1% of the airway wall area, whereas the adventitia averaged 83 vessels/mm2, and this comprised 8% of the wall area (43). Ultrastructural studies of the bronchial microvasculature reveal that capillaries as large as 8 mm in diameter are lined by continuous nonfenestrated endothelium (44,45). A basal lamina around the endothelium contains pericytes. In the rat, guinea pig, and hamster, fenestrated capillaries are present just beneath the bronchial epithelium, and bronchial venules contain many transendothelial channels that are permeable to such tracers as horseradish peroxidase and tannic acid (46). Comparison with the pulmonary microvasculature reveals that the bronchial microvasculature has thicker endothelium and more cytofibrils and pericytes. These characteristics enable rapid hypertrophy, regeneration, and angiogenesis in response to injury. Corrosion casting and SEM of the bronchial vessels has revealed large (50–400 mm in diameter), thin-walled, interconnecting sinuses within the submucosa of large airways in the sheep (Fig. 10) (47). Similar structures are present in other species, including the human (2,48,49). Most likely these vessels serve as a vascular reservoir, but they could play a role in the absorption of drugs from the airways. In sheep, resin casting and SEM of the bronchial vasculature revealed that intrapulmonary bronchial arteries 100–300 mm in diameter had sharp branches and deep focal constrictions and were markedly rugose (50). Such structures were labeled Sperrarterien (blockading arteries) by von Hayek (51) and are believed to be responsible for the increase in vascular resistance and consequent drop in pressure from systemic to pulmonary levels at the peripheral end of the bronchial vasculature.

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Figure 10 Scanning electron micrograph of a corrosion cast of the tracheal vasculature of the sheep viewed from the abluminal surface shows a network of sinuses running parallel to the long axis of the trachea. Subepithelial capillaries are seen deep to the sinuses. Source: From Ref. 73.

Function Normal Physiology

The main function of the bronchial circulation is to provide nutrients to the airways, nerves, glands, lymph nodes, and pulmonary vessels. In doing so, it also plays a role in host defense, fluid balance, and airway metabolism. The rich submucosal and peribronchial vascular network that is formed by the bronchial circulation (Fig. 1) enables rapid clearance of drugs administered into the airway lumen (53). Conversely, recovery from the effects of airway smooth muscle agonists is dependent upon the degree of bronchial blood flow and is delayed when blood flow is limited (54,55). Clearance of particulate matter, e.g., aerosolized 99mTc-labelled diethylenetriaminepentaacetate (DTPA), which has a particle size of !2 mm, is primarily via the pulmonary circulation; however, this role can be taken over by the bronchial circulation when the pulmonary artery is occluded as in embolism (56). Inspired air must be warmed and humidified before it reaches the alveoli so as to prevent desiccation of the airway mucosa and damage to the mucociliary escalator. This thermoregulatory function of bronchial blood flow is probably most critical in the dog. Animals respond to dry air hyperpnea with increased airway vascular permeability and vasodilatation (57–60). Neuropeptides and prostaglandins are believed to mediate the vasodilation (61). In the human, studies have shown that upon cold air inhalation, the airway is cooled during

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inspiration, and the pulmonary circulation primarily warms inspired air. During expiration, heat and moisture are recovered from the exhaled air by the airway mucosal vessels (62). Blood Flow

Measurement of airway blood flow is covered in detail by Hurtado and Wanner elsewhere in the book and is mentioned only briefly here. Measurement of bronchial blood flow has been a challenge because of the low flow rate, species variability, and anatomic variability in the number of bronchial vessels (40). The components of bronchial blood flow that are of interest to the clinician or investigator are the total flow to the airways and lung and the flow that is anastomotic with the pulmonary circulation. Flow probes have been used at the origins of different bronchial arteries to measure flow at that site, but accuracy is compromised because up to 30% of the measured flow is distributed to structures other than the airway (63,64). The minimally invasive technique of dye video-densitometry has overcome some of these problems (65,66). Angiographic contrast medium injected into the bronchial artery and into the ascending aorta is used as a flow indicator, and contrast density changes are monitored radiographically. The absolute bronchial artery flow is derived using a dilution curve, by calculating the relative flow of contrast in the bronchial artery as a percentage of the cardiac output, which is measured simultaneously by another method, e.g., thermal dilution. In sheep and dogs, blood flow in the tracheal mucosa was measured by laserDoppler flowmetry (67). This method has the advantage of being continuous and relatively noninvasive, but movement and mucus secretion affect it, and the measurement cannot be calibrated, so interpretation requires caution. Total and regional bronchial blood flow has been measured using left atrial injectates of radioactive microspheres and measuring radioactivity in the excised lung or samples of lung compared with that in a reference blood sample obtained from a peripheral artery at a known flow rate (68–72). Colored or fluorescent spheres may also be used in conjunction with spectrophotometric measurements of digested lung samples. The microspheres, 15 mm in diameter, lodge in precapillary sites. Although the simplicity and minimal invasiveness of this technique are attractive, it is fraught with potential problems including introducing changes in pressure gradients and flow because of bronchopulmonary communications and the use of a large number of spheres that are needed for statistical purposes; permitting only a limited number of discrete measurements because of energy overlap in the levels of radionuclides or color spectra; and contamination of data by spheres lodging in the pulmonary capillaries either because of recirculation or bronchopulmonary communications. About two thirds of the bronchial blood flow drains to the left heart. An invasive technique to measure this has been used during cardiopulmonary bypass by collecting pulmonary venous drainage under constant pulmonary vascular volume (73,74). Other techniques involve the uptake of a soluble inert gas, such as dimethyl ether (75).

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Regulation/Control

Experimental approaches have consisted largely of studies that determine the factors that control the bronchial circulation, and these will be considered under three broad headings—local humoral factors, neural, and mechanical (76). Humoral Factors

A large number of vasoactive agents have been shown to act on the bronchial vasculature and to bring about vasoconstriction or dilatation (1,2). The vasoconstrictors include vasopressin, systemic hypercarbia, and hypoxemia, whereas the vasodilators include bradykinin, histamine nitric oxide, and prostaglandin F2a and I2. Hypoxia also has a direct vasodilator effect on the bronchial circulation, probably by acting on bronchial vascular smooth muscle (77). The technique for producing hypoxemia or hypercarbia may alter the bronchial vascular response as was shown by a study in open-chest ventilated anesthetized dogs in whom acute hypoxemia caused a decrease in bronchial blood flow and an increase in vascular resistance; hypercarbia produced the opposite effect (78). Others have shown that both hypoxia and hypercarbia increase bronchial blood flow through bronchopulmonary anastomoses via a mechanism involving cyclooxygenase products of arachidonic acid (79,80). Neural Regulation

Efferent Mechanisms. A balance between alpha and beta adrenergic agonists must maintain resting bronchial blood flow, because infused or inhaled alpha-adrenergic agonists reduce blood flow (81–83), whereas beta-adrenergic agonists increase blood flow (75–82), but blockade of both alpha, and beta receptors does not affect blood flow (84). Infusion or inhalation of cholinergic agents or vagal stimulation results in dilatation of the bronchial vasculature and increased blood flow (85,86). A number of nonadrenergic, noncholinergic neurotransmitter peptides—such as vasoactive intestinal polypeptide, substance P, Neurokinin A, Neurokinin B, and calcitonin gene-related peptide—produce an increase in airway blood flow. Neuropeptide Y and bombesin produce vasoconstriction (87). Afferent Mechanisms. A number of respiratory irritants can stimulate sensory nerve fibers and increase airway blood flow. These include cigarette smoke, hypertonic solutions, O3, and water (88–92). Chemoreceptors in the carotid body respond to hypoxia with vasodilatation of the bronchial vasculature; however, chemical stimulation produces vasoconstriction (93,94). There are species differences in these responses, as well as differences in the responses of carotid body and aortic chemoreceptors (2). Mechanical Influences

The inflow pressure to the bronchial circulation is systemic, but it drains to the low-pressure pulmonary circulation. Therefore, the bronchial vasculature is subject to the mechanical effects of ventilation and changing lung volume due

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to inflation and to changes in pulmonary vascular pressure (95–99). At higher lung volumes systemic veins drain more bronchial arterial blood and less is shunted through anastomoses to the pulmonary circulation. With increased positive end-expiratory pressure (PEEP), there is a decrease in bronchial artery blood flow, which is not due to vasoconstriction but to a mechanical effect of lung inflation on bronchial blood vessels. An increase in pulmonary venous pressure can also decrease bronchial blood flow (100). Conversely, increased bronchial blood flow (300% of control) in sheep did not alter airway resistance or reactivity, suggesting that vascular engorgement does not play a primary role in producing airway obstruction (101). Similarly, bronchial vascular engorgement in sheep produced by elevating left atrial pressure only decreased airway luminal pressure in the presence of airway smooth muscle tone and of itself did not result in airway obstruction (102). Airway vascular resistance was measured in dogs, and under the influence of such drugs as bradykinin, histamine, and methacholine, airway vascular resistance decreased, and mucosal thickness increased, but other vasodilator drugs, such Substance P, vasoactive intestinal peptide, and prostaglandin E, produced only a small increase in airway mucosal thickness and hence no significant effect on airway resistance (103). B. In Disease

The bronchial circulation perfuses the airways and portions of the lung (via anastomoses and vasa vasorum) and therefore is a factor in a number of disease states. Only asthma, and chronic obstructive pulmonary disease (COPD) will be considered here. Asthma/Allergic Responses

In asthma, it is believed that an increase in bronchial circulation will produce airflow obstruction by engorgement or edema of the airway wall, reducing luminal diameter (104–106). In patients with fatal asthma, measurement of the peripheral airway walls revealed an increase in wall thickness and submucosal vascular volume compared to patients with nonfatal asthma, COPD or controls (43). Stromal cell-derived factor-1 (SDF-1) is believed to be responsible for the increased vascularity of the bronchial mucosa in asthmatics (107). A study of the vascular bed in bronchial biopsies from asthmatic, and nonasthmatic patients also showed an increase in the percentage vascular area and in the number of vessels in asthmatics and a correlation between the number of vessels and severity of asthma (108). Dry air challenge or methacholine will produce airflow obstruction, but this is attenuated if the patients are first treated with an alpha-agonist that constricts the bronchial circulation, indicating that the airway response is related to airway circulation (109,110). Inflammatory mediators may also play a role in airway hyperreactivity, as is evident in rats with viral infections (111). Delayed clearance of locally produced mediators could be a reflection of reduced bronchial blood flow, which would prolong the bronchoconstriction (53,112). This is evident

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in asthmatics who have a decrease in airway vasoactive intestinal peptide that reduces vasodilator responses and delays clearance of bronchoconstricting mediators (113). Bronchial blood flow also increases in the allergic response to an inhaled antigen, as has been shown in sheep sensitized to Ascaris antigen (114–116). This allergic vascular response involves prostaglandins and leukotrienes and is blocked by a leukotriene antagonist, such as SPL 55712 (116). The allergic response also increases vascular permeability permitting the leakage of albumin into the airways, as shown in guinea pigs (117). Chronic Bronchitis, Infection, and Emphysema

A variety of species, including rodents, and farm animals, and agents, such as noxious gases, smoke microbes, and drugs, have been used to produce experimental chronic bronchitis (118). The bronchial circulation is increased especially in bronchiectasis (119,120). Local chronic inflammation and pulmonary vascular obstruction both play a pathogenetic role in increasing bronchial vascularity. In emphysema both the bronchial and pulmonary arteries reveal narrowing and obliterative changes (121,122). In interstitial pulmonary fibrosis, the bronchial vessels proliferate, but the pulmonary vessels reveal obstructive changes and plexiform lesions. It appears as if the two circulations respond differently to lung inflammation—the pulmonary by reduction or complete cessation, perhaps secondary to local thrombosis, and the bronchial by proliferation. This difference reflects the different responses of the two circulations to inflammatory mediators. The host response also plays a role in the changes in the airway vasculature in response to chronic inflammation (123–125). In response to infection with Mycoplasma pulmonis, after 8 weeks, C57BL/6 mice had doubled the number of tracheal vessels because of a proliferation of capillaries and venules; in contrast, a different strain of mice, C3H, had a decrease in the number of capillaries, which had converted to venules with a larger vessel diameter. The blood vessel remodeling after Mycoplasma pulmonis infection is associated with microvascular leakiness and an abnormal sensitivity to substance P (126–129), and the angiogenesis is preceded by an influx of macrophages (130). Others have shown that despite marked increases in matrix metalloproteinase (MMP)-2 and MMP-9 expression, neither is essential for the microvascular remodeling that is induced by Mycoplasma pulmonis airway infection (131). IV.

Summary

For decades, the main focus of pulmonary biologists has been on the development and control of the pulmonary circulation, while the “other” lung circulation, the bronchial, has been comparatively ignored. Among other events, the advent of lung transplantation and the ever-increasing incidence of asthma has served to illustrate the critical role of the bronchial circulation in overall lung function: breakdown of airway anastomotic sites in lung transplant recipients and

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obstruction of small airways in both transplant recipients and asthmatics were major clinical hurdles prompting interest in the vascular supply to the airways. The tremendous advances in the fields of molecular biology and genetics today provide powerful tools with which to mine the as-yet-unexplored aspects of the bronchial circulation, such as the contributory developmental processes and their genetic control mechanisms. Now, finally, the bronchial circulation can take its rightful place at center stage. References 1. Wagner EM. Bronchial circulation. In: Crystal RG, West JB, Weibel ER, Barnes PJ, eds. The Lung: Scientific Foundations. 2nd ed. Philadelphia: Lippincott-Raven, 1997:1093–1105. 2. Deffebach ME, Widdicombe J. The bronchial circulation. In: Crystal RG, West JB, Barnes PJ, Neil SC, eds. The Lung: Scientific Foundations. New York: Raven Press Ltd. 1991:741–757. 3. Wanner A. Circulation of the airway mucosa. J Appl Physiol 1989; 67:917–925. 4. Baile EM. The anatomy and physiology of the bronchial circulation. J Aerosol Med 1996; 9:1. 5. Magno M. Comparative anatomy of the tracheobronchial circulation. Eur Respir J Suppl 1990; 12:557s–562s. 6. Boyden EA. The developing bronchial arteries in a fetus of the 12th week. Am J Anat 1970; 129a:357–368. 7. Fishman AP. Pulmonary Diseases and Disorders. New York: McGraw Hill, 1980:400. 8. Song JW, Im YS, Park JH, Yeon KM, Han MG. Hypertrophied bronchial artery at thin-section CT in patients with bronchiectasis: correlation with CT angiographic findings. Radiology 1998; 208:187–191. 9. Lakshminarayan S, Kowalski TF, Kirk W, Graham MM, Butler J. The drainage routes of the bronchial blood flow in anesthetized dogs. Respir Physiol 1990; 82:65–73. 10. Deffebach ME, Charan NB, Lakshminarayan S, Butler J. The bronchial circulation. Small, but a vital attribute of the lung. Am Rev Respir Dis 1987; 135:463–481. 11. Wagenvoort CA, Wagenvoort N. Arterial anastomoses, bronchopulmonary arteries and pulmobronchial arteries in perinatal lungs. Lab Invest 1967; 16:13–24. 12. Tobin CE. The bronchial arteries and their connections with other vessels in the human lung. Surg Gynecol Obstet 1952; 95:741–750. 13. Noden DM. Embryonic origins and assembly of blood vessels. Am Rev Respir Dis 1989; 140:1097–1103. 14. deMello DE, Sawyer D, Galvin N, Reid LR. Early fetal development of the mouse pulmonary vasculature. Am J Respir Cell Mol Biol 1997; 16:568–581. 15. deMello DE. Structural elements of human fetal and neonatal lung vascular development. In: Weir EK, Archer SL, Reeves JT, eds. The Fetal and Neonatal Pulmonary Circulations. New York: Futura Publishing Company, 2000:37–64.

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52. Csete ME, Chediak AD, Abraham WM, Wanner A. Airway blood flow modifies allergic airway smooth muscle contraction. Am Rev Respir Dis 1991; 144:59–63. 53. Kelly L, Kolbe J, Mitzner W, Spannhake EW, Bromberger-Barnea B, Menkes H. Bronchial blood flow affects recovery from constriction in dog lung periphery. J Appl Physiol 1986; 60:1954–1959. 54. Wagner EM, Mitzner WA. Bronchial circulatory reversal of methacholine-induced airway constriction. J Appl Physiol 1990; 69:1220–1224. 55. Rizk NW, Luce JM, Hoeffel JM, Price DC, Murray JF. Site of deposition and factors affecting clearance of aerosolized solute from canine lungs. J Appl Physiol 1984; 56:723–729. 56. White DA, Parsons GH. Tracheal blood flow during spontaneous and mechanical ventilation of dry gases in sheep. J Appl Physiol 1990; 69:1117–1122. 57. Garland A, Ray DW, Doerschuk CM, et al. Role of tachykinins in hyperpneainduced bronchovascular hyperpermeability in guinea pigs. J Appl Physiol 1991; 70:27–35. 58. Salonen RO, Webber SE, Deffebach ME, Widdicombe JG. Tracheal vascular and smooth muscle responses to air temperature and humidity in dogs. J Appl Physiol 1991; 71:50–59. 59. Baile EM, Dahlby RW, Wiggs BR, Pare PD. Role of tracheal and bronchial circulation in respiratory heat exchange. J Appl Physiol 1985; 58:217–222. 60. Baile Em, Godden DJ, Pare PD. Mechanism for increase in tracheobronchial blood flow induced by hyperventilation of dry air in dogs. J Appl Physiol 1990; 68:105–112. 61. Solway J, Pichurk O, Ingenito EP, et al. Breathing pattern affects airway wall temperature during cold air hyperpnea in humans. Am Rev Respir Dis 1985; 132:853–857. 62. Horisberger B, Rodbard S. Direct measurement of bronchial artery flow. Circ Res 1960; 8:1149–1156. 63. Bruner HD, Schmidt CF. Blood flow in the bronchial artery of the anesthetized dog. Am J Physiol 1947; 148:648–666. 64. Link DP, Parsons GH, Lantz BMT, Gunther RA, Green JF, Cross CE. Measurement of bronchial blood flow in the sheep by videodilution technique. Thorax 1985; 40:143–149. 65. Parsons GH, Kramer GE, Lind DP, et al. Studies of reactivity and distribution of bronchial blood flow in sheep. Chest 1985; 87:180–182. 66. Corfield DR, Deffebach ME, Erjefalt I, Salonen RO, Webber SE, Widdicombe JG. Laser-Doppler measurement of tracheal mucosal blood flow: comparison with tracheal arterial flow. J Appl Physiol 1991; 70:274–281. 67. Ashley KD, Herndon DN, Traber LD, et al. Systemic blood flow to sheep lung: comparison of flow probes and microspheres. J Appl Physiol 1992; 73:1996–2003. 68. Rudolf AM, Heyman MA. The circulation of the fetus in utero: methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ Res 1967; 21:163–184. 69. Hale SL, Alker KJ, Kloner RA. Evaluation of nonradioactive, colored microspheres for measurement of regional myocardial blood flow in dogs. Circulation 1988; 78:428–434.

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70. Glenny RW, Bernard S, Brinkley M. Validation of fluorescent labeled microspheres for measurement of regional organ perfusion. L Appl Physiol 1993; 74:2585–2597. 71. Bernard SL, Obermiller T, Polissar NL, Mendenhall JM, Butler J, Lakshminarayan S. Fifteen micrometer microspheres reflux up the pulmonary veins during pulmonary artery occlusion. Microvasc Res 1993; 45:262–268. 72. Modell HI, Beck K, Butler J. Functional aspects of canine bronchial-pulmonary vascular communications. J Appl Physiol 1981; 50:1045–1051. 73. Hill P, Goulding D, Webber SE, Widdicombe JG. Blood sinuses in the submucosa of the large airways of the sheep. J Anat 1989; 162:235–247. 74. Agostonin P, Arena V, Doria E, Susini G. Inspired gas relative humidity affects systemic to pulmonary blood flow in humans. Chest 1990; 97:1377–1380. 75. Onorato DJ, Demirozu MC, Breitenbucher A, Atkins ND, Chediak AD, Wanner A. Airway mucosal blood flow in humans. Am J Respir Crit Care Med 1994; 149:1132–1137. 76. McDonald DM. The ultrastructure and permeability of tracheobronchial blood vessels in health and disease. Eur Respir J Suppl 1990; 12:572s–585s. 77. Wagner EM, Mitzner WA. Effect of hypoxia on bronchial circulation. J Appl Physiol 1988; 65:1627–1633. 78. Baile EM, Pare PD. Response of the bronchial circulation to acute hypoxemia and hypercarbia in the dog. J Appl Physio 1983; 55:1474–1479. 79. Charan NB, Lakshminarayan S, Albert RK, Kirk W, Butler J. Hypoxia and hypercarbia increase bronchial blood flow through bronchopulmonary anastomoses in anesthetized dogs. Am Rev Respir Dis 1986; 134:89–92. 80. Charan NB, Albert RK, Lakshminarayan S, Kirk W, Butler J. Factors affecting bronchial blood flow through bronchopulmonary anastomoses in dogs. Am Rev Respir Dis 1986; 134:85–88. 81. Sanders EA, Gleed RD, Hackett RP, Dobson A. Action of sympathomimetic drugs on the bronchial circulation of the horse. Exp Physiol 1991; 76:301–304. 82. Barker JA, Chediak AD, Baier HJ, Wanner A. Tracheal mucosal blood flow responses. J Appl Physiol 1988; 65:829–834. 83. Salonen RO, Webber SE, Widdicombe JG. Effects of neurotransmitters on tracheobronchial blood flow. Eur Respir J Suppl 1990; 12:630s–636s. 84. Baile EM, Osborne S, Pare PD. Effect of autonomic blockade on tracheobronchial blood flow. J Appl Physiol 1986; 62:520–525. 85. Matran R, Alving K, Martling CR, Lacroix JS, Lundberg JM. Vagally mediated vasodilatation by motor and sensory nerves in the tracheal and bronchial circulation of the pig. Acta Physiol Scand 1989; 135:29–37. 86. Laitinen LA, Laitinen MVA, Widdicombe JG. Parasympathetic nervous control of tracheal vascular resistance in the dog. J Physiol 1987; 385:135–146. 87. Salonen RO, Webber SE, Widdicombe JG. Effects of neuropeptides and capsaicin on the canine tracheal vasculature in vivo. Br J Pharmacol 1988; 95:1262–1270. 88. Pisarri TE, Coleridge HM, Coleridge JCG. Reflex bronchial vasodilation in dogs evoked by injection of a small volume of water into a bronchus. J Appl Physiol 1993; 75:2195–2202.

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89. Matran R, Alving K, Lundberg JM. Cigarette smoke, nicotine and capsaicin aerosol-induced vasodilatation in pig respiratory mucosa. Br J Pharma 1990; 100:535–541. 90. Pisarri TE, Jonzon A, Coleridge HM, Coleridge JCG. Vagal afferent and reflex responses to changes in surface osmolarity in lower airways of dogs. J Appl Physiol 1992; 73:2305–2313. 91. Prazma J, Coleman CC, Shockley WW, Boucher RC. Tracheal vascular response to hypertonic and hypotonic solutions. J Appl Physiol 1994; 76:2275–2280. 92. Schlege ES, Gunther RA, Parsons GH, Colbert SR, Yousef MAA, Cross CE. Acute ozone exposure increases bronchial blood flow in conscious sheep. Resp Physiol 1990; 82:325–336. 93. Alsberge M, Magno M, Lipschutz M. Carotid body control of bronchial circulation in sheep. J Appl Physiol 1988; 65:1152–1156. 94. Sahin G, Webber SE, Widdicombe JG. Chemical control of tracheal vascular resistance in dogs. J Appl Physiol 1987; 63:988–995. 95. Agostini P, Arena V, Biglioli P, Doria E, Sala A, Susini G. Increase in alveolar pressure reduces systemic-pulmonary bronchial blood flow in humans. Chest 1989; 96:1081–1085. 96. Agostoni PG, Deffebach ME, Kirk W, Lakshminarayan S, Butler J. Mechanism by which end-expiratory pressure (PEEP) decreases anastomotic bronchial blood flow. Am Rev Respir Dis 1986; 133:A162. 97. Cassidy SS, Haynes MS. The effects of ventilation with positive end-expiratory pressure on the bronchial circulation. Respir Physiol 1986; 66:269–278. 98. Charan NB, Albert RK, Lakshminarayan S, Kirk W, Butler J. Factors affecting bronchial blood flow through bronchopulmonary anastomoses in dogs. Am Rev Respir Dis 1986; 134:85–88. 99. Wagner EM, Mitzner WA, Bleeker ER. Affects of airway pressure on bronchial blood flow. J Appl Physiol 1987; 62:561–566. 100. Wagner EM, Mitzner W. Active constriction of the bronchial vasculature in response to left atrial pressure elevation. Am Rev Respir Dis 1988; 137:370s. 101. Blosser S, Mitzner W, Wagner E. Effects of increased bronchial blood flow on airway morphometry, resistance, and reactivity. J Appl Physiol 1994; 76:1624–1629. 102. Wagner EM, Mitzner W. Effects of bronchial vascular engorgement on airway dimensions. J Appl Physiol 1996; 81:293–301. 103. Laitinen LA, Robinson NP, Laitinen A, Widdicombe JG. Relationship between tracheal mucosal thickness and vascular resistance in dogs. J Appl Physiol 1986; 61:2186–2193. 104. Wiggs BR, Bosken C, Pare PD, James A, Hogg JC. A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992; 145:1251–1258. 105. McFadden ER. Hypothesis: exercise-induced asthma as a vascular phenomenon. Lancet 1990; 335:880–883. 106. Wilson JW, Kotsimbos T. Airway vascular remodeling in asthma. Curr Allergy Asthma Rep 2003; 3:153–158. 107. Hoshino M, Aoike N, Takahashi M, Nakamura Y, Nakagawa T. Increased immunoreactivity of stromal cell-derived factor-1 and angiogenesis in asthma. Eur Respir J 2003; 21:804–809.

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108. Salvato G. Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non-asthmatic subjects. Thorax 2001; 56:902–906. 109. Cabanes LR, Weber SN, Matran R, et al. Bronchial hyperresponsiveness to methacholine in patients with impaired left ventricular function. N Eng J Med 1989; 320:1317–1322. 110. Dinh XAT, Chaussain M, Regnard J, Lockhart A. Pretreatment with an inhaled a1adrenergic agonist, methoxamine, reduces exercise-induced asthma. Eur Respir J 1989;409–414. 111. Piedimonte G, Umeno E, McDonald DM, Nadel JA. Sendai virus infection potentiates neurogenic inflammation in the rat trachea. Am Rev Respir Dis 1989; 139:A230. 112. Wagner EM, Mitzner WA, Bleecker ER. Role of the bronchial circulation in cold air induced bronchospasm. Am Rev Respir Dis 1986; 133:A174. 113. Ollerenshaw S, Jarvis D, Woolcock A, Sullivan C, Scheibner T. Absence of immunoreactive vasoactive intestinal polypeptide in tissue from the lungs of patients with asthma. N Engl J Med 1989; 320:1244–1298. 114. Long WM, Yerger LD, Codias EK, et al. Early and late antigen-induced changes in bronchial artery blood flow and bronchomotor tone in allergic sheep. Fed Proc 1986; 44:1756. 115. Baier H, Long WM, Wanner A. Bronchial circulation in asthma. Respiration 1985;199–205. 116. Long WM, Yerger LD, Martinez H, et al. Modification of bronchial blood flow during allergic airway responses. J Appl Physiol 1988; 65:272–282. 117. Persson CGA, Erjefalt I, Andersson P. Leakage of macromolecules from guinea pig tracheobronchial microcirculation. Effects of allergen, leukotrienes, tachykinins, and anti-asthma drugs. Acta Physiol Scand 1986; 127:95–106. 118. Reid L, Jones R. Experimental chronic bronchitis. Int Rev Exp Pathol 1983; 24:335–382. 119. Liebow AA, Hales MR, Lindskog GE. Enlargement of the bronchial arteries and their anastomoses with the pulmonary arteries in bronchiectasis. Am J Pathol 1949; 25:211–231. 120. Nakamura T, Katori R, Miyazawa K, et al. Bronchial blood flow in patients with chronic pulmonary disease and its influences upon respiration and circulation. Dis Chest 1961; 39:193–206. 121. Cudkowicz L. Bronchial arterial circulation in man: normalanatomy and responses to disease. New York: Marcel Dekker, 1979. 122. Cudkowicz L, Armstrong JB. The bronchial arteries in pulmonary emphysema. Thorax 1953; 8:46–58. 123. Thurston G, Murphy TJ, Baluk P, Russel Lindsey J, McDonald DM. Angiogenesis in mice with chronic airway inflammation. Am J Pathol 1998; 153:1099–1112. 124. Thurston G, Maas K, Labarbara A, Mclean JW, McDonald DM. Microvascular remodeling in chronic airway inflammation in mice. Clin Exp Pharmacol Physiol 2000; 27:836–841. 125. McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001; 164:39–45.

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126. Kwan ML, Gomez AD, Baluk P, Hashizume H, McDonald DM. Airway vasculature after mycoplasma infection: chronic leakiness and selective hypersenstitivity to substance P. Am J Physiol Lung Cell Mol Physiol 2001; 280:L286–L297. 127. Baluk P, Bowden JJ, Lefevre PM, McDonald DM. Upregulation of substance P receptors in angiogenesis associated with chronic airway inflammation in rats. Am J Physiol 1997; 273:L565–L571. 128. McDonald DM, Bowden JJ, Baluk P, Bunnet NW. Neurogenic inflammation. A model for studying efferent actions of sensory nerves. Adv Exp Med Biol 1996; 410:453–462. 129. Bowden JJ, Scoeb Tr, Lindsey JR, McDonald DM. Dexamethasone and oxytetracycline reverse the potentiation of neurogenic inflammation in airways of rats with mycoplasma pulmonis infection. Am J Respir Crit Care Med 1994; 150:1391–1401. 130. Dahlqvist K, Umemoto EY, Brokaw JJ, Dupuis M, McDonald DM. Tissue macrophages associated with angiogenesis in chronic airway inflammation. Am J Respir Cell Mol Biol 1999; 20:237–247. 131. Baluk P, Raymond WW, Ator E, Coussens LM, MsDonald DM, Caughey GH. Matrix metalloproteinase 2 and 9 expression increases in mycoplasma-infected airways but is not required for microvascular remodeling. Am J Physiol Lung Cell Mol Physiol 2004; 287:L307–L317. Apr 9 (Epub).

2 Noninvasive Measurement of Airway Blood Flow

ANDRES HURTADO

ADAM WANNER

Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, and Department of Biomedical Engineering, University of Miami College of Engineering, Miami, Florida, U.S.A.

Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, Miami, Florida, U.S.A.

I. Introduction Airway blood flow has (i) physiologic roles, including nourishment of the mucosa, conditioning of inspired air, and anastomotic blood supply to the lung, (ii) pathophysiological roles in airway inflammation and tissue repair after lung transplantation, and (iii) therapeutic roles by regulating the clearance of inhaled airway drugs. For the understanding of this circulation’s physiological and clinical implications, the measurement of bronchial blood flow is crucial. Measurement of blood flow to the airway in humans is a major challenge because of technical difficulties, as reflected by the large number of techniques developed. Most of these techniques are not only complicated but also invasive to the subject; therefore, they have been used sparingly, and physiologic data on the human airway circulation are scarce. Most of the available information about the airway circulation has been gathered from animals and assumed to be applicable to humans. However, there are interspecies anatomical and functional

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differences of the airway circulation. Noninvasive measurement of airway blood flow in humans has been achieved by the relatively noninvasive laser-Doppler velocimetry technique and the noninvasive soluble gas uptake technique. This chapter discusses the methodology, advantages, and disadvantages of and some results obtained with these techniques.

II.

Structural and Functional Basis of Noninvasive Techniques

Within the airway, bronchial blood flow is distributed to two compartments: the submucosa and the outer bronchial wall. For the purposes of this discussion, submucosal blood flow is called airway blood flow in distinction from, and as a portion of, bronchial (total) blood flow. The major portion of bronchial blood flow is distributed to the mucosa (airway blood flow) (1–7).

A. Anatomical Considerations

The normal lung contains two circulatory systems: the pulmonary and the bronchial. The pulmonary vasculature is a low-pressure, large-volume system, perfused by mixed venous blood from the right side of the heart; the pulmonary circulation is responsible for gas exchange. The bronchial circulation (and consequently, the airway circulation) is a high-pressure, small-volume system, that derives arterial blood from the systemic circulation. Its main function is to provide nutrition to the walls of the airway (trachea and bronchi), to the large pulmonary blood vessels, to hiliar structures, such as the lymph nodes, and to the visceral pleura (8–11). Moreover, most of the blood supply to the intraparenchymal airways, down to 1 mm in diameter, is from the bronchial circulation (12,13). In the intraparenchymal bronchi, the bronchial circulation anastomoses with the pulmonary circulation and is also known as “pulmonary collateral circulation” or “systemic blood supply to the lungs” (14,15). The origin of bronchial arteries varies greatly from species to species and also within species (16). In humans, bronchial arteries usually arise from the aorta (17). Other sources for the bronchial vasculature include the intercostal arteries, the internal mammary artery, and the coronary arteries (8,9,17,18). The arrangements of the bronchial vasculature in humans also vary, and somewhat different findings have been reported (17,19,20). The most common presentation consists of two posterior bronchial arteries to each lung, reported in 20 to 30% of cases (17,19). Another very likely configuration consists of two bronchial arteries to the left lung, and one to the right (17,20). Finally, it is not unusual to find one common trunk supplying both lungs (10). This variability has minor physiological relevance, because all roots arise directly or indirectly from the aorta, hence they are subject to systemic arterial pressure (1).

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27

Bronchial arteries cross the esophagus anteriorly and posteriorly to the mediastinum, esophagus, hilar lymph nodes, and the vagi. Upon reaching the main bronchus on each side, the vessels surround the airway in the form of an annulus forming the peribronchial plexus, which supplies branches to the bronchi and vasa vasorum. Smaller branches of the bronchial artery penetrate the bronchial muscular layer and form an extensive vascular plexus underneath the airway epithelium, i.e., the mucosal plexus (2,10,12). This architecture permits individual blood flow control through parallel resistors with the capability of shunting flow from one airway vascular bed to another (1,2). The two interconnected plexuses follow the airways as far as the terminal bronchiole. The terminal arterioles give rise to capillaries located just beneath the epithelial basement membrane, which in the bronchi tend to run parallel to the long axis of the airway; in the trachea the system appears more random (21). These bronchial capillaries anastomose freely with the pulmonary circulation at pre-, post-, and capillary levels (21–23). Thus, in the distal branches of the airways, blood is able to flow from the bronchial circulation via bronchial capillaries to pulmonary vessels (8,21,23). The tracheobronchial tree has an extensive subepithelial capillary network (Fig. 1) (21,24). In the human airway, the total number of vessels residing in the submucosa within 200 mm of the bronchial epithelial basement membrane has been counted using light microscopy. About 120 vessels per mm2 were reported by Beasley et al. (25). Where the bronchial venous system empties has a critical role in the control of microvascular pressures in the mucosa (1). Venous blood from the proximal tracheobronchial tree (first two or three divisions, mainly extrapulmonary) drains successively through bronchial veins, azygos, and hemiazygos, and superior vena cava to the right heart (2,9,26–29); therefore, this drainage is subject to right atrial pressure. In contrast, the remaining intrapulmonary bronchial venous blood

Epithelium

Subephitelial capillaries

Arteriole

Smooth muscle

Bronchial artery Venule

Autonomic nerve Submucosal gland

Figure 1 Cross-section of bronchial wall showing the subephitelial capillaries and its proximity to the airway lumen. Source: From Dr. AT Mariassy, Prof. of Anatomy at Nova University School of Osteopathic Medicine, Ft. Lauderdale, FL, U.S.A.

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Hurtado and Wanner BV

BV

PV

SVC

LA

PA

BA

TRACHEA

BRONCHUS

BRONCHIOLE

ALVEOLUS

Figure 2 Schematic representation of the bronchial circulation and venous drainage. Abbreviations: BA, bronchial artery; BV, bronchial vein; LA, left atrium; PA, pulmonary artery; PV, pulmonary vein; SVC, superior vena cava. Source: From Ref. 2.

drains through postcapillary pulmonary vessels into the left atrium (2,8,10,11,26), hence, it is subject to left atrial pressure (Fig. 2). Normally, approximately 66% to 80% of the bronchial venous flow empties into the left side of the heart (22,29–31). This arrangement suggests that alterations of right and left atrial pressures would produce different effects on vascular pressures at different levels of the tracheobronchial tree (2). In conclusion, a part of the bronchial circulation drains into the systemic venous return, while another part connects with the pulmonary venous drainage, thereby constituting a physiological right-to-left shunt (venous blood that mixes with the pulmonary end-capillary blood on the venous side of the pulmonary circulation) (32). B. Physiological Considerations

The mucosal vessels in the airways are ideally located to perform a number of functions, including nourishing of the bronchi, heating and humidifying the inspired air, and distributing and clearing of mediators or drugs (8,33,34). It also allows the bronchial circulation to play an important role in lung defense and in the pathogenesis of a number of airway diseases (35). The airway circulation may act as a capacitive system that could alter airway wall thickness and therefore airway caliber over short periods of time under the influence of a variety of mediators (36–38). Bronchial capillaries can hypertrophy, regenerate, and form new vessels (angiogenesis) very rapidly (8,11). Histological evaluation of the bronchial wall in asthmatics has revealed augmented vascularity, including increased number and size of microvessels (39–42). Nourishment of the Mucosa (Airway Blood Flow)

The main function of the rich subepithelial capillary network of the tracheobronchial tree is to provide adequate nourishment to the tissue that has one of the highest metabolic rates in the body (8,15,43). The epithelium is active both in ciliary beating and active transport of ions and macromolecules. The

Noninvasive Measurement of Airway Blood Flow

29

metabolic rate of cultured sheets of tracheal epithelia is high, corresponding to an oxygen consumption of 5.13 mmol minK1gK1. This value may be compared with 1.0 mmol minK1gK1 for liver and 4.5 mmol minK1gK1 for the beating heart (21). Under physiological conditions, total bronchial blood flow comprises 0.5–1% of cardiac output (2,11,15,33,44–48). The major part of bronchial blood flow, up to 80%, is distributed to the subephitelial tissue (airway blood flow), (2,4,5,34,49–51), where the microvasculature comprises 10% to 30% of tissue volume (7,36). Using different techniques, absolute values of airway mucosal blood flow, usually normalized for wet tissue weight, have been reported in different species, including the human (52–58). Tracheal airway blood flow has been reported to range from 30 to 95 ml.minK1$100 gK1 wet tissue in different animal models (3,5,13,51,59), with most values ranging between 30–50 ml.minK1$100 gK1. Air Conditioning

It is widely accepted that during quiet nasal breathing, the inspired air is conditioned, i.e., heated and humidified, inside the nose, becoming fully warmed and humidified before it reaches the main bronchi (9,60). As the air is warmed, its capacity to hold water increases, and it is humidified by evaporation from the airway lining. The net effect of the thermal exchanges on inspiration is to cool down the mucosa so that, on expiration, recovery of heat and water can occur (60). Complete air conditioning inside the nose might not be achieved during certain conditions, such as mouth breathing, hyperventilation, or during cold air breathing, when air conditioning also takes place inside the tracheobronchial tree (60,61). In contrast to the nasal circulation, arteriovenous anastomoses have not been identified in the bronchial circulation (9,21). By lacking a countercurrent mechanism to condition air, heat exchange and water control inside the airways must be accomplished by changes in airway mucosal blood flow. Despite the optimal location of the bronchial circulation for heat exchange, it represents such a small fraction of total circulation to the lung (pulmonary, and bronchial) that under physiological conditions, it can only be effective in heating the airway during a low thermal burden (61). Indeed, it has been shown that the response exhibited by the bronchial circulation depends on the magnitude of the thermal stress to which it is exposed (62–64). Under physiological conditions, the pulmonary circulation is more relevant in heat exchange, whereas bronchial circulation is more important in water transport to the bronchial mucosa (61). Gas Exchange in the Airway

Conducting airways of the lung play an important role in the exchange of highly soluble gases. Gases delivered via the airway circulation must cross the capillary walls, the layer of nonperfused tissues, and the mucus in order to reach the gas

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phase in the lumen of the airway. Moreover, any inhaled gas trying to reach the airway circulation must cross the same pathway in the opposite direction. The degree of exchange depends critically on the blood:gas partition coefficient (a), an index of solubility; the most soluble gases equilibrate more efficiently. For example water (a|20,000 at 378C) exchanges entirely within the airway tree as inspired air becomes fully humidified before entering the alveolar region during quiet breathing (65). The dynamics of soluble-gas exchange are similar to the dynamics of heat and water exchange, and gases with an aO3 participate in airway gas exchange (66–68). III.

Noninvasive Techniques for Airway Blood Flow Measurement

Berry and Daly (48) made the first measurements of bronchial blood flow in 1931 using isolated perfused dog lungs, a model described by Berry et al. (69). Since that time several techniques have been used to measure blood flow in the airways, including simultaneous determination of right and left ventricular output (70–73), formation of an aortic pouch from which bronchial arteries arise (29,30,74), placement of electromagnetic (75–79) or ultrasonic (13,80) flow probes around the bronchial artery, intravascular Doppler guide wires (81), video densitometry using contrast medium as the flow indicator (5,45,82), and intravascular injection of labeled microspheres (12,83–85). Many of these techniques are invasive and require extensive surgical preparation, which can affect the measurements by changing the physiological conditions. Moreover, some of them require postmortem examination of the airways. There is an excellent review about the invasive methods of measuring bronchial blood flow (44). A relatively noninvasive technique, laser-Doppler velocimetry (86–96), has been used to measure bronchial mucosal blood flow in animals; however, the method’s limitations (vide infra) have kept it from being used in humans, although some attempts have been made (97). The newest and least invasive technique developed is the measurement of airway blood flow with a soluble inert gas, dimethylether (DME). This technique is the only one currently used for noninvasive measurement of airway blood flow in humans. The methodology, applications, advantages, and disadvantages of these two techniques are discussed in detail below. An additional method, analysis of the expired air temperature profile, has been described in the recent past (98,99). Although the expired air temperature may be related to airway blood flow, it has not been validated, and it is not yet clear to what extent changes in expired air temperature profiles reflect changes in airway blood flow. A. Laser Doppler Velocimetry

Airway blood flow can be measured using a probe inserted through a fiberoptic bronchoscope and applying it to the membranous portion of the airway. A He-Ne

Noninvasive Measurement of Airway Blood Flow

31

laser beam (lZ632.8 nm) emitted from the tip of a side-firing fiber-optic probe penetrates into tissues. Photons are scattered randomly by stationary tissue cells and by moving red blood cells (RBCs); photons that strike moving RBCs inside capillaries are reflected with a frequency shift according to their velocities (Doppler shifting effect). An increase/decrease in the number of RBCs causes an increase/decrease in the number of shifted photons, whereas an increase/decrease in their velocity causes an increase/decrease in the magnitude of the frequency shift. Part of this scattered light is collected by returning optical fibers coupled to a photodetector that transduces the signal into an electrical one. After appropriate signal processing, the product of velocity and volume produces a signal that is proportional to blood flow (86,87). This method measures microvascular perfusion, providing relative values of airway blood flow (89). In order to obtain absolute flow values, an in vivo calibration by an independent method is required. The sampling volume of tissue depends on the probe used. In general, it is a semicircular area with a radius of 1 to 1.5 mm from the emitting tip (44,87,89,93,94). The main advantage of this technique resides in its potential to provide a continuous index of airway blood flow in humans. The most significant limitations and sources of error in this technique include: (i) motion artifacts, i.e., vibration of the bronchial mucosa by the heartbeat and by respiration, (ii) hematocrit changes, (iii) position of the tip: there is a great variability between sites (91), and individual vessels in the microvasculature can contribute as much as 70% of the total signal (89), (iv) the contact pressure of the tip, and (v) the effects of mechanical tissue-stress-induced hyperemia. Moreover, because it is a relative measurement, it requires a reference to analyze the data (86,87,90). Several studies have used this technique to investigate bronchial mucosal blood flow after lung allotransplantation in dogs (86–88,94,95). It has been demonstrated that bronchial mucosal blood flow is significantly reduced during acute rejection after lung transplantation (87,94,96). Evaluation of bronchial blood flow as a surveillance tool in patients who have undergone lung transplantation has been suggested (81,87,88,94). Baile et al. (97) measured airway blood flow using a laser Doppler flowmeter in two different groups. In the first group, the subjects (eight) were undergoing coronary artery bypass surgery, and the second group consisted of six awake voluntaries. Four to 15 laser Doppler measurements per subject were made. The authors found considerable inter subject, between regions, and within regions (site-to-site) variability in their measurements. Due to its limitations, this technique is not useful for the measurement of airway blood flow in humans unless improved laser Doppler flowmeters are developed (91). B. Soluble Gas Uptake

Using an inert soluble gas, DME, and based on a modification of Fick’s principle for the calculation of blood perfusion, airway blood flow can be measured (52). This technique allows calculation of airway blood flow in the mucosa

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(to a depth of !200 mm) from the steady-state uptake of DME from a defined airway segment. Theoretical Background

If a tissue containing capillaries with flowing blood is exposed to an inert gas soluble in that tissue and blood, the uptake of that gas is determined by the volume of tissue with which the gas equilibrates (transient state) and by the blood flow through those capillaries (steady state) (51,52,100). Over a defined segment of a conducting airway, the steady-state disappearance of the inert soluble gas from the lumen must therefore reflect capillary blood flow within the adjacent tissue (mucosa) (51,52). DME has a high solubility coefficient (101) and is therefore well suited for this purpose. It has been shown that when the airway is filled with the inert soluble gas DME, the gas initially diffuses to the tissue until it reaches equilibrium. Beyond this point, a steady state is reached, and the uptake of DME from the airway is entirely dependent on airway blood flow ðQ_ aw Þ (59). Q_ aw can be calculated by Bronstein’s modification of Fick’s principle: Q_ aw Z V_ DME =ða
FDME Þ (51,52), where V_ DME is the steady-state uptake of DME from the conducting airways, a is the Bunsen solubility coefficient for DME in tissue and blood, and FDME is the mean fractional concentration of DME in the airway. V_ DME is calculated from the slope of the mean expired DME concentration over time, multiplied by the volume of the defined airway segment in the conducting airways (VS): V_ DME Z ½VS !ðslope of mean expired DME concentrationÞ. Because the expired DME concentration is measured at room temperature (water vapor pressureZ24 mm Hg), it has to be corrected by a factor of ½ð760  47Þ=ð760  24Þ, or 0.97. However, because it is used both in the numerator ðV_ DME Þ and denominator ðFDME Þ, it cancels out. Nevertheless, VS, which is under body temperature, pressure, saturated with water vapor (BTPS) conditions, must also be converted to standard temperature, pressure, dry (STPD) with the factor ½ð760  47Þð273Þ=½ð760Þð273C 37Þ, or 0.83, therefore: Q_ aw Z V_ DME ð0:83Þ= ða
FDME Þ. If the inhaled volume of DME is large enough to fill the anatomical dead space (DS), its uptake from VS (e.g., to a depth of 50 ml from the proximal trachea) will occur in the mucosa lining that segment and by axial dispersion for uptake by the mucosa proximal and distal to that segment. By closing the airway opening, VS is sealed off at its proximal end, and no axial dispersion will occur; however, axial dispersion at its distal end will occur as long as the mucosal uptake of DME distal to the segment exceeds its uptake inside the segment of interest. Because the mucosal surface:DS ratio increases with increasing airway generations, (higher available area for gas uptake), axial dispersion of DME may indeed occur, and the “true segment” will be bigger than 50 ml. A “true segment” can be estimated by having the subject inhale a mixture of DME and helium (He), where He can be used as a reference gas to correct for dilutional effects.

Noninvasive Measurement of Airway Blood Flow

33

Onorato et al. (52) demonstrated in vitro that axial diffusion for He and DME is small and of similar magnitude, producing minimal changes of gas concentration for up to 20s (0.1% and 0.05% of initial concentration of DME and He, respectively). From the morphometric measurements of Weibel (102), the cumulative mucosal surface area can be calculated for different airway depths from the proximal end of the trachea. For example, the mucosal surface area would be 163 cm2 for a 50 ml anatomical DS extending distally from the proximal end of the trachea (51). For the calculation of V_ DME , the subject inhales a gas mixture containing DME and He at a fixed concentration ratio. The FDME and the mean helium fractional concentration ðFHe
Þ are measured over a desired volume segment in the expirate from different breath-hold times after inhalation of the gas mixture. From the slope of FDME =FHe
against time, multiplied by the expired volume segment in which these concentration are measured, (50 ml in this case), V_ DME is calculated. Although theoretically FDME =FHe is expected to decline
exponentially over time, a nearly linear curve is obtained between breathhold times of 5 and 20 sec. Because there is no appreciable or only a minimal difference in the calculation of V_ DME whether an exponential or linear fit is used, a linear fit is applied. The slope of the curve indicates that tissue equilibration with DME had already taken place within 5 sec and that the slope reflects the steady-state V_ DME . The algebraic mean of the absolute expired FDME for the shortest and longest breath-hold times is taken as the representative FDME for Fick’s equation. The mean FHe
between the shortest and the longest breath-hold times and the inspired helium fractional concentration ðFHe Þ are used to estimate the true airway volume in which the gases are distributed: in our example, true airway volumeZ 50ml !ðinspired FHe =expired FHe
Þ. The Bunsen solubility coefficient (a) for DME (9 ml/ml of tissue) was originally described for humans by Peterson et al. (101); it is defined as the volume of gas STPD dissolved in 1 ml of blood at 760 mm Hg atmospheric pressure measured at body temperature (378C). The equation used to calculate Q_ aw assumes that: (i) the diffusion limitation to the uptake of DME into the tissue during the transient state has no influence on the volume of DME dissolved in tissue and the tissue is fully saturated with DME at the beginning of the steady state; (ii) the DME concentration of capillary blood is the same in all capillaries in which flow is measured (single-layer capillary bed); (iii) the partial pressure of DME is the same in the tracheal DS, mucosal tissue, and end-capillary blood; and (iv) the DME concentration of incoming tracheal arterial blood is negligible (3). Methodology of Q_ aw Measurement in Humans

The subject is seated in front of a valve system that allows inhalation of room air through a mouthpiece (with the nasal passage occluded with a noseclip) or a gas mixture from a Teflon bag containing approximately 10% DME, 5% He and the

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Hurtado and Wanner

balance oxygen (O2), and exhalation into a rolling seal spirometer a (Fig. 3). The subject first inhales room air to total lung capacity (TLC) position, then exhales 500 ml, and subsequently inhales rapidly the same volume of gas mixture from the Teflon bag (53–58). The subject then holds the breath for a predetermined duration and then exhales into the spirometer through a critical flow orifice to standardize expiratory flow. The maneuver is performed with two breath-hold times, each of 5,

PC DAP

ANALOG INPUTS

DME, He, N2 GRASS POLYGRAPH

Volume

MASS SPECTROMETER

SPIROMETER

ENVIRONMENT CFO

BALLOON VALVE CONTROLLER

Compressed air

MOUTH UNIT

Compressed air

GAS TANKS

Test gases

TEFLON BAG: DME, He, O2

Figure 3 Block diagram for noninvasive measurement of airway blood flow in humans. Abbreviations: CFO, critical flow orifice; DAP, digital/analog processor; DME, dimethylether; PC, personal computer. a

Model 840; Ohio Instruments, Houston, TX.

Noninvasive Measurement of Airway Blood Flow

35

10, 15, and 20 seconds in random order. During exhalation, the instantaneous concentrations of DME, He, and nitrogen (N2) are measured at the airway opening with a mass spectrometer b along with the expired volume, measured with the spirometer. The mass spectrometer inlet is not heated, and no corrections are made for water pressure. The resulting overestimation of DME concentration by measuring it at the airway opening was considered negligible (w0.3%) (52,53,56). The mass spectrometer is also used to verify the concentration in the Teflon bag before inhalation of the gas mixture. The analog signals from the mass spectrometer and the spirometer are fed through analog-to-digital converters to a computer for the calculation of Q_ aw and the anatomic DS. From the expired N2 concentration curve, DS is calculated as described by Fowler et al. (103) The He-corrected decrease in DME concentration over time is obtained by least square fit using the two measurements per gas for each of the four breath-hold times. This is done in the expired volume fraction corresponding to the DS minus the most proximal 50 ml. From the He-corrected DME slope multiplied by the DS ðV_ DME Þ, the mean fractional DME concentration in the DS ðFDME Þ and the solubility coefficient for DME in blood and tissue (a), Q_ aw , is calculated using Fick’s principle: Q_ aw Z V_ DME ð0:83Þ=ða
FDME Þ. The airway caliber influences the measured value of Q_ aw (104); the mucosal surface to dead-space ratio increases with increasing airway generation. Therefore, the measured Q_ aw is lower in a dilated tracheobronchial tree than in a constricted one, where a given volume segment extends deeper into the bronchial tree. For this reason, Q_ aw is normalized for DS and expressed as microliters per minute per milliliter of DS (ml.minK1.mlK1) where ml.minK1 represents blood flow, and ml corresponds to the volume segment from which Q_ aw is measured (typically DS minus the proximal 50 ml) (53,56). The limitations of this technique are: (i) the necessity of an expensive mass spectrometer, and (ii) the requirement for multiple breath-holds to obtain DME uptake precludes instantaneous measurements of Q_ aw . The DME technique was first used to study the regulation of blood flow in a sealed tracheal chamber (created by a double-cuffed endotracheal tube) in sheep (3,6,50,59,75,105) before converting it to the noninvasive open airway technique described above for humans. Validation of the open airway technique was later accomplished by correlating the values of Q_ aw obtained with DME to those obtained by radioactive microspheres injection (Fig. 4) (51). This established the suitability of the technique for the noninvasive measurement of airway blood flow in humans (52–58,63,64,104). Experiments using this technique have reported the following values for Q_ aw (ml.minK1.mlK1) in normal subjects: 43.8G0.7 (nZ11) (53), 44.2G0.7 (nZ12) (55), and 44.2G1.1 (nZ10) (56).

b

Perkin Elmer 1100 Medical Gas Analyzer, Pomona, CA.

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Hurtado and Wanner 120

100

Qaw (DME), mL/min

80

60

40

20

r = 0.89, p = 0.01

0 0

20

40

60

80

100

120

Qaw (M), ml/min

Figure 4 Comparison of tracheal mucosal blood flow ðQ_ aw Þ as measured with dimethylether (DME) and microspheres (M) in sheep. Values are normalized for systemic arterial pressure and are expressed per 100 g wet tissue. R was calculated by SpearmanRank correlation test. Source: From Ref. 51.

Selected Observations

The initial set of experiments attempted to determine physiological responses. In the first measurement of airway blood flow in human subjects using this technique (52), the response of Q_ aw to vasoactive substances, i.e., the a1-adrenergic agonist methoxamine and the b2-adrenergic agonist albuterol, was evaluated. Inhaled methoxamine caused a dose-dependent transient decrease, and inhaled albuterol caused a dose-dependent transient increase in Q_ aw (52,53). Later on, the effects of thermal stress on Q_ aw were evaluated, and it was concluded that the response of the airway circulation depends on the magnitude of the thermal stress, with a decrease in Q_ aw at low levels of stress and massive increase at high levels of stress (63,64). The technique has also been used to determine Q_ aw in bronchial asthma and the effects of pharmacological therapies currently used for its treatment. Initially, Kumar et al. (54) demonstrated that Q_ aw is increased in stable asthmatics and is resistant to further increase by inhaled albuterol (Fig. 5). Brieva et al. (55) corroborated this increased baseline Q_ aw and the blunted beta-adrenergic

Noninvasive Measurement of Airway Blood Flow

Qaw

120

37

*

*

SGaw 100

% Change

80

60

* 40

20

*

0 GS NORMALS

no GS ASTHMATICS

Figure 5 Effect of inhaled albuterol on airway blood flow ðQ_ aw Þ and specific airway conductance (SGaw) in normal (nZ11) and asthmatic patients using (nZ13) or not using (nZ10) long-term inhaled GS. *p!0.05 vs. corresponding parameter in normal. Abbreviation: GS, glucocortico steroids. Source: From Ref. 54.

vasodilator response in asthma. In 19 glucocorticosteroid-naive patients, they found that after a 2-week course of fluticasone propionate (FP), Q_ aw decreased and the albuterol responsiveness was restored; both effects vanished by 2 weeks after cessation of the FP treatment. Kumar et al. (56) also reported a short-term transient vasoconstrictor effect of inhaled FP in the airway mucosa, with a greater responsiveness in subjects with asthma than in healthy subjects (Fig. 6). They suggested that by causing vasoconstriction and possibly mucosal decongestion, inhaled glucocorticosteroids (ICS) may have an immediate beneficial effect in asthma and could also enhance the action of inhaled bronchodilators by diminishing their clearance from the airway. In subsequent studies, Mendes et al. (57) first compared the efficacy of three ICS [FP, beclomethasone dipropionate (BDP), and budesonide (BUD)]; they found that inhaled FP and BUD cause greater vasoconstriction in the airway than BDP, again with a greater vasoconstriction in asthmatics than in healthy subjects. Later, Mendes et al. (58) compared the effects of two anti-inflammatory agents, the leukotriene modifier montelukast (MK) and the ICS FP on Q_ aw in 12 patients with mild intermittent

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Hurtado and Wanner 65

NORMAL ASTHMA

(**p = 0.01 vs BSL) (*p = 0.01 vs BSL)

60

Qaw (µl.min−1 .ml−1)

55 50 45

** *

40

*

35 30 BSL

60 30 Time post inhalation (min)

90

Figure 6 Effect of 880 mg of fluticasone propionate on airway blood flow normalized for dead space ðQ_ aw Þ in 10 subjects without asthma and 10 subjects with asthma over a 90-minute observation period. Mean valuesGSE. Abbreviation: BSL, baseline. Source: From Ref. 56.

asthma. The authors found that a 2-week treatment with FP, MK, or a combination of the two agents produces a transient decrease in Q_ aw , with no significant difference among them. Two weeks after discontinuing the treatment, Q_ aw returned to baseline.

IV.

Conclusions

Among the available techniques for noninvasive measurement of airway blood flow, the soluble gas uptake is the only one proven to be the most accurate and suitable for humans. Several studies have employed this technique to assess Q_ aw under physiological and pathological conditions. Future studies involving Q_ aw measurements are needed to better characterize airway blood flow, as in exercise-induced bronchoconstriction, chronic obstructive pulmonary disease, endobronchial tumors, and in response to pharmacologic agents among others. Due to its limitations, the Laser Doppler technique is not useful for the measurement of airway blood flow in humans at present, and several improvements will be required before this technology can be applied in humans.

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44. Baile EM, Pare´ PD. Methods of measuring bronchial blood flow. In: Butler J, ed. The Bronchial Circulation. New York: Marcel Deckker, 1992:101–180. 45. Link DP, Parsons GH, Lantz BMT, Gunther RA, Green JF, Cross CE. Measurement of bronchial blood flow in the sheep by video dilution technique. Thorax 1985; 40:143–149. 46. Salisbury PF, Weil P, State D. Factors influencing collateral blood flow to the dog’s lung. Circ Res 1957; 5:303–309. 47. Berne RM, Levy MN. Structure and Function of the respiratory system. 4th ed. Physiology. St Louis: Mosby, 1998: 517–533. 48. Berry JL, Daly IB. The relation between the pulmonary and bronchial vascular systems. Proc R Soc Lond Ser B 1931; 109:319–336. 49. Baile EM, Minshall D, Dodek PM, Pare´ PD. Blood flow to the trachea and bronchi: the pulmonary contribution. J Appl Physiol 1994; 76:2063–2069. 50. Scuri M, McCaskill V, Chediak AD, Abraham WM, Wanner A. Effect of inhaled and intravenous acetylcholine on bronchial blood flow in anesthetized sheep. J Appl Physiol 1996; 80:341–344. 51. Scuri M, McCaskill V, Chediak AD, Abraham WM, Wanner A. Measurement of airway mucosal blood flow with dimethylether: validation with microspheres. J Appl Physiol 1995; 79:1386–1390. 52. Onorato DJ, Demirozu MC, Breitenbucher A, Atkins ND, Chediak AD, Wanner A. Airway mucosal blood flow in humans: response to adrenergic agonists. Am J Respir Crit Care Med 1994; 149:1132–1137. 53. Brieva J, Wanner A. Adrenergic airway vascular smooth muscle responsiveness in healthy and asthmatic subjects. J Appl Physiol 2001; 90:665–669. 54. Kumar SD, Emery MJ, Atkins ND, Danta I, Wanner A. Airway mucosal blood flow in bronchial asthma. Am J Respir Crit Care Med 1998; 158:153–156. 55. Brieva JL, Danta I, Wanner A. Effect of an inhaled glucocorticosteroid on airway mucosal blood flow in mild asthma. Am J Respir Crit Care Med 2000; 161:293–296. 56. Kumar SD, Brieva JL, Danta I, Wanner A. Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma. Am J Respir Crit Care Med 2000; 161:918–921. 57. Mendes ES, Pereira A, Danta I, Duncan RC, Wanner A. Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids. Eur Respir J 2003; 21:989–993. 58. Mendes ES, Campos MA, Hurtado A, Wanner A. Effect of montelukast and FP on airway mucosal blood flow in asthma. Am J Respir Crit Care Med 2004; 169:1131–1134. 59. Barker JA, Chediak AD, Baier HJ, Wanner A. Tracheal mucosal blood flow responses to autonomic agonists. J Appl Physiol 1988; 65:829–834. 60. McFadden ER, Jr. Respiratory heat and water exchange: physiological and clinical implications. J Appl Physiol 1983; 54:331–336. 61. Serikov VB, Fleming NW. Pulmonary and bronchial circulations: contributions to heat and water exchange in isolated lungs. J Appl Physiol 2001; 91:1977–1985. 62. McFadden ER, Jr., Denison DM, Waller JF, Assoufi B, Peacock A, Sopwith T. Direct recordings of the temperatures in the tracheobronchial tree in normal man. J Clin Invest 1982; 69:700–705.

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63. Kim HH, Le Merre C, Demirozu CM, Chediak AD, Wanner A. Effects of hyperventilation on airway mucosal blood flow in normal subjects. Am J Respir Crit Care Med 1996; 154:1563–1566. 64. Le Merre C, Kim HH, Chediak AD, Wanner A. Airway blood flow responses to temperature and humidity of inhaled air. Resp Physiol 1996; 105:235–239. 65. George SC, Hlastala MP, Souders JE, Babb AL. Gas exchange in the airways. J Aerosol Med 1996; 9:25–33. 66. Hlastala MP. The alcohol breath test—a review. J Appl Physiol 1998; 84:401–408. 67. George SC, Babb AL, Hlastala MP. Modeling the concentration of ethanol in the exhaled breath following pretest breathing maneuvers. Ann Biomed Eng 1995; 23:48–60. 68. Jones AW. How breathing technique can influence the results of breath-alcohol analysis. Med Sci Law 1982; 22:275–280. 69. Berry JL, Brailsford JF, Daly IB. The bronchial vascular system in the dog. Proc R Soc Lond Ser B 1931; 109:214–228. 70. Cudkowicz L, Calabresi M, Nims RG, Gray FD, Jr. The simultaneous estimation of right and left ventricular outputs applied to a study of bronchial circulation in dogs. Am Heart J 1959; 58:732–742. 71. Cudkowicz L, Calabresi M, Nims RG, Gray FD, Jr. The simultaneous estimation of right and left ventricular outputs applied to a study of bronchial circulation in patients with chronic lung disease. Am Heart J 1959; 58:743–749. 72. Baile EM, Ling H, Heyworth J, Hogg JC, Pare´ PD. Bronchopulmonary anastomotic and noncoronary collateral blood flow in humans during cardiopulmonary bypass. Chest 1985; 87:749–754. 73. Agostoni PG, Arena V, Biglioli P, Doria E, Sala A, Susini G. Increase in alveolar pressure reduces systemic-to-pulmonary bronchial blood flow in humans. Chest 1989; 96:1081–1085. 74. Horisberger B, Rodbard S. Direct measurement of bronchial arterial flow. Circulation 1960; 8:1149–1156. 75. Elsasser S, Long WM, Baier HJ, Chediak AD, Wanner A. Independent control of mucosal and total airway blood flow during hypoxemia. J Appl Physiol 1991; 71:223–228. 76. Magno MG, Fishman AP. Origin, distribution, and blood flow of bronchial circulation in anesthetized sheep. J Appl Physiol 1982; 53:272–279. 77. Wells UM, Hanafi Z, Widdicombe JG. Osmolality alters tracheal blood flow and tracer uptake in anesthetized sheep. J Appl Physiol 1994; 77:2400–2407. 78. Long WM, Yerger LD, Martinez H, et al. Modification of bronchial blood flow during allergic airway responses. J Appl Physiol 1988; 65:272–282. 79. Long WM, Sprung CL, El Fawal H, et al. Effects of histamine on bronchial artery blood flow and bronchomotor tone. J Appl Physiol 1985; 59:254–261. 80. Gleed RD, Dobson A, Hackett RP. Pulmonary shunting by the bronchial artery in the anesthetized horse. Exp Physiol 1990; 75:115–118. 81. Norgaard MA, Hove JD, Efsen F, Saunamaki K, Hesse B, Pettersson G. Human bronchial artery blood flow after lung Tx with direct bronchial artery revascularization. J Appl Physiol 1999; 87:1234–1239. 82. Parsons GH, Link DP, Greenberg D, Lantz BM, Cross CE. Bronchial blood flow measured by video dilution technique in sheep. Invest Radiol 1987; 22:544–549.

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83. Kowalski TF, Guidotti S, Deffebach M, Kubilis P, Bishop M. Bronchial circulation in pulmonary artery occlusion and reperfusion. J Appl Physiol 1990; 68:125–129. 84. Baile EM, Nelems JMB, Schulzer M, Pare´ PD. Measurement of regional bronchial arterial blood flow and bronchovascular resistance in dogs. J Appl Physiol 1982; 53:1044–1049. 85. Prinzen FW, Bassingthwaighte JB. Blood flow distribution by microsphere deposition methods. Cardiovasc Res 2000; 45:13–21. 86. Yokomise H, Wada H, Inui K, et al. Application of laser Doppler velocimetry to lung transplantation. Transplantation 1989; 48:550–554. 87. Takao M, Katayama Y, Onoda K, et al. Significance of bronchial mucosal blood flow for the monitoring of acute rejection in lung transplantation. J Heart Lung Transplant 1991; 10:956–967. 88. Takao M, Katayama Y, Onoda K, et al. Significance of measurements of bronchial mucosal blood flow for the monitoring of acute rejection of transplanted lungs. Transplantation 1990; 50:345–348. 89. Corfield DR, Deffebach ME, Erjefa¨lt I, Salonen RO, Webber SE, Widdicombe JG. Laser-Doppler measurement of tracheal mucosal blood flow: comparison with tracheal arterial flow. J Appl Physiol 1991; 70:274–281. 90. Lin VW, Kramer GC, Parsons GH, Cross CE. Laser Doppler velocimetry of tracheal blood flow in sheep. Resp Physiol 1991; 85:341–354. 91. Godden DJ, Wagner EM, Pare´ PD, Mitzner W, Baile EM. Measurement of airway wall blood flow in sheep by laser-Doppler flowmetry: interpretation and problems. J Appl Physiol 1991; 70:641–649. 92. Baile EM, Godden DJ, Pare´ PD. Non-invasive, real-time measurement of tracheal blood flow in humans. Clin Invest Med 1988; 11:C110. 93. Agostoni P, Godden DJ, Baile EM. Measurement of bronchial blood flow in humans. In: Butler J, ed. The Bronchial Circulation. New York: Marcel Deckker, 1992:181–196. 94. Tanabe H, Takao M, Hiraiwa T, et al. New diagnostic method for pulmonary allograft rejection by measurement of bronchial mucosal blood flow. J Heart Lung Transplant 1991; 10:968–974. 95. Wada H, Hirata T, Inui K, et al. Laser Doppler velocimetry measurement of canine mucosal circulation response to bronchial artery reperfusion after lung transplantation. Thorac cardiovasc Surgeon 1992; 40:182–184. 96. Takao M, Katayama Y, Tanabe H, et al. Histologic changes in donor bronchi may explain the reduced mucosal blood flow seen during acute lung allograft rejection. J Heart Lung Transplant 1992; 11:994–1000. 97. Baile EM, Godden DJ, Pare´ PD. Effect of cold dry and warm humid air hyperventilation on tracheal wall blood flow in humans. Am Rev Respir Dis 1989; 139:A63. 98. Paredi P, Kharitonov SA, Barnes PJ. Faster rise of exhaled breath temperature in asthma: A novel marker of airway inflammation. Am J Respir Crit Care 2002; 165:181–184. 99. Paredi P, Caramori G, Cramer D, et al. Slower rise of exhaled breath temperature in chronic obstructive pulmonary disease. Eur Respir J 2003; 22:393–394. 100. Cander L, Forster RE. Determination of pulmonary parenchymal tissue volume and pulmonary capillary blood flow in man. J Appl Physiol 1959; 14:541–551.

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3 Molecular Mechanisms of Angiogenesis

EDWARD M. CONWAY, SASKIA APPELMANS, NELE KINDT, and PETER CARMELIET The Center for Transgene Technology and Gene Therapy, University of Leuven, and Flanders Interuniversity Institute for Biotechnology (VIB), Leuven, Belgium

I. Introduction Blood vessels provide essential nutrients to almost every organ system. It follows that abnormal blood vessel growth is implicated in many clinically relevant disease processes. Excess vessel growth is a feature of cancer, arthritis, psoriasis, diabetic retinopathy, infections, obesity, allergy, atherosclerosis, nasal polyps, and endometriosis, to name but a few. Inadequate vessel growth or excess vessel regression is most frequently linked to tissue ischemia, such as occurs during the evolution of a myocardial infarct, stroke, or limb ischemia. However, abnormal vessel regression also contributes to many other disorders including, for example, pre-eclampsia, age-dependent skin changes, nephropathies, bone loss, diabetes, macular degeneration, and such neurodegenerative disorders as amyotrophic lateral sclerosis. The lung provides a large diffusible interface with the circulation that is necessary for adequate gas exchange. It is not surprising, therefore, that defects in angiogenesis might impact on the development and progression of lung disease. It has long been recognized that patients suffering from severe asthma have airway mucosa that is heavily infiltrated with dilated, tortuous, and congested blood vessels (1–3). Even mild asthma is linked to increased vascularity of the bronchial mucosa, (2) and the extent appears to be correlated with enhanced lung 45

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airway expression of the potent angiogenic factor, vascular endothelial cell growth factor (VEGF) (4,5). In chronic obstructive lung disease with airflow limitation and alveolar destruction, levels of VEGF in sputum are decreased, (6) and excess pulmonary capillary regression is observed. Conversely in bronchitis, VEGF levels are elevated, likely contributing to increased airway vessel permeability, allowing plasma proteins to leak into the extravascular space. In pulmonary fibrosis, excess angiogenesis in the peribronchial regions of the lung may be caused by a deficiency of the angiostatic CXC chemokine, interferongamma-inducible protein-10 (IP-10) (7). These and other findings by many investigators provide strong evidence that molecular links exist between angiogenesis and the pathogenesis and progression of chronic lung diseases. Yet the relevant molecular pathways that orchestrate angiogenesis in the setting of pulmonary organogenesis and lung disease are only beginning to be elucidated. This knowledge will however, result in the development of novel strategies for the prevention and management of chronic lung diseases. This review provides an update on the basic molecular mechanisms governing how endothelial cells, smooth muscle cells, matrix molecules, and several critical receptors and their ligands interact to form blood vessels. Throughout the chapter, we provide some examples as to how these concepts impact on the pathogenesis and progression of asthma and chronic obstructive lung disease. It will become evident that our understanding of how vessels grow and regress is dynamic and rapidly evolving. Thus, this work should be viewed as only a step toward the goals of developing novel and effective therapies to treat and/or prevent disorders, such as asthma or emphysema, that are associated with defects in angiogenesis.

II.

Vessel Growth—In the Embryo and Adult

How do vessels grow, and what are the critical regulatory factors? In the earliest stages of embryonic development, a vascular system is not required, as the embryo receives its energy needs by diffusion (8). However, with rapid growth and increasing nutritional demands, the embryo transforms into a highly vascular organism, requiring a complex network of capillary plexuses, small blood vessels consisting only of endothelial cells, and larger ones enveloped by one or more layers of mural cells (pericytes or smooth muscle cells). Vessel growth may be most easily viewed as being comprised of three distinct molecular mechanisms (Figs. 1 and 2). Vasculogenesis refers to the initial events in embryonic vascular growth in which endothelial progenitor cells (angioblasts) provide the nidus for vessel growth in discrete locations, proliferating, differentiating, and assembling into endothelial cords and later into a plexus with endocardial tubes. Notably, it has more recently been recognized that this mechanism is not restricted to embryonic development but

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(A) SMC

SMC recruitment

capillary growth (angiogenesis)

(B)

(1) homing (2) capillary plexus

bone marrow

(3) mature network

(C) occlusion

shear stress Mφ cytokines matrix remodeling SMC growth

Figure 1 Mechanisms of vessel growth in the adult: (A) Angiogenesis refers to the growth, expansion, sprouting, and remodeling of new endothelial cell–lined vessels from preexisting vessels into a mature vascular network, whereas arteriogenesis describes the stabilization of vessels with mural cells (smooth muscle cells, SMC) (B) Vasculogenesis in the adult denotes the recruitment of endothelial progenitor cells from the bone marrow to distant sites in the body for de novo blood vessel formation or to stimulate new vessel growth via the release of angiogenic factors. (C) Collateral vessel growth refers to the formation, expansion, and remodeling of bridging vessels between existing arterial networks, particularly in response to an occlusive thrombus, and is dependent on shearstress induced recruitment of monocytes. Source: From Ref. 9.

may occur in adults, as endothelial progenitors may be recruited from the bone marrow to distant sites in the body for de novo blood vessel formation and/or to stimulate new vessel growth through the release of angiogenic factors. Subsequent growth, expansion, sprouting, and remodeling of primitive vessels into a mature vascular network is referred to as angiogenesis, whereas the term arteriogenesis is restricted to describe stabilization of the vessels with mural cells. Finally, collateral growth refers to the formation, expansion, and remodeling of

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Others ? Vascular Progenitor Cells

Endothelial Progenitors EMBRYO

Smooth Muscle Progenitors

PDGF-BB

VEGF

Pre-Arterial Angioblast VEGF Epicardial-derived Cells

Angioblast

Arterial Endothelial Cells Pre-Venous VEGF Angioblast

Hemangioblasts

Transdifferentation

Lymphatic Endothelium VEGF

ADULT

PDGF-BB

Circulating Endothelial Progenitor

Neural Crest Cells TGF-β1

PDGF-BB

? ?

Pericytes Smooth Muscle Cells

Venous Endothelial Cells

Hematopoietic cells Skeletal muscle LymphaticStem Cell

VEGF

VEGF

PDGFR-β+ progenitor

PDGF-BB

Common Vascular Progenitor

Mesenchymal Stem Cell

PDGF-BB

SMC Progenitor

Figure 2 Vascular progenitors in the embryo and adult. Within the embryo and in extraembryonic sites, blood vessels form from embryonic precursor cells. Endothelial, smooth muscle, and common vascular progenitors, each contribute to development of the vasculature. In the adult, angiogenic progenitor cells may be recruited from the circulation, the bone marrow, and other tissues. Abbreviations: PDGF-BB, platelet derived growth factor-BB; TGF-b1; transforming growth factor-b1; VEGF, vascular endothelial cell growth factor. Source: From Ref. 10.

bridging vessels between existing arterial networks, particularly in response to an occlusive event, such as a thrombus. III.

Vascular Endothelial Cell Growth Factor

Considering the enormous impact that VEGF has had in angiogenesis research, it is remarkable to consider that it is only in the last decade that VEGF has assumed the position of the key player in vessel growth. Indeed, the importance of this molecule justifies special attention. Although VEGF is a pleiotropic growth factor with multiple biologic functions in several organ systems, (11,12) it is best characterized in relation to its role in angiogenesis, its effects on vascular endothelial cells, and periendothelial cells, and its interactions with other angiogenesis-related factors (13–19). Originally described as a vascular permeability factor, (20) the critical role of VEGF in angiogenesis was first best illustrated in gene inactivation studies mice several years later. Lack of even one

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VEGF allele results in embryonic death in mice due to profound vascular developmental defects, with delayed differentiation of endothelial cells, impaired lumen formation, and inhibition of sprouting and branching of new vessels (21,22). Conversely, moderately elevated levels of VEGF also result in dramatic disturbances in angiogenesis (23), indicating the necessity for tight regulation of expression under a variety of pathophysiologic conditions. The human VEGF gene is located on chromosome 6p21.3, spans approximately 14 kb, and is organized into 8 exons. Alternative pre-message RNA (mRNA) splicing gives rise to six different gene products, resulting in VEGF isoforms of 121, 145, 165, 183, 189, and 206 amino acids (denoted VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206 respectively), VEGF165 being the most abundant and VEGF206 the least (24–27). The murine and rat VEGF genes are similar in size and structure to that of the human (28), but the isoforms are shorter by 1 amino acid. All VEGF isoforms are secreted proteins, but differences in heparin binding affinity of each influence the bioavailability, i.e., most of VEGF165 remains bound to the extracellular matrix, released by proteolytic enzymes, such as heparanase, whereas VEGF121 fails to bind to heparin and is freely diffusible. VEGF188 and VEGF206 are more basic and are almost completely sequestered in the extracellular matrix but also may be released in a soluble form by heparin or heparinase. These binding characteristics provide a means by which VEGF may act locally or at a distance. Beyond differences in patterns of expression, VEGF isoforms also have distinct biological properties that are relevant in delineating their role in health and disease, as well as for the development of therapies. Expression of VEGF is tightly regulated, predominantly at the level of transcription. Hypoxia is the major means by which VEGF transcription is enhanced, and it achieves this by facilitating the interaction of hypoxia-inducible transcription factors (HIF-1, HIF-2) with a hypoxia responsive DNA element (HRE) that resides in the promoter region of the VEGF gene (29,30). VEGF mRNA stability is also tightly regulated by hypoxia. The messenger RNA (mRNA) contains an adenylate-uridylate-rich element (ARE) in the 3 0 untranslated region, to which regulatory proteins may bind, thereby altering its susceptibility to degradation (31,32). Several growth factors, genes, and cytokines can also up-regulate VEGF mRNA accumulation. These include, for example, platelet derived growth factor (PDGF), tumor necrosis factor (TNF)-a, transforming growth factors (TGFa, TGFb), fibroblast growth factors (FGF-4), keratinocyte growth factor (KGF), nitric oxice (NO), insulin growth factor (IGF), and interleukins (IL1a, IL1b and IL6), c-Src, v-Raf, and Ras. Others, such as IL10, and IL-13, may inhibit VEGF release (25,31,33–36). The biological functions of VEGF are mediated via interactions with several specific tyrosine kinase receptors (Fig. 3), the most important ones being VEGF receptor-1 (VEGFR-1, or fms-like tyrosine kinase, Flt-1) and VEGFR-2 (fetal liver kinase, Flk-1 in mice; kinase insert domain containing receptor, KDR in humans) (25). Considerable evidence supports the concept that most

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VEGF-A PIGF VEGF-B

VEGF-A VEGF-C VEGF-D VEGF-E

VEGF165 Sema

VEGF165 VEGF145 Sema

NP-1

NP-2

VEGF-C VEGF-D

SS

sFlt1

Flt1 (VEGFR-1)

Flt1/KDR (VEGFR-2)

Flt4 (VEGFR-3)

Figure 3 VEGF and VEGF receptors. Three tyrosine kinase receptors of the VEGF family (VEGFR-1, VEGFR-2, and VEGFR-3), soluble VEGFR-1 (sFlt1), and the receptors neuropilin-1 (NP-1) and neuropilin-2 (NP-2) (not discussed further in the context of this chapter) are schematically shown. VEGF, its isoforms, and homologs, and the semaphorins (Sema) that bind to these receptors are also shown. As discussed in the text, the biological effects of the different VEGF isoforms and homologs are mediated through binding to the specific receptors, which in turn induce intracellular signaling. Thus, most angiogenic signals of VEGF-A are transmitted via VEGFR-2. PlGF and VEGFR-1 critically regulate pathologic angiogenesis in the adult. VEGF-C, VEGF-D, and VEGFR-3 play central roles in inducing lymphangiogenesis.

angiogenic signals by VEGF are transmitted via VEGFR-2. These include, for example, chemotactic, mitogenic, and prosurvival effects on vascular endothelial cells, and genetic studies in mice confirm the critical roles for both VEGF and VEGFR-2 in vascular development (21,22,37,38). Defining the role of VEGFR-1 was more problematic and still presents controversy. Inactivation of the VEGFR-1 gene in mice results in embryonic lethality with defects in vascular channel formation, (39) whereas mice lacking the intracellular signaling motif of Flt-1 have no vascular abnormalities (40), suggesting that this receptor might act as a “sink” for VEGF, thereby regulating the growth factor’s interaction with the more important VEGFR-2, particularly during development. With the identification of the angiogenic placental growth factor (PlGF), (41) much of the mystery of the role of VEGFR-1 has been solved. PlGF only binds to VEGFR-1. Although inactivation of the PlGF gene in mice did not affect physiologic angiogenesis (e.g., during embryonic development, placenta growth, pregnancy), PlGF, and VEGFR-1 were determined to critically regulate pathologic angiogenesis in the adult. Thus, the absence of PlGF results in, for example, subnormal angiogenic responses to myocardial or limb ischemia,

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retinal ischemia, and tumor growth. PlGF enhances the angiogenic properties of VEGF via several pathways. PlGF not only activates a unique program of angiogenic genes and directly upregulates expression of VEGF, but it also signals via VEGFR-1 and thereby induces intermolecular cross talk between VEGFR-1 and VEGFR-2, which enhances the response of VEGFR-2 to VEGF. Furthermore, PlGF stimulates angiogenesis by heterodimerizing with VEGF and inducing intramolecular phosphorylation reactions within VEGFR-1/VEGFR-2 dimers (42). Finally, due to the wide cell expression profile of VEGFR-1, PlGF recruits smooth muscle cells, inflammatory cells and hematopoietic precursor cells, which in turn contribute to pathologic angiogenesis (43–46). Overall, these unique, yet overlapping ligand-receptor systems are coordinated to respond to specific angiogenic requirements. VEGF/VEGFR-2 is crucially important for physiologic and pathologic angiogenesis, whereas PlGF/VEGFR-1 is more specifically designed to respond under pathologic conditions. Subsequent sections in this review will highlight the role of these and other angiogenic factors in different stages of vascular development and in response to a variety of stresses, paying particular attention to their potential impact in chronic lung disease.

IV.

Vasculogenesis

A. Angiogenic Progenitors in the Embryo

Within the embryo and in extraembryonic sites, blood vessels begin to form from embryonic precursor cells. Outside of the embryo—in the yolk sac— mesenchymal cells form aggregates, or so-called blood islands, within the mesoderm. Strong evidence supports the notion that the mesenchymal cell serves as a common precursor—the hemangioblast—for both the endothelial cell and the hematopoietic cell, although the molecular identity of the hemangioblast still remains elusive (Fig. 2) (47–49). In yolk sac blood islands, the hemangioblasts at the perimeter, differentiate into endothelial cells, while those at the center differentiate into hematopoietic precursors. Accumulating data indicate that vasculogenesis and hematopoiesis are closely linked functionally and via a common precursor. Several markers are common to both endothelial and hematopoietic precursors, including CD31, CD34 and VEGFR-2, deficiency of which results in embryonic hemangioblast differentiation arrest (37,38). Interestingly, although the potent VEGF promotes hemangioblast differentiation into endothelium, VEGF is not absolutely required (21,22), suggesting that there are other VEGFR-2 ligands or factors dependent on VEGFR-2 for endothelial differentiation (e.g., VEGF-C, VEGF-D). In that respect, the basic helix-loophelix transcription factor, Tal-1, in concert with VEGFR-2, appears to play an important role in regulating hemangioblast formation and differentiation into endothelial cells versus smooth muscle cells (50–52). The serine-threonine nuclear kinase, Pim1, similarly is critical for differentiation of VEGFR-2 positive precursors to endothelial and mural cells (53). But several additional genes and/or

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their protein products have been further implicated in modulating these early steps, including Ets-1, Hex, Vezf, Hox, Id-1-3, VEGFR-1,Tie-1, Tie-2, VE-cadherin, bFGF, cloche, and members of the GATA family, (49,50,54–63) underlining both the fine regulation and the complexity of the process. In the embryo, endothelial cell precursors migrate to discrete locations. The cues that direct angioblasts to migrate to the “right” location are not yet identified, although growth factors, such as VEGF, and granulocyte/monocytecolony stimulating factor (GM-CSF) play a role (64–66). However, it is well-recognized that angioblasts may migrate intraembryonically, circulate postnatally, and may be recruited for in situ vessel growth (67–70), where they differentiate and assemble into endothelial cords, subsequently forming a plexus with endocardial tubes, from which the dorsal aortae, the cardinal veins and embryonic portions of the yolk sac arteries and veins eventually arise. The capillary network in the lung of the mouse begins to form around E10 when lung mesenchymal cells undergo vasculogenesis, eventually making connections with the major pulmonary vessels (71–73). This is supported by studies using an ex vivo model of lung development, in which embryonic vessels developed from endothelial progenitor cells endogenous to the lung explants (74). Both vasculogenesis and hematopoiesis in the lung appear to be dependent, at least in part, on VEGF secreted by pulmonary epithelial cells, signaling via VEGFR-2 (37,75,76). However, mesenchymal-epithelial signaling, mediated by fibroblast growth factor (FGF), epidermal growth factor (EGF), PDGF, TGFb, and laminin, also contribute to vasculogenesis in the lung (77,78). B. Angiogenic Progenitors in the Adult

Recent evidence indicates that vasculogenesis may occur in adults (Fig. 2) (70). Angiogenic progenitor cells may be recruited from the circulation, the bone marrow, and other tissues, and these may cooperate in promoting new vessel growth during normal development and in response to injury (67,68,79). Endothelial progenitor cells (EPC) are highly proliferative, and express CD34, VE-cadherin, VEGFR-2 and AC133 (44,80). The origin of these cells is not fully understood, and in fact, they may be the products of differentiation of different cells, including, for example, mesoangioblasts (81) or multipotent adult progenitor cells (67,82). EPCs are mobilized predominantly from the bone marrow and migrate to sites of injury under the guidance of several factors, including VEGF, FGF-2, angiopoietin, stromal cell-derived factor-1, and IGF, whereupon they may release additional growth factors (e.g., VEGF, hepatocyte growth factor, GM-CSF), which in turn recruit more EPCs (83–86). Although the differentiation pathways of EPCs have not been fully elucidated, EPCs do acquire markers specific to the target tissue, thus accommodating to local environmental needs. Thus, EPCs contribute to vessel growth both by locally secreting pro-angiogenic factors and also by proliferating and maturing into endothelial cells that are incorporated into the nascent vessels.

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The existence of an endogenous source of angiogenic progenitors has naturally generated excitement in terms of therapeutic potential. Preclinical trials in animal models to enhance tissue revascularization of ischemic limbs, hearts, and retina with angiogenic progenitor cells have shown promise, with the contribution of EPCs to vessel growth varying from less than 1% to as high as 50% of new endothelial cells (87,88). The observation that embryo derived endothelial progenitor cells (eEPC) are less immunogenic—and when injected intravenously, home to hypoxic tumors—have prompted studies in which eEPCs are manipulated to carry suicide genes and are then infused in animals to target lung cancer metastases (89). There is controversy as to the role of progenitor cells in response to lung injury. After instillation of endotoxin into the lung airways of mice, bone marrow–derived progenitor cells were rapidly mobilized and differentiated into epithelial and endothelial cells (90). In contrast, in a model of lung growth and alveolization, bone marrow–derived progenitor cells did not significantly contribute to vascular growth or remodeling (91). These apparently conflicting results may be attributed to substantially different models and technical approaches, and thus further investigations are warranted. Overall, characterization of the molecules that regulate recruitment, differentiation, specialization, and incorporation of EPCs, will enhance the therapeutic utility of this novel technology in a wide array of disease processes. V. Angiogenesis Historically, the term angiogenesis was used solely to describe the growth of endothelial sprouts from preexisting postcapillary venules, i.e., sprouting angiogenesis. More recently, however, the term has been expanded to denote the growth and remodeling process of the primitive vascular system into a complex network. This necessarily also involves nonsprouting angiogenesis or intussusception. In sprouting angiogenesis, proteolytic degradation of the extracellular matrix (ECM) is followed by chemotactic migration and proliferation of endothelial cells, formation of a lumen and functional maturation of the endothelium. In nonsprouting angiogenesis, preexisting vessels are split longitudinally by the formation of transcapillary pillars of periendothelial cells or by transendothelial cell bridges, resulting in individual capillaries, a common feature of angiogenesis in the lung (92). A. Early Steps to Accommodate Endothelial Cell Proliferation and Migration

Angiogenesis is initiated by vasodilation, mediated in large part by NO, and accompanied by VEGF-mediated increases in vascular permeability, the latter by redistribution of intercellular adhesion molecules, such as PECAM-1, and VE-cadherin, and by alterations in cell membrane structure (93–96). Whereas VEGF is most prominent in promoting vascular permeability (97), angiopoeitin1, the ligand for the receptor Tie2, inhibits permeability, thereby providing a

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natural balance (98). Matrix metalloproteinases (MMPs) degrade ECM, facilitating the release and/or activation of additional growth factors (basic FGF, VEGF, IGF-1, TGFb, TNFa) that otherwise remain functionally sequestered in the ECM (18,99,100). The released angiogenic factors may then further modify the composition of the ECM. For example, VEGF directly stimulates secretion of fibronectin from airway smooth muscle cells via VEGFR-1 signaling pathways (101). Degradation of ECM by MMPs furthermore allows extravasation of plasma proteins that lay down a provisional scaffold for migrating endothelial cells. More than 20 MMPs have been implicated in playing direct or indirect roles in angiogenesis and cell proliferation (101,103). For example, MMPs 2, 3, and 9 facilitate angiopoietin-1 mediated vascular sprouting. MMPs 3, 7, and 9 promote angiogenesis in neonatal bones (104). MMPs 7 and 9 inhibit endothelial cell proliferation through the generation of angiostatin from plasminogen (102). Other proteinases, such as urokinase-type plasminogen activator (u-PA), and its natural inhibitor, plasminogen activator inhibitor (PAI)-1, also contribute to matrix degradation and have been shown to be critical for revascularization after myocardial infarcts (105). Not surprisingly, numerous natural MMP inhibitors (tissue-type inhibitors of MMPs, or TIMPs) exist to regulate their activity (99). Thrombospondin (TSP)-1 may also interfere with activation of MMPs 2 and 9, thus suppressing angiogenesis (106). The central role of the ECM in angiogenesis should not be overlooked: either inadequate or excessive breakdown of the ECM during angiogenesis will disrupt the support structure and upset the normal release of those guidance cues that are necessary for endothelial cell migration. This important concept is exemplified in mouse genetic studies by loss of PAI-1, which results in diminished tumor angiogenesis, and conversely in u-PA deficient mice by reduced angiogenesis post myocardial ischemia (105,107). It should also be emphasized that proteinases are not solely designed to enhance angiogenesis. By inactivating pro-angiogenic cytokines (e.g., stroma derived factor (SDF)-1), or releasing ECM-bound inhibitors (e.g., TSP-1, angiostatin, anti-thrombin, platelet factor 4) (108), proteinases may also interfere with endothelial cell growth and migration. Only through the coordinated and highly localized actions of these proteinases, inhibitors, growth factors and their respective receptors, interendothelial cell junctions and periendothelial cell support are relieved in the existing vessel to allow it to become destabilized, so that proliferating endothelial cells may migrate to distant sites. The sheer complexity of the multiple interactions inherent in the system underlines both the importance of tight regulation and redundancy and furthermore highlights the challenge in designing effective angiogenic and antiangiogenic therapies. B. Endothelial Cell Proliferation, Migration, and Vessel Sprouting

In these early steps of angiogenesis, the ECM has thus been tailored to facilitate vessel sprouting. The composition of the matrix has changed, with relative

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increases in fibronectin, fibrin, and fibrillar versus monomeric collagen, all of which provide a scaffold for endothelial cell guidance to their targets. Endothelial cell proliferation and migration are regulated by the interplay of many growth factors and their receptors, with considerable redundancy. VEGF has profound effects, modulated by angiopoietins, FGFs, and their receptors. VEGF homologues have their own unique effects that are dependent on site, developmental stage, and pathophysiologic situation. For example, VEGF-B is implicated in the regulation of coronary artery function. VEGF-C and VEGF-D are important for lymphangiogenesis, as is the receptor tyrosine kinase, VEGFR-3 (15,109,110). As noted previously, PlGF is redundant during embryonic vascular development but is essential in adults for the angiogenic response to pathologic stimuli (111,112). Regulated spatio-temporal expression of soluble and matrix bound forms of angiogenic factors facilitates local and distant angiogenic effects. For example, after vasculogenesis in the developing lung has been established, matrix-associated forms of VEGF (VEGF164) become restricted to the leading edge of branching airways from E13.5 to E15.5 in the mouse, accompanied by increased expression of VEGFR-1 and VEGFR-2 near budding components of distal airways. Thus, neovascularization and vascular sprouting, coordinated with airway branching, is promoted at that site (76), in concert with VEGF-mediated alterations in endothelial cell phenotype to increase expression of anticoagulant activity to prevent thrombosis and to ultimately attain properties of the mature alveolar capillary (113–115). Numerous other molecules facilitate sprouting. Time-lapse confocal microscopy studies show that VEGFR-1 is required for sprouting of vessels from the dorsal aorta during development (116). Through phosphorylation of Tie2, angiopoieitin-1 is chemotactic for endothelial cells, inducing sprouting and stimulating interactions with periendothelial cells. Angiopoieitin-2 has a complex role. In concert with VEGF, angiopoieitin-2 is angiogenic, but in the absence of VEGF, angiopoieitin-2 induces vessel regression (98,117–119). FGFs contribute to sprouting by recruiting mesenchymal and/or inflammatory cells, which in turn provide a steady source of angiogenic factors (62). FGFs themselves stimulate endothelial cell growth. PDGF promotes endothelial cell sprouting and recruits mural cells for vascular support (120). The cytokine TNFa may either stimulate or inhibit endothelial cell growth in various models (121). Monocyte chemotactic protein (MCP)-1, induced by VEGF, also promotes endothelial cell growth and migration (122). Other molecules recognized to play a role include integrins, PECAM-1, VE-cadherin, Eph/ephrin receptor-ligand pairs, IGF-1, nitric oxide, hepatocyte growth factor, and several interleukins (123–127). In the lung, signaling via the morphogen, sonic hedgehog (Shh), and modulated by Gli proteins is critical, not only for vasculogenesis in the lung and differentiation of mesenchymal cells into endothelium, but also for normal migration and coalescence of endothelial cells and vascular sprouting to form a capillary network (128). The more downstream target gene, Foxf1 (128), may further contribute by regulating epithelium-mesencyme interactions, thereby promoting bronchial smooth muscle,

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and endothelial cell proliferation, and migration. However, genetic studies in mice indicate that VEGF and/or TGFb1 are likely also involved (75,129,130). Many of these molecules are being explored as targets for the development of angiogenic and antiangiogenic therapies. C. Vascular Tube Formation

Within the ECM, the migrating endothelial cells first assemble as solid cords. Through intercalation and thinning of the cells, and fusion with preexisting vessels, these cords acquire a lumen that then undergoes further changes, increasing both in diameter and length. These events are tightly regulated by several factors but particularly by isoforms of VEGF. Whereas VEGF189 decreases lumen diameter, VEGF121, and VEGF165 act to increase all lumen dimensions. Other factors also contribute. For example, NO, which upregulates VEGF and is itself upregulated by VEGF, promotes lumen formation and increases lumen diameter (131). Angiopoieitin in combination with VEGF also increases lumen diameter, as do many of the integrins (132,133). Several other molecules affect lumen formation, including the transcription factor, myocyte enhancer binding factor 2C (MEF2C), and TSP-1, an endogenous inhibitor of lumen formation, and the recently identified EGF-like domain 7 (Egfl7), which facilitates vascular tubulogenesis by ensuring the proper spatial arrangement of angioblasts as they assemble into tubes (134). Coronary vessel tube formation during development coincides with lamin deposition and is closely followed by the appearance of collagen IV (135). Interestingly, bronchial airway mucosa angiogenesis, which is commonly associated with asthma, is characterized by vessels supported by a basement membrane expressing high levels of collagen IV (136). Particularly relevant to chronic inflammatory diseases of the lung, interactions between inflammatory leukocytes and endothelial cells cause the release of CXC chemokines (e.g., interleukin-8, MIP-2, growth-related gene product (GRO), monocyte induced by IFN-g, and IP-10) and CC chemokines [e.g., MCP-1, regulated on activation, normal T expressed and secreted (RANTES)], and these may regulate angiogenesis at several steps, including at the vascular tube formation stage (137). Indeed, the angiostatic function of IP-10, which counteracts the pro-angiogenic properties of IL-8, inhibits pulmonary fibrosis in a mouse model (7), underlining the clinical utility of delineating these complex pathways. D. Assembling the Vascular Network—The Role of Oxygen

With assembly of the endothelial cell cords into recognizable tubular structures, a dynamic process follows in which vessel tracts and intervessel connections appear and disappear until the target pattern of vessels has been established. The molecular mechanisms regulating patterning are being actively investigated. Best characterized is the role of oxygen. Regulation of oxygen homeostasis is a fundamental cellular mechanism that is widely conserved and that plays an

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important role in development and physiology (138). With lack of oxygen, vessel sprouting is induced, in part via activation of hypoxia-inducible factors. As briefly mentioned before, these are heterodimeric transcription factors, each containing an a- and a b subunit, that, under hypoxic conditions, are stabilized, binding to a consensus HRE in the promoter of target genes (e.g., VEGF) and leading to transcriptional upregulation and/or increased mRNA stability (139). Coactivators, such as p300/CBP, further optimize these protein–DNA interactions (140). During normoxia, HIFa is targeted for proteasomal degradation by the product of the gene that is responsible for von Hippel Lindau disease (VHL). Hydroxylation of a proline residue on hypoxia inducible transcription factor (HIF) is a requirement for VHL mediated degradation, and strong evidence supports the concept that the complex formation of prolyl hydroxylase, HIF, oxygen, and iron acts as a cellular oxygen sensor (141,142). Numerous genes involved in angiogenesis are upregulated via HIFs, including VEGF, the VEGF receptors VEGFR-1, VEGFR-2, neuropilin-1, neuropilin-2, angiopoieitin-2, Tie-2, nitric oxide synthase, transforming growth factor-b1 (TGFb1), MMP-2, MMP-3, uPAR, platelet derived growth factor-BB (PDGF-BB), endothelin-1, interleukin-6, interleukin-8, tissue factor, cathepsin D, fibronectin, and a-integrin (139,143). It is not surprising, therefore, that hypoxia, via activation of HIFs, affects not only vascular sprouting but also many facets of angiogenesis, including endothelial cell growth, migration, mural cell recruitment, vasoregulation, leukocyte attraction, ECM stability, etc. Dysregulation of HIF-dependent angiogenesis results in defects in organ growth due to altered and functionally incompetent vessel branching (144), whereas interfering with degradation of HIF enhances in vivo angiogenesis (145). Beyond its important role in angiogenesis, HIFs also are crucial for normal lung function. Interesting insights have been provided from evaluating genetically modified mice. Although embryonic lung development in HIF-2a knock out mice appeared normal, the lungs were partially or completely collapsed at birth, due to impaired thinning of the alveolar septae (146,147). Strong evidence suggests that this was caused by diminished levels of VEGF, resulting in inadequate differentiation of immature alveolar epithelium into surfactant producing type I and type II pneumocytes. These results define a novel role for HIF-2a induced VEGF expression in lung maturation and furthermore highlight the pleiotropic effects of VEGF and the intricate links between angiogenesis, lung development and pulmonary function (147). It also provides rationale for considering administering so-called “angiogenic” agents, such as VEGF, to treat specific developmental disorders involving the lung, including bronchopulmonary dysplasia or cystic fibrosis. E.

Assembling the Vascular Network—Guidance and Patterning

In concert with oxygen gradient-induced alterations in organization of the vasculature, vessel branching and network formation is also modulated by

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hemodynamic forces, which may induce capillary expansion or vessel sprouting and may alter branch angles and vessel morphology by shear stress release of angiogenic and antiangiogenic factors (148). Indeed, changes in flow can have dramatic effects, i.e., lack of flow results in regression of sprouts. These shear induced changes are effected by upregulation of transcription factors (e.g., c-Fos, Egr-1), enzymes (e.g., nitric oxide synthase and angiotensin converting enzyme), growth factors (e.g., PDGF-BB and TGF), and finally several molecules involved in signaling (e.g., integrins). Guidance and patterning of growing vessels requires extraordinarily tight regulation, and the responsible molecular pathways involved are only starting to be identified (149). In zebrafish and Xenopus, intersomitic vessels (between somites of the developing spinal column) are patterned in a precise manner, a process that is regulated in part by hypoxia but also by somite expression of repulsive signals (ephrinB2/B3) that guide vessels that express EphB3/4. Defects in the Eph/ephrin system result in deviations from the normal vessel pathways. Localization of VEGF isoforms, which may exist in a soluble form (VEGF120) or remain bound to the ECM (VEGF188), provides a gradient effect over different ranges, thereby facilitating regulated and directionally precise endothelial cell growth from the leading tips of growing vessels (150–152). Other vessel guidance and branching signals identified by genetic studies in small animal models include neuropilin-1, and out-of-bounce, FGF, renin, Sprouty, and Sema3A (153–156). Several of these are particularly interesting, as they highlight the similarities between branching in other organ systems and during angiogenesis. For example, reminiscent of what occurs during the growth of axons, specialized endothelial cells, referred to as tip cells, have been identified at the extremity of growing blood vessels, and these cells may act as sensors and motility devices, extending filopodia to assess the environment for vessel sprouting (155–159). The branching pulmonary vasculature parallels that of the lung airways, and similarly shares common pathways, elucidation of which will provide further insights into our understanding of diseases involving both angiogenesis and lung disease. PDGF-BB, known to affect airway branching, is reduced in the hearts of mice with impaired branching of myocardial vessels (151). In the renal vasculature, renin is a branching factor, whereas acidic FGF plays a similar role in the myocardium (160). Notably, FGF-related signaling pathways, regulated by Shh, bone morphogenic protein (BMP)-4, PDGF, and TGFb, appear to be key regulators in distal epithelial branching in the developing lung and are critical for postnatal alveolization (161). Tight regulation of expression of VEGF by developing respiratory epithelial cells is required for normal growth of pulmonary blood vessels and branching morphogenesis of the tubular structures of the lung (162). The FGF-like branchless enhances expression of Delta at the tips of tracheal branches. Delta is a ligand for Notch, which when activated, feeds back to downregulate branchless in adjacent cells, thereby localizing the effect of branchless to the sprouting tip of the tracheal branches. Genetic studies in mice and zebrafish have revealed that

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the Notch signaling pathway is similarly critical for vascular patterning and morphogenesis (163–166). Although only a few molecules have been identified that endow the growing vascular system with the necessary remarkable adaptive plasticity and precision in branching and patterning, the exciting parallels that are being drawn when comparing the vascular, neurologic, and pulmonary systems in terms of guidance signals required for formation of the respective networks, will result in the development of targeted, more effective therapies for a variety of diseases. F.

Long-Term Survival of Vascular Endothelium

With the assembly of a vascular network, endothelial cells have the remarkable capacity to attain a phenotype that is highly resistant to exogenous stresses, surviving for years under a variety of extreme pathophysiologic conditions. Endothelial cell apoptosis does, however, occur physiologically during and after development, characterized by vascular regression in the embryo, retina, and ovary. Many factors that regulate apoptosis of endothelial cells have been identified, although their specific roles at various stages in vasculogenesis/ angiogenesis, in different tissues during growth, and under various stresses have not been fully elucidated. Prominent among these is VEGF, which plays a critical role in all stages of vascular growth. VEGF stimulates angiogenesis in large part by promoting endothelial cell survival via interactions with VEGFR-2, PI3kinase, beta-catenin, and VE-cadherin, which in turn leads to activation of Akt and upregulation of antiapoptotic proteins, such as nitric oxide, Bcl-2, Bcl-XL, XIAP, and survivin (167). Withdrawal of VEGF, which occurs in premature babies exposed to hyperoxia, results in retinal vessel regression (168). Inactivation of a single VEGF allele in mice causes profound defects in angiogenesis, with vascular endothelial apoptosis (21). Whereas inadequate levels of VEGF severely affect the normal angiogenic response, excess levels are similarly dangerous. Modest increases in VEGF expression result in prominent developmental abnormalities in the heart and major vessels (23). The increased VEGF observed in patients with asthma contributes significantly to the generation of large, leaky airway mucosa vessels, with endothelial cells that express high levels of leukocyte adhesion molecules—a picture that is reminiscent of inflammation and tumor angiogenesis, both of which are also associated with, and indeed facilitated by, excess VEGF expression. The increased vascularity of the airway mucosa results in thickening of the airway walls. A vicious cycle ensues wherein the edema and increased permeability of the tracheobronchial vasculature leads to ready transit of inflammatory cells (169). Alveolar macrophages then release or facilitate the further expression of several angiogenic factors, including VEGF, PDGF, TGFb, and IGF-1, which can promote lung fibrosis, deposition of extracellular matrix, and vessel remodelling (170).

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Several other factors also modulate endothelial cell survival. Angiopoietin 1, via its cognate receptor Tie1, promotes endothelial cell survival (171,172), whereas angiopoietin 2 induces endothelial cell apoptosis (173). Inhibitors of angiogenesis, including for example, endostatin (174), thrombospondins (175), interleukin 12 (176), and cyclo-oxygenase-2 inhibitors (177), promote endothelial cell apoptosis, and although the precise mechanisms of action are incompletely understood, suppression of Bcl-2, and Bcl-XL may contribute. The growth arrest-specific gene 6 (Gas6), a polypeptide binding the receptor tyrosine kinases Tyro 3, Axl, and Mer, promotes survival of pulmonary artery endothelial cells (178) probably via PI3 kinase/Akt pathways (179). Remarkably however, transgenic studies in mice in which the genes encoding antiapoptotic proteins Bcl-2, Bcl-XL or XIAP have been inactivated (180–182) have not resulted in obvious vascular or angiogenic defects during development, nor have mice that are deficient in Gas6 or any of its receptors (183). Thus the in vivo significance of these biochemical pathways in angiogenesis and endothelial cell survival during embryogenesis come into question. PlGF and VEGF have been implicated in the pathogenesis of emphysemaassociated apoptosis of bronchial vascular endothelial cells and epithelial cells. Under normal conditions, PlGF mRNA is abundantly expressed in the lung. Whereas PlGF deficiency in knockout mice had no apparent effect on lung function (44), constitutive overexpression of PlGF in transgenic mice resulted in a phenotype consistent with chronic obstructive lung disease, with enlarged air spaces and increased lung compliance (184). Although the precise mechanism remains obscure, there were less endothelial cells in the lungs of the mice, which may have been due to proapoptotic effects of PlGF on type II pneumocytes in the alveolar septa, resulting in diminished secretion or availability of VEGF to support the viability of the endothelial cells. The loss of the prosurvival effects of VEGF via VEGFR2 on both lung epithelial and bronchial vascular endothelial cells might partly explain why these cells are noted to be apoptotic in emphysema, and that inhibition of VEGF receptors cause lung cell apoptosis and emphysema (185,186). In contrast to emphysema, the number of bronchial airway vessels and bronchial vascular endothelial cells, depending on the methods of quantitation, (187) is generally believed to be increased in asthma (3,136), and these findings are in part correlated with hypoxia and with increased local expression of VEGF, TGFb and PDGF-B, but may also be modulated by many other factors (188). VEGF not only induces endothelial cell growth, proliferation, and migration but also increases vascular permeability, allowing ready transit of inflammatory cells and plasma proteins into the lung parenchyma, exacerbating the tissue damage. Other angiogenic factors, such as basic fibroblast growth factor (bFGF) (189) and angiopoietin 1 also promote airway remodeling in asthma, increasing subepithelial fibrosis, airway smooth muscle cell proliferation, and angiogenesis (190). SDF-1 is a chemoattractant for leukocytes that

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can induce neovascularization and promote endothelial cell proliferation and survival. Bronchial biopsy specimens from patients with asthma revealed increased expression of SDF-1 by endothelial cells, macrophages, and T-lymphocytes that correlated with the extent of vascularity in the submucosa (191). The contribution of these vascular changes in asthma to manifestations and progression of the disease in terms of, for example, airway wall thickening due to edema, increased access of inflammatory cells and cytokines to the bronchiolo-alveolar network, effects on airway wall function and responsiveness, are under investigation and will have an impact on the identification of “angiogenic” targets for novel therapies (3). Furthermore, in spite of new insights into the mechanisms regulating the apoptotic pathway during vessel growth, the physiologic relevance of these pathways during embryonic and postnatal angiogenesis in health and disease remains poorly understood, largely due to a lack of in vivo genetic models in which regulators of apoptosis have specifically been inactivated in the endothelium. Delineating the in vivo role of endothelial cell apoptosis, however, may lead to the elucidation of autocrine and paracrine signal pathways during angiogenesis, information that could potentially impact on the development of both proangiogenic and antiangiogenic therapies for a variety of illnesses. G. Endothelial Cell Diversity—Meeting Local Demands

It has long been recognized that endothelial cells have diverse properties that vary according to tissue localization, stage of development, and pathophysiologic stress (192,193). For example, tumor vasculature is typically composed of vessels that are dilated, tortuous, and leaky (194). Brain microvascular endothelial cells have tight interendothelial cell junctions to protect the central nervous system from bacteria or toxins (195–197). Endothelial cells of the kidney and endocrine glands are discontinuous, with gaps (fenestrations) between the cells to allow efficient macromolecular transport (198,199). Factors that determine the fate of endothelial cells during proliferation, migration, and differentiation are being elucidated through a variety of means, including fate mapping studies and differential microarray techniques (200–203). Evidence supports the concept that the regulation of endothelial cell diversification and subsequent specificity is contributed to by a combination of intrinsic preprogramming of endothelial cell precursors, and extrinsic environmental elements. The importance of environmental cues is readily illustrated with several examples: An endocrine-gland specific angiogenic growth factor, EG-VEGF (199), has been identified that co-operates with VEGF to induce the formation of fenestrations. Bves is a novel cell adhesion molecule that specifically plays a role in coronary vasculogenesis (204). Endothelial cells of nonbrain origin can take on a blood-brain barrier phenotype when cocultured with astrocytes, a transformation that is triggered by the expression of PDGF-BB, Ang-1, Tie2, and N-cadherin by surrounding glial cells (205). On the other hand, genetic

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programming is also important. For example, although arteries, and veins were believed for many years to be formed in response to hemodynamic forces, recent studies revealed that factors to distinguish them are genetically programmed, established even prior to the start of blood flow in the embryo (206). Indeed, the molecular pathways have been deciphered to a large extent. Loss of notch signaling, which acts downstream of VEGF, disrupts normal differentiation of endothelial cell precursors into arteries and veins, leading to loss of artery specific markers (e.g., ephrinB2) and excess expression of venous markers in ectopic locations, such as the aorta (207). Notch mediates its effects via upregulation of the transcription factor, Hey2, which in turn augments expression of arterial specific genes (ADHA1, EVA1, keratin-7), and suppresses vein specific genes (GDF, lefty-1, lefty-2) (208–211). Arteriovenous specification may also be differentially regulated by different VEGF isoforms. Although VEGF164 is adequate for normal retinal vessel development, sole expression of the VEGF120 isoform is inadequate for normal retinal venular and arteriolar development, VEGF188 only supports venular development (152). Notably, even late in embryonic development, transdifferentiation from arterial to venous, or vice versa, may occur, further highlighting the extraordinary plasticity in the assembly of the vascular network and the interplay between environmental factors and cellular genetic preprogramming. The complex nature of the signals regulating vascular endothelial cell diversity are further revealed when one considers that vessels must be ultimately patterned differently, in terms of polarity, and temporal development. For example, establishment of the right-sided venous system is tightly regulated by expression of angiopoieitin 1 and Tie-1 (212,213). An additional illustrative example that has great clinical relevance is the organization of the great vessels of the heart. The outlet of the heart, the aortic arch, and its branches develop from the pharyngeal arch arteries (214). Defects in vessel patterning result in critical malformations, including for example, aortic arch interruptions and transposition of the great vessels (215). Mechanistic insights into the etiology of these and other disorders have been provided by combined genetic analyses of mice and zebrafish and correlating them with human population studies. In this respect, a common congenital syndrome characterized by defects in the great thoracic vessels and associated with craniofacial, thymic, and parathyroid abnormalities, is DiGeorge syndrome (216,217). Most affected individuals have a hemizygous chromosome deletion of 22q11, and mouse genetic studies implicate the transcription factor Tbx1 as playing a role. Dysregulation of VEGF expression at “hotspots” in the pharyngeal arch arteries during embryonic development appears to alter the normal pattern of appearance and regression of the left- and right-sided 4th pharyngeal arch arteries, giving rise to vascular malformations and birth defects found in DiGeorge syndrome (158,218). Zebrafish studies confirm that VEGF and Tbx1 functionally interact and thus have an impact on vessel polarity and patterning (158).

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H. Periendothelial Cells—Critical Role and Origins

To attain long-term structural and functional support of the vascular network, the endothelium-lined vessels must acquire a surrounding basement membrane and periendothelial cell layer comprised of either vascular smooth muscle cells or pericytes. These metabolically active cells provide stability to nascent vessels by inhibiting endothelial cell proliferation and migration, modulating blood flow and permeability, and providing molecular signals to matrix and endothelium (219,220). They thereby provide hemostatic control and protect new endothelium-lined vessels against rupture or regression. Indeed, vessels regress more easily when not covered by smooth muscle cells or pericytes. What are the origins of smooth muscle cells (221,222)? Recent in vitro studies support the existence of a common VEGFR-2 positive vascular progenitor cell (51,223), possibly derived from mesenchymal cells by the action of TGFb, that gives rise to smooth muscle cells upon exposure to PDGF-BB and to endothelial cells when exposed to VEGF (224–227). But smooth muscle cells may also differentiate from a variety of cells, including endothelial cells, macrophages, and bone marrow precursors (221,228). During embryonic development, dorsal aortic smooth muscle cells first arise from the endothelium (221). The smooth muscle cells that support the distal coronary arteries are derived from epicardial cells, whereas smooth muscle cells for the coronary veins come from the atrial myocardium (229). Cardiac neural crest cells are the source of smooth muscle cells of the great thoracic blood vessels and proximal coronary arteries (230). In the developing lung, mesenchymal cells may give rise to smooth muscle cells that express smooth muscle alpha-actin and smooth muscle myosin. However, the molecular mechanisms regulating their differentiation into smooth muscle cells that envelop the major pulmonary vessels are distinct from those surrounding the bronchi (128). In the adult, circulating bone marrow-derived progenitors may be an additional source of smooth muscle cells (231). This concept is supported by vessel transplant studies in mice in which neointimal smooth muscle cells of donor vessels were determined to be derived from the host and indeed in part from bone marrow-derived cells. Smooth muscle cell progenitors have also been shown to be present in peripheral blood of adult humans, but their contribution to neointima formation and reendothelialization in response to injury, remains controversial (232–234). Nonetheless, the existence of such a source of smooth muscle cells that may have importance in angiogenesis therapy provides an incentive for further investigation. I. Recruitment of Periendothelial Cells

The means by which periendothelial cells are recruited to and encase the endothelial tubes are complex and multifactorial, but genetic studies in mice and humans highlight the importance of this step to maintain vascular integrity. Platelet derived growth factor (PDGF-BB) is chemoattract for smooth muscle

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cells and, with its receptor, is crucial for investing capillary endothelial cells with pericytes, as is the transcription factor lung Kruppel-like zinc finger (LKLF) (235). Sphingosine-1-phosphate (SPP), its receptor, Edg-1, and downstream target Rac also promote periendothelial cell migration, thus enhancing vascular integrity during development (236). Disruption of genes encoding PDGF-B, its receptor, or Edg1 lead to microvessels that lack mural cell coverage and that are easily ruptured and bleed (237,238). TGFb1 (17) and its receptors and effectors, endoglin (239), activin-like receptor kinase (ALK) 1 (240), Smad5 (241), ERK (242), quaking (243), and connexin (244) collectively promote vessel maturation by enhancing stable mesenchymal differentiation and proliferation of periendothelial cells around nascent vascular endothelial cells. Indeed, patients with the inherited bleeding disorder, hereditary hemorrhagic telangiectasia, characterized by vascular malformations particularly in the lung and brain, have molecular defects of endoglin or ALK1 (245,246). In the same molecular pathway, suppression of expression of TGFb type I receptors reduces airway branching during lung morphogenesis, further demonstrating tight functional links in the development of alveoli and lung capillaries (77). In addition to facilitating branching and vascular remodeling, the ephrins and semA3 (247), via integrins (248) optimize pericyte-mesenchyme interactions to attain a complex and functional network. Recent studies have also shown that core 1-derived O-glycans in endothelial cells are required for normal angiogenesis, disruption of which result in defective association of endothelial cells with pericytes and extracellular matix and a bleeding diathesis in utero (249). N-cadherin seems to ‘glue’ endothelial and mural cells in close apposition. Endothelin-1, produced by endothelial cells of the thoracic blood vessels, is chemotactic for neural crest cells, both recruiting and transforming them into smooth muscle cells. Tissue factor also promotes pericyte recruitment, possibly through the generation of thrombin and/or a fibrinrich scaffold. Once mural cells have been recruited, they further “muscularize” the nascent vasculature by sprouting or by migrating alongside preexisting vessels, using these as guidance cues, such as in the retina or in the heart where smooth muscle cell coverage proceeds in an epicardial-to-endocardial direction. In mesenchyme-rich tissues, such as in the lung, in situ differentiation of mesenchymal cells contributes to muscularization (128). Although not as extensively studied, there is evidence to support the concept that, similar to endothelial cells, smooth muscle cells, even when derived from the same progenitors, have diverse and specific functions. Thus, for example, airway smooth muscle cells do not migrate in response to VEGF, which is in contrast to the response of pulmonary vascular smooth muscle cells; yet both are derived from lung mesenchyme (101,250). Characterization of the molecular signals that so tightly regulate smooth muscle cell differentiation, proliferation, and migration will aid in the development of therapeutic targets to ameliorate disease.

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Collateral Vessels

In contrast to capillaries that distribute blood to individual cells, arteries provide bulk flow to larger regions (251,252). When the arterial blood supply is critically jeopardized by an occlusion or diminished flow, the tissue becomes ischemic. Fortunately, arterial systems are often interconnected by collateral vessels that can compensate for the altered flow and thus protect the ischemic region from necrosis. The mechanisms of angiogenesis and collateral growth are significantly different. In response to an occlusive event due to, for example, thrombosis, the altered hemodynamics results in increased shear stress in the existing collateral vessel, thereby activating endothelial cells, which in turn, recruit monocytes from the circulation and bone marrow. Several secreted cytokines have been identified that are chemoattractant for monocytes and additionally protect them against apoptosis. These include, for example, MCP-1, GM-CSF, TGFb1 and TNFa (253–256). Monocytes are critical to collateral vessel growth and remodeling, as they are the source of essential growth factors and proteinases (e.g., MMPs) that faciliate endothelial cell and smooth muscle cell migration and proliferation. Indeed, depletion of monocytes impairs collateral growth, whereas augmentation in monocytes, enhances collateral growth (257,258). PlGF promotes collateral growth both by recruiting monocytes and by stimulating endothelial and smooth muscle cell growth (112,259). FGF administered with PDGF-BB has also been implicated in facilitating collateral growth, in part by upregulating PDGFR expression (260). Finally, whereas there is controversy as to the ability of VEGF alone to increase collateral vessel formation, in combination with one or more other angiogenic factors, such as PDGF, PlGF or Ang1, VEGF may be highly effective, thus providing an incentive to consider multimodal approaches to augment collateral vessel growth for ischemic disease (252,261). VI.

Summary

This review has provided a glimpse into the field of angiogenesis research, highlighting the major concepts, while underlining the complex interactions between endothelium, periendothelium, and extracellular matrix, mediated by a wide array of growth factors, cytokines, and receptors. In spite of the intricate and dense network of biochemical pathways involved in regulating vessel growth under a variety of stimuli, major insights continue to be uncovered. These will ultimately lead to a more clear understanding of the molecular links between angiogenesis and the pathogenesis and progression of lung diseases, with the expectation that this knowledge will facilitate the development of a host of safe and effective targeted therapies. Acknowledgments Dr. EM Conway thanks the Belgian Federation Against Cancer, the Belgian Fonds voor Wetenschappelijk Onderzoek (FWO), and the National Institutes of Health (NIH-NHLBI), U.S.A., for their support.

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4 Chemokine Regulation of Angiogenesis

SORACHAI SRISUMA

ELIZABETH M. WAGNER

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. and Division of Respiratory Physiology, Department of Physiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand

Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland, U.S.A.

I. Introduction Angiogenesis is increasingly being recognized for its role in promoting the pathogenesis of inflammatory diseases and tumorigenesis. Newly formed blood vessels have abnormal walls, causing them to be susceptible to disruption and extravasation of blood elements. Indeed, patients with inflammatory pulmonary diseases and lung cancer often develop a life-threatening massive hemoptysis (1). Hemoptysis in the vast majority of patients originates from systemic, rather than the pulmonary vasculature, and the bronchial vessels are almost universally involved (2,3). Numerous anatomical and radiological studies have shown that the

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bronchial vasculature, in contrast to the pulmonary vasculature, has a remarkable capacity for proliferation in various experimental and clinical lung disorders. Besides the bronchial vasculature, other systemic arteries surrounding the lung frequently contribute to the perfusion of lesions responsible for hemoptysis. The presence of systemic angiogenesis in the lung makes lung resection more difficult and increases the risks of major intraoperative hemorrhage. The mechanisms responsible for new vessel growth in the lung remain poorly understood. Defining the angiogenic process in the lung is important both for therapeutic promotion of new blood vessel growth in ischemic tissue and vascular developmental defects, as well as retardation of the process in tumors and other angiogenesis-related diseases. Generally, the occurrence of physiologic (e.g., wound healing) and pathologic angiogenesis (e.g., cancer, ischemia, infection) has coincided with the presence of infiltrating inflammatory cells (leukocytes, macrophages, mast cells) surrounding newly formed blood vessels (4,5). Concurrent inflammation and angiogenesis via activation of leukocytes and endothelial cells, respectively, can be mediated by common stimuli, such as chemokines. Chemokines or chemotactic cytokines are secreted proteins originally found to control leukocyte chemotaxis, transendothelial migration and tissue invasion (6). This group of proteins plays a role in virtually all inflammatory processes. In addition to their role in cell migration and angiogenesis, chemokines also stimulate leukocyte degranulation, control the movement of developing T and B lymphocytes during their maturation process, and facilitate T cell processing into TH1 or TH2 cells (7). This brief review will focus on the proangiogenic/antiangiogenic roles of chemokines in the context of various pulmonary disorders, with a special emphasis on the CXC chemokines.

II.

The Chemokines

A. Nomenclature, Identification, Chemical Structures

The chemokine superfamily consists of a group of small peptides (approximately 8- to 12-kDa). At least 40 chemokines identified to date have been classified into four distinct supergene families: CC, CXC, XC, and CX3C, according to the presence of four conserved cysteine residues in their primary amino acid sequence (8–10). CX3C, CXC, and CC chemokines contain three, one, and zero nonconserved amino acids, respectively between the first two cysteines, whereas XC chemokine lacks cysteines 1 and 3 of the typical chemokine structure. A standardized nomenclature has been proposed, using an L for ligand and an R for receptor after the family, and then followed by a number. Although, common names are also used that further complicate this field. Among the chemokine families, CXC chemokines seem to be of the greatest importance in angiogenesis and are the first chemokines that have been identified as regulators of angiogenesis (Table 1). Interestingly, most CXC chemokines are clustered on human chromosome 4 (q21) and exhibit 20–50% homology at the

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Table 1 Chemokine with Angiogenesis Regulation Chemokine CXC CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CC CCL1 CCL2 CCL11 CX3C CX3CL

Human ligand

Mouse ligand

Proangiogenic

Angiostatic

GRO-a GRO-b GRO-g PF-4 ENA-78 GCP-2 NAP-2 IL-8 MIG IP-10 I-TAC SDF-1a/b BCA-1

KC MIP-2 MIP-2 PF-4 LIX CKA-3 Unknown Unknown MIG IP-10 I-TAC SDF-1 BLC

CXCR2 CXCR2 CXCR2 Unknown CXCR2 CXCR1,2 CXCR2 CXCR1,2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR5

Yes Yes Yes – Yes Yes Yes Yes – – – Yes –

– Yes (high dose) – Yes – – – – Yes Yes Yes Yes Yes

I-309 MCP-1 Eotaxin

TCA-3 JE Eotaxin

CCR8 CCR2 CCR3

Yes Yes Yes

– – –

Fractalkine

Neurotactin

CX3CR1

Yes

–

Receptor

Abbreviations: BLC, B lymphocyte chemoattractant; TCA, T cell activation protein.

amino acid level (11). CXC chemokines can be further divided into two groups on the basis of a structure/function domain consisting of the presence or absence of the amino acids, glutamic acid-leucine-arginine (Glu-Leu-Arg; the ELR motif) preceding the first cysteine amino acid residue. Interestingly, the presence or absence of an ELR motif in their amino acid sequences seems to correlate with a proangiogenic or angiostatic activity, respectively (12). The ELRC CXC chemokines have been shown to be potent inducers of endothelial cell chemotaxis in vitro and of corneal neovascularization in vivo (12). The requirement of the ELR motif has been demonstrated by site-directed mutagenesis substitution of the ELR motif in the sequence of an ELRC CXC chemokine (CXCL8/common name: IL-8) with a non-ELR motif of the ELRK CXC chemokine (CXCL9/MIG). These mutant IL-8 molecules lacking the ELR motif demonstrated potent angiostatic effects in the presence of either ELRC CXC chemokines or other angiogenic growth factors, such as fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF). In contrast, a mutant of CXCL9/MIG containing the ELR motif behaves as a proangiogenic regulator. The switch in angiogenic properties of the mutated chemokines strongly supports the importance of the ELR motif as a structural domain for proangiogenic activity.

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The ELRC CXC chemokines are chemoattractants for neutrophils and act as potent angiogenic factors in the absence of preceding inflammation (13). Members of the CXC chemokine family that behave as angiogenic factors include CXCL1/2/3/growth-related oncogene (GRO)-a/b/g, CXCL5/epithelial neutrophil activating protein (ENA)-78, CXCL6/granulocyte chemotactic protein (GCP)-2, CXCL7/platelet basic protein (PBP), and CXCL8/IL-8 (8,9). The sequential N-terminal of PBP produces connective tissue activating protein (CTAP)-III, b-thromboglobulin (TG) and neutrophil activating protein (NAP)-2, after release from platelet granules and cleavage by monocyte-derived proteases, of which NAP-2 acts as proangiogenic mediator. Murine CXCL1/keratinocytederived chemokine (KC), (14) CXCL2/3/macrophage inflammatory protein (MIP)-2, (8) CXCL5/lipopolysaccharide-induced CXC chemokine (LIX), (15) and CXCL6/a-chemokine (Cka)-3 (16) are ELRC CXC structural homologs of human CXCL1/GRO-a, CXCL2/3/GRO-b/g, CXCL5/ENA-78 and CXCL6/ GCP-2, respectively, whereas CXCL8/IL-8 exists only in the human. In contrast, most of the ELRK CXC chemokines are chemoattractants for mononuclear leukocytes and potently inhibit angiogenesis. The angiostatic members of the CXC chemokine family, ELRK CXC, include CXCL4/platelet factor (PF)-4, CXCL9/monokine induced by interferon-g (MIG), CXCL10/interferon-g inducible protein 10 (IP-10), and CXCL11/interferon-inducible T cell chemoattractant (I-TAC). Interestingly, many members of ELRK CXC chemokines including CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC are induced by interferons, whereas the expression of ELRC CXC chemokines, such as CXCL8/IL-8, CXCL1/GRO-a, CXCL5/ENA-78, is downregulated by interferon-a,b and g (12). Treatment with interferons or inducers of interferons such as IL-12, can suppress tumor growth by inducing cells to generate antiangiogenic mediators (17). However, despite the findings that the ELR motif generally dictates a proangiogenic phenotype, exceptions have been demonstrated. The group of proangiogenic chemokines without the ELR motif in their sequence is growing and includes CXCL12/stromal cell-derived factor (SDF)-1, and members of the CC and CX3C chemokines as presented in Table 1. Despite being an ELRK CXC chemokine, CXCL12/SDF-1 has been shown to act as a proangiogenic factor in vivo (18). In addition, a pharmacological dose of CXCL2/GRO-b has been shown to suppress tumor growth-induced neovascularization in vivo (19). Several earlier reviews of chemokines and chemokine receptors, structure, function, and inhibition have been published previously (9,20). B. Chemokine Receptor Binding

The chemokine receptors are a subset of the guanine nucleotide-binding protein (G-protein)-coupled receptor (GPCR) superfamily, which is the seven-transmembrane receptor that binds G protein upon activation. Small molecule receptor antagonists might provide novel therapeutic agents that could interfere with

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specific cell recruitment (21). However, within the GPCR superfamily, chemokine receptors are unusual in that most of them are able to bind multiple ligands with similar affinity. For CXC chemokines, there are at least 5 types of CXCR (CXCR1-5). CXCL8/IL-8 shows similarly high-affinity binding to CXCR1 and CXCR2. Other ELRC CXC chemokines bind to CXCR2, although CXCR1 is selectively activated by CXCL8/IL-8 and CXCL6/GCP-2 only (9). The common characteristics among all chemokines that activate CXCR2 is an ELR motif in the amino terminus, which appears to serve as a recognition sequence for receptor binding and activation and was identified by scanning mutagenesis of CXCL8/IL-8 (22) and by amino-terminal truncated analogs (23). On the other hand, ELRK CXC chemokines bind to CXCR3, CXCR4, and CXCR5 (9). CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC are all angiostatic ELRK CXC chemokines and share the same receptor, which is CXCR3. CXCL12/SDF-1 binds only CXCR4, whereas CXCL13/B-cell activating chemokine (BCA)-1 binds only CXCR5. C. Chemokine Receptors on Endothelial Cells

With the ability of major production of specific monoclonal antibodies and probebased RNA expression assays, such as ribonuclease (RNase) protection assay and quantitative real-time polymerase chain reaction (PCR), the detection of receptor expression at the level of protein and transcript has been increasingly recognized (24–26). Human umbilical vein endothelial cells (HUVECs) have been shown to express CXCR1, CXCR2, CXCR4, CXCR5, CCR2, CCR3, CCR5, and CCR8, whereas human dermal microvascular endothelial cells (HMECs) express CXCR1, CXCR2, CXCR3, CXCR4, CCR2, and CCR3 (24–26). Further study is needed to confirm the heterogeneity of endothelial receptor expression and determine airway and lung endothelial cell specific expression. As a proangiogenic molecule, the ELRC CXC chemokines are able to induce chemotaxis of endothelial cells following interactions with specific receptors. The presence of CXCR1 and CXCR2 on endothelial cells and the ligand-receptor binding mechanism favored by the ELR motif as described earlier support the interaction of these ligand-receptor complexes (22,23). With the presence of specific antibody to CXCR1, endothelial cell chemotaxis in response to the ELRC CXC chemokines was not affected (27). On the other hand, in vitro and in vivo assays of angiogenic properties in response to ELRC CXC chemokines were inhibited in the presence of neutralizing antibodies to CXCR2 and in the CXCR2-deficient animals. Taken collectively, CXCR2 has been identified as the endothelial cell receptor responsible for ELRC CXC chemokinemediated angiogenesis. Interestingly, CXCR2 has the most significant sequence homology with Kaposi sarcoma herpes virus-G protein-coupled receptor (KSHV-GPCR) (28), which has been reported to mediate the pathogenesis of Kaposi sarcoma, a virusinduced angioproliferative malignancy occurring commonly in patients infected

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with HIV (29). KSHV-GPCR, the gene product of the human Herpesvirus 8, constitutively activates phosphoinositide-specific phospholipase in the absence of specific ligands, which are ELRC CXC chemokines (28,29). These common structural and signaling features suggest the characteristic mechanisms of action mediating proangiogenic properties following CXCR2 and KSHV-GPCR activation. CXCR4 has been proposed to be a critical CXC receptor expressed on endothelial cells, as mice deficient in either CXCR4 or its specific ligand, CXCL12 die in utero by exhibiting similar defects in cardiogenesis, gastrointestinal vascular development, hematopoiesis, and neuronal development (30). None of the other chemokine receptor or ligand knockout animals exhibits any defects in vascularization. The expression of CXCL12 is ubiquitously and highly detected in most normal tissues but not by leukocytes (31). Additionally, CXCL12 is not a cytokine-inducible gene and proinflammatory cytokines do not increase its expression, whereas other chemokines are produced in response to a variety of inflammatory stimuli, including the early response cytokines, tumor necrosis factors, complement 5a, leukotriene B4, interferons, and bacterial products (32). CXCL12 has thus been proposed to function as a homeostatic stabilizer of tissue architecture, especially the quiescent endothelium in adult and during vascular development (33). In addition to CXC chemokine receptors, several CC chemokine receptors, including CCR2, CCR3, CCR4, CCR5, and CCR8, have been shown to be expressed on endothelial cells (31). Some of their corresponding ligands, including CCL1/I-309 (via CCR8) (34), CCL2/JE/monocyte chemoattractant protein (MCP)-1 (via CCR2) (35), and CCL11/eotaxin (via CCR3) (36), have been shown to mediate angiogenic responses in endothelial cells.

D. Pro-/Antiangiogenic Functions

The process of angiogenesis occurs as an orderly series of events (37). The major changes take place mainly in endothelial cells and surrounding tissue. The proposed alterations of endothelial cells leading to angiogenesis are: vasodilatation and increased endothelial permeability, increased endothelial cell proliferation and survival, increased endothelial cell migration, endothelial cell assembly and lumen formation, and stabilization of the vascular network. Several studies have been conducted to investigate the proangiogenic or angiostatic role of chemokines on the activation of endothelial cells, focusing on endothelial cell proliferation and chemotaxis. CXCL8 has been reported to induce endothelial cell growth in a dosedependent manner (13), whereas other angiogenic chemokines, including CCL1, (38) CXCL12 (18) do not significantly increase endothelial cell growth. In contrast, endothelial cell proliferation can be blocked by ELRK CXC chemokines, such as CXCL4, CXCL9, and CXCL10 (39).

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Proangiogenic chemokines, including ELRC CXC chemokines (27), CXCL12/SDF-1 (31), CCL1/I-309 (34), CCL2/MCP-1 (35), and CCL11/ eotaxin (36), have been demonstrated to induce a dose-dependent increase in endothelial cell migration, whereas angiostatic chemokines, including ELRK CXC chemokines, can impair classical angiogenic growth factor- (e.g., FGFs, VEGF) or chemokine-induced endothelial cell chemotaxis (8,10). Furthermore, CXCL12/SDF-1 can inhibit ELRC CXC chemokine- and growth factor-induced endothelial cell chemotaxis (40). The ability of endothelial cells to migrate, differentiate, and ultimately form capillary-like structures has been determined predominantly by using the Matrigel assay for in vitro experiments and the corneal window angiogenesis assay or aortic ring sprouting assay for in vivo assays. ELRC CXC chemokines, CXCL12/SDF-1, CCL1/I-309, CCL2/MCP-1, and CCL11/eotaxin have all been shown to induce the formation of vascular structures in these in vitro and in vivo neovascularization assays. E.

Intracellular Signaling

Several signaling mechanisms of chemokine receptors have been extensively studied in leukocytes. Although chemokine-mediated neovascularization has been the focus of much research, information related to the downstream signaling leading to the activation of endothelial cells is very limited. Among all chemokinemediated effects, most studies have focused on endothelial cell chemotaxis via CXCR2. Upon ELRC CXC chemokine binding, CXCR2 has been shown to interact with G protein, leading to the dissociation of the a subunit from the b-g subunit. These G protein subunits can activate several major intracellular signaling pathways. A study in human intestinal microvascular endothelial cells (HIMEC) by Heidemann and his group showed that CXCL8/IL-8 mediates endothelial cell chemotaxis and tube formation by activation of phosphatidylinositol 3 0 -kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) 1/2 after engaging CXCR2 (41). Specifically, PI3K is essential for motility of endothelial cells, including actin reorganization and chemotaxis, in response to growth factors, such as VEGF (42,43). Recent investigation revealed that activation of CXCR4 by CXCL12/SDF-1 also mediates the physiological characteristics of angiogenesis in HIMEC, including endothelial cell proliferation, chemotaxis, and tube formation, through the ERK and PI3K signaling pathways (44). The downstream mechanisms after PI3K activation, however, have not been further investigated in endothelial cells. Another study revealed that ELRC CXC chemokines mediate cytoskeletal changes within endothelial cells (25). Endothelial cell contraction and migration are mainly governed by endothelial cytoskeleton and adhesive membrane components. Physiologically, there is a dynamic balance between the monomer of the actin cytoskeleton or globular (G)-actin and polymerized or filamentous (F)-actin in nonmuscle cells. The actin cytoskeleton is a dynamic structure that

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undergoes rearrangement under the control of various processes involved in the regulation of the cellular contractile status. Cortical rings of F-actin interacting closely with membrane adhesion proteins at the cell surface enhance the cell–cell barrier. On the other hand, the formation of cytoplasmic actin-myosin (nonmuscle type) stress fibers linked to the plasma membrane with adhesion proteins and focal adhesion complexes can induce several additional processes, including cell contraction, elevated intracellular tension, release of cell-cell contact, increased vascular permeability, and cell motility (45). Using human lung microvascular endothelial cells (HMVEC), Schraufstatter demonstrated that CXCL8/IL-8 leads to CXCR1-mediated formation of actin stress fibers via Rho activation (25). Ras homologue (Rho) is a member of the monomeric G proteins or small GTPase important for cell migration. Upon Rho activation, its major target, Rho kinase, is further activated and phosphorylates, as well as activates several downstream targets involved in increased actin polymerization, inhibition of the depolymerization of actin, increased interaction between actin and actinbinding protein, and increased phosphorylated myosin light chain (46). The ultimate effect of Rho activation is augmentation of actin-myosin interaction and formation of actin-myosin stress fibers, leading to cell contraction and gap formation (Fig. 1). Moreover, CXCL1/GRO-a and CXCL8/IL-8 were found to stimulate HMVEC migration via activation of Rac, another important small G protein, following CXCR2 binding (25). In contrast to Rho activation, activation of Rac results in the formation of lamellipodia, sheetlike structures consisting of a crosslinked meshwork of actin filaments at the leading edge of migrating cells. This is essential for cell migration and extension along a chemotactic gradient (46). Similar to CXCR1/2 activation, activation of VEGFR2 was recently shown to induce the contraction and migration of human umbilical vein endothelial cell via the activation of Rho and Rac, respectively (Fig. 1) (47). F.

Interaction with Other Chemokines/Growth Factors

CXC chemokines are characteristically heparin binding proteins (8). Like the growth factors, the ability of chemokines to bind to heparan sulfate proteoglycans (HSPG), which include heparin and heparan sulfate, with high affinity appears to be essential for their function. HSPG at the cell surface can capture the ligands and promote ligand dimerization by immobilizing and increasing local amounts of ligand, as is the case for the interaction between FGF or VEGF and its corresponding receptor (48). The multimerization of CXC chemokines is essential for maximal binding to their receptors and promoting biological functions, as shown by CXCL1/CXCR2 interactions and chemotaxis (49). ELRK CXC chemokines, including CXCL4/PF-4 and CXCL10/IP-10, are able to bind to HSPG (8,39). Moreover, CXCL4/PF-4, an ELRK CXC chemokine, can inhibit endothelial cell proliferation, endothelial cell migration, and the neovascularization in in vivo models in response to FGF-2 and VEGF 165

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VEGF-A plasma membrane

VEGFR2

CXCR2

G protein protein G

PI3K PI3K ? ? Rac

Rho

phosphorylation

phosphorylation

plasma membrane plasma membrane P

P actin-myosin stress fiber formation

cortical actin formation αβ

endothelial cell retraction

increased vascular permeability (initial step of angiogenesis)

integrin lamellipodia formation

endothelial cell migration

Figure 1 Schematic diagram showing the mechanisms of VEGF and ELRC CXC chemokine-induced endothelial cell retraction and migration. VEGF binds to VEGFR2, initiating tyrosine kinase activity and phosphorylating the downstream regulators, phosphatidylinositol 3 0 -kinase and p38 MAP kinase. Binding of ELRC CXC chemokines to seven transmembrane CXCR2 also stimulates the kinase enzyme via G-protein pathway. These kinases, in turn, induce phosphorylation of uncertain substances, leading to the stimulation of small GTPase molecules, Rho and Rac. Rho activation causes the formation of actin-myosin stress fibers throughout the cytoplasm and, ultimately, endothelial cell retraction, thereby allowing the cells to move freely. Rac activation induces actin polymerization along the cortical portion of cell, leading to formation of lamellipodia and promotion of cell migration. Abbreviations: G-protein, guanine nucleotide-binding protein; Rho, Ras homologue; VEGF, vascular endothelial growth factor.

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(heparin-bound form) by competitive binding to their corresponding receptors (8). Besides, CXCL4/PF-4 can block the oligomerization of FGF-2 and VEGF 165 by the formation of heterodimer complexes. Without the ability to block VEGF 121, the nonheparin binding isoform, CXCL4/PF-4 neither inhibits VEGF 121 binding to its corresponding receptor nor forms the heterodimers with VEGF 121. With the ability of both groups of CXC chemokines to bind to HSPG, this provides a possible mechanistic explanation how ELRK CXC chemokines inhibit ELRC CXC-induced angiogenesis. The study of the interaction between CXCL1 and CXCL10 on CXCL1 signaling in the CXCR2-expressing cells revealed that CXCL10 partially blocks CXCL1 binding to its receptor and completely inhibits the downstream signaling of CXCL1-mediated CXCR2 signaling (49). Occupying cell surface HSPG by CXCL10 may impair the CXCL1 binding activity of CXCR2, causing the inhibition of CXCR2 signaling, apart from ELRK CXC chemokines/CXCR3 activation. These findings provide a potential antagonistic mechanism for the different biological actions between ELRC and ELRK CXC chemokines, independent of distinct downstream signaling pathways. Regarding the interaction with established angiogenic growth factors, FGF-2 and VEGF induce CXCR4 expression in endothelial cells, thus increasing CXCL12/SDF-1 responsiveness, but do not induce the expression of other chemokine receptors (18). Salcedo and colleagues have shown that FGF-2 and VEGF mediate the augmentation of CXCR4 expression on endothelial cells via cyclooxygenase (COX)-2 induction, whose biochemical product, prostaglandin E2, promotes the upregulation of CXCR4 expression (26). Conversely, CXCL12/SDF-1 enhances the production of VEGF and FGF-2 in a positive feedback loop, therefore linking classical angiogenic factors to chemokine-induced angiogenesis. Moreover, VEGF has been found to significantly increase CCL2/MCP-1 expression, whereas both FGF-2 and VEGF are able to upregulate CXCL8/IL-8 levels in endothelial cells (50).

III.

Role of Chemokines in Angiogenesis

A. Systemic Organs

A survey of existing literature demonstrates the involvement of chemokines in the promotion of new vessel growth in virtually all organs studied. The activation of vascular endothelium by a variety of chemokines in liver (51,52), kidney (53), heart (54,55), brain (56,57), breast (58,59), and lung (60,61) demonstrates the importance as well as the ubiquitous nature of this class of proteins as growth factors. Many, although not all studies, implicate specific chemokines during tumor angiogenesis in each organ. However further study is required to unravel the unique properties of each tissue including the inflammatory cell profile and role of tissue hypoxia to better delineate the specific chemokines involved and their unique roles in the promotion of new vessel formation.

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B. Lung

Although chemokines have not been explicitly linked to neovascularization in asthma and chronic obstructive pulmonary disease (COPD), they have been shown to be associated with inflammation in situations where neovascularization may be incompletely described. Thus, there exists the potential for the balance between angiostatic and angiogenic chemokines to be altered. We describe below situations where enhanced chemokine expression may contribute to predicted vascular remodeling. Asthma

Through the release of cytokines and chemokines, CD4 TH2 lymphocytes are thought to orchestrate the recruitment and activation of the primary effector cells of the allergic response, namely mast cells and eosinophils. Other inflammatory cells, including plasma cells, macrophages, and neutrophils, are variably elevated in asthmatic airways compared with those of controls (62,63). Despite widespread use of anti-inflammatory agents, chronic persistent asthma still remains a major health problem with significant morbidity and mortality. Airway and vascular remodeling are common features of chronic severe asthma, although the molecular mechanisms regulating this response are not clearly defined. An increase in the size and number of blood vessels of the bronchial vasculature has been described in asthmatics (64,65). Vascular dilation and increased vascular density of the airway wall can be induced by various mediators that also cause plasma exudation and vascular congestion. These changes may cause a decrease in airway luminal area associated with increased airway wall thickness (64,65). The airway vasculature in asthma is not only involved in the formation of airway edema but also participates in the recruitment of leukocytes through the expression of a variety of adhesion molecules (64,66), the clearance of inflammatory mediators (67), and morphological changes in the airway wall (65). The expanded granulation tissue from wound healing after several episodes of asthmatic inflammation may cause subepithelial thickening of the airway walls, resulting in reduced luminal diameter and dampened radial tethering from surrounding expanded alveoli (68). This airway remodeling contributes to the airway hyperresponsiveness of asthma. A number of inflammatory cells, as well as airway smooth muscle cells (69), bronchial epithelial cells (70), endothelial cells (71), and lung fibroblasts (72), could be the source of proangiogenic chemokines. Chemokines significantly elevated in the asthmatic airways possibly contribute to asthma-related bronchial angiogenesis and vascular remodeling. However, few studies have evaluated directly increased vascularity in asthma and chemokine expression. Hoshino and colleagues, using biopsy specimens, demonstrated an increased vascularity in asthmatic subjects compared to normal volunteers, which was correlated with an increased number of SDF-1 positive cells within the submucosa (73). By immunohistochemistry, the major cellular sources of CXCL12/SDF-1 were

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shown to be endothelial cells, macrophages, and T-lymphocytes. Furthermore, the number of CXCL12/SDF-1 positive cells was inversely proportional to the level of airways hyperresponsiveness. This study provides strong suggestive evidence that CXCL12/SDF-1 may play a role in the angiogenesis observed in asthma. Yet, despite the proangiogenic properties of chemokines, these chemical mediators have diverse functions during asthmatic responses, which relate to cell recruitment, cellular activation, degranulation, differentiation, and directing the immune response (7). Identifying the specific chemokines responsible for bronchovascular remodeling will be essential to properly target specific ligand/ receptors for therapies. COPD

COPD is a chronic disease characterized by reduced expiratory airflow. COPD includes components of chronic bronchitis and pulmonary emphysema. Chronic bronchitis is associated with chronic inflammation of the airway associated with prolonged mucus hypersecretion, whereas emphysema exhibits progressive destruction of lung parenchyma, leading to airspace enlargement. Obstruction of airways and destruction of alveoli can impair the pulmonary circulation as well as gas exchange. Several studies have shown that there are at least two types of vascular pathology related to COPD: hypertrophy of systemic blood supply to the lung and pulmonary vascular remodeling (74,75). Studies using angiography showed enlarged bronchial arteries in all COPD patients (2,74). There are several stimuli for the hypertrophy of the systemic circulation that might be implicated in the COPD lung. First, the enlargement of bronchial circulation develops in response to chronic airway inflammation, such as that produced by cigarette smoking or chronic airway infection. Second, the pulmonary circulation supplying alveolar regions is destroyed in emphysematous lungs. Third, chronic alveolar hypoxia also results in pulmonary hypertension. Hypoxemia, due to either systemic or pulmonary causes, develops in COPD and contributes to pulmonary arterial vasoconstriction and, ultimately, a reduction in pulmonary perfusion. The latter two bring about an obliterative lesion in pulmonary vessels and development of pulmonary hypertension (76). Chronic hypoxia is the most important stimulus for vascular remodeling in patients with chronic lung diseases and pulmonary hypertension. General features of pulmonary vascular remodeling include excessive proliferation of endothelial cells, smooth muscle cells, and fibroblasts (75). Increased signaling by VEGF (77), FGF (78), thromboxane A2, (75) endothelin-1 (75), adrenomedullin (79), as well as decreased prostaglandin I2 (prostacyclin) (75), and peroxisome proliferator-activated receptor (PPAR)-g (80), have all been reported to relate to pulmonary hypertension-associated COPD. A detailed discussion of this topic is covered in the chapter by Morell and Pulimood.

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Chemokines play an important role in inflammatory cell recruitment associated with chronic inflammation in COPD. The accumulation of neutrophils, macrophages, and CD8 T lymphocytes in the airways and in the lung parenchyma has been shown to significantly correlate with signaling through the ELRC CXC chemokines—CXCR1/2, CCL2/MCP-1—CCR2, and ELRK CXC chemokines— CXCR3, respectively (81,82). However, no studies have focused specifically on the angiogenic role of these chemokines, which might relate to systemic vascular hypertrophy or vascular remodeling in COPD, independent of inflammatory cell recruitment.

IV.

Role of Chemokines in Other Lung Pathologies

A. Interstitial Pulmonary Fibrosis

Inflammation of the lung parenchyma may cause thickening of the alveolar septa or parenchymal filling with granulation tissue and subsequent fibrosis (68). The fibrotic response can be the consequence of a variety of lung injuries, including acute respiratory distress syndrome, infection, or rheumatological disorders. Pulmonary fibrosis is predominantly idiopathic. The pathology of pulmonary fibrosis demonstrates features of dysregulated and abnormal repair with exaggerated angiogenesis, fibroblast proliferation, and deposition of extracellular matrix, leading to a stiffened lung and impairment of gas exchange. The existence of neovascularization in the area of fibrosis was identified originally in idiopathic interstitial pulmonary fibrosis (83) and is associated with increased anastomoses between the systemic and pulmonary microvasculature. The source of neovascularization in pulmonary fibrosis in a rat model has been shown to derive from bronchial vessels (84). Measurement of bronchoalveolar lavage fluid and lung tissue from patients with idiopathic pulmonary fibrosis (IPF) revealed the overexpression of proangiogenic ELRC CXC chemokines CXCL8/IL-8 and CXCL5/ENA-78, compared with the relatively low levels of the angiostatic ELRK CXC chemokine, CXCL10/IP-10 (85,86). This imbalance would be predicted to favor augmented angiogenic activity. Immunolocalization of CXCL8/IL-8 revealed that the lung fibroblast was the main source of this chemokine and areas of CXCL8/IL-8 expression were essentially devoid of neutrophil infiltration in the IPF lung, whereas the predominant cellular sources of CXCL5/ENA-78 were alveolar macrophages and type II alveolar epithelium. Although the classical angiogenic factors may be involved in this process, no difference in the VEGF levels has been observed in IPF, as compared with normal lung tissue (87). A murine model of bleomycin-induced pulmonary fibrosis showed the same correlation as found in patients suffering with IPF, in that the imbalance of CXC chemokines favors the angiogenic ELRC CXC chemokine, CXCL2/MIP-2, which is associated with collagen deposition in the lung. The experimental depletion of CXCL2/MIP-2, with supplemental CXCL10/IP-10 during bleomycin

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exposure, results in attenuated lung angiogenesis and deposition of extracellular matrix, however, without a change in the presence of neutrophil infiltration, fibroblast proliferation, or collagen gene expression (88). All these findings substantiate the role of CXC chemokine-induced angiogenesis in promoting fibroplasia and collagen deposition in pulmonary fibrosis. B. Lung During Pulmonary Artery Obstruction

Perhaps the most extensively studied form of new systemic vessel growth in the lung is that which occurrs after pulmonary artery embolization. Virchow, in 1847, recognized that the bronchial circulation could proliferate and sustain lung tissue distal to a pulmonary embolism. Since that time, neovascularization of the systemic circulation into the lung after pulmonary artery obstruction has been confirmed and studied in humans (89), dog (90), pig (91), and rat (92). In these models, the importance of the bronchial circulation in supporting the ischemic parenchymal tissue has been confirmed, and both the morphology and physiology of the new vasculature studied. Bronchial arteriograms in patients with chronic thromboembolic disease demonstrate the unique capacity of systemic vessels to proliferate and to invade the ischemic lung parenchyma (93). Systemic blood flow to the lung has been shown to increase to as much as 30% of the original pulmonary blood flow after pulmonary artery occlusion (94). In addition to bronchial neovascularization, several intercostal arteries have been shown also to participate in the neovascularization of the ischemic lung (3). Both proliferation and hypertrophy are characteristic features that can be imaged by conventional angiography and by CT imaging, respectively. To further explore the mechanisms responsible for neovascularization after pulmonary embolism, we established a new model of pulmonary artery obstruction in the mouse (95). Although the bronchial vasculature extends from the carina to the terminal bronchioles in most species (96), we showed that mice do not have a bronchial vasculature beyond the mainstem bronchi. After left pulmonary artery ligation in the mouse, intercostal arteries provided a source for new vascularization of the lung. Subsequent work exploring the mechanisms responsible for angiogenesis in this model demonstrated that both lung ischemia and systemic wound healing (thoracotomy) in immediate proximity to the ischemic lung were essential for neovascularization. Furthermore, the CXC chemokines likely play a prominent role in the generation of new vessels to the lung (97). Using both gene array profiling and real time reverse transcriptionpolymerase chain reaction (RT- PCR), we showed a significant increase in three ELRC CXC chemokines—CXCL2/MIP-2, CXCL1/KC, and CXCL5/LIX—in the left upper lung (angiogenic) relative to the left lower lung (ischemic) early after left pulmonary artery ligation (4 hours to 3 days). Furthermore, we confirmed increased protein levels of these three chemokines and histological examination of tissue demonstrated CXCL2/MIP-2 positive macrophages and neutrophils within the lung. Thus, a role for the ELRC CXC chemokines in this model of lung

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angiogenesis is implicated. However, additional factors likely influence the cross-talk between chemokines within the ischemic lung and the thoracic wall tissue from which the intercostal vessels emanate. C. Lung Neoplasia

The growth and metastasis of a neoplasm is dependent on the formation of adequate vascular support (98). Bronchial adenomas and primary bronchogenic carcinomas receive a blood supply mainly from the bronchial circulation (96,99). Evidence showing that angiogenesis may occur early in lung carcinogenesis involves ingrowth of capillaries into the bronchial epithelium with formation of a microcapillary structure (100). The epithelium overlying the capillaries becomes squamous metaplastic and dysplastic with a high proliferative index; the mucociliary layer is replaced by nonciliated cells. This phenomenon, called angiogenic squamous dysplasia, occurs in non-small-cell lung carcinoma (NSCLC) subtypes, including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. The features of tumor-induced angiogenesis are proposed to be due to an imbalance in the expression of angiogenic factors as compared with angiostatic factors. In addition to classical angiogenic growth factors—e.g., VEGF, epidermal growth factor, platelet derived growth factor—several studies have shown the strong contribution of two ELRC CXC chemokines, CXCL8/IL-8 and CXCL5/ENA-78, to angiogenesis and tumor growth in a variety of human cancers, including NSCLC (40,101,102). These ELRC CXC chemokines are overexpressed in malignant tumors and are directly correlated with histologic assessment of tumor vascularity. Studies by Keane and associates revealed the significance of ELRC CXC chemokines and CXCR2 in neovascularizationrelated lung tumors (60). In this study, Lewis lung cancer tumors were implanted in CXCR2 wild-type and deficient animals. There was a direct correlation of the expression of endogenous ELRC CXC chemokines with tumor growth/ metastasis. In CXCR2-deficient mice, and anti-CXCR2 neutralizing antibodytreated wild-type mice, significantly reduced growth and metastasis were observed with increased areas of necrosis and reduced vascular density without any perturbation in the degree of neutrophil infiltration, compared to the nontreatment, wild-type animals. Moreover, ELRC CXC chemokines and neutralizing antibody to CXCR2 had no direct effect on tumor cell growth and intraneoplasia neutrophil infiltration per se. These studies provide strong evidence that CXCR2 mediates ELRC CXC chemokine-dependent angiogenic activity in a model of lung cancer. A potential association between the ELRC CXC chemokines and COX-2 has been due to the observation that COX-2 expression is constitutively enhanced in human NSCLC (103). Overexpression of COX-2 in NSCLC cell lines abundantly increased expression of both CXCL8/IL-8 and CXCL5/ENA-78 (104). Conversely, genetic ablation of COX-2 expression significantly reduced ELRC CXC chemokines expression in these cell lines. Additionally, proangiogenic

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activity of COX-2 overexpressing NSCLC cell lines was significantly reduced in the presence of anti-CXCR2 antibodies using an in vivo assay. In NSCLC-bearing severe combined immunodeficient (SCID) mice, enhanced tumor growth of COX-2 overexpressing tumors was impaired by neutralizing antibody to CXCL8/IL-8 and CXCL5/ENA-78 as well as pharmacological blockade of COX-2. Thus, COX-2 is able to promote the progression of NSCLC via induction of proangiogenic ELRC CXC chemokine expression. The expression of CXCL9/MIG and CXCL10/IP-10 was found to be higher in tumors with spontaneous regression and directly correlated to increased tissue necrosis and impaired neovascularization (105,106). CXCL10/IP-10 is found to be an endogenous angiostatic factor in human NSCLC (101). Although the level of CXCL10/IP-10 was significantly higher in NSCLC tumor specimens than in nearby normal lung tissue, a higher level of CXCL10/IP-10 was present in squamous cell carcinoma than in adenocarcinoma (8). This may be relevant to the differences in the course of diseases between two types of lung cancers. Lower survival rate, higher metastasis, and greater degree of angiogenesis-associated tumors are clinical and pathological features of adenocarcinoma, compared with squamous cell carcinoma in the lung. To study the expression of CXCL10/IP-10 on human NSCLC cell lines independent of lymphocyte involvement, SCID mice were inoculated with either adenocarcinoma or squamous cell carcinoma lines (101). CXCL10/IP-10 levels were significantly lower in adenocarcinoma than squamous cell carcinoma. Depletion of CXCL10/IP-10 using specific neutralizing antibody in SCID mice bearing squamous cell carcinoma enlarged the tumor size, whereas intratumor treatment of CXCL10/IP-10 in adenocarcinoma reduced tumor size, tumor metastasis, and tumor-associated neovascularization. D. Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a disease primarily involved in preterm newborns receiving long term oxygen therapy, causing hyperoxic- and ventilatorinduced lung injury. Such trauma, triggering inflammation and apoptosis- and necrosis-induced cell death, disrupts and potentially arrests the normal sequence of lung development. During embryogenesis, the pulmonary vascular network development follows the already formed airway branches and is considered the canalicular stage at 16–26 weeks in the human. The saccular or newborn stage occurs when lung volume, surface area, and vascularization dramatically increases during weeks 24–38. Septation and alveolar formation occurs in late fetal life and continues during the first couple years of life. Vascular growth continues with alveolarization. Perinatal lung injury in neonates who are born before the saccular or newborn stage (by 24th week gestation) results in the histological pattern of alveolar simplification and dysmorphic vascular growth. Histopathological features are characterized by an extremely immature lung with impaired alveolarization and vascular growth, resulting in enlarged airspaces and decreased surface area for gas exchange (107–109).

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Several mediators, e.g., VEGF, FGF, and angiopoietins, have been hypothesized to provide the signals linking distal airspace growth with vascularization (110–112). Among potential mechanisms leading to BPD, inflammation seems to play a key role. Various proinflammatory mediators, including cytokines and chemokines, are present in the lungs of preterm infants with BPD (109). Study of hyperoxia-induced lung injury in neonates has demonstrated that levels of ELRC CXC chemokines accompany the temporal pattern of neutrophil infiltration (113). Pretreatment with ELRC CXC chemokine antibodies or with a CXCR2 antagonist can prevent the hyperoxia-associated changes in BPD lungs (114). The close correlation of ELRC CXC chemokines to neutrophil accumulation and the preservation of normal alveolar architecture in their absence provide support for a proinflammatory role of chemokines in hyperoxiainduced neonatal lung injury, rather than a proangiogenic or anti-angiogenic function (115). A similar correlative observation of ELRC CXC chemokines and neutrophil sequestration playing an integral role in the pathogenesis of ventilatorinduced lung injury has been demonstrated (16).

V. Summary Numerous examples exist to demonstrate that specific CXC chemokines can both promote and inhibit new vessel growth. The systemic circulation to the lung, which has been shown to exhibit great proliferative capacity, is relatively unstudied. Yet with regard to several pathologic processes within the lung, knowledge of the mechanisms for new vessel growth will allow for new therapies targeted to promote or retard neovascularization. The need for further study identifying specific chemokine function that promotes vascular remodeling unique from general inflammatory responses is essential. References 1. Swanson KL, Johnson CM, Prakash UB, McKusick MA, Andrews JC, Stanson AW. Bronchial artery embolization: experience with 54 patients. Chest 2002; 121:789–795. 2. North LB, Boushy SF, Houk VN. Bronchial and intercostal arteriography in nonneoplastic pulmonary disease. Am J Roentgenol Radium Ther Nucl Med 1969; 107:328–342. 3. Keller FS, Rosch J, Loflin TG, Nath PH, McElvein RB. Nonbronchial systemic collateral arteries: significance in percutaneous embolotherapy for hemoptysis. Radiology 1987; 164:687–692. 4. Hunt TK, Hopf H, Hussain Z. Physiology of wound healing. Adv Skin Wound Care 2000; 13:6–11.

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55. Lakshminarayanan V, Lewallen M, Frangogiannis NG, et al. Reactive oxygen intermediates induce monocyte chemotactic protein-1 in vascular endothelium after brief ischemia. Am J Pathol 2001; 159:1301–1311. 56. Berger O, Gan X, Gujuluva C, et al. CXC and CC chemokine receptors on coronary and brain endothelia. Mol Med 1999; 5:795–805. 57. Rempel SA, Dudas S, Ge S, Gutierrez JA. Identification and localization of the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma. Clin Cancer Res 2000; 6:102–111. 58. Bachelder RE, Wendt MA, Mercurio AM. Vascular endothelial growth factor promotes breast carcinoma invasion in an autocrine manner by regulating the chemokine receptor CXCR4. Cancer Res 2002; 62:7203–7206. 59. Saji H, Koike M, Yamori T, et al. Significant correlation of monocyte chemoattractant protein-1 expression with neovascularization and progression of breast carcinoma. Cancer 2001; 92:1085–1091. 60. Keane MP, Belperio JA, Xue YY, Burdick MD, Strieter RM. Depletion of CXCR2 inhibits tumor growth and angiogenesis in a murine model of lung cancer. J Immunol 2004; 172:2853–2860. 61. Srisuma S, Biswal SS, Mitzner WA, Gallagher SJ, Mai KH, Wagner EM. Identification of genes promoting angiogenesis in mouse lung by transcriptional profiling. Am J Respir Cell Mol Biol 2003. 62. Wills-Karp M. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu Rev Immunol 1999; 17:255–281. 63. Blease K, Lukacs NW, Hogaboam CM, Kunkel SL. Chemokines and their role in airway hyper-reactivity. Respir Res 2000; 1:54–61. 64. Vrugt B, Wilson S, Bron A, Holgate ST, Djukanovic R, Aalbers R. Bronchial angiogenesis in severe glucocorticoid-dependent asthma. Eur Respir J 2000; 15:1014–1021. 65. Wilson JW, Robertson CF. Angiogenesis in paediatric airway disease. Paediatr Respir Rev 2002; 3:219–229. 66. Gallagher SJ, Shank JA, Bochner BS, Wagner EM. Methods to track leukocyte and erythrocyte transit through the bronchial vasculature in sheep. J Immunol Methods 2002; 271:89–97. 67. McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001; 164:S39–S45. 68. Walsh DA, Pearson CI. Angiogenesis in the pathogenesis of inflammatory joint and lung diseases. Arthritis Res 2001; 3:147–153. 69. McKay S, Sharma HS. Autocrine regulation of asthmatic airway inflammation: role of airway smooth muscle. Respir Res 2002; 3:11. 70. Xu H, Okamoto A, Ichikawa J, et al. TWEAK/Fn14 interaction stimulates human bronchial epithelial cells to produce IL-8 and GM-CSF. Biochem Biophys Res Commun 2004; 318:422–427. 71. Unger RE, Krump-Konvalinkova V, Peters K, Kirkpatrick CJ. In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMEC-ST1.6R. Microvasc Res 2002; 64:384–397. 72. Sato E, Haniuda M, Numanami H, et al. Histamine and serotonin stimulate eotaxin production by a human lung fibroblast cell line. Int Arch Allergy Immunol 2002; 128:12–17.

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5 The Role of the Extracellular Matrix in Angiogenesis

HORACE M. DELISSER Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.

I. Introduction In the adult, the endothelial cells of normal vessels maintain tight associations with each other and have a low mitotic index. This state of quiescence depends in part on cues from the extracellular matrix (ECM). However, signals are generated during the normal host response to injury, or from pathological processes, that activate the endothelia of preexisting microvessels to give rise to a new vasculature, with bone marrow–derived endothelial progenitor cells contributing to the cellular repertoire of the forming vessels. During this process of angiogenesis, the ECM, through interactions with cell surface integrins, plays critical roles in regulating functions of endothelial cells, including proliferation, migration, resistance to apoptosis, and tube morphogenesis, that are essential to formation of new vessels.

II.

The ECM of the Quiescent Endothelium

In mature, established vessels, the turnover of endothelial cells is low, with cell cycle entry occurring in only about 1 in 1000 cells (1). This “resting” state is not only the result of endothelial cell–cell interactions (2,3) and the presence of mural 105

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cells, such as pericytes (4,5), but is also mediated by the perivascular matrix. The ECM of the microvasculature from which new vessels arise is organized into two morphologically distinct compartments: a basolateral basement membrane that separates the endothelium from the more distant interstitial matrix (6,7). The basement membrane is a dense, sheetlike structure, the major components of which include type IV collagen, laminins, and heparan sulfate proteoglycans (HSPG). In contrast, the interstitial matrix is a porous, fibrillar network that allows the movement of cells through tissues, while also providing structural and mechanical support. Typical components of this interstitial compartment include fibrillar collagens and glycoproteins, such as fibronectin. Present within this entire matrix complex are protein and carbohydrate constituents [e.g., thrombospondins (TSPs), and hyaluronan], some which may be vessel and/or tissue specific [e.g., collagen XVII and pigment epithelium-derived factor (PEDF)], that promote quiescence. Also sequestered in the matrix are growth factors that can be mobilized for the formation of new vessels, but which may also mediate activities that contribute to the resting state of mature vessels. A. Thrombospondin-1

The TSPs are a family of five extracellular glycoproteins, composed of multiple, well-defined structural motifs, including epidermal growth factor repeats, calcium-binding motifs, and C-terminal domains that mediate cell binding (8). Among the TSPs, thrombospondin-1 (TSP-1) is the most extensively characterized with respect to the activity of these proteins in regulating endothelial cell function and angiogenesis (9). TSP-1 is a large 450-kD homotrimeric protein secreted by a variety of cell types, including endothelial cells, stromal fibroblasts, and immune cells. It inhibits the proliferation and migration of endothelial cells, prevents endothelial cell assembly into capillarylike structures, and induces apoptosis in endothelial cells that are forming new vessels. These direct effects on endothelial cell function are reportedly mediated by CD36 (10). Further, both in vitro and in vivo, TSP-1 makes endothelial cells refractory to activation by pro-angiogenic factors (11). Not surprisingly, it is a potent inhibitor of angiogenesis, suppressing angiogenic responses in experimental animals and slowing the growth of tumors (12,13). Evidence of its role as a mediator of endothelial cell quiescence is found in the fact that repression of TSP-1 accompanies the activation of oncogenes or inactivation of tumor suppressor genes and down-regulation of TSP-1 plays a critical role in the angiogenic switch in several tumors types (14). B. High Molecular Weight Hyaluronan

Hyaluronan (hyaluronic acid, HA) is an important constituent of the ECM (15,16). It consists of a polymer of repeating disaccharides of D-glucuronic acid and N-acetyl glucosamine linked together by alternating beta-1,4 and beta-1,3 glycosidic bonds. Most tissues in the body contain HA with 10,000 or more

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disaccharides reaching molecular masses of greater than a million Daltons (HMW-HA), where it functions as a scaffold to maintain tissue structure. HMWHA also downregulates cellular events, such as cell proliferation and locomotion, that are required for a variety of biological processes, including those required for vessel formation. Thus, in chick embryo limb buds, HMW-HA localizes to avascular areas, transplantation of HA-rich mesoderm from normally avascular to vascular areas causes avascularity, and exogenous administration of HA also results in a lack of vascular development (17). HMW-HA also either has no effect or inhibits endothelial cell migration, proliferation, and tube formation (18,19). Given its ubiquitous presence, these activities suggest that HMW-HA may provide some of the matrix-derived signals that promote the quiescence of mature vessels. C. Collagen XVIII/Endostatin

Collagen XVIII is an abundant heparan sulfate proteoglycan in vascular and epithelial basement membranes (20). Endostatin (ES), a proteolytic fragment of the C-terminal non-triple-helical (NC1) domain of collagen XVIII, has been identified as a potent inhibitor of angiogenesis (21) and has been localized in the elastic fibers of aorta and other large arteries (22). The reported in vitro effects of ES on endothelial cell function include activation of apoptosis and inhibition of proliferation and migration (21,23). The aorta represents one of the most abundant tissues sources of collagen XVIII and proteolytically released ES. Aortic explants from collagen XVII deficient mice showed a significantly higher number of long microvessel sprouts than explants from wild-type litter mates (24). This enhanced microvessel outgrowth could be inhibited to that of the wild type by treatment with ES. In vivo, collagen XVIII-deficient animals demonstrate a number of ocular abnormalities but little in the way of defects in the extraocular vasculature (25). However, collagen XVIII-null mice bred into the atherosclerosissusceptible apolipoprotein E-deficient strains demonstrate more extensive vasa vasorum and intimal neovascularization compared to heterozygote aortas (26), suggesting a vessel-type specific inhibitory activity. D. Pigment Epithelium-Derived Factor

There may well be tissue/organ specific factors that also promote vessel quiescence. An example of this is PEDF, a protein produced by retinal pigment epithelial cells and found at high concentrations within the matrix of the retina and vitreous of the eye (27). PEDF is a particularly potent antiangiogenic factor, whose levels are high and sustained when tissue oxygen concentrations are normal but which fall in the setting of tissue hypoxia (27,28). E.

Growth Factors Sequestered in the ECM

Angiogenic growth factors are present in the ECM surrounding quiescent vessels with their availability dependent in part on binding interactions with HSPG

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(29,30). For vascular endothelial growth factor (VEGF) several isoforms have been identified, all of which demonstrate identical biological activities but which differ in the presence of two heparin-binding domains and thus vary in their ability to bind HSPG (31). VEGF189 and VEGF206 have the two heparin-binding domains and are tightly associated with the matrix or endothelial cell surface through interactions with HSPG, whereas VEGF121 lacks heparin-binding capacity and is freely diffusible in the ECM. VEGF165 has one heparin-binding region and so retains the capacity for interactions with HSPG, while still being moderately diffusible. Basic fibroblast growth factor (bFGF), a mitogenic factor with multiple cellular targets is also stored within the basement membrane and subendothelial matrix (32,33). Although angiogenic factors are mobilized and activated with the onset of angiogenesis, low levels of these factors may have other functions in resting endothelia. Withdrawal of VEGF may lead to endothelial cell apoptosis and regression of established vessels (34). Further, in vitro studies have demonstrated that VEGF at low concentrations not only protects cultured microvascular endothelial cells from undergoing senescence but also restores the proliferative capacity of senescent endothelial cells, while returning them to a more normal morphology (35). Thus, during quiescence, the ECM may act as a reservoir of VEGF (and other factors), providing low levels of VEGF that maintain endothelial cell survival and preserve the proliferative capacity of the endothelium.

III.

The Angiogenic Matrix

The initiation of angiogenesis results in the transformation of the matrix from one that promotes endothelial cell quiescence to one that facilitates the proliferation, migration, and survival of angiogenic endothelial cells (29,36). This restructuring of the matrix results from the deposition of plasma-derived proteins, de novo protein synthesis/secretion and the activity of proteases that degrade the matrix, mobilize sequestered growth factors and liberate functionally active fragments from extravasated plasma or preexisting matrix proteins (37,38). Fragmentation of HMW species of HA may further contribute to the restructuring of the matrix. This angiogenic matrix is chaotic, with both pro- and antiangiogenic factors present, but is one that overall promotes the functions of endothelial cells required for angiogenesis. A. Extravasation of Plasma Proteins

Increased vascular permeability, with enhanced extravasation of plasma proteins, is an early and sustained feature of vessels undergoing angiogenesis (37). Extravasated plasma proteins include adhesive proteins, such as fibronectin and vitronectin, as well as components of the clotting cascade, including fibrinogen, which is rapidly converted to insoluble cross-linked fibrin. VEGF (or vascular

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permeability factor as it was first called) is the principal mediator of this enhanced microvessel permeability (39,40). Its activity in promoting vascular permeability involves the formation of transendothelial pores (41) as well the induction of interendothelial cell gaps and endothelial fenestrations (42). VEGF also upregulates the surface expression of endothelial cell integrins that bind to these extravsated proteins [avb3; (43)], as well as to collagens in the interstitial matrix [a1b1 and a2b1; (44)]. Other angiogenic factors my indirectly enhance vessel permeability by stimulating VEGF expression and/or activity (45). B. De Novo Protein Synthesis and Secretion

Upregulated expression and release of both inhibitory and stimulatory molecules contributes to the elaboration of the angiogenic matrix. Included among the secreted proangiogenic molecules is perlecan, a modular, heparan sulfate proteoglycan with a widespread tissue distribution that is synthesized by and associates with a variety of cell types, including endothelial and tumor cells (46,47). With respect to vessel formation, perlecan expression during murine development is prominent in tissues undergoing vasculogenesis where it is deposited along all endothelial-lined vascular beds (48). Importantly, in tumor xenografts induced by human prostate tumor cells, human perlecan is deposited along the basement membrane of newly formed tumor vessels (49). Significantly, antisense targeting of perlecan blocks tumor growth and angiogenesis (50). This suggests that pericellular perlecan, enriched along angiogenic vessels, might serve as a low-affinity coreceptor for delivering heparin-binding angiogenic factors, such as VEGF and bFGF, to their high affinity endothelial cell receptors. SPARC (secreted protein, acidic and rich in cysteine), on the other hand provides an example of an inhibitory matrix protein that is expressed by a number of cell types (e.g., macrophages, platelets, endothelial cells, fibroblasts, and tumor cells) and is enriched in the stroma of tumors and in the matrix of tissues responding to injury (51). Its importance is suggested by a variety of in vitro studies which have shown that SPARC disrupts cell adhesion, modifies the ECM, inhibits the cell cycle, and antagonizes the effects of growth factors. There is evidence that SPARC may be an inhibitor of angiogenesis. In vitro, SPARC inhibits VEGF-induced endothelial cell proliferation (52); triggers endothelial cell apoptosis (53); and inhibits bFGF-induced cell migration in a dose-dependent manner at concentrations ranging from 0.05 to 5 mg/ml, an effect that is lost at higher concentrations (53). Consistent with these observations are the findings that (1) SPARC inhibits rat corneal neovascularization and neuroblastoma tumor angiogenesis, and (2) SPARC-deficient mice demonstrate increased fibrovascular invasion of subcutaneous polyvinyl sponges compared to wild-type controls (53,54). The exact in vivo role of SPARC, however, is likely to be complex and context dependent, as tumor growth in SPARC-null was enhanced but was not accompanied by evidence of enhanced angiogenesis (55).

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DeLisser C. Proteolytic Processing of the ECM

Proteolytic processing of the matrix during angiogenesis is mediated principally by two distinct classes of extracellular proteolytic enzymes—the matrix metalloproteinases (MMPs) and the serine proteases of the plasminogen activator/plasmin system. MMPs are a large family of zinc-dependent endopeptidases that degrade components of the ECM as well other protein substrates, including chemokines, cytokines, and their receptors (56,57). They can be separated into two structurally distinct groups: secreted MMPs (including collagenases gelatinases, stromelysins and matrilysins) and membrane-type MMPs (MT-MMP), five of which have been identified (MMP-14, -15, -16, -17, -21). The secreted MMPs are produced as latent zymogens that are proteolytically activated in the pericelullar space by other MMPs or serine proteinases, such as plasmin, whereas activation of the MT-MMPs occurs in the sectretory pathway by furinlike enzymes. The activities of MMPs are also regulated by tissues inhibitors of metalloproteases (TIMPs). The generation of plasmin following the activation of plasminogen by urokinase-type and tissue-type plasminogen activators (uPA and tPA) results in a serine protease capable of cleaving a broad array of proteins, most notably fibrin (57,58). uPA binds with high affinity to a glycosyl-phosphatidyl-inositol (GPI)-linked cell surface receptor (uPA receptor or uPAR) and plasminogen associates with the plasma membrane. This colocalization of plasminogen and uPA enhances the efficiency of plasminogen activation, and thus plasmindependent proteolysis, and localizes proteolytic activity to the immediate vicinity of the cell surface. Physiological inhibitors of both plasminogen activators (PAI-1 and PAI-2) and plasmin (e.g., a2-macroglobulin and a2-antiplasmin) provide further regulation of plasmin proteolytic activity. The contributions of these proteases to the remodeling of the angiogenic ECM may be both stimulatory and inhibitory (38). Protease-mediated degradation of the basement membrane and controlled lysis of collagens and fibrin of the provisional matrix facilitate endothelial cell invasion of the ECM by removing physical impediments to movement, as well as exposing cryptic binding sites within matrix molecules that promote endothelial cell motility (59). Further, both bFGF and various forms of VEGF are immobilized in the matrix to heparan proteoglycans and can be released though the activity of proteases (31,60). In contrast to these proangiogenic effects, proteolytic activity results in an angiogenic microenvironment that is potentially enriched with a significant number of antiangiogenic peptide fragments derived from larger molecules that do not inhibit angiogenesis (Table 1). These include fragments from collagen type IV [arrestin, canstatin, oncothanin and tumstatin; (61–66)]; collagen XV [restin; (67,68)]; collagen type XVIII [ES; (21)]; perlecan [endorelipin; (69,70)]; fibronectin [anastellin and fibstatin; (71,72)], MMP-2 [PEX; (73)] or such

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Table 1 Anti-Angiogenic Peptide Fragments Derived from Larger Angiogenic-Inactive Molecules Type of inhibitor Inhibitors derived from ECM proteins

Inhibitor

Parent molecule

Anastellin Arrestin

Fibronectin a1 chain of type IV collagen a2 chain of type IV collagen Perlecan a1 chain of type XVIII collagen

Canstatin Endorelipin Endostatin

Fibstatin Oncothanin Restin Tumstatin Inhibitors derived from plasma proteins

Miscellaneous

Angiostatin Fibrinogen E-fragment Kringle 5 Cleaved antithrombin PEX Vasostatin

Fibronectin a3 chain of type IV collagen Type XV collagen a3 chain of type IV collagen Plaminogen Fibrinogen Plaminogen

Receptors N/A a1b1 aVb3, aVb5 a2b1 a1b5, HSPG, VEGFR, tropomyosin N/A aVb3 N/A aVb3 ATP synthase N/A

Antithrombin

Glucose-regulated protein 78 N/A

MMP-2 Calrecticulum

aVb3 N/A

Abbreviations: ATP, adenosine triphosphate; ECM, extracellular matrix; HSPG, heparan sulfate proteoglycans; MMP, matrix metalloproteinases; VEGFR, vascular endothelial growth factor receptor.

extravasated plasma proteins as plasminogen [angiostatin and kringle 5; (74–77)], fibrinogen [fibrinogen E-fragment; (78)], and high molecular weight kininogen [kininostatin; (79)]. Finally an NH2-termnal fragment of human calreticulin [vasostatin; (80)] and a cleaved form of antithrombin (81) have also been shown to have antiangiogenic activity. Some of these molecules may contribute to endothelial cell quiescence, but in the setting of active vessel formation they may help to regulate physiological angiogenesis or be a component of the host response to constrain pathological neovascularization. However, the complex and apparent chaotic nature of the angiogenic microenvironment is illustrated by the conflicting activities of plasmin on fibrinogen and fibrin. It acts on fibrinogen to generate fibrinogen E-fragment, a potent antiangiogenic peptide (78), while from fibrin it mediates the release of fibrin E-fragment, a related but proangiogenic peptide (82).

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DeLisser D. Low Molecular Weight Hyaluronan

At sites of inflammation and angiogenesis processes are active (e.g., oxidative/nitrating stresses and increased expression of hyaluronidases) that fragment HMW-HA into smaller low molecular species (LMW-HA) (83). In contrast to the inhibitory effects of HMW-HA, these LMW-HA (3-25 oligomers) species are potent and cell-specific stimulators of endothelial cell proliferation (18,19) and tube formation (84), as well as angiogenesis in chick chorioallantoic membranes (19) and in rat skin (85). Consistent with this is the observation that LMW-HA accelerates wound healing in a delayed revascularization model (86). Consequently, changes in the relative proportions of LMW-HA and HMW-HA provide may provide one of the switches that regulate the transition of quiescent endothelial cells to cells that are motile and proliferating.

IV.

Integrins and the ECM

Integrins are the principal cell surface adhesion receptors used by endothelial cells to interact with ECM and microenvironment and thus are intimately involved in the regulation of endothelial cell function by the ECM (87). Engagement of integrins by their matrix ligands results in the recruitment of intracellular signaling and cytoskeletal molecules and the formation adhesion complexes (focal adhesions and their variants) that anchor the cell to the matrix (88,89). Integrin binding induces an array of intracellular signaling responses that may include activation of focal adhesion kinase (FAK), Src kinase, Rho family GTPases, mitogen activated protein (MAP) kinase, protein kinase C (PKC), and the lipid kinase phosphatidylinosital 3-kinase (PI3K). These signaling cascades ultimately culminate in the modulation of gene transcription necessary for cell proliferation, survival and invasiveness, and/or activation of cytoskeletal machinery required for cell motility and morphogenesis. Of note, many of the signaling pathways and effectors activated following growth factor stimulation are also activated by the engagement of integrins. This suggests that integrins may regulate or amplify growth factor mediated cellular responses (90). The integrins are a family of heterodimeric proteins composed of a and b subunits. Currently, 18a and 8b subunits have been identified with at least 24 distinct receptors reported to date (87). As many as 10 different integrins have been described on endothelial cells, with patterns of expression dependent the tissue source and/or activation state of the endothelial cells (87,91). The integrins on quiescent endothelial cells are primarily those that bind to laminins and collagens of the BM (a1b1, a2b1, a3b1, a6b1 and a6b4). Also found on resting endothelial cells are a5b1 and avb5, integrins that respectively bind fibronectin and vitronectin, matrix proteins that are normally associated with a provisional matrix but which during quiescence may mediate other functions or ligand interactions.

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With the onset of angiogenesis, there is de novo or enhanced expression of integrins that bind to provisional matrix components, accompanied by an overall downregulation of collagen/laminin binding integrins (87). The most striking increase in expression is seen in avb3 (92–94), an integrin that binds to a number of ECM proteins, including fibronectin, fibrinogen, osteopontin, vitronectin, von Willebrand factor, and proteolyzed collagens. De novo expression of a4b1 and upregulated expression of a5b1, both receptors for fibronectin, have also been reported on angiogenic endothelial cells (95,96). With respect to the involvement of integrins upregulated during angiogenesis, studies of mice with targeted deletions of various integrin subunits have yielded both confirmatory and unexpected results. The loss of expression of receptors for fibronectin (a5b1 and a4b1) compromises vascular development. The a5 null mutation results in impaired vessel formation of yolk sac vessels, accompanied by leakage of primitive blood cells out these vessels, with lethality at E10-E11 (97), whereas a null mutation in the gene encoding a4 leads to significant embryonic lethality at E11.5-E14.5 and embryos lacking coronary blood vessels (98). In contrast, vascular development and angiogenesis remain essentially intact in mice lacking aV, b3, and b5. In aV-null mice, developmental angiogenesis is intact up to ED9.5 when the majority (80%) of embryos die from placental abnormalities (99). In the surviving animals, the only vascular-related phenotype is the presence of dilated vessels in the brain and gastrointestinal tract that rupture shortly after birth, causing death. b3-null mice demonstrate a bleeding disorder related to impaired platelet function, but no developmental vascular abnormalities are observed, and postnatal neovascularization in the retina is preserved (100). b5-null mice also develop normally without defects in wound healing (101). Further, mice lacking b3 or b3 and b5 unexpectedly have enhanced tumor growth and angiogenesis (102). All of these results are surprising given the large body of prior data demonstrating that antagonism of avb3 or avb5 with antibody reagents or small molecule inhibitors inhibited in vivo angiogenesis (92–94). Several explanations have been proposed for these findings based on aV integrins as potential inhibitors rather stimulators of angiogenesis (103,104). One model proposes that aV integrins in vivo may actually mediate ligand interactions that transduce signals that inhibit angiogenesis (103). Consequently, certain presumed antagonists may either mimic the binding of inhibitory ligands, such as TSPs, or displace activating ligands (e.g., vitronectin) and thus allow receptor access by negative stimuli. Unligated integrins promote apoptosis (105), and thus it has alternatively been proposed that blocking agents may disrupt ligand binding, resulting in enhanced “integrin-mediated death” from an increased number of unoccupied receptors. In turn, these unligated receptors, which normally constrain angiogenesis, are lost in the b3-null mice, resulting in enhanced tumor angiogenesis (104).

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DeLisser V.

ECM and Proliferation

A number of soluble factors have been identified with mitogenic activity for endothelial cells (106,107). Their effects are mediated through receptor tyrosine kinases in which growth factor binding receptor induces receptor dimerization and activation of intrinsic tyrosine kinase activity. The activated receptor subsequently undergoes autophosphorylation, which enables receptor association with and activation of intracellular signaling cascades (108). On a number of levels, the ECM is integral to the ability of these angiogenic factors to induce endothelial cell proliferation. First, the ECM serves as a reservoir from which growth factors may be mobilized (31,60); may act to protect them from proteolysis (109); and provide binding partners that enhance growth factor activity (110,111). Second, endothelial cells are anchoragedependent and so remain unresponsive to such growth factors as bFGF and VEGF when denied integrin ligation in vitro (112). However, when the cells are replated on an appropriate matrix (e.g., collagen or fibronectin), ERK1/2 is activated and proliferation occurs (113). Last, there appears to be “cross-talk” between integrin-mediated and growth factor–mediated cellular responses such that integrin engagement by matrix ligands may amplify the mitogenic signals of ligated growth factor receptors (114). Although a number of growth factors are able to activate the Ras-RafMEK-ERK–MAPK pathway to initiate endothelial cell proliferation, it appears that integrins may be differentially employed to do this. VEGF uses aVb5 and FAK to activate Ras, along with Src to activate cRaf, whereas bFGF employs aVb3, FAK, and PAK (p21 activated kinase) downstream of Ras to activate cRaf. Either of these pathways results in sustained ERK activation and subsequent angiogenesis (115).

VI.

ECM and EC Apoptosis

In quiescent vessels, interactions of a3b1 and a6b1 integrins with laminins of the basement membrane help to promote endothelial cell survival (116). As the basement membrane is degraded and the endothelial cells migrate and proliferate, these interactions are lost and as a result angiogenic endothelial cells are more susceptible to apoptosis than the endothelia of quiescent vessels (104). Cell death resulting from the loss of these integrin-mediated attachments is termed anoikis (117,118) and in endothelial cells has been associated with the activation of the extrinsic apoptotic pathway though a Fas/Fas Ligand (FasL)mediated mechanism (119). In addition to detachment from the matrix, the interplay between inhibitors and inducers of angiogenesis further increase the susceptibility of angiogenic endothelial cells to death-receptor mediated apoptosis. For angiogenic endothelial cells, proangiogenic factors (e.g., VEGF, bFGF, Il-8) induce the expression of Fas, while matrix-derived inhibitors of

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angiogenesis (e.g., TSP-1 and PEDF) upregulate FasL. As a result, nascent vessels are further targeted for apoptosis by Fas-mediated cell death (120). Resistance to this tendency toward endothelial cell death is provided by soluble angiogenic factors (121). For VEGF, its survival effects are mediated through activation of the AKT/PKB pathway and the expression of antiapoptotic proteins A1 and Bcl-2 (122). Another critical player preserving the survival of angiogenic endothelial cells is the integrin avb3. It is not detected on quiescent EC, but its expression increases markedly on angiogenic EC (92–94). The engagement of this integrin by proteins immobilized in the provisional angiogenic matrix promotes the survival of migrating and proliferating EC (93). Interestingly, when avb3 remains unligated or its ligation to matrix is prevented by the binding of soluble antagonists, integrin avb3 recruits caspase-8 to the cytoplasmic tail of its a subunit inducing apoptosis in a death receptor-independent manner (105). Included among these avb3 antagonists are soluble native proteins, such as the TSPs, and proteolytically-derived fragments from collagen (e.g., ES and tumstatin) or proteases (PEX and angiostatin) (105). The above suggests a model in which the endothelia of developing vessels are challenged with pro- and antiangiogenic signals that simultaneously trigger competing survival and apoptotic pathways. The outcome of these opposing influences determines whether endothelial cells live or die causing new vessels to persist or regress.

VII.

ECM and Endothelial Cell Migration

The ability of angiogenic endothelial cells to move into and through the perivascular matrix requires that they degrade the surrounding ECM (invasiveness) as they marshal the adhesive and cytoskeletal machinery needed to promote the morphological changes required for movement (motility). Cultured endothelial cells, as well as those in angiogenic tissues, have been demonstrated to express several MMPs and TIMPs (57,123). These include MMP1, -2, 3, -9, and -14 and TIMP-1 and -2, with the pattern of expressions varying with the EC type and the setting of angiogenesis. Evidence of the in vivo involvement of MMPs in angiogenesis comes from studies demonstrating inhibition of angiogenesis by naturally occurring and synthetic MMP inhibitors along with observation of an angiogenic phenotype in MMP-2- and MMP-9-deficient mice (124,125). Proteolysis by plasmin also appears to be an important invasive factor for endothelial cells, particularly as they penetrate the fibrin-rich angiogenic matrix. Although uPA, uPAR, and PAI-1 are not expressed on quiescent endothelial cells, they are readily detected on angiogenic endothelial cells in a variety of settings (126,127). It appears that plasmin-mediated matrix degradation may play a more significant role during tumor angiogenesis than in developmental or wound-associated angiogenesis (57). Of note, there is evidence of a pericellular

116

DeLisser

fibrinolysis system in endothelial cells that is independent of a PA-plasminogen cascade and is mediated by MMP-14, a membrane-bound MMP (128). Cell motility involves three coordinated steps: membrane protrusion, cell traction and deadhesion, and tail retraction (129). Adhesion at the leading edge (dependent on the formation of focal adhesions) and deadhesion at the rear portions of the cell (requiring the disassembly of focal adhesions) are essential for protrusion and tail retraction, respectively (130). Further, cell motility depends on a tightly regulated set of events involving the polymerization and depolymerization of actin and the exertion of force through actinomyosin-mediated contraction. Engagement of integrins by their matrix ligands initiate signaling cascades, similar to those observed for cell proliferation, that are intimately involved in the turnover of adhesion complexes and the cycling of actin polymerization/de-polymerization (131). As with cell proliferation, there is cross-talk between the growth factor and integrin-mediated responses that promote cell locomotion.

VIII.

ECM and Capillary Morphogenesis

An essential step in the formation of new vessels is the assembly of angiogenic endothelial cells into tubular networks as they migrate and proliferate into the perivascular interstitial matrix. A component of the matrix that may be required for this capillary morphogenesis is interstitial type collagen I (132). Addition of collagen I to monolayer cultures causes endothelial cells to retract, assume a spindle-shaped morphology, and align to form solid cords organized in a polygonal pattern. Over the course of several days in the continued presence of collagen I, these initial networks are subsequently remodeled into tubular structures with lumens, through the formation and then coalescence of intracellular vacuoles. This process in vitro is mediated by two collagen-binding integrins, a1b1 and a2b1, whose expression is also selectively induced by VEGF (44). Consistent with these in vitro observations is the finding that antibody antagonism of either integrin inhibits dermal and tumor angiogenesis (44,133). Vacuolation and lumen formation by endothelial cells in 3D fibrin gels requires both aVb3 and a5b1 integrins, suggesting that the multiple integrins may mediate endothelial cell morphogenesis depending on the composition of the matrix (134). With the conclusion of angiogenesis and restoration of the basement membrane, the endothelial cells are now once again sequestered from interstitial collagens. The maintenance of the patency and stability of the newly formed vessels now result in part from the interactions between a6 integrins (a6b1 and a6b4) (135) and laminin isoforms with the laminin a4 (laminin-8) and a5 (laminin-10) chains (136,137). This is supported by studies of laminin a4-null mice that demonstrate abnormal capillary basement membrane composition and structure, evidence of impaired blood vessel maturation, and vessels prone to hemorrhage (138). Further, the targeted inactivation of the gene encoding laminin

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a5 chain results in embryonic lethality with disturbed placental vessel formation and multiple brain and limb defects (139).

IX.

Summary and Conclusions

ECM is critical to vascular homeostasis and the formation of new vessels. It mediates signals that contribute to the resting state of established vessels, promoting the survival of endothelial cells as well as preserving their proliferative capacity. With the onset of angiogenesis, whether physiological or pathological, the ECM is remodeled to promote the proliferation, survival, and migration of angiogenic endothelial cells and their subsequent organization into patent tubes. These alterations in the matrix are mirrored by changes in the expression and activity of cell surface receptors such as the integrins. Given its critical roles in angiogenesis, it is not surprising that the ECM is emerging as an attractive therapeutic target. The development of these new treatment strategies will progress as we further our understanding of the complex and often competing nature of these matrix-dependent processes.

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6 Angiogenesis in the Asthmatic Airway

JOHN W. WILSON

TIFFANY BAMFORD

Department of Respiratory Medicine, Monash Medical School, and The Alfred Hospital, Prahran, Australia

Department of Medicine, Monash Medical School, and The Alfred Hospital, Prahran, Australia

I. Introduction A. Airway Remodeling

The airway wall in chronic inflammatory states is known to be the subject of tissue remodeling (1–3). Changes in the architecture of the bronchus result in airway wall thickening (4), with luminal narrowing, airflow obstruction, and hyperreactivity to inhaled stimuli. Studies using bronchial biopsies have shown this thickening to be due to changes in specific components of the airway wall, including epithelium (5), subepithelial collagen (6,7), submucosal collagen (8), submucosal vascularity (9), cellular infiltrate (10), and smooth muscle (11). Compared with asthma, the airway wall in chronic obstructive pulmonary disease (COPD) shows less evidence of eosinophil and mast cell infiltration, with glandular hypertrophy and mononuclear cell inflammation and small airway stenosis (12). These changes may underlie the progressive airflow obstruction, increased reactivity, and loss of airway wall distensibility in these conditions (13–15). The vascular supply of the airway is able to contribute significantly to long-term (and less reversible) changes in airflow through angiogenesis, as well

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as to respond to acute challenges through (reversible) vasodilatation and microvascular leakage. The bronchial circulation has been well studied since the time of Leonardo Da Vinci (16). It is composed of deep and superficial plexuses that are connected by capacitance vessels, possibly contributing to turgidity of the bronchial wall (17–19), with muscular protrusions into arterioles potentially regulating flow during asthma attacks (20). Recent studies of airway inflammation have focused on the role of the immune response in determining the continuation of inflammation and the remodeling response (3). The TH2 response, characteristic of asthma, may predispose to increased airway vascularity. IL-4 and IL-13 have been shown to be angiogenic (21), whereas mast cells produce angiogenic factors and cytokines, including histamine, TNF, IL-8 and bFGF (22), reinforcing views that they are commonly associated with neovascularization (23–26). B. Descriptions of Airway Vascularity

The increased vascularity of the asthmatic airway has been described qualitatively in postmortem and bronchial biopsy studies (9,27). There is some difference in published descriptions of vessel density, possibly relating to the marker used for identification (28). Increased vascularity occurs in parallel with severity of asthma and is associated with enhanced vascular ICAM-1 expression (20,29). The role of vessels cannot be separated from cellular infiltration, which is dependent on expression of cell and vessel adhesion glycoproteins (30). Interestingly, the blockade of ICAM-1 in animal models of asthma has resulted in abolition of both eosinophilic infiltration and airway hyperreactivity (31). However, anti-IL-5 therapy has been observed to induce a reduction in eosinophil numbers, without a significant change in lung function (32). IL-4 activationdependent VCAM-1 expression may also facilitate eosinophil migration (33). The complex relation between airway reactivity and eosinophil infiltration appears to be associated with vascular regulation of cell migration. There is discordance in vessel numbers and vascular representation within the airway wall between studies. Several studies have observed an increase (9,20,29,34–37), whereas some studies have observed no difference (10,38) in vascularity between asthma and nonasthmatic control within the airway wall. Review of these studies suggests that these observed discrepancies are likely to result from differences in the detection methods used to identify and quantify the airway vasculature. The techniques used to evaluate vascularity have evolved in conjunction with the methods used to sample the airways and range from the relatively nonspecific histological stains to the antibody specific immunohistochemical identification of vessels. The most common method used to assess airway vascularity and indicate its contribution to airway wall thickening is to identify vessels using collagen IV immunohistochemical staining, assess this staining using microscopic computer aided image analysis techniques, and express the

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results as the percentage of the airway wall occupied by vessels (9,35–37). The large number of methods cited in published reports indicates the difficulty with cross-comparison of measured values (Fig. 1). Standardization of techniques is important to enable meaningful comparisons to be made. Reliable measurements of airway blood flow can be made using an inert, soluble gas as described by Wanner and colleagues (39,40). The first reported studies of airway vasculature used nonspecific histological stains to identify vessels using morphological criteria alone (34,41), which has been confirmed by other studies using histological stains, such as Periodic Acid Schiff (42) and Azure-II-methylene blue basic function (10). More recently, studies have also incorporated specific markers to identify vessels using antibodies to the endothelial cell antigen CD31 (38) or EN4 (29) or components of the tissue wall surrounding vessels using antibodies to collagen IV (9,35–37), and Factor VIII (27,43). Factor VIII antigen (27) and CD31 identify fewer vessels and may be an indicator of activation or maturity (44–46). Endothelial cell replication can be detected in the airway wall using markers for cell proliferation and may correspond

Figure 1 Photomicrograph of collagen IV monoclonal antibody stain for basement membrane in (A) control airway, (B) mild asthma, (C) moderate asthma, (D) severe asthma (x400).

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with vessel numbers (45). Lymphatics may be distinguished from venules by the use of specific monoclonal antibodies such as PAL-E (47). Once identified in tissue sections, the vascularity of the airway can be assessed and quantified using microscopic analysis combined with image analysis techniques identifying three different indices: the percentage of the airway wall occupied by vessels (the total area occupied by vessels expressed as a percentage of the total airway wall) (9,27,35–37), the density of vessels (the total number of vessels expressed per unit area of total airway wall area) within the airway wall (9,10,29,35–38,43), and the mean vessel size (the number of vessels divided by the total area occupied by vessels) (9,35–37). Additionally, the use of fluorescent dyes has enabled mapping of subcutaneous vessels and descriptions of neovascular architecture (48). Vessels are three dimensional in nature, therefore making it difficult to distinguish between a single vessel folding in and out of the section several times and several individual vessels. A recently developed noninvasive bronchoscopic technique described by Tanaka et al. (49) visualizes vessels within the airway wall in situ by using a concentrated light source to illuminate the airway tissue, rendering it semitransparent, and then evaluating both vessel area and density. Thus, although limited to an approximate wall depth of 20 mm, the advantage of this technique is that it allows the discrimination between single but long and multiple vessels within the airway wall as individual vessel paths can be tracked in a three dimensional plane. Mechanisms capable of explaining increased vascularity include an increase in the number of vessels through the processes of neovascularisation (the de novo formation of the vasculature such as that which occurs during embryogenesis), angiogenesis (the growth of new vessels or vessel budding from existing vessels), or the increase in the proportion of the airway wall occupied by vessels due the increase in mass of the existing vessels via microvascular remodelling (vessel engorgement or dilatation) (50). Although tempting to assume angiogenesis underlies an observed increase in vascularity, the phenomenon of branching may be dependent on specific signaling mechanisms, in addition to simple endothelial replication (51). C. Angiogenesis

The response to inflammatory stimuli initiates and perpetuates an interwoven cascade of events that both directly and indirectly affect airway vascularity. In one study, controls showed a range in vascularity from 7.3–13.5%, whereas subjects with mild asthma had a range from 8.0–24.3% (9). The wide range of variation may reflect a genetic component to response, possibly associated with polymorphisms in genes for angiogenic factors or their receptors (52). The process of angiogenesis may have several dependent phases, which may be described as (1) microenvironmental, (2) angioblastic, (3) budding, and (4) proliferative. Clearly, angiogenesis will occur only in a specifically prepared field, to enable vascularity (vessel density) beyond that required for homeostasis,

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as indicated in the example above. Many factors and conditions have been described in association with angiogenesis in human disease and animal models (Table 1). In addition, underrepresentation of angiogenic factors may not enable homeostasis and hence be nonviable (86). Field conditions are likely to be crucial in the determination of both susceptibility to vascular remodeling and the extent of expression of angiogenesis. Limitation of the angiogeneic response may be by reduction of trophic factors or production of antiangiogenic agents (Table 2). As well as by replication of resident angioblasts, it is now known that human stem cells bearing CD34 are capable of seeding circulatory beds and establishing effective neovascularization (114–116). The likelihood of airway remodeling occurring after seeding by circulating mesenchymal stem cells, possibly to enhance airway smooth muscle (117), endothelium (118) and epithelium (119), is a further potential mechanism that may be amenable to therapeutic intervention. D. VEGF in Airway Vascularity

The typical changes of increased airway vascularity seen in asthma have now been shown to correlate closely with both increased vascular endothelial growth factor (VEGF) levels and increased receptor levels of flt-1 (VEGFR-1) and flk-1 (VEGFR-2) (36). VEGF binding to VEGFR-3 is associated with lymphangiogensis (120). This evidence of microenvironmental influences was pivotal in understanding the potentially vital role of VEGF isoforms in airways disease and has been shown for other factors, such as bFGF and angiogenin (60). The role of receptor expression and binding may be quite important, given that VEGF appears to be richly expressed in bleomycin lung, without angiogenesis (121). Additional work has indicated that VEGF induces eNOS and iNOS through activation of VEGFR-2 (122). In another vascular airway disorder (123), VEGF was found to be elevated in serum from cystic fibrosis patients and decreased after treatment of exacerbations (124). Where angiogenesis is not characteristic, such as in BALF derived from smokers and subjects with pulmonary fibrosis, VEGF is reduced (125). In an animal model of toluidine di-isocyanate TDI-induced airway inflammation, VEGF is associated with hyperreactivity and is reversed by VEGFR inhibition (126). Endothelial cells clearly have a unique interaction with VEGF, and their survival is enhanced through induction of anti-apoptotic Bcl-2 expression (127). To facilitate vessel formation, VEGF enhances vascular smooth muscle cell production of matrix metalloproteinase (MMP) (128). However, endothelial cells may become refractory to VEGF stimulation on contact with airway smooth muscle cells (Fig. 2) (129). E.

Vasodilatation and Microvascular Leakage

The increase in wall thickness mediated by vessels may occur with an increase in vessel numbers, vasodilatation, or with microvascular leakage. Until recently,

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Table 1 Angiogenic Factors and Conditions of Relevance to Airway Inflammation VEGF (36,53,54) aFGF (55) bFGF (56) TGFa (22) TGFb (57) HGF (58) TNFa (22) IGF-1 (59) Angiogenins (60) Angiopoietin-1 (61) Histamine (22,62) NO (63) LTC4 (64) Prostaglandins (65) PAF (66,67) SP (68) CGRP (69) NKA (70) VIP (71) IL-1 (72) IL-4 (21) IL-13 (21) IL-6 (73) ELRCCXC chemokines: IL-8 (22,74) ENA-78 (75) GCP-2 (76,77) Alpha v beta 3 integrin (78) VCAM-1 (21) MMP (79,80) ECM (81) Estrogens (82) Ephrin-B1 (83) Ephrin-B2 (66) LPS (84) Pulmonary arterial occlusion (85) Hypoxia and HGF-1 (58) Abbreviations: aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; CGRP, calcium gene-related peptide; ECM, extracellular matrix; ENA 78, epithelial cell derived and neutrophil-activating properties, 78 amino acids; GCP-2, granulocyte chemotactic protein-2; HGF, hepatocyte growth factor; IFGF-1, insulin-like growth factor-1; IL-1, interleukin 1; IL-4, interleukin 4; IL-6, interleukin 6; IL-8, interleukin 8; IL-13, interleukin 13; LPS, lipopolysaccharide; LTC4, leukotriene C4; NKA, neurokinin A; NO, nitric oxide; PAF, platelet activating factor; SP, substance P; TGFa, transforming growth factor a; TGFb, transforming growth factor b; TNFa, tumor necrosis factor a; VCAM-1, vascular cell adhesion molecule-1; VIP, vasoactive intestinal peptide.

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Table 2 Anti-Angiogenic Factors with Potential to Act in Airways Disease Antiangiogenic (87,88) Endostatin (89) Prolactin (anti-VEGF) (90) Suramin (91) TSP-1 (92) Thalidomide (93) Immunization to CM101 (94) Anti-VCAM-1 (21) Anti-IL-8 antisera (95) VEGFR2 blockade using VEGF-165 peptides (96,97) Monoclonal antibody blockade of VEGFR2 (98) PF-4 inhibits VEGF (99) Cancer therapy (100) Angiopoietin-2 (101–103) Thrombospondin (104,105) IL-2 with histamine (106) TIMP-1 (107) VEGFR-2 antibodies (108) Angiostatin (109) Corticosteroids (110) IL-12 (111,112) MMP inhibition (marimistat) (113) Vasostatin (112) Abbreviations: CM101, culture medium 101; IL-2, interleukin 2, IL-12, interleukin 12; PR-4, platelet factor-4; TIMP-1, tissue inhibitor of metalloproteinase-1; TSP-1, thrombospondin-1; VEGFR2, vascular endothelial growth factor receptor 2.

this has been difficult to quantify; however, by labeling plasma proteins in tissue sections, some estimate of leakage may be determined (45,130).Through the action of its VPF isoform, VEGF may modulate microvascular leakage and facilitate cell adhesion, potentiating diapedesis (131,132). Interestingly, vascular leakage dependent on this pathway may be mediated by platelet activating factor (PAF) (133) and may also be inhibited by VPF receptor-binding peptide fragments (134), as well as the plasma protein components that are leaked (135). This latter mechanism may act as a counterregulator of airway angiogenesis, limiting vessel growth. Many other factors control vessel permeability and integrity (136), some of which are listed in Table 3. Vessel diameter may well be crucially important in the role the bronchial circulation to increase tissue turgor, because of capacitance properties (155,156). Multiple inflammatory mediators are capable of causing vessel dilatation and may be considered in the regulation of vessel tone and airway wall thickening (Table 4) (18). Indeed, an important action of inhaled corticsteroids in airways disease may be through the Mackenzie vasoconstrictor effect (40,169), causing vessel shrinkage and reduction in leakage.

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M∅

Mast CD34+ CD3+

Eos vessel

smooth muscle

Figure 2 Potential sources of VEGF in airway diseases, resulting in endothelial cell proliferation and vessel leakage. Table 3

Factors Known to Regulate Microvascular Leakage

OK 2

(137) Histamine (66) Bradykinin (66) SP (138,139) CGRP (140) NKA (141) LTB4 (142) LTC4 (143) LTE4 (144) PAF (66) TNFa (145) VEGF (132,146) ET-1 (147) Mycoplasma (130) VPF (148) PGI2 (66) NO (66) Formoterol (149) Salmeterol (150) Terbutaline (151) PAF (152,153) Endotoxin (154)

Abbreviations: ET-1, endothelin-1; LTB4, leukotriene B4; NKA, neurokinin A; PGI2, prostaglandin I2; SP, substance P; OK 2 , superoxide ion; VPF, vascular permeability factor.

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Table 4 Factors Enabling Vasodilatation in Airway Vessels Histamine (69) Tryptase (157) Heparin (158) Angiogenin (159,160) TNFa (161) TGFb (162) PGD2 (163) bFGF (164) VEGF, via NO and PGI2 (165) PGD2 (69) LTD4 (166) PAF (167) Bradykinin (168) Methacholine (168) SP (168) VIP (168) Salbutamol (71) Abbreviations: LTD4, leukotriene D4; PGD2, prostaglandin D2; TGFb, transforming growth factor b.

F.

Stem Cells in Airway Vascularity and Vascular Angiogenesis

The potential role of bone marrow–derived stem cells has been described in the context of airway diseases for some time (170), principally from the perspective of hemopoietic cells and eosinophilic cellular responses. There is now clear evidence from both animal and human studies that bone marrow–derived stem cells contribute to neovascularization in disease. The observation that implantation of bone marrow cells into ischaemic myocardium facilitates an angiogenic response has initiated a number of studies primarily aimed at neovascularization of cardiac muscle (116,171). Reevaluation of the concept of vascularization in healthy tissues has provided alternative schemes for angioblastic progenitors (Fig. 3). The resident angioblast population may be stimulated to proliferate, given appropriate microenvironmental stimuli. A second possibility allows for migration of bone marrow–derived stem cells with the ability to home to “prepared fields” and differentiate into endothelial progenitors (172). Although required for homeostatic integrity of vessels, circulating stem cells are unlikely to use this second pathway without an appropriately prepared field (173). Multiple possible factors may act to mobilize CD34C progenitors, including erythropoietin, granulocyte stimulating factor (G-CSF), granulocytemacrophage stimulating factor (GM-CSF) and VEGF (174–177). This cell population is used as a target for human bone marrow transplantation and is rich in hemopoietic stem cells. These cells have the capacity to differentiate into endothelial cells (178), continuing to express CD34 and VEGF receptors (179). Alternatively, proliferation of other CD34C cells derived from the circulation,

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local CD34+

Inflammatory cell

Vessel budding

CD34+ CD34+ proliferation EC growth ??

smooth muscle

Figure 3 Alternative pathways for CD34C progenitor expansion, originating from either local angioblasts, CD34C circulating angioblasts, or circulating progenitors of unknown origin, resulting in endothelial cell proliferation.

but retaining a high level of plasticity, may occur in response to factor stimulation and result in angiogenesis (180). The role of CD34C cells may be to preferentially repair injury by restoring homeostasis (181), rather than promoting maladaptive excessive scar formation or angiogenesis. It is important to differentiate this population of cells from putative bone marrow derived mesenchymal stem cells, which are also capable of ameliorating an excessive response to injury (182), as well as having an immunosuppressive action in mixed lymphocyte reactions and the clinical state of graft versus host disease (183).

II.

Summary

Bronchial-wall changes characteristic of asthma are thought to include increased vascularity with vasodilatation, leading to thickening of the bronchial wall. This process may be mediated by such angiogenic factors as VEGF. Other factors may promote angiogenesis through recruitment of CD34C endothelial progenitor cells into the airway. Future strategies to ameliorate airflow obstruction in asthma may need to account for increased vascularity of the bronchial submucosa.

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167. Corfield DR, Webber SE, Widdicombe JG. Mechanisms of platelet activating factorinduced changes in sheep tracheal blood flow. Br J Pharmacol 1991; 103:1740–1744. 168. Laitinen LA, Robinson NP, Laitinen A, et al. Relationship between tracheal mucosal thickness and vascular resistance in dogs. J Appl Physiol 1986; 61:2186–2193. 169. Mendes ES, Campos MA, Hurtado A, et al. Effect of montelukast and fluticasone propionate on airway mucosal blood flow in asthma. Am J Respir Crit Care Med 2004; 169:1131–1134. 170. Denburg JA, Sehmi R, Saito H, et al. Systemic aspects of allergic disease: bone marrow responses. J Allergy Clin Immunol 2000; 106:S242–S246. 171. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7:430–436. 172. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85:221–228. 173. Davies JC, Potter M, Bush A, et al. Bone marrow stem cells do not repopulate the healthy upper respiratory tract. Pediatr Pulmonol 2002; 34:251–256. 174. Velders GA, Pruijt JF, Verzaal P, et al. Enhancement of G-CSF-induced stem cell mobilization by antibodies against the beta 2 integrins LFA-1 and Mac-1. Blood 2002; 100:327–333. 175. Heeschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003; 102:1340–1346. 176. Arora M, Burns LJ, Barker JN, et al. Randomized comparison of granulocyte colony-stimulating factor versus granulocyte-macrophage colony-stimulating factor plus intensive chemotherapy for peripheral blood stem cell mobilization and autologous transplantation in multiple myeloma. Biol Blood Marrow Transplant 2004; 10:395–404. 177. Rafii S, Avecilla S, Shmelkov S, et al. Angiogenic factors reconstitute hematopoiesis by recruiting stem cells from bone marrow microenvironment. Ann NY Acad Sci 2003; 996:49–60. 178. Young MR. Tumor skewing of CD34C progenitor cell differentiation into endothelial cells. Int J Cancer 2004; 109:516–524. 179. Salven P, Mustjoki S, Alitalo R, et al. VEGFR-3 and CD133 identify a population of CD34C lymphatic/vascular endothelial precursor cells. Blood 2003; 101:168–172. 180. Abuljadayel IS. Induction of stem cell-like plasticity in mononuclear cells derived from unmobilised adult human peripheral blood. Curr Med Res Opin 2003; 19:355–375. 181. Sivan-Loukianova E, Awad OA, Stepanovic V, et al. CD34C blood cells accelerate vascularization and healing of diabetic mouse skin wounds. J Vascular Res 2003; 40:368–377. 182. Ortiz LA, Gambelli F, McBride C, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA 2003; 100:8407–8411. 183. Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versushost disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363:1439–1441.

7 Bronchial Vascular Remodeling in Emphysema/Chronic Bronchitis

HARI S. SHARMA, ANDOR R. KRANENBURG, and VIJAY K. T. ALAGAPPAN Cardiopulmonary and Molecular Biology Lab, Department of Pharmacology, Erasmus Medical Center, University Medical Center, Rotterdam, The Netherlands

I. Introduction Chronic obstructive pulmonary disease (COPD) is global health problem with increasing morbidity and mortality (1). COPD is characterized by airflow limitation that is not fully reversible, usually progressive, and associated with an abnormal inflammatory response of the lungs following exposure to noxious particles and gases and inhaled cigarette smoke (2,3). One important pathological feature of COPD is airway inflammation, characterized by an influx of neutrophils, macrophages, and CD8CT-lymphocytes in the lumen and wall of bronchial and bronchiolar airways and parenchyma (4–6). Over time, alveolar destruction results in emphysema, and chronic bronchial inflammation leads to chronic bronchitis—which is why COPD is often called “emphysema and chronic bronchitis” (7). Interestingly, only 10% to 20% of all smokers develop symptomatic COPD; yet the causes of this variability in response of the airways and lung parenchyma to tobacco smoke exposure remain largely unclear. A. Emphysema

The development of emphysema and its different pathological patterns is likely the result of interactions between external risk factors and intrinsic host 147

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susceptibility factors. The parenchymal destruction seen in emphysema may be caused by an inflammatory process due to imbalances in protease-antiprotease and oxidant-antioxidant levels (8) and also by activation of innate and adaptive immune responses (9). The protease-antiprotease theory has been further refined to an elastase-antielastase theory of pathogenesis of emphysema, as neutrophil elastase and macrophage-derived proteases are primaily implicated (10,11). The role of cigarette smoke–induced free radicals in structural damage and inhibition of protease inhibitors also has been studied extensively (12–14). However, theories of antiprotease deficiency do not fully explain why only 10–20% of all smokers develop emphysema (14). One additional possibility is the vascular atrophy model of Leibow (15), as has been suggested (16,17). Leibow proposed that a reduction in the blood supply of the small precapillary blood vessels might induce the disappearance of alveolar septa and hence may result in the development of emphysema. B. Chronic Bronchitis

Mucous gland hypertrophy and goblet cell hyperplasia occur in chronic bronchitis and contribute to excess mucus production. Although the exact pathogenesis of chronic bronchitis remains unclear, bacterial colonization and the resulting inflammatory response are thought to be of central importance. The generation of proinflammatory cytokines and chemotactic stimuli by the airway epithelium are likely to play a central role in propagating the inflammatory response in patients with chronic bronchitis (18). Smokers with chronic sputum production have an increased infiltration of neutrophils and macrophages and an increased proportion of CD8CT-lymphocytes in their bronchial glands, supporting the important role of bronchial-gland inflammation in the pathogenesis of chronic bronchitis (19), which results in epithelial disruption, smooth muscle hypertrophy and fibrosis (20). It has been reported that there are increased macrophage counts in chronic bronchitis patients with airflow limitation, compared to patients without airflow limitation (21). In addition, neutrophils, compartmentalized in the mucosal surface of the airways and CD8CT cells, distributed along the subepithelial zone of the airways and lung parenchyma are consistently associated with the chronic airflow limitation found in COPD (22). The number of neutrophils further increases in the submucosa of patients with severe COPD, suggesting a role for these cells in the progression of disease (22).

II.

Airway Remodeling

Several studies have described changes consistent with airway remodeling in COPD, characterized by a thickened bronchiolar wall, an increase in airway smooth muscle (ASM) mass (5,23,24), and increased deposition of extracellular matrix (25), associated with peribronchiolar fibrosis. Airway remodeling and

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abnormal repair of small airways may explain the changes in small airways and parenchyma in these diseases (14). A. Injury and Repair

It is now well established that particles from cigarette smoke causes damage to the airways, particularly to the epithelial lining (26). Both nonsymptomatic smokers and patients with COPD show signs of damage and repair to the epithelial surface in the form of denuded epithelial lining and squamous metaplasia (4). The processes of normal and abnormal wound healing as a response to injury have been studied extensively (27–31). In general, wound healing involves a series of cellular and molecular events that initiates after injury of the epithelial lining and disruption of the underlying vasculature. This process is characterized by an influx of platelets and inflammatory cells, predominantly neutrophils, followed by macrophages and T-lymphocytes. These platelets and inflammatory cells release many growth factors and cytokines, as well as fibrin and fibronectin, that act to repair wounded tissue. The environment of cytokines and growth factors, (myo)fibroblast-derived extracellular matrix and adequate capillaries facilitate epithelial cell proliferation and migration, leading to wound closure (31). Within the airways, the bronchial epithelium, subepithelial myofibroblasts and ASM cells are the major cell types involved in tissue repair processes. Some of the important cellular and molecular events in the epithelial repair process and potential mechanisms leading to the remodeling of the airway are summarized in Figure 1.

III.

Vascular Remodeling in Chronic Obstructive Pulmonary Disease

Changes in the airway microvasculature have been described in many chronic respiratory diseases (34). Advances in staining methods and availability of vascular markers have improved the ability to visualize vessels in tissue specimens. The mechanisms and therapeutic implications of alterations in airway blood vessels are just beginning to be elucidated, and changes in the microvasculature still represent an important gap in the understanding of the pathophysiology of asthma and other chronic inflammatory airway disease (34). Patients with moderate to severe COPD display elevated pulmonary vascular pressures during exercise and pathological changes in the pulmonary circulation (4,35). Figure 2 shows a diagrammatic representation of two (medial or adventitial thickening) ways through which vascular remodeling could occur during chronic lung diseases. It has been postulated that emphysema actually may lead to loss of the pulmonary vascular bed and induce angiogenesis (36). Vascular abnormalities are associated with the development of COPD; conversely, advanced COPD leads to pathological changes in the pulmonary

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mucus

TNF-α Neu

TNF-α

VEGF, FGF-1, FGF-2

IL-8 MMP-9

+

CD8 T MMP-1,2, 9,12 MMP-8,9

Inflammation Mϕ

Mϕ

TGF-β1, VEGF FGF-1, FGF-2

Vessel

Myo-fibroblast

ECM Breakdown Tissue damage and repair Dysregulated repair

ECM deposition

Airway Remodeling

Figure 1 Cigarette smoke and airway injury leading to tissue remodeling. On exposure to tobacco smoke, epithelial cells are damaged. Epithelial cells and resident macrophages produce inflammatory mediators, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-8. In turn, inflammatory mediators stimulate migration of monocytes/macrophages, neutrophils, and CD8CT-lymphocytes to the airway. Both TNF-a and IL-8 cause degranulation of neutrophils with production and release of serine-proteinases, metalloproteinases (MMPs), as well as free radicals that can cause matrix and epithelial damage. In turn, TNF-a and growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF-1 and FGF-2), orchestrate epithelial repair. Ongoing inflammation and tissue breakdown trigger the release of such growth factors as transforming growth factor-b1 (TGF-b1), inducing extracellular matrix (ECM) production by myofibroblasts. Repetitive tissue damage and repair leads to excessive ECM deposition and subepithelial fibrosis, and ultimately to airway and vascular remodeling. Abbreviations: ECM, extracellular matrix; MMPs, matrix metalloproteinases; M4, macrophage; Neu, neutrophil. Source: Adapted from Refs. 32, 33.

circulation (35,37). This is likely due, in part, to alveolar hypoxia, which is well known to cause pulmonary vasoconstriction and, if the hypoxic stimulus persists, pulmonary vascular remodeling (36). With sustained vasoconstriction of pulmonary arteries, arterioles, and veins, the medial vascular smooth muscle (VSM) extends distally to vessels normally devoid of smooth muscle (36). Intimal thickening due to fibrosis and emergence of smooth muscle cells within the intima of small pulmonary arterial branches has also been reported (38). Several studies have commented on the importance of structural and functional abnormalities in the pulmonary vasculature of COPD patients. Hypoxic

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Adventitia

Lumen

Adventitial thickening

Normal vessel

Vascular Remodeling

Media

Medial thickening

Figure 2 Pulmonary vascular remodeling. Vessel remodeling can occur either through adventitial (predominantly through hyperplasia/hypertrophy of fibroblats) thickening or through the medial (mainly through vascular smooth muscle growth) thickening in different pathophysiological conditions of lungs.

vasoconstriction is considered to represent one of the major contributing factors of pulmonary hypertension and right-sided heart failure in COPD and other chronic pulmonary diseases (35,39). In addition, emphysema, accompanied by loss of elastic recoil, increased pulmonary pressure and destruction of part of the pulmonary microvasculature, may contribute to the increased vascular resistance observed in COPD (36,38). Thus, several phenomena acting in concert in COPD result in pulmonary vascular remodeling. Yet, little is known about the molecular mechanisms underlying these processes in the context of COPD. A. Angiogenesis

Mature endothelial cells are quiescent cells with an extremely low proliferative index. Smoke-induced injury with hypoxia, however, induces vascular endothelial growth factor (VEGF)-A mRNA expression via hypoxia inducible transcription factors (HIF 1 to 3), (40,41). This initiates angiogenesis by increasing endothelial permeability and stimulates endothelial cells to secrete several proteinases, such as matrix metalloproteinase’ (MMP’s), including collagens and elastin degrading MMP-1, MMP-2, MMP-3, and MMP-9, and heparinase acting on HSPGs (42). This, in turn, leads to extracellular matrix (ECM) breakdown and the liberation of additional growth factors, predominantly VEGF-A, as well as fibroblast growth factor-2 (FGF-2) and insulin-like growth factor-1 (IGF-1) sequestered within the surrounding matrix (40,42). Proliferating endothelial cells migrate to distant sites in wounded or inflamed tissue, guided by the actions of VEGF and FGF-2 in close contact with the collagen and heparansulfate proteoglycan matrix, thus resulting in endothelial tube formation (43).

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Of great importance is the recruitment of a stable vascular smooth muscle coating to newly formed vessels. This is initiated by VEGF in combination with angiopoietins (ANGs) produced by endothelial cells, of which currently four ligands are known (ANG-1 to ANG-4) that bind to two receptors expressed by endothelial cells, tie-1, and tie-2 (44). Binding ANG-1 to tie-2 induces endothelial cells to recruit fibroblasts or VSM, whereas ANG-2 binding to tie-2 inhibits this event (44). Transforming growth factor (TGF)-b1 and TGF-bR2 are involved in vessel maturation by inhibiting endothelial cell proliferation and inducing smooth muscle differentiation and stimulating of ECM deposition by VSM cells and fibroblasts, thereby solidifying the endothelial-mural connections (42). Pathological arteriogenesis involves hypoxia, tissue ischemia and increased sheer stress, which damage both endothelial and VSM cells (42). Inflammatory cells, such as monocytes, macrophages, and CD8CT-lymphocytes, infiltrate the vessel wall, causing additional damage to the vessel wall through release of mediators. Growth factors, such as FGF-2, platelet-derived growth factor (PDGF), and TGF-b1, are released by endothelial and VSM cells in response to inflammatory mediators. Eventually dysregulated repair leads to fibrosis and vascular remodeling (42). Taken together, vascular remodeling and angiogenesis in peripheral, as well as in central, airways could also be associated with the pathogenesis of COPD.

C. Bronchial Vessels

Intimal thickening and emergence of smooth muscle cells within the intima of small pulmonary arterial branches has been attributed to a chronic inflammatory process accompanied by fibrosis, analogous to arteriosclerosis in cardiovascular disease (45,46). In COPD, persistent alveolar hypoxia causes pulmonary vasoconstriction and increased muscularization of small arterial branches (36). With sustained vasoconstriction of pulmonary arteries, arterioles, and veins, the VSM extends distally to vessels normally devoid of smooth muscle (36). Recent observations indicate that muscular pulmonary and bronchiolar arteries have increased adventitial infiltration of CD8CT-lymphocytes and intimal thickening that correlates with collagen deposition (37,47). We studied pulmonary vascular remodeling, assessed as the ratio of a-smooth muscle actin staining and vascular wall (VW) area to lumen diameter (48). Vascular medial thickness, assessed by video image analysis, was significantly increased in pulmonary vessels of various sizes in patients with COPD. Others used expression of the smooth muscle marker a-SMA to investigate whether the ratio of smooth muscle (a-SMA/VW area) in the vascular wall had changed during the progression of COPD (49). Surprisingly, the ratio of a-SMA stained area to VW area remained unchanged. Approximately 42% of cells in all vessels stained positive for a-SMA, indicating that the increase in wall thickness could

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be attributed to the deposition of extracellular matrix proteins and medial accumulation of inflammatory cells and fibroblasts. Recently, we demonstrated expression staining for extracellular matrix proteins, such as fibronectin (Fig. 3D) and collagen subtypes (Fig. 3C), in the intimal vascular cells of these pulmonary vessels indicating for ongoing intimal fibrosis in COPD patients. Wall thickness of vessels 200 mm or more in diameter was increased in COPD (Fig. 3A and B). Our results on pulmonary vascular remodeling particularly in terms of intimal and medial thickening (Fig. 2) are in agreement with several earlier reports (35,38,47,50,51). Taken together, these data indicate that vascular remodeling in COPD could be a contributing event in the pathogenesis of pulmonary hypertension in these patients. Furthermore, the observed changes in the intimal fibrosis as well as medial thickening could narrow the vessel caliber and may eventually lead to more severe vascular obstruction in COPD patients. Additionally, an inverse correlation of FEV1 with medial thickening was found in pulmonary vessels of larger caliber, indicating that the degree of pulmonary vascular remodeling is related to the severity of obstructive lung function defect. Wright et al. (35,38) also demonstrated

Figure 3 Histopathology of airway and vascular remodeling. Photomicrographs of lung tissue sections from patients without chronic obstructive pulmonary disease (COPD) (A) and with COPD (B) showing a-smooth muscle actin staining in airway and vascular smooth muscle cells (ASM and VSM). Panel (C) and (D) represents the expression of Collagen III and Fibronectin respectively in a bronchial vessel of COPD patient depicting vascular intimal fibrosis and remodeling.

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increased wall thickness of small (!500 mm) pulmonary vessels in COPD subjects as compared to non-symptomatic smokers, which was correlated with the severity of the disease (as indicated by a decline in FEV1). Similar findings of vascular abnormalities in COPD were recently reported by Peinado and coworkers, who showed intimal but not medial thickening in the vasculature of mild COPD patients compared to nonsmoking controls (47,50). Furthermore, observations from the same group indicated that muscular pulmonary and bronchiolar arteries have increased adventitial infiltration of inflammatory cells, predominantly CD8C T-lymphocytes, and displayed VSM heterogeneity in relation to desmin as well as intimal thickening that correlated to the amount of total collagen deposition (37,47).

IV.

Growth Factors Involved in Vascular Remodeling

A variety of growth factors and cytokines released from various sites of airway and vascular walls have the potential to contribute to the pathogenesis of vascular remodeling in COPD. The major sources, target cells and effects for several growth factors implicated in chronic lungs diseases are listed in Table 1. The main families of growth factors include the VEGF family, FGFs, epidermal growth factor (EGF) family, TGF b family, IGF family, platelet derived growth factor (PDGF) family, and hepatocyte growth factor (HGF). A. Vascular Endothelial Growth Factor

One critical protein involved in vascular remodeling is VEGF. The VEGF family currently comprises six members (VEGF-A to F), of which the originally identified VEGF-A165 variant is the predominant form of five additional spliced variants (38). VEGFs are heparin-binding proteins and act via their high affinity, transmembrane receptors VEGF receptor (VEGFR)-1 (flt-1) and VEGFR-2 (KDR/ flk-1). The receptors belong to the family of tyrosine kinases and are predominantly expressed by endothelial and epithelial cells (40). VEGF promotes an array of responses in the endothelium, including hyperpermeability, endothelial cell proliferation, and angiogenesis with new vessel tube formation in vivo (40,55). VEGF is predominantly known as a paracrine-acting, angiogenic factor stimulating mitogenesis, migration, and permeabilisation of the vascular endothelium. Smooth muscle cells, fibroblasts, epithelial cells, macrophages, neutrophils, eosinophils, or dendritic cells can produce VEGF. Recent studies indicate that VEGF is expressed in the lung by bronchiolar, submucosal glandular and alveolar type I and II epithelial cells, alveolar macrophages, ASM, and VSM cells, as well as myofibroblasts (56–58). VEGFR-1 and -2 are present on endothelial, epithelial, and smooth muscle and inflammatory cells, including macrophages and T cells (59,60), whereas VEGFR-3 has been found on dendritic

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Table 1 Major Growth Factors in Vascular Remodeling Growth factor FGF-1

FGF-2

VEGF

TGF-ß

Source

Target

ECM Fibroblast

Fibroblast ASM VSM Epithelium

ECM Endothelial cell ASM VSM Macrophages Epithelium ASM Endothelial cells VSM Macrophages ECM ECM Platelets Macrophages Fibroblast ASM

Endothelial cell Fibroblast ASM VSM Epithelium Endothelial cell Epithelial cells Fibroblast Macrophages Fibroblast ASM VSM Endothelial cell

Epithelium

PDGF

IGF-1

IGF-2

Platelets Endothelial cell Macrophages Fibroblast ASM Epithelium ECM Fibroblast ECM Fibroblast

Neutrophil T-lymphocytes Monocyt/macrophage ASM Epithelium Fibroblast

Fibroblast ASM VSM Fibroblast

Function Proliferation Collagen production Proliferation Collagen production Proliferation Proliferation Proliferation Proliferation Proliferation Proliferation Migration Proliferation Proliferation Recruitment Recruitment ECM production Recruitment ECM production ECM production Differentiation ECM production Apoptosis Differentiation ECM production Chemotaxis

Proliferation Proliferation Recruitment Proliferation

Proliferation Differentiation Collagen synthesis Proliferation Differentiation Collagen synthesis

Abbreviations: ASM, airway smooth muscle; ECM, extracellular matrix; FGF, fibroblast growth factor; IGF, insulin-like growth factors; PDGF, platelet-derived growth factor; TGF-b, transforming growth factor beta; VEGF, vascular endothelial growth factor; VSM, vascular smooth muscle. Source: Adapted from Refs. 27, 28, 52–54.

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cells (61). The expression of VEGF can be induced under a variety of pathophysiological conditions, including pulmonary hypoxia and pulmonary hypertension with increased sheer stress (55,56). Hypoxia and pulmonary hypertension are pathological features often seen in advanced COPD patients and increased VEGF expression under the influence of HIFs may contribute to increased and abnormal proliferation of endothelial and VSM cells in pulmonary vessels leading to vascular remodeling (4). We postulated that VEGF and its receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1) could play an important role in the pathophysiology of COPD and compared their expression pattern in central and peripheral lung tissue from (ex)smokers with or without COPD (60). In this study we show that COPD is associated with an increased expression of VEGF in the bronchial, bronchiolar, and alveolar epithelium and in bronchiolar macrophages, as well as ASM and VSM cells in both bronchiolar and alveolar region. Interestingly, we observed a significant inverse correlation of VEGF with FEV1 in bronchial mucosal microvessels and ASM cells, bronchiolar epithelium, and medial VSM of larger pulmonary arteries associated with bronchiolar airways. Other members of the VEGF superfamily, such as placenta growth factor (PlGF), and other angiogenic factors, such as angiopoietin, may play an important role in the remodeling process. Recent data suggest that increased PlGF may modulate the availability/production of VEGF in emphysematous patients (62). The relative balance between proleakage (VEGF and Ang-2) and antileakage (Ang-1 and Ang-4) vascular growth factors also may be important in determining capillary integrity (63). B. Fibroblast Growth Factors

The FGF family consists of 23 members, and in view of their important role in chronic inflammation, fibrosis, and repair of various tissues, including the lung (9), they may well play a pivotal role in airway and vessel wall remodeling (64,65). FGFs exert their biological effects via binding to four high-affinity, transmembrane tyrosinekinase receptors designated FGFR-1 through FGFR-4 (66). Distinct FGF subtypes bind with different affinity to the various FGF receptors. Alternative splicing and regulated protein trafficking further modulate the intracellular events and resultant response initiated by FGF ligandreceptor interaction (66). In the lung, as well as in the vascular system, FGFs have been implicated in several pathological conditions; in addition FGF-1 and FGF-2 are also known angiogenic factors (67–69). Moreover, vascular remodeling in response to increased blood pressure is associated with elevated levels of basic FGF (70,71). In the normal pulmonary vasculature, FGF1, FGF-2, and FGFR1 are constitutively expressed in the media (vascular smooth muscle cells) of pulmonary vessels, and FGF-2 is also found in endothelial cells (72). Singh and colleagues demonstrated that increased expression of FGF2 in vascular

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smooth muscle and endothelium precedes arterial enlargement in response to increased arterial blood flow in vivo (70). Furthermore, Bryant et al. recently found that administration of FGF-2 could be protective against a decrease in vessel luminal area and wall thickening in response to altered blood flow and that this inhibitory effect could be blocked by anti-FGF2 neutralizing antibodies (71). Taken together, FGFs could therefore play an important role in airway and vascular remodeling in the development of COPD. In contrast, overexpression of PDGF resulted in airspace enlargement in an animal model (73). Although this result was somewhat surprising, the authors postulated that PDGF may act to upregulate protease expression with subsequent proteolysis of elastin fibers (73). C. Transforming Growth Factors

The TGF-b superfamily is important in cell development and differentiation and proliferative regeneration, although its actions are concentration- and cell-typespecific (27). In epithelial and endothelial cells TGF-b1 is usually associated with terminal differentiation, growth inhibition, and even apoptosis, but during wound healing it can be involved in regeneration (27). In (myo)fibroblasts, smooth muscle cells and other cells of mesenchymal origin, TGFb1 induces proliferation and synthesis of ECM proteins, including collagens, elastin, proteoglycans, and fibronectin (27,74,75).

V. Vascular Remodeling in Emphysema and Chronic Bronchitis A. Expression of Angiogenic Growth Factors in COPD

In patients with COPD, higher expression of VEGF is found in bronchial and alveolar epithelium and VSM as well as in alveolar macrophages, whereas higher VEGFR-1 and VEGFR-2 expression is found in the endothelium, compared to patients without COPD (60,76). These data suggest a role for VEGF in tissue and vascular remodeling seen in COPD. One intriguing possibility is that functional differences exist for VEGFRs in the pathogenesis of COPD. For example, activation of VEGFR-2 is involved in angiogenesis by stimulating mitogenesis of endothelial cells during vascular damage–repair processes (77). In contrast, VEGFR1 is involved in stimulating vascular smooth muscle expression of MMP and endothelial expression of plasminogen activator and its inhibitor, activities needed for blood vessel maturation (78,79). As VEGFR-1 has a higher affinity for VEGF than VEGFR-2, it is thought that VEGFR-1 is involved in resolving the angiogenic process (79,80). Pulmonary vascular smooth muscle expression of VEGF and VEGFR-2 are increased in smokers with COPD or chronic bronchitis, whereas VEGFR-1 expression did not differ between smokers with or without COPD in these compartments (37,60). In contrast to Kasahara et al. (81) who showed that VEGF and its receptor VEGFR-2 were decreased in total lung

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extracts of emphysematous lungs as measured by enzyme linked immunosorbent assay (ELISA) or western blot analysis, we found that the epithelial and endothelial cells in the alveolar spaces and in the most distal airways were intensely positive for VEGF and VEGFR-2 in patients with COPD (60). In our study the patients could be considered as having mild to moderate COPD, whereas the lungs studied by Kasahara et al. (81) were solely emphysematous. Likewise, in COPD, expression levels of FGF-1, FGF-2, and FGFR1 are increased in vascular and in epithelial compartments in patients with COPD compared to subjects without COPD (48,82). Table 2 summarizes the expression levels of angiogenic growth factors in COPD patients.

B. Role of VEGF and Its Receptors in Pathogenesis of Emphysema and Chronic Bronchitis

The destruction of lung tissue in emphysema may involve the progressive loss of capillary endothelial and epithelial cells through the process of programmed cell death, apoptosis. Several lines of evidence suggest that VEGF induces the expression of antiapoptotic proteins and thus acts as an endothelial survival factor (83–85). A recent study demonstrated that patients with emphysema had decreased levels of VEGF messenger RNA and protein, as well as decreased expression of VEGFR-2. Furthermore, decreased VEGF and VEGFR-2 expression were associated with endothelial and epithelial cell death in alveolar septa (81). Several recent animal studies have demonstrated that VEGFR-2

Table 2 Pulmonary Expression of Angiogenic Growth Factors in COPD, Chronic Bronchitis, and Emphysema Growth factors

Expressiona

Disease

EGF EGFR FGF-1 FGF-2 FGFR1 TGFb-1 VEGF VEGF VEGFR1 VEGFR2 VEGFR2

Increased Increased Increased Increased Increased Increased Increased Decreased Increased Increased Decreased

Chronic bronchitis COPD COPD COPD COPD COPD COPD Emphysema COPD COPD Emphysema

a

Pulmonary expression or circulating levels of various growth factors in comparison to age-matched controls. Abbreviations: COPD, chronic obstructive pulmonary disease; EGF, epidermal growth factor; EGFR, EGF receptor; FGF, fibroblast growth factor; FGFR, FGF receptor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

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blockade, in combination with chronic hypobaric hypoxia, destroys lung capillaries by inducing endothelial cell apoptosis and at the same time causes occlusion of precapillary pulmonary arteries by proliferating endothelial cells (55,86–88). Chronic treatment of animals with SU5416, a VEGFR-2 inhibitor, results in increased pulmonary artery pressure in both normoxic and hypoxic animals, widespread endothelial cell apoptosis, and emphysema, when compared with drug vehicle treated controls (17). Similar findings of emphysema have been reported in mice depleted of pulmonary VEGF by a Cre/LoxP-targeted deletion technique (89) and in animals treated with a soluble Flt-Fc chimeric protein (90). Thus, the expression of VEGF may be protective against signals leading to apoptosis, such as toxic agents from tobacco smoke. Cigarette smoke, possibly by inducing nitric oxide (91), may also act to decrease the expression of VEGF and VEGFR-2, thus resulting in lung septal endothelial cell death (81). In chronic bronchitis, increased VEGF levels may be due, in part, to ongoing repair processes (60,92). Small airway changes are associated with thickening of the airway wall and disruption of mucociliary clearance, resulting in the accumulation of inflammatory exudates in the airway lumen. In addition, Hogg et al. postulated that colonization and infection of the lower airways was associated with an adaptive immune response that accounts for the increase in lymphocytes and their organization into lymphoid follicles in patients with severe COPD (93). Thus, both the innate mucociliary clearance system (94) and the adaptive immune responses (93) appear to be important for the repair process. Kanazawa et al. (92) examined VEGF levels in the induced sputum of patients with emphysema, chronic bronchitis, and asthma and found correlations with both FEV1 and diffusing capacity of the lung for carbon monoxide (DLco). In emphysema patients, lower FEV1 or DLco directly correlated with lower VEGF levels in sputum. In contrast, patients with chronic bronchitis or asthma demonstrated an inverse correlation between VEGF levels and FEV1. These data suggest a positive association between the severity of airway inflammation and VEGF secretion in these two diseases. Given the scarcity of evidence, however, it is difficult to conclude that there is a differential role for VEGF in the pathogenesis of emphysema or chronic bronchitis. As Wagner rightly points out, it is unknown whether VEGF expression is below a critical threshold level and indeed the cause of emphysema or whether reduced expression is merely a marker of the disease process without functional importance (7). However, emerging evidences gives us a clear indication that there is modulation of angiogenic factors, such as VEGF, in COPD patients that appears to correlate with the underlying pathophysiology. Signaling through VEGFR-1/flt-1 may also be important to the pathogenesis of emphysema. A recent study of PlGF transgenic mice demonstrated enlarged airspaces without airway inflammation (62). Overexpression of PlGF resulted in apoptosis of type II pneumocytes, leading to decreased VEGF secretion and subsequent endothelial cell death. These authors hypothesize that a positive

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feedback loop exists between type II pneumocytes that secrete VEGF and the integrity of the septal microcirculation that promotes pneumocyte survival (62). C. Angiogenic Growth Factors as Potential Therapeutic Targets

VEGF and its receptors are critical regulators of angiogenesis, tumorigenesis, and metastasis. Several drugs have been developed to reduce tumour growth by impairing neovascularization. Among those in clinical trials are VEGF-Trap, bevacizumab (Avastin), CEP-7055, SU6668, and PTK787 (95). Avastin is a humanized monoclonal IgG1 antibody against VEGF that inhibits receptor binding. CEP-7055 is a small molecule, water-soluble indenopyrrolocarbazole derivate inhibitor of pan-VEGFR tyrosine kinase activity. It has been shown to inhibit pulmonary VEGFR-2 activation in vivo, capillary formation by human and rat endothelial cells in vitro and in vivo, granuloma formation and vascularization in a chronic inflammation model, and tumorigenesis (96). VEGF Trap is a soluble VEGFR constructed from VEGFR1 and VEGFR2 binding domains linked to an IgG1 constant region. In vivo animal models have demonstrated that VEGF Trap reduce hemangiogenesis, lymphangiogenesis, and tumor growth (97,98). SU6668 is an IgG2a fusion protein inhibiting the receptor tyrosine kinase activity of FGFR1, KDR, and PDGF-Rb. SU6668 has been shown to inhibit tumor growth, lung cancer metastasis, and tumor vascularization (99). Many other small molecule VEGFR inhibitors are being developed for clinical therapy (100). Recent studies support a role for VEGF/VEGFR in pulmonary and vascular remodeling and inflammation. For example, VEGF transgenic mice exhibit alveolar vascular and airspace remodeling as well as a Th2-type airway inflammation in a murine model of experimental asthma (101,102). In this study, the VEGF-R inhibitor SU1498, as well as VEGF-TRAP, decreased airway inflammation and airway hyperresponsiveness (101,102). Therefore, VEGF inhibitors may be of potential use for the treatment of inflammation and neovascularization seen in asthma or specific subtypes of COPD, such as chronic bronchitis. However, the redundancy of the VEGF-VEGFR system may limit the applicability and efficacy of VEGF/VEGFR inhibitors (103). In contrast, in patients with emphysema, therapy may need to be targeted more towards inhibition of apoptosis or stimulation of VEGF expression, in order to overcome endothelial and epithelial cell death and maintain alveolar septal integrity (104). Hence, treatment with VEGF/VEGFR inhibitors should be further investigated and may be restricted to specific chronic lung diseases. VI.

Conclusion

Current in vivo and in vitro data indicate that cross talk between smooth muscle cells, endothelium, myofibroblasts, and inflammatory cells via growth factors and cytokines are major contributing factors to vascular remodeling during different

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contraction

AIRWAY SMOOTH MUSCLE

PGE, Tx, PGD, PGF

PGE2

161

IL-11

FGF-2 PDGF IGF-2 TGF-b VEGF, Ang

IL-6

IL-5, IL-8, GM-CSF, LIF, RANTES, MCP, cotaxin

IL-1b

IFN-γ

IL-6, IL-12

Bronchial Vessel

Increased bronchial vascularity

• Airway edema • Transudation/exudation of mediators/cytokines • Increased trafficking of inflammatory cells • Increased blood supply to support hyperreactive and

Hyperplastic/hypertrophic airway smooth muscle

Figure 4 The potential role of airway smooth muscle cells in bronchial vascular remodeling. The expression and secretion of cytokines, chemokines and growth factors by airway smooth muscle cells can alter the proliferative response of vascular endothelial and smooth muscle cells. These complex interactions may contribute to the overall airway and vascular remodeling process by promoting increased vascularity, endothelial permeability and airway wall edema and trafficking of inflammatory cells during chronic lung diseases. Abbreviations: Ang, angiopoietin; FGF, fibroblast growth factor; GM-CSF, granulocyte macrophage colony stimulating factor; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; LIF, leukemia inhibitory factor; MCP, monocyte chemotactic protein; PDGF, platelet derived growth factor; PGD, prostaglandin D; PGE, prostaglandin E; PGF, prostaglandin F; RANTES, regulated upon activation normal T-cell expressed and presumably secreted; TGF-b, transforming growth factor; Tx, thromboxane.

pathophysiological conditions (49,105–107). Figure 4 summarizes the possible interactions between the ASM-derived mediators and the adjacent endothelial and vascular smooth muscle cell compartment in the bronchial blood vessels and how they contribute to the remodeling process. At present, our knowledge of airway and vascular remodeling in COPD is far from complete. Increased adventitial infiltration of inflammatory cells, predominantly CD8CT-lymphocytes, in

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muscular pulmonary and bronchiolar arteries has been reported (47,108). Many growth factors, among them VEGF and FGF, play an essential role in maintaining pulmonary and vascular viability and in tissue repair. Based on the available evidence, we hypothesize that in chronic bronchitis, expression of VEGF and VEGFR-2 expression leads to increased vascular remodeling, which is inefficiently compensated by the low expression of VEGFR-1. In subjects with emphysema, however, VEGFR2 expression is lower. Preferential activation of VEGFR-1 results in higher MMP activity, alveolar destruction, and endothelial apoptosis. Hence, the balance between VEGF, VEGFR-1, and VEGFR-2 are critical in the pathogenesis of COPD subtypes.

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8 Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease

NICHOLAS W. MORRELL and THOMAS B. PULIMOOD Division of Respiratory Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, U.K.

I. Introduction Pulmonary hypertension (PH) complicating chronic respiratory disease is defined as a mean pulmonary artery pressure (MPAP) greater than 20 mmHg at rest. Due to its prevalence, chronic obstructive pulmonary disease (COPD) is by far the commonest cause of PH and of cor pulmonale. In 1956 DW Richards in his Nobel lecture pointed out that, “a considerable proportion of patients suffering from chronic pulmonary disease with progressive pulmonary insufficiency will eventually develop cor pulmonale.” He observed that the principal factors inducing right ventricular strain, hypertrophy, and failure were: (1) PH, from one cause or another; and (2) secondary influences throwing a burden on the right heart, such as anoxia, hypercapnoea, increased blood volume, polycythaemia, increased cardiac output, and disordered breathing mechanics (1). Our understanding of the vascular remodeling process continues to advance, though our clinical understanding of the contribution of PH to exercise limitation in patients with COPD remains poor. In this chapter we have focused on the process of pulmonary vascular remodeling in patients with COPD, its pathophysiology, and potential therapy.

II.

The Normal Pulmonary Circulation

The pulmonary arteries and bronchi, with lymphatics run together in a single connective tissue sheath in the center of pulmonary segments and lobules. 169

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The airways and conventional arteries branch symmetrically and dichotomously. The arterial system also gives off extra branches perpendicularly, called supernumerary or short branches. The veins pursue a different course along the edge of the lobules in the interlobular septa but branch similarly. Cumming and coworkers (2) describe the “convergent approach” of naming pulmonary arteries. The most peripheral precapillary artery (nZ300 million |1 per alveolus) is numbered “order 1.” It has a diameter of 13 mM. This order increases accordingly until the main pulmonary artery, which is of the “order 17” with a diameter of 30,000 mM. The cross-sectional area of the pulmonary arterial tree expands, with an abrupt ninefold increase in area between order 1 and 2. Thus 90% of the arterial cross-sectional area is located in the precapillary vessels. Vascular conductance (w1/resistance) is greatest in the precapillary arteries and volume predominates in the larger vessels. The larger vessels are stiffer as they contain more elastic tissue. The vascular compliance (proportional change of volume per unit change of transmural pressure) correcting for vessel size is the same in large-and medium-sized vessels. There are grossly five types of arteries, based on their wall structure: (1) elastic arteries (orders 17–13), (2) muscular arteries (13–3), (3) partially muscular (5–3), (4) Nonmuscular (5–1), and (5) Supernumerary arteries that branch from orders 11–12, have sphincters at their origin provide and blood to alveoli adjacent to the conduit arteries and bronchi (3). The muscular pulmonary artery was initially defined by Brenner in 1935 but more recently refers to vessels with a diameter of 100–500 mm. This class of vessel has a thin media of circularly orientated smooth muscle and does not include any obvious longitudinal muscle in its wall. They have thicker muscle layers in relation to their external diameter (2–5%). The nonmuscular arteries or pulmonary arteriole, which is an arterial vessel with a diameter !80 mm and whose wall consists of a single elastic lamina, is normally devoid of a muscular media except at the site of its origin from a muscular pulmonary artery. In this region the smooth muscle cell is replaced by a pericyte whose basement membrane fuses with that of the endothelial cell lining the vascular lumen (3–5). A. Pulmonary Vascular Development

In fetal lung, the pulmonary vasculature develops via at least two processes that probably occur concurrently: vasculogenesis, in which new blood vessels form in situ from angioblasts, and angiogenesis, which involves sprouting of new vessels from existing ones. Large pulmonary arteries originate via the process of angiogenesis. A third process, fusion, is necessary to ultimately “connect” the angiogenic and vasculogeneic vessels (6). The thickness of the muscle coat of small arterial vessels decreases after birth from about 16% to 2%, most of which occurs in the first 4 months of life. This change is linked to the expansion of the lung with air and the fall

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of pulmonary arterial pressure from aortic levels in the fetus to low levels after birth (3).

B. The Capillary Network

About 300 million precapillary vessels lead into a network of alveolar septal capillaries. The capillary surface area is about 125 m2 (w86% of the alveolar surface area). Individual capillaries are not much wider than red blood cells, so the microvascular bed at normal pulmonary pressures is one red cell thick. Alveolar capillaries have a thick and thin side. The thin side consists of cytoplasmic extensions of the luminal endothelial cells and the alveolar epithelial cells with their fused basement membrane. The thick side has collagen, elastin, and fibroblast processes. The pulmonary vessels can be functionally separated into alveolar and extra-alveolar vessels. Alveolar vessels in the alveolar septum narrow as the lung expands and the septum stretches. Extra-alveolar vessels widen as lung expands. This precapillary segment of the pulmonary vascular bed, being the site of the greatest pressure decrease along the pulmonary circulation, contributes to the majority of pulmonary vascular resistance (PVR). It follows that small change in tone or wall structure at this level can lead to large elevations of pulmonary arterial pressure. For example, from Poiseuille’s law for steady flow: PVR a 8m$L=pD

(1)

where m is the viscosity of blood, L is vascular length, and D is vessel diameter. It can be seen that at constant length, the resistance in a tube doubles if D is decreased by 16%. The most distal segments of the precapillary arterioles contain an endothelial layer underlined by a single elastic lamina. Two smooth musclelike cell types are found in the more distal segments: (1) intermediate cells that, unlike smooth muscle cells, lie inside the internal elastic lamina and (2) pericytes that lie beneath the endothelium in small precapillary vessels that do not possess an elastic lamina (7). Pericytes are difficult to define as they constitute a heterogeneous population of cells, and their ontogeny is not well understood. They exhibit considerable plasticity and have the capacity to differentiate into other mesenchymal cells, such as smooth muscle cells and fibroblasts (8). Their function may also be the production and organization of the extravascular matrix and basement membranes. The main function of the pulmonary circulation is to deliver deoxygenated blood to the alveolar capillaries where gas exchange occurs. To achieve this vital role, the pulmonary circulation has this high flow, low resistance vascular bed (a distensible reservoir) to protect the thin blood gas barrier from high intravascular pressures that would otherwise promote alveolar oedema formation.

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On the basis of Ohm’s Law: Change in pressureZflow!resistance PpaKPpv Z Q !PVR Ppa Z ðQ !PVRÞ C Ppv

(2)

where Ppa and Ppv are the mean pulmonary arterial and venous pressures, Q is right-sided cardiac output, and PVR is already defined earlier. In clinical practice, the pulmonary venous pressure can be approximated by the pulmonary capillary wedge pressure (PCWP), which is measured via pulmonary artery catheter: PA mean Z ðQ !PVRÞ C PCWP

(3)

Thus, pulmonary artery pressure is determined by three variables: The volume of pulmonary blood flow (Q), resistance in the pulmonary vascular bed (PVR), pulmonary venous pressure (Ppv, which usually is correlated with PCWP). An abnormal increase in any of these variables will lead to PH and subsequent right ventricular overload. III.

Effects of COPD on Pulmonary Hemodynamics

Severe COPD is frequently complicated by PH, which decreases the 4-year survival rate of patients with COPD from 79% to 46% (9). PH generally results from an increased cardiac output, increased capillary wedge pressure or an increased PVR. In COPD the role of an increased cardiac output or increased capillary wedge pressure is thought to be negligible. Abnormally high cardiac output as well as capillary wedge pressure has been seen to be associated with exacerbations resulting in acute respiratory failure. Patients with COPD and severe PH usually have an increased PVR but a preserved cardiac output. The degree of PH does not appear to alter vascular structure consistently, although there is a trend towards an increase in the thickness of the muscular media in the smaller vessels (10). A number of factors contribute to the increased PVR observed in patients with COPD, including, for example, loss of the pulmonary capillary bed in emphysema. However, pulmonary vascular remodeling is the dominant process and one that is potentially preventable or reversible by therapeutic intervention (11). In patients undergoing right heart catheterization, an elevated pulmonary arterial pressure is reported in up to 40% of patients (12–14). Using Doppler-echocardiography it was shown that PH is common, affecting 55% of COPD patients attending a respiratory clinic and is often present in patients with only mild resting hypoxaemia (15). Opinion is divided as to whether the degree of PH in COPD is proportional to the severity of airflow limitation, hypoxia, or hypercapnoea (16). Although a reduced alveolar PO2 contributes to the pathogenesis of PH in patients with COPD, there is a poor correlation between arterial PO2 and pulmonary arterial

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pressure. Indeed, structural, and functional abnormalities in the pulmonary circulation are apparent at very early stages of COPD, when hypoxemia is not evident (17). PH in COPD is generally considered to be mild to moderate, characterized by an increase in mean Ppa of 20–35 mmHg (18). This increases further during exercise and more so in those more than 50 years of age (19). Exercise performance may be limited by right ventricular impairment in some patients with COPD (20). Before the development of significant hypercapnoea or hypoxemia, patients with mild COPD have a normal cardiac output (19,21). Changes in airway resistance may augment PVR in patients with COPD by affecting the alveolar pressure. The effect of airways resistance is particularly important when ventilation increases as in exacerbations of COPD. In patients with moderate to severe COPD, hyperventilation increases both the pulmonary arterial and pulmonary artery wedge pressures without changing the cardiac output. The same study has demonstrated a correlation between Ppa and FEV1 in COPD (22). Even small increases in pulmonary arterial flow in COPD during exercise significantly elevates Ppa (18,23,24). Similar changes are seen during nocturnal falls in oxygen saturation and during acute exacerbations of COPD (25,26). The abnormal increase in pulmonary arterial pressure on exercise is due to the relatively fixed PVR in these patients (27). In normal subjects the PVR falls with increasing cardiac output, and the pulmonary arterial pressure rises only modestly. Most importantly, the presence of PH in COPD patients is strongly associated with increased mortality and morbidity in these patients, independent of the degree of airflow obstruction (28,29). This increased morbidity and mortality seems to correlate with indices of right ventricular function in COPD. For example, electrocardiogram (ECG) evidence of right ventricular dysfunction was closely correlated with poor survival in a large cohort of COPD patients (30).

IV.

Effects of COPD on Pulmonary Vasculature

The mechanism(s) by which PH develops in COPD are numerous (Fig. 1). The contributory factors include (i) hypoxaemia with associated vasoconstriction (31,32); (ii) emphysematous destruction of the vascular bed (31); (iii) decreased vascular calibre and vessel distensibility, due to thickening of the intima and muscular media of the vessels (32); (iv) increased intrathoracic pressure secondary to airways obstruction (33); (v) cigarette smoke has been shown to cause changes to the pulmonary circulation (34); and (vi) inflammation. More recently some of these features have been challenged by Howard and others (35). These workers suggested that chronic hypoxia causes thickening of the walls of pulmonary arterioles, but these changes do not lead to structural narrowing of the lumen by encroachment. Moreover, hypoxia leads to new vessel formation within the pulmonary vasculature and not loss of vessels. Such neovascularization is thought to be a beneficial adaptation by increasing the area of the gas exchange

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Vascular remodeling

Exercise Sleep Acute exacerbations Age Hyperinflation

Inflammation

Pulmonary Hypertension Destruction of capillary bed

Genetic susceptibility

Thrombosis

Environmental factors Smoking Hypoxia Hypercapnia

Figure 1 Interacting factors which contribute to the pathogenesis of pulmonary hypertension in COPD.

membrane. These findings are supported by recent reports that inhibitors of the RhoA pathway can acutely reduce PVR in chronically hypoxic lungs to near normal values, suggesting that “fixed” structural changes may not be the dominant or only mechanism underling chronic PH. V.

Morphological Changes

Several groups of investigators have described quantitative differences in the pulmonary arteries and arterioles of patients with COPD (36–39). Pulmonary vascular abnormalities in patients with mild-to-moderate COPD mainly consist of thickening of the intima of muscular pulmonary arteries, which may reduce the lumen size and an increased proportion of muscularized arterioles (Fig. 1) (36,40–43). Changes in the radial muscular layer are less conspicuous, and the majority of studies have failed to consistently show muscular hypertrophy at this site. A. Muscular Pulmonary Arteries

Patients with end-stage COPD and cor pulmonale show changes in the intima and media of pulmonary muscular arteries and precapillary arterioles. Postmortem studies in these patients have revealed deposition of longitudinal muscle in the intima interwoven with a skein of elastic fibers, intimal elastosis, or intimal fibrosis. The longitudinal muscle layer was often thick, resulting in a reduction in the luminal diameter (10,44). There is abundant proliferation of SMCs and intense deposition of both elastin and collagen fibers in the intimal layer of pulmonary muscular arteries in patients with mild COPD and in smokers with normal lung function. There are no differences in the cellular and extracellular

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components of the intima between COPD patients and smokers. Remodeling of pulmonary arteries is not exclusive to patients with advanced disease as it has also been shown in patients with mild COPD (36,40–43). Hale and colleagues demonstrated an increase in the number of muscular arteries less than 200 mm in diameter (36). Histochemical and immunohistochemical studies conducted in pulmonary muscular arteries of patients with mild COPD indicate that the enlargement of the intima is produced by the proliferation of smooth muscle cells, some of which lose the contractile phenotype, and the deposition of both elastic and collagen fibers. The nature of cell proliferation and extracellular matrix protein deposition in patients with mild disease closely resembles that shown in patients with endstage COPD (45,46). Interestingly, studies conducted in smokers with normal lung function have also revealed intimal thickening in pulmonary muscular arteries (41), the characteristics of which do not differ from that shown in patients with mild COPD (46). In severe COPD the media of muscular pulmonary arteries was normal or atrophic (47). The adventitia has been shown to increase in total area with PH suggesting that all the three layers of the pulmonary vessel wall are affected by remodeling in COPD. Thirteen subjects from a cohort of patients studied by Wright showed that the adventitial changes correlated well with the MPAP measured at rest and in room air, which is an early feature of pulmonary venous hypertension. Their data suggested that pulmonary artery pressure increased on exercise in a group of mild to moderate COPD patients who were not hypoxic at rest, and that this was associated with structural changes in the muscular arteries (43). B. Nonmuscular/Partially Muscular Arteries (15–80 mm)

There is development of circular muscle layers in the vessels of this size bounded externally by an elastic lamina and internally by a less developed partially formed elastic lamina. The new muscle once again is thought to narrow the lumen and appears as a peripheral extension of the medial coat of muscular arteries. This often extends into the precapillary vessels (48). The changes in small pulmonary arteries of 15 patients with COPD have been investigated by light and electron microscopy. Image analysis once again found that the structural changes in the pulmonary arteries of the patients with COPD were characterized by muscularization of nonmuscular arterioles, media hypertrophy, longitudinal smooth muscle bundles in the intima and fibrosis in both the media and intima. In the course of time, these lesions resulted in thickening of the arterial wall and narrowing of the lumen. Clinically, the patients developed PH causing cor pulmonale. The structure of the arterial wall at different segments when compared showed significant differences between COPD and control groups. It is likely that these arteriolar changes are closely related to the development of PH (49).

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Cellular Changes

A. Endothelium

The endothelium forms the interface between hemodynamics and the underlying vascular wall and provides the antithrombogenic, semipermeable barrier between the vascular and extravascular fluid compartments. In addition to also fulfilling a variety of metabolic functions, the endothelium has profound effects on vascular tone, growth, and differentiation, as well as the vascular response to injury. The initiating injury in the context of PH in COPD may be hypoxia, increased flow (shear stress), inflammation, or the response to cigarette smoke on a background of genetic susceptibility. The endothelial cell may respond to specific forms of injury in various ways that can affect the process of vascular remodeling. As well as directly altering cell proliferation, injury may alter many of the normal homeostatic functions of the endothelium by altering endothelial permeability, metabolism, production of growth factors, and coagulation pathways. Dinh-Xuan and coworkers (50) showed endothelial dysfunction in pulmonary arteries of patients with end-stage obstructive lung diseases (bronchiectasis and emphysema). Peinado et al. (41) showed that in mild COPD (FEV1 72% predicted) patients who had undergone lung resection for lung cancer there was impaired relaxation to the NO (nitric oxide)-dependent vasodilator adenosine diphosphate (ADP), in pulmonary arteries of patients with mild COPD. Maximal relaxation of pulmonary artery rings induced by ADP, and acetylcholine to a certain extent, were reduced. Inhibition of NO synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) practically abolished the response to NO-dependent vasodilators. Overall, these results are consistent with endothelial dysfunction in pulmonary arteries of patients with mild COPD. So, endothelial dysfunction in pulmonary arteries of patients with mild COPD, is probably associated with an impaired release of endothelium-derived NO. Endothelium-dependent vasorelaxation is diminished in cigarette smokers and in the lungs of individuals with COPD and hypoxic cor pulmonale (50,51). It is presumed that the absorption of tobacco smoke constituents affects endothelial cell function (51), but the true mediator of these vascular diseases associated with smoking is not known. Vascular endothelial growth factor (VEGF) is abundantly expressed in cells of lung tissue. This molecule is mainly implicated in the maintenance of vascular endothelial cell function and in vascular cell proliferation. VEGF could play a role in the pathogenesis of the intimal cell proliferation shown in pulmonary arteries of smokers and patients with mild COPD. There is an increased expression of VEGF in muscular pulmonary arteries of patients with moderate COPD and also in smokers with normal lung function, as compared with nonsmokers. This expression is associated with hypertrophy of the arterial wall. In contrast, in patients with severe emphysema, the immunohistochemical

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expression of VEGF in pulmonary arteries and its protein content in lung tissue tends to be low, despite intense vascular remodeling (52). Congenital nitric oxide synthase (NOS3) deficiency in mice enhances hypoxic pulmonary vascular remodeling and hypertension and RV hypertrophy. NO production by NOS3 seems critical to counterbalance pulmonary vasoconstriction caused by chronic hypoxic stress (53). The pulmonary endothelium of heavy smokers shows a decreased expression of endothelial nitric oxide synthase (eNOS). They also show diminished endothelium dependent relaxation and medial hypertrophy in the pulmonary arteries similar to that seen in COPD patients (54). Smooth Muscle Cells and Pericytes

Though the media of the human pulmonary artery appears homogeneous by conventional histochemical staining techniques and light microscopy, heterogeneity is apparent using antibodies directed against contractile proteins (Fig. 2). In addition, heterogeneity in medial cell function is apparent in vitro, eg, in the release of adrenomedullin (ADM) (55) and expression of binding sites for ANG-II (56). Furthermore, heterogeneity in smooth muscle cells is also observed in cells isolated from different anatomic locations in the lung, i.e., proximal versus peripheral arteries. For example, human pulmonary artery smooth muscle cells from the peripheral pulmonary circulation (arteries 1 to 2 mm in diameter) proliferate more rapidly and are more sensitive to the antiproliferative effects of prostacyclin analogues than cells isolated from the main pulmonary artery (57). The process of extension of smooth muscle into normally nonmuscular arteries is probably brought about by differentiation and hypertrophy of intermediate cells and pericytes already present in the wall. Indeed pericytes have been shown to exhibit great plasticity in culture, being capable of differentiation into phagocytes, osteoblasts, and adipocytes (58). Fibroblasts

In some model systems, particularly in hypoxia models, the adventitial fibroblast appears to be the first cell activated to proliferate and to synthesize matrix proteins in response to the pulmonary hypertensive stimulus (59). The mechanisms that enable the adventitial fibroblast to migrate into the media (and ultimately the intima) are currently unclear, but there is good evidence to suggest that upregulation of matrix metalloproteinases (MMP2 and MMP9) occurs and that these molecules are involved in migration (Fig. 3). Similar to smooth muscle cells, phenotypic distinct subpopulations of fibroblasts have been described in the bovine pulmonary artery (60). Fibroblasts are also heterogeneous in their responses to hypertensive stimuli. For example, in response to hypoxia, some pulmonary artery fibroblasts proliferate, with increased activity of mitogen activated protein (MAP) kinases (61), whereas in others, no response is observed. It has been proposed in PH that there is selective expansion of subpopulations of fibroblasts within the adventitia (61). In fetal

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Figure 2 Heterogeneity of pulmonary artery smooth muscle cell protein expression within the human pulmonary arterial media. Immunofluorescence staining reveals homogeneous expression of smooth muscle alpha-actin (A), but desmin expression is heterogeneous and localized to the outer part of the media (B).

cells, the hypoxia-induced proliferation is related to the activity of specific isoforms of protein kinase C (62). VII.

Mechanisms of Vascular Remodeling in COPD

A. Hypoxia

In COPD regional alveolar hypoxia occurs due to local airflow obstruction. Systemic hypoxemia is due to an alveolar ventilation perfusion mismatch (63). Sustained local or global hypoxia within the lung leads to pulmonary vasoconstriction and ultimately vascular remodeling. There is a reduction in the response to hypoxic stimulus in the pulmonary arteries of COPD patients with moderate disease and mild hypoxemia. This reduction is associated with decline

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Figure 3 Normal small pulmonary artery (A) and remodeled artery in a patient from COPD (B), showing increased muscularization and the presence of a longitudinally oriented muscle bundle in the intima.

in endothelium-dependent relaxation, and probably reflects the reduced ability of the remodeled artery to readily constrict or dilate. Furthermore, there is a correlation between the contractile response to hypoxia in organ bath studies and the in vivo PaO2 (24). Studies performed in COPD patients have shown a wide variation in the individual responses of the pulmonary circulation to changes in inspired O2 concentration (40,64,65). In general, patients with end-stage COPD tend to exhibit a minimal vasodilator response to O2 breathing than patients with milder forms of the disease (10,42). When PH occurs associated with alveolar hypoxia, such as in COPD or from residence at high altitude, the remodeling observed in small pulmonary arteries is rather different from that seen in other forms of PH. Although distal neomuscularization still occurs, laying down of longitudinally orientated layer of smooth muscle within the intima of small (80 to 500 mm) pulmonary arteries and formation of “inner muscular tubes” is characteristic of hypoxia induced PH in humans (66).

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Chronic hypoxia can be induced by exposing animals to normal air at hypobaric pressures or to oxygen-poor air at normal pressure. Both vascular smooth muscle cells (VSMCs) and adventitial fibroblasts (67,68) proliferate under these conditions, but no significant endothelial cell proliferation occurs. The muscularization of the precapillary pulmonary arterioles in response to chronic hypoxia and vasoconstriction is slowly reversible when normal oxygen levels are restored. Pulmonary arterial muscularization is not specific to hypoxia. Increased pulmonary blood flow and the associated elevation in vascular shear stress can also produce muscularization, as can treatment with the plant alkaloid monocrotaline (69). B. Growth Factors Transforming Growth Factor-b (TGF-b) Family

The role of the transforming growth factor-b superfamily in pulmonary vascular remodeling was recently highlighted by the identification of heterozygous germline mutations in the bone morphogenetic protein type II receptor (BMPR-II) in familial primary PH. The TGF-b superfamily is composed of multifunctional mediators including activins, TGF-bs 1-3, bone morphogenetic proteins (BMP), and growth and differentiation factors (GDFs). At least 15 BMPs have been identified to date. The TGF-b superfamily has diverse roles in a wide variety of physiologic processes, including cell proliferation, differentiation, immunity, and inflammation (70,71). The contribution of these pathways to hypoxic PH at an early stage. Fibroblast Growth Factor (FGF)

In view of their important role in chronic inflammation and fibrosis, fibroblast growth factors (FGFs) may play an important role in vascular remodeling. FGFs exert their biologic effects via binding to four high-affinity FGF transmembrane tyrosine-kinase receptors (FGFR) (72). Distinct FGF subtypes bind with different affinity to the various FGFR. Alternative splicing and regulated protein trafficking further modulate the intracellular events and resultant response initiated by FGF ligand-receptor interaction (72). In the lung as well as in the vascular system, FGFs have been implicated in several pathologic conditions. FGF-1 and FGFR-1 were shown to be upregulated during the development of lung fibrosis (73). Moreover, vascular remodeling in response to increased blood pressure is associated with elevated levels of basic FGF (74). In COPD there is an increase in the expression of FGF-2 in small pulmonary vessels (!200 mm) and FGF-1 in large (O200 mm) pulmonary vessels, whereas FGFR-1 is increased in both vessel types. FGF-2 is localized to endothelial and VSMCs, and its expression is increased in pulmonary vessels with diameter !200 mm in patients with COPD, indicating a role for this growth factor in vascular remodeling.

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There is a significant inverse correlation of FEV1 with FGF-1 staining in the media of large vessels and with FGF-2 expression in both endothelium and vascular smooth muscle of small vessels (75). C. Vasoactive Mediators Endothelin 1

Endothelin-1 (ET-1) is a potent vasoconstrictor and mitogenic agent released by endothelial and smooth muscle cells and has been implicated in the pathogenesis of PH (76). BQ-123, an ETA-receptor antagonist, attenuates hypoxic PH in rats (77), and it is generally thought that hypoxia plays a major role, through the induction of ET-1, in the development of PH in patients with COPD (78). Cigarette smoke extract (CSE) stimulates ET-1 gene expression via protein kinase C (PKC) in pulmonary artery endothelial cells (PAECs). Platelets and CSE showed synergism in the stimulation of ET-1 gene expression, possibly through the activation of platelets by CSE. Further studies are needed to find the components of CSE that are responsible for this stimulating effect and for the mechanisms of synergism between platelets and CSE in the stimulation of ET-1 gene expression. PH in patients with COPD is associated with the increased expression of ET-1 in vascular endothelial cells (76). The plasma ET-1 level is not affected by acute progression of PH and hypoxemia during exercise, but persistent hypoxemia is associated with an increase in the plasma ET-1 level. In addition, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) may modulate the pulmonary vascular tone both directly and by inhibition of ET-1 release in these patients (79). Nitric Oxide

NO is an endogenous vasodilator that contributes to the low vascular resistance in the pulmonary circulation (80). NO is an important contributor to the regulation of pulmonary vascular tone in humans (81). In hypoxic lung disease the pulmonary endothelium appears to be less able to release NO than normal (41), probably as a result of reduced expression of constitutive nitric oxide synthase and a concomitant increase in such vasoactive mediators as ET-1 (79,82). Exposure to CSE results in a decrease in eNOS protein and eNOS mRNA contents, as well as in eNOS activity in PAECs (83). A reduction in NO production by cigarette smoke is presumed to be responsible, at least in part, for the increased risk of systemic and pulmonary vascular disease and dysfunction in cigarette smokers (84). Adrenomedullin

ADM is a hypotensive peptide that was originally isolated from human phaeochromocytoma. ADM consists of 52 amino acids with an intramolecular disulphide bond and has some similarity to calcitonin gene-related peptide (85). It has been demonstrated that VSMCs possess specific ADM receptors that are

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functionally coupled to adenylate cyclase (86). It has also been demonstrated that ADM is actively produced and secreted by vascular endothelial and smooth muscle cells (87). Plasma levels of ADM are elevated in patients with severe PH. These patients show a significant reduction of ADM concentration across the pulmonary circulation, suggesting that this substance is partly metabolized in the pulmonary circulation. Plasma levels of ADM are significantly correlated with right ventricular haemodynamics and those of ANP, and plasma levels of ADM increase with the elevation of PVR. These findings suggest that plasma levels of ADM increase in proportion to the extent of PH (88). Xu et al. (89) demonstrated that COPD patients with PH had higher plasma ADM levels than those without PH. ADM may thus play an important protective role as a local autocrine/paracrine factor in the development of COPD and PH. D. Thrombosis

In 1959 McLean (90) suggested that the intimal thickening of pulmonary arteries in COPD patients might be due to small vessel thrombosis secondary to peripheral airways inflammation. COPD patients exhibit increased levels of prothrombin F1C2 fragments, a marker of thrombin generation and fibrinogen. Thus COPD patients may manifest an ongoing prothrombotic state that could potentially account for thrombosis occurring in pulmonary vessels (91). Up to 10% of patients presenting with an acute exacerbation of COPD were found to have pulmonary emboli, using CT pulmonary angiography. An autopsy study showed that 28% of patients with chronic pulmonary disease and no previous pulmonary thromboembolism had central pulmonary artery lesions (92). A study looking at central pulmonary artery lesions using transeosophageal echocardiography in a population of stable COPD patients found that 12% had significant lesions suggestive of adherent or organized thrombus (93). The increase in viscosity caused by secondary polycythemia is thought to be one of the factors contributing to PH secondary to hypoxic COPD. Activation of the renin-angiotensin system is associated with the development of secondary erythrocytosis in chronically hypoxemic patients with COPD. The exact mechanism is not yet fully understood, but angiotensin II may be responsible for inappropriately sustained erythropoietin secretion or direct stimulation of erythroid progenitors (94). E.

Inflammation

Interestingly, in the lungs of cigarette smokers with mild COPD, intimal thickening is associated with a lymphocytic infiltrate, predominantly of CD8KT cells, and correlates with a loss of endothelium-dependent relaxation (95). Cigarette smoking causes an inflammatory reaction in the airways of patients with COPD. Peinado et al. (95) demonstrated the presence of an inflammatory infiltrate of CD8CT-lymphocytes in the adventitia of pulmonary arteries of patients with COPD; suggesting that an inflammatory process might be involved

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in the pathogenesis of the structural and functional alterations of the vascular bed in patients with COPD. The role of an inflammatory mechanism has been well established in several models of PH produced by toxic agents (e.g., monocrotaline), sepsis, and hyperoxia (96). Further, inflammatory cells (T and B lymphocytes) have been described surrounding the plexiform lesions in primary PH and in other forms of PH (97). The precise mechanism by which inflammatory cells may induce vascular remodeling and endothelial dysfunction remains unknown. Inflammatory cells may constitute a source of cytokines and growth factors, such as VEGF and transforming growth factor beta (TGF-b), that may target the endothelial cells and contribute to the development of structural and functional abnormalities of the vessel wall. Furthermore, some proinflammatory cytokines, such as interleukin-1 (IL-1), have also the capability to stimulate fibroblast growth and collagen synthesis, thus amplifying the vessel remodeling process (98). Taken together, these data suggest that inflammatory cells may participate in the pathogenesis of the structural abnormalities of pulmonary vessels in patients with mild COPD, likely through the release of both cytokines and growth factors. Under these conditions, it is not surprising that the degree of endothelium-dependent relaxation of pulmonary arteries was also inversely correlated with the severity of leukocytic infiltrate. Because endothelial cells play a key role in the regulation of vascular remodeling, their alteration by cytokines and growth factors released by inflammatory cells may not only promote the vessel remodeling but also impair the endothelial function. Alternatively, endothelial dysfunction of pulmonary arteries could result from a direct effect of tobacco smoking on endothelial cells. A study looking at morphometry of peribronchiolar and perivascular fibrosis outside the smooth muscle layer of bronchioles and outside the external elastic lamina of muscular pulmonary arteries in COPD patients shows that patients with chronic bronchitis had significantly thicker peribronchiolar fibrosis in bronchioles of 1 mm or less in diameter and also thicker perivascular fibrosis of the adjacent muscular pulmonary arteries than patients with emphysema or normal individuals. The extent of perivascular fibrosis was significantly correlated with peribronchiolar fibrosis only in the muscular pulmonary arteries adjacent to the bronchioles and not in those away from the bronchioles. These findings may have been due to direct extension of chronic inflammation from bronchioles to the adjacent muscular pulmonary arteries in chronic bronchitis but not in pulmonary emphysema. Such perivascular fibrosis might lead to sustained PH (99). F.

Effects of Cigarette Smoke on the Pulmonary Vasculature

Both animal (100–102) and human studies (42,43) have demonstrated that cigarette smoke can induce PH and is associated with structural alterations in the pulmonary vascular bed, with physiological evidence of a rigid vascular bed. Increased vascular resistance could be secondary to alterations of the capillary network or of the artery wall.

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Animal studies have shown that about two thirds of the lung have to be removed before a rise in pulmonary artery pressure occurs at rest (103). This would suggest that simple capillary loss is not an entirely adequate explanation. There is however evidence in animal models of an association of PH with emphysema. Animal models with papain induced emphysema have shown associated PH. The altered pressure-flow relationship found by Rubin and colleagues in their model of papain induced emphysema in dogs supports the idea that emphysema causes capillary loss (104). A significant correlation has been reported between air-space size and MPAP in papain-induced emphysema, and this correlation was again noted in a study in smoke-exposed guinea pigs (34). Furthermore, exposing guinea pigs to cigarette smoke for one month increased MPAP in the absence of air space enlargement. This suggests that there are other processes independent of capillary loss as a result of air-space enlargement causing PH in these animals and that the development of PH in these animals precedes emphysema (101). Santos and colleagues (46) demonstrated increased expression of VEGF protein by immunohistochemistry in the pulmonary arteries of smokers with normal lung function and patients with moderate COPD, as compared with nonsmokers. Increased levels of VEGF mRNA and protein were also found in lung tissue samples. This expression was associated with hypertrophy of the arterial wall. In contrast, in patients with severe emphysema, the immunohistochemical expression of VEGF in pulmonary arteries and its protein content in lung tissue tends to be low, despite intense vascular remodeling. Wright et al. reported that a single smoke exposure acutely but transiently upregulated gene expression of the vasoconstrictor/vasoproliferative agents ET and VEGF in pulmonary arteries from rat lungs. They then showed that in chronic smoke exposed animals, there were significantly elevated but variable increases in gene expression, with some animals demonstrating 30- to 50-fold increases. Increases in ET and VEGF expression occurred early and persisted through the exposure period, whereas increases in expression of the vasodilator, eNOS, developed more slowly. Protein levels of these mediators were also elevated as determined by immunohistochemistry and correlated with increases in gene expression levels. They concluded that in some animals cigarette smoke induces persisting and marked vascular production of mediators that control vascular muscularization and contraction/dilation. These changes may be important in the development of smoke-induced PH (105). Cigarette smoking results in a significant increase in plasma ET-1 levels (106), and the ETA-receptor antagonist BQ-610 blocks cigarette smoke-induced mitogenesis in rat airways and vessels (107). These findings suggest that ET-1 may contribute to the pulmonary vascular abnormalities associated with smoking and may be important in the eventual development of PH and cor pulmonale. However, the functional significance of these vascular abnormalities associated with smoking is unknown because smokers without development of COPD (i.e., without alveolar hypoxia) rarely develop overt PH.

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The serine-elastase inhibitor ZD0892 reduces smoke-induced cell proliferation and muscularization in the small pulmonary arteries and arterioles adjacent to the alveolar ducts (108). The correlation between measures of cell proliferation and muscularization indicates that muscularization is likely to be a direct consequence of smoke-driven cell division, a process shown to commence acutely after smoke exposure (109) and continues on a long-term basis during chronic exposures (110).

VIII.

Genetic Influences on Pulmonary Vascular Remodeling and Pulmonary Hypertension in COPD

Serotonin Transporter (5-HTT) Polymorphism

The serotonin transporter (5-HTT) is involved in the pulmonary artery smooth muscle hyperplasia that leads to PH. 5-HTT is encoded by a single gene expressed in several cell types, including neurons, platelets, pulmonary vascular endothelial cells, and pulmonary artery smooth muscle cells (PASMCs). Experimental and human studies showed that 5-HTT in PASMCs is a key determinant of pulmonary arterial remodeling. In addition to contributing to the uptake and subsequent inactivation of 5-HT passing through the lung, 5-HTT mediates PASMC proliferation. The ability of 5-HTT to cause PA-SMC proliferation is directly related to the level of 5-HTT gene expression, which is controlled by environmental and genetic factors. Hypoxia is a strong inducer of 5-HTT expression in vitro and in vivo (111). The level of 5-HTT expression is also determined genetically via an insertion/deletion polymorphism in the promoter region of the human 5-HTT gene. A study looking at 5-HTT gene polymorphism and PH in hypoxemic patients with advanced COPD showed that 5-HTT gene polymorphism appears to determine the severity of PH in hypoxemic patients with COPD. Patients carrying the LL genotype, which is associated with higher levels of 5-HTT expression in pulmonary artery smooth muscle cells than the LS and SS genotypes, had more severe PH than in LS or SS patients. Compared with control subjects, platelet 5-HTT protein is increased in COPD patients in proportion to the hypoxemia level, and strong 5-HTT immunostaining is observed in remodeled pulmonary arteries from COPD patients. eNOS Polymorphisms

A small study has examined the frequency of a polymorphism in the eNOS gene in patients with COPD (112). The frequency of the eNOS BB genotype was no different between COPD patients and controls. However, pulmonary artery pressures measured by echocardiography was significantly higher in patients with the BB genotype than in patients with the non-BB genotype. This interesting finding requires confirming in a larger study.

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Angiotensin converting enzyme (ACE) is responsible for the conversion of angiotensin I to angiotensin II and is present in very high concentrations in the lungs (113). Angiotensin II plays a role in the process of hypoxic PH (114,115). There is a suggestion from a small study that the deletion (D)/insertion (I) polymorphism in the ACE gene may be associated with PH evoked by exercise challenge in patients with COPD (116). Captopril, an ACE inhibitor, has been shown to influence mPAP, PVR, and lactate concentration after exercise in patients with COPD. Mean PAP, PVR, and lactate concentration after exercise are significantly lower with captopril than with placebo in patients with the II or ID genotypes but not in those with the DD genotype. In contrast, PvO2 after exercise has been shown to be significantly higher with captopril than with placebo in patients with the II genotype but not in those with the other genotypes (117). Lower ACE activity (whether defined by genotype or pharmacotherapy) is associated with lower exertional pulmonary artery pressure, lower PVR, higher mixed venous oxygen saturations, and lower blood lactate levels (118). One study showed a negative association in the ACE DD genotype and right ventricular hypertrophy in COPD patients with PH. The function of the renin angiotensin system may differ in primary PH from that in secondary PH in COPD (119).

IX.

Therapeutic Approaches

Long Term Oxygen Therapy

Studies have shown that continuous oxygen therapy (O15 hr per day) reduces mortality in hypoxic (arterial partial pressure of O2 !7.3 kPa) COPD patients, and stabilizes (but does not reverse) PH (120). Interestingly, the animal models of chronic hypoxic PH have shown reversibility of the pathologic changes with return to normoxia. Acute hypoxic pulmonary vasoconstriction in patients is relieved with administration of oxygen. Patients with high altitude, ambient hypoxia-associated PH are known to improve their condition as they acclimatize to sea level due to (1) relief of acute vasoconstriction, (2) likely regression of remodeling changes, and (3) decrease in polycythemia. It is less clear whether the pulmonary vascular remodeling changes associated with chronic hypoxia and lung disease are reversible simply with oxygen therapy. Two large clinical trials of long-term oxygen therapy (LTOT) in patients with COPD were reported in the early 1980s and showed an increased survival with the use of oxygen for more than 15 hour per day in hypoxemic patients. One study reported the mitigation of the progression of PH with the use of 15 or more hours of oxygen as compared with no oxygen therapy (120). The other study reported a mild decrease in PVR with near continuous oxygen therapy (19 hours) versus nocturnal (12 hours) administration (121). A separate study reported yearly decreases in PA pressure in severely hypoxemic patients who were treated with LTOT implicating some

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reversibility to the pulmonary vascular changes associated with the chronic hypoxia of COPD (122). Phosphodiesterase Inhibitors and Nitric Oxide

NO, a potent vasodilator, has an important role in pulmonary vasoregulation. NO has a diverse range of physiological effects in many tissues, including promoting relaxation of smooth muscle in the walls of blood vessels. NO signaling is mainly mediated by the guanylate cyclase/cyclic guanylate monophosphate pathway. The effects of NO effects on smooth muscle are mediated by cyclic guanosine monophosphate (cGMP). Inhaled NO supplementation is of benefit in the management of PH of the neonate (123) and is a recognized treatment for this condition. In chronic PH due to COPD, treatment for 3 months with combined inhalation of NO and oxygen caused a significant improvement in pulmonary haemodynamics, together with normal arterial oxygenation, which exceeded that of LTOT alone. In patients who received combination therapy the vasodilator response to NO after 3 months was larger than that observed during acute testing. Chronic inhaled NO has a greater effect than oxygen on the pulmonary vasculature (124). Recently, beneficial effects of the oral PDE-5 inhibitor sildenafil (originally approved for the treatment of erectile dysfunction) were reported for the treatment of PH. Sildenafil citrate enhances the effects of NO as a signaling molecule. The mechanism of action involves inhibition of an enzyme, cGMPspecific phosphodiesterase type 5 (PDE5), that degrades cGMP. Sildenafil, despite oral administration, displays some characteristics of a pulmonary selective vasodilator. In addition, evidence shows that sildenafil acts mainly in the vasculature of well-ventilated areas of the lung. However, to date, controlled randomized trials proving the efficacy of this approach for the treatment of PH in COPD are lacking. The results of such studies have to confirm the current encouraging findings before recommendations regarding the use of PDE-5 inhibitors as a new treatment can be made. Endothelin Antagonists (Bosentan)

ET-1 is a potent vasoconstrictor that is increased in the lungs of humans and animals with PH. ET-1 is released by endothelial cells, is a smooth muscle mitogen, and is proinflammatory. ET-1 binds to endothelin receptor A (ETA) and endothelin receptor B (ETB) to exert these effects. In patients with COPD, the plasma ET-1 level is not affected by acute progression of PH and hypoxemia during exercise, but persistent hypoxemia may be associated with an increase in the plasma ET-1 level. In addition, ANP and BNP may modulate the pulmonary vascular tone by interacting with ET-1 in these patients (79). Antagonism of PH in animal models with the dual ETA and ETB receptor antagonist bosentan was successful and led to successful human trials of bosentan in pulmonary arterial hypertension (PAH). In these trials, bosentan

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improved hemodynamics (125), dyspnea, and exertional capacity patients in patients with primary PH, scleroderma, and lupus (126,127). Although patients with parenchymal lung disease were not enrolled in these studies, physicians have begun to employ bosentan in these patients with PAH. Further prospective clinical trials need to be performed in specific patients groups due to the expense of this drug and the fact that up to 7% of patients in clinical trials developed a drug-related hepatitis that required dose reduction or cessation of therapy (128). Prostacyclin

Prostacyclin (prostaglandin PGI2) is an important endogenous pulmonary vasodilator acting through activation of the cyclic adenosine monophosphate (cAMP)-dependent pathways. PGI2 also inhibits the proliferation of VSMCs and decreases platelet aggregation. Prostacyclin synthesis is decreased in endothelial cells from PAH patients. In one study of acutely decompensated COPD patients with PH, PGI2 infusion failed to improve pulmonary haemodynamics and was associated with a deterioration in systemic arterial oxygen tension (129). An important concern with vasodilator therapy is the possibility of worsening ventilation perfusion (V/Q) matching, as blood is diverted through dilated vessels to less well ventilated alveoli. Lung delivery of PGI2 or one of its longer acting analogues may overcome this problem in COPD. ACE Inhibitors

Chronic inhibition of ACE activity, and thus angiotensin II generation, may have benefits in the long term treatment of patients with such chronic lung disease as COPD through (1) potential effects on pulmonary inflammation, architecture, and vasculature; (2) effects on respiratory drive and respiratory muscle function; (3) effects on the efficiency of peripheral use of oxygen; and (4) improvements in skeletal muscle functional capacity in the face of reduced oxygen delivery. The potential roles for ACE inhibitors and angiotensin II antagonists in the long term treatment of pulmonary disease and COPD needs further study (117,118). References 1. The Contributions of Right Heart Catheterisation to Physiology and Medicine, with Some Observations on the Physiopathology of Pulmonary Heart Disease, 1956. 2. Singhal S, Henderson R, Horsfield K, Harding K, Cumming G. Morphometry of the human pulmonary arterial tree. Circ Res 1973; 33:190–197. 3. Hughes J, Morrell N. Pulmonary circulation from basic mechanisms to clinical practice. 1st ed. London: Imperial College Press, 2001. 4. Wilkinson M, Langhorne CA, Heath D, Barer GR, Howard P. A pathophysiological study of 10 cases of hypoxic cor pulmonale. Q J Med 1988; 66:65–85.

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123. Roberts JD, Fineman JR, Morin FC, et al. Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. N Engl J Med 1997; 336:605–610. 124. Vonbank K, Ziesche R, Higenbottam TW, et al. Controlled prospective andomized trial on the effects on pulmonary haemodynamics of the ambulatory long-term use of nitric oxide and oxygen in patients with severe COPD. Thorax 2003; 58:289–293. 125. Williamson DJ, Wallman LL, Jones R, et al. Hemodynamic effects of bosentan, an endothelin receptor antagonist with pulmonary hypertension. Circulation 2000; 102:411–418. 126. Rubin LJ, Badesch DB, Barst RJ, et al. Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 2002; 346:896–903. 127. Channick RN, Simonneau G, Sitbon O, et al. Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet 2001; 358:1119–1123. 128. Maloney JP. Advances in the treatment of secondary pulmonary hypertension. Curr Opin Pulm Med 2003; 9:139–143. 129. Archer SL, Mike D, Crow J, Long W, Weir EK. A placebo-controlled trial of prostacyclin in acute respiratory failure in COPD. Chest 1996; 109:750–755.

9 Angiogenesis: Lessons Learned from Skeletal Muscle

PETER D. WAGNER and ELLEN C. BREEN Division of Physiology, Department of Medicine, University of California—San Diego, La Jolla, California, U.S.A.

I. Angiogenesis: Overview of a Rapidly Changing Field Angiogenesis can be defined as structural expansion of a microvascular bed occurring as outgrowth from the existing vasculature. This is distinct from vasculogenesis, which refers to the development of new vasculature, as most obviously occurs in development. Angiogenesis is a widely studied and very complex process. Almost daily, new twists are discovered as additional factors that initiate or regulate the process are discovered. The field is progressing very rapidly. Interestingly, there is as much interest in finding ways to suppress angiogenesis as there is in finding ways to augment it. Thus, because a tumor has difficulty growing without adequate vasculature, antiangiogenic approaches are being intensely studied as a potential method for treating cancer (1). On the other hand, in diseases caused by cellular ischemia, such as coronary artery disease, therapeutic approaches to augment angiogenesis are being developed as a logical way to restore O2 availability (2,3). Angiogenesis begins when an appropriate physiological stimulus is created. As discussed later in this chapter, local hypoxia has been thought to be a principal stimulus (4) (although there is increasing evidence that nonhypoxic stimuli may also be important) (5–18). The role of hypoxia can be understood in the 197

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context of cancer biology. For example, as a group of tumor cells grows, expanding outward from a cluster of just a few cells, the cells at the center of the cluster become farther and farther removed from sources of oxygen required to sustain them. As a result, local PO2 falls in the center of such clusters as the rate of diffusion of O2 into the cluster center is reduced. These cells are thus rendered hypoxic. Then, through probably a variety of cellular O2 sensing mechanisms (19), levels of hypoxia-sensitive transcription factors are increased in the hypoxic cells. These transcription factors, the most well known of which is Hypoxia Inducible Factor 1 (HIF-1a) (20), then bind to hypoxia responsive elements (HRE) in the promoter region of a number of genes. This binding in turn results in activation of their transcription, increased levels of their mRNA, and translation that increases levels of the encoded proteins. It is no accident that the genes that are activated in this way encode proteins that are well known to have functions that serve to protect or rescue cells from the effects of hypoxia. Prominent amongst the genes activated by hypoxia are those critical to angiogenesis, although many others are also upregulated. Included in the latter group, erythropoietin is activated by hypoxia (20). This is well-known to increase circulating red cell numbers and thus hemoglobin levels to augment O2 carrying capacity in the blood. The enzymes of the glycolytic pathway are similarly affected by hypoxia. This makes sense as a way to compensate anaerobically for loss of oxidative capacity due to hypoxia. In this way, ATP can be generated for cell survival even if O2 availability is limited. Genes for glucose transporters are similarly upregulated by hypoxia, as are many others. For angiogenesis, genes encoding a number of proteins that can positively or negatively affect the angiogenic process are so regulated. Analogous genes are being identified at a rapid pace. Not all are stimulated simply by hypoxia, and the signaling pathways and the complex interactions among these genes are under active and intense study at the present time. It should not be assumed that transcriptional activation of hypoxiasensitive genes is the only mechanism by which the levels of their mRNA’s and/or encoded proteins can be increased. Hypoxia has a second, general effect on mRNA levels of some genes: mRNA stabilization. For example, HuR binding of the mRNA for, vascular endothelial growth factor (VEGF) is increased in hypoxemia (and ischemia) (21). This reduces VEGF message breakdown, increasing its message levels and thus translation to protein. Hypoxia may also affect gene activation by interfering at the protein level with molecules that are essential to the hypoxic response. For the transcription factor HIF-1a, it is thought that hypoxia acts mostly posttranscriptionally. Here, whereas in normoxia HIF-1a protein is rapidly broken down by the proteasome, this breakdown is reduced in hypoxia (22,23). HIF degradation requires prolyl hydroxylase, which is O2 dependent. Hypoxia thus impairs this enzyme, and this stabilizes HIF-1a protein, increasing its concentration and allowing increased HRE binding to activate many genes as mentioned above.

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To this point, the word hypoxia has been used qualitatively to define conceptually a level of local PO2 low enough to activate the above pathways. It is actually quite problematic to determine quantitatively just what PO2 is required to initiate these events. It is likely better thought of in dose response terms and not as a threshold phenomenon. Furthermore, it may well be that while modest levels of hypoxia produce little or no gene response, and more severe hypoxia may produce a significant effect, very severe hypoxia may yet change the gene response again. At least at the phenotypic level, this can be seen as humans ascend to greater and greater altitudes. At, say, the altitude of Denver, about 5000 ft, little measurable response to hypoxia is evident. At intermediate altitudes, a variety of largely compensatory hypoxic responses occur that lead to greater tolerance of hypoxia, but at very high altitudes, that tolerance may be lost as hypoxia is so severe that, for example, body weight cannot be sustained, and long-term existence is threatened. The final common result of the activating and/or stabilizing effects of hypoxia and other angiogenic stimuli on angiogenic growth factors is to increase their concentrations at the tissue locations where an angiogenic response is required to restore cellular O2 and substrate availability. A number of such factors act together to guide angiogenesis itself. As described in many reviews of this complex process, such as (24,25), several well-defined events must take place to achieve capillary growth. The extracellular matrix supporting the capillary network is broken down, and endothelial cell proliferation is stimulated. The details of this process are well beyond the scope of this review, but the end result is increased numbers of capillaries in the tissue in question.

II.

Initiation and Regulation by Pro- and Antiangiogenic Growth Factors

A key concept in angiogenesis is that a variety of growth factors are involved in a manner that brings pro- and antiangiogenic molecules together. Thus, there appears to be a balance of such factors at work when angiogenesis occurs (26–29). Assuring adequate O2 and substrate availability is one of the most fundamental of cellular needs. It thus makes sense to have a process that has both positive and negative regulators, because compared to a simpler system of, say, a single positive regulator, there can be tighter control of the process. Perhaps this is also a protective mechanism against uncontrolled vascular growth. For angiogenesis, several well-known factors contribute to this process. Key proangiogenic molecules include VEGF, bFGF, TGFb, Angiopoietin 1 and 2, and, of course, their cellular receptors. The most commonly described negative regulators appear to be thrombospondin, ADAMTS1, serpinb5, TIMP3, endostatin, and angiostatin (30–36). Often these antiangiogenic molecules act by blocking or interfering with the ability of proteases to create a site for cell migration or invasion into the surrounding tissue.

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Scope—Focus on Skeletal Muscles: Which Genes Are Turned on, Which Are Important, What the Stimuli Are

Although this book focuses on pulmonary processes, this chapter is based on angiogenic processes in the skeletal muscle. Whether the pathways active in skeletal muscle can be transposed to the lungs is not clear, although it is likely that the major players are not dissimilar. However, the cells making up the lungs and the muscles are obviously very different, as are their respective functions. Angiogenesis in muscle represents a normal physiologic process in which capillary number is dynamically regulated to accommodate O2 needs of increases or decreases in the amount or power generation of muscle tissue. There is correlation between the formation of new tissue (myocyte size and/or number) and the required capillaries to supply the tissue with oxygen and nutrients. Of major significance is that in muscle, the angiogenic processes are turned on during exercise (4) when the intracellular PO2 is very low, about 3–4 mm Hg (37). Most pulmonary cells however are exposed to much higher PO2 values. Thus, epithelial cells and pulmonary venous endothelial cells see a PO2 of 100 mm Hg or higher most of the time. Even endothelial cells in the pulmonary arteries see PO2 values an order of magnitude higher than in exercising muscle, that is, about 40 mm Hg. However, in the conducting airway walls it is conceivable that wall tissue cells supplied by the bronchial arteries might be exposed to lower PO2 values. This chapter focuses on three elements of the skeletal muscle angiogenic process: 1. Which genes commonly thought associated with angiogenesis are activated by exercise 2. Which of these genes likely play the greatest functional role in angiogenesis 3. What exercise-related stimuli lead to their activation?

A. Angiogenic Genes Activated by Exercise

Exercise training is well known to result in increased muscle capillarity (38), and detraining conversely reduces capillarity. Thus, one would expect that exercise would lead to activation of angiogenic growth factors. Figure 1 shows mRNA levels of potentially angiogenic growth factors mRNA levels by Northern blot or RT-PCR in normal human subjects before and after exercise (39). These data come from quadriceps muscle biopsies following about 45 minutes of exercise at intensities that are about 50% of maximal. The biopsies were taken by needle aspiration within an hour of completing exercise. VEGF mRNA is increased several fold after exercise, with increases in every subject studied, while that of both bFGF and TGFb are not elevated. Time course studies of expression patterns are difficult to perform in humans, but time course information is important to

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Figure 1 Northern blots (for VEGF, bFGF, and TGFb1) in the top three panels and reverse transcription polymerase chain reaction RT-PCR results for VEGF (lower panel) show that compared to rest (R), one-hour exercise results in an upregulation of VEGF message. These data are from biopsies (quadriceps) taken from normal human subjects. Abbreviations: bFGF, basic fibroblast growth factors; INT, internal; RT-PCR, reverse transcription polymerase chain reaction; TGF-b1, transforming growth factors b1; VEGF, vascular endothelial growth factor.

obtain to understand the process. Such data from rats exercised just once on a treadmill at about 50–60% of maximal speed for an hour are shown in Figure 2 (4). The mRNA levels appear elevated several fold immediately after exercise has been completed. They remain elevated for about 4 hours before returning to baseline. Corresponding time course data for bFGF and TGFb-1 show little or no increases at any time over the 24 hours after the single exercise bout. RATS RUNNING AT 20 m/min, 21% O2 MUSCLE VEGF/18S RATIO

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Figure 2 Time course of VEGF mRNA in rats running on a treadmill for an hour at 20 meters/min, breathing room air. Compared to rest, there is a three- to fourfold increase in message level immediately after exercise, which gradually returns to normal after about four hours.

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In such rats, Thrombospondin mRNA levels are also increased immediately after an acute exercise bout by several fold. Angiopoietin-2 to angiopoietin-1 mRNA ratio was also found to be elevated after one day of exercise in rats with femoral artery ligation, suggesting a balance of factors involved in the destabilization of existing vessels and formation of new ones. In addition, the VEGF receptor mRNA levels in muscle are elevated in response to an acute exercise bout (40,41). By no means have all pertinent genes associated with angiogenesis been specifically studied before and after exercise, but gene expression profiling has revealed the time and intensity specific nature of the gene response that may lead to formation of new vessels (42–45). The increase in muscle VEGF mRNA after exercise is seen in a wide variety of circumstances. Thus, it occurs in all species studied to date—mouse, rat (4), dog (46), and humans (47). In human, it is seen not only in health but also in chronic diseases, including COPD (48), chronic heart failure (CHF) (49), and chronic renal failure (39). In these diseases, the response to exercise is almost as robust as in age-matched controls. In studies of rats of different ages, old age did not result in a reduced response to exercise (50). In contrast, the response of other putative angiogenic growth factors, such as TGFb and bFGF, are variable and when seen of much lesser degree. Of more direct consequence for the respiratory system, similar results have been found for rat diaphragm when that muscle is stressed by hypoxic and/or hypercapnic stimuli to hyperventilation (51). B. Which Angiogenic Genes Activated by Exercise Are Actually Important?

Establishing the biological importance of altered expression of any single gene is a difficult task. This is especially true in muscle after exercise because so many genes are affected by exercise, and we still do not know all of their specific functions let alone interactions with each other. Yet, until significance is clearly established, physiological understanding remains limited. It is true that transgenic mouse technology has revolutionized the study of individual gene significance, but even here there are many pitfalls to interpretation. This is especially true when a genomewide knockout is performed at the embryonic single cell level, because as the modified mice grow, they have the opportunity to develop compensatory mechanisms that make the adult phenotype appear normal, thus hiding the true importance of the gene. This problem was highlighted when the myoglobin gene was knocked out a few years ago (52). The initial description of this mouse reported normal exercise function, but later reports that examined O2 transport structure and function revealed elevated coronary perfusion, hemoglobin levels, and muscle capillarity (53,54). All of these compensations work to augment O2 availability and thus indicate that myoglobin does indeed have a significant role in normal O2 transport during exercise. Studies modifying potentially angiogenic genes in muscle are very limited. To date, only deletion of HIF (55) and VEGF (56) have been reported. Because both of

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these genes are essential to embryonic survival, conventional genome-wide embryonic knockout is not possible. Local deletion of VEGF using the Cre/LoxP strategy in a small region of adult mouse gastrocnemius muscle has shown substantial (65%) reduction in capillarity in cage-confined mice, as shown in Figures 3 and 4 (56). These findings were noted both at 4 and 8 weeks after the initial injection of Cre recombinase. That there was absolutely no recovery of vasculature by 8 weeks suggests that VEGF is essential to normal capillary maintenance, a finding underscored by the extensive apoptosis in the transfected region of muscle, also shown in Figure 3. It was surprising that no alternative rescue pathway, perhaps via bFGF or other known growth factors, apparently took place. It is also interesting that although the effects of VEGF were substantial as shown, about one-third of capillaries did remain viable in the transfected area. This could indicate residual VEGF expression, consistent with less than 100% efficiency of such gene deletion strategies. Alternatively, it could be interpreted as capillaries being controlled by more than one independent pathway—one accounting for the majority of capillaries being VEGF-dependent, and the other based on some VEGF-independent pathway not identified. This conditional knockout approach has advantages. Compensation pathways are less likely to be recruited than when the knockout is life-long, and as a

Figure 3 Application of the Cre/Lox strategy to delete VEGF in a small portion of mouse gastrocnemius. (Top left) Immunostaining for Cre recombinase protein in the bottom right half of the section. (Top right) Corresponding serial section showing VEGF protein staining, with much lower levels evident in the region corresponding to Cre recombinase expression. (Bottom left) Attenuation of capillaries detected by alkaline phosphatase staining in the Cre transfected and VEGF-deficient region. (Lower right) Extensive apoptosis evident by Tunel staining in another serial section in the region corresponding to VEGF reduction.

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2.5

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Figure 4 Quantitative reduction in capillary fiber ratio in the mice exemplified in Figure 3 showing 65–70% reduction in capillaries per fiber as a result of VEGF deletion. Note that the effects persist at least through eight weeks posttransfection.

“tracer” experiment, limited to a very small region of a single muscle, systemic consequences of gene deletion are also unlikely to occur. However, because the gene is deleted from such a small amount of muscle, overall exercise performance is not affected. Another, complementary way to examine the importance of VEGF in muscle is to produce transgenic mice that are bred to express VEGF in all tissues except muscle. This can be achieved by crossbreeding two specific mouse strains. One is a strain expressing Cre recombinase only in skeletal/cardiac muscle, a tactic achieved by coupling the Cre gene to a muscle-specific promoter, such as muscle creatine kinase, and then using standard procedures to raise such mice from embryos altered in this way. The second strain is a mouse expressing LoxP sequences in the VEGF gene, also created by embryonic transgenic procedures. This mouse displays these LoxP sequences in every cell, but the function of VEGF is preserved. Crossbreeding these two strains results in mice that have VEGF deleted only throughout their skeletal and cardiac musculature and for their entire lives (57). First, such mice do survive to adulthood. Second, preliminary findings show that their exercise endurance capacity is considerably limited, to about 20% of control animal values (57). Third, muscle capillarity is considerably reduced, to about 44% of control (57). Whether any compensatory pathways have developed remains to be investigated. Other potentially angiogenic genes could be studied by similar strategies, but such research remains to be done. C. What Are the Stimuli—Hypoxia or Nonhypoxic Phenomena?

The previous sections have established that skeletal muscles respond to exercise by increasing in particular VEGF activity and that VEGF is essential to normal muscle capillarity. What is it about exercise that turns on this critical gene?

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As mentioned above, hypoxia is the prime candidate. It is known that PO2 in muscle cytoplasm falls to about 3–4 mm Hg during heavy exercise (37), a value equivalent to about 0.5% O2. This has been shown by proton MRS in intact humans and occurs even when arterial PO2 remains at normal values around 100 mm Hg. It is well known that the VEGF promoter contains an HRE. It is also known that HIF-1a protein levels are increased in muscle during heavy exercise (18). Moreover, the fold increase in HIF-1a protein matches the fold increase in VEGF message in electrically stimulated rat gastrocnemius muscle (18). It may therefore be logical to suppose that low intracellular PO2 in muscle during exercise stabilizes HIF-1a protein, increasing its binding to the HRE in the VEGF promoter and thus activates VEGF transcription. In addition, hypoxia increases HuR binding to VEGF message, stabilizing it, and further contributing to increased VEGF message levels (21). That hypoxia is central to initiating angiogenesis has been the prevailing view, even more, it has been suggested that hypoxia increases production of H2O2 at the cytochrome complex III in the mitochondria (19). H2O2 is known to be involved in the stabilization of HIF-1a, providing an additional mechanism for activating VEGF. However, there is evidence emerging that hypoxia may not be the only trigger to angiogenic activation. HIF-1a can be stabilized by hypoxiaindependent pathways. In fact any agent that blocks mitochondrial respiration (i.e., NO) leads to a sequestration of the available oxygen to specific cellular targets. One such target is the prolyl-hydroxylase that rapidly degrades HIF-1a during normoxic conditions. Thus, in the presence of inhibitors of mitochondrial respiration HIF-1a is no longer stabilized by hypoxia (58). On the other hand, inflammatory molecules, such as LPS, can stabilize HIF-1a (59). Less directly, several physiological observations suggest that nonhypoxic regulation of VEGF may be occurring. Adult mammals taken to altitude generally do not develop angiogenesis in skeletal muscles, suggesting that some additional stimulus may be necessary. Transgenic mice created to express HIF in all tissues except skeletal muscles do not show impaired muscle capillarity (55). Human athletes after training express less VEGF mRNA in muscles in response to exercise than do their untrained counterparts (60). This is in spite of similarly low muscle cytoplasm PO2 values during exercise (61–65). Taken together, these findings allow one to hypothesize that muscle angiogenesis may only be in part a consequence of local hypoxia. It is tempting to speculate that local muscle inflammation and/or injury, which accompanies heavy exercise especially in the untrained subject (66–68), can initiate the angiogenic response independently of local hypoxia. As mentioned, VEGF can be activated by proinflammatory molecules and/or reactive oxygen species (5,10,13,14,69). And, exercise increases formation of cytokines and reactive oxygen species in muscle (67,70–81), which as also mentioned, can activate HIF stabilization. Another possibility might be that NO released locally during exercise can activate VEGF transcription in muscle. It is known that NO, when increased in normal resting rat muscle by arterial infusion of acetylcholine or

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nitroprusside into the femoral artery, significantly increases gastrocnemius VEGF mRNA levels (7). This entire research direction is in its early stages, remains speculative, and other hypoxia-unrelated pathways might be involved in VEGF activation during exercise. In summary, although the complete story of how exercise leads to increased muscle capillarity is far from solved, it does appear that the hypoxia/HIF/VEGF axis is likely involved, but nonhypoxic pathways may also be important. There is no question that VEGF itself is a critical growth factor without which capillarity is impaired and apoptosis occurs.

IV.

Relevance to the Lungs: Airways and Parenchyma, Focus on VEGF

Given that this book is about the lungs, some thoughts about angiogenesis in the lungs may be warranted. First, one can think of roles for VEGF (and other angiogenic growth factors) in several locations within the lungs. The lung contains an abundant level of VEGF, mainly expressed by the bronchial and alveolar epithelial cells with a lesser amount expressed by smooth muscle cells. However, unlike skeletal muscle in which capillary number is dynamically regulated to meet the change in muscle structure, the lung contains a very low percentage of muscularized vessels. Thus one may hypothesize that the vast amount of VEGF serves to maintain or protect the highly vascularized lung. In addition a change in the balance of angiogenic and antiangiogenic factors in the lung even for a short transient period appears to result in a disease state. This is true for VEGF in which lower levels lead to apoptotic loss of capillary structure (82–84), whereas overexpression of VEGF is associated with airway remodeling similar to that observed in asthma patients (85–87). In the alveolar wall, chemical blockade of the VEGF receptor or VEGF gene inactivation itself has striking effects in the mouse. These mice quickly develop an emphysematous phenotype (82,84), with lungs showing enlarged alveoli and increased compliance, and apoptosis is widespread. Furthermore, biopsies taken from human patients with emphysema also reveal decreased VEGF levels and both endothelial and epithelial cell apoptosis (83). Likewise, high levels are found in broncho-alveolar lavage fluid (BALF) and sputum from asthma patients, suggesting a role for VEGF in the inflammatory response (88,89). This fine control over the level of factors with angiogenic potential seems to be a common theme in the lung. For instance it is well known that overexpression of TGF-b leads to a fibrotic response, but yet inactivation of TGFb function results in an emphysema phenotype (90–93). These “angiogenic” factors appear to have an important role in protection and repair of the delicate lung architecture that provides an ample gas exchanging surface and are relatively intolerant to changes in these important regulators of lung structure.

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V. Summary This short review has examined the exercise-induced angiogenic process in skeletal muscle, focusing on which angiogenic molecules are likely key and what regulates them. The story is far from complete, but both hypoxic and possibly nonhypoxic inflammatory pathways of VEGF activation may be at the core of the angiogenic response. In at least three different compartments in the lung, there is provocative evidence that the angiogenic pathways involving VEGF may be found to be extremely important players in not just normal lung structural maintenance but also in disease processes in both the airways and the pulmonary vasculature. References 1. Folkman J. Angiogenesis inhibitors: a new class of drugs. Cancer Biol Ther 2003; 2:S127–S133. 2. Penny WF, Hammond HK. Clinical use of intracoronary gene transfer of fibroblast growth factor for coronary artery disease. Curr Gene Ther 2004; 4:225–230. 3. Prior BM, Lloyd PG, Yang HT, Terjung RL. Exercise-induced vascular remodeling. Exerc Sports Sci Rev 2003; 31:26–33. 4. Breen EC, Johnson EC, Wagner H, Tseng H-M, Sung LA, Wagner PD. Angiogenic growth factor mRNA responses in muscle to a single bout of exercise. J Appl Physiol 1996; 81:355–361. 5. Cohen T, Nahari D, Cerem LW, Neufeld G, Levi BZ. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 1996; 271:736–741. 6. Liu Y, Christou H, Morita T, Laughner E, Semenza GL, Kourembanas S. Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5 00 enhancer. J Biol Chem 1998; 273:15257–15262. 7. Benoit H, Jordan M, Wagner H, Wagner PD. Effect of NO, vasodilator prostaglandins and adenosine on skeletal muscle angiogenic growth factor gene expression. J Appl Physiol 1999; 86:1513–1518. 8. Gavin TP, Spector DA, Wagner H, Breen EC, Wagner PD. Effect of captopril on skeletal muscle angiogenic growth factor responses to exercise. J Appl Physiol 2000; 88:1690–1697. 9. Richard DE, Berra E, Pouyssegur J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1a in vascular smooth muscle cells. J Biol Chem 2000; 275:26765–26771. 10. Kosmidou I, Xagorari A, Roussos C, Papapetropoulos A. Reactive oxygen species stimulate VEGF production from C(2)C(12) skeletal myotubes through a P13K/Akt pathway. Am J Physiol 2001; 280:L585–L592. 11. Sandau KB, Zhou J, Kietzmann T, Brune B. Regulation of the hypoxia-inducible factor 1alpha by the inflammatory mediators nitric oxide and tumor necrosis factoralpha in contrast to desferroxamine and phenylarsine oxide. J Biol Chem 2001; 276:39805–39811.

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12. Maeno T, Tanaka T, Sando Y, et al. Stimulation of vascular endothelial growth factor gene transcription by all trans retinoic acid through Sp1 and Sp3 sites in human bronchioloalveolar carcinoma cells. Am J Respir Cell Mol Biol 2002; 26:246–253. 13. Page EL, Robitaille GA, Pouyssegur J, Richard DE. Induction of hypoxia-inducible factor-1alpha by transcriptional and translational mechanisms. J Biol Chem 2002; 277:48403–48409. 14. Valdembri D, Serini G, Vacca A, Ribatti D, Bussolino F. In vivo activation of JAK2/STAT-3 pathway during angiogenesis induced by GM-CSF. FASEB J 2005; 16:225–227. 15. Brown MD, Hudlicka O. Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: involvement of VEGF and metalloproteinases. Angiogenesis 2003; 6:1–14. 16. Kimura H, Esumi H. Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis. Acta Biochim Pol 2003; 50:49–59. 17. Schafer G, Cramer T, Suske G, Kemmner W, Wiedenmann B, Hocker M. Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1and Sp3-dependent activation of two proximal GC-rich promoter elements. J Biol Chem 2003; 278:8190–8198. 18. Tang K, Breen EC, Wagner H, Brutsaert TD, Gassmann M, Wagner PD. HIF and VEGF relationships in response to hypoxia and sciatic nerve stimulation in rat gastrocnemius. Respir Physiol Neurobiol 2004; 144:71–80. 19. Chandel NS, McClintock DS, Feliciano CE, et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1a during hypoxia. J Biol Chem 2000; 275:25130–25138. 20. Greijer A, van der Groep P, Kemming D, et al. Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1). J Pathol 2005; 206:291–304. 21. Tang K, Breen EC, Wagner PD. Hu Protein R (HuR) mediated post-transcriptional regulation of VEGF expression in rat gastrocnemius muscle. Am J Physiol 2002; 283:H1497–H1504. 22. Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science 2001; 294:1337–1340. 23. Epstein AC, Gleadle JM, McNeill LA, et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001; 107:43–54. 24. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol (Cell Physiol) 2001; 280:C1358–C1366. 25. Prior BM, Yang HT, Terjung RL. What makes vessels grow with exercise training? J Appl Physiol 2004; 97:1119–1128. 26. Iruela-Arispe ML, Dvorak HF. Angiogenesis: a dynamic balance of stimulators and inhibitors. Thromb Haemost 1997; 78:672–677. 27. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for tie2 that disrupts in vivo angiogenesis. Science 1997; 277:55–60. 28. Holash J, Wiegand SJ, Yancopoulos GD. New model of tumor antiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 1999; 18:5356–5362.

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46. Roca J, Gavin T, Jordan M, et al. Angiogenic growth factor mRNA responses to passive and contraction-induced hyperperfusion in dog skeletal muscle. J Appl Physiol 1998; 85:1142–1149. 47. Richardson RS, Wagner H, Mudaliar SRD, Henry R, Noyszewski EA, Wagner PD. Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise. Am J Physiol 1999; 277:H2247–H2252 (Heart Circ Physiol 46). 48. Richardson RS, Mudaliar SRD, Henry R, Wagner H, Wagner PD. VEGF mRNA response to acute exercise in skeletal muscle of patients with chronic obstructive pulmonary disease. Proceedings of the International Congress of Myology Nice, France, 2000:277. (Abstract) . 49. Gustafsson T, Bodin K, Sylven C, Gordon A, Tyni-Lenne R, Jansson E. Increased expression of VEGF following exercise training in patients with heart failure. Eur J Clin Invest 2001; 31:362–366. 50. Rossiter HB, Howlett RA, Holcombe HH, Entin PL, Wagner H, Wagner PD. Age is no barrier to muscle structural, biochemical and angiogenic adaptations to training up to 24 months in female rats. J Physiol 2005; 565:993–1005. 51. Siafakas NM, Jordan M, Wagner H, Breen EC, Benoit H, Wagner PD. Diaphragmatic angiogenic growth factor mRNA responses to increased ventilation caused by hypoxia and hypercapnia. Eur Respir J 2001; 17:681–687. 52. Garry DJ, Ordway GA, Lorenz JN, et al. Mice without myoglobin. Nature 1998; 395:905–908. 53. Grange RW, Meeson A, Chin E, et al. Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice. Am J Physiol Cell Physiol 2001; 281:C1487–C1494. 54. Schlieper G, Kim JH, Molojavyi A, et al. Adaptation of the myoglobin knockout mouse to hypoxic stress. Am J Physiol Regul Integr Comp Physiol 2004; 286:R786–R792. 55. Mason SD, Howlett RA, Kim MJ, et al. Loss of skeletal muscle HIF-1alpha results in altered exercise endurance. PLoS Biol 2004; 2:e288. 56. Tang K, Breen EC, Gerber HP, Ferrara NMA, Wagner PD. Capillary regression in vascular endothelial growth factor-deficient skeletal muscle. Physiol Genomics 2004; 18:63–69. 57. Olfert IM, Howlett RA, Breen EC, Wagner PD. Reduced endurance exercise capacity following skeletal muscle targeted VEGF gene deletion in mice. FASEB J 2005; 19:A132. 58. Hagen T, Taylor CT, Lam F, Moncada S. Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1apha. Science 2003; 302:1975–1978. 59. Blouin CC, Page EL, Soucy GM, Richard DE. Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1{alpha}. Blood 2004; 103:1124–1130. 60. Richardson RS, Wagner H, Mudaliar SRD, Saucedo E, Henry R, Wagner PD. Exercise adaptation attenuates VEGF gene expression in human skeletal muscle. Am J Physiol Heart Circ Physiol 2000; 279:H772–H778. 61. Ruscher K, Isaev N, Trendelenburg G, et al. Induction of hypoxia inducible factor 1 by oxygen glucose deprivation is attenuated by hypoxic preconditioning in rat cultured neurons. Neurosci Lett 1998; 254:117–120.

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10 Pharmacologic Modulation of Bronchial Vascular Remodeling: Current Therapies and Novel Approaches

ALFREDO CHETTA and DARIO OLIVIERI Section of Respiratory Diseases, Department of Clinical Sciences, University of Parma, Parma, Italy

I. Introduction Changes in bronchial microvasculature are present in chronic inflammatory airway disease and may contribute to airway remodeling in asthma. In addition to hypertrophy and hyperplasia of airway smooth muscle (1), increase in mucous glands (2), and thickening of the reticular basement membrane (3), some significant qualitative and quantitative changes in airway blood vessels may occur in asthma. Early studies on the pathology of asthma showed edematous bronchial mucosa with dilated and congested blood vessels in patients with fatal disease (4,5). More recent in vivo quantitative studies in asthmatic patients found an increase in the total number of vessels and in vascular area when compared to control subjects (6,7). Currently, it is assumed that the bronchial microcirculation in asthma may be involved via at least three different mechanisms: angiogenesis, dilatation, and permeability (8). The functional significance of bronchial vascular remodeling in asthma is not yet well known. However, the increase in the number and size of vessels can contribute to thickening of the airway wall, which in turn may lead to critical narrowing of the bronchial lumen, when bronchial smooth muscle contraction occurs (9). Additionally, the bronchial microcirculation may have an indirect role by acting as a gateway to the submucosa for inflammatory cells during the 213

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inflammatory response in asthma. Hence, pharmacological control of bronchial vascular remodeling may be crucial for symptom control in asthma patients. So far there has been little information specifically relating asthma medication to vascular remodeling. Moreover, with respect to antiasthma drugs, up to now the effects on bronchial vascular remodeling in humans have been studied only for corticosteroids and b2-agonists.

II.

Current Therapies

A. Corticosteroids

Corticosteroids are the most effective antiasthma drugs and therefore are recommended by the international guidelines as the mainstay for treatment of asthmatic patients (10). The important antiasthma effects of corticosteroids are related to their ability to inhibit inflammatory processes in the airways by downregulating several airway inflammatory cytokines (11), reducing cell infiltration into the bronchial wall (12), and reversing basement membrane thickening (13). In asthmatic patients, it has been also shown that corticosteroids can efficaciously act on airway wall vascularity, partly by altering airway mucosal blood flow and partly by changing chronic inflammation within the bronchial wall. An increase in airway blood flow is associated with airway inflammation in asthma (14), so inhaled corticosteroids can affect the airway mucosal blood flow by causing vasoconstriction and reducing edema (15). In asthmatic patients and normal controls, inhaled fluticasone propionate induced a vasoconstrictive action in the airway mucosa (15). Moreover, the vasoconstrictor response was greater in subjects with higher baseline airway mucosal blood flow irrespective of whether they were normal or asthmatic, and it was not associated with any change of forced expiratory volume in one second (FEV1) in subjects with or without asthma at any dose (15). The vasoconstrictive mechanism of corticosteroids is still unknown. Some reports support the hypothesis that steroid-induced vasoconstriction involves noradrenergic neurotransmission. Indeed, it has been reported that corticosteroids can enhance vasoconstrictor responses to adrenaline in the human hand (16) and to noradrenaline in rat mesenteric arterioles (17). Corticosteroids also increased the sensitivity of vascular smooth muscle to the vasoconstrictor effects of noradrenaline, an effect reversed by infusions of arachidonic acid and prostacyclin (18). These findings indicate that locally applied corticosteroids do not act by releasing noradrenaline from adrenergic nerve endings but rather by potentiating its physiological effect by upregulating postsynaptic adrenergic receptors, interfering with noradrenaline metabolism, or inhibiting presynaptic or postsynaptic noradrenaline uptake. The latter mode of action of steroids is supported by the finding that hydrocortisone was able to inhibit the uptake of noradrenaline by cultured vascular smooth muscle cells (19).

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Inhaled steroids can act not only on the flow but also on the qualitative and quantitative changes in airway blood vessels of asthmatic patients. A six-week treatment with fluticasone propionate 500 mg twice a day, but not 250 mg twice a day, was able to reduce the vascular component of airway remodeling in patients with mild to moderate asthma (Fig. 1) (20). After treatment with a high dose of fluticasone propionate, the total number of vessels and the vascular area in asthmatics were similar to those in healthy controls (Fig. 2) (20). In this study, the low dose was able to reduce the number of inflammatory cells in the bronchial mucosa, as well as the bronchial responsiveness to methacholine and asthma symptoms. Although the lowest dose of inhaled steroids that may be required to effectively treat vascular remodeling has yet to be established, it seems that only high doses of inhaled steroids can be effective on airway vascular remodeling in asthmatic patients. Orsida et al. (21) showed an effect of inhaled steroids on airway wall vascularity only above 500 mg of beclomethasone dipropionate. Moreover, another study by the same authors (22) failed to demonstrate any effect of a three-month treatment with a daily dose of 100 mg bid fluticasone propionate plus a background dose of inhaled steroids (200–500 mg/day of beclometasone or 200–400 mg/day of budesonide) on vascularity in asthmatic patients. Hoshino et al. (23) showed that 800 mg beclomethasone dipropionate for six months was able to reduce airway wall vascularity, evaluated as both vessel number and percent vascularity, in mild to moderate asthmatic patients. Additionally, in this study the change in percent vascularity was inversely related to both FEV1 and airway responsiveness (23). The mode of action of corticosteroids on the vascular components of airway remodeling in asthma is complex and not yet completely known. Corticosteroids n. s.

(A)

p < 0.05

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400

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Figure 1 (A) Individual values of number of vessels/mm2 in lamina propria measured before and after six weeks of treatment with FP 100 mg bid and FP 500 mg bid. Open squares indicate mean values. (B) Individual values of vascular area measured in asthmatic patients before and after six weeks of treatment with FP 100 mg bid and FP 500 mg bid. Open squares indicate mean values. Abbreviation: FP, fluticasone propionate. Source: From Ref. 20.

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Figure 2 Microphotograph showing immunostaining for collagen IV to identify vessels in the lamina propria of: an asthmatic patient (A); an healthy volunteer (B); an asthmatic patient before (C) and after (D) treatment with FP 100 mg bid; an asthmatic patient before (E) and after (F) treatment with FP 500 mg bid. Source: From Ref. 20.

may have antiangiogenic properties, because they act on mediators involved in the formation of new vessels and the remodeling of the existing ones. Vascular endothelial growth factor (VEGF), a specific mitogen for vascular endothelial cells, can also enhance vascular permeability and edema formation and is a major regulator of angiogenesis (24). Moreover, VEGF expression is increased in the airways of subjects with asthma and is correlated with vascularity (25). In cultures of aortic human vascular smooth muscle cells, hydrocortisone, cortisone, and dexamethasone inhibited the expression of the VEGF gene in a dose-dependent manner (26). Moreover, in airway and epithelial cells, budesonide reduced VEGF secretion and VEGF mRNA expression (27). These effects were inhibited by mifepristone, a glucocorticoid receptor antagonist, showing that budesonide acts through its glucocorticoid receptor-mediated action (27). Budesonide–mediated inhibition of VEGF mRNA was also time- and protein synthesis-dependent (27).

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Additionally, budesonide was able to inhibit airway smooth muscle cell production of VEGF (28). Corticosteroids could also affect bronchial microvascularity by inhibiting airway inflammatory mediators with pro-angiogenetic activity, such as interleukin (IL)-8 (29), granulocyte-macrophage-colony stimulating factor (GM-CSF) (30), tumor necrosis factor-a (30), and matrix metalloproteinases (31). Finally, the effect of corticosteroids on the vascular component of airway remodeling may also occur through its inhibitory effect on mast cells (20), which play an important role in inducing the neovascularization process in asthma through the release of proangiogenic factors. Histamine, the major preformed mast cell mediator, stimulates new vessel growth by acting through both H1 and H2 receptors (32). Heparin, the main glycosaminoglycan constituent of mast cells granules, also possesses proangiogenic activity (33). The evidence for a positive correlation between mast cells and the number of vessels in asthmatic patients (20) further supports these observations. Moreover, mast cells produce and secrete VEGF (34), which has been shown to stimulate mast cell migration at sites of angiogenesis (Table 1) (35). B. b2-Agonists

b2-agonists are considered primarily as bronchodilating drugs. They act by binding to b2 adrenergic receptors that, when activated, induce an increase in intracellular adenosine 3 0 ,5 0 -cyclic monophosphate (cAMP). The subsequent activation of cAMP-dependent protein kinase causes the phosphorylation of specific proteins, leading to smooth muscle relaxation (36). However, b2 adrenergic receptors are expressed not only on smooth muscle cells but also on some bronchial and inflammatory cells, and their activation might explain some pharmacological properties of b2-agonists, which may be useful to control airway inflammation (37). Several in vitro studies showed that b2-agonists were able to inhibit mediator release from inflammatory and airway cells, such as mast cells (38), lymphocytes Table 1 Inhibitory Effects on Proangiogenic Factors by Antiasthma Drugs Drug Corticosteroids

b2-agonists

Protein

Cell type

VEGF IL-8 GM-CSF TNF-a MMP-9 GM-CSF IL-8

Airway epithelial cells Airway smooth muscle Submucosa Submucosa Submucosa Airway epithelial cells Airway epithelial cells

Reference (27) (29) (30) (30) (31) (55) (54,55)

Abbreviations: GM-CSF, granulocyte-macrophage-colony stimulating factor; IL-8, interlukin-8; MMP-9, matrix metalloproteinase-9; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor.

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(39), eosinophils (40), monocytes (41), and smooth muscle cells (42). It has been also reported that b2-agonists inhibit the proliferation both of fibroblasts (43) and smooth muscle cells (44). Finally, long acting b2-agonists seem to have an effect on plasma exudation. Salmeterol has been to shown to inhibit vascular permeability induced by nasal allergen challenge (45), and formoterol was reported to have an inhibitory effect on histamine-induced plasma exudation in the lower airways (46). The inhibitory effect of long acting b2-agonists on plasma leakage is likely mediated by b2 adrenergic receptors, because it is blocked by prior administration of a b2 adrenergic receptor antagonist (47) and it is probably related to the inhibition of endothelial gap formation (48). Taken together, these findings support the view that the prolonged stimulation of the b2 adrenergic receptors by the b2-agonists could positively act on some pathways of chronic airway inflammation and bronchial wall remodeling. So far, in vivo studies have provided equivocal results. Short acting b2-agonists, used as the sole antiasthma therapy, increased airway inflammation in asthmatic patients (49), whereas long acting b2-agonists showed some antiinflammatory effects (30,50,51). Up to now, only two studies have specifically addressed the effect of long acting b2-agonists on some components of airway remodeling, such as basement membrane thickening (52) and the quantitative and qualitative changes in bronchial microvasculature (22). In the first study, after six weeks of treatment with inhaled salmeterol 50 mg twice a day, biopsy specimens from asthmatic patients showed no changes in the thickness of the basement membrane or in the number of inflammatory cells or mast cell degranulation (52). Conversely, the second study showed that in asthmatic patients treated with low dose inhaled corticosteroids, there was a positive effect on the vascular component of airway remodeling after a three-month treatment with inhaled salmeterol 50 mg twice a day, with a significant decrease in the number of vessels (22). However, the salmeterol treatment was not able to change the percentage of the vascular area, because of an overall dilatation of vessels likely due to a direct pharmacological effect of the b2-agonist on relaxing vascular smooth muscle (22). The effect of salmeterol on bronchial microvascularity may be considered as a consequence of interaction between b2-agonists and corticosteroids, with a magnification of the anti-inflammatory effects of the latter. The antiinflammatory actions of the corticosteroids follow the activation and translocation of ubiquitously expressed cytoplasmic corticosteroid receptors to the nuclei of cells. In vitro studies demonstrated that b2-agonists caused a propranol-sensitive, cAMP-dependent activation and nuclear translocation of corticosteroid receptors in human lung fibroblasts and vascular smooth muscle cells, providing the evidence for complementary anti-inflammatory cellular effects of b2-agonists and corticosteroids (53). Furthermore, a direct antiangiogenic effect of b2-agonists on endothelial hyperplasia may be hypothesized, because they act on some airway inflammatory mediators with proangiogenic activity. In vivo salmeterol treatment was associated with a decrease in bronchoalveolar lavage fluid levels of interleukin (IL)-8 (54), and in vitro it was

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effective in inhibiting in a dose-response manner both GM-CSF and IL-8 release from human bronchial epithelial cells (Table 1) (55). C. Other Antiasthma Drugs

In addition to corticosteroids and b2-agonists, among the currently used antiasthma drugs, theophyllines and leukotrienes receptor antagonists (LTRAs) have also provided some in vivo findings of their effects on chronic airway inflammation. Though theophyllines are primarily considered as bronchodilators and respiratory stimulants, they have been shown to positively interfere with some cellular inflammatory pathways. In atopic asthmatic patients, low-dose oral theophylline attenuated the airway inflammatory response to allergen inhalation, because it significantly reduced total and activated eosinophils beneath the epithelial basement membrane after allergen challenge (56). Moreover, chronic treatment with theophylline could affect T-lymphocyte populations both in the bronchial mucosa and in the blood, suggesting that theophylline prevents T-cell trafficking from blood into the airways (57,58). Despite these in vivo studies showing anti-inflammatory effects, there have been no studies on the effect theophylline effect on airway remodeling and bronchial microvasculature changes in asthma. Discordant results come from biopsy studies regarding the effect of LTRAs on mucosal inflammation. In a bronchial biopsy study, a four-week treatment with pranlukast was shown to reduce T cells, mast cells and activated eosinophils (59). On the other hand, the reduction of inflammatory cell number in the bronchial mucosa achieved with montelukast plus a low dose of fluticasone dipropionate was not significantly different from the reduction observed with a low dose of fluticasone dipropionate alone in patients with mild asthma (60). However, in an animal model reflective of the chronic airway inflammation and remodeling changes observed in patients with persistent asthma, montelukast significantly reduced the airway eosinophil infiltration, mucous plugging, smooth muscle hyperplasia and subepithelial fibrosis (61). Structural changes in bronchial microvasculature were not taken into account (61). Montelukast has recently been shown to positively affect airway mucosal blood flow in patients with mild intermittent asthma. Two-week treatments with montelukast 10 mg and two-week treatments with fluticasone propionate 440 mg daily were equipotent in reducing airway mucosal blood flow, and the magnitude of the response was not greater if the two drugs were combined (62).

III.

Novel Therapeutic Approaches

A. Potential Drug Targets

Angiogenesis is a complex physiological process, which is initiated by the release of matrix metalloproteases (MMPs) from activated endothelial cells. This leads

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to degradation of the extracellular matrix and basement membrane, migration of endothelial cells into the interstitial space, and the subsequent endothelial cell proliferation and differentiation into mature blood vessels. All these processes are tightly regulated through the interplay of several endogenous factors that induce or inhibit angiogenesis (Table 2). In addition to VEGF, the angiogenesis specific factors also include some members of the angiopoietin and ephrin families (24). VEGF is required to initiate the formation of immature vessels by vasculogenesis or angiogenic sprouting, during development as well as in the adult (24). Angiopoietin-1 (Ang-1) is subsequently required for maturation of this initially immature vasculature and then for maintaining vessel quiescence and stability (24). During vascular remodeling in the adult, VEGF and Ang-1 can reassume their roles. The former promotes formation of leaky, immature, and unstable vessels; conversely the latter protects vessels, making them resistant to the damage and leak induced by VEGF or inflammatory challenges (24). Several agents specifically targeting angiogenesis have been developed and may be grouped into a few categories based on their mechanism of action, such as the inhibitors of MMPs, the blockers of endothelial cell signaling, and the angiogenesis inhibitors. These compounds are currently under intense study to assess their efficacy as cancer treatments, and most of the information about these agents in humans comes from studies for the treatment of solid tumors (63). Several inhibitors of MMPs have been synthesized, including marimastat, prinomastat, BMS 275291, BAY 12-9566, and neovastat (64). So far, they have been intensively used as lung cancer treatments (64). Several drugs that block

Table 2

Endogenous Inducers and Inhibitors of Angiogenesis

Inducers Angiopoietin Angiogenin Angiotropin Fibroblast growth factor Granulocyte-colony stimulating factor Granulocyte-macrophage-colony stimulating factor Hepatocyte growth factor Heparin Histamine Interleukin-3, -8 Matrix metalloproteinases Placental growth factor Platelet-derived growth factor Tumor necrosis factor-a Vascular entothelial growth factor

Inhibitors Angiostatin Antithrombin Endostatin Interferon-a, -g Interleukin-4, -12 Platelet factor 4 Plasminogen activator inhibitor 1 Prolactin Thrombospondin-1 Tissue inhibitors of metalloproteinases-1, -2 Troponin I Vascular entothelial cell growth inhibitor

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endothelial cell signaling via VEGF and its receptor have been recently developed, and they included rhuMAB VEGF, SU5416, SU6668, ZD6474, CP-547,632 and ZD4190 (64). All these compounds are in the early stages of clinical trials as antitumor therapies (64). Using murine models, some angiogenesis inhibitors, such as angiostatin (65), endostatin (66), and antithrombin (67), have been identified and their antitumoral activity has been assessed. To date, only recombinant endostatin has entered phase I trials showing an acceptable toxicity profile and evidence of minor antitumor activity (68). B. Angiopoietin-1

Another possible new therapeutic strategy for reducing airway edema in chronic inflammatory airway diseases, such as bronchitis and asthma, may be based on the antileakage effect of Ang-1 (69). To date, there is no in vivo evidence of this effect. However, an in vitro study showed that Ang-1 not only supported the localization of proteins, such as platelet endothelial cell adhesion molecule-1 (PECAM-1), into junctions between endothelial cells and decreased the phosphorylation of PECAM-1 and vascular endothelial cadherin, but it also strengthened these junctions, as illustrated by a decrease in basal permeability and inhibition of permeability responses to thrombin and VEGF (70). Moreover, another report showed that acute administration of Ang-1 protected adult vasculature from leaking, countering the potentially lethal actions of VEGF and inflammatory agents (71). IV.

Conclusions

Despite the clinical and functional consequences of bronchial microvascular remodeling in asthma, up to now very few data have been published on its therapeutic approach. Among current antiasthma drugs, only corticosteroids act positively on the three aspects of bronchial vascular remodeling: angiogenesis, dilatation, and permeability. In particular, inhaled corticosteroids are able to affect the airway mucosal blood flow, by causing vasoconstriction and reducing edema, and at high doses they reduce the total number of vessels and the vascular area. So far, modest evidence of a positive effect on bronchial microcirculation changes has been reported with respect to b2-agonists and LTRAs treatment. Long acting b2-agonists seem to have an effect on plasma exudation; additionally salmeterol has been to shown to reduce angiogenesis. Moreover, montelukast has recently shown to reduce airway mucosal blood flow with the same magnitude as inhaled corticosteroids. To date, several endogenous inhibitors and promoters of angiogenesis have been detected, and accordingly several different classes of new agents that inhibit angiogenesis have been developed. These new compounds have been mainly studied as antitumor therapy and so far no data have been provided on their effect

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on bronchial microcirculation changes in patients with chronic inflammatory diseases. In the future, these compounds, especially Ang-1, could represent a novel approach for positively acting on bronchial microvascular changes in chronic inflammatory airway diseases, such as bronchitis and asthma.

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Index

Absorption of drugs, 10, 11 ACE. See angiotensin converting enzyme Adrenomedullin (ADM), 181–182 Adventitial thickening, 149, 151 Afferent regulatory mechanisms, 13 Air conditioning, 11, 29 Airway blood flow animal studies, 25, 30–31 definition, 26 invasive measurement techniques, 25, 30 laser Doppler velocimetry, 30–31 measurement, 25–44 noninvasive measurement techniques, 25–26, 30–38 soluble gas uptake measurement, 31–38 Airway circulation, use of term, 1–2 Airway remodeling, 127–128, 148–149, 150 Airway smooth muscle (ASM), 148–149, 153, 155, 156, 161 Air–blood barrier, 4–5, 8 Allergic responses, 14–15 See also asthma Altitude effects, 179, 199 Alveolar capillary dysplasia, 5–6 Anatomy, 1–3, 26–28 Angiogenesis asthma, 127–145, 213 cell proliferation/migration, 53–54 COPD, 151 definitions, 4, 47, 53 embryonic/fetal development, 4–9 endothelial cell localization, 61–62 endothelium survival, 59–61 extracellular matrix role, 105–125

[Angiogenesis] inducers, 220 inflammation, 131–132 inhibition, 83, 84, 86–87, 109, 110–111, 220 lung neoplasia, chemokine role, 94–96 molecular mechanisms, 45–80 periendothelial cells, 62–64 phases, 130 pulmonary artery obstruction, 94 regulation, 8–9, 81–104 reversibility, 9 skeletal muscle, 197, 200–207 vascular network assembling, 56–59 vascular tube formation, 56 VEGF role, 48–51 vessel sprouting, 53, 54–56 Angiogenic progenitors, 51–53 Angiopoietins (ANGs), 9, 53, 152, 202, 221 Angiostatic chemokines, 83, 84, 86–87 Angiostatin, 221 Angiotensin converting enzyme (ACE), 186, 188 ANGs. See angiopoietins Anoikis, 114 Antiangiogenetic agents, 216–217, 218, 220–221 Antiapoptotic proteins, 158 Antiinflammatory agents, 214, 218, 219, 221 Antileakage agents, 134, 221 Antithrombin, 221 Arterial pressure. See pulmonary hypertension 227

228 Arteriogenesis, 47, 62–64, 152 ASM. See airway smooth muscle Asthma abnormal angiogenesis, 45–46 airway blood flow measurement, 36–37 airway remodeling molecular signaling, 60–61 angiogenesis, 127–145 b2-agonists, 217–219 bone marrow-derived stem cells, 132, 134–136 bronchial circulation effects, 14–15 chemokine role, 91–92 corticosteroids, 37–38, 134, 214–217, 218 current therapies, 214–219 leukotrienes receptor antagonists, 219 microvascular leakage, 133–134, 221 novel therapeutic targets, 219–222 therapeutic approaches, 213–226 therapy assessment, 36–37 tissue remodeling, 128–130 vascularity evaluation, 128–130 vascular remodeling significance, 213–214 vasodilation, 133–134 VEGF effects, 132–133, 206 Basement membrane, 106, 107, 114 b2-Agonists, 217–219 Blood flow. See airway blood flow; bronchial blood flow Blood islands, 51 Blood lakes, 4, 7 Bone marrow-derived cells, 47, 48, 52–53, 134–136 Bosentan, 187–188 BPD. See bronchopulmonary dysplasia Bridging vessels, 47–48, 64–65 Bronchial adenomas, 94–95 Bronchial blood flow See also airway blood flow asthma, 14 circulation development, 1–23 circulation roles, 25, 26 COPD, 15 experimental study, 12–14

Index [Bronchial blood flow] measurement, 12, 25–44 regulation, 12–14 submucosal (airway)/outer wall compartments, 26 Bronchial-to-pulmonary arterial anastomoses, 3, 26, 27–28 Bronchial wall anatomy, 2 Bronchioscopy, 30 Bronchitis, 15, 46, 147, 148, 158–160 See also chronic obstructive pulmonary disease Bronchopulmonary dysplasia (BPD), 96–97 Bronchopulmonary shunts, 3 Bronstein’s modification of Fick’s principle, 32 Cancer, 94–96, 197–198 Capillary networks alveolar capillary dysplasia, 5–6 bronchial circulations, 10 embryonic development, 46 extracellular matrix, 116–117 pulmonary circulation, 8, 171–172 CC chemokines, 82, 83, 86, 87 CCR1-8 (chemokine receptors), 86 CD34C progenitors, 135 Cell migration, 53–55, 88, 89, 115–116 Chemokines angiogenesis regulation, 81–104 asthma, 91–92 bronchopulmonary dysplasia, 96–97 COPD, 92–93 endothelial cell effects, 85–86, 87–88 growth factor interactions, 88–90 interstitial pulmonary fibrosis, 93 intracellular signaling, 87–88 lung neoplasia, 94–96 nomenclature, 82–84 pulmonary artery obstruction, 94 receptor binding, 84–86 systemic organ angiogenesis, 90 Chemotactic cytokines. See chemokines

Index Chronic obstructive pulmonary disease (COPD) airway smooth muscle, 148–149, 153, 155, 156, 161 airway tissue remodeling, 148–149, 150 bronchial circulation effects, 14, 15 bronchial vascular remodeling, 147–168 bronchitis, 15, 46, 147, 148, 158–160 chemokine role, 92–93 emphysema, 15, 60, 147–148, 158–160, 184, 206 genetic polymorphisms, 185–186 growth factors, 154–162 hypoxia, 178–180 inflammation, 182–183 placental growth factor, 155, 156 pulmonary cellular changes, 176–178 pulmonary hemodynamics, 172–173 pulmonary hypertension, 169–196 pulmonary vascular remodeling, 173–186 pulmonary vascular resistance, 172–173 therapeutic approaches, 160, 186–188 thrombosis, 182 transforming growth factor-b, 157 vascular endothelial growth factor, 154–156, 157–162 vascular smooth muscle, 150, 152–153, 155, 156 Collagen I, 116 Collagen XVIII, 107 Collateral vessel growth, 47–48, 64–65 Conditional knockout experiments, 203–204 COPD. See chronic obstructive pulmonary disease Cor pulmonale, 169, 174, 175 Corticosteroids, 37–38, 134, 214–217, 218 Cre/LoxP strategy, 203–204 CX3C chemokines, 82, 83 CXC chemokines, 82–97 CXCR1-5 (chemokine receptors), 84–86, 87 Cytokines. See chemokines Cytoskeletal changes, 87–88

229 Damaged tissue repair, 149, 150 de novo protein synthesis/secretion, 109 Development. See embryonic/fetal development Dimethylether (DME), 31–38 Drugs absorption from airways, 10, 11 therapeutic approaches, 36–37, 160, 186–188, 213–226 ECM. See extracellular matrix Efferent regulatory mechanisms, 13 ELRC CXC chemokines, 82–87 ELRK CXC chemokines, 82, 84, 85, 86, 88 ELR motif, CXC chemokines, 82–84, 85 Embryonic/fetal development, 1, 4–9, 46 anatomical origins, 1–2 growth factors, 48–51 hypoxia-inducible factors, 57 preterm newborns, 96–97 pulmonary circulation, 170–171 vasculogenesis, 4–9, 46, 51–52 VEGF 48–49 Emphysema animal models, 184 bronchial circulation effects, 15 endothelial cell apoptosis, 60 pathogenesis, 147–148, 158–160 VEGF effects, 206 Endostatin (ES), 107, 221 Endothelial cells apoptosis, 59, 114–115 chemokine receptors, 85–86 cytoskeletal changes, 87–88 embryonic precursors, 51–52 extracellular matrix role, 105–125 long-term survival, 59–61 migration, 53–55, 88, 89, 115–116 molecular survival mechanisms, 59–60 proliferation, 53–55, 114 pulmonary vascular remodeling, 176–177 quiescent state, 105–108 signal blocking agents, 221 tissue localized diversity, 61–62

230 Endothelial progenitor cells (EPCs), 46–47, 48, 52–53 Endothelin-1 (ET-1), 181, 184, 187–188 eNOS gene/protein, 181, 185 Eosinophilic infiltration, 128 EPCs. See endothelial progenitor cells ERK. See extracellular signal-regulated protein kinase ES. See endostatin ET-1. See endothelin-1 Evaluation of vascularity, 128–130 Exercise effects, 200–207 Expiration, 11, 29 Expired air temperature profiling, 30 Extracellular matrix (ECM) angiogenesis role, 53–55, 56, 105–125 basement membrane, 106, 107 endothelial cell apoptosis, 114–115 endothelial cell migration, 115–116 endothelial cell proliferation, 114 interstitial matrix, 106 proteolytic processing, 110–111 quiescent endothelium, 105–108 Extracellular signal-regulated protein kinase (ERK), 87 Fetal development. See embryonic/fetal development Fetal liver kinase (Flk-1), 49 FGFs. See fibroblast growth factors Fibroblast growth factors (FGFs) chemokine interactions, 83, 88, 90 COPD 155, 156–157 exercise responses, 200–202 pulmonary vascular remodeling, 180–181 Fibroblasts, 176–178 Fibrosis, 148, 150, 153 Fick’s principle, 31, 32 Flk-1. See fetal liver kinase fms-like tyrosine kinase (Flt-1), 49, 159 Gas exchange, 29–30 Gas uptake blood flow measurement, 31–38 Gene expression exercise activation, 200–204

Index [Gene expression] growth factors in COPD, 157–158 hypoxia, 198–199, 204–206 ICAM-1 in asthma, 128 TGF-b in lungs, 206 Genetic influences on COPD, 185–186 Glucocorticosteroids. See inhaled corticosteroids G-protein-coupled receptor (GPCR), 84–86 G-protein (guanine nucleotide-binding protein), 84, 87 Growth factors angiogenesis control, 8–9, 199 COPD, 154–162 ECM sequestration, 107–108 extracellular matrix, 114 gene expression, 157–158 pulmonary vascular remodeling, 180–181 skeletal muscle, 200–207 therapeutic targets, 160, 216–217 Guanine nucleotide-binding proteincoupled receptor (GPCR), 84–86 Guanine nucleotide-binding protein (G-protein), 84, 87 HA. See hyaluronic acid Healing processes, 149, 150 Heat exchange, 29 Hemangioblasts, 51 Hematopoiesis, 51 Hemodynamics, 57–58, 64–65, 171–173 Hemoptysis, 81 Heparin, 88 Heparin sulfate proteoglycans (HSPG), 88, 90, 106, 107, 109 Herpes virus, 8, 85–86 HIFs. See hypoxia inducible factors High molecular weight hyaluron (HMW-HA), 106–107, 112 HRE. See hypoxia responsive elements HSPG. See heparin sulfate proteoglycans 5-HTT polymorphism, 185 Humoral regulatory factors, 13 Hyaluronic acid (HA), 106–107, 112

Index Hypercarbia, 13 Hypoxia angiogenesis, 151, 197–198 bronchial blood flow effects, 13 gene responses, 198–199 pulmonary vascular remodeling, 150, 178–180 skeletal muscle angiogenesis, 204–206 VEGF production, 49 Hypoxia inducible factors (HIFs), 49, 57, 198, 205 Hypoxia responsive elements (HRE) 49, 198 ICAM-1 expression, 128 ICS. See inhaled corticosteroids Idiopathic pulmonary fibrosis (IPF), 93 IL-8 (CXCL8), 83, 84–85, 86, 87 Infection, 15 Inflammation angiogenic factors, 131–132 asthma, 91, 213–214 bronchopulmonary dysplasia, 96–97 chemokines, 82, 91, 92, 97 COPD 92, 182–183 muscle angiogenesis, 205 Inhaled corticosteroids (ICS), 37–38, 134, 214–217 Injury responses, 135–136, 149, 150 Inspiration, 11, 29 Integrins, 112–113, 114, 116 Interstitial matrix, 106 Interstitial pulmonary fibrosis, 93 Intimal thickening, 152–154 Intracellular signalling, 87–88 Intussusception (nonsprouting angiogenesis), 53 Invasive blood flow measurement, 25, 30 IPF. See idiopathic pulmonary fibrosis Ischemia collateral vessels, 65 pathological angiogenesis, 54, 82, 152 PlGF, 50 pulmonary artery obstruction, 94 therapeutic angiogenesis, 53, 197 vascular regression, 45 VEGF, 9, 198

231 Kaposi sarcoma herpes virus-G protein-coupled receptor (KSHV-GPCR), 85–86 Kinase insert domain containing receptor (KDR), 49 KSHV-GPCR. See Kaposi sarcoma herpes virus-G protein-coupled receptor Laminins, 112–113, 116 Laser Doppler velocimetry, 30–31 Left atrial pressure, 27–28 Leukotrienes receptor antagonists (LTRAs), 219 Long term oxygen therapy (LTOT), 186 Low molecular weight hyaluron (LMW-HA), 112 LTOT. See long term oxygen therapy LTRAs. See leukotrienes receptor antagonists Lung neoplasia, 94–96 Lung transplantation, 31 Mass spectrometers, 35 Matrix metalloproteins (MMPs) angiogenesis role, 53–54, 57 cell migration, 115–116 COPD, 150, 151, 157, 162, 177 extracellular matrix degradation, 53–54 proteolysis, 110 therapeutic inhibitors, 220 VEGF role, 133, 151, 157, 162 Measurement of blood flow, 12, 25–44 Mechanical regulation factors, 13–14 Mesenchymal cells, 51, 135–136 Microscopic anatomy, 2–3, 10, 27 Microvascular leakage, 133–134, 221 Microvascular pressures, 27–28 MMPs. See matrix metalloproteins Molecular angiogenesis mechanisms, 45–80 Monocytes, 65 Mononuclear leukocyte chemoattraction, 84 Morphological changes in pulmonary vasculature, 174–175 Motility. See cell migration Mucosal plexus, 27

232 Mural cell coverage, 62–64 Muscularization, 64 Muscular pulmonary arteries, 170, 174–175 Nasal circulation, 29 Neoplasia, 94–96 Neural regulatory factors, 13 Neutrophil chemoattraction, 83–84 Nitric oxide (NO), 176, 181, 187, 205–206 Nonhypoxic angiogenesis, 204–206 Noninvasive blood flow measurement, 25–44 Nonmuscular pulmonary arteries, 170, 175 Nonsprouting angiogenesis, 53 Normal physiology, 11, 28–30 Nourishment of mucosa, 28–29 Ohm’s law, 172 Oxygen, 56–57, 186 See also hypoxia Partially muscular pulmonary arteries, 170, 175 PCWP. See pulmonary capillary wedge pressure PDE-5. See phosphodiesterase-5 PEDF. See pigment epithelium-derived factor Peribronchial plexus, 27 Pericytes, 63, 176 Periendothelial cells, 62–64 Perlecan, 109 PGI2. See prostacyclin PH. See pulmonary hypertension Pharmacological therapy, 36–37, 160, 186–188, 213–226 Phosphodiesterase-5 inhibitor (PDE-5 inhibitor), 187 Phosphoinositide 3 0 -kinase (PI3K), 87 Physiology, 11, 28–30 PI3K. See phosphoinositide, 3 0 -kinase Pigment epithelium-derived factor (PEDF), 107 Placental growth factor (PlGF) adult angiogenesis, 55 COPD 155, 156, 159

Index [Placental growth factor (PlGF)] endothelial cell apoptosis, 60 VEGF receptor interactions, 50–51 Plasma protein extravasation, 108–109 Poiseuille’s law, 171 Preterm newborns, 96–97 Primary bronchogenic carcinomas, 94–95 Proangiogenic chemokines, 83–84, 86–87 Prostacyclin (prostaglandin I2/PGI2), 188 Proteolytic processing, 110–111 Pulmonary arterial pressure, 169, 171–172 Pulmonary artery obstruction, 94 Pulmonary bronchial anastomoses, 3, 26, 27–28 Pulmonary capillary wedge pressure (PCWP), 172 Pulmonary circulation anatomy, 1, 3 bronchial relationship, 26–28 COPD effects, 150–151, 169–196 hemodynamics, 171–173 nomenclature, 170 normal, 169–170 smoking effects, 183–185 vascular remodeling, 9, 150–151, 169–196 Pulmonary collateral circulation, 26 Pulmonary fibrosis, 93 Pulmonary hypertension (PH), 169–196 Pulmonary vascular resistance (PVR), 171, 172–173 Radioactive microspheres, 35, 36 Repair processes, 149, 150 Revascularization, 53 Right atrial pressure, 27 Right ventricular dysfunction, 173 Saccular stage of development, 96 Serine proteases, 110–111 Serotonin transporter (5-HTT) polymorphism, 185 Skeletal muscle angiogenesis, 197, 200–207 Smoking bronchial vasculature, 149–151, 156–157, 159

Index [Smoking] COPD link, 92, 147, 148 pulmonary vasculature, 181, 182, 183–185 Smooth muscle cells, 47, 63, 175–176, 177, 178 Soluble gas uptake, 31–38 SPARC protein, 109 Sprouting angiogenesis, 53, 54–56 Steady state uptake of inert soluble gas, 32 Stem cells, 48, 132, 134–136 See also endothelial progenitor cells Submucosal blood flow. See airway blood flow Systemic blood supply to the lungs, 26 Systemic organ angiogenesis, 90 TGF-b. See transforming growth factor-b Therapeutic agents, 36–37, 160, 186–188, 213–226 Therapeutic angiogenesis, 53, 197 Thrombosis, 182 Thrombospondins (TSPs), 106, 202 Tissue-type inhibitors of MMPs (TIMPs), 54 Transcription factors, 198 Transforming growth factor-b (TGF-b), 155, 157, 180, 200–202, 206 Transgenic mice studies, 202–204, 205 Transplantation, 31 TSPs. See thrombospondins Tumors, 94–96, 197–198 Vascular endothelial growth factor receptors (VEGFRs), 49–51 embryonic vasculogenesis, 51–52 VEGFR-1, 49–51, 55, 132, 157, 160, 162 VEGFR-2, 49–51, 55, 132–133, 157–162 VEGFR-3, 50, 55, 132 Vascular endothelial growth factor (VEGF), 8–9, 48–51 angiogenesis, 48, 53–63 asthma effects, 132–133

233 [Vascular endothelial growth factor (VEGF)] chemokine interactions, 83, 86, 87, 88–90 COPD, 154–156, 157–162, 176 ECM sequestration, 107–108 endothelial survival, 59 function in lungs, 206 hypoxia response, 205 local deletion studies, 202–204 lung disease relationships, 46 pulmonary vessels, 176 skeletal muscle angiogenesis, 200–207 smoking effects, 184 upregulation with exercise, 200–202 vascular permeability, 53 Vascularity evaluation, 128–130 Vascular network assembly, 56–59 Vascular permeability, 53, 108–109 Vascular progenitors, 46–47, 48, 52–53 Vascular regression, 45–46, 59 Vascular smooth muscle (VSM), 150, 152–153, 155, 156 Vascular support cells, 62–64 Vascular tube formation, 56 Vasculogenesis, 4–9, 46–47, 51–53 Vasoactive substances, 13–14, 36, 181 Vasoconstriction, 134, 150–151, 214 Vasodilation angiogenesis, 53, 86 asthma, 14, 128, 133–134, 136 COPD 176, 179, 181, 184 VEGF. See vascular endothelial growth factor VEGF genes, 48–49 VEGF receptor proteins (VRP), 8–9 VEGFRs. See vascular endothelial growth factor receptors Vessel permeability, 133–134 Vessel sprouting, 53, 54–57 VRP. See VEGF receptor proteins VSM. See vascular smooth muscle XC chemokines, 82