Hypoxic pulmonary vasoconstriction: cellular and molecular mechanisms

Hypoxic Pulmonary Vasoconstriction: Cellular and Molecular Mechanisms

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TABLE OF CONTENTS List of Contributors Preface

ix xv

I. PHYSIOLOGY AND PATHOPHYSIOLOGY OF HYPOXIC PULMONARY VASOCONSTRICTION 1.

2. 3.

Physiological Function of Hypoxic Pulmonary Vasoconstriction Charles A. Hales Hypoxic Pulmonary Vasoconstriction: Heterogeneity Christopher A. Dawson The Physics of Hypoxic Pulmonary Hypertension and Its Connection with Gene Actions Yuan-Cheng Fung and Wei Huang

3 15

35

II. ROLE OF INTRACELLULAR AND SENSITIVITY IN HYPOXIC PULMONARY VASOCONSTRICTION 4.

5.

6.

7.

Sparks in Pulmonary Artery Smooth Muscle Cells: Implications for Hypoxic Pulmonary Vasoconstriction Wei-Min Zhang, Carmelle V. Remillard,

and James S.K. Sham Hypoxia-mediated Regulation on Transients in Pulmonary Artery Smooth Muscle Cells Jean-Pierre Savineau, Sébastien Bonnet,

and Roger Marthan Calcium Mobilization by Hypoxia in Pulmonary Artery Smooth Muscle A. Mark Evans and Michelle Dipp Critical Role of Sensitization in Acute Hypoxic Pulmonary Vasoconstriction Tom P. Robertson and Ivan F. McMurtry

53

67

81

103

vi III. ROLE OF ION CHANNELS IN HYPOXIC PULMONARY VASOCONSTRICTION 8.

9.

10.

11.

Regulation of Ion Channels in Pulmonary Artery Smooth Muscle Cells Sergey V. Smirnov Regulation of Channels by a Mitochondrial Redox Sensor: Implications for Hypoxic Pulmonary Vasoconstriction Rohit Moudgil, Evangelos D. Michelakis,

and Stephen L. Archer Hypoxic Regulation of Channel Expression and Function in Pulmonary Artery Smooth Muscle Cells Hemal Patel, Carmelle V. Remillard,

and Jason X.-J. Yuan Transient Receptor Potential Channels and Capacitative Entry in Hypoxic Pulmonary Vasoconstriction Alison M. Gurney and Lih-Chyuan Ng

121

135

165

199

IV. ROLE OF ENDOTHELIUM IN HYPOXIC PULMONARY VASOCONSTRICTION 12.

Endothelium-dependent Hypoxic Pulmonary Vasoconstriction Jeremy P.T. Ward and Philip I. Aaronson

217

V. MECHANISMS OF OXYGEN SENSING IN THE PULMONARY VASCULATURE 13. 14.

15.

Chemistry of Oxygen and Its Derivatives in the Lung Lisa A. Palmer Interaction of Oxidants with Pulmonary Vascular Signaling Systems Sachin A. Gupte and Michael S. Wolin Mitochondrial Oxygen Sensing in Hypoxic Pulmonary Vasoconstriction Navdeep S. Chandel

233

247

263

vii 16.

17.

18.

Redox Oxygen Sensing in Hypoxic Pulmonary Vasoconstriction Andrea Olschewski and E. Kenneth Weir Mitochondrial Diversity in the Vasculature: Implications for Vascular Oxygen Sensing Sean McMurtry and Evangelos D. Michelakis Hypoxia, Cell Metabolism, and cADPR Accumulation A. Mark Evans

277

293 313

VI. OXYGEN-SENSING MECHANISMS IN OTHER ORGANS AND TISSUES 19.

20.

21. 22.

Involvement of Intracellular Reactive Oxygen Species In the Control of Gene Expression by Oxygen Agnes Görlach, Helmut Acker, and Thomas Kietzmann Oxygen Sensing, Oxygen-sensitive Ion Channels and Mitochondrial Function in Arterial Chemoreceptors José López-Barneo, Patricia Ortega-Sáenz,

Maria García-Fernández, and Ricardo Pardal Oxygen Sensing by Adrenomedullary Chromaffin Cells Roger J. Thompson and Colin A. Nurse Oxygen-sensitive Ion Channels in Pheochromocytoma (PC12) Cells Laura Conforti and David E. Millhorn

341

361

375

389

VII. PATHOLOGY AND MECHANISMS OF HYPOXIA-INDUCED PULMONARY HYPERTENSION 23.

24.

25.

Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension Marlene Rabinovitch Rho/Rho-kinase Signaling in Hypoxic Pulmonary Hypertension Ivan F. McMurtry, Natalie R. Bauer, Sarah A. Gebb, Karen A. Fagan, Tetsutaro Nagaoka, Masahiko Oka, and Tom P. Robertson Hypoxia-sensitive Transcription Factors and Growth Factors

403

419

viii Hypoxia-sensitive Transcription Factors and Growth Factors Ari L. Zaiman and Rubin M. Tuder Heterogeneity in Hypoxia-induced Pulmonary Artery Smooth Muscle Cell Proliferation Maria G. Frid, Neil J. Davie, and Kurt R. Stenmark Persistent Pulmonary Hypertension of the Newborn: Pathophysiology and Treatment Steven H. Abman and Robin H. Steinhorn

25.

26.

27.

28.

Roles for Vasoconstriction and Gene Expression in Hypoxia-induced Pulmonary Vascular Remodeling Bernadette Raffestin, Serge Adnot, and Saadia Eddahibi Polyamine Regulation in Hypoxic Pulmonary Arterial Cells Mark N. Gillespie, Kathryn A. Ziel, Mykhaylo Ruchko, Pavel Babal, and Jack W. Olson

29.

30.

Strain Differences of Hypoxia-Induced Pulmonary Hypertension Mallik R. Karamsetty, James C. Leiter, Lo Chang Ou, Ioana R. Preston, and Nicholas S. Hill

437

449

471

497

511

523

VIII. EXPERIMENTAL MODELS FOR THE STUDY OF HYPOXIC PULMONARY VASOCONSTRICTION 31.

32.

33.

Index

Animal and In Vitro Models for Studying Hypoxic Pulmonary Vasoconstriction Jane A. Madden and John B. Gordon

545

Transgenic and Gene-Targeted Mouse Models in Hypoxic Pulmonary Hypertension Research Yadong Huang

559

Measurement of Ionic Currents and Intracellular Using Patch Clamp and Fluorescence Microscopy Techniques Carmelle V. Remillard and Jason X.-J. Yuan

569 583

List of Contributors Philip I. Aaronson, Ph.D., Reader, Department of Asthma, Allergy and

Respiratory Science, King’s College London, London, U.K. (Chapter 12)

Steven H. Abman, M.D., Professor, The Children’s Hospital, Pediatric Heart-

Lung Center, University of Colorado, Denver, Colorado (Chapter 27)

Helmut Acker, M.D., Professor, Facharzt für Physiologie, Max-Planck-Institut

für Molekulare Physiologie, Dortmund, Germany (Chapter 19)

Serge Adnot, Ph.D., Professor, INSERM U492, Département de Physiologie,

Hôpital Henri Mondor, Créteil, France (Chapter 28)

Stephen L. Archer, M.D., Professor and Director, Division of Cardiology,

University of Alberta Hospital, Edmonton, Alberta, Canada (Chapter 9)

Pavel Babal, M.D., Associate Professor, Department of Pathology, Comenius

University, Bratislava, Slovak Republic (Chapter 29)

Natalie R. Bauer, Ph.D., Fellow, Department of Medicine, University of

Colorado Health Sciences Center, Denver, Colorado (Chapter 24)

Sébastien Bonnet, Ph.D., Laboratoire de Physiologie Cellulaire Respiratoire,

Université Victor Ségalen Bordeaux2, Bordeaux, France (Chapter 5)

Navdeep S. Chandel, Ph.D., Assistant Professor, Department of Medicine,

Northwestern University, Chicago, Illinois (Chapter 15)

Laura Conforti, Ph.D., Assistant Professor, Department of Internal Medicine,

University of Cincinnati, Cincinnati, Ohio (Chapter 22)

Neil J. Davie, Ph.D., Fellow, Department of Pediatrics, University of Colorado

Health Sciences Center, Denver, Colorado (Chapter 26)

Christopher A. Dawson, Ph.D., Professor, Department of Physiology, Medical

College of Wisconsin, Milwaukee, Wisconsin (Chapter 2)

Michelle Dipp, Ph.D., Student, Worcester College, University of Oxford,

Oxford, U.K. (Chapter 6)

Saadia Eddahibi, Ph.D., INSERM U492, Faculté de Médecine de Créteil,

x Créteil, France (Chapter 28) A. Mark Evans, Ph.D., Lecturer, School of Biology, University of St. Andrews, Fife, U.K. (Chapters 6 and 18) Karen A. Fagan, M.D., Assistant Professor, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado (Chapter 24) Maria G. Frid, Ph.D., Instructor, Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado (Chapter 26) Yuan-Cheng Fung, Ph.D., Professor Emeritus, Department of Bioengineering, University of California, San Diego, La Jolla, California (Chapter 3) Maria García-Fernández, B.Sc., Student, Laboratorio de Investigaciones Biomédicas Edificio de Laboratorios, Hospital Universitario Virgen del Rocio, Sevilla, Spain (Chapter 20) Sarah A. Gebb, Ph.D., Instructor, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado (Chapter 24) Mark N. Gillespie, Ph.D., Professor and Chairman, Department of Pharmacology and Center for Lung Biology, University of South Alabama, Mobile, Alabama (Chapter 29) John B. Gordon, M.D., Associate Professor, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin (Chapter 31) Agnes Görlach, M.D., Department Leader, Abt. Experimentelle Kinderkardiologie, Deutsches Herzzentrum Muenchen, Muenchen, Germany (Chapter 19) Sachin A. Gupte, M.D., Ph.D., Fellow, Department of Physiology, New York Medical College, Valhalla, New York (Chapter 14) Alison M. Gurney, Ph.D., Professor, Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, U.K. (Chapter 11) Charles A. Hales, M.D., Professor and Chief, Pulmonary and Critical Care Medicine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (Chapter 1) Nicholas S. Hill, M.D., Professor, Tufts University School of Medicine-New

xi England Medical Center, Boston, Massachusetts (Chapter 30) Wei Huang, Ph.D., Associate Project Scientist, Department of Bioengineering, University of California, San Diego, La Jolla, California (Chapter 3) Yadong Huang, M.D., Ph.D., Assistant Professor, Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California (Chapter 32) Mallik R. Karamsetty, Ph.D., Assistant Professor, Department of Medicine, Brown University, Providence, Rhode Island (Chapter 30) Thomas Kietzmann, M.D., Assistant Professor, Institut fuer Biochemie und Molekulare, Goettingen, Germany (Chapter 19) James C. Leiter, M.D., Professor, Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire (Chapter 30) José López-Barneo, M.D., Ph.D., Professor, Laboratorio de Investigaciones Biomédicas Edificio de Laboratorios, Hospital Universitario Virgen del Rocio, Sevilla, Spain (Chapter 20) Jane A. Madden, Ph.D., Professor, Department of Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin (Chapter 31) Roger Marthan, M.D., Ph.D., Professor, Laboratoire de Physiologie Cellulaire Respiratoire, INSERM 256, Université Victor Ségalen Bordeaux2, Bordeaux, France (Chapter 5) Sean McMurtry, M.D., Fellow, Department of Medicine (Cardiology), University of Alberta, Edmonton, Alberta, Canada (Chapter 17) Ivan F. McMurtry, Ph.D., Professor, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado (Chapters 7 and 24) Evangelos D. Michelakis, M.D., Associate Professor, Department ofMedicine, University of Alberta, Edmonton, Alberta, Canada (Chapters 9 and 17) David E. Millhorn, Ph.D., Professor and Director, Genome Research Institute and Department of Genome Science, University of Cincinnati College of Medicine, Cincinnati, Ohio (Chapter 22) Rohit Moudgil, M.Sc., Student, Division of Cardiology, University of Alberta

xii Hospital, Edmonton, Alberta, Canada (Chapter 9) Tetsutaro Nagaoka, M.D., Fellow, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado (Chapter 24) Lih-Chyuan Ng, Ph.D., Fellow, Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada (Chapter 11) Colin A. Nurse, Ph.D., Professor, Department of Biology, McMaster University, Hamilton, Ontario, Canada (Chapter 21) Masahiko Oka, M.D., Assistant Professor, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado (Chapter 24) Andrea Olschewski, M.D., Staff Anesthesiologist, Department of Anaesthe­ siology, Justus-Liebig-University Giessen, Giessen, Germany (Chapter 16) Jack W. Olson, Ph.D., Professor, Department of Pharmacology and Center for Lung Biology, University of South Alabama, Mobile, Alabama (Chapter 29) Patricia Ortega-Sáenz, Ph.D., Fellow, Laboratorio de Investigaciones Biomédicas Edificio de Laboratorios, Hospital Universitario Virgen del Rocio, Sevilla, Spain (Chapter 20) Lo Chang Ou, Ph.D., Professor, Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire (Chapter 30) Lisa A. Palmer, Ph.D., Associate Professor, Department of Pediatrics, University of Virginia Health System, Charlottesville, Virginia (Chapter 13) Ricardo Pardal, Ph.D., Fellow, Laboratorio de Investigaciones Biomédicas Edificio de Laboratorios, Hospital Universitario Virgen del Rocio, Sevilla, Spain (Chapter 20) Hemal Patel, Ph.D., Fellow, Department of Pharmacology, University of California, San Diego, La Jolla, California (Chapter 10) Ioana R. Preston, M.D., Assistant Professor, Tufts University School of Medicine-New England Medical Center, Boston, Massachusetts (Chapter 30) Marlene Rabinovitch, M.D., Professor, The Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, California (Chapter 23)

xiii Bernadette Raffestin, M.D., Ph.D., INSERM U492, Département de Physiologie, Hôpital Ambroise Paré, Boulogne, France (Chapter 28) Carmelle V. Remillard, Ph.D., Fellow, Department of Medicine, University of California, San Diego, California (Chapters 4, 10 and 33) Thomas P. Robertson, Ph.D., Assistant Professor, Department of Physiology and Pharmacology, University of Georgia, Athens, Georgia (Chapters 7 and 24) Mykhaylo Ruchko, Ph.D., Instructor, Department of Pharmacology, University of South Alabama, Mobile, Alabama (Chapter 29) Jean-Pierre Savineau, Ph.D., Professor, Laboratoire de Physiologie Cellulaire Respiratoire, INSERM 356, Université Victor Ségalen Bordeaux2, Bordeaux, France (Chapter 5) James S.K. Sham, Ph.D., Associate Professor, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland (Chapter 4) Sergey V. Smirnov, Ph.D., Lecturer, Department of Pharmacy and Pharmacology, University of Bath, U.K. (Chapter 8) Robin H. Steinhorn, M.D., Professor, Department of Pediatrics, Northwestern University, Chicago, Illinois (Chapter 27) Kurt R. Stenmark, M.D., Professor, Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado (Chapter 26) Roger J. Thompson, Ph.D., Fellow, Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Colorado (Chapter 21) Rubin M. Tuder, M.D., Associate Professor, Departments of Pathology and Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland (Chapter 25) Jeremy P.T. Ward, Ph.D., Professor, Department of Asthma, Allergy and Respiratory Science, King’s College London, London, U.K. (Chapter 12) E. Kenneth Weir, M.D., Professor, Department of Medicine, University of Minnesota, Minneapolis, Minnesota (Chapter 16)

xiv Michael S. Wolin, Ph.D., Professor, Department of Physiology, New York Medical College, Valhalla, New York (Chapter 14) Jason X.-J. Yuan, M.D., Ph.D., Professor, Department of Medicine, University of California, San Diego, California (Chapters 10 and 33) Ari L. Zaiman, M.D., Ph.D., Fellow, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland (Chapter 25) Wei-Min Zhang, M.D., Ph.D., Fellow, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland (Chapter 4) Kathryn A. Ziel, Ph.D., Instructor, Department of Pharmacology and Center for Lung Biology, University of South Alabama, Mobile, Alabama (Chapter 29)

Preface Hypoxic pulmonary vasoconstriction (HPV) serves a regulatory function by matching perfusion to ventilation and shunting blood flow away from the poorly oxygenated regions of the lung. HPV is a critical physiological mechanism of the lung to ensure maximal oxygenation of the venous blood in the pulmonary artery. Persistent alveolar hypoxia, however, causes pulmonary hypertension which is characterized by sustained pulmonary vasoconstriction and pulmonary vascular remodeling. The hypoxia-mediated pulmonary hypertension causes right heart failure in patients with a variety of cardio­ pulmonary diseases, including chronic obstructive pulmonary disease, congenital heart disease, and mountain sickness. Over the last decade, considerable progress has been made in understanding the cellular and molecular mechanisms involved in HPV and hypoxia-induced pulmonary vascular remodeling. These significant findings provide an essential basis to specify the precise sequences of events of HPV, to identify the etiology of hypoxia-mediated pulmonary hypertension, and to develop new therapeutic approaches for patients with pulmonary hypertension. The major objective of this book is to provide a timely and long lasting guide for investigators in the fields of cardiovascular physiology and pathophysiology, pulmonary vascular disease, and high-altitude physiology and medicine. This will establish a solid scientific foundation for subsequent applications in clinical practice. The book is divided into eight sections: I. and Physiology and pathophysiology of HPV; II. Role of intracellular sensitivity in HPV; III. Role of ion channels in HPV; IV. Role of the endothelium in HPV; V. Mechanisms of oxygen sensing in the pulmonary vasculature; VI. Oxygen-sensing mechanisms in other organs and tissues; VII. Pathology and mechanisms of hypoxia-induced pulmonary hypertension; and VIII. Experimental models for the study of HPV. Subsections in each of the main sections address critical aspects related to hypoxia-induced pulmonary vasoconstriction and pulmonary hypertension. Section I highlights the physiological function (Chapter 1) and heterogeneity (Chapter 2) of HPV, as well as the physical principles of pulmonary circulation, gas exchange, and HPV and their correlation with gene actions (Chapter 3). Intracellular is not only a major trigger for smooth muscle contraction, but also an important signal transduction element that mediates gene expression, protein synthesis, cell migration, and cell proliferation. Section II discusses how intracellular signals are regulated by hypoxia to induce HPV and pulmonary vascular smooth muscle cell proliferation. Four chapters are devoted to aspects of recent findings on the roles of sparks (Chapter 4), agonist-mediated transients (Chapter 5), mobilization from the sarcoplasmic reticulum (Chapter 6), and sensitization (Chapter 7) in the development of HPV. How acute hypoxia regulates cytoplasmic, nuclear, and intracellularly-stored

xvi concentration in pulmonary artery smooth muscle cells is also discussed in this section. Section III is designed to explore the role of ion channels in HPV. Chapter 8 discusses the functionally expressed ion channels along with their regulation in the pulmonary vasculature. Two chapters focus on the regulation of channel activity by a mitochondrial redox sensor (Chapter 9) and by acute exposure to hypoxia (Chapter 10). The functional role of channels (especially voltage-gated channels) in regulating membrane potential and cytoplasmic concentration (via altering activity of voltage-gated channels) in pulmonary artery smooth muscle cells, the transcriptional regulation of channel genes by chronic hypoxia, and the role of dysfunctional channels in the development of hypoxia-induced pulmonary hypertension are also discussed extensively in Chapters 9 and 10. Furthermore, the contribution of transient entry to the development of receptor potential channels and capacitative HPV is reviewed in Chapter 11. Section IV includes an elegant discussion on the role of endothelium in HPV. Section V is designed to describe the putative and potential mechanisms of oxygen sensing in the pulmonary vasculature. It is focused on oxygen radicals (Chapters 13 and 14), mitochondrial oxidative phosphorylation chain (Chapters 15 and 17), cellular redox status (Chapter 16), and cADPR accumulation (Chapter 18). In addition to the pulmonary vasculature, there are many tissues and cells whose function is regulated by oxygen. Section VI is focused on the cellular mechanisms involved in oxygen-sensitive gene expression (Chapter 19), as well as the oxygen sensing mechanisms and oxygen-sensitive ion channels in arterial chemoreceptor (Chapter 20), chromaffin cells (Chapter 21), and pheochromocytoma cells (Chapter 22). Section VII discusses current knowledge on the etiology and pathological characterization of hypoxia-induced pulmonary hypertension and right heart failure. It is focused on the hypoxia-sensitive agonists, mitogens, and transcription factors found in animal and human lung tissues (Chapters 25, 28, and 29); the role of the heterogeneity in hypoxiainduced pulmonary vascular smooth muscle cells proliferation (Chapter 26); the potential mechanisms involved in pulmonary vascular remodeling and hypoxic pulmonary hypertension (Chapters 23, 24, and 28); the pathophysiology and treatment of persistent pulmonary hypertension in the newborn (Chapter 27); and the strain difference of hypoxia-induced pulmonary hypertension (Chapter 30). Section VIII includes three chapters on how to use animal and in vivo models (Chapter 31) and transgenic animal models (Chapter 32) for studying HPV and hypoxia-mediated pulmonary hypertension. A chapter on patch clamp and fluorescence microscopy techniques (Chapter 33) for measuring ion channel currents and intracellular is included. In summary, this book not only covers the current state-of-the-art findings relevant to cellular and molecular processes of hypoxic pulmonary vasoconstriction but also provides the underlying conceptual basis and knowledge regarding etiological mechanisms and experimental therapeutics for

xvii hypoxia-mediated pulmonary hypertension. I hope this book will be something of use not only to those who are experienced basic science investigators in the research fields of hypoxic cardiopulmonary physiology and pathophysiology and pulmonary vascular diseases, but also to a large community of clinicians or physician scientists whose primary subspecialty is in pulmonary and critical care medicine, cardiology, cardiothoracic surgery, environmental medicine, and sports medicine. Acknowledgment: The book is dedicated to Dr. Ayako Makino for continuously supporting me in pursuing an academic career and for her selfless love during the editing of the book, to my parents and grandparents who taught me how to overcome hurdles and difficulties, and to my mentors who guided me into the research field and taught me what HPV was. I would like to take this opportunity to thank all contributors for the excellent chapters and Ms. M. Ramondetta for her instruction in preparing and editing the text. I am indebted to Dr. C.V. Remillard for her diligence in preparing and editing the figures, to Dr. I.F. McMurtry for his suggestions in compiling this book, and to my colleagues and students at the University of California, San Diego for their dedication to sharing their knowledge with others. Finally, I would like to thank Drs. M.P. Blaustein and L.J. Rubin for their guidance and support throughout my career.

Jason X.-J. Yuan San Diego, California October, 2003

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I. PHYSIOLOGY AND PATHOPHYSIOLOGY OF HYPOXIC PULMONARY VASOCONSTRICTION

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Chapter 1 Physiological Function of Hypoxic Pulmonary Vasoconstriction Charles A. Hales Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A.

1. Introduction Hypoxic pulmonary vasoconstriction (HPV) clearly has a role in reducing perfusion through the lung in utero to enhance delivery to the systemic circulation. It may also have a role in adults to improve the balance of blood perfusion to ventilation in the lung to optimize gas exchange, although some have suggested that this is a vestigial response (17). This chapter will look at the physiological role of HPV in the fetus and the adult.

2. History of Hypoxic Pulmonary Vasoconstriction Investigators from as early as the 1930s were aware that men at altitude in the Andes mountains had an enlarged right heart as seen by electrocardiogram, chest X-ray and autopsy compared to men at sea level (29, 34). This was thought to be due to hypoxia, “anoxica anoxia”. How the right heart became hypertrophied was unclear until the recognition in 1946 that pulmonary vessels constricted to hypoxia (46). von Euler and Liljestrand were exploring the regulators of pulmonary blood flow in cats into which they had implanted a rigid tube and flange through the side of a pulmonary artery to measure pulmonary artery pressure. In 9 cats they measured a mean pulmonary artery pressure of 17 mmHg when the cats spontaneously inhaled air or were artificially ventilated. They noted that when the cats were ventilated with 10-11% that there was a distinct rise in pulmonary artery pressure. They did not measure cardiac output with hypoxia but did note only a small rise in pulmonary artery pressure with occlusion of one main pulmonary artery, causing double the blood flow to the remaining right lung, or only a modest rise in pulmonary artery pressure with muscular exercise. They felt the rise in pulmonary artery pressure when the cats received hypoxic ventilation was out of proportion to the rise seen with the flow­

4 induced change in pulmonary artery pressure, suggesting a direct effect of and found hypoxia to constrict the pulmonary vessels. They also examined may enhance HPV more than it constricts by itself (7). a lesser response. Subsequent to these classic studies, many other investigators have confirmed that there is a rise in pulmonary artery pressure in most cases in animals or humans with little or no rise in cardiac output caused by inhalation of 10% (31).

3. Physiologic Characteristics Vasoconstriction

of Hypoxic

Pulmonary

In humans and adult animals the alveolar oxygen tension needs to reach 60 mmHg or lower to initiate pulmonary vasoconstriction (5, 26). In newborn sheep, however, there is evidence of active hypoxic tone even when being ventilated with 30% This enhanced sensitivity to alveolar hypoxia in the newborn probably accounts for the well-known flip/flop of the circulation in the newborn when weaning from the ventilator during which suddenly pulmonary vascular resistance is high and the fetal shunts have opened again. At an alveolar oxygen of 60 mmHg or greater, there is little pulmonary vasoconstriction to hypoxemia even when the mixed venous is as low as 10 mmHg (26). Although as alveolar hypoxia gets more severe, mixed venous hypoxemia may become a more important stimulus to pulmonary vasoconstriction. Nevertheless, the greater responsiveness of lung vessels to alveolar than to vascular hypoxia has led to the assumption that lung vessels autoregulate flow in response to local alveolar ventilation so that poorly ventilated alveoli with low concentrations produce vasoconstriction to shift perfusion to better ventilated alveoli. HPV characteristically has an onset of action in seconds and can be sustained for hours if the hypoxia is regional (43). Diffuse hypoxia tends to reach an early peak rise in pulmonary artery pressure which then tails off over time (44). HPV can be as focal as a lobule (10) or in a larger area, including one lung or both lungs. The ability of the lung vessels to constrict and shift blood flow from one region to another depends on the size of the area made hypoxic (25). If the whole lung is hypoxic, then the lung vessels constrict diffusely and pulmonary artery pressure rises as the heart pumps harder to overcome the rise in pulmonary vascular resistance, allowing cardiac output and oxygen delivery to the tissues to stay as normal as possible. On the other hand, if the hypoxia is regional then the local vasoconstriction can effectively shift blood flow with only a very small rise in pulmonary artery pressure to other well ventilated areas of the lung which are compliant and can receive more flow. The anesthetized dog in Figure 1A shifted 54% of the perfusion from its left lung to the right well-ventilated lung in response to 100% for 7 mins to the left lung (13). The mean pulmonary artery pressure only increased from 14 to 15 mmHg to achieve this diversion and the stimulus to the HPV was alveolar hypoxia 25 mmHg) since the

5

arterial (from the oxygenated lung) was 89 mmHg and the mixed venous was near normal at 34 mmHg (13). The strength of the HPV in this dog exceeded that of most dogs and people where the reduction in blood flow in response to HPV of the lung is usually about 30% (13). The strength of HPV is nevertheless sufficient to divert blood flow from a dependent to a nondependent lung (Fig. 1B) in dogs and man (1, 8, 13).

The site of HPV seems to be the precapillary arterioles in pigs and dogs but perhaps as small as vessels in cats based on microventilatory puncture techniques and sophisticated x-ray arteriography (Fig. 2) (33, 38). The veins may constrict but this seems to be modest and diffuse. This is hard to measure, especially radiologically, because of upstream resistance in

6

the arteries causing less flow and distention of the veins during hypoxia. For years it was thought that the alveolar hypoxia sensor was in lung parenchyma. Now it is known from the work of Madden et al. that isolated pulmonary vessels in diameter) themselves can constrict to hypoxia and further that pulmonary artery smooth muscle cells can contract although weakly in culture in response to hypoxia (23, 32). There is still a possible role for factors exogenous to the vessels to amplify or depress the regional hypoxic vasopressor response as recently reviewed (22). For example, the hypoxic vasoconstrictor response to alveolar hypoxia is less after sympathectomy in lambs though not so in sheep (5) and endothelin may play a role in pigs (21). Thus, exogenous factors may amplify the strength of vasoconstriction endogenous to the pulmonary artery smooth muscle cells.

4. Fetal and Neonatal Pulmonary Circulation The placenta serves as the main source of oxygenation of fetal blood and it itself serves to autoregulate the distribution of blood flow through hypoxic fetoplacental vasoconstriction mediated via channels (15). Just prior to birth, blood flow to the airless lung is 8 to 10% of cardiac output and the of the oxygenated placental blood (17-20 mmHg) is insufficient to decrease resistance to perfusion in the fetal lung. At birth there is a sudden and drastic reduction in pulmonary vascular resistance, a rise in pulmonary venous blood flow with an increase in left atrial pressure so that the patent foramen ovale closes and there is closure of the patent ductus arteriosus such that there is an 8-10 fold increase in lung blood flow (35-37). The decrease in vascular resistance in the lung at birth is in part related to oxygenation of the lung but not entirely.

7 Hyperbaric oxygenation of the fetal lung in utero without expanding it, will increase blood flow (16), showing that HPV is active in decreasing lung perfusion. The increase in blood flow at most, though, rose only to 38% of cardiac output. However, the mixed venous was only 44 mmHg which is probably too low to stop all vasoconstriction. Inflation of the fetal lung with hypoxic gas mixtures will also increase lung blood flow showing physical features of the collapsed lung contribute to pulmonary vascular resistance, and as expected, adding inhaled air to the hypoxic distended lung further reduces the pulmonary vascular resistance (4, 19, 41). Thus, HPV contributes substantially to reducing perfusion to the lung in utero although mechanical factors do so as well.

5. Ventilation Distribution in Normal Subjects with Small Airways Dysfunction The adult lung has over 300,000,000 alveoli and the distribution of ventilation is not uniform. Airway closure occurs in dependent lung at the diaphragm if the subject is erect or at the back if supine. In subjects with small airways disease such as from asthma or smoking, the airway closure is more prominent and occurs earlier in life. Figure 3 shows a scintigram taken by the Massachusetts General Hospital positron camera of the distribution of tracer gas during inhalation in a 29-year old man (14). In this case inhalation of the tracer occurred from near residual volume as is necessary at that age to collapse airways. The image in Figure 3A (left) is taken at residual volume after equilibration with air and the tracer which is almost insoluble. The image in Figure 3A (right) is of a bolus of 250 ml of labeled tracer inhaled from

8 residual volume to total lung capacity. The tracer goes mainly to the apices of the lung. The image in Figure 3B (left) was made after the subject rebreathed the highly soluble gas nitrous oxide and then breath held at residual volume with an open glottis while the nitrous oxide streamed from the reservoir into the bloodstream. The image (Fig. 3B, left) was taken 8 seconds after injection of a in air into the mouth and showed the trachea and central airways. bolus of The image in Figure 3B (right) was 24 seconds later and showed a distribution of the gas similar to that seen with the inhaled bolus in the lower left. Waiting longer got no more tracer into the bases. Thus, the airways to the base were clearly closed, not allowing the inhaled gas to reach them even though a perfusion scan at residual volume showed persistent blood flow to the bases. This non-smoking young man had to exhale considerably to show airway closure. However, by age 44 even subjects with a normal forced expiratory volume in 1 second (FEV1) show airway collapse during tidal volume breathing while supine. Normals by age 65 show airway closure during tidal breathing even seated (18). These areas of low ventilation have the potential to decrease blood oxygenation considerably and it is in these areas that HPV could play a major role in reducing lung blood flow.

6. Extra-uterine Hypoxic Vasoconstriction Nitroglycerin is a pulmonary artery vasodilator. We wondered if it would inhibit HPV, letting us demonstrate whether HPV was important in improving gas exchange in humans. In a dog model similar to that shown with the double lumen endotracheal tube in Figure 1A, we found a mean decrease in perfusion of to the hypoxic lung of 28% in 8 animals (12). An infusion of nitroglycerin reduced the HPV so that only 9% of blood flow was diverted from in the dogs from 89 to 59 mmHg. the hypoxic lung resulting in a fall in Knowing that HPV was inhibited by nitroglycerin, we then gave 0.6 mg sublingual nitroglycerin to a supine 53-year old man admitted to the hospital with suspected coronary artery disease but found to have gastritis with no coronary artery disease. He was a former smoker with an of 99% predicted but a flow at low lung volumes of only 44% predicted, consistent with small airways dysfunction and susceptibility to increased airway collapse (Fig. fell from 80 to 65 mmHg six minutes after the nitroglycerin 3). The arterial pH, shunt or cardiac output tablet was taken with no significant change in (Fig. 4). Thus, the nitroglycerin tablet disturbed ventilation-perfusion (V/Q) balance since neither shunt nor ventilation was changed, consistent with a loss of HPV allowing increased blood flow to hypoxic areas of lung (12). We then looked at a series of individuals studied supine as it minimized any change in cardiac output by nitroglycerin, and maximized airway narrowing or closure since functional residual capacity falls by 15% in the supine position. Eight of the subjects had reduced airflow at low lung volumes consistent

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with small airways dysfunction and six were completely normal. Normal subjects by 9 mmHg and those with small airways dysfunction by 14 decreased their mmHg after nitroglycerin (Fig. 5). Shunt fraction was assessed in 13 of these individuals and it changed by less than 1%, thus not being responsible for the fall Subjects with advanced emphysema or pulmonary fibrosis were in in generally much less responsive to nitroglycerin (Fig. 5). The in these subjects was very low reflecting a likely significant loss of vascular bed. Perhaps in this state there is insufficient compliant vasculature anywhere in the lung for HPV to effectively divert blood. This display of the importance of HPV in humans with minimal or no lung disease has subsequently been shown by fell nitroprusside infusions in patients with congestive heart failure where in spite of an increase in cardiac output and a small rise in mixed venous from 31 to 33 mmHg (30).

7. Hypoxic Vasoconstriction in Pneumonia HPV appears to be ineffective in pneumonia. Light et al. measured blood flow with microspheres to the left lower lobe of the dog before and 3 days after inoculating the left lower lobe with Streptococcus pneumoniae (20). Using radiographs, they confirmed the presence of pneumonia confined to the left lower lobe. Perfusion to the left lower lobe showed a variable and nonsignificant decrease that was not affected by breathing. Utilizing blood samples from the lobar veins at terminal thoracotomy, they measured through the left lower lobe with pneumonia as 0.69 compared to 0.08 through the control right lower lobe. Alveolar ventilation approached zero in some of the pneumonia lungs.

10 They concluded that HPV was ineffectual in pneumonia so that the magnitude of shunt and low V/Q perfusion was increased causing marked hypoxemia. Other authors with other models (27) have reached similar conclusions and Sostman et al. have shown that perfusion scans in consolidated pneumonia cases in humans may be normal (40).

8. Hypoxic Vasoconstriction in Lobar Collapse As opposed to pneumonia where HPV does not seem to work well, there are abundant data in animals that HPV decreases blood flow through an atelectatic lobe. Benumof electromagnetically measured a 59% decrease in blood flow to the left lower lobes of dogs before and after atelectasis (3). Ventilation and reexpansion of that lobe with 95% and 5% failed to increase blood flow to fully restored perfusion. Others the left lower lobe whereas ventilation with have also supported a major role for HPV in animals for diverting lung blood flow from atelectatic lobes, improving systemic oxygenation (28, 42). The data in humans is sparse. Friedlander et al. inserted double lumen endotracheal tubes into anesthetized patients to separate ventilation between the

11 two lungs (9). They then positioned the patients in the lateral dicubitus position and ventilated the non-dependent lung and not the dependent lung after letting fell from 531±42mmHg when both lungs the dependent lung collapse. The were ventilated with to 285±42 mmHg when only the non-dependent lung was ventilated with An infusion of sodium nitroprusside increased cardiac from 29±6.3 to output from 2.5 to 3.2 L/min (P80%) of the channel agonist hypoxic constrictor response (Fig. 3C) whilst the BAYK8644 enhances HPV (8, 36, 38, 69, 70). This dependence on extracellular for activation of the sensor pathway is also true in the ductus arteriosus (73, 120), the carotid body (78, 111) and the adrenomedullary cell (62). Although hypoxia also causes release of from intracellular pools (Fig. 3D), the relative role of intracellular release of remains controversial (123). sensitivity While the voltage-sensitive channels have some intrinsic (33), they are largely responding to changes in membrane potential, as determined by channels (4). Lloyd demonstrated a contraction

140 of PAs treated with the channel blocker procaine (60). Subsequently, it was shown that hypoxia depolarizes PASMCs. The implied ability of hypoxia to inhibit channels was directly demonstrated in 1992 (93) and was subsequently confirmed by other groups (113, 134). channel inhibition depolarizes the plasma membrane and activates the voltage-gated channel to increase (126). Hypoxia and metabolic inhibitors cause constriction intracellular only in the pulmonary circulation, whereas they cause vasodilation in most systemic vascular beds (10, 61, 103). Indeed, the response of even the proximal PAs to hypoxia is predominantly vasodilation (11, 20). The localization of the hypoxic response appears to result from diversity in the local expression of channels, with these voltage-gate channels being predominantly functional in resistance (vs conduit) PAs (11), a finding recently confirmed by another group (112). Within the vasculature, hypoxia-sensitive current is specific to PASMC, and is not found in renal (93) or whole cell splanchnic (134) arterial SM. However, sensitive channels are found in all tissues (26, 64, 86, 93, 120, 130, 134). Progress has been made other recently in determining the molecular identity and regulatory pathways by which these channels respond to changes in In an interesting parallel to the carotid body, chronic hypoxia causes hypertrophy of PASMCs and diminishes the magnitude of the response to acute hypoxic ventilation. This is also true in PAs isolated from humans with chronic obstructive pulmonary disease (COPD). HPV was diminished in PAs from hypoxic patients (71), but was preserved in PAs from normoxic COPD patients (87). The magnitude of HPV in vitro was inversely related to the systemic in these chronically patients (87). Thus both PAs and carotid bodies downregulate their sensing functions in response to chronic hypoxia. This chapter will specifically deal with the pathway of how channels and the network of mitochondria that permeate the vascular SMC interact to generate HPV. We will explore an increasingly accepted redox mechanism for HPV (3, 7, 10, 17, 72, 74), which may have relevance most tissues. This pathway involves PASMC mitochondria acting as redox which sensors, producing activated oxygen species (AOS) in proportion to serve as diffusible mediators that modulate the activity ofseveral and ) (Fig. 4). These redox sensitive channels channel (e.g., control tone through their effects on membrane potential and the L-type channel. Before delving into the interaction of all three factors as a “functional hypoxic-sensing unit”, the characteristics of each component will be examined.

4. Mitochondria as Oxygen Sensors The mitochondrion’s role as the predominant site for consumption and ATP synthesis makes it an obvious candidate site for an sensor. The finding that inhibition of the mitochondrial electron transport chain (ETC) mimics

141 hypoxia further supports this hypothesis (4). Indeed ETC inhibitors cause pulmonary vasoconstriction, systemic vasodilation and carotid body activation, a set of responses elicited by few other stimuli save hypoxia (4).

In 1981, Rounds and McMurtry (103) reported that certain inhibitors of ETC and oxidative phosphorylation (including azide, cyanide, antimycin A, and rotenone) mimicked the HPV in isolated blood-perfused lungs. Furthermore, the same mitochondrial inhibitors which block cytochrome c oxidase (cyanide or azide), stimulated hypoxia-mediated activation of the carotid body. These early studies suggested that the basis for this mimicry of HPV was a decrease in ATP production due to inhibition of oxidative phosphorylation. In support of this “energy hypothesis” inhibition of glycolysis also had similar effects as hypoxia or ETC inhibitors (79, 115). However, it appears that it is the mitochondrion’s ability to alter cellular redox state and produce diffusible redox mediators, rather than its well-established role in producing ATP, that underlies its role as an sensor. Subsequent studies of HPV (induced by moderate hypoxia in perfused lungs) showed no association between HPV and depletion of ATP and adenylate charge (23). Although anoxia (