Endothelial Cells in Health and Disease

Endothelial Cells in Health and Disease

Endothelial Cells in Health and Disease Edited by

William C.Aird Harvard Medical School, Cambridge, Massachusetts, U.S.A. Beth Israel Deaconess Medical Center, Boston, Massachusetts, U.S.A.

Boca Raton London New York Singapore

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Contents Foreword Preface Contributors

1. The Endothelium as an Organ William C.Aird 2. Blood-Brain Barrier Eric V.Shusta 3. Lymphatic Endothelium Satoshi Hirakawa and Michael Detmar 4. High Endothelial Venules Jean-Marc Gauguet, Roberto Bonasio and Ulrich H.von Andrian 5. The Use of Proteomics to Map Phenotypic Heterogeneity of the Endothelium Johanna Lahdenranta, Wadih Arap and Renata Pasqualini 6. The Use of Genomics to Map Phenotypic Heterogeneity of the Endothelium Mary E.Gerritsen, Stuart Hwang, Constance Zlot, James Tomlinson and Michael Ziman 7. The Role of Genetic Predeterminants in Regulating the Phenotypic Heterogeneity of the Endothelium Brant M.Weinstein 8. The Use of Fate Mapping Studies to Follow Lineage Determination of the Endothelium David E.Reese and Takashi Mikawa 9. Oxygen Regulation of Endothelial Cell Phenotypes Yasushi Yoshikawa, Maksim Fedarau, Koichiro Iwanaga, Hiroaki Harada and David J.Pinsky 10. Fluid Mechanical Forces as Extrinsic Modifiers of Endothelial Function Johannes R.Kratz, Kush Parmar, Sripriya Natarajan and Guillermo GarcíaCardeña 11. Vascular Bed-Specific Signaling and Angiogenesis Napoleone Ferrara, Rui Lin and Jennifer LeCouter 12. Differential Regulation of Endothelial Cell Barrier Function Jeffrey R.Jacobson, Steven M.Dudek and Joe G.N.Garcia

viii x xv

1 38 74 87 119

134

150

168

187

212

228 243

13. Differential Regulation of Leukocyte-Endothelial Cell Interactions D.NeilGranger and Karen Y.Stokes 14. Vascular Biology of the Placenta Hartmut Weiler 15. Blood Endothelial Cells Robert P.Hebbel and Anna Solovey 16. Determination of Endothelial Heterogeneity by the Recruitment of Bone Marrow Derived Endothelial Progenitors Shahin Rafii and Jay Edelberg 17. Transcriptional Networks and Endothelial Lineage Peter Oettgen 18. The Diversity of Vascular Disease: A Clinician’s Perspective John P.Cooke 19. Molecular Targets of Tumor Vasculature Eleanor B.Carson-Walter and Brad St. Croix 20. The Role of the Endothelium in Severe Sepsis and Multiple Organ Dysfunction William C.Aird 21. The Hepatic Sinusoidal Endothelial Cell as a Primary Target of Disease Rimma Shaposhnikov and Laurie D.DeLeve 22. Endothelium and Hemostasis William C.Aird 23. Thrombotic Microangiopathies: Role of Microvascular Endothelium in Pathogenesis Thomas O.Daniel 24. Pulmonary Circulation and Pulmonary Hypertension Troy Stevens, Michael Kasper, Carlyne Cool and Norbert Voelkel 25. Endothelial Cell Phenotypes Associated with Organ Transplantation Simon C.Robson Index

257 276 301 320

335 356 383 403

420 437 451

476 506

551

Foreword As a clinical hematologist and vascular biologist, I have spent my career trying to understand the biology of the endothelium. In doing so, I have come to appreciate that this cell layer eludes traditional approaches to investigation, diagnosis, and treatment. The endothelium is distributed throughout the body and behaves as a multifunctional system—a communication network of sorts. The historical fragmentation of biomedicine into organ-specific disciplines has detracted from a full consideration and understanding of the endothelium. Simply put, the endothelium meets every criterion for an organ, and as such warrants attention in its own right. There exists a large gap between our knowledge of cultured endothelial cells and the intact endothelium, and an even wider gap between the bench and the bed-side. Future breakthroughs in endothelial cell biology will depend on the systematic integration of multiple disciplines, including—but certainly not limited to—cardiology, pulmonary medicine, nephrology, neurology, hematology, hepatology, molecular and cell biology, proteomics, genomics, complex systems biology, and molecular diagnostics. Endothelial Cells in Health and Disease represents a laudable first effort to synthesize—and indeed create—a new field in endothelial biomedicine. By weaving together a series of chapters from multiple disciplines, the Editor has succeeded in “bringing the endothelium to life.” After finishing the book, you will have a new-found appreciation for the robustness and versatility of this organ system and a much clearer insight into its important role in health and disease. This book marks the beginning of an exciting new era in “endotheliology.” Robert D.Rosenberg Department of Medicine, Division of Molecular Medicine and Hematology-Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, U.S.A. Department of Biology, Massachusetts Institute of Technology, Cambridge, U.S.A.

Preface Until the mid 19th century, the endothelium was considered to be little more than an inert layer of nucleated cellophane. In the 1950s and 1960s, the use of physiological assays and electron microscopy provided new insights into the role of the endothelium in inflammation. The development of cell culture techniques to harvest and grow endothelial cells from the human umbilical vein in the 1970s revolutionized the field of endothelial cell biology, leading to breathtaking advances in research and development. At the same time, there has been a growing awareness that the endothelium displays heterogeneous properties in vivo. Electron microscopy studies in the last century were the first to uncover site-specific differences in endothelial structure and function. Over the past two decades, additional avenues of research have uncovered previously unimaginable levels of complexity in the intact endothelium. It is now recognized that phenotypic heterogeneity is mediated, in large part, by the micro-environment. Thus, the very assay that helped to jumpstart the field of endothelial cell biology—namely, in vitro cell culture—has fallen short in providing insight into the spatial and temporal dynamics of this cell layer. This limitation has been circumvented by the recent development of novel assay systems to interrogate the endothelium in the context of its native environment, either in the living organism or in a reconstituted system in vitro. Such advances are beginning to paint a clearer picture of a cell layer that is teeming with life and every bit as active as other organ systems. When combined with imminent advances in diagnosis (e.g., molecular imaging and proteomics) and therapy (e.g., endothelial cell “attenuating” agents), the field of endothelial biomedicine has never looked so promising. Unfortunately, the exchange of information and ideas among vascular biologists has long been hampered by the existence of infrastructural barriers—intellectual, financial, cultural, and otherwise. Investigators who study endothelial cell biology in vascular beds outside the heart tend to interact with other members of their own organ-specific discipline—for example, blood-brain barrier experts with neuroscientists, pulmonary endothelium investigators with respiratory physiologists, molecular and cell biologists studying endothelium in sickle cell anemia with their hematology and oncology colleagues. There is little question that a full understanding of the endothelium in health and disease—including its potential as a therapeutic target—will require a more complete synthesis of the field. The purpose of this first edition of Endothelial Cells in Health and Disease is to begin the process of bringing together—for the first time—endothelial cell biologists from different disciplines to summarize recent progress in their respective fields. The book is divided broadly into five sections. The first section will provide an overview of the endothelium as an organ system followed by a consideration of historically recognized “specialized” endothelium, including the blood-brain barrier, lymphatics, and high endothelial venules. The second section highlights new and exciting proteomic and genomic techniques for mapping endothelial cell heterogeneity. The third

section is focused on epigenetic and environmental determinants of endothelial cell function, and includes chapters on genetic predetermination, fate mapping studies, oxygen tension, hemodynamics, and vascular bed-specific growth factor signaling. The fourth section covers a subset of phenotypes of the endothelium, including barrier function and leukocyte transmigration. The final section is devoted to a consideration of endothelium in different disease sates, including tumors, sepsis, sinusoidal obstruction syndrome, hypercoagulability, pulmonary hypertension, and transplantation. Additional chapters cover transcriptional networks, endothelial progenitors, circulating endothelial cells and, importantly, a clinician’s view of endothelial based-disease. A book such as this is necessarily limited in size and scope. As a result, several important topics have been excluded from the current edition. Perhaps the most conspicuous of these omissions is a discussion of coronary artery disease. This should not be construed as an oversight or lack of interest on my part. Rather, I have chosen to focus this book on other areas in vascular biology, with the assumption that those readers interested in pursuing the coronary bed have access to a wealth of excellent reviews in the literature. Many other important areas have been omitted, including a description of endothelium in the retina, the gastrointestinal tract, the skin, and the bone marrow; a consideration of angiogenesis; a summary of novel strategies for diagnosing endothelial-based diseases; and the application of evolutionary principles and complexity theory to an understanding of the endothelium. It is my goal to include these and related topics in subsequent editions of this book. I would like to thank all the contributors who worked hard to meet the deadlines and produced such excellent chapters. I am indebted to Mitchell Halperin, who gave me the courage to think and speak outside the box, to Bob Rosenberg who introduced me to the wonders of the endothelium, and to Michael Gimbrone who has been a constant source of inspiration. William C.Aird Department of Medicine, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, Massachusetts, U.S.A.

Contributors William C.Aird Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, U.S.A. Wadih Arap The University of Texas, M.D.Anderson Cancer Center, Houston, Texas, U.S.A. Roberto Bonasio The CBR Institute for Biomedical Research, Inc. and Department of Pathology, Harvard Medical School, Boston, Massanchusetts, U.S.A. Eleanor B.Carson-Walter Department of Neurosurgery, University of PittsburghPittsburgh, Pittsburgh, Pennsylvania, U.S.A. John P.Cooke Stanford University School of Medicine, Falk CVRC, Stanford, California, U.S.A. Carlyne Cool Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A. Brad St. Croix Tumor Angiogenesis Laboratory, National Cancer Institute-Frederick, Frederick, Maryland, U.S.A. Thomas O.Daniel Ambrx, Inc., San Diego, California, U.S.A. Laurie D.DeLeve USC Research Center for Liver Diseases, Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Michael Detmar Cutaneous Biology Research Center and Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A. Steven M.Dudek Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Jay Edelberg Cornell University Medical College, Ithaca, New York, U.S.A. Maksim Fedarau Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, U.S.A. Napoleone Ferrara Department of Molecular Oncology, Genentech Inc., South San Francisco, California, U.S.A. Joe G.N.Garcia Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Guillermo García-Cardeña Laboratory for Systems Biology, Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Jean-Marc Gauguet The CBR Institute for Biomedical Research, Inc. and Department of Pathology, Harvard Medical School, Boston, Massachusetts, U.S.A. Mary E.Gerritsen Department of Vascular Biology, Millennium Pharmaceuticals, Inc., South San Francisco, California, U.S.A. D.Neil Granger Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, U.S.A.

Hiroaki Harada Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, U.S.A. Robert P.Hebbel Department of Medicine, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota, U.S.A. Satoshi Hirakawa Cutaneous Biology Research Center and Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A. Stuart Hwang Department of Vascular Biology, Millennium Pharmaceuticals, Inc., South San Francisco, California, U.S.A. Koichiro Iwanaga Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, U.S.A. Jeffrey R.Jacobson Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Michael Kasper Department of Anatomy, Technische Hochschule Gustav Carus Universität, Dresden, Germany Johannes R.Kratz Laboratory for Systems Biology, Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Johanna Lahdenranta The University of Texas, M.D.Anderson Cancer Center, Houston, Texas, U.S.A. Jennifer LeCouter Department of Molecular Oncology, Genentech Inc., South San Francisco, California, U.S.A. Rui Lin Department of Molecular Oncology, Genentech Inc., South San Francisco, California, U.S.A. Takashi Mikawa Department of Cell and Developmental Biology, Cornell University Medical College, Ithaca, New York, U.S.A. Sripriya Natarajan Laboratory for Systems Biology, Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Peter Oettgen Division of Cardiology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, U.S.A. Kush Parmar Laboratory for Systems Biology, Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Renata Pasqualini The University of Texas, M.D.Anderson Cancer Center, Houston, Texas, U.S.A. David J.Pinsky Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, U.S.A. Shahin Rafii Cornell University Medical College, Ithaca, New York, U.S.A. David E.Reese Department of Cell and Developmental Biology, Cornell University Medical College, Ithaca, New York, U.S.A. Simon C.Robson Liver Center, Research North, Beth Israel Deaconess Hospital, Boston, Massachusetts, U.S.A. Rimma Shaposhnikov Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

Eric V.Shusta Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A. Anna Solovey Department of Medicine, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota, U.S.A. Troy Stevens Department of Pharmacology, University of South Alabama, Mobile, Alabama, U.S.A. Karen Y.Stokes Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, U.S.A. James Tomlinson Department of Vascular Biology, Millennium Pharmaceuticals, Inc., South San Francisco, California, U.S.A. Norbert Voelkel Pulmonary Hypertension Center and Pulmonary and Critical Care Medicine Division, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A. Ulrich H.von Andrian The CBR Institute for Biomedical Research, Inc. and Department of Pathology, Harvard Medical School, Boston, Massanchusetts, U.S.A. Hartmut Weiler The Blood Research Institute, Blood Center of Southeastern Wisconsin, Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, U.S.A. Brant M.Weinstein Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, Maryland, U.S.A. Yasushi Yoshikawa Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, U.S.A. Michael Ziman Department of Vascular Biology, Millennium Pharmaceuticals, Inc., South San Francisco, California, U.S.A. Constance Zlot Department of Vascular Biology, Millennium Pharmaceuticals, Inc., South San Francisco, California, U.S.A.

1 The Endothelium as an Organ William C.Aird Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, U.S.A.

1. INTRODUCTION The endothelium, which lines the blood vessels of the vascular tree, is a truly pervasive cell layer, weighing 1 kg in an average-sized human and covering a total surface area of 4000–7000 m2 (1). Endothelial cells from a single human, when lined end-to-end, would wrap more than four times around the circumference of the earth. The endothelium is not inert, but rather is highly active, participating in several physiological processes, including the control of vasomotor tone, the trafficking of cells and nutrients, the maintenance of blood fluidity, the regulation of permeability, and the formation of new blood vessels (2). According to the American Heritage Dictionary, an organ is defined as “a differentiated part of an organism, such as an eye, wing, or leaf that performs a specific function.” The Webster’s Revised Unabridged Dictionary defines an organ as a “natural part or structure in an animal or a plant, capable of performing some special action (termed its function), which is essential to the life or well being of the whole.” While the endothelium surely meets these criteria for an organ, it has yet to be widely accepted on these terms. In this chapter, I will argue that its membership into the “organ club” is long overdue.

2. THE BENCH-TO-BEDSIDE DISCONNECT If one peruses the table of contents and indexes of the more popular medical texts or oncall references, one finds little or no mention of the endothelium. The terms “endothelial cells” and “endothelium” are missing not only from the Merk Manual Index, but also from the 64-page July 2003 index of Scientific American Medicine. In the 15th edition of Harrison’s Principles of Internal Medicine, the index refers only to “endothelial injury, in sclerosis,” and “endothelial cell(s), interactions with lymphocytes; vascular proliferation” (3). In contrast to virtually every other conceivable organ, the endothelium lacks formal representation and organized support, that is, there is no subspecialty training in endothelial biomedicine, nor are there national or international societies for the endothelium. As physicians, few of us are attuned

Endothelial cells in health and disease

2

Table 1 Pub Med References According to Keyword and Yeara Endothe Endothe Hepato Hepato Cardio Cardiology lium liology cytes logy myocytes 1984 1987 1990 1993 1996 1999 2001 Total a

1,307 1,973 3,150 4,221 5,012 6,083 10,203 84,798

— — — — — 1 — 1

1,236 1,302 1,608 1,728 1,710 1,738 3,440 36,309

87 95 119 173 206 287 444 3,863

52 61 115 190 346 464 825 5,078

247 407 607 723 833 1,226 1,965 14,162

Accessed august 2003

to the health of this cell layer as we interview and examine our patients. Diarrhea, syncope, or jaundice equivalents do not presently exist for the endothelium. There is no “endothelial box” to circle or check off as we move through the review of systems. Moreover, the endothelium is not amenable to the traditional maneuvers of inspection, palpation, percussion, and auscultation. When it comes to laboratory testing, while renal function is readily assayed with urea and creatinine; liver function with transaminases and/or bilirubin; and hematological function with a complete blood count and peripheral smear, there are no convenient and reliable markers for endothelial cell dysfunction. In a recent Pub Med search, the keywords “endothelial cells” and “endothelium” yielded a total of approximately 55,000–85,000 articles, respectively (Table 1); the term “endotheliology,” a total of 1. This of course is no surprise, since current medical lexicon does not include the term “endotheliology” (nor for that matter any analogous term that embraces an endothelial-centric clinical discipline). But that is precisely the point. Contrast the above ratio with that in the liver or heart fields and one begins to see a curious disconnect (Table 1). In other words, despite the exponential growth of (largely basic science) studies over the years, endothelial disease continues to fly well below the clinical “radar screen.” There are several explanations for this bench-to-bedside gap, three of which are discussed below.

2.1. Out of Sight; Out of Mind One explanation for the under-appreciation of the endothelium as an organ relates to its hidden and enigmatic nature. The endothelium rarely “shows its hand,” at least in the classic ways that we, as physicians, are trained to detect. Like the hematological system, the endothelium is highly diffuse and spatially distributed, extending to all reaches of the human body. Yet unlike blood cells, the endothelial lining is tethered to the blood vessel wall and therefore inaccessible and poorly amenable to study. Although assays do exist for circulating markers of “activated” endothelium, many of these lack specificity, and as

The endothelium as an organ

3

single markers provide little in the way of useful information. Pathological specimens of the endothelium are not routinely available and even if they were, the findings would not necessarily correlate with function.

2.2. Historical Legacies A second factor that has paradoxically contributed to an under-appreciation of the endothelium as an organ relates to the traditional link between vascular biology (and by extension, endothelial cell biology) and cardiology. The connection is steeped in history, its roots dating back to William Harvey’s discovery of the circulation in the early 1600s. Following Harvey’s seminal work, the prevailing view of the cardiovascular system was that of a closed circulatory loop consisting of a pump and series of conduit vessels, with the singular role of delivering oxygen and nutrients to the various tissues of the body. Over the next 400 years, clinical and basic research focused largely on the pump itself, namely on the coronary arteries, the contractile apparatus, the conduction system, and the conduit vessels vis-à-vis their impact on the function of the heart (e.g., hypertension). These developments contributed to and were reinforced by the founding of a large clinical discipline (cardiology), powerful societal infrastructure (American Heart Association), highly successful public awareness campaigns, generous private and public funding, and enormous progress inresearch and development. The importance of these milestones cannot beoverestimated. They have led to vastly improved detection, prevention, andtreatment of coronary artery disease. Over the past 40 years, however, two seminal observations have revolutionized the field of vascular biology and, when taken together, argue for a more complete synthesis of the field. First was the recognition that the endothelium is not an inert barrier, but rather a highly active cell layer that is involved in a wide variety of homeostatic processes. The second important observation was that the endothelium, in traversing each and every organ, establishes a dialogue that is unique to the underlying tissue—in effect marching to the tune of the local microenvironment. This endothelial-tissue interface plays an important role not only in maintaining health of the organism, but also in dictating the focal nature of vascular disease states. Viewed from this perspective, the study of the endothelium transcends all clinical disciplines. While 20 years ago, one was hard pressed to identify more than a small handful of disorders in which the endothelium played a prominent role, today it may be argued that virtually every disease state involves the endothelium, either as a primary determinant of disease or as a victim of collateral damage (Table 2).

2.3. Complexity A final consideration that helps explain the bench-to-bedside chasm relates to the complexity of the endothelium. It was not that long ago that many investigators subscribed to the one gene-one enzyme-single function hypothesis. The goal of the human genome project was to develop a blueprint for human health and disease and to establish a menu list for selective drug targeting. One of the surprises arising from this project was a discovery that human genome contains a mere 30,000 genes—compared with an estimated 14,000 genes in Drosophila. These results indicate that complexity

Endothelial cells in health and disease

4

does not scale with the number of genes. The discordance is explained in part by differences in alternative splicing and variation in posttranslational modification. More importantly, complexity arises from the connections between components, that is, the regulatory network of protein-protein, protein-RNA, and protein-DNA interactions. Stated another way, all biological systems—including the endothelium—are non-linear and display emergent properties. Most investigators in vascular biology (this author included) tend to focus on specific aspects of endothelial cell function using in vitro assays. In doing so, we may overlook critical levels of organization that are essential to a full understanding of the system (Fig. 1). Just as one could never map the human mind by studying a

Table 2 Role of Endothelium in Disease

Disease

Number of Cross-References of Disease Selected Terms with “Endothelium”/“Endothelial References Cells” (Based on Pub Med Search 8/30/03)

Hemaatology-oncology Cancer

74,75

6,133/9,487

—

—

SSD

76–79

237/157

Thalassemia

80,81

29/35

82

—

Myeloproliferative diseases

83,84

68/73

Bone marrow transplantation

85,86

169/189

Transfusion medicine

87–90

282/297

TTP/HUS

91,92

125/100

Coagulation

40,93

2622/2016

Infection

94–96

2820//3141

Sepsis

97,98

891/741

Atherosclerosis

99–104

7318/3727

Congestive heart failure

105–108

570//228

Valvular heart disease

109,110

158/82

Hemoglobinopathies

Hemachromatosis

Infectious disease

Cardiology

The endothelium as an organ

5

Pulmonary Asthma

111,112

251/285

COPD

113,114

55/39

Pulmonary hypertension

115–117

771/397

38,118

182/159

Acute renal failure

119–121

139/125

Chronic renal failure

122–124

269/152

Peptic ulcer disease

125,126

64/74

Inflammatory bowel disease

127,128

147/162

Hepatitis

129,130

197/315

Cirrhosis

131,132

389/461

Pancreatitis

133,134

85/92

Rheumatoid arthritis

135–137

436/600

Scleroderma

138–140

250/204

141,142

2900/1693

Stroke

143–145

824/469

Multiple sclerosis

146,147

186/207

148,149

437/327

ARDS Nephrology

Gastroenterology

Rheumatology

Endocrinology Diabetes Neurology

Other Preeclampsia

Endothelial cells in health and disease

6

Figure 1 Levels of organization. As with all biological systems, the endothelium displays emergent properties. While each level of organization offers a unique platform for investigation, it is important to recognize that microlevel properties do not necessarily predict for macrobehavior. Indeed, as shown in this schematic, certain properties of the endothelium and vasculature are expressed only at higher levels of

The endothelium as an organ

7

organization. Representative examples of level-specific properties are shown. cultured monolayer of neurons, one cannot rely solely upon in vitro systems to predict and model the behavior of the intact endothelium. An important goal for the future, which will be expanded on below, is to learn how to harness the strength of reductionist and holistic approaches to better understand the algorithms that link individual endothelial cells to blood vessels, blood vessels to organs, and organs to the whole organism.

3. FALLING THROUGH THE CRACKS—A REAL-WORLD EXAMPLE Let us consider one example of how the conceptual gap in endothelial-based disease has had an impact on patient care. The case study is severe sepsis, defined as systemic inflammatory response to infection with secondary organ failure. Patients with severe sepsis are typically admitted to the intensive care unit. Since the syndrome is complicated by organ dysfunction, medical care is provided not only by critical care physicians but also by a team of organ-specific consultants, including nephrologists, neurologists, hematologists, and cardiologists. The pathophysiology of sepsis is complex and includes a non-linear interplay between multiple cell types and soluble mediators, including components of the inflammatory and coagulation pathway (4) (see Chapter 20). Over the past decade, enormous resources have been expended on sepsis trials, with more than 10,000 patients enrolled in over 20 placebo-controlled, randomized Phase 3 clinical trials. The vast majority of these therapies have failed to improve survival in patients with severe sepsis. A notable exception is a recombinant form of the natural anticoagulant activated protein C (rhAPC), which was shown in the PROWESS study to reduce 28-day all cause mortality in this patient population (5). The results of the PROWESS trial have created an identity crisis for intensivists and hematologists alike. The critical care field has finally come across an agent that saves lives in severe sepsis. However, the biological plausibility of its mechanism of action is mired in a maze of inflammatory and coagulation pathways. Hematologists have had their own struggles, in this case to make sense of the fact that after 20 years of failed trials in sepsis, the critical care world has embraced a molecule that is near and dear to their hearts as a life-saving measure in a patient population with which they are barely familiar with, and through a mechanism certainly more complicated than simple anticoagulation. In fact, recent evidence suggests that the common underlying thread in severe sepsis is endothelial cell dysfunction and the efficacy of aPC may ultimately be explained by its attenuation of adverse endothelial cell processes, such as apoptosis (6,7). However, the extent to which this is true will remain unknown as long as the intact endothelium continues to defy diagnostic interrogation. At a clinical level, an under-appreciation both for the non-linear nature of the host response to infection and the importance of the endothelium as an organ system may have an impact on patient care. In some cases, physicians who lack a full understanding of

Endothelial cells in health and disease

8

sepsis pathophysiology, as well as the potential mechanisms of action and risk-benefit profile of rhAPC, may avoid prescribing the agent for fear of the unknown. In other cases, poorly informed clinicians may administer the agent, unprepared for its potential complications. Finally, there exist a growing number of physicians genuinely interested in broadening their understanding of the complexities of the host response, and learning more about the endothelium as a component of this response. Owing to the bench-tobedside gap in the endothelial field, these individuals currently have limited resources. As long as an understanding of the role of the endothelium eludes the clinical mainstream, the potential for developing a new class of sepsis drugs, capable of attenuating endothelial cell dysfunction, will remain unrealized. Abetter understanding of the endothelium in health and disease and the development of new tools to assay the endothelium in vivo should help redirect research along more productive lines.

4. HISTORICAL PERSPECTIVES A consideration of time scales and history provides a valuable conceptual framework for approaching the endothelium. In the hospital setting, physicians are focused on stabilizing and discharging their patients as soon as possible—they work on a scale of minutes to days. Primary care physicians are concerned with the life cycle of the patient. With continued breakthroughs in biomedicine, including advances in robotics and information technology, medicine continues to evolve further from a healing art to a preventative science. It is difficult to predict how such a transition will alter the time scale of patient care. Many scientists, including chemists, molecular biologists, and cell biologists, are more concerned with events that occur over nanoseconds to minutes. At the other end of the spectrum is geological time, dating back to earth’s formation some 4.6 billion years ago (Fig. 2). Why should we be concerned with deep time? One of the features that separate biological from non-biological systems is the capacity to adapt and evolve. For billions of years, every one of our ancestors—on both our mother’s and father’s side—was able to attract a mate, and successfully reproduce, thus withstanding the most pressing test of natural selection. Perhaps, we can gain insights into human health and disease by examining our origins. Microfossil records indicate that life first evolved on the earth approximately 3.5 billion years ago. The earliest form was a unicellular prokaryote, similar (in some ways) to today’s photosynthesizing cyanobacteria. Eukaryotes evolved ≈1.8 billion years ago. The next major transition was the evolution of metazoan (multicellular) organisms, beginning 600 million years ago. The earliest forms resembled today’s simple segmented invertebrates. Vertebrates arose from a common ancestor approximately 500 million years ago. The first land vertebrates date back to 400 million years ago. The following

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Figure 2 Historical perspective. The earth formed 4.5 billion years ago. The first prokaryotic fossils date back to approximately 3.6 billion years (1). Shown are various milestones since that time drawn to scale. Eukaryotes appeared 1.8 billion years ago (2), multicellular organisms or metazoa 600 million years ago (3), and vertebrates 500 million years ago (4). The first land vertebrates evolved approximately 400 million years ago (not shown). The evolution of vertebrates marked the appearance of a closed circulation and endothelium (4). The human species is 150,000 years old (5). The transition from the Stone Age (Paleolithic) to the agricultural revolution (Neolithic) occurred a mere 10,000 years ago (6). The history of medicine (particularly as it relates to the cardiovascular system) is a mere blink of the eye in evolutionary terms (7).

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classes of animals appeared in relatively short order: amphibians, reptiles, birds, and mammals. The human species is relatively young, with its origins tracing back to 150,000 years ago. The endothelium appeared with the dawn of vertebrate evolution. Indeed, the vertebrate transition marked the appearance of several common design features, including the endothelium, a closed circulation, three types of circulating blood cells, a coagulation cascade, and acquired immunity. Remarkably, each of these systems evolved over a period of 50 million years, a mere “blink of the eye” in evolutionary terms. For 2.5 million years, our ancestors lived as hunter-gathers, using stones as tools, and building fires. Approximately 10,000 years ago, our ancestors had hunted most of the large species to extinction, and facing the law of diminishing returns, began to form communities, and to engage in farming and animal breeding. This Paleolithic-Neolithic boundary is perhaps the most important transition in the history of medicine and modern disease. The past 10,000 years—which span a mere 400 generations—have witnessed not only stunning advances in technology and civilization, but also the emergence of occupational, nutritional, and infectious epidemics. A consideration of time scales raises interesting questions about our future. It is sobering to consider that 99.99% of all species that have populated this planet are extinct and that the average metazoan survives only 2.5 million years. Regardless of our predictions about the future, as physicians and scientists, we are primarily focused on the present. Indeed, what separates us from every other species on earth is our consciousness and self-awareness. We are gifted with intelligence and innate curiosity and from this blend arises a desire to heal and alleviate human suffering. The history of “modern” western medicine is framed around three major figures. Hippocrates, a Greek physician, is known as the “Father of Medicine.” Hippocrates contributed to a large collection of medical works, termed the Hippocratic Corpus. Interestingly, Hippocrates and his contemporaries did not appreciate that the heart was a propulsive organ. The next major figure was Galen. Also a Greek physician, Galen was one of the most prolific investigators of all time, publishing some 2.5 million words in over 100 collections. Galen, too, did not recognize that blood circulated. He believed that blood was constantly produced in the liver and was distributed to the various tissues and organs via the veins, where it was consumed. Moreover, he hypothesized that the arteries carry a vital spirit from the lung to the peripheral tissues. Galen’s mistaken conclusions are not all that surprising when viewed in the context of the times. After all, arteries and veins differ in their thickness and pulse, arterial and venous blood differ in color, there is a time lag between the cardiac and arterial pulse, and the capillaries are invisible to the human eye. What is surprising is that 1500 years would pass before the fundamental errors of Galen’s theory were exposed. William Harvey, the third major figure in this discussion, discovered and reported that blood circulates in 1629 AD. Although he could not visualize the capillaries, Harvey did surmise their existence. In 1859, an upper-class English gentleman by the name of Charles Darwin made what may be one of the most important observations in the history of mankind. He proposed that all life evolved from a common beginning by the process of natural selection. Until that time, the prevailing view was that world was created 6000 years earlier. Virtually every intellectual scholar in biomedicine, including William Harvey—the discoverer of the circulation—was operating under the belief that they were studying God’s work.

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The change in paradigm is nicely captured by the landscape metaphor, as originally described by Richard Dawkins in his book, Climbing Mount Improbable (8). According to this metaphor, complex design rests at the peak of a mountain (Fig. 3). Dawkins called the mountain Mount Improbable because the species or organ could not have reached the summit by chance alone. There are two sides to the mountain. For centuries, mankind recognized only the side with the cliff. Reaching the summit depended on giant leaps through divine intervention or single generation macromutations, a process referred to as saltation. Darwin exposed the other side of the mountain. He proposed that the gradual incline was surmountable by the cumulative selection of chance mutations, a mechanism that came to be known as natural selection (9). The incline may not be gradual as Darwin once believed, but rather punctuated by “waves of innovation.” Once at the summit, the product—whether a species or an organ—carries the illusion of design (10). The evolutionary history and comparative biology of the endothelium may provide important insights into the endothelium in health and disease. For example, properties of the endothelium that are invariant between vertebrate species are more likely to represent fixed or core design features, whereas those that are not conserved may reflect new contingencies and thus be more amenable to therapeutic retooling. Human evolution has been driven in large part by an arms race with pathogens. Many behavioral properties of the endothelium can be explained by this process. Path dependence (also termed historical constraint or phylogenetic inertia) refers to the notion that a subsystem such as the endothelium is a product of an unbroken lineage of intermediates, and that the continuum over evolutionary time results in maladaptive or jury-rigged structural (and functional) designs. Defining these design

Figure 3 The Landscape metaphor. Imagine that complex design—for example, a species or an organ system (shown is human)—rests at the peak of a summit. Prior to 1859, mankind (preDarwin observer) recognized only the side with the cliff, ascribing the ascent

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to divine intervention. Darwin exposed the other side of the mountain—the one with a gradual incline, surmountable through step-by-step modification (natural selection of random mutations). Along a second axis is the complexity scale of modern day organisms, marked by unicellular organisms, open circulation (most invertebrates), and closed circulation (all vertebrates). “flaws” would provide novel insight into therapy. Evolutionary legacy refers to a mutation or design that may have been beneficial in an earlier time, but is no longer apparently adaptive. For example, we are likely to have evolved specific adaptations to species of pathogens which are now extinct. Finally, it is important to recognize that the endothelium evolved to maximize fitness in a far earlier era, approximately 30,000 years ago. A consideration of environmental changes provides important insight into the vulnerability of the endotheliumto disease.

5. WEAVING AN EVOLUTIONARY TALE An important challenge in studying evolutionary biology is to reconstruct the past. There are several approaches, including the study of fossils, molecular phylogeny, and comparative biology (with the assumption that what works for modern day creatures may have worked for ancestral forms). Unfortunately, the cardiovascular system does not fossilize. Thus, any rendition of the evolutionary history of the endothelium is, at best, speculative. Nevertheless, a consideration of the ancient past provides a powerful conceptual framework for understanding the endothelium in modern times. Single cell organisms obtain their oxygen through simple diffusion, a process that is defined by Fick’s law (the flow of oxygen is directly proportional to the pressure difference and surface area, and inversely proportional to distance oxygen must travel). In multicellular organisms, the cardiovascular system provides a means of overcoming the time-distance constraints of diffusion. As a general rule, the body plan of most multicellular organisms can be simplified according to the scheme shown in Fig. 4 (there are some interesting exceptions to this body plan, including insects). There are four basic elements to the scheme: (1) convection or bulk flow of oxygen from the environment to a highly vascularized surface, (2) oxygen diffusion from environment to blood, (3) convection or bulk flow to the various tissues of the body, and (4) diffusion across into the mitochondrial “sink” of each and every cell. Note that the laws of nature have not been defined:

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Figure 4 Body plan of multicellular organisms. This schematic simplifies oxygen transport according to four steps: (1) convection of oxygenated air or water from environment to a highly vascularized surface (skin, gills, or lungs), (2) diffusion of oxygen across the gas exchanger into the blood, (3) convection of oxygenated blood around to the various tissues of the body, and (4) diffusion of oxygen to the individual cells of the tissues. The circulation is closed in vertebrates, open in invertebrates. (Adapted from Ref. 73.) oxygen transport is still critically dependent on simple diffusion, both at the lung-blood interface and the blood-tissue interface. Oxygen is poorly soluble in water or plasma. Therefore, even in simple multicellular organisms, oxygen delivery is aided by the presence of a respiratory pigment, a molecule that essentially acts like a magnet to attract and carry oxygen. In invertebrates, the respiratory pigment (usually hemocyanin, but sometimes hemoglobin) circulates freely in solution. This observation provides the rationale for developing hemoglobin substitutes in transfusion medicine. In vertebrates, the hemoglobin is packaged inside red blood cells, where it is protected from oxidative stress of the environment and where oxygen binding may be finely regulated through a series of allosteric and cooperative interactions. Non-mammalian vertebrates (fish, amphibians, reptiles, and birds) contain nucleated red cells. In fact, the anulceate red blood cell is unique to mammals. It is interesting to speculate why the nucleus may have been “discarded” during recent evolution. There are many possible explanations. Perhaps the most compelling is that anucleate cells lack mitochondria and oxidative phosphorylation, and therefore do not consume oxygen. In this way, the mammalian red blood cell avoids the conflict of interest of being both a carrier and consumer of oxygen. Countering this argument is the observation that

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hummingbirds, which transport their oxygen in nucleated red cells, have a higher metabolic rate compared with most mammals. Whether from a 70-kg human, a 2-g shrew or a 150-ton blue whale, the mammalian red blood cell is remarkably similar in size and shape (there are some interesting exceptions which will not be discussed here). The high degree of invariance in cell size and shape is consistent with the notion that selection—acting upon the mammalian red blood cell—has led to the optimization of the variables in Fick’s equation. For example, the biconcave shape provides a high surface area-to-volume ratio and a short distance for oxygen to travel. During evolution, a solution to one problem tends to beget a new set of problems. As one example, the development of a cardiovascular system, while providing a means to overcome the time-distance constraints of diffusion (and thereby paving the way for the evolution of large animals), resulted in a highly pressurized system that is at risk for rupture and/or leakage, with potentially two life-threatening consequences: (1) exsanguination or loss of blood from the interior to the exterior (hence, the formation of the coagulation mechanism, consisting of “sticky” cells and a protein gel), and (2) the entry and dissemination of pathogens from the exterior to the interior (hence, the formation of the innate immune response, also composed of cells and a protein gel). In invertebrates, the heart pumps blood (termed hemolymph) into an open cavity (the hematocoele), which bathes the various tissues of the body. In vertebrates, blood is retained within a closed circulation, separated from the underlying tissues by an endothelial lining (11).

6. METAPHORICAL LEXICON An important challenge in promoting the endothelium as an organ is to describe what one cannot see, and to do so in a language that is understandable to the clinical mainstream. One approach is to fall back on the metaphor, otherwise described as a “rhetorical device for transporting knowledge by using a word that brings

Table 3 Metaphors for Describing the Endothelium Metaphor

Advantages

Disadvantages

Cellophane

Intact, delicate, thin; protective barrier property

Ignores living (active, non-inert) qualities; overlooks phenotypic heterogeneity

Gatekeeper

Active role in mediating selective transport Does not describe non-gatekeeping of gases, macromolecules, and white blood functions of the endothelium cells; especially applicable to postcapillary venules

Barometer

Senses biomechanical forces

Does not account for ability to sense biochemical signals; ignores capacity of endothelium to integrate and respond the signals

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Biosensor

Senses biomechanical and biochemical forces

Ignores capacity of endothelium to integrate and respond to the signals

Input-output device

Senses and responds to extracellular signals

Does not describe the intracellular transduction apparatus; fails to emphasize the non-linearity of the device; the study of a single device (e.g., endothelial cell) does not predict global behavior

Circuit board Represents multiple (trillions) of inputoutput devices Ant colony

Implies that the system is hardwired and fixed

Sophisticated, bottom-up organizations; emergence of properties; local rules generate global behavior; spatially and temporally distributed; both colonies (ant and endothelium) are hidden from view, and require special excavation tools for study

connotations from one field into play in another field.”a Metaphors are not literal and therefore must be chosen and interpreted carefully. The intention of this section is not to entrap the reader within the frame of any single example, but rather to provoke speculation and “jumpstart” the recognition process. One of the metaphors is outdated, others are admittedly fanciful, and still others may prove to be closer to the truth (Table 3). For all we know, the endothelium may itself be used one day as a metaphor to describe other biological or non-biological systems.

6.1. Endothelium as Nucleated Cellophane Under low-resolution light microscopy, the endothelium has the appearance of a layer of nucleated cellophane. Indeed, in the absence of more sophisticated a

http://www.santafe.edu/sfi/publications/Bulletins/bulletinFall99/insideSfi/language.html

diagnostic and investigative tools, the endothelium was considered to be little more than an inert barrier, separating flowing blood from underlying tissue. Virchow described the endothelium as “a membrane as simple as any that is ever seen in the body.” Today, the cellophane wrapper metaphor should be viewed for what it is: a historical relic that, while understandable in the context of 19th century biomedicine, fails to capture the living essence of the endothelium.

6.2. Endothelium as Gatekeeper The use of electron microscopy in the 1950s and 1960s provided a powerful new window into the endothelium. These studies demonstrated that the endothelium acts as a selective barrier to macromolecules (12,13). The repertoire of endothelial cell functions was

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subsequently expanded to include regulation of hemostasis, vasomotor tone and leukocyte trafficking. For example, in 1970, the endothelium was shown to express plasminogen activator activity and a cellular inhibitor of fibrinolysis (14), an observation that was later confirmed in cell culture studies (15,16). In 1973, Eric Jaffe demonstrated that endothelial cells express the procoagulant, von Willebrand factor (vWF) (17). Michael Gimbrone first reported that endothelial cells express inducible levels of prostaglandin E, providing additional evidence for the role for the endothelium in regulating hemostasis (18). In 1980, Furchgott (19) demonstrated that the endothelium plays an obligatory role in acetylcholine-mediated vasomotor relaxation via the release of a highly labile diffusible factor, originally termed endothelial derived relaxing factor (EDRF), and later identified as nitric oxide. In a series of elegant studies in the mid1980s, Gimbrone’s group provided compelling evidence that the endothelium actively mediates leukocyte adhesion and transmigration (20–22).

6.3. Endothelium as a Barometer or Biosensor As we have refined our molecular and cellular tools, we have come to appreciate the endothelium more as a barometer or biosensor of the local microenvironment. The advantage of these two metaphors is that they describe the capacity of the endothelial cell to sense biomechanical and biochemical changes in the microenvironment. The disadvantage is that they ignore the capacity of the endothelium to integrate and respond, either adaptively or non-adaptively, to these inputs.

6.4. Endothelium as an Input-Output Device Much of endothelial cell biology can be understood—at least conceptually—by considering each and every endothelial cell in the body as an adaptive input-output device (Fig. 5). The input arises from the extracellular milieu and may include biochemical or biomechanical signals. The output is manifested as the cellular phenotype and includes a number of structural and functional properties. Some of these properties are expressed at the level of individual cells or cell culture (e.g., protein, mRNA, proliferation, apoptosis, migration, and permeability), whereas other properties are expressed at higher levels of organization—for example, the blood vessel (e.g., leukocyte trafficking, vasomotor tone, fibrin deposition) or organism (e.g., redistribution of blood flow, vascular bed-specific phenotypes). Each endothelial cell may have unique intrinsic properties, the so-called “set point.” Differences in set

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Figure 5 Input-output device. Each endothelial cell may be considered an input-output device. Input arises from the extracellular environment and may include biochemical and biomechanical forces. Output is manifested by the cellular phenotype. This model provides a valuable framework for understanding the molecular basis of endothelial cell heterogeneity. If endothelial cells are intrinsically identical (top), then spatial and temporal differences in input signals (e.g., input A, B) will result in spatial and temporal differences in output (output A, B), resulting in heterogeneity. If endothelial cells are epigenetically modified (bottom), then they may display heterogeneous phenotypes (output A, C) at rest and/or in response an identical input (input A). Both mechanisms are operative in the intact organism and contribute to

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generation and maintenance of endothelial cell heterogeneity. point are brought about by environmentally induced epigenetic changes in the “hardwiring” of the cell. If one accepts the analogy of each endothelial cell representing its own input-output device, it is not a stretch to consider the endothelium as a circuit board—one that is hardwired (to some extent) to meet the demands of the tissue, and one that is highly vulnerable to short-circuiting as a mechanism of vasculopathic disease.

6.5. Endothelium as a Small-World, Scale-Free Network One way to approach the complexity of the host response is through the theoretical framework of a network. When we think of networks, we typically imagine neural connections within the brain. However, in a broader sense, networks may be viewed as any interconnected system in which constituent parts (called “nodes”) are linked to one another. Examples of networks that have been extensively studied and mapped include the web of Hollywood actors, the Internet, the World Wide Web, the structure of scientific collaborations, and certain cellular metabolic pathways (reviewed in Ref. 23). In each case, the topology reveals a scale-free behavior in which a relatively small number of nodes have an unusually high number of connections (called “hubs”), and in which no single node is typical of the others. Such topology predicts for two behavioral traits: (1) the network is resistant to accidental attacks—random removal of many nodes does not alter the overall architecture, since the hubs hold the network together, and (2) the network is vulnerable to coordinated attacks—provided that the hubs can be identified and destroyed. Although scale-free networks are pervasive and will likely prove ubiquitous in nature, the topology of most biological systems is not amenable to statistical analyses using current mathematical and molecular/cellular tools. Whether or not such networks actually exist in the endothelium is beside the point—network theory provides a valuable conceptual framework. For example, at the level of the individual endothelial cell, one might envision that various networks are at play, including metabolic and signaling pathways. At the level of the whole body, the endothelium is linked to multiple nodes including (but certainly not limited to) other endothelial cells, circulating cells, abluminal cells, and soluble mediators. In pathophysiology, there may be a dynamic change in the number and nature of both the nodes and links. For example, in sepsis there is induction of signal intermediate and transcription factor activity (nodes) while at a larger scale there is an increase in the interaction between the endothelium with leukocytes, platelets, and soluble mediators (links). An interesting feature of scale-free networks is what is called a “small-world” property—the distance between any two nodes is short. The concept is embodied in the popular expression “six degrees of separation,” a reference to studies showing that any two people in the world are only five handshakes away from each other. In sepsis, the

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increased number of links brought about by an “activation state” might be predicted to reduce the network diameter and increase robustness. A consideration of these principles may have a therapeutic payoff. For example, a robust and error-free sepsis network will be generally resistant to treatment. This of course is the case in sepsis—virtually all single-mediator trials (aimed at one or another “nodes”) have failed to put a dent in sepsis mortality. Based on network principles, there are two approaches to overcome the inherent resistance of the system. One is to destroy a sufficient number of nodes so as to induce collapse of the network (multimodality therapy or cluster bomb approach). The other is to identify and target the hubs in the network (smart bomb approach). The p53 transcription factor has been postulated to represent an intracellular hub in the context of cancer (24). In sepsis, the NF-κB transcription factor may be predicted to play a similar role.

6.6. Endothelium as a Social Colony Studies of ant behavior (entomology) and city life (sociology) provide the most unlikely, yet in many ways the most powerful, of metaphors for the endothelium. Both are examples of self-organizing complexity. The ant colony is a highly sophisticated, extraordinarily robust, and well-adapted biological system, which displays classic properties of emergence, that is, its behavior is understood at the level of the colony and not at the level of the individual ants. Ant colonies and the endothelium not only share this phenomenon of emergence, but also demonstrate a similar propensity to specialization—the ant colony is composed of soldiers, the queen, workers, and is geographically separated into distinct quarters, while the endothelium is similarly specialized in space and time. Both the ant colony and the endothelium represent a bottom-up society; there is no commander or pacemaker driving the system (contrary to popular belief, the queen ant is not in charge of the colony). Each member of society, whether an ant or an endothelial cell, goes about its business fully ignorant of the larger order, following local rules to generate complex global behavior. In reading the following passage from Deborah Gordon’s book, “Ants at Work,” one cannot help draw a comparison with the endothelium: The basic mystery about ant colonies is that there is no management…. There is no central control. No insect issues commands to another or instructs it to do things in a certain way. No individual is aware of what must be done to complete and colony task. Each ant scratches and prods its way through the tiny ant world of its immediate surroundings. Ants meet each other, separate, go about their business. Somehow these small events create a pattern that drives the coordinated behavior of colonies…. (25) A consideration of urban life provides striking parallels to the endothelium. Jacobs (1916–), who has no college degree and no formal training in urban planning, singlehandedly revolutionized the way people think about cities. She argued that variety, diversity, growth, and activity are key to their survival. Although cities are unlike ant colonies in that they involve some degree of central planning, there are certain features

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that capture the essence of spontaneous emergence and nonlinear dynamics, akin to ant colonies and the endothelium: Under the seeming disorder of the old city, wherever the old city is working successfully, is a marvelous order for maintaining the safety of the streets and the freedom of the city. It is a complex order. Its essence is intimacy of sidewalk use, bringing with it a constant succession of eyes. The order is all composed of movement and change, and although it is life, not art, we may fancifully call it the art form of the city and liken it to the dance-not to a simple-minded precision dance with everyone kicking up at the same time, twirling in unison and bowing off en masse, but to an intricate ballet in which the individual dancers and ensembles all have distinctive parts which miraculously reinforce each other and compose an orderly whole. (26) What is interesting about Jacob’s description of city life is that she recognized the importance of interactions, diversity, complexity, and emergent order. The (somewhat paradoxical) notion that a bustling sidewalk is good for the health of society is akin to the notion that a healthy endothelium is not quiescent but rather is highly active, feeding off the dynamic enterprise of cellular and soluble signals (27). Dampen the interactions (for example, remove all the circulating platelets, or reduce blood flow to a crawl) and the endothelium is no longer healthy.

7. ENDOTHELIAL CELL HETEROGENEITY The notion that endothelial cells are heterogeneous is by no means new. In the 1950s, several investigators—including Lord Florey, Guido Majno, George Palade, and Maia and Nicolae Simionescu—employed electron microscopy to demonstrate structural differences between capillaries in different organs. In 1958, Hibbs et al. wrote: Some variation in the structure of capillaries and arterioles normally occurs from one organ to another, and even among vessels of the same organ. (28) In 1961, Majno et al. (13) employed a “vascular labeling” technique in rat cremesteric muscle—involving systemic administration of black colloidal particles and transillumination under a light microscope—to show that histamine or serotonin resulted in differential leakage and deposit of black colloid particles on the venular side of the circulation. The growing recognition that not all endothelial cells are identical was articulated by Florey in 1966:

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Now it is recognized that there are many kinds of endothelial cells which differ from one another substantially in structure, and to some extent in function. (29) In 1967, Reese and Karnovsky (30) were the first to prove the existence of a functional blood brain barrier. In electron microscopic studies of mouse tissues, they demonstrated that exogenously administered horseradish peroxidase readily penetrated the endothelium of the heart, but not the brain. Significant breakthroughs in endothelial cell biology would wait until the early 1970s, when Jaffe and Gimbrone (31–33) independently reported the first successful isolation and primary culture of human endothelial cells from the umbilical vein. The cells, which were obtained by collagenase digestion, could be maintained in culture for weeks to months, and were identified as endothelium by the presence of Weibel-Palade bodies and vWF (VIII-associated antigen). Shortly thereafter, Gimbrone and Cotran (34) successfully isolated vascular smooth muscle cells from umbilical cords, demonstrating a clear distinction between these two cell types. These seminal findings provided the research community with a powerful new tool for dissecting endothelial cell biology and paved the way for breathtaking advances in the field. Indeed, most of our present-day knowledge about the endothelium—from cell surface receptors to signaling pathways, transcriptional networks, cytoskeleton, and cellular function—is directly attributable to our capacity to study endothelial cells in culture. While the cultured endothelial cell became a focal point for research in vascular biology, increasing evidence was pointing to the highly complex topology of the intact endothelium. In 1980s, several groups carried out systematic immunohisto-chemical analyses of the endothelium in various organs (35–37). These studies, which expanded on the earlier results of electron microscopy, revealed differential expression of lectins and antigens in vivo. In 1990s, and more recently in the new millennium, the use of novel genomic and proteomic techniques has uncovered a large array of site-specific properties of the endothelium, providing credence to the analogy of the endothelium as a circuit board, and supporting Gimbrone’s characterization of the endothelium as a dynamically modulatable multifunctional organ (2,38–40). The study of static cultures of one or another endothelial cell type has advantages and disadvantages. On the one hand, such an approach is essential for dissecting, mapping, and/or cloning certain properties of the cell. On the other hand, these studies fail to provide insight into the molecular basis and scope of phenotypic heterogeneity. This limitation was described by Auerbach in 1985: The concept that vascular endothelial cells are not all alike is not a new one to either morphologists or physiologists. Yet laboratory experiments almost always employ endothelial cells from large vessels such as the human umbilical vein or the bovine dorsal aorta, since these are easy to obtain and can be readily isolated and grown in culture. The tacit assumption has been that the basic properties of all endothelial cells are similar enough to warrant the use of the cells as in vitro correlates of endothelial cell activities in vivo. (41)

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According to Auerbach, a key to understanding structural and functional heterogeneity was to isolate and study microvascular endothelial cells from different organs. Unfortunately, site-specific properties of endothelial cells are not always retained in culture. Indeed, when removed from their native microenvironment, endothelial cells are uncoupled from critical extracellular cues and undergo phenotypic drift. While endothelial cells cultured from different sites of the vasculature have been shown to express different properties in vitro, the extent to which these in vitro phenotypes are representative of their in vivo counterparts remains largely unexplored. Michael Gimbrone not only described the reproducible isolation and culture of endothelial cells, thus setting the stage for virtually every breakthrough in endothelial cell biology over the next 30 years, but he was among the first to recognize the limitation associated with static cell cultures. Indeed, Gimbrone carried out many of his studies in more than one type of endothelial cell. More importantly, he pioneered approaches for manipulating the biochemical and biomechanical milieu of the endothelial cell in vitro as a means to study the spatial and temporal regulation of endothelial cell phenotypes (21,42−46). To return to an earlier analogy, endothelial cell heterogeneity may be readily understood from the perspective of an input-output device (Fig. 5B). At any given point in time, the net input of biochemical and biomechanical signals is certain to vary between endothelial cells—between and within different organs. Moreover, for any given endothelial cell, the input will vary from one moment to the next. The spatial and temporal variation in input is sufficient to explain structural and functional differences in properties (output). However, there is also evidence that some site-specific properties of the endothelium are mitotically hereditable and thus “locked in.” Epigenetic modification presumably occurs during development and/or in the postnatal period (and perhaps in disease and aging). Thus, endothelial cell heterogeneity arises from a combination of sitespecific differences in the microenvironment (“nature”) and epigenetic imprinting (“nuture”).

8. ENDOTHELIAL CELL ACTIVATION AND DYSFUNCTION When considering the role of the endothelium in disease, the two most common terms that are used are endothelial cell activation and endothelial cell dysfunction. Each of these terms is discussed below and qualified based on recent advances in the field. Early support for the role of the endothelium in pathophysiology is found in studies of inflammation. In the late 19th century, Cohnheim (47) described in detail the changes seen after injury to the tongue of the frog. He demonstrated that leukocytes adhere to the blood vessel wall of venules (so-called pavementing of leukocytes), many of which passed through the wall into the extravascular tissues (leukocyte emigration). These observations were later confirmed in mammalian species. Studies in an ear chamber model demonstrated that leukocytes adhere to the damaged side of a blood vessel, suggesting that the blood vessel wall—as distinct from the leukocyte—is primarily responsible for mediating adhesion (48). However, the mechanisms underlying inflammation-induced leukocyte adhesion remained elusive for decades. According to

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one theory, the endothelium secreted a gelatinous substance that traps leukocytes (49). Others claimed that electrostatic forces were responsible for mediating the endothelialleukocyte interactions (50). Like so many other aspects of endothelial cell biology, the elucidation of the molecular basis of leukocyte trafficking would wait until the successful culture of endothelial cells in the early 1970s. Pober and Gimbrone (51) were the first to demonstrate that a well-defined stimulus (lectin phytohemagglutinin) could induce the expression of an endothelial cell marker (Ia-like antigen). Through a series of elegant biochemical, molecular, and cellular studies, Gimbrone et al. (52–54) identified the first inducible endothelial cell-specific leukocyte adhesion molecule (ELAM-1; later designated E-selectin). Gimbrone’s group went on to show that numerous inflammatory mediators, including endotoxin, TNF-α and IL-1, induced the expression of new antigens (so-called “activation antigens”) on the surface of HUVEC, an effect that was correlated with the expression of proadhesive, antigenpresenting and procoagulant activities (21,22,43,55,56). Similar findings were reported by other labs (57,58). Pober and Cotran (59) proposed that “activation” reflects the capacity of endothelial cells to perform new functions without evidence of cell injury or cell division. In 1986, Cotran et al. (60) described activation of the endothelium in vivo. In the latter study, a murine monoclonal antibody, which had been shown to bind an antigen in IL-1stimulated HUVEC (52) (later identified as endothelial-leukocyte adhesion molecule (ELAM)-1 or E-selectin), was found to bind to the microvascular endothelium of human skin in delayed hypersensitivity reactions (DHR), but not normal skin (60). Contran concluded: To our knowledge, this is the only reported endothelial-specific mAB that fails to react with normal endothelium, but identifies activated endothelium in vivo… although evidence for active role of the endothelium in inflammation and the importance of inducible endothelium functions has come from recent work (in vitro studies— author’s italics), the notion of endothelial cell activation is a relatively old one. Early light and electron microscopic studies of DHR described plump, hypertrophied endothelium and increased numbers of intracellular organelles, and it was suggested that the endothelium was activated in these reactions. (Reference to Ref. 61). It now seems likely that these changes of activation reflect some of the alterations in structure, function, and growth induced in endothelium by specific monokines and lymphokines. (60) Though not intended as such, the initial observations by Cotran, Gimbrone and others have given way over the years to the notion of the endothelium as a toggle switch. According to this view, quiescent endothelial cells express an anticoagulant, antiadhesive and vasodilatory phenotype, whereas activated endothelial cells express procoagulant, proadhesive and vasoconstricting properties. However, the notion that endothelial cell activation is an all-or-none phenomenon is an over-simplification. For one, the phenotypic spectrum of an endothelial cell follows a continuum (one only has to look at dose-response studies to appreciate this point). Thus the endothelium is

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more analog in its behavior than it is digital.b Second, what constitutes activation for one cell type at a particular snapshot in time may not meet the definition of activation at another site or another moment in time. Third, as initially pointed out by Pober et al. (62), not all inflammatory mediators or endothelial cell activators are created equal. Commonly studied mediators such as TNF-α, thrombin, and lipopolysaccharide have overlapping, yet distinct effects on endothelial cell phenotypes (63). Finally, the terms “activation” and “activity” are not b

At the level of the genome, information is digital (not binary—0 and 1—like a computer but quaternary, represented by four nucleotides: A, C, G, and T). However, the more one moves through hierarchical levels of organization (gene→cell→organ→organism), the more one appreciates graded or analogue-like behavior. synonymous. Normal endothelium is by its very nature highly active—constantly sensing and responding to alterations in the local extracellular environment, as might occur in the setting of transient bacteremia, minor trauma, and other common daily stresses, most of which we are not consciously aware. Therefore, endothelial cell activation is not an allor-nothing response, nor is it necessarily linked to disease. Instead, endothelial cell activation represents a spectrum of response and occurs under both physiological and pathophysiological conditions. Early descriptions of endothelial cell dysfunction focused on structural changes or loss of anatomical integrity, particularly in the context of atherosclerosis. In 1966, Florey suggested that: …consideration of endothelial permeability may be of importance in elucidating the initial phases of the development of atherosclerosis. (64) In 1973, Ross and Glomset proposed a response-to-injury hypothesis to explain the lesions of atherosclerosis: The intact arterial endothelium normally acts as a barrier to some substance or substances present in plasma which upon exposure to vascular smooth muscle promote cell proliferation…the major effect of hemodynamic or other factors that injure the endothelium is to decrease this barrier. (65) Subsequent to Ross’s hypothesis, there was a growing appreciation that the intact endothelium may actively contribute to disease initiation and/or progression (66). The term endothelial cell dysfunction was first coined by Gimbrone (67) in 1980 to describe hyper-adhesiveness of the endothelium to platelets. In 1986, Ganz and colleagues demonstrated paradoxical vasoconstriction of coronary arteries induced by acetylcholine in early and advanced human atherosclerosis, suggesting that abnormal vascular response to acetylcholine may represent a defect in endothelial vasodilator function (68). Cybulsky and Gimbrone (69) were the first to hypothesize a pathophysiological link between inducible endothelial-leukocyte adhesion molecules and atherosclerosis (socalled athero-ELAMs). Using a combination of in vitro cell culture and monoclonal

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antibody strategy, they identified an inducible endothelial cell-specific antigen that binds predominantly to monocytes. Peptide sequencing revealed homology to the predicted sequence of human VCAM-1, which had been previously cloned as a cytokine-inducible protein in endothelial cells (70). In support of their hypothesis, Cybulsky and Gimbrone (69) localized VCAM-1 to the endothelium overlying atherosclerotic lesions in a hyperlipidemic rabbit model. These latter observations not only emphasized the role of endothelial dysfunction as a primary determinant of atherosclerosis, but also helped refocus research and development on the inflammatory nature of this disease process. Based on their findings, Cybulsky and Gimbrone amended the definition of endothelial cell dysfunction as follows: Endothelial cell dysfunction has been implicated in the vasospastic and thrombotic complications that are evident in advanced atherosclerosis. Induction of an adhesion molecule early in atherogenesis may also be considered a manifestation of endothelial dysfunction, in that it results in an abnormally hyperadhesive EC surface. (69) Given that the endothelium is multifunctional and highly distributed in space, it is safe to assume that endothelial cell dysfunction is not restricted anatomically to the heart, nor is it limited in disease scope to atherosclerosis. Endothelial cells residing in arteries, capillaries, and veins of every tissue and organ are prone to dysfunction. Gimbrone described endothelial cell dysfunction as: …nonadaptive changes in endothelial structure and function, provoked by pathophysiological stimuli, (resulting in) localized, acute and chronic alterations in the interactions with the cellular and macromolecular components of circulating blood and the blood vessel wall. (71) Indeed, the term endothelial cell dysfunction may be broadly applied to states in which the endothelial phenotype—whether or not it meets the definition of activation—poses a net liability to the host. Assigning liability scores is of course a subjective exercise. An evolutionary biologist might argue that endothelial cell dysfunction is most relevant in its effect on an individual’s reproductive capacity. A physician would surely expand the meaning of dysfunction to include a far broader spectrum of morbidity. An investigator interested in applying evolutionary principles to an understanding of endothelium in health and disease would point out that the endothelium evolved to a state of maximal fitness in the early ancestral environment, and is not adapted to withstand the rigors of high fat diet, epidemics associated with high density populations, sedentary lifestyle or old age.

9. THERAPEUTIC IMPLICATIONS The endothelium is an attractive therapeutic target for several reasons. First, it is strategically located between the blood and tissue; it is rapidly and preferentially exposed

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to systemically administered agents. Second, it is highly malleable and thus amenable to therapeutic modulation. Third, in establishing a dialogue with the underlying tissue, the endothelium provides the pharmacotherapist with a direct line of communication to the various organs of the body. Finally, if one believes, as I do, that the endothelium is involved in many if not most disease states, then the odds of endothelial-based treatments having a significant impact on human health and disease are high. When applying the concepts of endothelial cell activation and endothelial cell dysfunction to a consideration of therapeutics, it is important to recognize that endothelial cells may be activated—for example, they may express a phenotype that is characteristic of an inflammatory response—without being dysfunctional. Indeed, there are many instances in which endothelial cell activation is a welcome response, whether in wound healing, physiological angiogenesis, local defense against pathogens, and foreign bodies. Therapy is perhaps best reserved for cases in which the phenotype of the endothelium (whether or not it meets the definition of activation) represents a net liability to the host. The notion that endothelial cells resemble input-output devices and that their behavior is not binary, but continuous, has important therapeutic implications. The goal in treating the endothelium is not to reset the switch, but rather to fine-tune and recalibrate the cell, nudging it back to its ideal state. An important challenge is to learn how to determine the nature of that ideal state. Endothelial cell dysfunction usually arises from otherwise adaptive responses (or at least ones that were adaptive in the ancestral environment) that are now excessive, sustained, or spatially and/or temporally misplaced. The transition between endothelial cell function and dysfunction is not always clear. As more effective treatments become available for attenuating dysfunctional endothelium, it will be important to avoid overshooting the desired effect or “lobotomizing” the cells. In this respect, it will serve us well to remember that an active endothelium is a healthy endothelium. Finally, given that endothelial cell phenotypes vary according to time and location in the vascular tree—in both health and disease—it will be essential to target therapy to specific vascular beds.

10. REDUCTIONISM VS. HOLISM There is no question that future advances will rely heavily on continued studies at the cellular and molecular level. But it is equally important that we keep our eye on the larger perspective. Such considerations are by no means to unique to the endothelial cell biology or even to medicine at large. Deborah Gordon, a biologist from Stanford University, studies ants, because she is “interested in linking levels of organization.” The bottom-up emergence of ant colonies, embodied in following passages from her book, Ants at Work, is uncannily reminiscent of the endothelium: The most direct way to investigate animal behavior is to try to see it as a complete pattern, not to take it apart. The more we take it apart, the more work we have to do to put back together the conditions and other kinds of behavior it belongs with…there is some balance between the paralyzing contemplation of the complexity of everything, and a focus on

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components that can each be understood separately but are so isolated that they cannot be traced back to see how they fit into the whole system. (25) The emergence of complex systems poses a common set of challenges in many areas of investigation. How does one leverage the advantages that are inherent in each level of study (molecular, cellular, whole organisms, and colonies) for mechanistic, diagnostic, and therapeutic gain? In the case of the endothelium, advances at the reductionist (basic science) end of the spectrum have far outstripped those at the holistic (clinical) end. Indeed, as long as the field continues to elude the clinical mainstream and/or a platform for bridging the bench-to-bedside gap, it will remain entrenched in reductionism, its potential largely untapped, and unrealized. And herein lies perhaps the most powerful argument for recognizing the endothelium as a bona fide organ.

11. FUTURE DIRECTIONS 11.1. New Questions If we consider the endothelium as a dynamic organ, we may begin to ask new questions, which have gone largely unexplored. For example, if we accept that the endothelium displays emergent properties, what are the simple local rules that govern complex behavior? What determines task allocation in the “endothelial colony”? When viewed from the perspective of a network, the endothelium is a series of nodes that are connected to each other and to other nodes, including non-endothelial cells, soluble mediators, and extracellular matrix. Do these networks display scale-free properties? If so, which are the highly connected nodes (hubs)? And are these hubs vulnerable to attack from a therapeutic standpoint? How does one assign connection weights to the various nodes? How do the nodes and links change over time, and how do they differ in health and disease? As a non-linear system, does the endothelium display chaotic behavior? If so, can the butterfly effect explain the results of certain clinical trials and justify ongoing (largely fruitless) research and development based on linear reasoning? These and many other questions will be important to address as we move forward in understanding the endothelial organ.

11.2. Bridging the Bench-to-Bedside Gap Clinical progress in endothelial cell biology will depend on several factors. First, clinicians should begin to recognize the endothelium as an organ or subsystem—one that has pathophysiological, diagnostic, and therapeutic elements. Second, there must be coordinated efforts to teach and educate physicians about the endothelium, not simply to “bring them up to snuff” but also to train the next generation to develop and/or implement new diagnostic/therapeutic tools. Finally, there is an urgent need for improved technology for observing and tracking the endothelium. Several new diagnostic strategies may ultimately bear fruit in the field of endothelial biomedicine. First, the measurement of a panel of activation markers—as distinct from a

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single mediator—may yield previously unappreciated patterns in response that will aid in diagnosis and/or therapeutic monitoring. Further advances in proteomics will provide new technology platforms (e.g., protein-based chips) to simultaneously monitor dozens of biomarkers. One of the limitations of assaying blood from a peripheral vein or artery is that the sample represents the sum average of activity from multiple vascular beds, and may therefore overlook localized “hot spots” in specific sites of the vasculature. Thus, the use of catheters to sample blood from one or another vascular bed might provide a diagnostic window into these lesions. Refined protocols to isolate and interrogate the phenotype of circulating endothelial cells may yield insight into the function of their vascular-bed-of-origin. Perhaps a more comprehensive and systematic analysis of pathological specimens (e.g., skin biopsies) will provide data that correlate—at least to some extent—with endothelial cell function. Finally, imaging will play an increasingly important role in the clinic. Doppler measurements of blood flow, magnetic resonance angiography, and CT scanning are widely available in clinical practice and should be readily applicable to the study of the endothelial (dys) function in many disease states. Molecular imaging, which combines the power of proteomics and advanced labeling techniques, promises to revolutionize the diagnosis of endothelial-based disorders.

11.3. Should We Create a New Discipline? While we are standing on the cusp of a golden age in vascular biology—one that should see the endothelium promoted as a newly recognized organ—medicine, as it is currently structured, is poorly qualified to carry the field into the 21st century. In his book, “From Chaos to Care,” David Lawrence states the following: Strip away the professionals, the treatments, the equipment, and the institutions of modern medicine, and one finds that care is organized for an earlier, far different period of history…. Medicine today involves a vast, fragmented often isolated array of human, technical and institutional resources. (72) Lawrence is pointing out the limitations of the current infrastructure as an argument for overhauling the medical care system. However, the comments are also relevant to the present discussion. Medicine consists of series of highly fortified, largely historical, and in many cases outdated disciplines, which like cardiology have been cemented over the years by intellectual, financial, and administrative forces, not to mention fierce pride and loyalty. The American Board of Medical Specialties is an organization of 24 approved medical specialty boards. One of these, the American Board of Internal Medicine recognizes nine subspecialties, and provides certificates of added qualification for an additional seven disciplines—none of which systematically embraces endothelial-based diseases. Each of these fields has its own College, Association or Society. In many cases, basic and/or clinical advances are published in organ-specific biomedical journals. The resulting barriers discourage communication between disciplines and by way of mutual reinforcement create a culture that is highly resistant to change. The net result: there is little willingness to embrace the endothelium for what it is, namely a cell layer that is

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teeming with life, and every bit as active (if not more so) than most other organs in the body. There are at least two ways to begin moving the field of endothelial cell biology into the clinic at a pace that is commensurate with advances at the bench. One is to design a new clinical discipline in endothelial medicine. Training could constitute an added qualification to such specialties as cardiology, pulmonary or hematology, just to name a few. It may be argued that a new discipline in “endothelial biomedicine” would only add to the fragmented state of medicine. Or that it is too early—that there are insufficient diagnostic and therapeutic tools—to justify a separate field. The counter-argument, of course, is that a new discipline would in fact represent a synthesis of an otherwise highly scattered field, and would provide a necessary framework for bridging the bench-tobedside gap. An alternative approach is to improve the communication between and within existing clinical and basic disciplines. Endothelial cell investigators from different disciplines tend to represent a minority in their respective fields and have little opportunity to interact with one another. For example, a researcher studying the blood-brain barrier may spend a great deal of time interacting with neurologists and neuroscientists, but remarkably little time with endothelial cell biologists whose work focuses on other vascular beds. As another example, a clinician-scientist in pulmonary medicine interested in understanding the molecular basis of pulmonary hypertension and improving treatment for this condition is unlikely to cross paths with a hematologist who studies the role of the endothelium in thrombotic thrombocytopenic purpura. Both investigators are studying a common cell type and stand to gain from one another’s knowledge. Not only are present divisions antiquated and artificial, they also dampen cross-fertilization and progress. Transcending these barriers will require concerted effort on the part of many by way of collaborative basic research, interinstitutional and industry consortiums for developing novel diagnostic and therapeutic tools, and multidisciplinary team approaches to patient care. These and other efforts to synthesize the field are important first step toward tapping the endothelium for its full potential.

ACKNOWLEDGMENTS I thank Guillermo Garcia-Cardena and Susan Glueck for their critical review of the chapter. I thank Michael Gimbrone for his helpful comments. I am indebted to students in the 2004 Harvard MIT Division of Health Sciences and Technology course HST-527 for their feedback and fresh perspective. This work was supported in part by National Institutes of Health grants HL63609, HL65216, and HL36028.

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91. Mitra D, Jaffe EA, Weksler B, Hajjar KA, Soderland C, Laurence J. Thrombotic thrombocytopenic purpura and sporadic hemolytic-uremic syndrome plasmas induce apoptosis in restricted lineages of human microvascular endothelial cells. Blood 1997; 89(4):1224–1234. 92. Dong JF, Moake JL, Bernardo A, et al. ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor. J Biol Chem 2003; 278(32):29633– 29639. 93. Aird WC. Vascular bed-specific hemostasis: role of endothelium in sepsis pathogenesis. Crit Care Med 2001; 29(7):S28–S35. 94. Peters CJ, Zaki SR. Role of the endothelium in viral hemorrhagic fevers. Crit Care Med 2002; 30(suppl 5):S268–S273. 95. Kim KS. Strategy of Escherichia coli for crossing the blood-brain barrier. J Infect Dis 2002; 186(suppl 2):S220–S224. 96. Hotchkiss RS, Tinsley KW, Swanson PE, Karl IE. Endothelial cell apoptosis in sepsis. Crit Care Med 2002; 30(suppl 5):S225–S228. 97. Aird WC. The role of the endothelium in severe sepsis and the multiple organ dysfunction syndrome. Blood 2003; 23:23. 98. Reinhart K, Bayer O, Brunkhorst F, Meisner M. Markers of endothelial damage in organ dysfunction and sepsis. Crit Care Med 2002; 30(suppl 5):S302–S312. 99. Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclero tic risk. Arterioscler Thromb Vasc Biol 2003; 23(2):168–175. 100. Targonski PV, Bonetti PO, Pumper GM, Higano ST, Holmes DR Jr, Lerman A Coronary endothelial dysfunction is associated with an increased risk of cerebrovascular events. Circulation 2003; 107(22):2805–2809. 101. Quyyumi AA. Prognostic value of endothelial function. Am J Cardiol 2003; 91(12A):19H– 24H. 102. Stone PH, Coskun AU, Kinlay S, et al. Effect of endothelial shear stress on the progression of coronary artery disease, vascular remodeling, and in-stent restenosis in humans: in vivo 6-month follow-up study. Circulation 2003; 108(4):438–444. 103. Effect of nifedipine and cerivastatin on coronary endothelial function in patients with coronary artery disease: the ENCORE I investigators study (Evaluation of Nifedipine and Cerivastatin On Recovery of coronary Endothelial function). Circulation 2003; 107(3):422–428. 104. Chenevard R, Hurlimann D, Bechir M, et al. Selective COX-2 inhibition improves endothelial function in coronary artery disease. Circulation 2003; 107(3):405–409. 105. McNamara DM, Holubkov R, Postava L, et al. Effect of the Asp298 variant of endothelial nitric oxide synthase on survival for patients with congestive heart failure. Circulation 2003; 107(12):1598–1602. 106. Dixon LJ, Morgan DR, Hughes SM, et al. Functional consequences of endothelial nitric oxide synthase uncoupling in congestive cardiac failure. Circulation 2003; 107(13):1725–1728. 107. van den Berg BM, Vink H, Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res 2003; 92(6):592–594. 108. Ferrari R, Bachetti T, Agnoletti L, Comini L, Curello S. Endothelial function and dysfunction in heart failure. Eur Heart J 1998; 19(suppl G):G41–G47. 109. Leask RL, Jain N, Butany J. Endothelium and valvular diseases of the heart. Microsc Res Tech 2003; 60(2):129–137. 110. Poggianti E, Venneri L, Chubuchny V, Jambrik Z, Baroncini LA, Picano E. Aortic valve sclerosis is associated with systemic endothelial dysfunction. J Am Coll Cardiol 2003; 41(1):136–141. 111. Han B, Luo G, Shi ZZ, et al. Gamma-glutamyl leukotrienase, a novel endothelial membrane protein, is specifically responsible for leukotriene D(4) formation in vivo. Am J Pathol 2002; 161(2):481–490.

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112. Ulfman LH, Joosten DP, van Aalst CW, et al. Platelets promote eosinophil adhesion of patients with asthma to endothelium under flow conditions. Am J Respir Cell Mol Biol 2003; 28(4):512–519. 113. Churg A, Wang RD, Tai H, et al. Macrophage metalloelastase mediates acute cigarette smokeinduced inflammation via tumor necrosis factor-alpha release. Am J Respir Crit Care Med 2003; 167(8):1083–1089. 114. Cella G, Sbarai A, Mazzaro G, et al. Plasma markers of endothelial dysfunction in chronic obstructive pulmonary disease. Clin Appl Thromb Hemost 2001; 7(3):205–208. 115. Nagaya N, Kangawa K, Kanda M, et al. Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells. Circulation 2003; 108(7):889– 895. 116. Ameshima S, Golpon H, Cool CD, et al. Peroxisome proliferator-activated receptor gamma (PPARgamma) expression is decreased in pulmonary hypertension and affects endothelial cell growth. Circ Res 2003; 92(10):1162–1169. 117. Yeager ME, Golpon HA, Voelkel NF, Tuder RM. Microsatellite mutational analysis of endothelial cells within plexiform lesions from patients with familial, pediatric, and sporadic pulmonary hypertension. Chest 2002; 121(suppl 3):61S. 118. Muller AM, Hermanns MI, Cronen C, Kirkpatrick CJ. Comparative study of adhesion molecule expression in cultured human macro- and microvascular endothelial cells. Exp Mol Pathol 2002; 73(3):171–180. 119. Sutton TA, Mang HE, Campos SB, Sandoval RM, Yoder MC, Molitoris BA. Injury of the renal microvascular endothelium alters barrier function after ischemia. Am J Physiol Renal Physiol 2003; 285(2):F191–F198. 120. Sutton TA, Fisher CJ, Molitoris BA. Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int 2002; 62(5):1539–1549. 121. Brodsky SV, Yamamoto T, Tada T, et al. Endothelial dysfunction in ischemic acute renal failure: rescue by transplanted endothelial cells. Am J Physiol Renal Physiol 2002; 282(6):F1140–F1149. 122. Bennett-Richards K, Kattenhorn M, Donald A, Oakley G, Varghese Z, Rees L, Deanfield JE. Does oral folic acid lower total homocysteine levels and improve endothelial function in children with chronic reral failure? Circulation 2002; 105:1810–1815. 123. Cottone S, Mule G, Amato F, et al. Amplified biochemical activation of endothelial function in hypertension associated with moderate to severe renal failure. J Nephrol 2002; 15(6):643– 648. 124. Jacobson SH, Egberg N, Hylander B, Lundahl J. Correlation between soluble markers of endothelial dysfunction in patients with renal failure. Am J Nephrol 2002; 22(1):42–47. 125. Ma L, Elliott SN, Cirino G, Buret A, Ignarro LJ, Wallace JL. Platelets modulate gastric ulcer healing: role of endostatin and vascular endothelial growth factor release. Proc Natl Acad Sci USA 2001; 98(11):6470–6475. 126. Ma L, Wallace JL. Endothelial nitric oxide synthase modulates gastric ulcer healing in rats. Am J Physiol Gastrointest Liver Physiol 2000; 279(2):G341–G346. 127. Danese S, de la Motte C, Sturm A, et al. Platelets trigger a CD40-dependent inflammatory response in the microvasculature of inflammatory bowel disease patients. Gastroenterology 2003; 124(5):1249–1264. 128. Rijcken E, Krieglstein CF, Anthoni C, et al. ICAM-1 and VCAM-1 antisense oligonucleotides attenuate in vivo leucocyte adherence and inflammation in rat inflammatory bowel disease. Gut 2002; 51(4):529–535. 129. Breiner KM, Schaller H, Knolle PA. Endothelial cell-mediated uptake of a hepatitis B virus: a new concept of liver targeting of hepatotropic microorganisms. Hepatology 2001; 34(4 Pt 1):803–808. 130. Cacoub P, Ghillani P, Revelen R, et al. Anti-endothelial cell auto-antibodies in hepatitis C virus mixed cryoglobulinemia. J Hepatol 1999; 31(4):598–603.

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131. Yokomori H, Oda M, Ogi M, Sakai K, Ishii H. Enhanced expression of endothelial nitric oxide synthase and caveolin-1 in human cirrhosis. Liver 2002; 22(2):150–158. 132. Mouta Carreira C, Nasser SM, di Tomaso E, et al. LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis. Cancer Res 2001; 61(22):8079–8084. 133. Chen HM, Sunamura M, Shibuya K, et al. Early microcirculatory derangement in mild and severe pancreatitis models in mice. Surg Today 2001; 31(7):634–642. 134. Masamune A, Shimosegawa T, Fujita M, Satoh A, Koizumi M, Toyota T. Ascites of severe acute pancreatitis in rats transcriptionally up-regulates expression of interleukin-6 and -8 in vascular endothelium and mononuclear leukocytes. Dig Dis Sci 2000; 45(2):429–437. 135. Pablos JL, Santiago B, Galindo M, et al. Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis. J Immunol 2003; 170(4):2147– 2152. 136. Ferrell WR, Lockhart JC, Kelso EB, et al. Essential role for proteinase-activated receptor-2 in arthritis. J Clin Invest 2003; 111(1):35–41. 137. Klimiuk PA, Sierakowski S, Latosiewicz R, et al. Soluble adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and vascular endothelial growth factor (VEGF) in patients with distinct variants of rheumatoid synovitis. Ann Rheum Dis 2002; 61(9):804–809. 138. Apras S, Ertenli I, Ozbalkan Z, et al. Effects of oral cyclophosphamide and prednisolone therapy on the endothelial functions and clinical findings in patients with early diffuse systemic sclerosis. Arthritis Rheum 2003; 48(8):2256–2261. 139. Cerinic MM, Valentini G, Sorano GG, et al. Blood coagulation, fibrinolysis, and markers of endothelial dysfunction in systemic sclerosis. Semin Arthritis Rheum 2003; 32(5):285–295. 140. Marie I, Beny JL. Endothelial dysfunction in murine model of systemic sclerosis: tight-skin mice 1. J Invest Dermatol 2002; 119(6):1379–1387. 141. Wheatcroft SB, Williams IL, Shah AM, Kearney MT. Pathophysiological implications of insulin resistance on vascular endothelial function. Diabet Med 2003; 20(4):255–268. 142. Taylor AA. Pathophysiology of hypertension and endothelial dysfunction in patients with diabetes mellitus. Endocrinol Metab Clin North Am 2001; 30(4):983–997. 143. Cherian P, Hankey GJ, Eikelboom JW, et al. Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes. Stroke 2003; 34:2132–2137. 144. Cheng T, Liu D, Griffin JH, et al. Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med 2003; 9(3):338–342. 145. Brown RC, Davis TP. Calcium modulation of adherens and tight junction function: a potential mechanism for blood-brain barrier disruption after stroke. Stroke 2002; 33(6):1706–1711. 146. Greenwood J, Walters CE, Pryce G, et al. Lovastatin inhibits brain endothelial cell Rhomediated lymphocyte migration and attenuates experimental autoimmune encephalomyelitis. Faseb J 2003; 17(8):905–907. 147. Kuruganti PA, Hinojoza JR, Eaton MJ, Ehmann UK, Sobel RA. Interferon-beta counteracts inflammatory mediator-induced effects on brain endothelial cell tight junction molecules— implications for multiple sclerosis. J Neuropathol Exp Neurol 2002; 61(8):710–724. 148. Kauma S, Takacs P, Scordalakes C, Walsh S, Green K, Peng T. Increased endothelial monocyte chemoattractant protein-1 and interleukin-8 in preeclampsia. Obstet Gynecol 2002; 100(4):706–714. 149. Maynard SE, Min JY, Merchan J, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFltl) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003; 111(5):649–658.

2 Blood-Brain Barrier Eric V.Shusta Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.

1. INTRODUCTION AND BACKGROUND This chapter will describe the blood-brain barrier (BBB), a unique class of endothelium that separates the bloodstream from the neuronal milieu. The BBB has been studied quite extensively using classical pathology, physiology, cell biology, and biochemistry techniques. However, exciting opportunities afforded by genomics and proteomics technologies allow unparalleled access to the molecular constituents and mechanisms that endow the BBB with its functional characteristics. Combination of the various approaches mentioned above has led to the identification of functional and regulatory roles for the BBB in both health and central nervous system (CNS) disease. The BBB is functionally defined as the impermeable vasculature that is found throughout the entire brain. The large pial vessels associated with the meningeal membranes that envelop the brain have a lower permeability than that found in most peripheral vasculature but do not form an explicit barrier. However, advancing from the meninges into the brain matter, vascular permeability decreases rapidly and barrier-like impermeability (BBB) arises. The BBB impermeability is exhibited by brain vasculature of all sizes including, arterioles, capillaries, and venules as well as larger arteries and veins. Except for small regions of the brain, the impermeable vasculature extends throughout the entire brain (Sec. 2.1). The barrier is principally formed by the endothelial cell and functionally excludes blood-borne substances from entering the brain tissue. In addition to the endothelial barrier, several perivascular cell types on the brain side of the vessels intimately interact with the endothelial cell and help elicit the BBB phenotype. This endothelial cell-perivascular cell composite has been coined the “neurovascular unit” (Sec. 2). The perivascular cells include: vascular smooth muscle cells that line larger vessels, pericytes that share a basement membrane with endothelial cells, astrocytes which are glial cells that function in both endothelial cell differentiation and neuronal support, and neurons. The functional importance of these cell types on the neurovascular unit is discussed in detail throughout this chapter.

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2. BBB STRUCTURE AND FUNCTION The BBB is comprised of a specialized class of endothelium that forms a cellular barrier between the bloodstream and the interstices of the brain. The brain microvasculature is distinct from non-BBB vascular beds found in the periphery because it acts as a barrier to ions, small molecule solutes, peptides, and proteins that can pass freely in many other vascular beds. As a consequence of this barrier function, the endothelial cell acts as a regulatory interface for signaling and transport between the blood and the brain. By restricting non-specific flux of blood-borne constituents, the BBB plays an important role in maintaining parenchymal homeostasis and protecting the neuronal environment of the CNS from fluctuations in blood composition. The relative lack of permeability of brain capillaries mandates the presence of nutrient transport systems, especially those required to supply the brain with energy. Finally, the BBB also participates in immune surveillance mechanisms since the circulating lymphocytes do not have free access to the interstitial space of the brain. The main phenotypic attribute promoting barrier function is the presence of intercellular tight junctions between neighboring endothelial cells. These epithelial-like tight junctions regulate the paracellular flux of both metabolites and cells, and in essence render the brain impermeable to most circulating substances. In addition, the BBB displays only low levels of pinocytotic transport. It follows that the BBB must rely on the presence of specialized transporters and carrier systems to regulate the bi-directional flux of molecules between the bloodstream and the brain. The brain is a highly vascularized organ, presumably reflecting its demands for highlevel aerobic metabolism. The human brain has been estimated to contain over 400 miles of capillaries that are spaced at distances of approximately 40 µm. This high vascular density and surface area permit rapid and efficient transport of selected substrates across the BBB (1). The capillaries themselves range from 10 µm in diameter at the precapillary arteriole to about 5 µm at their smallest diameter (Fig. 1) (2,3). Because of the similarity in size, isolated capillary preparations invariably include arteriole and venule components and are often referred to as brain microvasculature preparations. Endothelial cells of the BBB are elongated and thin, measuring a mere 100–300 nm between luminal (vessel) and abluminal (brain) compartments (3–5) yielding rapid molecular transport. Finally, by asymmetric segregation of the luminal membrane constituents from those located in the abluminal membrane, the tight junctions render the BBB endothelium highly polarized (6–8). Although the endothelium is the principal determinant of barrier function, perivascular non-endothelial cells have also been shown to make significant contributions (Fig. 2). Vascular smooth muscle cells line the precapillary arterioles (Fig. 1B), while pericytes dot the abluminal side of capillaries (Fig. 1B and C). Pericytes share a common basement membrane with the endothelial cells (Fig. 2). The end feet of astroglial cells form a cagelike network encasing a large fraction of the endothelial cell surface (9) (Fig. 1D–F) and have been shown to play very important roles in eliciting the barrier phenotype in endothelial cells (see below). This set of cells then interacts with the brain microvascular endothelium to form the specialized, impermeable neurovascular unit.

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2.1 Molecular Architecture of Tight Junctions The barrier function of brain endothelial cells was confirmed in classical experiments where it was noted that intravenously injected horse radish peroxidase (40,000 Da)

Figure 1 The intimate relationship between brain endothelial cells and perivascular cells. (A) Light microscopy of isolated bovine brain capillaries stained with o-toluidine blue. (B) Light microscopy of isolated bovine brain capillaries immunostained with an anti-smooth muscle actin antibody. Note that the precapillary arteriole stains due to the presence of a smooth muscle lining, while the capillaries do not exhibit any staining for smooth muscle actin.

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Pericytes are indicated with arrowheads. (C) Light microscopy of isolated bovine brain capillaries stained with the endothelial cellspecific lectin, Griffonia simplicifolia agglutinin. The continuous staining of the microvessel is indicative of an antigen with endothelial cell origin, whereas the two pericytes (arrowheads) are unstained. Bars in A– C are 20 µm. (D and E) Confocal images of rat brain coronal sections immunostained with anti-GFAP antibodies. The rosette-like structure of the astrocyte endfeet form a cage around the blood vessels. Scale bars in D and E are 50 µm. L=lumen. (F) Drawing depicting interactions of astrocyte endfeet and the abluminal membrane of endothelial cells. (Panels D–F: From Ref. (9). Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc). was excluded from entering the brain parenchyma of the mouse (8). This was observed throughout the brain regardless of vessel size or presence of smooth muscle cells (8). It was subsequently observed that electron dense junctional contacts were present at each contact point between endothelial cells (7). Later, the barrier to low molecular weight substrates was confirmed as a microperoxidase, with a molecular weight of only 1800 Da, was also excluded from the brains of mice (6). These

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Figure 2 Schematic of brain capillary cross section. A=astrocyte foot process, N=nerve terminal, P=pericyte, EC=endothelial cell, TJ=tight junction. so-called tight junctions confer a high trans endothelial electrical resistance (TEER) of 500–3000 Ω cm2 (10,11). The barrier phenotype is present throughout the entire brain vasculature except in small regions near the ventricular system, known as circumventricular organs, that are perfused by capillaries without a BBB (12) and participate in neuroendocrine regulation. In a developing rodent, the brain vasculature is fenestrated until embryonic days E11–E13 during which time tight junctions develop as fenestrations typical of peripheral capillaries disappear (13,14). This process coincides with the impermeable phenotype found in the adult brain and restricts access to substances that normally diffuse freely into a developing embryonic brain.

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The tight junctions found in brain capillaries are composed of both integral membrane proteins that link adjacent cells and peripheral membrane proteins that link the junctions to the cytoskeleton. The first transmembrane tight junctional component identified was occludin (15). Occludin is an integral membrane protein that mediates cell-cell connections (Fig. 3). The cytoplasmic domain of occludin is highly phosphorylated when located in tight junctions (16) and this phosphorylation can regulate tight junctional permeability (17). Although the exact role of tight junction occludin has not been fully determined, existing data support a regulatory role rather than a barrier-establishing role (18). In contrast to occludin, another class of

Figure 3 Drawing of brain capillary cell-cell junction. Paracellular flux is not allowed by the tight junction composite of occudin, claudin, and JAM proteins. Cell-cell junctions are stabilized by cadherins at adherens

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junctions. Cell accessory proteins ZO, cingulin, 7H6, and AF6 are involved in coordinating the linkage of the transmembrane junctional proteins to the cytoskeleton and other cellular mediators. transmembrane proteins known as claudins appears to play the major role in forming the seal that restricts paracellular transport. There are at least 24 isoforms of claudins and this broad class confers junctional specificity to different cell types (19,20). Claudins-1 (21), 3 (22), and -5 (22–24) have been identified at the BBB. Claudin-5 has recently been shown to selectively regulate paracellular transport as evidenced by leakage of small molecule tracers, but not of larger molecules (1.9 kDa or greater) in claudin-5 knockout mice (25). Junctional adhesion molecules (JAM) are another type of transmembrane protein that localize to tight junctions (26). These proteins are members of the immunoglobulin superfamily and have the ability to increase transcellular resistance in cells not normally forming tight junctions (26,27) in addition to promoting occludin localization at intercellular boundaries (28). The JAM proteins are also involved in the extravasation of monocytes and leukocytes in vitro and in vivo (26,29,30). Endothelial cell-selective adhesion molecule (ESAM) is yet another transmembrane protein that is localized to tight junctional regions (31) and has enriched gene expression at the BBB (32). Platelet endothelial cell adhesion molecule (PECAM-1/CD31) has also been observed to localize at endothelial junctions (33,34). However, it is not entirely clear if PECAM-1 localizes to tight junctions at the BBB (35,36). Adherens junctions are also important components of the brain endothelial cell junctions (Fig. 3), but will not be discussed in detail here. In order to link the cell-cell junctional proteins to the cytoskeleton, brain endothelial cells enlist a variety of accessory proteins in the intracellular compartment. Among this subset of junctional proteins are members of the membrane-associated guanylate kinases (MAGUKs) known as zonula occludens or ZO-1, -2, and -3. These proteins are important in the anchoring of transmembrane proteins to the cytoskeleton and act as a binding scaffold for signaling proteins. ZO proteins consists of three domains, an SH3 domain which commonly binds signaling and cytoskeletal proteins (37), a PDZ domain that binds to the claudins (38), and a guanylate kinase domain that binds to occludin (39,40). Other accessory proteins include cingulin, a myosin-like protein which has been demonstrated to interact with ZO-1, ZO-2, ZO-3, myosin (41), occludin (42), and JAM (28) at tight junctions. 7H6 antigen is also located at tight junctions and confers resistance to paracellular transport of macromolecules and metastatic cancer cells (43). The formation and maintenance of the tight junctions are regulated by a variety of signaling cascades (reviewed in Ref. 44), and this allows the BBB tight junctions to respond to various stimuli and pathological conditions in a dynamic fashion (see Sec. 3).

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2.2. Molecular Transport Systems at the BBB To meet the high-energy demands of the brain, the relatively impermeable BBB modulates the influx and efflux of substances by way of highly regulated transport systems. Due to the polarized nature of the BBB endothelium, a transported molecule must travel serially through both the luminal and abluminal membranes. Thus, many transporters are located on both membranes and can demonstrate asymmetry, although some transport systems are localized exclusively at either the luminal or abluminal membrane. Transporter genes are estimated to occur at a frequency of approximately 3% in the human genome (45) and based on the predicted number of transcripts in the genome, this would yield ~1000 different transporters. Given the anatomical and functional topology of the brain microvasculature, one might expect the BBB to express an inordinately large number of transporter proteins. Indeed, recent genomics efforts indicate that transporters comprise between 10% and 15% of the expressed genome of the BBB (24,32,46). In many cases, the functional relevance of these transporters remains to be confirmed. One type of transport system, carrier-mediated transport, mediates the transBBB flux of small molecule energy sources, vitamins, and nutrients (Table 1). These carriers are highly stereospecific and operate on the millisecond timescale. The two

Table 1 Nutrient Carriers at the BBB Carrier

Example substrate BBB isoform Vmax (nmol/g min)

Hexose

Glucose

GLUT1

1420

Monocarboxylic acid

Lactate

MCT1

91

Large neutral amino acid Phenylalanine

LAT1

22

Basic amino acid

Arginine

CAT1

5

Amine

Choline

Unknown

11

Small neutral amino acid Alanine

Unknown

8

Nucleoside

Adenosine

Unknown

0.75

Acidic amino acid

Glutamic acid

Unknown

0.2

Adapted from Refs. 4 and 193 carrier-mediated facilitated transporters supporting the highest rate of saturable transport are the glucose (GLUT1) (47,48) and monocarboxylic acid (MCT1) transporters (49–51). Nearly all GLUT1 expression in the brain is restricted to the microvascular endothelium (48,52) and MCT1 expression is crucial for the transport of lactic acid and ketone bodies in the developing brain (50). Several nutrient transporters that import biosynthetic building blocks have also been localized to the BBB. The large neutral amino acid transporter type I isoform (LAT1) was demonstrated to be expressed at the BBB at 100 times the level found in any other tissue including the brain (53), in addition to having a much higher affinity than neutral amino acid carriers localized to other tissues (54). The

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basic amino acid transporter CAT1 has also been identified at the BBB (55) and in a recent genomics analysis was confirmed to be differentially expressed in the brain compared to liver and kidney tissues (32). Transport of choline as well as other molecules having quaternary ammonium groups occurs at the BBB (56). Adenosine transport, vitamin transport, and small neutral and acidic amino acid transport has been observed at the BBB, however, the genes for the proteins regulating this transport have yet to be cloned (Table 1). Receptor-mediated transport via the endosomal route is also prevalent at the BBB interface and occurs on the minute timescale. This transcytosis mechanism, analogous to that found at epithelial barriers, allows for the targeted uptake of circulating peptides and proteins, packaging into the endocytotic pathway, and deposition into the brain interstitium. Unlike endothelium elsewhere in the body, these proteinaceous substrates cannot be transported either by the paracellular route or by pinocytosis at the BBB and thus require the activity of the receptor-mediated transcytosis systems. Several receptormediated transcytosis systems have been identified at the brain microvasculature. The insulin receptor has been shown to mediate the receptor-mediated transport of insulin at the BBB and results in measurable brain insulin concentrations (57). Transferrin is also transported across the brain endothelial cells allowing coupled entry of iron into the brain (58). This process occurs via the transferrin receptor, which is highly expressed at the brain microvasculature (59). Leptin, a protein secreted by adipocytes, induces satiety when transported by an alternatively spliced short isoform of the leptin receptor present at the BBB (60). Similar to insulin, leptin is not produced in the brain (61) and must, therefore, enter from a peripheral source. In addition, the leptin receptor was specifically localized to the microvasculature in brain tissue (62). There is also evidence for saturable transcellular transport of low-density lipoprotein at the BBB (63,64). Another very interesting class of transporters at the BBB is the carrier-mediated efflux transporter. The protein product of the multidrug resistance gene (MDR1), or pglycoprotein, functions in the ATP-dependent efflux of many small molecule substrates and therapeutics (65) and has been localized to brain capillaries (66). This transport system has the ability to target lipid soluble drugs that diffuse through the luminal membrane and shuttle them back to the bloodstream (reviewed in Ref. 67). Evidence also suggests expression of other transporters in this family known as multidrug resistanceassociated proteins (MRP1 and MRP5) at the brain endothelium although the levels and species specificity is somewhat in question at this time (67). Ion transport and astrocyteregulated aquaporin-based transport of water at the BBB are important in maintaining homeostasis of the brain environment and have been reviewed elsewhere (68–70). Although the BBB could be described as a specialized transport interface, the number of cloned and characterized transporters is limited. Given the large percentage of transporter transcripts identified in functional genomics analyses as stated above, there is a large part of the BBB transport picture that remains to be resolved.

2.3. BBB Establishment and Maintenance—Role of the Microenvironment The endothelium of the BBB in vivo has unique characteristics when compared to the endothelium found in the periphery. The mechanism of specialized differentiation into

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BBB endothelial phenotype could be either genetically or environmentally based. It could be argued that the brain endothelial cell originates from a special mesodermal lineage or that these cells are genetically predisposed to becoming brain endothelial cells. On the other hand, it is possible that an endothelial cell subjected to a brain microenvironment may, by association, become a specialized BBB endothelial cell. A battery of experiments has given credence to the hypothesis that the local microenvironment is responsible for the unique differentiation of brain endothelial cells. Factors in the microenvironment that contribute to BBB function include the extracellular matrix, neighboring brain cells, and flow. These factors will be discussed in detail below. Early studies indicated that the BBB is not genetically predetermined, but rather is induced by the brain microenvironment. When neural tissue was grafted to peripheral sites, vessels invading the neural graft developed BBB properties (71,72); however, when brain vessels invaded peripheral tissue that had been grafted into the brain, no barrier characteristics were observed (72). Ultimately, direct in vivo evidence for the effects of astrocytes confirmed the barrier-inducing properties of this perivascular cell. Grafts of astrocytes can induce BBB-like properties in peripheral endothelium (73). In addition, when glial fibrillary acidic protein (GFAP) positive astrocytes are selectively ablated at the site of invasive CNS injury, the BBB remains impaired and permeable. This effect can be countered by grafting normal astrocytes to the site of the injury that foster the repair and restoration of BBB impermeability (74). Transgenic mice with GFAP-deficient astrocytes have an impaired BBB (75) and astrocytes from this animal were not able to induce a functional BBB in vitro compared to control astrocytes (76). Based on these data and the fact that GFAP is upregulated after brain injury, it has been speculated that GFAP may be an important factor for BBB induction, repair, and restoration. However, when cultured in vitro, brain endothelial cells tend to de-differentiate, lose many of their specialized characteristics and in essence resemble peripheral endothelial cells. The de-differentiation effects are extremely problematic when analyzing the permeability of pharmaceuticals in vitro or when studying basic biochemical or immunological interactions at the BBB. The de-differentiation phenomenon suggests that the BBB phenotype is critically dependent on signals residing in the local microenvironment. Indeed as will be discussed below, recent studies have demonstrated a role for perivascular cells, growth substrate, and culture conditions in eliciting BBB properties. Brain endothelial cells and pericytes share a basement membrane that is composed of collagen, laminin, fibronectin, entactin, heparin sulfate proteoglycans, and chondroitin sulfate proteoglycans (Fig. 2). This basement membrane forms a robust protective sheath around the capillaries that actually allows them to be purified intact by mechanical homogenization for subsequent dissociation and growth in primary culture or use as intact “in vivo-like” models. Perhaps not surprisingly then, brain microvascular endothelial cells have improved growth characteristics and are more representative of the in vivo situation in terms of permeability and other BBB properties when cells are plated on collagen (77–80), fibronectin (81,82), pronectin F (83), and extracellular matrix secreted by astrocytes (81). Clearly, the presence of certain physical contacts with the basement membrane as well as contact with astrocyte-derived matrix proteins are keys in the development of the in vivo phenotype.

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The physical situation encompassed by the extracellular matrix influences the phenotype of the brain endothelial cells. However, as the transplantation experiments suggest, soluble mediators and physical contacts with perivascular cells such as astrocytes and neurons can also play an important role in the establishment and maintenance of BBB barrier properties. The most substantial improvements for in vitro BBB models arise when the endothelial cell monolayers are cultured with the perivascular cells that help regulate the functionality of the BBB in vivo. This has been studied extensively for astrocytes whose foot processes are invested in nearly the entire surface of the BBB in vivo (Fig. 1D–F). Culturing with astrocyte conditioned medium (84), astrocyte plasma membranes (85), and astrocytes both in direct contact (86) on opposite sides of a permeable filter (78,87–89) or in indirect contact through a diffusion apparatus (84,90) are critical for the permeability, morphological, and biochemical characteristics of the in vitro models. Astrocyte influences can induce γ-glutamyl transpeptidase (γGTP) (88,89,91) and alkaline phosphatase activities (87,92), induce glucose transporter (GLUT-1) expression (93), stimulate growth and glucose utilization (83), and influence the polarity and expression of p-glycoprotein (94). In addition, astrocytes increase the TEER of endothelial cell monolayers (88,95), increase the level of peripheral vs. distributed actin (82,84), contribute to the complexity and extent of tight junction morphology in vitro (87), and decrease the passive diffusion of impermeable molecules such as sucrose, fluorescein, horse radish peroxidase and inulin (87,88,90). Interestingly, it has been observed that neuronal processes directly contact the basement membranes of endothelial cells in the brain (96,97), yet co-culture with neurons has been studied to a much lesser extent. Neurons can induce BBB-specific biochemical activity in the form of γGTP enzymatic activity, Na+−K+ ATPase activity (85) and induce endothelial cells to synthesize and sort occludin (98). In addition, endothelial cells were able to support a serotonergic phenotype in neurons co-cultured with astrocytes and endothelial cells (99). The effects that pericytes have on endothelial cell phenotype have not been thoroughly investigated although these two cell types share a common basement membrane. The nature of direct contacts between pericytes and endothelial cells is an important aspect of the microenvironment and research is needed in this area. Oftentimes, in vitro BBB models are static models, yet under physiological conditions, the brain microvasculature is subject to laminar flow conditions and associated shear stresses. Recent progress in eliciting the in vivo phenotype in vitro has been accomplished by Janigro and colleagues as they have pioneered a dynamic model of the BBB that takes into account the presence of flow-induced shear stress. When grown under dynamic flow conditions on permeable three-dimensional poly-propylene capillaries coated with pronectin F, brain microvascular endothelial cells have improved permeability and electrophysiological resistance properties when compared to static models (83,95). However, since the polypropylene capillaries are of large diameter (~150–600 µm), it is difficult to completely reproduce the in vivo situation where the capillary diameter is on the order of 5 µm. The effects of the soluble mediators released by the perivascular cells are evident, but the identities of these components are largely unknown. One protein released from astrocytes that has been definitively implicated in the development of barrier function is glial cell line-derived neurotrophic factor (GDNF) (100). Also, the use of forskolin to elevate cellular cAMP levels increases the tight junctional complexity and P-face

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association of the tight junction particles (101). Direct supplementation of cAMP has also been shown to increase barrier function (84) and alkaline phosphatase expression (92). Finally, the addition of hydrocortisone to cultures in the absence of serum has been demonstrated to increase the TEER by 10-fold and decrease the permeability to sucrose to near in vivo levels (102). Although the above discussion has focused on the effects of the microenvironment on endothelial BBB phenotype, cell-cell communication is likely to occur in both directions. However, at present, little is known about the effects that endothelial cells have on neighboring astrocytes, pericytes, and neurons.

2.4. Immunological Mechanisms at the BBB Historically, the brain has oftentimes been referred to as an immune-privileged organ. While it is true that levels of immunoglobulin and leukocytes are lower in brain parenchyma compared with other tissues, the brain is subject to immune surveillance, and the immune response may be upregulated during incidences of disease and inflammation. Given its strategic location, the BBB endothelium is likely to play a central role in regulating immune surveillance and leukocyte recruitment. The exact role of the BBB in regulating these responses is still in the process of being elucidated. However, significant evidence has been gathered that suggests an important immunoregulatory role for the BBB. In the absence of barrier disruption, blood to brain antibody transport does not occur to a significant extent (103). To date, no antibody transport systems have been demonstrated at the BBB that would facilitate import of these soluble immune components. Interestingly, however, rapid export of antibodies from the parenchyma to the bloodstream has been observed after intracerebral injection of IgG (104). Although preliminary evidence indicated the presence of an Fc receptor on brain endothelial cells (105), Fc receptors, FcR-I and FcR-II, are not located at the brain microvasculature while FcR-III is only expressed in some brain venular endothelium (106). Recently the neonatal Fc receptor, FcRn, was identified at the BBB of adult rats and has been implicated in IgG export from the brain (107). Therefore, under normal or pathological conditions, it is possible that the BBB may act in clearance of IgG from the brain in an attempt to attenuate local antibody-mediated immune response. Since the BBB is the first point of contact for circulating immune cells, it may be hypothesized that brain endothelial cells function as professional antigen presenting cells (APC) thereby facilitating T cell activation. Indeed, class II MHC can be induced on the surface of primary culture brain endothelial cells by inflammatory mediators such as interferon gamma (IFN-γ) (108,109). Treatment of endothelial cells with IFN-γ (109,110) or tumor necrosis factor alpha (TNFα) (110) in vitro can also stimulate the expression of T cell co-stimulatory molecule, B7. However, T cell proliferation assays with brain endothelium from a variety of species and under various pathophysiological conditions indicated that the endothelial cells are not able to fully activate proliferative T cell responses (111,112) and even the downregulation has been observed (109). While these data would argue against an antigen-presenting role for BBB endothelial cells, other studies have demonstrated an important role for the BBB in the regulation of leukocyte extravasation into the brain. Regulation of this process occurs via the expression of cell adhesion molecules in response to proinflammatory cytokines. For example, intercellular

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adhesion molecule-1 (ICAM-1), which serves as a docking receptor for circulating leukocytes, is upregulated on brain endothelium by IL-1α, IFN-γ, and TNF-α, and increases lymphocyte migration across brain endothelial cells (113). In addition to performing a basic tethering function, ICAM-1 binding can trigger a signaling cascade that may contribute to phenotypic modulation of the BBB and subsequent lymphocyte entry (reviewed in Ref. 114). TNF-α is a proinflammatory cytokine that is not normally produced in the CNS but has pronounced effects on brain endothelial cells. TNF-α can be produced locally at the site of inflammation or can originate from systemic sources and has been shown to increase the permeability of an in vitro model BBB (115) as well as enhancing the adherence and transmigration of leukocytes (116). When administered in vivo, TNF-α can also increase the flux of radiolabeled albumin from the cerebrospinal fluid (CSF) to the blood. This would then presumably allow surveillance of CNS constituents by the peripheral immune machinery (117). Thus, TNF-α may serve to enhance the surveillance of the CNS by allowing more immune cells into the brain and more brain-derived antigens out. In contrast to TNF-α, transforming growth factor-β (TGF-β) is produced constitutively in the CNS and has been demonstrated to downregulate adhesion molecule expression (118) and diminish leukocyte migration across the BBB in vitro and in vivo (116). TGF-β is also a candidate for suppression of T cell proliferation in the CNS as it is a component of the CSF (119). Therefore, while TNF-α and other proinflammatory molecules appear to enhance surveillance, TGF-β may act as a delicate counterbalance to regulate the immune response in the CNS. In the process of immune surveillance, leukocytes can migrate across an intact BBB in the absence of barrier compromise. For example, transendothelial transport of neutrophils across IL-1 treated human umbilical vein endothelial cell monolayers treated with astrocyte conditioned medium (as an in vitro BBB model) can occur without losing the characteristic TEER or the endothelial ultrastructure (120). Under these conditions, the tight junctional proteins occludin, ZO-1, and ZO-2 do not degrade, but rather remain localized at cellular junctions (120,121). One possible explanation for these findings is that leukocytes migrate preferentially through tricellular corners where three endothelial cells come together and the tight junctions are inherently discontinuous, although thus far this has only been observed in vitro (121). T-lymphocytes have been shown to express occludin (122), which in turn has been found to modulate transmigration across an intact epithelial barrier. These findings raise the intriguing possibility that T cells and endothelial cells may interact via occludin links, thereby limiting breaches in the BBB upon transmigration (123). Although there is an extensive literature relating to the stepwise progression of cellular transmigration across endothelium, it should be emphasized that the exact mechanism of transmigration across the BBB is not completely understood. Under pathological conditions there can be increases in the numbers of activated neutrophils, lymphocytes, and monocytes that cross the BBB due to loss of barrier function. This is especially prevalent in multiple sclerosis (MS), stroke, HIV encephalitis, Alzheimer’s disease, and brain tumors. These effects will be discussed in greater detail below.

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3. BBB INVOLVEMENT IN DISEASE 3.1. BBB Involvement in HIV Nearly two-third of patients with AIDS develop a neurological disorder such as HIV encephalitis or AIDS dementia complex (124). Progression of these diseases has been linked to migration of infected monocytes across the BBB into the brain (125,126). Evidence also supports the transendothelial passage of free HIV virus via adsorptive endocytosis (126). Regardless of the mode of entry, the incidence of HIV encephalitis and AIDS dementia correlates with the presence of monocyte infiltration and subsequent microglial activation rather than with the actual viral load (124). Vascular changes have also been noted in the brains of AIDS patients in the form of increased diameter of the cortical microvessels and thinning of the basal lamina of the blood vessels (127). In addition, HIV has been found to infect brain endothelial cells both in vivo (128,129) and in vitro (130,131). The microvascular endothelial cells of the brain are believed to play a role in the regulation of entry of the HIV virus and/or HIV infected monocytes into the parenchyma of the brain. HIV infected monocytes can stimulate the adhesion properties of the BBB through the release of cytokines and inflammatory mediators such as TNFα, IL-1β, and IFN-γ and can activate perivascular microglial cells (132–134). The activated endothelium exhibits upregulation of cell adhesion molecules E-selectin and vascular cell adhesion molecule-1, which can then support the binding and migration of HIV infected monocytes across the BBB into the brain (126,135). During the migration of these monocytes, HIV proteins stimulate Gelatinase B and promote the degradation of the basement membrane further compromising the integrity of the BBB (136). A coordinate decrease in the tight junction proteins of the BBB, occludin and ZO-1, is seen upon increased monocyte infiltration in AIDS patients (137,138). Once in the brain, the HIV virus and/or activated monocytes can initiate a cascade of detrimental responses through the release of cytokines and other toxic factors. Based on the mechanistic observations described above, the BBB appears to be intimately involved in the pathogenesis of neuroAIDS.

3.2 BBB Involvement in Stroke The integrity of the BBB is altered after stroke due to ischemic insult and hypoxia. Oxygen deprivation leads to a complicated series of events that ultimately results in increased BBB permeability, leakage of plasma components across the BBB and edema formation. Early ischemic events lead to the observed BBB dysfunction and include recruitment of leukocytes by increased expression of adhesion receptors E-selectin (139), P-selectin, and ICAM-1 (140). There is also an alteration in tight junctional content that contributes to the increase in permeability. In vitro BBB models composed of rat brain endothelial cells in co-culture with astrocytes were subjected to hypoxic conditions and a significant decrease in TEER of 25% after 4 hr of hypoxia and 95% after 8 hr was

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observed (141). This same set of experiments also demonstrated that the presence of astrocytes diminished the decrease in TEER after a 4 hr hypoxic insult (141) again implicating a role for this cell type in modulating BBB phenotype. Paracellular transport was also increased in bovine brain endothelial cells following long duration hypoxia (24– 48 hr) whether or not astrocytes were present (142,143). Tight junction occludin, ZO-1 and ZO-2 protein expression levels were unaltered but were relocated from cell junctions during hypoxia (143). Upon reoxygenation, a rapid increase in actin expression was observed along with increases in occludin, ZO-1, and ZO-2 protein and a coordinate decrease in the paracellular permeability. Finally, the basement membrane integrity is also impaired in ischemic conditions (144) and may be due in part to metalloproteinasebased degradation (reviewed in Ref. 145). Many of the pathological components of stroke are exacerbated by BBB opening. This important observation reinforces the need for molecular level understanding of this process so treatments that have the power to reverse BBB opening, and thus ameliorate the symptoms of stroke, can be developed.

3.3. BBB Involvement in MS and Experimental Autoimmune Encephalomyelitis MS is a chronic inflammatory and demyelinating disease of the CNS that is manifested by BBB disturbance, local edema, and demyelination (146). Damage to the BBB represents an early stage in lesion formation. Indeed, gadolinium MRI scans of MS patients have demonstrated that alterations in BBB permeability precede clinical onset of the disease (147,148). Much of our knowledge of the molecular processes underlying MS is based on an animal model for MS known as experimental autoimmune encephalomyelitis (EAE), which shares certain pathological and clinical features of the human condition. This disease can be induced by sensitization with CNS myelinassociated antigens and has been demonstrated to be T cell mediated (149). As in the pathogenesis of many other CNS diseases, ICAM-1 is upregulated at BBB endothelial sites in EAE and the levels of ICAM-1 correlate with disease activity (150). In addition to ICAM-1, the vascular cell adhesion molecule-1 (VCAM-1) was also observed to be expressed at high levels in chronic human MS lesions (151). These responses may be mediated by the release of proinflammatory cytokines (IFN-γ, TNF-α) from activated immune cells and as described earlier, the tight junctional integrity of the BBB can also be affected by cytokine release. As an example of the changes in tight junction integrity, abnormal levels and distributions of ZO-1 and occludin have been observed in brains of MS patients (152). Also in EAE models, claudin-3 expression is selectively downregulated in tight junctions compared to normal BBB (22). Such modulation of the BBB in EAE can lead to multiple modes of immune cell entry into the CNS including via the tight junction complex, via channels or pores formed in the tight junction complex, or by emperipolesis (153). Concomitant with the induction of adhesion and tight junction molecules, EAE progression correlates with an increase in vesicular trafficking that is more reminiscent of the peripheral vasculature and when the animals recover, capillary vesicular transport is reduced to near normal levels (154). Unexpectedly, recent genomics studies have identified expression of myelin basic protein at the BBB. Microvascular expression of myelin basic protein was confirmed by in situ hybridization with a myelin basic protein probe (155). Taken together, these findings

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raise the possibility that the BBB may play an even more important role in MS pathogenesis that was previously anticipated. For example, in addition to regulating the transendothelial migration process, the BBB may contribute to recruitment of T cells.

3.4. BBB Modulation in Brain Tumors In general, the vascular environment of low-grade gliomas is very similar to that found in normal brain (156). However, high-grade gliomas and metastatic brain tumors have significantly altered angio-architecture and permeability characteristics. Whereas normal brain capillaries have diameters around 5 µm, tumor capillaries are on average much larger (up to 40 (µm) and in contrast to normal brain capillaries, the larger tumor vessels are very tortuous and irregularly spaced (157–159). Microvascular permeability and loss of BBB function can be attributed to changes in the tight junctions, presence of fenestrae, and increases in vesicular trafficking. It has been well established that the tight junctions in brain tumors are abnormal when compared to the intact BBB. The overlap between adjoining endothelial cells is decreased or the tight junctions are substantially widened, suggesting increases in paracellular transport (160,161). Along these lines, the molecular composition of tight junctions in high-grade tumors is altered implying a role for tight junction regulation in tumor malignancy. Studies of tight junctions in human brain tumors determined that claudin-1 expression was lost, while claudin-5 and occludin expression were downregulated in hyperplastic vessels only, and ZO-1 expression was unaffected (162,163). Fenestrations are readily identified in brain metastases but are an extremely rare occurrence in glial tumors. Vesicular trafficking characteristics of brain tumor endothelium are much less clear and have been described as increased, decreased, or unchanged. The net result on the integrity of the blood-tumor barrier can be demonstrated by studies that confirmed a higher level of leakage of serum components from tumor vessels. Fluoresceinated molecules ranging in size from 376 to 500,000 Da leaked through the blood-tumor barrier (158) as did bovine serum albumin (164). However, it is important to note that a higher level of leakage does not imply absence of a barrier as it has been demonstrated that size-dependent leakage kinetics are operating at the compromised BBB (158,165). Also in some tumors, fluorescein leakage (376 Da) does not occur and this is indicative of a tight blood-tumor barrier (164). Vascular endothelial growth factor (VEGF), initially identified as vascular permeability factor, is a likely candidate for the regulation of tumor vessel permeability. In high-grade gliomas, VEGF is greatly upregulated and its receptors VEGFR-1 and VEGFR-2 are coordinately upregulated on tumor endothelium (166–168). Normal astrocytes assist in barrier formation; while in contrast, high-grade astrocytoma cells secrete VEGF that stimulates angiogenesis, vascular permeability, and redistribution of tight junction occludin expression (166,169). Glioma vessels continue to express the BBB-specific glucose transporter, GLUT-1 (170), and p-glycoprotein expression likely limits the efficacy of many anticancer agents (171) as would also be expected from normal brain endothelium. Finally, the microenvironment of brain tumors is critical to the ultimate phenotype of the tumor endothelium. As described above, metastatic brain tumors have presence of fenestrae and lack BBB properties. This perhaps is not surprising given the importance of perivascular cell influence on BBB properties, and the lack of these inductive factors in

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tumor cells arising from peripheral tissues. Another example is given by the transplantation of C6 rat astroglioma tumors into brain and muscle. The resulting tumor vasculature is identical in both sites (172) again suggesting that the brain cells provide inductive factors that determine the ultimate vessel phenotype. Although speculative, this may also explain the fact that low-grade gliomas have operational BBB while more transformed cells comprising a high-grade gliomas may lack the necessary inductive machinery to yield a working BBB. The molecular level understanding of brain tumor vessel diversity is crucial in the proper design of brain tumor drugs as efficacy could vary widely depending on local permeability characteristics.

3.5. BBB Involvement in Other CNS Diseases As documented above, the brain microvasculature participates heavily in the progression of a variety of CNS diseases. This list was by no means exhaustive, as the BBB may also have important roles in Alzheimer’s disease (173), hypertension (174), and the class of CNS complications due to infections by bacterial, fungal, and viral pathogens (175) among others.

4. GENOMICS AND PROTEOMICS OF THE BBB One of the major difficulties in discerning all of the important contributions to the BBB phenotype in health and disease is that many of the cells and soluble influences act synergistically in a dynamic fashion. A single soluble factor or cell-cell contact may influence one or more pathways in multiple cell types. In addition, the temporal and spatial relationships of disease progression are poorly understood, yet they are crucial for the development of appropriately targeted therapies. Genomics and proteomics analyses provide an opportunity to perform system-wide discrimination of healthy and diseased tissues in order to deconvolute complicated mechanistic networks that are involved in angiogenesis, aberrant proliferation, and altered cellular behavior. The information obtained by these approaches may facilitate the rapid identification of therapeutic targets and the discovery of novel BBB-specific transport systems having utility in non-invasive drug delivery.

4.1. Genomics of the BBB Oftentimes studies with the BBB are constrained by examining the expression and behavior of just a few genes or proteins of interest. This includes analyses of BBB disease involvement, validation of in vitro models, and identification of molecular determinants of in vivo phenotype. However, based on many of the phenotypic attributes of the BBB described throughout this chapter, it is apparent that a few genes do not adequately describe the underlying phenomena. The regulation of tight junctions, transcellular transport, cytoskeletal rearrangement, and immune responses unequivocally relies on intertwined networks of proteins derived from multiple cellular sources.

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Recently, BBB researchers have applied a variety of genomic techniques to the analysis of BBB function in health and disease. A major difficulty in performing genomics techniques using mRNA derived from total brain homogenates is that only the most highly expressed microvasculature transcripts would be identified above the background produced by non-endothelial cells. This is due to the fact that the brain blood vessels themselves constitute only about 1/1000 of the total brain volume (176) and coordinately a total brain mRNA sample would fractionally contain only a small amount of capillary mRNA. At the same time, microarray sensitivity can be in the neighborhood of 10−4 (177). Thus, a much more successful BBB profiling approach would consist of capillary purification (Fig. 1A) coupled with isolation of mRNA directly from this subset of cells. As mentioned earlier, this task can be readily accomplished using mechanical homogenization techniques due to the robust capillary basement membrane. Importantly when capillary isolation is performed within short times of sacrifice, this technique allows for recovery of in vivo-like mRNA samples with little change in the in vivo mRNA content (178). Armed with capillary-specific mRNA, genomics techniques such as gene microarray, subtractive suppression hybridization (SSH) (179), and serial analysis of gene expression (SAGE) (180) have been used to address questions regarding the functional attributes of the BBB in vitro and in vivo. Two main genomic strategies have been applied to the BBB in order to determine the gene expression profiles that contribute to its unique functional phenotype. The first methodology discussed takes advantage of the SSH technology that identifies differentially expressed BBB genes compared with peripheral tissue and the second is a comprehensive compilation (SAGE) of all expressed BBB transcripts (transcriptome). In order to determine the molecular origin of unique BBB functions, SSH has been applied to the analysis of both human and rat capillaries in a concurrent BBB genomics program (24,32,155). SSH is an excellent technique for determining genes that are differentially expressed among mRNA samples from different tissues (179). It is also a valuable technique for the analysis of low-abundance transcripts that may be present at the BBB and have important roles in regulation of BBB phenotype (179). In this study, genes common to the brain microvasculature, kidney tissue, and liver tissue were subtracted while identifying those genes that were by comparison upregulated at the BBB. Results from three rounds of SSH differential analysis [one round of human (24) and two rounds of rat (32,155)] indicate that between 51% and 55% of the differentially expressed genes at the BBB encode proteins with known function. Figure 4 is a compilation of these three studies with the genes encoding known proteins clustered by function. Of course, clustering into distinct categories is somewhat arbitrary given that many of these gene products have multiple functional roles in vivo. What immediately stands out is the high number of enriched genes that encode proteins involved in the control of angiogenesis, signaling, transport, and junctional structure. Importantly, the methodology proved to be effective in identifying genes previously implicated in mediating BBB phenotype. The balance of the genes identified in this study (45–49%) encode BBB-enriched proteins with unknown function. The elucidation of the functions of these proteins, though fraught with difficulty, will be critical to a full understanding of the BBB. Another genomics study utilized the SAGE method and resulted in a comprehensive analysis of gene expression at the rat brain microvasculature (46). The advantage of

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SAGE analysis compared with SSH or even cDNA microarray is that one can generate a

Figure 4 Functional clustering of BBB-enriched genes determined in a concerted genomics /proteomics program of the brain microvasculature. Transcripts identified in given genomics/proteomics studies are identified by: rat genomics study I denoted by * (155), rat genomics study II denoted by † (32), human genomics study denoted by ‡ (24), and bovine proteomic study denoted by # (190,191). PDGF-Rβ, platelet-derived growth factor receptor β subunit; IGF2, insulin-like growth factor 2; FGF19, fibroblast growth factor 19; HARP,

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heparin affinity regulatory peptide; IGF-BP-3, insulin-like growth factor binding protein 3; Gab2, Grb-2associated binder-2; LaAUF-1, AUrich RNA binding factor; Rgs5, Gprotein signaling regulator-5; Ptdgs, prostaglandin D synthase; VESP14, vascular endothelial cell-specific protein 14; hbrm, human homolog of yeast SW12 transcription factor; PC3, B-cell translocation gene-2; oatp2, organic anion transporting peptide type 2; MCT1, monocarboxylate transporter 1; BSAT-1, BBB-specific anion transporter type 1; CAT1, cationic amino acid transporter 1; FXYD5, FXYD domain-containing ion transport regulator 5; TfR, transferrin receptor; Cpe, carboxypeptidase E; Pgsg, secretory granule proteoglycan core protein precursor; APLP2, amyloid precursor-like protein 2; YWK-II, sperm membrane protein related to A4 amyloid protein; Itm2a, integral membrane protein 2a; Spi4, serine protease inhibitor 4; tPA, tissue plasminogen activator; MBP, myelin basic protein; PZR related, protein zero-related protein 1; PLP-1, proteolipid protein; PLTP, phospholipid transfer protein; Scd2, stearoyl-CoA desaturase 2; Flt-1, vascular endothelial growth factor receptor type 1; HIF-2α, hypoxia inducible factor 2α, VE-PTP, vascular endothelial receptor-type protein tyrosine phosphatase; MLC20, regulatory myosin light chain isoform C; ESAM, endothelial cell-selective

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adhesion molecule; Ro52, 52 kDa ribonucleoprotein; PECAM-1 platelet/endothelial cell adhesion protein. complete blueprint of the cellular transcriptome [estimated to encode around 30−40,000 proteins in humans (45,181)], and not simply a subset of genes that may be differentially expressed or present on a fabricated microarray chip. The SAGE analysis yields both the identity and the relative quantity of all expressed genes. One current drawback is that such a readout in the absence of SAGE analyses from other tissues limits the ability to assess what genes are differentially expressed at the BBB. However, SAGE data are being generated for different tissue components at a rapid pace and will allow for interesting molecular level comparisons. The SAGE analysis of the BBB identified the presence of nearly 11,000 transcripts, of which only 17% matched genes with known functions (46). When compared with SAGE libraries generated from cortex and hippocampus, BBB-enriched genes were identified and clustered into groups of transporters (11%), receptors (10%), vesicle trafficking (7%), structural proteins (12%), and signal transduction (18%). The distribution of enriched genes agrees quite well with that determined in the SSH genomics program emphasizing the functioning BBB as a “molecular switchboard” between the blood and brain. In addition to defining the molecular functionality of the BBB under normal conditions, both the SSH and SAGE analyses described above could be applied to compare and contrast molecular level details in pathological states. Genomics approaches have also been used to address several specific questions regarding the characteristics of the brain microvasculature. Lippoldt and co-workers (182) used SSH in order to identify potential contributions to stroke in hypertensive rats. They compared the gene expression profiles of cerebral capillaries from stroke-prone spontaneously hypertensive rats to stroke-resistant spontaneously hypertensive rats and identified several genes that were either upregulated or downregulated in capillaries from stroke-prone rats, including two encoded proteins with unknown function. The rat sulfonylurea receptor 2B was upregulated, while the G-protein signaling 5 regulator was downregulated in stroke-prone rats. In another study, vessels from epileptic tissue were compared to non-epileptic blood vessels by cDNA microarray to determine the classes of genes responsible for resistance to antiepileptic drugs. It was discovered that MDR1, MRP1, MRP2, MRP5, and cisplatin resistance-associated protein were all overexpressed in epileptic vessels helping to explain the origin of drug resistance (183). The effects of flow-induced shear stress on in vitro brain endothelial cultures were determined by cDNA microarray that compared static and dynamic in vitro models. This study concluded that flow induces cytoskeletal genes and contributes to the development of the antioxidant capacity of endothelial cells (184). Finally, the in vitro phenotype of human brain endothelial cells (HBEC) was compared to HUVEC cells to help identify genes crucial in conferring the BBB phenotype. Thirty-five genes were preferentially expressed in HBEC and included vasculogenic factors, immunoregulators, and growth factors (185).

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4.2. Proteomics of the BBB Although genomics approaches yield a wealth of molecular level information about what factors ultimately confer phenotype, it is important not to use these techniques exclusively. For one, the correlation between gene expression levels and protein expression levels is not necessary linear (186). Also, post-translational modifications including phosphorylation, glycosylation, and proteolysis that are critical to protein function are not addressed in genomics techniques. Thus, it is important to also understand the protein makeup of the BBB and generate proteomic profiles that can readily complement genomic profiles. The use of 2-dimensional gel electrophoresis in BBB research has been remarkably low to date. A recent study used 2-dimensional gel electrophoresis to analyze protein leakage from cells upon BBB damage. Damage was induced in vitro by physically distancing the astrocytes from endothelial cells in coculture. This condition upregulated the release of the protease inhibitor α2-macroglobulin from astrocytes and it was suggested this could be a protective mechanism to lessen BBB damage (187). In addition, 2-dimensional gel electrophoresis combined with mass spectrometry was used to study the protein expression profiles of rat brain endothelial cells during ischemic conditions (188). Total cellular protein was greatly reduced upon ischemia/reperfusion and around 30 different proteins were found to be differentially regulated. Many of the proteins that were upregulated were involved in the proteasome pathway whereas there was a coincident decrease in ribosomal proteins. These two factors were suggested as rationale for the observed decrease in total protein content. Enzymes involved in protection against reactive oxygen and nitrogen species as well as transcription factors involved in inflammation were also upregulated. Many of these alterations in protein expression were also confirmed by microarray experiments performed in parallel. Although 2-dimensional electrophoresis coupled with mass spectrometry is the current gold standard of proteomic technologies, it is difficult to analyze membrane proteins with this method due to solubility constraints (189). As described throughout this chapter, many of the unique features of the BBB can be ascribed to membrane proteins involved in signaling, junctions, adhesion, and transport. A novel proteomics methodology was developed to specifically analyze differential membrane protein expression at the BBB (190). This method, unlike gel electrophoresis, relies on probing protein expression in a native mammalian membrane environment so solubility is not an issue. In this method, a polyclonal antiserum raised against all bovine brain microvessel endothelial proteins is depleted with protein preparations derived from kidney and liver tissue. This depleted antiserum bereft of antibodies recognizing proteins common in other tissues was used as a probe for BBB-specific membrane proteins. The COS-1 cells transfected with a bovine BBB cDNA library were probed with the BBB-specific antiserum and those cells that expressed a BBB-specific protein were recovered and the identity of the protein was determined. The methodology was validated by identification of three membrane-bound proteins having enriched expression at the BBB. These included: Lutheran membrane glycoprotein (190), a basal cell adhesion molecule; the membrane cofactor protein CD46 (191), a complement regulator also found in astrocytes; and Ro52, an autoantigen implicated in Sjogren’s syndrome (192). The polyclonal antiserum also recognizes a host of other BBB-enriched proteins that will likely be identified in the future (190).

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The use of genomics and proteomics for the study of molecular level phenomena at the BBB is still in the early stages. However, as the preliminary studies above indicate, these tools should be integrated into existing BBB research programs. They clearly have the potential to add tremendous understanding to a variety of physiological processes that occur at the BBB in health and disease and will likely unearth additional clues on how to appropriately target therapeutic intervention to the brain.

5. CONCLUSION Throughout this chapter, I have attempted to demonstrate that the BBB has many specialized properties when compared to endothelium from other vascular beds. However, many questions remain to be answered. One particularly important goal is to determine whether there are BBB variations within the brain vascular bed itself. Many of the experiments described above focus on the vasculature-rich gray matter of the cerebral cortex. However, it is interesting to speculate about the potential diversity of BBB attributes in other brain regions such as the cerebellum and brain stem, or perhaps about local BBB variation within different sub-cortical domains. For example, are there BBB domains that exhibit locally unique function, or are there regions that could be specifically targeted in drug treatments? With advances in laser capture microdissection methods that allow for extraction of single blood vessels, proteomic and genomic analyses will likely be extended to address such questions on a molecular level. Although the BBB was referred to as a unique vascular bed throughout this chapter, it is important to acknowledge that other endothelial barriers exist in the body, and in general, these are much more poorly understood. The blood-retinal barrier of the vascularized inner retina of the eye has many properties similar to that of the BBB. A peripheral blood-nerve barrier exists although it is substantially more permeable than the BBB. The blood-testes barrier is endowed with impermeability characteristics by the Sertoli cells that surround a comparatively permeable endothelium. Finally, a blood-CSF barrier in the brain is clearly distinct from the BBB in its barrier and transport characteristics, and it is comprised of tightly apposed epithelial cells rather than endothelial cells. Research regarding blood barriers continues to progress, and development of drug targeting and delivery strategies that overcome these barriers is imperative for the effective treatment of disease.

ACKNOWLEDGMENTS I am grateful to colleagues Dr. Lester R.Drewes and Dr. Zsuzsanna Fabry for their critical review of this chapter. This chapter was supported in part by the National Institutes of Health (1R21-AA13834).

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3 Lymphatic Endothelium Satoshi Hirakawa and Michael Detmar Cutaneous Biology Research Center and Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.

1. INTRODUCTION The lymphatic vascular system consists of a network of thin-walled capillaries that drain protein-rich lymph from the extracellular spaces within most organs. Lymphatic capillaries are lined by a continuous single-cell layer of overlapping endothelial cell lines. They lack a continuous basement membrane and pericyte coverage, and are thus highly permeable. The larger lymphatic vessels also contain a muscular and adventitial layer. Lymphatic vessels are absent from avascular tissues such as the epidermis and the nails. Unlike blood vessels, lymphatics are also absent from the cartilage, the brain, and the retina. Lymphatics serve as a drainage system, returning interstitial fluid to the venous circulation via the larger lymphatic collecting vessels and the thoracic duct. Lymphatic vessels also play a major role in the afferent immune response by attracting anti-genpresenting cells—through secretion of chemokines such as secondary lymphoid chemokine—into the lymphatic vascular system and to the regional lymph nodes. Unfortunately, tumor cells can take advantage of these immune pathways to promote lymphatic tumor spread. Other components of the lymphatic system, including the lymph nodes, tonsils, Peyer’s patches, spleen, and thymus, play an important role in the immune response (1). Studies of the lymphatic system have been hampered by the inability to specifically stain lymphatic vessels and by the lack of known lymphatic-specific growth factors. The recent discovery of genes that specifically control lymphatic development, however, and the identification of lymphatic endothelium-specific markers have provided new insights into the molecular mechanisms that control lymphatic development and function (1,2). They have also enabled an improved understanding of the genetic causes of several hereditary diseases that are associated with lymphedema, and they have provided surprising evidence that malignant tumors can actively promote lymphangiogenesis and lymphatic metastasis (3).

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Figure 1 Schematic representation of specific growth factors and of their receptor expression by lymphatic and blood vascular endothelium.

2. LYMPHATIC GROWTH FACTORS Recently, new members of the vascular endothelial cell growth factor (VEGF) family have been discovered that predominantly promote the growth and maintenance of lymphatic vessels in the skin (Fig. 1). The first discovered member of this family, VEGF (also named VEGF-A or VPF, vascular permeability factor) binds to VEGFR-1 and VEGFR-2 predominantly on blood vascular endothelial cells and thereby preferentially promotes vascular endothelial cell proliferation, migration, and survival. However, under certain conditions, VEGF-A might also promote lymphangiogenesis via activation of VEGFR-2 on lymphatic endothelium (4). In contrast, placental growth factor/PlGF selectively acts on VEGFR-1 but not on VEGFR-2 and, because VEGFR-1 is absent from lymphatics, does not promote lymphatic vessel growth. VEGF-C and VEGF-D are the only known ligands of VEGFR-3 (also known as Flt4) that is exclusively expressed by lymphatic endothelium in normal tissues (Fig. 1). Transgenic mice with targeted overexpression of VEGF-C or VEGF-D in the epidermis show enhanced numbers of lymphatic vessels, confirming their role as lymphatic growth factors (5). Moreover, overexpression of a soluble VEGFR-3 in the skin of transgenic mice resulted in a dramatic reduction of cutaneous lymphatic vessels (6).

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3. LYMPHATIC-SPECIFIC MARKER GENES AND THEIR POTENTIAL FUNCTION Vascular endothelial growth factor receptor-3 is expressed in developing venous and lymphatic endothelia during early embryonic development; however, its expression becomes largely restricted to the lymphatic endothelium in adult organs (7). Recent studies have identified nonsense mutations in the VEGFR-3 gene in several patients with hereditary lymphedema that are characterized by dilated lymphatic capillaries and interstitial accumulation of lymph fluid (8). Vascular endothelial growth factor receptor-3 mutations have also been identified in Chy mutant mice, which are manifest by cutaneous lymphedema (9). These findings indicate that VEGFR-3 plays an essential role in the development and function of the lymphatic system (10). However, VEGFR-3 is also expressed by some blood capillaries during tumor neovascularization and in wound granulation tissue (11,12) and, therefore, VEGFR-3 alone might not be considered as a sufficiently specific marker for lymphatic vessels. The homeobox gene Prox1 represents the most specific marker for lymphatic endothelium at present (Table 1). Inactivation of Prox1 in mice results in embryonic lethality and completely prevents the development of the lymphatic vasculature (13,14), whereas heterozygote Prox1 deficient mice develop chylous ascites. Among endothelial cells, Prox1 is exclusively expressed in embryonic lymphatic endothelial cells and in lymphatic vessels of adult tissues and tumors (1). During embryonic development, Prox1 plays a key role in the formation of lymphatic progenitor cells from embryonic veins. Beginning at E9.5 of mouse development, Prox1 starts to become specifically expressed in a subpopulation of endothelial cells located on one side of the anterior cardinal vein (13). At this stage, the venous endothelium also expresses the hyaluronan receptor LYVE-1 and VEGFR-3; the expression of both of these receptors later becomes restricted to lymphatic endothelium (7) (Fig. 2). This is followed by polarized budding and migration of Prox1/LYVE-1/VEGFR-3-positive lymphatic progenitor cells (13) that progressively down-regulate the expression of blood vascular genes such as CD34 and laminin (14), and that express increasing levels of lymphatic markers such as VEGFR-3, LYVE-1, and secondary lymphoid chemokine (CCL21) (15). It remains at present unknown whether modulation of specific ephrins and Eph receptors is also involved in mediating the budding of lymphatic progenitor cells from embryonic veins. The lymphatic endothelium hyaluronan receptor LYVE-1, a CD44 homolog, has been identified as a specific cell surface protein of lymphatic endothelial cells and activated macrophages (16,17). LYVE-1 and the blood vascular markers PAL-E and CD34 exhibit mutually exclusive vascular expression patterns in the skin (18). The biological function of LYVE-1 at present remains unclear. Secondary lymphoid chemokine (SLC; also known as CCL21) is released by the lymphatic endothelium, but not by blood vascular endothelium, and interacts

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Table 1 Differential Expression of Established Vascular Markers in Lymphatics vs. Blood Vessels Marker

Blood Vessels

Lymphatics

CD 31 (PECAM-1)

++

+

CD34

++

−

PAL-E

++

−

VEGFR-1

+

−

VEGFR-2

+

+

VEGFR-3

−

+

Type IV collagen

++

(+)

Type XVIII collagen

++

(+)

Laminin

++

(+)

Prox1

−

++

LYVE-1

−

++

Podoplanin

−

++

SLC/CCL21

−

+

Figure 2 Proposed model for the steps involved in the embryonic development of the mammalian lymphatic system. (Adapted from Ref 1.)

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with the CC chemokine receptor 7 (CCR7) on mature dendritic cells, leading to their attraction toward the lymphatic vessels (15). Podoplanin, a mucin-type transmembrane glycoprotein, is expressed by lymphatic vasculature but not by blood vessels. Mice lacking podoplanin exhibit congenital lymphedema and impairment of lymphatic vessel formation and function (19). Podoplanin is also known as T1alpha, OTS-1 and PA2.26, and it is also expressed by some other cell types including lung alveolar type I cells, choroid plexus cells, osteocytes and kidney podocytes. Its function has remained unclear but preliminary evidence suggests a role in actin cytoskeleton reorganization. Desmoplakin mediates the attachment of intermediate filaments to the plasma membrane in epithelial cells and is also expressed by lymphatic endothelium, but not in blood vascular endothelium (20). Neuropilin-2 (NRP-2) is a coreceptor for VEGF-C (Fig. 1) and also mediates axonal guidance during neuronal development. Neuropilin-2 is expressed by lymphatic endothelium and neuropilin-2 deficient mice have reduced numbers of small lymphatic vessels and capillaries (21). Angiopoietin-2 is also required for the proper development of the lymphatic vasculature (22). It binds to the endothelialspecific Tie2 receptor and regulates vascular remodeling that is important for vessel sprouting and vessel regression. At present, it remains unclear whether different segments of the lymphatic system or lymphatics in different organs are characterized by expression of distinct sets of marker genes.

4. LINEAGE-SPECIFIC DIFFERENTIATION OF LYMPHATIC ENDOTHELIUM IS MAINTAINED IN VITRO Recent studies have shown that it is possible to selectively isolate and expand human microvascular lymphatic endothelial cells (LEC). These studies revealed that the widely used method for the isolation of human dermal microvascular endothelial cells (HDMEC) in fact yields mixed cell populations of blood vascular endothelial cells and lymphatic endothelial cells, and that commercially available HDMEC cultures also contain variable mixes of both cell types. This may explain some of the previous reports on the different molecular and cellular behavior of HDMEC as compared to human umbilical vein endothelial cells (HUVEC) that are of pure blood vascular origin. Three different approaches for LEC purification have been successfully used: LEC were isolated based on their expression of the lymphatic-specific glycoprotein podoplanin (23,24) or of the lymphatic-specific VEGFR-3 (25). Alternatively, LEC were directly purified from neonatal foreskin cell suspensions as CD34-negative/CD31-positive cells (26). In contrast, pure populations of blood vascular endothelial cells (BEC) were isolated as CD45-negative, podoplanin-negative and CD31-positive cells (23) or as CD45-negative, CD34-positive and CD31-positive cells (26). Importantly, isolated LEC maintain strong and specific expression of LYVE-1, podoplanin and Prox1, whereas BEC do not express significant amounts of podoplanin, LYVE-1 or Prox1 even after multiple passages in vitro. These results indicate that the phenotype of lymphatic endothelium is predominantly genetically driven and stable in vitro, and that the lineage-specific

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lymphatic differentiation is not dependent upon microenvironmental factors. It is of interest that LEC and BEC—when mixed in culture—stay separated and form capillary tubes that wind around each other, indicating that both vascular cell lineages possess mechanisms for lineage-specific cell recognition (23). Gene array analyses of LEC vs. BEC have been successfully used to identify previously unknown lineage-specific genes that are expressed either by lymphatic or by blood vascular endothelium in the skin (24,26). LEC-specific genes include macrophage mannose receptor, desmoplakin, adducin, plakoglobin and CCL20/ MIP3alpha, whereas BEC-specific genes include versican, N-cadherin, endoglin/CD105, integrin 5, ICAM-1, CD44, CXCR4 and VEGFR-1/Flt-1 (24,26); see Table 2. The molecules specifically expressed by LEC and BEC likely play important roles in the specific functional regulation and physiological maintenance of the two types of vasculature, and they also mediate the trafficking of leukocytes into and out of the skin, as well as the exit of tumor cells to form lymphatic or hematogeneous metastases.

5. NEW INSIGHTS INTO THE GENETIC BASIS OF LYMPHEDEMA Lymphedema is caused by insufficient lymph transport due to lymphatic hypoplasia, impaired lymphatic function, or obstruction of lymph flow (27). Primary lymphedema has been classified as Milroy disease when present at birth, or as Meige disease when it develops predominantly after puberty. Milroy disease is linked, in some families, to the VEGFR-3 locus on distal chromosome 5q (28,29), and missense mutations of the VEGFR-3 gene have been identified in several cases of hereditary, early-onset lymphedema (8). As discussed earlier, a gene mutation in the VEGFR-3 tyrosine kinase domain has also been identified in Chy mutant mice (9)—characterized by chronic lymphedema of the extremities and by hypoplastic lymphatic vessels in the skin. In lymphedema-distichiasis, an autosomal-dominant disorder with congenital lymphedema and double rows of eyelashes (distichiasis), inactivating mutations in the FOXC2 gene, a member of the forkhead/winged-helix family of transcription factors, were identified in several families (30). Mutations of the SOX18 gene on chromosome 20q13, an SRYrelated transcription factor, cause recessive and dominant forms of hypotrichosislymphedema-telangiectasia syndrome. Amino acid substitutions in the DNA-binding domain of SOX18 have been found in the recessive form of the disease whereas a heterozygous nonsense mutation

Table 2 Lineage-Specific Expression of Blood Vascular Endothelial Cell (BEC) and Lymphatic Endothelial Cell (LEC) Genes Revealed by Microarray Analysis Blood Vascular Endothelial Cells

Lymphatic Endothelial Cells

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Growth factors and chemokines

VEGF-C Placental growth factor

CCL20 (MIP-3alpha) Fibroblast growth factor-12

Receptors

GPR 39 Chemokine receptor X VEGFR-1/Flt1 CD44 CXCR4 Endoglin

Macrophage mannose receptor Membrane glycoprotein gp130 Coxsackievirus/adenovirus rec. Intracellular hyaluronic acid binding protein

Extracellular matrix molecules

Versican Type III collagen, α1 chain Type VI collagen, α1 and α3 chains SPARC/osteonectin

Reelin MFAP3

Adhesion molecules

Integrin alpha4 Integrin alpha5 Integrin beta3 N-cadherin ICAM-1

CEA-CAM Desmoplakin Galectin 8 Integrin alpha9 Plakoglobin

Miscellaneous

Endothelial cell-specific molecule 1 Interferon alpha-inducible protein 27 uPA

Intestinal trefoil factor Down syndrome critical region gene 2

of the transactivation domain causes the dominant hereditary form of the disease (31). SOX18 mutations have also been identified to be responsible for the phenotype of ragged (Ra) mice that show characteristic abnormalities of the hair coat and also develop lymphedema (31). Integrin 9 appears to be specifically expressed by some lymphatic endothelial cells, and mice lacking integrin α9 develop fatal bilateral chylothorax, lymphedema, and lymphocytic infiltration in the chest wall (32). Lymphedema has also been detected in a number of other genetic mouse models, revealing important roles of distinct genes in lymphatic development and function (Table 3). Based on its potent lymphangiogenic effect, VEGF-C has been tested for gene and protein therapy of lymphedema in animal models. Adeno-associated virus-mediated VEGF-C gene therapy promoted lymphatic vessel generation in the skin of Chy mice (9). Furthermore, VEGF-C156S, a mutant form of VEGF-C that selectively activates VEGFR-3, induced regeneration of cutaneous lymphatic vessels without blood vessel growth or vascular leakiness, side effects observed with VEGF-C gene therapy due to activation of VEGFR-2 (33). Successful regeneration of a lymphatic network was also achieved by injection of VEGF-C protein in a surgical lymphedema model in the rabbit ear, indicating the potential use of VEGF-C for the treatment of secondary lymphedema (34).

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Table 3 Genetic Mouse Models with Abnormalities of the Lymphatic Vascular System Gene

Model References

Phenotypes

Transcription factors Prox1

KO

13

No lymphatic vasculature

FOXC2

KO

43

Heterozygote mice exhibiting lymphatic hyperplasia

Net

KO

44

Chyle in the thoracic cage

SOX18

Ragged mice

45

Generalized edema and chyle in the peritoneum; cardiovascular and hair follicle defects

Angiopoietin-2 KO

22

Chylous ascites and peripheral edema; abnormal patterning

KO

46

Lack of sprouting of first lymphatic vessels from embryonic veins

TG

47

Hyperplastic lymphatic vessels

KO

48

Cardiovascular failure and defect remodeling of vascular networks

VEGFR-3

Chy mice

9

Lymphedema

Neuropilin-2

KO

21

Reduction of small lymphatic vessels and capillaries during development

32

Respiratory failure caused by pleural fluid

Growth factors/receptors

VEGF-C

Adhesion molecules Integrin alpha9 KO Miscellaneous Podoplanin

KO

19

Lymphedema, impaired lymphatic patterning and diminished lymphatic transport

SLP-79 and Syk

KO

49

Abnormal blood vessel-lymphatic connections

6. TUMOR LYMPHANGIOGENESIS Tumor metastasis to regional lymph nodes frequently represents the first step of tumor dissemination and serves as a major prognostic indicator for cancer progression. However, little is known about the mechanisms by which tumor cells gain entry into the lymphatic system. A widely held view has suggested that lymphatic endothelium only plays a passive role during this process (35) and that lymphatic invasion only occurs once stroma-infiltrating tumor cells happen upon preexisting peritumoral lymphatic vessels.

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The recent identification of lymphatic growth factors and receptors together with the discovery of lymphatic-specific markers have provided important new insights into the formation of tumor-associated lymphatic vessels and their active contribution to lymphatic tumor spread (3). Several studies in animal tumor models have now provided direct experimental evidence that increased levels of VEGF-C or VEGF-D promote tumor lymphangiogenesis and lymphatic tumor spread to regional lymph nodes, and that these effects can be suppressed by blocking VEGFR-3 signaling (36–40). A large number of clinicopathological studies found a direct correlation between tumor expression of the lymphangiogenesis factors VEGF-C or VEGF-D and metastatic tumor spread in many human cancers, providing indirect evidence for the involvement of lymphangiogenesis in tumor progression (41). Our recent studies in human cutaneous malignant melanomas demonstrated—for the first time—the presence of both intratumoral and peritumoral lymphangiogenesis in cutaneous melanoma (42). They also showed that primary melanomas that later metastasized were characterized by increased lymphangiogenesis—as compared to nonmetastatic tumors—and that the degree of tumor lymphangiogenesis can serve as a novel predictor of lymph node metastasis and overall patient survival, independently of tumor thickness (42). Further studies involving larger numbers of cases are needed to confirm these findings.

7. CONCLUSION Due to a number of recent discoveries in the field of vascular biology, some of the mechanisms controlling the normal and pathological development of the lymphatic vasculature are now being established, and several genetic defects have been identified in patients with lymphedema. The identification of specific markers and growth factors for lymphatic vessels and the establishment of cultured lymphatic endothelial cells have been instrumental in this advance. The recent concept of tumor lymphangiogenesis and its role in tumor metastasiss are of particular importance for the progression of malignant tumors. Further progress in this field will likely lead to better diagnosis and treatment of a variety of lymphatic disorders and of certain types of cancer.

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23. Kriehuber E, Breiteneder GS, Groeger M, Soleiman A, Schoppmann SF, Stingl G, Kerjaschki D, Maurer D. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J Exp Med 2001; 194:797–808. 24. Petrova TV, Maekinen T, Maekelae TP, Saarela J, Virtanen I, Ferrell RE, Finegold DN, Kerjaschki D, Ylae-Herttuala S, Alitalo K. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J 2002; 21:4593–4599. 25. Maekinen T, Veikkola T, Mustjoki S, Karpanen T, Catimel B, Nice EC, Wise L, Mercer A, Kowalski H, Kerjaschki D, Stacker SA, Achen MG, Alitalo K. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J 2001; 20:4762–4773. 26. Hirakawa S, Hong YK, Harvey N, Schacht V, Matsuda K, Libermann T, Detmar M. Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells. Am J Pathol 2003; 162:575–586. 27. Witte MH, Bernas MJ, Martin CP, Witte CL. Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology. Microsc Res Tech 2001; 55:122–145. 28. Ferrell RE, Levinson KL, Esman JH, Kimak MA, Lawrence EC, Barmada MM, Finegold DN. Hereditary lymphedema: evidence for linkage and genetic heterogeneity. Hum Mol Genet 1998; 7:2073–2078. 29. Evans AL, Brice G, Sotirova V, Mortimer P, Beninson J, Burnand K, Rosbotham J, Child A, Sarfarazi M. Mapping of primary congenital lymphedema to the 5q35.3 region. Am J Hum Genet 1999; 64:547–555. 30. Fang J, Dagenais SL, Erickson RP, Arlt MF, Glynn MW, Gorski JL, Seaver LH, Glover TW. Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am J Hum Genet 2000; 67:1382–1388. 31. Irrthum A, Devriendt K, Chitayat D, Matthijs G, Glase C, Steijlen PM, Fryns JP, Van Steensel MA, Vikkula M. Mutations in the transcription factor gene SOX18 underlie recesive and dominant forms of hypothrichosis-lymphedema-teleangeactasia. Am J Hum Genet 2003; 72:1470–1478. 32. Huang XZ, Wu JF, Ferrando R, Lee JH, Wang YL, Farese RV Jr, Sheppard D. Fatal bilateral chylothorax in mice lacking the integrin alpha9beta1. Mol Cell Biol 2000; 20:5208–5215. 33. Saaristo A, Veikkola T, Tammela T, Enholm B, Karkkainen MJ, Pajusola K, Bueler H, YlaHerttuala S, Alitalo K. Lymphangiogenic gene therapy with minimal blood vascular sideeffects. J Exp Med 2002; 196:719–730. 34. Szuba A, Skobe M, Karkkainen MJ, Shin WS, Beynet DP, Rockson NB, Dakhil N, Spilman S, Goris ML, Strauss HW, Quertermous T, Alitalo K, Rockson SG. Therapeutic lymphangiogenesis with human recombinant VEGF-C. FASEB J 2002; 16:1985–1987. 35. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249–257. 36. Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, Riccardi L, Alitalo K, Claffey K, Detmar M. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001; 7:192–198. 37. Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA, Prevo R, Jackson DG, Nishikawa S, Kubo H, Achen MG. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med 2001; 7:186–191. 38. Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D, Prevo R, Banerji S, Huarte J, Montesano R, Jackson DG, Orci L, Alitalo K, Christofori G, Pepper MS. Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J 2001; 20:672–682. 39. Karpanen T, Egeblad M, Karkkainen MJ, Kubo H, Yla-Herttuala S, Jaattela M, Alitalo K. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res 2001; 61:1786–1790.

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40. He Y, Kozaki K, Karpanen T, Koshikawa K, Yla-Herttuala S, Takahashi T, Alitalo K. Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling. J Natl Cancer Inst 2002; 94: 819–825. 41. Stacker SA, Baldwin ME, Achen MG. The role of tumor lymphangiogenesis in metastatic spread. FASEB J 2002; 16:922–934. 42. Dadras SS, Paul T, Bertoncini J, Brown LF, Muzikansky A, Jackson DG, Ellwanger U, Garbe C, Mihm MC, Detmar M. Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival. Am J Pathol 2003; 162:1951–1960. 43. Kriederman BM, Myloyde TL, Witte MH, Dagenais SL, Witte CL, Rennels M, Bernas MJ, Lynch MT, Erickson RP, Caulder MS, Miura N, Jackson D, Brooks BP, Glover TW. FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum Mol Genet 2003; 12:1179–1185. 44. Ayadi A, Suelves M, Dolle P, Wasylyk B. Net-targeted mutant mice develop a vascular phenotype and up-regulate egr-1. EMBO J 2001; 20:5139–5152. 45. Pennisi D, Gardner J, Chambers D, Hosking B, Peters J, Muscat G, Abbott C, Koopman P. Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. Nat Genet 2000; 24:434–437. 46. Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova TV, Jeltsch M, Jackson DG, Talikka M, Rauvala H, Betsholtz C, Alitalo K. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat Immunol 2004; 5:74–80. 47. Jeltsch M, Kaipainen A, Joukov V, Kukk E, Lymbousssaki A, MX, Lakso M, Alitalo K. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 1997; 276: 1423–1425. 48. Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola K, Breitman M, Alitalo K. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 1998; 282:946–949. 49. Abtahian F, Guerriero A, Sebzda E, Lu MM, Zhou R, Mocsai A, Myers EE, Huang B, Jackson DG, Ferrari VA, Tybulewicz V, Lowell CA, Lepore JJ, Koretzky GA, Kahn ML. Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk. Science 2003; 299:247–251.

4 High Endothelial Venules Jean-Marc Gauguet, Roberto Bonasio, and Ulrich H.von Andrian The CBR Institute for Biomedical Research, Inc. and Department of Pathology, Harvard Medical School, Boston, Massachusetts, U.S.A.

1. INTRODUCTION Secondary lymphoid organs, including peripheral lymph nodes (PLNs), mesenteric lymph nodes (MLNs), Peyer’s patches (PPs), appendix, tonsils, and spleen are essential components of the immune system. These organs are specialized to collect antigen (Ag) and Ag presenting cells (APCs) from distinct anatomical regions. Their extravascular environment is designed to optimize lymphocyte recognition of and subsequent responses to cognate Ag. A remarkable property of secondary lymphoid organs is their ability to recruit vast numbers of circulating B and T lymphocytes. With the exception of the spleen, secondary lymphoid organs contain specialized post-capillary and small collecting venules, called high endothelial venules (HEVs), which serve as the principal site of lymphocyte entry from the blood (1,2). High endothelial venules express organspecific patterns of lymphocyte traffic molecules that are not found in other microvascular beds. These molecules coordinate the recruitment of circulating lymphocytes by promoting multi-step adhesion cascades involving selectins, chemokines, integrins, and their respective ligands (3,4). In this chapter, we will review our current understanding of the functional, structural, and molecular characteristics of HEVs that allow them to function as the gateway to secondary lymphoid organs. We will examine the development of HEVs in normal and pathologic settings and how these unique microvessels respond to environmental cues. In addition, we will discuss techniques to study these structures as well as emerging technologies that may pave the way for future discoveries.

2. HISTORICAL PERSPECTIVE High endothelial venules have been the subject of scientific scrutiny since the 19th century. Because of the unusual cuboidal shape of the endothelial lining, histological cross-sections of these microvessels resemble those of exocrine gland ducts (the misnomer “lymph gland” for LNs probably derived from erroneous interpretations of HEVs). During the following decades, their physiological function was a topic of

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controversy, even after it was firmly established that HEVs are part of the circulatory system where large numbers of lymphocytes traverse the vessel wall. The prevalent initial interpretation was that lymphocytes were generated in lymphoid organs and secreted via HEVs into the blood (5,6). A much clearer picture emerged only in the early 1960s when Gowans and Knight (7) demonstrated that small (i.e., naïve) lymphocytes recirculate from the blood into lymph nodes and via the thoracic duct back into the blood. These authors injected radio-labeled lymphocytes i.v. into rats and recorded the recovery of labeled cells in the thoracic duct lymph. The recirculating cells first appeared 2–4 hr after injection and peaked in the lymph after 24 hr. More than 90% of the injected cells were recovered from chronic thoracic duct fistulas within four days, indicating that virtually all lymphocytes migrate actively and continuously (7). The fundamental importance of HEVs in the recirculation process was revealed when Marchesi and Gowans (1) used electron microscopy to demonstrate that these vessels serve as the entry site for circulating lymphocytes into secondary lymphoid organs. In this landmark study, i.v. injected lymphocytes could be “seen penetrating the endothelium of the venules” in LNs; lymphocytes were found within the vessel lumen, between endothelial cells and at the basement membrane. Labeled cells that had reached the LN parenchyma were in close proximity to HEVs, suggesting that the new arrivals had just crossed the microvascular barrier. While in most mammals the chief direction of lymphocyte migration is from the blood across HEVs into lymphoid tissues, some cells may also migrate in the opposite direction. In sheep and rat LNs (8,9), lymphocytes use HEVs predominantly to migrate into lymphoid organs, while in pig MLNs, lymphocyte migrate from the lymphoid tissue into the bloodstream (10). Despite these apparent species differences, HEVs are thought to be essential vascular determinants of lymphocyte traffic in all mammals.

3. HOW TO STUDY HEVs The study of HEVs has relied heavily on microscopic examination of tissue sections and, as a result, knowledge of these structures has deepened with advances in microscopy technology. The use of electron microscopy (EM) allowed investigators to peer into high endothelial cells (HECs) to examine their intracellular components and architecture (11). Electron microscopy studies also proved to be pivotal for the first description of how lymphocytes use HEVs to gain entry into lymphoid organs (1). More recent studies have employed EM to examine the transport of soluble lymph-borne factors across HEVs to the vessel lumen (12). The first in vitro tool to study interactions between lymphocytes and HEVs was described by Stamper and Woodruff (13). In this assay, frozen sections of lymphoid organs are incubated with a lymphocyte suspension, gently washed, and fixed with glutaraldehyde. Using dark-field microscopy, it is then possible to visualize and enumerate lymphocytes that interact with HEVs. A later modification of the StamperWoodruff assay reduced the number of lymphocytes needed for this technique (14). Since lymphoid organs rapidly recruit and transiently sequester circulating lymphocytes, a homing assay can be used to measure the number of transfused lymphocytes (distinguished from endogenous lymphocytes by labeling with radioisotopes, fluorescent

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dyes, or genetically encoded markers) that enter into a lymphoid organ within a certain period of time (typically 1–24 hr) (14). By combining homing experiments with the Stamper-Woodruff assay, it was discovered that the cells that showed tropism for secondary lymphoid organs in vivo, also adhered most avidly to HEVs in vitro, and that this binding required an active metabolism and a calcium flux (15). Investigators also used these assays to test the role of specific traffic molecules, e.g., by treating lymphocytes, tissue sections or animals with antibodies or with an enzyme such as neuraminidase (16–19). A more recent development is intravital microscopy, which uses epifluorescence techniques to visualize lymphocyte interactions with HEVs in a living animal. The first intravital analysis of lymphocyte interaction with HEVs was performed in murine PPs (20). More recently, intravital microscopy techniques to examine murine subiliac (also called inguinal) (21,22) and popliteal PLNs were developed (23). Immunohistochemistry and affinity chromatography with HEVs-specific monoclonal antibodies (mAbs) and in situ hybridization have been among the key technologies used to identify and characterize molecules produced by HEVs that are essential for lymphocyte trafficking. For example, mAb MECA-79 was critical for the identification of peripheral node addressin (PNAd), a group of sulfated glycans that are expressed in both human and mouse PLNs HEVs (18). In situ hybridization was instrumental to identify the chemokine CCL21 (SLC/TCA-4/6Ckine/exodus 2), which induces chemotaxis of naïve lymphocytes and is expressed in the T cell area of PLNs, especially in HEVs (24). In addition to studying the function of HECs in vivo, there have been several attempts to establish and study primary HEC-like cell lines (25,26). These lines preserve some of the properties of bona fide HECs, including a certain ability to bind lymphocytes (27), and expression of some proteins and antigens characteristic of primary HECs (28,29). However, none of the cultured HEVs lines express all of the traffic molecules that are found on HECs in vivo and, therefore, they do not fully recapitulate the unique biology of these cells (28,29).

4. MORPHOLOGY AND ANATOMY OF HEVs 4.1. Functional Anatomy of LNs and PPs Lymph nodes are strategically located along lymphatics, which drain peripheral tissues. The prototypical LN is a lentil- or bean-shaped parenchymatous organ, composed of loosely associated lymphoid and myeloid leukocytes and stromal cells, surrounded by a fibrous capsule that confers structural integrity. Peripheral lymph nodes are subdivided into two main areas: cortex and medulla (Fig. 1a). The cortex comprises the superficial B cell area that contains B follicles and germinal centers and the deeper T cell area or paracortex. The structural unit of the deep cortex is the paracortical cord, a column of T cells and antigen presenting cells, delimited by lymph-draining sinuses (30). At the center of each cord is an HEVs, which merge into incrementally larger collecting venules while draining blood toward the medulla. The medulla contains a highly developed system of sinuses that receive lymph fluid from the subcapsular and cortical sinuses and coalesce at the LN’s hilus into an efferent lymphatic vessel (31). The paracortical cords extend into

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the medulla, where they are referred to as medullary cords, which contain many plasma cells and macrophages (32). Peyer’s patches are organized lymphoid organs in the wall of the small intestine. In contrast to LNs, PPs do not receive afferent lymph. Their interface with the gut lumen is formed by M cells, a specialized type of epithelial cell, which continuously transcytose antigenic material from the intestinal cavity into the subepithelial

Figure 1 Functional anatomy of lymph nodes. (A) A schematic representation of a mouse LN is provided with relevant features highlighted. The feeding artery and the venous microcirculation are shown in red and blue, respectively. The large venule that drains blood from the medulla is marked as order I. To improve clarity, only a segment of the intranodal venular tree is shown. The lymphdraining compartments are shown in light green. Arrows indicate the direction of lymph flow. (B) Crosssection of an HEVs with FRC that drain lymph fluid and lymph-borne small molecules such as chemokines (shown as small red circles) from lymphatic sinuses toward the HEV.

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Electron microscopy studies suggest that the FRC conduits drain lymph into a narrow Peri-Vascular Channel (PVC) surrounding each HEVs. Upon delivery to HEVs, chemokines are transcytosed for presentation in the lumen. An adherent lymphocyte is shown inside the HEVs. (C) Architecture of a venular tree showing the characteristic branching orders in mouse inguinal LNs. dome, an area that is rich in DCs (33,34). The PP parenchyma is dominated by large B follicles, whereas T cells are confined to the interfollicular area.

4.2. Microvascular Organization of PLNs and PP The microvasculature organization varies considerably between different PLNs, but has a number of shared features. We distinguish a main feeding artery and a main collecting vein, both of which access the organ at the hilum (Fig. 1A). The arterial tree is not specialized and does not support interactions with circulating leukocytes; on the other hand, the degree of organization on the venular side is remarkable. In mouse inguinal LNs, starting from the main collecting venule, a distinct hierarchy of branches can usually be identified (22) (Fig. 1C). The endothelium of the first and second branching orders in the medulla has a flat appearance similar to endothelium in non-lymphoid tissues. In contrast, endothelium in higher order venules in the paracortex and deep cortex has a cuboidal, cobble-stone like shape. Thus, only microvessels with a higher branching order are HEVs. Ultrastmctural studies indicate that endothelial cells (ECs) in the transition region between HEVs and flat-walled venules are often arranged in an overlapping plate pattern (35). However, in immunohistochemical terms, the transition from HEVs to non-HEVs is typically very abrupt, from one EC to the next. This has been

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Figure 2 MECA-79 antigen expression is restricted to HEVs in PLNs. (A) A false-color intravital micrograph shows MECA-79 coated fluorescent microspheres (yellow) bound to small diameter venules (order III–V) within the venular tree (blue) of a mouse inguinal LN. Beads do not accumulate in arterioles (red), large diameter venules (order I and order II) or capillaries. (B) Intravital micrograph showing the distribution of CFSElabeled MECA-79 after i.v. injection (top panel). A schematic diagram (lower panel) identifies the venular orders and the superficial epigastric artery (SEA) and vein (SEV) in this preparation. (C) Immunohistochemistry reveals MECA-79 staining of a PLN HEVs illustrating the presence of MECA-79 on the lumenal (Lu) and abluminal [basement membrane (BM)] surface of the HECs. (Panels (B) and (C): Modified from Refs. 50,97).

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shown by staining with antibodies against HEVs-specific traffic molecules, such as the mAb MECA-79 against the peripheral node addressin or PNAd (Fig. 2A–C). This transition in immunoreactivity is located at the cortico-medullary boundary, and can be so accurate that a vessel running parallel to the juncture can have a HEVs phenotype on the side that faces the cortex and flat endothelium on the medullary side (36). While HEVs in PLNs are restricted to the T cell area, HEVs in PPs originate in B follicles. From here, they drain blood toward the interfollicular T cell zone where they merge into larger collecting venules. While there are no apparent structural differences between HEVs in PLNs and PPs (37), biochemical, immunohistochemical, and functional studies have uncovered a number of tissue-specific differences, which manifest in the selective recruitment of distinct lymphocyte subsets (see below).

4.3. Lymph Flow in PLNs Lymph is drained to LNs by a system of blind-ending vessels lined by specialized lymphatic endothelial cells. Afferent lymphatic vessels perforate the capsule of the LN on its convex aspect, distal to the hilum, and drain into the subcapsular sinus (Fig. 1A). Lymph-borne cells, particulate material, and soluble molecules have different fates after entering a LN. Dendritic cells (DCs) and effector memory lymphocytes invade the subcapsular sinus floor and migrate to their proper location in the cortex (38,39). Particulate material and large molecules are excluded from the parenchyma (40) and are phagocytosed by subcapsular phagocytes or drain to the

Figure 3 Characteristic ultrastructural and molecular features of HECs in PLN. The vessel lumen and stromal spaces are indicated in the figure. Posttranslational modifications of sialomucins within the prominent Golgi apparatus generates L-selectin ligands and the MECA-79 antigen. efferent lymphatic vessel through the sinus system. Low molecular weight molecules with a radius of less than 4 nm are channeled into the parenchyma along a network of

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collagen fibers (41,42) that are ensleeved by fibroblastic reticular cells (FRCs). This sizing column-like drainage system is called the “FRC conduit” (30,43). Fibro-blastic reticular cells also encircle HEVs, and EM studies have suggested the existence of a narrow perivascular gap between them, the perivenular channel (PVC) that is thought to receive lymph fluid via the FRC conduit (Fig. 1B).

4.4. Ultrastructure of High Endothelial Cells The most prominent ultrastructural feature of HECs, besides their characteristic shape, is a prominent Golgi apparatus, which is often oriented toward the luminal aspect of the cell (11) (Fig. 3). In addition, HECs have abundant mitochondria and ribosomes, with sparse rough endoplasmic reticulum and several multi-vesicular bodies (11,44,45).This ultrastructural appearance of HECs is atypical for conventional ECs and was interpreted as indicative of a cellular machinery with high metabolic activity geared toward the biosynthesis of glycoproteins (11). Further support for this notion came from metabolic studies showing that PLNs HECs incorporate substantial amounts of sulfate (46). The importance of these two observations will be examined below. The apical surface of HECs is dotted with shallow pits (9) and a glycocalyx that appears thicker than the glycocalyx on non-HEVs endothelium (47). The abluminal surface rests on a thin basement membrane and macular tight junctions join juxtaposed cells at their apical and basal surfaces (44). These discontinuous junctions, unlike the continuous tight junctions observed in flat endothelium, are thought to allow lymphocytes to squeeze between adjacent endothelial cells without causing vascular leakiness.

5. LEUKOCYTE RECRUITMENT VIA HEVs It is now widely accepted that tissue- and subset-specific leukocyte migration is governed by a sequence of molecularly distinct adhesion and signaling events (3,4,48). Adhesion cascades are initiated by a tethering step that allows leukocytes to bind loosely to endothelial cells. Once attachment has occurred, marginated cells are pushed forward by the blood stream resulting in a slow rolling motion along the vascular wall (step 1). Subsequently, rolling cells encounter chemotactic stimuli on or near the EC surface that can bind to specific leukocyte receptors (step 2). Chemoattractant binding, in turn, induces rapid intracellular signaling and triggers activation-dependent adhesion steps that allow leukocytes to stick firmly (step 3) and, eventually, to emigrate through the vessel wall. While microvessels in most non-lymphoid tissues can only support substantial leukocyte traffic upon exposure to inflammatory mediators, HEVs must recruit large numbers of lymphocytes in the absence of inflammation. Thus, HEVs constitutively express a unique pattern of traffic molecules that are fundamental for the normal function of the immune system.

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5.1. The Multi-Step Adhesion Cascade in PLNs Detailed intravital microscopy analyses have defined the specific adhesion cascades that mediate T and B cell homing to LNs (Fig. 4A) (49–52). Tethering and rolling of both subsets are mediated by L-selectin (CD62L). The endothelial L-selectin ligand is the peripheral node addressin (PNAd), an O-linked carbohydrate moiety, the major components of which are recognized in humans and mice by the monoclonal antibody MECA-79 (18,53). The structure of the MECA-79 antigen and HEV-s-expressed Lselectin ligands will be discussed below. Firm arrest of rolling T cells in LN HEVs is mediated by LFA-1 (49,54,55). Lymphocytes deficient in LFA-1 home poorly to LNs, although the observed magnitude of the homing defect was somewhat variable between two independently generated knockout strains (55,56). In one strain, residual LFA-1-independent homing was found to be mediated by another integrin, α4β7, which was shown to interact with VCAM-1 in LN HEVs (56). The agent that activates integrins on naive and central memory T cells is the chemokine CCL21 (also called SLC, TCA-4, 6Ckine, or exodus 2) (50,51). CCL21 is constitutively expressed by HECs and binds to CCR7 (57–59). A second CCR7 agonist, CCL19 (also called ELC or Mip-3β), is expressed by lymphatic endothelium and interstitial cells within LNs, but not by HEVs. However, lymph-borne CCL19 can be transported to the luminal surface of HEVs where it induces integrin activation on rolling T cells (12). The physiologic role of CCL 19 in lymphocyte homing remains to be determined. Mice have two isoforms of CCL21; CCL21ser is expressed in HEV, whereas CCL2Leu is generated in lymphatic endothelium (60). A mutant strain called plt/plt (paucity in lymph node T cells) is deficient in CCL21ser and CCL19 (60,61). Lymph nodes of plt/plt mice and CCR7 deficient mice contain few naïve T cells, but the B cell compartment is much less affected and LNs in these mutant animals also contain substantial numbers of central memory T cells (62,63). This indicates that B cells and central memory T cells may respond not only to CCR7 agonists, but also to another integrin activating signal in HEVs. Indeed, a recent study has shown that rolling B cell can be stimulated to arrest in HEV by CXCL12 (SDF-1α), the ligand for CXCR4 (52). CCR7 and CXCR4 can independently maintain B cell homing (albeit at some-what lower levels than in wildtype animals where both function simultaneously). Interestingly, although CXCL12 potently induces integrin activation on rolling naïve T cells in vitro (58,64), it does so very poorly in vivo (65). Thus, despite low-level expression of CXCL12 in wildtype murine HEVs, naïve T cells require CCR7 signals, at least in the Balb/c and DDD/1 genetic background (50,52). On the other

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Figure 4 Multi-step adhesion cascades in HEVs. Schematic representations of the multi-step cascade in PLNs (A, B) and in PPs (C, D) are shown. In (A) a naïve T lymphocyte undergoes tethering and rolling via L-selectinPNAd (step 1); chemoattractant stimulation by CCR7-CCL21 (step 2); and firm adhesion via activated LFA-1 (step 3); eventually permitting the arrested cell to diapedese. The selectivity of this recruitment cascade is illustrated in (B); L-selectin negative cells (e.g., effector memory T cells) and CCR7 negative leukocytes (e.g., granulocytes) fail to undergo each of the prerequisite steps. Peyer’s patches display different “ZIP codes” in HEV segments within the interfollicular T cell area (C) and in upstream B

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follicles (D). MAdCAM-1 is expressed in all PP HEVs and mediates rolling interactions with both L-selectin and α4β7 integrins. For step 2, HEVs in different areas display distinct chemokine patterns: T cells are activated by CCL21, while in HEVs associated with B follicles present CXCL12 and CXCL13 to stimulate B cell-expressed CXCR4 and CXCR5, respectively. Firm arrest of both B and T cells in PPs is thought to be mediated by activated α4ß7 and/or LFA-1 integrins. hand, CXCL12/CXCR4 has a modest role in naïve T cell homing in C57BL/6 mice, indicating that some chemokine pathways may be regulated in a strain-dependent manner (52). Despite the ability of B cells to respond to two distinct integrin activation signals in HEVs, homing of B cells to LNs is significantly less efficient than that of T cells. This is because the L-selectin levels on B cells are ~50% lower than on T cells (66). T cells from L-selectin+/− heterozygous mice express similar L-selectin levels as L-selectin+/+ B cells, and these two lymphocyte populations home equivalently to LNs (66). In experiments with pre-B cell clones stably transfected with human L-selectin, we observed that at least 50000 L-selectin molecules/cell were needed for efficient rolling in LN HEVs (21,67 and unpublished data). Thus, it is important to keep in mind that the mere presence of Lselectin on a leukocyte does not necessarily predict its potential to home to LNs, even when this cell expresses all other prerequisite traffic molecules (i.e., CCR7 and LFA-1). A leukocyte may be deemed L-selectin+ based on flow cytometric criteria, but this would be of little consequence if the expressed copy number is substantially lower than that on naïve T cells (~70 000–100 000 molecules/cell). The fact that a sequence of three distinct molecular steps must be successfully engaged in HEVs explains why some leukocytes home to PLNs, whereas others do not. For example, granulocytes express L-selectin and LFA-1, but not CCR7 or CXCR4; mature myeloid DCs express CCR7 and LFA-1, but not L-selectin; and effector CTL lose both L-selectin and CCR7. Consequently, granulocytes roll, but fail to arrest, whereas DCs and effector cells cannot tether or roll in LN HEVs (49,51,68) (Fig. 4B). L-selectin-independent homing via HEVs can be induced by i.v. injections of activated platelets (69,70). Circulating activated platelets express P-selectin on their surface, which mediates platelet binding to PNAd in HEVs and, simultaneously, to PSGL-1 on circulating leukocytes (69). This platelet bridge can transiently restore lymphocyte homing in L-selectin deficient mice. Indeed, L-selectin deficient animals mount poor cutaneous hypersensitivity (CHS) responses when they are sensitized by painting of a hapten antigen on the skin because naïve T cells do not migrate into skin draining LNs

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(71). When the homing cascade is restored during the sensitization phase by infusing activated platelets for several hours, the CHS response is restored (70). Of note, a small fraction of leukocytes roll in a P-selectin-dependent manner in PLNs HEVs of L-selectindeficient mice even when no platelets are being infused (70). However, it is not yet clear if this minor rolling pathway contributes to physiologic L-selectin-independent homing and whether the relevant source of P-selectin in this setting are circulating activated platelets or endothelial cells or both.

5.2. The Multi-Step Adhesion Cascade in PP and MLN The homing cascade discussed above applies primarily to skin-draining LNs. High endothelial venules in mucosa-associated LNs, such as MLNs, express not only PNAd, but also the mucosal addressin cell adhesion molecule (MAdCAM)-1, a ligand for the α4β7 integrin (17,72). The α4β7/MAdCAM-1 pathway can mediate selectin-independent lymphocyte tethering and rolling in HEVs (73). Thus, L-selectin deficiency compromises lymphocyte homing to PLNs more severely than to MLNs (74), whereas β7 integrin deficiency results in reduced homing to MLNs, but has no effect in PLNs (75). Payer’s patches HEVs express only MAdCAM-1, but not PNAd, on their luminal surface however, there is MECA-79-reactive material at the abluminal side of PP HEV (18). The binding site for α4β7 resides within the first Ig domain of MAdCAM-1 (76). In addition, MAdCAM-1 contains a mucin domain that can be decorated with L-selectin ligands. Indeed, affinity-purified MAdCAM-1 from MLNs is recognized by the mAb MECA-79 (77). By contrast, MAdCAM-1 in PP HEVs is not detected by MECA-79, but nevertheless supports lymphocyte tethering and rolling via both L-selectin and the α4β7 integrin (73). When α4β7 becomes functionally activated, it can also mediate firm arrest (78). However, naïve lymphocytes express relatively little α4β7 (79) and require additional engagement of LFA-1 for firm arrest, while α4β7 is primarily critical to slow the rolling cells (73) (Fig. 4C). Intravital microscopy experiments have shown that the integrin-activating chemokines in PP HEVs are segmentally presented (52,80). In B follicles, HEVs present CXCL12 and CXCL13, the ligands for CXCR4 and CXCR5, respectively (Fig. 4D). Signals through these two receptors induce integrin activation on rolling B cells, but not T cells (52). Conversely, as soon as HEVs enter into the interfollicular T cell area, they express CCL21 and promote preferential T cell arrest (80). Thus, HEVs are not only distinct between different lymphoid tissues, but there is even segmental specialization within individual microvessels. The factors that orchestrate this remarkable endothelial subspecialization remain to be identified.

5.3. Remote Control An additional mechanism for leukocyte recruitment to PLNs operates during inflammation (50,81). Chemokines, such as CCL2 (MCP-1), produced at a peripheral site of inflammation are drained via the lymph to the subcapsular sinus. Here they enter the FRC conduit and are channeled toward HEVs in the cortex (42). Chemokines that reach

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the abluminal surface of HEVs are then transcytosed within vesicles to the luminal side and presented to rolling leukocytes (12,81). The nature of the chemokine transport vesicles in HEVs has not been determined, but might involve caveolae, which have been shown to mediate chemokine transcytosis in dermal microvessels (82). This mechanism, termed “remote control,” allows the rapid recruitment of circulating monocytes, which express CCR2, the receptor for CCL2. Monocytes do not express CCR7 or CXCR4, and are therefore excluded from resting PLNs. However, by discharging CCL2 into the lymph, inflamed peripheral tissues project a potent monocyte chemoattractant signal onto HEVs in draining LNs, which triggers integrin activation (81). Once in the lymph node, monocytes may differentiate into macrophages or dendritic cells to participate in the ensuing immune response. In addition, inflammation induces mRNA for CXCL9 (MIG) in draining LNs and presentation of this chemokine on a subset of HEVs, which support monocyte adhesion in vitro (83). It is not known whether CXCL9 is produced by the inflamed HEVs themselves or by other intra- (or extra-) nodal cells from where the chemokine might have been transported to the HEVs. The ability to remotely modulate the multi-step adhesion cascade in HEVs by discharging chemokines into the lymph enables peripheral tissues to control the composition and function of leukocytes in draining LNs. However, recent work suggests that there may be also counter-regulatory mechanisms. Lymphatic endothelial cells express the non-signaling serpentine receptor D6, which binds promiscuously to a number of chemokines, including CCL2 (84). Upon ligation, D6 triggers the rapid internalization and degradation of its cargo and may thus have a role as a gatekeeper to avoid uncontrolled seepage of chemokines into the lymph (85). Interestingly, HEVs express high levels of a similar non-signaling chemokine receptor, the Duffy antigenrelated receptor for chemokines (DARC) (86). It seems plausible that DARC, too, has a function in the transportation, presentation, or turnover of chemokines in HEVs. However, the mechanisms that control the expression and function of either D6 or DARC are still poorly understood.

5.4. Transendothelial Migration The last step of the adhesion cascade, extravasation, is also the least understood at the molecular level. Adhesion molecules that play a role in transendothelial migration of inflammatory cells in non-lymphoid tissues, such as PECAM-1 (CD31) and CD99, have not been shown to contribute to lymphocyte diapedesis across HEVs. One candidate molecule is JAM-C, an immunoglobulin superfamily member that is concentrated at intercellular junctions between HECs (87) and forms homoand heterotypic interactions with other JAM family members (88). JAM-B (VE-JAM) is also found at intercellular boundaries in HEVs and normal endothelium (89), and interacts with leukocytes (88), but its role in transmigration has not been demonstrated. The reader is referred to recent reviews for further details (90–92).

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6. HEV-SPECIFIC LYMPHOCYTE TRAFFIC MOLECULES 6.1. The MECA-79 Antigen in PLNs HEVs MAb MECA-79 immunoprecipitates a set of glycans on the surface of HEVs in both murine and human lymphoid tissues (18,53,93). Affinity-purified MECA-79-reactive glycoproteins support L-selectin binding in vitro (53), and MECA-79 blocks lymphocyte recruitment to PLNs (18,94) as well as rolling on HEVs in vivo (21,95). Immunohistochemical staining of tissue sections with MECA-79 reveals reactive material on both the lumenal and abluminal surface of PLNs HEVs (Fig. 2C), but only on the abluminal side of PP HEVs (18,96). The physiological role of abluminal MECA-79 antigen is not understood, but it is clear that it is biosynthetically distinct from the lumenal material (96). Intravital microscopy studies of the localization of fluorescent MECA-79 or MECA-79-conjugated fluorescent latex beads have shown that MECA-79 antigen is highly restricted to cortical HEVs within the PLNs vasculature (97,98) (Fig. 2A, B). In the HEV lumen, MECA-79 localizes to microvillouslike protrusions, which may facilitate L-selectin-dependent tethering of circulating lymphocytes (86). Several sialomucins have been identified that can be decorated with the MECA-79 epitope. These include GlyCAM-1 (Sgp50), a secreted glycoprotein (99,100); CD34 (Sgp90) (101); podocalyxin (102); and one (or more) large molecular species termed Sgp200, which remain(s) to be identified (103). Two additional proteins that may support lymphocyte recruitment to PLNs include endomucin (104) and endoglycan (105,106). Endomucin can carry the MECA-79 epitope and has been detected on HEVs, whereas endoglycan functions as an L-selectin ligand through tyrosine sulfation and glycosylation, but is not recognized by MECA-79. As far as we can tell, these glycoproteins probably serve as interchangeable scaffolds that are functionally redundant, at least in the context of L-selectin ligand presentation. For example, CD34 is a major component detected by MECA-79 in human tonsils (107), but CD34 deficient mice have normal lymphocyte migration to lymphoid tissues (Table 1) (108). GlyCAM-1 expression is restricted to HEVs and mammary epithelial cells (109), podocalyxin was originally described in the kidney, and CD34 is expressed on many different cells types including non-HEV endothelium and cells of hematopoietic origin (110). However, on non-HEV cell types, none of these molecules react with MECA-79 or support L-selectin binding. Indeed, L-selectin ligand presenting sialomucins undergo extensive HEV-specific post-translational modifications that are essential for their function in lymphocyte migration.

6.2. Post-translational Modification of HEV Proteins The importance of HEV-specific carbohydrate modifications for lymphocyte trafficking was first suggested by observations that specific mono- and polysaccharides

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Table 1 Effect of Disrupting Genes Involved in Lymphocyte Recruitment to Lymphoid Organs and Expressed by HEV Gene

Leukocyte Trafficking in Knockout

Reference(s)

L-selectin (CD62L)

Hypocellular PLNs and impaired lymphocyte trafficking. Impaired DTH responses.

74,163

CD34

Defects in early hematopoietic progenitor development. No defects in lymphocyte trafficking.

108

CD43

Increased lymphocyte migration to secondary lymphoid organs.

97

CD44

Delayed or subtle defect in entry of CD44 deficient lymphocytes to PLNs.

164,165

LFA-1 (CD11aCD18/αLβ2)

Impaired lymphocyte trafficking to PLNs, MLNs, and PPs.

55,56

β7 Integrin

Impaired lymphocyte trafficking to MLNs and PPs and impaired adhesion to PP HEVs.

75

ICAM-1 (CD54)

Defects in DTH response and neutrophil recruitment 166,167 to inflammatory sites. No defect in lymphocyte recruitment to lymphoid organs.

ICAM-2 (CD 102)

No defect in lymphocyte recruitment to lymphoid organs.

168

CCR7

Impaired lymphocyte and DC trafficking to PLNs and PPs.

62

CCL21ser/CCL19(plt/plt mice)

Impaired T lymphocyte trafficking to PLNs and PPs. 50,52,80,169

CXCR5 CXCL13 (BLC)

Impaired B lymphocyte homing and migration to B follicles in Peyer’s patches and spleen.

52,170

CXCR4 CXCL12 (SDF1α)

Impaired T and B lymphocyte migration to lymphoid organs in the absence of CCR7 ligands.

52

DARC

Potential defect in inflammatory chemokine transport or presentation on endothelium.

135,171

Knockout Phenotype Lymphocyte Homing Enzymes

MECA-79 L-selectin IgM Staining of HEV Staining of Reference(s) HEV

C2GlcNAcT-Ia

Normal/decreased

Normal

Absent

127,127a,131.

HEC-GlcNAc6ST 50%

Reduction

Abluminal

Abluminal

95,96

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FucT-VII FucT-IV FucT-VII/FucT-IV

80% Reduction Increased

staining only

staining only

Normal

Absent

119

b

Normal

98,120

b

b

NA

>95% Reduction

102

NA

NA

120

a

Abbreviations: C2GlcNAcT-I, Core2 β-1,6-N-acetylglucosaminyltransferase-I; HECGlcNAc6ST, HEC- GlcNAc-6-sulfotransferase; FucT-VII, α(1,3)fucosyltransferase-VII; FucT-IV, α(1,3)fucosyltransferase-IV. b NA: Data not available. inhibit lymphocyte binding to LN HEVs (111–113). After it was discovered that Lselectin functions as a lectin-like adhesion molecule that binds the HEV-specific MECA79 antigen (53,93), detailed biochemical studies of affinity-purified MECA-79-reactive material (or PNAd) were performed (114–116). Today, we know that most HEVexpressed L-selectin ligands are sialylated, fucosylated and sulfated O-linked carbohydrates that decorate a select group of sialomucins in HEVs (Table 2).

6.2.1 Sialic Acid Rosen et al. (117) demonstrated that removal of sialic acid from PLNs sections with sialidase blocked lymphocyte binding to HEVs, and intravenous injection of sialidase inhibited lymphocyte homing to PLNs (19). Although the mAb MECA-79 does not require the presence of sialic acid for binding, the HEV-expressed glycoproteins that react with MECA-79 must be modified with sialic acid to function as L-selectin ligands (118). Indeed, the major capping group that binds L-selectin is a sulfated form of sialyl LewisX (sLeX) (i.e., Siaα2,3Galβ1,4-[Fucα1,3]GlcNAc) (115,116). Sialic acid is not essential for lymphocyte migration to PPs, since treatment of PP HEVs with sialidase does not affect lymphocyte binding or homing to PPs (19,117). However, treatment of purified MAdCAM-1 with sialidase blocked in vitro L-selectin binding to MAdCAM-1 (77), suggesting that the interaction of α4β7 integrins with MAdCAM-1 does not depend upon sialylation, while L-selectin binding to MAdCAM-1 requires it. Which of the many α2,3 sialyltransferases is responsible for the biosynthesis of L-selectin ligands in PLNs HEVs remains to be determined.

6.2.2. α(1,3)-Fucosylation The presence of α(1,3)-linked fucose is essential for the activity of virtually all physiologic selectin ligands, including those in HEVs. The two fucosyltransferases (FucT) implicated in the generation of selectin ligands on leukocytes and endothelial cells are FucT-IV and FucT-VII (Table 1) (119,120). FucT-VII deficient mice have ~80% reduced lymphocyte homing to PLNs (119). FucT-IV-VII double knockout mice have a significantly more complete defect in lymphocyte homing to PLNs than FucT-VII deficient mice, indicating that both FucTs can generate L-selectin ligands in HEVs (120). However, FucT-IV deficient mice have moderately increased lymphocyte homing to PLNs and MLNs (98).

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To explain this counter-intuitive observation, one must consider the acceptor preferences of FucT-IV and FucT-VII (121,122). FucT-VII requires an α(2,3)-sialylated terminal lactosamine to generate sLeX-like selectin ligands. FucT-IV can also use the sialylated lactosamine substrate, albeit less efficiently than FucT-VII. However, unlike FucT-VII, FucT-IV additionally fucosylates internal GlcNAc residues in the polylactosamine (121) and uses a non-sialylated lactosamine to generate LewisX-like structures (120), which do not interact with selectins. Thus, FucT-IV may compete with α(2,3)-sialyltransferase(s) and/or FucT-VII for terminal lactosamine as a shared precursor substrate on O-linked glycans. Consequently, FucT-IV may divert some terminal lactosamine acceptor moieties toward the LewisX pathway that does not yield L-selectin ligands, and thus away from the α(2,3)sialyltransferase/FucT-VII synthetic route. In wildtype mice, FucT-IV thus attenuates FucT-VII-dependent production of sLeX-based L-selectin ligands. Without FucT-IV, increased acceptor availability for α(2,3)sialyltransferase/FucT-VII permits increased selectin ligand production, whereas FucT-VII deficiency has a lesser impact, since sLeX-based L-selectin ligands could still be generated if FucT-IV is abundantly expressed.

Table 2 Structure of Sialyl Lewisx Derivatives and Enzymatic Modification Carried Out by HEV Expressed Enzymes

Enzyme C1GlCNACT

Enzyme reaction a

C2GlcNAcT-I

HECGlcNAc6ST

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FucT-VII (FucT-IV)

FucT-IVb

Biantennary glycans arise from a corel branch at R1 or a core2 branch at R2. * 6-Sulfated corel glycans are sufficient to generate the MECA-79 epitope found on the lumenal surface of HEVs. a Abbreviations: C1G1cNAcT, Corel-β1,3-N-acetylglucosaminyltransferase; C2GlcNAcT-I, Core2 β-1, 6-N-acetylglucosaminyltransferase-I; HEC-GlcNAc6ST, HEC-GlcNAc-6-sulfotransferase; FucT-VII, α(1,3)fucosyltransferase-VII; FucT-IV, α(1,3)fucosyltransferase-IV. b Unlike FucT-VII, FucT-IV fucosylates non-sialyated carbohydrates and internal GlcNAc residues on lactosamines. In addition, FucT-IV fucosylates glycolipids (not depicted) (172,173). Experimental evidence in support of this scenario has been provided recently (98). This work has revealed additional complexity, because FucT-IV and FucT-VII are not uniformly expressed within the venular tree in PLNs. The larger collecting venules in the medulla of mouse subiliac LNs express a functional L-selectin ligand that is spatially and antigenically distinguishable from MECA-79-reactive PNAd (98), which is restricted to para-and subcortical HEVs and requires primarily FucT-VII (97). By contrast, the Lselectin ligand(s) in medullary venules are MECA-79⎯ and are regulated largely by FucTIV, which is more highly expressed in this microvascular bed than elsewhere in PLNs.

6.2.3. Sulfation The critical role of sulfation of HEV proteins was first suggested by the unique ability of HEVs amongst vascular endothelium to incorporate large amounts of sulfate (46). Indeed, metabolic inhibition of sulfation reduces L-selectin ligand activity in PLNs (114). There are two sulfated sLeX moieties in PLNs, one containing Gal-6-SO4 (6′-sulfo Lewisx) and the other containing GlcNAc-6-SO4 (6-sulfo Lewisx) (Table 2) (116), but only 6-sulfation of sLeX is thought to enhance L-selectin binding (123). The GlcNAc-6O-sulfotransferases responsible for the generation of L-selectin ligands in HEVs are GlcNAc6ST (124) and HEC-GlcNAc6ST/LSST (125,126). Mice deficient in HECGlcNAc6ST/LSST have hypocellular PLNs, impaired (~50%) lymphocyte homing to PLNs, reduced L-selectin binding to HEVs, and a near absence of MECA-79-reactive

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material in the lumen of HEVs (96,127). The latter is not surprising, since the epitope recognized by MECA-79 requires 6-sulfation of sLex (103,128). However, in HEVs of HEC-GlcNAc6ST knockout mice, lumenal MECA-79 antigen is absent, yet lymphocytes still roll in an L-selectin-dependent manner, but at a higher velocity (95,96). These findings indicate that some of the carbohydrates recognized by L-selectin are distinct from that recognized by MECA-79. Moreover, abluminal MECA-79 staining is preserved around HEVs of HEC-GlcNAc6ST knockout mice, suggesting that additional sulfotransferases are involved.

6.2.4. O-linked Carbohydrates O-linked glycans in a core-2 linkage (i.e., G1cNAcβ1,6[Galβ1,3]-GalNac) represent a major component of L-selectin ligands in HEVs (129) (Table 2). Thus far, only the contribution of core2 β-1,6-N-acetylglucosaminyltransferase (C2GlcNAcT-I) (130) and corel-β1,3-N-acetylglucosaminyltransferase (C1G1cNAcT) (128) to the generation of Lselectin ligands has been studied. While C2GlcNAcT-I was found to be critical for the generation of P- and E-selectin ligands, no defect was observed in lymphocyte trafficking to peripheral lymphoid organs of mice deficient for this enzyme (131), although more recent data suggest that these animals have subtle defects in lymphocyte trafficking to PLNs (127,127a) (Table 1). Residual L-selectin ligand activity in C2GlcNAcT-1 deficient mice may be due to additional core2 β-1,6-N-acetylglucosaminyltransferases and/or the activity of C1G1cNAcT, which has been shown in vitro to be critical in the generation of MECA-79-reactive glycans and contribute to the synthesis of L-selectin ligands (128) (Table 2).

6.3. MAdCAM-1 MAdCAM-1 has two distal regions homologous to the immunoglobulin (Ig) domains found on other adhesion molecules of the immunoglobulin superfamily, including ICAM1 and VCAM-1 (72,76), and a mucin-like region rich in serine and threonine, which can bear O-linked carbohydrate modifications necessary for L-selectin binding (132). MECA79 can immunoprecipitate MAdCAM-1 from MLNs, but not PPs, and MAdCAM-1 from MLNs supports L-selectin binding in vitro (77). However, MAd-CAM-1 in PP HEVs can also present L-selectin ligands (that are presumably not recognized by MECA-79) and support L-selectin tethering and rolling in vivo (73). Therefore, MAdCAM-1 represents a unique multi-functional adhesion molecule capable of interacting with both α4β7 integrin and L-selectin.

6.4. Chemokine Presentation on HEV Chemokines presented on the endothelial surface induce integrin activation and firm arrest of rolling leukocytes. The immobilization and display of chemokines are thought to be mediated by glycosylaminoglycans (GAGs). Glycosylaminoglycans are proteoglycans that carry negatively charged sulfate and carboxyl groups, which permits electrostatic interactions with basic peptide motifs present in most chemokines (133). Heparin and

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heparan sulfate are highly expressed on endothelial cells and are the likely candidates to bind and present chemokines. The Duffy antigen-related chemokine receptor (DARC) is a non-signaling seventransmembrane receptor that binds both CC and CXC chemokines. The DARC is highly expressed in HEVs (86) and has been suggested to play a role in chemokine transport. The DARC has been identified in endothelial caveolae (134), which have been shown to function as a conduit for chemokine translocation from the abluminal to the lumenal surface of venules (82) (Fig. 3). Studies in knockout mice suggest that DARC does not appear to play a role in regulating constitutive lymphocyte trafficking to PLNs (135). However, DARC can scavenge inflammatory chemokines including CCL2, which suggests a role for DARC in inflammation, possibly during “remote control” of monocyte recruitment to PLNs (see above).

7. PLASTICITY OF ENDOTHELIUM AND HEV 7.1. Perinatal Switch In the developing mouse, the main addressin expressed in both LN and PP HEVs is MAdCAM-1 (136). It is thought that a population of CD4+CD3−IL7Rαhi cells is required to provide the signals necessary for the further development of LNs and PPs (137). These rare cells (1–2% of total PBLs in the mouse fetus) express the α4β7 integrin and must bind to MAdCAM-1 to migrate to LNs (136).On the other hand, studies in rats found almost no HEVs in LNs prenatally (138). Endothelial cells in cortical vessels acquired a cuboid morphology only gradually after birth, indicating that HEVs commitment may be differentially regulated in different species.

7.2. Lymph-borne HEV Differentiation Factors Once developed, the mature HEVs phenotype requires constant maintenance signals. Hendriks et al. (139) reported that HEVs from PLNs that had undergone occlusion of their afferent lymphatics became flat and lost their characteristic morphology. Subsequent studies showed that lymphocytes do not adhere to the lymph-deprived HEVs (140), and genes that contribute to the generation of L-selectin ligands are turned off (141,142). The nature of the HEV-sustaining lymph-borne signal is still mysterious. Lymph flow per se is probably not essential, since HEVs are also found in PPs, which do not possess afferent lymphatics.

7.3. Ectopic HEV Normal endothelium has the potential to differentiate into HEVs when given the appropriate stimuli. During chronic inflammation, such as in certain infections and autoimmune diseases, the cellular infiltrate organizes itself into lymph node-like

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structures containing naïve T cells, B cell follicles and dendritic cells, in a process termed “lymphoid neogenesis” (reviewed in Refs. 143–145). Post-capillary venules in these aggregates assume an HEV-like morphology, express PNAd and/or MAd-CAM-1 as well as CCL21, and recruit naïve T cells from the blood (65). The ectopic expression of CCL19, CCL21 or CXCL13 is sufficient to cause lymphoid neogenesis, at least in certain organs such as the pancreas (146–148). How the recruitment of naïve lymphocytes induces the differentiation of ectopic HEVs is not yet clear. A likely player is lymphotoxin (LTα), a member of the TNF family that is expressed by activated CD4 and CD8T cells (149), and also by the above mentioned CD4+CD3−IL7Rαhi cells that seed nascent LNs. Indeed LTα and its receptors, coordinate the genesis of secondary lymphoid organs (150,151), and expression of LTα1β2 is sufficient and necessary to induce lymphoid neogenesis (145,147).

8. CONCLUSION AND FUTURE DIRECTIONS 8.1. Unanswered Questions Although the past two decades have seen considerable progress in our understanding of the functions and molecular and biochemical underpinnings of HEVs, many fundamental questions remain to be answered. What does it take to make an HEV? As we have seen, factors in lymph and at sites of chronic inflammation are required to maintain HEVs and to turn regular endothelial cells into HECs, respectively. What are the mechanisms that induce and sustain this conspicuous differentiation? Can we interfere with these signals for therapeutic purposes? Can we use them to grow and study HECs in vitro? How many different subtypes of HEVs are there and what are the signals and the transcriptional programs that control their distinct phenotype? Another gray area is the question of how lymphocyte transmigration across HEVs is regulated. Is leukocyte recruitment also regulated at this last step of the adhesion cascade, or is diapedesis a default process? To answer these questions, novel technologies have begun to be employed together with the established techniques. We will briefly discuss some of these promising new approaches in this last section.

8.2. Genomics The unique features of HEVs likely derive from a specific set of expressed genes. Since bona fide HECs cannot be grown in vitro, material for expression profiling must rely on the cumbersome purification of relatively few cells from single-cell suspensions of mammalian lymphoid tissues. For example, quantitative 3′-cDNA libraries have been generated for both murine PLNs (152) and PPs (153) HEVs. These studies have identified several known and unknown genes that are expressed in HEVs, but not in flat endothelial cells. Human tonsil HEVs have been compared to other human endothelial cells by subtractive hybridization and similar molecular techniques (86,154,155). This work has identified several HEV-specific surface molecules, enzymes, molecules involved in sulfate metabolism, and a nuclear transcription factor, NF-HEV, which is

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much more abundantly expressed in HEVs compared to other vascular beds (156). It remains to be determined whether and to what extent NF-HEV is necessary and/or sufficient to induce or regulate HEV function or phenotype.

8.3. Multi-Photon Microscopy Intravital microscopy has been useful to dissect the multi-step adhesion cascades for lymphocyte homing via HEVs (43). However, traditional epifluorescence-based intravital microscopy cannot generate three-dimensional images, which restricts its usefulnes for studies of post-adhesion events, such as diapedesis or migration within the parenchyma. Multi-photon microscopy (157) is beginning to revolutionize this field by allowing investigators to generate optical section deep within intact lymphoid tissues (158). Recent studies have analyzed the behavior of lymphocytes in intact, freshly excised LNs (159– 161); and, more recently, in intravital settings using anesthetized mice (23,162).

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5 The Use of Proteomics to Map Phenotypic Heterogeneity of the Endothelium Johanna Lahdenranta, Wadih Arap, and Renata Pasqualini The University of Texas, M.D.Anderson Cancer Center, Houston, Texas, U.S.A.

1. INTRODUCTION The inner lining of the vasculature consists of a heterogeneous population of endothelial cells. Phenotypes of these cells vary between different organs, between different parts of the vasculature in a given organ, and even between neighboring endothelial cells of the same organ and the same blood vessel type. Every single endothelial cell of the body is subjected to a seemingly infinite array of signals, including soluble factors, such as growth factors and chemokines, cell-cell and cell-basement membrane interactions, and other variables, such as pH, pO2, sheer stress from blood flow, stretch, and temperature, to name a few. All these variables in the endothelial cell microenvironment will influence the phenotype—thus function—of the cell, to the extent that its predetermined genetic makeup allows. Together, these diverse phenotypes (structural and functional) lead to vascular heterogeneity (e.g., at the level of the organ, tissue, and blood vessel). Hopefully, this phenomenon will become increasingly recognized in the clinical practice of medicine. Structural and functional heterogeneity of the endothelium has been a subject of study for decades, but only more recently has the focus of studies in endothelial cell biology shifted to the molecular heterogeneity of the endothelium. The exploration of the molecular diversity of blood vessels is a rapidly expanding research area that is driven by the vast potential of discoveries in molecular heterogeneity to contribute to the development of targeted diagnostics and therapeutics. It is now recognized that a complex system of ligand-receptor pairs exists within the vasculature of tissues. The expression levels and activation states of these addresses are modulated in blood vessels during tumor progression, and can also be altered in the context of other pathological conditions involving abnormal blood vessel development and function, such as retinopathies, inflammation, and atherosclerosis.

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Many therapeutic targets may be expressed in very restricted—but highly specific and accessible—locations in the vascular endothelium. High-throughput DNA sequencing or gene arrays are typically carried out on endothelial cells that have been removed from their tissue of origin, and in neglecting the anatomical and functional context of the endothelium, may readily overlook potential targets for intervention. Moreover, such approaches yield an immense amount of data, adding to the difficulty in interpreting all the information in a meaningful way. Instead, identification of vascular bed specific ligand-receptor pairs and knowledge about their cellular distribution and accessibility will be requisite for the development of endothelium-targeted therapies. Proteomics may be defined as the systematic analysis of the proteins in biological samples that aims to document the overall distribution of proteins in cells, identify and characterize individual proteins of interest, and ultimately to elucidate their relationships and functional roles. Vascular proteomics is the molecular phenotyping of cells forming blood vessels at the protein-protein interaction level. Exploiting the molecular diversity of cell surface receptors expressed in the human endothelium may lead to a ligandreceptor-based molecular map of the blood vessels in the body, a so-called “vascular map.” Standard proteomic tools—two-dimensional protein gels combined with mass spectrometry—have uncovered a large repertoire of differentially expressed proteins on endothelial cells. Two-dimensional protein gels have been used to separate out several thousand proteins from different endothelial cell lines of differentially treated/stimulated endothelial cells. Mass spectrometry has then been employed to identify the differentially expressed proteins. In our laboratory, we have been developing integrated, combinatorial library platform technologies whose goal is to enable the identification, validation, and prioritization of functional molecular targets in human blood vessels. This methodology will allow drug development based on targeting the differential protein expression in the vasculature associated with normal tissues or diseases with an angiogenesis component. These include cancer, arthritis, diabetes, and cardiovascular diseases. Our long-term goal is to translate a functional map of molecular targets and biomarkers into clinical applications.

2. LARGE-SCALE FUNCTIONAL VASCULAR PROTEOMICS A major goal in drug development has long been to develop technology for targeting therapeutics more effectively to their intended disease site and to improve their therapeutic index by limiting the systemic exposure of other tissues to untoward or toxic effects. The methods described here have two main applications. First, they may lead to identification of vascular targeting ligands. Second, they may enable the construction of a molecular map of human vascular receptors. In theory, targeted delivery of drugs, liposomes, peptide sequences, gene therapy vectors, and biological therapies can be achieved in clinical applications. Ultimately, it may be possible to guide imaging or therapeutic compounds to the target site in real clinical situations. In the future, the determination of molecular profiles of blood vessels in specific conditions may also lead to the discovery of disease-specific vascular targets. Early identification of targets, optimized regimens tailored to the molecular profile of individual patients, and

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identification of new vascular addresses may provide a rationale for revisiting or salvaging otherwise ineffective or toxic drug candidates. Below, we review several phage display targeting strategies that may enable the construction of a molecular map outlining vascular diversity in each organ, tissue, or pathological condition.

3. PHAGE DISPLAY TARGETING STRATEGIES Phage display technology allows presentation of large peptide and protein libraries on the surface of filamentous phage permitting the selection of peptides and proteins, including antibodies, with high affinity and specificity to almost any target. This technology has had a major influence on the work and discoveries made in the fields of immunology, cell biology, drug discovery, and pharmacology. The power of phage display lies in the ability to propagate selected peptide/protein ligands through multiple rounds of selection and the direct link of the phenotype of the antigen/receptor binding ligand to the genotype of the phage particle presenting the ligand. Phage display technology was first introduced as an expression vector (“fusion phage”) capable of presenting a foreign amino acid sequence accessible to binding antibody (1). Since then, large numbers of phage displayed peptide and protein libraries have been constructed (2–4, reviewed in Ref. 5), leading to various techniques for screening such libraries. Peptide display technology has since then been applied to a wide range of protein interaction studies with purified/recombinant proteins, cells, and intact tissues in situ as well as in vivo. A vast body of work has been done using phage displayed antibody libraries for diagnostic and therapeutic applications (reviewed in Refs. 5 and 6). Phage display involves genetically manipulating bacteriophage so that peptides or antibodies can be expressed on their surface. Random peptide libraries consist of large random collection of peptides, displayed as recombinant proteins on the surface of a filamentous bacteriophage. Random peptide libraries usually contain up to 109 individual phage clones. Here, we review the recent progress that has been made in using phage displayed random peptide libraries as a proteomics tool to map heterogeneity of the endothelium.

3.1. In Vitro Targeting 3.1.1. Cell-Free Screening on Isolated Receptors Phage display of random peptide libraries is a powerful system for obtaining small peptide ligands for virtually any protein of interest. A high proportion of isolated peptide ligands interact with the natural binding site of the target protein acting as antagonists or agonists of the natural protein functions (reviewed in Ref. 7). This is likely due to the hydrophilic nature of the protein surface, which itself has a role in prevention of

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unspecific interactions with other molecules. However, the binding sites of proteins will generally allow water molecules to be displaced by the binding of specific ligands, such as selected peptides (8). It is a common observation that the binding motif of a targeting peptide is a tripeptide motif appearing several times in different sequence contexts. A motif consisting of three amino acids seems to provide the minimal framework for structural formation and protein-protein interaction (9). Examples of tripeptide recognition units and receptor binding ligand motifs include RGD, LDV, and LLG to integrins (10,11), GFE to membrane dipeptidase (12,13) and NGR to CD13/aminopeptidase N (APN) (14). Considerable progress has been made in the construction of phage display random peptide libraries and in screening methods. Ligands can be selected and isolated by “biopanning,” a process in which phage that bind to a target molecule are eluted and amplified in a host bacteria. Besides proteins, peptides affecting biologically significant protein-DNA interactions (15), peptides binding to carbohydrates (16,17), carbon nanotubes (18), and small chemical compounds like taxol (19) have been isolated from phage display random peptide libraries. In general, the affinity selection of ligands from a phage display random peptide libraries involves the following fundamental steps: (i) preparation of a primary library or amplification of an existing library, (ii) exposure of the phage particles to a target (immobilized protein/cell surface protein/vascular endothelium) for which specific ligands are planned to be identified, (iii) removal of nonspecific binders (washing/perfusion), (iv) recovery of the target bound phage by elution or direct bacterial infection and amplification of the recovered phage, (v) repetition of the steps (i)–(iv), usually three to six rounds, until an enriched population of binders is obtained, and finally, (vi) sequencing the peptide inserts of the enriched phage clones. Enriched peptide inserts are then analyzed, and desired peptides can be synthesized as recombinant or synthetic peptides for further analysis of the ligand-target interaction. Phage displayed random peptide libraries were used early on in the mid-1980s to map antigen recognition sites of antibodies (1). However, such an approach has not been employed to probe antibodies associated with cancer. Cancer patients can mount a humoral immune response (i.e., make antibodies) against vascular receptors that are also found in or presented by the nonmalignant cells of tumor blood vessels. We have developed a phage display-based screening to select peptide sequences recognized by the repertoire of circulating tumor-associated antibodies. We isolated peptides recognized by antibodies purified from serum of cancer patients. Consensus peptide motifs showed marked selective binding to circulating antibodies from cancer patients over control antibodies from blood donors. We next validated such motifs by showing that serum reactivity to the peptide can be specifically linked to disease progression and to patient survival. Finally, we isolated a corresponding tumor antigen eliciting the immune response. These results show that it is possible to identify tumor antigens by fingerprinting the pool of antibodies from the serum of cancer patients. Exploiting the differential humoral response to cancer through such approach may lead to the discovery of new molecular addresses (20). In addition, targeting tumor antigens may lead to the development of improved vaccines against tumors.

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3.1.2. Screening the Molecular Diversity of Cell Surfaces Identification of the endothelial cell surface receptor fingerprint is required for the development of vascular targeted therapies. Several features of the biology of cell surface receptors point to rationality of receptor-ligand identification for intact receptor molecules embedded in cell membranes instead of isolated receptors. As opposed to purified receptors, membrane-bound proteins are more likely to preserve their active conformation, which can be lost upon purification and immobilization once proteins are removed from their natural environment on the cell membrane. In addition, many cell surface receptors are active as homodimers or heterodimers, whose formation may require the cell membrane environment; these interactions further contribute to the ligand specificities of some receptors. Combinatorial approaches for probing the molecular heterogeneity of cell surfaces allow the identification of cell membrane ligands in an unbiased functional assay and without any predetermined notions of the cell surface receptor repertoire; thus, unknown receptors can be targeted. Nonetheless, the great complexity of cell surface molecules still presents a challenge for the isolation of highly specific ligands for a given cell population. In recent years, a number of successful cell biopannings done with phage display have been reported. Examples include cells expressing the urokinase receptor and the melanocortin receptor, fibroblasts and myoblasts, endothelial cells, neutrophils, T cells, head and neck carcinoma cells, and others (21). This relative success notwithstanding, cell surface selection of phage libraries has been plagued by technical difficulties. First, a high number of nonbinder and nonspecific binder clones are recovered when phage libraries are incubated with cell suspensions or monolayers. Moreover, removal of the background by repeated washes is both labor-intensive and inefficient. Finally, cells and potential ligands are frequently lost during the many washing steps required. We have developed a new approach for the screening cell surface-binding peptides from phage libraries. To circumvent some of the practical difficulties in probing the cell surface, i.e., the recovery of nonspecific clones and the loss of cells and subsequent specific phage clones, we developed a method called Biopanning and Rapid Analysis of Selective Interactive Ligands (termed BRASIL) for probing the cell surface with phage display libraries (22). The BRASIL method is based on differential centrifugation in which cells of interest incubated with a phage library in an aqueous upper phase are centrifuged through an organic phase separating the unbound phage from the specific phage-cell complex. The BRASIL method is an efficient and convenient technique for the selection of phage that binds specifically to a given cell surface. Since multiple samples and several subtraction rounds can be done in a relatively short amount of time, applications for high-throughput screenings are apparent. We have recently performed a screening of the NCI 60-cell panel using the BRASIL method creating a ligand “fingerprint” for the NCI 60-cell panel demonstrating the applicability of the BRASIL method for high-throughput analysis (Bover et al., in progress). Furthermore, the BRASIL method is also more sensitive and more specific than phage selection techniques relying on washing of the cells, representing a significant improvement over conventional cell-panning methods. The use of the BRASIL method is not limited to random peptide

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libraries or mammalian cells, but can be used to screen antibody fragment displaying phage libraries and as well as other cell types. Since our longstanding interest has been targeting the vascular endothelium, we screened a phage display random peptide library on activated, VEGF165-stimulated endothelial cells after a library subtraction step with quiescent endothelial cells (22). We subsequently isolated a peptide ligand for VEGF receptor. This VEGF-receptor ligand appears to be a chimera between overlapping binding sites on different VEGF-B isoforms, since part of it resembles a neuropilin-1-binding site found in VEGF-B167 and a part of it resembles a neuropilin-1-binding epitope of VEGF-B186 (23). Our chimeric peptide ligand interacts specifically with VEGF receptors in a pattern consistent with VEGF-B-type ligands (24) as confirmed by binding assays with individual phage on a panel of purified targets. We further examined the ability of the synthetic VEGF-receptor ligand to block phage binding to VEGF receptors in vitro and found that the isolated peptide ligand is about 100-fold more efficient in blocking phage binding to VEGFR-1 than to neuropilin-1. Because of the observed differential interaction of the chimeric peptide ligand with its receptors, it is tempting to speculate that there are differences in the number of peptide-binding sites on VEGFR-1 and neuropilin-1, or alternatively in the affinity of the interaction at each binding site. The isolation and further elucidation of vascular receptor-ligand interaction attest to the belief that vascular targets can be found on endothelial cell membranes in vitro. Important application for the BRASIL method can be foreseen in both targeting and identification of ligand-receptor pairs in cell populations derived from patient samples. The method may be used in tandem with fluorescence-activated cell sorting of leukemic cells obtained from bone marrow aspirates from patients or even with circulating endothelial progenitor cells from periferal blood. Tumor- and inflammatory cells from ascites or fine-needle aspirates of solid tumors also seem like ideal clinical material for the identification of novel tumor antigens with the BRASIL method. Moreover, because multiple samples and several selection rounds can be performed in a short amount of time, automation for high-throughput clinical applications is likely to follow.

3.2. In Vivo Targeting 3.2.1. Vascular Targeting Technology We have developed a technology for the identification of protein-protein interactions specific to a given site and functional status of vascular endothelium. Vascular targeting technology is used to identify molecular differences in blood vessels of different organs and tissues, as well as differences between normal blood vessels and angiogenic blood vessels (12,25–27), and is based on the isolation and identification of peptides from random displayed phage display libraries homing to specific vascular beds after an intravenous administration of the library. The ability of individual peptides to target a tissue can also be analyzed by this method (12,25,26). This work has uncovered a ligandreceptor-based molecular signature of the vasculature that allows both tissue-specific targeting of normal blood vessels as well as targeting of angiogenic blood vessels in tumors and other pathological conditions.

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We have been using animal models of human cancer to discover addresses that are present in tumor blood vessels but not in blood vessels of normal tissues. Based on our previous work, we have found vascular receptors in tumor-bearing mice, and have been able to show that they are also present in human tumors by staining human tissues with antibodies against the molecules (14,25,28). Thus, it is possible to use animal models to find molecular markers of human blood vessels. However, it is unknown whether targeted delivery will always be achieved in humans by using mouse-derived probes. Extrapolation of the results from mouse experiments to human biology requires that the molecules of interest be expressed and regulated similarly in both species. It has recently become evident that this is not always the case, offering one likely explanation for the difficulties translating information derived from mouse models into clinical applications. Several examples of cross-species variation of gene expression patterns within the vascular network have recently surfaced. For example, the prostate-specific membrane antigen (PSMA) shows notably different expression pattern in human and mice. In prostate PSMA in human is expressed; in mouse on the other hand, the expression of PSMA is limited to the brain and kidney (29). Additionally, PSMA is an endothelial cell marker of human tumor blood vessels (30), whereas mouse tumor blood vessels do not have a detectable endothelial expression of PSMA (W.D.Heston, personal communication). Another example of cross-species variation is the TEM7 gene, which is highly and selectively expressed in the endothelium of human colorectal adenomas (31). In mouse, TEM7 gene is expressed in Purkinje cells of the cerebellum, while the tumor blood vessels show no mTEM7 expression (32). There are also species-specific differences in the induction of protein expression by cytokines. For example, tumor necrosis factor-α (TNF-α) and oncostatin M function cooperatively to induce vascular expression of P- and E-selectin in mice, but diverge significantly in their effects on expression of P- and E-selectin in humans or nonhuman primates (33). These prominent species-specific differences in protein expression patterns and ligand-receptor accessibility prompt us to carefully evaluate the information obtained from animal studies before directly applying it to clinical studies. Clearly, the construction of a human vascular map will be of essence in the successful translation of vascular targeting into clinical practice.

3.2.2. Vascular Addresses in Blood Vessels Since 1996, when in vivo phage display methodology was first described (26), numerous murine tissue-specific endothelial cells markers and their peptide ligands have been identified this way by our laboratory and others (reviewed in Refs. 21 and 34). These include peptide ligands targeting brain and kidney (26), lung, skin, pancreas, intestine, uterus, adrenal gland and retina (12), muscle (35), prostate (36), normal and malignant breast tissue (37), lymph nodes (38,70), and placenta (39). In vivo phage display approach has also revealed a vascular address system in tumor blood vessels (14,25,27,28,40–43) as well as in tumor lymphatic vessels (44). Furthermore, it has been possible to identify unique molecular signatures by in vivo phage display that are specifically expressed at different stages of tumor progression from a highly proliferative and angiogenic dysplastic lesion to invasive phase in transgenic mouse models (42,43). By using transgenic mouse models for multistage tumorigenesis involving the pancreatic

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islet of Langerhans, peptides discriminating between the vasculature of the premalignant angiogenic islets and fully developed tumors were identified (43). A similar study has also been carried out with a transgenic model of squamous cell carcinoma, further confirming the findings of a tumor stage-specific vascular signature (42). Peptide ligands homing to atherosclerotic regions of blood vessel walls have been identified by using in vivo phage display in mice deficient in low-density lipoprotein (45). For isolation of peptide ligands to receptors in human synovium vasculature, in vivo phage display has been used in conjunction with mice transplanted with human synovium tissue (46). Since the blood vessels from human synovium grafts form functional anastomosis with murine subdermal vessels and support the adhesion and extravasation of human leukocytes into the grafts (47), it is likely that the peptide ligands isolated from human synovium xenografts target human vasculature supporting human tissue (46). We have recently taken the first steps to construct the human molecular vascular map (48). A patient with Waldenström macroglobulinemia, who after massive intracranial bleeding remained comatose with progressive and irreversible loss of brainstem function until meeting the formal criteria for brain-based determination of death (49), received an intravenous infusion of a CX7C (C=cysteine, X=any amino acid) phage random peptide library. In order to recover phage from various tissues, samples were obtained from bone marrow, prostate, liver, fat-tissue, skeletal muscle, and skin. Phage isolated from each tissue were processed for sequencing of the peptide inserts. To analyze the distribution of inserts from the random peptide library, we designed a high-throughput pattern recognition software for the analysis of short amino acid residue sequences. This analysis was applied for phage recovered from each target tissue and for the unselected CX7C random phage display peptide library. Briefly, we compared the relative frequencies of every tripeptide motif from each target tissue (47, 160 tripeptide motifs in total) to those of the motifs from the

unselected library to test for randomness of distribution. Comparisons of the motiffrequencies in a given organ relative to those frequencies in the unselected libraryshowed a nonrandom nature of the peptide distribution; such a bias is remarkablegiven that only a single round of in vivo screening was performed. Of the tripeptide motifs recovered from tissues, some were preferentially found in a single site, whereas others were recovered from multiple sites. This indicates that some of the recovered peptides home in a tissue-specific manner binding to differentially expressed endothelial cell markers in a given tissue, whereas others bind to ubiquitous endothelial cell surface molecules. Further analysis of the original phage peptide inserts revealed four to six amino acid residue motifs that were shared among multiple peptides isolated from a given organ. Each of these motifs were searched for similarities to known proteins in online databases, and found that some of the enriched peptide motifs appeared within known human proteins. Since our screening method isolates ligands for differentially expressed vascular receptors, our recovered peptide motifs are likely to mimic epitopes present in circulating ligands interacting with endothelial cell surface molecules. These circulating ligands may be either secreted proteins or surface receptors present on circulating cells interacting with the target tissue. We were able to identify a panel of candidate human proteins potentially mimicked by selected peptide motifs. For

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example, one of the peptide motifs identified from the bone marrow is contained within bone morphogenetic protein-3B, which is a growth factor known to regulate bone development (50). It is reasonable to expect that the isolated peptide ligand mimics this protein. Similarly, we also identified interleukin 11 (IL-11) as a ligand mimicked by a peptide specifically enriched in the prostate tissue. Interleukin 11 has been previously shown to signal through the IL-11 receptors within endothelium and prostate epithelium (51,52). This IL-11 mimicking peptide specifically bound to the endothelium and to the epithelium of normal prostate in phage overlay assay with human tissue sections, on the other hand, IL-11 mimetope failed to bind other organs, such as skin. In contrast, a phage isolated from the skin did not bind to prostate or other tissues; instead, this phage specifically identified blood vessels in the skin. The binding of the IL-11 mimetope peptide to IL-11Rα was also verified in vitro. Validation of the ligand-receptor interaction has confirmed that our high-throughput identification of circulating peptide ligands does provide us with functional information in vascular biology in addition to organ homing ligands useful by themselves for vascular targeting. Additional support for the use of combinatorial screenings in patients (48) for the development of anticancer targeted therapies comes from our studies, where we show the potential of IL11Rα as a target for intervention in human prostate cancer (53). Expression of IL-11Rα was increased in primary and metastatic prostate cancer and its associated blood vessels in a stage-specific manner during disease progression. Furthermore, a peptide guided by the IL-11 mimetope peptide linked to the well-established proapoptotic peptide D(KLAKLAK)2 (54) was specifically targeted and internalized into prostate cancer cells resulting in apoptosis (53). Integration of the isolation of ligands from the in vivo screenings to proteomic strategies to identify the receptors for these ligands has produced an array of tissuespecific vascular receptors. Complementary genetic and biochemical approaches have been used to identify receptors for tissue homing peptides. Membrane dipeptidase on lung endothelium was identified as the receptor for GFE-peptide (13), aminopeptidase N on angiogenic vasculature as the receptor for the tumor homing NGR-peptide (14), aminopeptidase P on both normal and malignant breast tissue as the receptor for CPGPEGAGC-peptide (37) and FcRn/β2-microglobulin as a target for the TPKTSVTpeptide in placenta (39). Moreover, the ligands themselves may be used as either drug discovery leads or for therapeutic modulation of their corresponding receptor(s). Since the early days of mapping addresses in the blood vessels of normal and diseased organs (26), we have now moved to methodologies that enable us to identify more vascular addresses (48) in even more refined time and space restricted expression patterns. In order to proceed from the ligands identified by phage display to receptors in the blood vessels, sophisticated array technology and state-of-the-art proteomics are needed. Array technology allows the simultaneous analysis of thousands of molecular parameters within a single experiment. DNA array technologies evolved to allow smaller sample volumes, more efficient analyses and higher throughput. Since proteins are more complex and diverse biomolecules than nucleic acids, development of similar platforms in the protein field has proved more difficult. We plan to explore current techniques used in the generation and development of protein arrays (55) and their application in proteomics to identify receptors targeted by peptides and antibodies of interest in humans. Ultimately, our goal is to identify a large-scale panel of receptors in human

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blood vessels. In addition, antibodies raised against each target could be arrayed and used to profile the proteins present in, for example, a cell or tissue. Besides offering a way of identifying endothelial cell surface markers accessible to the circulation and providing novel tools for selective vascular targeting, in vivo phage display studies further our understanding of organ and tumor endothelium specificity and define the role that endothelial cell markers play in angiogenesis. It is recognized that a tumor cannot get larger than 1 mm in diameter without new blood vessel formation. The rules of the normal blood vessel formation do not seem to apply to tumor blood vessels and there is a tremendous amount of functional and structural irregularity and molecular heterogeneity in tumor/angiogenic blood vessels when compared with normal blood vessels in the same tissue environment. Some of the vascular markers found in tumor blood vessels are vascular proteases that not only serve as receptors for circulating ligands but also modulate angiogenesis (14,28,41). We have identified by in vivo phage display CD13/amiriopeptidase N (APN) and aminopeptidase A (APA) as functional targets in angiogenic vasculature that may contribute to an important regulatory proangiogenic pathway (14,28). These peptidases that are expressed in both the endothelial and periendothelial cell compartment of angiogenic blood vessels are accessible to circulating ligands. Their enzyme activities regulate the angiogenesis process, since either genetic or biochemical ablation of the activity of these enzymes significantly reduces the formation of new blood vessels in several pathological states, such as cancer and retinopathies. The function of CD13/APN appears to depend on the availability of its substrates, thus its location: in synaptic membranes, it metabolizes enkephalins and endorphins; in the intestinal brush border, it degrades regulatory peptides and scavenges amino acids; in lymphocytes, its activity is associated with mitotic activation, antigen processing, cell adhesion, and migration (reviewed in Ref. 56). Aminopeptidase A also appears to be a molecule that has different functions, according to the organ and time period examined. A broad spectrum of tissues expresses APA (57), but its only well understood role is the conversion of angiotensin II to angiotensin III in the rennin-angiotensin system (58). An intriguing feature of CD13/APN is that its expression and enzymatic activity can be physiologically regulated. Its activity and substrate specificity depend on conformational changes induced by various stimuli, including proliferative signals. Studies using monoclonal antibodies indicate that CD13/APN undergoes regulatory intramolecular alterations that result in exposure of cryptic sites and regulation of enzyme activity (59). The immunoreactivity patterns obtained with cultured cells and tissue sections from kidney, breast, and prostate carcinomas suggest that different CD13/APN forms are expressed in myeloid cells, epithelia, and tumor-associated blood vessels (60). Association with proteins or factors present only in the tumor microenvironment might cause differential reactivity or accessibility to different CD13/APN ligands, such as the tumor vasculature targeting NGR-peptide ligand. This example of CD13/APN clearly demonstrates the importance of utilizing a functional proteomics method, such as in vivo phage display, for the identification of ligands that target molecules with complex regulatory mechanisms and expression patterns in time and space. Following the identification of the CD13/APN peptide ligand homing to angiogenic vasculature, it has been successfully used to target an array of therapies to tumor vasculature ranging from cytotoxic drugs to proapoptotic moieties (see Sec. 3 of this chapter).

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4. TARGETED THERAPIES Many malignant, cardiovascular, and inflammatory diseases have a marked angiogenic component. In cancer, tumor vasculature is a suitable target for intervention because the vascular endothelium is composed of nonmalignant cells that are genetically stable but epigenetically diverse. Cancers appear to put an epigenetic molecular signature on their own blood vessels that targeted probes can use as a homing signal to deliver a drug into the vascular receptors. The biological basis for such molecular signature of the tumor vasculature is still largely unknown. However, peptides selected by homing to blood vessels have been used to guide the delivery of cytotoxic drugs (25), proapoptotic peptides (36,54), metalloprotease inhibitors (41), cytokines (61), genes (62,63, Hajitou et al., in progress), and liposomes (64) to receptors in the angiogenic vasculature showing marked therapeutic efficacy in tumor-bearing mouse models. Tumor targeting peptide ligands can also deliver imaging agents to tumor vasculature (65). Generally, coupling to homing peptides appears to yield a targeted compound with a better therapeutic index than the untargeted parental compound. Clearly, there is also an advantage in the pharmacokinetics of a drug being targeted to the vascular endothelium, which is directly accessible upon intravenous administration, rather than tumor cells. Caveolae has also surfaced as an interesting new target for vascular drug delivery. Caveolae is specialized distinct plasma membrane microdomains and the associated noncoated plasmalemmal vesicles (66,67) on several cell types, including the cells of the continuous microvascular endothelium. Many proteins have been found enriched in caveolae, including cell surface receptors such as platelet-derived growth factor receptors, epithelial growth factor receptors, basic fibroblast growth factor receptor, and endothelin receptors (reviewed in Ref. 68). Even though the molecular differences of caveolae between endothelial cells derived from different tissues remain unknown, recent data suggest that caveolae can contain tissue-specific cell surface molecules. A lung endothelial cell specific antibody targeting the caveolae has been generated using an antibody and subfractionation strategy. Upon intravenous administration, this antilung caveolae antibody localized to the microvascular endothelium of rat lungs. In addition, targeting caveolae increased trasendothelial transport of the anticaveolae antibody (69). These preclinical data suggest that targeting the vasculature of normal and pathological tissues may be the basis of a new pharmacology for the treatment of malignant and inflammatory diseases by delivering therapeutic agents to blood vessels.

5. CONCLUSION A major goal in drug development has long been to develop technology for targeting therapeutics more effectively to their intended disease site and to improve their therapeutic index by limiting the systemic exposure of other tissues to untoward or toxic effects. The technologies reviewed here have two main applications. First, they may identify vascular targeting ligands. Second, they may enable the construction of a molecular map of human vascular receptors. In theory, targeted delivery of drugs,

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liposomes, peptide sequences, gene therapy vectors, and biological therapies can be achieved in clinical applications. Ultimately, it may be possible to guide imaging or therapeutic compounds to the target site in real clinical situations. In the future, the determination of molecular profiles of blood vessels in specific conditions may also lead to vascular targets. Early identification of targets, optimized regimens tailored to molecular profile of individual patients, and identification of new vascular addresses may lead to revisiting or salvaging of ineffective or toxic drug candidates.

ACKNOWLEDGMENTS Our work has been funded in part by grants from NIH (CA088106, CA078512, CA090270, CA082976, CA051134 and R14101-7200003 to R.P.; CA0103042, CA090270, CA090810, DR06783, CA103030 and CA103086 to W.A.), Juvenile Diabetes Research Foundation (to W.A.), and awards from the Gilson-Longenbaugh Foundation and Angelworks (to R.P. and W.A.). J.L. received fellowships from the Susan G.Komen Breast Cancer Foundation and the Cancer Society of Finland.

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57. Li L, Wu Q, Wang J, Bucy RP, Cooper MD. Widespread tissue distribution of amino-peptidase A, an evolutionarily conserved ectoenzyme recognized by the BP-1 antibody. Tissue Antigens 1993; 42:488–496. 58. Jackson EK. Renin and angiotensin. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics. Hardman JG, Limbird LE, Goodman Gilman A, eds. McGraw-Hill Medical Publishing Division, 2001:809–841. 59. Xu Y, Wellner D, Scheinberg DA. Cryptic and regulatory epitopes in CD13/amino-peptidase N. Exp Hematol 1997; 25:521–529. 60. Curnis F, Arrigoni G, Sacchi A, Fischetti L, Arap W, Pasqualini R, Corti A. Differential binding of drugs containing the NGR motif to CD13 isoforms in tumor vessels, epithelia, and myeloid cells. Cancer Res 2002; 62:867–874. 61. Curnis F, Sacchi A, Borgna L, Magni F, Gasparri A, Corti A. Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nat Biotechnol 2000; 18:1185–1190. 62. Grifman M, Trepel M, Speece P, Gilbert LB, Arap W, Pasqualini R, Weitzman MD. Incorporation of tumor-targeting peptides into recombinant adeno-associated virus cap-sids. Mol Ther 2001; 3:964–975. 63. Muller OJ, Kaul F, Weitzman MD, Pasqualini R, Arap W, Kleinschmidt JA, Trepel M. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotechnol 2003; 21:1040–1046. 64. Pastorino F, Brignole C, Marimpietri D, Cilli M, Gambini C, Ribatti D, Longhi R, Allen TM, Corti A, Ponzoni M. Vascular damage and anti-angiogenic effects of tumor vessel-targeted liposomal chemotherapy. Cancer Res 2003; 63:7400–7409. 65. Hong FD, Clayman GL. Isolation of a peptide for targeted drug delivery into human head and neck solid tumors. Cancer Res 2000; 60:6551–6556. 66. Palade GE. The fine structure of blood capillaries. J Appl Physiol 1953; 24:1424. 67. Peters KR, Carley WW, Palade GE. Endothelial plasmalemmal vesicles have a characteristic striped bipolar surface structure. J Cell Biol 1985; 101:2233–2238. 68. Zajchowski LD, Robbins SM. Lipid rafts and little caves. Compartmentalized signalling in membrane microdomains. Eur J Biochem 2002; 269:737–752. 69. McIntosh DP, Tan XY, Oh P, Schnitzer JE. Targeting endothelium and its dynamic caveolae for tissue-specific transcytosis in vivo: a pathway to overcome cell barriers to drug and gene delivery. Proc Natl Acad Sci USA 2002; 99:1996–2001. 70. Kolonin MG, Saha PK, Chan L, Pasqualini R, Avap W. Reveusal of obesity by targeted ablation of adipose tissue. Nat Med 2004; 10:625–632.

6 The Use of Genomics to Map Phenotypic Heterogeneity of the Endothelium Mary E.Gerritsen, Stuart Hwang, Constance Zlot, James Tomlinson, and Michael Ziman Department of Vascular Biology, Millennium Pharmaceuticals, Inc., South San Francisco, California, U.S.A.

1. INTRODUCTION The vascular endothelium lines the blood vessels of the body, and in man, the estimated number of individual cells comprising this line is somewhere in the order of 1–6×1013. The magnitude of the surface area of this lining is also impressive, in the order of 719m2 (1). Although scientists initially thought this cell layer was essentially an inert barrier between the blood and the tissue, this extensive “organ” is now known to carry out a diverse array of specialized functions. Moreover, these functions vary markedly from one vascular bed to another. Quiescent endothelium presents a non thrombogenic surface and plays an important role in the regulation of the transit of the solutes, proteins, and cellular components of the blood. Perturbation of the cells by various stimuli, be they physical, biochemical, or mechanical, can result in the marked shift in the endothelial phenotype; a transition achieved by numerous mechanisms including changes in gene expression, protein expression, and protein phosphorylation. It is important to recognize the fact that there is no “generic endothelial cell,” there are actually many different populations of cells with remarkable heterogeneity in structure and biology. At an ultrastructural level, early electron microscopic studies demonstrated remarkable differences in the anatomy of endothelial cells (2,3). As of Majno (2) so aptly stated “Electron microscopy has revealed that there are almost as many varieties of ‘capillaries’ as there are organs and tissues.” One of the more obvious differences is in the continuity of the endothelial cells of the capillaries, leading to the definition of several broad categories of endothelial cells: continuous, discontinuous (sinusoidal), and fenestrated. Endothelium of the continuous type is the most common, and can be found in the walls of arterioles, capillaries, and venules of skeletal, smooth, and cardiac muscle, the mesentery, skin, connective tissue, lung, brain and eye, as well as lining the major conduit vessels (large arteries, veins). These endothelial cells are characterized by

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occluding (tight) junctions. However, although structurally similar, continuous endothelia exhibit remarkable differences in their relative permeability to different solutes. Fenestrated endothelium is found in the exchange vessels of secretory and excretory organs (i.e., exocrine and endocrine glands), the gastric mucosa, the kidneys (glomerulus and peritubular capillaries, synovium, and choroid plexus). Numerous small “windows” (i.e., small transcellular openings ranging from 50 to 80 nm in diameter, as the name implies) characterize fenestrated endothelia. At the ultrastructural level, differences in the fine structure of the fenestrae are revealed. In some organs, the fenestrae appear open; in others, the fenestrae are “closed” by thin membranous diaphragm like structures. Discontinuous endothelia may be thought of as an extreme form of “fenestrated endothelium” and can be found in the sinusoids of the liver, spleen, and bone marrow. These cells are found in thin-walled blood vessels with irregular outlines and calibers, and highly fenestrated structures. In the liver, the fenestrae are often clustered to form sieve plates, and none of the fenestrae are closed with diaphragms. There are additional endothelial phenotypes; for example, another highly specialized endothelial structure is found in the high endothelial venules of peripheral lymph nodes. These cells are characterized by a cuboidal morphology with particularly well-developed Golgi complex, rough endoplasmic reticulum, and poly-ribosomes. The cell-cell junctions are generally discontinuous with few and then poorly organized tight junctions where they do occur (reviewed in Ref. 4). At a functional level, heterogeneity in endothelial cell biology is now widely recognized. Different vascular beds demonstrate a wide range of solute and protein permeabilities; although some of this is dictated by the aforementioned structural features of the endothelium regional differences in the expression of plasmalemmal vesicles, different receptors, transporters, and basement membrane also contribute (5,6). Exquisite differences in the composition of the endothelial glycocalyx have been revealed by elegant lectin binding studies (7–11). Endothelial cells at different vascular sites also appear to have specialized responses to specific stimuli such as cytokines, shear forces, or growth factors adding an additional layer of complexity to the concept of endothelial heterogeneity. Indeed, the expression of unique endothelial membrane proteins under basal or perturbed conditions provides the opportunity for vascular targeting, i.e., the ability to target antibodies or drugs to specific vascular beds. Vascular targeting offers exciting new therapeutic opportunities for the treatment of cancer, chronic inflammatory diseases, and other disorders (12).

2. GENOMICS AND ENDOTHEUAL DIVERSITY What dictates the phenotype of an endothelial cell? At least part of the heterogeneity derives from interactions of the endothelium with the organ or tissue environment, through soluble factors, cell-cell interactions, or cell-matrix interactions. An interesting question, now addressable at a whole genome level, is whether or not there are differences in the “genetics” of endothelial cells. Various approaches to address this issue are available; laser-capture microdissection of endothelial cells in situ, or isolation and culture of the cells from different organs. These cells can be used to generate RNA

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samples that can be further used for transcriptional analysis. Another approach is to map proteomic mapping proteomic differences using phage display. When endothelial cells are removed from the tissue and grown in culture, some of the differentiated features appear to be lost. For example, endothelial cells derived from the cerebral cortex lose many of their blood-brain barrier properties (13). Endothelial cells from endocrine organs lose their fenestrations (14), and endothelial cells from peripheral lymphatic tissues lose their high endothelial morphology (15). However, other differentiated features appear to be well preserved. The endothelial markers VE-cadherin, CD31 and intercellular adhesion molecule (ICAM)-2 are expressed in virtually all endothelial cells in vivo, and are expressed in virtually all properly identified cultured endothelial cells (16). Like their in vivo counterparts, cultured endothelial cells also respond to cytokines such as interleukin (IL)-1, lipo-polysaccharide (LPS), and tumor necrosis factor (TNF)α, upregulating the expression of adhesion molecules such as vascular cell adhesion molecule (VCAM)-1 and ICAM-1. Various technologies have been used to identify vascular bed specific antigens or markers, including differential lectin binding, raising antibodies to endothelial cells or membranes from specific vascular beds, suppression subtractive hybridization, and differential display. Podograbinska et al. (17) utilized Affymetrix oligonucleotide arrays to probe the molecular features of cultured human lymphatic endothelial cells (LEC) and what these authors defined as blood microvascular endothelial cells (BECs) (note, these might actually be better defined as “nonlymphatic” microvascular endothelial cells to avoid confusion with blood-outgrowth endothelial cells or circulating progenitor cells). These authors purified lymphatic endothelial cells using a magnetic bead approach and an antibody to a lymphatic endothelial cell marker (lymphatic vessel hyaluronic receptor, LYVE-1). After depletion of the LEC, BECs were isolated from the remaining cells using the vascular endothelial cell marker, CD31. Although it is not clear from the methods of this study whether different isolates of LEC and BEC were profiled, or if the data are derived from a single comparison, these authors reported remarkable differences in the molecular signature of LECs vs. BECs. The LECs expressed high levels of genes implicated in protein metabolism, sorting and trafficking, including proteins of the SNARE family, rab GTPases, and sec-related proteins. Shusta et al. (18) used suppressive subtractive hybridization to evaluate differential gene expression within the human brain microvasculature. These authors used isolated microvessels, which are comprised of endothelial cells, pericytes, smooth muscle cells and astrocyte foot processes; thus, the genes identified in this approach are not from purified endothelial cells. The human brain capillary tester cDNA was subtracted with driver cDNA obtained from human liver and kidney RNA. This study identified 20 known genes, 12 genes encoding proteins of unknown function and five novel genes. Several genes participating in the regulation of the endothelial tight junction and cytoskeleton were identified, including claudin 5; additional genes encoding for nutrient or peptide transporters were also identified including ATPase subunit α2, a monomer in a family of subunits that form heterodimers to catalzye the active transport of Na+ and K+ across the cell membrane. Kallman et al. (19) compared the gene expression of primary human cerebral endothelial cells (HCEC) (predominantly microvascular in origin) with human umbilical vein endothelial cells (HUVEC) using a cDNA array of 375 genes. These authors identified 35 genes selectively expressed in the HCEC and 20 in the

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HUVEC under basal conditions. Cerebral endothelial specific genes (basal conditions) included VEGF, insulin growth factor (IGF) binding proteins 1, 5, and 6, follistatin, decorin, a number of chemokines (macrophage migration inhibitory factor (MIF-1), epithelial-derived neutrophil-activating peptide 78 (ENA-78), growth regulated protein (GRO)-α, eotaxin), c-kit ligand, macrophage colony stimulating factor (M-CSF), pleiotrophin, interferon (IFN) -γ receptor 1, c-kit, oncostatin M, platelet derived growth factor receptor subunits α and β, erythroblastic leukemia viral homolog 1 (erbB1), acidic and basic fibroblast growth factor (FGF), FGF5, integrins α1 and (35, IL-1β, IL-6, glial derived neurotrophic factor (GFRα3), semaphorin F, brain derived neurotrophic factor (BDNF), nerve growth factor (NGF) receptor, tissue inhibitor of metalloproteinase (TIMP)-3 transforming growth factor (TGF) β2, activin like kinase (ALK) 3, osteoprotegerin, and HVEM (herpes virus entry mediator). HUVEC specific genes included cadherin 8, CD31, cadherin 5 (VE-Cadherin), ICAM-2, endothelial specific tyrosine kinase receptor 2 (Tie-2), angiopoietin-2 (Ang-2) kinase insert domain receptor (KDR), interleukin 1 receptor-like 1 precursor (ST2 protein), CXC chemokine receptor 4 (CXCR4), ephrin (Eph) A4, EphB2, integrin β8, midkine, endothelial nitric oxide synthase (eNOS), the chemokine orphan receptor RDC-1, matrix metalloproteinase (MMP) 10, MMP13, MMP8, and the TNF superfamily members, Tumor necrosis factor receptor superfamily member 11A precursor (Receptor activator of NF; RANK) and Tumor necrosis factor (ligand) superfamily, member 4 (OX40L). The protein expression of several of the genes identified as HCEC specific (VEGF, follistatin, supercoiling factor (SCF), TGFβ2, IL6, BDNF and monocyte chemoattractant protein (MCP) -1) was confirmed by ELISA. Immunoflourescence studies also confirmed the selective expression of decorin in the cerebral microvasculature in situ (compared to umbilical cord). Arap et al. (20) reported the first in vivo screening of a peptide library in a human patient, surveying 47, 160 motifs that localized to different organs. Peptides that home to specific vascular beds were selected after intravenous administration of a phage-display random peptide library, revealing a vascular address system that could allow tissuespecific targeting of normal blood vessels. These authors identified specific peptide homing motifs that were preferentially identified in bone marrow (GGG, GFS), fat (EGG, LSP), skeletal muscle (LVS), prostate (AGG), skin (GRR, GGH, GTV) as well as motifs that were found in multiple organs (GVL, EGR, FGV, FGG, GER, SGT). Although a lung “specific” receptor was isolated from similar phage studies in mice (membrane dipeptidase) (21), the specific receptors for the aforementioned peptides have not yet been elucidated from the human studies. Several groups have generated genetically modified mice using different promoters coupled to reporter genes, and have identified vascular bed specific expression of certain transgenes. Knock-in approaches allow the use of endogenous promoter and locus for a specific gene whereas transgenic approaches use predefined promoter length and a heterologous genomic locus. For example, Koop et al. (22) generated receptor protein tyrosine phosphatase mu (RPTPmu) -LacZ mice knock-in mice that expressed the beta galactosidase (LacZ) reporter gene under the control of the endogenous RPTPmu promoter. These authors found LacZ expression in endothelial cells of arteries and capillaries, but expression was virtually absent in endothelial cells of veins and in the discontinuous endothelial cells of the adult liver and spleen. Aird et al. (23) generated

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transgenic mice using a fragment of the von Willebrand factor (vWF) gene (2182 bp of 5′ flanking sequence, the first exon and first intron) coupled to the LacZ reporter genes. Under the control of this promoter, β-galactosidase expression was detected within endothelial cells of the heart, brain, and skeletal muscle, suggesting that the vWF gene is regulated by vascular bed specific pathways, in response to signals derived from the local milieu. Many other transgenic studies have been carried out with endothelial cell-specific promoters. In virtually every case (with the possible exception of a long fragment of the Tie-2 gene), the promoter was shown to direct expression in a unique and limited subset of endothelial cells.

3. A GENOME-WIDE APPROACH TO EVALUATE HETEROGENEITY OF CULTURED HUMAN ENDOTHELIAL CELLS Recent technological advances have made genome-wide comparisons of the cellular transcriptome feasible and rapid. We have used the Affymetrix oligonucleotide arrays to profile endothelial gene expression in response to various stimuli, and have validated the genes identified by multiple independent methods, demonstrating that this approach is reliable, and robust (24–27). Ideally, obtaining the transcriptome of endothelial cells in situ would be the most desirable. However, although individual cells can be isolated by laser dissection, considerable amplification (and the inherent potential errors of amplification) of the RNA is required to obtain sufficient material for transcriptional profiling. Another approach is to use isolated, cultured endothelial cells from different organs. To determine potential genetic heterogeneity of human endothelial cells, we selected a number of commercially available endothelial cells, all of the so-called “continuous” type. All cell types had been well characterized by the manufacturer (Clonetics); in addition, we confirmed endothelial identity in independent studies assessing the expression of the endothelial marker CD31 and uptake of Di-I-Ac-LDL (>99% purity). Two of the cell preparations were termed microvascular endothelium, and can be considered to contain a mixture of arterial, venous, and capillary endothelial cells (human lung microvessel endothelial cells, HLMVEC and human dermal microvessel endothelial cells, HDMVEC). Two of the cell preparations were derived from large conduit vessels of the arterial tree (human aortic endothelial cells, HAEC and human coronary artery endothelial cells, HCAEC). We also evaluated HUVEC, which, while not necessarily representative of a venous phenotype, are the most widely studied endothelial cell type, facilitating the comparison of our results with the literature. At least two independent cell “lines” (i.e., from different donors) were evaluated of each representative “endothelial cell type”; this approach helps to rule out “individual differences” in gene expression, which could be misinterpreted as heterogeneity per se. To the best of our knowledge, none of the different EC types came from the same donor. To improve the fidelity of the results, all cells were grown under identical conditions, with great care to maintain identical lots of serum, media, plasticware, growth factors, and cytokines. All cells were used at similar “passage” number (5 or 6) and RNA was

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extracted and labeling performed under rigid and identical conditions. A summary of the cell preparation information is provided in Table 1. The profiling experiments were performed on nearly confluent (90–95%) cells that had been incubated in Medium 199 containing 1X ITS (Insulin-transferrin-Selenium-A), 2mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (all components from Invitrogen Corp.; Carlsbad, CA) and 1% fetal bovine serum (FBS, Tissue Culture Biologicals; Tulare, CA) for 18 hr. Use of this “Basal Medium” enables the identification of cytokine or growth factor induced genes without the complication of high background caused by high concentrations of FBS. At the initiation of experiments, the media were removed and fresh Basal Medium added with or without (Basal) addition of TNFα (10 ng/mL) or VEGF (10 ng/mL). The cells were incubated for 4 hr. At the termination of experiments, media were removed

Table 1 Summary of Endothelial Cell Preparations Used in the Affymetrix Array Analysis of Gene Expression Description

Passage

Biowhittaker ID

Lot Number

HUVEC (pool of five individual isolates)a

6

CC-2519

2F0132

HUVEC (pool of five individual isolates)a

6

CC-2519

2F0237

HUVEC (pool of five individual isolates)a

6

CC-2519

2F0332

HDMVEC (single isolate)

5

CC-2543

2F0047

HDMVEC (single isolate)

5

CC-2543

1F0928

HLMVEC (single isolate)

6

CC-2527

1F1554

HLMVEC (single isolate)

6

CC-2527

1F1643

HAEC (single isolate)

5

CC-2535

F1335

HAEC (single isolate)

5

CC-2535

F1340

HCAEC (single isolate)

5

CC-2585

1F2058

HCAEC (single isolate)

5

CC-2585

2F0239

a None of the individual isolates in this sample are present in either of the other two HUVEC lots, thus providing sampling of 15 individual isolates across each HUVEC treatment.

and 15 mL of Tri reagent (Molecular Research Center Inc.; Cincinnati, OH) were added directly to the endothelium in each T75 flask; lysates were removed and stored frozen at −80°C. RNA was subsequently extracted following manufacturer’s instructions. Total RNA isolated this way was further purified using RNeasy Mini kits as described in the product manual (Qiagen Inc., Valencia, CA). DNAse treated total RNA, 5 µg, was

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converted to cRNA and fragmented cRNA hybridized to arrays (U133A) per manufacturer’s suggested protocol (Affymterix, Santa Clara, CA). Data were analyzed with the MASv5 (Affymetrix) and Rosetta Resolver (Rosetta Biosoftware, Bothwell, WA). Array results that met with manufacturer’s (Affymetrix) recommended quality criteria were imported into Rosetta Resolver (28) which uses an additive and multiplicative noise based error model to increase confidence in gene expression measurement and a statistical method for combining replicates (29). Figure 1 shows a ratio-analyzed agglomerative cluster of the transcriptosomes of the five endothelial cell types under basal, TNFα and VEGF stimulated conditions. We used a fold change cutoff of 1.5-fold, and a p-value of 0.05 and included sequences where at least one combined intensity experiment met the aforementioned thresholds. It is clear from this figure that there was considerable variability in the basal expression of the different mRNAs (Fig. 1A), but when the cells were stimulated with either TNFα (Fig. 1B), many of the same genes were up- and downregu-lated in all five types of cultured endothelia. The more highly upregulated, common genes include ephrin A1, interleukin 8, the adhesion molecules ICAM-1, VCAM-1, and E-selectin, the membrane bound chemokine fractalkine, and the anti-apoptosis gene, B Cell CLL/Lymphoma 2 (Bcl-2). Two of the common downregulated genes were CD97 and BDNF. VEGF stimulated responses were somewhat more variable, but there are clear clusters of commonly upand downregulated genes (Fig. 1C). Examples of common upregulated genes include follistatin, bone morphogenic protein 2, E-selectin, CD55, Dual specificity phosphatase 6 (DUSP6), IL-6, IL-8, the VEGF receptor fms like tyrosine kinase 1 (flt-1), stanniocalcin 1, and Ang-2. Common downregulated genes included death associated protein kinase 3, placental growth factor, Bcl-3 and TNF receptor associated factor (TRAF) 5. For both TNFα and VEGF, many, if not all of these aforementioned upregulated genes, have been

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Figure 1 Agglomerative clustering of (A) basal-, (B) TNF-, (C) and VEGFinduced gene expression in different endothelial cells. HUVE human umbilical vein endothelial cells,

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HAEC, human aortic endothelial cells, HLMVEC human lung microvessel endothelial cells, HDMVEC human dermal microvessel endothelial cells, HCAEC human coronary artery endothelial cells, HDMVEC human dermal microvascular endothelial cells. Red: upregulated; green: downregulated; black: not detected or did not meet statistical significance. previously documented to be upregulated at both the protein and RNA level (27,30–48). To identify possible cell-type specific sequence, we applied the following criteria. A given sequence had to increase or decrease more than twofold (log ratio 0.3) at a p-value of <0.05 in one cell type, but in all the other cell types, the p-value had to exceed 0.1. This enabled the identification of putative cell-type specific sequences which were then grouped into one “Cell-specific” bioset; we then reclustered the expression data using only those sequences in the cell-specific bioset. Genes not meeting the thresholding criteria are greyed out. Figure 2 clearly suggests that there may be selective genes expressed by different endothelial cells under basal, TNFα and VEGF stimulated conditions. It is interesting to note that the majority of these genes are “uncharacterized” often defined only as hypothetical “proteins”, genes with names based on homology but little other known information, KIAA sequences, or ESTs; thus follow-up studies on the potential selective expression of these genes will require considerable additional efforts and reagent development. Nonetheless, this in vitro approach, coupled with the breadth and power of transcriptional profiling, offers considerable promise towards the rapid identification of novel endothelial cell-type specific genes.

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Figure 2 Selection of putative celltype specific sequences and agglomerative clustering to illustrate endothelial cell-type specific gene expression. (A), basal, (B), TNF (C), VEGF treated cells. Red: upregulated;

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green: downregulated; grey: not detected or did not meet cutoffs for statistical significance. While this chapter was in preparation, a study by Chi et al. (49) was published using a very similar approach to the one we used. These authors used human DNA microarrays containing 43,000 elements representing 32,275 unique Unigene clusters. These authors analyzed mRNA from 53 different cultured endothelial cells, sampling coronary artery, pulmonary artery, aorta, umbilical artery, iliac artery, microvascular endothelial cells from a number of sources (skin, lung intestine, uterus myometrium, nasal polyps, bladder, and myocardium), and venous endothelial cells from umbilical vein and saphenous vein. In this study, cells were also cultured under consistent conditions, but all cells were exposed to 5% fetal serum, VEGF, bFGF, epidermal growth factor (EGF), hydrocortisone, and ascorbic acid as components of the culture media. These authors found that endothelial cells from different blood vessels and microvascular endothelial cells from different tissues have distinct and characteristic gene expression profiles. Using a Wilcoxon rank-sum test to identify genes with the most consistent different levels of expression between endothelial cells from large vessels vs. microvessels, a hierarchical cluster was generated. This resulted in the observation that, with the exception of the saphenous vein endothelial cells, all large vessel endothelial cells clustered into one branch, separated from all of the microvascular endothelial cells. Some genes differentially expressed by large vessels included genes involved in the biosynthesis and remodeling of extracellular matrix, such as fibronectin, collagen 5α1, collagen 5α2, and osteonectin. Macrovascular endothelial cells also expressed many genes associated with neuronal cells, such as robo-1, neuron navigator 1, and neuron navigator 3, neuroligin, neurogranin, and neuroregulin and its receptor ErbB. Microvascular endothelial cells, on the other hand, expressed a number of genes encoding basement membrane proteins such as laminin, collagen 4α1, collagen 4α2 and collagen 4α binding protein. Several extracellular matrix interacting proteins such as CD36, α1, α4, α9, and β4 integrin were also selectively expressed by microvascular endothelial cells. Microvascular cells also expressed secreted factors involved in the promotion of the survival and differentiation of neuroglial cells such as transforming growth factor a, glial maturation factor γ, stromal cell derived factor 1 and spinal cord derived growth factor. Since this study also sampled several arterial vs. venous endothelial cell populations, it also provided the opportunity to identify gene expression pattern differences between these two populations. Vein-specific genes included EphB4, in agreement with the published literature (50). Venous endothelial specific genes also included smoothened, growth differentiation factor 1, lefty-1 and lefty-2. The arterial specific gene cluster included cell surface proteins such as Notch 4, epithelial V-like antigen (EVA) 1, CD44, Ephrin B1; metabolic enzymes (aldehyde dehydrogenase) and notably, the transcription factor Hey2 (Hairy/Enhancer of split-related basic helix loop helix protein 2). Although this study did not confirm the putative selective gene clusters by a second independent means, they were able to show that Hey2 was capable of activating the expression of arterial specific genes. Putative organ specific microvascular genes were also identified, including sine oculus homobox homolog 3 (SIX3), a homeodomain protein, specific to nasal polyp endothelial cells. Basic FGF, squalene epoxidase, 24-dehydrocholesterol

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reductase, stearoyl-CoA desaturase, fatty acid desaturase, and 3-hydroxy-3methylglutaryl-CoA synthase 1 were selectively expressed in skin endothelial cells. Intestinal microvascular cells specifically expressed biotinase, and lung endothelial cells, phospholipase A2 group XII, an enzyme involved in surfactant secretion. Interestingly, myometrial endothelial cells expressed the calcitonin receptor and gallinin; calcitonin is important for embryo implantation (51) and gallinin is a peptide hormone that stimulates uterine contraction (52). Follow-up studies to validate these array data are now needed to confirm these fascinating observations.

4. SUMMARY AND FUTURE DIRECTIONS The intricate and highly specialized features of the vascular endothelium will continue to enchant investigators for the foreseeable future. With each new technology, a new layer of the “onion” is exposed and the tempo of discovery accelerates. The very recent studies using the new molecular approaches have now suggested a level of endothelial diversity beyond our wildest imagination. However, the majority of these studies have used cell cultures, which, while convenient and rapid, may or may not provide accurate predictions of endothelial genomics in situ. Some, many or all of these apparent differences derived from cell culture studies could reflect a drift in phenotype; alternatively some, many or all may authentic differences. The answer probably lies somewhere between; fortunately many of the observations from cell cultures have translated to the in vivo situation. Moreover, many of the observed differences in endothelial culture requirements, biochemical properties, metabolism, and function have stood the test of time, and have, moreover, survived the test of “different laboratories” and the even more stringent test of “different countries.” Although followup of these molecular studies will require extensive validation, development of new reagents and tools, these new observations should now stimulate many future investigations. Additional studies which incorporate technologies such as laser capture microdissection, coupled with amplification, to assess and validate genes identified from these in vitro studies to permit more global, but accurate, analyses of the endothelial transcriptome in its native environment will also provide additional insights into the diversity of the vascular system. What are the controls for this diversity? What “dials and switches” turn on the expression of some genes, and turn off the expression of others? How are the varied inputs integrated? Are endothelial cells are genetically preprogrammed to express certain genes or phenotypes? Nature or Nurture? The best is yet to come. Viva le difference!

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43. Tu Z, Kelley VR, Collins T, Lee FS. I kappa B kinase is critical for TNF-alpha-induced VCAM1 gene expression in renal tubular epithelial cells. J Immunol 2001; 166: 6839–6846. 44. Read MA, Whitley MZ, Gupta S, Pierce JW, Best J, Davis RJ, Collins T. Tumor necrosis factor alpha-induced E-selectin expression is activated by the nuclear factor-kappaB and c-JUN Nterminal kinase/p38 mitogen-activated protein kinase pathways. J Biol Chem 1997; 272:2753– 2761. 45. Guerrini L, Casalino L, Corti A, Blasi F. NF-kappa B-mediated regulation of urokinase gene expression by PMA and TNF-alpha in human A549 cells. FEBS Lett 1996; 393: 69–73. 46. Murakami T, Mataki C, Nagao C, Umetani M, Wada Y, Ishii M, Tsutsumi S, Kohro T, Saiura A, Aburatani H, Hamakubo T, Kodama T. The gene expression profile of human umbilical vein endothelial cells stimulated by tumor necrosis factor alpha using DNA microarray analysis. J Atheroscler Thromb 2000; 7:39–44. 47. Park SM, Kim HS, Choe J, Lee TH. Differential induction of cytokine genes and activation of mitogen-activated protein kinase family by soluble CD40 ligand and TNF in a human follicular dendritic cell line. J Immunol 1999; 163:631–638. 48. Yoshida S, Ono M, Shono T, Izumi H, Ishibashi T, Suzuki H, Kuwano M. Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol 1997; 17:4015−4023. 49. Chi J-T, Chang H, Haraldsen G, Jahnsen F, Troyanskaya O, Chang D, Wang Z, Rockson S, van de Rijn M, Botstein D, Brown P. Endothelial cell diversity revealed by global expression profiling. Proc Natl Acad Sci USA 2003; 100:10623–10628. 50. Gerety SS, Wang HH, Chen ZF, Anderson D. Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol Cell 1999; 4:403−414. 51. Zhu L, Cullinan-Bove K, Polihronis M, Bagchi M, Bagchi I. Calcitonin is a progester-oneregulated marker that forecasts the receptive state of endometrium during implantation. Endocrinology 1998; 139:3923–3924. 52. Niiro N, Nishimura J, Hirano K, Nakano H, Kanaide H. Mechanisms of galanin-induced contraction in rat myometrium. Br J Pharmacol 1998; 124:1623–1632.

7 The Role of Genetic Predeterminants in Regulating the Phenotypic Heterogeneity of the Endothelium Brant M.Weinstein Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, Maryland, U.S.A.

1. INTRODUCTION The complex and intricate network of blood and lymphatic vessels that make up the circulatory system of vertebrates plays a vital functional role, supplying oxygen and nutrients, removing wastes, and serving as the conduit for transport of immune and hormonal cells and factors. As might be expected, the formation, remodeling, and regression of blood and lymphatic vessels is a complex process exquisitely controlled in adults by signals from the adjacent tissues that these vessels serve and their surrounding extracellular matrix. However, during early development, the patterning of major blood and lymphatic vessels and in particular the specification and differentiation of the cells that comprise these vessels are regulated by defined genetic programs. In this chapter, we review current understanding of the origins of the different cellular building blocks of blood and lymphatic vessels and genetic programs that regulate their emergence.

2. THE DIVERSITY OF THE VASCULATURE The circulatory system is comprised of a heterogenous set of blood cells enclosed by a system of tubular blood and lymphatic vessels. These vessels are composed of two basic cell types, vascular endothelial cells (VEC) and vascular smooth muscle cells (VSMC). The vessel lumen is lined by a single-cell thick epithelium of endothelial cells. The endothelial epithelium is surrounded by supporting pericytes or smooth muscle cells embedded in a thicker vascular wall, in larger vessels. There are two fundamental types of blood vessels, arteries and veins, that in general carry blood away from and towards the heart, respectively. Larger arteries and veins are morphologically distinguishable; higher-pressure arteries generally have thicker medial smooth-muscle containing layers

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while larger veins have thinner, more elastic walls and valves to prevent backflow. Even at the level of the smallest capillaries, however, arterial and venous blood vessels are distinguishable from one another by their differential expression of molecular markers, as discussed further below. In addition to the blood vasculature, an additional important but often-overlooked component of the circulatory system is the lymphatic vasculature (see Chapter 3). Lymphatic vessels form a separate, parallel vascular network linked to the blood vasculature that is critical for tissue fluid homeostasis, waste removal, and immune functions. Lymphatic vessels are lined by lymphatic endothelial cells (LEC), which as described further below are most likely derived from venous VEC. Later in development and during postnatal life vessels further specialize and acquire distinct functional and regional identities. The extensive diversity of endothelial cell types in adult vertebrates has perhaps been best illustrated by studies using phage display to reveal a remarkable array of differentially expressed luminal surface molecules on endothelial cells (1). Many of these further endothelial cell specializations or re-specializations are controlled by emergent epigenetic influences, and are the subject of other chapters in this volume. The initial establishment of distinct molecular and functional identities for arterial, venous, and LEC occurs during early development, however, and is regulated by defined genetic programs. In the rest of this chapter, we describe what is known about the emergence of these fundamental vascular cell types and the genetic programs that regulate this.

3. EMBRYONIC ORIGINS OF THE VASCULATURE A useful heuristic distinction has been made between two major processes used to generate blood vessels during development and postnatally. The earliest, major embryonic vessels form by the coalescence of individual endothelial progenitor cells or “angioblasts” that arise de novo from extraembryonic and embryonic lateral mesoderm (2). These progenitors form vesicles and cords of attached VEC which undergo further morphogenesis to form epithelial tubes. This process, called “vasculogenesis,” was thought to be restricted to early vascular development but recent evidence has shown that vasculogenesis also occurs later during development and even postnatally. Most later developmental and postnatal blood vessel formation, however, occurs via “angiogenesis,” or the sprouting and elongation of new vessels from preexisting vessels or remodeling of preexisting vessels. In later developments and adult life, these two types of vessel formation processes often occur together and the distinction between them is frequently not so clear. In addition to the earliest extraembryonic and lateral mesoderm, angioblasts also arise de novo at later stages of development from other mesodermal tissues such as the paraxial/somitic mesoderm, mesodermal mesenchyme, and even, as recently described, during adult life from hematopoietic stem cells. Vessels generally form initially as unlined endothelial tubes but soon acquire supporting pericytes or VSMC. The acquisition of these supporting cells is critical for proper morphogenesis, stability, and survival of nascent blood vessels (for comprehensive review of vascular smooth muscle development, see Ref. 3). The origins of pericytes or VSMC are in many if not most cases unclear. The ectodermal cranial neural crest contributes VSMC to the aortic arches and cardiac septa. The coronary vasculature is constructed from cells derived from the

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epicardium. Most other VSMC are believed to be of mesodermal origin, but further specific information on their ontogeny is largely lacking. Recent work suggests that VSMC may in at least some cases share common origins with cells contributing to the endothelial and hematopoietic lineages and that there may be a multipotent common progenitor for these lineages at least transiently during development (see below). The origins of the LEC that line lymphatic vessels have been somewhat controversial (reviewed in Ref. 4). Studies performed with mammalian embryos (5,6), as well as more recent studies, have suggested that the LEC in the earliest primitive lymph sacs originate from endothelial cells that were in veins earlier in development, in particular in larger veins such as the subclavian and anterior cardinal veins. These primitive lymphatic sacs then form sprouts which grow and extend into surrounding tissues and organs to form the peripheral lymphatic system. This “centrifugal” model for formation of the lymphatic system has been supported by recent characterization of molecular markers of LEC that have revealed budding of LEC from veins to form primitive lymph sacs exactly as described by Sabin (see below). The centrifugal model has, however, been challenged by an alternative model (7) in which the LEC comprising the primary lymph sacs originate from mesenchyme, independently from veins, and then secondarily establish connections to venous vessels such as the cardinal and subclavian veins. This “centripetal” model for lymphatic vessel formation has also received some recent support from lineage studies of the origin of LEC of the avian wing bud. These studies have suggested that wing bud LEC are derived from somitic or paraxial mesoderm (8,9). The two models for LEC genesis are of course not mutually exclusive, and it may be that while the primitive lymph sacs and central lymphatic vessels arise largely by budding from veins, some progenitors for LEC in distal lymphatic vessels and capillaries also arise de novo from mesenchyme in a manner analogous to that proposed for the emergence of additional VEC progenitors. The formation of the earliest vessels by vasculogenesis occurs initially in the absence of blood flow, and in many vertebrates, particularly fish and amphibians, defined endothelial cellular cords closely prefigure the final positions of the eventual functional vessels and the emergence of this pattern is relatively insensitive to oxygen or other environmental factors sensed by the embryo. Moreover, in fish and amphibians, many early vessels form de novo as distinct, single tubes, suggesting that vascular plexus formation and its subsequent remodeling are not obligatory steps in vessel formation. A recent study of the formation of the trunk vasculature in the zebra fish using time-lapse imaging of transgenic zebra fish with EGFP-fluorescent endothelial cells showed that these vessels form in a highly stereotypic way along defined pathways, and that this initial patterning occurs even in the absence of blood circulation (10). Furthermore, the anatomy of early vessels in the trunk and other locales is quite well conserved across vertebrate species, as highlighted in a detailed anatomical study of the anatomy of the developing zebra fish vasculature carried out using confocal microangiography (11). All of this suggests that genetic programs regulate not only the identity of the first cellular components of embryonic blood vessels, but also the patterning of these earliest vessels. The nature of the cues mediating this programmed patterning is at present largely unknown, however, and is not the subject of this chapter.

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4. THE HEMANGIOBLAST During gastrulation, vertebrate embryos are divided into three basic tissue layers; endoderm, mesoderm, and ectoderm. The blood and vessels of the embryo proper are thought to be mostly of mesodermal origin, with the earliest blood and endothelium of the embryo originating from closely juxtaposed if not identical pools of cellular progenitors in the lateral mesoderm. The existence of a common progenitor for both vascular and hematopoietic lineages has been debated for many years. The idea was first proposed by Sabin (12) based on the observed common origin of these cell types in yolk sac blood islands, but until recently there was little definitive evidence that such a common progenitor cell actually exists. A variety of recent reports have now demonstrated that a common hemangioblast progenitor is at least a transient intermediate in the development of some but probably not all endothelial and hematopoietic cells (for a recent review see Ref. 13). The possible lineage relationships of hematopoietic and endothelial cells have also been expanded further to include smooth muscle and possibly other cell types (see below and Ref. 14). With the advent of molecular biology and characterization of genes expressed in hematopoietic and endothelial cells, it has became apparent that blood cells and endothelial cells share expression of many genes during their early development, including Flk1, Flt1, SCL/Tall, Tie1, Tie2, Runx1. Targeted disruption of some of these genes in mice has revealed their functional importance for both lineages as well. Mice homozygous for knockouts of Flk1/VegfR2 (15) or Flt1/VegfR1 (16,17) have severe defects in both blood and vascular development. Flk1 knockouts have a severe reduction in both blood and vascular progenitors (15), while Flt1 has increased number of endothelial cells in abnormally organized vessels. The SCL gene is expressed in vascular, hematopoietic, and neural tissues (18). The SCL knockout lacks both primitive and definitive hematopoietic lineages, but endothelium is still present (19–22). However, SCL−/− mice in which hematopoietic expression of SCL is “rescued” by expression of an SCL transgene display defective angiogenic remodeling of yolk sac vessels (23) and ectopic expression of SCL in zebra fish embryos results in an increase in the numbers of both hematopoietic and vascular cells (24), suggesting that SCL promotes the specification of both lineages. The zebra fish cloche mutant (25) has also provided suggestive genetic evidence in support of the existence of a hemangioblast. The cloche mutants lack virtually all endothelial and blood cells and are deficient in an as-yet unidentified gene functioning early in both lineages, or in a common progenitor (25–27). Experimental studies suggest that cloche acts upstream of flk (26) and downstream of or in parallel to hhex (28) and fli1 (29). Transplantation experiments indicate that cloche is required cell-autonomously for both the formation of vascular endothelium (25) and for the differentiation and/or survival of red blood cells (30), although there is evidence it also plays a cell-non-autonomous role in hematopoietic development, perhaps as a result of a requirement for interaction with endothelium in order to generate blood cells (30). These data on shared expression and function of vascular and hematopioetic genes are highly suggestive, but in vitro experiments using cultured ES cells have provided perhaps the best evidence to date for the existence of a hemangioblast. ES cells that have been differentiated into embryoid bodies in vitro give rise to clonal “blast colonies” containing both hematopoietic and endothelial cells (31). These “BL-CFC” cells are or are derived

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from cells coexpressing both VegfR2 and SCL (32,33). These results and a number of additional recent studies (34,37) have led to a proposed model in which a multipotential VegfR2-positive mesodermal precursors are directed into hematopoietic, endothelial, and smooth muscle lineages based on their expression of additional factors including SCL/Tall and Runx1 (Fig. 1, for a more detailed review of this model and the data in support of it see Ref. 14). The existence of common multipotential progenitor for all three lineages including smooth muscle is a particularly interesting and novel idea that has also

Figure 1 A model for specification of various cell lineages from Flk1+ mesodermal precursors (modified after Ref. 14). The presence or absence of SCL/Tall, Runx1, and as yet undetermined transcription factor expression determines whether Flk1+ cells produce endothelial cells (VEC), primitive or definitive hematopoietic precursors, or other cell types including smooth muscle cells (SMCs). VEC differentiate into arterial VEC under the direction of a molecular pathway including shh,

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vegf, and notch signaling. Venous differentiation is likely the default identity of VEC. Venous VEC can also differentiate into LEC under the direction of Prox1 and other factors. Transdifferentiation of VEC to smooth muscle also may occur. See text for details. VEC, vascular endothelial cells; LEC, lymphatic endothelial cells; VSMC, vascular smooth muscle cells; HSC, hematopoietic stem cells. gained some support from a number of other recent in vitro and in vivo findings. Clonal ES cell-derived Flk1+ cells can differentiate in vitro into both endothelial and mural (VSMC) cells and form organized vascular networks (34). Interestingly, this fate choice can be modulated by SCL/Tall levels; ES cells with increased Tall expression show reduced smooth muscle differentiation, while loss of Tall promotes smooth muscle formation (37). Mice with cre recombinase “knocked in” to the Flk locus crossed to a cre reporter line have shown that Flk+ cells can give rise to various muscle cell types in addition to vascular and hematopoietic cells (35), suggesting a common progenitor may also be present during normal development in vivo. A variety of additional evidence has arisen to suggest that adults also possess multipotential mesodermal progenitors and/or cells transdifferentiating from one lineage to another, including reports that adult human or hematopoietic stem cells can contribute to both blood and vascular cell types (38,39) and a number of different studies that suggest VEC can transdifferentiate into vascular smooth muscle cells, at least under certain circumstances (reviewed in Ref. 3).

5. ARTERIAL-VENOUS FATE DETERMINATION Most fundamentally, the vasculature is divided into two types of blood vessels, arteries and veins. The classical view of arterial-venous (A–V) identity is that it follows from physiological parameters such as differences in blood flow and pressure that emerge in otherwise naive vascular plexi as blood circulation begins. However, recent work has shown that in the early embryo genetic programs regulate the initial specification of arterial and venous endothelial cell types (Fig. 1, reviewed in Ref. 40–42). Until recently, it was not even clear that there were significant molecular or functional distinctions between endothelial cells lining arteries and veins. In the past few years, a variety of reports have made it apparent that there are genes specifically or preferentially expressed in arterial and venous endothelium (Fig. 2). Probably the first conclusive evidence for a functionally important molecular distinction between arterial and venous endothelial cells came from work with ephrin and Eph genes in mice (43). Wang et al. (43) described the

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expression of ephrin B2, a member of the ephrin family of membrane ligands. Ephrin B2, a member of the ephrin family of membrane ligands, is expressed in arterial endothelial cells and is absent in venous endothelial cells, while the ephrinB2 receptor EphB4 is preferentially expressed in veins. The arterial- and venous-restricted expression of these genes is apparent very early in vascular development, before onset of circulation or even formation of lumenized vascular tubes,

Figure 2 Arterial and venous endothelial cells have molecularly defined identities that are evident prior to circulatory flow or even tubulogenesis. In the zebra fish, the expression of artery markers such as ephrinB2 (C) and vein markers such as flt4 (D) is evident by in situ hybridization of 25 somite stage embryos several hours before circulation begins in the trunk. In fact, expression of ephrinB2 within the dorsal aorta begins just as the endothelial cells that have migrated from the lateral mesoderm are aggregating into a cord of cells at the trunk mid-line. Expression of the panendothelial marker fli1 is shown for comparison (B). Box in upper diagram (A) shows approximate location of in

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situ images, for reference. Red arrows: dorsal aorta; blue arrows: posterior cardinal vein. (Panel A modified after Ref. 74.) showing clearly that these restricted expression patterns do not depend on flow. Targeted gene deletion of each member of this ligand-receptor pair resulted in similar cardiovascular abnormalities, demonstrating that they are necessary for normal vascular development and likely directly interact with one another (43,44). Interestingly, ephrin B2 mutant mice generated with the LacZ “knocked in” to the ephrin B2 locus continue to express LacZ appropriately in the arterial compartment. Thus, while expression ephrinB2 and EphB4 expression highlight a functionally important molecular distinction between arterial and venous endothelium, initial specification of arterial or venous fate must involve additional factors upstream of ephrin B2 (43). A number of recent studies, in particular studies carried out in the zebra fish, have helped to uncover and dissect the functional roles of these upstream factors, resulting in the identification of a signaling cascade for arterial fate determination consisting of sequential hedgehog, vascular endothelial growth factor (vegf), and notch signaling (Fig. 3A). A variety of studies in mammals and other vertebrates have revealed the specific expression of Notch signaling genes (Notch, Delta, Jagged, etc.) in arterial but not in venous endothelial cells, and murine knockout studies showed that these molecules play an important functional role in the vasculature (reviewed comprehensively in Ref. 40,41,45). Although the nature of this functional role was not determined in the murine studies, the arterial-specific expression of these genes suggested that they might be playing a role in artery formation. A number of recent studies in the zebra fish (46–48) showed that Notch signaling acts to promote arterial differentiation at the expense of venous differentiation during vascular development. Repression of notch signaling in zebra fish embryos was accomplished using either a Notch signaling-deficient neurogenic called mindbomb (mib), or by intracellular injection of mRNA encoding a dominantnegative DNA binding mutant of Xenopus suppressor of hairless protein (48). In either case this resulted in loss of ephrinB2 expression from arteries and ectopic expansion of normally venous-restricted markers such as ephb4 and flt-4 into the arterial domain. Conversely, activation of Notch signaling was effected using either ubiquitous or endothelial-specific transgenic expression of the activated notch intracellular domain. In both cases this suppressed expression of vein-restricted markers and promoted ectopic expression of ephrinB2 and other arterial markers in venous vessels. In a subsequent study, Lawson et al. (47) further dissected the A–V differentiation signaling hierarchy by demonstrating that sonic hedgehog (shh) and vegf act upstream of Notch during arterial differentiation. Embryos lacking shh or vegf also fail to express ephrin-B2 within their blood vessels, like embryos deficient in Notch signaling. Overexpression of shh promotes ectopic arterial vessel formation in the trunk, while overexpression of vegf via injection of vegf mRNA suppresses expression of veinrestricted markers and results in expression of ephrinB2 and other arterial markers in venous vessels. By combining different methods for activating or repressing each of these signaling pathways in “molecular epistasis” experiments, Lawson et al. were able to demonstrate that shh activity induces expression of vegf in the somites, and that vegf then

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activates notch signaling in the endothelial cells of the developing dorsal aorta, promoting arterial differentiation (see Fig. 3B and C for an example of these epistatic analyses). Genetic screening methods were used to identify a zebra fish mutant deficient in both angiogenesis and arterial differentiation as a result of a defect in phospholipase C gamma-1 (plcg1) (49). Phospholipase C-γ genes are known effectors of signaling via receptor tyrosine kinases such as the vegf receptor Flk1. The vascular expression of plcg1 and vascular-specific phenotype of

Figure 3 A molecular pathway for A– V fate determination (A). Zebrafish studies have revealed that vascular endothelial growth factor (vegf) acts down-stream of sonic hedgehog (shh) and upstream of the Notch pathway to determine arterial cell fate. Various methods were used to increase (left side) or decrease (right side) the levels and/or activities of each of these signaling pathways in order to assay effects on arterial-venous fate. Loss of Notch, vegf, or shh signaling results in loss of arterial identity while exogenous activation or overexpression of these factors causes ectopic expression of arterial markers. “Molecular epistasis” experiments were performed by combining

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different methods in order to assemble these components into an ordered pathway (B, C). For example, microinjection of vegf mRNA results in increased and ectopic expression of the artery marker ephrinB2 in wild type embryos but does not in notchdeficient embryos homozygous for the mindbomb mutation (B). In contrast, ubiquitous heat-shock inducible expression of activated notch osclerotic intra”) results in robust induction of ephrinB2 expression even in vegf-deficient embryos injected with a morpholino oligonucleotide to “knock down” vegf protein synthesis (C). Together these results indicate that vegf is acting upstream of notch to promote arterial differentiation. (For additional details on the zebrafish studies used to derive this pathway, see Refs. 47,48.) the mutant in this gene suggested that it might be functioning downstream of vegf signaling. Indeed, it was found that plcg1 mutants are insensitive to both angiogenic and arterial differentiation responses to vegf overexpression, demonstrating that this gene plays a major role in signaling downstream from vegf in vivo. Recent studies in mice have also implicated shh and vegf signaling in regulating blood vessel growth and arterial differentiation. The Shh induces expression of all three vegf-1 isoforms, angiopoietin 1, and angiopoietin 2 in ischemic limbs, and induces new blood vessel growth without affecting endothelial cell migration or proliferation (50). Mukouyama et al. (51) evaluated the influence of the nervous system on blood vessel development, and found that peripheral nerves express vegf and influence vascular patterning and arteriogenesis in embryonic skin. They demonstrated that vegf expressing neurons and Schwann cells induced ephrinB2 expression in endothelial cells when cocultured, and that exogenously added vegf had a similar effect. They also demonstrated that a vegf-blocking antibody could abrogate this response. Two additional studies performed in adult animals suggest that vegf also plays a role in postnatal arterial differentiation. Visconti et al. (52) showed αMHC::VEGF transgenic mice expressing VEGF-A in the heart had an increased percentage of arterial (ephrin B2+) vessels in adult cardiac tissue compared to wild type mice. In another study, Springer et al. (53) demonstrated an increase in arterial concentration in adult skeletal muscle in response to

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VEGF-A expression. Transplantation of myoblasts expressing VEGF-A into nonischemic skeletal muscle resulted in an increased capillary density in the region of the implanted cells and a region of arteriogenic growth immediately adjacent to the implanted cells. The authors noted that this type of arteriogenic growth is distinct from that typically seen as a result of collateral arteriole formation because of its proximity to the site of vegf delivery in a region of tissue that has few if any preexisting arteriolar vessels. Some of the links in the pathway shown in Fig. 3A have also been further confirmed by other recent in vitro experiments. Notch4 activation by Delta4 (vascular expression of both of these genes is restricted to arteries) in cultured primary human dermal micro-vascular endothelial cells (HMVECd) upregulates expression of ephrinB2 and hairy-related factors HES1, HERP1, and HERP2 (54). Vegf (but not FGFs) induces Notch4 and Delta4 expression in cultured arterial endothelial cells (55). This induction requires functional Vegf receptors VEGFR1 and VEGFR2 as well as PI3-kinase pathway. Constitutive activation of Notch in these cells inhibits proliferation and promotes survival and network formation, while Notch blockade may partially inhibit network formation. Although the results above indicate that the A–V identity of early blood vessels precedes circulatory flow or even angioblast assembly, other recent work suggests that this fate choice is not irreversible and that maintenance of differentiated A–V identity might require components of the vascular wall. Two separate groups recently performed quailchick grafting experiments to examine the plasticity of A–V endothelial cell fate (56,57). Portions of embryonic arteries or veins were grafted from quail donors at various stages of development into chick hosts, and the A–V identity of donor cells contributing to different host vessels was assessed using artery- or vein-specific molecular markers. Up until approximately E7 donor cells populate both types of vessels and assume the appropriate molecular identity, but after E7 this plasticity is progressively lost. However, isolated endothelial cells or isolated dissected endothelia were still plastic even in older vessels, suggesting that components of the vascular wall are necessary to maintain, or are sufficient to redirect, the A–V identity of adjacent endothelial cells.

6. LYMPHATIC FATE DETERMINATION As noted above, a separate but interconnected network of lymphatic vessels comprised of LEC rather than VEC parallels the blood vascular network. Molecular characterization of LEC and VEC has shown that these cell types share most of their genes in common, a fact that has also made isolation and functional characterization of LEC difficult. In the past few years, however, a number of reasonably specific or at least diagnostic markers of LEC have been uncovered that have helped us begin to understand the specification and differentiation of these cells. These include the lymphatic endothelial hyaluronan receptor LYVE-1 (58,59), podoplanin (60), VEGFR3, a receptor for VEGF-C and VEGF-D (61,62), and Prox-1 (63) (see Chapter 3). Interestingly, many LEC markers are also transiently expressed in developing VEC, particularly in the venous blood vessels. This might be expected if, as proposed by Florence Sabin, LEC are derived from venous VEC (Fig. 1). Immunohistochemical studies using LEC markers to examine successive stages of development have in fact shown that the primitive lymphatic sacs, at least, form by

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budding off from the cardinal veins and other major venous vessels, validating Sabin’s “centrifugal” model for formation of the lymphatic vasculature (see above). Many of the genes used as markers for LEC have also been shown to have important functional roles in these cells, and analysis of their null phenotypes has led to a model for specification and differentiation of these cells from venous VEC (Fig. 4; see Ref. 4 for a recent comprehensive review of lymphatic development and LEC specification). The homeobox gene Prox-1 appears to be a critical “master regulator” of LEC specification from VEC. Prox-1 is a very early marker of prospective LEC progenitors in veins such as the cardinal and subclavian veins. It localizes to a subpopulation of the venous VEC that subsequently sprout and bud off to give rise to lymphatic vessels. In Prox1-null mice this sprouting and budding becomes arrested (63) and the cells that do bud off have a VEC rather than LEC phenotype as

Figure 4 A proposed model for lymphatic emergence from venous endothelium (after Ref. 4). Lymphatic endothelium emerges from “competent” venous endothelium expressing LYVE-1. Polarized expression of Prox-1 on a subset of venous endothelial cells “biases” these cells towards producing lymphatic endothelium and induces or allows continued maintenance of the expression of a number of different LEC genes including VEGFR3, the receptor for the lymphangiogenic factor VEGF-C. (See Ref. 4 for a more

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comprehensive discussion of this model.) determined by expression of various marker genes (64). Ectopic expression of Prox-1 in cultured endothelial cells induces genes specific for LEC such as VEGFR3 and suppresses expression of VEC-specific genes (65,66). The VEGFR3 (Flt4) receptor is another important regulator of lymphangiogenesis together with its ligands Vegf-C and Vegf-D. Like most lymphatic-specific genes, VEGFR3 is initially expressed in VEC early in development but later becomes restricted to LEC (61,62). The VEGFR3 null mice die from cardiovascular failure at E9.5 due to defects in blood vessel formation and morphogenesis (61), precluding assessment of the lymphatic phenotype of loss of VEGFR3 function. However, some patients with hereditary lymphedema have non-sense mutations in VEGFR3 (67) and transgenic expression of soluble VEGFR3 in the skin of adult mice causes extensive loss of lymphatic vessels, while the rest of the circulatory system is normal (68). Furthermore, transgenic overexpression of the VEGFR3 ligands vegfC or vegfD in skin results in lymphatic hyperplasia (69,70). A number of other genes have also been shown to be important for proper development and function of the lymphatic vasculature. Podoplanin is not required for specification of the lymphatic vasculature (its expression is also regulated by Prox-1) but rather for the proper morphogenesis and/or function of lymphatic vessels. Podoplanin null mice have lymphatic, vessels, but there is diminished lymphatic transport, congenital lymphedema, and dilation of lymphatic vessels (60). Angiopoietin-2 signaling through Tie-2 is required for lymphangiogenesis and for proper angiogenic remodeling of blood vessels (71). This ligand is dispensable for embryonic vascular development despite its proposed important role as an antagonist of angiopoietin-1 signaling in VEC (72). Interestingly, the lymphatic but not vascular defects in angiopoietin-2 knockout mice can be rescued by angiopoietin-1 suggesting that in the lymphatic system, at least, angiopoietin-2 is functioning as a Tie-2 agonist rather than an antagonist (71). The Neuropilin-1 and Neuropuilin-2 genes are believed to act as VEGFR cofactors in the vasculature. Neuropilin-2 null mice display loss of smaller lymphatic vessels and capillaries, although larger vessels and lymph sacs still form, suggesting a specific role in branching morphogenesis of smaller lymphatics (73).

7. CONCLUSION During adult life lymphatic, arterial, and venous vessels respond to the needs of local tissues and to hemodynamic changes by remodeling, initiating new vessel growth, or inducing preexisting vessel regression. In the past few years, however, it has become clear that more “hard-wired,” genetic programs guide the initial specification of the endothelial progenitors for these vessels during early development. A wide array of important molecular regulators of early A–V and lymphatic fate determination have now been identified, and the functions of many of these regulators have been studied in detail in both mammalian and non-mammalian developmental models. Despite this wealth of new information, our understanding of how these factors work together in defined

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molecular pathways is still relatively limited. Achieving this understanding is an important goal, not only from a basic research but also from a clinical standpoint. Learning how to properly regulate blood vessel formation and differentiation is crucial to developing effective new pro- and antiangiogenic therapies for ischemia and cancer and to developing useful methods for bioengineering tissues and organs. Since the evidence suggests that many of the molecular pathways regulating endothelial differentiation and heterogeneity during embryogenesis are almost certainly “reused” to help direct vessel formation during later development and adult life, a greater understanding of these pathways is critical in the future development of these new therapies.

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8 The Use of Fate Mapping Studies to Follow Lineage Determination of the Endothelium David E.Reese and Takashi Mikawa Department of Cell and Developmental Biology, Cornell University Medical College, Ithaca, New York, U.S.A.

1. INTRODUCTION The development of a vasculature is critical to the survival of the higher vertebrate embryo. Recent work has further shown that signals from vascular endothelial cells play an inductive role in differentiation of the Purkinje fiber network from cardio-myocytes (1) and the formation of pancreas from gut endoderm (2). Endothelial cells line all vascular structures including the endocardium, both venous and arterial beds, and lymphatic vessels. Endothelial cell precursors or angioblasts arise in most mesoderm including both intra and extraembryonic mesoderm, where they coalesce to form primitive vascular chords de novo, a vasculogenic mechanism. Some angio-blasts are highly migratory in nature originating far from their eventual location (3). Later elaboration of the vascular structure is achieved primarily through the sprouting of new blood vessels from the existing vasculature via angiogenesis (4–7). While endothelial cells all originate from mesoderm, it is unclear at what point they are determined to take on a specific phenotype amongst several endothelial cell types. By tracking the migratory patterns and lineage decisions made by endothelial precursors, the mechanisms involved in endothelial cell commitment and diversity can be more precisely defined. The strategies employed in fate mapping and lineage determination of endothelial cells are the subject of this chapter. Construction of fate maps allows us to follow a precise cell or groups of cells throughout development (Fig. 1). These studies provide clues as to what potential tissue-tissue interactions are required for the development of a given cell type. While fate maps provide powerful information about the origin and migration of a given group of cells, they do not provide high enough resolution for lineage analysis. To determine the timing of lineage segregation, methods must be employed to target single cells followed by clonal analysis. This chapter describes the techniques used for both fate mapping and lineage analysis in both genetic and experimental systems with particular attention paid to the use of retroviruses in lineage analysis. The use of these methods is illustrated with examples of studies that have determined the ontogeny of endothelial cells.

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Figure 1 The fate maps of various vertebrate species. The relative position of progenitor cells for cardiogenic mesoderm (Cm—red), paraxial mesoderm (Pxm—green), lateral plate mesoderm (Lpm—purple), and notochord (Nc—dark grey) is given. In both Xenopus and zebra fish, the axes are the animal pole (An), vegetal pole (Ve), ventral (V) and dorsal (D). In the avian fate map, the anterior (A) and posterior (P) are shown with the primitive streak (Ps) designated at midline.

2. TAGGING METHODS The basic principles of cell labeling for fate mapping or lineage analysis are (a) to tag a defined group of cells and (b) to restrict the tag to its progeny without horizontal spread. Several approaches have been developed, including direct labeling of cells with a vital dye, genetic tagging, or the production of chimeric animals. These techniques are briefly discussed below.

2.1. Vital Dye A widely used technique for fate mapping is the labeling of the cell population of interest with vital dyes. Lipophilic tracers, such as DiI (1,1′-dioctadecy1-3,3,3′,3′-tetramethylindocarbocyanie●perchlorate) and DiO (3,3′-hexadecyloxacarboyanine ●perchlorate), are commonly used in this procedure due to their tight association with cell membranes with little transfer to the cells adjacent to the labeled cell. These lipophilic fluorescent dyes

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serve as tracers well suited for fate maps because they can be loaded at relatively high concentrations into the plasma membrane without significant toxicity. While these dyes are only weakly fluorescent in an aqueous environment, they are highly fluorescent once incorporated into the lipid bilayer of the cell membrane. Within the membrane, they are relatively photostable and therefore lend themselves to multiple observations under epifluorescent illumination. Because of the association with the cell membrane, the lipophilic dye is passed to the progeny of the labeled parental cells unless cell fusion or phagocytosis by nonlabeled cells occurs. Vital dyes can be delivered to the targeted cells either extracellularly or intracellularly through electrical or pressure-assisted microinjection. Direct injection of fluorescein-conjugated dextran particles into a cell is an alternative method often used in tagging a large cell such as a frog blastomere (11). Laser-assisted activation of a caged fluorescent dye in a single cell or a group of cells is another cell labeling method that has been increasingly used in fate mapping and cell lineage studies in zebra fish and Caenorhabditis elegans embryos (12), although this approach is only applicable for tagging cells within a few cell layers of the embryonic surface. In all cases, the fate map and lineage analysis data are obtained by (a) tracing migration patterns and/or morphogenetic movement of fluorescently labeled cells within a given developmental window, and (b) determining cell types represented within a tagged cell population. However, since the concentration of dye per cell exponentially declines as cells proliferate, these methods are limited to a short-term tracing of cell fate and lineage segregation.

2.2. Chimera Long-term tracing of cell fate and lineage segregation necessitate a stable tag for all daughter cells of the originally labeled parental cells without dilution by cell proliferation. Cell transplantation from a donor tissue carrying a recognizable trait to a host is one such approach. In avians, the most frequent method used is orthotopic implantations between quail and chick embryos (13,14). Since the cells replace those in an identical region of the host embryo, the implanted quail cells are expected to follow the same developmental potential during chick embryogenesis. Quail cells are detected in the host chick embryo by the more euchromatic appearance of quail nuclei (15) and the quail-specific marker QCPN (16). In some cases, quail-derived cells are detected by pigmentation (17). While the chick/quail chimera approach has been powerful in fate mapping studies during and after gastrulation, the avian embryo is not suited for mapping the fate of early blastodermal cells. Xenopus chimeras have been a powerful model system for fate mapping individual blastomeric cells (18). Mouse chimeras can also be generated by mixing cells of the blastocyst or inner cell mass from two strains, each of which carries distinguishable traits (19). A broad range of mosaicity can be obtained simply by changing the ratio of cells from each strain. Therefore, this approach has the potential to serve as a model for cell lineage study. However, since it is currently difficult, if not impossible, to define the position of individual cells either in the blastocyst or inner cell mass, this method has not yet been applicable for general fate mapping studies.

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2.3. Genetic Tagging While chimeric approaches assure stable tagging for fate mapping, it requires a skill in microsurgical techniques, which is not always applicable for all researchers. The genetic introduction of a tracer, which is less technically demanding, has been developed. Genetic tagging can be achieved via a number of techniques including the direct injection of DNA, an activation of tracer expression via random mutation or Cre-lox knock-in, and viral mediated gene transfer.

2.3.1. Direct Introduction of DNA Cells can be genetically tagged by the direct introduction of an expression vector plasmid DNA encoding a tracer such as the bacterial β-galactosidase (β-gal) (20). This tagging method is frequently used in Xenopus laevis in which individual cells are readily injectable. Large numbers of embryos can be injected in a reproducible manner. Application of this approach is, however, generally confined to early embryonic cell fate decisions. As development proceeds, this advantage is lost as cell size decreases and the ability to inject single cells diminishes. Furthermore, most injected plasmid DNAs remain as an episome without integrating into the host genome, resulting in an uneven distribution between daughter cells. Some daughter cells completely lose the tracer gene. The mosaic nature of tracer gene expression between daughter cells often complicates the interpretation of the experimental data.

2.3.2. Cre-Lox Knock-in and Intragenic Recombination The Cre-lox recombination approach has been used for fate mapping in the mouse embryo (21). This strategy employs a recombination induced by Cre recombinase through which constitutive expression of a tracer gene, usually β-gal or GFP is retained in all daughter cells upon recombination. However, Cre-lox strategies for fate mapping depend upon the availability of a promoter/enhancer that is active only briefly at a specific time and location during embryogenesis. A rarely occurring intragenic recombination has been used for retrospective clonal analysis in the mouse embryo (22,23). The approach requires a mouse line that carries a tandem-duplicated β-gal gene driven by a tissue-specific promoter. The translated product is enzymatically inactive. However, if an intragenic recombination occurs between the duplicated sequences of the transgene, β-gal activity is restored. Since intragenic recombination is a rare event, cells positive for β-gal are likely a clonally related population within a defined genetic program. Obviously, this tissue-specific promoter-driven reporter gene expression is not applicable for studying cell lineage diversification. Another limitation is the uncertainty in the timing of an intragenic recombination. For example, it can occur long before cells become permissive for the promoter function. Therefore, this method is not suited for studying the growth rate of clonal populations.

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2.3.3. Viral Infection The use of replication-defective retroviruses and adenovimses to trace cell fate and lineage is based on the introduction of tracer gene via viral infection. In both cases, the virus is engineered to lack the necessary genes for viral production from host cells so that only progeny from infected cells will express the marker gene of interest. Attractive features of adenoviral mediated gene transfer include that cloning and production of a high titer of viral particles are relatively straightforward and that cells can be infected regardless of proliferative state. There are disadvantages that limit the utility of adenoviruses in the study of cell fate and lineage. First, the copy number of the introduced gene cannot be controlled and may provide drastically different levels of marker expression. Second, because the DNA introduced to the host is maintained as a linear episome, often the gene is not stable and is exponentially diluted over time by subsequent cell divisions. This loss of material does not permit the adenoviral system to be used for long-term lineage studies. The value of replication-defective retroviruses for cell lineage analysis was first demonstrated in the central nervous system and the eye (24,25). Retroviruses are RNA viruses whose genome is reverse transcribed to a DNA intermediate, which then stably integrates into the host DNA. The integration process results in a single copy of viral DNA per genome, which is inherited in every subsequent cell division. The fact that the integration is stable makes retroviral gene delivery ideal for long-term lineage study, as marker expression does not decline over time. Since retroviruses have been the major tool used in the study of endothelial cell lineage, they will be discussed in detail in the next section.

2.4. Benefits and Limitations of Approaches No one technique described above is exclusively superior to others for cell fate and cell lineage analysis. While vital dye labeling and chimeric animal production are the most widely used methods for fate mapping, their spatial resolution is often not high enough to make lineage analyses possible. Cre-lox approaches offer the benefit of reproducibility and the ability to maintain the mouse lines indefinitely. However, the use of Cre-lox lines is currently limited by the leakiness of promoter activity and the availability of a suitable promoter for the study of a given group of cells. Viral mediated gene transfer can be very efficient but depends on the accessibility of the tissue for microinjection. At the center of these questions is the determination of when the timing of commitment to a given cell fate is made. While all these methods have benefits and limitations, by combining approaches one can ask fundamental questions regarding cell ontogeny and fate. The purpose of this chapter is to illustrate the diverse nature of endothelial cells in biology and how the described methods are used to study these cells.

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3. RETROVIRAL CELL LINEAGE ANALYSIS 3.1. Principle Background Viruses are obligate intracellular parasites that replicate by the invasion of a host cell and utilization of the host cell machinery to generate progeny virion. The development of replication-defective viral vectors for cell lineage analysis has involved the optimization of viruses for the introduction of genetic material into host cells without replication or horizontal spread of viruses themselves. Amongst these, retroviruses are most frequently used in cell lineage analysis because the retroviral gene stably integrates into the host genome, assuring precise inheritance of the integrated viral genome and high level of expression in daughter cell population. Wild-type viruses are composed of a central core containing two copies of the single stranded RNA genome associated with nucleoproteins and reverse transcriptase molecules (Fig. 2). The core component is enveloped by the plasma membrane that contains specific viral glycoproteins. Viral particles bind to a cell surface receptor, then enter the cell via endocytosis. Viral genomic RNA is released into the cytoplasm and is converted into a linear double-stranded cDNA by the viral reverse transcriptase. Upon mitosis of host cells, linear viral cDNA integrates into the host cell chromosomal DNA (Fig. 2). The integrated viral cDNA, or provirus, contains long terminal repeats (LTRs) at both the 5′ and 3′ ends, which flank three intervening genes, gag, pol, and env. A part of the 5′ LTR encodes an enhancer and promoter for the transcription of the viral genes that are required for viral propagation (Fig. 2). A portion of the full-length transcript from the provirus is packaged into viral particles as the retroviral genome. The packaged virions are then released from the host cell by exocytosis resulting in horizontal transmission to other host cells. A replication-defective virus is engineered to retain vertical but not horizontal transmission from the primary infected cells (26–28). Construction of replicationdefective retroviruses involves: (1) transfer of the proviral sequence to a plasmid DNA, and (2) replacement of the structural genes (gag, pol, and env) needed for viral

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Figure 2 Comparison of the life cycle of replication competent (A) and incompetent (B) retroviruses. The retroviral life cycle involves the binding of the viral particle to membrance receptors of the host cell, which intiates endocytosis of the viral particle followed by the uncoating of the RNA genome and associated polymerase. Once a double-stranded DNA is made from the RNA template via reverse transcription, a viral integrase mediates the formation of the provirus via integration of the viral cDNA into a random site of the host genome. Upon integration, all necessary retroviral mRNA and proteins are proteins are produced via the host’s cellular machinery and used to package new virions. Once packaged, the viral particle exits the cell as a fully infectious particle. In replication-incom-petent retroviruses, the genes required for replication are replaced with a β-gal cassette which constitutively expresses the enzyme allowing for visualization of infected

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cells and their progeny. Since the virus lacks replication machinery, infection of neighboring cells does not occur. replication with a reporter gene, such as β-gal, retaining elements required for viral packaging and transcription. This viral plasmid vector is introduced into a packaging cell line, which is engineered to constitutively express the viral structural genes. The transcripts from the provirus sequence can then be encapsulated with the transexpressed structural proteins (Fig. 2). Recombinant viral particles released from the packaging cells are then harvested for cell lineage studies. In an infected host, the viral sequence encoding a reporter gene, but lacking structural genes, is converted to DNA and inserts at a random site in the host genome. Transcripts from the provirus can serve as a template for the translation of the reporter protein, but these are not encapsulated as viral genomic RNA since there are no trans-expressed structural proteins required for capsid and envelope assembly. Thus, without horizontal spread, the reporter gene is inherited and expressed only by the descendants of the originally infected cells. Although retroviral tags offer many attractive features, they are hampered by two potentially significant problems. First, they cannot be used to analyze postmitotic cell populations. Second, the integration of the provirus may potentially alter the expression of an endogenous gene if integration occurs in an important locus. Even with these reservations, replication-defective retrovimses have proven to be very useful for analyzing several tissues and organ systems (1,24,29–39). Below are examples that illustrate the power of replication-incompetent retroviruses in resolving questions related to endothelial cell ontogeny.

3.2. Application to Endocardial Endothelial Cells The development of a given organ’s vasculature can occur either via the local coalescence of individual angioblasts to form a primary vascular network de novo (vasculogenesis) or via recruitment from an existing network (angiogenesis) (3,6,40). The endocardial endothelia form via a vasculogenic mechanism as does the early vascular network formation including the dorsal aortae. Fate map studies on a group of cells in the chick gastrula have established that endocardial and cardiomyocyte precursors both arise from a rostral portion of the Hamburger and Hamilton (HH) stage 3 (41) primitive streak (42). By HH stage 4, the precursors have migrated out of the primitive streak and are continuing their migration rostrocaudally, forming bilateral heart fields (41,44–46). During the lateral body fold of the neurulation stage embryo, the bilateral cardiac fields fuse and form a single tubular heart (46). As this stage, only two cell types comprise the tubular heart, endocardial endothelia and cardiomyo-cytes (47). During the fusion of the bilateral heart fields, the majority of cardiogenic cells become epithelialized and differentiate into presumptive myocytes. A minor population segregates from the original epithelial layer to form the presumptive endocardial cells (47,48). Endocardial progenitor cells begin expression of endothelial markers, such as Flk-1 and QH1, as they segregate from the epithelialized cardiogenic mesoderm (48).

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Thus, both myocyte and endocardial endothelial cell lineages arise from the cardiogenic mesoderm and begin to function before any other organ system is established. The terminal differentiation and morphogenesis of these two cardiogenic cells depend on the underlying endoderm (49,50). Cardiomyocyte gene expression requires endodermderived paracrine factors, such as FGFs (51–53), BMPs (53,54), IGF (55), and Activin (56,57), while endothelial cell differentiation is promoted by angiogenic peptides (58), such as VEGF (59,60) and FGFs (61,62). However, since these signaling molecules are expressed broadly in the endoderm, how they instruct individual cells of the heart field mesoderm to undergo terminal differentiation to form either the cardiomyocyte lineage or the endocardial cell lineage remains paradoxical. Further, while both endocardial and myocyte cell lineages arise from the cardiogenic mesoderm (42,63), the primitive heart tube, as in the adult heart, consists of a greater proportion of myocardial cells than endocardial cells (47). It was also unclear how myocardial cells more than endocardial cells segregate from the cardio genic mesoderm. Two potential models existed for the generation of endocardial endothelia and myocytes from the cardiogenic mesoderm. The first model predicts that the cardiogenic field contains bipotent progenitor cells capable of forming either myogenic or endocardial cells, based on the expression patterns of myocardial and endocardial cell markers in cardiogenic mesoderm (48) and marker coexpression in an immortalized myogenic cell line (64). The second model proposes that the separation of lineages has already taken place within the cardiogenic field, because of morphological heterogeneity within the cardiogenic mesoderm (65–68). These two models have been tested by the retroviral tagging and tracing of individual progenitor cells within the heart field of the chicken embryo. Individual cells in the heart field give rise to a clone consisting only of one cell type, either endocardial or myocardial (62). No clones containing both of these two cell types have been found. Interestingly, these same studies revealed that 95% of the cells within the cardiogenic field contribute to the myocardium, whereas only 5% of cells generate the endocardial endothelia. These data support the model in which the endocardial and myocardial lineages are distinct within the cardiogenic field, with the vast majority of these cells giving rise to the myocardial portion of the avian heart tube. The results also suggest that the segregation of two cardiac lineages, endocardial endothelia and cardiomyocytes, occurs at or prior to heart field formation. This possibility has been tested by tracing the fate of individual cells in the HH-stage 3 primitive streak, from which the cardiogenic mesoderm arises. Individual virally tagged cells of the rostral half of HH-stage 3 primitive streak have been found to generate a daughter population that proliferates and migrates into the heart field, differentiating into either endocardial or myocardial cells, but not both cell types (69). Thus, the rostral portion of the primitive streak at HH-stage 3 consists of at least two distinct subpopulations, entering either the cardiomyocyte lineage or the endocardial cell lineage. Retroviral single cell fate tracing studies have demonstrated that these two cell lineages of the heart are already segregated within the primitive streak, significantly prior to their migration to the heart field. Therefore, it is likely that the precardiomyocytes and preendocardial cells may be already permissive to inductive signal(s) from underlying endoderm to initiate their terminal differentiation into either muscle or endothelial cells.

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Consistent with this model, it has been suggested that endothelial cell commitment may occur before and independent of gastrulation, while myocyte commitment has not yet occurred at the prestreak stage (70). Single cell marking and tracing studies in zebra fish (71) have identified a blastomere population that generates only endocardial or myocardial cells, indicating that the separation of these two lineages can occur at blastula stage prior to the formation of mesoderm. Thus, in both zebra fish and chicken, segregation of cell lineages occurs prior to formation of mesoderm. Similarly, work in mouse has demonstrated that endocardial and myocardial cells arise from bilateral clusters of cells in the distal epiblast (72). It remains to be seen whether, like in avians, these two cell lineages are already segregated at this early stage of murine embryonic development.

3.3. Origin of Coronary Endothelia In the heart, there are three other endothelial cell types, coronary arterial, venous, and capillary. Coronary endothelial cells exhibit distinct phenotypes from endocardial endothelia. For example, coronary endothelial cells establish a typical vessel network throughout the myocardium, whereas endocardial cells never develop vessels that penetrate into the myocardium. It is also interesting that some coronary vessel branches run through the subendocardial space and yet do not fuse with the endocardium or open to the heart chamber. While the exact mechanism for these distinct phenotypes is not known, the series of retroviral cell lineage studies described below have identified the distinct ontogeny for these two cardiac endothelial cell populations. While the endocardium develops as the heart tube forms, coronary vessels are absent from the embryonic heart, until the myocardium begins to thicken by the fusion of trabeculae and myocyte proliferation (73,74). It was once thought that the coronary vessel network is established by angiogenic sprouting from the aorta (75–78). However, discontinuous channels of endothelial cells that are not linked with either the aorta or endocardium have been identified in the E6 avian embryo (79,80). Furthermore, the establishment of a closed coronary vessel network does not occur until E14 (73,74). Therefore, the prevalent model predicted de novo formation of the coronary arteries. However, the embryonic origin and mechanism of vascular tube formation of the coronary cell types were unproven. To determine the embryonic origin and mechanism responsible for the formation of the coronary arteries, retroviral cell tagging approaches have been employed. Cells that give rise to the embryonic heart have been known to originate from both the cardiogenic field and a subset of neural crest cells termed the cardiac neural crest. Since these are the two main sources of embryonic heart cells, the possibilities that the coronary arteries originated from the heart field or from neural crest derivatives were attractive models to explain the origins of coronary arteries. Two lines of evidence, however, argued against these models. First, injection of replication-incompetent retroviruses to the heart tube never gave rise to cells within the coronary system (36). Also, cardiac neural crest cells retrovirally labeled with β-gal were only observed within the tunica media of the aortic and pulmonary trunks and not within the coronary arterial system (38). While these results demonstrated that the coronary endothelia did not arise from cardiac neural

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crest or from cells of the heart tubes, they did not provide any information as to the location of the coronary endothelial precursors. In addition to cardiogenic mesoderm and neural crest cells, there is a third population of cells that migrates to the heart. The proepicardium (81,82) is a transient embryonic structure located at the septum transversum and migrates over the embryonic heart at E3. Upon contact with the heart, the proepicardium-derived cells unsheathe the outer myocardium forming an epithelial layer called the epicardium (82). Since the arrival of coronary cell types occurs after the formation of the epicardium, this embryonic structure was an attractive candidate as the source of coronary cell precursors (Fig. 3). This possibility was tested by the retroviral tagging of cells that form the epicardium. Coronary vessels containing β-gal+ endothelial cells, smooth muscle, or cardiac endothelium were observed in the resulting hearts (37). The same result was obtained when the proepicardium was microinjected with the β-gal virus prior to migration to the heart (38). These studies have provided direct proof for the proepicardial origin of coronary vasculature, including endothelial cells. This conclusion was further confirmed using both adenoviral tagging and (83) chick/quail chimera analysis of the proepicardium (84). Consistent with this, chick/quail chimera experiments demonstrated that the proepicardium contained cells which could differentiate into coronary endothelial cells (85). Importantly, individual β-gal+ clones contained only one cell type, either endothelial cells, smooth muscle, or fibroblasts, suggesting that these lineages are already determined within the proepicardium. Furthermore, regardless of the smooth muscle or endothelial cell clone, each β-gal+ clone occupied only a segment of a coronary vessel and in no case did it populate the entire length of a vessel (37,38). Taken together, it was concluded that the coronary vasculature forms via a vasculogenic mechanism (86,87). Thus, the retroviral cell lineage studies have identified that the proepicardium is a source of coronary endothelia distinct from those of the endocardium. They also indicate that the coronary arteries form de novo rather than sprouting from the aorta. Equally important is that the coronary endothelial cell lineage is established and segregated from other cardiac cell types within the proepicardium even before their arrival at the heart. These data may provide a foundation for defining the

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Figure 3 The generation of multiple endothelial cell types from mesoderm. As the progression from a zygote to an avian embryo, three germ layers form via gastrulation, ectoderm (Ec), mesoderm and endoderm (En). Two endothelial cell types first differentiate from the mesoderm, including endocardial (blue) and the hemangioblast (red). The hemangioblast further gives rise to blood cell lineages (gray) or arterial (red), venous (purple), capillary (orange), and lymphatic (green) endothelial cell populations. Neural tube (Nt), notochord (Nc), somites, and lateral plate mesoderm (Lpm).

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timing and mechanism that induces and/or specifies the coronary endothelial cell fate within proepicardial cell populations.

4. OTHER LINEAGE ANALYSIS ON ENDOTHELIAL CELLS 4.1. Vascular Endothelia Classic embryological studies in the avian, suggested that the embryonic vasculature arose from the invasion of extraembryonic blood vessels into the embryo (88,89). However, when the area pellucida (embryo proper) is experimentally separated from the area opaca (extraembryonic) prior to blood vessel formation, vascular development proceeds in the embryo proper (90). Thus, the embryonic germ layers provide the precursor cells for the embryonic vasculature prior to joining the extraembryonic vessels later in development. The precise origin of embryonic endothelial cells was further narrowed with data that showed that only the mesoderm of avians could give rise to endothelial cells (3). All mesoderm with the exception of prechordal mesoderm has the potential to produce angioblasts and avian embryonic vascular development occurs throughout the embryonic disc with the exception of the midline region (67). Indeed, the endothelia of the dorsal aorta originates in both the paraxial and lateral plate mesoderm (91). Fate mapping experiments in Xenopus and zebra fish demonstrate that endothelial cells of the dorsal aorta arise from the lateral plate mesoderm (92,93). In contrast to endocardial endothelial cells, where the precursor field is well defined, less in known about the origin of endothelial precursors of the major blood vessels.

4.2. Arterial vs. Venous Cell Identity Understanding the precise origin of vascular endothelial precursors in the embryo will also aid in determining when different endothelial cell identities diverge. Two possibilities exist for the differentiation of arterial and venous vascular endothelial cells. The first suggests that commitment to either an arterial or venous cell phenotype occurs prior to blood vessel formation. Recent evidence from fate mapping in zebra fish suggests that endothelial cell populations may be committed to a given fate within the lateral plate mesoderm before vascular assembly (93). Angioblasts from zebra fish labeled with a caged dye were activated by laser and followed during development. Using this method, individually labeled endothelial cells were found in either arterial or venous vascular beds. Interestingly, however, data from avian experiments suggest exactly the opposite possibility: that the local environment controls arterial or venous identity and that cell fate is only determined upon recruitment into the vascular network. Donor embryonic quail venous endothelial cells were capable of incorporating into the arteries of the host and expressed ephrin-B2, a marker of arterial cells (94,95). Conversely, arterial endothelial cells from donors were able to incorporate into veins of host embryos and lost expression of ephrin-B2. These results suggest that plasticity exists during embryonic

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development, at least in avians. When identical experiments were performed with E7 embryos, donor endothelial cells failed to incorporate into any blood vessels suggesting that plasticity is only maintained during a certain developmental window. It remains to be seen whether the differences in mechanisms for arterial and venous identity are simply due to species variation.

4.3. Lymphatic Endothelia While much emphasis has been placed on the origin of endocardial and vascular endothelia, less is known about the ontogeny of the lymphatic endothelial cell. Early work suggested that the lymphatics of the forelimbs were formed from the sprouting of the jugulo-axillary lymph sacs into the limb buds (96). However, using markers for the lymphatic endothelia, it has been shown that by E3.5, the wing buds already contain lymphangioblasts (97). The jugulo-axillary lymph sacs do not form until E4–5, and therefore, the origin of these lymphangioblasts was unclear. Recent chimera studies in the quail show that cells from the dorsal half of the somites at the wing bud level migrate into the limb and differentiate into lymphatic endothelia (98). These data are interesting because the vascular endothelial cells of wing blood vessels can also arise from somatic mesoderm (99–101). The question remains as to when these endothelial cells, originating from the same tissue adopt their diverse functional and molecular fates in the avian wing.

Figure 4 Model of the clonal ontogeny of coronary endothelial cells arising from the proepicardium (38). The propepicardium located at the septum transversum contains cells heterogeneous endothelial precursor clones (represented by gray and white) which migrate over the heart tube. Retroviral cell labeling (black) results in individual clonal growth in the developing coronary vascular cords. This clonal growth results in the

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formation of a discontinuous coronary epithelium resulting from individual cells of the proepicardium.

5. SUMMARY AND REMAINING QUESTIONS Fate mapping and single cell lineage analyses have been used to define both the timing and location of embryonic vascular patterning and commitment to a given vascular cell fate (Fig. 4). By understanding the microenvironment of endothelial precursors when lineage determination takes place, we can more precisely understand potential cell-cell and molecular interactions required for endothelial cell differentiation and diversity. The questions that arise from these studies include: (1) How do endocardial precursors differentiate while exposed to the same signals as cardiomyocyte precursors? (2) What mechanisms govern the patterning of the coronary endothelia and the establishment of a connection with the proximal aorta? (3) Do vessels established via vasculogenesis and angiogenesis fundamentally differ based on different developmental histories? (4) How plastic are early endothelial cells to their arterial, venous, capillary, or lymphatic identities? Understanding the mechanisms that govern these processes will substantially further our understanding of the vascular system and the endothelial cells upon which it is based.

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9 Oxygen Regulation of Endothelial Cell Phenotypes Yasushi Yoshikawa, Maksim Fedarau, Koichiro Iwanaga, Hiroaki Harada, and David J.Pinsky Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, U.S.A.

1. INTRODUCTION For maximal generation of the ATP, energy “currency” required to support cellular functions, modern eukaryotic organisms respire. Even with abundant energy-rich fuel such as glucose or fatty acids, metabolic fires simply smolder without available oxygen. During the course of evolution, organisms have evolved complex cellular and organ-level mechanisms to both sense and respond to diminished levels of oxygen. Multicellular organisms have developed cardiovascular systems to deliver the required oxygen and metabolic substrates to respiring tissue. Among the various cells that sense and control the flow of oxygen to tissues, endothelial cells play an essential role. Under quiescent conditions, during which oxygen is plentiful and danger signals are absent, endothelial cells maintain blood fluidity by preventing thrombosis, maintain vascular tone in a somewhat vasodilated state so as not to impede flow, and provide a selectively permeable barrier to the flow of macromolecules from the luminal to the abluminal surface. When oxygen becomes scarce, however, complex mechanisms are set into play, which result in significant alterations in the endothelial cell phenotype. At an organismal level, there are carotid baroreceptors, which can control pulmonary ventilation. From an organ’s vantage point, autoregulatory loopscontrol vascular tonus, and state of red cell mass through the regulated synthesis of erythropoietin. At a cellular level, alterations in calcium homeostasis or other signaling pathways can result in altered cell shape, or changes in cellular trafficking of organelles or molecular species. Metabolic pathways involving substrate uptake, utilization, or respiratory function can also be significantly modulated by conditions of oxygen scarcity. At a subcellular level, transcriptional mechanisms of cellular activation represent a hallmark of the cellular response to oxygen deprivation. As the chief orchestrators of blood flow, and hence the transit of fluids and substrates towards and into tissues, endothelial cells play a pivotal role in both sensing and delivering oxygen. In wounded tissue, in which tissues are severed from existing blood supply, endothelial cells must grow into a relatively hypoxic environment in order to form new vessels which are the basis for tissue repair. They are, therefore, considered to

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be a relatively “hardy” cell, in that they do not die under conditions of extreme oxygen deprivation, but rather, adapt by developing a prototypical phenotype which ultimately allows them to re-establish vascular homeostasis. Some of the mechanisms that are triggered by oxygen deprivation may at first blush seem unlikely to be useful. Consider, for example, the thrombotic response. At first glance, this process might seem detrimental, in that fibrin plugs would limit delivery of oxygen and nutritive substances to tissue at risk. In fact, the mitogen-rich thrombi provide a critical architectural stroma on which intact new vessels can grow. From a theoretical point of view, one would predict that mechanisms responsible for sensing oxygen tension and which trigger the adaptive response would be activated by low levels (or absence) of oxygen. This indeed is the case. For instance, the transcription factor hypoxia-inducible factor (HIF)-1α under normal oxygen-plentiful conditions undergoes rapid degradation. Under conditions of low oxygen tension, however, the degradative (hydroxylation) processes are aborted, and levels rapidly accumulate, allowing its heterodimerization with HIF-1β, nuclear translocation, and transcriptional activation of multiple gene programs (see below). This is just one of several adaptive mechanisms by which the endothelial cell can both sense and respond to reduced levels of oxygen. This chapter is intended to describe several of the homeostatic features of the vasculature which are perturbed under conditions of hypoxia or ischemia. Although ischemia per se has several components, such as stasis of blood flow, acidic pH, reduced glucose availability, accumulation of lactate or other metabolic waste products, which may have independent or additive effects, the primary regulation of the endothelial phenotype under ischemic conditions is likely due to vanishing levels of oxygen. Most of the endothelial adaptations observed in ischemic vessels can be observed in experimental endothelial cell culture systems in which hypoxic exposure represents the sole environmental perturbation. Therefore, this chapter will focus on the responses of endothelial cells to hypoxia, which can either represent physiological adaptations or pathological disruption of vascular homeostasis. In addition, where appropriate, we discuss the potential role for reperfusion injury in accentuating the hypoxia insult.

2. CLINICAL RELEVANCE Most studies describing modulation of endothelial cell phenotypes under hypoxic and/or ischemic conditions show that oxygen deprivation causes an explosive cascade of inflammatory events and tips the natural anticoagulant/procoagulant balance of the endovascular wall towards one with a preponderance of coagulation. Postischemic suppression of blood flow is generally associated with neutrophil plugging, with enhanced adhesion receptor expression and microvascular thrombosis, leading to the deterioration of organ function. Therefore, rapid re-establishment of vascular homeostasis following the ischemic or hypoxic insult represents one of the most important elements for limiting damage in various disorders such as myo-cardial infarction, stroke, and solid organ transplantation. Understanding the clinical manifestations of hypoxia and/or ischemia may lead to novel approaches to protect tissue against ischemic and/or reperfusion injury, and could lead to new treatment strategies in a wide array of clinical settings.

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Experiments using a range of agonists and antagonists in various hypoxic or ischemic organ models have indicated that some agents have the potential to effectively improve preservation of organ function after the hypoxia and/or ischemia has passed, that is, after there has been a restoration of oxygenated blood flow.

2.1. Cardiac Ischemia The mortality and morbidity of ischemic cardiovascular disease remain the greatest health problem in the Western world. Despite significant therapeutic advances such as angioplasty and thrombolysis, myocardial injury associated with ischemia-reperfusion is still a major unsolved problem. Ischemic myocardial injury, which ensues within minutes following an abrupt cessation of blood flow, is characterized by a central region of cellular necrosis, surrounded by lesions wherein cell loss is believed to occur more through increasing apoptosis (1). Therapeutic strategies have evolved around improving flow, reducing inflammation, decreasing energy utilization or provision of alternative fuels, and maintaining key cellular ionic gradients. Calvillo et al. (1) reported that erythropoietin may coordinate the local response to injury by maintaining vascular autoregulation, and also by attenuating both apoptotic and inflammatory causes of cell death. In this recent work, administration of recombinant erythropoietin was shown to reduce cardiomyocyte loss under ischemic conditions. Additionally, a significant proportion of the delayed injury that occurs does so as a result of recruitment of inflammatory cells into the infarct area, which then release cytokines and other inflammatory mediators. There are also suggested anti-inflammatory strategies that may squelch the explosive cascade of inflammatory events following cardiac ischemia. One of the early and central cytokine mediators of ischemia, interleukin (IL)-1, is synthesized by endothelial cells which have been placed in a hypoxic environment (2) as well as by vascular endothelial cells and the cardiac myocytes themselves in an ischemic zone (3). Administration of a recombinant form of an endogenous peptide that is a competitive inhibitor of IL-1/IL-1 receptor interaction represents a potentially new pharmacological strategy to reduce the expression of intercellular adhesion molecule-1 (ICAM-1) in transplanted cardiac grafts which have undergone an obligatory ischemic period during harvest and transit. This approach was shown to reduce the occurrence of primary cardiac graft failure in an murine transplantation model (3). In various other experimental model systems, adhesion receptor expressions (P-selectin, E-selectin, ICAM-1) have all been shown to be deleterious mediators of ischemic myocardial injury, the blockade of which could theoretically prove beneficial as adjunctive therapy with thrombolysis. To date, however, no purely anti-inflammatory strategy has been shown to be beneficial in the setting of human myocardial infarction. There has been a great deal of recent excitement, again only in the laboratory setting, for the potential use of “cellular therapies” (CD34+ endothelial stem cells) to improve outcomes following myocardial infarction (4). These studies are part of a burgeoning belief that growth of new blood vessels (neoangiogenesis) may be particularly beneficial in the ischemic myocardium. Along these lines, Torry et al. (5) reported that the lack of expression of vascular endothelial growth factor (VEGF) 164 and VEGF188 isoforms in mice produces deficient myocardial angiogenesis that results in ischemic

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cardiomyopathy, suggesting that VEGF is an important endogenous regulator of vascular integrity in heart tissue. Consequently, these authors have proposed a therapy that can promote the expression of endogenous VEGF, which may provide a new means for therapeutic angiogenesis in human heart tissue. Luttun et al. (6) reported that placental growth factor (PIGF) stimulated myocardial revascularization as efficiently as did VEGF, however, PIGF did not elicit edema or hypotension, recognized to be common untoward side effects of VEGF therapy. Direct VEGF administration studies, at least in the setting of peripheral limb ischemia, have recently been disappointing in several clinical trials of claudication, yet the jury is still out as to whether VEGF protein or gene transfer may find their way into the clinical treatment of ischemic heart disease.

2.2. Lung Ischemia The lung represents a rich vascular plexus, with nearly one-third of its cellular mass comprised of endothelium. Pulmonary microvascular endothelial cells are unique in that they are normally exposed to high atmospheric concentrations of oxygen. Therefore, it is not surprising that the lungs are among the most sensitive of solid organs to periods of ischemia or oxygen deprivation, as may occur in major pulmonary embolism with pulmonary infarction, high altitude sickness, or following lung transplantation, in which case the donor lungs are severed from their native blood supply, hypothermically preserved, and re-perfused in the recipient. In a lung transplant model, Abraham et al. (7) discovered that unventilated hypoxia increases vascular permeability in lung grafts. Similar to the aforementioned studies with myocardial ischemia, selective VEGF antagonism during graft preservation might be of benefit to counteract edema formation. Although there are potentially vascular-bed specific differences in the phenotypes of endothelial cells and the patterns of gene expression in response to hypoxic or ischemic injury, there are probably more similarities than differences. Our own studies of lung graft inflammation and thrombosis following lung transplantation show that a transcription factor, early growth response gene-1 (Egr-1), can trigger expression of multiple families of deleterious genes, such as cytokines (e.g., IL-1), adhesion receptors (e.g., ICAM-1), prothrombotic triggers (e.g., tissue factor, TF), antifibrinolytic mediators (e.g., plasminogen activator inhibitor-1, PAI-1), and so on, leading to the dysfunction of transplanted organs (8). In several experimental model systems, including cardiac and pulmonary ischemia, these pathological events can be diminished by a simple strategy of Egr-1 suppression using a phosphorothioate antisense oligodeoxyribonucleotide added to the organ flush/preservation solution (9). In addition, stimulation of the peroxisome proliferator-activated receptor-gamma (PPAR-γ), whose glitazone-class agonists are currently in widespread clinical use as insulin sensitizers in diabetes, suppresses the activation of Egr-1 expression, leading to improved preservation of lung graft function following ischemic preservation and transplantation (10). Just as IL-1 receptor antagonist represents an endogenously synthesized peptide which places inflammatory cascades in check, so too it appears that IL-10 and heme oxygenase (Hmox)-1 do likewise. In a lung ischemia–reperfusion model, for instance, it has been shown that IL-10 expression is profoundly increased during the ischemic period, and that this is associated with augmentation of endogenous fibrinolytic mechanisms in vivo (11). IL-10 gene null (−/−) mice showed poor post-ischemic lung function and survival after

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I/R compared with IL-10 (+/+) mice. In addition, recombinant IL-10 given to IL-10 (−/−) mice reduced pulmonary vascular fibrin deposition, and rescued mice from lung injury (11). In large part, it appeared that the presence of IL-10 reduced ischemia-driven expression of PAI-1, so that some of the accrued benefit may have been accounted for by reduced thrombotic burden in ischemic lung microvessels. It should also be added that, functionally, the presence of Hmox-1 might be a key survival factor in the setting of lung ischemia and reperfusion. Ho-1-deficient mice exhibit lethal ischemic injury, but were rescued from death by inhaled carbon monoxide (CO). CO activates soluble guanylate cyclase and thereby suppresses hypoxic induction of the gene coding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes. These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to depress the fibrinolytic axis (12).

2.3. Brain Ischemia Despite advances in the treatment of hypertension and hypercholesterolemia, stroke remains the third leading cause of death and the primary cause of permanent disability in the United States (13). Clinical adoption of laboratory-based strategies to improve outcomes following presentation with stroke has been abysmal, primarily because of repeated failures of clinical trials with neuroprotective or anti-inflammatory strategies. To the community of scientists and physicians dedicated to improving stroke outcomes, the discordance in outcome between animal studies and clinical trials has led to much puzzlement and discourse. To date, the only therapy that has proven benefit in humans is thrombolysis, albeit with a narrow therapeutic window. Nevertheless, anti-inflammatory strategies, such as anti-P/E selectin (14) or anti-ICAM-1, may yet find their way into the therapeutic arsenal, perhaps in combination with thrombolytic therapy. ICAM-1 and its integrin counterligand are upregulated in ischemic cerebral microvascular endothelial cell and leukocytes, respectively. Tumor necrosis factor (TNF)-α, IL-1, and platelet-activating factor (PAF, an inflammatory lipid) also participate in leukocyte accumulation and subsequent activation in the setting of ischemic stroke. During cerebral ischemia, angiogenesis occurs inside and around the infarcted area, and reperfusion injury after focal cerebral ischemia is also accompanied by expression of a number of potentially pathological mediators. Therapy to block the function of one more of these factors might possibly reduce reperfusion injury and infarct extension. Like ischemic myocardial injury, erythropoietin appears to play a major protective role in the brain, and studies have shown that locally administered recombinant human erythropoietin prevents ischemic-induced neural death (15,16). In other studies, Renner et al. (17) found that platelet-derived growth factor receptor (PDGF-R)-β is a key factor driving vascular remodeling in stroke; it is conceivable that therapeutic modulation of the PDGF system might improve angiogenesis, cellular protection, and/or edema inhibition (17). CD39, the main ectopyrase in the brain responsible for the catabolism of extracellular nucleotides ATP and ADP, has recently been shown to participate in the pathogenesis of ischemic microvascular thrombosis in stroke (18). A recombinant form of soluble CD39 decreased postischemic platelet and fibrin deposition and increased perfusion without increasing intracerebral hemorrhage in a murine model of stroke. In contrast, aspirin did not increase postischemic blood flow or reduce infarction volume,

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but did increase intracerebral hemorrhage (18). These studies represent just a sampling of potential new and exiting therapeutics that are based on understanding how cerebral vessels modulate their phenotype under conditions of oxygen deprivation or ischemia, and how pharmacologic strategies may act to more rapidly normalize vascular homeostasis to reduce the devastating clinical consequences of stroke.

2.4. Kidney Ischemia Ischemic acute renal failure represents a significant cause of morbidity and mortality in humans, affecting 2–5% of all hospitalized patients and 30–50% of patients in intensive care units. Ischemia-reperfusion injury of the kidney is mediated by diverse cytokines and chemokines produced by renal tubular epithelial cells and infiltrating cells. There is also evidence indicating that tissue-injury mechanisms in the setting of ischemic acute renal failure and renal transplantation (19,20) are quite similar to pathological ischemic vascular processes in other organs.

2.5. Limb Ischemia Peripheral arterial occlusive disease, which occurs predominantly due to athero-sclerosis in people over 60 years old, represents an important clinical problem which affects approximately 7.4 million people in the United States. One potentially exciting therapeutic avenue for the treatment of limb ischemia, manifested clinically as claudication, is the use of VEGF. Chang et al. (21) found that an intra-arterial injection of adeno-associated virus overexpressing VEGF normalized muscle oxygen tension and induced increased arteriogenesis. Despite the limited success with angiogenic therapy seen in the TRAFFIC trial (22,23), subsequent angiogenic studies have been negative (24). Nevertheless, Luttun et al. (6) reported that compared with VEGF, PIGF better stimulated functional recovery of the ischemic limb, primarily by enhancing growth of collateral side branches in mice. Additionally, PIGF and its receptor Flt1 stimulate arteriogenesis by affecting smooth muscle cells, and the PlGF-induced mature vessels persisted for prolonged periods (>1 year), even well after the arteriogenic stimulus had dissipated. Other growth factors may also play a role in the reparative response following ischemic tissue injury. Emanueli et al. (25) reported that the neurotrophin nerve growth factor (NGF) participates in the functional neovascularization of tissue following injury. Supplementation with this growth factor appears to promote angiogenesis through a VEGF-Akt-NO-mediated signaling pathway. As these studies focused on graft survival, function, and rejection following ischemic preservation/transplantation, it is conceivable that these strategies might be able to improve current techniques for organ transplantation. For example, if a harvested organ can be maintained for a prolonged period in spite of hypoxic and ischemic conditions, current limitations in time and distance in transplantation will diminish in importance and potentially expand the donor pool by increasing the likely use of “marginal” donor organs. Another theoretical advantage of improving organ preservation revolves around the putative link between organ rejection and ischemia-reperfusion injury. Suppressing the latter may lead to reduced acute (and hence, chronic) rejection, which currently

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represents the main limitation to the survival of patients who receive a transplanted organ. Overall, it is apparent that elucidating the changes in the vasculature during hypoxia may reveal potential for improving survival in patients who suffer from many types of ischemic organ injury.

3. MODULATION OF ENDOTHELIAL BARRIER FUNCTION BY HYPOXIA Endothelial cells form a metabolically active and barrier separating the contents of the vascular lumen from surrounding tissue and organs. Under normal physiological conditions, the endothelium comprises a selectively permeable monolayer, allowing macromolecules of various sizes to pass through, depending on their Stokes’ atomic radii. This process is referred to as restricted diffusion, and means that solutes of smaller diameter can easily migrate through the endothelial barrier, whereas larger molecules, such as proteins and cellular elements of circulating blood are confined within the vascular lumen. After a protracted period of severe hypoxia, however, the phenotype of this selectively permeable barrier changes quite remarkably, as it becomes characterized by impairment of endothelial barrier function manifest as increased vascular permeability. The physiological basis for this dramatic change in endothelial phenotype in low oxygen environment has been largely elucidated. Endothelial cells, exposed to oxygen-deficient environment in vitro, develop changes in their actin-based cytoskeleton with subsequent formation of 1–3 µm intercellular gaps between adjacent cells (26). This is due to internal reorganization of the internal cytoskeleton, which is connected at adherens junctions with the endothelial plasma lemma, resulting in a retraction of the edges of apposing endothelial cells (26). With the appearance of intercellular gaps, the endothelial layer loses its ability to restrict passage of molecules based on size, and unrestricted diffusion or even mass movement of fluids and solutes can transpire. At a tissue level, these changes are manifest as increased vascular permeability with subsequent perivascular interstitial edema due to massive paracellular leakage of solutes, proteins, and cellular components (27). This condition is clinically recognized as the capillary leak syndrome, which has been commonly accepted as pathophysiological evidence of impaired vascular barrier function in response to different stimuli (28), including hypoxia. It can be observed in various clinical situations and pathological conditions, such as high altitude exposure or ischemic brain stroke, which can be accompanied by acute pulmonary and cerebral edema, respectively (29,30). As a model to study the effect of prolonged hypoxic exposure on endothelial barrier function, a common experimental method relies on growing endothelial cells to confluence on special filters which restrict the passage of macromolecules and lower molecular weight solutes (31). This experimental model has been successfully used to investigate the role of different receptor agonists, such as thrombin, histamine, or TNF-α on vascular barrier permeability, and reflects pathophysiological processes occurring in vivo, although cultured endothelial monolayers in this model are somewhat more permeable than vessels from which they are originally derived (32,33). The increased passage of tracer solutes, such as [3H]-insulin, across cultured pulmonary artery or aortic monolayers has been noticed after prolonged (24 hr) exposure to hypoxia (12–14 mm

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Hg). This hypoxia-induced increase of vascular barrier permeability occurs in a timedependent manner and was dependent on absolute level of hypoxia as well (26), but there is a restoration of normal endothelial function within 48 hr upon reoxygenation that points to the reversible character of the barrier function perturbation driven by hypoxic stress (26). What is the key regulatory link affected during hypoxia/ischemia which causes such remarkable perturbations in endothelial barrier function? Multiple studies show that the cAMP second messenger system is an important determinant of many critical vascular functions, including permeability, coagulation balance, and leukocyte/blood cell interaction (26,34,35). A decreased level of intracellular cAMP is one of the principal mechanisms driving vascular dysfunction under conditions of decreased oxygen tension. In endothelial cells subjected to hypoxia, the activity of adenylate cyclase, both basal and stimulated, is diminished, which contributes to the decline in intracellular cAMP (26,36). A similar decline in cAMP synthesis and increased permeability have been observed following exposure of endothelial monolayers to TNF-α (36), an inflammatory cytokine whose effects in large part mimic those seen with hypoxic exposure. In vascular smooth muscle cells subjected to hypoxia, the level of intracellular cAMP also drops precipitously, but in this case it is mainly due to specific increases of types III and IV phosphodiesterase activity (37). Under subsequent reoxygenation following the hypoxic period, the extensive formation of reactive oxygen species (ROS) leads to activation of phosphodiesterase types II, III, and IV in endothelium, thus further contributing to the rapid decline in intracellular cyclic nucleotide level (38,39). Administration of membrane permeable cAMP analogs such as dibutyryl (db)-cAMP has been shown to reverse the hypoxia-mediated enhancement of vascular permeability. These same effects were elicited by treatment of hypoxic endothelial cell cultures with stimulators of type I (8-[4chlorophenylthio] adenosine 3′,5′-phosphate) and type II (N6-benzoyladenosine 3′,5′cyclic monophosphate) protein kinase A (26), but not adenosine or adenosine monophosphate. Taken together, these studies show that a decline in intracellular cAMP content, driven by hypoxia and/or redox stress and brought about either by a reduction in cAMP synthesis or an increment in cAMP catabolism, can lead to reversible changes in cytoskeletal architecture and permeability characteristics. Another cyclic nucleotide second messenger pathway which is also quite important in maintaining homeostatic physiological and biochemical processes, including barrier properties of endothelium, and which is disrupted by hypoxic exposure, is the nitric oxide (NO)/cGMP system (40). Levels of NO are markedly decreased, especially during reoxygenation, because of the scavenging effects of ROS on NO bioavailability (41). Administration of the NO donor S-nitroso-N-acetylpenicillamine or superoxide dismutase (which increases bioavailable levels of NO) can effectively attenuate hypoxia-induced increases in vascular permeability (42). Taken together, these studies show not only the common signaling mechanisms involved in hypoxic regulation of endothelial permeability characteristics, but show that pharmacological modulation of these pathways can normalize barrier function in ischemic tissue.

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4. INFLAMMATION Of the many homeostatic properties that are modulated by hypoxia, one of the most important is inflammation. Hypoxia clearly evokes an inflammatory response, marked by initial endothelial cell activation, which triggers local inflammation in the context of recruitable effector leukocyte populations in the blood and the organismal level. Inflammatory reactions, initially triggered by hypoxic modulation of the endothelial phenotype, lead to self-propagating and auto-amplifying regulatory loops in which inflammation itself perpetuates various local changes of communication between endothelial cells and leukocytes.

4.1. Proinflammatory Cytokines Proinflammatory cytokines play an important role in mediating hypoxic or ischemicrelated tissue injury. During hypoxia, proinflammatory cytokines are induced and secreted by endothelial cells as well as leukocytes and subsequently influence both local vascular function as well as function and gene expression of tissues/organs in remote locations, to which these cytokines are carried by flowing blood. IL-1 and TNF-α are critical mediators of the inflammatory response during hypoxia stress. These cytokines are synthesized de novo within hypoxic endothelial cells and secreted extracellularly during hypoxic stress (or reoxygenation/reperfusion). Hypoxia increases IL-1 mRNA in cultured endothelial cells, which may then act in an autocrine manner to induce E-selectin and ICAM-1 expression (2). Another key mediator of inflammatory stress is IL-8, which is a member of the CXC family of chemokines originally identified as a neutrophil activating peptide and now recognized to be a potent neutrophil chemotactic factor. IL-8 is induced and secreted extracellularly from endothelial cells during hypoxia stress (43,44). IL-8 production is regulated at a transcriptional level and hypoxia-mediated induction of the IL-8 gene involves increased binding of nuclear factor κB (NF-κB) binding to the upstream promoter region. IL-8 also promulgates the general inflammatory milieu because it induces transcription of monocyte chemotactic protein (MCP-1) in endothelial cells during hypoxia. MCP-1, in turn, serves to recruit monocytes during hypoxic stress. IL-8 also increases expression of inducible protein-10 (IP-10), a CXC chemokine family member which is chemotactic for T cells (43,44). In human coronary sinus samples, an increase in IL-8 was detected following the period of aortic cross-clamping, with a direct correlation between IL-8 levels, cardiac ischemic duration, and release of myoglobin, a marker of myocyte injury (43). There is conflicting information as to the physiological or pathological role for another cytokine, IL-6, which is synthesized and released extracellularly from endothelial cells exposed to hypoxic stress (45). There are data to suggest that IL-6 work may actually act as an anti-inflammatory cytokine, to quell inflammation during the hypoxic reaction to stress. IL-6 has been shown to suppress production of IL-1 and TNF-α (46), and to induce expression of IL-1 receptor antagonist and TNF-α receptor antagonist (47).

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4.2. Adhesion Receptors Adhesion molecules are activated and expressed during hypoxia on both endothelial cells and leukocytes, increasing their cognate interactions and promoting inflammation. Leukoadhesion to endothelial cells is a multistep process that involves the following sequence: (1) initial rolling deceleration of the leukocyte along the activated endothelium; (2) more highly adhesive interactions with other adhesion receptors; (3) activation of endothelial cell and tight attachment, and (4) emigration. It is useful to consider the various stages of leukocyte adhesion, because each may be modulated in different ways by the hypoxic environment. The initial tethering and rolling of leukocytes on endothelial cells are mediated by carbohydrate-containing glycoprotein adhesion receptors called selectins. P-selectin, which exists embedded in the membranes of Weibel-Palade bodies, is rapidly translocated to the plasmalemma during the process of Weibel-Palade exocytosis due to increased calcium influx during hypoxic stress; (48). Using cycloheximide to block protein synthesis, these experiments showed that very little (if any) active protein synthesis was required for hypoxia to trigger surface expression of P-selectin. The other hypoxia-inducible selectin, E-selectin, requires transcription and translation of message de novo following hypoxic stress, a process that is dependent on NF-κB (49). The next phase of leukocyte adhesion to activated endothelium is mediated by members of the immunoglobulin family of adhesion receptors, of which ICAM-1 is a primary member. ICAM-1 expression is regulated through various signal transduction pathways that are coupled to activation of a transcription factor such as Egr-1 and NF-κB, and hence is both hypoxia- and redox-sensitive. Once translated and expressed on the endothelial surface, ICAM-1 tightly engages β2-integrin on the leukocyte surface. ICAM1 is also upregulated secondarily by inflammatory cytokines such as IL-1 and TNF-α, which are also induced during hypoxia stress. The ICAM-1 adhesion molecule plays a critical role in ischemia-induced inflammation during and after heart transplantation and lung transplantation, the reduced expression or blockade of which may serve as a useful target for therapeutic intervention to mitigate ischemia-induced tissue injury (3,50).

4.3. Activation of Endothelial Cells and Leukocyte Emigration Adhesion of leukocytes to the endothelial cell surface activates the endothelial cells themselves, potentially through conformational changes in ICAM-1 during receptor engagement which are transduced into the cell to affect signaling. Some of these signals may be mediated by an increase of intracellular calcium, phosphorylation of myosin light chain kinase, and F-actin structural changes (51). Emigration of leukocytes from the vascular lumen is accompanied by endothelial cell activation, consisting of discrete structural changes of the endothelial cell-cell junctions. These structural alterations facilitate leukocyte emigration. ICAM-1 and other integral membrane proteins such as PECAM-1 are important signal transduction intermediaries which elicit cytoskeletal and junctional changes in endothelial mono-layers which promote leukocyte diapedesis (51,52). ICAM-1, which induces calcium signaling through PKCs, mediates phosphorylation of actin-associated protein and cytoskeletal rearrangement in brain

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endothelial cells (53). E-selectin also associates with elements of the actin cytoskeleton leading to cytoskeletal reorganization. Concomitantly cytoplasmic domain of E-selectin leads MAPK activation and subsequent changes in gene transcription (54). By changing endothelial cytoskeletal properties and increasing expression of various membrane proteins, it can easily be seen how hypoxia or ischemia can facilitate the pathways leading to leukocyte emigration.

5. HYPOXIA-DRIVEN REGULATION OF GENE TRANSCRIPTION In order to survive in the absence of sufficient oxygen, cells have developed specific physiologic adaptations consisting of discrete metabolic alterations. In many cases, these adaptations are regulated at the level of transcription by transcription factors binding to the promoter regions of target genes. Several transcription factors, such as Egr-1 and HIF-1 are activated during hypoxic stress; other genes, such as NF-κB and activation protein-1 (AP-1), are activated in the presence of redox stress, such as occurs during reoxygenation accompanied by the formation of reactive oxygen intermediates. When these transcription factors are induced and translocate to the nucleus, they promote expression of many downstream target genes, some of which can propagate or exacerbate local tissue injury.

5.1. Egr-1 Egr-1 belongs to a family of related transcription factors (zinc finger proteins), and is induced rapidly following hypoxic stress. Activation of protein kinase C II-β (PKC-IIβ) represents a proximal trigger for the induction of Egr-1. Induction of Egr-1 increases transcription of downstream target genes, including proinflammatory cytokines such as IL-1, chemokines such as MIP-2 and MCP-1, adhesion receptors such as ICAM-1, and procoagulant molecules such as PAI-1 and TF. Egr-1 can, therefore, be viewed as a critical pathological trigger for hypoxia- or ischemic-related inflammation (8,55). (Fig. 1).

5.2. HIF-1 As an essential survival mechanism, mammalian cells have evolved the ability to sense changes in O2 concentration. The transcription factor HIF-1 represents one of the most important of several O2 sensing mechanisms. HIF-1 plays a pivotal role in cell adaptation to hypoxic stress, as it facilitates metabolic alterations during hypoxia. HIF-1 influences downstream target genes such as erythropoietin, VEGF, insulin-dependent glucose transporter (GLUT-1), Hmox-1, and the inducible NO synthase (NOS), iNOS (56,57). Quite recently, the mechanism by which mammalian cells “sense” O2 by upregulating HIF-1 activity has been elucidated. Under normoxic conditions, HIF-1 is continually degraded through inactivation of the HIF-1α subunit by prolyl-hydroxylase, an enzyme that catalyzes hydroxylation of proline residues using oxygen as a substrate. In contrast,

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under anaerobic conditions, HIF-1α remains undegraded because the prolyl-hydroxylase is inactive, and hence HIF-1 heterodimer can migrate to and accumulate within the nucleus where it activates promoters to elicit transcription of downstream target genes (58). Asparaginyl-hydroxylase also regulates activity of this important transcription factor. Under hypoxic conditions, hydroxylation of asparagine residues of the COOHterminal of HIF is abrogated, which ultimately results in activation of downstream target genes (59). Both prolylhydroxylase and asparaginyl-hydroxylase hence serve as direct sensors of intracellular oxygen tension.

Figure 1 Hypoxia increases Egr-1 mRNA expression in lungs of mice. H: hypoxia; N: normoxia. Panel A: Northern blot; panel B: Western blot. (From Ref. 55.)

6. COMPLEMENT The complement cascade plays an important role in hypoxia-reoxygenation injury, and this system too can be modulated by a hypoxic-reoxygenated environment. Hypoxia leads to complement activation and deposition of C3 on the endothelial cell surface, with reoxygenation further augmenting C3 deposition. This activation is mediated by both the classical and lectin pathways of complement activation (60–62). Moreover, there is further amplification of this cascade in that deposition of C5b-9 on endothelial cells elicits expression of other endothelial cell-leukocyte adhesion molecules such as ICAM-1 and VCAM-1, predominantly via an NO/cGMP-regulated NF-κB translocation mechanism (63). Brain ischemia induces accumulation of C1q on neurons, and this in turn influences endothelial-leukocyte-platelet interactions. Accumulated C1q activates GPIIb–IIIa fibrinogen binding sites and the expression of P-selectin, which is likely to contribute to thrombotic events and in turn influences EC-PLT axis (64). A bifunctional

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complement inhibitory pro tein such as sLex-glycosylated sCR1 has been shown to inhibit both leukocyte and platelet accumulation in stroke (65). These data suggest that ischemia-reperfusion triggers a number of inflammatory cascades at the endothelial surface, which are linked at multiple levels.

7. VASODILATATION AND VASOCONSTRICTION Hypoxia/ischemia strongly modulates vascular tonus. For example, under hypoxic conditions, vessels exhibit a rapid but transient vasoconstriction followed by relaxation during the first few minutes. If the hypoxic exposure is sustained, however, the vasoconstriction too becomes more sustained. De-endothelialization of blood vessels in an experimental apparatus abolishes hypoxia-induced vasoconstriction, while at the same time enhancing vasodilatation. Under basal conditions, there is a dynamic balance between vasoconstrictive and vasodilatatory substances produced by endothelial cells, which modulate each other through multiple feedback mechanisms (66,67). Some factors are very important for control of vasodilatation [NO and prostacyclin (PGI2)], whereas others modulate vasoconstriction (endothelin) (68).

7.1. Endogenous Vasodilators NO is formed from L-arginine by one of three NOS isoenzymes (69). Activity of the predominant endothelial isoform, NOS III (also called eNOS), depends on ambient levels of intracellular concentration of Ca2+ (70). Shear stress, cellular proliferation, and various receptor agonists (such as bradykinin, serotonin, adenosine, ADP/ATP, histamine, or thrombin) increase eNOS enzymatic activity by increasing intracellular calcium (71). Experimentally, the calcium ionophore A23187 similarly increases intracellular calcium and hence its application results in a pulse of NO liberation. During hypoxia there is a decrease in basal e-NOS gene expression, leading to a substantial decrease in basal endothelial NO production (72). As NO activates vascular soluble guanylate cyclase (sGC) to form intracellular cGMP in vascular smooth muscle cells, leading to their relaxation, diminished levels of NO are expected to promote vasoconstriction. In the pulmonary vascular bed, this decrease may contribute in part to the sustained vasoconstrictor response to hypoxia. During reperfusion, NO (and hence, cGMP) levels plummet due to the quenching of NO by superoxide (41), and hence, reperfusion is typically characterized by vasoconstriction. Other vasodilators also contribute to the quintessentially vasodilated state of normal vessels. These include PGI2, formed in endothelial cells by the action of cyclooxygenase (COX) and arachidonic acid, a process that is similar to NO formation in that its release is modulated by physical and humoral stimuli (73). Shear stress and many agonists, which are also agonists leading to production of NO, contribute to the release of PGI2 (74). Agonist-induced release of PGI2 seems to be predominantly regulated by Ca2+ release from intracellular storage pools (75). PGI2 causes relaxation of vascular smooth muscle by activating adenylate cyclase resulting in an increased production of cAMP

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(70). Therefore, even if not directly regulated by hypoxia, the recognized modulation of cAMP synthesis and catabolism by hypoxia makes it likely that the downstream PGI2 vasodilatory signal would be dampened under hypoxic or ischemic conditions owing to effects of hypoxia on VSMC cAMP. A third endogenous vasodilator, endothelial-derived hyperpolarizing factor (EDHF), is also an important physiological regulator of vascular tone. Electrophy-siological studies in various arterial preparations demonstrate that acetylcholine and other receptor agonists elicit an endothelial-dependent hyperpolarization (and subsequent vascular smooth muscle cell relaxation), which is due to a discrete, diffusible EDHF that is distinct from NO and PGI2. EDHF-induced hyperpolarization of smooth muscle is inhibited by blocking the Na/K ATPase, as well as by blocking the inwardly rectifying K+ channel currents (76). These data indicate that EDHF works by activating a K+ channel in vascular smooth muscle (77). There is little information available as to how or whether hypoxia or ischemia modulates EDHF activity. Another endogenous vasodilator, whose physiological importance is becoming increasingly clear, is CO, a byproduct of the action of the enzyme Hmox. Heme is cleaved by Hmox to yield equimolar amounts of ferric iron (Fe3+), CO, and biliverdin. Biliverdin is subsequently converted to bilirubin by biliverdin reductase (78). The Hmox system consists of three enzymatic isoforms. Hmox-1 is the stress-induced isoform; Hmox-2 is the constitutive isoform, which is the dominant isoform under physiological conditions (79); and Hmox-3, also a constitutive isoform closely related to Hmox-2 (80). Hmox-1 is an inducible isoform whose expression skyrockets in many cell types, including endothelial cells, in response to oxygen deprivation or oxidant stress (67). In a manner similar to NO, CO activates sGC, causing an increase in cGMP content within neighboring vascular smooth muscle cells (81). Induction of Hmox-1 protects tissues against inflammatory stress and leukocyte adhesion (79,82). CO also functions as an inhibitor of platelet aggregation by activating platelet cGMP levels (83), and a mitogenic inhibitor. CO decreases the expression of mitogens such as ET-1 and PDGF-B in response to hypoxia. The Hmox-1/CO pathway may serve as a back-up mechanism for vascular homeostasis under nonphysiological conditions, such as hypoxia or ischemiareperfusion (84).

7.2. Endogenous Vasoconstrictors Endothelial-derived contracting factors (EDCFs) fall into one of three major categories, including vasoconstrictor peptides, eicosanoid metabolites of arachidonic acid, and free radicals. Various agonists such as norepinephrine, acetylcholine, and serotonin acting on endothelial surface receptors stimulate the production of vasoconstrictor metabolites such as endothelin, thromboxane A2, leukotrienes, and superoxide anions (71). One of the most potent EDCFs is endothelin (ET), which consists of peptides comprised of 21 amino acids (85). There are three ET isoforms; ET-1 is synthesized predominantly in endothelial cells, where it can act in a paracrine manner on ET-A receptors located on the surface of vascular smooth muscle cells, resulting in vasoconstriction. ET-1 also activates endothelial ET-B receptors that subsequently elicit release of vasodilators such as NO and PGI2 (77). Shear stress, hypoxia, and ischemia profoundly stimulate endothelial production of endothelins (84). In addition to the depressed expression of vasodilators,

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the production of vasoconstrictors by hypoxia quite strongly tips the vascular phenotype towards a vasoconstricted state.

8. COAGULATION/FIBRINOLYSIS Because of its strategic location juxtaposed between flowing blood and surrounding tissues, the endothelium bears an essential responsibility for maintaining blood flow by preventing thrombus formation and promoting degradation/fibrinolysis of preformed thrombus. Pathologic states accompanied by decreased oxygen tension can distinctly modulate this function of endothelium to shift the natural anticoagulant/ procoagulant balance to favor activation of coagulation. This activation of coagulation is encountered in diverse clinical situations and syndromes, including cerebral, cardiac, pulmonary, and vascular ischemic disorders. Another clinically relevant problem is the preservation of donor organs for transplantation, which is likewise associated with severe hypoxemia within the preserved vascular bed (see Chapter 25). Postischemic restoration of flow (reperfusion) results in prominent fibrin deposition that induces vascular dysfunction. It is essential to understand the role played by oxygen deprivation in triggering the procoagulant endothelial phenotype, and to understand the exact mechanisms underlying pathogenesis of thrombus accrual in situations in which the vasculature is subjected to severe hypoxic stress. One of the mechanisms by which the anticoagulant phenotype of vascular endothelium is achieved involves a cell surface thrombin-binding protein, thrombo-modulin, an integral endothelial protein which greatly enhances the ability of thrombin to convert inactive protein C to activated protein C. Activated protein C then functions as an anticoagulant by proteolytic degradation of two critical coagulation cofactors, Va and VIIIa (86). When cultured bovine endothelial cells are subjected to hypoxia, consisting of a pO2 of approximately 12–14 mm Hg (similar to levels seen in ischemic tissue), there is a marked decrease in expression of the thrombomodulin gene (31,87). This downregulation of both mRNA and protein levels leads to a parallel suppression of thrombomodulin functional activity by approximately 80–90% over a 48–72 hr hypoxic exposure. A potential signaling mechanism which contributes to the downregulation of thrombomodulin levels is the hypoxia-mediated suppression of cAMP second messenger levels. Repletion of intracellular cAMP by the addition of membrane-permeable cAMP analogs (such as dibutyryl-cAMP and 8-bromo-cAMP) abrogates the hypoxia- or TNF-αinduced decline in endothelial cell thrombomodulin expression (36,38). Another mechanism likely to be important in hypoxia-mediated alteration of the anticoagulant phenotype of vascular endothelium is the observed upregulation of TF expression, which serves as one of the strongest prothrombotic stimuli and as such which is typically excluded from the intravascular space. Expression of TF, which interacts with factor VIIa to activate the extrinsic pathway of coagulation, increases markedly in vascular wall cells following hypoxic exposure. In in vivo studies of mice exposed to normobaric hypoxia (6–8% oxygen), there was a profound increase in TF expression along with fibrin accrual in the hypoxic vs. normoxic control lung tissue, shown by histological evidence of fibrin deposited in pulmonary microvessels (89). In this model, it was apparent that mononuclear phagocytes recruited to hypoxic vascular foci, rather than

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endothelial cells themselves, were largely responsible for fibrin accumulation. This observation is concordant with other studies which have shown that hypoxia per se does not induce the significant endothelial expression of TF in vitro (31). In other pathologic conditions leading to abrupt intravascular thrombosis, such as atherosclerosis or massive vascular injury, the immediate exposure of subendothelial TF to blood contents leads to rapid coagulation with massive thrombus formation. Together with hypoxia-induced lung injury, these conditions share a common pathological feature, namely the recruitment of mononuclear phagocytes as a rich synthetic pool of TF. Although theoretically, polymorphonuclear leukocytes could also contribute to the pathological accumulation of fibrin under ischemic conditions, there are contrary data in that immunodepletion of polymorphonuclear leukocytes had no effect on fibrin accumulation (89). In contrast, the analogous immunologic depletion of monocytes did have a considerable reducing effect on fibrin deposition in lungs subjected to hypoxia (89). The analysis of TF expression in hypoxic murine lungs revealed a ~20-fold increase of TF transcripts in hypoxic lungs compared to the normoxic controls (90). In parallel immunohistochemical studies showed colocalization of increased TF in hypoxic lung with mononuclear phagocytes (90). These data make it clear that monocytes serve as a primary source of TF expression in hypoxia-driven thrombosis, after becoming arrested and activated at sites of hypoxic vascular injury. One of the signals by which monocyte/macrophages are drawn to ischemic vasculature is the MCP-1 whose expression is also upregulated by hypoxic endothelium (44). More detailed exploration of molecular mechanisms underlying hypoxic induction of TF expression in monocytes led to the identification of a key role for Egr-1 transcription factor, which increases rapidly following mechanical vascular injury (91,92) and which appears to be the primary force driving TF expression in hypoxia. In vitro studies show hypoxic mononuclear phagocytes to exhibit substantial upregulation of Egr-1 expression associated with induction of TF (55). Further evidence that Egr-1 expression is the main trigger for hypoxia-driven TF expression is based on experimental data obtained from mice lacking the Egr-1 gene. These Egr-1−/− mice showed significantly decreased intravascular fibrin deposition after ischemia-reperfusion lung injury and a minimal increment in TF mRNA, with absence of actual changes in TF antigen activity (90). In contrast, wild-type mice subjected to an otherwise equivalent normoxic environment expressed TF and exhibited significant fibrin deposition. The discussion of vascular perturbations during the hypoxia would be incomplete without emphasizing the role of the fibrinolytic system in the net accrual of fibrin following hypoxic exposure. PAI-1 is a 52 kDa serine protease inhibitor which serves as the primary inhibitor of the fibrinolytic cascade, thus promoting the net accrual of fibrin by retarding its dissolution. Under hypoxic conditions, plasma levels of PAI-1 antigen increase as early as 4 hr after the start of hypoxic stress in hypoxic mononuclear phagocytes (93) (Fig. 2). In contrast, mRNA levels for both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) decrease under hypoxic conditions (93). One can envision how these two

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Figure 2 Hypoxia (H) increases expression of PAI-1 mRNA (A) and antigen (B) compared with normoxic (N) conditions. (From Ref. 93.) mechanisms—decreased expression of plasminogen activators and increased expression of a potent suppressor of plasminogen activation—will synergize to potently suppress fibrinolysis. Experiments using mice deficient in PAI-1, t-PA, or u-PA provide strong evidence for the relevance of hypoxia-induced suppression of fibrinolysis in mediating fibrin accrual. Mice lacking PAI-1 showed significantly decreased intravascular fibrin deposition in hypoxia-exposed lungs compared to wild type mice. In contrast, t-PA−/− and u-PA−/− mice demonstrated a strong potentiation of fibrin formation on exposure to hypoxia (Fig. 3). Immunohistochemical analysis of cells in hypoxic lungs once again identified mononuclear phagocytes as the prime source of increased PAI-1 expression under conditions of low oxygen tension. As PAI-1 expression is increased by Egr-1 transcription factor (8), these data support the conceptual framework indicating that Egr-1 acts as a master switch regulating a range of effector mechanisms underlying hypoxic

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stress. Together these data lend support to the notion that the net fibrinolytic activity during the hypoxia is decreased, and that PAI-1 overexpression is an important factor in orchestrating this process. There are also other mechanisms that can contribute to the prothrombotic diathesis of hypoxia. As previously described, hypoxia sets in motion a cascade of events leading to progressive decrease in cAMP content and increase in intracellular Ca2+ concentration. This triggers the release of von Willebrand factor (vWF) from preformed storage pools in endothelial Weibel-Palade bodies, via Ca2+-dependent exocytosis (48). vWF has an important role in coagulation because it fosters platelet adhesion and aggregation, especially under conditions of high shear stress; thus, its

Figure 3 Relative 125I fibrinogen deposition (A–C), a surrogate marker for fibrin formation, is increased in mice lacking key components of the fibrinolytic system (uPA or tPA deficient). Mice lacking the fibrinolytic inhibitor (PAI-1) exhibit diminished fibrin accrual. (D) Similar results are seen on an immunoblot, which detects a neoepitope revealed in cross-linked fibrin. (From Ref. 93.)

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release under conditions of hypoxia can be viewed as an additional mechanism promoting the procoagulant phenotype of the endothelium. Interestingly, administration of a NO donor (3-morpholinosydonimine, SIN-1), or the cGMP-analog 8-bromo-cGMP decreased the hypoxia-induced release of P-selectin and vWF (94) but inhibition of endotheliumderived NO has the opposite effect on Weibel-Palade bodies exocytosis (95). NO also has other very important anticoagulant functions which are perturbed in states of ischemia or reperfusion, in which its synthesis is suppressed or it rapidly dissipates in a superoxide-rich environment. NO maintains the anticoagulant endothelial phenotype by inhibiting platelet aggregation and adhesion (96–98), and by decreasing retraction of lateral margins of endothelial cells thereby preventing exposure of subendothelial TF and collagen to direct contact with blood contents. The procoagulant aspects of NO dissipation are particularly prominent during the postischemic period in which restoration of blood flow (reoxygenation) induces ROS production in neutrophils that have accumulated during the hypoxic period. These ROS are highly reactive and rapidly react with NO quenching it and decreasing the bioavailability of NO (99). It has been shown that supplementation with the NO precursor L-arginine improves vascular function in the setting of lung reperfusion (100), and cardiac preservation is likewise enhanced in a cardiac transplantation model by administration of the NO donor nitroglycerin (101). There is one additional mechanism whose perturbation may contribute to a generalized hypoxic prothrombotic diathesis. Endothelial CD39 (nucleoside triphosphate diphosphohydrolase 1, NTPDase-1) is a recently recognized transmembrane protein, whose extracellular portion exhibits ectoapyrase activity. It hydrolyzes ADP released from activated platelets, thus strongly inhibiting ADP-induced platelet aggregation (102). It has been shown that administration of soluble CD39 has a major influence on maintaining blood fluidity and reduces microvessels thrombosis in different models such as brain ischemia (18). The regulation of CD39 expression under hypoxic conditions is currently under study by several groups.

9. CONCLUSION Endothelial cells, as well as other cells of the vascular wall, including mononuclear phagocytes and vascular smooth muscle cells, are prime movers in the delicate balance that preserves nutritive flow while maintaining immune, barrier, and hemostatic functions. Powerful and redundant mechanisms have evolved to protect this critical homeostatic balance. For instance, several primordial and exquisitely sensitive subcellular oxygen-sensing mechanisms have evolved as adaptive or protective mechanisms. In the absence of oxygen or in the presence of ROS generated during reperfusion, these mechanisms are triggered to unleash a torrent of cascades designed to restore homeostasis. Depending on the exuberance of the response and the specific backdrop, these cascades are often, however, frankly pathological. Hypoxia thereby causes the endothelial phenotype to become procoagulant and proinflammatory, the barrier to lose its selectivity, and various mediators to be released, leading to vasoconstriction. Although in the setting of an infectious process or a traumatic wound, these mechanisms may be adaptive and lead to healing, in other settings these changes in endothelial phenotype can lead to vascular compromise and organismal demise. Understanding the molecular basis

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for the hypoxic modulation of the endothelial phenotype may ultimately lead to new treatments as myocardial infarction, stroke, and a litany of other ischemic disorders.

ACKNOWLEDGMENTS/DISCLOSURES This work was supported in part by grants from the National Institutes of Health; R01 NS41460, R01 HL59488, R01 HL55397, R01 HL69448, and R01 HL060900. Dr. Pinsky has served as a consultant for and received research funds from Aga-LindeHealthcare and has an equity position in St. Camillus Medical.

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10 Fluid Mechanical Forces as Extrinsic Modifiers of Endothelial Function Johannes R.Kratz, Kush Parmar, Sripriya Natarajan, and Guillermo García-Cardeña Laboratory for Systems Biology, Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A.

1. INTRODUCTION From the moment the heart starts beating and blood flow is first established for first time in the developing vertebrate embryo, the cardiovascular system is constantly exposed to fluid mechanical forces. The pulsatile nature of blood flow generates a complex interplay of three distinct types of fluid mechanical forces: wall shear stresses, cyclic strains, and hydrostatic pressures (1). These hemodynamic factors act on the cells that comprise the vascular wall, in particular the endothelium, influencing their structure and function (2). There is increasing evidence that mechanical stimulation plays an important role in the development of the vasculature, the maintenance of vascular integrity and homeostasis, and the development of vascular diseases (2–5). The endothelial lining of the heart and vasculature comprise a dynamic interface with the blood and acts as an integrator and transducer of both humoral and mechanical stimuli (3). This single-cell-thick layer is able to rapidly sense changes in blood flow and respond by secreting or metabolizing potent vasoactive substances (e.g., nitric oxide) that contribute to pressure/flow homeostasis. In face of chronic flow changes, a more deliberate structural remodeling of the vessel wall also can occur via endothelium-dependent mechanisms (6,7). These adaptive responses reflect rapid changes in protein function, enzymatic activity, and transient or long-lasting effects on endothelial gene expression. Moreover, multiple studies in in vitro model systems have confirmed that fluid shear stresses, comparable to those generated by the frictional force of blood flow on the endothelial lining in vivo, can directly influence protein function, enzymatic activities (1), and transcriptional events in cultured endothelial monolayers influencing their functional phenotype (8–11). It remains a central question in the field of vascular biology how these mechanical forces are sensed by the cells of the blood vessel wall and then translated into pathophysiologically relevant phenotypic changes. Activation of various signaling cascades and transcription

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factor systems have helped to provide insight into the cellular mechanisms linking shear stress stimuli and genetic regulatory events. It is now clear that endothelial cells have the capacity not only to sense fluid mechanical forces, but also to discriminate among distinct types of forces (8,10) Collectively, these observations strongly suggest that fluid mechanical forces can act as local “extrinsic modifiers” of endothelial functions within the vascular tree. This chapter focuses on the emerging areas where the role of fluid mechanical forces, in particular shear stress, is being explored with exquisite experimental approaches that allow us to unveil the molecular links between mechanics and biology.

2. CHARACTERIZING THE FLUID MECHANICAL STIMULI AND THE ENDOTHELIAL MECHANOSENSING SYSTEM 2.1. Blood Flow Patterns in the Human Circulatory System Although one central organ, the human heart, generates the force to pump blood throughout the entire vascular network, the different location with respect to the heart, diameters, curvatures, and surface characteristics of blood vessels lead to distinct blood flow patterns in different regions of the human vasculature. As a result of these differences, a complex set of fluid mechanical forces (e.g., shear stress, pressure, cyclic strain) are continuously imposed on the endothelial lining of these vessels. Thus, a current challenge in this area is to characterize with high accuracy the distinct flood patterns experienced by the endothelium along the circulatory system in order to simulate these flow profiles in vitro, and to be able to link hemodynamics with in vivo measurements of endothelial function. For large and medium scale vessels, the cells in blood are so small relative to the vessel diameter that the blood can be approximated as a Newtonian fluid. Thus, meaningful measurements of blood flow velocities can be made, and the shear stress that the endothelial cells sense is linearly related to the change in velocity profile from the center to the wall of the vessel. The most common technique for measuring blood velocity is Doppler ultrasound. In this method, the change of frequency in sound waves reflecting from inside the vessel is used to determine the velocity of the blood flowing within (12). Magnetic resonance (MR) imaging can also be used to measure blood velocities (13). In addition, structural MR or CT images of the vessels can be combined with ultrasound measurements of blood velocities to mathematically compute the blood flow patterns as they change through the length of a vessel (14). These techniques have demonstrated several distinct patterns of blood flow. Along the straight portions of medium and large arteries, e.g., the internal carotid artery or the abdominal aorta, the direction of blood flow is parallel to the vessel wall. There is a pulse of fast flow (peak shear stress from 10–70dyn/cm2) (15), followed by a deceleration to a

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slower flow rate, and then a more gradual acceleration to a steady velocity that is maintained for the rest of the cardiac cycle (shear stress of 0–10 dyn/cm2). In some vessels, blood may transiently flow backwards during the deceleration phase. This pulsatile nature of the flow, characterized by the rate at which blood accelerates to its peak speed, is a hallmark feature of arterial flow. In large and medium-caliber straight veins (e.g., saphenous veins), blood flow is still predominantly one-dimensional, but is much slower (peak shear stress of approximately 1–2dyn/cm2). Blood travels at this speed in the forward direction for approximately twothirds of the cardiac cycle, and in the reverse direction, at a similar speed, for the rest of the time. The acceleration and deceleration of the blood to reach its peak velocities are much slower than they are for arterial flow. In the branch points and curvatures of vessels, these flow patterns no longer hold. In arteries, disturbed flow occurs at these points—three-dimensional whorls of flow that often have a very low time-average speed in any one given direction (14). The aortic arch, carotid bulb, and branch points of the aorta are all regions that experience disturbed flow. Although different flow patterns have been established for several vessels, there are still many more regions that remain to be well characterized. The coronary arteries in particular are of great clinical interest, due to the high prevalence of ather-osclerosis in these vessels, but their smaller diameter (in comparison to the aorta or carotid artery) and constant motion with the beating heart has made measuring the velocity within the coronary vasculature especially challenging. The shear stress that endothelial cells experience in capillaries has also been challenging to characterize since whole blood can no longer be considered a Newtonian fluid owing to the small diameter of vessel.

2.2. Modeling Blood Flow Patterns In Vitro Much insight into the responses endothelial cells display when exposed to flow comes from in vitro experiments, where the effect of flow can be studied in a well-defined and controlled fashion and in isolation from other hemodynamic factors, such as blood pressure and cyclic strain (1). Thus, modeling more realistic flow patterns seen that better approximate those seen in vivo has been a constant challenge of such experiments (Fig. 1). The first in vitro flow experiments exposed endothelial cells to a uniform laminar shear stress with a value equal to physiological time-average values. Such laminar flows can be generated using either parallel plate chambers (16,17) or a cone-plate apparatus (18). With the latter device, the rotation of the cone can be controlled to generate onedimensional waveforms that may be closer to the cyclic nature of physiological flows— sinusoidal waveforms or oscillating square waves (19). More recently, a cone-plate apparatus controlled by a computerized motor has been able to reproduce most onedimensional physiological flow waveforms as captured by Doppler ultrasound (20). Another approach to mimic more physiological flows has been to grow endothelial cells on small tubes and pump fluid through. Pumping fluid through these tubes in a cyclic fashion exposes the endothelial layer to a pulsatile flow pattern (21). However, the precise definition of the several hemodynamic components present in these systems remains to be defined.

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Mimicking the two-and three-dimensional disturbed flow patterns seen in sites of pathology—such as the atherosclerosis-prone carotid bulb—has been another important in vitro effort. Cone-plate devices with sufficiently large angles between the cone and plate will generate turbulent (i.e., randomly varying, both temporally and spatially) instead of laminar flow over endothelial cells (19). Another model places a square step in the lower plate of a parallel plate chamber, establishing disturbed (i.e., spatial-varying) flow patterns at the junction of the step and plate (22). All these models have been extremely helpful for increasing our understanding of the influence of shear stress on the structure and function of endothelial cells.

2.3. Endothelial Mechanotransduction The capacity displayed by endothelial cells to sense and discriminate distinct flowinduced shear stresses raises the question of how these cells sense these mechanical

Figure 1 Modeling blood flow patterns in vitro. Different characteristics of the complex flow patterns seen in the vasculature have been captured in flow patterns used for in vitro experiments. Typical flows have been modeled as steady laminar flow, where the shear stress is constant over time and maintained at a physiologic average level, or as oscillatory flow with either a square wave or sinusoidal patterns.

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Nonlaminar flows have been modeled as turbulent (varying randomly with time) or disturbed (varying over space). Recently Doppler ultrasound measurements of flow have been used to develop flow patterns that more closely mimic physiological patterns, such as one-dimensional abdominal aortic flow. forces. Although the true nature of the mechanosensing and mechanotransduction systems in endothelial cells remains poorly understood, several plausible and promising hypotheses have been put forward in recent years. Donald Ingber and colleagues have proposed that the main mechanism of force-sensing by cells comes from changes to the cytoskeletal structure as a cell is deformed by mechanical forces. According to this model (termed, tensegrity), flow applies a shear stress-derived force to the entire cytoskeleton and some transmembrane molecules (e.g., integrins) that are linked to the cytoskeleton. This cytoskeletal association allows such molecules to exert a force through cytoskeletal components, transducing the force to a different region of the cell—perhaps altering nuclear structure and affecting transcription, or changing the physical conformation of a protein to activate it. In support of such a theory, the stiffness of the cell cytoskeleton has been demonstrated to increase in response to selectively applying a shear force to certain cell surface integrin receptors. In addition, these forces immediately cause changes at a distance, in the arrangement of molecular assemblies in the cell nucleus (23). The tensegrity model does not exclude the possibility of additional discrete molecules that serve to sense shear stress and transduce a signal independently of the cell cytoskeleton. Mathematical models of a molecule comprised of viscoelastic elements show that such a sensor on the surface of an endothelial cell would deform drastically at the onset of laminar flow, then approach a steady state behavior, which is the pattern seen in many endothelial responses to flow (24). In addition, such an element’s deformation would be different upon exposure to distinct types of shear stress (25). In the past several years, a number of putative flow sensors have been proposed. Ion channels are a major class of molecules that may behave in such a manner. Within seconds after cessation of flow over pulmonary endothelial cells that have been exposed to flow for 24 hr, the cells experience a membrane depolarization of approximately 20 mV, which is mediated by an inwardly rectifying K+ ATP channel (26). This change in membrane potential can cause a Ca2+ flux into the cell, triggering a number of signaling events (26,27). Na+ and Cl−channels may also be involved in sensing flow-induced shear stress (28,29). Other cell surface molecules have also been suggested to function as endothelial flow sensors. For example, under flow-induced shear stress, the VEGF receptor 2 (VEGF-R2, Flk-1) can be activated independently of its ligand (30). VEGF-R2 has been shown under flow to form a complex with the endothelial adherens junction proteins VE-cadherin and β-catenin. In endothelial cells derived from VE-cadherin −/− mice, several flowdependent phosphorylation events downstream of VEGF-R2 activation and several gene regulation changes were absent (31). Furthermore, αvβ3 and α1 integrins have been

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shown to be necessary for the rapid tyrosine phosphorylation of the VEGF-R2 in response to flow (32). Blocking antibodies against another candidate flow sensor, caveolin-1, which is one of the major structural proteins of caveolae, prevent the phosphorylation of ERK-1/2 that is normally induced in endothelial cells by flow (33). Interestingly, bovine aortic endothelial cells exposed for 24–72 hr to flow-induced shear stress exhibit a shift of caveolin-1 localization from the Golgi complex to the cell surface and increase the number of their caveolae (34). These results are consistent with the hypothesis that caveolae may serve as flow sensing organelles (35) and support the concept that endothelial cells may increase the concentration of mechanosensitive receptors in response to flow in order to become more sensitive to this stimulus. Additional molecules have recently been shown to be more directly affected by flow. Glycosaminoglycans and the proteoglycans to which they bind are promising candidates as flow sensors. The molecules form an often-overlooked apical extracel-lular layer known as glycocalyx. Selective removal of the heparan sulfate component of glycocalyx from endothelial cell membranes greatly abrogates their NO production in response to flow. Meanwhile, NO production in response to bradykinin, histamine, or acetylcholine remains largely unaffected (36,37). NMR studies have proposed a mechanism for this mechanotransductive effect, showing that proteoheparan sulfate changes from a random coil to a filamentous form when exposed to flow, enabling it to bind Na+ and triggering vasodilation (38). Platelet endothelial cell adhesion molecule-1 (PECAM-1) is phosphorylated in response to flow-induced shear stress and in turn promotes ERK phosphorylation. Using antibody-coated magnetic beads, PECAM-1 was selectively exposed to a tangential force, triggering its phosphorylation (39). G-proteins have been implicated in many of the pathways triggered by flow-induced shear stress, but they have also been proposed as a sensor of the flow itself. The application of 0–30 dyn/cm2 of flow-induced shear stress to phospholipid vesicles containing purified G-proteins, activated the G-proteins, with increased activation at higher levels of shear stress (40). Although these approaches to selectively shearing or removing molecules do not entirely exclude cytoskeletal transmission of forces, they do indicate that heparan sulfate, PECAM-1, and G-proteins are highly plausible candidates as endothelial flow sensors. It is thus clear that several endothelial cell molecules respond to fluid mechanical forces, and these responses may or may not be directly related to cytoskeletal changes determined by cellular tensegrity. Future challenges will be not only to uncover the integration of these various mechanosensors at the cellular systems level, but also to probe the selective importance of these sensors for specific cellular processes, which will ultimately help us better understand their dysregulation in disease and develop targeted therapeutics.

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3. FLUID MECHANICAL FORCES AND VASCULAR DEVELOPMENT 3.1. Historical Perspective The modern era of cardiovascular developmental biology dates to the major experimental and conceptual breakthroughs of the latter part of the 19th century. The German embryologist Wilhelm Roux postulated vascular development to proceed in three phases: a stage of primary differentiation governed entirely by “hereditary principles,” followed by a transitional stage wherein the genetic element is gradually supplanted by functional adaptation, and finally a stage where further vascular development is completely regulated by mechanical forces acting through the circulation (41). As we hope to convey in this section, the experimental knowledge we have amassed to date appears to confirm and extend Roux’s visionary paradigm of vascular development. Near the end of the 19th century, Richard Thoma penned the three biomechanical laws that would shape the study of cardiovascular development for the decades to follow (42). These laws, translated into English by Bruce, state the following: (1) “The increase in size of the lumen of the vessel wall depends on the rate of the blood current”; (2) “The growth in thickness of the vessel wall is dependent on its tension”; and (3) “Increase in the blood-pressure in the capillary areas leads to new formation of capillaries.” These laws, made by careful observation and deductive logic, were to set the stage for contemporary experimental biologists in the field of cardiovascular development. Elegant work by Thoma and Sabin had already established the presence of an extensive vascular network at the time circulation begins in the chick embryo, including the paired dorsal aortae, the duct of Cuvier, and cardinal veins (42). Given the existence of this primitive vascular plexus, subsequent studies were the first to explore the interrelationship of cardiac function and cardio-vascular development. In his landmark paper, Chapman studied later development of the vascular system after “eliminating most of the mechanical factors mentioned by Thoma.” He did this by surgical removal of the entire heart before the onset of circulation, and noticed a marked failure to form the major omphalomesenteric arteries and veins (43). This result, he concluded, confirmed the existence of Roux’s second stage of development. Indeed, other studies by Loeb and Stockard using potassium chloride to abrogate cardiac contractility came to similar conclusions (44). A study by Knower noted the irregular course of vessels and impaired formation of glomeruli after removal of the heart in frog embryos (45). These latter studies suggested that later stages of vascular development were subject to regulation by blood flow. There were several drawbacks to the pioneering work described above, including relatively primitive experimental manipulation of living embryos and possibly confounding effects of hypoxia and nutrient delivery. The limitations notwith-standing, they were to foreshadow the current era of molecular underpinnings of biological processes as it applies to vascular development.

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3.2. Cardiovascular Development: Genetics and Epigenetics The interplay of genetic and epigenetic influences on cardiovascular development is still poorly understood, but experimental verification of the importance of this relationship has recently been very informative. One of the most important advances to enable the understanding of these processes has been the identification of molecular markers of distinct endothelial identities. For example, vascular structures have been physiologically distinguished as arteries or veins since the time of William Harvey, but the elucidation of a molecular distinction only came with the recent discovery of the expression pattern of Ephrin-B2 in mouse embryos (46). Ephrin-B2 is expressed specifically in arterial endothelium and smooth muscle cells (47). Mice homozygous null for the EphrinB2 gene display vascular defects specifically in the arterial vessels (46). Analogous studies the zebra fish Danio rerio first identified Hey2, a transcriptional target of the canonical Notch pathway, as exhibiting arterial-specific expression (48). Homozygous hey2 mutants completely fail to fashion the dorsal aorta and have hearts reminiscent of coarctation defects commonly seen in human congenital heart disease (49). The number of molecular markers of arteries and veins has grown since these first series of studies were reported. They include Notch family members, Ephrins and their receptors, Neuropilins, and Connexin 40 among others (see Chapter 7) (50). The identification of these markers, in conjunction with the development of the zebra fish as an experimental system, has allowed for some of the most revealing in vivo studies probing the role of fluid forces in cardiovascular development. The power of this model system to ask questions about development lies primarily in the fact that the zebra fish embryo is perfectly viable with oxygen delivered by diffusion only, with no need for erythrocytes for at least five days, well past the development of a complete cardiovascular system. The first study to identify the relationship between cardiac function and peripheral vascular developmental in zebra fish was through a mutagenesis screen for defects in kidney development (50). It was noted that most of the embryos with impaired renal development also exhibited severe cardiac defects. To ask whether there was a functional relationship, the group examined embryos mutant for a known cardiaospecific gene required for contractility. Indeed, the authors of this study discovered in these cardiac mutants, a marked impairment in formation of the glomerular capillary network, which norm ally migrates to form an intimate plexus with the primordial renal tubular network (51). It was found that the endothelium of these heart mutants had dramatically reduced levels of MMP-2 expression, an important metalloproteinase for glomerular development. Importantly, reduction of MMP-2 levels could phenocopy the renal defects of the heart mutants. Lastly, in a careful control for the confounding issue of tissue perfusion, complete replacement of blood with saline in normal embryos had no effect on formation of the glomeruli. Thus, the mechanical force of blood flow is critical for proper glomemlar capillary development in zebra fish, and this was related to an induced change in gene expression in glomerular endothelium. It is interesting to note that the study a century before by Knower came to this same conclusion in frog embryos (44). Like the endothelium of vessels, the endocardial lining of the heart readily senses shear stress, and the role of these forces during cardiogenesis has recently been elegantly

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probed in zebra fish. Hove et al. used in vivo imaging to reveal the flow vortices inside the atria and ventricle of the developing zebra fish heart (52). Importantly, calculated shear stresses from these data were substantial (~2.5dyn/cm2 at 37 hr, ~76 dyn/cm2 at 4.5 days), well within the limits detectable by non-endocardial endothelial cells (minimum of ~1 dyn/cm2). This indicates that, at least in zebra fish, endocardial cells could transduce this mechanical force into altered gene expression and consequent cardiac morphogenesis. Indeed, Hove et al. observed severely disrupted cardiogenesis after occlusion of either the sinus venosus or the outflow tract in the early embryo. Because these two manipulations would produce opposing effects on intracardiac pressure, it follows that the commonality of abrogated shear stress is the underlying factor critical for proper cardiogenesis. In these occluded hearts, Hove et al. observed failure to form the third chamber, the bulbus cordis, and also impaired cardiac looping, a conserved mechanism that shifts the atrio-ventricular alignment from cephalocaudal to lateral positions. Lastly, they noted the collapse of both the inflow and outflow tracts. Intracardiac fluid forces are thus an essential component of proper cardiac development, and could possibly underlie the large percentage of congenital heart diseases of idiopathic nature. The role of the fluid forces in the formation of the vascular network and the identity of arteries and veins during zebra fish development has also recently been examined. In silent heart embryos, which have no heart beat, endothelial-specific transgenic expression of GFP reveals that the overall pattern of the vascular system is intact, just as the embryologists mentioned above noticed in their primitive experiments. However, careful utilization of this experimental system revealed that the fluid forces of circulation are in fact necessary to determine the functional relationship of angiogenic sprouts and the original vessel tree (53). Fluid forces thus determine the arterial or venous fate of endothelium within the context of the fully developed circulatory plexus in the zebra fish trunk. The field of vascular development, at a time of sophisticated methods and complex hypotheses, thus comes full circle with the visionary postulates by Roux.

3.3. The Lymphatic System A far less studied aspect of vascular development is the lymphatic circulation. Despite the dearth of molecular insights into lymphatic endothelium, some important work has implicated lymphatic flow as a critical mediator of lymphangiogenesis. Boardman et al. (54) replaced a small band of dermis from mouse tails with a type I collagen gel, allowing for flow of interstitial fluid and unobstructed visualization of lymphangiogenesis. Importantly, flow of lymphatic fluid preceded formation of the lymphatic vessels, which were identified with surface markers Flt4 and LYVE-1 (lymphatic endothelial hyaluronan receptor). Fluid channels in the collagen gel were observed well before the lymphatic endothelial cells formed proper tubes, and appeared to guide the precise tracks taken subsequently by the migrating endothelial cells. Furthermore, an important link was found between the expression pattern of VEGF-C, a major lymphangiogenic factor, and the direction of growth of these vessels. The pattern of VEGF-C expression suggested a role for fluid flow in the expression and transport of this molecule, as it first appeared at the sites of fluid channels. The proposed model implicates a central role for interstitial fluid flow in the guidance of subsequent migration

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and lumenization of lymphatic endothelium. This is in contrast to the vasculature, where lumen patency precedes fluid flow, rendering the role of flow more limited to regulation of the endothelium in the already formed vessel. The above studies provide strong evidence that fluid mechanical forces are critical for a number of aspects of cardiovascular development. However, they leave us with little understanding of the molecular mechanisms underlying this fascinating paradigm. From the question of mechanosensing by the endothelium, to the translation of these signals to changes in gene expression, to cell-cell signaling within the microenvironment, there lays an enormous gap in understanding between fluid forces and ultimate organismal development. This evolutionary conserved role of fluid flow is probably specifically adapted to each process, but some commonality might also exist. Even from the cursory understanding we are privy to now, it is apparent that changes in MMP expression are implicated in the role of fluid forces in both glomerular and lymphatic development. Surely unbiased investigation of endothelial and endocardial genomic expression patterns in response to shear stresses will aid to bridge the gulf between mechanical forces and complex developmental processes. Furthermore, the genetic specialization of a particular endothelial bed (e.g., endocardial, glomerular, cerebral) will add a further level of complexity in the genomic response to fluid forces. It is this interaction of microenviromental epigenetic influences with classical genetic predetermination that must confer an endothelial phenotype befitting its context.

4. FLUID MECHANICAL FORCES AND DISEASE 4.1. Atherogenesis The involvement of vascular endothelium in disease processes such as atherosclerosis has been recognized since the time of Virchow (55). However, mechanistic insight into the pathobiology of this tissue has developed only recently, largely as a result of the application of modern cellular and molecular biological techniques (56). We now appreciate that the single-cell thick lining of the circulatory system is, in fact, a vital organ whose health is essential to normal vascular physiology and whose dysfunction can be a critical factor in the pathogenesis of vascular disease. For example, the nonrandom distribution of early lesions of atherosclerosis in human subjects and experimental animals remains one of the most consistent, intriguing, and incompletely understood aspects of this disease process. Lesions typically develop in the vicinity of branch points and areas of major curvature within the arterial vasculature. Physical and computational models have identified these vascular regions as having low time-average shear stress, a high oscillatory shear index, and steep temporal and spatial gradients in shear stress. In contrast, unbranched arterial geometries that are exposed to more uniform laminar flows appear relatively protected from lesion development (57,58). In an effort to unveil mechanistic links between hemodynamics and atherogenesis, research over the last 20 years has focused on emulating these flow characteristics in vitro (as described in Sec. 1 of this chapter) to delineate the signaling pathways and downstream changes at the level

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of gene expression and cell structure-/function. The development and use of these in vitro models in several laboratories has allowed investigators to demonstrate direct effects of biomechanical forces on the expression of pathophysiologically relevant endothelial genes (3). In addition, differential upregulation of certain “atheroprotective” genes (i.e., eNOS, Mn-SOD, and COX-2) in endothelial cells exposed to laminar shear stress, but not to nonlaminar shear stress has been documented (59). These observations led to a guiding hypothesis for the field developed by Gimbrone and colleagues (59), namely, that the steady laminar shear stresses characteristic of lesion-protected areas elicit expression of “ather-oprotective” genes, whereas the altered shear stresses generated by disturbed laminar flows in lesion-prone areas elicit the expression of “atheropathogenic” genes (and/or suppress “atheroprotective” genes). To test this hypothesis at a fundamental cellular level, the Gimbrone laboratory and others used transcriptional profiling to capture the gene expression programs of endothelial cells exposed to distinct types of biomechanical stimuli (8,10). Results from these experiments clearly demonstrated that endothelial cells can differentially sense and transduce distinct biomechanical input stimuli into different patterns of gene expression. Moreover, using these profiles as predictors of function, these groups were able to demonstrate that endothelial cells can translate these input stimuli into distinctive functional phenotypes. Critical testing of Gimbrone’s “atheroprotective gene hypothesis” will depend upon refinement of both in vitro and in vivo fluid mechanical models and a validation of candidate atheroprotective genes in the setting of human vascular pathobiology. The development of reliable methods for linear amplification of transcripts from small numbers of cells and their analysis by cDNA microarrays or analogous genome scale technologies holds much promise in this regard as recently demonstrated by Peter Davies’s group (60). Application of these comprehensive and relatively unbiased methods of molecular analysis to endothelial cells subjected to experimentally defined flow conditions will add significantly to our understanding of the dynamic range of biomechanically induced phenotypic modulation. Ultimately, the extension of this method of analysis to endothelial phenotype in the natural disease context should provide valuable new insights into the links between fluid mechanical forces and atherogenesis.

4.2. Coronary Artery Bypass and Graft Failure While several medical therapies have been developed to prevent coronary artery disease, such as antihypertensive, cholesterol-lowering, and diabetes-controlling agents, surgical and interventional therapy remain the definitive treatment for advanced disease. Today, coronary artery bypass graft (CABG) surgery is performed on over 400,000 patients each year in the United States with a significant reduction in morbidity and mortality. Unfortunately, within the first year after surgery, 15% of venous grafts occlude, and after 10 years, only 60% of vein grafts remain patent. Subsequent revascularization, either reoperative surgery or percutaneous intervention, is required in 4% of patients after 5 years and 19% of patients after 10 years (61). The clinical impact of saphenous vein graft disease is currently increasing, and efforts to reverse this trend mandate an improved understanding of the molecular mechanisms of venous arterialization and the pathogenesis of failed bypass grafts.

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The remodeling of vessels in response to changes in environmental cues (e.g., blood flow, oxygen tension) as occurs during CABG surgery may initially be adaptive, but eventually becomes pathologic with the development of thrombosis followed by a cascade of events leading to graft failure (62). Arterialized venous bypass grafts demonstrate accelerated arteriosclerosis compared to normal vessels in spite of the fact that veins in their endogenous sites rarely develop atherosclerotic lesions. Thus, the biomechanical environment of the arterial milieu seems to play a critical role in vessel remodeling and subsequent graft failure. The venous-to-arterial shift of the local environment engenders reorganization of the venous vascular architecture, such that over time, the venous graft acquires an arterylike structure. Early anatomical adaptive changes include an elevation in cellular mass mainly caused by an increase in the number of smooth muscle cells in the vessel wall (medial thickening). Several studies have demonstrated that cell proliferation and apoptosis play an important role in this process (63). Insights into the molecular mechanisms of venous arterialization have come from studies in which the expression of individual molecules was characterized. These studies have demonstrated the dynamic expression of genes involved in extracellular matrix formation and turnover in the course of the adaptive changes displayed by the vein during its arterialization (62,64,65). In addition, the expression of several growth factors has been shown to be modulated during the arterialization process including TGF-β, PDGF, and VEGF (62,63). Direct insights into the functional role of specific cells of the vessel wall in vascular remodeling have been mainly derived from studies of arterial remodeling. Studies conducted in models of arterial remodeling demonstrated the obligatory role of vascular endothelium in this process (7). More recently, specific endothelial derived molecules (e.g., nitric oxide) have been shown to be responsible for this endothelial-dependent response (66). These studies strongly suggest that the vascular endothelium plays a prominent role in vessel remodeling by sensing changes in the biomechanical environment and responding to them by changes in gene expression and the production of bioactive molecules. Clearly, the molecular mechanisms of venous arterialization remain to be elucidated. The recent development of a mouse model of venous arterialization in mice (67) should help us to define such mechanisms. The extent to which the phenotype of the endothelium changes in response to the new biomechanical environment (venous-to-arterial), and how these changes orchestrate the remodeling process in the context of venous arterialization in health and disease remain central questions in the field.

5. CONCLUSIONS Our ability to couple in vitro models of pathophysiologically relevant hemodynamic environments with systems biology approaches will continue to yield molecular insights into the mechanoactivated programs in vascular endothelium. Validation of these programs in the context of intact blood vessels should mechanistically link endothelial mechanobiology, vascular development, and cardiovascular disease.

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11 Vascular Bed-Specific Signaling and Angiogenesis Napoleone Ferrara, Rui Lin, and Jennifer LeCouter Department of Molecular Oncology, Genentech Inc., South San Francisco, California, U.S.A.

1. INTRODUCTION The cardiovascular system is the first organ system to develop and reach a functional state in an embryo (1). “Vasculogenesis,” the in situ differentiation of endothelial cell precursors, and “angiogenesis,” the growth from the endothelium of existing vessels cooperate in achieving the mature vasculature (2). Recent studies have complemented this view, suggesting that incorporation of bone marrow-derived endothelial progenitor cells (EPC) into the growing vessel contributes to normal and abnormal angiogenesis (3– 5). Blood vessel growth is also implicated in the pathogenesis of a variety of proliferative disorders, including tumors, intraocular neovascular syndromes, rheumatoid arthritis, and psoriasis (6–8). The endothelial cells that comprise the vascular beds of specific tissues display unique phenotypes, growth properties, and functions (9). Recent studies have shown that endothelial cells induce differentiation of liver and pancreas (10,11), suggesting that gut endothelium may have unique paracrine properties, independent on their role in providing a blood supply. This diversity extends also to the vasculature of tumors. Some of the earliest pioneering work in the field of tumor angiogenesis resulted in the seminal observation that the tumor vascular architecture is different depending on the tumor type, leading to the far-reaching hypothesis that the microenvironment has a major influence on the growth and morphologic characteristics of tumor vessels (12). Although distinct endothelial features have been noted to reflect functional requirements imposed by the tissue context, the mechanisms that determine these properties have not been well defined. In vivo (13,14) and ex vivo (15) experiments that have assessed endothelial cell phenotypic and functional development have indicated the existence of endothelial cell-extrinsic signals. These may include secreted peptides, extracellular matrix, or cell membrane components. Recent work has assessed in vivo selective binding of peptides displayed on phage, or assignment of so-called “vascular address” (16). In the adult, angiogenesis is restricted to and required for reproductive function and wound healing. Several angiogenic factors with otherwise pleiotropic

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activities have been reported including angiogenin, hepatocyte growth factor (HGF), acidic and basic fibroblast growth factor (FGF), and interleukin (IL)-8 (17–20). The requirements for the endothelial cell specific, angio-genic factors, vascular endothelial growth factor (VEGF) (21) and the angiopoietins Ang-1 (22) and Ang-2 (23) and their cognate receptors, were demonstrated in a series of genetic studies in mouse (24,25). Although VEGF and the angiopoietins are largely selective for endothelial cells, they are widely expressed (20,26). Therefore, it has been difficult to reconcile endothelial cell phenotypic diversity with the action of these ubiquitous factors. However, recent studies have provided evidence for certain vascular bed-specific response to VEGF and other angiogenic factors such as bFGF. The physiological properties of vessels induced by these factors were mainly determined by the micro-environment (14). Furthermore, a recent study has shown that liver sinusoidal endothelial cells strongly up-regulate HGF in response to VEGFR-1 agonists, while this response is not observed in other endothelial cell types (27). Therefore, it is conceivable that morphological and functional diversity among endothelia is achieved by several mechanisms, including vascular bed-specific response to ubiquitous mediators and the existence of unique mitogens/differentiation factors with a tissue-restricted expression pattern.

2. EG-VEGF EG-VEGF was identified as a novel human endothelial cell mitogen, through a bioassay assessing the ability of a library of purified human secreted proteins to promote the growth of primary bovine adrenal cortex capillary endothelial (ACE) cells (28). EGVEGF does not belong to the VEGF family or other known families of endothelial mitogens but instead is a member of a structurally related class of peptides including the digestive enzyme colipase, the Xenopus head-organizer, dickkopf (29), venom protein A (VPRA) (30) or Mamba intestinal toxin-1, “MIT-1” (31), a nontoxic component of Dendroaspis polylepis polylepis venom, and the secreted proteins from Bombina variegata designated Bv8 (32). The distinguishing structural motif is 10 cysteine residues that form five disulfide bridges within a conserved span, designated a colipase-fold (33). EG-VEGF (80% homologous to VPRA) and VPRA are most closely related (83% and 79% homology, respectively) to the Bv8 peptide. Interestingly, Bv8 and EG-VEGF share the first four amino acids of the mature protein, the sequence “AVIT” that, therefore, appears to be distinctive feature of this protein family (34). Mouse and human orthologues of Bv8, also known as prokineticin-2 (35), have been recently described. Both VPRA/MIT-1 and Bv8 were shown to induce gastrointestinal motility (31,32). More recently, the human orthologues of these proteins were shown to have the same motility-enhancing activity and the denomination of “prokineticins” was proposed to designate this property (35).

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3. EFFECTS OF EG-VEGF ON THE VASCULAR ENDOTHELIUM Significantly, EG-VEGF selectively promoted proliferation, survival, and chemotaxis of endothelial cells isolated from steroidogenic tissues. The mitogenic and prosurvival activities of EG-VEGF correlate with the ability of this peptide to induce phosphorylation of the mitogen activated protein kinases, ERK1/2, and the Akt serine/threonine kinase of the PI3K survival pathway (36). Nitric oxide production influences vascular tone and permeability (37). Endothelial nitric oxide synthetase (eNOS) is a downstream effector of Akt (37). Lin et al. (36) demonstrated the sequential phosphorylation of Akt and eNOS in EG-VEGF-treated ACE cultures, indicating that EG-VEGF may influence vessel permeability in vivo. Indeed, exogenous EG-VEGF in the ovary (28) or testis (38) can dramatically affect vascular leakage. A specialized ultrastructural feature of the endothelial cells of certain capillary beds (e.g., liver sinusoids, and most endocrine glands) is the presence of small plasma membrane discontinuities, referred to as fenestrae (39). These 63–68 nm openings facilitate fluid and solute exchange. Like VEGF (40,41), EG-VEGF can induce the formation of fenestrae in ACE cells, and may in part be an important mediator of this endothelial phenotype in select sites, namely the steroidogenic tissues. In vivo, delivery of adenovirus encoding EG-VEGF resulted in tissue-specific angiogenesis. Although no response was elicited by the administration of EG-VEGF in the skin or skeletal muscle, a potent angiogenic response was apparent within the ovary (28). These data confirmed the tissue-specificity of EG-VEGF response and supported the existence of endothelial-specific receptors with a restricted capillary bed expression.

4. EG-VEGF G-PROTEIN COUPLED RECEPTORS Biochemical studies of ACE cells pretreated with pertussin toxin indicated that the EGVEGF interacts with a Gai-coupled type receptor (36). Two small, highly identical 80– 90% G-protein coupled receptors of the neuropeptide Y (NPY) receptor class have been identified as the cognate receptors for EG-VEGF and the related peptide, Bv8 (42,43). In this context, it is noteworthy that NPY has both neurotropic and angiogenic activities (44,45). These receptors were designated EG-VEGFR-1/ZAQ and EG-VEGFR2/GPCR73 (43) or prokineticin receptor-1 (PKR-1) and PKR-2 (42). Transcripts, and presumably both proteins, for EG-VEGFR-1 and EG-VEGFR-2 are expressed in ACE cultures (43). Importantly, we demonstrated restricted expression of these receptors in endothelial cells residing in the testis interstitial tissue (38). Therefore, similar to VEGF and its receptors, the EG-VEGF/EG-VEGFR system represents a paracrine system, in which the ligand is produced by nonendothelial cells, and the receptors are selectively expressed in the vascular endothelium.

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5. EXPRESSION OF HUMAN EG-VEGF IS PREDOMINANTLY IN STEROIDOGENIC GLANDS Northern blot analysis of a panel of RNAs from a variety of human tissues revealed EGVEGF expression in ovary, testis, adrenal, and placenta, while a consideraly lower signal was noted in a nonsteroidogenic organ like the prostate (28). No significant hybridization signal was detected in other organs by this technique, indicating that steroidogenic glands are the predominant site of EG-VEGF expression (28). In agreement with this conclusion, Zhang et al. have recently reported that, while a very low-abundance signal can be detected in some organs using the highly sensitive Taqman analysis, a dramatically higher EG-VEGF expression signal is measured in steroidogenic organs (46). Among these, the ovary and testis expresses the highest level of the EG-VEGF transcript. In situ hybridization analysis demonstrated that steroidogenic cells within these glands are the source of EG-VEGF (28). Within the testis, the hybridization signal was essentially restricted to the testosterone-producing Leydig cells. The ovary is noted for its dynamic, cyclical growth that is accompanied by a high rate of angiogenesis and thus it has been a major focus for our analyses.

6. DIFFERENTIAL EXPRESSION OF EGVEGF AND VEGF IN THE NORMAL HUMAN OVARY SUGGESTS COMPLEMENTARY FUNCTIONS IN FOLLICULAR AND LUTEAL ANGIOGENESIS We initially reported the expression of EG-VEGF in the ovarian stroma and in the early follicles, restricted to cells of the theca interna (28). Recently, we expanded our earlier analysis of EG-VEGF expression in human and primate ovarian follicles to include a wider range of human preovulatory and atretic follicular stages, and a range of corpus luteum (CL) stages (47). Expression of VEGF and EG-VEGF mRNA was detected by in situ hybridization in all of the specimens examined. Granulosa cells in primordial and primary follicles express EG-VEGF strongly, whereas VEGF expression is very weak or undetectable. VEGF expression is more uniformly detectable but still weak in secondary follicles with 2–3 layers of granulosa cells. As preovulatory follicles mature, VEGF expression appears to progressively increase, so that antral follicles show intense granulosa cell signal that is often associated with moderate or weak VEGF expression in the adjacent thecal layers. As the secondary follicle matures, EG-VEGF expression in granulosa cells declines. Atretic follicles at different stages of their evolution strongly expresses EG-VEGF in the residual thecal cells surrounding the dense hyaline remnant of the follicular basal lamina. VEGF is only weakly expressed in a subset of these cells immediately adjacent to the follicular basal lamina.

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These studies have revealed that the generally complementary expression pattern of EG-VEGF and VEGF also extends to the luteal phase (47). The CL is a hormoneregulated, transient endocrine gland that develops following ovulation and produces progesterone, required for the maintenance of early pregnancy. Much evidence indicates that the VEGF transcript is highly expressed, especially in the early CL (48–51). Administration of VEGF inhibitors early in the luteal phase dramatically suppress luteal angiogenesis (52–54). Such treatment may also delay follicular development (55) in rodents and primates. However, it is debated whether VEGF is highly expressed by midearly-late stage and some studies have pointed out that the VEGF signal is significantly reduced by mid-stage (56). VEGF immunoneutralization studies performed in primates at mid-stage have shown a significant reduction in progesterone levels, but the magnitude of the reduction was considerably smaller than that induced when the anti-VEGF treatment was initiated early in the luteal phase (57). Furthermore, VEGF blockade during early pregnancy in the marmoset reduced progesterone levels, but had little effect on pregnancy rates (58). These findings suggest that, while VEGF-dependent angiogenesis is a critical rate-limiting step for the development of an early capillary plexus, later events may be less dependent on VEGF, raising the possibility that additional factors are implicated. CL derived from ovulatory follicles mature in a canonical 14-day pattern (59). We examined EG-VEGF and VEGF expression in a series of CL representing time points approximately 2–14 days postovulation. At approximately 2–3 days postovulation (time points are inferred, according to the histological criteria of Corner (59)) the EG-VEGF and VEGF expression resembles the pattern seen in the late preovulatory follicle: granulosa cells are intensely VEGF-positive, but lack significant EG-VEGF expression. At approximately 5 days postovulation, both VEGF and EG-VEGF are strongly expressed in a portion of granulosa lutein cells (theca lutein cells are not clearly distinct histologically at this stage; they may also express EG-VEGF and VEGF). At approximately 8 days postovulation, EG-VEGF expression is intense in the theca lutein cells, while VEGF expression has diminished to the point where only weak signal remains in the peripheral thecal cells. The functional significance of “granulosa” versus “theca” lutein cells is still object of debate and may involve compartimentalization of steroidogenic activities (60). However, the finding that VEGF is predominantly associated with granulosa and EG-VEGF with theca lutein cells, suggests an even greater level of specialization than previously realized as well as a differential contribution, both spatially and temporally, to vascular remodeling events in the CL. In agreement with previous findings, peak VEGF expression was found at early-stage, associated with the initial development of a capillary plexus within the human CL. However, EG-VEGF expression was not detectable until early-mid stage CL but persisted throughout the mid luteal phase, at a time when VEGF expression was much reduced or undetectable (47). Taken together, these observations support the notion that VEGF activity is rate-limiting for the creation of the capillary plexus within the CL. Additionally, EG-VEGF may stimulate the angiogenesis that accompanies early-mid CL development and be especially important for the formation of a more mature vascular bed that includes arterioles and thus for the persistence and adequacy of luteal function (47). These hypotheses can be tested directly by assessing follicular, and CL, development and function in primates treated with selective EG-VEGF inhibitors.

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As previously noted, EG-VEGF expression is consistently detected in the ovarian stroma (28). Furthermore, a particularly high expression of EG-VEGF (but not VEGF) mRNA was demonstrated in “hilus” cells (47). These cells are thought to be the functional equivalent of Leydig cells in the ovary (61) and hyperplastic or neoplastic changes affecting them are known to result in a masculinizing syndrome (62,63). These findings corroborate the association of EG-VEGF mRNA expression with a steroidogenic phenotype. Also, the intimate relationship of hilus cells with blood vessels and nerve terminals was noted even in the earliest studies (62,63). Intriguingly, as noted later in this chapter, the EG-VEGF homologue Bv8 has been shown to have neurotrophic (64) and neuromodulator (65) functions. While Bv8 mRNA is undetectable in the human ovary, it is tempting to speculate that EG-VEGF may play both an angiotrophic and neurotrophic role in this context.

7. EXPRESSION OF EG-VEGF IN PCOS AND POTENTIAL ROLE IN ANGIOGENESIS ASSOCIATED WITH CHRONIC ANOVULATION Angiogenesis is also a prominent feature of the polycystic ovary syndrome (PCOS), a leading cause of infertility affecting as many as 5–10% of women of reproductive age. PCOS was originally described as a disorder characterized by the association of hirsutism, obesity, reduced fertility, and enlarged, polycystic, ovaries (66). Hyperplasia of the theca interna and stroma, with excessive production of androgens, are hallmarks of PCOS (for review see Ref. 67). Indeed, the ultrasonographic assessment of stromal area (68) and blood flow (69) is currently used as diagnostic test. Although PCOS was described over 50 years ago, its etiology has remained largely unclear. However, increased LH/FSH ratio, defective selection of a dominant follicle, and anovulation are considered to be key aspects of the pathogenesis. Recent evidence also indicates that PCOS is a part of a complex endocrine/metabolic disorder, where insulin resistance plays a major role (70). Both VEGF and EG-VEGF are expressed in all PCOS ovaries examined, but with an almost mutually exclusive expression pattern (47). Somewhat surprisingly, expression of VEGF mRNA is largely limited to the cyst walls, with little or no expression in the stroma. Cysts appear to express only VEGF, only EG-VEGF, or VEGF in an inner rim surrounded by an outer rim of EG-VEGF. Some VEGF expression was seen in theca interna, although not as consistently as in granulosa cells. EG-VEGF expression was strongest in theca interna of follicles in various stages of atresia. As above noted, intense signal, albeit of lower magnitude than that detected in the theca, occurs in the stroma. Importantly, thecal and stromal tissue expressing EG-VEGF maintain an abundant vascular supply, despite lacking significant VEGF expression. Endothelial immunostaining with anti-CD34 demonstrates persistent vascularity in these areas. Such a pattern is consistent with the establishment of a proangiogenic gradient directing new vessel growth toward the EG-VEGF expressing cells. Therefore, at least in terms of mRNA expression, EG-VEGF shows a particularly strong correlation with vascularity in

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PCOS specimens. These findings raise the possibility that, while VEGF is an essential player in normal cycling ovaries, EG-VEGF might be particularly significant for the acyclical angiogenesis occurring during chronic anovulation. Additional studies are clearly needed to verify this hypothesis.

8. REGULATION OF HUMAN EG-VEGF GENE EXPRESSION Initially, we characterized the hypoxic regulation of human EG-VEGF gene (28). Adrenal carcinoma cell line exposed to low oxygen displays a strong up-regulation of both VEGF and EG-VEGF genes (28). This EG-VEGF response is potentially mediated by HIF-1 (71). In agreement with this hypothesis, several putative hypoxia response elements are present within a 4kb region in the EG-VEGF promoter. Importantly, hypoxia-induced angiogenesis is primarily an adaptive physiological response to an increase in metabolic demands associated with proliferative processes, including those occurring during the cyclical ovarian changes (72). Transcriptional regulation of the EG-VEGF gene is also of particular interest, given the selective expression of EG-VEGF mRNA within steroidogenic cells. Pro minently, a consensus binding site for the NR5A1 orphan nuclear receptor is present within the human EG-VEGF promoter (73). NR5A1 is considered to be a key regulator of endocrine development and function. It regulates multiple target genes involved in gonadal and adrenal determination and development; steroidogenesis; and reproduction (for review see Ref. 74). Gene-targeting studies revealed a critical role for NR5A1 in adrenal and gonad development (75,76). In the adult ovary, NR5A1 protein expression is recognized at the onset of follicular development and is strongly induced in antral follicles in theca and granulosa cells (77). The expression profiles of NR5A1 and the highly related NR5A2, another orphan nuclear receptor, within the ovarian follicles and CL are an intriguing aspect of target gene regulation. Although NR5A1 expression is extinguished at the onset of the conversion to CL (77), NR5A2 is induced in the mid-early stage (78) and may target common genes. The parallels between the known expression and activities of NR5A1/2 and the expression profile of human EG-VEGF indicate that these factors are potentially important regulators of EG-VEGF transcription and provide a plausible explanation for the restricted expression pattern of EG-VEGF. Since EG-VEGF expression is not associated with ovarian epithelial cells, which give rise to the majority of malignancies, this factor is not expected to contribute to initial growth and progression of ovarian carcinomas. In agreement with this hypothesis, Zhang et al. (46) recently reported that EG-VEGF mRNA is not detectable in ovarian surface epithelium and in ovarian epithelial malignancies. However, EG-VEGF may potentially be a mediator of angiogenesis in ovarian or testis tumors that arise from steroidogenic cells, such as thecomas, granulosa cell or Leydig cell tumors.

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9. THE EG-VEGF MOUSE ORTHOLOGUE: A DIFFERENT EXPRESSION PATTERN BUT RELATED FUNCTIONS To further characterize the function and biology of EG-VEGF, the mouse orthologue was cloned and its expression pattern and activity examined (73). The predicted mature mouse EG-VEGF (mEG-VEGF) peptide is 88% identical to the human sequence. mEGVEGF maps to a region of chromosome 3 syntenic with human chromosome 1p13.1, the locus for hEG-VEGF, providing further evidence that these genes are orthologues. The gene organization is also highly conserved; however, the promoter sequences have diverged reflecting unique transcriptional regulation. For example, the putative NR5A1 consensus site present in the human is absent from the mEG-VEGF promoter sequence (73). Apparently, the divergence of promoter sequence between human and mouse accounts for the selective and unique expression patterns. Strikingly, mEG-VEGF is expressed in a distinct expression pattern from its human counterpart. The transcript is predominantly restricted to the liver and kidney. In the adult kidney, the specific hybridization signal is restricted to the epithelial tubule cells. The fetal liver expresses a very high level of EG-VEGF transcript, with signal restricted to hepatocytes. The EG-VEGF mRNA level is reduced in the adult liver. Interestingly, the sinusoidal endothelial cells within the liver (79) and peritubular capillary plexuses in the kidney (80) are similar to those of the endocrine glands in that they are fenestrated. Hence, similar to human EG-VEGF, mouse EG-VEGF may influence the phenotype and growth properties of endothelial cells in distinct tissue compartments enriched in fenestrated endothelium. The expression of EG-VEGFR-2 was associated with purified liver endothelial cells or the endothelial cell-enriched fraction derived from kidney. EGVEGFR-1 expression was undetectable in these samples. Conversely, the transcript for the ligand was detected in RNA samples derived from the hepatocyte fraction, or the endothelial cell-depleted kidney fraction. mEG-VEGF expression was virtually undetectable in purified endothelial cells. These findings are consistent with the hypothesis that mEG-VEGF functions as a paracrine growth and survival factor for liver and kidney endothelial cells. Recombinant mEG-VEGF peptide stimulated the growth and survival of primary mouse liver sinusoidal endothelial cells (73). Therefore, although mEG-VEGF is expressed at distinct tissue sites relative to the human orthologue, the biological activities appear to be analogous, due to the selective coexpression of an EG-VEGF receptor.

10. Bv8 IS A CLOSELY RELATED EG-VEGF HOMOLOGUE WITH A DISTINCT EXPRESSION PATTERN As above noted, EG-VEGF is highly related to a peptide that was designated Bv8. This molecule was originally purified from the skin secretions of the yellow-bellied toad, B. variegata. This amphibian peptide was initially characterized as an inducer of

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gastrointestinal motility and hyperalgesia in the rat (32). The function of Bv8 as a neuropeptide was further evaluated with the mouse peptide. In addition to localizing mouse Bv8 transcript in the CNS, Melchiorri et al. (64) reported that Bv8 stimulated neuronal survival in cultures of primary granular cells. Negri et al. (81) have also described the ability of the Bv8 peptide to induce nociceptive sensitization. Also, the denomination of “prokineticin-2” has also been used for its human orthologue (35). This protein has been proposed to represent an output signal from the suprachiasmatic nucleus (SCN) that regulates circadian rhythm activity. The Bv8 expression pattern within the SCN is rhythmic, and Bv8 transcription is perturbed in circadian mutant mouse lines (65). Exogenous Bv8 peptide delivered into the rat brain in the dark phase inhibited the normal nocturnal locomotor activity. Importantly, endogenous Bv8 levels within the SCN are lowest in the active, dark phase (65). The primary structures of predicted mouse and human Bv8 isoforms have been described (35,38,82). Several putative hypoxia regulatory elements exist in the Bv8 promoter regions, and similar to EG-VEGF (28), Bv8 transcript is induced by hypoxic treatment in cell culture (38). Overall, the Bv8 human and mouse promoter sequences are highly conserved, indicating a related transcriptional regulation in these species. Purified Bv8, like EG-VEGF, induced proliferation, migration, and survival of ACE cells (38). As previously mentioned, these activities are attributed to the ability of both Bv8 and EGVEGF to bind and activate the same G-protein coupled receptors (43,42). The predominant site of Bv8 expression in both human and mouse is the seminiferous tubules of the testis (38,82). Within the tubules, the Bv8 transcript is largely restricted to the primary spermatocytes. Bv8 mRNA is not detected at this tissue site prior to spermatocytic development. Within the testis, the EG-VEGF/Bv8 receptors are expressed in vascular endothelial cells within the interstitial space (38). Delivery of exogenous Bv8, EG-VEGF, or VEGF to the testis via the direct injection of recombinant adenoviruses resulted in a potent, indistinguishable angiogenic response. Taken together, these results suggested that Bv8 or EG-VEGF function as angiogenic mitogens/survival factors in the testis. Interestingly, the testis exhibits the highest endothelial cell turnover among the noncyclic tissues (83). Additionally, Bv8 and EG-VEGF might have functions unrelated to angiogenesis within the male reproductive tract. In this context, it is noteworthy that overexpression of VEGF in the mouse testis resulted in an arrest of spermatogenesis (84). It remains to be established whether disregulation of Bv8/EG-VEGF may also contribute to the pathophysiology of male infertility. The availability of specific antibodies and the Bv8 gene-targeted mouse should permit further studies of the molecular functi on in the testis and during sperm maturation and fertilization.

11. CONCLUSION The identification of EG-VEGF suggests a novel view of the regulation of angiogenesis. Steroidogenic glands appear to have developed highly specific local mechanisms, apparently to complement the action of the ubiquitous VEGF. Interestingly, such an acquisition seems to be, at least in part, a late event in evolution and may reflect a greater functional/morphological complexity of organs like the ovary. In this context, the species

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differences in the expression pattern of human and mouse EG-VEGF are particularly intriguing. The association of human EG-VEGF expression with steroidogenic cells is compelling. However, the mouse orthologue has a different expression pattern. Although rodents have served as models for endocrinology and ovarian physiology, clear differences exist between the rodent and human ovary. A fundamental difference is the selection and development of a single ovarian follicle in humans and other monovular species. The process of selection of the dominant follicle has been associated with angiogenesis, as there is evidence that selected follicles possess a more elaborate microvascular network (85). The length of the ovarian or luteal cycle also distinguishes the human or primate from the rodent. In humans, the cycle is 28 days and in rodents the cycle is completed every 4 days (86). The primate CL is functional for 2 weeks prior to its regression in the infertile cycle, whereas the rodent CL is active for less than a day (87). Both the selection of the dominant follicle and the length of the cycle impose distinct regulatory functions in the human (primate system), including a more complex regulation of growth and maintenance of the vascular endothelium. It is tempting to speculate that EG-VEGF represents one growth factor component that contributes to these evolutionary changes. While at an earlier point in evolution EG-VEGF performs analogous function but at distant sites, this gene appears to have been “coopted,” by virtue of a tissue-specific transcriptional regulation, into the ovary, coincident with a greater length and complexity of the ovarian cycle. In this context, it will be interesting to determine when, on an evolutionary scale, EG-VEGF expression first became associated with the ovary and other steroidogenic tissues. Furthermore, as previously observed, human EG-VEGF is highly expressed in PCOS (47). This disorder occurs in humans but not in rodents. While a focus of this chapter has been the potential role of EG-VEGF in the cyclic ovarian angiogenesis, this molecule might play an important role in the pathophysiology of other steroidogenic organs such as adrenal and placenta, which remains to be investigated. The existence of restricted, local mediators of angiogenesis and endothelial cell survival and function permit novel approaches to salvage or regenerate tissue. Several attempts have been made to promote new vessel growth using angiogenic factors such as VEGF and bFGF. However, the success of these efforts have been limited by systemic side-effects, including hypotension, edema, and accelerated atherosclerosis. Moreover, these efforts have generally not achieved functional and mature blood vessels. Mitogens selective for the endothelium of specific tissues like cardiac or skeletal muscle would potentially offer major advantages. A principle benefit of tissue-specific angiogenic therapeutics could be the reduction of undesired side-effects associated with the broadspectrum molecules. Endothelial cell stimulation, even in the absence of angiogenesis, promoted salvage and regeneration of the tissue parenchyma by stimulating the release of paracrine factors (27). Thus, the EG-VEGF/Bv8 family not only has therapeutic implications per se, but also raises the possibility that other selective regulators of angiogenesis within specific tissue or organs exist and therefore presents novel potential therapeutic targets.

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12 Differential Regulation of Endothelial Cell Barrier Function Jeffrey R.Jacobson, Steven M.Dudek, and Joe G.N.Garcia Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

1. INTRODUCTION The endothelium serves as a semipermeable barrier separating the circulation from the surrounding interstitium. Its luminal surface is coated with a negatively charged glycocalyx comprised of membrane-bound proteoglycans and glycoproteins. The tight apposition of individual endothelial cells (ECs) with neighboring cells via intercellular junctions acts as a significant determinant of basal endothelial barrier function. Separately, focal adhesions, the integrin-based linkages between the extracellular matrix and the endothelial cytoskeleton, provide strong tethering of the endothelium to the vessel wall, a process that also contributes to barrier integrity. Long thought to be a passive cellular barrier, the endothelium is now recognized to be highly dynamic and responsive to various effectors of both barrier enhancement and disruption. Perturbations of cell-cell and cell-matrix interactions can result in marked effects on barrier integrity and, subsequently, vascular permeability. In this respect, key elements of the cytoskeleton and its regulators have recently been identified and characteriz ed as important determinants of endothelial barrier function. In this chapter, we will discuss the mechanisms of endothelial barrier regulation. We will explore various effectors of endothelial barrier function, both biochemical and biophysical, focusing on differences in EC phenotype along the vascular tree leading to differential barrier regulation and its significance with respect to various disease states.

2. OVERVIEW OF ENDOTHELIAL CELL BARRIER REGULATION Derangements in endothelial cell barrier function are now recognized as a critical determinant of the morbidity and mortality associated with disease states characterized by

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inflammation and increased vascular permeability, including sepsis, acute lung injury, and acute respiratory distress syndrome (1).a More precisely, vascular permeability is defined by two general pathways that determine the movement of fluids and solutes from the vascular space to the surrounding environment. The transcellular pathway utilizes a tyrosine kinase-dependent, gp60-mediated transcytotic albumin route, which is thought to be a minor contributor to inflammatory vascular permeability although its regulation and function remain unclear (2,3). Conversely, the paracellular pathway, characterized by the formation of paracellular gaps in response to various inflammatory mediators, is acknowledged as the primary determinant of vascular permeability, and its regulation has been more fully detailed (4). Intercellular adhesion occurs via the participation of several proteins involved in both tight junction and adherens junction complexes. Via engagement of the actin cytoskeleton, these complexes promote mechanical stability and are involved in transduction of extracellular signals into the cell (5). Tight junctions consist of the transmembrane proteins occludins, claudins, and junctional adhesion molecules (JAMs) coupled to cytoplasmic proteins such as the zona occludens (ZO) family (6). Adherens junctions are composed of cadherins which link adjacent cells via homotypic interactions (7). These proteins attach to catenins (α, β, γ) intercellularly, which in turn anchor to the actin cytoskeleton. Importantly, the primary adhesive protein of EC adherens junctions is vascular endothelial (VE) cadherin (8). Although evidence for a significant functional role of tight junctions in paracellular permeability is limited, adherens junctions are recognized as playing a central role in this setting, largely via their association (and disassociation) with the actin cytoskeleton (9,10). Regulation of paracellular gap formation can be thought of as a balance of competing intracellular contractile forces and adhesive cell-cell and cell-matrix tethering forces (Fig. 1). These forces are regulated through dynamic activation of the actin-based cytoskeleton, the complexities of which have only recently been brought to light. The EC cytoskeleton is comprised of three key elements: actin microfilaments, intermediate filaments, and microtubules. While the roles of microtubules and intermediate filaments in EC barrier regulation remain to be fully defined, the critical importance of actin microfilaments is demonstrated by increased EC permeability in response to cytochalasin D (11), a known actin disrupter. Conversely, phallacidin, an actin stabilizer, decreases sensitivity to agonist-induced EC barrier disruption (12). Through multiple focal linkage sites to various membrane adhesive proteins, glycocalyx components, functional intercellular proteins of the zona occludens and zona adherens, and focal adhesion complex proteins, the actin microfilament system is fully engaged in determining barrier integrity. At the same time, however, actin is also largely responsible for the generation of tensile intracellular forces via an actomyosin motor. Dynamic actin rearrangement is stimulated by the coordinate activities of the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) (13) and the kinase effector of the small GTPase Rho (14,15), Rho kinase, which combine to elevate levels of myosin light chain (MLC) phosphorylation and subsequent stress fiber formation (Fig. 2). Focal or spatially defined variability in levels of MLC phosphorylation accounts for the phenotypic-specific contracted or relaxed state of the cell. Actin rearrangement is further dependent on numerous actin binding, capping, nucleating,

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a

Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The acute respiratory distress syndrome network.

Figure 1 Regulation of endothelial cell barrier function. The endothelial cell (EC) monolayer forms a semipermeable barrier between the vasculature and underlying extracellular matrix. The barrier enhancing agent sphingosine 1phosphate (S1P) promotes EC cytoskeletal changes that strengthen cell-cell and cell-matrix interactions as outlined in the cells on the left. S1P binds the G-protein coupled Edg (endothelial differentiation gene), initiating downstream signaling including Rac activation. Rac stimulates PAK-dependent cortical actin ring formation and cortactin translocation to the actin ring where it interacts with MLCK. S1P also induces adheren junction protein assembly (cadherins, catenins, etc.)

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and focal adhesion rearrangement (see text) that strengthen cell-cell and cellmatrix contacts. Various inflammatory stimuli (e.g. thrombin) induce EC cytoskeletal changes resulting in intercellular gap formation and subsequent passage of luminal contents into the interstitium and surrounding tissues (e.g. produces alveolar edema in the lungs). As outlined in the cell on the right, thrombin ligation of the PAR receptor increases intracellular Ca2+, which through Ca2+/calmodulin interaction activates MLCK to phosphorylate myosin light chains, producing actomyosin interaction, actin stress fiber formation, and cell contraction. Thrombin also elevates Rho activity, which further promotes this sequence of events by inhibiting (through Rho kinase) myosin dephosphorylation. Furthermore, thrombin disrupts adherens junction contacts and rearranges focal adhesion interaction to sites at the ends of stress fibers. and severing proteins which contribute to the overall complexity of its regulation. For example, caldesmon is an actin-binding protein that, in its unphosphorylated state, inhibits cell contraction via inhibition of actomyosin ATPase (16,17). However, this effect of caldesmon is attenuated by the actin-severing protein gelsolin resulting in increased stress fiber-dependent contraction (18,19).

3. AGONIST-MEDIATED ENDOTHELIAL BARRIER REGULATION The mechanisms by which various agonists are able to induce either EC barrier disruption or enhancement have been the focus of extensive investigation. We have employed several models in our own investigations including barrier disruption

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Figure 2 Transendothelial monolayer electrical resistance (TER) measurements represent a sensitive in vitro assay of barrier regulation. Human pulmonary artery ECs were grown to confluence on evaporated gold microelectrodes, and TER measurements were performed in the authors’ laboratory using an electrical cell-substrate impedance sensing system (ECIS) (Applied Biophysics, Troy, NY) as previously described (21). Panel A illustrates the barrier disrupting effects of thrombin (1 U/mL) on EC permeability (increased permeability is reflected in decreased electrical resistance across the monolayer), while panel B shows the barrier enhancing effects of S1P (1µM). Arrows indicate when agonists were added. induced by thrombin (20) as well as barrier enhancement evoked by sphingosine 1phosphate (S1P) (Fig. 2), a product of platelets (21), and by hepatocyte growth factor (HGF) (22). These and other agonists demonstrate unique effects on EC cytoskeletal rearrangement and permeability and have provided important insights into EC barrier regulation (Table 1). The serine protease thrombin produces numerous EC responses which regulate hemostasis, thrombosis, and vessel wall pathophysiology and is recognized as a potentially important mediator in the pathogenesis of acute lung injury. We have characterized the ability of thrombin to activate the endothelium directly and to increase albumin permeability across endothelial cell monolayers in vitro (23).

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Table 1 Agonist-mediated Endothelial Cell Cytoskeletal Rearrangement and Barrier Regulation. The effects of specific agonists may be categorized as either Ca2+-dependent or independent. Agonist-induced cytoskeletal rearrangements, characterized by alterations in stress fiber formation and cortical actin, are intimately linked to EC barrier function and permeability. Ca2+

EC Permeability

Thrombin (26,35)

↑

↑

↑

↓

HGF (22)

↑

↓

↓

↑

↑

↓

↓

↑

Sheat stress (42)

↑

↓

early ↑ / late ↓

↑

VEGF (52,61)

↑

↑

↑

↓

LPS (62,63)

—

↑

↑

↓

TNF-α (38)

↑

↑

↑

↓

Agonist

a

S1P (21) b

Stress Fibers Cortical Actin

a

Higher concentrations associated with barrier disruption (>1 µM). Produces increased actin stress fibers at early times points (15 min, 10 dynes/cm2) whereas few fibers are apparent late (24 h). b

Thrombin induced a concentration-dependent increase in I125-albumin clearance that was independent of its interaction with fibrinogen and appeared to be due to a reversible change in EC shape through the formation of intercellular gaps. This observation provided a blueprint for a mechanistic examination of EC barrier properties. We subsequently reported that the direct activation of EC by thrombin is dependent upon its ability to proteolytically cleave the extracellular NH2-terminal domain of the PAR-1 receptor, a member of the family of proteinase-activated receptors (PARs) (24– 27). The cleaved NH2-terminus, acting as a tethered ligand, then activates the receptor. This event initiates a number of downstream effects including the activation of phospholipases A2, C, and D, increased cytosolic Ca2+, and increased permeability (27,28,26,29–31). Activation of the EC thrombin receptor also induces the release of various products including von Willebrand factor, endothelin, NO, and PGI2 (32–34). Specific components of the contractile apparatus are now recognized as targets for thrombin-mediated barrier regulatory signaling pathways. For example, activation of the thrombin receptor induces rapid activation of a Gq protein-coupled phospholipase C leading to inositol 1,4,5-triphosphate3(IP3)-mediated increases in cyotsolic Ca2+. In turn, increased cytosolic Ca2+ leads to the coordinate activation of Rho and MLCK (15,35,13,36,37), with the latter protein directly phosophorylating MLC at Thr-18 and Ser-19. Activation of the thrombin receptor also induces G12/13-mediated Rho activation

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which, via its target effector Rho kinase, inhibits MLC phosphatase activity by direct myosin phosphatase (MYPT1) phosphorylation thus attenuating dephosphorylation of MLC (35). This potent increase in MLCK/Rho kinase-mediated MLC phosphorylation results in a dramatic increase in intracellular force development and tension, a diminution of the cortical actin ring, and a prominent increase in F-actin stress fibers that traverse the cell (Fig. 1). These intracellular events correspond to increases in EC monolayer permeability and are associated with morphologic cellular changes including the formation of intercellular gaps with the disruption of adherens junctions and reorganization of focal adhesion plaques. Notably, investigations of other agonists have implicated mechanisms of EC barrier disruption independent of Rho Kinase and MLCK activation. For example, we have identified an important role for microtubule rearrangement in EC barrier dysfunction by tumor necrosis factor (TNF)-α (38). In contrast to thrombin’s barrier-disrupting effects, the platelet-derived phospholipid, S1P, produces significant EC barrier enhancement in vitro (21). Originally characterized as a potent angiogenic factor (39), S1P ligates G-protein coupled Edg receptors on the surface of EC to initiate a series of cytoskeletal and adhesive protein rearrangements that result in decreased EC permeability (Fig. 1). Activation of the small GTPase Rac and its downstream target, PAK, is critical for formation of a prominent cortical actin ring that accompanies S1P-induced EC barrier enhancement (21). Moreover, Rac activation stimulates translocation of the multidomain actin-binding protein cortactin to the cortical actin ring where its ability to activate and possibly inhibit other cytoskeletal proteins appears to be essential for maximal S1P-mediated barrier enhancement (S.Dudek, personal communication, 2003). In addition to inducing actin cytoskeletal rearrangement, S1P alters cell–cell and cellmatrix contacts in association with EC permeability reduction. S1P dramatically increases localization of VE-cadherin, α-, β-, and γ-catenin at EC cell-cell junctions (40) while increasing interaction of these adherens junction proteins with the cortical actinbased cytoskeleton (K.Schaphorst, personal communication, 2003). Whereas the barrier disrupting agent thrombin rearranges cell–matrix contacts so that focal adhesion proteins assemble at the ends of newly formed massive actin stress fibers, S1P induces focal adhesion protein rearrangement in association with cortical actin ring formation (41). Stimulation of FAK phosphorylation and p60src activation may partially account for the differential focal adhesion distribution observed in S1P compared to thrombin-treated EC (41). Continued mechanistic evaluation of these models of EC barrier disruption and enhancement hopefully will provide further insights into potential targets for therapeutic modulation of vascular permeability.

4. EFFECTS OF MECHANICAL FORCES ON ENDOTHELIAL BARRIER FUNCTION Experiments investigating the effects of physiologically relevant mechanical forces have yielded additional insights into endothelial barrier regulation. Two forces are of particular interest in endothelial barrier function: shear stress, resulting from the effects of blood flow across EC monolayers, and cyclic stretch, resulting from either the pulsatile

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distention of blood vessels or from mechanical ventilation, specifically with respect to the lung vasculature. We previously reported that exposure of lung EC to physiologic levels of laminar shear stress (10 dyn/cm2) results in rapid actin stress fiber formation associated with increased levels of MLC phosphorylation in the cortical actin ring and marked translocation of cortactin, an actin-binding protein involved in peripheral actin polymerization (42). This increase in MLC phosphorylation is dependent on both MLCK and Rho signaling. Moreover, both cortactin translocation and cytoskeletal rearrangement are prevented by inhibition of the small GTPase Rac, known to be involved in membrane ruffling and lamellapodia formation. Cytoskeletal rearrangement occurs within minutes and persists in the presence of sustained shear stress (24 hr). These cortical actin changes are consistent with those seen in other models of barrier enhancement, including S1P (21), and suggest that endothelial adaptation to physiologic shear stress represents a protective response. Separately, we have employed the Flexercell® Tension plus (FX-4000T ) system to study EC subjected to cyclic stretch. This apparatus allows for EC to be grown in a monolayer overlying a flexible substrate under which a vacuum can be applied thus inducing stretch proportional to the degree of vacuum pressure. We previously reported that ECs exposed to 18% radial stretch for 48 hr do not demonstrate any breach in monolayer integrity evaluated histologically or by measurements of transmonolayer electrical resistance under basal conditions (43). However, cyclic stretch-preconditioned EC demonstrated greater paracellular gap formation with increased gap surface area in response to thrombin that correlated with increased levels of MLC phosphorylation. Likely determinants of this heightened response include increased intracellular signaling events via mechanical transduction, priming of the activated cytoskeleton, and the resultant concurrent effects of increased contractile and decreased tethering forces. Additionally, our findings were associated with significant changes in the expression of genes related to regulation of the cytoskeleton (including MLCK and Rho family signaling genes) as determined by Affymetrix microarray experiments. We believe this model is particularly relevant to the clinical condition of ventilator-associated lung injury.

5. DIFFERENTIAL ENDOTHELIAL CELL PHENOTYPES AND BARRIER REGULATION Although the endothelium is often regarded as a singular, uniform entity, it is now abundantly clear that there are notable phenotypic and functional differences of EC at various sites along the vasculature. These differences likely account for the physiologic variability in vascular function appreciated in health as well as the heterogeneous nature of many diseases characterized by increased vascular permeability. Morphologic differences between rat pulmonary artery EC monolayers and rat lung microvascular ECs are appreciable by electron microscopy, with tighter inter-cellular connections and fewer visible gaps in microvascular cells (44). Furthermore, examination of cultured bovine lung EC from microvessels, pulmonary vein, and pulmonary artery reveals cell-specific attributes with respect to the expression of surface proteins, cell

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morphology, and permeability (45). In particular, microvascular ECs have a ~4-fold larger surface area than pulmonary artery EC (46), while electron microscopy of microvascular ECs demonstrates increased plasmalemmal vesicles, thought to be involved in the transcelluar transport of macromolecules across the cell, as well as a significant increase in focal adhesion sites relative to pulmonary vein or pulmonary artery EC (45). Evidence of a more abundant array of intercellular junctional complexes in microvascular ECs, determinants of small solute transport across cell monolayers, correlates with the observed decreased permeability in these cells under basal conditions. Moreover, despite the increased plasmalemmal vesicles noted in microvascular ECs, there is evidence that they serve as a more restrictive barrier to macromolecules as well (40). In fact, cultured human microvascular ECs exhibit barrier integrity that is approximately 10-fold higher than human macrovascular ECs as measured by electrical resistance across monolayers (47). This likely represents the interaction of several factors including site-specific glycocalyx protein profiles, the extent of junctional protein expression, and alterations in the cytoskeletal profile in a cell-specific manner. In addition to the notable differences between micro- and macrovascular ECs from the lung, significant phenotypic variability also exists in blood vessels from different sites or organs. For example, morphologic differences are noted in rat EC derived from aorta relative to pulmonary artery (48). Similarly, rabbit inferior vena cava ECs have been described as larger than rabbit aorta ECs (49). With respect to cell surface glycoproteins, cell-cell interactions, and protein and mRNA expression, EC variability throughout the vasculature is the rule. The functional significance of this variability remains a highly important area of study. Although data relying on a direct comparison of the response of various EC phenotypes to specific agonists are limited, these investigations have yielded curious results. For the most part, microvascular ECs are more resistant to agonists that induce increased permeability. For example, cultured bovine and sheep pulmonary artery ECs have been characterized as more sensitive to endotoxin than lung micro vascular ECs in terms of cell contraction and loss of barrier function (50). Likewise, investigation of bovine EC has demonstrated increased cell detachment of pulmonary artery EC relative to microvascular ECs in response to TNF-α (51). However, a more dramatic effect of thrombin on bovine EC permeability has been described in microvascular ECs compared to pulmonary artery ECs (46). These findings suggest that this differential response is characterized by specific variations in intracellular signaling yet to be fully defined and is dictated, in part, by distinct receptor expression profiles. It is readily apparent that inflammation and coagulation are tightly linked which may explain high thrombomodulin expression in the vasculature. Importantly, however, the qualitative effects of various agonists with respect to barrier regulation and permeability have proven largely consistent across EC phenotypes. Our own experiments have characterized the effects of vascular endothelial growth factor (VEGF) on large and small-vessel bovine pulmonary endothelial cells (52). Although VEGF induces chemotaxis and barrier disruption in both cell types, a differential concentration dependence is seen. In particular, barrier disruption is more pronounced in macrovascular ECs with the administration of low concentrations of VEGF (1 ng/mL), whereas microvascular ECs are more responsive with respect to chemotaxis. These differences, however, are less apparent with higher concentrations of VEGF (10–100 ng/mL).

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As for potential mechanisms underlying differential EC barrier regulation, there is evidence to indicate that Ca2+-dependent events do not play as significant a role in microvascular ECs. Specifically, studies employing rat pulmonary EC demonstrate a blunted response by microvascular cells to increases in intracellular Ca2+ relative to macrovascular cells with respect to changes in permeability (44). Additionally, microvascular ECs are characterized by increased levels of intracellular cAMP, a determinant of the resting contractile state and focal adhesion complex formation, which is regulated by mechanisms distinct from that of macrovascular ECs (53). Specifically, despite increased cAMP, there is decreased ATP-to-cAMP conversion in microvascular ECs. Furthermore, these cells are less responsive to β-adrenergic stimulation—relied on in the clinical setting to increase cAMP and decrease vascular permeability—likely due to increased phosphodiesterase activity relative to macrovascular ECs. Finally, differences in junctional protein expression may account for some of the differential permeability observed in endothelial subpopulations. Microvascular ECs express more VE-cadherin than macrovascular cells, and perhaps as a result, infusion of anti-VEcadherin antibodies increases permeability primarily in alveolar capillaries (9).

6. ROLE OF DIFFERENTIAL ENDOTHELIAL BARRIER FUNCTION IN DISEASE Recognition of the unique response of the endothelium at different sites along the vasculature in various disease states is crucial to understanding their underlying pathophysiology. The observation that the vast endothelial network is comprised of cells with a spectrum of phenotypes accounts, in part, for this variability. Evidence of this is provided by the differential production of nitric oxide by rat lung microvascular and aorta EC in response to TNF-α and LPS, two clinically relevant agonists (54). Models of acute lung injury also support this hypothesis as the systemic administration of lipopolysaccharide (LPS) to mice is associated with differential expression of von Willebrand factor in different organs (55). Separately, our finding of a differential response to VEGF in bovine pulmonary artery and lung microvascular cells (52) is relevant to models of ischemic lung injury which have demonstrated increased VEGF expression in this setting (56,57). One further example is provided by evidence of notable differences in protein expression and permeability between coronary and pulmonary EC exposed to human sera from patients with thermal injuries (58). In addition to specific cell differences, however, variability in the local extracellular environment may also contribute to a differential endothelial response. This is certainly the case in focal disease processes such as lobar pneumonia, typically associated with increased lung vascular permeability in the area of involvement, but is also true for more diffuse processes, such as sepsis and acute lung injury, in which the concentrations of various cytokines and inflammatory mediators may vary widely at different vascular sites. Moreover, variability in the extracellular environment is in particular relevant to the effects of mechanical forces on the endothelium. For example, an appreciation of the effects of shear stress on the lung vasculature must take into account regional differences in blood flow, specifically a decrease at the apices relative to the bases. Further differences in regional blood flow, and thus the degree of shear stress, may be affected by

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a multitude of clinically relevant factors including changes in blood volume, oxygenation, positional changes, as well as the administration of mechanical ventilation. In fact, it is precisely because of the potentially favorable effects on regional blood flow and lung stretch that prone positioning is used in some mechanically ventilated patients with acute respiratory distress syndrome (59,60).

7. CONCLUSION Differential endothelial barrier regulation continues to be a focus of active investigation. Although a complete picture remains elusive, the increasing availability of new technologies provides hope that significant advances in this area may be readily forthcoming. While better methods to quantitatively image the vascular barrier in a dynamic way are lacking, the application of both genomic and proteomic tools may yield invaluable insights into the intricacies of EC barrier regulation with respect to individual EC phenotypes and their specific mRNA and protein profiles. Ultimately, these findings may be critical to future investigations into novel and highly specific therapeutic targets with respect to various vascular pathobiologies.

ACKNOWLEDGMENTS This work acknowledges support by the Center for Translational Respiratory Medicine, grants from the NHLBI (HL 71411, HL, 70013, HL 58064, and HL 66583) and the Dr. David Marine Endowment.

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8. Dejana E, Bazzoni G, Lampugnani MG. Vascular endothelial (VE)-cadherin: only an intercellular glue? Exp Cell Res 1999; 252:13–19. 9. Corada M, Mariotti M, Thurston G, Smith K, Kunkel R, Brockhaus M, Lampugnani MG, Martin-Padura I, Stoppacciaro A, Ruco L, McDonald DM, Ward PA, Dejana E. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo. Proc Natl Acad Sci USA 1999; 96:9815–9820. 10. Hordijk PL, Anthony E, Mul FP, Rientsma R, Oomen LC, Roos D. Vascular-endothelialcadherin modulates endothelial monolayer permeability. J Cell Sci 1999; 112(Pt 12):1915– 1923. 11. Shasby DM, Shasby SS, Sullivan JM, Peach MJ. Role of endothelial cell cytoskeleton in control of endothelial permeability. Circ Res 1982; 51:657–661. 12. Phillips PG, Lum H, Malik AB, Tsan MF. Phallacidin prevents thrombin-induced increases in endothelial permeability to albumin. Am J Physiol 1989; 257:C562–C567. 13. Goeckeler ZM, Wysolmerski RB. Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation. J Cell Biol 1995; 130:613–627. 14. Chrzanowska-Wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol 1996; 133:1403–1415. 15. Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K. Formation of actin stress fibers and focal adhesions enhanced by rho-kinase. Science 1997; 275:1308– 1311. 16. Vorotnikov AV, Gusev NB, Hua S, Collins JH, Redwood CS, Marston SB. Phosphorylation of aorta caldesmon by endogenous proteolytic fragments of protein kinase c. J Muscle Res Cell Motil 1994; 15:37–48. 17. Stasek JE Jr, Patterson CE, Garcia JG. Protein kinase c phosphorylates caldesmon 77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. J Cell Physiol 1992; 153:62–75. 18. Arora PD, Janmey PA, McCulloch CA. A role for gelsolin in stress fiber-dependent cell contraction. Exp Cell Res 1999; 250:155–167. 19. Gusev NB, Pritchard K, Hodgkinson JL, Marston SB. Filamin and gelsolin influence Ca(2+)sensitivity of smooth muscle thin filaments. J Muscle Res Cell Motil 1994; 15:672–681. 20. Garcia JG, Pavalko FM, Patterson CE. Vascular endothelial cell activation and permeability responses to thrombin. Blood Coagul Fibrinolysis 1995; 6:609–626. 21. Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, English D. Sphingosine 1-phosphate promotes endothelial cell barrier integrity by edg-dependent cytoskeletal rearrangement. J Clin Invest 2001; 108:689–701. 22. Liu F, Schaphorst KL, Verin AD, Jacobs K, Birukova A, Day RM, Bogatcheva N, Bottaro DP, Garcia JG. Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta. FASEB J 2002; 16:950–962. 23. Garcia JG, Siflinger-Birnboim A, Bizios R, Del Vecchio PJ, Fenton JW II, Malik AB. Thrombin-induced increase in albumin permeability across the endothelium. J Cell Physiol 1986; 128:96–104. 24. Vogel SM, Gao X, Mehta D, Ye RD, John TA, Andrade-Gordon P, Tiruppathi C, Malik AB. Abrogation of thrombin-induced increase in pulmonary microvascular permeability in par-1 knockout mice. Physiol Genomics 2000; 4:137–145. 25. Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64:1057– 1068. 26. Garcia JG, Patterson C, Bahler C, Aschner J, Hart CM, English D. Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and

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13 Differential Regulation of Leukocyte-Endothelial Cell Interactions D.Neil Granger and Karen Y.Stokes Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, U.S.A.

1. INTRODUCTION The adhesion of leukocytes to vascular endothelium is a hallmark of the inflammatory process. The requirement for and participation of specific adhesion glycoproteins in the binding of leukocytes to endothelial cells has been elegantly demonstrated using a variety of experimental approaches (1,2). However, our current understanding of the molecular basis for leukocyte-endothelial cell adhesion is largely based on data generated from studies utilizing isolated leukocytes and monolayers of cultured endothelial cells. Much of this information was derived from relatively few endothelial cell models, most notably human umbilical cord endothelial cells (HUVEC). The importance of the HUVEC monolayer model to the field of inflammation research cannot be overstated (1,2). It led to the discovery and characterization of a number of key cell adhesion molecules (CAMs) expressed on endothelial cells and/or leukocytes that are now known to mediate adhesive interactions such as leukocyte rolling, firm adhesion, and emigration. This in vitro model has also provided quantitative insights into the modulating role of physical factors such as shear stress on leukocyte-endothelial cell adhesion. Similarities in the time-course and magnitude of CAM expression following activation of different endothelial cell populations grown in culture have led to the general perception that endothelial cells distributed throughout the body are relatively homogeneous in their responses and contributions to inflammatory stimuli. Significant progress in our understanding of regional differences in CAM expression and the subsequent recruitment of leukocytes has resulted from the development of technologies that allow for quantification of endothelial CAMs as well as leukocyteendothelial cell adhesion in a variety of vascular beds. Studies employing these methodologies have revealed considerable quantitative differences between regional vascular beds and within different segments of the same vascular bed. The overall objective of this chapter is to summarize evidence that addresses the issue of intra- and inter-organ variations in leukocyte-endothelial cell adhesion.

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2. OVERVIEW OF LEUKOCYTEENDOTHELIAL INTERACTIONS Leukocyte-endothelial interactions can be divided into three main steps: rolling, firm adhesion, and emigration (1,3). The initial tentative contact between circulating white blood cells and the vessel wall, known as rolling, is mediated by the binding of selectin molecules on the endothelium (E-selectin and P-selectin) or leukocytes (L-selectin) to their corresponding ligands (e.g., sialyl Lewis X and PSGL-1) on the other cell. This slows the leukocytes down, essentially increasing the likelihood that they will firmly adhere to the vessel wall via other adhesion molecules, immunoglobulins such as ICAM1 on the vascular endothelium, and integrins such as CD11/18 on the leukocytes. These two families of CAMs are also responsible for the subsequent transmigration of the leukocytes through the vessel wall into the interstitium where they can promote further tissue damage.

3. INTRA-ORGAN DIFFERENCES IN LEUKOCYTE-ENDOTHELIAL CELL ADHESION Direct visualization of the microvasculature in tissues that are either acutely or chronically inflamed has revealed that leukocytes selectively bind to endothelial cells that line postcapillary venules. While leukocyte recruitment into capillaries frequently accompanies inflammation of the lung and, to a lesser extent, other vascular beds, steric hindrance of less deformable circulating leukocytes within long, narrow capillaries, rather than cell-cell interactions mediated by CAMs, is often offered as an explanation for the leukocyte sequestration in this segment of the vasculature (4). Endothelial cell swelling within capillaries as well as compression of the capillary lumen by an elevated interstitial fluid pressure caused by accumulation of edema fluid have also been implicated in the entrapment of leukocytes within capillaries of inflamed tissues (5). In addition, endothelial cells lining venules, when compared to other microvascular segments, appear to sustain most of the leukocyte trafficking that occurs in inflamed tissues. For example, in the rat mesenteric microcirculation, 39% of all leukocytes passing through venules are rolling, while only 0.6% of leukocytes roll in the upstream arterioles (6). The basis for the preferential binding of leukocytes to venular endothelial cells during inflammation has been the focus of much speculation. Relatively higher shear rates in arterioles and higher endothelial CAM expression in venules are two explanations that are most often provided for the differences in leukocyte-endothelial cell adhesion between these two microvascular segments.

3.1. Shear Rates Shear rates generated by the movement of blood within the microvasculature are generally higher in arterioles than in downstream venules. Whether leukocytes adhere to

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vascular endothelium in microvessels depends on the balance between the proadhesive forces generated by adhesion glycoproteins expressed on the surface of leukocytes, endothelial cells, or both, and the anti-adhesive forces generated by hydrodynamic factors such as wall shear stress or shear rate (6). Hence, vessels exhibiting a low spontaneous shear rate (low blood flow) would tend to exhibit more leukocyte adhesion than vessels with high shear rates. In 1973, Atherton and Born (7) suggested that the reason leukocyte rolling and adhesion are rarely observed in arterioles is because the higher shear forces exceeded the adhesive forces in these vessels. Based on their proposal, one might predict that reductions in arteriolar shear

Figure 1 Relationship between number of adherent leukocytes and wall shear rate in venules and arterioles in cat mesentery (8). Over the same range of shear rates, leukocytes preferentially adhere in venules, with minimal adhesion noted in arterioles, even at low shear rates. rate to levels experienced by venules should promote leukocyte adhesion in arterioles. To address this issue, cat mesenteric arterioles and venules of the same size were exposed to

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the same range of shear rates (100–1250 sec−1) (8). Leukocyte rolling and adhesion were observed in arterioles but only at low (<200 sec−1) shear rates. At each shear rate studied, far less adhesion of leukocytes was observed in arterioles than venules (Fig. 1). Leukocyte adherence was never observed in arterioles at shear rates >385 sec−1, whereas in venules, leukocyte adherence persisted at shear rates up to 900 sec−1. Based on these observations, it was concluded that hemodynamic differences between arterioles and venules cannot explain the predilection for leukocyte rolling and adherence in venules. Another approach to elucidate the reason for these observations has been to perform retrograde perfusion of the microcirculation. In the mesentery, this is associated with a reduced flux of rolling leukocytes in venules and increased leukocyte rolling in arterioles (9). Despite this change in responses, more leukocytes still rolled in venules during normograde perfusion than rolled in arterioles during retrograde flow. These findings, coupled to the evidence summarized above, suggest that a more probable explanation for the greater adhesive interactions between leukocytes and venular endothelium is that the counter-receptors (ligands) for leukocyte adhesion molecules are more densely concentrated on venular endothelium.

3.2. CAM Expression CAM expression has been evaluated in the microvasculature using immunohistochemical methods. This approach yields results that consistently show a preferential expression of endothelial CAMs in postcapillary venules. Endothelial cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM1), E-selectin, and P-selectin can be detected on the surface of activated endothelial cells in arterioles and occasionally capillaries; however, the density of these adhesion molecules is far greater on venular endothelium (3). In the liver, for example, P-selectin and E-selectin are highly expressed on arterial and venular endothelium, but not on capillary (sinusoidal) endothelium during acute or chronic inflammation (10). While immunohistochemical localization of endothelial CAMs has clearly demonstrated preferential distribution of these adhesion glycoproteins in venules, this approach has yielded relatively little quantitative information concerning the density of CAM expression within vascular beds. Laser confocal microscopy has been employed to localize and quantify the constitutive expression of ICAM-1 in arterioles, capillaries, and venules in the mesentery and liver of rats, using an FITC-labelled antirat monoclonal antibody (11). In the mesentery, venules exhibited a 10-fold higher density of ICAM-1 than in the upstream arterioles and capillaries (Fig. 2A). While the level of ICAM-1 expression in liver venules was comparable.to that detected in mesenteric venules, there was a surprising lack of difference between ICAM-1 density for liver sinusoids (capillaries)

Differential regulation of leukocyte-endothelial cell interactions

Figure 2 Laser confocal microscopic determinations of endothelial ICAM-1 expression in different segments of the rat mesenteric and hepatic microcirculation (A), and in different sized venules of rat mesentery (B). Data are expressed as apparent concentration, which is derived from the intensity of the fluorescence and the predetermined binding ratio between the fluorescence and the

261

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immunoglobulin to which it is attached. (Based on data from Ref. 11.) vs. venules, suggesting the absence of a capillary-venule expression gradient of ICAM-1 within the liver microcirculation. However, the authors could not exclude the possibility that hepatocytes per se, rather than endothelial cells, express the high levels of ICAM-1 seen within the sinusoids (12), since the FITC-labelled ICAM-1 antibody can readily diffuse into the space of Disse that separates the porous sinusoidal wall and hepatocyte membrane. Laser confocal microscopic assessment of ICAM-1 expression in mesenteric venules has also revealed heterogeneity of ICAM-1 expression within different sized venules (Fig. 2B). Venules with diameters of 25 µm appear to exhibit the greatest density of ICAM-1 on the endothelial cell surface, while 15 and 35−40 µm diameter venules exhibit the lowest constitutive expression of the adhesion molecule (10). This venule sizedependent distribution of ICAM-1 is consistent with functional evidence demonstrating that 25–30 µm diameter venules sustain the most intense leukocyte adhesion responses to proinflammatory mediators (11). It remains unclear whether other endothelial CAMs exhibit a similar size-dependent distribution within postcapillary venules, or indeed whether ICAM-1 displays comparable size-dependent distribution in organs other than the mesentery. Endothelial cells isolated and cultured from major arterial and venous vessels have also been used to compare the levels of constitutive and induced CAM expression between the arterial and venous segments of the vasculature. Generally, these studies demonstrate that endothelial cells isolated from both ends of the vascular tree have the capacity to express the major CAMs that participate in the recruitment of leukocytes (14– 16). Some studies show that different cytokines and endotoxin elicit qualitatively and quantitatively similar responses in the expression of ICAM-1, VCAM-1, and E-selectin between cultured human arterial and venous endothelial cells, with stimulants like oxidized low density lipoprotein (oxLDL) serving as a more effective inducer of the endothelial CAMs on arterial endothelial cells (14). A limitation of some of these comparisons is that the arterial and venous endothelial cells were not derived from the same vascular bed or donor. When such an analysis is performed, clear differences between arterial and venous endothelial cells can be demonstrated. For example, a comparison of donor-matched human iliac venous and arterial endothelial cells has revealed that both arterial and venous endothelial cells express tumor necrosis factoralpha (TNF-α)-inducible ICAM-1 and E-selectin and that these molecules are functional in mediating leukocyte adhesion (16). VCAM-1, on the other hand, was only inducible on the cultured venous endothelial cells, where it was functional in binding leukocytes.

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4. INTER-ORGAN DIFFERENCES IN LEUKOCYTE-ENDOTHELIAL CELL ADHESION 4.1. In Vivo CAM Expression In vivo studies based on immunohistology have rather consistently demonstrated a localized expression of different endothelial CAMs in venules of normal and/or inflamed tissues and the intensity of the immunostaining for certain endothelial CAMs in these venules appears to be functionally correlated with the amount of leukocyte infiltration (3). Differences in tissue processing and immunostaining procedures, imprecise localization of antigen to the lumenal membrane, the subjective nature of scoring the intensity of antibody staining, and variations in the binding affinity of antibodies employed between laboratories make it difficult to draw conclusions about inter-organ differences in CAM expression. An approach that has been developed to overcome many of the limitations of immunohistochemistry for quantification of endothelial CAM expression involves monitoring the accumulation of a radiolabelled monoclonal antibody (mAb) that is directed against a specific endothelial CAM. This novel approach was originally developed by Haskard and associates (17) to quantify vascular lumen expression of E-selectin in porcine skin following systemic or local injection of interleukin-1. We have modified this radiolabelled mAb method to provide quantitative measurements of endothelial CAM expression in rat and mouse models of acute and chronic inflammation (18–20). Our dual radiolabelled mAb method determines the relative accumulation, in any regional vascular bed, of a binding mAb to a specific endothelial surface epitope (e.g., P-selectin or CD40) and an isotype-matched nonbinding mAb, the latter of which is used to compensate for non-specific accumulation of the binding mAb. We have successfully employed this method to quantify the expression of different endothelial CAMs [P-selectin, E-selectin, VCAM-1, ICAM-1, ICAM-2, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1)] in different vascular beds of the mouse (18–23). The method yields values that are expressed as either % injected dose (%ID) or nanograms of mAb per gram of tissue. In our previous studies employing the dual radiolabelled mAb technique, it was demonstrated that: (1) the radiolabelling procedure does not alter the ability of the mAb to block leukocyte-endothelial cell adhesion in postcapillary venules (18); (2) accumulation of the radiolabelled binding (but not the non-binding) mAb in a vascular bed can be inhibited in a dose-dependent fashion by progressively increasing concentrations of the cold (unlabelled) binding mAb (but not the non-binding mAb) (18); (3) expression of the targeted endothelial cell surface epitope on non-binding circulating cells (e.g., CD40 on T-cells) does not interfere with the assay since all residual blood is flushed from the vasculature prior to tissue sampling; and (4) application of this technique to the relevant adhesion molecule-deficient mice yields expression values that are essentially 0, both under basal and stimulated conditions (19,20). It has also been

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determined that the accumulation of binding mAb, relative to the non-binding mAb, is essentially unchanged after a 2–5 min intravascular mixing period (18–20). This is an advantage of the technique since the rapid accumulation of a mAb due to engagement of its ligand allows for an estimate of mAb binding before significant immunoglobulin extravasation can occur. The distributional half-lives of the binding and non-binding mAbs are on the order of several hours while the endothelial CAM measurement technique quantifies the mAb binding that occurs within a few minutes. Our initial studies of ICAM-1 expression using the dual radiolabelled mAb technique were undertaken in rats (18). Constitutive and endotoxin-induced ICAM-1 expression was monitored in different regional vascular beds. The binding of the radiolabelled ICAM-1 mAb varied widely among organs. The constitutive level of ICAM-1 expression appeared to be significant in all vascular beds, which is consistent with the effectiveness of ICAM-1 neutralizing mAbs in blocking the rapid leukocyte–endothelial cell adhesion elicited by certain mediators and conditions (3). Endotoxin induced an increased ICAM-1 expression in virtually every organ, with vascular beds normally exhibiting a low constitutive expression of ICAM-1 (heart and skeletal muscle) showing the greatest increases and lung (which has the highest constitutive expression of ICAM-1) showing the smallest increment. The profound differences in predicted expression of basal ICAM-1 between vascular beds like the lung, heart, and skeletal muscle may reflect corresponding differences in endothelial surface area in these tissues, rather than a higher density of ICAM-1 per endothelial cell. This possibility is supported by experiments showing that the relative accumulation of a mAb directed against angiotensin converting enzyme, which is constitutively expressed on the surface of endothelial cells, in different organs follows a pattern similar to that obtained basally with an anti-ICAM-1 mAb (lung>heart>skeletal muscle) and it is positively and significantly (r=0.98, p<0.001) correlated with published estimates of endothelial surface area for the same organs (24). This suggests that a meaningful comparison of the density of endothelial CAM expression between vascular beds must take into consideration inter-organ differences in endothelial surface area. Two approaches have been used to normalize the endothelial CAM expression data to vascular surface area: (1) normalize the expression data to published estimates of endothelial surface area for each specific vascular bed, providing a value expressed as µg mAb per cm2 (25), and (2) normalize the level of expression for an inducible CAM (e.g., P-selectin or ICAM-1) to a constitutively expressed and non-inducible CAM, such as PECAM-1 or ICAM-2 (22,23). Large differences in the constitutive expression of PECAM-1 (22) and ICAM-2 (23) have been noted for a variety of vascular beds, although we have demonstrated in the mouse that PECAM-1 and ICAM-2 levels greatly exceed the constitutive and induced expression of all other endothelial CAMs (Fig. 3B). As can be seen in Fig. 4, the level of PECAM-1 expression in tissues is positively and highly significantly correlated with endothelial cell surface area. Thus, since PECAM-1 expression is not altered by cytokine stimulation in murine tissues (22), the expression of other (cytokine-responsive) endothelial CAMs can be normalized for regional variations in vascular surface area using PECAM-1 expression (22,23). Interestingly, the invariance of PECAM-1 expression within vascular beds has led some investigators to use the accumulation of radiolabelled PECAM-1 mAb in tissues as a measure of microvessel proliferation (26,27).

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4.1.1. Selectins Selectins are expressed on the surface of activated endothelial cells in a variety of tissues of the mouse (Table 1). Significant constitutive expression of P-selectin can be demonstrated in the intestine and, to a lesser extent, lung of wild-type mice, but not in mice that are genetically deficient in P-selectin (19,25). Constitutive expression of Eselectin is also highest in the intestine, followed by the heart. The high constitutive levels of both selectins in the gut are consistent with the view that this organ is normally in a state of controlled inflammation. In contrast, relatively low levels of E- and P-selectin are normally noted in the brains of wild-type mice. The expression of both P- and E-selectin is increased in a time-dependent manner in different tissues of wild-type mice treated with endotoxin. P-selectin expression reaches a maximum between 4 and 8 hr after endotoxin challenge, while E-selectin requires 3–5 hr for peak expression. The largest increments in both P- and E-selectin expression are seen in the lung, small intestine and heart after endotoxin challenge, with the brain and skeletal muscle exhibiting the smallest responses. In all instances, however, selectin expression increased at least 10-fold. Histamine also significantly increases the expression of P-selectin, but not E-selectin, in most vascular beds (19). However, unlike the transcription-dependent response elicited by endotoxin challenge, histamine produces an elevation in P-selectin within 5 min and the adhesion molecule

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Figure 3 Kinetics of endothelial cell adhesion molecule expression in the mouse intestine after challenge with endotoxin (A) and cytokines (B). Also shown is the response of P-selectin expression to histamine challenge in panel A (first peak). (Based on data from Refs. 19–23.)

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Figure 4 Relationship between published estimates of endothelial cell surface area and PECAM-1 expression in different tissues of the mouse. (Based on data from Refs. 22,24,44−46.) Table 1 Regional Differences in the Constitutive Expression of Endothelial Cell Adhesion Molecules in Murine Tissues Endothelial CAM Expression (Molecules×107 per cm2) Organ

P-selectin

E-selectin

ICAM-1

VCAM-1

Lung

2.66

0.11

26.81

2.37

Heart

0.57

4.65

9.25

8.18

25.49

8.78

17.10

3.27

Muscle

1.02

0.00

8.90

3.50

Brain

0.48

0.15

0.90

4.29

Small intestine

Data derived from Refs. (19,20,25) and corrected for PECAM-1. remains elevated for as long as one hour. The rapid expression of P-selectin after histamine likely represents mobilization of a preformed pool of the adhesion molecule from Weibel-Palade bodies (3). The brain vasculature is unresponsive to histamine, while

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the stomach responds most vigorously to the autacoid, followed by the heart, lung, and intestine. The histamine-induced P-selectin expression in all vascular beds is inhibited by a histamine-H1- (but not an H2) receptor antagonist, indicating that histamine engagement of H1-receptors on endothelial cells results in the mobilization of preformed P-selectin to the endothelial cell surface (19). The time-related changes in P-selectin expression after histamine treatment as well as the temporal responses of P- and E-selectin to endotoxin are shown for the intestinal microvasculature in (Fig. 3A). Tissue-specific responses of the selectins to a variety of inflammatory stimuli have been investigated in other animal models. For example, the dual radiolabelled mAb technique has been used to address the possibility that endotoxin can infer tolerance of Eand P-selectin expression to a subsequent dose of endotoxin (28). While the responses of intestinal P- and E-selectin to endotoxin challenge are significantly reduced following an initial priming dose of endotoxin, the selectins in other organs such as lungs and heart do not exhibit an endotoxin-induced preconditioning response. Interestingly, the constitutive expression of selectins, particularly E-selectin, is much higher in microvessels of solid tumors than other (normal) vascular beds, when corrected for endothelial cell surface area. Furthermore, the expression of E-selectin, but not P-selectin, in tumor vessels is refractory to TNF-α stimulation (23). Similarly high constitutive levels of the selectins and a blunted response to TNF-α have been demonstrated in neovascularized tissue and skin (29). Selectin-deficient mice have also proven useful in defining the role of E- and Pselectin in the recruitment of leukocytes in different models of inflammation. An inherent assumption in the use of such mutants is that gene targeting in embryonic stem cells results in either the deletion or an attenuated expression of the targeted glycoprotein in the resultant mutant mouse. This assumption is generally supported by the absence of the gene encoding for the targeted adhesion molecule. However, a criticism frequently levied by physiologists is whether gene-targeted mice exhibit an altered expression of only those proteins that were targeted by gene deletion or whether chronic deletion of one gene results in a compensatory change in other genes. These concerns appear justified relative to endothelial cell expression of the selectins in CD18−/−, ICAM-1−/− and P-selectin−/− mice, all of which exhibit an altered expression of E- and P-selectin under basal conditions and/or following TNF-α challenge. These altered responses are often vascular bed specific (30). Since the pattern of selectin expression in endothelial cells cultured from wild-type and mutant mice is similar to endothelium in vivo (31), it would appear that the endothelial cells per se (rather than surrounding cells/environment) undergo phenotypic changes that affect multiple CAMs even when only one gene is deleted or disrupted. These responses of alternate endothelial CAMs in gene-targeted mice should be considered when interpreting functional data derived from the same animals.

4.1.2. Intercellular Adhesion Molecule-1 ICAM-1 has been implicated as a mediator of the firm adhesion and transendothelial migration of leukocytes in different vascular beds. Significant constitutive levels of this endothelial CAM have been measured in most vascular beds of the rat and mouse (18,20). When constitutive ICAM-1 expression levels in wild-type mice are normalized for endothelial surface area, endothelial cells in the lung exhibit the highest density of

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ICAM-1, followed closely by the small intestine (Table 1). Heart and skeletal muscle express comparable constitutive levels of ICAM-1, with the brain vasculature expressing 1/10th the density of the muscle circulations and 1/30th the density of lung endothelial cells. Nonetheless, the density of constitutively expressed ICAM-1 in all of these organs is 2–250 times greater than that observed for P- or E-selectin in the same tissues. As would be expected, ICAM-1 is undetectable by the dual radiolabelled mAb technique in mutant mice that are genetically deficient in ICAM-1 (20). Interestingly, constitutive ICAM-1 expression is significantly lower in the vascular beds of the gastrointestinal tract, liver and skin, but not the lung, heart or brain, of germ-free mice compared with their conventional counterparts (32), suggesting that indigenous gastrointestinal microflora are responsible for a significant proportion of the basal ICAM-1 expression detected in both intestinal and extra-intestinal tissues. Tumor necrosis factor-α (administered i.p.) produces a dose-dependent increase in ICAM-1 expression in virtually all tissues of the mouse (20). Significant increases above constitutive levels are seen as early as 2 hr and plateau levels are reached at 5–9 hr, where they remain elevated for at least 24 hr (Fig. 3B) and are comparable to levels observed in endotoxin-treated mice. Tissue-specific differences are observed, although these are blunted when normalization for PECAM-1 is performed. While TNF-α elicits an elevated ICAM-1 expression that is sustained for several hours in most vascular beds, the changes in circulating soluble ICAM-1 (sICAM-1) concentration elicited by the same systemic stimulus are only transient (33). The transient surge in plasma sICAM-1 after an inflammatory stimulus is consistent with rapid, massive shedding of membrane-bound ICAM-1 from endothelial cells throughout the vasculature and the eventual clearance of shed protein from the circulation. The dissociation between plasma sICAM-1 concentration and endothelial ICAM-1 expression in most vascular beds suggests that sICAM-1 may not be a useful surrogate marker for the intensity of ICAM-1 expression and severity of inflammation (33). The high density of ICAM-1 expression in the lung vasculature has been exploited by some investigators for selective drug delivery to this tissue (34,35). Catalase conjugated with mAbs against ICAM-1 accumulates preferentially in the lung and confers protection against oxidative injury. ICAM-1-dependent immunotargeting has also been used to deliver anti-thrombotic drugs to pulmonary vascular endothelium (35). Tissue-type plasminogen activator (tPA) conjugated with an anti-ICAM-1 mAb has been shown to accumulate in rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. The available quantitative data for ICAM-1 expression would suggest that immunotargeting based on constitutive ICAM-1 density may be an effective therapeutic strategy for diseases affecting the splanchnic as well as the pulmonary circulation.

4.1.3. Vascular Cell Adhesion Molecule-1 VCAM-1 is another member of the immunoglobulin supergene family that contributes to the firm adhesion and endothelial transmigration of leukocytes, particularly lymphocytes and monocytes. VCAM-1 is constitutively expressed in a number of vascular beds in wild-type mice (20). When normalized to endothelial surface area, the basal level of VCAM-1 does not vary dramatically between lung, heart, intestine, muscle, and brain.

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VCAM-1 density on resting endothelial cells is generally lower than that of ICAM-1, except in the heart where the two CAMs exhibit comparable surface levels and in the brain where VCAM-1 density is about four times higher than ICAM-1 density (Table 1). A consequence of the high basal expression of VCAM-1 in some vascular beds is a relatively small increment (1.5- to 3.5-fold increase) in expression levels upon cytokine stimulation. Peak expression of VCAM-1 after TNF-α stimulation can occur as early as 2 hr but generally requires up to 5 hr, and this is followed by a sustained elevation that lasts for at least 24 hr (Fig. 4). Organ-specific differences in VCAM-1 responses to stimulation are obvious even after normalization for PECAM-1 expression (20).

4.1.4. Mucosal Addressin Cell Adhesion Molecule-1 MAdCAM-1 is an excellent example of an endothelial CAM that is found in relatively few vascular beds. This adhesion molecule plays an important role in mediating the adhesion of lymphocytes to vessels within the lymphoid tissues of the intestine, i.e., Peyer’s patches and lymph nodes (36). The density of constitutive MAdCAM-1 expression on endothelial cells in the small intestine is roughly one-third the density detected for ICAM-1 in the same tissue (21). While the mesenteric lymph nodes express more MAdCAM-1 than the gut proper, negligible levels are seen in the brain, heart, lungs, and most other organs. Only in those tissues that exhibit significant constitutive expression of MAdCAM-1, are 2- to 3-fold increases in expression observed after TNF-α administration (21). Peak expression of MAdCAM-1 generally occurs 18–20 hr after TNF-α administration and it remains elevated for at least 48 hr (Fig. 3B).

4.2. In Vitro CAM Expression In vitro studies have been of limited value in addressing regional differences in endothelial CAM expression. This is largely due to the emphasis given to isolation and culture of endothelial cells from large vessels (human umbilical vein, iliac artery and vein, pulmonary artery, etc.). However, recent progress in the isolation and culture of microvascular endothelial cells from regional vascular beds of wild-type and mutant mice (37) suggests that an analysis of phenotypic differences between endothelial cells from different vascular beds is now possible. Primary cultures of microvascular endothelial cells have been recently generated from different organs of mice whose tissues harbor a temperature-sensitive SV40 large T antigen (H-2Kb-tsA58; ImmortoMice) (38). A selection strategy that targeted cell populations expressing E-selectin and VCAM-1 proved successful in generating microvascular endothelial cell lines from a number of different organs, including lung, colon, kidney, heart, bone, brain, uterus, ovary, bladder, liver, prostate, and pancreas. The pattern of constitutive expression of E-selectin, VCAM1, and ICAM-1 was similar for most of the lines derived from different organs, exhibiting very low expression levels of E-selectin and VCAM-1 and more pronounced expression of ICAM-1. However, the highest expression of VCAM-1 was found in the heart, which is in agreement with in vivo findings using the dual radiolabelled mAb method (Table 1).

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4.3. Intravital Microscopy Intravital microscopy has permitted the visualization and quantification of the rolling, firm adhesion, and transendothelial migration of leukocytes in different regional vascular beds (3). In all vascular beds studied to date, the adhesive interactions between leukocytes and endothelial cells are largely confined to postcapillary venules. Blocking mAbs to specific endothelial CAMs and the use of mice that are genetically deficient in CAMs have been effectively employed to define the molecular determinants of leukocyte-endothelial cell adhesion in different vascular beds (1,3). While these studies have revealed that there are many similarities in the molecular determinants of leukocyteendothelial cell adhesion between vascular beds, some notable differences have been reported. For example, antigen challenge following systemic ovalbumen sensitization (type 1 hypersensitivity response) induces leukocyte-endothelial cell adhesion in postcapillary venules of the skin and cremaster (39). However, in cremaster muscle, leukocyte recruitment is entirely dependent upon P-selectin, while both P- and E-selectin expression must be blocked in order to prevent leukocyte interactions with dermal venular endothelium. The dual radio-labelled mAb technique revealed that only Pselectin (not E-selectin) expression is increased following antigen challenge in the cremaster, while only E-selectin is upregulated in the skin microcirculation. Additional evidence has been provided that suggests unique recruitment paradigms in the microcirculation of the liver and brain (40). In the brain, for example, where venular shear rates are high and adhesion molecule expression is low, platelets may play an important role as a bridge between leukocytes and endothelium (41). Recent evidence in the brain microcirculation supports the possibility that leukocytes may also act as a bridge between platelets and cerebral endothelial cells (42). This ability of leukocytes to serve as a platform for the adhesion of platelets in venules is not unique to the brain, however, since a similar process has been demonstrated in mesenteric venules (43). Overall, the results obtained from intravital microscopic studies of leukocyte-endothelial cell adhesion are consistent with endothelial CAM expression data that predict organ-and microvascular segment-specific mechanisms of leukocyte recruitment.

5. CONCLUSIONS AND IMPLICATIONS Technological advances over the past two decades have allowed for quantitative estimates of endothelial CAM expression in different regional vascular beds as well as within specific segments of a single vascular bed. The results generated from these studies indicate that there are significant intra- and inter-organ differences in CAM expression and this is reflected in the organ specificity of mechanisms for leukocyte recruitment. The assumption that isolated cultured endothelial cells derived from major blood vessels provide information that is applicable to many or most regional vascular beds should be viewed with caution. The revelation that there are unique profiles of constitutive and induced expression of endothelial CAMs in different regional vascular beds demonstrates the diversity of endothelial cell activation responses that can occur in

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health and disease, and it offers hope for targeted delivery of drugs to endothelial cells in specific tissues during periods of localized or systemic inflammation.

ACKNOWLEDGMENT This work was supported by grants from the National Institutes of Health (HL26441 & DK 43785).

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14 Vascular Biology of the Placenta Hartmut Weiler The Blood Research Institute, Blood Center of Southeastern Wisconsin, Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, U.S.A.

1. INTRODUCTION The placenta is a gender-specific transitory organ with the sole purpose of enabling the growth and development of the fetus. It mediates the efficient transfer and exchange of nutrients and metabolites between maternal and fetal circulation, suppresses the mother’s immune response to the genetically distinct fetal “allograft,” and releases mediators that adapt the mother’s physiology to the specific requirements of pregnancy (1). The focus of this chapter is the vascular bed of the placenta. Establishment and maturation of the placental vascular bed require the functional integration of fetal and maternal circulations, coordination of maternal and fetal blood vessel formation, and involves unique processes of vascular remodeling. The blood-tissue interface in the placenta possesses characteristic features not found anywhere else in the body. It is genetically heterogeneous, involves both fetal and maternal blood vessels, and is formed by two different cell types, namely the endothelium and trophoblast. Due to their anatomical localization at the blood-tissue interface, trophoblast cells must acquire endothelial-like functions. This allows them to replace endothelium in the maternal arteries, and to increase blood flow to the placenta, and to participate in the regulation of hemostasis at the feto-maternal interface. Much of the interest in placental vascular biology has been fueled by the lack of knowledge about the pathogenesis of a single, but exceedingly common disease of pregnancy, pre-eclampsia (PE), which is caused by the dysfunction of the placental vascular bed. During mammalian embryogenesis, the vasculature of the placental yolk sac is the first fetal vascular bed to be established, and mutations that disrupt blood vessel formation and maturation in the yolk sac cause intrauterine death. The goals of this chapter are to: (1) provide an overview of placental development and anatomy of the placental vasculature; (2) review the unique aspects of the placental vasculature, i.e., the remodeling of spiral arteries, its functional specialization, and the adoption of an endothelial-like phenotype by trophoblast cells; (3) briefly discuss current hypotheses about the pathogenesis of PE; and (4) summarize the current knowledge about the striking importance of hemostasis for the development of the placental yolk sac vasculature.

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2. DEVELOPMENT AND FUNCTIONAL ANATOMY OF THE PLACENTA Many of the more recent insights into the molecular mechanisms of placental development and pathology have been gathered from analyses of genetically modified laboratory mice (2,3). Fortunately, findings from these studies may have particular relevance to the human placenta since both species share a particular kind of placentation (hemochorial placenta), in which specialized placental cells of fetal origin—the trophoblast—are directly bathed in maternal blood. Following is a brief review of placental development in mice, and an anatomical comparison of the mature mouse and human placenta. Implantation of the embryo in the uterine wall triggers a thickening of the uterine wall due to differentiation and proliferation of uterine stromal cells. This process of decidualization is accompanied by the formation of new blood vessels in the uterus that deliver maternal blood to the embryo. Mice, as well as humans, display a hemochorial type of placentation, in which the continuity of maternal blood vessels emanating towards the implantation site is breached and embryonic tissue (trophoblast) is placed in direct contact with maternal blood. During the first 9 days of mouse development, the exchange of nutrients, gases, and waste products between mother and embryo occurs across an avascular layer of extraembryonic membrane, the parietal yolk sac that surrounds the embryo proper. This membrane consists of an outer layer of trophoblast giant cells attached to a thick extracellular matrix (Reichert’s membrane) that is produced by underlying parietal endoderm cells (see Fig. 1 for an overview of placental anatomy). Underlying the parietal yolk sac is another layer of extraembryonic membranes, the visceral yolk sac. The latter structure contains an outer layer of visceral endoderm that mediates adsorption and secretion of metabolites. Based on the spectrum of products produced by these cells, and the specific gene expression profile, visceral endoderm likely functions as an extraembryonic liver-like organ. Attached to the embryonic aspect of the visceral endoderm layer is the highly vascularized mesoderm. Blood vessels of the visceral yolk sac are the first region of the embryonic vasculature to become functional, i.e., to exhibit pulsatile blood flow powered by the embryonic heart. The parietal and visceral yolk sac together constitute the early or yolk sac placenta. Approximately 8 days following fertilization (embryonic day 8.5, E8.5), the allantois with associated embryonic blood vessels makes contact with the chorionic plate, and initiates formation of the chorioallantoic placenta, which functionally supplants the yolk sac placenta. Fetal blood vessels of the allantois invade the chorionic plate, and induce the proliferation and differentiation of trophoblast stem cells in the ectoplacental cone. The concerted expansion of the fetal vasculature into the placenta, the emergence and migration of differentiated and specialized trophoblast cell populations derived from the trophoblast stem cell pool, and the continued remodeling of the maternal vasculature give rise to the chorio-allantoic placenta, which remains the major route of exchange between maternal and fetal circulations through the end of gestation. The functional and anatomical aspects of the relation between maternal and fetal circulation in the mouse have been described in detail (4). The main artery supplying maternal blood to the uterus branches into several spiral arteries that converge again at the fetal trophoblast giant cell layer into large arterial canals delivering the blood to the base of the placenta. The

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mother’s blood then passes again upwards through the intervillous space of the placental labyrinth containing the fetal capillary bed of the placenta, and is eventually drained into venous sinuses in the decidua. Within the labyrinth, fetal syncytial trophoblast cells contact

Figure 1 Anatomy of the fetoplacental unit. Schematic representation of mouse embryos at ~7.5 days after conception (E7.5; equivalent to first trimester human development) and at midgestation (E10–12). In the E7.5 embryo, maternal blood is in direct contact with the embryo’s outer shell of polyploid trophoblast giant cells. The yolk sac has not yet fully developed. Fetomaternal exchange occurs through the trophoblast and parietal endoderm layers. Fusion of the allantois with the chorionic plate initiates formation of the chorio-allantoic placenta, in which fetal capillary networks are embedded in maternal blood sinuses. Expansion

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of the visceral yolk sac and a rotating movement of the embryo wrap the fetus in an additional layer of extraembryonic membranes, the visceral yolk sac. For details, see text. maternal blood on one side, and appose fetal capillary endothelium on the other side. The afferent maternal spiral arteries undergo a remodeling process of the arterial wall that results in the loss of vascular wall smooth muscle cells and a substantial increase of vessel caliper to increase blood flow into the placenta. Maternal arteries and the venous segments of the maternal vascular bed are lined by maternal endothelial cells. In contrast, the interface between maternal blood and fetal tissue in the placental labyrinth, in the central arterial canals, and in segments of the spiral arteries proximal to the trophoblast giant cell layer consists of embryonic trophoblast cells (one continuous layer of trophoblasts lining this segment of the vessel). Invasion of the spiral arteries by embryonic trophoblast is a late event in mice (after ~E12), and remains limited to a short distance from the trophoblast giant cell layer. Human embryos also form a rudimentary, yet functional yolk sac. Of the extraembryonic membranes, human placenta does not elaborate a parietal endoderm layer. In spite of anatomical differences, the structure and function of the mature chorioallantoic placenta are largely equivalent in humans and mice (5). In both species, the barrier between maternal and fetal blood consists of a single layer of fetal endothelium lining embryonic blood vessels and one ore more layers of fetal

Figure 2 Cyto-architecture of the interface between maternal and fetal blood in the mouse and human placenta. The mouse placenta forms a

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labyrinth-type meshwork of trophoblast with embedded fetal capillaries. Maternal blood penetrates the sponge-like trophoblast syncytium. In humans, the trophoblast is consolidated into columns (villi) floating in maternal blood sinuses. In both species, the trophoblast layers contacting maternal blood form a multi-nucleated syncytium. Fetal blood vessels are always lined by an intact endothelium. trophoblast (Fig. 2). In the mouse, intermingling of fetal and maternal vascular structures creates the convoluted, sponge-like structures of the placental labyrinth. The human trophoblast is organized in more defined cytotrophoblast columns in which fetal capillaries are embedded. The trophoblast columns are distinguished as anchoring villi attached to the decidua, and floating villi. The surface of either villus type is covered by a continuous shell of syncytiotrophoblast cells bathed in blood draining from maternal arteries. Mouse syncytiotrophoblast cells in the labyrinth layer resemble the human syncytiotrophoblast and underlying villous cytotrophoblast. The mouse trophoblast giant cell is likely the counterpart of the human extra-villous cytotrophoblast, and both cell types share characteristic features such as polyploidy as a result of endoreduplication. The human placenta does not elaborate a specific spongiotrophoblast layer, but trophoblast of the anchoring villi may be analogous to the mouse spongiotrophoblast. Invasive endo/perivascular and interstitial trophoblast populations have been described in mice and humans, but invasion of the mouse endo/perivascular trophoblast into maternal spiral arteries is much more shallow than in humans, where it can extend into the outer, muscular layer (myometrium) of the uterus.

3. FUNCTIONAL SPECIALIZATION OF THE PLACENTAL ENDOTHELIUM Placental endothelium is a rather heterogeneous cell population with respect to origin (maternal vs. fetal), location (placenta vs. visceral yolk sac), and structure of the vascular bed. A common feature of the both the fetal and the maternal vascu-lar endothelium in the placenta is its transitory and dynamic nature: it is established de novo during development of the placenta and its lifespan is limited to the duration of pregnancy. This defining property sets the placental vascular bed apart from other vascular beds of the organism, and is reflected in a high proliferative index of placental endothelium. As a consequence, the proper regulation of vasculogenesis, angiogenesis, and vessel maturation is critical for adequate placental function, maintenance of embryonic growth,

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and successful pregnancy out-come (6,7). Indeed, the primary reason for the embryonic lethality seen in many experimental mouse models with defects in the molecular pathways controlling the formation and maintenance of blood vessels is a failure to establish the proper function of the placental vascular bed, in particular the elaboration of blood vessels in the placental yolk sac (8,9). A peculiar—and as of yet unexplored— aspect of the defects in these mouse models is that they appear to affect blood vessels in the placental yolk sac in a more serious manner than blood vessels in other organs of the developing fetus. Consistent with the overall theme of endothelial cell heterogeneity, certain genes are preferentially expressed in the placenta compared with other vascular beds, and in some cases genes are differentially expressed between different segments of the placental vasculature. For example, expression of the Glut-3 glucose transporter in the endothelium is limited to the placenta, blood-retina and blood-brain barrier (10). Likewise, maternal vessels in the decidua are distinguished by expressing high levels of the HDL-receptor SR-B1 (11). Other markers of placental endothelium such as the expression of integrins usually associated with sprouting or activated endothelium, probably reflect the dynamic, “vasculogenic” state of the placental vascular bed, rather than its functional specialization (12,13). The de novo establishment/expansion of placental blood vessels, as well the acquisition of vascular bed-specific characteristics is likely controlled in an epigenetic manner by the local microenvironment. The predominant cell type in this microenvironment is the fetal trophoblast at the interface between fetal and maternal circulation. Trophoblast cells produce and release a variety of factors regulating placental angio/vasculogenesis, and changes in blood supply/perfusion associated with hypoxia elicit compensatory responses of the trophoblast population (14–19). In addition, the placenta contains at any stage of development significant numbers of lymphocyte populations thought to be involved in controlling local cytokine production, facilitating endovascular trophoblast invasion and cytokine-dependent remodeling of spiral arteries, and modulating placental development (20–25). Uterine NK cells (uNK; CD56bright/CD16negative) are distinct from other cells of the NK lineage, and are recruited to the placenta by a specific homing process mediated in part by interaction of the chemokine receptor CXCR4 (expressed by NK cells) with CXCL12 produced by extravillous trophoblast cells (26).

4. REMODELING OF MATERNAL SPIRAL ARTERIES: A UNIQUE PROCESS OF VASCULAR MODIFICATION IN THE PLACENTA Blood supply into the placental area where exchange between the fetal capillary network and maternal blood occurs is delivered via coiled spiral arteries, which originate from radial arteries emanating from the arcuate blood vessels of the uterine wall (27). Over the first 4–5 months of human pregnancy, these arteries undergo a

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Figure 3 Remodeling of maternal arteries in the human uterus. Maternal arteries (spiral arteries) delivering blood to the placenta undergo a remodeling process, i.e., an erosion of vascular smooth muscle cells, and dilation of the blood vessel. Fetal cytotrophoblast cells originating from villi anchored to the decidua invade the arterial lumen, likely from the outside in (intravasation), resulting in the partial replacement of maternal endothelium by fetal trophoblast. remodeling process that results in the dilation of these vessels, loss of elasticity and vasoraotor control, loss of vascular wall smooth muscle cells, and invasive replacement of maternal endothelial cells by fetal trophoblast (Fig. 3). The latter process gives rise to a functionally specialized population of endovascular trophoblast lining the terminal segments of the spiral arteries. It is thought that these alterations enhance maternal blood

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flow to the placenta, and uncouple the regulation of blood flow into the placental vascular bed from maternal vasomotor control. The cellular and molecular mechanisms underlying the loss of an organized smooth muscle cell layer, vasodilation, and the invasion of the arterial lumen and media by fetal trophoblast cells are just beginning to emerge (for review see Refs. 28–30). The initial dilation and erosion of smooth muscle cells does not appear to depend on direct trophoblast-vessel interactions, but could be triggered by nitric oxide and/or other mediators released from uterine lymphocytes or remote trophoblast cells. The population of spiral artery lumen and wall with endovascular trophoblast may occur via two hypothetical routes, extravasation and intravasation: extravasation describes the “insideout” migration of trophoblast cells of unknown origin from within the villous blood space upwards into the spiral arteries, and from there into the wall of the vessels. The concept of intravasation is somewhat better supported by observation, and assumes that trophoblast cells located at the tip the trophoblastic villi migrate into the decidual interstitium and invade the spiral artery from the outside in. As in the case of the initial vasodilation, the molecular details of this interaction between trophoblast and vessel are unclear at present. One hypothesis (trophoblast-endothelial conversion) postulates that the acquisition of an endothelial-like adhesion phenotype [for example, PECAM-1, VEcadherin, vascular cell adhesion molecule (VCAM)-1] would allow trophoblast cells to engage in cell-cell interactions that are otherwise restricted to homotypic interactions between endothelial cells (31). Alternatively, the trophoblast might rely on receptorligand interactions mediating leukocyte-endothelial adhesion under conditions of inflammation. Invasive trophoblast has been reported to express sialyl-LewisX carbohydrate moieties, which might engage E- and P-selectins present on resident endothelium (32,33). The mobilization and survival of invasive trophoblast has tentatively been linked to trophoblast-derived endothelial nitric oxide synthase (NOS) and iNOS production (34,35). Such a role for nitric oxide could serve to coordinate the (at least in part) NO-dependent vasodilation with the consecutive invasion of dilated vessels by trophoblast. The continued interest in this particular and unique aspect of placental vascular biology stems from the potential importance of this phenomenon for the pathogenesis of PE and intrauterine growth retardation, since both disease entities are associated with abnormal remodeling of maternal uterine arteries (see below).

5. ADOPTION OF A VASCULAR PHENOTYPE BY TROPHOBLAST CELLS: THE CONCEPT OF “ENDOTHELIAL MIMICRY” Trophoblast cells that are in direct contact with maternal blood express a battery of gene products usually associated with endothelial cells in other vascular beds. The adoption of an endothelial cell-like phenotype has been termed “endothelial mimicry” (36,37). This concept of “epithelial-endothelial transformation” describes the fact that fetal placental trophoblast cells, although derived from a different embryonic cell lineage, in many respects resemble vascular endothelial cells. Such a differentiation-dependent acquisition

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of an endothelial-like gene expression program was first described in human endovascular trophoblast cells, which downregulate specific epithelial adhesion markers (E-cadherin), but turn on the expression of endothelial cell-like integrins and adhesion receptors (i.e., PECAM/CD31; VE-cadherin, VCAM-1, α4 and αvβ3 integrins) as they invade the mother’s blood vessels in the uterus and interdigitate with endothelial cells of maternal origin (31). Studies of the mouse placenta show a similar expression of endothelial-specific adhesion molecules and differentiation antigens in trophoblast cells (38). In addition, trophoblast cells express a comprehensive profile of gene products that mediate intravascular homeostasis, including regulators of blood coagulation [thrombomodulin (TM), endothelial protein C receptor (EPCR), tissue factor pathway inhibitor (TFPI-1), ecto-ADPase/CD39; tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), protease nexin-1, and annexin V], protease activated receptors (PAR) for coagulation factors and other extracellular proteases (PAR1, -2, and -4), as well as regulators of vascular tone and leukocyte interactions (including iNOS, prostacyclin synthase, Cox-2, and E-selectin). The remarkable overlap in function between trophoblast cells and vascular endothelium suggests that concepts devooped to describe endothelial dysfunction in thrombosis and inflammation can probably be adopted to describe pathogenic mechanisms underlying diseases of pregnancy.

6. DYSFUNCTION OF THE VASCULAR BED IN PLACENTAL DISEASE: PRE-ECLAMPSIA The interest in placental biology, trophoblast function, and vascular biology of the placenta has been fueled by the desire to gain insights into the pathogenesis of a severe and potentially fatal disease of pregnancy, PE (for an overview of pathology and pathogenesis, see Refs. 39–42). The disease occurs only in pregnancy, and affects in excess of 1 in 20 women. Severe PE is manifest as pregnancy-induced or pregnancyaggravated hypertension due to increased sensitivity of the vasculature to pressor agents, and a resulting generalized vasospasm. Pre-eclampsia affects multiple organs and leads to proteinuria, oliguria, hepatocellular dysfunction, and edema, in particular pulmonary edema. It is accompanied by serious neurological complications, and so-called HELLP syndrome develops (Hemolytic anemia, ELevated liver enzymes, severe potentially fatal eclamptic seizures. In about 10% of preeclamptic women, the and Low Platelet count), in which exaggerated hemostatic activation leads to consumption of platelets. The unique manifestations of PE and the underlying pathogenic principles are based on the peculiar properties of the vascular bed in the placenta; the disease is the consequence of disrupted or de-regulated feto-maternal communication. Pre-eclampsia only occurs in the presence of a placenta, and is resolved by delivery of the fetus. The roots of the disease must, therefore, reside in a defect in the placenta, which then affects multiple other organs, likely via release of soluble mediators from the placenta. A current working concept of the pathogenesis of PE invokes a two-stage model, in which reduced perfusion of the placenta (first stage) triggers an adaptive response on the part of the fetus to compensate for the reduced blood supply (second stage). Pre-eclampsia occurs if the fetal response is excessive or aberrant, or if the response of the mother to the fetal signals is inappropriate. An attractive feature of this hypothesis is that it is conceptual rather than mechanistic,

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and therefore, can accommodate the known multitude of risk factors and modifiers of PE, which might affect different, yet convergent aspects of the disease mechanism. Attempts to explain the pathophysiology of PE have concentrated on two specific aspects: (1) the pathogenesis of reduced placental perfusion, and (2) the nature of the circulating factors triggering maternal hypertension and vascular dysfunction. Clinical evidence of altered endothelial function in the mother has led to the notion of PE as an endothelial-based disease. Even before the onset of clinical disease, the mother exhibits increased levels of circulating markers of endothelial damage, elevated activity of the coagulation system and platelets, and reduced endothelium-dependent vasomotor function (reviewed in Refs. 41 and 43). These findings suggested that the endothelium is the relevant target of the hypothetical placenta-derived factor(s) and that altered endothelial function is responsible for at least some of the syndrome’s manifestations. An exciting recent study has indeed identified a soluble factor, soluble flt-1, which fulfils several, if not all criteria of the elusive PE factor (44,45): it is present at elevated levels in the blood of preeclamptic women, it is produced in the placenta, and induces hypertension and renal microvascular disease. Soluble flt-1 is a soluble isoform of the membrane-bound receptor tyrosine kinase flt-1 that can capture and thereby inhibit the activity of plasma vascular endothelial growth factor (VEGF) and placental growth factor (PIGF). Increased sflt-1 plasma levels may deprive endothelial cells of necessary survival signals, thereby inducing microvascular dysfunction in the brain, liver and kidney, which then may lead to edema, impaired glomerular filtration, and possibly deregulated hemostasis and platelet activation. These findings are biologically plausible andconsistent with a two-stage hypothesis of PE in that reduced placental perfusion would be predicted to trigger the VEGF/PIGF system that controls placental vascularization. While it is clear how soluble flt-1 might destabilize blood vessels and thereby negatively affect vascular function outside the placenta, it remains to be investigated why this would be beneficial for restoring placental perfusion, and what factors regulate the level of flt-1 expression in the placenta in response to altered perfusion. Much less is known about the causes of reduced placental perfusion per se. There is general agreement that PE is characterized by a failure of the utero-placental vasculature to undergo the above adaptations to increase the blood flow to the placenta. Endovascular trophoblast invasion is reduced and shallow, and spiral artery remodeling remains rudimentary. Accordingly, hypotheses to explain this association assume alterations of endovascular trophoblast function, for example, a failure of trophoblast cells to undergo endothelial transformation and assume an endothelial-like adhesion repertoire (46,47), or reduced survival of endovascular trophoblast cells secondary to increased apoptosis (48,49). Altered expression of VEGF ligands and receptors by trophoblast may affect both endothelial transformation and survival of cytotrophoblasts (50), suggesting a potential mechanistic link between endothelial damage caused by soluble VEGF receptor flt-1 (see above) and altered trophoblast function. A large number of additional parameters affecting the incidence of PE have been described, including a genetic component, an immune component (adverse response to allogenic paternal antigens), preexisting hypertension, and thrombophilia (for overview, see Refs. 41 and 42). These observations clearly attest the complex pathogenesis of PE, in which the endothelium might constitute the primary target organ of the initial pathogenic stimulus.

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7. ROLE OF COAGULATION PATHWAYS IN THE PLACENTA—EVIDENCE FROM THE STUDY OF MOUSE MODELS Studies of genetically altered mice with deficits in hemostatic system function have revealed that some, but not all coagulation factors serve an essential role in mammalian embryogenesis and pregnancy (see Fig. 4 for an overview of the relevant coagulation pathways). The genetic abrogation of these factors appears to specifically affect either the blood vessels of the placental yolk sac or the proper development of the chorio-allantoic placenta. Elimination of factors with an essential function in thrombin generation [i.e., the primary initiator of the coagulation cascade, tissue factor (TF), factors V and factor X, and prothrombin itself] causes lethal developmental defects that lead to termination of pregnancy in midgestation. The common defect in these hemostatic factor-deficient mice is a failure of blood vessel maturation and/or integrity in the fetal yolk sac. Abnormal development or function of the definite (chorio-allantoic) placenta occurs in mice with a deficiency of the blood coagulation initiator TF, the blood clotting inhibitor TM, and the thrombin substrate fibrinogen. In summary, these studies of the placental defects in mutant mice have delineated a novel mechanism by which the blood coagulation system participates in the formation and maturation of blood vessels, and in the development of the placenta.

7.1. Thrombin Generation and Thrombin-Receptor Engagement Mediate Blood Vessel Integrity in the Fetal Yolk Sac Disruption of the embryo’s ability to efficiently generate thrombin leads to abnormal development of fetal blood vessels in the visceral yolk sac. The development of mice lacking either (1) the initiator of blood clotting, TF, (2) factor V, the essential cofac-tor of the thrombin generating enzyme complex, (3) prothrombin itself, or (4) the thrombin receptor, PAR-1, is compromised between 9 and 10 days after fertilization (embryonic day E9–10) (51). The wall of blood vessels in the yolk sac contains reduced numbers of pericytes, maturation of the capillary plexus into a vascular network of small and large vessels is impaired, and the loss of vascular integrity may be associated with bleeding. The elimination of the thrombin receptor PAR-1 thus recapitulates the vascular abnormalities caused by impaired thrombin generation, and the selective restoration of PAR-1 expression in the endothelium of otherwise PAR-1-deficient mice restores normal development (52). This suggests that thrombin generation and PAR-1 engagement on endothelial cells are two critical steps in a common pathway that controls the establishment and/or maintenance of fetal blood vessel integrity. Surprisingly, disruption of the gene encoding the inhibitor of the TF (TFPI) produces similar abnormalities of yolk sac blood vessel development as reported for embryos devoid of TF (53). Reducing the expression of coagulation factor VII improves the survival rate of TFPI-null mice, but massive hemorrhage is still a problem shortly after birth of these doubly deficient mice (54). Conversely, the

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Figure 4 (Caption on facing page) Coagulation system and inhibitory pathways affecting placental function. TF is the membrane-associated initiator of blood clotting. In the systemic vasculature, TF is not present on cells in contact with blood, such as blood vessel endothelium. Coagulation is triggered by loss of the endothelial barrier between blood-borne factor VII and cells expressing TF. The ensuing formation of proteolytically active TF/VIIa complexes results in mobilization of coagulation factors IX and X, thereby initiating localized

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thrombin generation. TFPI regulates the proteolytic activity of the TF/VIIa complex via formation of a ternary complex between TF, VIIa, Xa, and TFPI. The inhibitory action of TFPI depends on the presence of activated factor X. This mechanism allows only for a short-lived triggering of the coagulation cascade that effects an initial burst of thrombin generation. The subsequent amplification and maintenance of thrombin production occurs in a TF-independent manner via the Tenase-complex (composed of Ca2+, phosphoplipid, factor IXa, factor VIIIa) and pro-thrombinase complex (Ca2+, phospholipid, factor Va, factor Xa). The activity of the coagulation system is balanced by various natural anticoagulant mechanisms, including antithrombin, and the protein Cpathway. The protein C pathway is initiated by formation of a complex between thrombin and endothelial membrane-bound TM. TM-associated thrombin converts circulating protein C into enzymatically active activated protein C (APC), which then together with the nonenzymatic cofactor protein S degrades activated coagulation factors Va and VIIIa. Destruction of these key components of the thrombin generating Tenase and prothrombinase complexes causes a downregulation of thrombin production. Pathways critical for embryonic development are underlaid in grey, components essential for embryonic survival or placental integrity are shown in red.

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Within the embryonic vascular bed, thrombin generation via the TF pathway, and subsequent PARengagement are required for the establishment or maintenance of vascular integrity in the yolk sac. TM and EPCR are expressed by fetal trophoblast cells in contact with maternal blood, and must act at the feto-maternal interface to prevent the tissue-factor initiated abortion of embryos. Fibrinogen is required only in the maternal blood compartment to prevent fatal bleeding from the placenta. combination of the prothrombotic factor V Leiden mutation with heterozygous TFPI deficiency results in perinatal lethality (55). Thus, it appears that the loss of TFPImediated anticoagulant function can at least in part be balanced by a reduction of the procoagulant potential, and, vice versa, be exaggerated by enhancing the potential for thrombin generation. One possible explanation for the paradoxically similar phenotype caused by two mutations that exert opposite effects on thrombin generation is the fact that unfettered coagulation activation in TFPI-null mice might lead to a consumptive depletion of coagulation factors, thereby in effect reproducing the effect of congenital factor deficiency. The above complete loss-of-function mutations have not been observed in humans, and might conceivably be incompatible with survival of the human embryo, either by causing failure of the—in humans only mdimentary—yolk sac circulation, or by compromising vascular function in general.

7.2. Partial Tissue Factor Deficiency Causes Structural Abnormalities in the Placental Labyrinth As described in the previous section, the complete absence of TF disrupts the establishment or maintenance of vascular integrity in the mouse yolk sac circulation. Expression of a transgenic TF “minigene” encoding human tissue factor rescues TFdeficient embryos, although the human TF transgene conveys less 1% of that present in normal mice (56). Although the transgene prevents the defects in yolk sac blood vessels, these “low TF” mice exhibit two distinct types of placental defects at later developmental stages (57): one-half of the “low TF” females develop fatal midgestation hemorrhage from the placenta, but only, if the mother carries offspring that are similarly deficient in TF expression (i.e., low TF). Placentae of “low TF” embryos growing in a “low TF” mother show structural abnormalities in the labyrinth trophoblast layer, enlarged maternal

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blood lacunae, free blood pools and subsequent placental hemorrhage. The structural abnormalities in the labyrinth of “low TF“embryos occur irrespective of the maternal genotype, but the consequences of this defect appear exacerbated by the lack of TF in maternal tissue, leading to lethal midgestation hemorrhage. This shows that hemostasis in the maternal blood space is coordinately and cooperatively regulated by both maternal and embryonic TF, the latter being expressed at the trophoblast surface. A second defect causes fatal postpartum uterine bleeding of “low TF” females, and occurs regardless if the embryo does or does not express normal levels of TF. This finding demonstrates that normal levels of TF expression in the maternal decidua and uterine epithelium are required and sufficient to prevent the massive hemorrhage associated with parturition. The cause-effect relationship between structural abnormalities in the placenta and the mechanism of TF function in this context are still under investigation. In particular, it is unknown whether the structural abnormalities in the labyrinth reflect a defect in trophoblast function per se, or are secondary to a defect in (maternal and/or embryonic) blood vessel integrity, similar to the defects seen in the yolk sac vessels of completely TF-deficient embryos.

7.3. Maternal Fibrinogen Is Necessary for Maintenance of Pregnancy Development of the embryo and placenta is not affected by the complete elimination of embryonic fibrinogen or platelets, strongly suggesting that the formation of fibrin-platelet aggregates within the fetal circulation is not essential for the integrity of the fetal vascular bed (58,59). Indeed, even mice that lack both fibrinogen and platelets develop normally to term (author’s unpublished observations), but afibrinogenemic and/or platelet-deficient mice may experience fatal bleeding complications after birth. On the other hand, fibrinogen deficiency is incompatible with female reproduction (59,60). While afibrinogenemic mice are fertile, all pregnant females succumb to fatal uterine bleeding originating in the placenta at or before E10. These complications coincide with the formation of maternal blood sinuses in proximity to the embryonic trophoblast and result in profuse bleeding where the placenta attaches to the decidua. By E9, the spongiotrophoblast appears underdeveloped, begins to detach from the decidua, and shortly thereafter complete placental abruption occurs. These findings in mice are entirely congruent with clinical observations in pregnant women with congenital afibrinogenemia who suffer from vaginal bleeding and miscarriage in the first trimester of gestation (61,62). Similar anomalies in the architecture of the labyrinth layer of the placenta associated with necrosis, narrowing of vascular spaces, and hemorrhage are seen in mice lacking the β3-integrin constituting part of the platelet fibrinogen receptor (63). In contrast, no placental abnormalities or spontaneous hemorrhage of the mother during pregnancy has been found in mice with a defective platelet GP1b-receptor (modeling the Bernard-Soulier syndrome) (64) or in mice lacking the GPlb-ligand necessary for platelet adhesion, von Willebrand factor (65). Indeed, platelet-deficient NFE2-knockout mice (58) that survive the postnatal bleeding complications are capable of supporting even multiple pregnancies to term (author’s unpublished data). The sum of these observations is consistent with the notion that maternal fibrinogen and/or fibrin are required to prevent fatal uterine bleeding and for firm attachment of the fetal placenta to the maternal

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decidua. Neither attachment nor prevention of the progression from placental bleeding to fatal uterine hemorrhage are strictly dependent on the interaction of fibrin(ogen) with platelets, since mice lacking platelets due to elimination of the transcription factor NF-E2 are able to carry pregnancy to term, and produce multiple litters without succumbing to lethal hemorrhage. This suggests that the defects seen in platelet receptor knockout mice are not caused by the disruption of platelet function, but by the absence of these receptors from decidual or trophoblast cells. It is interesting to note in this context, that the fibrincrosslinking factor XIII has been implicated in placental function (66,67), but, as to date, no data have been reported on factor XIII knockout mice.

7.4. The Protein C Pathway Is Essential for Placental Development The TM-protein C pathway attains a dynamic feedback inhibition of sustained thrombin generation. The pathway’s anticoagulant activity is mediated by interaction of thrombin with the endothelial transmembrane protein TM and protein C (reviewed in Ref. 68–72). This interaction results in formation of the natural antic-oagulant activated protein C (APC), which prevents the amplification of thrombin generation via proteolysis of activated coagulation factors Va and VIIIa. The APC not only curtails thrombin generation, but also exerts anti-inflammatory and cytoprotective (antiapoptotic) effects in monocytes and endothelial cells (73–75). Candidate pathways mediating the cytoprotective and anti-inflammatory effects of APC on endothelial cells—i.e., engagement of PAR-1 by the EPCR-APC complex (76) and possibly nuclear translocation of the EPCR-APC complex (77)—are initiated by activation of protein C via the TM-thrombin complex. This suggests that TM is an important gatekeeper and modulator of coagulation protease signaling through PARs. Ablation of the mouse TM gene causes intrauterine embryonic death (78). TMdeficient embryos do not survive beyond day 8.5 postcoitum (day 8.5 p.c. total length of gestation in mice is approximately 18 days), and are completely degraded within 24 hr thereafter. The abortion of TM−/− embryos must be caused by a loss of TM function from placental trophoblast cells, since mutant embryos develop normally as TM expression is selectively restored in the placenta (79). TM deficiency is not associated with placental thrombosis, but causes a complete failure of diploid trophoblast cells to maintain proliferation and elicits apoptotic cell death of poly-ploid giant trophoblast cells in contact with maternal blood (80). Anticoagulation therapy of pregnant mothers or elimination of maternal fibrinogen (by breeding experiments with fibrinogen-deficient mice) prevented trophoblast cell death and rapid resorption of TM-deficient embryos, but did not overcome the growth defect. Treatment of pregnant females with the inhibitor of fibrinolysis tranexamic acid was as effective as the complete elimination of fibrinogen in suppressing trophoblast cell death, yet again did not prevent the growth arrest of TM-null embryos. In contrast, genetic “anticoagulation” by crossing TM-deficient mice with animals either completely lacking TF (which is constitutively produced by mouse trophoblast cells (80)), or expressing only minimal TF activity (56) (see above) showed that the absence of significant TF activity rescues completely TM-deficient mice from early midgestation (i.e., E9) lethality (80). The death of TM-null embryos therefore appears to be the consequence of two distinct processes that are both initiated by TF (Fig.

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5). First, tissue factor expressed by trophoblast cells initiates thrombin formation which leads to fibrin deposition and the accumulation of fibrin split products that induce

Figure 5 Function of the protein C anticoagulant system at the fetomaternal interface. (A) Control of trophoblast proliferation by coagulation receptors. Trophoblast cells constitutively activate coagulation (via tissue factor interaction with maternal coagulation factors), sense the presence of activated coagulation factors (via

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PARs), and regulate the generation of active coagulation proteases. TM, conceivably in concert with the EPCR, regulates the overall response of this receptor system to the contact with maternal blood. PAR-1 engagement via thrombin or the EPCR-aPC complex augments proliferation of trophoblast cells. In contrast PAR-2 or 4 engagement inhibits trophoblast growth in vitro. TM’s only function is to inhibit a process initiated by tissue factor, since TM is dispensable in the absence of tissue factor. See text for details. (B) Thrombomodulindependent inhibition of cell death and thrombosis. Like the growth arrest, cell death and thrombosis in TM-deficient mice are initiated by TF. Cell death requires thrombin-mediated conversion of fibrinogen to fibrin, and subsequent fibrin degradation by plasmin. Cell death has been shown to occur in the early (E8.5) lethality of TM-deficient mice. Excessive fibrin deposition may in addition lead to placental thrombosis and infarction. Fibrinolysis inhibition mediated by the thrombinactivated fibrinolysis inhibitor, TAFI, is greatly augmented by TM, providing an additional mechanism to prevent trophoblast cell death. apoptotic cell death in trophoblast giant cells. Second, TF also initiates a process that leads to the growth arrest of diploid trophoblast cells that normally give rise to the differentiated trophoblast populations of the mature placenta. The mechanism of TFinduced growth arrest remains unclear, but in vitro experiments suggest that activated coagulation factors can inhibit the proliferation of diploid trophoblast cells, likely by engagement of PAR-2 and/or PAR-4. Interestingly, engagement of PAR-1 has the opposite effect and inhibits trophoblast growth. It has recently been shown that the APC-

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EPCR complex can activate PAR-1, and elicit intracellular responses that are different from those triggered by thrombin. Generation of APC by the TM-thrombin complex, and activation of PAR-1 via complex could therefore enhance placental growth as the contact of the fetus with maternal blood is established. Elimination of TM would, therefore, simultaneously disrupt the PAR-1-mediated growth stimulating effect, and suppress trophoblast proliferation in a PAR-2/4-dependent manner (80). Disruption of the mouse EPCR gene also produces intrauterine lethality (81), and EPCR is abundantly expressed in mouse trophoblast (82). However, embryonic development in EPCR-deficient mice proceeds somewhat further, and—in striking contrast to TM-null mice—anticoagulation of the mother prevents the developmental defects of mutant embryos. The latter finding strongly implies placental thrombosis as the dominating pathogenic mechanism underlying the lethality of EPCR-null embryos. Moreover, reducing TM’s ability to support APC formation to an even greater extent than predicted from a complete loss of EPCR function does not interfere with placental function and growth (83). These discrepancies strongly suggest that TM and EPCR have common, but also unique functions in development.

8. CONCLUSION Due to its unique anatomical, developmental and functional aspects, analysis of the placental vascular bed in normal pregnancy, and attempts to unravel the mechanisms of disease associated with pregnancy have been, and continue to be, a true inter-disciplinary effort. This effort requires integration of concepts developed in such diverse fields as developmental biology, angio- and vasculogenesis, hemostasis, or immunology, and this list certainly is far from complete. Study of the placenta, and of the vascular bed of the placenta also holds another, very interesting promise: proper formation and function of the placenta is an absolute prerequisite for normal embryogenesis. Failure to pass this critical checkpoint in fetal development either causes termination of pregnancy, or compromises fetal health. From an evolutionary point of view, one would, therefore, have to argue that the placenta is precisely the place where genes and gene variants must reveal their true face with respect to their effect on overall reproductive performance. As the placenta is in terms of evolution a comparably “late” addition to the inventory of reproductive strategies, the molecular and cellular pathways required for building a functional placenta are very likely adopted from other developmental programs. It is therefore no simple coincidence that investigators studying the effect of gene knockouts in mice quite frequently find themselves analyzing a defect in the function of the placenta. Overall, the synthesis of data derived from human patients on the one hand, and basic studies of laboratory animals, such as the mouse, on the other hand will continue to provide novel concepts for the function of these genes in other vascular beds.

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8.1. Key Points Placental Vascular Bed Fetal tissue (trophoblast) is in direct contact with maternal blood (hemochorial type of placentation). Blood-tissue interface consists of genetically distinct maternal endothelium and fetal trophoblast.

Remodeling of Maternal Uterine Blood Vessels Spiral arteries loose smooth muscle cells and dilate. Uncoupling of placental blood supply from the mothers vasomotor control. Mediated by a specialized population of lymphocytes (uterine natural killer cells; uNK). Specific recruitment of uNK cells to placenta via homing receptors.

Endovascular Trophoblast Invasion Trophoblast cells invade and replace endothelium of maternal arteries.

Endothelial Mimicry by Trophoblast Cells Differentiation-dependent acquisition of an endothelial cell-like adhesion receptor repertoire by endovascular trophoblast. Expression of an endothelial-like repertoire of anticoagulant mediators.

Pre-Edampsia Associated with defective remodeling and invasion of maternal arteries by fetal trophoblast. De-regulated fetal and maternal response to reduced placental perfusion. Angiogenic and vasoactive regulators released from placenta lead to systemic vascular dysfunction in the mother.

Coagulation Factors Regulate Placental Development/Function Thrombin generation regulates blood vessel formation by engagement of endothelial cell thrombin receptor (PAR). Protein C system receptors TM and EPCR on trophoblast cells are essential for pregnancy maintenance. Initiator of coagulation, tissue factor, regulates placental development.

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43. Savvidou MD, Hingorani AD, Tsikas D, Frolich JC, Vallance P, Nicolaides KH. Endothelial dysfunction and raised plasma concentrations of asymmetric dimethylarginine in pregnant women who subsequently develop pre-eclampsia. Lancet 2003; 361:1511–1517. 44. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003; 111:649–658. 45. Luttun A, Carmeliet P. Soluble VEGF receptor Flt1: the elusive preeclampsia factor discovered? J Clin Invest 2003; 111:600–602. 46. Zhou Y, Damsky CH, Fisher SJ. Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective endovascular invasion in this syndrome? J Clin Invest 1997; 99:2152–2164. 47. Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ. Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J Clin Invest 1993; 91:950–960. 48. Genbacev O, DiFederico E, McMaster M, Fisher SJ.Invasive cytotrophoblast apoptosis in preeclampsia. Hum Reprod 1999; 14(suppl 2):59–66. 49. Leach RE, Romero R, Kim YM, Chaiworapongsa T, Kilburn B, Das SK, Dey SK, Johnson A, Qureshi F, Jacques S, Armant DR. Pre-eclampsia and expression of heparin-binding EGF-like growth factor. Lancet 2002; 360:1215–1219. 50. Zhou Y, McMaster M, Woo K, Janatpour M, Perry J, Karpanen T, Alitalo K, Damsky C, Fisher SJ. Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am J Pathol 2002; 160:1405–1423. 51. Connolly AJ, Ishihara H, Kahn ML, Farese RV Jr, Coughlin SR. Role of the thrombin receptor in development and evidence for a second receptor. Nature 1996; 381: 516–519. 52. Griffin CT, Srinivasan Y, Zheng YW, Huang W, Coughlin SR. A role for thrombin receptor signaling in endothelial cells during embryonic development. Science 2001; 293:1666–1670. 53. Huang ZF, Higuchi D, Lasky N, Broze GJ Jr. Tissue factor pathway inhibitor gene disruption produces intrauterine lethality in mice. Blood 1997; 90:944–951. 54. Chan JC, Carmeliet P, Moons L, Rosen ED, Huang ZF, Broze GJ Jr, Collen D, Castellino FJ. Factor VII deficiency rescues the intrauterine lethality in mice associated with a tissue factor pathway inhibitor deficit. J Clin Invest 1999; 103:475–482. 55. Eitzman DT, Westrick RJ, Bi X, Manning SL, Wilkinson JE, Broze GJ, Ginsburg D. Lethal perinatal thrombosis in mice resulting from the interaction of tissue factor pathway inhibitor deficiency and factor V Leiden. Circulation 2002; 105:2139–2142. 56. Parry GC, Erlich JH, Carmeliet P, Luther T, Mackman N. Low levels of tissue factor are compatible with development and hemostasis in mice. J Clin Invest 1998; 101:560–569. 57. Erlich J, Parry GC, Fearns C, Muller M, Carmeliet P, Luther T, Mackman N. Tissue factor is required for uterine hemostasis and maintenance of the placental labyrinth uring gestation Proc Natl Acad Sci USA 1999; 96:8138–8143. 58. Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, Jackson CW, Hunt P, Saris CJ, Orkin SH. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 1995; 81:695–704. 59. Suh TT, Holmback K, Jensen NJ, Daugherty CC, Small K, Simon DI, Potter S, Degen JL. Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice. Genes Dev 1995; 9:2020–2033. 60. Iwaki T, Sandoval-Cooper MJ, Paiva M, Kobayashi T, Ploplis VA, Castellino FJ. Fibrinogen stabilizes placental-maternal attachment during embryonic development in the mouse. Am J Pathol 2002; 160:1021–1034. 61. Dube B, Agarwal SP, Gupta MM, Chawla SC. Congenital deficiency of fibrinogen in two sisters. A clinical and haematological study. Acta Haematol 1970; 43:120–127.

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15 Blood Endothelial Cells Robert P.Hebbel and Anna Solovey Department of Medicine, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota, U.S.A.

1. INTRODUCTION It has been known for some time that endothelial cells can be found circulating in blood. Only recently, however, have studies begun to explore the utility of their enumeration and examination. An exciting related area pertains to the presence of endothelial progenitors in blood. Unfortunately the literature on this subject is replete with cell descriptions that are often ambiguous and sometimes simply incorrect. This chapter will utilize precise terminology (Table 1) to distinguish between the different endothelial cells found in blood, circulating endothelial cells (CEC) and endothelial progenitor cells (EPC), and for the endothelial-like cells that grow out from a culture of blood, blood outgrowth endothelial cells (BOEC) and spindle cells (SpC). We here will critically examine the literature on blood endothelial cells and attempt to correct the several misapprehensions that currently exist.

2. CIRCULATING ENDOTHELIAL CELLS Circulating endothelial cells are found in fresh, uncultured peripheral blood and express the multiple markers typical of mature, differentiated endothelial cells.

Table 1 Types of Blood Endothelial Cellsa Terminology

Location

Morphology

Phenotypic Markers

Reference

CEC—circulating EC

Found in fresh blood

Variable

EC+Mo−AC133−

1

BOEC—blood outgrowth EC

Grow out from blood

Cobblestone

EC+Mo−AC133−

22

Elongated

EC+Mo−AC133−

28

Elongated

EC+Mo+AC133−

25,29

SpC—spindle cells

Grow out from

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blood EPC—endothelial progenitor cell a

Found in blood (and marrow)

Mononuclear

EC+Mo−AC133+

43,46

EC: endothelial cells; Mo: monocytic or myelomonocytic cells.

Our studies have shown that the cells identified as CEC using monoclonal antibody P1H12 (which labels nothing else in blood) are also positive for von Willebrand factor (vWF), flk1, integrin α0β3, CD34, thrombomodulin, and (if activated) VCAM-1 and Eselectin and ICAM-1 (1). Over the last few decades, investigators have documented elevated numbers of CEC in various disease states, sometimes in experimental animals but mostly in humans. These conditions are listed in Table 2, in which they are clustered into two groups. The “early studies” were generally done before the 1990s and identified endothelial cells in blood simply by morphology, or by morphology plus staining for vWF (which is not strictly endothelial-specific). In 1991, two groups independently published watershed papers which utilized antiendothelial monoclonal antibodies to unambiguously identify endothelial cells in blood of normal humans, antibodies S-Endo-1 (2) and CLB-HEC19 (3). Thereafter, observations of CEC may be considered to be reliably endothelial, and these are indicated as “modern studies” in Table 2. A brief glance at this table reveals that this diverse list of conditions tends to have in common a component of vascular injury or expectation of injury, whether by instrumentation, infarction, infection, inflammation, or malignancy. Notably represented are states of cardio-vascular disease and risk.

2.1. Information Obtainable from CEC Recent studies of sickle cell anemia have illustrated that CEC can not only be enumerated but also be phenotyped in an attempt to gain insight into the endothelial biology of the vessel wall.

Table 2 Conditions Reported to Have Elevated Numbers of CECa Early Studies

Modern Studies

Cancer

Heart catheterization

Endotoxin administration

Vascular injury

Citrate infusion

Lupus

Coagulation activation

Acute myocardial infarction

Anoxia

Unstable angina

Streptokinase

Cerebral thrombophlebitis (Bechet’s)

Methionine challenge

Septic shock

Acute myocardial infarction

CMV infection

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Angina

Thrombotic thrombocytopenic purpura

Hypercholesterolemia

Cancer

Peripheral artery disease

Vasculitis

Tobacco smoking

Renal allograft rejection

Diabetes

Cor pulmonale

Hypertension

ARDS

Anaphylactic shock

Thalassemia

Sickle disease

Sickle cell disease

Postsurgery

Chronic venous insufficiency

Aortic insufficiency a

This list is derived from a Pub Med literature search conducted in May, 2003.

2.1.1. Enumeration Because CEC are rare cells, their enumeration has relied on tedious microscopic examination of buffy coat or peripheral blood mononuclear cell preparations. Such enumeration shows that normal donors have an average of 2.6 CEC per mL of peripheral blood, with a range up to 5 per mL, the practical limit of detection being about 0.1 per mL (1–4). In contrast, sickle cell anemia patients have elevated numbers of CEC, 13±12 per mL when in “steady state” between acute events, and 23±18 per mL at onset of acute vaso-occlusive crisis, a tendency also evident in longitudinal examination of individual subjects (Fig. 1) (1). Examination of Fig. 1 reveals an important caveat if enumeration of CEC is used to infer clinical information: at least in sickle subjects, number of CEC fluctuates chaotically. If this is true in other conditions, single readings may be misleading. It should be noted that number of CEC in some of the conditions listed in Table 2 are alleged to reach surprisingly high levels. Some of this is explained by choice of methods. Our studies (1,5–7) counted as CEC only cells actually having a nucleus. In contrast, the extreme numbers reported by Mancuso et al. (8) (with “CEC” values reaching tens of thousands per milliliter) were based only on FACS determination of particles marking with endothelial surface markers; i.e., they almost certainly were actually measuring cell fragments and endothelial-derived microparticles. A significant uncertainty, in terms of the utility of CEC enumeration, derives from the fact that the half-life of CEC in the blood is not known. We have found that infusion of autologous BOEC (see below) into dogs results in removal of the artificially induced CEC with single pass kinetics (Solovey and Hebbel, unpublished data). Also, arguing that their circulatory half-life is short is the fact that CEC fluctuations in sickle patients can be large from day to day. Nonetheless, in no case is it yet proven whether a change in CEC number reflects a change in circulatory residence time or a change in rate of CEC generation. It does not appear that splenic function plays a large role, as a study of two

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nonhemoglobinopathic individuals with hemolytic anemia and splenectomy did not reveal an increase in CEC number (1).

Figure 1 Number of CEC in three subjects with sickle cell anemia. Number fluctuates chao tically, and tends to be greater at onset of acute vaso-occlusive crisis (arrows). (From Ref. 1.) 2.1.2. Phenotype Our studies of CEC in sickle disease indicate the range of phenotypic assessments that can be applied to CEC (Fig. 2). Surface protein expression (e.g., adhesion molecules and tissue factor) was detected by immuno-fluorescence microscopy (1,5,6). In situ hybridization was used to document that CEC expressing tissue factor antigen also contained message for that protein (5). Single cell binding and biochemical assays were used to document that expressed tissue factor was actually functional (5). Internal proteins can be monitored by immuno-fluorescence for expression, as Nath et al. (9) did for hemeoxygenase-1. Nuclear structure and TUNEL staining can be combined to assess whether or not CEC are undergoing apoptosis (6). One limitation is that we find it is impossible to prepare a pure population of CEC, as there is always heavy contamination by monocytes. Therefore, PCR can be reliably utilized only if endothelial specific genes are being examined. In aggregate, these studies indicate that sickle CEC have an abnormally activated phenotype (proadhesive, procoagulant, oxidatively stressed), which also has been influenced by enhanced antiapoptotic signaling. The crucial question, of course, is whether such cells are truly informative regarding phenotype of the endothelial cells remaining in situ in the vessel wall. Studies to date have been predicated on the assumption that this is likely to be true, since CEC and vessel wall endothelium would be exposed to the same phenotype-influencing blood milieu. However, it is unknown to

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what extent CEC reveal (or have lost) any tissue-specific phenotypic characteristics of the vessel wall endothelium. In any case, the phenotype of CEC is the same whether the cells are live or dead or whether they are apoptotic or not, arguing that they acquired their phenotype before detaching (or at least before dying or undergoing apoptosis) (1). In addition, sickle transgenic mice have activated CEC and corresponding activation of tissue vessel endothelium (7).

Figure 2 Examples of CEC analysis using fluorescence methods. (A) A single cell stained with P1H12 (green) to identify it as endothelial. (B) The same cell in A is here stained for tissue factor (TF) antigen (red). (C) A single CEC stained for P1H12 (blue) shows a blush (yellow) of positive hybridization signal for TF mRNA. (D) Providing a control for (C), a P1H12 positive CEC (blue) fails to show hybridization signal with the control, sense probe. (E) A CEC stains positive for (added) factor VIIa (red), indicating binding ability of the CEC TF. (F) Providing a control for (E), binding of factor VIIa is prevented when a blocking antibody to TF was used. (G) A CEC develops green positivity in an enzymatic assay that tests for presence of functional TF. (H) Providing a control for (G), a blocking antibody to TF prevents development

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of most (green) signal. (I) A CEC that expresses TF (blue) develops positive hybridization signal for TF mRNA. (J) Providing a control for (I) hybridization does not occur in the TF positive CEC (blue) for the control sense probe. (From Ref. 6.) 2.1.3. Endothelial Heterogeneity Study of sickle transgenic mice illustrated what we believe to be a very important aspect of endothelial biology. The phenotype of tissue endothelial cells in sickle mice varies from organ-to-organ and between large and small vessels, and expression pattern is different for different adhesion molecules (Fig. 3) (7). If this heterogeneity extends to humans, as we believe it does, the endothelial biology of a given disease may be exceedingly complicated and vary considerably from region to region. Also notable, is the fact that phenotypic correlation between CEC and tissue endothelium in the sickle mouse can only be said to be present in the most general sense because of this heterogeneity. This, in turn, means that extraction of information that is truly clinically useful from CEC phenotype will probably require development of tissue-specific endothelial markers so observers can know which organ a given CEC is derived from. Fortunately, recent years have seen some promising developments in this regard, as in vivo phage display is used to identify peptides that exhibit endothelial binding in an organ-specific fashion (10,11). It seems likely that inclusion of organ-specific markers in the assessment of CEC would be of benefit in identifying locations of endothelial activation or even regional differences in the endothelial biology of systemic disease. And it is conceivable that liberation of endothelial cells from the vessel wall would be greater in

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307

Figure 3 Heterogeneity of adhesion molecules by tissue endothelial cells in sickle mice. Illustrated are expression scores (for VCAM, ICAM, and Eselectin) for endothelial cells from heart (H), liver (L), spleen (S) and kidney (K), for large vs. small vessels. (From Ref. 7.) areas of biological disease or stress, so that examination of blood CEC would advantageously tend to reflect over-sampling from such areas. These concepts have not yet been tested in animal models.

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2.2. Medical Applications of CEC Phenotyping The usefulness of CEC studies is only beginning to be explored, but some promising observations have been made. In the area of microbiology, CEC can be used to document presence of infection with CMV (12,13) or Rickettsia (14,15). Several studies suggest that number of CEC may be informative in certain diseases. Higher numbers are said to indicate greater severity of CMV disease (13) and poorer prognosis in septic shock (16). Conversely, a decrease in (the elevated) number of CEC is reported to accompany therapeutic response of Rickettsial infection (17) and successful treatment of thrombotic thrombocytopenic purpura (18) and cerebral thrombosis in Bechet’s disease (19). CEC number reportedly correlates with activity of lupus (20). Percent of apoptotic CEC is said to increase in response to antiangiogenic therapy with endostatin (21). Solovey et al. (6) observed an inverse correlation between CEC apoptosis and plasma level of the endothelial survival factor VEGF (vascular endothelial growth factor) in subjects with sickle cell anemia (Fig. 4). Finally, Solovey found that activated phenotype of CEC in sickle disease was down-regulated in response to anti-inflammatory therapy with sulfasalazine (Fig. 5) (7). Thus, it may be that assessment of CEC can be of value in monitoring therapeutics targeted at the endothelial cell.

2.3. Origin of CEC Using FISH to genotype cells (for XX or XY), Lin et al. (22) examined CEC in fresh blood of “normal” humans without acute disease but who had previously undergone bone marrow transplantation for malignancy. Virtually all CEC were found to be of recipient origin and, therefore, likely to be from the vessel wall. Nonetheless, it is not known for sure if the increased number of CEC in disease states is from vessel wall.

Figure 4 Inverse correlation between CEC apoptosis and plasma level of

Blood endothelial cells

309

Vascular Endothelial Growth Factor. Compared to normals, subjects with sickle cell anemia have lower level of CEC apoptosis and higher VEGF levels. (From Ref. 6.)

Figure 5 Sulfasalzine diminished CEC adhesion molecule expression. Data are shown for three sickle patients studied longitudinally. Percentage of CEC expressing ICAM-1, VCAM-1, E-selectin, and tissue factor is always 50–100% (indicated by hatched box) in absence of a treatment effect. When subjects take sulfasalazine (solid bar at bottom of each graph), adhesion molecule expression (but not that of TF) is decreased. Addition of salsalate (open bar at bottom) has no additional benefit. (Reproduced from Ref. 7.)

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Theoretically, they could derive from marrow endothelial progenitors. Also, endothelial precursor cells may reside in peripheral tissues. For example, in skeletal muscle, there is a ‘side population’ (based on light scatter properties of dispersed cells) of stem cells that are derived from marrow but can differentiate into endothelial cells (23). Nonetheless, a vessel wall origin seems likely, as a number of pathobiologic stresses can be imagined to be capable of dislodging vessel wall endothelial cells to form CEC. This process could be physiologic, e.g., a response to thrombin stimulation or proteases or other biological modifiers that cause alteration of adhesion molecules on the EC. It could involve mechanical factors such as shear stress or thrombus. It may be an injury or apoptotic event. Perhaps arguing for an injurious cause, very high CEC counts tend to be seen in cases of likely widespread vascular damage (4), and CEC numbers increase in sickle disease in association with acute vaso-occlusive crisis (1). It is said that morphology of CEC varies from disease to disease, with intact cells of varying size seen postangioplasty and in sickle disease, necrotic cells in Rickettsia infections, and sheets of cells in acute coronary syndromes (4). Perhaps this reflects different geneses of CEC formation in different disease states. It has been noted that CEC in CMV infection fail to express the β3 partner of αvβ3 integrin (12), perhaps suggesting that altered adhesion could be etiologic. On the other hand, CEC in sickle disease were noted to express an increased amount of αvβ3 (6). Apoptosis is a characteristic of two-thirds of CEC from normal subjects (6), either indicating that apoptosis may have triggered departure from the vessel wall, or simply revealing changes after departure that are due to the endothelial cell’s notorious intolerance of loss of anchorage. Interestingly, in sickle subjects CEC apoptosis is markedly lower than in normals (6), so endothelial apoptosis is unlikely to underlie formation of CEC in this disease. A significant implication of CEC being in blood is that they presumably have left behind an area of bared subendothelial matrix. For example, 50 CEC per mL (seen in sickle patients) would correspond to a total of about 250,000 CEC in the adult. In turn, this would correspond to approximately 10 cm2 of surface area laid bare, which is a substantial and alarming amount of denudation. There are no data about the fate of sites of CEC origination, but this implied loss of endothelialization may contribute to propensity for thrombosis in a number of disease states associated with increased CEC (Table 2).

3. ENDOTHELIAL-LIKE CELLS GROWN FROM BLOOD: BOEC AND SPC Many laboratories have now successfully grown endothelial-like cells from peripheral blood samples, including from human (22), sheep (24), mice (25), rabbit (26), and dog (Hebbel, unpublished data). Since blood contains CEC and since endothelial cells derived from vessel walls clearly have proliferative capacity (as evidenced by their wide use for experimentation), outgrowth of endothelial cells cannot constitute prima facie evidence for presence of an endothelial progenitor in blood. Nevertheless, some investigators have erroneously used the term “endothelial progenitor cell (EPC)” for endothelial-like cells grown from peripheral blood. In some reports, the outgrowth progeny are, in fact, derived

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from an endothelial progenitor, but there is little evidence that the progenitors per se actually expand in number in culture. In other cases, however, it appears likely that neither the source cell in blood nor the outgrowth progeny are true endothelial cells. For clarity, we use the term BOEC for true blood outgrowth endothelial cells, i.e., cells having endothelial morphology (typically cobblestone) and expressing markers of mature differentiated endothelial cells, but not myelomonocytic markers such as CD14 or CD45, and not the stem cell marker AC133. In contrast, cultures of peripheral blood can also yield SpC, spindle-shaped cells that share endothelial and myelomonocytic markers. The literature on blood-derived endothelial progenitors has often misidentified SpC as being EPC.

3.1. Blood Outgrowth Endothelial Cells Of the several reports of true endothelial outgrowth from blood, by far the most robust outgrowth apparently is derived from the method of Lin et al. (22) from which a culture of a peripheral blood sample can yield 1019 BOEC. In this method, peripheral blood mononuclear cells (not further subfractionated) were plated onto collagen I and grown with 10% fetal bovine serum and multiple endothelial growth factors in a commercial kit that contains other additives such as heparin and hydrocortisone. On the second day of culture, nonadherent cells were discarded. Subsequently, endothelial growth showed two phases, the nature of which was revealed by genotypic examination (for XX vs. XY) of the BOEC grown out from blood in humans who had previously received a gendermismatched marrow transplant. The delayed, second phase consisted of robust outgrowth from a transplantable marrow-derived cell in peripheral blood, a putative EPC. In contrast, the early outgrowth phase was slower and also included a limited expansion of CEC derived from the vessel wall. From onset of the second phase and onwards, morphology was cobblestone, and cells displayed multiple endothelial markers (vWF, thrombomodulin, CD34, CD31, CD36, VE-cadherin, αv integrin, flk-1, P1H12) and Weibel-Palede bodies. Importantly, these BOEC were negative for CD14 (a monocyte marker), CD45 (hematopoietic cells), and AC133 (stem cell marker). Thus, BOEC have markers of differentiated endothelial cells, do not share myelomonocytic markers, and have cobblestone morphology. Other descriptions of apparently true BOEC include the report by Shi et al. (27) who studied initially nonadherent CD34+ cells from G-CSF mobilized peripheral blood, and the paper by Nieda et al. (28) who studied CD34+ cells from umbilical cord blood. A critical point is illustrated by the fact that the outgrowth cells described by Nieda were elongated, not cobblestone. The morphology of endothelial cells can be affected by many factors, including the specific proteins that are included in substratum, whether the whole cell or only a single surface makes adhesive contact, by the presence of cytokines or cAMP, by serum concentration and source (animal vs. human), and by concentration of growth factors. Therefore, elongated cells obtained from endothelial culture conditions can still be endothelial. However, there is no evidence that assumption of an elongated shape by true endothelial cells is accompanied by acquisition of positivity for CD14 or other myelomonocytic markers. In fact, in our experience, such cells are consistently negative for CD14, for example. The elongated BOEC reported by Nieda were, in fact, CD14

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negative (28). Therefore, if a culture produces elongated cells that are significantly positive for CD14, it should be suspected that they are spindle cells rather than true endothelial cells.

3.2. Spindle Cells Spindle Cells (SpC) are a characteristic cell in Kaposi’s sarcoma, and can be obtained by culturing blood (29). The origin of the blood SpC is hypothesized to be from cells lining sinuses, e.g., in the spleen. Their culture is classically promoted by cytokines (29), but it is likely that this requirement is simulated by the extraordinarily high concentrations of mitogens used by some groups to culture “EPC,” e.g., serum concentrations as high as 20–25%. In any case, SpC grown from blood are elongated, and they exhibit both endothelial and myelomonocytic markers (e.g., CD14) (29). Such cells at best can be regarded only as “endothelial-like cells,” as several groups have carefully done. For example, Pujol et al. (30) reported culture of CD14 positive peripheral blood monocytes on fibronectin with endothelial growth factors (the conditions used by several groups to obtain cells alleged to be EPC). The outgrowth cells were mixed morphology, including spindle shapes, and were called “endothelial-like” because they exhibited not only endothelial markers but also CD14 and CD68 (30). Consistent with this, culture of CD34−/CD14+ monocytes on fibronectin and with endothelial growth factors (31) or bovine brain extract (32) yields spindle shaped cells that share endothelial and macrophagocytic lineage markers. Notably, SpC derived from CD34− cells can be incorporated into vessel walls as endothelial-like cells, as long as there is help from cocultured CD34+ positive cells (32). It is important to note that outgrowth SpC exhibit endothelial functions such as tube formation in vitro, as well as uptake of acetylated LDL (acLDL) and binding of Ulex lectin, markers sometimes used with the unwarranted assumption that they are endothelial specific.

3.3. Spindle Cells Misidentified as Endothelial Progenitor Cells Asahara et al. (25) established cultures of CD34+ cells (but only at 16% purity in a population of mononuclear cells) from blood and obtained outgrowth cells they referred to as “EPC” (25). Unfortunately, no evidence was provided to support the designation of these cells as progenitors. Moreover, morphology was spindleshaped, and the cells expressed rather low levels of endothelial markers and CD34, and very high levels of myelomonocytic markers, e.g., 27% CD45 positive and ~90% CD14 positive (25,33,34). It would seem that these outgrowth “EPC” are more likely to have been SpC, absent evidence to the contrary. Subsequently, the term EPC has been repeatedly applied to cells within short-term cultures of peripheral blood mononuclear cells that exhibit dual-positivity for uptake of acLDL and Ulex lectin binding. We regard this as insufficient evidence for an EPC, since neither of these markers is specific for endothelial cells and, in fact, both also identify monocytes (35,36), the cell population within which the so-called EPC reside. In our experience, short-term cultures of peripheral blood mononuclear cells are dominated by monocytes. In fact, Rehman et al. (36) used the Asahara method but found that majority

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of “EPC” expressed monocyte/macrophage markers, and that the dual labeled acLDL/Ulex positive cells are derived form monocytes/macrophages. In our opinion, the term EPC has been inappropriately applied in a number of recent publications to cells that are actually SpC (e.g., Refs. 25,33,34,37–41) or even BOEC (24).

4. ENDOTHELIAL PROGENITOR CELLS We use the convention of calling putative endothelial progenitors in either peripheral blood or marrow EPC. The putative circulating EPC and marrow EPC may well be one and the same, as there is ample precedent for spillage of marrow stem cells into the peripheral circulation. Unfortunately, the literature on EPC is greatly confused due to application of inappropriate terminology and due to unproven assumptions, as noted above.

4.1. True Endothelial Progenitor Cells Nevertheless, the evidence for presence of EPC in blood and marrow is actually quite strong (22,42–46). Phenotyping needs to take into account that few markers are endothelial specific. In aggregate, the few solid papers on this subject suggest that the true EPC has phenotype CD34+, flk-1+, FGF-R1+, AC133+. The population of blood and marrow cells that carries these defining markers tends to also express c-kit, tie-2, P1H12, and VE-cadherin, but to be negative for vWF and uptake of acLDL. The relevant cell may reside in the c-kit bright subpopulation (44). It should be noted that all studies of EPC to date have been performed on bulk populations of cells and have not yet been critically tested by studies using single-cell culture. Some critical aspects of this putative progenitor are not known. A stem cell should be self-renewing and transplantable. True endothelial progenitors can be transplanted in humans (22,47) and animals (48), but whether the true EPC has self-renewing capacity has not been defined. It could be that the EPC is a committed progenitor rather than a self-renewing or pluripotent progenitor cell. There has been considerable interest in the possibility that the source of EPC is an hemangioblast, analogous to that seen in early development (49,50). Intriguingly, evidence suggests that adult mice do have a hemangioblast, able to be transplanted as a single cell and able to generate both endothelial and hematopoietic linage cells (48). A possibly supportive report from human studies remains to be confirmed (51). There are additional possibilities. The enormous plasticity of some marrow cells has been noted (52), and multipotent adult progenitor cells (MAPC) have recently been identified in marrow and is capable of generated endothelial cells (53). Phenotype data suggest that MAPC and EPC are not one and the same, although it remains possible that EPC are derived from MAPC rather than an hemangioblast.

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4.2. Can EPC Be Counted? Various stimuli are said to mobilize EPC, including VEGF (33,37,54), statins (38,39), GM-CSF (55), burns and surgical manipulation (56), acute myocardial infarction (57), and erythropoietin (58). However, some of these papers used an insufficiently rigorous definition of EPC (dual positivity of acLDL uptake and Ulex lectin binding) and, therefore, require confirmation. Hill et al. (59) cultured blood mononuclear cells short term on fibronectin, performing a preadhesion step to supposedly eliminate chance of outgrowth from mature CEC (although there are no data to document that this works, and some that argue it does not). Interestingly, they found that endothelial cell colony counts correlated inversely with Framingham cardiovascular risk score and directly with brachial artery flow in response to nitroglycerin. Unfortunately, it was not demonstrated whether the outgrowth colonies were from vessel derived CEC or from a transplantable EPC, and apparently some endothelial colonies were identified only by acLDL and Ulex labeling.

5. BIOLOGICAL ROLE OF BLOOD ENDOTHELIAL CELLS Existence of a biological role for blood endothelial cells (CEC and EPC) is not proven for humans, but animal studies have produced intriguing results. Either CEC or their progenitors seem to be capable of establishing endothelial chimerism (meaning addition of blood-borne endothelial cells to the endothelium in a vessel wall). This has been observed after renal transplantation and allograft rejection (60–62), and after marrow (63–65) or liver (66,67) transplantation. These observations establish the precedent for a blood-borne cell to contribute to endothelialization. It may be speculated that CEC can function to patch denuded areas of vessel wall, although the high proportion of CEC that are undergoing apoptosis (6) would seem to make this unlikely. Of great interest, it appears that true EPC derived from marrow can contribute to endothelialization in the context of neovascularization during postnatal vasculogenesis in tumors, wounds, the ovary, and in ischemic tissue (25,34,44,48 55,68,69). This may even result in improved function, e.g., of the heart after myocardial infarction (44,70,71). Intriguingly, it has been suggested, based on murine studies, that marrow-derived endothelialization is necessary for the process of angiogenesis (72). In terms of biomedical applications, marrow cells have been used for graft endothelialization (73,74), as have BOEC (24,75). And BOEC have some interesting potential uses, such as performing as carriers of gene expression vectors after ex vivo gene transfer. A particularly robust expression of factor VIII was observed using stably transfected BOEC in mice (76). Engineered BOEC have also been used to inhibit angiogenesis (77) or for attempted vascular wall protection after balloon injury (78).

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6. CONCLUSION Notwithstanding ambiguities and confusion in the relevant literature, it seems clear that blood contains not only CEC but also EPC. The utility of these cells for diagnostic purposes and for therapeutics, respectively, is only beginning to be explored. Hopefully, analysis of CEC can be a valuable tool in defining the heterogeneity of endothelial biology of human disease.

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16 Determination of Endothelial Heterogeneity by the Recruitment of Bone Marrow Derived Endothelial Progenitors Shahin Rafii and Jay Edelberg Cornell University Medical College, Ithaca, New York, U.S.A.

1. INTRODUCTION Adult bone marrow (BM) is a rich reservoir for stem cells capable of generating an array of lineage-specific cell types, including hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs) (1) and endothelial progenitor cells (EPCs) (2–7). Several lines of evidence have demonstrated that incorporation of mobilized stem and progenitor cells in end organs contributes to the formation of functional vasculature and sets the stage for tissue regeneration or tumor growth. Vascular endothelial growth factor (VEGF) family of angiogenic factors through interaction with their receptors VEGFR1, VEGFR2, and VEGFR3 expressed on endothelial and hematopoietic cells promotes the mobilization and recruitment of vascular progenitor cells into neo-angiogenic sites, thus accelerating the revascularization process (Fig. 1). VEGF-A regulates angiogenesis and vasculogenesis (Figs. 1 and 2) by inducing proliferation, migration, and recruitment of endothelial cells (8–13). VEGF-A through interaction with its cognate tyrosine kinase receptor, VEGFR2 (KDR, Flk-1), supports survival and motility of endothelial cells. Another VEGF tyrosine kinase receptor, VEGFR-1, is not only expressed on endothelial cells, but also by HSCs and subsets of myeloid HPCs, conveying signals that support their motogenic potential (5,14). VEGFR3 expression is critical for the regulation of lymphangiogenesis. Angiogenic factor-mediated mobilization of EPCs and HSCs is a dynamic process and requires sequential release of specific stem cell-active chemokines and proteases that facilitate egress of primed cells from the BM to the peripheral circulation. Activation of metalloproteinases results in the release of stem cell-active cytokines, thereby enhancing their motility and mobilization to the peripheral vascular beds. Angiogenic factors recruit subsets of proangiogenic HSCs and HPCs facilitating the revascularization processes (5,14). Corecruitment of BM-derived HSCs/HPC along with

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EPCs contributes to the initiation and sustenance of postnatal neoangiogenic processes and organ vascular patterning. Furthermore, recruited

Figure 1 VEGF family plays a critical role in the regulation of angiogenesis and hematopoiesis. VEGF-A is the most widely expressed angiogenic factor that supports migration, survival, and proliferation of endothelial cells through interaction with its tyrosine kinase receptors; VEGFR2 and VEGFR1. PIGF and VEGF-B exert their effect exclusively through interaction with VEGFR1. VEGFR1 is not only expressed on endothelial cells, but also on subsets of “hematopoietic cells, including myelomonocytic and HSCs. VEGF-E, which is expressed primarily by the Pox viruses, exert its effect only on VEGFR2.

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Figure 2 Chemokine-mediated mobilization of HSCs and EPCs is a multistep dynamic process and requires the sequential activation of proteases, adhesion molecules, and cytokines that enhance stem cell motility. Vascular trauma, ischemia, or tumor growth induces the release of specific chemokines or cytokines, including VEGF and SDF-1. These factors upregulate adhesion molecules and induce activation of metalloproteinases (MMPs) resulting in the release of stem cell active cytokines promoting the cycling, thereby increasing the motility of otherwise quiescent sessile VEGFR2+ endothelial and VEGFR1+ HSCs. Rapid incorporation into injured tissue or growing malignant tumor accelerates the revascularization process. Reciprocal interaction between comobilized HSCs and their myeloid progenitors with EPCs is necessary for function incorporation of the EPCs into newly formed vessels.

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hematopoietic cells provide cellular instructions for the selective incorporation of subsets of EPCs, thereby modulating organ-specific vascular heterogeneity (15,16). In this chapter, the mechanism whereby recruitment of vascular progenitors may contribute to endothelial heterogeneity will be discussed.

2. CONTRIBUTION OF EPCS TO POSTNATAL ANGIOGENESIS At the site of tissue vascularization, endothelial cells can originate from either adjacent pre-existing blood vessels (11,17) and/or recruitment of a unique population of BMderived EPCs from the circulation (3,4,6,18–23). Endothelial Progenitor cells may be considered embryonic angioblasts that proliferate and migrate in the BM, circulate in the bloodstream, and promote tissue vascularization at neo-angiogenic sites in ways that may differ from one end organ to the next (24–26). Under steady-state physiological conditions, VEGFR2+ EPCs represent less than 0.01% of circulating mononuclear cells in the peripheral circulation. Plasma elevation of VEGF-A in intact adult mice results in the mobilization of EPCs (27). VEGF-A also induces mobilization of a large numbers of VEGFR1+ HPCs. These data raise the possibility that comobilization of VEGFR1+ hematopoietic cells and VEGFR2+ EPCs is essential for neo-vascularization. Vascular trauma induced by burn injury or surgical manipulation results in rapid mobilization of CD133+ VEGFR2+ EPCs (28). Six hours after vascular trauma, EPCs can be detected in the peripheral circulation. Remarkably, 24 hr after vascular injury, 12% of the total circulating peripheral mononuclear cells are comprised of CD133+ VEGFR2+ cells. Mobilization of EPCs was paralleled by plasma elevation of VEGF-A, suggesting that VEGF-A released as a result of vascular injury promotes mobilization of EPCs. Several studies have shown that BM-derived EPCs functionally contribute to neoangiogenesis during wound healing and limb ischemia (3,7,21–23,29–32), postmyocardial infarction (33–36), endothelialization of vascular grafts (20,37,38), atherosclerosis (39), retinal and lymphoid organ neovascularization (40−42), organ vascularization during neonatal growth (43) and tumor growth (4,21,44–51). However, whether EPCs contribute temporally to normal, physiological turnover of endothelial cells is not known. These studies have advanced the novel concept that either vascular trauma or organ regeneration results in release of specific angiogenic factors with chemokinetic activity, including VEGF-A and fibroblast growth factor (FGF)-2, that promote recruitment of EPCs to the site of active neo-angiogenesis. Rapid recruitment of circulating EPCs accelerates vascular healing and prevents potential vascular complications secondary to thrombosis or hypoxia. Tissue ischemia results in upregulation of angipgenic factors including VEGF-A, which, through interaction with its receptors VEGFR2 (KDR, Flk-1) and VEGFR1 (Flt-1) expressed on EPCs and HSCs/HPCs, promote migration of these cells to the site of injury (Fig. 1) (8–13).

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3. POTENTIAL PATHWAYS WHEREBY TISSUE-SPECIFIC INCORPORATION OF SUBSETS OF MOBILIZED EPCs CONTRIBUTE TO VASCULAR HETEROGENEITY Endothelial cell phenotypes vary between different sites of the vascular tree. Vascular heterogeneity may be dictated by the factors released locally at the target organ

Figure 3 Selective mobilization of vascular progenitors is directed by specific chemokines and cytokines. Specific release of angiogenic factors as a result of tissue injury, organ regeneration, and angiogenic switch during oncogenic transformation results in selective mobilization of stem cells from the marrow microenvironment. Specific activation of proteases may also provide for

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another level specificity to stem cell mobilization. where the EPCs are delivered. Although VEGF-A may play a predominant role in the recruitment of endothelial cells, other organ-specific proangiogenic factors released in conjunction with VEGF-A may contribute to vascular bed-specific recruitment and phenotype. For example, EPCs play a critical role in the maintenance/reconstitution of cardiac vascular functional patterning through induction of platelet-derived growth factor (PDGF) pathways (Fig. 3). PDGF isoforms are integral to cardiac and vascular development and function (52,53). The physiological effects of PDGF extend beyond the direct regulation of the developing blood vessels in the heart and are critical in (re)establishing and/or maintaining a cardioprotective vascular environment in the adult heart (54,55). The senescent impairment in cardiac myocyte induction of endothelialPDGF-B expression pathways (56) could diminish the capacity to generate myocardial cells from local and/or circulating stem cells for the aging heart, potentially contributing to the increased pathogenesis of cardiovascular disease in older individuals. The longterm restoration of the PDGF-mediated cardioprotective pathway may be achieved through delivery of young BM-derived EPCs (56). Endothelial progenitor cells from 3-, but not 18-, month-old mice induced cardiac endothelial expression of PDGF-B, in vitro and in vivo. Transplantation of BM cells from young, but not older, adult mice supports prolonged restoration of cardiac angiogenic activity in the aging host. The young cells populate the BM of intact, unirradiated senescent mice and EPCs derived from the transplanted young BM cells are subsequently recruited to sites of cardiac angiogenesis, restoring critical endothelial cell populations. Clinical approaches based on transplantation of genetically matched young BM-derived EPCs or restoration of the angiogenic function of senescent EPCs may allow for long-term cardioprotection of the aging heart. Another proangiogenic factor that may promote selective survival of cardiac vasculature is brain-derived nerve growth factor (BDNF) (57). BDNF deficient mice have vascular defects not only in the central nervous system but also myocardium. These defects result in diffuse hemorrhage within the myocardium, suggesting that selective organ-specific expression of BDNF and its tyrosine kinase Trk-receptors may play a role in cardiac angiogenesis. Whether BDNF also recruits a specific subset of marrow-derived endothelial progenitors is not known. Taken together, the above data suggest that PDGF and BDNF may collaborate with VEGF-A to provide for selective assembly of functional myocardial vasculature, in part through recruitment of endothelial progenitors. However, whether organ-specific recruitment of progenitors is dictated locally at the site of organ regeneration or through increased mobilization of selected subsets of BM-derived progenitors or a combination of these two complex processes is not known.

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4. SELECTIVE RECRUITMENT OF EPCS FROM BM NICHES MAY CONTRIBUTE TO ENDOTHELIAL HETEROGENEITY Despite recent advances in the phenotypic identification of various subsets of tissuespecific stem cells (58–60), the molecular mechanisms and cellular mediators involved in the mobilization of stem cells from BM niches have just recently been elucidated. The concept of HSC niche was introduced by Schofield (61), who hypothesized that HSCs are fixed within a specific microenvironment providing conditions that are conducive to the survival and differentiation of HSCs. Subsequently, it was demonstrated that the HSC niche may be physically localized primarily to the endosteal region of the BM (62). Repopulating HSCs, as represented by CFU-S, were shown to reside at high concentration in the proximity of the endosteal region (63,64). Subsequently, it was demonstrated that transplanted HSCs home to the endosteal region of the BM (65–67). Based on these studies, it has become apparent that BM may be functionally compartmentalized into defined niches. Expression of chemokines, tethered cytokines, or of yet unrecognized factors, may provide “road maps” for the localization of stem cells within their respective BM microenvironment (Fig. 4). Several lineage-specific stem cells, including VEGFR1+ HSCs and VEGFR2+ EPCs, have been shown to reside in the osteoblastic region of the BM (68). Other VEGFR3+ lymphatic endothelial precursor cells. VEGFR3 is expressed on a subset vascular-specific progenitors that may also reside in this microenvironment are of CD34+ hematopoietic cells (69). Incubation of CD34+ VEGFR3+ cells with VEGF-C that signals specifically through VEGFR3 results in differentiation of subsets of these cells into lymphatic endothelial cells (69). VEGFR3 is expressed on the subsets of CD34+ that are destined to differentiate into lymphatic endothelial cells suggesting that expression of VEGF-C within the BM microenvironment may play a role recruitment of lymphatic endothelial progenitors to the peripheral circulation (Figs. 1 and 2). Collectively, these data suggest that in the initial phases of organ regeneration mobilization and recruitment of VEGFR2+ or VEGFR3+ endothelial progenitor cells may also contribute to the vascular lymphatic neo-vessel formation

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Figure 4 Comobilization of hematopoietic cells as myogenic progenitors guides incorporation of vascular cells into regenerating myocardium. Tissue-specific release of PDGF may direct specific mobilization of subset of EPCs and PDGFR+ myogenic stem cells to the regenerating myocardium. PDGF in collaboration with VEGF-A released by hematopoietic cells sets up the molecular platform for the incorporation of subsets of EPCs into the regenerating myocardium. (Fig. 2). However, it remains to be determined whether BM-derived lymphatic progenitors play any physiological role in supporting postnatal lymphangiogenic processes.

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5. STEM CELL MOBILIZATION IS MEDIATED THROUGH ACTIVATION OF SPECIFIC MATRIX METALLOPROTEINASE (MMP-9) Clues to the mechanism whereby angiogenic factors promote mobilization of vascular progenitors originated from the finding that marrow suppression results in upregulation of MMP-9 expression (68). MMP-9 modulates recruitment of HSCs from the osteoblast niche by releasing soluble Kit ligand (sKitL) (Figs. 2 and 3). The majority of sKitL is believed to be the product of the membrane-bound Kit ligand (mKitL), which is expressed on the surface of various stromal cells within the BM. The rapid increase in plasma sKitL levels in MMP-9+/+ mice, and the relative deficiency of sKitL at baseline or after myelosuppression in MMP-9−/− mice, suggest that MMP-9 plays a physiological role in releasing sKitL setting up the stage for hematopoietic reconstitution. PIGF and VEGF-A promote the release of sKitL, an essential factor in the recruitment of HSCs from the endosteal region, through activation of MMP-9 (68). Activation of VEGFR1 results in activation of MMP-9 and mobilization of VEGF-A to the peripheral circulation. Similarly activation of VEGFR2 induces MMP-9 expression thereby supporting the recruitment of VEGFR2+ cells from the BM microenvironment. Recently, it has been demonstrated that recruitment of EPCs is impaired in eNOS−/− mice resulting in disruption of neo-angiogenesis (70). NO production was linked to activation of MMP-9 and release of sKitL resulting in mobilization of EPCs. Whether activation of other types of proteases, including heparinases, or endopeptidases direct mobilization of subsets of organ-specific VEGFR2+ or VEGFR3+ EPCs is not known and needs further investigation.

6. CORECRUITMENT OF PROANGIOGENIC HEMATOPOIETIC CELLS MAY DIRECT ORGAN-SPECIFIC INCORPORATION OF MOBILIZED EPCS Several lines of evidence suggest that hematopoietic cells contribute to postnatal angiogenic processes, and that they do so in collaboration with BM-derived VEGFR2+ endothelial progenitors. Hematopoietic cells support angiogenesis via direct cellular contact or release of angiogenic factors, including VEGF-A, VEGF-C, and metalloproteinases (5). In support of these data, lack of hematopoietic reconstitution in the transcription factor AML-1 deficient mice results in profound impairment in angiogenesis (71). Moreover, VEGFR1+ hematopoietic cells have been shown to contribute to tumor angiogenesis (44). Collectively, these data suggest that collaboration of VEGFR1+ hematopoietic cells with VEGFR2+ EPCs enables the incorporation of

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endothelial cells into neo-angiogenic vessels. However, whether hematopoietic cells may also direct the separation of blood and lymphatic blood vessels has only recently been studied. Revascularization of injured or regenerating organs requires a balanced formation of blood as well as lymphatic vessels. Lymphangiogenesis is regulated by expression of angiogenic factors including VEGFR3/VEGF-C signaling pathways (72,73) (Fig. 1). Activation of certain factors including the homeobox gene Prox-1, and specific surface markers including Podoplanin and Lyve-1 have been used to assess lymphangiogenesis (74). One report has underscored the critical role of hematopoietic cells in mandating and sustaining the heterogeneity of the organ-specific vasculature. In this study, it was demonstrated that the adaptor protein SLP-76 (75) and the tyrosine kinase, Syk (76), expressed primarily in the hematopoietic cells, contribute to anatomical separation of lymphatic from blood vasculature (15,16). Mice lacking either of these two factors exhibit a profound disorganization of blood and lymphatic vessels, resulting in arteriovenous-lymphatic shunts. These abnormal connections lead to loss of vascular architecture, enlargement of the heart and mixing of blood with chylous fluid. This study also introduces the intriguing possibility that hematopoietic cells expressing SLP-76 and Syk are the major determinants of separation of these two vascular systems (Fig. 2). This concept was borne out from the observation that endothelial cells apparently do not express SLP-76 and Syk, suggesting that recruitment of SLP-76+ and Syk+ hematopoietic cells is sufficient to dictate separation of lymphatic and blood vessels. These studies suggest that it is the signaling through Syk and SLP-76 within the hematopoietic cells that provides molecular instructions for vascular and lymphatic separation thereby dictating some aspects of vascular heterogeneity.

7. CONCLUSIONS Vascular heterogeneity is determined in part by the organ-specific microenvironment. Stromal cells within each organ may express factors that confer tissue specificity to the vasculature. Although as of yet not fully validated, accumulating evidence suggests that recruitment of BM-derived cells may influence the phenotype of the regenerating vessels in the adult organs. Most likely this process is regulated by the release of specific proangiogenic cytokines and chemokines by the physiologically stressed organ. Based on the available evidence, it is possible that angiogenic factors released by the ischemic or regenerating tissue may induce mobilization of generic type of EPCs, that differentiate into organ-specific endothelial cells after settling down within the microenvironment of that particular organ. According to this scenario, vascular heterogeneity is dictated by the tissue-specific microenvironment. Alternatively, EPCs preprogrammed to differentiate into an organ-specific vascular bed may be electively mobilized from the BM. However, the precise phenotype of organ-specific EPCs, and their location within BM niches have yet to be identified. The evidence for organ-specific recruitment of BM-derived endothelial progenitors is best studied in cardiac revascularization. Conceivably, CD133+ VEGFR2+ PDGF-R+ EPCs may selectively home and contribute to cardiac vasculature, while VEGFR2+

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CXCR4+ EPCs may selectively home to hematopoietic microenvironments. However, extensive additional studies are in order to define the precise mechanism whereby organspecific progenitors are recruited from BM niches and selectively incorporated into various organs. Mobilization and recruitment of EPCs from specific BM niches, including osteoblastic or vascular niches, may also provide another level complexity to the determination of the vascular heterogeneity. High levels of VEGF-A produced by the injured tissue or regenerating organ result in the mobilization of BM-derived EPCs to the peripheral circulation accelerating revascularization process. Given that EPCs may also contribute to vascular healing, overexpression of various tissue-specific VEGFs in conjunction with as of yet unrecognized chemokines or cytokines may promote the peripheral blood mobilization and incorporation of EPCs into the injured vascular bed thereby setting up the stage for organ-specific vessel formation. Identifying the organ-specific chemokines, cytokines and proteases that are released or activated by injured or regenerating tissues will provide the platform to identify subsets of BM-derived cells that are recruited to specific organ, thereby dictating vascular heterogeneity.

ACKNOWLEDGMENTS S.Rafii is supported by National Heart, Lung, and Blood Institute (NHLBI) grants R01s HL-58707, HL-61849, HL-66592, HL-67839, Translational Research Award from The Leukemia & Lymphoma Society, Research Scholar Grant from American Cancer Society (RSG-01-091-01). J.Edelberg is supported by National Institute on Aging (NIA) grants R01s AG19738 and AG20918.

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17 Transcriptional Networks and Endothelial Lineage Peter Oettgen Division of Cardiology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, U.S.A.

1. INTRODUCTION Until recently, little was known about the transcription factors that are necessary for regulating vascular development, endothelial differentiation, and endothelial-specific gene expression. This is in sharp contrast to other developmental processes such as hematopoiesis and myogenesis where several cell or tissue-specific transcription factors have been identified. Progress in elucidating the molecular mechanisms underlying the transcriptional control of vascular development has lagged considerably, in large part due to the fact that the model systems for studying blood vessel development are more limited. The identification of several vascular-specific genes involved in vasculogenesis and evaluation of the genomic regulatory regions required for directing their expression over the past decade has facilitated the identification of the transcriptional mechanisms required for vascular-specific gene expression. Vascular defects associated with targeted disruption of genes encoding additional transcription factors, previously unsuspected to be involved in regulating vascular-specific gene expression, have also led to the identification of role for these factors in vascular development or vascular-specific gene expression. The purpose of this chapter is to summarize the recent approaches and advances made in our understanding of the transcriptional regulation of vascular development, angiogenesis, and vascular-specific gene expression.

2. ORIGINS OF ENDOTHELIAL DIFFERENTIATION AND DIVERSITY IN THE DEVELOPING MOUSE EMBRYO The first site of endothelial differentiation in the developing mouse embryo is in the yolk sac. Mesodermal cells migrate through the primitive streak into a region designated to be the yolk sac, where they rapidly differentiate to give rise to cells of the hematopoietic and endothelial lineage. Further differentiation of these lineage-specific precursors leads to

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the formation of blood islands, that consist of a central focus of hematopoietic cells, principally primitive erythrocytes, surrounded by a layer of developing endothelial cells. The differentiation of pluripotent embryonic stem cells along the endothelial and hematopoietic lineages is associated with a distinct temporal expression pattern of specific cell surface markers (Fig. 1). The close proximity of cells of the hematopoietic and endothelial cells has suggested the existence of a bipotential cell, the so-called “hemangioblast” with the ability to differentiate into cells of the hematopoietic and endothelial lineages. These cells are thought to express the vascular endothelial growth factor (VEGF) receptor Flk-1 (1). Further differentiation and full commitment along the endothelial lineage initially leads to the formation of the angioblast, the earliest endothelial precursor cell and eventually to the fully differentiated endothelial cell. This process is associated with the sequential expression of specific cell surface markers (Fig. 1). Significant progress has been made in identifying the transcription factors that promote the development of the hemangioblast, and further differentiation along the hematopoietic lineage. These include stem cell leukemia (SCL)/tal-1, GATA-1, and HOXB4 genes (2–5). In contrast, very little is known about the transcription factors that promote commitment of the hemangioblast along the endothelial lineage. Endothelial differentiation is associated with the temporal expression of several tyrosine kinases including Flt-1, Tie1, and Tie2. Following extraembryonic differentiation in the yolk sac, differentiation of the hematopoietic and endothelial cells also begins in the developing embryo. The first site of hematopoiesis in the developing mouse embryo is in the para-aortic

Figure 1 Differentiation of embryonic stem cells along the hematopoietic and endothelial lineages. Cell surface

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markers at various stages of differentiation are listed with an approximate time line in days (days postcoitum) for differentiation during murine embryogenesis listed below. splanchnopleural region (pSp) at E8.5-9.5, and then in the AGM (aorta-gonadomesonephros) region at E10.5, where endothelial as well as hematopoietic progenitors have also been identified (6,7). Definitive hematopoiesis then occurs independently in the fetal liver, and eventually the bone marrow, while vascular development continues in the embryo proper. During the development of the primary vasculature, endothelial cells that incorporate into newly formed blood vessels continue to differentiate during blood vessel maturation as is evidenced by the sequential expression of additional endothelial-specific markers (8). This pattern of sequential expression of endothelial-specific markers is similar to what is observed in the yolk sac (Fig. 1). The formation of mature blood vessels in the developing embryo requires the recruitment and migration of endothelial cells to sites of active blood vessel development (Fig. 2). This begins with the formation of a simple tube followed by vessel stabilization, with the recruitment of surrounding pericytes and their differentiation into vascular smooth muscle cells. This process is facilitated by the local release of several growth factors including platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF) -β, and angiopoietin-1. Further differentiation of the vasculature leads to the formation of arteries and veins, a process mediated in part by ephrin B2 and ephrin B4 (9). Many differences exist in the genes expressed in arterial vs. venous endothelial cells. For example, neuropilin-1, one of the VEGF receptors, is predominantly expressed on arterial endothelial cells whereas neuropilin-2 is expressed predominantly on the venous side (10). Ultimately endothelial cell function and expression become highly specialized with regard to function and structure in particular vascular beds. In addition to genetically determined differences in endothelial phenotype, local environmental factors may also play an important role in determining the expression of selected proteins in specific endothelial cells in specific vascular beds. These differences in expression of particular genes are at least in part mediated by paracrine interactions with tissue-specific cell types and endothelial cells in the particular vascular beds. Factors in the microenvironment that may promote local differences in endothelial function and gene expression include the release of soluble factors, direct cell-cell interactions, and differences in the organization of the surrounding matrix proteins.

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Figure 2 The role of transcription factors during different stages of vascular development.

3. MOLECULAR MECHANISMS OF ENDOTHELIAL-SPECIFIC GENE EXPRESSION A number of strategies have been used to identify the particular transcription factors that are critical for regulating the process of endothelial differentiation and heterogeneity in the expression of specific genes in different vascular beds. One approach, for example, includes the identification of critical genetic regulatory elements required to direct the expression of particular candidate genes. These studies have been greatly facilitated by transgenic approaches using marker genes such as β-galactosidase (LacZ) to allow for determination of the regulatory elements that are required for expression of particular genes in specific vascular beds or at a various stages of development. In this section, a variety of strategies will be reviewed toward identifying the transcription factors involved in regulating vascular-specific gene expression.

3.1. Candidate Gene Approach One common approach to identifying the transcription factors required for a particular process is to examine the transcriptional regulation of specific genes that are known to be critical for the process. This approach has been successful in identifying several of the transcription factors required for T- and B- cell differentiation and has similarly been employed to facilitate the identification of the transcription factors required for vascularspecific gene expression. Several receptor tyrosine kinases, including the Flk-1, Flt-1, Tie1, and Tie2 genes are known to be critical mediators of endothelial differentiation and vascular development. Targeted disruption of all of these genes leads to defects in vascular development and early embryonal lethality (11–13).

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The genomic regulatory regions required for directing vascular-specific gene expression have been identified for several of these genes that has been verified in vivo by their ability to direct the expression of the LacZ gene in transgenic experiments. Interestingly, for the Tie1 gene, the promoter alone is sufficient to direct LacZ expression in an endothelial cell-specific fashion (14). In contrast, for both the Tie2 and Flk-1 genes, an intronic enhancer is required to direct complete vascular-specific gene expression (15,16). Comparison of the mouse and human DNA sequences within the regulatory regions of these genes has facilitated the identification of conserved binding sites for different classes of transcription factors. Conservation of these sites suggests that the binding of family members of these transcription factors may be required to direct vascular-specific gene expression. Further support for this concept comes from the fact that point mutations in these sites lead to marked reductions in the vascular-specific gene expression of LacZ in transgenic animals. The Tie1 gene promoter contains conserved binding sites for ETS factors and AP2 (14). Mutations in most of these conserved binding sites lead to marked reductions in the ability of the Tie1 promoter to direct LacZ gene expression in transgenic animals (16). A similar approach has been used to identify the transcription factor binding sites that are necessary for directing the vascular-specific gene expression of the Flk-1 gene (16). Conserved binding sites for the ETS factors, SCL/tal-1 factor, and GATA factors were identified. Point mutations in some of these binding sites also lead to marked reductions in the vascular-specific expression directed by the Flk-1 regulatory regions in transgenic studies. Conserved ETS binding sites also exist in the Tie2 and Flt-1 genes (17–19). The results of these studies strongly support that members of certain transcription factors are involved in the regulation of vascular development. One powerful tool to facilitate the evaluation of these promoters is Hprt-targeting of promoter fragments coupled to LacZ in transgenic animals. This approach has been successfully used to define important regulatory elements that are required to direct vascular-specific gene expression (20–22). Hprt-targeting overcomes the problem of variable expression associated with random integration often observed with standard transgenic approaches. This approach has also led to the identification of specific modules that promote the expression of LacZ in certain vascular beds, suggesting that binding sites for specific transcription factors are contained within these elements, which may ultimately lead to the identification of the particular transcription factors that are required to direct the expression of these genes to particular vascular beds.

3.2. Transactivation Studies Although studies utilizing marker genes such as LacZ in tandem with the genomic regulatory elements for a particular gene have led to the identification of potential classes of transcription factors that may be required for the direction of vascular-specific gene expression, this has not resulted in the identification of the specific transcription factors within each of these classes. One approach toward identifying the specific transcription factors that are responsible for the regulation of these genes is to perform transactivation studies and compare the ability of the different members of a particular family of factors to transactivate the gene. This strategy was used to test the ability of different members of the ETS factor family to transactivate the Flt-1, Tie1, and Tie2 genes. Whereas, the ETS

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factors ETS-1 and ETS-2 most potently transactivated the Flt-1 gene promoter, they were only able to mildly transactivate the Tie1 and Tie2 gene promoters (14,18). In contrast, the ETS factors NERF and ELF-1 more potently transactivated the Tie1 and Tie2 genes (14,18). The ability of specific ETS factors to transactivate the specific gene targets also correlates with their ability to bind to specific conserved ETS binding sites. This suggests that different subsets of the ETS factors may regulate different vascular-specific genes.

3.3. Animal Models of Vascular Development One of the major difficulties in identifying the specific transcription factors involved in regulating vascular-specific gene expression, particularly as it relates to blood vessel development, is the difficulty in isolating either embryonic or extraembryonic blood vessels during mouse embryogenesis. Because blood vessel development is a highly conserved process over evolution, the use of alternate model systems has permitted easier access to studying blood vessel development. Two animal models that have been particularly useful for these studies include the developing zebrafish and chicken. Both have the advantages of allowing direct visualization of blood vessels. Two genes that have been identified in zebrafish and appear to be critical early regulators for initiating vascular development are cloche and spade tail (23). Similarly, the SCL transcription factor was also shown to promote vasculogenesis, hematopoiesis, and endothelial differentiation when expressed ectopically in zebrafish mesoderm (24). The ETS transcription factor Fli-1 has also been shown to be enriched in the developing blood vessels of zebrafish embryos (25). As an alternative model of blood vessel development, several investigators have used the developing chicken as a model to study blood vessel development, because of the easier access to developing blood vessels, particularly in the extraembryonic chorioallantoic membrane. These blood vessels can be microdissected at different stages of development facilitating the determination of whether specific genes are upregulated or enriched in developing blood vessels. This approach was used to identify which of the members of the ETS transcription factor family are upregulated during blood vessel development. A novel role for the ETS factor ELF-1 in vascular development was identified using this approach (26). In situ hybridization and immunohistochemical experiments confirmed the enriched expression of this factor in extraembryonic and embryonic blood vessels of the developing chicken embryo (26). The ETS factor ETS-1 has also been shown to be enriched in the developing blood vessels of the chicken and antisense oligonucleotides have been shown to inhibit angiogenesis when delivered to the chicken chorioallantoic membrane (27). One potential criticism of using nonmammalian models to identify the transcription factors involved in regulating blood vessel development is that the same factors may not necessarily be evolutionarily conserved. Arguing against this is the fact that studies in the chicken and in zebrafish have demonstrated that not only are the factors conserved with regard to protein sequence, but also show a similar enriched expression pattern during vascular development. For example, the helix-loop-helix transcription factor, SCL is expressed in developing blood vessels and in the vasculature of both the developing mouse and zebrafish (24,28). The ETS factor, ELF-1 which has previously been identified for its role for T-cell specific gene expression has also been shown to be a strong transactivator of the Tie1 and Tie2 genes and is highly enriched in developing

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blood vessels of the developing chicken embryo. The overall homology between the chicken and human ELF-1 protein is 80% (26). Similarly, the ETS factor Fli-1 has recently been shown to be a critical regulator of blood vessel development not only in zebrafish but also in the mouse (25,29). In situ hybridization studies of the developing mouse have also demonstrated that ETS-1 is expressed in developing blood vessels associated with tumor angiogenesis (30). Targeted disruption of Fli-1 in mice results in a loss of vascular integrity accompanied by bleeding and embryonic lethality at day 11.5 (29). Expression of the Tie2 gene is also down regulated in these mice. The expression of two of the GATA factors, GATA-2 and GATA-3 has been examined in developing mouse and human fetal tissues. Both factors are enriched in the developing dorsal aorta (31,32).

3.4. Defining Potential Overlapping Roles of Selected Transcription Factors in Hematopoietic and Endothelial Lineages Perhaps one of the most interesting recent findings regarding the transcriptional regulation of vascular development has been the determination that the transcription factor SCL/tal-1, which was originally thought to play a role strictly in hematopoeisis, also appears to be critical for embryonic blood vessel development. Targeted disruption of this gene leads to embryonic lethality by day 9.5, due to an absence of yolk sac erythropoiesis (33). However, it was unclear whether this gene might also contribute to nonhematopoietic pathways at later stages of development. By performing transgenic experiments in which the GATA-1 promoter is used to restore SCL gene expression in hematopoeitic lineages in SCL −/− mice, the mice develop striking abnormalities in yolk sac angiogenesis (28). This suggests that certain transcription factors may be critical for both the normal development of hematopoietic cells and blood vessels, and that there may be a common stem cell precursor for both lineages. The most striking defects were a disorganized array of capillaries, and absence of normal vitelline blood vessel formation. Although the larger vitelline blood vessels were not present, a smaller network of interconnecting vessels did exist. The architecture of these vessels revealed normal appearing endothelial cells as well as the smooth muscle cells or pericytes that constituted the outer lining of the blood vessels. The expression of a number of vascularspecific genes including Tie-1, Tie-2, Flk-1, and Flt-1 also appeared normal. Members of the ETS transcription factor family that were originally described for their role in lymphoid development have now also been shown to regulate vascular-specific genes. The ETS factor NERF was originally identified for its role in regulating the expression of B-cell specific genes such as the tyrosine kinase blk (34). The NERF gene is expressed as at least three isoforms, NERF1a, NERF1b, and NERF2. Whereas NERF2 is a potent transactivator, the NERF1 isoforms have a truncated transactivation domain and act as natural dominant negative forms of NERF2. These isoforms are differentially expressed in different cell types. Whereas NERF1a and 1b are expressed in B-cells, NERF2 is highly expressed in endothelial cells and is a strong transactivator of the endothelialspecific Tie1 and Tie2 genes (18). Similarly, the related ETS factor ELF-1, which was originally shown to regulate T-cell specific genes, was also shown to be enriched in

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developing blood vessels of the chicken (26). The ETS factor Tel was originally identified for its role as a protooncogene in the development of human leukemias. Interestingly, targeted disruption of this factor led not only to defects in hematopoiesis, but also to defects in extraembryonic angiogenesis (35).

3.5. Determinants of Vascular Specificity in the Absence of Vascular-Specific Factors Although several of the transcription factors that are important for blood vessel formation have been identified, many of them are not particularly endothelial or vascular cellspecific in their distribution. Several of the factors appear to be involved in both hematopoiesis and vasculogenesis. What ultimately determines vascular-specific gene expression and the formation of blood vessels? Either additional vascular cell-specific proteins (e.g., adapter proteins or newly identified transcription factors) are required to promote the regulation of a vascular-specific phenomenon, or it is possible that combinatorial mechanisms involving the expression of a specific group of proteins that in and of themselves are not individually cell-specific, but their coexpression in particular cells results in cell-specific expression. One such additional protein which was recently described factor is the zinc finger transcription factor Lmo2, a member of the LIM family of proteins (36). Lmo2 serves as a bridging molecule between GATA factors and E-box proteins, which does not require its direct binding to DNA (37). Attempts to identify the DNA binding specificity of the Lmo2 protein with “CASTing” experiments in which double stranded DNA sequences with random internal sequences were coincubated with nuclear extracts from the erythroid lineage MEL cell. Antibodies directed against Lmo2 were used to isolate the oligonucleotides binding to Lmo2 protein. Evaluation of nucleotide sequence of these oligonucleotides demonstrated consensus binding sites for GATA and E-box factors. Further analysis suggested that Lmo2 was not directly binding to the oligonucleotides but was acting to bridge the two proteins. Interestingly, overexpression of Lmo2 inhibits erythroid differentiation (38). Furthermore, targeted disruption of Lmo2 demonstrates a role for this transcription factor in vascular remodeling during blood vessel development (39). This suggests that these bridging proteins may actually be just as important in determining differentiation from one lineage to another. Lmo2 belongs to a subset of the LIM proteins called “LIM only” that lack a homeodomain and hence do not bind to DNA. A subset of LIM only proteins, the cysteine-rich proteins (CRP), have been shown to be important regulators of muscle development. CRP1, for example, is expressed in gut smooth muscle and has been implicated in muscle differentiation (40). CRP3/MLP is expressed in the heart and skeletal muscle. Overexpression of CRP3/MLP in cultured myoblasts augments differentiation. Further support of a role for CRP3 in muscle development comes from CRP3-deficient mice that exhibit profound defects in cardiac as well as skeletal muscle (41). CRP2/SmLIM is principally expressed in vascular smooth muscle cells, with preferential expression in arterial vs. venous blood vessels (42). Another family of proteins that function primarily through protein-protein interactions with transcription factors are the ID proteins. These are HLH proteins that lack a DNA binding domain and function by forming heterodimers with bHLH transcripition factors (43). Mice deficient in Id1 and Id3 exhibit defects in endothelial differentiation and angiogenesis (44). In

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summary, these data suggest that these adapter proteins may be just as important in determining tissue-specific expression of certain genes and directing developmental processes. Similar properties are exhibited by the SCL/tal-1 transcription factor that does not require DNA binding for its function (45). Mutant forms of the SCL gene, lacking the putative DNA binding domain, were able to rescue hematopoietic defects of the phenotype of SCL −/− embryos, as well as the hematopoietic and vascular defects of the cloche zebrafish mutants, which are thought to be upstream of SCL. Another mechanism for providing cell-type specificity, even though the particular factor may be expressed in several cell types, is through differential expression of functionally different isoforms of the transcription factor in different cell types. The ETS transcription factor NERF, for example, that was originally identified as being important in B-cell function by regulating the B-cell specific tyrosine kinase blk, has also subsequently been shown to regulate the Tie2 tyrosine kinase in endothelial cells (18,34). The NERF gene has multiple isoforms that are differentially expressed in B-cells compared to endothelial cells (18).

3.6. Transcriptionaly Mediated Hypoxia Responses During Blood Vessel Development and Angiogenesis After the development of a primary vascular network, the developing embryo requires the formation of additional blood vessels or angiogenesis. This process is largely driven by hypoxia which serves as a stimulus for the release of angiogenic growth factors. One of the main classes of transcription factors that promote this process is the basic helix-loophelix PAS domain family. A prototype member of this family is the arylhydrocarbonreceptor nuclear translocator (ARNT) (46). ARNT forms a heterodimeric complex with another PAS transcription factor hypoxia-inducible factor (HIF-1α) (47). In response to oxygen deprivation, these transcription factors stimulate the expression of such angiogenic factors as VEGF (48). Targeted disruption of the ARNT gene results in embryonic lethality by day 10.5 (49). Although a primary vascular network forms, the predominant defective angiogenesis occurs in the yolk sac and branchial arches, and overall growth of the embryos is stunted. These defects are similar to those observed in VEGF or tissue factor deficient mice (50,51). Thus, although the primary vascular network developed, the angiogenic responses to hypoxia are severely impaired. Similar findings are observed in HIF-1α knockout mice in which embryonic lethality occurs by day E10.5 as a result of cardiac and vascular malformations (52). Although neither of these transcription factors is expressed in a vascular-specific way, their role in angiogenesis and vascular development is primarily related to their ability to stimulate the production of angiogenic factors such as VEGF in response to hypoxia. A third member of this family of transcription factors, endothelial PAS domain protein 1 (EPAS1) was recently identified (53). EPAS is predominantly expressed in endothelial cells, and can also heterodimerize with ARNT. Targeted disruption of the EPAS gene has been evaluated by two different groups resulting in two different phenotypes (54,55). Tian et al. detected abnormalities in catecholamine homeostasis in EPAS −/− mice and no distinct abnormalities in blood vessel formation, whereas Peng et al. identified vascular defects at later stages of embryogenesis during vascular remodeling in their EPAS −/− mice (55). The differences in the phenotype cannot be attributed to differences

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in targeting construct, since both groups disrupted the expression of the bHLH domain, but were more likely attributed to differences in the strain of the mice or subtle differences in the ES cells used. Although the formation of a primary vascular network or vasculogenesis occurs, later defects in vascular remodeling are observed during large vessel formation associated with hemorrhage and inability of the vessels to fuse properly. This suggests that all three of these PAS family members play a similar role in facilitating later stages of vascular remodeling and angiogenesis in the developing embryo. Modulation of the function of HIF-1α is also achieved by interaction with other proteins. The transcriptional adapter proteins p300 and CREB-binding protein (CBP) form a multiprotein/DNA complex together with HIF-1α on the promoters of the VEGF and erythropoietin genes to promote expression of these genes in response to hypoxia (56). Interestingly, CBP deficient mice exhibit abnormalities in both vasculogenesis and angiogenesis (57). In contrast, the von Hippel-Lindau tumor suppressor protein (pVHL) has been shown to promote proteolysis of HIF-1α through ubiquitination under normoxic conditions. Defective VHL function is associated with cancers that exhibit dysregulated angiogenesis and upregulation of hypoxia inducible genes (58). The signaling mechanisms by which hypoxia activates HIF-1α are beginning to be elucidated. The catalytic subunit of PI 3-kinase, p110, plays a pivotal role in the induction of HIF-1α activity in response to hypoxia (59). Both induction of VEGF gene expression and HIF-1α activity in response to hypoxia could be blocked by the addition of a PI3-kinase inhibitor. Further support of this concept comes from experiments in which VEGF gene expression and HIF-1α activity are inducedby cotransfection of p110. Other studies have recently demonstrated that HIF-1α activity may also be modulated by the mitogen-activated protein kinases p42 and p44 (60).

3.7. Targeted Disruption and Overexpression Studies of Additional Transcription Factors An alternative approach that has resulted in the identification of other transcription factors that are required for blood vessel development is through targeted disruption. In many cases, this has unexpectedly resulted in determining a novel role for a particular factor in blood vessel development. An example is targeted disruption of the AP-1 transcription factor family member Fra1, which leads to abnormalities in extraembryonic vascularization (61). The zinc finger transcription factor LKLF is expressed in a variety of vascular and nonvascular cell types. However, targeted disruption of this transcription factor leads to abnormalities in later stages of blood vessel development (62). Although the early events of both angiogenesis and vasculogenesis were normal in LKLF-deficient mice, they develop abnormalities in the smooth muscle architecture of the tunica media, leading to aneurysmal dilatation of the blood vessels, with eventual blood vessel rupture. Diminished numbers of endothelial cells, pericytes, and extracellular matrix deposition are also seen. The transcription factor Tfeb, a basic helix-loop-helix transcription factor, was recently shown to be required for vascularization of the placenta (63). The homeobox gene Hox D3 is induced in endothelial cells in response to basic fibroblast growth factor (bFGF), and antisense oligonucleotides to Hox D3 block the ability of bFGF to induce urokinase plasminogen activator (uPA). Overexpression of Hox D3 increases integrin

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expression in endothelial cells (64). Another homeobox transcription factor that may contribute to both hematopoeisis and endothelial differentiation is hhex. Overexpression of this factor in zebrafish embryos leads to enhanced endothelial and erythroid differentiation (65).

4. OVERVIEW OF TRANSCRIPTION FACTORS AT DIFFERENT STAGES OF VASCULAR DEVELOPMENT The development of the vascular system requires the carefully timed and spatially oriented series of events that is orchestrated by distinct sets of transcription factors (Fig. 2, Table 1). This begins with the differentiation of pluripotent stem cells into endothelial cells, their migration to sites of active blood vessel development, the formation of simple endothelial tubes, followed by distinct types of blood vessels and endothelial cells, in particular tissues. The purpose of this section is to give an overview of the transcription factors that are known to play a role in the various stages of vascular development.

4.1. Endothelial Differentiation One of the first steps during vascular development is the differentiation of endothelial cells from pluripotent stem cells. This process initially involves the expression of other endothelial-specific markers such as CD31(PECAM-1); VE-cadherin is associated with the differentiation of these cells into mature endothelial cells. The specific transcription factors that mediate these events have not yet been identified. However, because there are conserved binding sites for several of the transcription factors involved in hematopoiesis in the regulatory regions of vascular-specific genes, this suggests that members of the same transcription factor families may also involved in the process of endothelial differentiation. Several studies have recently suggested the existence of a common precursor for both endothelial cells and cells of hematopoietic origin. The possible existence of a common precursor was originally suggested because of the close association of hematopoietic cells and endothelial cells in the developing embryos in the so-called blood islands. Hematopoietic and endothelial cells coexpress a number of genes. One of the earliest markers expressed on cells of endothelial and hematopoietic origin is the VEGF receptor Flk-1. Further support for the existence of the hemangioblast comes from differentiation of pluri-

Table 1 Transcription Factor

Family

Role

AML-1 (83)

CBF

Angiogenesis

ELF-1 (26)

ETS (wHTH)

Tie2 gene regulation

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Ets-1 (27)

ETS (wHTH)

Angiogenesis

Fli-1 (29)

ETS (wHTH)

Vascular development, Tie2 gene regulation

NERF2 (18)

ETS (wHTH)

Tie2 gene regulation

TEL (35)

ETS (wHTH)

Yolk sac angiogenesis

MEF2 (75)

MADS box

Vascular development, smooth muscle cell differentiation

SMAD5 (76)

MADS box

Smooth muscle differentiation, angiogenesis

SCL/tal-1 (28)

bHLH

Vascular development

DHAND (77)

bHLH

Vascular smooth muscle differentiation

Tfeb (63)

bHLH-Zip

Placental vascularization

HESR1, gridlock (69,70)

bHLH

Aorta development, endothelial tube formation

EPAS (54,55)

PAS-bHLH

Angiogenesis

HIF-1α (52)

PAS-bHLH

Angiogenesis

ARNT (49)

PAS-bHLH

Angiogenesis

Fra1 (61)

bZip

Endothelial differentiation

Vezf1 (68)

Zinc finger

Endothelial differentiation

LKLF (62)

Zinc finger

Vascular smooth muscle differentiation

HOXD3 (64)

Homeobox

Endothelial response to angiogenic factors

COUP-TFII (84)

Nuclear receptor

Yolk sac angiogenesis

potent embryonic stem cells along endothelial and hematopoietic lineages (1,66). When individual blast colonies are allowed to differentiate further they form adherent cells that expressed more endothelial-specific markers such as PECAM-1 and Tie2, whereas many of the nonadherent, cells, presumed to be hematopoietic origin, expressed genes such as “hemoglobin” consistent with cells derived from the erythroid lineage. Furthermore, when Flk-1 positive cells were isolated from ES cells and allowed to differentiate in vitro, they could be sorted into cells of both endothelial and hematopoietic origin by flow cytometry using surface markers specific for endothelial or hematopoietic cells (67). Some of the specific transcription factors that may be required for endothelial differentiation have recently been identified. The vascular defects seen in mice with targeted disruption of the immediate-early gene Fra1 were partially attributed to a marked reduction in the number of endothelial cells. The defects were mainly seen in the placenta with severely impaired vascular development leading to embryonic lethality between E10.0 and E10.5 (61). The zinc finger transcription factor Vezf1 is expressed solely in vascular endothelial cells and their precursors (68). Endothelial-specific expression of Vezf1 was also observed in endothelial cells of the developing dorsal aorta, the branchial arch artery, and endocardium and colocalized with Flk-1 expression.

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4.2. Endothelial Tube Formation Following their differentiation from pluripotent stem cells, endothelial cells migrate and form primitive tubes. The basic HLH transcription factor HESR1 has recently been shown to be upregulated during endothelial tube formation (69). Overexpression of this gene in endothelial cells results in down regulation of Flk-1 which may result in inhibiting endothelial cell proliferation by diminishing endothelial responsiveness to VEGF. Antisense oligonucleotides directed against HESR1 were able to block the formation of capillary tubes. The homologue of this factor in zebrafish is called gridlock and is a critical mediator of the development of arteries such as the aorta but not of veins (70). The homeobox gene HOX B3 has recently been shown to be involved in facilitating capillary morphogenesis (71). Overexpression of this factor in the chicken chorioallantoic membrane leads to increased capillary vascular density and antisense oligonucleotides inhibit endothelial tube formation of microvascular endothelial cells cultured on extracellular matrix. Another transcription factor involved in endothelial tube formation is nuclear receptor PPAR-gamma. In contrast to HESR1 and HOX B3, ligands activation of this transcription factor blocks endothelial tube formation and endothelial proliferation (72).

4.3. Smooth Muscle Cell Differentiation After initial endothelial tube formation, vessel maturation requires the subsequent recruitment of surrounding mesenchymal cells and their differentiation into vascular smooth muscle cells. This process has been shown to involve the interaction of endothelial cells with mesenchymal cells and the release of specific growth factors such as PDGF (73,74). A number of transcription factors have also recently been shown to be critical for smooth muscle differentiation (Table 1). One family of transcription factors that is crucial for muscle development, in general, is the MADS-box transcription factor family. Two members of this family, SMAD5 and MEF2C, have recently been shown to be important in vascular development and in smooth muscle cell differentiation (75,76). Targeted disruption of SMAD5 leads to vascular defects resulting in embryonal lethality at day 10.5–11.5. The defects include enlarged blood vessels with diminished numbers of vascular smooth muscle cells. The absence of SMAD5 results in apoptosis of mesenchymal cells and marked reduction in the differentiation of mesenchymal cells into vascular smooth muscle cells (76). Similarly, the targeted disruption of MEF2C leads to abnormalities in smooth muscle cell differentiation and the inability of endothelial cells to form into vascular structures (75). LKLF is a member of the krueppel-like family of zinc finger transcription factors. Targeted disruption of this gene leads to vascular defects. Most notably there is a reduction in the number of differentiated smooth muscle cells and pericytes. These defects result in aneurysmal dilatation of the large vessels and eventual rupture with intraamniotic hemorrhage (62). A similar phenotype was recently reported for mice lacking the cytoplasmic domain of Ephrin B2, suggesting that signaling through ephrin B2 may involve activation of LKLF or similar transcription factors during later stages of blood vessel development (9). The basic helix-loop-helix transcription factor dHAND has recently been shown to be crucial for yolk sac vascular development. In dHAND, null mice endothelial cell differentiation and recruitment of surrounding

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mesenchymal cells occurs normally. However, the mesenchymal cells fail to differentiate into vascular smooth muscle cells (77). One of the genes that was shown to be down regulated in these mice was the VEGF165 receptor neuropilin, suggesting that dHAND may be a critical mediator of the VEGF signaling pathway.

4.4. Development of Arterial and Venous Vascular Systems Further maturation of the vascular system ultimately leads to the formation of a broad array of blood vessel types, including arterial, veinous, and capillary vessels. Arteries and veins can be distinguished by important structural as well as functional differences. Whereas arteries function predominantly to carry blood away from the heart, veins transport blood from tissues back to the heart. Arteries are exposed to higher pressure and flow than veins and are characterized by a thick medial layer consisting of vascular smooth muscle cells. In contrast, veins have less surrounding smooth muscle cells and have valves to promote unidirectional flow of blood. A variety of growth factors and receptors have recently been implicated in the differentiation of arterial vs. venous system (9,10,78,79). Molecular differences between arteries and veins have been demonstrated in a variety of species during embryonic development. The transmembrane ligand ephrin B2 is expressed in endothelial cells lining only arteries. The tyrosine kinase receptor for ephrin B2, EphB4, is expressed predominantly in venous endothelial cells. Signaling through the Notch pathway is critical for the expression of artery-specific genes and the repression of these markers in developing veins. The morphogen sonic hedgehog (shh) has recently been identified for its role in arterial differentiation. Shh appears to stimulate the expression of VEGF and thereby activate the Notch pathway to induce arterial differentiation (78). Shh can stimulate not only the expression of VEGF, but also the angiopoietins Ang-1 and Ang-2 that are also involved in vascular development (79). A variety of transcription factors have been shown either to be downstream mediators of some of the factors mentioned above or are expressed in particular types of endothelial cells and the target genes for some of these transcription factors remain to be determined. The HMG box factor, Sox-13 is expressed in embryonic arteries but not veins (80). A relative of HIF-1α, EPAS-1, has an arterial endothelial-specific expression pattern. Members of a novel family of Hairy-related bHLH transcription factors, including HRT13, are exclusively expressed in arterial cells during embryonic blood vessel development (81). The zebrafish bHLH transcription factor gridlock is another member of the Hairyrelated bHLH transcription factors. During vascular development, expression of gridlock is similarly restricted to the formation of the arterial vs. the venous system (70). Expression of gridlock is induced by activation of Notch. During vascular development, gridlock acts tosuppress the expression of Ephrin B4, thereby promoting arterial differentiation (82).

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4.5. Temporal and Spatial Aspects of Vascular Development One of most intriguing aspects of any developmental process is how differentiating cells migrate to the proper location in the correct spatial and temporal organization to form specific structures such as organs or tissues. Blood vessel development similarly involves the correct spatial organization of differentiating endothelial and vascular smooth muscle cells. Endothelial differentiation is an early event followed by the formation of primitive tubes. The subsequent recruitment of surrounding mesenchymal cells and their differentiation into vascular smooth muscle cells is a later event leading to the formation of stable blood vessels. Growth factors including PDGF, bFGF, VEGF, angiopoietin-1, and TGF-β are key mediators of these events promoting the proliferation and migration of cells. Several of the transcription factors described above are key regulators of the expression of either the growth factors, their receptors, or mediators of the cellular responses to these growth factors. A summary of the temporal role for these transcription factors is shown in Fig. 2. One of the earliest transcription factors required for the differentiation of a pluripotent stem cell into a hemangioblast is SCL/tal-1 (45). Knockout studies suggest that two transcription factors that may be required for differentiation or survival of endothelial cells early in development are Fra1 and Vezf1 (61,68). Another early step in the differentiation of endothelial cells is the expression of VEGF receptors that promote not only the differentiation but also the proliferation of endothelial cells. Regulation of the VEGF receptors gene expression is mediated by the Ets transcription factors, GATA factors, and bHL H factor dHAND (16,17,77). The expression of VEGF is largely mediated by the PAS domain family of transcription factors, including HIF-1α, EPAS, and ARNT in response to hypoxia. The next stage of blood vessel development involves the proliferation and migration of the endothelial cells and their formation into primitive tubes. Endothelial tube formation is regulated at least in part by the transcription factors HESR1 and PPARγ (69,72). Maturation of primitive endothelial tubes into mature blood vessels requires the recruitment of surrounding mesenchymal cells or pericytes and their differentiation into vascular smooth muscle cells. This process is largely mediated by the angiopoietins and the Tie2 receptor. Tie2 gene expression has been shown to be regulated by the ETS factors NERF, ELF-1, and Fli-1 (18,26,29). One of the key regulators of angiopoietin-1 expression is the transcription factor AML1. Targeted disruption of this factor led to abnormalities in angiogenesis that could be rescued by administration of angiopoietin-1 (83). Another transcription factor that also appears to regulate Ang-1 levels is the nuclear receptor COUP-TFII (84). Targeted disruption of this gene is associated with angiogenic defects and marked reductions in the level of angio-poietin-1. The differentiation of mesenchymal cells into vascular smooth muscle cells is also a highly orchestrated process. Members of the MADS-box factors such as SMAD5 and MEF2C mediate the effects of TGF-β thereby promoting endothelial mesenchymal interactions and smooth muscle cell differentiation. Crucial gaps in our understanding of the role of specific transcription factors in this process include the identification of the transcriptional mediators that mediate endothelial responses to growth factors such as VEGF and angiopoietin-1. The list of factors mentioned above likely represent only a small subset of

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the factors required for vascular development. Several additional factors likely exist for the different stages of vascular development.

5. CONCLUSION Endothelial-specific gene expression is a tightly regulated process that is not onlycritical for normal vascular development, but also required for the determination of functional differences in the endothelium in specific tissues and blood vesseltypes in the fully developed adult. The results of several recent studies described above suggest that gene expression in endothelial cells is not simply governed by a unique subset of endothelialspecific transcription factors but rather, a consequence of a complex coordinated interplay between multiple transcription factors as well as additional protein-protein interactions. The identification of all of these factors will not only provide a better understanding of normal vascular development and endothelial-specific gene expression in a variety of tissues in the adult, but alsoprovide an important framework for determining how alterations in endothelial function may occur, and thereby contribute to the pathogenesis of a number of human diseases.

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18 The Diversity of Vascular Disease: A Clinician’s Perspective John P.Cooke Stanford University School of Medicine, Falk CVRC, Stanford, California, U.S.A.

1. INTRODUCTION Passing through the portals of any vascular medicine clinic is an assortment of poor souls that manifest diverse signs and symptoms of vascular disorders and coexisting medical conditions. In the Vascular Medicine Clinic at Stanford University, the majority of these individuals are sent to us by their physicians with a presumptive diagnosis of “peripheral vascular disease,” a general term providing little information about the patient’s condition. Under the rubric of peripheral vascular disease fall a great variety of ailments that include arterial, arteriolar, venous, and lymphatic disorders. Each of these disorders may include developmental and/or acquired abnormalities of vascular function or structure, often complicated by coexisting derangements of hemodynamics, immune function, metabolism, or coagulation. A particular vascular disease may manifest in a variety of guises depending upon the age and gender of the afflicted, the chronicity of the condition, the concomitant medical disorders, and the interaction with environmental, behavioral, cultural, and genetic influences specific to the individual patient. Relevant to the focus of this volume, it is likely that the anatomic distribution, progression, and severity of vascular diseases are determined in part by phenotypic heterogeneity of the endothelium. The most common of the peripheral vascular disorders is arterial occlusive disease secondary to atherosclerosis affecting the aorto-iliac and/or infrainguinal arteries. A more concise descriptor is peripheral arterial disease (PAD), a term which has gained acceptance in the vascular community. However, the vascular specialist knows that PAD comes in many forms. There is a multiplicity of etiologies, with heterogeneous presentations. Accordingly, in this chapter, we will consider the single entity of PAD, as a useful paradigm for the diversity of vascular disease.

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2. THE DIVERSE ETIOLOGIES OF PERIPHERAL ARTERIAL DISEASE The term peripheral artery disease can refer to pathology in any noncoronary artery, including carotid, renal, mesenteric, and occasionally subclavian. Most usually, however, the term PAD is taken to mean arterial occlusive disease involving the aorto-iliac and/or infrainguinal arteries. This disease is widely prevalent in Western societies, with about 10–12 million Americans affected (1). The PARTNERS study revealed that at least 25% of individuals over the age of 70 have evidence of PAD as demonstrated by an anklebrachial index (ABI) of less than 0.90 (2). A similar percentage of diabetics or smokers over the age of 55 have a reduced ABI.

2.1. Atherosclerosis The most common cause of PAD is atherosclerosis. Atherosclerosis is accelerated by tobacco use, diabetes, hypertension, dyslipidemia, aging and/or sedentary state (3). The patient with PAD of the lower extremities is typically afflicted by several of these cardiovascular risk factors. In patients with premature PAD (i.e. under the age of 60) or those with PAD out of proportion to their known risk factors, it is not unusual to find elevated plasma levels of homocysteine or lipoprotein (a) contributing to the pathophysiology (4,5). Each of these risk factors is known to induce a characteristic change in endothelial phenotype, which initiates the pathophysiological process (see below). Early lesions of atherosclerosis are known as fatty streaks. On gross inspection, these lesions are slightly raised yellowish blemishes of the luminal surface, typically occurring at bifurcations of large to medium-sized conduit vessels. These lesions are present in childhood, and are presaged by mononuclear infiltrate in the intima of vulnerable arterial regions (6). In Western societies, where there is a high prevalence of dyslipidemia, diabetes, hypertension, and tobacco use, these lesions gradually progress over time, developing into large complex lesions that can impede blood flow through the vessel lumen. Complex lesions typically are characterized by a fibrous cap composed of vascular smooth muscle cells, fibroblasts and extracellular matrix, a lipid-rich core, and variable amounts of mononuclear infiltrate, medial calcification, and neovascularization. By the time the patient is symptomatic, the disease is quite advanced, with diffuse involvement of the aorta, iliac and infrainguinal vessels, and lesions in various stages of development. Nevertheless, hemodynamically significant lesions are often discrete, and typically located at bends or branches, such as the aorto-iliac bifurcation, or the trifurcation of the popliteal artery. Because atherosclerosis is the most common cause of PAD, its pathophysiology and the role of endothelial heterogeneity are discussed in detail below. However, there are two rare causes of PAD that are worthy of brief mention in this context, because their unique distribution is likely a reflection of endothelial heterogeneity.

Endothelial cells in health and disease

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2.2. Buerger’s Disease Buerger’s disease, also known as thromboangiitis obliterans, is a disease of young male smokers that is very different from atherosclerosis in its clinical presentation, anatomic distribution, and pathophysiology (7). The patient typically presents with pain and ulceration of the toes, due to occlusive disease of the digital arteries. With more progressive disease, the infrapopliteal arteries may become involved, and the patient may develop ischemic pain in the foot. The disease is strikingly different from atherosclerosis in its anatomic distribution. Whereas atherosclerosis involves the large and medium-sized conduit arteries of the lower extremity, Buerger’s disease rarely affects vessels above the knee. In these patients, the lower extremity vessels proximal to the popliteal artery are typically pristine, even in the presence of severe obliterative disease of the infrapopliteal and digital arteries (Fig. 1). Furthermore, whereas atherosclerosis rarely causes hemodynamically significant lesions in the upper extremity (subclavian artery), Buerger’s disease frequently obliterates digita