Mechanisms of Proliferative and Obliterative Vascular Diseases
Insights from the Pulmonary and Systemic Circulations

ALFRED P. FISHMAN, MARK C. FISHMAN, BRUCE A. FREEMAN, MICHAEL A. GIMBRONE, MARLENE RABINOVITCH, DAVID ROBINSON, and DOROTHY BERLIN GAIL

Department of Rehabilitation Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Cardiovascular Research Center and the Vascular Research Division, Harvard University, Boston, Massachusetts; Department of Anesthesiology, University of Alabama, Birmingham, Alabama; Division of Cardiovascular Research, University of Toronto, Toronto, Canada; and Division of Heart and Vascular Disease and Division of Lung Disease, National Heart, Lung, and Blood Institute, Bethesda, Maryland

	INTRODUCTION

TOP INTRODUCTION DEVELOPMENTAL GENETICS OF VESSEL PATHOPHYSIOLOGIC STIMULI IN INFLAMMATORY RESPONSE TO INJURY BRINGING VASCULAR BIOLOGY INTO RECOMMENDATIONS

Pulmonary vascular biologists and systemic vascular biologists have tended to orient their respective researches in different ways. This dichotomy is readily understood in light of their predominantly different interests. Those working on the systemic circulation have traditionally focused on the pathogenesis of atherosclerosis, a common disease that affects large arteries and in which lipid metabolism features prominently; more recently, systemic vascular biologists have explored mechanisms involved in restenosis after angioplasty. Oppositely, those engaged in research on the pulmonary circulation have often centered their attention on primary pulmonary hypertension, a rare disease of small muscular arteries and arterioles in which disturbances in lipid metabolism are not implicated.

These divergent pathways converge on proliferative and obliterative disease of the affected vessels. Moreover, the two pathways include certain similar elements. Some are involved in the interactions between the vessel wall and components of the perfusing blood. Others entail interplay between the endothelial lining and the smooth muscle of the media. In both, although perpetuating and aggravating mechanisms have been identified, the original injury/insult that initiates the proliferative process remains speculative. Finally, similar cytokines, enzymes, mediators, receptors, inflammatory cells and products, and cellular reactions to injury seem to be involved in both.

On September 2 and 3, 1997, a group of investigators who approach proliferative and obliterative diseases of arteries from different vantages met on the campus of the National Institutes of Health (NIH) to compare notes. This report draws on presentations of the participants, using material they provided, and the recommendations as synthesized by the organizers and discussants.

	DEVELOPMENTAL GENETICS OF VESSEL PATTERNING AND PERTURBATION

TOP INTRODUCTION DEVELOPMENTAL GENETICS OF VESSEL PATHOPHYSIOLOGIC STIMULI IN INFLAMMATORY RESPONSE TO INJURY BRINGING VASCULAR BIOLOGY INTO RECOMMENDATIONS

Proliferative and obliterative vascular diseases are characterized by disruption of the normal patterning of vessels and of their cellular constituents. The pathophysiologic pathways that lead to these end-stages are rarely discernible. Because they may trace the normal assembly of vessels during development, developmental biology, which combines embryologic analyses of cellular origins, lineage, and cell-cell interactions with genetic studies, holds the prospect of revealing the role of individual genes in pathogenesis.

How developmental biology can shed light on the underlying process can be illustrated by reference to the hypoxic pulmonary arterial pressor response. For example, simple genetic organisms can be used to study the influence of low oxygen levels along three lines: (1) the morphogenetic structures which are generated as units, or "modules" of vessel assembly and which may be perturbed by injury; (2) the genes responsible for decisions about cell fate and patterning; and (3) the biochemical hierarchy within which the genes work. Another promising avenue for exploration is directed at uncovering how the molecular nature of the signaling systems is conserved in metazoan evolution. In Drosophila, oxygen is delivered through its tracheal system, a network which branches internally from surface ectoderm. The generation of the trachea begins with the trachealess gene, a transcription factor closely related to vertebrate hypoxia inducible factor 1 (HIF-1); this transcription factor regulates oxygen-sensitive genes. Branching depends on branchless, a fibroblast growth factor (FGF)- like molecule, which surrounds the growing trachea and its tyrosine kinase receptor on the tracheal cells.

However, Drosophila lack endothelium. The simplest genetic system with endothelium is zebrafish, in which endothelial assembly can be visualized directly because the embryos are transparent. Genetic screens of zebrafish have revealed single genes that are needed for the growth and development of components of the vascular tree, such as generation of the hemangioblast, side-to-side fusion of the dorsal aortae, angiogenesis, and determining which vessels become arteries or veins.

Evolutionary conservation is also evidenced by the pathways that generate smooth muscle. For example, in Drosophila, a single myocyte enhancer factor-2 (MEF2) gene is needed to specify muscle cells of all types. Four MEF2 genes divide the role in vertebrates, each critical for the development of particular types of muscle, one of which is smooth muscle. The embryonic lineage of vascular constituents is not understood but is relevant to vascular pathology. For example, there are several populations of vascular smooth muscle cells which are distinguished by cellular structure and growth responsiveness. An important issue will be to determine the lineage relationships of the cells during development, their clonality, and their ability to change fate, for example in response to hypoxia or other stimuli. There are several approaches to this problem. One might be lineage labeling during development.

These genes, which are critical for development, may relate to adult disease of blood vessels. For example, it is conceivable that insufficiency of a developmental gene can predispose to vascular injury later in life. Oppositely, it may be possible to take advantage of genetic pathways involved in vascular assembly to identify targets for therapy.

	PATHOPHYSIOLOGIC STIMULI IN PROLIFERATIVE AND OBLITERATIVE VASCULAR DISEASES

TOP INTRODUCTION DEVELOPMENTAL GENETICS OF VESSEL PATHOPHYSIOLOGIC STIMULI IN INFLAMMATORY RESPONSE TO INJURY BRINGING VASCULAR BIOLOGY INTO RECOMMENDATIONS

The endothelium plays a pivotal role in considerations of proliferative and obliterative disease. A central premise of modern vascular biology is that the endothelial lining is a dynamically mutable interface. This interface is locally responsive to various stimuli originating from the circulating, emigrating cells, and/or neighboring tissues and, thus, can actively participate in the physiologic adaptation or pathophysiologic dysfunction of a given region of the vasculature. From a teleological standpoint, the endothelium appears ideally suited to function in this capacity, given its unique anatomic position between blood and tissues, and its ability to generate an impressive repertoire of biological effectors (e.g., nitric oxide, eicosanoids, cytokines, growth stimulators and inhibitors, vasoactive peptides, pro- and anticoagulants, and fibrinolytic factors).

Imbalances in the production and/or interactions of these diverse mediators appear to influence various vital functions of the endotheliumin particular, its role as a nonthrombogenic container for blood, and antithrombotic, anti-inflammatory, and growth inhibitory activities that help maintain the integrity of the vascular wall, in the face of multiple disease risk factors and injurious stimuli.

Perhaps the best studied examples of activation of vascular endothelium are the effects of proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and bacterial products such as endotoxins. These have provided a robust experimental model which has been exploited at the cellular level (from receptors to signaling pathways to genetic regulatory events) and at the tissue level (in various animal models and diseased human tissues). More recently, a second paradigm of vascular cell activation has emergedthe effects of the biochemical forces generated by pulsatile blood flow (wall shear stresses, pressures, cyclic strains) on endothelial structure and function. The ability of laminar shear stresses to upregulate putative "atheroprotective" endothelial genes and, in general, to influence significantly the functional phenotype of endothelial cells indicates that this paradigm of activation may be playing a significant (patho)physiologic role.

Considerable progress has been made in identifying some of the proximal "sensing" mechanisms involved in biomechanical stimulation of vascular endothelium. These include: ionic channels (K+, Na+) that have both positive and negative effects; cytoskeletal/integrin/focal adhesion complex components; mitogen-activated protein (MAP) kinase and related signaling-cascades; as well as recently discovered members of the Smad family of the transforming growth factor-beta (TGF-)- receptor associated signaling molecules. An important immediate, as well as more delayed, effect of laminar shear stimulation is upregulation of nitric oxide production, with its multifunctional implications for leukocyte adhesion and inflammation, smooth muscle contractility, permeability, and gene regulation. The interplay of humoral and biomechanical stimuli in endothelial nitric oxide production may be central to vascular homeostasis at the local level.

Immune-mediated endothelial injury, as evidenced by the accelerated arteriosclerosis that typically occurs in transplant allografts (e.g., heart, kidney), reflects a complex interplay of humoral endothelial activation and contact-mediated "cytotoxic" T-lymphocyte damage. New insights into the molecular mechanisms of novel animal models (e.g., "humanized severe combined immunodeficiency" [SCID]) mice bearing transplanted human vessel segments) should aid in the development of rational therapeutic interventions for this important clinical problem that limits the long-term effectiveness of organ transplantation.

The permeability barrier afforded by endothelium is a key target in inflammatory and thrombotic processes. Recent studies have begun to elucidate the molecular mechanisms that link input stimuli, such as leukocyte adhesion, thrombin, cytokines, growth factors (e.g., vascular endothelial growth factor/vascular permeability factor [VEGF/VPF]), with lateral junction integrity. In particular, endothelial cytoskeletal contractile function, as regulated by myosin light chain kinase, appears to play a pivotal role. Further studies in this area should yield valuable insights into the treatment of endothelial barrier dysfunctiona key component of pulmonary and systemic vascular diseases.

Chemokines and their receptors represent a current area of intense investigation in vascular biology. Major strides have been made in the structure/function analysis of the two primary chemokine receptor families. These have yielded a basic understanding of their immediate coupling mechanisms and downstream biologic consequences. In addition to the well-established roles of chemokines such as monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in leukocyte recruitment in atherosclerosis and inflammation, the recent discovery that certain chemokine receptors can function as "coreceptors" in human immunodeficiency virus (HIV) infection of T lymphocytes and monocytes provides an exciting example of the potential serendipitous dividends of basic research investment.

	INFLAMMATORY RESPONSE TO INJURY

TOP INTRODUCTION DEVELOPMENTAL GENETICS OF VESSEL PATHOPHYSIOLOGIC STIMULI IN INFLAMMATORY RESPONSE TO INJURY BRINGING VASCULAR BIOLOGY INTO RECOMMENDATIONS

Many proliferative and obliterative vascular diseases are characterized by an underlying inflammatory process. Examples include atherosclerosis, primary pulmonary hypertension, restenosis after angioplasty, and transplant vasculopathy. The pathogenesis of these lesions includes proliferation of intimal smooth muscle cells and a variable increase in the numbers of macrophages and lymphocytes. This fibroproliferative response can be attributed to abnormalities in the signal transduction events that mediate inflammation. The net result is release of chemokines and generation of reactive oxygen and/or nitrogen species which in turn lead to cell proliferation, aberrant vascular plasticity, and altered antithrombotic properties.

Although the underlying mechanisms are complex, abnormalities in ^·NO metabolism may provide a mechanism for progression of vascular lesions and loss of endothelial-dependent vascular function. Indeed, inflammation or oxidative stress stimulates gene expression of inducible nitric oxide synthase (iNOS) which results in increased local production of ^·NO. ^·NO is now known to provide a variety of salutary signals to the cell that are important for regulation of normal function and tissue viability. These effects of ^·NO extend far beyond the well-recognized cyclic guanosine monophosphate (cGMP)-mediated vessel relaxation and platelet aggregation, and include profound effects on reactions involving oxygen-derived radicals and associated proinflammatory signal transduction mechanisms.

Oxygen-derived radicals play an important role in regulating gene expression, inflammatory reactions, and vascular tone associated with proliferative and obliterative vascular diseases. These radicals include both superoxide anion as well as lipid hydroperoxides derived predominantly from pathways directed to eicosanoid synthesis. ^·NO regulates the tissue concentration of oxygen-derived reactive species through chemical reaction. An example is the interaction of ^·NO with superoxide which consumes both reactants to produce peroxynitrite. In the absence of ^·NO, evaluated superoxide concentrations could generate and react with H₂O₂ to form the highly reactive and toxic hydroxyl radical. Scavenging of superoxide by ^·NO decreases the tissue concentration of both superoxide and H₂O₂, thereby blunting the rate of hydroxyl radical generation.

While ^·NO regulates superoxide concentration, the converse is also relevant, namely that oxygen-derived radicals regulate ^·NO concentration. Thus, increased superoxide will decrease ^·NO concentration, thereby blunting ^·NO signaling and possibly resulting in impaired vascular relaxation. As an example of this effect, administration of superoxide dismutase or of allopurinol, an inhibitor of xanthine oxidase activity, has an antihypertensive effect in many forms of vascular disease, presumably by decreasing tissue levels of superoxide thereby preventing the removal of ^·NO through reaction with superoxide. It is worth noting that the production of peroxynitrite has its own consequences. Peroxynitrite has the capacity to nitrate proteins and lipids which serve as a footprint for ^·NO-mediated oxidative reactions. The physiologic effects of these nitration reactions and their role in vascular injury are currently under investigation.

In addition to the acute effects of ^·NO on blood vessels and cells, ^·NO-associated oxidative reactions can have delayed manifestations through their profound effects on gene regulation. Gene regulation by ^·NO is mediated through the DNA binding activity of redox-regulated gene transcription factors such as NF-B and AP-1. These transcription factors regulate expression in vascular cells of proinflammatory molecules such as IL-1, tumor necrosis factor-alpha (TNF-), MCP-1 and -2, intercellular adhesion molecule-1, and others. ^·NO, by interacting with superoxide, can blunt the upregulation of these genes that normally occurs during oxidative stress. There are several interesting clinical examples where this activity may be important in inflammatory vascular diseases. For example, upregulation of iNOS has been demonstrated transiently with balloon dilatation of an artery and, at a more sustained level, during transplant vasculopathy and at sites of aortic allograft. Of potential therapeutic relevance, preclinical studies show that adenoviral vector-mediated gene transfer of iNOS inhibits intimal hyperplasia in allografts or following balloon injury. On the toxicology side, the immunosuppressive agent cyclosporine inhibits iNOS expression and accelerates intimal hyperplasia. This result is compatible with a role for ^·NO in preventing the superoxide-mediated upregulation of transcription factors associated with cell proliferation. These varied observations provide evidence for the critical regulatory action of ^·NO toward vasoproliferative processes.

The discussion so far has described the interaction of reactive oxygen species and ^·NO in regulating tissue oxidant concentrations and associated effects of vascular tone and cellular proliferation. In this context, it is also important to consider the family of latent matrix metalloproteinases, another component of vascular remodeling mechanisms associated with inflammation. These enzymes are stored in an inactive form in vascular smooth muscle cells and can be activated by reactive oxygen and nitrogen species produced during inflammation. Activation of these proteinases can lead to degradation of matrix associated with advanced atherosclerotic plaques resulting in instability of the plaques and possible precipitation of clinical cardiovascular events. Because the metalloproteinases can be activated by reduced oxygen species, regulation of the tissue concentration of oxidants by ^·NO or other means may play an important role in the pathogenesis of atherosclerotic-mediated tissue injury. These observations infer that administration of scavengers for oxygen-derived radicals may promote stability of atherosclerotic plaques and curb the vascular inflammation associated with atherosclerosis.

Based on our understanding of the basic mechanisms of inflammatory injury in vascular lesions, several potential strategies for limiting vascular inflammatory injury provide exciting prospects for clinical benefit. As mentioned previously, transfection of iNOS with nitric oxide synthase (NOS) overexpression in two different models has had profound effects in limiting occlusive proliferative disease. In the first, iNOS overexpression dramatically inhibits intimal hyperplasia following balloon injury in rats or pigs. In the second, iNOS overexpression completely inhibited intimal hyperplasia in a rat model of transplant vasculopathy. As described earlier, the iNOS effect may be mediated through ^·NO effects on oxygen radical-mediated signaling, smooth muscle proliferation, and regrowth of endothelium. In addition, hypoxic pulmonary vasoconstriction has been inhibited by transfection with either the endothelial or the inducible form of NOS.

Additional studies are currently being conducted with genes that influence other elements of the inflammatory cascade. Examples include cyclooxygenase-1, prostacyclin synthase, and both extracellular and cytoplasmic forms of superoxide dismutase. As an interesting result, transfection of stem cells resulting in stable expression of IL-1 receptor antagonist and the soluble TNF receptor can modulate the vascular and inflammatory response to bacterial lipopolysaccharide. It seems likely that numerous additional targets for gene therapy will become apparent as new information appears concerning modulation and mediators for the vascular inflammatory response.

Although gene therapy would appear to hold greater promise, several major problems must be resolved before this approach is feasible for clinical application. A principal dilemma is that the intrinsic inflammatory response may be triggered by gene therapy vectors acting synergistically with preexisting inflammation. Further, preexisting inflammation may block DNA translocation or messenger RNA (mRNA) transcription, thereby limiting efficacy of gene transfer. In essence, a paramount issue is the continuing search for effective but nontoxic vectors. But these are potentially soluble problems and the prospect of gene therapy remains a beacon as a we approach the 21st century.

	BRINGING VASCULAR BIOLOGY INTO THE CLINICAL ARENA

TOP INTRODUCTION DEVELOPMENTAL GENETICS OF VESSEL PATHOPHYSIOLOGIC STIMULI IN INFLAMMATORY RESPONSE TO INJURY BRINGING VASCULAR BIOLOGY INTO RECOMMENDATIONS

Research on the pathogenesis of proliferative and obliterative disease has suggested possibilities for therapeutic interventions at critical junctions in the proliferative process. Experiments in rats suggest that increased activity of an endogenous vascular elastase (EVE) may be pivotal in the development and progression of vascular changes associated with pulmonary hypertension. This enzyme is a serine proteinase of 20 kD, the natural inhibitor of which appears to be elafin. Cell culture studies have shown that apoliprotein A1 might induce this elastase activity in smooth muscle cells by a process which involves induction of tyrosine kinase activity, extracellular signal-related kinase-1 (ERK1) phosphorylation, and induction of nuclear expression of a transcription factor AML1.

The structural changes in the arterial wall evoked by EVE relate to the ability of the enzyme to release mitogenic growth factors (such as FGF) from the extracellular matrix and, either directly or via activation of matrix metalloproteinases (MMP), induce 3-integrin-dependent production of the glycoprotein tenascin. This glycoprotein can cluster 3-integrin receptors and induce tyrosine kinase activity and the phosphorylation of a growth factor receptor upon its ligation. In organ culture, withdrawal of tenascin by elastase or MMP inhibitors induces smooth muscle cell apoptosis and regression of hypertrophied arteries in organ culture. This strategy remains to be tested in whole animals.

Smooth muscle cell migration is associated with upregulation of fibronectin by a post-transcriptional mechanism that may be induced by elastin peptides. The post-transcriptional mechanism involves regulation of the mRNA by a microtubule-associated protein, LC3. One possible approach to retarding the migration of smooth muscle cells that accompanies neointimal formation may be the sequestration of LC3, using in vivo gene transfer techniques.

The Marfan syndrome, which is associated with one or more mutations in the fibrillin-1 gene, was cited as an example of how the discovery of a genetic mutation might direct therapy. Targeted disruption of fibrillin-1 in mice homogeneous for the gene has been used experimentally to induce the "Marfan mouse" in which the vascular lesions included calcification of the media, cellular hyperplasia of the intima and adventitia, and vasculitis. Strategies have been developed to enable destruction of abnormal gene and replacement of the abnormal gene by a normal gene and gene product.

A search currently under way for the gene mutation that is believed to underlie the development of primary pulmonary hypertension involves families with large numbers of affected individuals. A single dominant gene seems to be involved. Women are affected twice as frequently as men; once the disease is established, outcome is the same in both sexes. Genetic anticipation in these families suggests trinucleotide repeat expansion. In six families containing affected individuals, a microsatellite marker has identified linkage to a 30-megabase region of chromosome 2q31 with a logarithm of the odds (LOD) score greater than 7. As yet, however, candidate genes which segregate in that locus do not appear to be mutated.

Current therapeutic interventions center on the use of pulmonary vasodilators. Experience with these agents in children is much more limited than in adults. However, evidence is mounting that vasodilator therapy in children improves both quality of life and survival. As in adults, outcome is better in those who undergo pulmonary vasodilatation during acute testing with a pulmonary vasodilator; vasodilatation is manifest by a decrease in pulmonary vascular resistance. In these children, calcium channel-blockers, taken orally, improve outcome. For those who fail to respond to acute testing, continuous infusion of epoprostenol (prostacyclin) often improves survival and the quality of life. The continuous infusion of epoprostenol may serve either as a bridge or even, in some patients, as an alternative to lung or heart-lung transplantation.

Appetite suppressants have been implicated in the pathogenesis of pulmonary hypertension that mimics, both clinically and pathologically, primary pulmonary hypertension. Inordinate concentrations of circulating serotonin have been involved in the pathogenesis of the pulmonary vascular lesions. The fawn hooded rat is being explored for its relevance to the clinical disorder because high levels of serotonin are associated in this species with pulmonary hypertension. Humans with primary pulmonary hypertension have also been reported to have high levels of circulating serotonin. However, it seems likely that some factor other than serotonin sets the stage for the development of primary pulmonary hypertension.

	RECOMMENDATIONS

TOP INTRODUCTION DEVELOPMENTAL GENETICS OF VESSEL PATHOPHYSIOLOGIC STIMULI IN INFLAMMATORY RESPONSE TO INJURY BRINGING VASCULAR BIOLOGY INTO RECOMMENDATIONS

We have derived a series of recommendations from the above considerations of the pathogenesis of proliferative and obliterative vascular diseases. These can be categorized as: (1) the developmental genetics of vessel patterning and perturbation, (2) the pathophysiologic stimuli in proliferative and obliterative vascular disease, (3) the inflammatory response to injury, and (4) applying the lessons of vascular biology in the clinical arena.

Developmental Genetics

1. The molecular pathways that fashion normal vessels during development. Insights along this line may shed light on how normal branching patterns and patency are established, and how endothelial and smooth muscle cells differentiate, especially in contact with each other; the insights into normal development may also promote understanding of the processes that go away during anomalous vascular growth and occlusion that culminates in obstructive vascular disease. Model organisms, such as the mouse or zebrafish, afford the opportunity to study these processes by indicating single gene mutations.
2. The genes involved in the vascular response to hypoxia. This stimulus is known to be relevant to the development of certain types of pulmonary vascular hypertrophy in the adult. These studies can take advantage of organisms with defined hypoxic responses. The receptor for oxygen and many components of the cascade are still elusive and may be part of the cascades set into motion during obliterative vascular disease.
3. The lineage and embryonic origin of cells that constitute vessels. This approach may define the regulatory genes involved in the maturation of such cells and indicate whether such genes are normally or abnormally activated during pathologic vascular responses in the adult.

Pathophysiologic Stimuli

1. The cellular and molecular mechanisms that determine endothelial responses to biomechanical stimulation. These include shear stress, pressure fluctuations, and cyclic strains. Among the areas to be explored are definition of the most significant genes regulated by these various biomechanical forces from the standpoint of disease pathogenesis or disease resistance and their patterns of expression in human vascular tissues in health and disease.
2. Signal transduction mechanisms in vascular endothelium in health and disease. A key question along this line is whether there are unique aspects of pulmonary versus systemic endothelium, especially with respect to signal transduction pathways for pathophysiologic stimuli.
3. The effects of environmental toxins (e.g., appetite suppressants) on endothelial function in the pulmonary versus systemic circulations.
4. The role of chemokines and their receptors in vascular disease. These diseases include atherosclerosis (especially MCP-1), transplant-associated arteriosclerosis, allergic airway hyperresponsiveness, and granulomatous lung disease.
5. The role of cytokines, growth factors, and cytoskeletal elements in endothelial barrier function and disruption. The pathways that regulate interactions between junctional proteins and the cytoskeleton need clarification.
6. The various roles played by hypoxia in physiologic and pathophysiologic processes. Among these are vascular remodeling, gene expression, intracellular signaling, and vascular development.
7. The ideal vectors and routes for delivery of key anti-inflammatory genes to the vasculature.
8. The key pharmacologic targets for treating proliferative and obliterative pulmonary vascular diseases by applying the concepts and lessons of genetics to the pathologic process of remodeling that follows injury to vessels.

Inflammatory Response to Injury

1. The events involved in signal transduction when the vessel wall is stimulated by an injury that prompts alterations in the production and expression of inflammatory mediators. Among such events are hyperlipidemia, mechanical forces (shear and pressure), and xenobiotics (viruses and environmental toxins).
2. The reactive species responsible for mediating redox-sensitive gene expression of inflammatory molecules along with the chemical reactions that affect specific target molecules.
3. The interdependence of chemokine growth factor (e.g., basic fibroblast growth factor, platelet-derived growth factor) and reactive species in their control of vascular cell remodeling (e.g., cell proliferation, apoptosis, and necrosis). In particular, how cell-cell interactions between migration inflammatory cells, smooth muscle cells, and endothelium influence these processes.
4. The molecular mechanisms underlying the regulation of vascular cell function by NOS-derived products and how they are influenced by local oxygen tension and scavengers of reactive species.
5. Identifying key pharmacologic targets for treating proliferative and obliterative pulmonary vascular diseases by applying the concepts of molecular genetics to the pathologic process of remodeling that follows injury to vessels.

Bringing Vascular Biology to the Clinical Area

1. Application of cellular and molecular approaches to the study of functional alterations in endothelial cells, smooth muscle cells, and fibroblasts and their interactions with matrix components, growth factors, proteolytic enzymes, and circulating blood cells.
2. Genetic studies of familial primary pulmonary hypertension. Efforts should continue to map the locus of familial primary pulmonary hypertension and to screen for mutations in genes that segregate in this region. Experimental models, involving mutated genes for familial pulmonary hypertension, should be developed.
3. Continuing research on pulmonary vasodilators and antiplatelet agents, particularly on alternative methods for administering prostacyclin, its analogs and similar compounds.
4. Animal models to investigate how the continuous infusion of prostacyclin relieves primary pulmonary hypertension in individuals who do not manifest acute vasodilatation in response to this agent.
5. The role of the immune system in vascular diseases is not understood. Among the possible areas for investigation are the in vitro responses of lymphocytes to allogenic vessel wall cells from the pulmonary and systemic circulations. If differences do exist, it is important to determine why lung transplants do not develop graft arteriosclerosis with the same frequency as do other vascular allografts. Also awaiting clarification are the contributions to immune-mediated vascular lesions to other forms of vascular disease, such as to the complications of atherosclerosis.

	Footnotes

Correspondence and requests for reprints should be addressed to Dorothy Berlin Gail, Ph.D., Director, Lung Biology and Disease Program, Division of Lung Diseases, National Heart, Lung, and Blood Institute, 2 Rockledge Centre, Suite 10018, 6701 Rockledge Drive, MSC 7952, Bethesda, MD 20892.

Acknowledgments: The authors gratefully acknowledge contributions to the manuscript of Dr. Timothy Billiar and Dr. Aron Fisher.