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Blood, Vol. 91 No. 10 (May 15), 1998: pp. 3527-3561

REVIEW ARTICLE

Endothelial Cells in Physiology and in the Pathophysiology of Vascular Disorders

By Douglas B. Cines, Eleanor S. Pollak, Clayton A. Buck, Joseph Loscalzo, Guy A. Zimmerman, Rodger P. McEver, Jordan S. Pober, Timothy M. Wick, Barbara A. Konkle, Bradford S. Schwartz, Elliot S. Barnathan, Keith R. McCrae, Bruce A. Hug, Ann-Marie Schmidt, and David M. Stern

From the Department of Pathology and Laboratory Medicine and Department of Medicine, University of Pennsylvania, Philadelphia, PA; the Wistar Institute, Philadelphia, PA; the Department of Medicine, Boston University School of Medicine, Boston, MA; the Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT; the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK; the Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT; the School of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA; the Cardeza Foundation, Jefferson Medical College of Thomas Jefferson University School of Medicine, Philadelphia, PA; the Department of Medicine, University of Wisconsin School of Medicine, Madison, WI; the Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA; and the Departments of Physiology and Cell Biophysics, Columbia University College of Physicians and Scientists, New York, NY.

    PART I

THE ENDOTHELIUM has long been viewed as an inert cellophane-like membrane that lines the circulatory system with its primary essential function being the maintenance of vessel wall permeability. Shortly after the first description of circulating blood by William Harvey in 1628, the existence of a network of vessels arose from studies of Malphigi, who described the physical separation between blood and tissue.1 In the 1800s, von Reckingausen established that vessels were not merely tunnels bored through tissues but were lined by cells. The strength of Starling's experiments and his law of capillary exchange proposed in 1896 served to solidify the belief that the endothelium was principally a selective but static physical barrier, not withstanding Heidenhahn's description in 1891 of the endothelium as an active secretory cell system. However, electron microscopic studies of the vessel wall by Palade in 1953 and physiological studies by Gowan in 1959 describing the interaction between lymphocytes and endothelium of postcapillary venules stimulated numerous subsequent studies that led to the current view of the endothelium as a dynamic, heterogeneous, disseminated organ that possesses vital secretory, synthetic, metabolic, and immunologic functions.1

The endothelial cell (EC) surface in an adult human is composed of approximately 1 to 6 × 1013 cells, weighs approximately 1 kg, and covers a surface area of approximately 1 to 7 m2.2 ECs line vessels in every organ system and regulate the flow of nutrient substances, diverse biologically active molecules, and the blood cells themselves. This gate-keeping role of endothelium is effected through the presence of membrane-bound receptors for numerous molecules including proteins (eg, growth factors, coagulant, and anticoagulant proteins), lipid transporting particle (eg, low-density lipoprotein [LDL]), metabolites (eg, nitrous oxide and serotonin), and hormones (eg, endothelin-1), as well as through specific junctional proteins and receptors that govern cell-cell and cell-matrix interactions.

The endothelium also plays a pivotal role in regulating blood flow. In part, this results from the capacity of quiescent ECs to generate an active antithrombotic surface that facilitates transit of plasma and cellular constituents throughout the vasculature. Perturbations, such as those that may occur at sites of inflammation or high hydrodynamic shear stress, disrupt these activities and induce ECs to create a prothrombotic and antifibrinolytic microenvironment. Blood flow is also regulated, in part, through secretion and uptake of vasoactive substances by the endothelium that act in a paracrine manner to constrict and dilate specific vascular beds in response to stimuli such as endotoxin.

Detailed study of endothelial function first became feasible with the development in the 1970s of techniques to culture ECs in vitro.3-5 Limitations of this approach have become apparent recently with the realization that cell culture perturbs ECs from their quiescent in vivo state (0.1% replications per day) to an activated phenotype (1% to 10% replications per day) with loss of specialized functions associated with diverse vessels and organ systems. More complex analytic systems now exist that incorporate changes in EC properties imparted by plasma and cellular blood elements, by rheologic factors, and by cell-cell interactions that occur within the vessel wall. Genetic recombination studies in mice are likely to advance understanding of ECs in both their physiologic and pathologic roles in thrombosis, atherosclerosis, tumor metastasis, and organ rejection.

The purpose of this review is to provide a broad overview of EC participation in several biological processes judged to be relevant to clinical hematologists and investigators of vascular biology. Part I will principally examine the known physiologic roles of the endothelium, whereas Part II will discuss the interactions between ECs and blood cells and emphasize the contribution of the EC to the pathogenesis of specific diseases. Because of the introductory nature of this review, many topics and important contributions have been omitted, including the involvement of ECs in hematopoiesis, neuroendocrinology, cell aging, cellular integrins and matrix interactions, vascular permeability, lipid metabolism, the lymphatic vasculature, and the endothelium as a target for gene therapy. We hope that the abbreviated bibliographies will serve as an introduction to readers who wish to further investigate EC biology.

    VASCULOGENESIS AND ANGIOGENESIS

Overview of early vascular development.   Recently developed techniques that permit alteration of genomic sequences and manipulation of developing embryonic tissues have provided important insights into molecular and genetic elements that regulate vascular development.6 These studies show that the cardiovasculature is the first system to form in the gastrulating embryo. The de novo organization of ECs into vessels in the absence of any pre-existing vascular system is referred to as vasculogenesis and only occurs in the early embryo. Angiogenesis, the continued expansion of the vascular tree as a result of ECs sprouting from existing vessels, occurs in avascular regions of the embryo and is repeated many times in the mature animal, most commonly during wound healing and tumor metastasis7 (Fig 1). It remains uncertain how the pattern of the vascular tree is established or which factors govern the site of sprouting or the route taken by migrating ECs during angiogenic expansion.


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Fig 1. The formation of new vessels during vasculogenesis and angiogenesis. Vasculogenesis, the de novo organization of ECs into vessels in the absence of preexisting vascular structures, takes place during embryogenesis in the blood islands of the yolk sac (pictured) and in the embryo through expression of growth factors, in particular fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). The tyrosine receptor kinases, VEGFR-1 (flk-1) and VEGFR-2 (flt-1), are expressed on mesenchymal cells and newly formed ECs, respectively, and are essential for the generation and proliferation of new ECs and the formation of tubal EC structures. Angiogenesis, the continued expansion of the vascular tree, is mediated through the expression of additional tyrosine kinase receptors, tie-2 (tek), which binds to Ang1 and Ang2 (angiopoietins), resulting in the maintenance of mature vessels, the development of new vessels, and the regression of formed vessels in processes dependent on a combination of factors, most notably the presence or absence of growth factors.

Origin of the vascular endothelium.   Molecular events involved in EC differentiation from the early mesoderm remain uncertain. Vascular and hematopoietic tissues develop together, beginning shortly after implantation with the formation of blood islands within the primitive yolk sac8 (Fig 1) composed of two cell types: (1) angioblasts that form the outer layer of ECs encasing the blood island; and (2) hematopoietic stem cells, in the inner cluster, from which the first embryonic blood cells develop. Angioblasts committed to EC differentiation are found primarily in embryonic mesoderm,9 whereas the early epiblast also contains a subpopulation of hematopoietic stem cells.10 Recently, angioblasts have been identified in the adult as well.11 The endoderm is the initial stimulus for angioblast formation.8,9 Within the embryo proper, the first angioblasts arise from the lateral mesodermal plate and cardiac crescent8,9; some cells migrate into the forming brain, whereas others assemble into the endocardium of the early heart tube. Other angioblasts form a plexus of ECs at the base of the primitive heart tube that assemble into the vitelline vessels, allowing blood cells from the yolk sac to circulate within the body of the embryo.8 The vasculature of the viscera is formed from ECs that differentiate directly from the surrounding mesenchyme incorporated into the angiogenic extensions of invading vessels.12 For example, as the airway of the developing lung expands, endodermally derived cells of the branching airway provide directional queues for advancing branchial arteries and induce formation of angioblasts that become part of the pulmonary vasculature.13 Vasculogenic activity of early organ rudiments was shown through engraftment experiments using pieces of early quail lung and chick embryos. These experiments lead to the hypothesis that endoderm, but not ectoderm, induces vasculogenesis but that both endoderm and ectoderm can support angiogenesis14; recent experiments suggest additional sources of endothelial growth factors may exist.15

Genetic programs regulating EC differentiation and early vascular development.   The best insight into molecular events required to initiate and maintain vascular development has come from detailed analyses of mouse embryos in which the genes for specific polypeptide growth factors or their transmembrane receptor tyrosine kinases (RTKs) have been inactivated. Such experiments show that initiation of vascular development requires both basic fibroblast growth factor (beta FGF) and vascular EC growth factors (VEGF; see Beck and D'Amore16 for a detailed discussion of growth factors and vascular development). Three alternatively spliced isoforms of VEGFs, members of the platelet-derived growth factor family (VEGF, VEGF-B, and VEGF-C17), interact with specific tyrosine kinase receptors. The growth factor-receptor interactions include VEGFR-1 (also known as flt-1 or fms-like tyrosine kinase-1) with VEGF and a related placenta growth factor (PlGF); VEGFR-2 (known alternatively as flk-1, fetal liver kinase-1; or Kdr, kinase-inserted domain containing receptor) with both VEGF and VEGF-C; and VEGFR-3 (originally designated flk-4) with VEGF-C. All VEGFs stimulate receptor autophosphorylation and EC replication and migration. The crucial role of this ligand in early vasculogenesis is demonstrated by the fact that loss of the VEGF gene results in embryonic death. Subsequent assembly of ECs into vessels requires activation of VEGFR-1 on the surface of the newly differentiated cells.18,19 The decision for a vessel to become a vein or an artery appears to be under the control of yet another growth factor, VEGFR-3,20 that is expressed later in development only on ECs that will become veins or lymphatic vessels.

Expansion of the vascular tree, continued endocardial and ventricular development, and formation of the vascular wall is controlled by two members of a second family of RTKs and their ligands,19 tie-1 (tyrosine kinase with Ig and epidermal growth factor homology domains) and tie-2 or tek (tunica interna EC kinase). Two ligands, termed angiopoietin-1 and angiopoietin-2,21,22 are specific for tie-2 and are synthesized by cells surrounding the developing vessels. Ligand binding results in autophosphorylation of tyrosine residues in the intracellular domain of tie-2, but does not lead to EC replication or tube formation, as is the case for other endothelium-associated receptor-ligand interactions. Interestingly, angiopoietin-2 appears to function as an antagonist for angiopoietin-1, blocking its binding to tie-2. Targeted mutations of the genes for either tie-2 or angiopoietin-1 result in embryos with abnormal hearts and vessels with poorly formed walls.23 This has led to the suggestion that angiopoietin-1 acts via its receptor on ECs to stimulate the production of growth factors that, in turn, stimulate the differentiation of surrounding mesenchyme into pericytes or smooth muscle cells required for vessel wall formation.24 This is consistent with the phenotype of a Tie-2 mutation in humans that leads to smooth muscle deficiencies around small vessels and microaneurysms.25 These observations suggest that carefully regulated activity of tie-1 and 2 is required for continued vascular branching and vessel remodeling. Thus, the assembly of the early vascular tree depends on the programmed expression of at least two sets of RTKs and their ligands, one set for EC differentiation and initiation of vessel formation and the other for subsequent branching, establishment of capillary beds, and vessel wall formation.

Although genetic manipulations now possible in the mouse have provided important insights, there is much that we do not know about vascular development. Random genetic mutations introduced into the zebrafish have also generated many surprising and fascinating cardiovascular anomalies.6,26 For example, zebrafish can be induced to develop with hearts that do not contain an endocardium although the remainder of the vascular system appears functional. Thus, it is likely that a combination of genetics and developmental biology, unencumbered by previous assumptions, will continue to show new genes and suggest new paradigms that will advance our understanding of vascular development.

Extracellular matrix and matrix adhesion receptors in vascular development.   The ability of ECs to form capillary-like tubes is regulated not only by specific cytokine/receptor combinations, but also by the extracellular matrix. For example, human umbilical vein ECs (HUVECs) exposed to transforming growth factor-beta (TGF-beta ) grow as a rapidly dividing monolayer if cultured on a flat surface coated with type I collagen.27 However, under similar TGF-beta exposure, but within a type I collagen gel, ECs spontaneously organize into capillary-like tubules and continue to divide. Several kinds of molecules on the EC surface act together to mediate cell-extracellular matrix (ECM) interactions, including proteoglycans as well as proteins. The best studied family of receptors that mediate cell-matrix interactions is the integrins,28 which serve both a tethering and an information transfer function. Integrin-ligand binding triggers cytoskeletal organization at specific sites on the surface membrane to facilitate cell movement or maintain tissue stability. Binding also activates intercellular pathways that can result in either cell replication or programmed cell death.29

Cells express more than one integrin and the combination of integrins expressed during embryonic development is constantly changing, suggesting that specific combinations are required as development proceeds. Experimental results in the developing mouse embryo suggest that functional compensation by integrins can occur during embryogenesis. It is also possible that receptors required for angiogenesis in early development may differ from those required for collateral vessel formation or tumor angiogenesis or that both gene inactivation and the introduction of inhibitory agents have unknown secondary effects. Similarly, mouse embryos continue to develop normally when genes for certain ECM components have been inactivated, whereas inactivation of other genes, such as the abundant fibronectin, results in early embryonic lethality.30 Similarly, knocking out the gene for a fibronectin receptor subunit (alpha 5beta 1) also results in a poorly developed heart and vascular system and early embryonic death.30 Again, other integrins, most likely alpha vbeta 3, appear to compensate for the absence of alpha 5beta 1 during preimplantation development and early gastrulation, but are unable to do so as development proceeds.

Endothelial cell-cell interactions and vessel formation.   Angioblasts and ECs must contact like cells for vessels to sprout and lengthen. Such cell-cell adhesion is mediated by a distinct series of cell surface receptors that includes platelet EC adhesion molecule (PECAM-1),31 a member of the Ig superfamily, and vascular endothelial (VE)-cadherin.8 ECs express two isoforms of PECAM that mediate cell adhesion that differ in their requirement for divalent cations and sulfated proteoglycans. VE-cadherin, also known as cadherin-5, found almost exclusively on ECs, promotes cell-cell adhesion by a calcium-dependent homotypic mechanism,32 ie, in the presence of calcium, VE cadherin from one cell binds to the VE-cadherin expressed on an adjacent cell. As the vessel matures, more classic junctional complexes, such as tight junctions and gap junctions, form depending on the function of the particular vascular bed. Thus, during vascular development, junction formation initially involves rather weak adhesion complexes, likely required for cell-cell recognition, that facilitate the assembly of additional junctional complexes. However, the factors that determine the organ-specific nature of junction formation remain unknown.

Proteinases and vascular development.   Vascular development may be regulated by some of the same factors that are involved in the control of blood clotting and capillary formation. EC movement through the ECM is tightly regulated and requires integrin mediated cell-matrix adhesion complex formation and subsequent disassembly. This involves repetitive cycles of reversible integrin/matrix binding, assembly, and disassembly of cytoskeletal elements as well as matrix degradation restricted to the advancing edge of the moving cell.33 This is accomplished through the organization of active molecular complexes that approximate integrins and integrin ligands with matrix metalloproteinases and plasminogen activators with their respective substrates and inhibitors at sites of cell-matrix interaction.34 It has long been hypothesized that, when ECs are exposed to angiogenic stimuli, plasminogen activation is initiated through the binding of plasminogen and urokinase to their receptors. This leads to the formation of plasmin that activates prometalloproteases, degrades noncollagenous components of the matrix, provides a path for cell migration, and releases peptides that can promote or inhibit continued angiogenesis.35 Two peptides, angiostatin and endostatin,36,37 are currently being evaluated for clinical use in reversing tumor angiogenesis.36 However, much of the dogma regarding the role of plasmin in angiogenesis may have to be revisited in light of recent data indicating that transgenic animals with targeted disruption in the genes for tissue-type plasminogen activator (t-PA), urokinase (u-PA), the urokinase receptor (u-PAR), and plasminogen appear to develop a normal vasculature in the absence of trauma or other stressors (see below).

Perspective.   The induction of embryonic angioblasts to differentiate into ECs, organize into a vascular network, and subsequently populate the specialized vascular bed of an organ results from a complex genetic program, the details of which are only now emerging. This program is not only sensitive to the composition and structure of the ECM but is influenced by cell-cell contact as well as angiogenic and angiostatic growth factors and peptides generated by vascular expansion itself. Many of these same events are recapitulated after injury or as part of an inflammatory response and, if allowed to proceed unchecked (eg, tumor angiogenesis and diabetic retinopathy), can have serious consequences for the organism. Insight into the molecular and genetic programs involved in vascular differentiation may suggest better approaches to minimize ischemic tissue damage, avoid tissue rejection, stimulate wound healing, and inhibit tumor growth. This section has focused on the development of the vascular endothelium; however, the formation of the basement membrane, the induction and differentiation of vascular smooth muscle cells, and the complex process of assembling the elastic lamina are all essential to vessel formation and are under separate regulation. Ultimately, prevention of devastating effects from congenital abnormalities and facilitation of normal vascular function will, in a large part, be influenced by our ability to manipulate molecular mechanisms involved in vascular development.

    EC HETEROGENEITY

Many human vascular diseases are exquisitely restricted to specific types of vessels. For example, the contribution of platelets to the pathogenesis of arterial and venous thrombosis differs as does the susceptibility of these two types of vessels to atherosclerosis. It is also common for vasculitis to show marked predilection for specific arteries, veins, or capillaries or for certain organs. Tumor cells may show similar predilection to metastasize through particular vascular beds.38 Even when systemic risk factors are clearly evident, such as is the case with inherited disorders of lipoprotein metabolism or proteins that control coagulation, there is marked regional variation in disease expression. Furthermore, clinical events such as thromboses are generally episodic and often localized to single vessels. The basis for variation is poorly understood, but may lie, in part, in the heterogeneity of ECs themselves (see Augustin et al2 and McCarthy et al39 for reviews).

To date, appreciation of EC function has been largely based on the behavior of cultured umbilical vein ECs (HUVECs). Indeed, it is remarkable that so many concepts in vascular biology have been predicated on the repertoire of umbilical ECs studied under such potentially unphysiologic in vitro conditions; this is especially true considering their derivation from a type of vessel that rarely, if ever, is affected by the most common human vascular disorders. More recently, there has been greater appreciation that EC heterogeneity may contribute both to the maintenance of adaptive processes and to the development of disorders restricted to specific vascular beds.

EC heterogeneity among and within tissues.   Variation in the appearance of capillary endothelium from different vascular sites has long been recognized and appears well suited to postulated differences in function (Fig 2). For example, the brain and retina are lined by continuous ECs connected by tight junctions that help to maintain the blood-brain barrier; the liver, spleen, and bone marrow sinusoids are lined by discontinuous ECs that allow cellular trafficking between intercellular gaps; while the intestinal villi, endocrine glands, and kidneys are lined by fenestrated ECs that facilitate selective permeability required for efficient absorption, secretion, and filtering (see Dejana32 for review). ECs from diverse tissues are also heterogeneous with respect to their surface phenotype and protein expression. For example, von Willebrand factor (vWF), used commonly as a marker for ECs, is not expressed uniformly on cells from all types of vessels,40,41 the expression of tissue type plasminogen activator is limited in vivo to approximately 3% of vascular ECs,42 and the constitutive expression of u-PA is reportedly confined to renal ECs,43,44 which are also uniquely susceptible to injury by verotoxin.45 Microvascular ECs also differ in their susceptibility to undergo apoptosis induced by plasma from patients with thrombotic thrombocytopenic purpura.46 The induction of tissue factor after infusion of cytokines or endotoxin is similarly restricted to specific vessels,47 among many other examples of heterogeneity at the level of protein expression.


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Fig 2. EC heterogeneity. (A) Electron micrograph showing the junction between two capillary ECs in a guinea pig pancreas (micrographs reprinted with permission from R.F. Bolender, The Journal of Cell Biology, 1974, vol. 61, p. 269). (B) Electron micrograph demonstrating the diversity of ECs from two types of capillaries: (1) vesicular invaginations (arrow) on both luminal and abluminal plasma membrane of a muscle capillary EC; (2) fenestrated capillary from the lamina propria of the colon with thin diaphragms (arrow) covering the plasma membrane pores (micrographs reprinted with permission from E. Weihe, Textbook of Histology, (ed 12), 1994, p. 391, courtesy of Chapman and Hall).

One of the clearest examples of EC heterogeneity lies in the expression of homing receptors involved in cell trafficking. In the mouse, Lu-ECAM-1 (lung-specific EC adhesion molecule) is exclusively expressed by pulmonary postcapillary ECs and some splenic venules,48 whereas Mad-CAM-1 (mucosal addressin cell adhesion molecule-1) is expressed primarily on high endothelial venules in Peyer's patches of the small intestine.49 Microvascular ECs derived from the bone marrow show an affinity for binding megakaryocytes and CD34+ progenitor cells and constitutively secrete hematopoietic stimulating factors such as Kit-ligand, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6 (IL-6), which help control trafficking, proliferation, and hematopoietic lineage-specific differentiation.50 Tumor cells may show clear preferential adhesion to the endothelium of specific organs paralleling their in vivo metastatic propensities (see McCarthy et al39 for review).

Microvascular ECs cultured from the brain, liver, and other organs each express distinct patterns of cell surface markers, protein transporters, and intracellular enzymes.51 These tissue-specific phenotypic differences can be maintained for some time under identical tissue culture conditions (eg, Grau et al52). Distinct subsets of ECs often exist within a single organ. In situ studies of adult human liver show two distinct sinusoidal EC phenotypes: hepatic periportal vessels express PECAM-1 and CD34, whereas sinusoidal intrahepatic ECs do not.53 During the development of the human liver, ECs progress from a phenotype closely resembling adult hematopoietic sinusoidal bone marrow ECs, which supports fetal intrahepatic hematapoiesis, to one resembling adult hepatic sinusoidal ECs, including the expression of the T-lymphocyte marker CD4.54

Environmental and genetic regulation of EC phenotype.   There is extensive evidence to indicate that heterogeneity develops in part as a result of variation in exposure of EC to environmental stimuli, some of which act only over short distances or even require cell-cell contact to effect change. Numerous exogenous factors affect EC phenotype, including mechanical forces, soluble growth promoters and inhibitors, cytokines, plasma lipids and proteins [eg, thrombin, plasmin, antibodies, Lp(a), etc], and contact with circulating and tissue-based cells (eg, smooth muscle cells and pericytes) and with the ECM, microbes, and their soluble products.

There are numerous examples of how the microenvironment can regulate the endothelial phenotype, a phenomenon that has been referred to as transdifferentiation.2 For example, aortic ECs cultured on extracellular matrix derived from the lung are induced to express Lu-ECAM-1,48 whereas the cells develop fenestrae when cultured on matrix derived from kidney-derived MDCK cells.55 Transplantation studies in the chick-quail system illustrate that ECs can take on the characteristics of the tissue into which they are transplanted in vivo,56 whereas other examples show that ECs acquire a different phenotype ex vivo.57 Studies in transgenic mice expressing the Lac Z reporter gene under control of 2,182 bp of the 5' flanking sequence and the first exon and intron of the vWF gene suggest that expression is regulated by signals derived from the local microenvironment that influence pathways specific for particular vascular beds.58

EC heterogeneity can thus arise as a consequence of local concentrations of exogenous effectors or due to intrinsic variations in responsiveness (reviewed in McCarthy et al39). ECs grown on extracts of basement membrane from different organs have been observed to develop preferential adhesivity for tumor cells prone to metastasize to that organ.59 ECs derived from saphenous vein have been reported to synthesize less prostaglandin I2 (PGI2) than those from the internal mammary artery, a finding that may contribute to the rapidity with which pathogenic changes may develop in venous bypass grafts placed under arterial pressure.60 Possible genetic bases for EC diversity have only recently been considered and have not been studied in depth. Microvascular and macrovascular ECs differ in the fastidiousness of their growth, propsensity to form capillary-like structures, synthesis of PGI2, and expression of adhesion receptors for lymphocytes, among other properties (see Ades et al61 for review). Tissue-specific transcription factors or signal transduction molecules responsible for activating and/or de-repressing transcription apparati in a tissue-specific manner are only beginning to be understood. Identification of these control factors will be important in the design of vectors that will enable expression of EC proteins in a tissue-specific manner.

The effects of cell culture.   Only in the past few years has the technology become available that permits in situ study of EC behavior. These studies indicate that the constitutive phenotype of ECs is unstable and their behavior can change rapidly once explanted. Commonly used culture conditions may activate or otherwise alter the endothelial phenoytpe (eg, Grant et al62). There is, as yet, no model for generating the resting EC in vitro. Thus, all the information described in subsequent sections should be considered in the context of the cell source as well as the ex vivo culture conditions, including passaging, the presence/omission of shear forces, and factors released into blood that alter the behavior of the endothelium from that which occurs in healthy blood vessels in vivo. Thus, much may be gained in the future by a more critical consideration of EC heterogeneity both in terms of understanding homeostasis and vascular pathology, as well as in targeting the delivery of gene therapy, antithrombotic agents, and antitumor agents to an anatomically or functionally distinct endothelial region.63

    VASOREGULATION

The endothelium not only provides a structural barrier between the circulation and surrounding tissue, but ECs also secrete mediators that influence vascular hemodynamics in the physiologic state (Table 1). ECs contribute to the regulation of blood pressure and blood flow by releasing vasodilators such as nitric oxide (NO) and prostacyclin (PGI2), as well as vasoconstrictors, including endothelin (ET) and platelet-activating factor (PAF). These chemically diverse compounds are not stored in intracellular granules. Rather, their major biologic effects are regulated by localization of specific receptors on vascular cells, through their rapid metabolism, or at the level of gene transcription. NO is constitutively secreted by ECs, but its production is modulated by a number of exogenous chemical and physical stimuli, whereas the other known mediators (PGI2, ET, and PAF) are synthesized primarily in response to changes in the external environment.

 
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Table 1. Vasoregulatory Substances Synthesized by the Endothelium

NO.   ECs elaborate NO, a heterodiatomic free radical product generated through the oxidation of L-arginine to L-citrulline by NO synthases.64 One isoform, eNOS or the Nos3 gene product, is constitutively active in ECs but is stimulated further by receptor-dependent agonists that increase intracellular calcium and perturb plasma membrane phospholipid asymmetry.65 Receptor-dependent agonists that stimulate eNOS include thrombin, adenosine 5'-diphosphate, bradykinin, substance P, and muscarinic agonists, in addition to shear stress66 and cyclic strain.67 The increase in eNOS activity evoked by shear stress contributes to the phenomenon of flow-mediated vasodilatation, an important autoregulatory mechanism by which blood flow increases in response to exercise.68 This is in part a result of shear-induced transcriptional activation due to the presence of a shear response consensus sequence, GAGACC, in the promoter of the Nos3 gene.69 In addition to eNOS, cytokines have been shown to stimulate bovine microvascular endothelium in culture to express an inducible isoform of NO synthase, iNOS, or the Nos2 gene product.70 EC-derived NO has several important effects on the vasculature. First, NO maintains basal tone by relaxing vascular smooth muscle cells71 through the binding of NO to the heme prosthetic group of guanylyl cyclase. Endothelial-derived NO also inhibits platelet adhesion, activation, secretion, and aggregation and promotes platelet disaggregation, in part through a cyclic GMP-dependent mechanism.72 PGI2, which does not affect platelet adhesion,73 acts synergistically with NO to inhibit other steps in the platelet activation cascade.74 NO also inhibits expression of P-selectin on platelets and, by inhibiting the agonist-dependent increase in intraplatelet calcium,72 suppresses the calcium-sensitive conformational change in the heterodimeric integrin glycoprotein alpha IIbbeta 3 (GP IIb-IIIa) required for fibrinogen binding.75 Additionally, NO appears to promote platelet disaggregation indirectly by impairing the activity of phosphoinositide 3-kinase, which normally supports conformational changes in alpha IIbbeta IIIa, rendering its association with fibrinogen effectively irreversible.76

In addition to these effects on the vasculature, endothelial-derived NO inhibits leukocyte adhesion to the endothelium77,78 and inhibits smooth muscle cell migration79 and proliferation.80 These latter effects serve to limit neointimal proliferation that occurs after vascular injury and, combined with its stimulatory effect on EC migration and proliferation,81 suggest that NO helps to sustain vascular reparative mechanisms.

ECs also produce a less well-characterized compound known as endothelium-derived hyperpolarizing factor (EDHF) that promotes vascular smooth muscle relaxation (see Garland et al82 for review). Muscarinic agonists stimulate ECs to release EDHF, causing a transient hyperpolarization of the cell membrane. It has been proposed that EDHF exerts its vascular effects by activating ATP-sensitive potassium channels, smooth muscle sodium-potassium ATPase, or both,83 but its role in vascular (patho)physiology requires further study.

ET.   Remarkably, ECs produce not only the potent vasodilator NO, but also synthesize endothelin-1 (ET),84 the most potent vasoconstrictor identified to date. Endothelins comprise a family of 21-amino acid peptides produced by many cell types.84 ET-1 is not stored in granules85 but is formed after transcription of the gene encoding preproendothelin-1, the inactive precursor of ET-1, after stimulation by hypoxia, shear stress, and ischemia. ET-1 released from ECs binds to the abundant G-protein-coupled ET-A receptor expressed on vascular smooth muscle cells, which results in an increased intracellular calcium concentration and, in turn, increases vascular smooth muscle cell tone.86 Of interest, this effect of ET-1 persists after the hormone dissociates from its receptor through longer-lived effects on intracellular calcium. NO shortens the duration of these effects by accelerating the restoration of intracellular calcium to basal levels.87 The interplay between ET-1 and ET-A receptors likely contributes to basal vascular tone as well. ET-1 potentiates the vasoconstrictor actions of catecholamines, which, in turn, potentiate the actions of ET-1. In states of endothelial dysfunction, such as atherosclerosis, in which concentrations of bioactive NO are reduced, the relatively unopposed actions of ET-1 promote vasoconstriction and smooth muscle proliferation.88

Prostacyclin (PGI2) and PAF.   The contribution of ECs to the regulation of vasomotor tone is even more finely regulated as evidenced by the production of additional vasoactive compounds such as prostacyclin (PGI2) and PAF. Prostacyclin and PAF factor provide an interesting contrast. Both are intercellular signaling molecules synthesized by stimulated ECs in vitro and in vivo.89 Both are lipids: PGI2 being an eicosanoid and PAF being a phospholipid.90,91 Neither is constitutively present in resting human ECs nor stored within the cell. The synthesis of each is induced rapidly by humoral and mechanical stimuli via discrete, regulated pathways.90,91 Once formed, PGI2 and PAF have relatively short half lives, one of several features that limits the magnitude of their signals and exerts control over their biologic activities.92,93

However, a major difference between the two factors lies in the range over which they exert their effects: PAF acts in a juxtacrine fashion, whereas PGI2 acts as a paracrine signaling molecule. PAF, expressed on the surface of the endothelium, remains cell-associated even in the presence of physiologic concentrations of albumin or other acceptor molecules91 and binds to and activates its receptor on leukocytes,94 fulfilling critical criteria of a juxtacrine signaling molecule. Consistent with this notion, PAF synthesized by cultured human ECs acts in concert with P-selectin (see below) to promote leukocyte adhesion.94

In contrast, PGI2 is rapidly released from ECs,95 although the export mechanism has not been precisely defined. Thus, PAF and PGI2 have spatially differentiated realms of signaling, even though both derive from a common precursor and are synthesized concurrently.91,96 This feature may contribute to differences in their actions at the endothelial interface with the blood: PAF is specialized to signal leukocytes at the cell surface, whereas PGI2 acts primarily in solution to retard platelet aggregation and deposition. Both PGI2 and PAF also elicit autocrine effects on ECs,91,92 which may be important in modulating angiogenesis and controlling the synthesis of EC-derived mediators.

PGI2 was the first endothelial-derived vascular smooth muscle relaxing factor to be identified. PGI2, which was generated locally, and PGI2 or its analogs, which were infused systemically, caused vasodilatation and altered regional blood flow.93 A receptor for PGI2, the IP receptor, is present on vascular smooth muscle as well as on platelets,97 consistent with early experimental observations, indicating that PGI2 acts principally to modulate the function of these two cell types.98 Although IP receptors are present in the arterial vascular wall, PGI2 is not constitutively produced and does not appear to regulate basal systemic vascular tone.99 Rather, PGI2 synthesis is induced at sites of vascular perturbation, where it may regulate vasoconstriction and platelet deposition.66 Because of its effects on blood flow and relevant cell-cell interactions, PGI2 may influence local inflammatory responses as well. An important recent advance has been the identification of prostaglandin H synthase-II (PHS-II), an inducible form of a key enzyme in PGI2 formation providing a mechanism by which the production of PGI2 and other eicosanoids can be sustained in chronic states of inflammation and vascular injury.

The receptor for PAF, the first receptor characterized at a molecular level that recognizes a biologically-active lipid, is a member of the serpentine G-protein-linked family (reviewed in Whatley et al91). Intravascular infusion of PAF causes either vasodilatation or vasoconstriction, depending on the concentration administered, the time, and the specific vascular bed studied.93 Some hemodynamic effects of PAF in vivo are indirect and depend on the generation of eicosanoids or leukotrienes or mediators derived from activated leukocytes or platelets and on cardiac effects.91-93 In shock and other in vivo pathologic states, PAF acts concomitantly or sequentially with other classes of mediators, including leukotrienes and tumor necrosis factor-alpha (TNF-alpha ).90,91,100 As with PGI2, it is unlikely that PAF is a circulating regulator of blood pressure under basal conditions, despite early studies suggesting that PAF-like activity is released from kidneys.92

    THE ROLE OF THE ENDOTHELIUM IN COAGULATION

A crucial physiologic function of the endothelium is to facilitate blood flow by providing an antithrombotic surface that inhibits platelet adhesion and clotting. However, when the endothelium is perturbed by physical forces or by specific chemical factors, the cells undergo programmatic biochemical changes that culminate in their transformation to a prothrombotic surface. A dynamic equilibrium exists between these two states, modulated both at the level of gene transcription and at the level of the intact cell, that often permits the injured endothelium to return to its unperturbed state once the procoagulant stimulus has dissipated (Table 2; see Bombeli et al101 for review). Although the fibrin clots formed as a consequence of procoagulant activity may serve a protective organ function by limiting vascular damage induced by trauma, infection, and inflammation, the loss of anticoagulant activity may predispose to several common thrombotic disorders discussed in the sections that follow.

 
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Table 2. Regulation of Hemostasis and Thrombosis by the Endothelium

Anticoagulant mechanisms.   Control of thrombin generation is a pivotal step in the balance between the natural antithrombotic and the induced procoagulant activities of the endothelium. Thrombin, a serine protease, serves diverse functions in coagulation, including the activation of platelets, several coagulation enzymes, and cofactors. Thrombin also stimulates procoagulant pathways on the ECs themselves. Therefore, it is not surprising that several highly regulated pathways have evolved to constrain the generation and activity of thrombin (see Rosenberg and Rosenberg102 for review), such that little enzyme activity is found in the plasma of healthy individuals.103 The matrix surrounding the endothelium contains heparan sulfate and related glycosaminoglycans (GAGs) that promote the activity of cell/matrix associated antithrombin III (AT-III)104; the subendothelium contains dermatan sulfate, which promotes the antithrombin activity of heparin cofactor II.105 ECs also prevent thrombin formation through the expression of tissue factor pathway inhibitor (TFPI), which binds to factor Xa within the tissue-factor/VIIa/Xa complex (see Broze106 for review). TFPI is released from its EC stores by heparin. TFPI and AT-III both contribute to physiologic hemostasis and can be depleted in acquired thrombotic states.107,108

The endothelium also helps to contain thrombin activity through the expression of thrombomodulin (see Esmon and Fukudome109 for review). Binding of thrombin to TM facilitates the enzyme's ability to activate the anticoagulant protein C. In turn, the activity of activated protein C (APC) is enhanced by its cofactor protein S, which is synthesized by EC, among other cell types.110 ECs also express receptors for APC111 that regulate the activity of this pathway. APC, in turn, promotes the inactivation of activated factors V and VIII. Binding of thrombin to TM also dampens the enzyme's ability to activate platelets, factor V, factor XIII, and fibrinogen and promotes EC fibrinolytic activity (see below). TM also inhibits prothrombinase activity indirectly by binding factor Xa.112 Thrombin bound to TM is rapidly endocytosed and degraded.113 Various inflammatory cytokines downregulate TM gene transcription and accelerate TM internalization114,115 while at the same time promote tissue factor expression (see below). Soluble TM is also shed into plasma and elevated plasma levels have been identified in various disorders associated with EC injury (see Cucurull and Gharavi116 and below).

Procoagulant mechanisms.   The pivotal step in transforming the EC membrane from an anticoagulant to a procoagulant surface is the induction of tissue factor (TF). TF dramatically accelerates factor VIIa-dependent activation of factors X and IX, so it is not surprising that TF is not expressed by unperturbed endothelium, at least in the adult organism.117 Interruption of the gene for TF is associated with impaired vascular development and lethal embryonic bleeding,118-120 but the source and function of TF during development have not been elucidated. Synthesis of TF is induced in vitro by diverse agonists, including thrombin, endotoxin, several cytokines, shear, hypoxia, oxidized lipoproteins, and many other provocations (see Rapaport and Rao121 and Nemerson122 for reviews). Procoagulant activity is accelerated by exposure of anionic phospholipids that may occur as a consequence of apoptosis.108 TF is localized primarily beneath and between cultured ECs,123 although some evidence for expression on the cell surface has been presented.124 TF mRNA and protein levels decline despite continued exposure to agonists, a mechanism that may help contain the extent of fibrin formation. Cells in culture also shed microvesicles containing TF,125 and plasma levels of TF are elevated in patients with disseminated intravascular coagulation,126 although the cellular source has not been established. TF expression is rapidly induced after vascular injury,127 and TF is found associated with ECs within atherosclerotic plaque128,129 and in tumor-derived vessels.130 TF may also contribute to the regulation of angiogenesis and tumor metastases through mechanisms independent of coagulation.131,132 Yet, it has been difficult to demonstrate expression of TF by ECs in vivo even in response to potent provocations where expression was expected, another example of the dissociation between the behavior of these cells in culture and that seen in the whole organism.47

Once ECs expressing TF are exposed to plasma, prothrombinase activity is generated and fibrin is formed on the surface of the cells.133 This implies that ECs express binding sites for factors IX, IXa, X, and Xa; thrombin; and fibrin.134 Yet, the identity and location of most of these binding sites is unknown, as is their role in either physiologic hemostasis or in thrombosis. Factor IX has recently been shown to bind type IV collagen in the EC matrix,135 although its cellular association site promoting assembly of the intrinsic FX activation complex has not been identified. Several candidate FX/Xa binding sites have been reported,136,137 whereas others may be induced as a result of exogenous stimuli.138 ECs also express receptors for proteins of the contact factor pathway,139 but their role in hemostasis is uncertain.

The most thoroughly characterized EC binding site for a coagulation protein is the thrombin receptor, also termed the protease-activated receptor-1 (PAR1). The thrombin receptor is a high-affinity G-protein-coupled protein140 that is activated when a fragment derived from the amino terminus of the protein, formed as a result of cleavage by thrombin, binds to the remaining cell-associated receptor fragment. Binding of thrombin leads to a wide array of changes in expression of prothrombotic and antithrombotic molecules by cultured ECs, including TF, PAI-1, NO, PAF, ET, and PGI2, among others (see Kanthou and Benzakour141 for review) and disruption of cell-cell contacts (see Garcia et al142 for review). Thrombin is also mitogenic for ECs, fibroblasts, and smooth muscle cells and is chemotactic for monocytes.

The seemingly normal phenotype of surviving adult mice with targeted disruptions in the thrombin receptor gene143 was unanticipated and raised questions about the physiologic role of this protein as well as other proteinase activated receptors expressed on ECs.144 The subsequent discovery of two additional protease-activated G-protein-coupled receptors, PAR-2 and PAR-3, helped to explain this observation.145 Both PAR-1 and PAR-2 are present on some human ECs, whereas PAR-1, but not PAR-2, is expressed on human platelets. PAR-3 is expressed by human bone marrow and mouse megakaryocytes, but its expression on ECs has not been established. Notable tissue- and species-specific differences in expression and cellular distribution of PARs have been described,145,146 making it difficult at present to relate the phenotype of the various murine knockouts to human physiology.

ECs also express several receptors for fibrin and specific fibrin degradation products,147 including a 130-kD glycoprotein,148 a tissue transglutaminase,149 and the alpha vbeta 3 integrin, although evidence regarding their expression in vivo is only now emerging.150,151 Binding of fibrin promotes EC adhesion, spreading, proliferation, and migration; cell retraction; leukocyte adhesion; and inhibition of PGI2 synthesis. Cultured ECs also express glycoprotein Ib, which binds vWF secreted constitutively by ECs and, presumably, the ultralarge vWF multimers released from Weibel-Palade bodies in response to a number of agonists (see Wagner and Bonfanti152 for review). Expression of GPIbalpha by ECs is enhanced by TNF-alpha ,153 but whether this glycoprotein participates in the physiologic or pathophysiologic binding of vWF in vivo requires additional study. The alpha vbeta 3 integrin also binds vWF in the EC substratum. In vitro, conditions that decrease alpha vbeta 3 expression (TNF-alpha plus interferon-gamma [IFN-gamma ] or arterial shear stress) increase GPIbalpha expression,154 suggesting that the EC state may affect the availability of adhesion receptors, although these finding require confirmation in vivo.

Undoubtedly, additional receptors for coagulation proteins with distinct functions will be characterized in the future. Genetic and acquired alterations in the structure, expression, and function of these EC receptors may contribute to hitherto unexplained hemorrhagic and thrombotic disorders. However, despite rather extensive study of EC procoagulant function in culture, the extent to which platelet adherence or fibrin formation actually occurs on the surface of the intact endothelium in vivo (as opposed to subendothelial matrix exposed to blood) remains unclear. Perhaps the best indirect evidence comes from animal models of Escherichia coli sepsis and cytokine infusion in which TF- and contact factor-dependent intravascular coagulation and multiorgan ischemic injury occurs in the absence of overt EC disruption.155 Nevertheless, the contribution of endothelium, platelets, monocytes, and other cell types in these models will require further study.

    ECs AND FIBRINOLYSIS

Experiments using cultured ECs have yielded a concept that the endothelial surface is profibrinolytic and thus helps maintain blood in its fluid state.156 However, experiments using animal models have shown this conceptually satisfying hypothesis does not accurately reflect the situation in vivo.42,157 It has also become clear that the contribution of ECs to fibrinolysis varies with their metabolic status (ie, quiescent or activated), their vascular derivation, and the concentration of other hemostatically active molecules in the local plasma milieu.

Plasminogen activators.   Studies with ECs cultured from various tissues have led to the widely held inference that t-PA production and secretion is a property of all ECs.156 However, a few studies have gone largely unnoticed in which PA activity, demonstrated by fibrin zymography, was observed only in association with the adventitia and not with lumenal ECs.157 More recent studies using in situ hybridization and immunohistochemistry have demonstrated t-PA antigen and mRNA only in a distinct subset of quiescent microvascular ECs of both primates and mice.42,158 Hence, contrary to assumptions based on work with cultured ECs, t-PA is associated only with a distinct subpopulation of the microvasculature, even after provocation. In both murine brain and lung, the percentage of microvascular ECs producing t-PA increases markedly upon exposure to pertinent stimuli; however, in both cases, t-PA production remains an exclusive property of microvascular ECs.42 Hence, invoking local EC production of t-PA in large vessels as a mechanism of maintaining blood flow may not adequately describe the in vivo situation.

t-PA production by cultured ECs is regulated by a variety of external stimuli at the level of gene transcription and cellular release.159 Measurements of plasma t-PA levels suggest such regulated production/secretion occurs in vivo as well.160 Intracellular signaling pathways operative in stimulated t-PA release have been described in vitro.161 The mechanisms that control EC t-PA production in vivo are less well understood, but clearly such regulation occurs.162 Humans exhibit higher plasma levels of t-PA after exercise or venous compression, but the cellular source of this increase has not been established.161

The other mammalian PA, u-PA, appears not to be produced by most quiescent ECs.43 Rather, it is expressed by ECs involved in wound repair or angiogenesis,163 consistent with the hypothesized importance of u-PA in cell migration and tissue remodeling. Yet, u-PA is obviously important to vascular homeostasis, because mice genetically deficient in u-PA develop inflammation induced thrombi164 and manifest thrombotic tissue injury in response to lipopolysaccharide (LPS).165 However, the extrarenal source of u-PA in physiologic states has not been established.

PA receptors.   The presence of EC receptors for t-PA has been reported by several groups.166-171 Binding of t-PA to ECs has been reported to promote its fibrinolytic activity166,172 and to stimulate cell proliferation.173 Recently, one such t-PA binding site has been identified as annexin II, which is expressed on ECs174 and binds t-PA in a specific and saturable manner in vitro.172 However, the expression of annexin II on the endothelium in vivo has not yet been demonstrated.

The u-PA receptor (u-PAR) expressed by ECs appears identical to that expressed on other cell types.175,176 u-PAR is a three-domain protein linked to cell surfaces by a glycerophosphatidyl inositol anchor. Single-chain u-PA (the form found in plasma177) bound to cells via u-PAR exhibits increased plasminogen activating efficiency175,178 and is relatively protected from inhibition by PAI-1 and PAI-2.179,180 u-PAR may be expressed primarily on the surface of migrating ECs participating in angiogenesis, rather than on quiescent ECs lining normal vessels.181 Mice genetically lacking u-PAR develop normally and do not exhibit spontaneous vascular occlusion.182 Hence, u-PAR has yet to be shown to participate in maintaining physiologic blood fluidity, although it may be important in vascular repair.

Cells express diverse binding sites for plasminogen, among which are proteins that exhibit a carboxyterminal lysine (see Plow et al35 for review). Plasminogen binds to ECs in vitro with an affinity that would predict receptor occupancy at physiologic plasma concentrations.183 Cell-associated plasmin may be relatively protected from inhibition by alpha 2-plasmin inhibitor.184 However, the exact identity of these EC plasminogen binding sites remains uncertain