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Next Article 
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.
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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.
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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.
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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 ( 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- (TGF-) grow as a rapidly dividing monolayer if cultured on a flat surface coated
with type I collagen.27 However, under similar TGF- 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 ( 51) also results in a poorly developed
heart and vascular system and early embryonic death.30
Again, other integrins, most likely v3,
appear to compensate for the absence of
51 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.
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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).
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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
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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.
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 IIb3 (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 IIbIIIa, 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-
(TNF-).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.
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 v3 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 GPIb by ECs
is enhanced by TNF-,153 but whether this glycoprotein
participates in the physiologic or pathophysiologic binding of vWF in
vivo requires additional study. The v3
integrin also binds vWF in the EC substratum. In vitro, conditions that
decrease v3 expression (TNF- plus interferon- [IFN-] or arterial shear stress) increase GPIb 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 2-plasmin
inhibitor.184 However, the exact identity of these EC
plasminogen binding sites remains uncertain |
