Vascular Endothelium

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The vascular endothelium is responsible for vessel responsiveness and thromboresis-tance. It is a multifunctional organ system composed of metabolically active and physiologically responsive component cells that meticulously regulate blood flow and myocardial perfusion.

From: Contemporary Cardiology: Management of Acute Coronary Syndromes, Second Edition Edited by: C. P. Cannon © Humana Press Inc., Totowa, NJ

Basic Anatomy

Vascular endothelial cells form a single layer of simple squamous lining cells. The cells themselves are polygonal in shape, varying between 10 and 50 ^m in diameter, and elongated in the long axis, orienting the cellular longitudinal dimension in the direction of blood flow. The endothelial cell has three surfaces: nonthrombogenic (luminal), adhesive (subluminal), and cohesive. The luminal surface is smooth and devoid of electron-dense connective tissue. Its luminal membrane or glycocalyx adds significantly to the vessels' thromboresistant properties, carrying a negative charge that repels similarly charged circulating blood cells. The subluminal (abluminal) surface adheres to connective tissue within the subendothelial zone. Small processes penetrate a series of internal layers to form myoendothelial junctions with subjacent smooth muscle cells. The cohesive surface of the vascular endothelium joins adjacent cells to one another by cell junctions of two basic types, occluding (tight) and communicating (gap).

Intrinsic Thromboresistance

As an active site of protein synthesis, endothelial cells synthesize, secrete, modify, and regulate connective tissue components, vasodilators, vasoconstrictors, anticoagulants, procoagulants, fibrinolytic proteins, and prostanoids. The intrinsic thromboresistant properties of a normally functioning vascular endothelium include three distinct yet integrated systems that entenuate platelets, fibrin and coagulation factor- mediated thrombotic processes.

Platelet-Directed Cell Surface Proteins

Prostacyclin

Prostacyclin (PGI2) is a potent vasodilating substance released locally in response to biochemical and mechanical mediators. PGI2, by increasing intracellular cyclic adeno-sine monophosphate, also inhibits platelet aggregation.

Nitric Oxide

Furchgott and Zawadski (1) first discovered that acetylcholine-mediated vasodilation requires an intact vascular endothelium (i.e., it is endothelium-dependent). Nitric oxide is an L-arginine derivative that relaxes smooth muscles by increasing intracellular cyclic guanosine monophosphate. It is released locally in response to a number of biochemical mediators, including thrombin, bradykinin, thromboxane A2, histamine, adenine nucleotides, shear stress, and aggregating platelets. In addition to vasoactive properties, nitric oxide is also a potent inhibitor of platelet adhesion and aggregation. Moreover, nitric oxide and PGI2 appear to have synergistic antiaggregatory properties (Fig. 1).

Fibrin-Directed Cell Surface Proteins

Plasminogen Activators

Vascular endothelial cells synthesize and release activators that are capable of converting plasminogen to the serine protease plasmin, an enzyme that proteolytically degrades fibrin (and fibrinogen). Tissue plasminogen activator (tPA) and urokinase-type plasminogen activator generate plasmin locally; therefore, fibrinolysis is limited to the immediate environment. Stimuli for the release of vascular plasminogen activators include epinephrine, thrombin, heparin, interleukin-1 (IL-1), venous occlusion, aggre-

Platelet inhibition

mmmmmmmw Endothelial ce" membrane

Fig. 1. The attenuation of platelet aggregation is a vital component of natural vascular thromboresis-tance. Nitric oxide and PGI2 are particularly important.

gating platelets, and desamino-8-D-arginine vasopressin. (Plasminogen activators and their role in atherosclerosis and thrombosis will be discussed in a section to follow.)

Coagulation Factor-Directed Cell Surface Proteins Heparin-Like Molecules

Endothelial cells are capable of synthesizing heparin-like molecules with anticoagulant properties (2). Thus, vascular thromboresistance is mediated, at least in part, through the interaction of heparin-like substances with antithrombin and heparin cofac-tor II (both located on the endothelial surface), thus accelerating the neutralization of hemostatic (procoagulant) proteins.

Heparin cofactor II, a potent inhibitor of thrombin, is secreted by the liver into circulating blood, where it is present at a concentration of1.0-2.0 ^m/L. Unlike antithrombin, heparin cofactor II is activated predominantly by dermatan sulfate; however, under high-shear stress conditions, heparan sulfate can stimulate its inhibiting action as well. In vivo, thrombin inhibition by heparin cofactor II appears to be mediated by the interaction of dermatan sulfate with the vessel wall, predominantly in the extracellular matrix (3,4). At least four distinct subspecies have been identified in endothelial cells: two high-molecular-weight complexes, a heterodimeric form bound to fibronectin, and two small molecules referred to as decorin and biglycan (5).

Antithrombin

Antithrombin is a 58,000-Dalton plasma glycoprotein that circulates at a concentration of 2.3 mmol/L and is capable of neutralizing the coagulation proteins thrombin and factors IXa, Xa, XIa, and XIIa by covalent binding at their active sites.

Protein C and Protein S

Protein C is synthesized in the liver and is secreted into plasma as a two-chain disul-fide-bonded glycoprotein. It acts as an important anticoagulant (activated protein C [APC]) by selectively deactivating the activated forms of factor V and factor VIII (principally by cleaving their heavy chains). Protein S facilitates the anticoagulant function of APC by promoting its interaction with factors Va and VIIIa. Because protein S enhances APC-mediated factor Va inactivation by only twofold, the existence of an APC-independent anticoagulant effect has been suggested (6). Indeed, protein S is able to inhibit both the prothrombinase complex and the intrinsic tenase complex. Protein S can also interact directly with factor Va and factor VIIIa.

Both protein C and protein S are found on the vascular endothelial surface. Thrombomodulin, an integral membrane protein located on the luminal surface of most endothelial cells, forms a 1:1 complex with thrombin. In this complex, thrombin activates protein C (while at the same time thrombin is neutralized). Accordingly, thrombomodulin is able to inhibit thrombin-catalyzed fibrinogen clotting, factor V activation, and platelet activation.

Tissue Factor Pathway Inhibitor-1

Tissue factor pathway inhibitor (TFPI)-1 is located on the endothelial surface. It acts against the combined action of tissue factor and factor VII in the presence of factor Xa. The proposed mechanism for inhibition involves the formation of a quaternary complex with TFPI and factor X in a two-step reaction: factor Xa generated by tissue factor-factor VIIa complex binds reversibly with TFPI, and the binary complex formed binds, in a calcium-dependent manner, to membrane-bound tissue factor-factor VIIa (7). In essence, TFPI prevents the extrinsic coagulation cascade from activating the prothrom-binase complex; however, it has also been recognized that TFPI inhibits the intrinsic coagulation cascade, supporting the role of tissue factor on factor VIIIa and factor IX-mediated clotting (8). The presence of factor IX also impairs TFPI-mediated inhibition of tissue factor VIIa.

In the presence of glycosaminoglycans, including heparin, heparan sulfate, and dex-tran sulfate, the release of TFPI from endothelial cell storage sites is increased by several fold (9).

Tissue Factor Pathway Inhibitor-2

TFPI-2 is found within human umbilical vein endothelial cells, the liver, and the placenta and has been shown to inhibit tissue factor VIIa, kallikrein, factor XIa, and factor X activation by factor IXa (10,11). It does not independently (in the absence of heparan sulfate) inactivate factor Xa or thrombin.

Annexin v

The annexins are unique family of nonglycosylated proteins that bind to negatively charged phospholipids, including phosphatidylserine and phosphatidylethanolamine (12). One of the 13 recognized annexins, annexin V is a potent endothelial surface-based anticoagulant, which can displace phospholipid-dependent coagulation factors. It also impairs platelet adhesion Fig. 2.

ATHEROSCLEROSIS Endothelial Cell Performance

Coronary atherosclerosis is diffuse in nature, primarily involving the vessel intima (composed of the endothelium, the underlying basement membrane, and a layer of myointimal cells). A structurally and functionally normal coronary artery vasodilates in response to acetylcholine, physical exercise, or mechanical provocation. In contrast, an atherosclerotic coronary artery undergoes paradoxic vasoconstriction when exposed to acetylcholine, and a progressive decrease in cross-sectional luminal area follows rapid ventricular pacing. The failure to vasodilate prevents an increase in physiologic blood flow and, in addition, subjects the endothelial surface to excessive shear stress and injury.

Fig. 2. The modulation of coagulation is a vital component of natural vascular thromboresistance. Protein C (PC) binds to surface thrombomodulin (TM) and, in the presence of protein S (PS), forms C APC, which then neutralizes two coagulation proteases—factor V and factor VIII. TFPI, antithrom-bin (AT), heparin cofactor II (HCII), and annexin V are also important components of thromboresistance.

Fig. 2. The modulation of coagulation is a vital component of natural vascular thromboresistance. Protein C (PC) binds to surface thrombomodulin (TM) and, in the presence of protein S (PS), forms C APC, which then neutralizes two coagulation proteases—factor V and factor VIII. TFPI, antithrom-bin (AT), heparin cofactor II (HCII), and annexin V are also important components of thromboresistance.

It has become apparent that hypercholesterolemia adversely effects endothelial cell function (even before the development of atherosclerosis), and although morphologically intact, the vascular endothelium in regions of atherosclerosis fails to release nitric oxide (13). Hypercholesterolemia has been shown to impair endothelium-dependent vascular relaxation in coronary resistance vessels—the vascular bed responsible for regulating myocardial tissue perfusion (14).

Vascular endothelial cells are strategically positioned to play an important role in the regulation of local clotting processes. The cells are also ideally positioned to promote thrombosis, when needed, following vascular injury. Damaged and dysfunctional endothelial cells, however, quickly lose their ability to maintain thromboresistance and can, in fact, promote pathologic thrombosis. Indeed, assembly of the prothrombinase complex can take place on the endothelial surface of atherosclerotic vessels. Moreover, impaired local fibrinolytic activity attenuates clot dissolution.

Even the earliest stages of coronary atherosclerosis are associated with decreased endothelium-dependent dilation of the microvasculature, which may impair epicardial blood flow and increase cell-vessel wall interactions (15).

In addition to losing its thromboresistant capabilities, the dysfunctional vascular endothelium can become, in essence, prothrombotic. Following vascular injury, endothelial cells amplify the coagulant response through the synthesis and expression of factors VIII, IX, and X (16,17). Moreover, an abnormal endothelium can produce tissue factor, impair fibrinolytic activity, and decrease the effectiveness of the APC-medi-ated anticoagulant pathway (by impairing thrombomodulin-thrombin interactions on the endothelial surface) (18).

Endothelial Cell Responses to Thrombotic Stimuli

Comprehensive thromboresistance includes an appropriate response to thrombotic stimuli, preventing thrombus growth. Unfortunately, dysfunctional endothelial cells lose their ability to synthesize and secrete proteins capable of inhibiting platelets and coagulation proteins. A prime example is the response to thrombin. Under normal circumstances thrombin stimulates platelet-mediated vasoconstriction (caused by thromboxane A2 release), which is prevented by the simultaneous thrombin-induced release of prosta-

Fig. 3. Atherosclerosis is the end result of numerous risk factors, acting either alone or in combination. The overall impact of any given risk factor(s) is likely determined by genetic regulatory mecha-

cyclin and nitric oxide from endothelial cells. In atherosclerotic vessels, the response to thrombin is almost entirely vasoconstrictive (and prothrombotic) (19).

Atherogenesis

Macroscopic Pathology

Coronary atherosclerosis, the most common underlying condition among patients with acute coronary syndromes, has been described macroscopically over the past century and a half by removed pathologists and clinicians ranging from Von Rokitansky and Virchow to Osler. The pathologic sequence of events includes an initiating step, defined as the fatty streak, followed by plaque maturation and transition, setting the stage for intravascular thrombosis. The progression of coronary atherosclerosis varies widely among individuals, as does the time course and influence of recognized risk factors (Fig. 3).

Microscopic Pathology

Observations at the microscopic and cellular levels have contributed substantially to unraveling several of the mysteries that surround human atherosclerosis and have fostered clear view of the mechanisms leading to intravascular thrombosis. It is now evident that the atherosclerotic plaque and its cellular components represent an ideal substrate for thrombus formation. Thus, the term "atherothrombosis" appears fitting.

Developmental Anatomy and Cellular Biology

In experimental animals, focal sites of predilection for either spontaneous or dietary-induced atherosclerosis can be determined reliably prior to plaque development. These areas are delineated by their in vivo uptake of the protein-binding azo dye Evans blue. Salient features of these lesion-prone areas include increased endothelial permeability to an intimal accumulation of plasma proteins, including albumin, fibrinogen, and low-density lipoproteins (LDL). There is also increased endothelial cell turnover. Overall, the prelesion area within endothelial cells takes on a unique appearance, and the surface glycocalyx is two- to fivefold thinner than normal endothelial cells (20).

Lesion-prone areas within blood vessel walls exhibit a unique property of blood monocyte recruitment, followed by accumulation of these cells in the subendothelial space, a process that is accelerated in the presence of hyperlipidemia. Based on the available information, it appears that at least two processes are pivotal in the initiation of atherosclerosis: (i) an enhanced focal endothelial transcytosis of plasma proteins, including LDL, which accumulate in the widened proteoglycan-rich subendothelial space; and (ii) the preferential recruitment of blood monocytes to the intima, a process that is markedly augmented by even a short period of hyperlipidemia. Thus, the lesion-prone suben-dothelial space has two key participants in atherosclerosis, namely, the monocyte (macrophage) and LDL.

Monocyte recruitment in the intimal space of lesion-prone areas is thought to be mediated by an enhanced generation of chemoattractants of which monocyte chemoat-tractant protein-1 (MCP-1), a cationic peptide synthesized and secreted by both arterial smooth muscle cells and endothelial cells, is of particular importance. It is also recognized that the production of MCP-1 is stimulated by minimally modified (oxidized) LDL, whereas oxidized LDL itself is chemotactic (21).

Atheromatous Plaque Growth, Evolution, and Ultrastructure

After monocytes attach to the morphologically intact but dysfunctional endothelium (receptive stage), there is a net directed migration of monocytes through the endothe-lium to the subendothelial space, where they undergo differentiation. The phenomenon of monocyte activation-differentiation plays an important role in atherosclerosis, particularly with regard to plaque remodeling and lesion progression. This complex process proceeds by means of at least two mechanisms: (i) the generation of reactive oxygen species (free radicals); and (ii) the phenotypic modulation of expression of the scavenger receptor or family of receptors. The chemical modification of LDL results in its avid uptake by monocytes (now considered macrophages), and the subsequent transformation to foam cells follows. The specific receptor responsible for the uptake of modified LDL fails to down-regulate; as a result, a substantial amount of intracellular LDL cholesterol accumulates. When the influx of LDL particles exceeds the capacity of the macrophage scavenger receptors to remove them from the intracellular space, oxidized LDL particles accumulate within the arterial intima (Fig. 4A). These particles are cytotoxic, causing both injury and death to endothelial cells, smooth muscle cells, and macrophages. The net result is disruption of the relatively fragile macrophage-derived foam cells, leading to release of their intracellular lipid into the extracellular compartment of the intima; this sequence of events gives rise to the origin of the pultaceous cho-lesteryl ester-rich core of the atherosclerotic plaque (Fig. 4B) (22-25).

Fig. 4. The initial step in atherosclerosis involves monocyte and LDL binding to a dysfunctional endothelial surface (receptive stage). Monocyte activation and chemical modulation (oxidation) of LDL (modified LDL) results in avid uptake and transformation to macrophages (foam cells). Transformed smooth muscle cells synthesize and secrete MCPs that participate in monocyte recruitment and migration within the intimal layer. The influx of modified LDL exceeds the capacity of macrophage surface receptors (impaired down-regulation), allowing accumulation of potentially cytotoxic LDL particulars in the extracellular space. This step is a pivotal step in the development of a lipid core.

Fig. 4. The initial step in atherosclerosis involves monocyte and LDL binding to a dysfunctional endothelial surface (receptive stage). Monocyte activation and chemical modulation (oxidation) of LDL (modified LDL) results in avid uptake and transformation to macrophages (foam cells). Transformed smooth muscle cells synthesize and secrete MCPs that participate in monocyte recruitment and migration within the intimal layer. The influx of modified LDL exceeds the capacity of macrophage surface receptors (impaired down-regulation), allowing accumulation of potentially cytotoxic LDL particulars in the extracellular space. This step is a pivotal step in the development of a lipid core.

Lipid (Necrotic) Core

The release of copious foam cell lipids to the extracellular compartment induces a second cascade of inflammatory responses within the vascular intimal layer. In particular, granulomatous foci involving macrophages, lymphocytes, and multinucleate giant cells surround and invade the extracellular lipid.

Besides foam cell death, what other mechanisms can account for the formation of extracellular lipid deposits? New lines of evidence suggest that lipoproteins, particularly LDL, aggregate and then fuse with one another in the extracellular space to form microscopically evident lipid deposits (26-32). Structures resembling lipoprotein aggregates have been visualized in human atherosclerosis by electron microscopy, and lipid aggregates containing apolipoprotein B (apo B) have also been isolated.

A number of proteins and peptides have been detected in relative abundance within or near the atherosclerotic core. Many of the proteins found in this region are relatively hydrophobic, including the apolipoproteins, C-reactive protein (CRP), and the 70- and 60-kDa heat shock proteins. A list of proteins and peptides detected by immunologic methods in the atherosclerotic lipid core is given in (Table 1).

Cells that border and penetrate the atherosclerotic core not only participate in the deposition (or removal) of core lipids but can also be influenced by the accumulating lipids

Table 1

Proteins and Peptides Found in the Lipid Core of Atheromatous Plaques

Myeloperoxides Hyaluronectin Albumin CRP

Heat-shock protein 60

Heat-shock protein 70

Fibrinogen

Tissue factor

Apolipoproteins

Apo B

Apo A

Apo E

Complement factor C3 C56-9 neoantigen and proteins. Complement components have been found in relative abundance in the core, and both toxic and chemotactic responses may be generated via activation of complement. Antigenic markers of complement activation, including C3D and the terminal C5B-9 neoantigen, have been found in the atherosclerotic core, and terminal C5B-9 has been detected coincident with the cholesterol-rich vesicles in the subendothelium

Infection and Atherogenesis

The link between infection and atherosclerosis has been investigated with great enthusiasm given the potential for widely implementable therapies (Table 2).

Chlamydia pneumonia titers are increased among some patients with atherosclerosis, and the organism has been isolated from atheromatous plaques (39,40). Although the pathobiologic relationships have not been elucidated fully, C. pneumoniae accelerates LDL uptake in monocytes and facilitates their transition to foam cells. Infected endothe-lial cells also become prothrombotic with decreased synthesis of tissue factor pathway inhibitor, plasminogen activator, and increased tissue factor expression (41).

Cytomegalovirus (CMV) exhibits atherogenic effects through the synthesis of one of more proteins that stimulate smooth muscle cell proliferation (42) and LDL uptake within monocytes. CMV also impairs fibrinolysis, increases production of lipoprotein-a (Lpa), and increases platelet adhesion.

The available evidence supports chronic rather than acute infection as a potential pro-atherogenic factor in genetically susceptible individuals. Infection has been linked to hypertriglyceridemia, hyperfibrinogenemia, reduced high-density lipoprotein levels, anticardiolipin antibodies, and elevated CRP levels, suggesting both direct and indirect effects on both atherogenesis and thrombogenesis.

Plaque Rupture

The clinical expression of atherosclerotic disease activity is determined by pathologic events leading to coronary thrombosis. In this regard, there are two key factors: (i) the propensity of plaques to rupture, and (ii) the thrombogenicity of exposed plaque components.

The morphologic characteristics of plaques that determine their propensity to rupture have been determined. Pathology based-studies using necropsy and atherectomy tissue samples have shown convincingly that plaques associated with intraluminal thrombosis

Thrombogenesis

Table 2

The Potential Link Between Infectious Agents and Atherosclerotic Vascular Disease

Infectious agent

Association suggested in animal models

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Association suggested in humans

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Cytomegalovirus

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