The biology of the artery wall in atherogenesis

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THE BIOLOGY OF THE ARTERY WALL IN ATHEROGENESIS Kevin D. O'Brien, MD, and Alan Chait, MD

Atherosclerosis is the cause of death in more than 50% of people in Western societies.21 In addition, it results in significant cardiac morbidity, such as anginal syndromes, myocardial infarction, ischemic cardiomyopathy, and sudden cardiac death, and in noncardiac morbidity, such as cerebrovascular accidents and peripheral vascular disease. Thus the ability to prevent the development of atherosclerotic lesions or, alternatively, to induce a decrease in the severity of established atherosclerotic plaques (often referred to as regression) would have major implications for the public health. Also, the ability to stabilize lesions is likely to prevent ulceration, rupture, hemorrhage, or thrombosis of plaques, all of which may result in acute ischemic syndromes, such as unstable angina, myocardial infarction, or stroke. Fortunately recent advances have been made in our understanding of the factors involved in the initiation, progression, and regression of atherosclerosis and in the mechanisms by which commonly recognized risk factors might influence atherogenesis. These insights have identified potential avenues for interventions that may retard, prevent, or even reverse the process of atherosclerosis. In addition, some factors associated with the development of plaque instability also have been identified, thus allowing a greater understanding of how interventions might Some of the research cited in this article was supported in part by NIH grants HL 02788, HL 30086, and DK 02456 and by a Research Fellowship and a Grant-in-Aid from the American Heart Association, Washington Affiliate

From the Division of Cardiology (KDO), and the Division of Metabolism, Endocrinology, and Nutrition, and the Clinical Nutrition Research Unit (AC), University of Washington, Seattle, Washington MEDICAL CLINICS OF NORTH AMERICA VOLUME 78 • NUMBER 1 • JANUARY 1994

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decrease the risk of the acute ischemic syndromes. This article focuses on the complex interplay between cells and noncellular factors, including lipoproteins, during atherogenesis. Further, various proteins involved in mediating cellular recruitment, attachment, migration, proliferation, and synthesis of matrix are reviewed. The roles of commonly accepted risk factors, such as diabetes and hypertension, are discussed as well as potential interventions that may retard the atherosclerotic process. PROCESS OF ATHEROGENESIS

The hallmarks of atherosclerosis are the deposition of lipid in the arterial intima, recruitment of inflammatory cells (predominantly monocytes27 and T lymphocytes32 ) into the intima, smooth muscle cell accumulation, and the elaboration of collagen and matrix proteins by smooth muscle cells. 65 In more advanced plaques, necrosis also develops in the central portion of the lesion. 69 In many lesions, an ingrowth of small vessels (i.e., neovasculature) from the vasa vasorum of the adventitia also is present.54 Epidemiologic studies, such as the Framingham Study,3 have demonstrated an association of increased total plasma cholesterol with increased risk of atherosclerotic events. Genetic disorders of lipoprotein metabolism, such as familial hypercholesterolemia, which result in increases in plasma low-density lipoprotein (LDL) cholesterol also are associated with an increased risk of atherosclerosis.13 Other risk factors, including diabetes mellitus, hypertension, smoking, and low levels of high-density lipoprotein (HDL) are less atherogenic if lipids and lipoproteins are low.lO These findings suggest that intimal lipid deposition is the initial inciting event in atherosclerosis. Further evidence supporting this point of view comes from the observation that atherosclerosis-prone regions of the artery include branch points or flow-dividers, where increased lipid deposition can be documented, both by staining for the lipid components of lipoproteins and by immunostaining for the apolipoprotein components of lipoproteins?8 DEFECTS IN LIPOPROTEIN METABOLISM THAT FAVOR DEPOSITION IN THE ARTERY WALL

Cholesterol and triglycerides are fundamental components of many important biologic processes, including membrane and hormone synthesis, intercellular and intracellular signaling, and energy metabolism. IO,13 Because they are hydrophobic, however, they must be transported in the plasma in lipoprotein particles that are composed of an outer coat that contains phospholipids; unesterified cholesterol; and one or more proteins, termed apolipoproteins or apoproteins, which are specific for each particle type. This outer coat surrounds a central core containing the hydrophobic esterified cholesterol and triglycerides. The major classes of lipoproteins include the triglyceride-rich lipo-

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proteins, chylomicrons, and very-Iow-density lipoprotein (VLDL) and their remnants, and the cholesterol-rich lipoproteins, LDL and HDL. Chylomicrons are synthesized by intestinal cells in response to ingestion of dietary fat and cholesterol and are metabolized by lipoprotein lipase (LPL) to chylomicron remnants. LPL is an enzyme that is noncovalently attached to the luminal surface of endothelial cells and that hydrolyzes the core triglycerides of lipoproteins. HDL provides the chylomicron remnants with apolipoprotein E (apo E), which allows the remnants to be removed from the plasma by two classes of receptors present on hepatocytes, the LDL or apo B/E receptor, and the LDL receptor-related protein (LRP). The liver synthesizes and secretes the endogenous triglyceride-rich lipoprotein, VLDL, which is then metabolized in the plasma through interaction with LPL into VLDL remnants, which are rapidly converted in the circulation, probably by the action of lipases, into LDL. Genetic disorders in which binding to receptors of apo E is defective or absent may result in accumulation of chylomicron and VLDL remnants in plasma termed remnant removal disease. Remnant accumulation is associated with increased atherosclerosis of the coronary and peripheral arteries. 49 LDL is typically cleared from the plasma by hepatocyte LDL receptors. LDL receptors are expressed ubiquitously by most cell types and are an important mechanism by which cells take up exogenous cholesterol. Patients with familial hypercholesterolemia have defective LDL receptors and thus impaired hepatic clearance of LDL from the circulation.13 This results in marked elevations of plasma LDL cholesterol and can result in deposition of LDL in extrahepatic tissues, such as the arterial intima. In particular, LDL deposition occurs in atherosclerosis-prone areas of the arterial intima, where hemodynamic forces impair endothelial function and increase endothelial permeability to LDL.78 LDL also may be modified in the plasma by the action of an hepatic enzyme, hepatic lipase, which processes the lipoprotein into smaller particles. Small, dense LDL particles are typical of patients with familial combined hyperlipidemia (FCH), in which there is overproduction of the apo B-containing lipoproteins and also are present in insulin-resistant states. Small, dense LDL and FCH have been associated with an increased risk of atherosclerosis. 4 FCH is characterized by elevated plasma triglycerides or cholesterol; increased apo B and small, dense LDL; and low plasma HDL levels. HDL mediate removal of cholesterol from the circulation. They interact with cells in peripheral tissues by binding to cell surface receptors via one of HDL's apolipoproteins, apo A-I. During this interaction, HDL may take up excess cholesterol, reenter the circulation, and be targeted for removal by binding to LRP or LDL receptors in the liver. This process of HDL-mediated removal of cholesterol from tissues has been termed reverse cholesterol transport. Low HDL levels have been associated with an increased risk of atherosclerosis/ possibly owing to impaired clearance of cholesterol from tissues such as the arterial wall. lO Thus, net deposition of lipoproteins in arterial tissues may occur through (1) accumulation of excessive amounts of lipoproteins in the

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plasma, as in familial hypercholesterolemia, familial combined hyperlipoproteinemia, or remnant removal disease; (2) increased endothelial permeability to lipoproteins as a result of damage to endothelial cells by hemodynamic forces or oxidative damage or as a result of increased ease of entry of small, dense lipoprotein particles; or (3) defective removal of lipoproteins from arterial tissues as a result of HDL deficiency. Also, lipoprotein deposition in plaques may be favored by elaboration of matrix components, such as chondroitin sulfate proteoglycans or fibronectin, which may bind LDL/ 6• 71 thereby favoring their retention in the artery walPo RECRUITMENT OF INFLAMMATORY CELLS INTO THE

ARTERY WALL

The next step in the atherosclerotic process is the attachment of inflammatory cells to arterial endothelium and migration of these cells into the intima. Although lipoprotein deposition has been thought to be a necessary precursor to inflammatory cell recruitment, lipoproteins in their native form do not appear to incite an inflammatory reaction. Recently a form of LDL has been described9 that has undergone minimal oxidative modification such that recognition of the lipoprotein by LDL receptors is retained but that stimulates (1) elaboration of a monocytespecific adhesion protein, as yet undefined;9 (2) expression of a monocyte-specific chemoattractant, monocyte chemotactic protein-l (MCP-l);19 and (3) expression of monocyte and granulocyte colony-stimulating factors. 66 Taken together, these findings raise the possibility that minimally oxidized LDL could initiate inflammatory cell recruitment into the developing atherosclerotic lesion, as illustrated in Figure 1. Immunoglobin Superfamily Adhesion Molecules

Recent studies have documented the expression by arterial endothelial cells of several inducible adhesion molecules that may recruit inflammatory cells into plaques. The first two adhesion molecules detected in plaques were intercellular adhesion molecule-l (ICAM_l)62.64 and vascular cell adhesion molecule-l (VCAM-l).2o. 46, 54 Both are members of the immunoglobulin gene superfamily of adhesion receptors, so named because of the presence of immunoglobulinlike domains in both molecules?7 ICAM-l is constitutively expressed by endothelial cells, but its expression is markedly up-regulated by the exposure of endothelial cells to bacterial lipopolysaccharide (LPS) and to cytokines (cell-secreted peptides that affect cell activation or protein synthesis in both paracrine and autocrine fashions), such as interleukin-l (IL-l) or tumor necrosis factorex (TNF-ex).77 ICAM-l binds monocytes and lymphocytes via the lymphocyte function-associated molecule-l (LFA-l) receptor77 and is thus a good candidate molecule for recruitment into the intima of both of the predominant inflammatory cell types present in plaques. Further evidence for the role of ICAM-l in atherogenesis is the detection of ICAM-l pro-

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Lumen

0

Mildly Oxidized LDL

Intima

MONOCt;!E

----:.~I~

MACROPHAGE

Figure 1. Proposed effects of mildly oxidized LDL. LDL that has entered the artery wall via the arterial lumen or neovasculature could become mildly oxidized so that some lipid peroxidation has occurred but the protein component of the particle has not yet been oxidized to the extent that LDL receptor binding is lost. This mildly oxidized LDL has three pro-atherogenic properties in that it stimulates endothelial cells (EC) to synthesize (1) MCP-1, a monocyte chemoattractant that could attract monocytes to the artery wall, (2) a monocytespecific adhesion molecule that mediates binding of these leukocytes to endothelium, and (3) colony stimulating factors that promote monocyte differentiation into macrophages. MCP1 = monocyte chemotactic protein-1 , CSFs = colony-stimulating factors.

tein in human atherosclerotic tissue by immunohistochemistry.62, 64 VCAM-l, an adhesion molecule that binds to the very late activation antigen-4 (VLA-4) present on monocytes and lymphocytes, is expressed by endothelial cells in response to stimulation with LPS, TNF-(X, IL-l, and interferon-), (IFN_)').46,77 VC AM-l has been detected immunohistochemically on arterial endothelial cells in both Watanabe heritable hyperlipidemic WHHUO and cholesterol-fed46 rabbits and in humans. 54 In cholestrol-fed rabbits expression of VCAM-l preceded the presence of mononuclear inflammatory cells in the arterial intima,46 consistent with the hypothesis that elevated plasma cholesterol results in intimal mononuclear inflammatory cell recruitment. Recently it has also been demonstrated that lysophosphatidyl-choline, an early product of LDL oxidation, can induce VCAM-l expression on endothelial cells. 40 In addition, the presence of both ICAM-l and VCAM-l proteins has been observed on macrophages and smooth muscle cells in the arterial intima,54, 62, 64 suggesting that these adhesion molecules may have a broader range of functions than previously appreciated. Finally, expression of VCAM-l on the endothelium of neovasculature penetrating into the base of plaques from the adventitial vasa vasorum54 suggests that the arterial luminal surface may not be the only route by which inflammatory cells enter the plaque. Selectins

Another group of adhesion molecules are termed select ins, owing to their high affinity binding to carbohydrate groups, a characteristic shared

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with a class of proteins called lectins. Selectins that could play roles in atherogenesis include E-selectin (formerly termed ELAM-l) and P-selectin. E-selectin has been detected on endothelial cells overlying atherosclerotic lesions.84 E-selectin may bind sialyl Lewis X, a carbohydrate normally found on monocytes, antigen-stimulated T lymphocytes, and polymorphonuclear cells (PMN).23 P-selectin is constitutively expressed but normally sequestered in ex-granules of platelets and in Weibel-Palade bodies of endothelial cells until these cells are activated, at which point the selectin is expressed on the cell surface.23 The putative role of selectins is to mediate transient attachment, or rolling, of leukocytes along the endothelial surface, thus slowing their velocity sufficiently to allow attachment to VCAM-l or to ICAM-l. 23 Also, several factors implicated in atherogenesis have been shown in vivo to act directly on monocytes to increase their adhesiveness, such as exposure to high concentrations of lipoproteins (e.g., LDL, minimally modified LDL, or I3-VLDL), growth factors (GM-CSF), cytokines (IL-3), or chemotactic factors (C5a).23 Figure 2 illustrates the proposed mechanisms by which selectins and immunoglobulin superfamily adhesion molecules mediate leukocyte recruitment into the vessel wall. MECHANISMS OF CELLULAR LIPOPROTEIN UPTAKE AND REMOVAL Cellular Lipoprotein Uptake

As discussed earlier, nearly all cells take up exogenous cholesterol via LDL receptors. Increases in cell cholesterol content, however, result in down-regulation of LDL receptor number, thereby protecting cells from excessive accumulation of cholesterol by way of this pathway. Brown and Goldstein13 have shown that chemical modification of LDL via acetylation results in increased uptake of the modified lipoprotein by way of another cell surface receptor, termed the scavenger receptor, present on macrophages and endothelial cells. Scavenger receptor expression is not affected by cell cholesterol content. 13 Acetylated LDL, however, does not exist in vivo. Thus several other potential mechanisms for cell cholesterol uptake have been proposed. Oxidatively Modified Lipoproteins

Unmodified LDL do not cause appreciable lipid accumulation in cultured arterial cells. Therefore there has been considerable interest in modifications of lipoproteins that might facilitate their cellular uptake and render them more atherogenic?9 Oxidatively modified lipoproteins have many biologic effects that may modulate atherogenesis. LDL can be oxidized by the three major cell types in the artery wall-endothelial

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Lumen

Intima

Endothelial Cell Activation

S = Selectln (eg. E-selectln, P-selectln) I

=Immunoglobulin Supertamlly (eg. VCAM-1, ICAM-1)

Figure 2. Leukocyte recruitment into vessels. Circulating leukocytes come into random contact with endothelium, but their attachment to endothelial cells (EC) is mediated by a two· step process. In the first step, termed rolling, leukocytes are slowed down by a series of transient attachments to selectins, e.g., P-selectin and E-selectin, expressed on stimulated EC. In the second step, rolling leukocytes then attach via integrins, e.g., to immunoglobulin superfamily adhesion molecules ICAM-1 and VCAM-1, that mediate leukocyte binding to and transmigration across the endothelium. Leukocyte binding to integrins is enhanced by exposure of the cells to lipoproteins, growth factors, cytokines, or chemotactic factors. Selectins and integrins mediate leukocyte recruitment across endothelium of arteries, veins, and smaller vessels such as plaque neovasculature.

cells, monocyte-macrophages, and arterial smooth muscle cells?9,92 Both water-soluble and lipid-soluble antioxidants are important in protecting LDL from oxidative modification, which proceeds when these antioxidants become depleted. 37 Lipid-soluble antioxidants, such as probucol and vitamin E, are incorporated into lipoprotein particles and can protect them from oxidative modification, but antioxidants in the aqueous milieu of LDL, e.g., vitamin C, must first be consumed before the lipoproteins are oxidized. 25 ,37 Biologic Effects of Oxidized Low-Density Lipoproteins

Biologic consequences of oxidized LDL that have been demonstrated in vitro include uptake by macrophage scavenger receptors leading to foam cell formation, stimulation of monocyte and inhibition of macrophage chemotaxis, and cytotoxicity?9,92 In addition, mildly oxidized LDL stimulates the expression of several endothelial cell genes, including monocyte chemotactic protein-l,19 adhesion molecules,9 and colony stimulating factors,66 and of procoagulant factors, such as tissue factor and plasminogen activator inhibitor-l (PAI-l).92 When LDL has become sufficiently oxidized to trigger scavenger receptor recognition, foam cell formation can ensue. The cytotoxic properties of oxidized LDL may damage endothelium or may lead to plaque necrosis later during atherogenesis. Figure 3 illustrates some of the proposed ways in which oxidized LDL stimulates atherogenesis.

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Lumen tTissue Factor

o --+ 0

Extensively MOil~dlYed LDL Oxidized LDL XIIZ

Intima

~

t PAI-!

Cytotoxicity

~~

ASMC

\

cytokine . . and growth factor release

~--~.

MACROPHAGE

~

cytokine and growth factor release

FOAM CELL

Figure 3. Proposed biologic effects of extensively oxidized LDL. LDL that has been oxidized extensively so that LDL receptor binding is abolished can have various proatherogenic effects such as (1) promoting thrombosis by inducing endothelial cell (EC) expression of tissue factor and plasminogen activator inhibitor-1 (PAI-1). (2) damaging EC through direct cytotoxicity. (3) induction of foam cell formation through uptake by macrophages and arterial smooth muscle cells (AS MC) in a manner unregulated by increased cell cholesterol content. and (4) modulation of proatherogenic growth factor and cytokine synthesis by both macrophages and ASMC.

Evidence for Oxidized Lipoproteins In Vivo

Lines of evidence that oxidized LDL occurs in vivo and is important in atherogenesis include the demonstration of a fraction of circulating LDL with properties similar to oxidized LDL, similarities in the properties of oxidized and native LDL eluted from the arterial wall, the presence of oxidation-specific epitopes in atheromatous lesions, oxidationspecific epitopes in lipoproteins eluted from atherosclerotic plaques, and circulating autoantibodies against oxidized LDL.79,92 Recently an association between the presence of autoantibodies to an oxidatively modified form of LDL and risk of progression of carotid atherosclerosis has been shown.72 Thus considerable evidence suggests that oxidatively modified forms of LDL occur in vivo and may play a role in atherogenesis. Effects of Oxidized Low-Density Lipoprotein on Cytokine Release

Oxidized LDL has been shown to stimulate macrophages to release a cytokine implicated in atherogenesis, IL-l/ but to decrease their synthesis of a growth factor also implicated in atherogenesis, platelet-derived growth factor (PDGF) B-chain. 65 Oxidized LDL does stimulate release of another isoform of PDGF, PDGF A-chain by smooth muscle

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cells. SO When cocultures of monocytes and T lymphocytes are exposed to oxidized LOL, the T lymphocytes are activated, as determined by expression of HLA-OR antigens and interleukin-2 receptors?6 These activated T lymphocytes could then release other cytokines, such as TNF-u and IFN-,,{, although these cytokines could be either proatherogenic or antiatherogenic. Additional Mechanisms for Cell Cholesterol Accumulation

Several additional potential mechanisms for cellular uptake of cholesterol have been described. 1. Aggregated LOL, which may form through binding of multiple LOL particles to matrix components such as fibronectin or proteoglycans,36, 71, 87 may be taken up phagocytically by macrophages. 2. Immune complexes of lipoproteins,39 such as oxidized or glycosylated LOL,IO,92 may be internalized via the Fc receptor of macrophages. 3. LPL may also enhance uptake of cholesterol by arterial wall cells by binding of lipoproteins containing LPL to extracellular matrix so they are retained in the artery walli73 by binding of LPLcontaining lipoproteins to cell-surface glycosaminoglycans, which· then brings them into close approximation to LOL receptors or LRPi7 by mediating uptake of lipoprotein particles via an LOL receptor-independent mechanism91 or by depleting lipoproteins of triglycerides, which increases their uptake by cells.5 Synthesis of LPL by macrophages in human atherosclerotic plaques is most typically associated with lipid-laden foam cells,56, 93 consistent with the hypothesis that LPL plays a role in lipid accumulation by these cells. Lipoprotein(a)

Lipoprotein(a) is a lipoprotein particle similar in composition to LOL but that has an additional apoprotein, apo(a), covalently linked to apo B. Lipoprotein(a) has been firmly linked with an increased risk of atherosclerosis,43 although the mechanisms by which the molecule may increase risk are not known. Immunohistochemical studies have documented that lipoprotein(a) is preferentially deposited in tissues in many different inflammatory conditions, including Crohn's disease, granulomatous lymph nodes, and pericarditis.53 Similarly, preferential deposition of lipoprotein(a) as compared with LOL has been shown in human atherosclerotic tissue. 53 Because lipoprotein(a) has extensive sequence homology with plasminogen but lacks plasminogen's enzymatic activity, it has been proposed that lipoprotein(a) may act as a competitive inhibitor of plasminogen, thus acting to increase thrombosis.43, 53 Also, lipoprotein(a)

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binds avidly to fibrin unmasked by partial degradation, and lipoprotein(a) colocalizes with fibrin in atherosclerotic plaques, where it might act as a "scaffold" for smooth muscle cell (SMC) recruitment. 43 Lipoprotein(a) has been shown to stimulate smooth muscle cell proliferation by inhibiting transforming growth factor [3/9 which usually inhibits mitogenesis. Thus lipoprotein(a) could promote wound healing but affect an increase in plaque mass in arteries. Mechanisms of Cholesterol Removal from Plaques

Both HOL and apo E may play roles in cholesterol removal from plaques. Binding of HOL to the surface of cells may stimulate translocation of intracellular cholesterol to the cell surface for desorption to HOL particles/6 which can then act as an acceptor for excess cellular cholesterol. Apo E is secreted by both macrophages and SMC in culture49 and can be secreted into culture medium as apo E and cholesterol-containing particles. Experiments with an apo E-defective, monocyte-like cell line have shown that insertion of a functional apo E gene makes these cells much more effective at decreasing their intracellular cholesterol content in response to cholesterol-loading than cells without a functional apo E gene. 51 Finally, apo E secreted by macrophages or SMC can bind to HOL particles and target them for removal from the circulation by the liver. Synthesis of apo E by macrophages in plaques, particularly lipid-laden foam cell macrophages, has been documented. 55 Mouse Models of Lipoprotein Metabolism in Atherogenesis

Several experiments using mice in which human genes are overexpressed or mouse genes are inactivated have provided further evidence to support roles for lipoproteins in atherogenesis. Overexpression of apo A-I/o the major apoprotein of HOL, and of LOL receptors35 can protect susceptible mouse strains from the development of atherosclerotic lesions. In contrast, overexpression of apo(a), the apoprotein unique to lipoprotein(a) can markedly increase the susceptibility of mice to atherosclerosis. 44 Finally, the importance of apo E as an antiatherogenic factor has been shown in mice in which inactivation of the apo E gene (socalled knockout mice) results in a marked increase in atherosclerosis. 6o,96 Because these mice have a fivefold increase in circulating cholesterol levels, however, it is not clear whether the increase in atherosclerosis is due to increased lipid deposition in tissues as a result of decreased removal of apo E-containing lipoproteins from the circulation, to impaired apo E-mediated cholesterol removal from cholesterol-loaded cells, or to a combination of both factors. INFLAMMATORY FACTORS IN ATHEROGENESIS

The previous sections have described ways in which net lipoprotein deposition in the arterial wall may result in endothelial cell activation and inflammatory cell recruitment into plaques. This section focuses on

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current knowledge of specific inflammatory cell types present in atherosclerotic tissue and how they may interact with other inflammatory cells, endothelial cells, SMC, and lipoproteins in atherogenesis. Macrophages represent the dominant inflammatory cell type in atherosclerotic intima,27. 69 whereas T lymphocytes represent a significant minority?2 PMN are exceedingly rare, constituting less than 0.1% of inflammatory cells.32 Immunoglobulins are present in atherosclerotic intima, often in association with lipoproteins, but B lymphocytes are uncommon, suggesting that most immunoglobulins in plaques enter from the plasma. 47 Collections of B lymphocytes and plasma cells, however, often are present in the adventitia surrounding advanced atherosclerotic plaques, along with oxidized lipoproteins. It has been suggested that these adventitial B lymphocyte collections represent an autoimmune response to oxidized lipoproteins from the plaque. 32 Monocyte/Macrophages

Monocyte entry into plaques may be mediated by adhesion molecules expressed on endothelial cells in response to minimally oxidized LOU or to cytokines, such as IL-l, INF-)" or TNF-cx. 32,47 Monocytes may be recruited to enter the subendothelial space by cytokines, such as MCPl.94 MCP-l expression by endothelial cells, monocyte/macrophages, and SMC can be induced by several cytokines, including IL-l and TNF-cx, whereas monocyte/macrophages can also be stimulated to secrete MCP1 by IFN-)' and M-CSF.23 Lysolecithin present in oxidized LOL also is chemotactic for monocytes. 92 Once in the intima, macrophages can then accumulate excessive amounts of cholesterol to become lipid-laden foam cells through several mechanisms. Macrophages can be stimulated by oxidized LOL to release IL-l/ which could act on endothelial cells to up-regulate their expression of ICAM-l, VCAM-l, and E-selectin, thereby recruiting more mononuclear cells into the plaque. POGF, which can be synthesized by macrophages, also is a potent chemoattractant for and stimulator of proliferation in SMC. 65 Because the presence of both IL-l and POGF has been demonstrated in plaques,88 these cytokines may be responsible for SMC recruitment and proliferation. IL-l may stimulate high-level expression of IL-6 by both macrophages and SMC,47 and IL-6 activates T lymphocytes. 47 The expression of M-CSF, which may stimulate macrophage proliferation, also has been demonstrated in lesions. 16, 68 T Lymphocytes

T lymphocytes are detectable in atherosclerotic lesions of all ages. Most of the T lymphocytes in plaques have cell surface markers, which indicate that they have been antigen stimulated, possibly by local antigens. Studies have also demonstrated that these antigen-stimulated T

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lymphocytes, or memory T cells, have heterogeneous antigen specificities, suggesting that T lymphocytes that have already been antigen stimulated may be recruited into plaques. 32 E-selectin, an adhesion molecule that has been detected in atherosclerotic tissue, specifically binds memory T cells and therefore may mediate their recruitment into plaques.84 Oxidized lipoproteins could also play a role in T lymphocyte activation, as it has been shown that exposure of cocultures of monocytes and T lymphocytes causes secretion of T lymphocyte cytokines. 26 Activated T lymphocytes can then secrete a variety of cytokines that may affect other cells in the plaque, such as IFN-'Y, which inhibits cell proliferation.32 In addition, IFN-'Y has several effects that could limit excessive cholesterol accumulation, including down-regulation of Fc and scavenger receptors/2 LRP,41 and LPL. 33 These effects suggest that IFN-'Y may inhibit atherogenesis locally, a point of view borne out by studies showing that intravenous IFN-'Y inhibits plaque growth in a rabbit carotid injury model of atherogenesis. 33 SMOOTH MUSCLE CELLS IN ATHEROSCLEROTIC LESIONS

Classically SMC proliferation has been described as a characteristic of atherosclerotic lesions.69 In animal models of balloon-induced injury, medial SMC proliferation is seen, followed by migration of SMC to the mediaY The situation in human atherosclerosis may be different, however, because SMC proliferative rates are quite low in human atherosclerotic lesions. 28 This has been interpreted as consistent with atherosclerosis being a chronic disease that develops over the course of several years. A characteristic of adult human coronary arteries, however, is the presence of diffuse intimal thickening, consisting primarily of SMC, proteins, and proteoglycans,28,78 so in humans, in contrast to many animal models, some SMC present in atherosclerotic lesions could be of intimal rather than medial origin. Vascular SMC have a range of phenotypes. 65, 86 The phenotype characteristic of most medial SMC is often termed contractile because it contains large numbers of myofilaments. Another phenotype, more typical of intimal SMC, has fewer myofilaments (and thus may stain less well with SMC-specific antibodies directed against actin) and contains welldeveloped Golgi apparatus and rough endoplasmic reticulum and is often refe~red to as the synthetic phenotype. The specific types of matrix synthesized, such as collagen and proteoglycans, may differ markedly among cells. In addition, many intimal and some medial SMC have many characteristics that indicate immunologic activation, such as expression of major histocompatibility complex class 11 molecules and synthesis of growth factors and cytokines.32, 65, 86 Some SMC in the intima accumulate large amounts of lipid and assume a foam cell morphology. Several mechanisms exist by which SMC might accumulate lipid. LDL may be taken up by the LDL receptor.

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LPL modifies LDL to a form with an increased rate of uptake by LDL receptors. Apo E present in the plaque could bind to extracellular lipid and then act as a ligand for LDL receptors. 49 Similar to macrophages, however, SMC down-regulate their surface expression of LDL receptors in response to cholesterol loading, so additional mechanisms for cholesterol uptake must be operative in intimal SMC. Although it has not yet been demonstrated for human cells, rabbit SMC in culture express scavenger receptors22 and so may take up modified forms of cholesterol, such as oxidatively modified cholesterol, by this route. SMC can oxidatively modify LDL. 79, 92 In some cell types, cholesteryl esters can be taken up from lipoproteins without particle endocytosis through a process termed selective uptake.30 Finally, studies of cultured human vascular SMC have shown that, when present in high concentrations, lipoproteins can cholesterol load SMC by transfer of free cholesterol from the surface of the lipoproteins to SMC membranes.?6 Table 1 summarizes proposed mechanisms for lipid uptake and removal from both macrophages and SMC. As noted earlier, several cytokines and growth factors are present in atherosclerotic plaques. The PDGF isoform secreted by macrophages, PDGF-B chain, is present in all stages of plaque developmen~5 and can stimulate SMC chemotaxis, proliferation, and synthesis.65 SMC proliferation or protein synthesis can be stimulated by IL-l, TNF-a, and TGF-(3, and the effects of these cytokines on SMC are probably mediated by autocrine secretion of the SMC PDGF isoform, PDGF A-chain. 65 Collagen Synthesis in Atherosclerosis

Different types of collagen are expressed at different stages of lesion development. 38 Type IV collagen, presumably associated with basement Table 1. PROPOSED MECHANISMS FOR CELLULAR LIPOPROTEIN METABOLISM Macrophages Lipoprotein uptake Endocytosis LDL receptor LRP Scavenger receptor Fc receptor Oxidized LDL receptor Phagocytosis LDL receptor Fc receptor Scavenger receptor Receptor-independent uptake LDL-mediated binding to heparan sulfate Lipid removal HDL-mediated efflux Secretion of apo E-lipid vesicles LDL = Low-density lipoprotein; LRP lipoprotein.

=

Smooth Muscle Cells Endocytosis LDL receptor Scavenger receptor Free cholesterol transfer Selective uptake of cholesteryl esters

HDL-mediated efflux Secretion of apo E-lipid vesicles

lipoprotein receptor-related protein; HDL

=

high-density

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membranes, is the only collagen type detected in the intima of infants. As diffuse intimal thickening develops, type I and type III collagens accumulate, in addition to type IV collagen associated with basement membranes. In atherosclerotic lesions, type V and type VI collagen are detectable, and type IV collagen also is more prevalent. Type I collagen synthesis may be stimulated by a variety of factors, including TGF-[3.67 Type I collagen binds lipoproteins, so its accumulation in the intima may promote lipoprotein accumulation. Type IV collagen is seen in SMC that are senescing and may be associated with calcium deposition in tissues. Decrease in the amount of type IV collagen surrounding SMC also may be a necessary precursor to SMC migration. Type V collagen is expressed in a variety of processes with progressive fibrosis, but its function is unknown. The function of type VI collagen is also unknown, but it is found in association with proteoglycan complexes and may therefore play a role in cell-cell and cell-matrix adhesion. Role of Proteoglycans in Atherogenesis

The three major families of proteoglycans are (1) chondroitin sulfate proteoglycans, characteristically synthesized by SMC; (2) heparan sulfate proteoglycans, the predominant proteoglycans of endothelial cells (EC) and an important component of basement membranes but also present to a lesser extent in association with elastin fibers and on SMC and macrophage plasma membranes; and (3) dermatan sulfate proteoglycans, synthesized by both EC and SMC, and which associate with collagen fibrils.90 Proteoglycans constitute a minor part of normal arterial intima but may accumulate in the intima of atherosclerosis-prone regions 78 and are present in relatively larger amounts in atherosclerotic plaques. Proteoglycans accumulate in the reendothelialized intima of balloon-injured arteries of animals with diet-induced hypercholesterolemia but not in the deendothelialized intima. The excess proteoglycans produced are predominantly chondroitin sulfates, whereas levels of heparan sulfates may be reduced. 90 Chondroitin sulfate is present in increased amounts in atherosclerotic tissue. Lipoproteins, especially LDL, have an affinity for chondroitin sulfate,36 so this proteoglycan may be important in retaining lipoproteins in atherosclerotic tissue. LDL-proteoglycan complexes may be taken up phagocytically by cultured macrophages,36, 71. 87 raising the possibility that binding of LDL to chondroitin sulfate in the artery wall could facilitate lipoprotein uptake. Heparan sulfate proteoglycans have many proposed roles in vascular wall biology. Heparan sulfates on the luminal surface of EC bind LPL, where the enzyme then hydrolyzes triglycerides in circulating lipoproteins. It has been demonstrated that LPL can mediate uptake of LDL and lipoprotein(a) by a receptor-independent mechanism involving binding of LPL to heparan sulfates on the surface of cells. 91 Heparan sulfates inhibit cellular migration90 and can abolish the stimulatory effects of

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thrombospondin and epidermal growth factor on SMC proliferation. 90 Heparan sulfates also play important roles in hemostasis at the endothelial cell surface, as they bind and markedly increase the activity of antithrombin III, thereby enhancing the inactivation of thrombin and inhibiting thrombosis. 90 Finally, heparan sulfates may be important in sequestering growth factors, such as basic fibroblast growth factor (bFGF) in basement membranes, so basement membrane injury could release heparan sulfate-sequestered bFGF to stimulate SMC proliferation. 65

INTIMAL NEOVASCULATURE AND ADVENTITIAL VESSELS IN ATHEROSCLEROSIS

A characteristic of many advanced human atherosclerotic plaques is the presence of neovasculature, or small vessels that have infiltrated into the plaque from the vasa vasorum of the adventitia. The factors that stimulate growth of these vessels and their relative roles as potential sources for lipid deposition and inflammatory cell infiltration into plaques has received little attention. It has been shown, however, that the mononuclear cell-specific adhesion molecule, VCAM-l, is expressed on neovascular endothelial cells in the intima of human atherosclerotic lesions. 54 These regions often contain inflammatory cell infiltrates, suggesting that the intimal neovasculature may be a route for recruitment of inflammatory cells into plaques. 54 The relative importance of these vessels, however, as compared with the arterial luminal surface as a route for lipid deposition and inflammatory cell recruitment is not known. In the same study, it was also noted that VC AM-l was present on endothelial cells of some adventitial vessels in the majority of plaques but not control arteries, suggesting active recruitment of inflammatory cells in the adventitia of plaques. 54 This observation is consistent with the finding that collections of inflammatory cells may be present in the adventitia of advanced plaques, possibly representing an autoimmune inflammatory reaction to oxidized lipoproteins. 32 The implications of these findings for plaque growth are not known. It is also not known which factors regulate the ingrowth of neovasculature into the base of plaques. One possibility is that as the intima increases in thickness, regions near the base of the intima that are relatively distant from the arterial lumen become hypoxemic. SMC in these regions could release bFGF, which is angiogenic, from intracellular stores. Also, macrophages could contribute to local hypoxemia owing to their high rates of oxygen consumption. The demonstration of heat shock proteins, which can be released in response to hypoxemia, in macrophage-containing regions of plaques8 would be consistent with this possibility. Finally, macrophages themselves might release angiogenic factors, such as TNF-a 24 ,45 or TGF-j3.67

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EFFECTS OF ATHEROSCLEROTIC RISK FACTORS ON THE ARTERY WALL Dyslipidemia

Several genetic or acquired defects in lipoprotein metabolism may lead to increased lipid deposition in tissues or to decreased lipid removaL Patients with familial hypercholesterolemia have defective LDL receptors and thus impaired hepatic clearance of LDL from the circulation. Small, dense LDL particles are typical of patients with FCH, in which there is overproduction of the apo B-containing lipoproteins. Small, dense LDL might have an increased rate of entry into the artery wall and accumulate in insulin-resistant states. They are more easily oxidized and bind more avidly to proteoglycans than LDL of normal density. FCH also is characterized by elevated plasma cholesterol, triglycerides, or both; increased apo B; and low plasma HDL levels. Potentially atherogenic lipoprotein remnants accumulate in remnant removal disease. Low HDL levels have been associated with an increased risk of atherosclerosis, possibly owing to impaired clearance of cholesterol from tissues such as the arterial wall. Diabetes

The multiple potential mechanisms through which diabetes affects atherogenesis have been extensively and elegantly reviewed.lO Many proatherogenic effects of diabetes may be related to lipid modification. Diabetics have a pattern of lipoprotein abnormalities, referred to as diabetic dyslipidemia: increased VLDL remnants; increased apo E content in lipoproteins; small, dense LDL; and decreased HDL. The first three abnormalities may increase cellular cholesterol uptake, whereas the last may decrease cell cholesterol removaL Also, lipoproteins characteristic of diabetics, such as small, dense LDL and VLDL remnants, are more susceptible to oxidative modification than those of nondiabetics. Thus the whole range of proposed biologic effects of oxidized lipoproteins, including adhesion receptor expression, increased cytokine and growth factor production, and increased cellular uptake of cholesterol, would be amplified. Diabetes may have several procoagulant effects. The hypertriglyceridemia may increase thrombosis through increased activity of factor VII, factor X, and PAl-I. Diabetes may also increase platelet aggregability. In addition, a correlation has been demonstrated between proteinuria in diabetics with renal disease and levels of lipoprotein(a). High circulating insulin levels in diabetics also may be proatherogenic. A correlation between fasting insulin levels and atherosclerotic disease has been demonstrated in both diabetics and nondiabetics. In vitro high insulin levels stimulate SMC proliferation and matrix production. High insulin levels also have been shown to increase LDL receptor

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activity, increase endogenous cholesterol synthesis, increase intracellular cholesterol esterification, and impair HDL-mediated cholesterol removal. It is more likely, however, that hyperinsulinemia is a marker of other risk factors, such as dyslipidemia and hypertension, which frequently coexist. Cigarette Smoking

Cigarette smoking may influence atherosclerosis in several ways. In animal models, cigarette smoking induces ultrastructural changes, which may be associated with increased endothelial permeability, including blebbing, and an increase in the number and size of plasmalemmal vesicles. 59 In a canine model using 125I-labeled fibrinogen, exposure to carbon monoxide has been shown to increase arterial endothelial permeability to plasma proteins, whereas nicotine increases their retention in the artery walU Increased platelet adhesion has also been observed, without detectable loss of endothelial cells. 59 Platelets isolated from smokers also have an increased rate of aggregation to adenosine diphosphate, and fibrinogen levels of smokers are higher than those of nonsmokers and fall after smoking is discontinued. 52 Exposure to cigarette smoke or oral nicotine has been shown to increase platelet production of the vasoconstricting prostaglandin thromboxane A2?4 Therefore cigarette smoking could increase the risk of thrombosis in several ways. Tobacco constituents are mitogenic for SMC in culture. 6 Cigarette smoke unfavorably affects lipoprotein profiles in rats by decreasing HDL and increasing LDL and VLDL levels. 42 , 50 Further direct exposure to cigarette smoke modifies LDL, in part through a superoxide anion-dependent mechanism, to a form taken up more avidly by macrophages. 95 Hypertension

There are several potential mechanisms by which hypertension interacts with the arterial wall to accelerate atherosclerosis. Studies using scanning electron microscopy have shown increased monocyte adherence to arterial endothelium in rats made hypertensive by treatment with deoxycorticosterone acetate (DOCA),82 and increased subendothelial infiltration of macrophages has been demonstrated in both rat18 and rabbit'l4 models of hypertension. The factors that mediate these processes may include endothelial injury as a result of increased shear forces or secretion of chemotactic factors by damaged SMC. Hypertension may also be associated with changes in immune function. As compared with macrophages from normotensive Wistar-Kyoto rats, macrophages from spontaneously hypertensive (SHR) rats have decreased proliferation in response to the mitogen concanavalin A,58 and peripheral blood mononuclear cells exhibit decreased ~ agonist-induced cyclic adenosine monophosphate production,85 an effect that can be mim-

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icked by exposure to GM-CSF or interleukin-2. Whether these changes occur in a therosclerosis is unclear. Angiotensin 11 can have several effects on cells of the artery wall that may have relevance to atherosclerosis, Cultured SMC from SHR rats hypertrophy in response to angiotensin 11 exposure, and this effect appears to be mediated by binding of angiotensin 11 to the ATl but not AT2 angiotensin receptors,14 Angiotensin 11 increases synthesis of several proteins by vascular SMC, including a-actin. 57 Angiotensin 11 has been shown to induce expression of PDGF A-chain and of TGF-[3 in vascular SMC. 31 Also angiotensin 11 has been shown to have effects on macrophage cytokine expression because it induces macrophage expression of the SMC mitogen, heparin binding-epidermal growth factor (HB-EGF),81 In light of these experimental manifestations of angiotensin 11 exposure, it might be predicted that treatment with angiotensin converting enzyme inhibitors, such as cilazapril or captopril, could reduce atherosclerosis, Cilazapril treatment has been shown to decrease subendothelial macrophage infiltration18 and to reduce intimal hyperplasia in a rat carotid balloon injury mode1. 63 Captopril treatment has been shown to decrease aortic atherosclerosis in the Watanabe heritable hyperlipidemic rabbit model of familial hypercholesterolemia,15 Whether these beneficial effects are due to inhibition of direct angiotensin 11 effects at the level of cells of the arterial wall, or to their blood pressure lowering effects, or to both are not known, Finally, in addition to inducing SMC hypertrophy, hypertension has been shown to be associated with increased production of matrix proteins, as extracellular matrix is increased in the media of both SHR48 and DOCA-treated Wistar83 rats, In the case of the DOCA-treated Wistar rats, it was found that diabetes had a synergistic effect on the amount of matrix produced. 82 Several mechanisms by which dyslipidemia, diabetes, cigarette smoking, and hypertension may interact with the artery wall to promote atherogenesis are summarized in Table 2, CHARACTERISTICS OF THE ARTERY WALL ASSOCIATED WITH CLINICAL EVENTS

Most clinical events in atherosclerosis are associated with a defect in the integrity of the plaque resulting in thrombosis or an acute increase in plaque mass,21 Thrombosis occurs as a result of loss of endothelial integrity owing to endothelial cell damage or death or to rupture of the fibrous cap, resulting in exposure of a thrombogenic surface, Thrombus also may form on intact endothelium, which has increased expression of tissue factor in response to cytokine activation,61 An acute increase in plaque mass can occur as the result of rupture of the fibrous cap and hemorrhage into the plaque from the arterial lumen or from intraplaque hemorrhage from the intimal neovasculature. Some factors have been identified that predispose to rupture of the fibrous cap or to intraplaque

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Table 2. PROPOSED EFFECTS OF ATHEROSCLEROTIC RISK FACTORS ON ATHEROGENESIS Dyslipidemia Endothelial damage Induction of endothelial gene expression by mildly oxidized lipoproteins Increased susceptibility to oxidation of small, dense lipoproteins Lipid accumulation Intracellular accumulation of modified lipoproteins Extracellular lipid binding to matrix Procoagulant effects Induction of tissue factor and PAI-1 by oxidized LDL i Activity of factor VII and X and PAI-1 in hypertriglyceridemia Reduced cholesterol efflux from cells in presence of low HDL Diabetes Lipoproteins Dyslipidemia ( i triglyceride; 1 HDL; small, dense LDL) resulting in increased lipid disposition i Susceptibility to oxidation Procoagulant effects t Activity of factors VII and X and PAI-1 t Platelet aggregability Hyperinsulinemia-mediated effects t SMC proliferation and matrix synthesis t LDL receptor activity t Endogenous cholesterol synthesis t Intracellular cholesterol esterification 1 HDL-mediated cholesterol removal Cigarette Smoking Endothelial damage t Endothelial permeability t Intimal plasma protein retention Procoagulant effects t Platelet aggregation t Fibrinogen levels t Thromboxane A2 production Lipoproteins 1 HDL t LDL, VLDL t Lipoprotein oxidation Hypertension Endothelium t Monocyte adherence, infiltration Immune dysfunction 1 Macrophage proliferation 1 Lymphocyte [3-receptor responsiveness Angiotensin II-mediated effects t SMC matrix protein synthesis (e.g., a-actin) t SMC cytokine synthesis (e.g., PDGF A-chain, TGF-[3) t Macrophage cytokine expression (e.g., HB-EGF) PAI·1 = Plasminogen activator inhibitor-1; LDL = Iow-density lipoprotein; HDL = high-density lipoprotein; SMC = smooth muscle cells; VLDL = very-low-density lipoprotein; PDGF = platelet-derived growth factor; TGF-13 = transforming growth factor-l3; HB-EGF = heparin binding-epidermal growth factor.

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hemorrhage. Macrophages secrete many proteolytic enzymes, including collagenase and stromelysin, which weaken the plaque and increase its propensity to rupture. 21 It has been demonstrated that those fibrous caps that have ruptured contain higher densities of macrophages and have less tensile strength than those that have not. 21 Computer modeling studies have shown that the presence of a soft load bearing surface in the base of plaques, as could be created by lipid deposition or infiltration of proteolytic enzyme-releasing inflammatory cells, increases the mechanical stress on fibrous caps and thus their propensity to rupture. 21 Thus the development of strategies designed to decrease lipid deposition or macrophage infiltration should decrease clinical events. A significant reduction in clinical event rates has been demonstrated in two angiographic trials of lipid-lowering therapies, the Familial Atherosclerosis Treatment Study (FATS)l1 and the St. Thomas Atherosclerosis Regression Trial (STARS).89 Both of these studies had high proportions of patients with symptomatic coronary artery disease at trial entry, but intensive lipid lowering was associated with decreases in clinical event rates of 70% to 80%. One proposed mechanism for this reduction in events is that lowering of serum cholesterol may decrease plaque lipid, as has been documented in animal models, thus stabilizing those lipidrich plaques that are most prone to rupture. 12 APPROACHES TO ATHEROSCLEROSIS THERAPY

Based on our current understanding of atherogenesis, the effects of various strategies to reduce the progression of the disease can be grouped into the following categories: 1. Decreased accumulation of lipid in the arterial wall. Strategies to accomplish this goal would include decreasing levels of atherogenic lipoproteins, such as LDL, through dietary changes and pharmacologic therapy, or increasing levels of antiatherogenic lipoproteins, such as HDL. Modification of factors that can damage endothelial integrity, such as cigarette smoking, diabetes, and hypertension, would also decrease lipid deposition. 2. Reduction of physical/hemodynamic damage to the arterial wall. This goal can be met through treatment of hypertension, preferably with agents that do not adversely affect lipid profiles. 3. Decreased oxidant stress through treatment of diabetes and cessation of smoking and possibly through the use of antioxidants. 4. Decrease in central obesity and its accompanying insulin resistance, which may result in hypertension, diabetes, and increased prevalence of small, dense LDL. FUTURE DIRECTIONS

Future directions worthy of investigation include (1) reduction in levels of lipoprotein(a); (2) prevention of the range of atherogenic effects

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ascribed to oxidized lipoproteins through therapy with antioxidants such as vitamins C, E, and beta-carotene, either alone or in combination, or pharmacologic antioxidants such as probucol; (3) prevention of inflammatory cell recruitment into plaques through application of antiadhesion agents; (4) limiting lipoprotein deposition in the artery wall by inhibition of proteoglycan synthesis or inhibition of lipoprotein/proteoglycan interactions; (5) inhibiting expression of proatherogenic cytokines or growth factors within the artery wall; (6) scavenger receptor inhibitors; and (7) anti-inflammatory drugs. If atherogenesis proceeded through an orderly sequence of events, intervention at anyone step might retard or prevent the process. Atherogenesis, however, involves the complex interplay of multiple factors, as illustrated in Figure 4. The challenge is to find a factor or set of factors that are relatively specific to atherogenesis and that can be targeted for intervention without adversely affecting the beneficial roles of these factors in other biologic processes. For example, prevention of adhesion molecule expression would inhibit recruitment of mononuclear cells into the artery wall and retard atherogenesis, but if adhesion molecule expression were inhibited in all vascular tissue, the ability to fight infection or to maintain immunologic surveillance against malignancy would be impaired. Likewise, if altering the expression of a particular cytokine retarded atherogenesis but interfered with other important biologic processes, the net effect could be deleterious. Also, attempts to target a particular process, such as scavenger receptor-mediated lipoprotein uptake, VCAM-l-mediated mononuclear cell attachment, or PDGF-mediated SMC proliferation, may not be effective if there are other mechanisms by which cells can accumulate lipoproteins, other molecules that may mediate mononuclear cell attachment, or other cytokines or growth factors that stimulate SMC proliferation. Thus the most effective antiatherogenic strategies are those that intervene either at earlier points in the process or at multiple points. For example, if atherogenesis represents a response to vascular injury, it would make the most sense to prevent the initial injury, as might be accomplished through some combination of modification of lipid deposition, treatment of hypertension, discontinuation of cigarette smoking, and treatment of diabetes. Also, treatment of factors that could amplify the inflammatory response to injury would be important. In contrast, intervention at later points that may represent reparative responses, such as SMC migration or protein synthesis, could in fact be harmful if they decreased plaque stability. An example of an intervention that might retard atherogenesis at several steps is inhibition of lipoprotein oxidation. Decrease in the oxidative modification of lipoproteins could have multiple effects, including (1) decreased endothelial cell expression of monocyte chemoattractants, adhesion molecules, and growth factors; (2) decreased endothelial cell damage; (3) decreased uptake of oxidatively modified lipoproteins by arterial wall cells; and (4) decrease in the stimulation of inflammatory cytokine and growth factor production by macrophages and SMC.

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O'BRIEN & CHArT

Lumen

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Figure 4. An overview of events involved during atherogenesis. Lipoprotein deposition in the

intima may result from an excess of lipoproteins such as low density lipoprotein (LDL) in the circulation and occurs preferentially at sites of endothelial injury. These lipoproteins then undergo mild oxidative modification in the subendothelial space to form mildly oxidized LDL which can then induce endothelial cell (EC) expression of chemotactic factors which attract monocytes, and of mononuclear cell adhesion molecules by which monocytes and T Iympho· cytes enter the subendothelial space. Colony stimulating factors (CSFs) secreted by EC in response to stimulation by mildly oxidized LDL induce the differentiation of monocytes into macrophages. Cytokines secreted by T Iymphocytes activate macrophages, resulting in the release of several growth factors and cytokines which act on both intimal and medial arterial smooth muscle cells (AS MC) to stimulate proliferation, migration, and synthesis of matrix proteins. Macrophages and ASMC secrete angiogenic factors which stimulate neovascular ingrowth from the vasa vasorum. This neovasculature then provides routes for further lipoprotein deposition and mononuclear cell infiltration. Macrophages then differentiate into foam cells through ingestion of large amounts of lipid in the form of extensively oxidized LDL, lipoprotein-antibody complexes and/or complexes of proteoglycans secreted by ASMC and LDL. Efflux of excess cholesterol from foam cells could be mediated by HDL and by secretion of apo E-lipid vesicles.

SUMMARY

A great deal of progress has been made in the past few years in our understanding of the processes involved in atherogenesis and in mechanisms by which commonly accepted risk factors may affect these processes. These insights have allowed us to understand how various interventions may retard atherogenesis and decrease clinical events by improving plaque stability. The identification of new risk factors, such as lipoprotein(a), and of particular molecules that can be identified in atherosclerotic tissue, such as adhesion molecules, growth factors, cyto-

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kines, and proteins that regulate cholesterol uptake and removal, have identified several potential new targets for therapeutic intervention. Advances in molecular biologic techniques, induding transgenic techniques, have markedly increased the types of potential interventions available. A major challenge for the future will be to determine which among this plethora of therapeutic possibilities holds the most promise for decreasing the morbidity and mortality associated with this disease. ACKNOWLEDGMENT The authors thank Lisa Anne Billings for typing the manuscript.

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Address reprint requests to Kevin D. O'Brien, MD Division of Cardiology, RG-22 University of Washington Seattle, WA 98195