Roles of lipoproteins in the initiation and development of atherosclerosis

Pharmac. Ther. Vol. 31, pp. 237 to 255, 1985 Printed in Great Britain. All fights reserved

0163-7258/85 $0.00+0.50 Copyright © 1987 Pergamon Journals Ltd

Specialist Subject Editors: W. L. HOLMESand D. K.RITCI-IEVSKY

ROLES OF LIPOPROTEINS IN THE INITIATION AND DEVELOPMENT OF ATHEROSCLEROSIS ALAN DAUGHERTY a n d GUSTAV SCHONFELD

Cardiovascular Division and Lipid Research Center, Departments of Medicine and Preventive Medicine, Washington University School of Medicine, St Louis, MO 63130, U.S.A.

1. INTRODUCTION Atherosclerosis is directly implicated in the majority of deaths in the Western world. Several possible mechanisms that initiate and enhance the growth of atherosclerotic lesions have been identified. Of greater importance to patients is that with proper medical management, using diets and drugs, atherosclerotic lesions can be halted in their progression and they may even regress. Nevertheless, more effective regimens to retard or reverse plaque formation are needed. However, these are difficult to develop because the disease is multifactorial in etiology, lesions are complex consisting of several cell types, interstitial fibers and glycosaminoglycans and they are extremely slow in their development and regression. Results of preventive or curative interventions are also difficult to assess because there is no simple, reliable, precise methodology to assess atherosclerosic lesions non-invasively on a sequential basis. Consequently, the assessment of atherosclerosis in patients commonly relies either on expensive, somewhat risky and uncomfortable invasive angiographic techniques or on the assessment of indirect end-points or indexes of the disease, such as clinical symptoms and the measurement of coronary risk factors. Many risk factors for development of atherosclerotic disease have been documented; they include plasma lipids and lipoproteins (Kannel et al., 1971a; Matthews et al., 1977), arterial blood pressure (Kannel et al., 1971b), smoking (Herbert, 1975), a 'Type A personality' profile (Jenkins, 1976), obesity (Keys et al., 1972), diabetes mellitus (Keen, 1976) and many others (Hopkins and Williams, 1981). Among the most consistent prognosticators of clinically manifest atherosclerosis are the plasma concentrations of certain lipoproteins. It is our purpose to review the evidence that supports this association, including epidemiological studies and clinical trials. However, most of this review will deal with the experimental evidence linking lipoproteins and atherosclerosis. 2. ATHEROSCLEROSIS--PATHOLOGY AND MORPHOLOGY The term atherosclerosis is derived from the Greek words 'athera' and 'skeratic', which refer to the gruel-like center of the complicated lesions and the hardness of the vessel wall, respectively. Morphologically atherosclerotic lesions have been divided into three classes: fatty streaks, fibrolipid plaques and complicated lesions. The current concept is that these different forms represent stages in the natural progression of lesion formation and maturation. The hypothesis of the transition of fatty streaks to fibrolipid plaques is supported by pathologic examination of lesions in humans of different ages (McGill, 1974), and of animal models (Faggiotto and Ross, 1984; Faggiotto et al., 1984), including non-human primates, and by following the progressive changes of the morphology of lesions and the physiochemical characteristics of deposited lipids (Small, 1977; Katz et al., 1976). Fatty streaks are characterized by excessive lipid deposition in both the intracellular and extraceUular compartments (Geer et al., 1961; Smith and Slater, 1972; Lang and Insull, Supported by NIH Grants HL15308, HL32000, HL17646 Specialized Center of Research in Ischemic Heart Disease and the Lipid Research Clinics Contract NHLBI N01-HV2-2916. 237

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A. DAUGHERTVand G. SCHONFELD

1970). Although the endothelium covering fatty streaks is generally intact, the cells tend to have a swollen appearance. Foam cells predominate in the intima. Foam cells probably originate from monocyte-derived macrophages but lipid-laden myocytes are also present (Schaffner et al., 1980; Fowler et al., 1979). Fibrolipid plaques have fibrous caps on their endothelial surfaces and consist of proliferating myocytes, some of which are of monoclonal origin (Benditt and Benditt, 1973) and some macrophage-monocytes. Extracellular lipids, fibrosis and some products of cellular necrosis are present also in the centers of the lesions. Complicated lesions are larger, they may encroach on blood flow significantly, they may contain even more fibrotic material and their centers are necrotic and to a large extent calcified. The surfaces of such lesions may be ulcerated and, if there has been dissection of the plaque by blood, the centers may be hemorrhagic. 3. THEORIES OF ATHEROSCLEROSIS The events which are thought to lead to the initiation and progression of atherosclerosis have been grouped under several 'theories', including the 'infiltration' (Watts, 1971), 'incrustation' (Duguid, 1946), 'monoclonal proliferation' (Benditt and Benditt, 1973) and 'response-to-injury' theories (Ross and Glomset, 1976). The latter is by far the most popularly accepted concept because it encompasses many elements of the others and reflects the multifactorial nature of the atherosclerotic process. According to this theory, the initiating event in atherogenesis is endothelial damage caused by chemical, viral immunologic or mechanical means. At the site of endothelial damage, platelets adhere and release platelet-derived growth factor (PDGF) which penetrates into the intima and media (Ross et al., 1974). Macromolecules, such as lipoproteins, which ordinarily pass through the endothelium only in very small amounts, also penetrate unhindered. PDGF initiates migration and proliferation of smooth muscle cells to the site of injury, where the smooth muscle cells modulate from the 'contractile' to the 'synthetic' state and proliferate (Campbell and Campbell, 1985). Synthetic cells produce and deposit interstitial fibers, such as collagen, elastin, and glycosaminoglycans. Endothelial cells proliferate and migrate to cover any areas denuded of endothelial cover. Macrophages migrate into the injured area to scavenge cell debris and macromolecules. As a result of this activity, a few days to weeks after injury, a plaque is present which on the luminal side is partially covered by endothelium and partially by smooth muscle cells. Inside the plaque there are smooth muscle cells and macrophages containing lipid droplets and interstitial fibers lined by lipoproteins. The discontinuity in the cell wall has been repaired. During a routine cycle of injury and repair the clump of cells and fibrolipid material that has accumulated at the site of injury is 'remodelled' and the plaque gradually disappears. After several months, the artery wall re-assumes its normal state. However, when the injury is sustained or repeated, attempts at repair continue and larger amounts of cells and fibrotic material continue to be deposited at the sites of injury. Alternatively, if regression of the plaque is incomplete, abnormal vessel morphology persists. Lipoproteins continue to accumulate in plaques between cells complexed with glycosaminoglycans, and within cells the products of lysosomal degradation of lipoprotein accumulate as cholesteryl esters. Uptake of lipoproteins by smooth muscle cells is mediated by low density lipoproteins (LDL) (apolipoprotein B, E) receptors. Macrophages endocytose native unmodified LDL and fl-very low density lipoproteins (fl-VLDL) via LDL receptors, modified LDL via scavenger receptors, and fl-VLDL via specific fl-VLDL receptors. 4. EPIDEMIOLOGICAL AND CLINICAL TRIALS EVIDENCE OF LIPOPROTEIN INVOLVEMENT IN ATHEROSCLEROSIS The association between total plasma cholesterol concentrations and the prevalence and incidence of atherosclerotic disease is well established in free-living populations from both

Roles of lipoproteins in atherosclerosis

239

cross-sectional and prospective longitudinal studies of human societies in many parts of the world (Conner and Conner, 1972; Keys et aL, 1958; Westlund and Nicolaysen, 1972; Kannel et al., 1971a; Pooling Project Research Group, 1978; Table 1). There is also a positive univariate correlation between hypertriglyceridemia and the prevalence and incidence of coronary heart disease in virtually all populations, but in most studies that used multivariate analysis, other factors were found to be more predictive than triglycerides (Rosenman et al., 1976; Heyden et al., 1980). However, in the Stockholm Prospective Study, total plasma triglycerides were elevated in survivors of acute myocardial infarction when compared with those of a control group (Carlson and Lindstedt, 1969), and subsequent studies confirmed that triglycerides were independent coronary risk factors even on multivariate analysis (Carlson and Bottiger, 1972; Carlson et al., 1979). There are also strong correlations between particular classes of lipoproteins and clinical manifestations of atherosclerosis. As early as 1954, Gofman et al., demonstrated that mean concentrations of LDL were higher in patients with coronary heart disease. This LDL-atherosclerosis connection has been amply confirmed in many cross-sectional and prospective epidemiological studies of free-living populations (Castelli et aL, 1977; Wilson et al., 1980), as well as in patients undergoing coronary angiography. Concentrations of both plasma LDL cholesterol and apoliproprotein B (the sole protein component of human LDL) have predictive value (Albers et al., 1984). A very strong link between LDL and atherosclerosis has been provided by the findings of the Lipid Research Clinics Coronary Primary Prevention Trial (1984a,b) and by the NHLBI Trials (Brensike et al., 1984). Lowering of plasma LDL cholesterol concentrations in hypercholesterolemic middle-aged men resulted in a significantly lower incidence of coronary heart disease events, and lesion progression, respectively. In both studies, the degree of lowering of risk was directly related to falls in LDL cholesterol or the LDL: high density lipoproteins (HDL) cholesterol mass ratio. By contrast, an inverse relation exists between plasma HDL cholesterol concentrations and coronary heart disease (Brunner and Lobl, 1958; Gofman et al., 1966). Numerous other reports have confirmed this effect in population surveys (Miller and Miller, 1975; Gordon et al., 1977; Goldbourt and Medalie, 1979) and in patients with angiographicallydefined coronary artery disease (Tan et al., 1980; Miller et al., 1981). Sequential arteriographic assessment of patients demonstrated that atherosclerotic lesion growth was retarded in the group which had high HDL cholesterol concentrations when compared with total plasma cholesterol (Arntzenius et al., 1985). It appears that the HDL 2 subfraction is more strongly predictive than HDL3 of the severity of disease (Miller et al., 1981). Plasma concentrations of apolipoprotein AI the major apolipoprotein of HDL are also correlated with the incidence of coronary artery disease (Kukita et al., 1984), and the severity of angiographically detectable disease is closely related to the ratio of apolipoprotein AI to apolipoprotein B in plasma (Sedlis et al., 1986). 5. GENETICALLY DETERMINED LIPOPROTEIN DISORDERS ASSOCIATED WITH ATHEROSCLEROSIS Several genetically determined disorders of lipoproteins are associated with the presence of atherosclerosis (Brunzell and Miller, 1981; Schonfeld, 1983). Homozygous patients with familial hypercholesterolemia (Goldstein and Brown, 1975) have 5-8 times higher than normal concentrations of plasma LDL (Table 1). In these patients, atherosclerosis develops rapidly, with myocardial infarction frequently occurring before the age of 20 years. Heterozygous patients have LDL cholesterol concentrations approximately 2.5 times higher than normal. Myocardial infarction commonly occurs 20 years earlier than in non-affected individuals of the same kindreds, frequently during the fifth decade of the patients' lives. The genetic deficiency of these patients is traceable to defects in the high-affinity LDL (apolipoprotein B, E) receptor which is present on virtually all mammalian cells and is primarily responsible for mediating the endocytosis, and hence the subsequent intracellular catabolism of LDL particles. Mutations of the LDL receptor gene

240

A. DAUGHERTY and G. SCHONFELD TABLE 1. Lipid and Lipoprotein Plasma Concentrations in Fasted American Causacian Males on the Lipid Research Clinics Population Studies (1979) Age (yr) 5-9 11~16 20-24 35 39 45~,9 55-59 65-69 70 M=mean,

Total cholesterol (mg/dl)

Total triglycerides (mg/dl)

LDL cholesterol (mg/dl)

HDL cholesterol (mg/dl)

M 155 160 162 200 213 215 221 210

M 52 63 89 144 143 134 139 133

M 93 97 103 133 144 146 150 143

M 56 55 45 43 45 48 51 55

90 183 188 197 248 258 260 275 253

90 70 94 146 250 218 210 217 202

90 117 122 138 176 186 191 199 183

10 42 40 32 31 33 31 33 33

10 and 9 0 = 10th and 90th percentiles, respectively.

can result in the complete absence of receptor protein or the production of dysfunctional receptor proteins (Tolleshaug et al., 1982). An animal model of familial hypercholesterolemia also exists. The Watanabe hereditable hyperlipidemic (WHHL) rabbit (Watanabe et al., 1977; Watanabe, 1980; Bilheimer et al., 1982) carries most of its cholesterol in LDL, d 1.019-1.063 g/ml (Havel et al., 1982), and its atherosclerotic lesions closely resemble the complex lesions observed in humans at autopsy (Buja et al., 1983). However, it should be noted that conditions are not identical in rabbits and man; patients with familial hypercholesterolemia frequently have normal triglyceride levels. In addition to hypercholesterolemia, W H H L rabbits also have marked hypertriglyceridemia (Wakasugi et al., 1984). Consequently, although the presence of atherosclerotic lesions in man appear to be related to isolated elevations of LDL, the rabbit lesions are not necessarily related solely to elevated plasma LDL concentrations. Dysbetalipoproteinemia (Fredrickson, 1971) is characterized by the presence of very low density lipoproteins (VLDL), which have fl-migration on electrophoresis (rather than the usual prefl-migration) and enhanced contents of cholesteryl esters. Plasma LDL cholesterol concentrations may be normal, high or low. These patients either lack apolipoprotein E or have any of several mutant dysfunctional forms of apolipoprotein E. This mutation is important because a domain of apolipoprotein E serves as a recognition site on lipoproteins for several cellular membrane receptors, including the LDL receptor, the chylomicron-remnant receptor on hepatocytes and the fl-VLDL receptor on macrophages. Defective apolipoprotein E also delays the lipoprotein-lipase-catabolized hydrolysis of VLDL and chylmicron triglycerides. Thus, defects result in the accumulation of partially catabolized products of chylomicrons and VLDL, such as fl-VLDL and intermediate density lipoproteins (IDL) in plasma (Hui et al., 1984; Ehnholm et al., 1984). These lipoproteins contain apolipoprotein E and ordinarily they are removed from plasma or converted to LDL. Greatly elevated concentrations of fl-VLDL and IDL (type III hyperlipoproteinemia) occur when, in addition to the apolipoprotein E defect, a second metabolic defect is present, such as obesity, hypothyroidism or heterozygosity for hypertriglyceridemia or hypercholesterolemia. Such hyperlipidemic patients exhibit greatly accelerated rates of clinically manifest atherosclerotic disease. In addition to these (and other) lipoprotein disorders characterized by elevations of 'atherogenic' plasma lipoproteins, decreased plasma concentrations of 'antiatherogenic' HDL are also associated with increased progression of atherosclerosis. Patients with Tangier disease, whose H D L concentrations are extremely low due to a defect in the metabolism of apolipoprotein AI, manifest enhanced susceptibilities to atherosclerosis in both the homozygous and heterozygous states (Schaefer et al., 1980). Patients with combined apoliprotein AI/apoliprotein CIII deficiency have exceedingly low plasma HDL concentrations and severe coronary heart disease (Norum et al., 1975). Genetic defects resulting in the presence of dysfunctional lecithin :cholesterol acyltransferase (LCAT) molecules or in the absence of LCAT are also associated with premature atherosclerosis

Roles of lipoproteins in atherosclerosis

241

(Gjone, 1974). Conversely, familial hyperalphalipoproteinemia is associated with longevity in affected individuals (Glueck et al., 1976). 6. EXPERIMENTAL ASSOCIATIONS OF LIPOPROTEINS AND ATHEROSCLEROSIS Studies of lipoprotein-fiber matrix interactions have been reviewed elsewhere (Camejo, 1982). In the following sections, we shall review the roles of the major lipoprotein classes in atherogenesis. The lipoprotein receptors that are present in arterial tissue are summarized in Table 2. 6.1. CHYLOMICRON REMNANTS Despite the known correlation between elevated plasma cholesterol concentrations and atherosclerosis, coronary artery disease frequently occurs in individuals who have apparently normal fasting plasma lipid concentrations. To resolve this apparent paradox, it was proposed that intestinally derived lipoproteins may be atherogenic, but they are present only transiently after a fat-containing meal and ordinarily they are not detected in plasma collected after 12-14 hours of fasting (Zilversmit, 1979; Redgrave, 1984). Some of the evidence in support of this position has been derived from rabbits fed cholesterol-enriched diet. Within a few days after initiation of cholesterol feeding, rabbits develop gross hypercholesterolemia (Camejo et al., 1974) and after 2-3 weeks virtually all the plasma lipoproteins are present in the d < 1.019 g/ml ultracentrifugal density fraction (Roth et al., 1983). Originally, this lipoprotein fraction was thought to consist primarily of partially catabolized intestinal lipoproteins, i.e. chylomicron remnants (Ross and Zilversmit, 1977; Thompson and Zilversmit, 1983; Redgrave et al., 1976). Therefore, it was postulated that the chylomicron remnants in plasma were responsible for the very rapid appearance of atherosclerotic lesions in the vasculature of cholesterol-fed rabbits. The chylomicron remnant-atherosclerosis connection is disputed on two grounds. First, the dominant lipoproteins in plasmas of cholesterol-fed rabbits appear to be not chylomicron remnants but liver-derived fl-VLDL (Daugherty and Schonfeld, unpublished observations). Second, the penetration of chylomicron remnants into intact arterial wall and their presence there has yet to be demonstrated. In fact, uptake of cholesterol derived from chylomicrons or chylomicron remnants by the arterial wall in vivo is negligible in hypercholesterolemic rabbits (Stender and Zilversmit, 1981). Although constituents of chylomicron remnants, such as apolipoproteins C and E and cholesteryl esters, are present in atheromatous lesions (Onitri et al., 1976; Hoff and Goubatz, 1977), these constituents are also present in other lipoproteins, which could have acted as the primary carriers into the arterial wall. Thus, few data are available to support delivery of cholesterol into arterial wall by chylomicron remnants in vivo. Furthermore, although chylomicron remnants do interact with cultured fibroblasts and smooth muscle cells in culture (Redgrave et al., 1982; Floren et al., 1981), probably via LDL receptors, it is not clear that remnants can stimulate lipid accumulation in aortic myocytes (Kenagy et al., 1982). 6.2. VERY LOW AND INTERMEDIATE DENSITY LIPOPROTEINS There is little evidence linking freshly secreted normal VLDL to atherogenesis since these VLDL contain only small amounts of cholesteryl ester and do not interact very avidly with cellular receptors when incubated with cultured cells. Studies in vivo to examine deposition of normal VLDL in arterial tissue are not available. By contrast, VLDL obtained from hypertriglyceridemic patients (HTG-VLDL) induced marked triglyceride synthesis and accumulation in cultured mouse peritoneal macrophages (Gianturco et al., 1982). HTGVLDL also suppresses fl-hydroxy-fl-methylglutaryl-CoA-reductase activity, and stimulates oleate esterification to cholesterol in cultured human fibroblasts (Gianturco et al., 1978; Gianturco et al., 1980; Krul et al., 1985). Dogs that respond to cholesterol-enriched diets with a marked hypercholesterolemia ( > 750 mg/dl) have VLDL present in their plasma which is cholesteryl ester-enriched and migrates in the fl-position on electrophoresis (fl-VLDL; Mahley et al., 1974), whereas

Cell

Myocytes Endothelial Monocyte-macrophages

ApoE ApoB-48 Modified ApoB- 100

fl-VLDL MDA-LDL ENDO-LDL GAG-LDL HDL 3 ApoAI

ApoB-100 ApoE

LDL fl-VLDL HDLc

Ligand

Upregulated by increase in cellular cholesterol

Unregulated

Upregulated by increased cholesterol

Downregulated by increased cellular cholesterol

Receptor regulation

TABLE 2. Lipoprotein Receptors of Arterml Tissue*

Lipoprotein bound

Removes excess cholesterol from cells

Promotes formation of foam cells

Promotes formation of foam cells

Not known

Role in atherosclerosis

*Adapted from Mahley and lnnerarity (1983). Abbreviations are: apolipoprotein B-100, ApoB-100; Apolipoprotein B-48, ApoB-48; Apolipoprotein E, ApoE; apolipoprotein AI, ApoAI; malondialdehyde-modified LDL, MDA-LDL; endothelial-modified LDL, ENDO-LDL; glycosaminoglycan-coupled,GAG-LDL.

HDL

LDL (apoB,E) Myocytes Fibroblasts Endothelial Monocyte-macrophages /~-VLDL Monocyte-macrophages Endothelial Modulated myocytes Scavenger Monocyte-macrophage Endothelial

Receptor

.¢'~

>

tO

tO

Roles of lipoproteins in atherosclerosis

243

VLDL obtained from normolipidemic animals are triglyceride-enriched and migrate in the prefl-position (~t-VLDL). The diet-induced persistence over weeks to month of fl-VLDL in plasma is followed by the appearance of aortic atherosclerosis. Cholesterol-fed rabbits (Camejo et al., 1974), rats (Mahley and Holcombe, 1977; Cole et al., 1984), Patas monkeys (Mahley et al., 1976) and swine (Mahley et al., 1975) also accumulate excessive concentrations of cholesteryl ester-rich fl-VLDL in plasma in association with the appearance of atherosclerotic lesions (Anitschkow, 1913; Mahley, 1982). Direct evidence linking fl-VLDL to atherosclerotic lesion was produced in cholesterolfed rabbits intravenously injected with 1~SI-labeled fl-VLDL. The fl-VLDL was localized selectively in atheromatous lesions of aortas (Daugherty et aL, 1985). In aortae of normal rabbits, ~25I-labeled fl-VLDL also appear to be deposited more avidly than the prefl-migrating VLDL harvested from normolipidemic plasma (Rodriguez et aL, 1976). fl-VLDL interact with several cells in culture (Mahley et al., 1980b; Hui et al., 1984). In fibroblasts and smooth muscle cells, fl-VLDL are endocytosed via LDL (apolipoprotein B, E) receptors. Apolipoprotein B-100 and/or apolipoprotein E on the lipoprotein particles serve as recognition sites for these receptors. Macrophages, the cell type thought to be the precursor of foam cells (Fowler et al., 1979; Klurfeld, 1985), also interact with fl-VLDL. The foam cells in explants of atheromatous aortae of hypercholesterolemic rabbits selectively accumulate fl-VLDL (Pitas et aL, 1983), and fl-VLDL are taken up by macrophages in primary culture, causing massive accumulation of cholesteryl esters and the appearance of lipid droplets in the cell (Goldstein et aL, 1980). These lipid-laden cells have morphological similarities to the foam cells of arterial lesions. Macrophages endocytose fl-VLDL primarily via a specific fl-VLDL receptor. Using competitive binding studies of fl-VLDL against postprandial human d < 1.006 g/ml lipoproteins on human monocyte-macrophages, Van Lenten et al. (1983) concluded that apolipoprotein B-48 served as the recognition marker for the fl-VLDL receptor. Also in competitive binding studies, where fl-VLDL and apolipoprotein E-phospholipid microemulsions competed with each other for receptor occupancy, Wang-Iverson et al. (1985) concluded that apolipoprotein E mediated the uptake and degradation of fl-VLDL. These disparate interpretations are due to the inherent limitations of competitive binding assays. Apolipoproteins can exchange between fl-VLDL and the competing particle, thus changes in the cellular metabolism of fl-VLDL not related to apolipoprotein content of the native particle could be produced. Another factor that complicates interpretation of results obtained with cultured macrophages is that these cells secrete apolipoprotein E (Basu et aL, 1983; Werb and Chin, 1983), lipoprotein lipase (Khoo et al., 1981; Mahoney et aL, 1982) and a variety of hydrolytic enzymes into the medium (Werb, 1983). These secreted products may alter the fl-VLDL incubated with the cells, so that the particle ingested by macrophages may not necessarily represent the particle initially presented to the cells. Mahley's group has overcome some of these difficulties in experiments where human fl-VLDL recombined either with normal apolipoprotein E 3 or dysfunctional apolipoprotein E2 were presented to macrophages. Only fl-VLDL, which contained functional apolipoprotein E, were taken up, suggesting that apolipoprotein E served as the recognition marker for the fl-VLDL receptor of macrophages (Innerarity et aL, 1986). Regulation of the activity of the cellular fl-VLDL receptor has been studied in mouse peritoneal macrophages and human monocyte/macrophage (Soutar and Knight, 1984; Van Lenten et aL, 1983). The consensus from these studies is that the activity of the fl-VLDL receptor on macrophages is downregulated when the cholesterol concentration of media is reduced. A further consideration in the atherogenic mechanism of fl-VLDL relates to the structural and metabolic heterogeneity of these lipoproteins (Fairnaru et aL, 1982). fl-VLDL derived from cholesterol-fed dogs can be fractionated according to size into two distinct subpopulations. The larger subpopulation has characteristics consistent with chylomicron remnants. The smaller is similar in size to VLDL harvested from normolipidemic plasma; it is greatly enriched in cholesteryl esters and apolipoprotein E and thought to be hepatic in origin. Similar structural and metabolic heterogeneity has also

244

A. DAUGHERTYand G. SCHONFELD

been described for fl-VLDL of rabbits (Daugherty et al., 1986a) and rhesus monkeys (Lusk et al., 1982). When incubated with cultured mouse peritoneal macrophages (Fainauru et al., 1982), the larger fl-VLDL subfraction is a more powerful stimulant of the intracellular synthesis of cholesteryl esters than the smaller. These differences were not accounted for by differences in cholesterol contents of the fl-VLDL subfractions, suggesting that some structural features of the two subfractions were distinguished from each other by macrophages. In summary, experimental studies favor atherogenic roles for hepaticderived lipoproteins such as fl-VLDL and HTG-VLDL (Gianturco et al., 1982). Chylomicron remnants isolated from cholesterol-fed animals and IDL probably also are atherogenic. 6.3. Low DENSITY LIPOPROTEINS Dietary-induced hypercholesterolemia is associated with an elevation of plasma LDL concentrations in many species (Mahley, 1978), including man (Cole et al., 1983; Cole et al., 1985). Consequently, considerable attention has centered on the role of LDL in the development of atherosclerotic plaques. Heterogeneities of LDL structure and compositions are known to exist (Fisher, 1983), and changes in the physical and chemical composition occur on induction of hypercholesterolemia. During diets high in fat and cholesterol, LDL cholesteryl ester contents and particle sizes increase in several species of animals, including rat (Mahley and Holcombe, 1977), dog (Mahley et al., 1974), swine (Pownall et al., 1980; Mahley et al., 1975) and monkey (Mahley et al., 1976; Rudel et al., 1977; Fless et al., 1982; St. Clair et al., 1980). Hypercholesterolemic LDL promotes more cholesteryl ester deposition in cells than LDL harvested from normolipidemic subjects, because of the increased content of cholesteryl ester in the lipoprotein core (St. Clair and Leight, 1978) which results in an increased influx of lipid (St. Clair and Leight, 1983). Also, diet-induced increases in LDL concentrations and particle size are positively correlated with the incidence and severity of atherosclerosis in monkeys (Rudel et al., 1979). Although LDL concentrations do increase modestly in man on cholesterol feeding, the compositions and sizes of the particles are altered only slightly (Schonfeld et al., 1982; Cole et al., 1983; Cole et al., 1985). The most extensive studies of lipoprotein arterial uptake in vivo have been performed using LDL, usually radiolabeled in the protein moiety. In studies of normolipidemic rabbits by Bratzler et al. (1977), rabbits were killed at various times following the injection of radioiodinated human LDL, and the distribution of radioactivity across the wall of descending aorta determined. For periods up to 4 hr post-injection, radioactivity was greatest at the intimal surface, with gradually decreasing amounts being present between intima and the adventitial layers. At later intervals, relatively more radioactivity was present in the medial layers. It was concluded that the major site of entry of LDL into the vessel was via the lumen of the artery rather than the vasa vasorum. Anatomically, normal vascular endothelium forms a continuous unbroken monolayer and the endothelium acts as a retarding barrier to the flow of the macromolecules of plasma into the arterial wall. However, since LDL do penetrate normal vessels via the luminal surface (Bratzler et al., 1977; Hoff and Gaubautz, 1977), they must pass either between endothelial cells without disturbing the monolayer sufficiently to be detected by electron microscopy or through the cells by vesicular transport. Current morphological evidence favors the latter. This form of vesicular transport appears to operate independently of the LDL receptor, as native and acetylated LDL are transported into arterial wall in vivo at similar rates (Vasile et al., 1983; Wiklund et al., 1985). Damage to the endothelium sufficient to cause focal denudation permits large masses of LDL to accumulate in vascular tissue and may promote atherosclerotic lesion formation (Alavi and Moore, 1984). Indeed, elevated plasma concentrations of LDL are cytotoxic to endothelium and may compromise its integrity (Henriksen et al., 1979). Recent pathologic techniques in which vascular tissue is pressure-fixed prior to morphological examination demonstrate that discernable loss of vascular endothelium does not occur in

Roles of lipoproteins in atheroselerosis

245

early stages of atherosclerosis (Faggiotto et al., 1984; Faggiotto and Ross, 1984). Yet excessive LDL deposition in arterial wall occurs early in atherosclerosis thus suggesting enhanced transport. Therefore, either there are subtle functional changes in the endothelium not detectable by morphological methods that permit excessive influx of LDL or their are changes in LDL that enhance their rates of penetration into the artery wall. Attempts have been made to quantify accumulation of LDL in 'healthy' areas of arteries and atherosclerotic lesions using ~25I-labeled LDL and radioactive counting of tissues and autoradiography. Lesioned areas accumulate more radioiodinated LDL than unlesioned areas and aortae of control animals (Slater et al., 1982), suggesting that LDL is clearly a vehicle for delivering cholesterol into lesions. However, the quantitative accuracy of studies of arterial (or other tissue) accumulation of LDL by means of LDL radioiodinated by conventional techniques may be questioned when one considers the manner in which LDL is metabolized by cells. Following interaction with the LDL (apolipoprotein B, E) receptor, LDL is endocytosed (Goldstein and Brown, 1974), and transported to the lysosome where its components are degraded enzymatically. Apolipoprotein B is cleaved to its constituent amino acids, which leak rapidly from the lysosome. Radioiodination techniques commonly radiolabel the tyrosines of proteins (e.g. the iodine monochloride technique of McFarlane, 1958). The iodotyrosine, which leaks from lysosomes cannot be re-utilized for protein synthesis, presumably because of the steric hindrance of iodide which is large when compared with the amino acid. Consequently, breakdown products of radioiodinated LDL are lost rapidly from the cell. Thus, measurement in vivo of tissue uptake of 125I-labeled LDL at early intervals after intravenous injection will quantify a large component of radioactivity present in the extravascular space, leading to an overestimation of the actual uptake by tissues. The measurement of tissue radioactivity later will yield underestimates of tissue uptake. The slow rates of endocytosis relative to rates of intracellular transport and lysosomal degradation appear to be generally true for proteins. Therefore, conventional radioiodination leads to similar artifacts in the quantitation of catabolism of a variety of proteins. Quantitation of uptake in arteries is even more complex because not only are appreciable amounts of VLDL and LDL taken up and metabolized by cells, many particles are also trapped and complexed with fibers. To permit more accurate quantitation of tissue accumulation of proteins, radiolabeled 'residualizing' molecules covalently coupled to proteins have been developed (Van Zile et al., 1979; Baynes and Thorpe, 1981; Pittman et al., 1979). Residualizing molecules are mono- or oligosaccharides, which, because they are linked covalently to proteins, are delivered to lysosomes as part of the protein to which they are linked. But in contrast with iodotyrosine which rapidly leaks out after hydrolysis of the protein, radiolabeled saccharides remain entrapped within the organelle with residence times many times greater than that of iodotyrosine. Initially, the radionuclided saccharides were beta-emitters resulting in 'residualizing labels', such as 3H-raffinose (Van Zile et al., 1979) and t4C-sucrose (Pittman et al., 1979; Pittman et al., 1982). However, these beta emitting isotopes have limited application in vivo. Recently, residualizing molecules labeled with radioiodine have yielded products of considerably higher specific radioactivities. The schema for conjugation of these residualizing labels is shown in Fig. 1. These are much more useful in studies of lipoprotein catabolism (Pittman et al., 1983; Strobel et al., 1985; Daugherty et al., 1985b). Using LDL labeled with radioiodinated tyramine-cellobiose, Carew et aL (1984) demonstrated that LDL degradation in the intima (particularly in the endothelium of rabbit aortae) was over 40 times greater than in the media. The involvement of LDL receptors in the intimal catabolism of LDL was assessed by comparing the accumulation of ~25I-tyramine-cellobiose-labeled native LDL with that of similarly ~3q-labeled methylated LDL. Approximately 5 0 0 of intimal degradation was mediated by the LDL receptor, suggesting that penetration of LDL into arterial tissue is largely via receptor-independent transendothelial vesicular transport mechanisms, but LDL receptors greatly influence rates of degradation of LDL by the vessel wall (Wiklund et al., 1985). J.P.T. 31/~-F

246

A. DAUGHERTYand G. SCHONFELD

~H2OH LH2OH ~HOH O ~O--CH--(CHOH)=--CH2NH2 I ,H OOH / [,~ LACTITOL TYRAMINE OH ] SOLIDPHASERADIOIODINATION /

H,o. CH20H

t

~HOH

~J-~o r--O--CH-(CHOH)2--CH2NH

!

HCOOH CH=

°x'7-" GALACTOSE/

o .

-.,..

H,o.

OH

/ OI.--O--CH--ICHOHI J --CH-NH[ 125I~ry,

~ PROTE,N.NHCH 2

i.,L- O r--o- CH-ICHO%--CH~--NH

H2OH

~HOH

CI~ jN~_ .CI T("~T CYANURIC Nk""JN CHLORIDE C~I ~ ~PROTEIN

,~-

125 I

125I

O N...~.N

/7

- -

PROTEIN-NH

FIG. 1. Scheme of the conjugation of recently developed radioiodinated residualizing protein labels. The residualizing label shown is lactitol tyramine. The principal labels in present use are dilactitol tyramine and tyramine ceUobiose. Conjugation of labels to protein by reductive amination initially involves treating the label with galactose oxidase to form aldehyde residues. Conjugation to the protein of interest occurs in the sodium cyanoborohydride. The coupling of residualizing label to proteins by means of cyanuric chloride is represented on the right.

It is difficult to understand how arterial smooth muscle cells, which express apolipoprotein B, E receptors as their only identifiable lipoprotein receptors, can accumulate cholesterol during atherosclerosis in the presence of elevated concentration of extracellular cholesterol, since LDL receptor activity is downregulated under such conditions (Goldstein and Brown, 1977). One possible explanation is that as the smooth muscle cells of atherosclerotic lesions are modulated from the contractile to the synthetic state they lose their abilities to downregulate LDL receptors adequately. In fact, cultured contractile smooth muscle cells do not become overloaded with cholesterol after incubation with normal native LDL, whereas synthetic cells show marked lipid deposition (Campbell and Chamley-Campbell, 1981; Campbell et al., 1983). Of course, lipid accumulation could also be due to alterations of the post-receptor pathway that lead to cholesterol esterification, or the removal of cholesterol from cells could be impaired. Some combination of the above also is possible. An alternative explanation for the mechanism of atherogeneity of elevated concentrations of LDL is that, as a consequence of the longer residence time of LDL in plasmas of subjects with hypercholesterolemia, there is a greater propensity for chemical modification of the apolipoprotein of LDL, which could direct catabolism away from the LDL receptor-mediated pathways to the alternate catabolic scavenger pathways involving macrophages. Macrophages become even more cholesteryl ester-laden during atherogenesis than smooth muscle cells. The LDL receptors of macrophages are effectively downregulated by

Roles of lipoproteins in atherosclcrosis

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exogenous cholesterol (Soutar and Knight, 1984), but as mentioned, macrophages also possess scavenger receptors which do not recognize native LDL but do recognize and mediate the endocytosis of chemically modified LDL. In contrast with LDL receptors, the activities of scavenger receptors are not downregulated by exogenous cholesterol. The first reported chemical modification of LDL was acetylation, which produces a negatively charged particle (Basu et al., 1976). This negatively charged LDL is not recognized by cells such as fibroblasts and smooth muscle cells, but is avidly internalized by macrophages resulting in a dramatic increase in cellular cholesteryl ester contents (Goldstein et al., 1979; Brown et al., 1980a,b). Sufficient modification of lysine residues also redirects the metabolism of LDL to scavenger receptors (Mahley et al., 1979). However it is unlikely that LDL is acetylated in vivo. A physiologically more likely possibility discovered by Fogelman et al. (1980) is that exposure of LDL to malondialdehyde, a byproduct of arachidonate catabolism, redirects the recognition of LDL away from the LDL receptor towards the scavenger receptor. Whether malondialdehyde modifies lipoproteins under physiological conditions at the surface or in the intima of the vessel wall remains to be determined. LDL also is modified during incubation with cultured endothelial cells by a process not involving malondialdehyde (Henriksen et al., 1981). LDL isolated from 'conditioned media' of endothelial cells is avidly taken by cultured macrophages probably via the macrophage receptor. The modification involves the cleavage of the phospholipid moiety of LDL particles by the action of an endothelial cell-derived phospholipase A2, followed by proteolytic cleavage of apolipoprotein B. Proteolysis of apolipoprotein B may 'mask' or destroy recognition sites for the LDL receptor and 'uncover' new sites for the scavenger receptor (Parthasarathy et al., 1985). Steinberg and his coworkers have hypothesized that this modification of LDL in vivo may occur during the transendothelial transport of LDL from the lumen of the artery to the interstitial space, resulting in increased foam cell formation. In support of this concept, particles which display the metabolic characteristics ARTERIAL LUMEN I ENDO- t I THELIUM ,j

INTIMA

i I MACROPHAGE

MEDIA

MYOCYTE

F

EXTRACELLULAR MATRIX

)

LDL receptor Scavengerreceptor

Lysosome

FIG. 2. Potential sites for interaction of LDL with cells in arterial tissue. At the endothelium, LDL may be catabolized by a mechanism mediated by the LDL receptor. Alternatively, LDL in plasma may be modified by malondialdehyde (MDA) released from platelets and subsequently be recognized by scavenger receptors. LDL may pass through the endothelial barrier via a transendocytotic mechanism and may enter the subendothelial layer in either its native form or a modified form after the action of phospholipase A2 (PLA2). In the subendothelial layer, the native form of LDL may be catabolized by myocytes or interact with components of the extracellular matrix such as glycosaminoglycans. LDL which is modified by either the endothelium or components of the extracellular matrix may be recognized by the scavenger receptors present or monocyte-derived macrophages.

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A. DAUGHERTYand G. SCHONFELD

of modified LDL have been found in peripheral lymph (which should be representative of the interstitial fluid bathing cells of the arterial wall) and in extracts of atheromatous plaques (Hoff and Gaubautz, 1977; Reichl et al., 1978; Goldstein et al., 1981). In summary, elevated plasma concentrations of LDL may contribute to atherogenesis by entering the arterial wall through endothelium, complexing with glycosaminoglycans and entering modulated smooth muscle cells which are unable to downregulate their receptors. In addition, LDL modified by transendothelial transport are endocytosed by macrophages leading to excessive accumulation of cholesteryl esters by these cells as well (Goldstein and Brown, 1977; Fig. 2). 6.4. HIGH DENSITY LIPOPROTEINS

As noted earlier, negative correlations between incidence of clinical atherosclerosis and plasma H D L cholesterol concentrations are well established in humans. This correlation has not been studied in populations of animals, but many diet-induced atherosclerotic models in fact have reduced plasma H D L cholesterol concentrations, including rabbits (Roth et al., 1983), non-human primates (Faggiotto et al., 1984) and dogs (Mahley et al., 1974). The protective role of H D L is postulated to be due to its ability to inhibit cellular uptake of cholesterol from other lipoproteins (Carew et al., 1976) and its ability to facilitate the removal of cholesterol from cells directly or indirectly (Glomset, 1968; Miller and Miller, 1975; Miller et al., 1985). To exert its effects, H D L must penetrate into arterial tissue. It has been calculated that H D L accumulates more avidly in the aorta in vivo than larger lipoproteins (Stender and Zilversmit, 1981) and these authors propose that the rates of accumulation lipoproteins within aortic tissue are directly related to the diameters of the particles. However, dietary-induced hypercholesterolemia greatly decreases aortic accumulation of H D L and increases the accumulation of the larger/~-VLDL particles (Daugherty et al., 1985). Thus, the access of H D L particles to components of arterial tissue in vivo has not been delineated, particularly in the presence of atherosclerosis where they would be most needed. HDL reduces cholesteryl ester synthesis and accumulation in cultured mouse peritoneal macrophages (Innerarity et al., 1982), and removes cholesterol through a receptormediated retroendocytosis mechanism (Schmitz et al., 1985). Mature spherical HDL particles also facilitate removal of cholesterol from several other types of cultured cells (Miller et al., 1977; Tauber et al., 1981; Oram et al., 1983; Oram, 1983; Schmitz et al., 1985; Miller, 1978; Daerr et al., 1980; Biesbrock et al., 1983). However, typical H D L is not the only lipoprotein implicated in cholesterol removal, other mediators have been proposed. One indicator is a complex thought to consist of apolipoprotein AI, lecithin, LCAT enzyme and apolipoprotein D (cholesteryl ester transfer protein) (Fielding and Fielding, 1980). The cholesterol emuxing from cells transfers to the complex where it is esterified by lecithin:cholesterol acyltransferase; a reaction requiring the presence of apolipoprotein AI and during which phosphatidylcholine is converted to lyophophatidycholine. Cholesterol efflux per se does not require the presence of LCAT activity (Stein et al., 1978). But, removal of apolipoprotein AI from plasma abolishes the transport of cholesterol from cultured fibroblasts (Fielding and Fielding, 1981). Thus, apolipoprotein AI and phospholipids appear to be optimal requirements for the transfer of cholesterol away from cells. Newly esterified cholesterol is subsequently transferred by the cholesteryl ester transfer protein to the cores of nascent vesicular HDL; converting them first to the relatively small H D L 3particles and then to the larger HDL 2. The cholesteryl esters of H D L are distributed further to other lipoproteins by an action of the cholesterol ester transfer protein (Fig. 3). According to this concept, H D L are responsible for removal of cholesterol from cells but are not direct participants in the process. Another pathway for cholesterol removal may involve the budding of the phospholipid membranes of chylomicrons and VLDL (Tall and Small, 1978). The buds are thought to be pinched off to form monolayer vesicular structures consisting of phospholipids, cholesterol and apolipoprotein AI. These vesicles enter the lymph and eventually the blood stream where they serve as substrates for LCAT.

Roles of lipoproteins in atherosclerosis Cholesterol ~ PERIPHERAL CELLS

249

NASCENT HDL CHOLESTEROLESTERIFICATION (MediatedbyAPOAI+LCAT)

~11 ApoD / ~ ' ~ ApoAI ~ MATURE HDL /~ LCAT [ ] Cholesterol ~' CholestorylEsters / Phospholipid_d ~ CHOLESTERYL ESTERTRANSFER

FIG. 3. Potential mechanism by which HDL produces 'reverse cholesterol transport' from peripheral cell for delivery to hepatocyteswhere the sterol may be stored or catabolized. In the course of cholesterol esterification the esters move into the cores of vesicles converting them to spherical HDL-like particles. HDL], or HDL~ (see below) may be formed by this process. In man, approximately 65% and 25% of HDL protein (when all subfractions are pooled) consists of apolipoprotein AI and apolipoprotein AII, respectively. Apolipoprotein C comprise ,~ 10% and apolipoprotein E ~ 2%. It is generally considered that for typical H D L most of the metabolic activity is mediated through apolipoproteins AI or AII. However, during dietary-induced hypercholesterolemia, a form of H D L is present in plasma which is considerably enriched in apolipoprotein E (some preparations could have apolipoprotein E as their sole apolipoprotein). This lipoprotein, designated as HDLc or HDLI (Weisgraber and Mahley, 1978; Reitman and Mahley, 1979; Mahley et al., 1980a), is isolated from plasma by ultracentrifugation with flotation between the densities of 1.040-1.090 g/ml. HDL~ epitomizes the semantic difficulties of defining lipoproteins strictly operationally on the basis of their densities since the typical H D L density range is 1.063-1.21 g/ml. ElectrophoreticaUy, HDLc migrates at the 0t~ position and has a higher content of cholesteryl ester than typical HDL. HDLc may represent the mature cholesteryl ester filled form of the apolipoprotein E-phospholipid-cholesterol vesicles initially secreted by cells and thus may be an adaptation for removing cholesterol from overloaded peripheral cells.

7. CONCLUSION This review has outlined the strong associations between atherosclerosis and abnormalities of lipid and lipoprotein metabolism. While indicators such as total plasma cholesterol concentration initially highlighted this association, it is becoming increasingly obvious that the intricacies of lipoprotein metabolism and arterial biology need to be appreciated. Expansion of knowledge is required particularly in vivo. Important areas include defining subtle changes of endothelial integrity which influence rates of lipoprotein penetration into the artery during which lipoproteins may be modified. The importance of in vivo interactions of lipoproteins with the extracellular matrix in influencing extracellular retention of lipoproteins and intracellular cholesterol loading is largely undetermined. Also, although defined in cultured cell systems, the absolute and relative quantitative importance of the various possible pathogenetic mechanisms involving lipoprotein interactions are not defined in vivo. The development of drugs with well-defined modes of action may help to quantitate some of the relationships and lead to more effective prevention of atherosclerosis.

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Acknowledgements--We are grateful for the help of Dr Thomas G. Cole, Dr Elaine S. Krul and Lily Lo, and to Phyllis Anderson for typing this manuscript.

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