The role of altered lipoproteins in the pathogenesis of atherosclerosis

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30. Miller NE, Rao SN, Alaupovic P, et al. Familial apolipoprotein C-II deficiency: plasma lipoproteins and apolipoproteins in heterozygous and homozygous subjects and the effects of plasma infusion. Eur J Clm Invest 1981;11:69. 31. Catapano AL, Mills GL, Roma P, LaRosa M, Capurso A. Plasma lipids, lipoproteins and apoproteins in a case of apo C-II deficiency. Clin Chim Acta 1983;130:317. 32. Reardon MF, Sakai H, Steiner G. The roles of lipoprotein lipase and hepatic triglyceride lipase in the catabolism of

The rote of altered lipoproteins pathogemmis of atherosclerosis

Plasma lipolytic

deficiencies

triglyceride rich lipoproteins. Circulation 1980;62(suppl 3):194. 33. Musliner TA, Herbert PN, Kingston MJ. Lipoprotein substrates of lipoprotein lipase and hepatic lipase from human post heparin plasma. Biochim Biophys Acta 1979;575:277. 34. Groot PHE, Scheek LM, Jansen H. Liver lipase and high density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase. Biochim Biophys Acta 1983;751:393.

in the

Margaret E. Haberland, Ph.D., and Alan M. Fogelman, M.D. Los Angeles, Calif.

The concept that altered lipoproteins, particularly low-density lipoproteins (LDL), contribute to the initiation or propagation of the atherosclerotic reaction has emerged as a consequence of recent advances in our understanding of the metabolism of cholesteryl ester-rich lipoproteins. In vitro studies have implicated lipoprotein alteration as a prerequisite to producing the massive deposition of cholesteryl esters within the foam cells of the atherosclerotic lesion.’ Altered lipoproteins producing this effect include LDL altered by chemical or cellular modification or complexed with other molecules, as well as #I-migrating very low-density lipoproteins @VLDL).’ The scientific interest aroused by these investigations is best understood in the context of the temporal and biochemical events associated with the development of the atherosclerotic lesion. There is now substantial evidence that blood monocytes play an early role in the pathogenesis of atherosclerosis and are progenitors of many of the foam cells of the arterial lesion.2-11Recent morphologic studies4-6,g*lo have systematically documented the sequential cellular events occurring in lesions during the development of atherosclerosis induced by chronic, diet-induced hypercholesterolemia in the Division of Cardiology, Department of Medicine, University of California, Los Angeles, School of Medicine. Supported in part by US Public Health Service Grants HL30568 and RR%%, a grant from the American Heart Association, greater Los Angeles affiliate (64915), the Laubiacb Fund, and the M. K. Grey Fund. Reprint requests: Dr. Margaret E. Haberland, Division of Cardiology, Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, CA 90024.

From

animal models. Early events in atherosclerotic lesions in these animals include adherence of blood monocytes to the endothelium, transendothelial migration of the monocytes, and subsequent accumulation of monocyte-derived, lipid-laden macrophages (foam cells) in the aubendothelium.4~5~e The relatively rapid appearance of subendothelial macrophages and continued entry of monocytes into the intima during the progression of lesions of atherosclerosis may occur in response to changes in the surface of the endothelial cell or the production of chemotatic factors12-14at the sites of the atherosclerotic reaction. The progression of atherosclerosis in hypercholesterolemic pigtail monkey@ includes continuing subendothelial accumulation of the macrophage-derived foam cells and increasing distortion and, ultimately, disruption of the endothelial cell layer to expose the growing population of underlying foam cells; endothelial denudation is accompanied by adherence of platelets to the exposed foam cells and areas of exposed subendothelial connective tissue. The continued advancement of atherosclerosis to formation of fibrous plaques includes participation of the underlaying smooth muscle cells, as well as a number of factors produced by the cells of the arterial wall.’ The development of foam cells of atherosclerotic lesions characteristically includes the massive deposition of cholesteryl esters in lipid droplets within the cytoplasm.lbsl6 Although the molecular mechanisms involved in this cellular transition have yet to be defined, a major portion of the cholesterol in atherosclerotic plaques is derived from plasma lipo573

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protein~.‘~~l8 The major route of delivery of lipoprotein cholesterol to cells occurs by the high-affinity uptake of LDL: as described in the elegant studies of Goldstein and Brown,‘g internalization of LDL mediated by the LDL receptor and delivery of the lipoprotein to the lysosomesfor hydrolysis provides both hepatic and extrahepatic cells with an extracellular source of cholesterol for use in membrane assembly and steroid synthesis. Goldstein and Brown have further demonstrated that the delivery of LDL to the cell mediated by the LDL receptor is exquisitely controlled by the intracellular cholesterol concentration. Studies from our laboratories% have shown that the LDL receptor activity is so closely regulated that even prolonged incubation of human monocyte macrophages in vitro with high concentrations of LDL does not produce deposition of cholesteryl ester in these cells. It has long been appreciated that high levels of plasma LDL are associated with accelerated atherosclerosis.Hypercholesterolemia is the most common risk factor associated with atherosclerosis in Western society.21In homozygous familial hypercholesterolemia (type II hyperlipoproteinemia), in which functional LDL receptors are absenttg onset of atherosclerosis occurs in the first decade with death between the ages of 1 and 30 yeamz2These clinical 6ndings suggest that alternative receptor mechanisms may mediate the delivery of cholesterol-rich lipoproteins to macrophages; poor regulation or lack of regulation of these receptor activities by intracellular cholesterol levels may account for the massive deposition of cholesteryl ester in the macrophagederived foam cells of the atherosclerotic reaction. Brown and Goldstein’ provided the initial demonstration that alteration of lipoproteins can produce both recognition by several such alternative receptor pathways in macrophages and the consequent deposition of cholesteryl ester typically associated with the formation of the foam cells of the atherosclerotic lesion. These altered lipoproteins include LDL modified by chemical derivatization or cellular oxidation, which are internalized by the scavenger cell receptor; lipoprotein-antibody immune complexes, which are taken up by the F, receptor; complexes of LDL with dextran sulfate, which are internalized by the dextran sulfate-LDL receptor; and #?-VLDL, naturally occurring plasma lipoproteins present in the plasma of humans with the genetic disease familial dysbetalipoproteinemia, which are taken up by the @-VLDL receptor. The early involvement of the blood monocyte in atherosclerosis,2-” together with the relative ease with which the cell can be isolated from the blood of selected human donors,23

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has resulted in a number of studies directed toward elucidation of the role of alteration of lipoproteins in inducing cholesteryl ester deposition in macro&w=. CELLULAR UPTAKE CONSEQUENCES

OF ALTERED

LDL AND ITS

A number of investigations have focused on those mechanisms producing conversion of LDL into a ligand recagnized by the scavenger cell receptor. These findings in vitro have shaped our concepts of the roles of both altered lipoproteins and monocytes in atherogenesis in vivo. As a direct consequence of these studies, additional roles of not only altered LDL but also altered albumin have been proposed that may contribute to the pathogenesis of atherosclerosis. These additional events include suppression or induction of leukocyte chemotaxis,24,25cytotoxic effects,%and triggering of protease secretion in murine macrophages.27*28The following studies, based primarily on investigations of ligands of the scavenger cell receptor, describe the evidence for these proposed roles of altered lipoprotein in atherogenesis. The scavenger cell receptor was first described in 1979 by Goldstein et al29 in studies of the interaction of acetyl LDL with murine macrophages. It mediates the endocytosis of modified LDL, which typically are anionic in character.’ The receptor activity is selectively present on cells participating in the atherosclerotic lesion: human monocytes and macrophages,23+30 including cells from patients with homoxygous familial hypercholesterolemia31; endothelial cells of bovine origin32v33;and foam cells obtained from explants of rabbit atherosclerotic aorta.” Via et al.35have recently isolated the scavenger cell receptor from murine P386D, macrophages and have identified the receptor as a glycoprotein of approximately 260,006 dalton. The activity of the scavenger cell receptor, in contrast to the activities of the LDL and ,&VLDL receptors,36p 31is not subject to down regulation by the intracellular content of cholestero1.23~2Q Thus continued endocytosis of modified, anionic LDL by the scavenger cell receptor of macrophages and subsequent lysosomal hydrolysis generate large quantities of intracellular cholesterol, of which equal amounts are either reesterified in the cytoplasm or secreted into the medium.% As a consequence, incubation of in vitro macrophages with modified, anionic lipoproteins produces massive accumulation of esterified cholesterol stored in cytoplasmic droplets. In the absence of extracellular acceptors for cholesterol, the esterified

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cholesterol stored in these droplets undergoes a continual futile cycle of hydrolysis and adenosine triphosphate-dependent reesterification.38 These lipid-laden cells produced in vitro have some of the characteristics of foam cells of atherosclerotic lesions.39 Anionic LDL that are internalized by the scavenger cell receptor have been produced by a variety of methods. In 1978 Weisgraber et al.‘(’ demonstrated that chemical modification of the lysine residues of LDL to produce modified, anionic LDL abolishes interaction of the lipoprotein with the LDL receptor. In related in vivo studies conducted in animals, Mahley et al.4’ demonstrated that sinusoidal cells of the reticuloendothelial system rapidly clear modified, anionic LDL. Subsequent investigations have shown that in vitro chemical derivatixation of the lysine residues of the LDL proteinl* 29,3gv 41-43or in vitro transition metal-dependent, cellular oxidation of LDL24,11rl-46 produces lipoproteins that are recognized by the scavenger cell receptor of macrophages. The finding that cholesteryl ester-rich particles extracted from atherosclerotic aorta produce partial recognition by the scavenger cell receptor” has further implicated lipoprotein alteration as an event associated with the pathogenesis of atherosclerosis. BIOLOGIC

MODIFICATION

OF LDL

However, those processes producing the in vivo conversion of LDL to a form recognized by the scavenger cell receptor have yet to be elucidated. The potential role of lipid peroxidation in the in vitro conversion of LDL has been investigated by several groups. In 1980 Fogelman et al.% first proposed malondialdehyde as an example of a lipid product capable of directly modifying LDL and demonstrated that incubation of human monocyte macrophages with malondialdehyde-LDL produced accumulation of cholesteryl esters accounting for >!50% of the total cellular cholesterol content. Malondialdehyde is produced physiologically by the metabolism of arachidonic acid by platelets during aggregation@and by the peroxidative decomposition of unsaturated lipids by blood monocytes during phago~ytosis.~~ Haberland et al.Q have demonstrated that malondialdehyde produces threshold recognition of LDL by the scavenger cell receptor after modification of 16% of the lysine residues of apolipoprotein (ape) B4z*43; threshold recognition of malondialdehyde-LDL by the scavenger cell receptor occurs with concomitant loss of recognition by the LDL receptor on human monocyte macrophages. Quinn et al.,24 Henricksen et al.,” and Parthasarathy et al.& have demonstrated that con-

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version of LDL to a form recognized by the scavenger cell receptor can be accomplished by incubation of LDL with rabbit endothelial cells. This cellular alteration of LDL is dependent on both transition metal-dependent lipid peroxidation and hydrolysis of the LDL phosphatidylcholine mediated by phospholipase A2 activity intrinsic to LDL. Addition of butylated hydroxytoluene to prevent free radicalmediated peroxidation or of p-bromophenacyl bromide to specifically inhibit phospholipase A,,activity intrinsic to LDL prevents these changes.‘6Heinecke et al.& have demonstrated that arterial smooth muscle cells also convert LDL in a transition metalmediated, superoxide-dependent reaction to lipoprotein recognized by the scavenger cell receptor of murine macrophages and human monocyte macroPhages. Haberland et al.42*“3have proposed from studies of chemically derivatixed LDL that a conformational change in the tertiary structure of the apo B protein of LDL occurs in response to charge modification of specific, critical lysine residues; this conformational alteration may result in the steric display of f’unctional amino acyl groups forming the binding determinant(s) recognized by the scavenger cell receptor. It is of interest that a decline in the available +amino groups of lysine residues of cellular oxidized LDL has been renorted’? whether oxidation of LDL results in chemical modification of the lysine residues has yet to be established. However, Jurgens et alw have recently demonstrated that 4-hydroxy2,3-transnonenal, a product of transition metaldependent lipid peroxidation, readily modifies the lysine residues of the apo B protein of LDL. These findings2’s5o suggest that transition metal-dependent, cellular oxidation of LDL may generate reactive lipid products. These reactive lipid products, like malondialdehyde, may directly modify the LDL protein to produce a form recognized by the scavenger cell receptor. Whether the direct conversion of LDL to modified, anionic lipoprotein or complex formation of LDL with other molecules occurs in vivo as a prerequisite to producing receptor-mediated, cholesteryl eater deposition in macrophages in the arterial wall has yet to be established. It is clear that identification of such mechanism(s) in vivo would significantly advance our concepts of the pathogenesis of atherosclerosis. POSSIBLE PROMOTE

PATHWAYS BY WHICH MODIFIED ATHEROGENESIS

LDL MAY

Effects of altered lipoproteins other than cholestory1 ester deposition that may contribute to the

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initiation or propagation of the atherosclerotic reaction have also been described. These effects include the ability of modified proteins, such as maleylalbumin and acetyl LDL, to induce the secretion of neutral proteases, plasminogen-activating factor, and tumor cytolytic factors by primed murine peritoneal macrophages.nv28 In other studies, Morel et aP have demonstrated that free radical-mediated peroxidation induced by transition metals in the presence of endothelial cells and smooth muscle cells produces LDL that is cytotoxic to cell growth. These cellular effects of modified proteins could contribute to the formation of atheromatous lesions, either directly by causing tissue damage or indirectly by mediating prolonged inflammation. The ability of endothelial cell-oxidized LDL to arrest migration of murine peritoneal macrophagesz4 and of maleylalbumin to induce chemotaxis in human monocytesz5 has also been described. The report by Berliner et alI4 that p-VLDL increases the production of mom&e-specific chemotactic factor by human aortic endothelial cells adds yet another important perspective to consideration of the role of altered lipoproteins in recruitment of monocytes. Finally, Van Lenten et al.61p52have demonstrated that LDL forms a complex with bacterial lipopolysaccharide; internalization of the complex, mediated by the LDL receptor, affects the expression of the scavenger cell-receptor activity during monocyte differentiation in vitro. These results suggest that LDL complexed to other molecules may deliver factors, such as lipopolysaccharide, that subsequently influence cellular functions. These in vitro studies have implicated altered lipoproteins in a number of important events associated with the initiation or propagation of the atherosclerotic reaction. Macrophages, identified as progenitors of many of the lipid-laden foam cells of the early lesions of atherosclerosis, internalize altered cholesterol-rich lipoproteins by receptor mechanisms, which could account for the massive deposition of cholesteryl ester droplets in macrophagederived foam cells. Altered lipoproteins may trigger other cellular responses contributing to the pathogenesis of atherosclerosis, including monocyte chemotaxis, macrophage secretory events, regulation of metabolic functions in endothelial cells and differentiating monocytes, and cytotoxicity. The potential role of altered lipoproteins in the pathogenesis of atherosclerosis awaits identification of in vivo physiologically altered LDL or those processes demonstrated to operate in vivo that produce physiologically altered LDL.

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February 1987 Heart Journal

REFERENCES

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23. Fogelman AM, Hal&and ME, Seager J, Hokom M, Edwards PA. Factors regulating the activities of the low density lipoprotein receptor and the scavenger receptor on human monocyte-macropahges. J Lipid Res 1981;22:1131. 24. Quinn MT, Parthasarathy S, Steinberg D. Endothelial cellderived chemotatic activity for mouse peritoneal macrophages and the effecta of modified forms of low density lipoprotein. Proc Nat1 Acad Sci USA 1985;82:5949. 25. Haberland ME. Rasmussen RR. Fonelman AM. Receutor recognition of maleyl-albumin induces chemotaxis in human monocytes. J Clin Invest 1986;78:827. 26. Morel DW, Hessler JR, Chisohn GM. Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid. J Lipid Res 1983;24:1070. 27. Johnson WJ, Pizza SV, Imber MJ, Adams DO. Receptors for maleylated proteins regulate secretion of neutral proteases by murine macrophages. Science 1982;218:574. 28. Hartnug HP, Kladetzky RG, Hennerici M. Chemically modified low density lipoproteins as inducers of enzyme release from macrophages. FEBS Lett 1985;186:211. 29. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Nat1 Acad Sci USA 1979;76:333. 30. Fogelman AM, Shechter I, Seager J, Hokom M, Child JS, Edwards. PA. Malondialdehyde alteration of low density lipoprotein leads to cholesteryl ester accumulation in human mono&e-macroohaaes. Proc Nat1 Acad Sci USA 1980: 77~2214. - ” 31.. Knight BL, Soutar AK. Changes in the metabolism of modified and unmodified low density lipoproteins during the maturation of cultured blood monocytes from normal and homozygous familial hypercholesterolaemic subjects. Eur J Biochem 1982;125:407. 32. Stein 0, Stein Y. Bovine aortic endothelial cells display macrophage-like properties toward acetylated ‘Z1-labelled low density lipoprotein. Biochim Biophys Acta 1980; 620~631. 33. Baker DP, Van Lenten BJ, Fogelman AM, Edwards PA, Kean C, Berliner JA. LDL, scavenger and j3-VLDL receptors on aortic endothelial cells. Arteriosclerosis 1984;4:248. 34. Pitas RE, Innerarity TL, Mahley RW. Foam cells in explants of atherosclerotic rabbit aortas have receptors for &very low density lipoproteins and modified low density lipoproteins. Arteriosclerosis 1983;3:2. 35. Via DP, Dresel HA, Cheng SL, Gotto AM Jr. Murine macrophage tumors are a source of a 260,000-dalton acetyllow density lipoprotein receptor. J Biol Chem 1985; 26oz7379. 36. Van Lenten BJ, Fogehnan AM, Hokom MM, Benson L, Haberland ME, Edwards PA. Regulation of the uptake and degradation of 6-VLDL in human monocyte-macrophages. J Biol Chem 1983;258:5151. 37. Goldstein JL, Ho YK, Brown MS, Innerarity TL, Mahley RW. Cholesteryl ester accumulation in macrophagee resulting from receptor-mediated lipoproteins. J Biol Chem 1980; 255:1839. I

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38. Brown MS, Ho YK, Goldstein JL. The cholesteryl ester cycle in macrophage foam cells: continual hydrolysis and reesterification of cvtonlasmic cholestervl esters. J Biol Chem 1980;255:9344. - 39. Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RGW. Reversible accumulation of cholestervl esters in macrophages incubated with acetylated linoproieins. J Cell Biol 1979;82:597. 40. Weisgraber KH, Innerarity TL, Mahley RW. Role of the lvsine residues of nlasma linonroteins in hieh affmitv binding to cell surface receptors on human fibroblasts. J Biol Chem 197&253X)53. 41. Mahley RW, Innerarity TL, Weisgraber KH, Oh SY. Altered metiboliam (in vivo and in vitro) of plasma lipoproteins after selective chemical modification of Iysine residues of the apoprotein. J Clin Invest 1979;64:743. 42. Haberland ME, Fogehnan AM, Edwards PA. Specificity of receptor-mediated recognition of malondialdehyde-modified low density lipoproteins. Proc Nat1 Acad Sci USA 1982; 791712. 43. Haberland ME, Olch CL, Fogelman AM. Role of lysines in mediating interaction of modified low density lipoproteins with the- scavenger receptor of human mono&¯onhaees. J Biol Chem 1984z25911305. 44. *He&i&en T, Mahoney EM, Steinberg D. Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: recognition by receptors for acetylated low density lipoproteins. Proc Nat1 Acad Sci USA 1981;78:6499. 45. Parthasarathy S, Steinbrecher UP, Bamett J, W&turn JL, Steinberg D. Essential role of phospholipase A, activity in endothehal cell-induced modification of low density lipoprotein. Proc Nat1 Acad Sci USA 1985:82:3000. 46. Heinecke JW, Baker L, Rosen H, Chait A. Superoxidemediated modification of low density lipoprotein by arterial smooth muscle cells. J Clin Invest 1986;77:757. 47. Goldstein JL, Hoff HF, Ho SK, Brown MS. Stimulation of cholesteryl ester synthesis in macrophages by extracts of atherosclerotic human aortas and complexes of albumin/ cholesteryl esters. Arteriosclerosis 1981;1:210. 48. Smith JB, Ingerman CM, Silver MJ. Malondialdehyde formation as an indicator of prostaglandin production by human platelets. J Lab Clin Med 1976;8&167. 49. Stossel TP, Mason RJ, Smith AL. Lipid peroxidation by human blood phagocytes. J Clin Invest 1974;54:638. 50. Jurgens G, Lang J, Esterbauer H. Modification of human low density lipoprotein by the lipid peroxidation product 4hydroxynonenal. Biochim Biophys Acta 1986;875:103. 51. Van Lenten BJ, Fogelman AM, Seager J, Ribi E, Haberland ME, Edwards PA. Bacterial endotoxin selectively prevents the expression of scavenger receptor activity on human monocyte-macrophages. J Immunol 1985;134:3718. 52. Van Lenten BJ, Fogehnan AM, Haberland ME, Edwards PA. The role of lipoproteins and receptor-mediated endocytosis in the transnort of bacterial linowlvsaccharide. Proc Nat1 Acad Sci USA 1986;83:2704. - - -