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BackgroundThe fact that low density lipoprotein (LDL) 1 is extremely susceptible to oxidative damage has been known for some time (1, 2), but until quite recently this was primarily a nuisance for the student of lipoprotein metabolism. It now appears that oxidation of LDL plays a significant role in atherogenesis.Beginning in the 1980s evidence began to accumulate that cholesterol accumulation in the developing atherosclerotic lesion was probably not due to the uptake of native LDL by way of the Brown/Goldstein LDL receptor but instead due to the uptake of some modified form of LDL (then still unidentified) by way of one or more alternative receptors (also then unidentified). This conclusion grew from two well accepted observations. First, patients and animals totally lacking the LDL receptor nevertheless accumulate cholesterol in foam cells much the same way as do patients and animals with normal LDL receptors; second, the two cell types in lesions that give rise to cholesterol-laden foam cells (the monocyte/ macrophage and the smooth muscle cell) do not accumulate cholesterol in vitro even in the presence of very high concentrations of native LDL (3, 4). This paradox could be resolved if circulating LDL underwent some form of modification and if the modified form, rather than native LDL itself, then served as the ligand for delivery of cholesterol to developing foam cells. Acetylation of LDL in vitro generated a modified LDL that could induce cholesterol accumulation in macrophages (3). The uptake of this acetylated LDL was by way of a new receptor designated the acetyl LDL receptor (later cloned and renamed scavenger receptor A (SRA) (5). SRA, unlike the LDL receptor, is not down-regulated when the cholesterol content of the cell increases. Thus, acetyl LDL could, in principle, account for foam cell formation. However, there was (and still is) no evidence that acetylation of LDL occurs to any extent in vivo. Another modified form of LDL emerged as a candidate when it was shown that simply incubating LDL overnight with a monolayer of arterial endothelial cells converted it to a form that was taken up much more rapidly by macrophages and capable of increasing their cellular cholesterol content (6 -8). The uptake was specific and saturable, and it occurred in part by way of the acetyl LDL receptor. Incubation with smooth muscle cells could also modify LDL in much the same way (7,8). This cell-mediated modification turned out to be, very simply, oxidative modification (9, 10). The addition of antioxidants to the culture medium completely blocked cellinduced modification, and the changes induced by the cells could be duplicated by incubating LDL in the presence of transition metals in the absence of cells. Thus, oxidative modification induced by cells appeared to be a biologically plausible modification of LDL that could account for foam cell formation and the initiation, or at least acceleration, of the atherosclerotic process.

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, proerties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was de--graded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

Low density lipoprotein (LDL) incubated with cultured endothelial cells from rabbit aorta or human umbilical vein is altered in several ways (EC-modified): (i) It is degraded by macrophages much faster than LDL similarly incubated in the absence of cells or incubated with fibroblasts.(ii) Its electrophoretic mobility is increased. (iii) Its density is increased. We report here that antioxidants completely prevent these changes. We also report that these changes do not take place if transition metals in the medium are chelated with EDTA. During EC-modification as much as 40% of the LDL phosphatidylcholine is degraded to lysophosphatidylcholine by a phospholipase A2-like activity. When incubation conditions in the absence of cells were selected to favor oxidation-for example, by extending the time of incubation of LDL at low concentrations, or by increasing the Cu2+ concentration-LDL underwent changes very similar to those occurring in the presence of cells, including degradation of phosphatidylcholine. Hence, some phospholipase activity appears to be associated with the isolated LDL used in these studies. The results suggest a complex process in which endothelial cells modify LDL by mechanisms involving generation of free radicals and action of phospholipase (s).The lipid-laden foam cells in atherosclerotic lesions are derived largely or in part from monocyte/macrophages (1, 2). These cells have only low levels of the classical low density lipoprotein (LDL) receptor and take up native LDL in vitro at relatively low rates, insufficient to cause lipid accumulation to the extent found in vivo (3). It has been suggested that this paradox may be explained if the LDL particle is in some way altered in vivo to a form taken up more readily than native LDL. Certain chemically modified forms of LDL, including acetylated LDL, are indeed taken up much more rapidly than native LDL by macrophages, and this uptake involves a different receptor, designated the acetyl-LDL receptor (3-5). Incubation of LDL with cultured endothelial cells generates a modified form (or forms) of LDL (endothelial cell-modified LDL; EC-modified LDL) that is taken up 3-to 10-fold more rapidly by macrophages and at least in part by way of the acetyl-LDL receptor (6-8). The modification in biological properties is accompanied by a marked increase in electrophoretic mobility and hydrated density but the mechanisms involved are still poorly understood.LDL is highly sensitive to metal-catalyzed oxidation (9-11) and oxidized LDL has been shown to be toxic to some cultured cells (12,13). Endothelial cells in culture have been shown to be capable of oxidizing LDL (14). Since the modification of LDL by endothelial cells involves long incubation under aerobic conditions, we examined the possible role of oxidative changes in the process. In the present paper, we report that generation of EC-modified LDL is associated with lipid peroxidation and with extensive hydrolysis of LDL phosphatidylcholine (PtdCho) to lysophosphatidylcholine (lyso-PtdCho).MATERIALS...

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can give rise to foam cells, the key component ofthe fatty streak lesion of atherosclerosis. Oxidation of LDL probably generates a broad spectrum of conjugates between fragments of oxidized fatty acids and apolipoprotein B. We now present three mutually supportive lines of evidence for oxidation of LDL in vivo: (i) Antibodies against oxidized LDL, malondialdehyde-lysine, or 4-hydroxynonenal-lysine recognize materials in the atherosclerotic lesions of LDL receptor-deficient rabbits; (ii) LDL gently extracted from lesions of these rabbits is recognized by an antiserum against malondialdehyde-conijugated LDL; (iii) autoantibodies against malondialdehyde-LDL (titers from 512 to >4096) can be demonstrated in rabbit and human sera.A growing body of evidence suggests that oxidative modification of low density lipoprotein (LDL) enhances its atherogenicity (for review see ref. 1). Monocyte-derived macrophages, the precursor of most foam cells in early atherosclerotic lesions, cannot take up native LDL rapidly enough to cause lipid loading (2). Oxidative modification converts LDL to a form recognized by the macrophage acetyl-LDL receptor (1, 2) and possibly by other receptors as well (3). This is true whether the oxidation is effected by incubation under appropriate conditions with cultured cells or by autooxidation catalyzed by Cu2+ ions in the absence of cells. Oxidative modification of LDL is accompanied by extensive degradation of its polyunsaturated fatty acids, generating a complex array of shorter chain-length fragments (4). During the oxidation, some of these fragments become covalently linked to apolipoprotein B (5), and much ofthis conjugation involves the E-amino groups of lysine residues. This chemically modified form of apolipoprotein is recognized by the acetyl-LDL receptor (6). Thus we can generate models for oxidized LDL by conjugating the apolipoprotein with single compounds generated during oxidation. Fogelman et al. (7) demonstrated that malondialdehyde (MDA)-conjugated LDL is so recognized. If oxidized LDL contains lysine residues conjugated with a variety of fatty acid fragments of different chain lengths, it should react with antibodies against a variety of such lysine derivatives. We previously showed that immunization of animals with autologous LDL modified by conjugation of lysine groups with glucose yields antisera directed specifically against glucitollysine (8) and that antibodies generated by injection of carbamoylated autologous LDL generates antisera that recognize the carbamoyllysine-not only in LDL but in other conjugated proteins as well (9). In other words, the specificity of these antisera is for very narrowly defined "X"-lysine adducts.The present studies, which use immunochemical methods, offer three lines of evidence that oxidation of LDL occurs in vivo.

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The recruitment of monocyte-macrophages into the artery wall is one of the earliest events in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte chemoattractant secreted by many cells in vitro, including vascular smooth muscle and endothelial cells. To test whether it is expressed in the artery in vivo, we used Northern blot analysis, in situ hybridization, and immunocytochemistry to study the expression of MCP-1 in normal and atherosclerotic human and rabbit arteries. Northern blot analysis showed that MCP-1 mRNA could be isolated from rabbit atherosclerotic lesions but not from the intima media of normal animals. Furthermore, MCP-1 mRNA was extracted from macrophage-derived foam cells isolated from arterial lesions of ballooned cholesterol-fed rabbits, whereas alveolar macrophages isolated simultaneously from the same rabbits did not express MCP-1 mRNA. MCP-1 mRNA was detected by in situ hybridization in macrophage-rich regions of both human and rabbit atherosclerotic lesions. No MCP-1 mRNA was found in sublesional medial smooth muscle cells or in normal arteries. By using immunocytochemistry, protein was demonstrated in human lesions, again only in macrophage-rich regions. Immunostaining of the serial sections with an antiserum against malondialdehyde-modified low density lipoprotein indicated the presence of oxidized low density lipoprotein and/or other oxidation-specific lipidprotein adducts in the same areas that contained macrophages and MCP-1. We conclude that (a) MCP-1 is strongly expressed in a small subset of cells in macrophage-rich regions of human and rabbit atherosclerotic lesions and (ii) MCP-1 may, therefore, play an important role in the ongoing recruitment of monocyte-macrophages into developing lesions in vivo.The earliest grossly visible atherosclerotic lesion is the fatty streak, characterized by the accumulation of lipid-loaded foam cells in the subendothelial space (1). Many of these foam cells are derived from circulating monocytes (2-4) that have penetrated into the subendothelial space and presumably taken up excess native and/or oxidized low density lipoprotein (LDL) (5-8). Thus, one of the important early events in the pathogenesis of atherosclerosis is the adherence of monocytes to the endothelium, followed by their migration into the subendothelial space (1, 3). The entry between endothelial cells presumably is in response to a gradient of one or more chemotactic factors. Several monocyte chemotactic factors have been described-produced by endothelial cells, by smooth muscle cells, or by macrophages (9-14)-but there is almost no information on which of these are important in vivo. Chemotactic activity may also be derived from the extracellular components of the artery wall. For example, proteolytic peptide fragments from several connective tissue matrix proteins are chemotactic for leukocytes (15)(16)(17). In addition, in vitro-oxidized LDL is chemotactic for circulating monocytes (18) and oxidatively modified LDL isolated ...

Previous studies in this laboratory established that low density lipoprotein (LDL) incubated with cultured endothelial cells, smooth muscle cells, or macrophages undergoes a free radical-catalyzed oxidative modification that generates lipid peroxides and extensive structural changes in the LDL molecule. The oxidatively modified LDL strongly inhibited chemotactic responses of the mouse resident peritoneal macrophage. The present studies show that this oxidized LDL does not inhibit the motility of mouse monocytes and actually exhibits a chemotactic activity for human monocytes; the chemotactic activity of the oxidized LDL resides in the lipid fraction. These findings allow us to propose a pathogenetic sequence by which elevated plasma LDL levels, followed by oxidative modification in the arterial wall, could sufficiently account for the generation of the lipid-laden foam cells and the initiation of the fatty streak, the earliest well-defined lesion in atherogenesis.Accumulation of lipid in the arterial intima is central to the development of atherosclerosis. Intimal lipid accumulates intracytoplasmically in foam cells, which are derived both from medial smooth muscle cells (1, 2) and monocyte-derived macrophages (3)(4)(5), the latter probably being quantitatively more important (3-5). Exactly how monocytes are recruited and retained in the artery wall remains unclear, but probably the initial event is adhesion to the endothelial surface (3) followed by penetration that is influenced by a chemotactic factor(s). Many different factors chemotactic for monocytes have been described (6); the relative importance of these remains uncertain. Crude extracts of whole aorta contain chemotactic activity (7), and cultured arterial smooth muscle cells and macrophages release chemotactic activity into the culture medium (8). We recently described release of chemotactic activity for mouse resident peritoneal macrophages from cultured aortic endothelial cells (9) and showed that oxidatively modified low density lipoprotein (LDL) inhibited the chemotactic response of the macrophage. In the present studies we confirm the finding of Berliner et al. (10) that endothelial cell-conditioned medium is chemotactic also for human monocytes. However, oxidatively modified LDL, instead of inhibiting the motility of monocytes actually enhances their motility. We further show that the chemotactic activity resides in the lipid fraction of the modified LDL, presumably in one or another peroxidized lipid component. Thus oxidative modification of LDL, in addition to favoring the accumulation of cholesterol stores in developing foam cells (11), could play a role in recruitment and retention of monocyte/macrophages into the subendothelial space and, finally, may contribute to atherogenesis through injury to endothelial cells. MATERIALS AND METHODSHam's F-10 medium and fetal bovine serum were from HyClone (Logan, UT); female Swiss Webster mice were from Simonsen Laboratories (Gilroy, CA); Ficoll/Hypaque, bovine serum albumin, zymosan A, fucoidi...