No abstract

Serum cholesterol is carried by several lipoprotein parti-Medicine and Atherosclerosis cles that perform the complex physiologic tasks of * Department of Medicine transporting dietary and endogenously produced lipids Division of Endocrinology and Metabolism (reviewed in Witztum and Steinberg, 1995). Chylomicrons † Department of Cellular and Molecular Medicine provide the primary means of transport of dietary lipids, University of California, San Diego while very low density lipoproteins (VLDL), low density 9500 Gilman Drive lipoproteins (LDL), and high density lipoproteins (HDL) La Jolla, California 92093 function to transport endogenous lipids. Triglyceriderich VLDL particles containing apolipoprotein B-100 (apo B-100) and apolipoprotein E (apo E) are synthesized by the liver and function to transport fatty acids to adi-Complications of atherosclerosis are the most common pose tissue and muscle. After triglyceride removal in causes of death in Western societies. In broad outline, peripheral tissues, a portion of the remaining VLDL rematherosclerosis can be considered to be a form of nants are metabolized to LDL particles by further rechronic inflammation resulting from interaction between moval of core triglycerides and dissociation of apolipomodified lipoproteins, monocyte-derived macrophages, proteins other than apo B-100. In humans, the majority T cells, and the normal cellular elements of the arterial of serum cholesterol is carried by LDL particles. wall. This inflammatory process can ultimately lead to While LDL has an essential physiological role as a the development of complex lesions, or plaques, that vehicle for the delivery of cholesterol to peripheral tisprotrude into the arterial lumen. Plaque rupture and sues, increased LDL cholesterol levels are associated thrombosis results in the acute clinical complications of with increased risk of cardiovascular disease. LDL is myocardial infarction and stroke (Navab et al., 1996; taken up by cells via LDL receptors that recognize an Ross, 1999; Steinberg and Witztum, 1999). Among the N-terminal domain of apo B-100. The circulating level many genetic and environmental risk factors that have of LDL is determined in large part by its rate of uptake been identified by epidemiologic studies (Table 1), elevated levels of serum cholesterol are probably unique through the hepatic LDL receptor pathway, as eviin being sufficient to drive the development of atherodenced by the fact that lack of functional LDL receptors sclerosis in humans and experimental animals, even in is responsible for the massive accumulation of LDL in the absence of other known risk factors. The elucidation patients with homozygous familial hypercholesterolof molecular mechanisms that control cholesterol bioemia (Goldstein and Brown, 1977). The expression of synthesis and serum cholesterol levels (reviewed in LDL receptors is subject to feedback control by intracel-Goldstein and Brown, 1977) led to the development of lular cholesterol levels. Low levels of intracellular choles-"statins," a poten...

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.

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-snglycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acidcontaining phospholipids may be important initiators of atherogenesis.

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...