Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.

IntroductionIn vitro and in vivo studies support the hypothesis that macrophages are key early mediators of atherogenesis (1-3) and their impaired recruitment and activation protects against lesion development (4-6). A significant amount of research also supports the hypothesis that subendothelial modified LDLs provide the initiating ligands for the macrophage (7,8), and these are recognized by scavenger receptors (9-13).Oxidized LDL is a ligand for the class A scavenger receptors type I and II (SRA-I/II), MARCO, the class B scavenger receptor, CD36, and the class D receptor, CD68. This modified lipoprotein has been considered the most important atherogenic LDL (14-16). In vitro studies have provided evidence that SRA-I/II and CD36 are the major oxidized LDL receptors mediating lipid accumulation and foam cell formation, whereas MARCO and CD68 play a more minor role (17-21). Absence of SRA-I/II in atherogenic murine models has had a variable impact on atherosclerosis (22-24). Thus, an essential contribution of scavenger receptors to the pathogenesis of atherosclerosis in vivo remains unresolved.CD36 has been shown to be highly regulated in monocytes/macrophages during differentiation (25,26) and to be present in lipid-laden macrophages in atherosclerotic lesions (27,28). This scavenger receptor is upregulated by IL-4 (25), macrophage colony stimulating factor (26), modified LDL (17, 18), cellular cholesterol content (29), and peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands (30, 31). Unlike SRA I/II, CD36 is more broadly expressed (32-35) and has been shown to play a strategic role in lipoprotein and lipid metabolism (36, 37). The phenotype of CD36-null mice generated in our laboratory included increased plasma levels of cholesterol, triacylglycerol, and fatty acids and supported a major role for CD36 in fatty acid uptake and lipid metabolism in vivo (36). In transgenic mice, overexpression of CD36 in muscle enhanced fatty acid oxidation during stimulation/contraction and also had significant influence on plasma lipoprotein and fatty acid levels (37). Absence of CD36 was implicated recently in insulin resistance in the spontaneous hypertensive rat by using genetic analysis (37, 38). These studies point to an essential role for CD36 not only in uptake of lipid but in determination of cellular lipid stores.To determine if CD36 is a major macrophage scavenger receptor responsible for early lipid accumulation and foam cell formation, which can predispose animals to the development of fatty streaks and ultimately more advanced atherosclerotic lesions, we generated CD36-apo E double-null mice and evaluated aortic lesions on normal chow and Western diets. Macrophage scavenger receptors have been implicated as key players in the pathogenesis of atherosclerosis. To assess the role of the class B scavenger receptor CD36 in atherogenesis, we crossed a CD36-null strain with the atherogenic apo E-null strain and quantified lesion development. There was a 76.5% decrease in aortic tree lesion area (Western diet) a...

The macrophage scavenger receptor CD36 plays an important role in the uptake of oxidized forms of low density lipoprotein (LDL) and contributes to lesion development in murine models of atherosclerosis. However, the structural basis of CD36 lipoprotein ligand recognition is unknown. We now identify a novel class of oxidized phospholipids that serve as high affinity ligands for CD36 and mediate recognition of oxidized forms of LDL by CD36 on macrophages. Small unilamellar vesicles of homogeneous phosphatidylcholine (PC) molecular species were oxidized by the myeloperoxidase (MPO)-H 2 O 2 -NO 2 ؊ system, and products were separated by sequential LC/ESI/MS/MS. In parallel, fractions were tested for their ability to bind to CD36. Four major structurally related phospholipids with CD36 binding activity were identified from oxidized 1-palmitoyl-2-arachidonyl-PC, and four corresponding structural analogs with CD36 binding activity were identified from oxidized 1-palmitoyl-2-linoleoyl-PC. Each was then synthetically prepared, its structure confirmed by multinuclear NMR and high resolution mass spectrometry, and shown to possess identical CD36 binding activity and LC/ESI/MS/MS characteristics in both native and derivatized forms. Based upon the structures of the active compounds identified, and structure-function studies with a variety of synthetic analogs, we conclude that the structural characteristics required for high affinity binding of oxidized PC species to CD36 are a phospholipid with an sn-2 acyl group that incorporates a terminal ␥-hydroxy(or oxo)-␣,␤-unsaturated carbonyl (oxPC CD36 ). LC/ESI/MS/MS studies demonstrate that oxPC CD36 are formed during LDL oxidation by multiple distinct pathways. Formation of this novel class of oxidized PC species contributes to CD36-mediated recognition of LDL oxidized by MPO and other biologically relevant mechanisms. The present results offer structural insights into the molecular patterns recognized by the scavenger receptor CD36 and provide a platform for the development of potential therapeutic inhibitory agents.CD36 is a heavily glycosylated, single chain, integral plasma membrane protein that belongs to an evolutionarily conserved family of proteins that serve as scavenger and lipid receptors (1, 2). It is expressed on the surface of adipocytes, microvascular endothelial cells, macrophages, platelets, and specialized epithelial cells (1, 2). CD36 functions in vivo in scavenger recognition of oxidized lipoproteins and senescent or apoptotic cells, fatty acid transport, cell-matrix interactions, and antiangiogenic actions (3-5). Its deficiency in humans has been correlated with alterations in myocardial fatty acid uptake, hypertrophic cardiac myopathy, and insulin resistance (6 -8). Recent studies (3, 4, 9 -11) have focused attention on CD36 as a participant in the atherosclerotic process because of its ability to recognize oxidized forms of LDL (oxLDL).1 CD36 mediates lipid accumulation and macrophage foam cell formation in vitro and in vivo (3,12,13). It is heavily expre...

The macrophage scavenger receptor CD36 plays an important role in binding and uptake of oxidized forms of low-density lipoprotein (LDL), foam cell formation, and lesion development during atherosclerosis. The structural basis of CD36-lipoprotein ligand recognition is an area of intense interest. In a companion article we reported the characterization of a structurally conserved family of oxidized choline glycerophospholipids (ox-PC CD36 ) that serve as novel high affinity ligands for cells stably transfected with CD36, mediating recognition of multiple oxidized forms of LDL (Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P., Shan, L., Gugiu, B., Fox, P. L., Hoff, H. F., Salomon, R. G., and Hazen, S. L. (July 8, 2002) J. Biol. Chem. 277, 10.1074/ jbc.M203318200). Here we use macrophages from wildtype and CD36 null mice to demonstrate that CD36 is the major receptor on macrophages mediating recognition of oxPC CD36 species when presented (؉/؊ plasma) in pure form, within PC bilayers in small unilamellar vesicles, and within liposomes generated from lipid extracts of native LDL. We also show that oxPC CD36 promote CD36-dependent recognition when present at only a few molecules per particle, resulting in macrophage binding, uptake, metabolism, cholesterol accumulation, and foam cell formation. Finally, using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), we demonstrate that oxPC CD36 are generated in vivo and are enriched in atherosclerotic lesions. Collectively, our data suggest that formation of this novel family of oxidized phospholipids participates in CD36-mediated recognition of oxidized lipoproteins and foam cell formation in vivo.CD36 is a multifunctional cellular receptor with broad ligand specificity (1, 2). It is expressed in a number of cells including microvascular endothelial cells, platelets, adipocytes, striated muscles, macrophages, and some vascular smooth muscle cells (1, 2). CD36 regulates cellular adhesion and angiogenesis, serving as a receptor for thrombospondin; it also serves as a scavenger receptor in macrophages, mediating uptake of apoptotic cells and modified lipoproteins, and participates in carbohydrate and lipid metabolism, modulating insulin resistance and long chain fatty acid transport (3-8). CD36 has recently been implicated in a variety of pathologic conditions, including atherosclerosis, diabetes, and cardiomyopathy. Perhaps the most compelling data on the role of CD36 in atherosclerosis are from studies of CD36-deficient mice, which show a 70 -80% reduction in aortic lesion size (9). In vitro experiments demonstrate that macrophages from CD36-deficient mice take up different forms of oxidized LDL 1 poorly and are resistant to foam cell formation, providing a mechanism for the atheroprotection observed in CD36 null mice (9, 10). Given the potential clinical significance of this receptor, it is important to know the nature of the ligand(s) in oxidized LDL (oxLDL) that are recognize...

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