A Novel Family of Atherogenic Oxidized Phospholipids Promotes Macrophage Foam Cell Formation via the Scavenger Receptor CD36 and Is Enriched in Atherosclerotic Lesions

Abstract

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...

CD36: Implications in cardiovascular disease

“…In terms of modified LDL, the exact nature of the CD36 ligand has been recently defined as a truncated phosphatidylcholine species, with a specific sn-2 esterified γ-hydroxy (or oxo) -α,β-unsaturated carbonyl containing fatty acid (Podrez, et al, 2002a,Podrez, et al, 2002b. These modifications can occur as a result of the action of reactive oxygen or nitrogen species.…”

Oxidized Phospholipids as Endogenous Pattern Recognition Ligands in Innate Immunity

“…The structures of oxPC CD36 identified were established using a combination of cell binding studies and multiple distinct chromatographic and MS methods in conjunction with (i) results of numerous derivatization strategies to ascertain functional groups on isolated products, (ii) inference of structures of products that appeared plausible based upon MS results and known mechanisms of lipid oxidation and fragmentation, and (iii) de novo synthesis of each oxidized lipid. The identities of the oxidation products and synthetic standards were confirmed by demonstration that synthetic species recapitulate all biological, chemical, MS, and chromatographic characteristics of the lipids isolated from oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-PC, oxidized 1-palmitoyl-2-lineoyl-sn-glycero-3-PC, and oxidized 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-PC (19,20,22,40,41). Liquid chromatographic/electrospray ionization/tandem MS analyses have demonstrated the generation of these species in lipoproteins oxidized by multiple distinct pathways in vitro (40), in atherosclerotic lesions in vivo (41), in retina following light exposure in dark-adapted rodents (20), and in cerebral tissues following ischemia-reperfusion injury (42).…”

“…Podrez et al (40,41) reported the first systematic study aimed at identifying the structures of specific oxidized lipids that serve as ligands for the scavenger receptor CD36. Structure/function analyses with multiple synthetic species defined critical structural elements of endogenous ligands for CD36 that promote high affinity binding to the scavenger receptor: a phospholipid with a truncated sn-2 acyl group that incorporates a terminal ␥-hydroxy(or oxo)-␣,␤-unsaturated carbonyl (oxPC CD36 ) (40,41). The structures of the oxidized lipid ligands of CD36 are illustrated in Fig.…”

“…Although initially defined using PC molecular species, as outlined below, subsequent studies have shown that all classes of phospholipids examined thus far (PC, phosphatidylethanolamine, PS, and phosphatidic acid) harboring the high affinity motif for CD36 promote CD36-mediated recognition and phagocytosis (19,20,22,40,41). The structures of oxPC CD36 identified were established using a combination of cell binding studies and multiple distinct chromatographic and MS methods in conjunction with (i) results of numerous derivatization strategies to ascertain functional groups on isolated products, (ii) inference of structures of products that appeared plausible based upon MS results and known mechanisms of lipid oxidation and fragmentation, and (iii) de novo synthesis of each oxidized lipid.…”

“…1B illustrates structures of additional oxidized glycerophospholipids detected in the above in vivo models that have been shown to be generated by further oxidative fragmentation of oxPL CD36 . Although many have been reported to possess pro-inflammatory biological activities (32) or when adducted to proteins may mediate scavenger receptor recognition (34), they do not bind with high affinity to CD36 when present in membranes as free oxPL (40,41).…”