David P. Hajjar

Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice

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) and a 45% decrease in aortic sinus lesion area (normal chow) in the CD36-apo E double-null mice when compared with controls, despite alterations in lipoprotein profiles that often correlate with increased atherogenicity. Macrophages derived from CD36-apo E double-null mice bound and internalized more than 60% less copper-oxidized LDL and LDL modified by monocyte-generated reactive nitrogen species. A similar inhibition of in vitro lipid accumulation and foam cell formation after exposure to these ligands was seen. These results support a major role for CD36 in atherosclerotic lesion development in vivo and suggest that blockade of CD36 can be protective even in more extreme proatherogenic circumstances.

CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism

CD36, identified more than a quarter of a century ago as a platelet integral membrane glycoprotein (glycoprotein IV), was until recently best known as a receptor for thrombospondin-1 (TSP-1). TSP-1 is found in ECMs and platelet α granules, and it participates in cell attachment, motility, and proliferation, as well as in modulation of protease activity, TGF-β activation, neurite outgrowth, and angiogenesis (1). Initially, this receptor-ligand pair was shown to mediate interactions between platelets and monocytes, tumor cells, and matrix. Since then, CD36 has been implicated in multiple biological processes that define it as a multiligand scavenger receptor (see ref. 2 for review). These ligands appear remarkably diverse: In addition to TSP-1, they include long-chain fatty acids, modified LDL, retinal photoreceptor outer segments, Plasmodium falciparum malaria-parasitized erythrocytes, sickle erythrocytes, anionic phospholipids, apoptotic cells, and collagens I and IV. The biology of CD36 can be broadly divided in terms of functions that it mediates with or without TSP-1, but it is probable that it acts in concert with other proteins, such as fatty acid–binding proteins, caveola-associated proteins, integrins, cytoskeletal proteins, and signaling molecules, to effect its diverse functions.

A Molecular Redox Switch on p21ras STRUCTURAL BASIS FOR THE NITRIC OXIDE-p21ras INTERACTION

We have identified the site of molecular interaction between nitric oxide (NO) and p21ras responsible for initiation of signal transduction. We found that p21ras was singly S-nitrosylated and localized this modification to a fragment of p21ras containing Cys118. A mutant form of p21ras, in which Cys118 was changed to a serine residue and termed p21rasC118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21ras, resulting in an active form, but not on p21rasC118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21rasC118S. These data indicate that Cys118 is a critical site of redox regulation of p21ras, and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.

Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species

The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.

Inflammation in Atherosclerosis and Implications for Therapy

Atherosclerosis is now understood to be a disease characterized by inflammation that results in a host of complications, including ischemia, acute coronary syndromes (unstable angina pectoris and myocardial infarction), and stroke. Inflammation may be caused by a response to oxidized low-density lipoproteins, chronic infection, or other factors; and markers of this process, such as C-reactive protein, may be useful to predict an increased risk of coronary heart disease. Thus, we believe that inflammatory processes may be potential targets of therapy in preventing or treating atherosclerosis and its complications.

Native and Modified Low Density Lipoproteins Increase the Functional Expression of the Macrophage Class B Scavenger Receptor, CD36

The uptake of oxidized low density lipoprotein (OxLDL) by macrophages is a key event implicated in the initiation and development of atherosclerotic lesions. Two macrophage surface receptors, CD36 (a class B scavenger receptor) and the macrophage scavenger receptor (a class A scavenger receptor), have been identified as the major receptors that bind and internalize OxLDL. Expression of CD36 in monocyte/macrophages in tissue culture is dependent both on the differentiation state as well as exposure to soluble mediators (cytokines and growth factors). The regulatory mechanisms of this receptor in vivo are undetermined as is the role of lipoproteins themselves in modulating CD36 expression. We studied the effect of lipoproteins, native LDL and modified LDL (acetylated LDL (AcLDL) and OxLDL) on the expression of CD36 in J774 cells, a murine macrophage cell line. Exposure to lipoproteins resulted in a marked induction of CD36 mRNA expression (4–8-fold). Time course studies showed that maximum induction was observed 2 h after treatment with AcLDL and at 4 h with LDL and OxLDL. Increased expression of CD36 mRNA persisted for 24 h with each treatment group. Induction of CD36 mRNA expression was paralleled by an increase in CD36 protein as determined by Western blot with the greatest induction by OxLDL (4-fold). In the presence of actinomycin D, treatment of macrophages with LDL, AcLDL, or OxLDL did not affect CD36 mRNA stability, implying that CD36 mRNA was transcriptionally regulated by lipoproteins. To determine the mechanism(s) by which lipoproteins increased expression of CD36 we evaluated the effects of lipoprotein components on CD36 mRNA expression. ApoB 100 increased CD36 mRNA expression significantly, whereas phospholipid/cholesterol liposomes had less effect. Incubation of macrophages with bovine serum albumin or HDL reduced expression of CD36 mRNA in a dose-dependent manner. Finally, to evaluate the in vivo relevance of the induction of CD36 mRNA expression by lipoproteins, peritoneal macrophages were isolated from mice following intraperitoneal injection of lipoproteins. Macrophage expression of CD36 mRNA was significantly increased by LDL, AcLDL, or OxLDL in relation to mice infused with phosphate-buffered saline, with OxLDL causing the greatest induction (8-fold). This is the first demonstration that exposure to free and esterified lipids augments functional expression of the class B scavenger receptor, CD36. These data imply that lipoproteins can further contribute to foam cell development in atherosclerosis by up-regulating a major OxLDL receptor.

The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling.

Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression.

Infection and Atherosclerosis Potential Roles of Pathogen Burden and Molecular Mimicry

Infection has been implicated as a cause of atherosclerosis since the first half of the 19th century. Over the years, sporadic publications have appeared in the literature reflecting a persistent but relatively low level of research activity in this area. In the last decade, however, publications relating to this topic have increased markedly. And very recently, new epidemiological and mechanistic data relating infection to several different diseases, including atherosclerosis, have appeared, stimulating the emergence of important paradigm shifts in how we think about the causes of chronic disease. The following article reviews some of these newer concepts as they relate to a possible role of infection in atherosclerosis.

Transforming Growth Factor-β1 (TGF-β1) and TGF-β2 Decrease Expression of CD36, the Type B Scavenger Receptor, through Mitogen-activated Protein Kinase Phosphorylation of Peroxisome Proliferator-activated Receptor-γ

CD36, the macrophage type B scavenger receptor, binds and internalizes oxidized low density lipoprotein, a key event in the development of macrophage foam cells within atherosclerotic lesions. Expression of CD36 in monocyte/macrophages is dependent on differentiation status and exposure to soluble mediators. In this study, we investigated the effect of transforming growth factor-β1 (TGF-β1) and TGF-β2 on the expression of CD36 in macrophages. Treatment of phorbol ester-differentiated THP-1 macrophages with TGF-β1 or TGF-β2 significantly decreased expression of CD36 mRNA and surface protein. TGF-β1/TGF-β2 also inhibited CD36 mRNA expression induced by oxidized low density lipoprotein and 15-deoxyΔ12,14 prostaglandin J2, a peroxisome proliferator-activated receptor (PPAR)-γ ligand, suggesting that the TGF-β1/TGF-β2 down-regulated CD36 expression by inactivating PPAR-γ-mediated signaling. TGF-β1/TGF-β2 increased phosphorylation of both mitogen-activated protein (MAP) kinase and PPAR-γ, whereas MAP kinase inhibitors reversed suppression of CD36 and inhibited PPAR-γ phosphorylation induced by TGF-β1/TGF-β2. Finally, MAP kinase inhibitors alone increased expression of CD36 mRNA and surface protein but had no effect on PPAR-γ protein levels. Our data demonstrate for the first time that TGF-β1 and TGF-β2 decrease expression of CD36 by a mechanism involving phosphorylation of MAP kinase, subsequent MAP kinase phosphorylation of PPAR-γ, and a decrease in CD36 gene transcription by phosphorylated PPAR-γ.

Prostacyclin modulates cholesteryl ester hydrolytic activity by its effect on cyclic adenosine monophosphate in rabbit aortic smooth muscle cells.

We tested the hypothesis that prostacyclin (PGI2), 6-keto-prostaglandinF1 alpha(6-keto-PGF1 alpha), and several E series prostaglandins (PG) may affect the activity of cholesteryl ester (CE) hydrolase since our previous experiments indicated that smooth muscle cells (SMC) in neointima of injured rabbit aorta (a) acquire the capacity to produce PGI2 and (b) have increased lysosomal CE hydrolytic (acid cholesteryl ester hydrolase [ACEH])activity. Using cultured SMC from rabbit thoracic aorta, we demonstrated that PGI2, 6-keto-PGF1 alpha, and 6-keto-PGE1 enhanced ACEH activity fourfold. No significant effects on ACEH activity were observed with PGE1 or PGE2. Preincubation of SMC with an inhibitor of adenylate cyclase activity (dideoxyadenosine) abolished the effect of these PG on CE hydrolytic activity. Addition of dibutyryl cAMP to these SMC significantly increased ACEH activity. Although concentrations of PGI2 used significantly increased cAMP levels, proliferation of these SMC was not observed. In related experiments, we determined if the addition of PGI2, 6-keto-PGF1 alpha, or 6-keto-PGE1 to cultured aortic SMC would enhance the egress of unesterified cholesterol and CE from these SMC. A significant loss of total cholesterol from PG-treated SMC was observed at the end of 14 d. Results suggest that increased synthesis of PGI2 by neointimal SMC in the injured aortic wall may, at least in part, explain the changes in CE catabolism and accumulation following injury. These PG may also be important in CE metabolism and accumulation in human arteries.