Jihong Han

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.

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.

Role of CD36, the Macrophage Class B Scavenger Receptor, in Atherosclerosis

Abstract: Recent work in the field of atherosclerosis has greatly expanded our knowledge of the pathogenesis of this disease. Scavenger receptors, including CD36, are thought to be most important early in the disease progression during macrophage uptake of modified LDL and foam cell formation. Genetically engineered murine models have been used to elucidate the contribution of the different scavenger receptors, to identify specific ligands related to LDL modifications, and to assess the possible therapeutic ramifications of targeting scavenger receptors. We have demonstrated a major role for CD36 in macrophage foam cell development and subsequent lesion development in vivo. Absence of CD36 in an atherogenic Apo E null background resulted in a 70% decrease in total lesion area in Western diet‐fed mice. We have also made significant progress in our understanding of the regulation of expression of CD36 and have demonstrated that OxLDL can stimulate its own uptake by induction of CD36 gene expression. The mechanism by which OxLDL upregulates CD36 involves activation of the transcription factor, PPAR‐γ.

Pitavastatin Downregulates Expression of the Macrophage Type B Scavenger Receptor, CD36

Background—Pitavastatin (NK-104) is a novel inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme for cholesterol biosynthesis. In clinical trials, pitavastatin has been shown to significantly decrease serum LDL cholesterol and triglyceride levels and increase HDL cholesterol. Scavenger receptor-mediated accumulation of oxidized LDL (OxLDL)-derived cholesteryl ester is considered to be a critical step in the development of atherosclerotic foam cell formation. We studied the effect of pitavastatin on CD36 (a class B scavenger receptor) expression by murine macrophages. Methods and Results—Treatment of J774 cells and murine peritoneal macrophages with pitavastatin decreased CD36 mRNA expression in a dose-dependent manner. Decreased CD36 mRNA was associated with decreased CD36 cell surface protein expression in human THP-1 cells and human monocyte-derived macrophages. Pitavastatin also reduced the increase in CD36 mRNA, cell surface protein, and binding/uptake of OxLDL induced by peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) ligands and/or OxLDL. Pitavastatin did not alter the half-life of CD36 mRNA, which suggests pitavastatin downregulates CD36 expression by reducing CD36 transcription. In addition, pitavastatin significantly decreased PPAR&ggr; mRNA and protein expression. Finally, pitavastatin increased p44/42 mitogen-activated protein kinase activity and PPAR&ggr; phosphorylation and increased the ratio of phosphorylated PPAR&ggr; to nonphosphorylated PPAR&ggr;. Conclusions—The present data demonstrate that pitavastatin prevents OxLDL uptake by macrophages through PPAR&ggr;-dependent inhibition of CD36 expression and suggest that pitavastatin could modulate CD36-mediated atherosclerotic foam cell formation.

CD36 in Atherosclerosis: The Role of a Class B Macrophage Scavenger Receptor

Abstract: CD36, an 88 kD transmembrane glycoprotein, is an important receptor for oxidized lipoproteins. Unlike the LDL receptor, expression of CD36 is upregulated by this pro‐atherogenic particle, and binding and uptake perpetuates a cycle of lipid accumulation and receptor expression. This effect is, in part, mediated by the transcription factor, peroxisome proliferator activated receptor‐γ (PPARγ), and its ligands. We have found that specific inhibitors of protein kinase C (PKC) reduce basal mRNA expression of CD36 and block induction of CD36 mRNA and protein by oxidized LDL (OxLDL) and a PPARγ ligand. In addition, PKC inhibitors block both PPARγ mRNA and protein expression. These results suggest that activation of CD36 gene expression by OxLDL involves activation and translocation of PKC with subsequent PPARγ activation. More recently, we have generated a mouse null for CD36, and crossed it with the atherogenic Apo E null strain. Evaluation of lesion development in these animals will allow us to assess the in vivo contribution of CD36 to the pathogenesis of atherosclerosis.

Regulation of peroxisome proliferator-activated receptor-γ-mediated gene expression a new mechanism of action for high density lipoprotein

Cellular cholesterol content reflects a balance of lipid influx by lipoprotein receptors and endogenous synthesis and efflux to cholesterol acceptor particles. The beneficial effect of high density lipoprotein (HDL) in protecting against the development of cardiovascular disease is thought to be mediated predominately through its induction of cellular cholesterol efflux and “reverse cholesterol transport” from peripheral tissues to the liver. We tested the hypothesis that HDL could inhibit cellular lipid accumulation by modulating expression of peroxisome proliferator-activated receptor-γ (PPARγ)-responsive genes. To this end, we evaluated expression of two PPARγ-responsive genes, CD36, a receptor for oxidized low density lipoprotein, and aP2, a fatty acid-binding protein. HDL decreased expression of macrophage CD36 and aP2 in a dose-dependent manner. HDL also decreased aP2 expression in fibroblasts, reduced accumulation of lipid, and slowed differentiation of fibroblasts into adipocytes. HDL stimulated mitogen-activated protein (MAP) kinase activity, and inhibition of CD36 expression was blocked by co-incubation with a MAP kinase inhibitor. HDL increased expression of PPARγ mRNA and protein, induced translocation of PPARγ from the cytoplasm to the nucleus, and increased PPARγ phosphorylation. Our data demonstrate that despite induction and translocation of PPARγ in response to HDL, MAP kinase-mediated phosphorylation of PPARγ inhibited expression of PPARγ-responsive genes and suggest mechanisms by which HDL may inhibit cellular lipid accumulation.

Functional Interplay Between the Macrophage Scavenger Receptor Class B Type I and Pitavastatin (NK-104)

Background—Scavenger receptor class B type I (SR-BI), a receptor for high-density lipoprotein (HDL), plays an important role in the bidirectional cholesterol exchange between cells and HDL particles and the atherosclerotic lesion development. Enhancement of SR-BI expression significantly reduces, whereas lack of SR-BI expression accelerates, the atherosclerotic lesion development in proatherogenic mice. Statins, a class of inhibitors for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, significantly suppress de novo cholesterol synthesis and reduce the incidence of coronary heart disease. Statins also display multiple pleiotropic effects independently of cholesterol synthesis in the vascular cells. Here, we investigated the effects of pitavastatin (NK-104), a newly synthesized statin, on macrophage SR-BI expression. Methods and Results—We found that pitavastatin significantly increased SR-BI mRNA and protein expression in a macrophage cell line in a concentration- and time-dependent manner. It also increased SR-BI expression in both mouse peritoneal and human monocyte-derived macrophages. Associated with increased SR-BI expression, pitavastatin enhanced macrophage HDL binding, uptake of [14C]cholesteryl oleate/HDL, and efflux of [3H]cholesterol to HDL. Pitavastatin abolished the inhibition of macrophage SR-BI expression by cholesterol biosynthetic intermediates. It also restored SR-BI expression inhibited by lipopolysaccharide and tumor necrosis factor-α through its inactivation of the transcription factor nuclear factor-&kgr;B. Conclusions—Our data demonstrate that pitavastatin can stimulate macrophage SR-BI expression by reduction of cholesterol biosynthetic intermediates and antiinflammatory action and suggest additional pleiotropic effects of statins by which they may reduce the incidence of coronary heart disease.

Pitavastatin alters the expression of thrombotic and fibrinolytic proteins in human vascular cells

In addition to lowering blood lipids, clinical benefits of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG Co‐A; EC 1.1.1.34) reductase inhibitors may derive from altered vascular function favoring fibrinolysis over thrombosis. We examined effects of pitavastatin (NK‐104), a relatively novel and long acting statin, on expression of tissue factor (TF) in human monocytes (U‐937), plasminogen activator inhibitor‐1 (PAI‐1), and tissue‐type plasminogen activator (t‐PA) in human aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC). In monocytes, pitavastatin reduced expression of TF protein induced by lipopolysaccharide (LPS) and oxidized low‐density lipoprotein (OxLDL). Similarly, pitavastatin also reduced expression of TF mRNA induced by LPS. Pitavastatin reduced PAI‐1 antigen released from HUVEC under basal, OxLDL‐, or tumor necrosis factor‐alpha (TNF‐α)‐stimulated conditions. Reductions of PAI‐1 mRNA expression correlated with decreased PAI‐1 antigen secretion and PAI‐1 activity as assessed by fibrin–agarose zymography. In addition, pitavastatin decreased PAI‐1 antigen released from OxLDL‐treated and untreated SMC. Conversely, pitavastatin enhanced t‐PA mRNA expression and t‐PA antigen secretion in untreated OxLDL‐, and TNF‐α‐treated HUVEC and untreated SMC. Finally, pitavastatin increased t‐PA activity as assessed by fibrin–agarose zymography. Our findings demonstrate that pitavastatin may alter arterial homeostasis favoring fibrinolysis over thrombosis, thereby reducing risk for thrombi at sites of unstable plaques. J. Cell. Biochem. 90: 23–32, 2003.

Anti-adipogenic action of pitavastatin occurs through the coordinate regulation of PPARγ and Pref-1 expression

Adipocyte differentiation in vitro is coordinately activated by two transcription factors, peroxisome proliferator‐activated receptor γ (PPARγ) and CCAAT enhancer binding protein α (C/EBPα), but it is inhibited by preadipocyte factor‐1 (pref‐1). Statins, inhibitors of HMG‐CoA reductase and de novo cholesterol synthesis, can have pleiotropic effects which influence adipocyte phenotype by ill‐defined mechanisms. We investigated the effects of pitavastatin (NK‐104) on adipocyte differentiation and the transcriptional pathways involved.

Activation of peroxisome proliferator-activated receptor-α in mice induces expression of the hepatic low-density lipoprotein receptor

Mutations in the low‐density lipoprotein receptor (LDLR) gene cause familial hypercholesterolaemia in humans and deletion of the LDLR induces lesion development in mice fed a high‐fat diet. LDLR expression is predominantly regulated by sterol regulatory element‐binding protein 2 (SREBP2). Fenofibrate, a peroxisome proliferator‐activated receptor α (PPARα) ligand, belongs to a drug class used to treat dyslipidaemic patients. We have investigated the effects of fenofibrate on hepatic LDLR expression.