Xiaoye Zhou

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.

Inhibition of ERK1/2 and Activation of Liver X Receptor Synergistically Induce Macrophage ABCA1 Expression and Cholesterol Efflux

ATP-binding cassette transporter A1 (ABCA1), a molecule mediating free cholesterol efflux from peripheral tissues to apoAI and high density lipoprotein (HDL), inhibits the formation of lipid-laden macrophage/foam cells and the development of atherosclerosis. ERK1/2 are important signaling molecules regulating cellular growth and differentiation. The ERK1/2 signaling pathway is implicated in cardiac development and hypertrophy. However, the role of ERK1/2 in the development of atherosclerosis, particularly in macrophage cholesterol homeostasis, is unknown. In this study, we investigated the effects of ERK1/2 activity on macrophage ABCA1 expression and cholesterol efflux. Compared with a minor effect by inhibition of other kinases, inhibition of ERK1/2 significantly increased macrophage cholesterol efflux to apoAI and HDL. In contrast, activation of ERK1/2 reduced macrophage cholesterol efflux and ABCA1 expression. The increased cholesterol efflux by ERK1/2 inhibitors was associated with the increased ABCA1 levels and the binding of apoAI to cells. The increased ABCA1 by ERK1/2 inhibitors was due to increased ABCA1 mRNA and protein stability. The induction of ABCA1 expression and cholesterol efflux by ERK1/2 inhibitors was concentration-dependent. The mechanism study indicated that activation of liver X receptor (LXR) had little effect on ERK1/2 expression and activation. ERK1/2 inhibitors had no effect on macrophage LXRα/β expression, whereas they did not influence the activation or the inhibition of the ABCA1 promoter by LXR or sterol regulatory element-binding protein (SREBP). However, inhibition of ERK1/2 and activation of LXR synergistically induced macrophage cholesterol efflux and ABCA1 expression. Our data suggest that ERK1/2 activity can play an important role in macrophage cholesterol trafficking.

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.

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.

Genetic Deletion of Low Density Lipoprotein Receptor Impairs Sterol-induced Mouse Macrophage ABCA1 Expression A NEW SREBP1-DEPENDENT MECHANISM

Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR-/-) on ABCA1 expression. LDLR-/- macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR-/- macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR-/- and wild type macrophages, whereas expression of LXRα or LXRβ was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR-/- cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR-/- macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR-/- and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR-/- macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR-/- cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR-/- cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions.