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

BACKGROUND AND PURPOSE: 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 alpha (PPARalpha) ligand, belongs to a drug class used to treat dyslipidaemic patients. We have investigated the effects of fenofibrate on hepatic LDLR expression. EXPERIMENTAL APPROACH: The effects of fenofibrate on hepatic LDLR expression (mRNA and protein) and function were evaluated by both in vitro (with AML12 cells) and in vivo experiments in mice. KEY RESULTS: Fenofibrate increased LDLR expression and LDL binding in a mouse hepatoma cell line, AML12 cells. Fenofibrate restored sterol-inhibited hepatocyte LDLR expression. Mechanistic studies demonstrated that induction of LDLR expression by fenofibrate was dependent on PPARalpha and sterol regulatory elements (SRE). Specifically, fenofibrate induced LDLR expression by increasing maturation of SREBP2 and phosphorylation of protein kinase B (Akt) but had no effect on SREBP cleavage-activating protein. In vivo, a high-fat diet suppressed LDLR expression in mouse liver while elevating total and LDL cholesterol levels in plasma. However, fenofibrate restored LDLR expression inhibited by high-fat diets in the liver and reduced LDL cholesterol levels in plasma. CONCLUSIONS AND IMPLICATIONS: Our data suggest that fenofibrate increased hepatic LDLR expression in mice by a mechanism involving Akt phosphorylation and LDLR gene transcription mediated by SREBP2.

Mandibular appliance modulates condylar growth through integrins.

Functional orthopedic therapy corrects growth discrepancies between the maxilla and mandible, possibly through postural changes in the musculature and modulation of the mandibular condylar cartilage growth. Using Wistar rats, we tested the hypothesis that chondrocytes respond to forces generated by a mandibular propulsor appliance by changes in gene expression, and that integrins are important mediators in this response. Immunohistochemical analyses demonstrated that the use of the appliance for different periods of time modulated the expression of fibronectin, alpha5 and alphav integrin subunits, as well as cell proliferation in the cartilage. In vitro, cyclic distension of condylar cartilage-derived cells increased fibronectin mRNA, as well as Insulin-like Growth Factor-I and II mRNA and cell proliferation. A peptide containing the Arginine-Glycine-Asparagine sequence (RGD), the main cell-binding sequence in fibronectin, blocked almost all these effects, confirming that force itself modulates the growth of the rat condylar cartilage, and that RGD-binding integrins participate in mechanotransduction.

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

BACKGROUND AND PURPOSE: Adipocyte differentiation in vitro is coordinately activated by two transcription factors, peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT enhancer binding protein alpha (C/EBPalpha), 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. EXPERIMENTAL APPROACH: The effects of pitavastatin on adipocyte differentiation were evaluated by the formation of oil droplets, content of cellular triglyceride and expression of adipocyte-specific genes. Regulatory mechanisms were assessed by analysis of PPARgamma, C/EBPalpha and pref-1 expression. KEY RESULTS: Pitavastatin significantly inhibited adipocyte differentiation of 3T3-L1 preadipocytes in response to adipogenic inducers. Evidence for inhibition included fewer Oil Red O positive droplets, less cellular triglyceride and decreased expression of adipocyte-specific genes, including fatty acid binding protein (aP2), CD36, adipsin and glucose transporter 4 (GLUT4). The inhibitory effects of pitavastatin on adipocyte differentiation of 3T3-L1 preadipocytes were time and concentration dependent. Pitavastatin significantly blocked induction of PPARgamma expression, but not C/EBPalpha expression or DNA binding activity of PPARgamma. Also, pitavastatin induced pref-1 expression in preadipocytes and maintained expression of pref-1 at high levels in differentiated cells. CONCLUSIONS AND IMPLICATIONS: Our data suggest that pitavastatin inhibits adipocyte differentiation by blocking PPARgamma expression and activating pref-1 expression. These studies may have implications in the regulation of adipogenesis in response to statins.

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I.

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.

Role of CD36, the macrophage class B scavenger receptor, in atherosclerosis.

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-gamma.

Recruitment and activation of Raf-1 kinase by nitric oxide-activated Ras.

Nitric oxide (NO) and related species serve as cellular messengers in various physiological and pathological processes. The monomeric G protein, Ras, transduces multiple signaling pathways with varying biological responses. We have previously reported that NO triggers Ras activation and recruitment of an effector, phosphatidylinositol 3'-kinase (PI3K) and Ras-dependent activation of mitogen-activated protein (MAP) kinases which include extracellular signal regulated kinases (ERKs), c-Jun NH(2)-terminal kinase (JNK), and p38 MAP kinase. In this study, we further defined NO-activated Ras signaling pathways. We have identified Raf-1 as another effector recruited by NO-activated Ras in T lymphocytes. NO activation results in association of Ras and Raf-1 and is biologically significant, as we observe an NO-induced increase in Raf-1 kinase activity. Downstream to Raf-1 kinase lie MAP kinases and their subsequent downstream targets, transcription factors. We found that treatment of T lymphocytes with NO yielded phosphorylation of the transcription factor, Elk-1. This phoshorylation is dependent on NO binding to the cysteine 118 residue of Ras. By further delineating the pathway with pharmacological inhibitors, Elk-1 phosphorylation was also found to be dependent on PI3K and ERK. Moreover, NO triggered an increase in mRNA levels of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), which was ERK dependent. Thus, we have defined an NO-induced signaling pathway in T lymphocytes arising at the membrane where NO-activated Ras recruits Raf-1 and culminating in the nucleus where Elk-1 is phosphorylated and TNF-alpha messenger RNA is induced. This NO-activated Ras-mediated signaling pathway may play a critical role in Elk-1-induced transcriptional activation of T lymphocytes, host defense and inflammation.

CD36 in atherosclerosis. The role of a class B macrophage scavenger receptor.

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-gamma (PPAR gamma), 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 PPAR gamma ligand. In addition, PKC inhibitors block both PPAR gamma mRNA and protein expression. These results suggest that activation of CD36 gene expression by OxLDL involves activation and translocation of PKC with subsequent PPAR gamma 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.

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.

Induction of CD36 expression by oxidized LDL and IL-4 by a common signaling pathway dependent on protein kinase C and PPAR-gamma.

CD36, a class B scavenger receptor, is a macrophage receptor for oxidized low density lipoprotein (OxLDL) and may play a critical role in atherosclerotic foam cell formation. We have previously demonstrated that OxLDL, macrophage-colony stimulating factor (M-CSF), and interleukin-4 (IL-4) enhanced expression of CD36. The effect of OxLDL on CD36 is due, in part, to its ability to activate the transcription factor, PPAR-gamma (peroxisome proliferator activated receptor-gamma). Other PPAR-gamma ligands (15-deoxyDelta(12,14) prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinedione class of antidiabetic drugs) also increase CD36 expression. We have now evaluated signaling pathways involved in the induction of CD36. Treatment of RAW264.7 cells (a murine macrophage cell line) with protein kinase C (PKC) activators (diacylglycerol and ingenol) up-regulated CD36 mRNA expression. Specific inhibitors of PKC reduced CD36 expression in a time-dependent manner, while protein kinase A (PKA) and cyclic AMP agonists had no effect on CD36 mRNA expression. PKC inhibitors reduced basal expression of CD36 and blocked induction of CD36 mRNA by 15d-PGJ(2), OxLDL and IL-4. In addition, PKC inhibitors decreased both PPAR-gamma mRNA and protein expression and blocked induction of CD36 protein surface expression by OxLDL and 15d-PGJ(2) in human monocytes, as determined by FACS. 15d-PGJ(2) had no effect on translocation of PKC-alpha from the cytosol to the plasma membrane. These results demonstrate that two divergent physiological or pathophysiological agonists utilize a common pathway to up-regulate of CD36 gene expression. This pathway involves initial activation of PKC with subsequent PPAR-gamma activation. Defining these signaling pathways is critical for understanding and modulating expression of this scavenger receptor pathway.

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.

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.

Transforming growth factor-beta1 (TGF-beta1) and TGF-beta2 decrease expression of CD36, the type B scavenger receptor, through mitogen-activated protein kinase phosphorylation of peroxisome proliferator-activated receptor-gamma.

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-beta1 (TGF-beta1) and TGF-beta2 on the expression of CD36 in macrophages. Treatment of phorbol ester-differentiated THP-1 macrophages with TGF-beta1 or TGF-beta2 significantly decreased expression of CD36 mRNA and surface protein. TGF-beta1/TGF-beta2 also inhibited CD36 mRNA expression induced by oxidized low density lipoprotein and 15-deoxyDelta(12,14) prostaglandin J(2), a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, suggesting that the TGF-beta1/TGF-beta2 down-regulated CD36 expression by inactivating PPAR-gamma-mediated signaling. TGF-beta1/TGF-beta2 increased phosphorylation of both mitogen-activated protein (MAP) kinase and PPAR-gamma, whereas MAP kinase inhibitors reversed suppression of CD36 and inhibited PPAR-gamma phosphorylation induced by TGF-beta1/TGF-beta2. Finally, MAP kinase inhibitors alone increased expression of CD36 mRNA and surface protein but had no effect on PPAR-gamma protein levels. Our data demonstrate for the first time that TGF-beta1 and TGF-beta2 decrease expression of CD36 by a mechanism involving phosphorylation of MAP kinase, subsequent MAP kinase phosphorylation of PPAR-gamma, and a decrease in CD36 gene transcription by phosphorylated PPAR-gamma.

Herpesviruses and thrombosis: activation of coagulation on the endothelium.

Vascular injury is an initiating event in the development of atherosclerosis and herpesviruses have been proposed as potential mediators of vascular injury. The demonstration that an avian herpesvirus could induce atherosclerosis in chickens [Fabricant CG, Fabricant J, Litrenta MM, Minick CR. Virus induced atherosclerosis. J Exp Med 1978;148:335-340; Fabricant CG, Fabricant J, Minick CR, Litrenta MM. Herpes virus induced atherosclerosis in chickens. Fed Proc 1983;42:2476-2479; Minick CR, Fabricant CG, Fabricant J, Litrenta MM. Atheroarteriosclerosis induced by infection by herpesvirus. Am J Pathol 1978;96:673-706] suggested the potential of these viral agents to cause similar lesions in humans. In addition, epidemiological evidence linking herpesvirus infection and atherosclerosis [Cunningham MJ, Pasternak RC. The potential role of viruses in the pathogenesis of atherosclerosis. Circulation 1988;77:964-996; Melnick JL, Adam E, DeBakey ME. Cytomegalovirus and atherosclerosis. BioEssays 1995;17:899-903; Adam E, Melnick JL, Probesfield JL et al. High levels of cytomegalovirus antibody in patients requiring vascular surgery for atherosclerosis. Lancet 1987;2:291-293] adds further credence to their role as possible etiologic agents. However, the link between herpesviruses and vascular thrombosis is more tenuous. In this review, we highlight some recent advances in this field, from our laboratory and others, to support the hypothesis that herpesviruses act as prothrombotic agents by activating the coagulation cascade.

Regulation of prostaglandin H2 synthase activity by nitrogen oxides.

Nitric oxide and its derivatives have been shown to both activate and inhibit prostaglandin H(2) synthase 1 (PGHS-1). We set out to determine the mechanisms by which different nitrogen oxide derivatives modulate PGHS-1 activity. To this end, we show that 3-morpholinosydnonimine hydrochloride (SIN-1), a compound capable of generating peroxynitrite, activates purified PGHS-1 and also stimulates PGE(2) production in arterial smooth muscle cells in the presence of exogenous arachidonic acid. The effect of SIN-1 in smooth muscle cells was abrogated by superoxide and peroxynitrite inhibitors, which supports the hypothesis that peroxynitrite is an activating species of PGHS-1. Indeed, authentic peroxynitrite also induced PGE(2) production in arachidonic acid-stimulated cells. In contrast, when cells were exposed to the nitric oxide-releasing compound 1-hydroxy-2-oxo-3-[(methylamino)propyl]-3-methyl-1-triazene (NOC-7), PGHS-1 enzyme activity was inhibited in the presence of exogenous arachidonic acid. Finally, in lipid-loaded smooth muscle cells, we demonstrate that SIN-1 stimulates arachidonic acid-induced PGE(2) production; albeit, the extent of activation is reduced compared to that under normal conditions. These results indicate that formation of peroxynitrite is a key intermediary step in PGHS-1 activation. However, other forms of NO(x)() inhibit PGHS-1. These results may have implications in the regulation of vascular function and tone in normal and atherosclerotic arteries.

A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism.

A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination. These mice produced no detectable CD36 protein, were viable, and bred normally. A significant decrease in binding and uptake of oxidized low density lipoprotein was observed in peritoneal macrophages of null mice as compared with those from control mice. CD36 null animals had a significant increase in fasting levels of cholesterol, nonesterified free fatty acids, and triacylglycerol. The increase in cholesterol was mainly within the high density lipoprotein fraction, while the increase in triacylglycerol was within the very low density lipoprotein fraction. Null animals had lower fasting serum glucose levels when compared with wild type controls. Uptake of 3H-labeled oleate was significantly reduced in adipocytes from null mice. However, the decrease was limited to the low ratios of fatty acid:bovine serum albumin, suggesting that CD36 was necessary for the high affinity component of the uptake process. The data provide evidence for a functional role for CD36 in lipoprotein/fatty acid metabolism that was previously underappreciated.

Cellular cholesterol regulates expression of the macrophage type B scavenger receptor, CD36.

CD36, the macrophage type B scavenger receptor, binds and internalizes oxidized low density lipoprotein (OxLDL), and may potentially play a role in the development of atherosclerosis. We reported that the native and modified low density lipoproteins increased CD36 mRNA and protein ( J. Biol. Chem. 272: 21654-21659). In this study, we investigated the effect of alterations of cellular cholesterol content on macrophage expression of CD36. Depletion of cholesterol by treatment with beta-cyclodextrins (beta-cyclodextrin [beta-CD] and methylated beta-cyclodextrin [MebetaCD]) significantly decreased CD36 mRNA and 125I-labeled OxLDL binding. Conversely, loading macrophages with cholesterol or cholesteryl ester (acetate) with MebetaCD:cholesterol complexes increased CD36 mRNA, 125I-labeled OxLDL binding, and CD36 surface expression as determined by fluorescence activated cell sorting. Thus, CD36 expression paralleled cellular cholesterol levels after removal of cholesterol with beta-cyclodextrins or addition of cholesterol with MebetaCD:cholesterol complexes. Neither cholesterol depletion nor loading altered expression of type A scavenger receptor mRNA. Kinetics studies showed that changes in CD36 mRNA occurred after changes of cellular cholesterol. Neither beta-cyclodextrins nor MebetaCD:cholesterol altered CD36 mRNA half-life in the presence of actinomycin D, suggesting that alterations in CD36 expression by cholesterol occur at the transcriptional level. These experiments demonstrate that CD36 expression is enhanced by cholesterol and down-regulated by cholesterol efflux, and imply that macrophage expression of CD36 and foam cell formation in atherosclerotic lesions may be perpetuated by a cycle in which lipids drive expression of CD36 in a self-regulatory manner.