PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.

Association between liver X receptor alpha gene polymorphisms and risk of metabolic syndrome in French populations.

CONTEXT: The metabolic syndrome is a complex and multifactorial disorder often associated with type 2 diabetes mellitus and cardiovascular diseases. The liver X receptor alpha (NR1H3) plays numerous roles in metabolic pathways involved in metabolic syndrome. OBJECTIVE: In the search for susceptibility genes to metabolic syndrome, we hypothesized that common genetic variation in NR1H3 gene influences metabolic syndrome susceptibility. DESIGN: Two large French population-based studies (n=1130 and 1160) including overall 664 individuals with and 1626 individuals without metabolic syndrome were genotyped for three polymorphisms (rs12221497, rs11039155 and rs2279239) of NR1H3. RESULTS: We found that the -6A allele of rs11039155 was consistently associated with a 30% reduction in risk of metabolic syndrome in the two independent population samples (adjusted OR (95% CI)=0.68 (0.53-0.86), P=0.001 for the combined sample). Moreover, it was associated with an increase in plasma HDL-cholesterol concentrations (P=0.02 for the combined sample). Neither rs12221497 nor rs11039155, both polymorphisms located in the 5' region of NR1H3, had significant influence on NR1H3 and ATP-binding cassette transporter A1 (ABCA1) gene expression in primary human macrophages. CONCLUSIONS: These results suggest that NR1H3 plays an important role in the HDL-cholesterol metabolism and in the genetic susceptibility to metabolic syndrome.

IL-1ß- and IL-4-polarized macrophages have opposite effects on adipogenesis of intramuscular fibro-adipogenic progenitors in humans.

Intramuscular fat deposition represents a negative prognostic factor for several myopathies, metabolic diseases and aging. Fibro-adipogenic progenitors (FAPs) are considered as the main source of intramuscular adipocytes, but the mechanisms controlling their adipogenic potential are still not elucidated in humans. The aim of this study was to explore the regulation of human FAP adipogenesis by macrophages. We found that CD140a-expressing FAPs were located close to CD68 positive macrophages in muscles from patients with Duchenne muscular dystrophy (DMD). This strongly suggests a potential interaction between FAPs and macrophages in vivo. Isolated human primary FAPs were then differentiated in the presence of conditioned media obtained from primary blood monocyte-polarized macrophages. Molecules released by IL-1ß-polarized macrophages (M(IL-1ß)) drastically reduced FAP adipogenic potential as assessed by decreased cellular lipid accumulation and reduced gene expression of adipogenic markers. This was associated with an increased gene expression of pro-inflammatory cytokines in FAPs. Conversely, factors secreted by IL-4-polarized macrophages (M(IL-4)) enhanced FAP adipogenesis. Finally, the inhibition of FAP adipocyte differentiation by M(IL-1ß) macrophages requires the stimulation of Smad2 phosphorylation of FAPs. Our findings identify a novel potential crosstalk between FAPs and M(IL-1ß) and M(IL-4) macrophages in the development of adipocyte accumulation in human skeletal muscles.

Peroxisome proliferator-activated receptor alpha reduces cholesterol esterification in macrophages.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acid derivatives and hypolipidemic drugs of the fibrate class. PPARalpha is expressed in monocytes, macrophages, and foam cells, suggesting a role for this receptor in macrophage lipid homeostasis with consequences for atherosclerosis development. Recently, it was shown that PPARalpha activation promotes cholesterol efflux from macrophages via induction of the ABCA1 pathway. In the present study, the influence of PPARalpha activators on intracellular cholesterol homeostasis was investigated. In human macrophages and foam cells, treatment with fibrates, synthetic PPARalpha activators, led to a decrease in the cholesteryl ester (CE):free cholesterol (FC) ratio. In these cells, PPARalpha activation reduced cholesterol esterification rates and Acyl-CoA:cholesterol acyltransferase-1 (ACAT1) activity. However, PPARalpha activation did not alter ACAT1 gene expression, whereas mRNA levels of carnitine palmitoyltransferase type 1 (CPT-1), a key enzyme in mitochondrial fatty acid catabolism, were induced. Finally, PPARalpha activation blocked CE formation induced by TNF-alpha, possibly due to the inhibition of neutral sphingomyelinase activation by TNF-alpha. In conclusion, our results identify a role for PPARalpha in the control of cholesterol esterification in macrophages, resulting in an enhanced availability of FC for efflux through the ABCA1 pathway.

CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors.

BACKGROUND: The scavenger receptors are cell-surface receptors for native and modified lipoproteins that play a critical role in the accumulation of lipids by macrophages. CLA-1/SR-BI binds HDL with high affinity and is involved in the cholesterol reverse-transport pathway. Peroxisome proliferator-activated receptors (PPARs) are transcription factors regulating the expression of genes implicated in lipid metabolism, cellular differentiation, and inflammation. Here, we investigated the expression of CLA-1/SR-BI in macrophages and its regulation by PPARs. METHODS AND RESULTS: CLA-1 is undetectable in human monocytes and is induced upon differentiation into macrophages. Immunohistological analysis on human atherosclerotic lesions showed high expression of CLA-1 in macrophages of the lipid core colocalizing with PPARalpha and PPARgamma staining. Activation of PPARalpha and PPARgamma resulted in the induction of CLA-1 protein expression in monocytes and in differentiated macrophages. Finally, SR-BI expression is increased in atherosclerotic lesions of apoE-null mice treated with either PPARgamma or PPARalpha ligands. CONCLUSIONS: Our data demonstrate that CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and induced by PPAR activation, identifying a potential role for PPARs in cholesterol homeostasis in atherosclerotic lesion macrophages.

A truncated human peroxisome proliferator-activated receptor alpha splice variant with dominant negative activity.

The peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARalpha activators. This incited us to screen for PPARalpha variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARalpha mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPARalpha protein (PPARalphatr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARalphatr mRNA is expressed in several human tissues and cells, representing between 20-50% of total PPARalpha mRNA. By contrast, PPARalphatr mRNA could not be detected in rodent tissues. Western blot analysis using PPARalpha-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARalphatr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARalphatr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARalphawt was mainly nuclear localized, the majority of PPARalphatr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARalphatr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARalphawt transactivation function, PPARalphatr interfered with PPARalphatr transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARalphawt by PPARalphatr, suggesting that the dominant negative effect of PPARalphatr might occur through competition for essential coactivators. In addition, PPARalphatr interfered with transcriptional activity of other nuclear receptors such as PPARgamma, hepatic nuclear factor-4, and glucocorticoid receptor-alpha, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARalpha splice variant that may negatively interfere with PPARalphawt function. Factors regulating either the ratio of PPARalphawt vs. PPARalphatr mRNA or the nuclear entry of PPARalphatr protein should therefore lead to altered signaling via the PPARalpha and, possibly also, other nuclear receptor pathways.

Fibrates.

Atherosclerosis of the large arteries is the main origin of cerebro- and cardiovascular diseases, the leading causes of mortality and morbidity in industrialized countries. The pathophysiology of coronary and cerebrovascular atherosclerosis is multifactorial and complex. Fibrates are hypolipidemic drugs that lower progression of atherosclerotic lesions mainly through activation of the nuclear receptor peroxisome-proliferator activated receptor-alpha. In addition, fibrates exert pleiotropic and anti-inflammatory actions. In this chapter, we will focus on the different effects of fibrates impacting on the development of atherosclerosis.

Peroxisome proliferator-activated receptor (PPAR) agonists decrease lipoprotein lipase secretion and glycated LDL uptake by human macrophages.

Lipoprotein lipase (LPL) acts independently of its function as triglyceride hydrolase by stimulating macrophage binding and uptake of native, oxidized and glycated LDL. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors expressed in monocyte/macrophages, where they control cholesterol homeostasis. Here we study the role of PPARs in the regulation of LPL expression and activity in human monocytes and macrophages. Incubation of human monocytes or macrophages with PPARalpha or PPARgamma ligands increases LPL mRNA and intracellular protein levels. By contrast, PPAR activators decrease secreted LPL mass and enzyme activity in differentiated macrophages. These actions of PPAR activators are associated with a reduced uptake of glycated LDL and could influence atherosclerosis development associated with diabetes.

Peroxisome proliferator-activated receptor gamma activators inhibit interleukin-12 production in murine dendritic cells.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily. They are divided into three subtypes (alpha, beta or delta, and gamma) and are involved in lipid and glucose homeostasis and in the control of inflammation. In this study, we analyzed the expression of PPARs in murine dendritic cells (DCs), the most potent antigen presenting cells. We find that immature as well as mature spleen-derived DCs express PPARgamma, but not PPARalpha, mRNA and protein. We also show that the PPARgamma activator rosiglitazone does not interfere with the maturation of DCs in vitro nor modifies their ability to activate naive T lymphocytes in vivo. Finally, we present evidence that PPARgamma activators down-modulate the CD40-induced secretion of interleukin-12, a potent Th1-driving factor. These data suggest a possible role for PPARgamma in the regulation of immune responses.

Transcriptional regulation of macrophage cholesterol trafficking by PPARalpha and LXR.

PPARs (peroxisome-proliferator-activated receptors) and LXRs (liver X receptors) are ligand-activated transcription factors that control lipid and glucose metabolism, as well as the inflammatory response. Since the macrophage plays an important role in host defence and immuno-inflammatory pathologies, particular attention has been paid to the role of PPARs and LXRs in the control of macrophage gene expression and function. Altered macrophage functions contribute to the pathogenesis of many infectious, immunological and inflammatory disease processes, including atherosclerosis. Research over the last few years has revealed important roles for PPARs and LXRs in macrophage inflammation and cholesterol homoeostasis with consequences in atherosclerosis development. This review will discuss the role of these transcription factors in the control of cholesterol trafficking in macrophages.

Peroxisome proliferator-activated receptors and inflammation: from basic science to clinical applications.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family. After activation by specific ligands, they regulate the transcription of genes involved in lipid and lipoprotein metabolism, glucose and energy homeostasis, as well as cellular differentiation. Recent studies have identified expression of the three PPARs in all cells of the arterial wall, where they control cholesterol homeostasis as well as the inflammatory response and, as a consequence, modulate atherogenesis. More generally, PPARs influence cell proliferation as well as the immune and inflammatory response in different tissues and cells. In this review, we will summarize the evidence indicating that PPARs are modulators of the inflammatory response with potential therapeutic applications not only in atherosclerosis, but potentially also in other inflammation-related diseases, such as hepatic inflammation and inflammatory bowel disease.

Peroxisome proliferator-activated receptors (PPARs): nuclear receptors at the crossroads between lipid metabolism and inflammation.

Peroxisome proliferator-activated (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor family. PPARs function as regulators of lipid and lipoprotein metabolism and glucose homeostasis and influence cellular proliferation, differentiation and apoptosis. PPARalpha is highly expressed in tissues such as liver, muscle, kidney and heart, where it stimulates the beta-oxidative degradation of fatty acids. PPARgamma is predominantly expressed in intestine and adipose tissue. PPARgamma triggers adipocyte differentiation and promotes lipid storage. The hypolipidemic fibrates and the antidiabetic glitazones are synthetic ligands for PPARalpha and PPARgamma, respectively. Furthermore, fatty acids and eicosanoids are natural PPAR ligands: PPARalpha is activated by leukotriene B4, whereas prostaglandin J2 is a PPARgamma ligand. These observations suggested a potential role for PPARs not only in metabolic but also in inflammation control. The first evidence for a role of PPARalpha in inflammation control came from the demonstration that PPARalpha deficient mice display a prolonged response to inflammatory stimuli. It was suggested that PPARalpha deficiency results in a reduced beta-oxidative degradation of these inflammatory fatty acid derivatives. More recently, PPAR activators were shown to inhibit the activation of inflammatory response genes (such as IL-2, IL-6, IL-8, TNFalpha and metalloproteases) by negatively interfering with the NF- kappaB, STAT and AP-1 signalling pathways. PPAR activators exert these anti-inflammatory activities in different immunological and vascular wall cell types such as monocyte/macrophages, endothelial, epithelial and smooth muscle cells in which PPARs are expressed. These recent findings indicate a modulatory role for PPARs in the control of the inflammatory response with potential therapeutic applications in inflammation-related diseases, such as atherosclerosis and inflammatory bowel disease.

Expression of adiponectin receptors in human macrophages and regulation by agonists of the nuclear receptors PPARalpha, PPARgamma, and LXR.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors expressed in macrophages where they control cholesterol homeostasis and inflammation. In an attempt to identify new PPARalpha and PPARgamma target genes in macrophages, a DNA array-based global gene expression profiling experiment was performed on human primary macrophages treated with specific PPARalpha and PPARgamma agonists. Surprisingly, AdipoR2, one of the two recently identified receptors for adiponectin, an adipocyte-specific secreted hormone with anti-diabetic and anti-atherogenic activities, was found to be induced by both PPARalpha and PPARgamma. AdipoR2 induction by PPARalpha and PPARgamma in primary and THP-1 macrophages was confirmed by Q-PCR analysis. Interestingly, treatment with a synthetic LXR agonist induced the expression of both AdipoR1 and AdipoR2. Furthermore, co-incubation with a PPARalpha ligand and adiponectin resulted in an additive effect on the reduction of macrophage cholesteryl ester content. Finally, AdipoR1 and AdipoR2 are both present in human atherosclerotic lesions. Moreover, AdipoR1 is more abundant than AdipoR2 in monocytes and its expression decreases upon differentiation into macrophages, whereas AdipoR2 remains constant. In conclusion, AdipoR1 and AdipoR2 are expressed in human atherosclerotic lesions and macrophages and can be modulated by PPAR and LXR ligands, thus identifying a mechanism of crosstalk between adiponectin and these nuclear receptor signaling pathways.

Peroxisome proliferator-activated receptors (PPARs): nuclear receptors with functions in the vascular wall.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors which function as regulators of lipid and lipoprotein metabolism and glucose homeostasis and influence cellular proliferation, differentiation and apoptosis. PPAR alpha is highly expressed in liver, muscle, kidney and heart, where it stimulates the beta-oxidative degradation of fatty acids. PPAR gamma is predominantly expressed in intestine and adipose tissue, where it triggers adipocyte differentiation and promotes lipid storage. Recently, the expression of PPAR alpha and PPAR gamma was also reported in cells of the vascular wall, such as monocyte/macrophages, endothelial and smooth muscle cells. The hypolipidemic fibrates and the antidiabetic glitazones are synthetic ligands for PPAR alpha and PPAR gamma, respectively. Furthermore, fatty acid-derivatives and eicosanoids are natural PPAR ligands: PPAR alpha is activated by leukotriene B4, whereas prostaglandin J2 is a PPAR gamma ligand, as well as some components of oxidized LDL, such as 9- and 13-HODE. These observations suggested a potential role for PPARs not only in metabolic but also in inflammation control and, by consequence, in related diseases such as atherosclerosis. More recently, PPAR activators were shown to inhibit the activation of inflammatory response genes (such as IL-2, IL-6, IL-8, TNF alpha and metalloproteases) by negatively interfering with the NF-kappa B, STAT and AP-1 signalling pathways in cells of the vascular wall. Furthermore, PPARs may also control lipid metabolism in the cells of the atherosclerotic plaque. In addition, different clinical trials (such as the LOCAT, BECAIT and VA-HIT) as well as animal studies indicate that PPAR activators may have anti-atherogenic properties by reducing the progression of atherosclerotic lesions. In this review, we summarize the evidence indicating that PPAR alpha and PPAR gamma directly modulate vessel wall functions, and its consequences in the control of cardiovascular disease.

Peroxisome proliferator-activated receptors: from transcriptional control to clinical practice.

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors that control energy homeostasis through genomic actions. Over the past few years significant advances have been made in unravelling the pathways that are modulated by PPARs. Gene targeting experiments in mice and genetic studies in humans have demonstrated a physiological role for these receptors in adipocyte function, glucose homeostasis, and lipid and lipoprotein metabolism. Recent data indicate that PPARs enhance the reverse cholesterol transport pathway by regulating genes that control macrophage cholesterol efflux, cholesterol transport in plasma and bile acid synthesis. Clinical and experimental evidence suggest that PPAR activation decreases the incidence of cardiovascular disease not only by correcting metabolic disorders, but also through direct actions at the level of the vascular wall. Thus, dysregulation of PPAR activity modulates the onset and evolution of metabolic disorders such as dyslipidaemia, obesity and insulin resistance, predisposing to atherosclerosis.

The OSBP-related protein family in humans.

Oxysterols are oxygenated derivatives of cholesterol that have a number of biological effects and play a key role in the maintenance of the body cholesterol balance. In this study, we describe the cDNA sequences and genomic structures of the recently identified human oxysterol-binding protein (OSBP)-related protein (ORP) family (Laitinen, S. et al. 1999. J. Lipid Res. 40: 2204-2211). The family now includes 12 genes/proteins, which can be divided into six distinct subfamilies. The ORP have two major structural features: a highly conserved OSBP-type sterol-binding domain in the C-terminal half and a pleckstrin homology domain present in the N-terminal region of most family members. Several ORP genes are present in S. cerevisiae, D. melanogaster, and C. elegans, suggesting that the protein family has functions of fundamental importance in the eukaryotic kingdom. Analysis of ORP mRNA levels in unloaded or acetylated LDL-loaded human macrophages revealed that the expression of ORP genes was not significantly affected by the loading, with the exception of ORP6, which was up-regulated 2-fold. The present study summarizes the basic characteristics of the OSBP-related gene/protein family in humans, and provides tools for functional analysis of the encoded proteins.

PPARalpha agonists inhibit tissue factor expression in human monocytes and macrophages.

BACKGROUND: Monocytic tissue factor (TF) expression may contribute to thrombogenicity associated with plaque rupture and may propagate thrombus formation at the site of vascular lesions. Induction of monocytic TF expression by endotoxin is mediated by the activation of transcription factors such as AP-1 and NF-kappaB. Both these signaling pathways are modulated by peroxisome proliferator-activated receptor-alpha (PPARalpha). Therefore, we have studied the effects of fibrates and other PPARalpha agonists on the expression of TF. METHODS AND RESULTS: We show that PPARalpha protein, like primary human monocytes, is also expressed in the human monocytic THP-1 cell line. Fenofibric acid, WY14643, and GW2331 inhibited TF mRNA upregulation after stimulation of THP-1 cells with lipopolysaccharide or interleukin-1ss. In primary human monocytes and macrophages, the lipopolysaccharide- or interleukin-1ss-mediated induction of TF activity was also inhibited by fenofibric acid, WY14643, or GW2331. CONCLUSIONS: These data indicate that activation of PPARalpha results in the downregulation of the TF gene. Our results suggest a novel role for PPARalpha in the control of atherosclerotic plaque thrombogenicity through its effects on TF expression in monocytes and macrophages.

Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway.

Endothelin-1 (ET-1), a 21-amino acid vasoactive peptide mainly produced by vascular endothelial cells, is involved in the regulation of vascular tone and smooth muscle cell proliferation. Peroxisome proliferator-activated receptors (PPARs), key players in lipid and glucose metabolism, have been implicated in metabolic disorders that are predisposing to atherosclerosis. Because of the potential role of ET-1 in vascular disorders such as hypertension and atherosclerosis, we investigated the regulation of ET-1 expression by PPAR activators. Western blot and reverse transcription-polymerase chain reaction analyses demonstrated that both PPARalpha and PPARgamma are expressed in human coronary artery endothelial cells as well as in endothelial cell lines such as HMEC-1 and ECV304. In bovine aortic endothelial cells and HMEC-1 cells, both PPARalpha and PPARgamma ligands inhibited thrombin-induced ET-1 secretion, whereas basal ET-1 secretion was only slightly suppressed. Reverse transcription-polymerase chain reaction experiments showed that this inhibition of ET-1 production occurs at the gene expression level. Using transient transfection assays, we demonstrated that PPARs downregulate thrombin-activated transcription of the human ET-1 promoter. Transactivation studies with c-Jun and c-Fos expression plasmids indicated that PPARs negatively interfere with the activator protein-1 signaling pathway, which mediates thrombin activation of ET-1 gene transcription. Furthermore, electrophoretic mobility shift assays demonstrated that PPAR activators reduce the thrombin-stimulated binding activity of bovine aortic endothelial cell nuclear extracts as well as c-Jun binding to an activator protein-1 consensus site. Taken together, these data indicate that (1) both PPARalpha and PPARgamma are expressed in human vascular endothelial cells and (2) PPAR activators inhibit thrombin-induced ET-1 biosynthesis, indicating a novel role for PPARs in vascular endothelial function.