Giulia Chinetti

Activation of human aortic smooth-muscle cells is inhibited by PPARα but not by PPARγ activators

Peroxisome proliferator-activated receptors (PPARs) are key players in lipid and glucose metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as dyslipidaemia and diabetes. Whereas PPARγ promotes lipid storage by regulating adipocyte differentiation, PPARα stimulates the β-oxidative degradation of fatty acids. PPARα-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARα is also a modulator of inflammation. Hypolipidaemic fibrate drugs are PPARα ligands that inhibit the progressive formation of atherosclerotic lesions, which involves chronic inflammatory processes, even in the absence of their atherogenic lipoprotein-lowering effect,. Here we show that PPARα is expressed in human aortic smooth-muscle cells, which participate in plaque formation and post-angioplasty re-stenosis. In these smooth-muscle cells, we find that PPARα ligands, and not PPARγ ligands, inhibit interleukin-1-induced production of interleukin-6 and prostaglandin and expression of cyclooxygenase-2. This inhibition of cyclooxygenase-2 induction occurs transcriptionally as a result of PPARα repression of NF-κB signalling. In hyperlipidaemic patients, fenofibrate treatment decreases the plasma concentrations of interleukin-6, fibrinogen and C-reactive protein. We conclude that activators of PPARα inhibit the inflammatory response of aortic smooth-muscle cells and decrease the concentration of plasma acute-phase proteins, indicating that PPARα in the vascular wall may influence the process of atherosclerosis and re-stenosis.

ACTIVATION OF PROLIFERATOR-ACTIVATED RECEPTORS ALPHA AND GAMMA INDUCES APOPTOSIS OF HUMAN MONOCYTE-DERIVED MACROPHAGES

Peroxisome proliferator-activated receptors (PPARs) have been implicated in metabolic diseases, such as obesity, diabetes, and atherosclerosis, due to their activity in liver and adipose tissue on genes involved in lipid and glucose homeostasis. Here, we show that the PPARα and PPARγ forms are expressed in differentiated human monocyte-derived macrophages, which participate in inflammation control and atherosclerotic plaque formation. Whereas PPARα is already present in undifferentiated monocytes, PPARγ expression is induced upon differentiation into macrophages. Immunocytochemistry analysis demonstrates that PPARα resides constitutively in the cytoplasm, whereas PPARγ is predominantly nuclear localized. Transient transfection experiments indicate that PPARα and PPARγ are transcriptionally active after ligand stimulation. Ligand activation of PPARγ, but not of PPARα, results in apoptosis induction of unactivated differentiated macrophages as measured by the TUNEL assay and the appearance of the active proteolytic subunits of the cell death protease caspase-3. However, both PPARα and PPARγ ligands induce apoptosis of macrophages activated with tumor necrosis factor α/interferon γ. Finally, PPARγ inhibits the transcriptional activity of the NFκB p65/RelA subunit, suggesting that PPAR activators induce macrophage apoptosis by negatively interfering with the anti-apoptotic NFκB signaling pathway. These data demonstrate a novel function of PPAR in human macrophages with likely consequences in inflammation and atherosclerosis.

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

Abstract: Peroxisome proliferator-activated receptors (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. PPARα is highly expressed in tissues such as liver, muscle, kidney and heart, where it stimulates the β-oxidative degradation of fatty acids. PPARγ is predominantly expressed in intestine and adipose tissue. PPARγ triggers adipocyte differentiation and promotes lipid storage. The hypolipidemic fibrates and the antidiabetic glitazones are synthetic ligands for PPARα and PPARγ, respectively. Furthermore, fatty acids and eicosanoids are natural PPAR ligands: PPARα is activated by leukotriene B4, whereas prostaglandin J2 is a PPARγ 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 PPARα in inflammation control came from the demonstration that PPARα deficient mice display a prolonged response to inflammatory stimuli. It was suggested that PPARα deficiency results in a reduced β-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, TNFα and metalloproteases) by negatively interfering with the NF-κB, 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.

Sorting out the roles of PPARα in energy metabolism and vascular homeostasis

PPARα is a nuclear receptor that regulates liver and skeletal muscle lipid metabolism as well as glucose homeostasis. Acting as a molecular sensor of endogenous fatty acids (FAs) and their derivatives, this ligand-activated transcription factor regulates the expression of genes encoding enzymes and transport proteins controlling lipid homeostasis, thereby stimulating FA oxidation and improving lipoprotein metabolism. PPARα also exerts pleiotropic antiinflammatory and antiproliferative effects and prevents the proatherogenic effects of cholesterol accumulation in macrophages by stimulating cholesterol efflux. Cellular and animal models of PPARα help explain the clinical actions of fibrates, synthetic PPARα agonists used to treat dyslipidemia and reduce cardiovascular disease and its complications in patients with the metabolic syndrome. Although these preclinical studies cannot predict all of the effects of PPARα in humans, recent findings have revealed potential adverse effects of PPARα action, underlining the need for further study. This Review will focus on the mechanisms of action of PPARα in metabolic diseases and their associated vascular pathologies.

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.

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.

Peroxisome Proliferator-Activated Receptor (PPAR) α and PPARβ/δ, but not PPARγ, Modulate the Expression of Genes Involved in Cardiac Lipid Metabolism

Abstract— Long-chain fatty acids (FA) coordinately induce the expression of a panel of genes involved in cellular FA metabolism in cardiac muscle cells, thereby promoting their own metabolism. These effects are likely to be mediated by peroxisome proliferator-activated receptors (PPARs). Whereas the significance of PPAR&agr; in FA-mediated expression has been demonstrated, the role of the PPAR&bgr;/&dgr; and PPAR&ggr; isoforms in cardiac lipid metabolism is unknown. To explore the involvement of each of the PPAR isoforms, neonatal rat cardiomyocytes were exposed to FA or to ligands specific for either PPAR&agr; (Wy-14,643), PPAR&bgr;/&dgr; (L-165041, GW501516), or PPAR&ggr; (ciglitazone and rosiglitazone). Their effect on FA oxidation rate, expression of metabolic genes, and muscle-type carnitine palmitoyltransferase-1 (MCPT-1) promoter activity was determined. Consistent with the PPAR isoform expression pattern, the FA oxidation rate increased in cardiomyocytes exposed to PPAR&agr; and PPAR&bgr;/&dgr; ligands, but not to PPAR&ggr; ligands. Likewise, the FA-mediated expression of FA-handling proteins was mimicked by PPAR&agr; and PPAR&bgr;/&dgr;, but not by PPAR&ggr; ligands. As expected, in embryonic rat heart-derived H9c2 cells, which only express PPAR&bgr;/&dgr;, the FA-induced expression of genes was mimicked by the PPAR&bgr;/&dgr; ligand only, indicating that FA also act as ligands for the PPAR&bgr;/&dgr; isoform. In cardiomyocytes, MCPT-1 promoter activity was unresponsive to PPAR&ggr; ligands. However, addition of PPAR&agr; and PPAR&bgr;/&dgr; ligands dose-dependently induced promoter activity. Collectively, the present findings demonstrate that, next to PPAR&agr;, PPAR&bgr;/&dgr;, but not PPAR&ggr;, plays a prominent role in the regulation of cardiac lipid metabolism, thereby warranting further research into the role of PPAR&bgr;/&dgr; in cardiac 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 role of PPARs in atherosclerosis

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors that regulate lipid and lipoprotein metabolism, glucose homeostasis and inflammation. The PPAR family consists of three proteins, alpha, beta/delta and gamma. Recent data suggest that PPAR alpha and gamma activation decreases atherosclerosis progression not only by correcting metabolic disorders, but also through direct effects on the vascular wall. PPARs modulate the recruitment of leukocytes to endothelial cells, control the inflammatory response and lipid homeostasis of monocytes/macrophages and regulate inflammatory cytokine production by smooth muscle cells. Experiments using animal models of atherosclerosis and clinical studies in humans strongly support an anti-atherosclerotic role for PPAR alpha and gamma in vivo. Thus, PPARs remain attractive therapeutic targets for the development of drugs used in the treatment of chronic inflammatory diseases such as atherosclerosis. Future research will aim for the development of more potent drugs with co-agonist activity on PPAR alpha, PPAR beta/delta and/or PPAR gamma as well as tissue and target gene-selective PPAR receptor modulators (SPPARMs).

PPARα 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-κB. Both these signaling pathways are modulated by peroxisome proliferator–activated receptor-α (PPARα). Therefore, we have studied the effects of fibrates and other PPARα agonists on the expression of TF. Methods and Results—We show that PPARα 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-1β. In primary human monocytes and macrophages, the lipopolysaccharide- or interleukin-1β–mediated induction of TF activity was also inhibited by fenofibric acid, WY14643, or GW2331. Conclusions—These data indicate that activat...