Selective PPARα Modulator Pemafibrate and Sodium-Glucose Cotransporter 2 Inhibitor Tofogliflozin Combination Treatment Improved Histopathology in Experimental Mice Model of Non-Alcoholic Steatohepatitis.

Ballooning degeneration of hepatocytes is a major distinguishing histological feature of non-alcoholic steatosis (NASH) progression that can lead to cirrhosis and hepatocellular carcinoma (HCC). In this study, we evaluated the effect of the selective PPARα modulator (SPPARMα) pemafibrate (Pema) and sodium-glucose cotransporter 2 (SGLT2) inhibitor tofogliflozin (Tofo) combination treatment on pathological progression in the liver of a mouse model of NASH (STAM) at two time points (onset of NASH progression and HCC survival). At both time points, the Pema and Tofo combination treatment significantly alleviated hyperglycemia and hypertriglyceridemia. The combination treatment significantly reduced ballooning degeneration of hepatocytes. RNA-seq analysis suggested that Pema and Tofo combination treatment resulted in an increase in glyceroneogenesis, triglyceride (TG) uptake, lipolysis and liberated fatty acids re-esterification into TG, lipid droplet (LD) formation, and Cidea/Cidec ratio along with an increased number and reduced size and area of LDs. In addition, combination treatment reduced expression levels of endoplasmic reticulum stress-related genes (Ire1a, Grp78, Xbp1, and Phlda3). Pema and Tofo treatment significantly improved survival rates and reduced the number of tumors in the liver compared to the NASH control group. These results suggest that SPPARMα and SGLT2 inhibitor combination therapy has therapeutic potential to prevent NASH-HCC progression.

Pemafibrate, a selective PPARα modulator, prevents non-alcoholic steatohepatitis development without reducing the hepatic triglyceride content.

Non-alcoholic steatohepatitis (NASH) is characterized by macrovesicular steatosis with ballooning degeneration of hepatocytes, diffused lobular inflammation, and fibrosis. PPAR ligands are promising therapeutic agents in NASH; accordingly, we evaluated the effects of the first clinically available selective PPARα modulator, pemafibrate. We found that pemafibrate improves F4/80-positive macrophage accumulation, ballooning degeneration of hepatocytes, and the non-alcoholic fatty liver disease (NAFLD) activity score without affecting triglyceride (TG) accumulation in the liver of a mouse model of NASH (STAM). A global gene expression analysis indicated that pemafibrate enhances TG hydrolysis and fatty acid ß-oxidation as well as re-esterification from dihydroxyacetone 3-phosphate and monoacylglycerol to TG. These changes are accompanied by the induction of genes involved in lipolysis and lipid droplet formation, along with an increased number and reduced size of lipid droplets in pemafibrate-treated livers. Pemafibrate reduced the expression of the cell adhesion molecule Vcam-1, myeloid cell markers, and inflammation- and fibrosis-related genes in STAM mice. Furthermore, pemafibrate significantly reduced VCAM-1 expression induced by high glucose in cultured human umbilical vein endothelial cells. These results suggest that pemafibrate prevents NASH development by reducing myeloid cell recruitment via interactions with liver sinusoidal endothelial cells, without altering hepatic TG accumulation.

Apolipoprotein A-V modulates insulin secretion in pancreatic beta-cells through its interaction with midkine.

Apolipoprotein A-V is an important determinant of plasma triglyceride level in both humans and mice. This study showed the physiological impact of apoA-V on insulin secretion in rat pancreatic beta-cells (INS-1 cells). In order to precise the mechanism of action, binding experiments coupled to mass spectrometry were performed to identify a potential membrane receptor. Results showed an interaction between apoA-V and midkine protein. Confocal microscopy confirmed the plasma membrane co-localisation of this two-proteins after the treatment of INS-1 cells with the apo-AV recombinant protein and indicated that the cell surface midkine could be involved in apoA-V endocytosis, since these two proteins were co-translocated at the plasma membrane or in the cytosol compartment. This co-localisation is correlated with an increase in insulin secretion in a dose dependant manner during short incubation period. Reduction of midkine expression by small interfering RNA duplexes revealed a decrease in the ability of these transfected cells to secrete insulin in presence of apoA-V. Competition experiments for the apoA-V-midkine binding at the cell surface using antibody directed against midkine is able to influence INS-1 cell function as insulin secretion. Our results showed apoA-V ability to enhance insulin secretion in beta-cells and provide evidence of an internalization pathway involving the midkine as partner.

Liver X receptor activation induces the uptake of cholesteryl esters from high density lipoproteins in primary human macrophages.

OBJECTIVE: Liver X receptors (LXRs) are oxysterol-activated nuclear receptors regulating reverse cholesterol transport, in part by modulating cholesterol efflux from macrophages to apoAI and HDL via the ABCA1 and ABCG1/ABCG4 pathways. Moreover, LXR activation increases intracellular cholesterol trafficking via the induction of NPC1 and NPC2 expression. However, implication of LXRs in the selective uptake of cholesteryl esters from lipoproteins in human macrophages has never been reported. METHODS AND RESULTS: Our results show that (1) selective CE uptake from HDL(3) is highly efficient in human monocyte-derived macrophages; (2) surprisingly, HDL(3)-CE uptake is strongly increased by LXR activation despite antiatherogenic effects of LXRs; (3) HDL(3)-CE uptake increase is not linked to SR-BI expression modulation but it is dependent of proteoglycan interactions; (4) HDL(3)-CE uptake increase is associated with increased expression and secretion of apoE and LPL, two proteins interacting with proteoglycans; (5) HDL(3)-CE uptake increase depends on the integrity of raft domains and is associated with an increased caveolin-1 expression. CONCLUSIONS: Our study identifies a new role for LXRs in the control of cholesterol homeostasis in human macrophages. LXR activation results in enhanced dynamic intracellular cholesterol fluxes through an increased CE uptake from HDL and leads to an increased cholesterol availability to efflux to apoAI and HDL.

Glucose regulates the expression of the apolipoprotein A5 gene.

The apolipoprotein A5 gene (APOA5) is a key player in determining triglyceride concentrations in humans and mice. Since diabetes is often associated with hypertriglyceridemia, this study explores whether APOA5 gene expression is regulated by alteration in glucose homeostasis and the related pathways. d-Glucose activates APOA5 gene expression in a time- and dose-dependent manner in hepatocytes, and the glycolytic pathway involved was determined using d-glucose analogues and metabolites. Together, transient transfections, electrophoretic mobility shift assays and chromatin immunoprecipitation assays show that this regulation occurs at the transcriptional level through an increase of USF1/2 binding to an E-box in the APOA5 promoter. We show that this phenomenon is not due to an increase of mRNA or protein expression levels of USF. Using protein phosphatases 1 and 2A inhibitor, we demonstrate that d-glucose regulates the APOA5 gene via a dephosphorylation mechanism, resulting in an enhanced USF1/2-promoter binding. Last, subsequent suppressions of USF1/2 and phosphatases mRNA through siRNA gene silencing abolished the regulation. We demonstrate that the APOA5 gene is up regulated by d-glucose and USF through phosphatase activation. These findings may provide a new cross-talk between glucose and lipid metabolism.

The nuclear receptor Rev-erbalpha is a liver X receptor (LXR) target gene driving a negative feedback loop on select LXR-induced pathways in human macrophages.

A role of the nuclear receptor Rev-erbalpha in the regulation of transcription pathways involving other nuclear receptors is emerging. Indeed, Rev-erbalpha is a negative regulator of transcription by binding to overlapping response elements shared with various nuclear receptors, including the peroxisome proliferator-activated receptors and the retinoid-related orphan receptor alpha (RORalpha). Here, we show that Rev-erbalpha is expressed in primary human macrophages and that its expression is induced by synthetic ligands for the liver X receptors (LXRs), which control cholesterol homeostasis, inflammation, and the immune response in macrophages. LXRalpha binds to a specific response element in the human Rev-erbalpha promoter, thus inducing Rev-erbalpha transcriptional expression. Interestingly, Rev-erbalpha does not influence basal or LXR-regulated cholesterol homeostasis. However, Rev-erbalpha overexpression represses the induction of toll-like receptor (TLR)-4 by LXR agonists, whereas Rev-erbalpha silencing by short interfering RNA results in enhanced TLR-4 expression upon LXR activation. Electrophoretic mobility shift, chromatin immunoprecipitation, and transient transfection experiments demonstrate that Rev-erbalpha represses human TLR-4 promoter activity by binding as a monomer to a RevRE site overlapping with the LXR response element site in the TLR-4 promoter. These data identify Rev-erbalpha as a new LXR target gene, inhibiting LXR-induction of TLR-4 in a negative transcriptional feedback loop, but not cholesterol homeostasis gene expression.

Liver X receptor activation potentiates the lipopolysaccharide response in human macrophages.

Macrophages play a central role in host defense against pathogen microbes by recognizing bacterial components, resulting in the activation of an arsenal of anti-microbial effectors. Toll-like receptor (TLR)-4 mediates the recognition of lipopolysaccharide, a pathogen-associated molecular pattern from gram-negative bacteria. Activation of the TLR-4 signaling pathway by lipopolysaccharide increases antibacterial effects by inducing secretion of cytokines that activate an immune inflammatory response and by generating bactericidal reactive oxygen species via the NADPH oxidase system. Liver X Receptors (LXRs) are nuclear receptors controlling cholesterol homeostasis and inflammation in macrophages. In addition, LXRs are critical for macrophage survival and play a role in the innate immune response in the mouse. In this study, we investigated whether LXR activation also regulates host defense mechanisms in human macrophages. In primary human macrophages, oxidized LDL and synthetic LXR ligands increased TLR-4 gene expression. Transient transfection assays, gel shift and chromatin immunoprecipitation analysis indicated that LXRs induce human TLR-4 promoter activity by binding to a DR4-type LXR response element. LXR induction of TLR-4 mRNA was followed by an induction of TLR-4 protein expression. Moreover, although short-term pretreatment with LXR agonists significantly reduced the inflammatory response induced by lipopolysaccharide, pretreatment of macrophages for 48 hours with LXR agonists resulted in an enhanced lipopolysaccharide response. Finally, LXR activation increased reactive oxygen species generation by enhancing the expression of NADPH oxidase subunits. These data provide evidence for an immunomodulatory function of LXRs in human macrophages via mechanisms distinct from those previously identified in mouse macrophages.

Safety issues and prospects for future generations of PPAR modulators.

Because of their wide range of actions on glucose homeostasis, lipid metabolism and vascular inflammation, peroxisome proliferator-activated receptors (PPARs) are promising targets for the development of new drugs for the treatment of metabolic disorders such as diabetes, dyslipidemia and atherosclerosis. In clinical practice, PPARalpha agonists, such as the already available fibrates, improve dyslipidemia, while PPARgamma agonists, such as thiazolidinediones, improve insulin resistance and diabetes. The complementary action of simultaneous activation of each PPAR in patients suffering from metabolic syndrome and type 2 diabetes has led to new pharmacological strategies focused on the development of agonists targeting more than one receptor such as the dual PPARalpha/gamma agonists. However, despite the proven benefits of targeting PPARs, safety concerns have recently led to late stage development failures of various PPAR agonists including novel specific PPARgamma agonists and dual PPARalpha/gamma agonists. These safety concerns include potential carcinogenicity in rodents, signs of myopathy and rhabdomyolysis, increase in plasma creatinine and homocysteine, weight gain, fluid retention, peripheral edema and potential increased risk of cardiac failure. Although the discontinued compounds shared common side effects, the reason for discontinuation was always compound specific and the toxicological or adverse effects which have motivated the discontinuation could be either due to the activation of PPARgamma, PPARalpha or both (class effect) or due to a PPAR unrelated effect. Thus, the risk evaluation of each adverse effect should be viewed on a case by case basis considering both the PPAR profile of the drug, its absorption/distribution profile, the nature of the side effect and the putative PPAR-related mechanism of action. This review mainly focuses on the preclinical and clinical adverse events of PPAR agonists that could be of concern when considering the development of new PPAR agonists. The selective modulation of PPAR activities is a promising approach to develop new drugs with preserved efficacy but diminished adverse effects.

[4-(2H-1,2,3-benzotriazol-2-yl)phenoxy]alkanoic acids as agonists of peroxisome proliferator-activated receptors (PPARs).

A series of [4-(2H-1,2,3-benzotriazol-2-yl)phenoxy]alkanoic acids has been synthesized and tested as agonists of Peroxisome Proliferator-Activated Receptor (PPAR) alpha, gamma, and delta. Three compounds displayed 56 to 96% of maximal activity of the reference drug Wy-14643 on PPARalpha, and two of these, i.e., 1 and 5, exhibited also moderate activity on either PPARgamma or delta with efficacy equal to 50% and 46% of that of rosiglitazone and GW 501516, respectively. Thus, compounds 1 and 5 represent interesting starting point for preparing novel agents for the treatment of dyslipidemia or of dyslipidemic type-2 diabetes.

The RXR agonist bexarotene improves cholesterol homeostasis and inhibits atherosclerosis progression in a mouse model of mixed dyslipidemia.

OBJECTIVE: The activity of the antitumoral agent bexarotene (Targretin, Bexarotene) depends on its binding to the nuclear retinoid-X receptor (RXR) and subsequent transcriptional regulation of target genes. Through RXR activation, bexarotene may modulate numerous metabolic pathways involved in atherosclerosis. Here, we investigated the effect of bexarotene on atherosclerosis progression in a dyslipidemic murine model, the human apolipoprotein E2 knockin mouse, that develops essentially macrophage-laden lesions. METHODS AND RESULTS: Atherosclerotic lesions together with different metabolic pathways involved in atherosclerosis were investigated in mice treated or not with bexarotene. Bexarotene protects from atherosclerosis development in mice, at least in part by improving the circulating cholesterol distribution profile likely via a marked decrease of dietary cholesterol absorption caused by modulation of intestinal expression of genes recently identified as major players in this process, Niemann-Pick-C1-Like1 (NPC1L1) and CD13. This atheroprotection appears despite a strong hypertriglyceridemia. Moreover, bexarotene treatment only modestly modulates inflammatory gene expression in the vascular wall, but markedly enhanced the capacity of macrophages to efflux cellular lipids. CONCLUSIONS: These data provide evidence of a favorable pharmacological effect of bexarotene on atherosclerosis despite the induction of hypertriglyceridemia, likely via a beneficial action on intestinal absorption and macrophage efflux.

Adipophilin increases triglyceride storage in human macrophages by stimulation of biosynthesis and inhibition of beta-oxidation.

Lipid accumulation alters macrophage biology and contributes to lipid retention within the vessel wall. In this study, we investigated the role of adipophilin on triglyceride accumulation and lipid-droplet formation in THP-1-derived macrophages (THP-1 macrophages). In the presence of acetylated low-density lipoprotein, macrophages infected with an adenovirus expressing human adipophilin showed a 31% increase in triglyceride content and a greater number of lipid droplets compared with control cells. Incubation of macrophages with very low-density lipoprotein (VLDL) dramatically increased cellular triglyceride content similarly in control and adipophilin-overexpressing cells. By itself, VLDL increased adipophilin expression, which explains the lack of effect of adipophilin overexpression on cellular triglyceride content in macrophages loaded with VLDL. The lipid-droplet content of macrophages was increased by overexpression of adipophilin and/or loading with VLDL. In contrast, inhibition of adipophilin expression using siRNA prevented lipid-droplet formation and significantly reduced intracellular triglyceride content. Using inhibitors of beta-oxidation and acyl-coenzyme A synthetase, results were obtained which suggest that adipophilin elevates cellular lipids by inhibition of beta-oxidation and stimulation of long-chain fatty acid incorporation into triglycerides. Adipophilin expression in THP-1 macrophages altered the cellular content of different lipids and enhanced the size of lipid droplets, consistent with a role for adipophilin in human foam cell formation.

Apolipoprotein E knockout mice over-expressing human tissue inhibitor of metalloproteinase 1 are protected against aneurysm formation but not against atherosclerotic plaque development.

AIMS: We investigated the effect of plasma levels of human tissue inhibitor of metalloproteinase (hTIMP)-1 on arterial lesion development and aneurysm formation in apolipoprotein-E-deficient mice (ApoE(-/-)). METHODS: Control and transgenic mice were fed either a chow diet or a high-fat diet for 90 and 180 days. RESULTS: hTIMP-1 has a tendency to decrease atherosclerotic lesions, but did not attain significance (approximately 6% reduction in hTIMP-1(+/+), p = 0.075, and approximately 4% in hTIMP-1(+/0), p = 0.088 vs. control). Immunohistological and histological analyses revealed a reduction in macrophage accumulation (23% of control in hTIMP(+/0), p = 0.065, and 49% of control in hTIMP(+/+), p < 0.05) but not in collagen degradation within the lesion in transgenic mice. Moreover, elastin degradation in sites of pseudo-microaneurysms was reduced in transgenic mice (37% of control in hTIMP-1(+/0), p < 0.05, and 50% of control in hTIMP-1(+/+), p < 0.05). DNA array analysis of matrix metalloproteinase (MMP) expression followed by real-time PCR quantification revealed a significant up-regulation of MMP-3, MMP-12 and MMP-13 in arterial lesions of ApoE(-/-) mice fed a high-fat diet in comparison with the same mice fed a chow diet. CONCLUSION: These data show that hTIMP-1 reduces aneurysm formation in ApoE(-/-) mice but does not protect them against the development of arterial lesions.

Both hepatic and extrahepatic ABCA1 have discrete and essential functions in the maintenance of plasma high-density lipoprotein cholesterol levels in vivo.

BACKGROUND: Extrahepatic tissues have long been considered critical contributors of cholesterol to nascent HDL particles in the reverse cholesterol transport pathway, in which ABCA1 plays the crucial role. Recent studies, however, including both overexpression and deletion of ABCA1 selectively in the liver, have highlighted the primary role of the liver in the maintenance of HDL levels in vivo. METHODS AND RESULTS: The availability of mice with complete deletion of ABCA1 (total knockout [TKO]) and with liver-specific deletion of ABCA1 (LSKO) has enabled us to dissect the discrete roles of hepatic relative to extrahepatic ABCA1 in HDL biogenesis. Delivery of adenoviral ABCA1 resulted in selective expression of physiological levels of ABCA1 in the livers of both LSKO and TKO mice, resulting in increased HDL cholesterol (HDL-C). Expression of ABCA1 in the liver of LSKO mice resulted in plasma HDL-C levels that were similar to those in wild-type mice and significantly above those seen in similarly treated TKO mice. HDL particles from ABCA1-expressing LSKO mice were larger and contained significantly increased cholesterol compared with TKO mice. Infusion of human apolipoprotein A-I/phospholipid reconstituted HDL particles normalized plasma HDL-C levels in LSKO mice but had no effect on HDL-C levels in TKO mice. CONCLUSIONS: Although hepatic ABCA1 appears crucial for phospholipid transport, extrahepatic tissues play an important role in cholesterol transfer to nascent HDL particles. These data highlight the discrete and specific roles of both liver and extrahepatic ABCA1 in HDL biogenesis in vivo and indicate that ABCA1 shows lipid cargo selectivity depending on its site of expression.

Glucose regulates LXRalpha subcellular localization and function in rat pancreatic beta-cells.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which have been implicated in lipid homeostasis and more recently in glucose metabolism. Here, we show that glucose does not change LXRalpha protein level, but affects its localization in pancreatic beta-cells. LXRalpha is found in the nucleus at 8 mM glucose and in the cytoplasm at 4.2 mM. Addition of glucose translocates LXRalpha from the cytoplasm into the nucleus. Moreover, after the activation of LXR by its synthetic non-steroidal agonist (T0901317), insulin secretion and glucose uptake are increased at 8 mM and decreased at 4.2 mM glucose in a dose-dependent manner. Furthermore, at low glucose condition, okadaic acid reversed LXRalpha effect on insulin secretion, suggesting the involvement of glucose signaling through a phosphorylation-dependent mechanism.

Paullinia pinnata extracts rich in polyphenols promote vascular relaxation via endothelium-dependent mechanisms.

Paullinia pinnata L. (Sapindaceae) is an African tropical plant whose roots and leaves are used in traditional medicine for many purposes, especially for erectile dysfunction, but its action mechanism is unknown. P. pinnata root and leaf methanolic extracts are rich in phenolic compounds. This study shows that both extracts are highly antioxidative and induce a slight transcriptional activity of peroxisome proliferator activated receptor-alpha. They also increased and decreased endothelial nitric oxide synthase and endothelin-1 mRNA levels in bovine aortic endothelial cells, respectively. In this study P. pinnata methanolic extracts in cumulative doses elicited in a dose-dependent manner the relaxation of phenylephrine precontracted isolated rat aortic rings. N-nitro-L-arginine methyl ester significantly attenuated the capacity of both extracts to induce arterial relaxation, indicating that this arterial relaxation was mediated by endothelial nitric oxide release. It could be suggested that the arterial relaxation induced by both extracts could be mainly linked to their capacities to inhibit nitric oxide oxidation through their antioxidant properties.

Modulation of hepatic inflammatory risk markers of cardiovascular diseases by PPAR-alpha activators: clinical and experimental evidence.

Atherosclerosis is a long-term chronic inflammatory disease associated with increased concentrations of inflammatory hepatic markers, such as CRP and fibrinogen, and of peripheral origin, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. Peroxisome proliferator-activated receptor (PPAR-)-alpha is a ligand-activated transcription factor that regulates expression of key genes involved in lipid homeostasis and modulates the inflammatory response both in the vascular wall and the liver. PPAR-alpha is activated by natural ligands, such as fatty acids, as well as the lipid-lowering fibrates. PPAR-alpha agonists impact on different steps of atherogenesis: (1) early markers of atherosclerosis, such as vascular wall reactivity, are improved, (2) however, reduced expression of adhesion molecules on the surface of endothelial cells, accompanied by decreased levels of inflammatory cytokines, such as TNF-alpha, IL-1, and IL-6, leads to a decreased leukocyte recruitment into the arterial wall; (3) in later stages of the atherosclerotic process, PPAR-alpha agonists may promote plaque stabilization and reduce cardiovascular events, via effects on metalloproteinases, such as MMP9. Moreover, PPAR-alpha activation by fibrates also impairs proinflammatory cytokine-signaling pathways in the liver resulting in the modulation of the acute phase response reaction via mechanisms independent of changes in lipoprotein levels. Effective coronary artery disease (CAD) prevention requires the use of agents that act beyond low-density lipoprotein cholesterol-lowering. PPAR-alpha agonists appear to comprehensively address some of the abnormalities of the most common clinical phenotypes of the high CAD risk patient of the 21st century such as in the metabolic syndrome and type 2 diabetes: low high-density lipoprotein cholesterol, high triglycerides, small, dense low-density lipoprotein, and a proinflammatory, procoagulant state.

Perilipin, a potential substitute for adipophilin in triglyceride storage in human macrophages.

Abnormal lipid deposition in human arteries leads to the formation of fatty streaks due to the accumulation of a large number of macrophage derived-foam cells. The formation and catabolism of intracellular lipid droplets is regulated by droplet-associated proteins. Among such proteins, the role of perilipin in human macrophages was unknown. In this study, we first showed that perilipin expression was increased during differentiation of human monocytes to macrophages. Interestingly, cellular perilipin content was unaffected by treatment of cells with OxLDL, AcLDL, VLDL or sterol esters. Moreover, its expression was not dependent on the presence of adipophilin, another lipid droplet-associated protein, since it was not affected by transfection of macrophages with siRNA-adipophilin. Perilipin overexpression in macrophages with an expression vector resulted in significant lipid droplet formation and TG accumulation and this was unaffected by decreasing adipophilin levels using siRNA. Consequently, perilipin, like adipophilin, might play an important role in the conversion of macrophages into foam cells and contribute to lesion formation. Therefore, inhibition of adipophilin might not be sufficient to prevent lesion formation as previously suggested, and perilipin inhibition might be additionally required.

Plasma cystatin-C and development of coronary heart disease: The PRIME Study.

The pathogenesis of ischemic coronary events involves degradation of the extracellular matrix in atherosclerotic lesions. The cysteine protease inhibitor cystatin-C may be involved in this phenomenon. The association of plasma cystatin-C with the incidence of myocardial infarction-coronary death and angina, was examined in a nested case-control (two controls per case) design within the prospective cohort study (Prospective Epidemiological Study of Myocardial Infarction (PRIME Study)) which included 9,758 men aged 50-59 years who were free of coronary heart disease (CHD) on entry and followed for a 5-year period. Three hundred and thirteen participants suffered myocardial infarction or coronary death (n = 159) or angina pectoris (n = 154) during follow-up. Cystatin-C was positively correlated with body mass index (BMI), low-density lipoprotein (LDL)-cholesterol, triglycerides and several inflammatory markers such as fibrinogen (r = 0.18), C-reactive protein (CRP) (r = 0.24), interleukin-6 (= 0.20), tumor necrosis factor-alpha (TNFalpha) (r = 0.27) and two TNFalpha receptors: TNFR1A (r = 0.43) and TNFR1B (r = 0.41); and negatively with high-density lipoprotein (HDL)-cholesterol (r = -0.25). After adjustment for traditional risk factors (age, diabetes, smoking, hypertension, BMI, triglycerides, LDL- and HDL-cholesterol), cystatin-C was significantly associated with the occurrence of the first ischemic coronary event. However, this association was no longer significant when CRP was included in the analysis. A decrease in glomerular filtration rate did not explain higher cystatin-C in cases than in controls. Cystatin-C appears to participate in the inflammatory phenomenon observed in the atherosclerotic process. Cystatin-C is not a more predictive risk marker of CHD than CRP or interleukin-6, but could be useful in detecting moderate chronic renal disease.

Extracellular human thioredoxin-1 inhibits lipopolysaccharide-induced interleukin-1beta expression in human monocyte-derived macrophages.

Oxidative stress plays an important role in atherosclerotic vascular disease, and several recent studies were focused on thioredoxin-1 (Trx-1) and its potential protective role against oxidative stress. Since human monocyte-derived macrophages (HMDM) are important cells in several inflammatory diseases including atherosclerosis, we conducted this study to evaluate the impact of extracellular recombinant human Trx-1 (rhTrx-1) on gene expression in lipopolysaccharide-activated HMDM. Our results showed that rhTrx-1 was capable of reducing interleukin (IL)-1beta mRNA and protein synthesis in a dose-dependent manner. This effect was partly mediated through a reduction of NF-kappaB activation as analyzed by transient transfection and gel shift assays. In addition, we showed that the attenuation of NF-kappaB activity was the result of the reduction of both p50 and p65 subunit mRNA and protein synthesis on one hand and of the induction of I-kappaBalpha mRNA and protein expression on the other hand. Moreover, inhibition of endogenous Trx-1 mRNA was also observed, suggesting a contribution to the diminution of NF-kappaB activity since endogenous Trx-1, in contrast to the exogenous Trx-1, activates the NF-kappaB system. Finally, H2O2-oxidized rhTrx-1 reduced IL-1beta mRNA synthesis in lipopolysaccharide-activated HMDM. This result highly suggested that the rhTrx-1 used in this study could be oxidized in the culture medium and, in turn, reduced IL-1beta mRNA and protein synthesis. Taken together, these data indicated a potential new mechanism through which extracellular rhTrx-1 exerts an anti-inflammatory function in HMDM.

PPAR alpha inhibits vascular smooth muscle cell proliferation underlying intimal hyperplasia by inducing the tumor suppressor p16INK4a.

Vascular SMC proliferation is a crucial event in occlusive cardiovascular diseases. PPARalpha is a nuclear receptor controlling lipid metabolism and inflammation, but its role in the regulation of SMC growth remains to be established. Here, we show that PPARalpha controls SMC cell-cycle progression at the G1/S transition by targeting the cyclin-dependent kinase inhibitor and tumor suppressor p16(INK4a) (p16), resulting in an inhibition of retinoblastoma protein phosphorylation. PPARalpha activates p16 gene transcription by both binding to a canonical PPAR-response element and interacting with the transcription factor Sp1 at specific proximal Sp1-binding sites of the p16 promoter. In a carotid arterial-injury mouse model, p16 deficiency results in an enhanced SMC proliferation underlying intimal hyperplasia. Moreover, PPARalpha activation inhibits SMC growth in vivo, and this effect requires p16 expression. These results identify an unexpected role for p16 in SMC cell-cycle control and demonstrate that PPARalpha inhibits SMC proliferation through p16. Thus, the PPARalpha/p16 pathway may be a potential pharmacological target for the prevention of cardiovascular occlusive complications of atherosclerosis.