Jean-Louis Paul

Inhibition of MicroRNA-92a Prevents Endothelial Dysfunction and Atherosclerosis in Mice

Rationale for Study: MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein expression at post-transcriptional level. We hypothesized that a specific pool of endothelial miRNAs could be selectively regulated by flow conditions and inflammatory signals, and as such be involved in the development of atherosclerosis. Objective: To identify miRNAs, called atheromiRs, which are selectively regulated by shear stress and oxidized low-density lipoproteins (oxLDL), and to determine their role in atherogenesis. Methods and Results: Large-scale miRNA profiling in HUVECs identified miR-92a as an atheromiR candidate, whose expression is preferentially upregulated by the combination of low shear stress (SS) and atherogenic oxLDL. Ex vivo analysis of atheroprone and atheroprotected areas of mouse arteries and human atherosclerotic plaques demonstrated the preferential expression of miR-92a in atheroprone low SS regions. In Ldlr−/− mice, miR-92a expression was markedly enhanced by hypercholesterolemia, in particular in atheroprone areas of the aorta. Assessment of endothelial inflammation in gain- and loss-of-function experiments targeting miR-92a expression revealed that miR-92a regulated endothelial cell activation by oxLDL, more specifically under low SS conditions, which was associated with modulation of Kruppel-like factor 2 (KLF2), Kruppel-like factor 4 (KLF4), and suppressor of cytokine signaling 5. miR-92a expression was regulated by signal transducer and activator of transcription 3 in SS- and oxLDL-dependent manner. Furthermore, specific in vivo blockade of miR-92a expression in Ldlr−/− mice reduced endothelial inflammation and altered the development of atherosclerosis, decreasing plaque size and promoting a more stable lesion phenotype. Conclusions: Upregulation of miR-92a by oxLDL in atheroprone areas promotes endothelial activation and the development of atherosclerotic lesions. Therefore, miR-92a antagomir seems as a new atheroprotective therapeutic strategy.

HDL Phospholipid Content and Composition as a Major Factor Determining Cholesterol Efflux Capacity From Fu5AH Cells to Human Serum

The relationships of cell cholesterol efflux to HDL phospholipid (PL) content and composition in human serum were analyzed in two groups of subjects selected on the basis of their HDL cholesterol (HDL-C) levels: a norm-HDL group (1.10 mmol/L < HDL-C < 1.50 mmol/L) and a high-HDL group (HDL-C > 1.75 mmol/L). In the high-HDL group, the relative fractional efflux was significantly higher than in the norm-HDL group, and in both groups, fractional efflux was correlated with a number of lipoprotein parameters, the best correlation and the only one that remained significant after multivariate analysis being with HDL phospholipid (HDL-PL). Analysis of the HDL-PL subclasses revealed that HDL in the high-HDL sera was enriched with phosphatidylethanolamine (HDL-PE) and relatively deficient in sphingomyelin (HDL-SM) compared with norm-HDL sera. Moreover, the fractional efflux values in the high-HDL group were negatively correlated with the proportion of HDL-PE (r = -.64, P < .0001) and positively correlated with the proportion of HDL-SM (r = .43, P < .01). Thus, this study provides evidence that HDL-PL concentration can be used to predict the capacity of serum to accept cellular cholesterol. Among the differences described between norm-HDL and high-HDL sera, the variability in PE to SM ratio might reflect changes in serum cholesterol acceptors that modulate the first step of reverse cholesterol transport.

Phosphatidylinositol 3-Kinase and Calcium-Activated Transcription Pathways Are Required for VLDL-Induced Smooth Muscle Cell Proliferation

Abstract— Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca2+ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose- and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca2+ release was inhibited and the IP3-sensitive Ca2+ release induced by ATP (10 &mgr;mol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3&bgr; in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca2+-induced Ca2+ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype.

Human ApoA-IV Overexpression in Transgenic Mice Induces cAMP-Stimulated Cholesterol Efflux From J774 Macrophages to Whole Serum

The role of apolipoprotein A-IV (apoA-IV) in lipoprotein metabolism has not been established. The aim of the present study was to investigate the role of apoA-IV in reverse cholesterol transport by comparing cellular cholesterol efflux to serum or serum fractions from control mice and from mice transgenic for human apoA-IV (HuA-IVTg mice). When Fu5AH hepatoma cells were used, the cholesterol efflux to serum from either control or transgenic mice was similar. When control J774 macrophage cells were used, a comparison of efflux to serum or lipoprotein-deficient serum (LPDS) failed to demonstrate any differences between control and transgenic mice. In contrast, when the J774 cells were pretreated with cAMP, there was a stimulation of efflux to whole serum or LPDS from HuA-IVTg mice. cAMP treatment had no effect on efflux to serum or LPDS from control mice. Pretreatment of the cells with cAMP did not enhance the efflux response to high density lipoprotein isolated from HuA-IVTg mouse serum. Our results suggest that apoA-IV, unassociated with high density lipoprotein particles, is responsible for enhanced cholesterol efflux. This study illustrates the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux with use of a biological fluid and is potentially of physiological relevance, especially in apolipoprotein-rich extravascular fluids.

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Background Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra). Methodology/Principal Findings Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia. Conclusions/Significance GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the β-cell microenvironment and contribute to progressive type 2 diabetic β-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent β-cell dysfunction in type 2 diabetes.

Effects of red wine polyphenolic compounds on paraoxonase-1 and lectin-like oxidized low-density lipoprotein receptor-1 in hyperhomocysteinemic mice

Hyperhomocysteinemia, or abnormally high plasma homocysteine (Hcy) concentration, has often been associated with vascular thrombosis and the development of premature atherosclerosis. Many studies have shown that moderate wine consumption has potential beneficial effects related to the prevention of atherosclerosis, in part attributed to the biological properties of polyphenolic components, mainly flavonoids. The aim of the present study is to determine the effects of a red wine polyphenolic extract (PE) administration on hyperhomocysteinemia due to cystathionine beta-synthase (CBS) deficiency and on the associated biochemical markers of hepatic and endothelial dysfunctions in mice. Red wine PE was added for 4 weeks to the drinking water of heterozygous CBS-deficient mice fed a high-methionine diet, a murine model of hyperhomocysteinemia. Red wine PE supplementation at low dose significantly reduced plasma Hcy levels and restored the hepatic and plasma-decreased paraoxonase-1 activity induced by chronic hyperhomocysteinemia. Moreover, aortic expression of proinflammatory cytokines and adhesion molecules and levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 were reduced in hyperhomocysteinemic mice fed the red wine PE supplementation. These findings suggest that red wine PE administration in low quantities has beneficial effects on biochemical markers of endothelial dysfunction due to hyperhomocysteinemia.

Analysis of the relationship between triglyceridemia and HDL-phospholipid concentrations: consequences on the efflux capacity of serum in the Fu5AH system.

The high triglyceride/low HDL-cholesterol trait is a common finding in the general population. The aim of the present study was to analyze and interpret the relationships between triglycerides (TG), HDL-related parameters and serum cholesterol efflux potential in an asymptomatic population including both normo- and hyperlipidemic individuals. In a large sample (n = 1143) of this population, there was a negative correlation between TG and HDL-cholesterol (HDL-C) (r = -0.49, P<0.0001) whereas the negative correlation between TG and HDL-phospholipid (HDL-PL) (r = -0.29, P<0.0001) was weaker, leading to a strong positive correlation between TG and HDL-PL/C ratio (r = 0.58, P<0.0001). Thus, increased TG concentrations were associated with an enrichment of HDL with PL. Since we have demonstrated previously that HDL-PL is the major determinant for cholesterol efflux potential from Fu5AH rat hepatoma cells, we determined the effect of the variations in HDL lipid composition on the cholesterol efflux capacity in a subsample of 198 subjects. Compared with normolipidemic subjects (NLP) (TG< or = 1.7 mmol/l; LDL-C< or = 4.1 mmol/l, n=58), hypertriglyceridemic subjects (HTG) (TG>1.7 mmol/l, n=63) exhibited lower HDL-C levels (1.08+/-0.21 vs. 1.25+/-0.32, P=0.0003) whereas they showed similar HDL-PL concentrations (1.25+/-0.21 vs. 1.25+/-2.7) and, thus, higher HDL-PL/C ratio (1.17+/-0.15 vs. 1.02+/-0.14, P=0.0001). The relative efflux capacity of serum measured in the Fu5AH system (5% serum, 4 h incubation at 37 degrees C) was on average identical in the HTG and NLP groups. Thus, this study provides evidence that despite decreased HDL concentrations, as determined routinely by the HDL-C assay, some HTG subjects maintained serum cholesterol efflux capacity thanks to the enrichment of HDL with PL.

Erythrocyte antioxidant status in asymptomatic hypercholesterolemic men.

An imbalance between antioxidant and oxidant-generating systems leading to an oxidative stress has already been proposed in the pathogenesis of atherosclerosis. In the present study we investigated the antioxidant status in 60 asymptomatic hypercholesterolemic (HC) men compared with 48 normocholesterolemic (NC) men. Hypercholesterolemic subjects had a significantly lower red blood cell vitamin E (vit E-RBC) content in spite of their normal total plasma and HDL vitamin E concentrations. Activities of erythrocyte superoxide dismutase and glutathione peroxidase were not significantly different between groups. We also determined the resistance of RBCs to an oxidative stress by determining the extent of hemolysis induced by a water-soluble azo-compound. This resistance was significantly decreased in HC men compared with NC subjects. These results demonstrate an altered antioxidant status of RBC in asymptomatic HC men associated with an increased erythrocyte susceptibility to an oxidative stress. The measure of the vitamin E content in RBC might be the most sensitive parameter for evidencing early oxidative stress which does not need an adaptation of enzymatic protective systems.

Opposite Effects of Plasma From Human Apolipoprotein A-II Transgenic Mice on Cholesterol Efflux From J774 Macrophages and Fu5AH Hepatoma Cells

Overexpression of human apolipoprotein A-II (hapo A-II) in transgenic mice (hAIItg mice) induced marked hypertriglyceridemia and low levels of plasma high density lipoprotein (HDL) with a high hapo A-II content. We sought to determine whether cholesterol efflux to plasma and HDL from these mice would be affected. In the Fu5AH cell system, plasma from hAIItg mice induced a markedly lower cholesterol efflux than did control plasma, in accordance with the dependence of efflux on HDL concentration. Moreover, HDLs from hAIItg mice were less effective acceptors than were control HDLs. In the J774 macrophage cell system, pretreatment with cAMP, which upregulates ATP binding cassette transporter 1, induced a marked increase in the efflux to hAIItg plasma as well as to purified hapo A-I and hapo A-II, whereas it had no effect on cholesterol efflux to control plasma. A strong positive correlation was established between percent cAMP stimulation of efflux and plasma hapo A-II concentration. The cAMP stimulation of efflux to hAIItg mouse plasma may be linked to the presence of pre-&bgr; migrating HDL containing hapo A-II. Thus, despite lower HDL and apolipoprotein A-I contents, the increased ability of plasma from hAIItg mice to extract cholesterol from macrophage-like cells may have an antiatherogenic influence.

Liver microRNA-21 is overexpressed in non-alcoholic steatohepatitis and contributes to the disease in experimental models by inhibiting PPARα expression

Objective Previous studies suggested that microRNA-21 may be upregulated in the liver in non-alcoholic steatohepatitis (NASH), but its role in the development of this disease remains unknown. This study aimed to determine the role of microRNA-21 in NASH. Design We inhibited or suppressed microRNA-21 in different mouse models of NASH: (a) low-density lipoprotein receptor-deficient (Ldlr−/−) mice fed a high-fat diet and treated with antagomir-21 or antagomir control; (b) microRNA-21-deficient and wild-type mice fed a methionine-choline-deficient (MCD) diet; (c) peroxisome proliferation-activator receptor α (PPARα)-deficient mice fed an MCD diet and treated with antagomir-21 or antagomir control. We assessed features of NASH and determined liver microRNA-21 levels and cell localisation. MicroRNA-21 levels were also quantified in the liver of patients with NASH, bland steatosis or normal liver and localisation was determined. Results Inhibiting or suppressing liver microRNA-21 expression reduced liver cell injury, inflammation and fibrogenesis without affecting liver lipid accumulation in Ldlr−/− fed a high-fat diet and in wild-type mice fed an MCD diet. Liver microRNA-21 was overexpressed, primarily in biliary and inflammatory cells, in mouse models as well as in patients with NASH, but not in patients with bland steatosis. PPARα, a known microRNA-21 target, implicated in NASH, was decreased in the liver of mice with NASH and restored following microRNA-21 inhibition or suppression. The effect of antagomir-21 was lost in PPARα-deficient mice. Conclusions MicroRNA-21 inhibition or suppression decreases liver injury, inflammation and fibrosis, by restoring PPARα expression. Antagomir-21 might be a future therapeutic strategy for NASH.