79th EAS Congress Atherosclerosis Supplements 12, no. 1 (2011) 13–184 95 444 ROLES OF LXRa AND LXRb IN THE REGULATION OF INTESINAL CHOLESTEROL ABSORPTION: DISTINCTIVE EFFECTS ON BILE ACID METABOLISM IN MICE X. Hu 1 , K. Steffensen 2 , Z.-Y. Jiang 3 , P. Parini 1 , J.- ˚ A. Gustafsson 2 , M. G˚ afvels 1 , G. Eggertsen 1 . 1 Department of Laboratory Medicine, 2 Department of Biosciences and Nutrition, Karolinska Institutet - Huddinge, Stockholm, Sweden, 3 Department of Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Introduction: Activation of the liver X receptors (LXRs) has been shown to modulate cholesterol absorption in the small intestine, although the individual effects of LXRa and LXRb have not been fully understood. Our study aimed to investigate how cholesterol absorption is modulated by the two isoforms in the small intestine. Methods: Wild type (WT), LXRa-/- or LXRb-/- mice were subjected either to standard diet or 0.2% cholesterol-enriched diet. Results: No differences of cholesterol absorption were observed in all genotypes of mice under normal diet. However, LXRb-/- mice showed a significant lower cholesterol absorption rate than WT mice when fed 0.2% cholesterol diet. This profile was altered when GW3965 was added to the 0.2% cholesterol diet, while the absorption rate increased significantly only in LXRa-/- mice. Analysis of gallbladder bile demonstrated that 0.2% cholesterol diet significantly lower the ratios of CA/b-muricholic acid (b-MCA) in LXRb-/- and WT mice compared to LXRa-/-, even when GW3965 was added. Additionly, hepatic Cyp8b1 mRNA was reduced in WT fed with 0.2% cholesterol ± GW3965, which agreed with the CA% in the bile. Similar patterns were seen in LXRb-/- mice, but not in LXRa-/-. The Cyp7a1 mRNA in LXRa-/-was repressed by the 0.2% cholesterol treatment, and remained low when adding GW3965. Fecal analysis showed a strong reduction of bile acid excretion in LXRa-/- mice subjected to 0.2% cholesterol ± GW3965. Conclusion: Our data indicate that LXRa and LXRb affect the synthesis of individual bile acids in different ways upon activation, leading to alterations in the intestinal cholesterol absorption. 445 PPARA GENE LEVEL DIFFERENTLY AFFECTS LIPID METABOLISM AND INFLAMMATION IN APOLIPOPROTEIN E2 KNOCK-IN MICE F. Lalloyer 1 , K. Wouters 1 , E. Vallez 1 , J. Vanhoutte 1 , M. Baron 1 , R. Shiri- Sverdlov 2 , M. Hofker 3 , B. Staels 1 , A. Tailleux 1 . 1 Inserm U1011-Institut Pasteur de Lille-Universit´ e Lille Nord de France, Lille, France, 2 Department of Molecular Genetics, Nutrition and Toxicology Research (NUTRIM) and Cardiovascular Research (CARIM) Institutes of Maastricht, University of Maastricht, The Netherlands, Maastricht, 3 Department of Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Objective: PPARa is a ligand-activated transcription factor which controls lipid metabolism and inflammation. PPARa is activated by fibrates, hypolipidemic drugs used in the treatment of dyslipidemia. Previous studies assessing the influence of PPARa agonists on atherosclerosis in mice yielded conflicting results and the implication of PPARa therein has not been assessed. The human apoE2 knock-in (apoE2-KI) mouse is a model of mixed dyslipidemia, atherosclerosis and non-alcoholic steatohepatitis (NASH). The aim of this study was to analyze the consequences of quantitative variations of PPARa gene levels and its response to the synthetic PPARa agonist fenofibrate, on NASH and atherosclerosis in apoE2-KI mice. Methods and Results: Wildtype (+/+), heterozygous (+/-) and homozygous (-/-) PPARa-deficient mice in the apoE2-KI background were submitted to a western diet supplemented or not with fenofibrate. Western diet-fed PPARa-/- apoE2-KI mice displayed an aggravation of steatosis and hepatic inflammation compared to PPARa+/+ and PPARa+/- apoE2-KI mice, indicating a role of PPARa in liver protection. Moreover, PPARa expression was required for the fenofibrate-induced protection against NASH. Interestingly, fenofibrate treatment induced a similar response on hepatic lipid metabolism in PPARa+/+ and PPARa+/- apoE2-KI mice, whereas, for a maximal anti-inflammatory response, both alleles of the PPARa gene were required. Surprisingly, atherosclerosis development was not significantly different between PPARa+/+, PPARa+/- and PPARa-/- apoE2-KI mice. However, PPARa gene level determined both the anti-atherosclerotic and vascular anti- inflammatory responses to fenofibrate in a dose-dependent manner. Conclusions: These results demonstrate a quantitatively different role of PPARa in the modulation of liver metabolism, inflammation and atherogenesis. 446 ATHEROSCLEROTIC LESION REMODELLING IN REV-ERBa-DEFICIENT MICE E. Bauge, C. Comte, C. Duhem, J. Vanhoutte, B. Staels, H. Duez. Inserm U1011, Univ Lille Nord de France, Institut Pasteur de Lille, Lille, France Rationale: Rev-erba is a nuclear receptor which has been shown to influence numerous physiological processes such as circadian rhythm, lipid, glucose, bile acid metabolism and adipogenesis. Rev-erba is expressed in different cell types of the vascular wall where it regulates the expression of genes involved in the inflammatory and thrombotic response. Finally, Rev-erba in vitro dampens oscillation of PAI-1, an important inhibitor of fibrinolysis cascade which may influence the development of atherothrombosis. Howewer, implication of Rev- erba in atherosclerotic processes has never been investigated. Objective: Here, we investigated the effect of Rev-erba deficiency on atherosclerotic lesion formation in apoE-deficient mice. Methods and Results: We demonstrate that Western diet-fed Rev- erba-deficient mice on the apoE-/- background display a significant increase in total aortic atherosclerotic lesion area as compared to apoE-/- Rev-erba+/+ mice fed the same diet. Plaque immunostaining examinations showed that apoE-/- Reverba-/-mice display atherosclerotic lesion characterized by a decreased content in macrophages and increased collagen deposition. Conclusion: These results demonstrate that Rev-erba plays an important role in atherosclerosis and demonstrate for the first time a role of a clock machinery component in atherosclerosis development. This suggests that Rev-erba may play a role in circadian disorder related cardiovascular disease. 447 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS REGULATE NADPH OXIDASE EXPRESSION AND FUNCTION IN HUMAN AORTIC SMOOTH MUSCLE CELLS A. Manea 1,2 , S.A. Manea 2 , I.M. Fenyo 2 , M. Raicu 2 . 1 Petru Poni Institute of Macromolecular Chemistry, Iasi, 2 Molecular Biology and Pharmacology, Nicolae Simionescu Institute of Cellular Biology and Pathology, Bucharest, Romania Objective: Elevated NADPH oxidase (Nox) activity promotes oxidative injury of the vascular cells. Peroxisome proliferator-activated receptors (PPARs) play key roles in the regulation of energy homeostasis, fatty acid metabolism and inflammation. Evidence exists that PPARs control vascular redox state. Hitherto, the molecular mechanisms are scantily elucidated. In this study, we investigated the role of PPARs in the regulation of Nox in human aortic smooth muscle cells (SMCs). Methods and Results: Cultured SMCs were exposed to various PPAR agonists or 4-hydroxynonenal (4-HNE) up to 24h. Dihydroethidium and dichlorofluorescein assays, real-time PCR, and Western-blot analysis, revealed that PPAR agonists and 4-HNE induce a dose-dependent up-regulation of Nox activity and Nox1/4/5 expression. Knock-down of PPARa/d/g partially diminished the 4-HNE-dependent induction of Nox. In silico analysis indicated the presence of typical retinoid X receptor (RXR)a elements within human Nox1/4/5 and p22phox promoters. The promoter activities of the Nox1/4/5 and p22phox genes were significantly enhanced in SMCs overexpressing hPPARa/d/g or hRXRa; a finding that indicates the presence of functionally PPRE elements. The transcriptional activities of each component were significantly up-regulated by PPAR agonists. Conclusions: These findings support a new concept whereby Nox-derived ROS elicit anti-inflammatory activities via PPARs. Thus, autogeneration of endogenous PPAR ligands (4-HNE) may represent an important mechanism of Nox regulation in the vasculature. Work supported by an EFSD New Horizons grant. Adrian Manea acknowledges the financial support of European Social Fund - “Cristofor I. Simionescu” Postdoctoral Fellowship Programme (ID POSDRU/89/1.5/S/55216), Sectoral Operational Programme Human Resources Development 2007–2011. 448 PLASMA PHOSPHOLIPID TRANSFER PROTEIN (PLTP) IS A PROCOAGULANT FACTOR IN VIVO C. Desrumaux 1,2 , V. Deckert 1,2 , S. Lemaire-Ewing 1,2,3 , C. Mossiat 2,4 , A. Athias 1,2 , D. Vandroux 2,3 , L. Dumont 2,3 , S. Monier 1,2 , J.-P. Pais de Barros 1,2 , A. Klein 1,2 , E. De Maistre 2,3 , D. Blache 1,2 , A. Beley 2,3 , C. Marie 2,4 , P. Garnier 2,4 , L. Lagrost 1,2,3 . 1 INSERM UMR866, 2 Universit ´ e de Bourgogne, 3 CHU, Hˆ opital du Bocage, 4 INSERM U887, Dijon, France Objective: Plasma phospholipid transfer protein (PLTP) is a determinant of lipoprotein metabolism and atherogenesis. Although earlier in vitro studies suggested a putative role for PLTP in the modulation of blood coagulation, the pathophysiological relevance of this effect is currently unknown in vivo. To address this question, blood coagulation was compared in the present study in wild-type (WT) and PLTP-deficient (PLTP-/-) mice under both basal and oxidative stress conditions. Methids and Results: Under basal conditions, PLTP deficiency was associated with a significant, 45% increase in tail bleeding time (P< 0.05) despite a reduction in vascular alpha-tocopherol content and an impairment of endothelial function. When acute oxidative stress was generated in vivo in the brain vasculature, the sequestration of alpha-tocopherol in blood of PLTP-/- mice was associated with a significant reduction in steady state levels of oxidized lipid derivatives (-22%, P< 0.01), decreased blood vessel occlusion (-52%, P< 0.05), and decreased volume of ischemic lesions (-39%, P< 0.05) compared to those measured in WT mice. Conclusion: In addition to its recognized hyperlipidemic, proinflammatory and proatherogenic properties, PLTP increases blood coagulation and worsens the extent of ischemic lesions in response to acute oxidative stress. Thus,