Reduced clusterin (apoJ) content of low density lipoproteins contribute to endothelial dysfunction in adiponectin-deficient mice

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Abstracts / Atherosclerosis 241 (2015) e1ee31

EAS-0811. IS FATTY LIVER AN INDEPENDENT RISK FACTOR FOR SUBCLINICAL ATHEROSCLEROSIS? A. Di Costanzo 1, L. D'Erasmo 1, L. Polimeni 1, L. Loffredo 1, P. Coletta 1, M. Del Ben 1, F. Angelico 2, A. Montali 1, G. Girelli 3, B. De Masi 3, M. Maranghi 1, M. Arca 1. 1 Internal Medicine and Clinical Specialties, Sapienza University, Roma, Italy; 2 Department of Public Health and Infectious Disease, Sapienza University, Roma, Italy; 3 Immunohematology and Transfusion Medicine, Sapienza University, Roma, Italy Background. NAFLD has been reported to be a risk factor for atherosclerosis. However, this association might be biased by the high prevalence of pro-atherogenic metabolic abnormalities in NAFLD. It has been reported that carriers of the M148M variant in the patatin like phospholipase 3 (PNPLA3) gene develop NAFLD without metabolic abnormalities. Aims and methods. To clarify whether fatty liver itself promotes vascular damage, we compared subclinical atherosclerosis in 3 groups: 1) subjects with NAFLD due to metabolic syndrome, but not carrying the PNPLA3 variant (metabolic NAFLD group) (n¼ 89), 2) subjects with NAFLD due to the PNPLA3 M148M (genetic NAFLD) (n¼31) and 3) normal controls (n¼ 16). Fatty liver was demonstrated by ultrasounds. Carotid intima-media thickness (CIMT) was measured as marker of subclinical atherosclerosis. Comparisons were adjusted for age, gender and smoke by GLM. Results. Age and gender were not different among groups. Subjects with metabolic NAFLD had significantly higher values of anthropometric variables, lipids, glucose and transaminases as compared to those with genetic NAFLD and controls (p<0.05). Overall, CIMT of subjects with metabolic NAFLD (0,85±0,18 mm) was significantly higher than that of subjects with genetic NAFLD (0,69±0,21 mm), which in turn was similar to controls (0,71±0,13 mm) [adjusted P¼0.001]. These differences persisted when comparisons were performed according to the degree of liver steatosis. Conclusions. We showed that subjects with metabolic NAFLD have increased subclinical vascular damage as compared to those with genetic NAFLD, thus suggesting that fatty liver per se might not be a risk factor for atherosclerosis.

EAS-0817. CETP INHIBITORS TORCETRAPIB, DALCETRAPIB, AND ANACETRAPIB INDUCE ADIPOCYTE-DERIVED ALDOSTERONE PRODUCTION THROUGH NOX AND STAT3 ACTIVATION 1

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F.J. Rios , K.B. Neves , A.Nguyen Dinh Cat , S. Even , R. Palacios , C. Jenkins 1, A. Carswell 1, A.C. Montezano 1, R.M. Touyz 1. 1 BHF-GCRC, Institute of Cardiovascular and Medical Sciences, Glasgow, United Kingdom; 2 Department of Pharmacology, University of Sao Paulo, Ribeirao Preto, etica Molecular Brazil; 3 Departamento de Bioquímica Fisiología y Gen Facultad de CC. de la Salud, Universidad Rey Juan Carlos, Madrid, Spain Clinical trials indicated that CETP inhibitors increase HDL levels, but had unexpected side effects, such as hyperaldosteronism and hypertension. Some CETP inhibitors appear to accumulate in adipocytes, which are a source of aldosterone (Aldo). Here, we questioned whether CETP inhibitors influence Aldo production in adipocytes and assessed the role of reactive oxygen species (ROS) and STAT3 in this process. Human adipocytes (SW872), CETP-positive, were studied and compared to mouse adipocytes (3T3-L1), CETP-negative. Cells were treated with torcetrapib (TOR), dalcetrapib (DAL), or anacetrapib (ANA). To evaluate the role of ROS, cells were pre-treated with N-acetylcystein (NACeROS scavenger), ML171 and GKT136901 (Nox1/4 inhibitor, respectively), and Rotenone (Rot, mitochondrial ROS-inhibitor). ROS were measured by lucigenin and amplexred. Aldo production, measured by ELISA, was induced by TOR (668pg/mL), DAL (348pg/mL), and ANA (434pg/mL) (p<0.05 vs control 196pg/mL); an effect blocked by NAC. TOR, DAL, and ANA increased superoxide (2-fold) and H2O2 (1.5-fold) production, and STAT3-phosphorylation (p<0.05). In TOR treated cells, Aldo production was inhibited by GKT (62%), ML171 (62%), Rot (45%), and S3I-201 (STAT3-inhibitor, 66%). DAL-induced Aldo

production was blocked by ML171(62%) and S3I-201 (74%). ANA-induced Aldo production was inhibited by GKT (94%), ML171 (83%), and Rot (66%). In mouse adipocytes, TOR, DAL, and ANA induced Aldo (453pg/mL, 375pg/ mL, and 445pg/mL vs cont 253pg/mL,p<0.05) and ROS production (1.7fold, p<0.05). In conclusion, CETP-inhibitors induced aldosterone production in human and mouse adipocytes through redox-related mechanisms involving STAT3, Noxs and mitochondria. These findings may explain, in part, the hyperaldosteronism and hypertension reported in CETP clinical trials.

EAS-0858. EFFECT OF MODERATE EXERCISE ON POSTPRANDIAL TRIGLYCERIDE RICH LIPOPROTEINS (TRL) K. Ghafouri, J. Gill, M. Caslake. Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom Atherogenic effects of high postprandial triglyceride (TG) concentrations have been well documented, therefore reducing TG concentrations should lower the risk of developing atherosclerosis. Exercise has been used to reduce postprandial TG, by increasing triglyceride-rich lipoprotein (TRL) catabolism. We hypothesise that might cause compositional changes in TRL which increase their affinity to lipoprotein lipase (LPL). We investigated the effect of exercise on the affinity of postprandial TRL for LPL. Ten overweight men underwent 2 oral fat tolerance tests (OFTT), in random order. On the afternoon prior to OFTT, subjects either walked on a treadmill for 90min at ~50% maximal oxygen uptake or performed no exercise. Multiple blood samples were taken chylomicrons(S f >400) and VLDL1(Sf60-400) were separated. Lipoprotein fractions, standardised to TG 0.6 mmol.l-1, were incubated with a standard amount of LPL and affinity was calculated as the rate of NEFA release. In the postprandial state in the control trial, the affinity of chylomicrons for LPL was 11.3 (6.0 to 21.6) fold greater (p ¼ 0.0001) than the affinity of VLDL1, Whereas in the exercise trial the affinity of chylomicrons for LPL was 6.0 (3.0 to 12.0) greater (p ¼ 0.0007) than the affinity of VLDL1. Moreover, the affinity of VLDL1 for LPL-mediated TG-hydrolysis relative to the affinity of chylomicrons was 2.6(1.3 to 5.4) fold greater in the exercise compared to the control trial (p ¼ 0.03).In conclusion, exercise increased the affinity of both chylomicron and VLDL1 for LPL which will result in more rapid removal of TRL from the circulation. Endothelial cells communication 2

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EAS-0489. REDUCED CLUSTERIN (APOJ) CONTENT OF LOW DENSITY LIPOPROTEINS CONTRIBUTE TO ENDOTHELIAL DYSFUNCTION IN ADIPONECTIN-DEFICIENT MICE H.B. Deng 1, B. Huang 1, C. Xu 1, S. Park 2, A. Xu 3, I.K. Lee 2, Y. Wang 1. 1 Dept of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, Hong Kong, China; 2 Dept of Internal Medicine, Kyungpook National University, Kyungpook, Korea; 3 Dept of Medicine, The University of Hong Kong, Hong Kong, Hong Kong, China Aim: Low adiponectin and clusterin (apoJ) levels are risk factors of atherosclerosis. The present study aims to investigate the relationships of adiponectin with clusterin in lipoproteins and endothelial dysfunction. Methods: Wild type and adiponectin-deficient mice were fed high fat diet for at least 12 weeks. The recombinant adenoviruses encoding mouse adiponectin or luciferase (as control) were administrated into wild type and adiponectin-deficient mice through tail vein injection. Cholesterol and protein levels were measured in low and high density lipoproteins (LDL and HDL) separated by density gradient ultracentrifugation. Clusterin was detected in LDL and HDL by Western blotting using specific antibody. Vascular function were assessed in wild type and adiponectin-deficient mice using wire myography. Results: Circulating levels of total and LDL-cholesterols were significantly increased in adiponectin-deficient mice by 50.7% and 89.1%, respectively,

Abstracts / Atherosclerosis 241 (2015) e1ee31

compared with wild type mice (P < 0.05). Protein/cholesterol ratio in LDL particles was significantly lower in adiponectin-deficient mice than that in wild type mice (0.58±0.10 vs. 0.79±0.02, P < 0.05). Clusterin content in LDL particles were significantly decreased in adiponectin-deficient mice (fold change vs. wild type: 0.35±0.04, P < 0.05). The endothelium-dependent contraction was significantly enhanced in arteries from the adiponectindeficient mice compared to the wild type controls. The endotheliumdependent relaxation was significantly attenuated by adiponectin deficiency. Treatment of recombinant adenoviruses encoding mouse adiponectin increased clusterin content in LDL by nearly 90% and ameliorated the endothelium-dependent vascular function in adiponectin-deficient mice. Conclusions: Adiponectin may exert anti-atherogenic effects and prevent endothelial dysfunction via its interactions with clusterin in LDL.

EAS-0659. ARGINASE-II INDUCES ENDOTHELIAL AUTOPHAGY SUPPRESSION AND SMOOTH MUSCLE CELL MITOCHONDRIAL DYSFUNCTION CONTRIBUTING TO ATHEROSCLEROTIC PLAQUE VULNERABILITY Y. Xiong, Y. Yu, M. Forbiteh, J.P. Montani, Z. Yang, X. Ming. Department of Medicine, University of Fribourg, Fribourg, Switzerland Aim: Emerging evidence demonstrates the potential role of arginase, particularly arginase (Arg-II) as a therapeutic target in cardiovascular diseases such as atherosclerosis. Since endothelial cells and vascular smooth muscle cells (VSMC) play an important role in plaque vulnerability, we investigated the hypothesis that Arg-II may suppress endothelial autophagy and promote smooth muscle cell mitochondrial dysfunction, contributing to unstable atherosclerotic plaque formation. Methods: Endothelial cells and smooth muscle cells were isolated from human umbilical veins (abbreviated as HUVEC and HUVSMC). Cells of passage 1e2 (P1eP2) were used as young cells. The young cells were further split continuously till replicative senescence (P9eP12) was reached. Male atherosclerosis-prone apolipoprotein E-deficient (Apo E/  Arg-II+/+) mice and ApoE/ Arg-II/ mice were fed a high fat diet for 10 weeks to as atherosclerosis animal models. Results: In young endothelial cells, overexpression of Arg-II leads to activation of the mTOR cascade, which suppresses autophagy. Genetic ablation of Arg-II in the aortas of ApoE/ mice reveals a more stable plaque feature in the aortic roots as evidenced by decreased necrotic core and less inflammation. On the other hand, overexpression of Arg-II induces young HUVSMC mitochondrial dysfunction as monitored by the decreased mitochondrial membrane potential (Djm) and enhanced mitochondrial ROS production. Furthermore, ApoE/ mice with Arg-II deficiency show less atherosclerotic lesion formation and reveal characteristics of stable plaques and less apoptotic VSMCs as compared to the ApoE/ mice. Conclusions: Arg-II impairs endothelial autophagy and promotes mitochondrial dysfunction triggering VSMC apoptosis and senescence, contributing to the atherosclerotic plaque vulnerability.

EAS-0724. THE ITINERARY OF ENDOTHELIAL CELLS

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D. Perisa, L. Rohrer, A. von Eckardstein. Institute for Clinical Chemistry, University Hospital Zürich, Schlieren, Switzerland Atherosclerosis is a progressive accumulation of lipids in the arterial intima leading to plaque formation. Epidemiological studies show an inverse association of high density lipoprotein (HDL) cholesterol with cardiovascular events. HDL has multiple anti-atherogenic functions, some of which take place in the vessel wall. To get access to the intima and the lipid-laden macrophages, HDL has to pass the endothelial barrier. To elucidate the itinerary of HDL through endothelial cells (ECs), we investigated localisation and distribution of fluorescently and 1.4 nm

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Nanogold labelled HDL in fluorescent (FL) as well as electron microscopy (EM). Colocalisations of HDL with dextran were negative excluding fluid phase uptake and transferrin and albumin show only slight colocalisation indicating an atypical uptake mechanism. Further experiments showed that HDL is neither targeted to lysosomes nor to the Golgi or the endoplasmatic reticulum. Only small amounts can be detected in early endosomes (Rab5) and endosome to trans-golgi network (Rab9), but not at all with late endosomes (Rab7), the recycling endosomes (Rab11a) or vesicles involved in trans-Golgi network sorting (Stx6). EM ultrastructure shows HDL to be located mainly in multivesicular bodies. Interestingly, HDL was found to stimulate its own uptake into ECs. We identified several HDL and/or ApoA-I induced changes in the phosphorylation of signaling molecules, which however still remain to be linked to HDL endocytosis. In conclusion HDL transport into and through ECs seems to follow a nonclassical trafficking route. It appears to be regulated by positive rather than negative feedback.

EAS-0733. ENDOTHELIAL DYSFUNCTION, A MAJOR EVENT IN ATHEROSCLEROSIS: PPIS IMPAIR ENDOTHELIAL FUNCTION THROUGH ACTIVATION OF PAI-1 G. Yepuri, R. Sukhovershin, T.Z. Nazari-shafti, Y.T. Ghebremariam, J.P. Cooke. Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, USA Aim: Endothelial dysfunction is a major risk factor for cardiovascular disease(CVD) and atherosclerosis. Proton pump inhibitors(PPIs) like Esomeprazole(Nexium) are widely used drugs for the treatment of gastroesophageal reflux disease. Recently, our laboratory discovered that PPIs inhibit a key enzyme dimethylarginine dimethylaminohydrolase(DDAH), which may lead to endothelial dysfunction. In addition, our clinical databases indicate that PPI use is associated with cardiovascular risk. We believe that PPIs pose a major risk for CVD by impairing endothelial function. The aim of my project is to investigate the mechanistic basis by which PPIs increase endothelial dysfunction that may lead to development of CVDs including atherosclerosis. Methods: Human microvascular endothelial cells(HMVECs) were treated with clinically relevant concentration of esomeprazole and passaged over time prior to assessing the markers that indicate endothelial dysfunction. Results: Long term incubation of endothelial cells(ECs) with PPIs accelerate oxidative stress and impair endothelial function. Specifically, PPIs impaired the angiogenic and proliferative capacity of ECs as confirmed by Matrigel tube formation and cell proliferation assays. Investigation of molecular pathways involved in PPI-induced endothelial dysfunction revealed that plasminogen activator inhibitor-1(PAI-1); a gene commonly associated with endothelial dysfunction, was strongly up-regulated by PPI treatment. This data was confirmed using several orthogonal assays. Conclusion: We provide molecular evidence that PPIs pose a risk for CVDs and atherosclerosis by accelerating endothelial dysfunction through, at least in part, activation of PAI-1 protein. Given the widespread use of PPIs in the absence of medical supervision, our data raises a concern for people who are on long-term PPI therapy.

EAS-0843. OXIDISED LDL ACTIVATES BLOOD PLATELETS THROUGH CD36-NADPH OXIDASE-MEDIATED INHIBITION OF THE CGMP/PROTEIN KINASE G SIGNALLING CASCADE C. Woodward 1, S.G. Magwenzi 1, K.S. Wraith 1, N. Yuldasheva 2, S. Wheatcroft 2, M. Kearney 2, M. Febbriao 3, K. Naseem 1. 1 Centre for Cardiovascular and Metabolic Research, Hull York Medical School, Hull, United Kingdom; 2 Division of Cardiovascular and Diabetes Research, University of Leeds, Leeds, United Kingdom; 3 School of Dentistry, University of Alberta, Edmonton, United Kingdom