High plasma lipid levels inhibit transgene expression in adenovirus-mediated gene therapy

e60

Abstracts / Atherosclerosis 241 (2015) e32ee71

Conclusions: Our observational data supports that increased POA plasma levels are associated to MS and NAFLD. Experimental cardiovascular medicine II EAS-0131. SIMVASTATIN PREVENTS CARDIAC HYPERTROPHY IN A RAT MODEL VIA MODULATION OF THE JAK/STAT PATHWAY N. Alrasheed 1, M.M. Al-Oteibi 1, R.Z. Al-Manee 1, S.A. Al-Shareef 1, N.M. Alrasheed 2, I.H. Hasan 1, M.A. Al-Amin 1, H.N. Al-Ajmi 1. 1 Pharmacology and Toxicology, King Saud University, Riyadh, Saudi Arabia; 2 Pharmacology and Toxicology, King Saud University and Princess Nora Bint Abdulrahman, Riyadh, Saudi Arabia Aim: Cardiac hypertrophy is a complex inflammatory disease that develops viadysregulation of various signal transduction pathways. Therefore, this study was designed to test the hypothesisthat statins could mediate a cardioprotective effect by reducing theproinflammatory profile in cardiac tissue and this protective effect iscorrelated with reduced JAK2/ STAT3 expression in a hypertrophic rat model. Methods: Cardiac hypertrophywas induced in rats by isoproterenol (5 mg kg-1, i.p.) for 7consecutive days. Rats treated with simvastatin (10 mg kg-1, p.o) for30 days, and on 21st day, isoproterenol was administered for 7consecutive days. Cardiac biomarkers, lipid profiles, and inflammatory marker wereassessed. The expression of phosphorylated JAK2 and STAT3 in cardiac tissue wasstudied using immunohistochemistry. The protocol of this study was approved byExperimental Animals Ethics Committee in King Saud University. Results: Treatment of rats withsimvastatin significantly reduced the heart weight (0.92±0.02 vs. 1.28±0.05 mg)(p<0.05), heart/body weight ratio (0.48±0.02 vs. 0.59±0.02 mg g-1)(p<0.01) and ameliorated the altered cardiac biomarkers [troponin-T(49.67±4.67 vs. 65.4±5.02 ng ml-1) (P<0.01); CK (35.3±0.98vs. 45.5±1.2 U ml-1) (P<0.001); lipid profile (P<0.01)]and inflammatory marker [IL-6 levels (370±9.6 vs. 816.7±40.9 pg ml-1)(P<0.01)] compared to isoproterenol group. Moreover,immun-ohistochemical and histopathological examinations revealed that simvastatinadministration reduced JAK2/STAT3 expression and confirmed the protective effectsof simvastatin against isoproterenol-induced alteration of cardiac structure. Conclusions: Simvastatin exerted a marked protective effect against isoproterenol-inducedcardiac hypertrophy via modulation of JAK/STAT signaling pathway. Thus,simvastatin could be an important therapeutic agent for cardiac hypertrophy.

EAS-0422. PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 DEFICIENT MICE ARE PROTECTED FROM NEOINTIMA FORMATION IN CAROTID ARTERY INJURY MODEL N. Ferri 1, N. Ferri 1, G. Tibolla 1, R. Baetta 2, A. Corsini 3, A.L. Catapano 3. 1 Scienze Farmacologiche e Biomolecolari, University of Milan, Milan, Italy; 2 Cardiology Center, Monzino Hospital IRCCS, Milan, Italy; 3 University of Milan, Scienze Farmacologiche e Biomolecolari, Milan, Italy We have previously shown that PCSK9 is expressed in cultured smooth muscle cells (SMCs) and it is detectable in human carotid atherosclerotic plaques. The aim of the present study was to compare the vascular changes induced by periadventitial placement of a non-occlusive constrictive silicone collar for 9 weeks around the common carotid artery of WT and PCSK9 de?cient mice. Collared carotids of the PCSK9-/mice (n¼12 per group) showed a less marked intimal thickening compared WT mice (15408±5333 um2 vs. 27529±6312 um2; *P¼0.05), a decreased intimal media ratio (1.476±0.232 vs. 0.641±0.08 *P¼0.05) and higher lumen area (13947±4619 um2 vs. 24981±5619 um2). Carotid lesions of WT mice had an elevated content of SMCs (21.0±7.56% vs. 10.7±1.97% P¼0.05) and collagen (18.38±7.90% vs. 10.45±9.11%; **P¼0.01). PCSK9 KO SMCs showed higher levels of the contractile

marker smooth muscle a-actin (2.24±0.36 fold; **P<0.01 ) and decreased levels of synthetic markers h-caldesmon (-54±14%; **P<0.01) and Col1a1 mRNA (-31±11% *P¼0.05) compared to WT cells. PCSK9 KO cells showed a slower proliferation rate (doubling time 106.3±4.5 h vs. 57.3±2.1 h), and impaired migratory response to PDGF-BB. Morphological changes in response to PDGF-BB of PCSK9 KO mSMCs, measured by iCelligence monitoring, was significantly impaired compared to WT cells, indicating a defect in PDGF-BB signaling pathway. The present results suggest a favorable action of PCSK9 on neointima formation probably facilitating the phenotypic switch of SMCs from contractile to synthetic phenotype and improving cell proliferation and migration in response to PDGF-BB. This work was supported by the grant 2012-0549 from Fondazione Cariplo, ITALY

EAS-0526. SELECTIVE CHOLESTEROL LOWERING GENE TRANSFER ATTENUATES THE DEVELOPMENT OF PRESSURE OVERLOAD-INDUCED CARDIOMYOPATHY IN MICE I. Muthuramu 1, N. Singh 1, R. Amin 1, A. Postnov 2, T. Dresselaers 2, O. Gheysens 2, B. De Geest 1. 1 Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, Leuven, Belgium; 2 Department of Imaging & Pathology, Katholieke Universiteit Leuven, Leuven, Belgium Background and objectives: Epidemiological studies support a strong association between plasma cholesterol levels and heart failure incidence. The effect of selective cholesterol lowering gene transfer on cardiac remodelling induced by transverse aortic constriction (TAC) was evaluated. Methods: Female C57BL/6 low density lipoprotein receptor deficient (LDLr-/-) mice were fed a 0.2% cholesterol 10% coconut oil diet. Gene transfer with 5 x 1010 genome copies of an adeno-associated viral serotype 8-low density lipoprotein receptor (AAV8-LDLr) vector was performed 2 weeks before TAC. Results: Selective cholesterol lowering gene transfer resulted in an 82.8% (p<0.0001) reduction of plasma cholesterol levels compared to controls. Mortality rate was significantly higher in TAC control mice than in TAC AAV8-LDLr mice during a follow-up period of 8 weeks (hazard ratio for mortality 2.19, 95% CI 1.13 to 4.22). Left ventricular hypertrophy (p<0.001) and interstitial myocardial fibrosis (p<0.05) were significantly reduced in TAC AAV8-LDLr mice compared to TAC control mice. These structural differences were associated with improved systolic and diastolic function in TAC AAV8-LDLr mice. The stroke volume in TAC AAV8-LDLr mice was 26.5% (p<0.05) higher compared to TAC control mice and was preserved compared to sham groups. Total myocardial uptake of [18F]-FDG was 56.1% (p<0.01) lower in TAC AAV8-LDLr mice compared to TAC control mice. This was paralleled by a decrease of the maximal standardized uptake value (SUV), SUV 50%, and SUV 75%. Conclusion: Cholesterol lowering gene therapy potently inhibits the development of pressure overload-induced cardiomyopathy. Differences in insulin signalling may contribute to these pronounced effects.

EAS-0652. HIGH PLASMA LIPID LEVELS INHIBIT TRANSGENE EXPRESSION IN ADENOVIRUS-MEDIATED GENE THERAPY €, J. Huusko, E. Gurzeler, M.H. Dijkstra, A. Laine, S. Yl€ A. Kivela aHerttuala. A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland Aim: Adenoviruses are the most efficient vectors in preclinical and clinical trials. We previously reported differences in transgene expression after systemic adenoviral VEGF-A gene transfer in hyperlipidemic mouse models. The aim of this study was to find out whether high lipid levels and

Abstracts / Atherosclerosis 241 (2015) e32ee71

specific lipoproteins could decrease adenoviral transduction efficiency and potentially relate to insufficient transgene expression after adenoviral gene transfer in clinical trials. Methods: Adenoviral transduction efficiency was studied in C57Bl/6 mice and four different hyperlipidemic mouse models (ApoE-/-, LDLR-/-, LDLR-/-ApoE-/- and LDLR-/-ApoB100/100 mice) after systemic adenoviral gene transfers of hVEGF-A165 via tail vein. Mice were fed with regular chow or HFD for five weeks. Plasma hVEGF-A levels were studied four days after gene transfer. In addition, different tissue samples were collected for mRNA and histology. Results: Plasma levels of hVEGF-A on HFD were lower in LDLR-/- and LDLR-/-ApoB100/100 mice compared to C57Bl/6 and apoE-deficient mice. In addition, hVEGF-A levels on regular chow were higher compared to levels on HFD in all LDLR-/- based models. Similar results were seen when the liver hVEGF-A mRNA levels were compared between different strains and diets. Conclusions: High plasma lipid levels, especially apoE-containing lipoproteins, are protective against adenoviral infection in mice. Higher cholesterol levels also in humans could be beneficial against viral infections leading to insufficient transgene expression after adenoviral gene transfer. Therefore, high lipid levels as an inhibiting factor of transgene expression is an important aspect that should be taken in to account when interpreting results of preclinical and clinical trials.

EAS-0790. CD68GFP/APOE-/- MOUSE: A NOVEL MODEL TO STUDY MACROPHAGE BIOLOGY IN ATHEROSCLEROSIS A.J. Iqbal 2, D. Jones 1, D.R. Greaves 2, K.M. E. McNeill 1, Channon 1. 1 Cardiovascular Medicine, University of Oxford, Oxford, United Kingdom; 2 Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom To determine the fate of recruited monocytes entering sites of atherosclerosis we produced CD68GFP/ApoE-/- mice. These mice express high levels of GFP under control of the hCD68 promoter in myeloid cells, particularly in monocytes and macrophages.

e61

Aim: Obesity is associated with insulin resistance, dyslipidemia, and an increased risk of cardiovascular disease. The role of imatinib, an inhibitor of PDGF receptor tyrosine kinase, on intimal hyperplasia after arterial injury remains unclear, especially in insulin-resistant and dyslipidemic states. Methods: Male C57BL/6J mice were fed a high-fat diet (HFD, 60%) or a regular rodent chow for 6 weeks (n ¼ 12; each group). On day 22, both groups of mice were subjected to left femoral artery injury using a guide wire. Imatinib (25 mg / kg body weight) or vehicle control was injected intraperitoneally on the day before arterial injury and daily for the following 3 weeks. Body weight, blood pressure, blood glucose, plasma insulin and lipids were determined. In addition to performing glucose tolerance test, HOMA-IR (an index of insulin resistance) was determined. Intimal hyperplasia was evaluated by measuring intima/media ratio and smooth muscle a-actin expression. Results: Imatinib treatment for 3 weeks abrogated the weight gain (~33%) seen in HFD-fed mice. While there were no significant changes in blood pressure (SBP, DBP, and MAP) under these conditions, imatinib lowered the fasting hyperglycemia and hyperinsulinemia and prevented glucose intolerance and insulin resistance in HFD-fed mice. In addition, imatinib significantly diminished the increases in plasma total cholesterol and triglycerides. Importantly, imatinib treatment led to a marked inhibition of exaggerated intimal hyperplasia and smooth muscle a-actin expression after femoral artery injury in HFD mice. Conclusion: Imatinib has the potential to ameliorate restenosis after angioplasty in obese subjects with metabolic syndrome. EAS-0879. SKI-II, A SPHINGOSINE KINASE 1 INHIBITOR, EXACERBATES ATHEROSCLEROSIS IN LOW-DENSITY LIPOPROTEIN RECEPTORDEFICIENT (LDL-R-/-) MICE ON HIGH CHOLESTEROL DIET F. Potì 1, U. Ceglarek 2, R. Burkhardt 2, M. Simoni 1, J.R. Nofer 3. 1 Department of Biomedicine Metabolism and Neural Sciences, University of Modena and Reggio Emilia AUSL Modena, Modena, Italy; 2 Institute for Laboratory Medicine Clinical Chemistry and Laboratory Diagnostics, University of Leipzig, Leipzig, Germany; 3 Center for Laboratory Medicine, University Hospital Münster, Münster, Germany

Examination of foam cells shows GFP expression is maintained in mature foam cells expressing macrophage markers. Co-localization studies show a high degree of correlation between GFP expression and plaque macrophage content, as assessed using anti-CD68 and anti-Gal3 immunofluorescence in both chow-fed and high fat diet fed animals. Co-staining with markers of endothelial cells, smooth muscle cells and neutrophils showed no co-localization with the GFP bright cell population. GFP expression within plaque macrophages is sufficient to allow en-face imaging of macrophages within the whole descending aorta and allows both luminal and adventitial cell accumulation to be visualized in one z-series of images. Monocyte adoptive transfer, using CD68GFP/ApoE-/- donor cells, into ApoE-/- animals allows the identification and analysis of recruited monocytes by both flow cytometry and immunofluorescence microscopy. Monocyte recruitment can be observed during both acute vascular inflammation in response to angiotensin II infusion and during high fat diet feeding protocols. 72 hours following adoptive transfer of monocytes by iv injection into high fat diet fed ApoE-/- animals, monocytes are recruited to both the plaque adventitia and luminal atherosclerotic plaque. This novel tool will allow study of the kinetics of monocyte recruitment, differentiation and retention within sites of vascular inflammation, as well offering an efficient method to quantify macrophage content in sites of atherosclerotic plaque development.

Background and aim: Sphingosine 1-phosphate (S1P) is a lysosphingolipid associated with high-density lipoproteins (HDL) that partly accounts for their anti-atherogenic potential. We here investigated whether a reduction in S1P plasma levels affects atherosclerosis in low-density lipoprotein receptor (LDL-R-/-) mice.

EAS-0832. IMATINIB ATTENUATES EXAGGERATED INTIMAL HYPERPLASIA AFTER ARTERIAL INJURY AND IMPROVES GLYCEMIC CONTROL AND LIPID PROFILE IN HIGH FAT DIET-FED MICE

EAS-0919. DEVELOPMENT OF A NOVEL CLASS OF ANTI-ATHEROGENIC AGENTS: ELECTROPHILIC NITROALKENE-VITAMIN E (ALPHA-TOCOPHEROL) ANALOGS

L. Segar 1, P. Pichavaram 1, N. Shawky 1, J. Jun 2. 1 Clinical and Administrative Pharmacy, University of Georgia, Augusta, USA; 2 Department of Medicine, Pennsylvania State University, Hershey, USA

 pez 4, C. J. Rodriguez 1, G. Galliussi 1, G. Ferrer-Sueta 2, H. Botti 3, G.V. Lo Batthy any 5. 1 Biochemistry and Proteomic Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay; 2 Biological Physicochemical laboratory,

Methods and results: LDL-R-/- mice on Western diet containing low (0,25% w/w) or high (1,25% w/w) cholesterol were treated for 16 weeks with SKI-II, a sphingosine kinase 1 inhibitor that reduced plasma S1P by ~30%. At the end of the treatment, blood, internal organs and aortas were collected and atherosclerotic plaque area was analyzed by Oil Red O staining of the “en face” prepared aortas. SKI-II significantly increased atherosclerotic lesions in the thoracic aorta in mice on high but not low cholesterol diet (29,12±7,65 vs 20,75±6,12 % plaque/total area; p<0,05). This compound failed to affect body weight, blood cell counts and plasma total and HDL cholesterol, but decreased triglycerides. In addition, mice on high cholesterol diet receiving SKI-II showed elevated plasma levels of tumor necrosis factor-a and endothelial adhesion molecules (sICAM-1, sVCAM-1). Conclusion: Prolonged lowering of plasma S1P produces pro-atherogenic effects that are evident only under condition of pronounced hypercholesterolemia.