- Email: [email protected]
298 RELATION OF MATRIX METALLOPROTEINASES (MMPS)-POSITIVE MACROPHAGES TO CORONARY PLAQUE FRAGILITY AND CORONARY ARTERIAL OUTWARD REMODELING
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binding of uPA to its receptor, uPAR, and was mediated via activation of SREBP-1 through PI3K and MEK signal transduction pathways. Furthermore, uPA increased macrophage oxidative stress by enhancing NADPH oxidase activation. In turn, uPA initiated an oxidative stress-response by upregulating the expression of macrophage paraoxonase 2 (PON2) through the PI3KNADPH oxidase-ROS-MEK-SREBP2 signaling cascade activation. In addition, we have shown that uPA binding to uPAR on hepatocytes stimulates PPARg nuclear export, resulting in down-regulation of paraoxonase 1 (PON1) gene expression, and decreased PON1 secretion. Reduced levels of PON1 in serum may lead to reduced protection of LDL and of HDL from oxidation, reduced HDL-mediated cholesterol efflux from macrophages, and increased oxidative stress in atherosclerotic lesions. Conclusions: Taken together, our work represents a new paradigm by which uPA is implicated in atherogenic processes beyond its role in the fibrinolytic system, and provides new insight into the relationship between uPA and vulnerable plaque. 294 ACCELERATED ATHEROSCLEROSIS IN FEMALE MICE LACKING THE ANDROGEN RECEPTOR J. Bourghardt1 , A. Wilhelmson1 , C. Alexanderson1 , K. De Gendt2 , G. Verhoeven2 , A. Holmang ¨ 3 , A. Krettek4,5 , S.-O. Olofsson1 , C. Ohlsson6 , ˚ Tivesten1 . 1 Wallenberg Laboratory, University of Gothenburg, Gothenburg, A. Sweden, 2 Katholieke Universiteit Leuven, Leuven, Belgium, 3 Institute of Neuroscience and Physiology, Unversity of Gothenburg, 4 Nordic School of Public Health, 5 Institute of Medicine, University of Gothenburg, 6 Center for Bone and Arthritis Research, University of Gothenburg, Gothenburg, Sweden Objective: In women, elevated androgen levels have been reported to associate with the metabolic syndrome and atherosclerosis. However, the physiological role of androgens in metabolism and cardiovascular health in females is unclear. We evaluated the impact of physiological, androgen receptor (AR)-dependent actions of androgens on atherosclerosis using female ARdeficient (AR−/−) mice on an apolipoprotein E-deficient background. Methods and Results: After 8 weeks on high-fat diet, atherosclerosis assessed en face was increased by 59% (P < 0.01) in female AR−/− mice compared with control mice. Female AR−/− mice had increased body weight and relative fat mass (+62%, P < 0.001), increased hepatic triglyceride levels (+33%, P < 0.05) and reduced insulin sensitivity. In addition, the female AR−/− mice had elevated serum levels of cholesterol (+52%, P < 0.001) and triglycerides (+44%, P < 0.05) compared with control mice. Conclusions: Female AR−/− mice display accelerated atherosclerosis associated with obesity, hepatic steatosis, insulin resistance and dyslipidemia. These results suggest that AR-mediated effects of androgens are crucial for metabolism and cardiovascular health in females. 295 ATHEROSCLEROSIS REGRESSION IN A MOUSE MODEL WITH HUMAN-LIKE HYPERCHOLESTEROLEMIA 1 , S. Hagg ¨ 1 , S. Maleki1 , M. Shang1 , T. Michoel2 , J. Skogsberg1 . J. Bjorkegren ¨ 1 Karolinska Institutet, Stockholm, Sweden, 2 University of Freiburg, Freiburg, Germany Atherosclerosis is a lifelong, progressive disease that becomes clinically significant in a large fraction of the population, leading to myocardial infarction (MI) and stroke. Statin based lipid-lowering regimens reduce morbidity and mortality from both MI and stroke. To fully exploit the beneficial effects of lipid lowering, we need a better understanding of the transcriptional changes induced by lowering plasma lipoproteins. We have recently performed transcriptional profiling at 10-week intervals in atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic switch to lower plasma lipoproteins (Ldlr−/− Apob100/100 Mttpflox/flox Mx1-Cre). Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed. Analysis of lesion expression profiles indicated that accumulation of lipid-poor macrophages reached a point that led to the rapid expansion phase with accelerated foam-cell formation and inflammation, an interpretation supported by lesion histology. In the current study, we preformed genetic lowering of plasma cholesterol (i.e. lipoproteins) at the rapid lesion expansion start point which prevented the formation of advanced plaques. In addition, lowering of plasma cholesterol in plaques that already had been formed not only halted the atherosclerosis lesion development it also substantially regressed the lesion size. By performing transcriptional profiling of the atherosclerotic arterial wall in the plasma cholesterol lowered mice important gene networks responsible for atherosclerosis regression were identified. These networks will be validated in relevant cell culture systems and in human association studies. The identified networks should be of interest for the development of novel atherosclerosis therapies.
Poster presentations
296 TRANSCRIPTOMIC ANALYSIS OF MACROPHAGE POLARIZATION: ROLE OF PPARY IN ALTERNATIVE ACTIVATION E. Derlindati, D. Ardigo, ` I. Zavaroni. Internal Medicine, University of Parma, Parma, Italy Introduction: Human macrophages (HM) undergo different forms of activation in response to cytokines. Classical activation (M1) displays pro-inflammatory properties, and can be induced by IFNg and/or LPS. Alternative activation (M2a) shows immuno-regulatory properties and is promoted by IL-4, whereas IL-10 produces a “deactivated” state, with immunosuppressing characteristics (M2c). The PPARg system appears to be implicated in M2a activation enhancing their anti-inflammatory properties. Aim: Evaluation of the biological processes involved in the different types of HM activation compared to the effects induced by exposure to PPARg-agonists. Methods: HM were stimulated with INFg+LPS (M1), IL-4 (M2a), IL-10 (M2c), or a PPARg-agonist (GW1929-Sigma). Phenotypic changes for each experimental condition were investigated through whole-genome transcriptomic analysis by microarrays (Agilent-Technologies). Gene ontology (GO) analysis was performed using Gorilla tool. Validation was obtained by RT-PCR on the most differentially expressed genes. Results: After characterization of M1, M2a, and M2c genomic profiles, we investigated the differences between PPARy-stimulated HM and M1, observing a significant decrease in immunitary and inflammatory processes (corresponding GO-term’s p-value = 2.00×10−35 and 1.20×10−20 ) and a great increase in cell cycle activity (p = 1.51×10−35 ). Similar, but quantitatively lower, results were observed also in comparison with M2a and M2c, although these two subpopulations are usually regarded as anti-inflammatory. Discussion: Exposure of HM to a PPARg-agonist induces a transcriptomic phenotype closer to M2a/M2c than to M1. Nevertheless, PPARg-agonist activation decreases inflammatory cytokine production and increases cellcycle gene expression also compared to cytokine-induced M2a and M2c activation, suggesting a specific role of PPARg in the regulation of macrophage differentiation. 297 DARBEPOETIN ENHANCES ENDOTHELIUM-DEPENDENT VASOMOTOR FUNCTION IN PATIENTS WITH STABLE CORONARY ARTERY DISEASE ONLY AFTER PRECEDING ISCHAEMIA-REPERFUSION L. Tilling, J. Hunt, A. Donald, B. Clapp, P. Chowienczyk. King’s College London, British Heart Foundation Centre, London, UK Objective: To examine whether the erythropoietin analogue darbepoetin improves flow mediated dilatation (FMD), a measure of endothelium-derived NO, and whether this is influenced by preceding ischaemia-reperfusion. Background: Vasoprotective effects of erythropoietin in animal models are mediated by endothelium-derived nitric oxide (NO) and/or mobilization of endothelial progenitor cells (EPC) and may be enhanced by ischaemia. Whether they are present in humans is unknown. Methods: 36 patients (50−75 years) with stable coronary artery disease were randomized to receive a single dose of darbepoetin (300 mg) or saline placebo. FMD was measured at the brachial artery using high resolution ultrasound before and at 24h, 72h and 7 days after darbepoetin/placebo. At 24h FMD was repeated after 20 minutes ischaemia-reperfusion of the upper limb. A further group of 11 patients were studied according to the same protocol, all receiving darbepoetin, with omission of forearm ischaemia-reperfusion at 24h. Results: Immunoreactive erythropoietin peaked at 24h and remained elevated at approximately 500 fold baseline at 72h. FMD did not differ significantly between groups at 24h (before ischaemia-reperfusion). At 72h, (48h after ischaemia-reperfusion) FMD was greater (by 2.3±0.5% in the darbepoetin compared to the placebo group, a 66% increase over baseline, P < 0.001) and greater than FMD at the same time point without preceding ischaemiareperfusion (P < 0.01). There was no increase in CD133+ /CD34+ /VEGFR2+ cells after darbepoetin treatment. Conclusions: Preceding ischaemia-reperfusion is required for darbepoetin to enhance endothelial function, possibly by increasing expression of the erythropoietin receptor and by a mechanism likely to involve Akt/NO rather than circulating EPC. 298 RELATION OF MATRIX METALLOPROTEINASES (MMPS)-POSITIVE MACROPHAGES TO CORONARY PLAQUE FRAGILITY AND CORONARY ARTERIAL OUTWARD REMODELING T. Ito1 , S. Yamada1 , N. Hirayama-Kuniyoshi1 , M. Shiomi1,2 . 1 Institute for Experimental Animals, Kobe Univesity Graduate School of Medicine, 2 Section of Animal Models for Cardiovascular Diseases, Division of Cardiovascular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan Objectives: The purpose of this study is to examine the influence of MMPspositive macrophages on coronary plaque fragility and coronary outward remodeling. Methods: Coronary arteries of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits aged 15−24 months were fixed with 10%
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neutral buffered formalin solution and were embedded in paraffin. Coronary segments were sectioned serially and stained immunohistochemically using antibodies specific for rabbit macrophages (RAM-11), MMP-1, MMP-12, and for a-actin (1A4). Sections were also stained with elastic van Gieson staining and Azan-Mallory staining. Results: Various types of atherosclerotic lesions were observed in the coronary arteries, such as plaque with large lipid core covered with thin fibrous cap, fibroatheroma, fibromuscular lesion, macrophage-rich lesion, and plaque with calcification or intraplaque hemorrhage. Macrophages were observed beneath the fibrous cap and at a bottom of the intimal plaques where the tunica media was attenuated. At a plaque surface, macrophages were positive for MMPS; the density of SMCs and collagen fibers were decreased in the fibrous cap; the fibrous cap was attenuated partly. At a bottom of the intimal plaque where the tunica media was attenuated, macrophages was positive for MMPs; the elastic lamina was disappeared, the layer of SMCs in the tunica media was reduced. Conclusions: These observations suggest that MMP-positive macrophages at the plaque surface may relate to plaque fragility and those observed at a bottom of the intimal plaques may relate to arterial outward remodeling. 299 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2-INHIBITION PREVENTS ENDOTHELIAL SUSCEPTIBILITY TO TNF-a UNDER CHRONIC NON-UNIFORM SHEAR STRESS K. Urschel, W.G. Daniel, C.D. Garlichs, I. Cicha. Department of Cardiology and Angiology, University of Erlangen-Nuremberg, Erlangen, Germany Introduction: Non-uniform shear stress contributes to the development of atherosclerotic lesions by activating inflammatory pathways in endothelial cells (ECs). VEGFR2 is a key receptor for endothelial mechanotransduction. We investigated the role of VEGFR2 in ECs activated by non-uniform shear stress, with focus on monocyte recruitment and endothelial adhesion molecule expression. Methods: ECs seeded in bifurcating flow-through cell culture slides were exposed to shear stress overnight, followed by 2 hours stimulation with TNF-a. Subsequently, ECs were perfused for 1 hour with medium containing THP-1 monocytes to study cell adhesion. During flow, cells were incubated with a specific VEGFR2-antagonist (ZM 323881). For siRNA approach, cells were transfected with siRNAs knocking down VEGFR2 prior to shear stress exposure. Endothelial protein expression was determined by immunofluorescence and cell adhesion by light microcopy. Results: siRNA-mediated VEGFR2 knockdown resulted in a decrease of VEGFR2 protein expression by 40%. This knockdown was accompanied by a marked reduction of TNF-a-induced NFú-B translocation from cytoplasm to the nucleus in ECs. Either siRNA-mediated receptor knockdown, or treatment with the specific inhibitor, significantly reduced TNF-a-induced monocytic cell recruitment to endothelium under non-uniform shear stress conditions. The decrease in monocytic cell adhesion via inhibition of VEGFR2 signaling was accompanied by a reduction of E-selectin and VCAM-1. Conclusion: The inhibition of pro-atherogenic mechanical signaling by blocking VEGFR2 decreased endothelial activation and monocytic cell recruitment in response to inflammatory cytokines under in vivo-like non-uniform shear stress conditions. Localized targeting of VEGFR2 can therefore represent a useful approach in the treatment of atherosclerosis. 300 THE GUT MICROBIOTA MODULATES ATHEROSCLEROSIS PROGRESSION IN MALE APOE−/− MICE 2 1 F. Fak ˚ 1 , C. Reinhardt2 , F.-P. Martin3 , F. Backhed ¨ . Dept. of Medicine, Gothenburg University, 2 University of Gothenburg, Gothenburg, Sweden, 3 Nestle´ Research Center, Lausanne, Switzerland
Introduction and Objective: Bacterial infections appear to correlate with cardiovascular disease but the precise role of bacteria in disease initiation or progression has yet to be elucidated. Design: To investigate the role of bacteria in atherosclerosis, we re-derived atherosclerosis-prone apolipoprotein E-deficient mice (Apoe/−) as germ free. The mice were kept on a high fat, Western diet supplemented with cholesterol for 12 weeks and were sacrificed at 20 weeks of age. Atherosclerosis was assessed in the aortic root region of the heart and entire aortas were pinned and stained with Oil Red O. In addition, blood cholesterol, triglycerides, lipid metabolites and cytokines were analyzed. Results: Male germ free Apoe/− mice exhibited significantly decreased lesion area in the aortic root along with lower blood cholesterol and TG triglyceride levels, as compared to conventionally raised mice. Female mice, however, developed lesions of the same size as the control mice. Conclusions: Our results indicate that bacteria are important for atherosclerosis progression through a gender-dependent mechanism.
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301 ALCOHOL INHIBITS NOTCH SIGNALING IN VASCULAR SMOOTH MUSCLE CELLS AT THE LEVEL OF g-SECRETASE D. Morrow1 , J. Cullen1 , P. Cahill2 , E. Redmond1 . 1 Surgery, University of Rochester, Rochester, NY, USA, 2 Vascular Health Research Center, Dublin City University, Dublin, Ireland Moderate alcohol consumption is associated with reduced cardiovascular disease. We recently reported an inhibitory effect of alcohol (ethanol, EtOH) on Notch signaling and subsequently on vascular smooth muscle (SMC) proliferation, a key process in atherosclerotic plaque development. The object of this study was to test the hypothesis that EtOH inhibits Notch signaling in SMC at the level of g-secretase, a protease that catalyzes the release of the intracellular domain of Notch (NICD) necessary for signaling. Human coronary artery SMC were treated with a recombinant Notch ligand, DLL4 (2 mg/ml), or transfected with/without constitutively active Notch 1ICD (N1ICD), in the absence or presence of EtOH (25 mM). Ethanol treatment inhibited DLL4-stimulated CBF-1/RBP-Jk dependent promoter activity (determined by luciferase assay) and DLL4-stimulated downstream target gene HRT3 mRNA expression. In contrast, ethanol had no effect on N1ICD-driven CBF1/RBP-Jk dependent promoter activity. These data suggest that EtOH inhibits Notch signaling at, or prior to, NICD generation. g-secretase activity was determined in solubilized membrane preparations from SMC treated with/without EtOH (25 mM) or the g-secretase inhibitor DAPT (20 mM) using (i) a fluorometric assay kit and (ii) western blot detection of cleavage products using a Flag-tagged Notch based substrate, N100Flag. In both assays, Ethanol inhibited g-secretase activity, to the same extent as DAPT. In contrast, ethanol had no effect on a-secretase (TACE/ADAM 17) activity, determined using a fluorometric assay kit. In conclusion, EtOH inhibits Notch signaling in SMC via inhibition of the g-secretase mediated intramembrane proteolysis necessary for NICD generation and subsequent transcriptional activation. 302 CB2 RECEPTOR SIGNALING DOES NOT MODULATE ATHEROGENESIS IN MICE F. Willecke, K. Zeschky, D. Wolf, M. Moser, I. Hilgendorf, C. Bode, A. Zirlik. ¨ Universitatsklinik Freiburg, Freiburg im Breisgau, Germany Strong evidence supports a protective role of the cannabinoid receptor 2 (CB2 ) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. The present study aimed to characterize the role of the CB2 receptor in Murine atherogenesis. Methods and Results: Low density lipoprotein receptor-deficient (LDLR−/− ) mice subjected to intraperitoneal injections of the selective CB2 receptor agonist JWH-133 or vehicle 3x/week consumed a high cholesterol diet for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots (0.316±0.038 mm2 vs. 0.312±0.044 mm2 , P = 0.94) and arches (0.091±0.024 mm2 vs. 0.066±0.013 mm2 , P = 0.41), suggesting that CB2 activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen was also −/− mice developed lesions of similar in both groups. Accordingly, CB−/− 2 /LDLR similar size in roots (0.261±0.038 mm2 vs. 0.223±0.023 mm2 , P = 0.40) and −/− arches (0.095±0.022 mm2 vs. 0.075±0.022 mm2 , P = 0.54) as CB+/+ 2 /LDLR controls consuming HCD for 16 weeks. With exception of an increase in lipid and macrophage content, plaque composition was also similar between these two groups. Neither genetic deficiency nor pharmacologic activation of the CB2 receptor altered inflammatory cytokine expression or peritoneal leukocyte recruitment in vivo or inflammatory cell adhesion in the flow chamber in vitro. Conclusion: Our study demonstrates that activation and deletion of the CB2 receptor do not modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB2 in other inflammatory processes. However, in the context of atherosclerosis, CB2 does not appear to be a suitable therapeutic target. 303 MICRORNA 143–145 DEFICIENCY IS ASSOCIATED WITH IMPAIRED VASCULAR FUNCTION G.D. Norata1 , C. Pinna1 , F. Zappella1 , L. Elia2 , G. Condorelli3 , A. Catapano1 . 1 University of Milan, Milan, Italy, 2 University of California San Diego, La Jolla, CA, USA, 3 Consiglio Nazionale delle Ricerche, Segrate, Italy Background: MicroRNAs are necessary for vascular smooth muscle growth, differentiation and function. MiR143–145 modulate cytoskeletal dynamics and acquisition of the contractile phenotype by smooth muscle cells. Loss of this miRNA cluster results in deregulated blood pressure and reduced vasoconstriction. As all these observations point to a key role for miR143–145 in the vasculature, we therefore investigated whether miR143–145 deficiency is associated with impaired vascular tone. Methods and Results: Vasocontraction was assessed in isolated aortic rings from miR143–145 KO and wild type animals incubated with increasing concentrations of phenylephrine (10-9 M to 10-5 M) or KCl 0.3M. In both cases, aortic vessel contraction was dramatically reduced in miR143–145 KO animals compared to controls. Next, aortic rings were pre-contracted with phenylephrine
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binding of uPA to its receptor, uPAR, and was mediated via activation of SREBP-1 through PI3K and MEK signal transduction pathways. Furthermore, uPA increased macrophage oxidative stress by enhancing NADPH oxidase activation. In turn, uPA initiated an oxidative stress-response by upregulating the expression of macrophage paraoxonase 2 (PON2) through the PI3KNADPH oxidase-ROS-MEK-SREBP2 signaling cascade activation. In addition, we have shown that uPA binding to uPAR on hepatocytes stimulates PPARg nuclear export, resulting in down-regulation of paraoxonase 1 (PON1) gene expression, and decreased PON1 secretion. Reduced levels of PON1 in serum may lead to reduced protection of LDL and of HDL from oxidation, reduced HDL-mediated cholesterol efflux from macrophages, and increased oxidative stress in atherosclerotic lesions. Conclusions: Taken together, our work represents a new paradigm by which uPA is implicated in atherogenic processes beyond its role in the fibrinolytic system, and provides new insight into the relationship between uPA and vulnerable plaque. 294 ACCELERATED ATHEROSCLEROSIS IN FEMALE MICE LACKING THE ANDROGEN RECEPTOR J. Bourghardt1 , A. Wilhelmson1 , C. Alexanderson1 , K. De Gendt2 , G. Verhoeven2 , A. Holmang ¨ 3 , A. Krettek4,5 , S.-O. Olofsson1 , C. Ohlsson6 , ˚ Tivesten1 . 1 Wallenberg Laboratory, University of Gothenburg, Gothenburg, A. Sweden, 2 Katholieke Universiteit Leuven, Leuven, Belgium, 3 Institute of Neuroscience and Physiology, Unversity of Gothenburg, 4 Nordic School of Public Health, 5 Institute of Medicine, University of Gothenburg, 6 Center for Bone and Arthritis Research, University of Gothenburg, Gothenburg, Sweden Objective: In women, elevated androgen levels have been reported to associate with the metabolic syndrome and atherosclerosis. However, the physiological role of androgens in metabolism and cardiovascular health in females is unclear. We evaluated the impact of physiological, androgen receptor (AR)-dependent actions of androgens on atherosclerosis using female ARdeficient (AR−/−) mice on an apolipoprotein E-deficient background. Methods and Results: After 8 weeks on high-fat diet, atherosclerosis assessed en face was increased by 59% (P < 0.01) in female AR−/− mice compared with control mice. Female AR−/− mice had increased body weight and relative fat mass (+62%, P < 0.001), increased hepatic triglyceride levels (+33%, P < 0.05) and reduced insulin sensitivity. In addition, the female AR−/− mice had elevated serum levels of cholesterol (+52%, P < 0.001) and triglycerides (+44%, P < 0.05) compared with control mice. Conclusions: Female AR−/− mice display accelerated atherosclerosis associated with obesity, hepatic steatosis, insulin resistance and dyslipidemia. These results suggest that AR-mediated effects of androgens are crucial for metabolism and cardiovascular health in females. 295 ATHEROSCLEROSIS REGRESSION IN A MOUSE MODEL WITH HUMAN-LIKE HYPERCHOLESTEROLEMIA 1 , S. Hagg ¨ 1 , S. Maleki1 , M. Shang1 , T. Michoel2 , J. Skogsberg1 . J. Bjorkegren ¨ 1 Karolinska Institutet, Stockholm, Sweden, 2 University of Freiburg, Freiburg, Germany Atherosclerosis is a lifelong, progressive disease that becomes clinically significant in a large fraction of the population, leading to myocardial infarction (MI) and stroke. Statin based lipid-lowering regimens reduce morbidity and mortality from both MI and stroke. To fully exploit the beneficial effects of lipid lowering, we need a better understanding of the transcriptional changes induced by lowering plasma lipoproteins. We have recently performed transcriptional profiling at 10-week intervals in atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic switch to lower plasma lipoproteins (Ldlr−/− Apob100/100 Mttpflox/flox Mx1-Cre). Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed. Analysis of lesion expression profiles indicated that accumulation of lipid-poor macrophages reached a point that led to the rapid expansion phase with accelerated foam-cell formation and inflammation, an interpretation supported by lesion histology. In the current study, we preformed genetic lowering of plasma cholesterol (i.e. lipoproteins) at the rapid lesion expansion start point which prevented the formation of advanced plaques. In addition, lowering of plasma cholesterol in plaques that already had been formed not only halted the atherosclerosis lesion development it also substantially regressed the lesion size. By performing transcriptional profiling of the atherosclerotic arterial wall in the plasma cholesterol lowered mice important gene networks responsible for atherosclerosis regression were identified. These networks will be validated in relevant cell culture systems and in human association studies. The identified networks should be of interest for the development of novel atherosclerosis therapies.
Poster presentations
296 TRANSCRIPTOMIC ANALYSIS OF MACROPHAGE POLARIZATION: ROLE OF PPARY IN ALTERNATIVE ACTIVATION E. Derlindati, D. Ardigo, ` I. Zavaroni. Internal Medicine, University of Parma, Parma, Italy Introduction: Human macrophages (HM) undergo different forms of activation in response to cytokines. Classical activation (M1) displays pro-inflammatory properties, and can be induced by IFNg and/or LPS. Alternative activation (M2a) shows immuno-regulatory properties and is promoted by IL-4, whereas IL-10 produces a “deactivated” state, with immunosuppressing characteristics (M2c). The PPARg system appears to be implicated in M2a activation enhancing their anti-inflammatory properties. Aim: Evaluation of the biological processes involved in the different types of HM activation compared to the effects induced by exposure to PPARg-agonists. Methods: HM were stimulated with INFg+LPS (M1), IL-4 (M2a), IL-10 (M2c), or a PPARg-agonist (GW1929-Sigma). Phenotypic changes for each experimental condition were investigated through whole-genome transcriptomic analysis by microarrays (Agilent-Technologies). Gene ontology (GO) analysis was performed using Gorilla tool. Validation was obtained by RT-PCR on the most differentially expressed genes. Results: After characterization of M1, M2a, and M2c genomic profiles, we investigated the differences between PPARy-stimulated HM and M1, observing a significant decrease in immunitary and inflammatory processes (corresponding GO-term’s p-value = 2.00×10−35 and 1.20×10−20 ) and a great increase in cell cycle activity (p = 1.51×10−35 ). Similar, but quantitatively lower, results were observed also in comparison with M2a and M2c, although these two subpopulations are usually regarded as anti-inflammatory. Discussion: Exposure of HM to a PPARg-agonist induces a transcriptomic phenotype closer to M2a/M2c than to M1. Nevertheless, PPARg-agonist activation decreases inflammatory cytokine production and increases cellcycle gene expression also compared to cytokine-induced M2a and M2c activation, suggesting a specific role of PPARg in the regulation of macrophage differentiation. 297 DARBEPOETIN ENHANCES ENDOTHELIUM-DEPENDENT VASOMOTOR FUNCTION IN PATIENTS WITH STABLE CORONARY ARTERY DISEASE ONLY AFTER PRECEDING ISCHAEMIA-REPERFUSION L. Tilling, J. Hunt, A. Donald, B. Clapp, P. Chowienczyk. King’s College London, British Heart Foundation Centre, London, UK Objective: To examine whether the erythropoietin analogue darbepoetin improves flow mediated dilatation (FMD), a measure of endothelium-derived NO, and whether this is influenced by preceding ischaemia-reperfusion. Background: Vasoprotective effects of erythropoietin in animal models are mediated by endothelium-derived nitric oxide (NO) and/or mobilization of endothelial progenitor cells (EPC) and may be enhanced by ischaemia. Whether they are present in humans is unknown. Methods: 36 patients (50−75 years) with stable coronary artery disease were randomized to receive a single dose of darbepoetin (300 mg) or saline placebo. FMD was measured at the brachial artery using high resolution ultrasound before and at 24h, 72h and 7 days after darbepoetin/placebo. At 24h FMD was repeated after 20 minutes ischaemia-reperfusion of the upper limb. A further group of 11 patients were studied according to the same protocol, all receiving darbepoetin, with omission of forearm ischaemia-reperfusion at 24h. Results: Immunoreactive erythropoietin peaked at 24h and remained elevated at approximately 500 fold baseline at 72h. FMD did not differ significantly between groups at 24h (before ischaemia-reperfusion). At 72h, (48h after ischaemia-reperfusion) FMD was greater (by 2.3±0.5% in the darbepoetin compared to the placebo group, a 66% increase over baseline, P < 0.001) and greater than FMD at the same time point without preceding ischaemiareperfusion (P < 0.01). There was no increase in CD133+ /CD34+ /VEGFR2+ cells after darbepoetin treatment. Conclusions: Preceding ischaemia-reperfusion is required for darbepoetin to enhance endothelial function, possibly by increasing expression of the erythropoietin receptor and by a mechanism likely to involve Akt/NO rather than circulating EPC. 298 RELATION OF MATRIX METALLOPROTEINASES (MMPS)-POSITIVE MACROPHAGES TO CORONARY PLAQUE FRAGILITY AND CORONARY ARTERIAL OUTWARD REMODELING T. Ito1 , S. Yamada1 , N. Hirayama-Kuniyoshi1 , M. Shiomi1,2 . 1 Institute for Experimental Animals, Kobe Univesity Graduate School of Medicine, 2 Section of Animal Models for Cardiovascular Diseases, Division of Cardiovascular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan Objectives: The purpose of this study is to examine the influence of MMPspositive macrophages on coronary plaque fragility and coronary outward remodeling. Methods: Coronary arteries of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits aged 15−24 months were fixed with 10%
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neutral buffered formalin solution and were embedded in paraffin. Coronary segments were sectioned serially and stained immunohistochemically using antibodies specific for rabbit macrophages (RAM-11), MMP-1, MMP-12, and for a-actin (1A4). Sections were also stained with elastic van Gieson staining and Azan-Mallory staining. Results: Various types of atherosclerotic lesions were observed in the coronary arteries, such as plaque with large lipid core covered with thin fibrous cap, fibroatheroma, fibromuscular lesion, macrophage-rich lesion, and plaque with calcification or intraplaque hemorrhage. Macrophages were observed beneath the fibrous cap and at a bottom of the intimal plaques where the tunica media was attenuated. At a plaque surface, macrophages were positive for MMPS; the density of SMCs and collagen fibers were decreased in the fibrous cap; the fibrous cap was attenuated partly. At a bottom of the intimal plaque where the tunica media was attenuated, macrophages was positive for MMPs; the elastic lamina was disappeared, the layer of SMCs in the tunica media was reduced. Conclusions: These observations suggest that MMP-positive macrophages at the plaque surface may relate to plaque fragility and those observed at a bottom of the intimal plaques may relate to arterial outward remodeling. 299 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2-INHIBITION PREVENTS ENDOTHELIAL SUSCEPTIBILITY TO TNF-a UNDER CHRONIC NON-UNIFORM SHEAR STRESS K. Urschel, W.G. Daniel, C.D. Garlichs, I. Cicha. Department of Cardiology and Angiology, University of Erlangen-Nuremberg, Erlangen, Germany Introduction: Non-uniform shear stress contributes to the development of atherosclerotic lesions by activating inflammatory pathways in endothelial cells (ECs). VEGFR2 is a key receptor for endothelial mechanotransduction. We investigated the role of VEGFR2 in ECs activated by non-uniform shear stress, with focus on monocyte recruitment and endothelial adhesion molecule expression. Methods: ECs seeded in bifurcating flow-through cell culture slides were exposed to shear stress overnight, followed by 2 hours stimulation with TNF-a. Subsequently, ECs were perfused for 1 hour with medium containing THP-1 monocytes to study cell adhesion. During flow, cells were incubated with a specific VEGFR2-antagonist (ZM 323881). For siRNA approach, cells were transfected with siRNAs knocking down VEGFR2 prior to shear stress exposure. Endothelial protein expression was determined by immunofluorescence and cell adhesion by light microcopy. Results: siRNA-mediated VEGFR2 knockdown resulted in a decrease of VEGFR2 protein expression by 40%. This knockdown was accompanied by a marked reduction of TNF-a-induced NFú-B translocation from cytoplasm to the nucleus in ECs. Either siRNA-mediated receptor knockdown, or treatment with the specific inhibitor, significantly reduced TNF-a-induced monocytic cell recruitment to endothelium under non-uniform shear stress conditions. The decrease in monocytic cell adhesion via inhibition of VEGFR2 signaling was accompanied by a reduction of E-selectin and VCAM-1. Conclusion: The inhibition of pro-atherogenic mechanical signaling by blocking VEGFR2 decreased endothelial activation and monocytic cell recruitment in response to inflammatory cytokines under in vivo-like non-uniform shear stress conditions. Localized targeting of VEGFR2 can therefore represent a useful approach in the treatment of atherosclerosis. 300 THE GUT MICROBIOTA MODULATES ATHEROSCLEROSIS PROGRESSION IN MALE APOE−/− MICE 2 1 F. Fak ˚ 1 , C. Reinhardt2 , F.-P. Martin3 , F. Backhed ¨ . Dept. of Medicine, Gothenburg University, 2 University of Gothenburg, Gothenburg, Sweden, 3 Nestle´ Research Center, Lausanne, Switzerland
Introduction and Objective: Bacterial infections appear to correlate with cardiovascular disease but the precise role of bacteria in disease initiation or progression has yet to be elucidated. Design: To investigate the role of bacteria in atherosclerosis, we re-derived atherosclerosis-prone apolipoprotein E-deficient mice (Apoe/−) as germ free. The mice were kept on a high fat, Western diet supplemented with cholesterol for 12 weeks and were sacrificed at 20 weeks of age. Atherosclerosis was assessed in the aortic root region of the heart and entire aortas were pinned and stained with Oil Red O. In addition, blood cholesterol, triglycerides, lipid metabolites and cytokines were analyzed. Results: Male germ free Apoe/− mice exhibited significantly decreased lesion area in the aortic root along with lower blood cholesterol and TG triglyceride levels, as compared to conventionally raised mice. Female mice, however, developed lesions of the same size as the control mice. Conclusions: Our results indicate that bacteria are important for atherosclerosis progression through a gender-dependent mechanism.
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301 ALCOHOL INHIBITS NOTCH SIGNALING IN VASCULAR SMOOTH MUSCLE CELLS AT THE LEVEL OF g-SECRETASE D. Morrow1 , J. Cullen1 , P. Cahill2 , E. Redmond1 . 1 Surgery, University of Rochester, Rochester, NY, USA, 2 Vascular Health Research Center, Dublin City University, Dublin, Ireland Moderate alcohol consumption is associated with reduced cardiovascular disease. We recently reported an inhibitory effect of alcohol (ethanol, EtOH) on Notch signaling and subsequently on vascular smooth muscle (SMC) proliferation, a key process in atherosclerotic plaque development. The object of this study was to test the hypothesis that EtOH inhibits Notch signaling in SMC at the level of g-secretase, a protease that catalyzes the release of the intracellular domain of Notch (NICD) necessary for signaling. Human coronary artery SMC were treated with a recombinant Notch ligand, DLL4 (2 mg/ml), or transfected with/without constitutively active Notch 1ICD (N1ICD), in the absence or presence of EtOH (25 mM). Ethanol treatment inhibited DLL4-stimulated CBF-1/RBP-Jk dependent promoter activity (determined by luciferase assay) and DLL4-stimulated downstream target gene HRT3 mRNA expression. In contrast, ethanol had no effect on N1ICD-driven CBF1/RBP-Jk dependent promoter activity. These data suggest that EtOH inhibits Notch signaling at, or prior to, NICD generation. g-secretase activity was determined in solubilized membrane preparations from SMC treated with/without EtOH (25 mM) or the g-secretase inhibitor DAPT (20 mM) using (i) a fluorometric assay kit and (ii) western blot detection of cleavage products using a Flag-tagged Notch based substrate, N100Flag. In both assays, Ethanol inhibited g-secretase activity, to the same extent as DAPT. In contrast, ethanol had no effect on a-secretase (TACE/ADAM 17) activity, determined using a fluorometric assay kit. In conclusion, EtOH inhibits Notch signaling in SMC via inhibition of the g-secretase mediated intramembrane proteolysis necessary for NICD generation and subsequent transcriptional activation. 302 CB2 RECEPTOR SIGNALING DOES NOT MODULATE ATHEROGENESIS IN MICE F. Willecke, K. Zeschky, D. Wolf, M. Moser, I. Hilgendorf, C. Bode, A. Zirlik. ¨ Universitatsklinik Freiburg, Freiburg im Breisgau, Germany Strong evidence supports a protective role of the cannabinoid receptor 2 (CB2 ) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. The present study aimed to characterize the role of the CB2 receptor in Murine atherogenesis. Methods and Results: Low density lipoprotein receptor-deficient (LDLR−/− ) mice subjected to intraperitoneal injections of the selective CB2 receptor agonist JWH-133 or vehicle 3x/week consumed a high cholesterol diet for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots (0.316±0.038 mm2 vs. 0.312±0.044 mm2 , P = 0.94) and arches (0.091±0.024 mm2 vs. 0.066±0.013 mm2 , P = 0.41), suggesting that CB2 activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen was also −/− mice developed lesions of similar in both groups. Accordingly, CB−/− 2 /LDLR similar size in roots (0.261±0.038 mm2 vs. 0.223±0.023 mm2 , P = 0.40) and −/− arches (0.095±0.022 mm2 vs. 0.075±0.022 mm2 , P = 0.54) as CB+/+ 2 /LDLR controls consuming HCD for 16 weeks. With exception of an increase in lipid and macrophage content, plaque composition was also similar between these two groups. Neither genetic deficiency nor pharmacologic activation of the CB2 receptor altered inflammatory cytokine expression or peritoneal leukocyte recruitment in vivo or inflammatory cell adhesion in the flow chamber in vitro. Conclusion: Our study demonstrates that activation and deletion of the CB2 receptor do not modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB2 in other inflammatory processes. However, in the context of atherosclerosis, CB2 does not appear to be a suitable therapeutic target. 303 MICRORNA 143–145 DEFICIENCY IS ASSOCIATED WITH IMPAIRED VASCULAR FUNCTION G.D. Norata1 , C. Pinna1 , F. Zappella1 , L. Elia2 , G. Condorelli3 , A. Catapano1 . 1 University of Milan, Milan, Italy, 2 University of California San Diego, La Jolla, CA, USA, 3 Consiglio Nazionale delle Ricerche, Segrate, Italy Background: MicroRNAs are necessary for vascular smooth muscle growth, differentiation and function. MiR143–145 modulate cytoskeletal dynamics and acquisition of the contractile phenotype by smooth muscle cells. Loss of this miRNA cluster results in deregulated blood pressure and reduced vasoconstriction. As all these observations point to a key role for miR143–145 in the vasculature, we therefore investigated whether miR143–145 deficiency is associated with impaired vascular tone. Methods and Results: Vasocontraction was assessed in isolated aortic rings from miR143–145 KO and wild type animals incubated with increasing concentrations of phenylephrine (10-9 M to 10-5 M) or KCl 0.3M. In both cases, aortic vessel contraction was dramatically reduced in miR143–145 KO animals compared to controls. Next, aortic rings were pre-contracted with phenylephrine