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299 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2-INHIBITION PREVENTS ENDOTHELIAL SUSCEPTIBILITY TO TNF-a UNDER CHRONIC NON-UNIFORM SHEAR STRESS
79th EAS Congress
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
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
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
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