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INFLUENCE OF HEMODYNAMICS ON VASCULAR NITRIC OXIDE METABOLISM
S190
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
P541 INFLUENCE OF HEMODYNAMICS ON VASCULAR NITRIC OXIDE METABOLISM. Veronica Gambillara, Gabriela Montorzi, Christelle Haziza, Paolo Silacci, Nikos Stergiopulos. LHTC, EPFL, Lausanne, Switzerland. Introduction: Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. Indeed, plaques are initiated and promoted in specific areas corresponding to branching points and curvatures of vessels where the forces acting on the wall are different from the rest of the arterial tree. It has been established that a disturbed flow characterized by a cyclic reversal flow is directly associated to the plaque-prone areas. Nitric oxide (NO) plays an essential role in mediating many effects of flow, including vasorelaxation, inhibition of apoptosis, inhibition of platelets aggregation, and anti-inflammatory action. The aim of the study was to analyze endothelial function and smooth muscle cell contractility in intact arteries exposed to different flow pattern cultured in an ex-vivo perfusion system (EVASS). Methods: Arterial segments of the left common porcine carotid were mounted on the EVASS and perfused for one and three days under three different flow conditions: unidirectional high shear stress (6 ± 3 dynes/ cm2), mimicking the plaque-free flow conditions, unidirectional low shear stress (0.3 ± 0.1 dynes/cm2), and oscillatory shear stress (0.3 ± 3 dynes/ cm2), mimicking the plaque-prone flow conditions. At the end of perfusion period, endothelial function, eNOS expression, and smooth muscle cell function were assessed. Results and Conclusion: After three days of perfusion, endothelial function was drastically decreased in arteries exposed to the oscillatory shear stress. Impaired NO-mediated vasorelaxation was correlated to lower expression of eNOS gene but SMC contractility, phenotype and proliferation were not affected by the different flow patterns, suggesting that the oscillatory shear stress affects endothelial but not smooth muscle function along this period of perfusion. These data suggest that endothelial dysfunction triggered by cyclic reversal flow is directly mediated by a down-regulation of eNOS gene expression and support the hypothesis that flow plays an active role on the pathophysiology of atherosclerosis.
P542 MODULATION OF HUMAN CORONARY ARTERY SMOOTH MUSCLE CELLS BY SHEAR STRESS: THE ROLE OF TGF BETA1. Jonathan Ghosh, David Murray, Cay Kielty, Michael Walker. UK Centre for Tissue Engineering, Manchester Medical School, Manchester, UK. Introduction: Elucidation and manipulation of the effects of shear stress on vascular smooth muscle cells has applications in the prevention of intimal hyperplasia and as a tool for vascular tissue engineering. This investigation aims to characterise the influence of shear stress on human coronary artery smooth muscle cell (HCASMC) proliferation, extracellular matrix production, cytoskeleton organisation and to investigate the role of TGFBeta1. Methods: HCASMC (passage 5 – 7) cultured under physiological conditions were subjected to degrees of shear stress. Expression of extracellular matrix molecules collagen I, collagen IV, collagen VIII, elastin and fibrillin 1 was determined by semi-quantitative RT-PCR and immunofluorescence. Expression of cytoskeleton components alpha-actin, smooth muscle myosin heavy chain, SM22alpha and smoothelin was determined by semiquantitative RT-PCR. Organisation of alpha-actin filaments was examined by immunofluorescence. The proportion of cells undergoing mitosis was identified by co-staining with DAPI and FITC-conjugated antibodies against Ki67 nuclear protein.
Semi-quantitative RT-PCR and ELISA determined expression and release of TGFBeta1, PDGFbb and latent TGFBeta binding proteins (LTBP)-1 and -3. Effects of shear stress on extracellular matrix synthesis, proliferation, cytoskeletal expression and organisation following blockade of TGFBetareceptor 2 by neutralising antibodies was also investigated. Results: In static culture, HCASMC expressed collagen I and IV. Shear stress induced a rapid upregulation in fibrillin 1, collagen VIII and elastin synthesis, with continued collagen I and IV expression. Upregulated extracellular matrix expression ceased 24 hours after termination of fluid flow. Expression of cytoskeletal molecules was downregulated during shear stress, reversing 24 hours after termination of fluid flow. Alphaactin filament orientation was random in static culture but displayed a parallel arrangement whilst subjected to shear stress, perpendicular to direction of fluid flow. The rate of reorganisation was dependent upon degree of shear stress. A significantly higher proportion of cells were actively undergoing mitosis under shear stress than in static culture. Increased expression and release of TGFBeta1, but not PDGFbb or LTBPs-1 and -3, occurred following shear stress stimulation. HCASMC with TGFBeta1 supplementation in static culture showed similar enhanced proliferation and extracellular matrix production as observed during shear stress without growth factor supplementation. Blockade of TGFBeta receptor-2 resulted in attenuation of matrix production and cell proliferation under shear stress although cytoskeletal remodeling was unchanged. Conclusions: Under shear stress, HCASMC produce increased amounts of elastomeric extracellular matrix, display a higher level of cytoskeleton organisation and are stimulated into active cell division. There is evidence that increased proliferation and matrix production, but not cytoskeleton remodeling, occurs by means of a TGFBeta1 autocrine or paracrine pathway.
P543 SHEAR STRESS MODULATES CHROMATIN STRUCTURE AND ACTS AS A VASCULAR PHENOTYPE DETERMINANT IN MOUSE EMBRYONIC STEM CELLS. Barbara Illi, Alessandro Scopece, Simona Nanni, Antonella Farsetti, Maurizio C. Capogrossi, Carlo Gaetano. Istituto Cardiologico Monzino, Milano, Italy, Istituto Dermopatico dell’ Immacolata, Roma, Italy, Istituto Regina Elena, Roma, Italy, Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma, Italy. It has been recently reported that shear stress (SS) may play an important role in embryonal heart development. However, it is still unknown whether this phenomenon is limited to the heart or may influence the entire cardiovascular tree.We recently reported that SS modifies the acetylation and phosphorylation status of core histones and modulates DNA structure, providing the molecular basis for SS-dependent gene expression, in human endothelial cells. In the present study it was investigated the effect of SS on chromatin remodeling and gene expression in mouse embryonic stem cells (ES), as an in vitro model of vascular differentiation. ES were cultured in the absence of Leukemia Inhibitory Factor (LIF) for 24 hours before exposure to laminar SS of 10 dyne/cm2 either in the presence or absence of the histone deacetylase inhibitor Trichostatin A (TSA). SS alone enhanced histone H3 acetylation (4 fold) and cooperated with TSA to stimulate phosphorylation and phosphoacetylation of histone H3 and H4. These molecular events were paralleled by a strong induction of the Vascular Endothelial Receptor-2 (VEGFR-2) which, in ES, represents an early vascular differentiation marker. RT-PCR and western blot analyses revealed a rapid increase in VEGFR-2 mRNA and protein in ES cells exposed to SS. In addition, in a series of transient transfection assays, SS and TSA cooperatively induced Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) promoter activity,
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
P541 INFLUENCE OF HEMODYNAMICS ON VASCULAR NITRIC OXIDE METABOLISM. Veronica Gambillara, Gabriela Montorzi, Christelle Haziza, Paolo Silacci, Nikos Stergiopulos. LHTC, EPFL, Lausanne, Switzerland. Introduction: Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. Indeed, plaques are initiated and promoted in specific areas corresponding to branching points and curvatures of vessels where the forces acting on the wall are different from the rest of the arterial tree. It has been established that a disturbed flow characterized by a cyclic reversal flow is directly associated to the plaque-prone areas. Nitric oxide (NO) plays an essential role in mediating many effects of flow, including vasorelaxation, inhibition of apoptosis, inhibition of platelets aggregation, and anti-inflammatory action. The aim of the study was to analyze endothelial function and smooth muscle cell contractility in intact arteries exposed to different flow pattern cultured in an ex-vivo perfusion system (EVASS). Methods: Arterial segments of the left common porcine carotid were mounted on the EVASS and perfused for one and three days under three different flow conditions: unidirectional high shear stress (6 ± 3 dynes/ cm2), mimicking the plaque-free flow conditions, unidirectional low shear stress (0.3 ± 0.1 dynes/cm2), and oscillatory shear stress (0.3 ± 3 dynes/ cm2), mimicking the plaque-prone flow conditions. At the end of perfusion period, endothelial function, eNOS expression, and smooth muscle cell function were assessed. Results and Conclusion: After three days of perfusion, endothelial function was drastically decreased in arteries exposed to the oscillatory shear stress. Impaired NO-mediated vasorelaxation was correlated to lower expression of eNOS gene but SMC contractility, phenotype and proliferation were not affected by the different flow patterns, suggesting that the oscillatory shear stress affects endothelial but not smooth muscle function along this period of perfusion. These data suggest that endothelial dysfunction triggered by cyclic reversal flow is directly mediated by a down-regulation of eNOS gene expression and support the hypothesis that flow plays an active role on the pathophysiology of atherosclerosis.
P542 MODULATION OF HUMAN CORONARY ARTERY SMOOTH MUSCLE CELLS BY SHEAR STRESS: THE ROLE OF TGF BETA1. Jonathan Ghosh, David Murray, Cay Kielty, Michael Walker. UK Centre for Tissue Engineering, Manchester Medical School, Manchester, UK. Introduction: Elucidation and manipulation of the effects of shear stress on vascular smooth muscle cells has applications in the prevention of intimal hyperplasia and as a tool for vascular tissue engineering. This investigation aims to characterise the influence of shear stress on human coronary artery smooth muscle cell (HCASMC) proliferation, extracellular matrix production, cytoskeleton organisation and to investigate the role of TGFBeta1. Methods: HCASMC (passage 5 – 7) cultured under physiological conditions were subjected to degrees of shear stress. Expression of extracellular matrix molecules collagen I, collagen IV, collagen VIII, elastin and fibrillin 1 was determined by semi-quantitative RT-PCR and immunofluorescence. Expression of cytoskeleton components alpha-actin, smooth muscle myosin heavy chain, SM22alpha and smoothelin was determined by semiquantitative RT-PCR. Organisation of alpha-actin filaments was examined by immunofluorescence. The proportion of cells undergoing mitosis was identified by co-staining with DAPI and FITC-conjugated antibodies against Ki67 nuclear protein.
Semi-quantitative RT-PCR and ELISA determined expression and release of TGFBeta1, PDGFbb and latent TGFBeta binding proteins (LTBP)-1 and -3. Effects of shear stress on extracellular matrix synthesis, proliferation, cytoskeletal expression and organisation following blockade of TGFBetareceptor 2 by neutralising antibodies was also investigated. Results: In static culture, HCASMC expressed collagen I and IV. Shear stress induced a rapid upregulation in fibrillin 1, collagen VIII and elastin synthesis, with continued collagen I and IV expression. Upregulated extracellular matrix expression ceased 24 hours after termination of fluid flow. Expression of cytoskeletal molecules was downregulated during shear stress, reversing 24 hours after termination of fluid flow. Alphaactin filament orientation was random in static culture but displayed a parallel arrangement whilst subjected to shear stress, perpendicular to direction of fluid flow. The rate of reorganisation was dependent upon degree of shear stress. A significantly higher proportion of cells were actively undergoing mitosis under shear stress than in static culture. Increased expression and release of TGFBeta1, but not PDGFbb or LTBPs-1 and -3, occurred following shear stress stimulation. HCASMC with TGFBeta1 supplementation in static culture showed similar enhanced proliferation and extracellular matrix production as observed during shear stress without growth factor supplementation. Blockade of TGFBeta receptor-2 resulted in attenuation of matrix production and cell proliferation under shear stress although cytoskeletal remodeling was unchanged. Conclusions: Under shear stress, HCASMC produce increased amounts of elastomeric extracellular matrix, display a higher level of cytoskeleton organisation and are stimulated into active cell division. There is evidence that increased proliferation and matrix production, but not cytoskeleton remodeling, occurs by means of a TGFBeta1 autocrine or paracrine pathway.
P543 SHEAR STRESS MODULATES CHROMATIN STRUCTURE AND ACTS AS A VASCULAR PHENOTYPE DETERMINANT IN MOUSE EMBRYONIC STEM CELLS. Barbara Illi, Alessandro Scopece, Simona Nanni, Antonella Farsetti, Maurizio C. Capogrossi, Carlo Gaetano. Istituto Cardiologico Monzino, Milano, Italy, Istituto Dermopatico dell’ Immacolata, Roma, Italy, Istituto Regina Elena, Roma, Italy, Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma, Italy. It has been recently reported that shear stress (SS) may play an important role in embryonal heart development. However, it is still unknown whether this phenomenon is limited to the heart or may influence the entire cardiovascular tree.We recently reported that SS modifies the acetylation and phosphorylation status of core histones and modulates DNA structure, providing the molecular basis for SS-dependent gene expression, in human endothelial cells. In the present study it was investigated the effect of SS on chromatin remodeling and gene expression in mouse embryonic stem cells (ES), as an in vitro model of vascular differentiation. ES were cultured in the absence of Leukemia Inhibitory Factor (LIF) for 24 hours before exposure to laminar SS of 10 dyne/cm2 either in the presence or absence of the histone deacetylase inhibitor Trichostatin A (TSA). SS alone enhanced histone H3 acetylation (4 fold) and cooperated with TSA to stimulate phosphorylation and phosphoacetylation of histone H3 and H4. These molecular events were paralleled by a strong induction of the Vascular Endothelial Receptor-2 (VEGFR-2) which, in ES, represents an early vascular differentiation marker. RT-PCR and western blot analyses revealed a rapid increase in VEGFR-2 mRNA and protein in ES cells exposed to SS. In addition, in a series of transient transfection assays, SS and TSA cooperatively induced Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) promoter activity,