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1P-0181 Changes in gene expression of endothelial cells stimulated by lysophosphatidylcholine
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Monday September 29, 2003: Poster Session Endothelial cells
Phorbol 12-myristate-13-acetate, a protein kinase C (PKC) agonist, induced activation of ASK1 activity and up-regulation of PAI-1 expression, and an inhibitor of PKC suppressed both ASK1 activation and PAI-1 expression induced by high glucose. Importantly, overexpression of the constitutive active form of ASK1 by using an adenovirus vector induced up-regulation of PAI-1 expression in EC and infection of adenoviral construct including the dominant negative form of ASK1 gene suppressed high glucose-induced PAI-1 expression, suggesting that activation of ASK-1 is directly related with up-regulation of PAI-1. We conclude that activation of ASK1 may be involved in the underlying mechanism of endothelial PAI-1 expression induced by hyperglycemia in ECs. 1P-0181
Changes in gene expression of endothelial cells stimulated by lysophosphatidylcholine
A. Sheikh, H. Ochi, J. Masuda. Department of Laboratory Medicine, Shimane Medical University, Japan Objective: Lysophosphatidylcholine (lyso-PC), a phospholipid enriched in oxidized LDL, has been implicated to activate endothelial cells and modulate the development of atherosclerosis. In this study, we searched lyso-PC-regulated genes using cDNA macroarray to investigate roles of lyso-PC-activated endothelial cells in atherogenesis. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured. Macroarrays of 540 cDNA were used to compare gene expression patterns between HUVEC stimulated by lyso-PC for 8 hours and HUVEC treated with medium alone. Some of the highly regulated genes determined by macroarray were further verified by real-time PCR assays. This approach demonstrated that gene expression of at least 5 molecules, including tissue plasminogen activator (tPA), cyclooxygenase-1, integrin-α 6 and small G proteins Rac-2 and Rab-11, was up-regulated. Real-time PCR assay also showed that at least tPA was dose-dependently up-regulated by lyso-PC. Conclusion: We newly identified some molecules of which gene expression was modulated by lyso-PC in endothelial cells. Further analysis of these molecules may provide new insights on roles of lyso-PC in atherogenesis. 1P-0182
The effect of integrin linked kinase (ILK) overexpression in anchorage-dependent endothelial cells
Y. Seock-Won, K. Hyo-soo, P. Young-Bae. Cardiovascular Laboratory, Clinical Research Center, Seoul National University Hospital, Republic of Korea Objective: Cell adhesion to the extracellular matrix (ECM) is an essential process that controls proliferation, migration and survival of anchoragedependent cells such as endothelial cell (EC). Integrin-linked kinase (ILK) has been reported in epithelial cells to internalize cell survival signals. The implication of ILK, however, has not been investigated in EC. In this study, we generated a new adenoviral construct for wild type ILK and evaluated the role of ILK in anchorage-dependent and independent proliferation of EC. Methods and Results: (1) Construction of Adv-ILK: Human wild type ILK plasmid was subcloned into a shuttle vector (pADTrack-CMV). The recombinant shuttle vector was co-transfected with adenoviral genome (pAdEasy-1) into E.coli(BJ5183) where homologous recombination occurred. The recombinant Adeno-ILK was transfected to 293cell to get the viral particles which were purified by CsCl ultracentrifugation and dialysis. (2) Confirmation of ILK expressioin: When HUVECs were deprived of anchorage by culturing them on a dish coated with polyHEMA (adhesion inhibitor), the protein expression of ILK significantly decreased on western blot analysis. (3) Effect of ILK overexpression: In the attached condition, ILK overexpression did not affect proliferatinve activity of EC. However, in the detached condition, ILK overexpression significantly increased serum stimulated proliferation of EC. Conclusion: ILK overexpression provided anchorage-dependent EC with anchorage-independence, which suggests that ILK may be a good candidate gene to potentiate angiogenesis by protecting EC from stress when ECs are inevitably, although temporily, detached from ECM during angiogenesis.
1P-0183
Global analysis of RNA expression profile in human vascular cells and hepatocytes treated with HMG-CoA reductase inhibitors
S. Morikawa 1 , T. Kohro 1 , Y. Saito 2 , T. Hamakubo 1 , T. Kodama 1 . 1 Research Center for Advanced Science and Technology, the University of Tokyo; 2 Chiba University, Japan In addition to their effect on cholesterol metabolism, the 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors (statins) have an effect on the expression levels of many genes. In order to elucidate the range of this effect as comprehensively as possible, by means of DNA microarrays we investigated the changes in gene expression profiles brought about by atorvastatin or pitavastatin in cultured human umbilical vein endothelial cells (HUVEC), cultured human coronary artery smooth muscle cells (HCASMC) and cultured human hepatocarcinoma Hep G2 cells. Among the 6146 genes in the array, the statins affected the expression levels of genes involved in coagulation, vascular constriction and cell growth in a cell-type specific manner. In HUVEC, they induced integrin β4 and thrombomodulin profoundly, and profoundly suppressed pentraxin 3 (PTX3) both at 8 and 24 hour. In HCASMC, the statins induced thrombomodulin and urokinase inhibitor, and potently suppressed the cysteine-rich angiogenic inducer 61 (Cyr61) and cyclin B. In contrast, the expression level of these genes did not significantly change in Hep G2 cells. Many genes related to the cell cycle and/or growth were also regulated in HUVEC and HCASMC by the statins. These results indicate that many aspects of pleiotropic effect can be mediated by the transcriptional control by statins. Genes newly identified by this study may be related to beneficial effect of statin therapy. 1P-0185
Normal ranges for flow-mediated dilatation of the brachial artery in different age groups
L. Ryliskyte, A. Laucevicius, Z. Petrulioniene, J. Badariene, S. Janaviciene. Vilnius University Hospital Santariskiu Klnikos, Heart Clinic, Lithuania Ojectives: High-frequency ultrasonographic imaging of the flow-mediated dilatation (FMD) in the brachial artery is the main non-invasive technique used for clinical evaluation of endothelial function (EF). Since interpretive limitations of this technique exist our aim was to study normative ranges for FMD in subjects of different age groups. Methods: Detailed clinical assessment of cardiovascular (CV) risk factors (including evaluation of lipid profile and glycemic parameters) was performed in 227 subjects free of coronary heart disease and it’s equivalents. After the calculation of global CV risk for each patient EF was evaluated by FMD test in 115 low CV risk subjects aged 44.19±12.23 (26 to 83). Results: The mean FMD was 8.23±4.51% (0 to 20.9). In univariate analysis FMD was inversely correlated with resting vessel diameter (FMD=19.883.2xresting vessel diameter, r=-0.66, p=0.003) and high density lipoprotein (HDL) cholesterol concentration (r=-0.57, p=0.013). Multivariate analysis revealed that there are only two independent predictors of FMD - resting vessel diameter (r=-0.45, p<0.0001) and age (r=-0.55, p<0.0001). The multiple regression equation for this data was FMD=25.5-0.17xage-2.6xresting vessel diameter. Glycerol nitrate induced vessel dilatation was 20.53±9.27% (0.91 to 53.60). It was inversely correlated with the age (r=-0.39; p<0.0001), resting vessel diameter (r=-0.54, p<0.001) and FMD (r=0.51, p<0.0001). However, the ratio of FMD and glycerol nitrate induced dilatation was not significantly correlated with FMD (r=0.26, p=0.05). Conclusion: Our study demonstrates that FMD in low cardiovascular risk patients is inversely correlated with the age as well as brachial artery diameter. According to the model established different normal ranges for FMD could be calculated for each age group. 1P-0186
Shear flow attenuates interleukin-6-induced STAT3 activation in endothelial cells
L. Wang. Institute of Biomedical Sciences, Academia Sinica, Taiwan Cells exposed to interleukin-6 (IL-6) induce Stat3 activation that is mediated via the phosphorylation at tyrosine 705 (Tyr705), which is required for nuclear translocation, DNA binding and gene induction. Studies indicate that Stat3 activation is involved in endothelial growth. Endothelial cells (ECs) are constantly exposed to shear flow. ECs subjected to shear flow triggering growth arrest have been reported. In the present study, we have examined the effects of shear flow on bovine ECs exposed to IL-6. ECs at static condition, IL-6 treatment induced a transient phosphorylation at tyr705 with an IL-6 dose-dependent manner. ECs when subjected to shear flow, a shear
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
Monday September 29, 2003: Poster Session Endothelial cells
Phorbol 12-myristate-13-acetate, a protein kinase C (PKC) agonist, induced activation of ASK1 activity and up-regulation of PAI-1 expression, and an inhibitor of PKC suppressed both ASK1 activation and PAI-1 expression induced by high glucose. Importantly, overexpression of the constitutive active form of ASK1 by using an adenovirus vector induced up-regulation of PAI-1 expression in EC and infection of adenoviral construct including the dominant negative form of ASK1 gene suppressed high glucose-induced PAI-1 expression, suggesting that activation of ASK-1 is directly related with up-regulation of PAI-1. We conclude that activation of ASK1 may be involved in the underlying mechanism of endothelial PAI-1 expression induced by hyperglycemia in ECs. 1P-0181
Changes in gene expression of endothelial cells stimulated by lysophosphatidylcholine
A. Sheikh, H. Ochi, J. Masuda. Department of Laboratory Medicine, Shimane Medical University, Japan Objective: Lysophosphatidylcholine (lyso-PC), a phospholipid enriched in oxidized LDL, has been implicated to activate endothelial cells and modulate the development of atherosclerosis. In this study, we searched lyso-PC-regulated genes using cDNA macroarray to investigate roles of lyso-PC-activated endothelial cells in atherogenesis. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured. Macroarrays of 540 cDNA were used to compare gene expression patterns between HUVEC stimulated by lyso-PC for 8 hours and HUVEC treated with medium alone. Some of the highly regulated genes determined by macroarray were further verified by real-time PCR assays. This approach demonstrated that gene expression of at least 5 molecules, including tissue plasminogen activator (tPA), cyclooxygenase-1, integrin-α 6 and small G proteins Rac-2 and Rab-11, was up-regulated. Real-time PCR assay also showed that at least tPA was dose-dependently up-regulated by lyso-PC. Conclusion: We newly identified some molecules of which gene expression was modulated by lyso-PC in endothelial cells. Further analysis of these molecules may provide new insights on roles of lyso-PC in atherogenesis. 1P-0182
The effect of integrin linked kinase (ILK) overexpression in anchorage-dependent endothelial cells
Y. Seock-Won, K. Hyo-soo, P. Young-Bae. Cardiovascular Laboratory, Clinical Research Center, Seoul National University Hospital, Republic of Korea Objective: Cell adhesion to the extracellular matrix (ECM) is an essential process that controls proliferation, migration and survival of anchoragedependent cells such as endothelial cell (EC). Integrin-linked kinase (ILK) has been reported in epithelial cells to internalize cell survival signals. The implication of ILK, however, has not been investigated in EC. In this study, we generated a new adenoviral construct for wild type ILK and evaluated the role of ILK in anchorage-dependent and independent proliferation of EC. Methods and Results: (1) Construction of Adv-ILK: Human wild type ILK plasmid was subcloned into a shuttle vector (pADTrack-CMV). The recombinant shuttle vector was co-transfected with adenoviral genome (pAdEasy-1) into E.coli(BJ5183) where homologous recombination occurred. The recombinant Adeno-ILK was transfected to 293cell to get the viral particles which were purified by CsCl ultracentrifugation and dialysis. (2) Confirmation of ILK expressioin: When HUVECs were deprived of anchorage by culturing them on a dish coated with polyHEMA (adhesion inhibitor), the protein expression of ILK significantly decreased on western blot analysis. (3) Effect of ILK overexpression: In the attached condition, ILK overexpression did not affect proliferatinve activity of EC. However, in the detached condition, ILK overexpression significantly increased serum stimulated proliferation of EC. Conclusion: ILK overexpression provided anchorage-dependent EC with anchorage-independence, which suggests that ILK may be a good candidate gene to potentiate angiogenesis by protecting EC from stress when ECs are inevitably, although temporily, detached from ECM during angiogenesis.
1P-0183
Global analysis of RNA expression profile in human vascular cells and hepatocytes treated with HMG-CoA reductase inhibitors
S. Morikawa 1 , T. Kohro 1 , Y. Saito 2 , T. Hamakubo 1 , T. Kodama 1 . 1 Research Center for Advanced Science and Technology, the University of Tokyo; 2 Chiba University, Japan In addition to their effect on cholesterol metabolism, the 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors (statins) have an effect on the expression levels of many genes. In order to elucidate the range of this effect as comprehensively as possible, by means of DNA microarrays we investigated the changes in gene expression profiles brought about by atorvastatin or pitavastatin in cultured human umbilical vein endothelial cells (HUVEC), cultured human coronary artery smooth muscle cells (HCASMC) and cultured human hepatocarcinoma Hep G2 cells. Among the 6146 genes in the array, the statins affected the expression levels of genes involved in coagulation, vascular constriction and cell growth in a cell-type specific manner. In HUVEC, they induced integrin β4 and thrombomodulin profoundly, and profoundly suppressed pentraxin 3 (PTX3) both at 8 and 24 hour. In HCASMC, the statins induced thrombomodulin and urokinase inhibitor, and potently suppressed the cysteine-rich angiogenic inducer 61 (Cyr61) and cyclin B. In contrast, the expression level of these genes did not significantly change in Hep G2 cells. Many genes related to the cell cycle and/or growth were also regulated in HUVEC and HCASMC by the statins. These results indicate that many aspects of pleiotropic effect can be mediated by the transcriptional control by statins. Genes newly identified by this study may be related to beneficial effect of statin therapy. 1P-0185
Normal ranges for flow-mediated dilatation of the brachial artery in different age groups
L. Ryliskyte, A. Laucevicius, Z. Petrulioniene, J. Badariene, S. Janaviciene. Vilnius University Hospital Santariskiu Klnikos, Heart Clinic, Lithuania Ojectives: High-frequency ultrasonographic imaging of the flow-mediated dilatation (FMD) in the brachial artery is the main non-invasive technique used for clinical evaluation of endothelial function (EF). Since interpretive limitations of this technique exist our aim was to study normative ranges for FMD in subjects of different age groups. Methods: Detailed clinical assessment of cardiovascular (CV) risk factors (including evaluation of lipid profile and glycemic parameters) was performed in 227 subjects free of coronary heart disease and it’s equivalents. After the calculation of global CV risk for each patient EF was evaluated by FMD test in 115 low CV risk subjects aged 44.19±12.23 (26 to 83). Results: The mean FMD was 8.23±4.51% (0 to 20.9). In univariate analysis FMD was inversely correlated with resting vessel diameter (FMD=19.883.2xresting vessel diameter, r=-0.66, p=0.003) and high density lipoprotein (HDL) cholesterol concentration (r=-0.57, p=0.013). Multivariate analysis revealed that there are only two independent predictors of FMD - resting vessel diameter (r=-0.45, p<0.0001) and age (r=-0.55, p<0.0001). The multiple regression equation for this data was FMD=25.5-0.17xage-2.6xresting vessel diameter. Glycerol nitrate induced vessel dilatation was 20.53±9.27% (0.91 to 53.60). It was inversely correlated with the age (r=-0.39; p<0.0001), resting vessel diameter (r=-0.54, p<0.001) and FMD (r=0.51, p<0.0001). However, the ratio of FMD and glycerol nitrate induced dilatation was not significantly correlated with FMD (r=0.26, p=0.05). Conclusion: Our study demonstrates that FMD in low cardiovascular risk patients is inversely correlated with the age as well as brachial artery diameter. According to the model established different normal ranges for FMD could be calculated for each age group. 1P-0186
Shear flow attenuates interleukin-6-induced STAT3 activation in endothelial cells
L. Wang. Institute of Biomedical Sciences, Academia Sinica, Taiwan Cells exposed to interleukin-6 (IL-6) induce Stat3 activation that is mediated via the phosphorylation at tyrosine 705 (Tyr705), which is required for nuclear translocation, DNA binding and gene induction. Studies indicate that Stat3 activation is involved in endothelial growth. Endothelial cells (ECs) are constantly exposed to shear flow. ECs subjected to shear flow triggering growth arrest have been reported. In the present study, we have examined the effects of shear flow on bovine ECs exposed to IL-6. ECs at static condition, IL-6 treatment induced a transient phosphorylation at tyr705 with an IL-6 dose-dependent manner. ECs when subjected to shear flow, a shear
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan