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Complement C3 is involved in adventitial fibroblast phenotypic differentiation induced by angiotensin-II and TGF-beta 1
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Abstracts
function or insufficiency which means myocardium apoptosis. ACEI can improve or reverse myocardium apoptosis. doi:10.1016/j.ijcard.2009.09.247 EX000682 Complement C3 is involved in adventitial fibroblast phenotypic differentiation induced by angiotensin-II and TGF-beta 1 S.J. GUO, P.J. GAO, L.Y. WU, D.L. ZHU Shanghai Institute of Hypertension, China Objectives: The differentiation of vascular adventitial fibroblasts (AFs) to myofibroblasts (MFs) is a key step in vascular remodeling. Our previous study demonstrated that TGF-β1 and angiotension II (Ang II) were able to induce this differentiation in vitro. The aim of the study was to identify the molecules which might be responsible for the cell phenotypic change and investigated the role of complement C3, one of the related molecules, in differentiation of MFs. Design and methods: Cultured rat AFs were treated with TGF-beta 1 (10 ng/ml) and Ang II (10− 7 M). TGF-beta 1induced gene expression profiling was studied using Affimetrix oligonucleotide microarrays. Expression profile of complement C3 was verified by real-time RT-PCR. Then, the role of complement C3 in differentiation of AFs to MFs induced by Ang II was investigated using Western-blot, MTT array and Thymidine-Incorporation Assay. Results: Microarray analysis identified 2121 genes with a 2-fold change or above post-TGF-β1 stimulation. Among 1231 genes with known function, the gene expression profile of secreted phosphoprotein 1(APP1) and Rho-associated coiled-coil forming kinase 2 (ROCK2) was same as α-smooth muscleactin, a MF differentiation mark, and genes of potassium voltage gated channel, Shal-related family and member 2(KCND2) were up-regulated. Furthermore, endothelin 1(EDN1), complement C3, NADPH oxidase 4 (NOX4) and NAD(P)H dehydrogenase, quinone 1 (Nqo1) were also found to be involved in MF differentiation. These results were confirmed by realtime quantitative RT-PCR. For the very first time, complement C3 was found to express in vascular adventitial fibroblasts from SHR, WKY and SD rats and increased by angiotension II. Inhibition of complement C3 decreased the expression of α-smooth muscle-actin and reduced the proliferation of AFs from SD rats and WKY rats post treatment of Ang II. Exogenous C3 stimulated the growth of adventitial fibroblasts. Conclusions: Complement C3 contributes to adventitial fibroblast phenotypic differentiation and may be a target of vascular remodeling therapy. doi:10.1016/j.ijcard.2009.09.248 EX000707 Intervention study of rat cardiomyocytes β1-adrenergic receptor gene methylation modification H. YUANa,b, X. DUa,b, Z.J. HUANGb, X.W. XINGb, Q.X. JIANGa, Y. LIa, G.P. YANGa,b a The department of cardiology, The Third Xiang-ya Hospital of Central South University, China b Hypertension Research Center of Hunan Province, China Objections: Intervention in methylation modification of β1-AR gene of rat myocardial cells to explore the influence of DNA methylation on β1-AR gene expression. Methods: H9c2 rat myocardial cells were routinely cultured and randomly divided into two groups: 5-aza-2′-deoxycytidine (5-aza-dC) groups in which cells were treated by different concentration of 5-aza-dC-0.5 μmol/L, 2.0 μmol/L, 5.0 μmol/L and 10.0 μmol/L and control group in which cells were treated by DMSO. All the cells were treated for 72 h. AO/EB staining was applied to observe the cell morphology and apoptosis. Genomic DNA was isolated for bisulfite genomic sequencing and methylation-sensitive PCR (MSP) analysis to explore the methylation
status of β1-AR gene promoter. Total RNA was extracted for fluorescence-quantitative RT-PCR analysis to determine the expression of β1-AR gene mRNA. Results: Methylation online analysis shows that the β1-AR gene promoter of H9c2 miocardial cells was methylated. The methylated site of the β1-AR gene promoter region was verified by sodium sulfite. Compared to the other group, the methylation status of β1-AR gene promoter of H9c2 cells treated by 5.0 μmol/L 5-aza-dC was significantly down-regulated. While, the expression of β1-AR gene mRNA was up-regulated in all groups, which showed the significantly statistical difference (P < 0.05), and 5.0 μmol/L group β1-AR gene mRNA expression levels of the highest. Conclusions: β1-AR gene promoter had many methylated positions. The down-regulated methylation of the promoter would result in the up-regulated expression of β1-AR gene mRNA. So, it was possible that DNA methylation regulated the expression of β1-AR gene mRNA, this would be the epigenetic molecular mechanism of different antihypertension efficacy of metoprolol in β1-AR gene-directed therapy. doi:10.1016/j.ijcard.2009.09.249 EX000723 High salt diet alters NF-κB and CREB DNA binding activities in the heart, kidney and hypothalamus of spontaneously hypertensive rats Q.H. SHANGa,b, H.W. WANGa, A.F.R. STEWARTa, F.H.H. LEENENa Hypertension Unit, University of Ottawa Heart Institute, Ottawa, Ontario, Canada b Department of Cardiology, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou, China
a
Objectives: In Dahl salt-sensitive or spontaneously hypertensive rats (SHR) on high-salt diet, activation of nuclear factor-kappa B (NF-κB) contributes to cardiac hypertrophy and renal damage, whereas activation of cAMP responsive element binding protein (CREB) may offset cardiac effects of NF-κB. The brain RAAS mediates the salt-induced hypertension, possibly by NF-κB, in the paraventricular nucleus (PVN). We assessed effects of high-salt diet on the DNA-binding activities of NF-κB and CREB in heart, kidney and PVN of WKY rats and SHR. Design and methods: 4-5 week-old WKY rats and SHR were placed on high- (8%) or regular- (0.6%) salt diets for 2 or 4 weeks (n=6). Electrophoretic mobility shift assays were employed to determine NF-κB and CREB DNA-binding activities. The protein-DNA-binding specificity was confirmed by supershift assays using phosphorylated CREB and NF-κB p65 antibodies. Results: In WKY rats, high salt increased NF-κB DNA-binding activity by ~30% in PVN. High salt had no effect on CREB DNA-binding. In SHR on regular salt diet, NF-κB DNA-binding activity was higher in the left ventricle (LV) and PVN than in WKY while CREB DNA-binding activity was higher in all regions. High salt increased NF-κB DNA-binding activity by 25-30% in LV and kidney, but decreased CREB DNA-binding activity by ~30%, 20% and 40% in LV, kidney and PVN, respectively. There were negative correlations between renal NFκB and CREB, kidney weight and renal CREB, whereas there were positive correlation between LV weight and LV NF-κB in SHR on a high salt diet. Conclusions: High salt causes an inverse relationship between NF-κB and CREB in LV, kidney and PVN which may contribute to salt-induced sympathetic hyperactivity, LV and renal hypertrophy in SHR. doi:10.1016/j.ijcard.2009.09.523 EX000726 Application of ultrasonic integrated backscatter technique in the evaluation of the regression of myocardial fibrosis in the spontaneously hypertensive rats after valsartan therapy XIAOJUN BI Tongji Hospital of Tongji Medical College, China
Abstracts
function or insufficiency which means myocardium apoptosis. ACEI can improve or reverse myocardium apoptosis. doi:10.1016/j.ijcard.2009.09.247 EX000682 Complement C3 is involved in adventitial fibroblast phenotypic differentiation induced by angiotensin-II and TGF-beta 1 S.J. GUO, P.J. GAO, L.Y. WU, D.L. ZHU Shanghai Institute of Hypertension, China Objectives: The differentiation of vascular adventitial fibroblasts (AFs) to myofibroblasts (MFs) is a key step in vascular remodeling. Our previous study demonstrated that TGF-β1 and angiotension II (Ang II) were able to induce this differentiation in vitro. The aim of the study was to identify the molecules which might be responsible for the cell phenotypic change and investigated the role of complement C3, one of the related molecules, in differentiation of MFs. Design and methods: Cultured rat AFs were treated with TGF-beta 1 (10 ng/ml) and Ang II (10− 7 M). TGF-beta 1induced gene expression profiling was studied using Affimetrix oligonucleotide microarrays. Expression profile of complement C3 was verified by real-time RT-PCR. Then, the role of complement C3 in differentiation of AFs to MFs induced by Ang II was investigated using Western-blot, MTT array and Thymidine-Incorporation Assay. Results: Microarray analysis identified 2121 genes with a 2-fold change or above post-TGF-β1 stimulation. Among 1231 genes with known function, the gene expression profile of secreted phosphoprotein 1(APP1) and Rho-associated coiled-coil forming kinase 2 (ROCK2) was same as α-smooth muscleactin, a MF differentiation mark, and genes of potassium voltage gated channel, Shal-related family and member 2(KCND2) were up-regulated. Furthermore, endothelin 1(EDN1), complement C3, NADPH oxidase 4 (NOX4) and NAD(P)H dehydrogenase, quinone 1 (Nqo1) were also found to be involved in MF differentiation. These results were confirmed by realtime quantitative RT-PCR. For the very first time, complement C3 was found to express in vascular adventitial fibroblasts from SHR, WKY and SD rats and increased by angiotension II. Inhibition of complement C3 decreased the expression of α-smooth muscle-actin and reduced the proliferation of AFs from SD rats and WKY rats post treatment of Ang II. Exogenous C3 stimulated the growth of adventitial fibroblasts. Conclusions: Complement C3 contributes to adventitial fibroblast phenotypic differentiation and may be a target of vascular remodeling therapy. doi:10.1016/j.ijcard.2009.09.248 EX000707 Intervention study of rat cardiomyocytes β1-adrenergic receptor gene methylation modification H. YUANa,b, X. DUa,b, Z.J. HUANGb, X.W. XINGb, Q.X. JIANGa, Y. LIa, G.P. YANGa,b a The department of cardiology, The Third Xiang-ya Hospital of Central South University, China b Hypertension Research Center of Hunan Province, China Objections: Intervention in methylation modification of β1-AR gene of rat myocardial cells to explore the influence of DNA methylation on β1-AR gene expression. Methods: H9c2 rat myocardial cells were routinely cultured and randomly divided into two groups: 5-aza-2′-deoxycytidine (5-aza-dC) groups in which cells were treated by different concentration of 5-aza-dC-0.5 μmol/L, 2.0 μmol/L, 5.0 μmol/L and 10.0 μmol/L and control group in which cells were treated by DMSO. All the cells were treated for 72 h. AO/EB staining was applied to observe the cell morphology and apoptosis. Genomic DNA was isolated for bisulfite genomic sequencing and methylation-sensitive PCR (MSP) analysis to explore the methylation
status of β1-AR gene promoter. Total RNA was extracted for fluorescence-quantitative RT-PCR analysis to determine the expression of β1-AR gene mRNA. Results: Methylation online analysis shows that the β1-AR gene promoter of H9c2 miocardial cells was methylated. The methylated site of the β1-AR gene promoter region was verified by sodium sulfite. Compared to the other group, the methylation status of β1-AR gene promoter of H9c2 cells treated by 5.0 μmol/L 5-aza-dC was significantly down-regulated. While, the expression of β1-AR gene mRNA was up-regulated in all groups, which showed the significantly statistical difference (P < 0.05), and 5.0 μmol/L group β1-AR gene mRNA expression levels of the highest. Conclusions: β1-AR gene promoter had many methylated positions. The down-regulated methylation of the promoter would result in the up-regulated expression of β1-AR gene mRNA. So, it was possible that DNA methylation regulated the expression of β1-AR gene mRNA, this would be the epigenetic molecular mechanism of different antihypertension efficacy of metoprolol in β1-AR gene-directed therapy. doi:10.1016/j.ijcard.2009.09.249 EX000723 High salt diet alters NF-κB and CREB DNA binding activities in the heart, kidney and hypothalamus of spontaneously hypertensive rats Q.H. SHANGa,b, H.W. WANGa, A.F.R. STEWARTa, F.H.H. LEENENa Hypertension Unit, University of Ottawa Heart Institute, Ottawa, Ontario, Canada b Department of Cardiology, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou, China
a
Objectives: In Dahl salt-sensitive or spontaneously hypertensive rats (SHR) on high-salt diet, activation of nuclear factor-kappa B (NF-κB) contributes to cardiac hypertrophy and renal damage, whereas activation of cAMP responsive element binding protein (CREB) may offset cardiac effects of NF-κB. The brain RAAS mediates the salt-induced hypertension, possibly by NF-κB, in the paraventricular nucleus (PVN). We assessed effects of high-salt diet on the DNA-binding activities of NF-κB and CREB in heart, kidney and PVN of WKY rats and SHR. Design and methods: 4-5 week-old WKY rats and SHR were placed on high- (8%) or regular- (0.6%) salt diets for 2 or 4 weeks (n=6). Electrophoretic mobility shift assays were employed to determine NF-κB and CREB DNA-binding activities. The protein-DNA-binding specificity was confirmed by supershift assays using phosphorylated CREB and NF-κB p65 antibodies. Results: In WKY rats, high salt increased NF-κB DNA-binding activity by ~30% in PVN. High salt had no effect on CREB DNA-binding. In SHR on regular salt diet, NF-κB DNA-binding activity was higher in the left ventricle (LV) and PVN than in WKY while CREB DNA-binding activity was higher in all regions. High salt increased NF-κB DNA-binding activity by 25-30% in LV and kidney, but decreased CREB DNA-binding activity by ~30%, 20% and 40% in LV, kidney and PVN, respectively. There were negative correlations between renal NFκB and CREB, kidney weight and renal CREB, whereas there were positive correlation between LV weight and LV NF-κB in SHR on a high salt diet. Conclusions: High salt causes an inverse relationship between NF-κB and CREB in LV, kidney and PVN which may contribute to salt-induced sympathetic hyperactivity, LV and renal hypertrophy in SHR. doi:10.1016/j.ijcard.2009.09.523 EX000726 Application of ultrasonic integrated backscatter technique in the evaluation of the regression of myocardial fibrosis in the spontaneously hypertensive rats after valsartan therapy XIAOJUN BI Tongji Hospital of Tongji Medical College, China