权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular Mechanisms of Vasa Vasorum Angiogenesis and Dysregulation of Vascular Smooth Muscle Cell Differentiation

血管血管生成和血管平滑肌细胞分化失调的分子机制

基本信息

批准号：
16390216
负责人：
KURABAYASHI Masahiko
金额：
$ 9.09万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390216/
关键词：
Vascular smooth cell Hypoxia Atherosclerosis Mitochondria Artery Gene expression Signal transduction チロシンキナーゼ HIF-1α 活性酸素種血管新生 PAI-1

项目摘要

Thickened atherosclerotic plaques are prone to be hypoxic because of poor perfusion. In this study, we tested the hypothesis that hypoxia produces reactive oxygen species (ROS) and activates c-Src tyrosine kinase in vascular smooth muscle cells (VSMC). Treatment of VSMCs with redox-sensitive probes, dihydroethidium and 2',7'-dihydrodichlorofluorescein diacetate, revealed that hypoxia increased intracellular superoxide anion and hydrogen peroxide (H_2O_2). Northern blot analysis showed that hypoxia-induced PAI-1 and VEGF genes expression was significantly inhibited by c-Src inhibitor PP1. Western blot analysis showed that hypoxia rapidly elicited tyrosine phosphorylation of c-Src. Induction of the PAI-1 and VEGF genes expression was significantly attenuated in c-Src-, Yes-, and Fyn-negative cells as compared with c-Src-overexpressing cells. Hypoxia-inducible HIF-1α protein expression was significantly attenuated by PP1, diphenyleneiodonium (DPI), and N-acetyl 1-cysteine. Rotenone and an … More timycin A, which inhibit the mitochondrial electron transport chain, abolished the HIF-1α induction by hypoxia. Treatment of VSMC with H_2O_2 induced HIF-1α protein even under normoxia. These results suggest that ROS and c-Src play a critical role in hypoxia-induced HIF-1α protein and PAI-1 and VEGF gene expression in VSMC. Our data also demonstrate that mitochondria act as the O_2 sensor mediating c-Src phosphorylation during hypoxia.In addition, we determined the role of Notch target gene HERP1 in the regulation of phenotypic modulation of vascular SMC. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression. Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC of the neointima after balloon injury of rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was markedly elevated in cultured VSMC compared with medial SMC. Overexpression of HERP1 dramatically inhibited the myocarin-induced SMC-marker gene expression. Immunoprecipitation assays showed that HERP1 physically interacts with SRF. Thus, we conclude that HERP1 may play a role in promoting the phenotypic modulation of VSMC during vascular injury and atherosclerotic process by interfering with SRF binding to CArG box through physical association between HERP1 and SRF. Less

增厚的动脉粥样硬化斑块由于灌注不良，容易缺氧。在这项研究中，我们测试了缺氧会产生活性氧 (ROS) 并激活血管平滑肌细胞 (VSMC) 中的 c-Src 酪氨酸激酶的假设。用氧化还原敏感探针、二氢乙锭和2',7'-二氢二氯荧光素二乙酸盐处理VSMC，发现缺氧会增加细胞内超氧阴离子和过氧化氢(H_2O_2)。 Northern blot分析显示缺氧诱导的PAI-1和VEGF基因表达被c-Src抑制剂PP1显着抑制。蛋白质印迹分析表明，缺氧迅速引发 c-Src 酪氨酸磷酸化。与 c-Src 过表达细胞相比，c-Src、Yes 和 Fyn 阴性细胞中 PAI-1 和 VEGF 基因表达的诱导显着减弱。 PP1、二亚苯基碘 (DPI) 和 N-乙酰基 1-半胱氨酸显着减弱缺氧诱导的 HIF-1α 蛋白表达。鱼藤酮和季霉素 A 可以抑制线粒体电子传递链，从而消除缺氧引起的 HIF-1α 诱导。用H_2O_2处理VSMC，即使在常氧条件下也能诱导HIF-1α蛋白的产生。这些结果表明ROS和c-Src在缺氧诱导的VSMC中HIF-1α蛋白以及PAI-1和VEGF基因表达中发挥着关键作用。我们的数据还表明，线粒体在缺氧过程中充当介导c-Src磷酸化的O_2传感器。此外，我们还确定了Notch靶基因HERP1在调节血管SMC表型调节中的作用。本研究描述了正常血管和患病血管中 HEP1 的表达，并检验了 HEP1 抑制 SRF/心肌素依赖性 SMC 基因表达的假设。免疫组织化学显示，HERP1 和心肌素表达定位于大鼠主动脉球囊损伤后和人冠状动脉粥样硬化病变中新内膜的 SMC。与内侧 SMC 相比，培养的 VSMC 中 HERP1 和心肌素的表达均显着升高。 HEP1 的过表达显着抑制了心肌素诱导的 SMC 标记基因表达。免疫沉淀分析表明，HERP1 与 SRF 存在物理相互作用。因此，我们得出结论，HERP1可能通过HERP1和SRF之间的物理关联干扰SRF与CArG盒的结合，从而在血管损伤和动脉粥样硬化过程中促进VSMC的表型调节中发挥作用。较少的