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而容易出现低氧。在这项研究中，我们检验了低氧产生活性氧（ROS）并激活血管平滑肌细胞（VSMC）中的C-SRC酪氨酸激酶的假设。用氧化还原敏感的探针，二氢二硫二二苯甲酸和7'-二氢二氯氟乙酸酯二乙酸酯处理VSMC，发现缺氧增加了细胞内的超氧阴离子和过氧化氢（H_2O_2）。北印迹分析表明，缺氧诱导的PAI-1和VEGF基因表达受到C-SRC抑制剂PP1的显着抑制。蛋白质印迹分析表明，缺氧迅速引起C-SRC的酪氨酸磷酸化。与C-SRC过表达的细胞相比，在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。 PP1，二苯基二元（DPI）和N-乙酰基1-半胱氨酸可显着减弱缺氧诱导的HIF-1α蛋白表达。烤面包酮和一个……更多的蛋白霉素A，抑制线粒体电子传输链，通过缺氧废除了HIF-1α蛋白。使用H_2O_2处理VSMC，即使在常氧下也诱导了HIF-1α蛋白。这些结果表明，ROS和C-SRC在VSMC中缺氧诱导的HIF-1α蛋白以及PAI-1和VEGF基因表达中起关键作用。我们的数据还表明，线粒体充当缺氧期间介导C-SRC磷酸化的O_2传感器。此外，我们确定了Notch靶基因HERP1在调节血管SMC表型调节中的作用。本研究表征了HERP1在正常和解散的血管中的表达，并检验了HERP1抑制SRF/肌动蛋白依赖性SMC基因表达的假设。免疫组织化学表明，在大鼠主动脉和人类冠状动脉粥样硬化病变中，HERP1和心肌表达位于新内膜的SMC中。与媒体SMC相比，在培养的VSMC中，HERP1和心肌的表达显着升高。 HERP1的过表达急剧抑制了肌醇诱导的SMC标记基因表达。免疫沉淀测定表明HERP1与SRF物理相互作用。这就是我们包括HERP1通过通过HERP1和SRF之间的物理关联来干扰SRF结合在血管损伤和动脉粥样硬化过程中在血管损伤和动脉粥样硬化过程中的表型调节中发挥作用。较少的