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

增厚的动脉粥样硬化斑块由于血流灌注不良而容易缺氧。在这项研究中，我们验证了低氧产生活性氧(ROS)并激活血管平滑肌细胞(VSMC)c-Src酪氨酸激酶的假说。用氧化还原敏感的探针二氢乙锭和2‘，7’-二氢二氯荧光素二乙酸酯处理VSMC，发现缺氧使细胞内超氧阴离子和过氧化氢(H_2O_2)增加。Northern印迹分析表明，c-Src抑制剂PP1可显著抑制缺氧诱导的PAI-1和VEGF基因表达。Western印迹分析表明，低氧可迅速诱导c-Src酪氨酸磷酸化。与c-Src过表达细胞相比，c-Src、Yes和Fyn阴性细胞对PAI-1和VEGF基因表达的诱导作用显著减弱。低氧诱导的缺氧诱导的HIF-1α蛋白表达可被二苯基碘离子、N-乙酰-1-半胱氨酸显著降低。鱼藤酮和…更多抑制线粒体电子传递链的TIMYCIN A可阻断缺氧诱导的HIF-1α。常氧条件下H_2O_2诱导血管内皮细胞HIF-1α蛋白表达。这些结果提示，ROS和c-Src在缺氧诱导的VSMC中HIF-1α蛋白和PAI-1、VEGFmRNA的表达中起重要作用。此外，我们还研究了Notch靶基因HERP1在血管SMC表型调控中的作用。本研究鉴定了HERP1在正常和病变血管中的表达，并验证了HERP1抑制SRF/myocardin依赖的SMC基因表达的假说。免疫组织化学结果显示，HERP1和myocardin在大鼠主动脉球囊损伤后的新生内膜和人冠状动脉粥样硬化病变的SMC中均有表达。与中膜SMC相比，培养的VSMC中HERP1和myocardin的表达均显著升高。HERP1过表达可显著抑制肌钙蛋白诱导的SMC标志物基因表达。免疫沉淀实验表明，HERP1与SRF发生物理相互作用。因此，我们认为HERP1可能通过HERP1和SRF之间的物理结合干扰SRF与Carg box的结合，从而在血管损伤和动脉粥样硬化过程中促进VSMC的表型改变。较少

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