Genetic Modulation of Vascular Smooth Muscle Phenotype and Intimal Hyperplasia

描述（由申请人提供）：冠状动脉和外周循环中的动脉粥样硬化闭塞通常通过血管成形术/支架术或静脉旁路移植术治疗。这些干预可能会因内膜增生而随着时间的推移而失败，内膜增生是血管壁中导致狭窄和闭塞的过度愈合反应。病变由具有增殖和去分化表型的血管平滑肌细胞（VSMC）组成。了解调节这种损伤反应的分子途径对于开发新的治疗方法至关重要。生存素（Survivin，SVV）是一种新的调控细胞凋亡和增殖的蛋白质。因此，我推测SVV在控制VSMC表型中起着至关重要的作用。我推测SVV在活化的VSMC中的表达以一种失调的方式组成性升高。该转化研究项目的主要目标是验证SVV作为内膜增生的治疗靶点。 我的第一个目的是描述SVV基因靶向对VSMC细胞周期进程的影响。将对SVV敲减转染的VSMC进行增殖试验，并使用流式细胞术定量细胞周期各期的细胞分数。我推测SVV基因敲除将抑制VSMC的细胞增殖，并减弱对血小板衍生生长因子（PDGF）等外源性生长因子的增殖反应。 我的第二个目的是研究SVV基因敲低对VSMC生长因子信号通路的影响。将SVV敲低的VSMC暴露于激动剂，并通过条件培养基的定量RT-PCR、Western印迹和ELISA检测各种生长因子的表达。我假设SVV基因敲低将减少VSMC中的自分泌/旁分泌信号，特别是PDGF通路，这与内膜增生相关。 我的第三个目标是确定局部靶向SVV基因抑制在小鼠动脉损伤模型中的作用。靶向SVV的siRNA将被局部递送到动脉，然后在不同的时间点通过免疫染色和形态测定分析来检查增殖和凋亡的变化。我假设经处理的血管将表现出VSMC增殖减少，凋亡增加，并导致体内新生内膜形成减少。 公共卫生相关性：冠状动脉和外周循环中的动脉粥样硬化闭塞目前用血管成形术、支架植入术或静脉旁路移植术治疗。然而，这些干预随着时间的推移而失败，因为血管壁中的内膜增生导致再闭塞。了解存活素（一种调节细胞凋亡和增殖的新型蛋白质）在内膜增生发展中的作用，对于开发治疗这种重大公共卫生疾病的新疗法至关重要。

DESCRIPTION (provided by applicant): Atherosclerotic occlusions in coronary and peripheral circulations are commonly treated with angioplasty/stinting or vein bypass graft surgery. These interventions can fail over time due to intimal hyperplasia, an exaggerated healing response in the vessel wall that leads to narrowing and occlusion. The lesion consists of vascular smooth muscle cells (VSMC) with a proliferative and de-differentiated phenotype. Understanding the molecular pathways which regulate this injury response is critical for the development of new therapeutics. Survivin (SVV) is a novel protein that regulates both apoptosis and proliferation. Therefore I hypothesize that SVV has a crucial role in controlling VSMC phenotype. I hypothesize that SVV expression in activated VSMC is constitutively elevated in a deregulated fashion. The primary goal of this translational research project is to validate SVV as a therapeutic target in intimal hyperplasia. My first aim is to characterize the effects of SVV gene targeting on cell cycle progression in VSMC. Proliferation assays will be performed on VSMC transuded with SVV knockdown and flow cytometry will be used to quantitative the fraction of cells in each phase of the cell cycle. I hypothesize that SVV knockdown will be cytostatic in VSMC and will blunt the proliferative response to exogenous growth factors such as platelet-derived growth factor (PDGF). My second aim is to examine the effects of SVV gene knockdown on growth factor signaling pathways in VSMC. VSMC transuded with SVV knockdown will be exposed to agonists and the expression of various growth factors will be examined by quantitative RT-PCR, Western blot, and ELISA of conditioned media. I hypothesize that SVV knockdown will reduce autocrine/paracrine signaling in VSMC, specifically the PDGF pathway, which is of established relevance to intimal hyperplasia. My third aim is to determine the effects of locally targeted SVV gene inhibition in a mouse model of arterial injury. siRNA targeting SVV will be delivered to the artery locally, which will then be examined at various time points for changes in proliferation and apoptosis by immunostaining and morphometric analysis. I hypothesize that treated vessels will exhibit reduced VSMC proliferation, increased apoptosis, and a resulting reduction in neointimal formation in-vivo. PUBLIC HEALTH RELEVANCE: Atherosclerotic occlusions in the coronary and peripheral circulation are currently treated with angioplasty, stenting, or vein bypass graft surgery. However, these interventions fail over time due to intimal hyperplasia in the vessel wall that leads to reocclusion. Understanding the role of survivin, a novel protein that regulates apoptosis and proliferation, in the development of intimal hyperplasia is critical for the development of new therapeutics to treat this disease of major public health magnitude.