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的动脉粥样硬化性闭塞通常通过血管成形术/穿刺或静脉搭桥手术治疗。随着时间的推移，这些干预措施可能由于内膜增生而失效，内膜增生是血管壁的过度愈合反应，导致狭窄和闭塞。病变由血管平滑肌细胞（VSMC）组成，具有增殖和去分化表型。了解调节这种损伤反应的分子途径对新疗法的发展至关重要。存活素（SVV）是一种调节细胞凋亡和细胞增殖的新型蛋白。因此，我假设SVV在控制VSMC表型中起着至关重要的作用。我假设SVV的表达在激活的VSMC中以不受管制的方式组成性地升高。这个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。我的第一个目标是表征SVV基因靶向对VSMC细胞周期进展的影响。增殖试验将在SVV敲除的VSMC上进行，流式细胞术将用于定量细胞周期每个阶段的细胞比例。我假设SVV敲低会抑制VSMC的细胞生长，并会减弱对外源性生长因子（如血小板衍生生长因子（PDGF））的增殖反应。我的第二个目标是研究SVV基因敲低对VSMC中生长因子信号通路的影响。将SVV敲低后的VSMC暴露于激动剂中，并通过条件培养基的定量RT-PCR、Western blot和ELISA检测各种生长因子的表达。我假设SVV敲低会减少VSMC中的自分泌/旁分泌信号，特别是与内膜增生相关的PDGF通路。我的第三个目标是确定局部靶向SVV基因抑制在动脉损伤小鼠模型中的作用。靶向SVV的siRNA将被局部递送到动脉，然后通过免疫染色和形态计量学分析在不同的时间点检测其增殖和凋亡的变化。我推测，经过处理的血管会表现出VSMC增殖减少，细胞凋亡增加，从而导致体内新生内膜形成减少。

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.