Alternative splicing in the vascular response to pathological shear stress

描述(由申请人提供)：血流对血管内皮细胞施加的摩擦力在动脉粥样硬化、动脉瘤生长和病理性血管重塑中发挥重要作用。在所有这些反应中，内皮细胞表达相似的动脉粥样硬化和促炎基因，以响应低和振荡的血流，这表明了基本的分子相似性。虽然已经确定了一些即刻剪切反应信号通路，但协调血管反应的下游信号仍不清楚。由于血管疾病是美国发病率和死亡率的最大原因，主要是由动脉粥样硬化斑块的形成引起的，因此了解血管对血流变化的反应的介质是至关重要的。内皮下的细胞外基质蛋白决定了对血流改变的炎症反应。一种特殊形式的分泌型纤维连接蛋白，由构成转录本的选择性剪接产生，通常在静止的成人血管中缺失，在动脉粥样硬化病变和其他炎症性血管病变中高度表达，如动脉瘤和新生内膜形成。阻止这种异构体形成的基因突变会损害小鼠的动脉粥样硬化，但这种剪接是如何调节的，其生物学后果尚不清楚。我假设内皮中纤维连接蛋白的选择性剪接增加是对动脉粥样硬化血流的反应，并且选择性外显子的包含通过促进炎症细胞的募集和激活来促进动脉粥样硬化的进展。为了测试这一点，我将(1)通过对实验中暴露于血流增加或减少的颈动脉进行分子分析，检测体内纤维连接蛋白对动脉粥样硬化血流的反应。然后，我将(2)测试缺陷纤维连接蛋白剪接对小鼠颈动脉单核细胞募集/分化的影响。 产生特异的(EIIIA和EIIIB)纤维连接蛋白亚型。最后，我将(3)通过使用条形码shRNA在体外筛选表达荧光FN剪接报告的内皮细胞来确定内皮细胞对流的反应中选择性剪接的调节。

DESCRIPTION (provided by applicant): Frictional forces exerted by blood flow on the vascular endothelium play an essential role in atherosclerosis, as well as aneurysm growth and pathological vascular remodeling. The endothelium expresses a similar profile of atheroprone, pro-inflammatory genes in response to low and oscillatory flow in all of these responses, suggesting fundamental molecular similarities. Although some of the immediate shear responsive signaling pathways have been identified, the downstream signaling which coordinates the vascular response remains unclear. Since vascular disease, the greatest cause of morbidity and mortality in the United States, is primarily caused by the formation of atherosclerotic plaques, understanding the mediators of the vascular response to altered blood flow is of critical importance. The extracellular matrix proteins underlying the endothelium dictat the inflammatory response to altered flow. A specific form of the secreted fibronectin protein, generated by alternative splicing of the constitutive transcript and normally absent in quiescent adult vasculature, is highly expressed in atherosclerotic lesions and other inflammatory vascular pathologies, such as aneurysm and neointima formation. Genetic mutations which prevent the formation of this isoform impair atherosclerosis in mice, but how this splicing is regulated and it biological consequences remain unclear. I hypothesize that alternative splicing of fibronectin in the endothelium is increased in response to atheroprone blood flow, and that the inclusion of alternative exons promotes atherosclerosis progression by promoting inflammatory cell recruitment and activation. To test this I will (1) Examine alternative splicing of fibronectin in response to atheroprone flow in vivo by molecular analysis of carotid arteries experimentally exposed to increased or decreased blood flow. I will then (2) Test the effect of deficient fibronectin splicing on monocyte recruitment/differentiation in the carotid arteries of mice unable to produce specific (EIIIA and EIIIB) fibronectin isoforms. Finally, I will (3) Identify the regulaors of alternative splicing in the endothelial response to flow by using barcoded shRNA to screen endothelial cells expressing a fluorescent FN splicing reporter in vitro.