Alternative splicing in the vascular response to pathological shear stress

血管对病理剪切应力反应中的选择性剪接

基本信息

批准号：
8312032
负责人：
Patrick Andries Murphy
金额：
$ 4.92万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-03 至 2015-04-02
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8312032
关键词：
Adult Affect Alternative Splicing Aneurysm Antibodies Apolipoprotein E Area Arterial Fatty Streak Atherosclerosis Bar Codes Biological Blood Vessels Blood flow Carotid Arteries Cells Collagen Complex Deposition Endothelial Cells Endothelium Event Exons Extracellular Matrix Extracellular Matrix Proteins Fibronectins Gene Mutation Genes Genetic Transcription Growth In Situ In Vitro Inflammation Inflammatory Inflammatory Response Knockout Mice Leukocytes Ligation Link Liquid substance Measures Mediator of activation protein Modeling Molecular Molecular Analysis Morbidity - disease rate Mus Pathology Pattern Phenotype Play Process Protein Isoforms Proteins RNA Splicing Regulation Reporter Role Signal Pathway Signal Transduction Spliced Genes Spliceosomes Testing Transcript United States Variant Vascular Cell Adhesion Molecule-1 Vascular Diseases Vascular Endothelium Vascular remodeling biological adaptation to stress in vivo leukocyte activation macrophage monocyte mortality neointima formation prevent protein expression protein function receptor response restenosis shear stress small hairpin RNA

项目摘要

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. PUBLIC HEALTH RELEVANCE: Through alternative splicing, genes may encode diverse and even opposing protein functions. Thus, the regulation of alternative splicing by spliceosome complexes enables global regulation of protein function. We propose to test the regulation and function of alternative splicing of fibronectin, a critical component of extracellular matrix underlying the vascular endothelium in inflammatory vascular disease processes, such as atherosclerosis, aneurysm and restenosis.

描述（由申请人提供）：血流在血管内皮上施加的摩擦力在动脉粥样硬化以及动脉瘤生长和病理血管重塑中起着至关重要的作用。内皮在所有这些响应中对低和振荡流的促炎基因表达了类似的促炎基因，这表明基本的分子相似性。尽管已经确定了一些直接剪切的响应信号通路，但协调血管反应的下游信号尚不清楚。由于血管疾病是美国发病率和死亡率的最大原因，因此主要是由动脉粥样硬化斑块的形成引起的，因此了解血液流动改变的血管反应的介体至关重要。内皮的基础的细胞外基质蛋白决定对流动改变的炎症反应。由本构型转录本的替代剪接产生的分泌纤连蛋白蛋白的特定形式，通常在静态成人血管中不存在，在动脉粥样硬化病变和其他炎症性血管病理中高度表达，例如动脉瘤和新的炎症。防止这种同工型形成的遗传突变会损害小鼠的动脉粥样硬化，但是如何调节这种剪接以及IT生物学后果尚不清楚。我假设纤连蛋白在内皮中的替代剪接会因动脉op子血流量而增加，并且包含替代外显子通过促进炎症细胞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）通过对颈动脉的分子分析实验暴露于血液流量增加或减少的颈动脉，检查纤连蛋白在体内的替代剪接。然后，我将（2）测试不足的纤连蛋白剪接对小鼠颈动脉中单核细胞募集/分化的影响产生特定（EIIIA和EIIIB）纤连蛋白同工型。最后，我将（3）通过使用条形码shRNA在体外表达荧光FN剪接报告基因的筛选内皮细胞，以确定内皮对流的替代剪接的调节。公共卫生相关性：通过替代剪接，基因可以编码多种甚至相反的蛋白质功能。因此，通过剪接体复合物调节替代剪接可以使蛋白质功能的全球调节。我们建议测试纤连蛋白的替代剪接的调节和功能，纤连蛋白的替代剪接是炎性血管疾病过程中血管内皮基础细胞外基质的关键成分，例如动脉粥样硬化，动脉粥样硬化和再肾上腺炎。