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）：血流对血管内皮施加的摩擦力在动脉粥样硬化以及动脉瘤生长和病理性血管重塑中起着重要作用。在所有这些反应中，内皮细胞在响应低流量和振荡流量时表达相似的易发生动脉粥样硬化的促炎症基因，这表明基本的分子相似性。尽管已经确定了一些直接剪切响应信号传导途径，但协调血管反应的下游信号传导仍不清楚。由于血管疾病是美国发病率和死亡率的最大原因，它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改变的反应的介质至关重要。内皮细胞外基质蛋白决定了对血流改变的炎症反应。分泌型纤连蛋白的一种特定形式是由组成型转录物的选择性剪接产生的，通常在静止的成人脉管系统中不存在，在动脉粥样硬化病变和其他炎症性血管病变（例如动脉瘤和新内膜形成）中高度表达。阻止这种亚型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及其生物学后果仍不清楚。我推测，内皮细胞中纤连蛋白的选择性剪接会随着容易发生动脉粥样硬化的血流而增加，并且选择性外显子的包含会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）通过对实验暴露于增加或减少的血流的颈动脉进行分子分析，检查纤连蛋白对体内动脉粥样硬化流的选择性剪接。然后，我将（2）测试纤连蛋白剪接缺陷对小鼠颈动脉中单核细胞募集/分化的影响产生特定的（EIIIA 和 EIIIB）纤连蛋白亚型。最后，我将 (3) 通过使用条形码 shRNA 筛选体外表达荧光 FN 剪接报告基因的内皮细胞，鉴定内皮细胞对血流反应中选择性剪接的调节因子。公共健康相关性：通过选择性剪接，基因可以编码多种甚至相反的蛋白质功能。因此，剪接体复合物对选择性剪接的调节能够实现蛋白质功能的全局调节。我们建议测试纤连蛋白选择性剪接的调节和功能，纤连蛋白是炎症性血管疾病过程中血管内皮细胞外基质的关键成分，例如动脉粥样硬化、动脉瘤和再狭窄。