Investigation of alternative splicing in response to low and disturbed flow

 项目简介(申请人提供)：我的长期目标是在一流学术机构领导一个研究小组，致力于我的兴趣：血管发育和疾病的基因调控，重点是内皮和血流动力学激活。我对血流对血管系统的影响很感兴趣，在过去的10年里，我一直专注于内皮功能的转录调控。我相信，体内遗传学和成像将是了解内皮、血流和招募的血细胞之间复杂相互作用的关键。反过来，这对于了解血管发育和疾病，如动脉瘤和动脉粥样硬化，是至关重要的，这些疾病是由血流动力学障碍驱动的。我致力于在这项工作中度过我的一生。这一奖项将提供关键的培训和发展时间，以确保我成功过渡到独立的教职员工职位。在理查德·海因斯的实验室里，在我的F32奖下，我研究了纤维连接蛋白转录本对动脉内皮细胞低和紊乱血流的调节，发现了一个令人惊讶的剪接开关，导致了两个广泛保守的替代外显子的包含。纤维连接蛋白是动脉瘤中含量最丰富的细胞外基质蛋白之一。利用遗传模型，我证明了这种剪接开关在低流量和扰动的情况下可以防止动脉血管壁的出血性剥离。尽管许多关键的内皮蛋白是选择性剪接的，并且选择性剪接程序正在成为其他发育过程中的关键调控机制，但对于选择性剪接在动脉内皮细胞中的调节和功能几乎一无所知。为了解决这一缺陷，我将(目标1)研究更详细的机制(S)，通过纤维连接蛋白的选择性剪接防止夹层出血，(目的2)利用我们收集的数据库，在扰乱的血流下巨噬细胞募集诱导的内皮细胞选择性剪接事件，以确定关键剪接因子及其在血流驱动的炎症中的功能。理查德·海因斯(细胞外基质和纤维连接蛋白生物学专家)和克里斯·伯奇(基因组-选择性剪接的规模分析专家)将在K99奖项阶段指导我的工作。麻省理工学院剪接领域的人才聚集将是一个额外的好处；菲尔·夏普30年前在这里首次发现了剪接，它仍然是他的实验室和附近其他实验室的重点。这些目标的完成将为血流--激活的动脉内皮细胞的遗传调控增加一个新的维度，并通过对基础生物学的更深入了解，使诊断和治疗血流驱动的血管疾病的发展成为可能。

 DESCRIPTION (provided by applicant): Project Summary My long term goal is to lead a research group in a top tier academic institution in the pursuit of my interest: the genetic regulation of vascular development and disease, with a focus on the endothelium and hemodynamic activation. I am fascinated by the effects of blood flow on the vasculature and have focused on the transcriptional regulation of endothelial function for the past 10 years. I believe that in vivo genetics and imaging will be the key to understanding the complex interaction between the endothelium, blood flow, and recruited blood cells. This, in turn, is critical to understanding vascular development and diseases, such as aneurysms and atherosclerosis, which are driven by hemodynamic disturbance. I am committed to spending my life in this work. This award will provide critical training and development time to ensure my successful transition to an independent faculty position. In Richard Hynes' lab, under my F32 award, I examined the regulation of the Fibronectin transcript in response to low and disturbed flow in the arterial endothelium and uncovered a surprising splicing switch that resulted in the inclusion of two widely conserved alternative exons. Fibronectin is one of the most abundant extracellular matrix proteins in aneurysms. Using genetic models I showed that this splicing switch protects against hemorrhagic dissection of the arterial vessel wall under low and disturbed flow. Although a number of critical endothelial proteins are alternatively spliced, and alternative splicing programs are emerging as pivotal control mechanisms in other developmental processes, almost nothing is known about the regulation and function of alternative splicing in the arterial endothelium. To address this deficit, I will (Aim 1) examine i greater detail the mechanism(s) through which alternative splicing of Fibronectin protects against dissecting hemorrhage, and (Aim 2) exploit a database we have amassed, of alternative splicing events induced in the endothelium by macrophage recruitment under disturbed flow, to identify critical splicing factors and their functions in flow-­--driven inflammation. Richard Hynes (expert in extracellular matrix and Fibronectin biology) and Chris Burge (expert in genome-­--scale analysis of alternative splicing) will mentor my work during the K99 phase of the award. The congregation of talent in the splicing field at MIT will be an added benefit; Phil Sharp first discovered splicing here 30 years ago and it remains a focus of his lab and others nearby. The completion of these aims will add a new dimension to the genetic regulation of the flow-­--activated arterial endothelium, and enable the development of diagnostics and treatments for flow-­--driven vascular diseases through a deeper understanding of the basic biology.