Mechanisms of Basement Membrane Regulation During Angiogenesis

项目摘要 在血管生成过程中，内皮细胞分泌多种蛋白质，这些蛋白质构成平面蛋白质网络， 包封血管，统称为基底膜。在这一关键过程中的失误， 表现为危及生命的可遗传形式的脑内出血以及促成中风， 动脉瘤这种与疾病的联系突出了血管基底膜作为一种免疫调节剂的重要性。 不可或缺的大分子支架，以承受血液循环的持续机械应变。到 迄今为止，生物医学界对基底膜蛋白是如何被 在血管生成期间由血管内皮调节。我们的初步调查显示 Rab10内体介质直接调节多个基底膜的分泌和储存的情况 血管形成过程中的膜蛋白。我们的研究结果表明Rab10可能是一个前所未有的 公认的共同管道基膜蛋白，很容易从极化分泌切换到 降解，取决于刺激。我们的假设是内皮细胞 主要使用Rab10运输途径来调节基底膜沉积， 直接维持血管稳定性。我们将从三个方面来检验这一一般假设。一是 机械地探索Rab10如何协调多个关键基底膜的分泌， 内皮细胞中的蛋白质。在这个体外的目的，使用生化，二维和三维静态 和实时成像分析，我们将深入探讨Rab10及其相关效应物如何影响内皮细胞 基底膜沉积其次，我们将使用转基因斑马鱼来评估Rab10在以下方面的作用： 血管生成和血管基底膜调节，勒韦林的先天活体成像 能力的第三，我们将采用金标准转基因小鼠模型来条件性消融Rab10， 探讨Rab10如何影响血管形态发生以及成人血管基底 膜完整性我们相信，对这种血管基底膜生物学的理解， 这是确定疾病状态如何破坏这一过程并表现为 危及生命的血管疾病，以及我们对血管生成的理解的重大进展， 整个过程。

PROJECT SUMMARY During angiogenesis, endothelial cells secrete a variety of proteins that compose a planar protein network that encapsulate blood vessels, collectively termed basement membrane. Missteps in this crucial process can manifest as a life-threatening heritable form of intracerebral hemorrhage as well as contributing to stroke and aneurysm. This association with disease highlights the importance of the vascular basement membrane as an indispensable macromolecular scaffold to withstand the continuous mechanical strain of blood circulation. To date, the biomedical community knows extraordinarily little about how basement membrane proteins are regulated by the vascular endothelium during angiogenesis. Our preliminary investigations suggest a novel scenario in which a Rab10 endosome-mediator directly regulates secretion and storage of multiple basement membrane proteins during blood vessel formation. Our results indicate that Rab10 may be a never-before recognized common pipeline for basement membrane proteins, easily switching from polarized secretion to degradation, depending on the stimulus. Our general working hypothesis is that endothelial cells predominately use a Rab10 trafficking pathway to regulate basement membrane deposition, contributing directly to maintenance of vascular stability. We will test this general hypothesis in three aims. First, we will mechanistically explore how Rab10 orchestrates the secretion of multiple, critical basement membrane proteins in endothelial cells. In this in vitro aim, using biochemical, 2-dimensional and 3-dimensional static and live-imaging assays, we will thoroughly explore how Rab10 and its related effectors influence endothelial basement membrane deposition. Second, we will used transgenic zebrafish to evaluate Rab10's role in angiogenesis and vascular basement membrane regulation, levering zebrafish's innate live-imaging capabilities. Third, we will employ a gold-standard transgenic mouse model to conditionally ablate Rab10 in the vasculature to probe how Rab10 affects blood vessel morphogenesis as well as adult blood vessel basement membrane integrity. We believe an understanding of this vascular basement membrane biology would constitute an important first-step toward determining how disease states disrupt this process and manifest into life-threatening vascular disorders, as well as a significant advancement of our understanding of the angiogenic process as a whole.