Genes regulating capillary morphogenesis and apoptosis

描述(由申请人提供)：在这份更新方案中，我们继续我们的工作，展示Rho GTP酶，CDC42，在血管形成过程中内皮细胞(EC)管腔形成的早期血管组装的分子控制中的关键作用。我们的总体假设是，CDC42是EC管腔形成和管状形态发生的中心信号分子，因为它在细胞极性途径以及协调细胞信号途径控制囊泡运输和涉及细胞内空泡的融合事件中发挥关键作用，而细胞内空泡是这一过程的基础。在这最后一次资助期间，我们在展示cdc42在管腔形成中的功能以及识别这些事件所需的关键下游cdc42效应器方面取得了重要进展。此外，我们还表明，在斑马鱼发育过程中，细胞内空泡的形成和融合是管腔形成的关键调节因素(与Brant Weinstein博士合作)。我们能够使用GFP-CDC42在体外和体内标记和可视化细胞内的空泡，这进一步表明了CDC42在这些事件中的功能作用。此外，我们还开发了基本的实验方法，如siRNA技术及其在我们的形态发生系统中的功能应用，3D胶原基质中EC管腔形成的实时图像分析，管形态形成过程中的蛋白质组捕获技术，以确定调控管腔形成的Cdc42相关蛋白，以及确定控制这些事件的关键信号转导级联。我们提出了三个特定的目标，即：特定的目标1：确定在3D胶原基质中，CDC42效应器Pak2和Pak4控制EC管腔和网络形成的分子机制。具体目标#2：明确细胞极性信号通路涉及CDC42、PAR3、PAR6和PKC Zeta如何控制3D胶原基质中EC管腔的形成机制。具体目标#3：研究JAMC和JAMB在EC管腔形成中的作用，并鉴定和表征上游的CDC42GEF激活CDC42来控制这些事件。与公共健康相关：这项工作侧重于血管衬里细胞在三维环境中形成管状结构的能力，例如胶原蛋白，这是组织的主要结构成分。这些衬里细胞携带一组蛋白质，控制它们组装成细胞衬里管的能力，这些蛋白质是组装血管细胞网络以发展我们的血管系统所必需的。在心血管疾病或癌症等各种人类疾病的背景下，对血管形成机制的基本了解对于努力刺激或抑制这一过程至关重要。

DESCRIPTION (provided by applicant): In this renewal proposal, we continue our work demonstrating the critical role of the Rho GTPase, Cdc42, in the molecular control of endothelial cell (EC) lumenogenesis in early blood vessel assembly during vasculogenesis. Our overall hypothesis is that Cdc42 represents a central signaling molecule in EC lumen formation and tube morphogenesis because of its key role in cell polarity pathways as well as coordinating cell signaling pathways controlling vesicular trafficking and fusion events involving intracellular vacuoles that are fundamental to this process. During this last funding period, we have made essential progress in demonstrating the function of Cdc42 in lumen formation as well as the identification of critical downstream Cdc42 effectors required for these events. Also, we have shown that intracellular vacuole formation and fusion are critical regulators of lumenogenesis both in vitro and in vivo during Zebrafish development (collaborative work with Dr. Brant Weinstein). We are able to label and visualize the intracellular vacuoles in vitro and in vivo using GFP-Cdc42 which further suggests a functional role for Cdc42 during these events. Also, we have developed essential experimental approaches such siRNA technology and its functional application in our morphogenic systems, real-time image analysis of EC lumenogenesis in 3D collagen matrices, proteomic capture techniques during tube morphogenesis to identify Cdc42-associated proteins which regulate lumen formation and identification of critical signal transduction cascades that control these events. We propose three specific aims which are; Specific Aim #1: To determine the molecular mechanisms by which the Cdc42 effectors, Pak2 and Pak4, control EC lumen and network formation in 3D collagen matrices. Specific Aim #2: To define how cell polarity signaling pathways involving Cdc42, Par3, Par6 and PKC zeta control the EC lumen formation mechanism in 3D collagen matrices. Specific Aim #3: To investigate the role of JamC and JamB in EC lumen formation and identify and characterize upstream Cdc42 GEFs which activate Cdc42 to control these events. PUBLIC HEALTH RELEVANCE: This work focuses on the ability of blood vessel lining cells to tube-like structures within three- dimensional environments such as collagen, which is a major structural component of tissue. These lining cells carry a group of proteins that control their ability to assemble into a cell-lined tube and which are required to assemble cellular networks of vessels to develop our blood vascular system. A basic understanding of the mechanisms underlying how blood vessels form is critical in efforts to stimulate or inhibit the process in the context of various human diseases such as cardiovascular disease or cancer.