Genes regulating capillary morphogenesis and apoptosis

描述（由申请人提供）：在本次更新申请中，我们继续我们的工作，证明Rho GTPase Cdc42在血管形成过程中早期血管组装过程中内皮细胞（EC）管腔形成的分子控制中的关键作用。我们的总体假设是，Cdc42代表了EC管腔形成和管形态发生的中心信号分子，因为它在细胞极性通路中起关键作用，并协调细胞信号通路，控制囊泡运输和融合事件，涉及细胞内液泡，这是这一过程的基础。在最后的资助期内，我们在证明Cdc42在管腔形成中的功能以及鉴定这些事件所需的关键下游Cdc42效应物方面取得了重要进展。此外，我们已经证明细胞内液泡形成和融合是斑马鱼发育过程中体外和体内放光发生的关键调节因子（与Brant Weinstein博士合作）。我们能够在体外和体内使用GFP-Cdc42标记和可视化细胞内液泡，这进一步表明Cdc42在这些事件中的功能作用。此外，我们已经开发了必要的实验方法，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，在管形态发生期间的蛋白质组学捕获技术，以鉴定调节管腔形成的cdc42相关蛋白，并鉴定控制这些事件的关键信号转导级联。我们提出三个具体目标：具体目标#1：确定Cdc42效应物Pak2和Pak4在3D胶原基质中控制EC管腔和网络形成的分子机制。特定目标#2：定义包括Cdc42、Par3、Par6和PKC zeta在内的细胞极性信号通路如何控制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，并识别和表征激活Cdc42以控制这些事件的上游Cdc42 gef。公共卫生相关性：这项工作的重点是血管内膜细胞在三维环境中形成管状结构的能力，如胶原蛋白，这是组织的主要结构成分。这些内衬细胞携带一组蛋白质，这些蛋白质控制着它们组装成内衬细胞管的能力，这些蛋白质是组装血管细胞网络以形成我们的血管系统所必需的。对血管形成机制的基本理解对于刺激或抑制各种人类疾病（如心血管疾病或癌症）的过程至关重要。

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.