Endothelial Cell Lumen Formation Requires Rho GTPases

描述(由申请人提供)：本研究的长期目标是 为了确定新血管形成所需的机制(例如 血管生成)。内皮细胞(ECs)经历的基本形状变化 在血管生成过程中，包括液泡和管腔的形成以及分支 形态发生是需要更多研究的领域。使用体外系统 我们实验室开发的人BC再悬浮在胶原和纤维蛋白中 母体经历形态发生过程。这个过程涉及到液泡的形成。 然后随着时间的推移，这些分支形成相互连接的网络。这些戏剧性的 形状的改变需要肌动蛋白的细胞骨架。Rho家族的GTP酶 已被报道调节由肌动蛋白控制的细胞形状变化 细胞骨架。该提案的第一个目的是研究 RhoA、Rac1和Cdc42在EC管腔形成中的三维表达。要确定 每种GTP酶在液泡和管腔形成中的单独作用，重组 构建了腺病毒来操纵内皮细胞中的基因表达。致信地址 这个问题，腺病毒表达的显性阴性和结构性 构建了RhoA、Rac1和Cdc42的活性形式。这些人的能力 病毒阻止液泡和管腔的形成，以及分支形态发生 要下定决心。此外，腺病毒将运送GFP-Rho GTP酶嵌合体 为了确定这些分子在ECs中的靶点在不同的步骤 形态发生。基于这些发现，我们将筛选出新奇和已知的 利用酵母双杂交文库研究Rho GTP酶在内皮细胞中的结合伙伴 从内皮细胞和免疫亲和层析。此外，过度表达 Rho GTP酶的主要负向下游效应子将用于 进一步剖析EC中控制不同 形态发生的步骤。发现EC液泡和管腔的基本机制 三维胶原蛋白的形成和分支形态发生 纤维蛋白基质可能有助于揭示血液所需的基本机制 内皮细胞的血管形成。

DESCRIPTION (provided by the applicant): The long-term goal of this research is to identify mechanisms required for new blood vessel formation (e.g. angiogenesis). The basic shape changes that endothelial cells (ECs) undergo during angiogenesis, including vacuole and lumen formation, and branching morphogenesis are areas that need more investigation. Using in vitro systems developed in our laboratory, human BC resuspended in both collagen and fibrin matrices undergo morphogenesis. This process involves the formation of vacuoles that then branch to form interconnected networks over time. These dramatic shape changes require the actin cytoskeleton. The Rho family of GTPases have been reported to regulate cell shape changes controlled by the actin cytoskeleton. The first aim of the proposal is to study the involvement of RhoA, Rac 1 and Cdc42 in EC lumen formation in three-dimensions. To determine the individual roles of each GTPase in vacuole and lumen formation, recombinant adenoviruses were constructed to manipulate gene expression in ECs. To address this question, adenoviruses expressing dominant negative and constitutively active forms of RhoA, Rac 1 and Cdc42 were constructed. The ability of these viruses to block vacuole and lumen formation, and branching morphogenesis will be determined. In addition, adenoviruses will deliver GFP-Rho GTPase chimeras to determine where these molecules target in ECs at the distinct steps of morphogenesis. Based on these findings, we will screen for both novel and known binding partners of Rho GTPases in ECs using a yeast two-hybrid cDNA library from ECs and also immunoaffinity chromatography. Further, overexpression of dominant negative downstream effectors for the Rho GTPases will be used to further dissect downstream signaling events in ECs that control the different steps in morphogenesis. Discovering the basic mechanism of EC vacuole and lumen formation, and branching morphogenesis within three-dimensional collagen and fibrin matrices may help uncover fundamental mechanisms required for blood vessel formation by endothelial cells.