Endothelial Cell Lumen Formation Requires Rho GTPases

描述（由申请人提供）：本研究的长期目标是 为了识别新血管形成所需的机制（例如， 血管生成）。内皮细胞（EC）经历的基本形状变化 在血管生成过程中，包括空泡和管腔形成，以及分支 形态发生是需要更多研究的领域。使用体外系统 在我们的实验室开发，人BC重悬在胶原蛋白和纤维蛋白 基质经历形态发生。这一过程包括液泡的形成 然后随着时间的推移形成分支以形成互连的网络。这些戏剧性 形状的改变需要肌动蛋白细胞骨架。GTP酶的Rho家族具有 据报道，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、Rac 1和Cdc 42在EC管腔形成中的三维表达。以确定 每个GT3在空泡和管腔形成中的单独作用，重组 构建腺病毒以操纵EC中的基因表达。解决 这个问题，腺病毒表达显性阴性和组成型 构建了RhoA、Rac 1和Cdc 42的活性形式。这些能力 病毒阻止液泡和管腔的形成，分支形态发生将 下定决心。此外，腺病毒将递送GFP-Rho GT3嵌合体， 以确定这些分子在不同步骤中在EC中的靶向位置， 形态发生根据这些发现，我们将筛选新的和已知的 利用酵母双杂交cDNA文库筛选内皮细胞Rho GTPases的结合伴侣 以及免疫亲和层析。此外，过度表达 Rho GTP酶的显性负性下游效应物将用于 进一步剖析了EC中控制不同细胞的下游信号事件， 形态发生的步骤EC空泡和管腔形成的基本机制 形成和三维胶原内的分支形态发生， 纤维蛋白基质可能有助于揭示血液 内皮细胞形成血管。

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.