Blood vessel tubulogenesis: Rasip1-directed GTPase signaling and cell polarity.

描述（由申请人提供）：在血管中心形成载血通道（管腔形成或“管生成”）是心血管发育过程中最关键的步骤之一。迄今为止，这一过程的分子机制尚不清楚。了解并最终控制血管生长是许多临床方法的关键目标，从阻断肿瘤血管生成到促进伤口愈合的血管。研究人员对胚胎内皮细胞（ECs）进行了转录谱分析，以发现调节血管形成的因素，并鉴定出一种名为Rasip1的gtpase相互作用蛋白。研究发现，Rasip1在胚胎ECs中特异性表达，在小鼠体内切除Rasip1可阻断胚胎血管小管的形成和血管的形成。我们发现这些带有小gtpase的Rasip1复合物及其效应物能够促进Cdc42和Rac1，抑制RhoA。Rasip1-/-索（E8.0-8.5）的关键缺陷包括：成血管细胞极性的丧失和极性决定因子Par3的适当定位，以及1整合素介导的ECM和细胞骨架粘附的破坏。主要的假设是，Rasip1调节不同的下游GTPase信号通路，如Rho， Rac和Cdc42，它们调节不同的细胞过程，协调驱动血管小管形成。本研究旨在探讨Rasip1介导的细胞极性、收缩性和粘附性如何影响内皮小管形成，并利用小鼠遗传学、体外模型和生物化学方法解剖Rasip1的下游通路。对于小鼠突变体的分析，将评估简单的参数来研究E8.0-8.5小鼠背主动脉的管腔形成（成血管细胞形态和组织，以及极性和粘附标志物）。具体目标是：1.；研究rasip1依赖性细胞极性的作用和血管管腔形成过程中根尖/管腔膜形成的机制（Par3和Crb3）。2. 确定不同的Rasip1调控的GTPase信号通路Rho， Rac1和Cdc42在血管管腔形成过程中的细胞结果，并评估哪些途径可以挽救或加剧Rasip1无效的管腔衰竭。3. 通过鉴定管腔形成“信号复合体”成分来阐明依赖于rasip1的管腔形成机制。这些研究的短期目标是阐明Rasip1调控途径，并进一步了解心血管的发展。长期目标是找到新的分子靶点，通过阻断管腔形成来阻断疾病中的血管生长，比如正在生长的肿瘤。！

DESCRIPTION (provided by applicant): Formation of blood-carrying channels at the heart of blood vessels (lumen formation or 'tubulogenesis') is one of the most critical steps during cardiovascular development. To date, the molecular mechanisms underlying this process remain unclear. Understanding and ultimately controlling blood vessel growth are key goals of many clinical approaches, ranging from blocking vessels in tumor angiogenesis to promoting vessels in wound healing. Transcriptional profiling of embryonic endothelial cells (ECs) was carried out to discover factors that regulate blod vesel formation, and identified a GTPase-interacting protein called Rasip1. Rasip1 was found to be expressed specificaly in embryonic ECs, and ablation of Rasip1 in mice blocks embryonic vascular tubulogenesis and blood vessel formation. We showed those Rasip1 complexes with small GTPases and their effectors, to promote Cdc42 and Rac1, and suppress RhoA. Key defects in Rasip1-/- cords (E8.0-8.5) include: loss of angioblast cell polarity and of proper localization of the polarity determinant Par3, as well as disruption of 1integrin-mediated adhesion to ECM and of the cytoskeleton. The main hypothesis is that Rasip1 regulates distinct downstream GTPase signaling pathways, such as Rho, Rac and Cdc42, which regulate distinct cellular processes that coordinate to drive vascular tubulogenesis. This proposal asks how Rasip1-mediated cell polarity, contractility and adhesion influence endothelial tubulogenesis, and dissects pathways downstream of Rasip1 using mouse genetics, in vitro models and biochemistry. For analysis of mouse mutants, simple parameters will be assessed to study lumen formation in E8.0-8.5 mouse dorsal aortae (angioblast morphology and organization, as well as polarity and adhesion markers). Specific aims are: 1. To examine role of Rasip1-dependent cell polarity and mechanisms of apical/luminal membrane formation during vascular lumen formation (Par3 and Crb3). 2. To identify the cellular outcomes of the different Rasip1-regulated GTPase signaling pathways, Rho, Rac1 and Cdc42, during vascular lumen formation, and assess which pathways can rescue or exacerbate the Rasip1 null lumen failure. 3. To elucidate mechanism of Rasip1-dependent lumen formation via identification of lumen formation 'signaling complex' components. The short-term objective of these studies is to elucidate Rasip1 regulated pathways and further our understanding of cardiovascular development. The long-term objective is to find new molecular targets to block blood vessel growth in disease, such as in growing tumors, by blocking lumen formation. !