Ras/Rap Signaling and Endothelial Morphogenesis

内皮细胞在发育和稳定过程中通过细胞间的相互作用进行交流 船只。粘着连接是连接个体肌动蛋白细胞骨架的一个重要结构。 细胞通过基于钙粘附素的相互作用。尽管内皮细胞黏附连接在血管中很重要 发展，令人惊讶的是，人们对它们的形成和调控知之甚少。在成熟的血管中，黏附 连接需要形成一个屏障，以控制流体和大分子在 血液和组织部位。第二信使二酰甘油(DAG)促进血管通透性和 破坏内皮屏障功能，佛波酯模拟DAG活性。这项提议的目标是 确定我们最近发现的一种新的内皮DAG/佛波酯受体RasGRPS如何影响 在成体血管中黏附连接处并影响血管通透性。RasGRPS 激活小的GTP酶RAS和/或RAP，我们的初步数据表明RasGRPS起着至关重要的作用 IRF指发育中的血管对DAG/佛波酯的反应，其机制与渗透性相似 成年血管的反应。因此，我们假设RasGRPS介导的RAS/Rap信号是一个关键的 在细胞信号转导的情况下，调节黏附连接的内皮控制点 通过DAG占主导地位。我们通过外源性给药佛波醇酯来建立这个实验，但是 我们假设这种情况是在以DAG水平升高为特征的疾病状态下重现的， 比如糖尿病。我们认为成人和发展中的糖尿病的血管并发症 阻断RasGRPS活性可以改善胎儿的发育。这些假说将在三个方面进行检验 旨在充分表征发育中的血管和内皮细胞的RasGRPS依赖反应 在细胞和分子水平上对DAG/佛波酯，并检查遗传的后果 RasGRPS对糖尿病血管并发症及内皮屏障功能的影响 胚胎和成体。

Endothelial cells communicate with each other via cell-cell interactions during development and in stable vessels. One important structure is the adherens junction, which links the actin cytoskeleton of individual cells via cadherin-based interactions. Although endothelial adherens junctions are important in vascular development, surprisingly little is known about their formation and regulation. In mature vessels, adherens junctions are required to form a barrier that controls the movement of fluids and macromolecules between blood and tissue sites. The second messenger diacylglycerol (DAG) promotes vascular permeability and disrupts endothelial barrier function, and phorbol esters mimic DAG activity. The goal of this proposal is to determine how a novel endothelial DAG/phorbol ester receptor we recently identified, RasGRPS, affects adherens junctions developmentally and impacts on .vessel permeability in adult vessels. RasGRPS activates the small GTPases Ras and/or Rap, and our preliminary data point to a crucial role for RasGRPS irf the response of developing vessels to DAG/phorbol esters, via a mechanism similar to the permeability response of adult vessels. Thus we hypothesize that RasGRPS-mediated Ras/Rap signaling is a critical endothelial control point for the regulation of adherens junctions, in situations where cellular signaling through DAG is dominant. We set this up experimentally by exogenous administration of phorbol esters, but we posit that this scenario is recapitulated in disease states that are characterized by elevated DAG levels, such as diabetes. We propose that the vascular complications of diabetes in both adults and developing fetuses can be ameliorated by blockade of RasGRPS activity. These hypotheses will be tested in three aims that fully characterize the RasGRPS-dependent response of developing vessels and endothelial cells to DAG/phorbol esters at the cellular and molecular levels, and examine the consequences of genetic manipulation of RasGRPS on endothelial barrier function and the vascular complications of diabetes in embryos and adults.