Ras/Rap Signaling and Endothelial Morphogenesis

描述（由申请人提供）：内皮细胞在发育和稳定血管中通过细胞间相互作用相互交流。一个重要的结构是粘附连接，它通过钙粘蛋白相互作用将单个细胞的肌动蛋白细胞骨架连接起来。尽管内皮粘附连接在血管发育中很重要，但令人惊讶的是，人们对其形成和调控知之甚少。在成熟血管中，粘附连接需要形成一个屏障来控制血液和组织部位之间液体和大分子的运动。第二信使二酰基甘油（DAG）促进血管通透性和破坏内皮屏障功能，而佛波酯模拟DAG的活性。本提案的目的是确定我们最近发现的一种新的内皮DAG/phorbol酯受体RasGRPS如何影响成人血管粘附连接的发育和血管通透性。RasGRP3激活小GTPases Ras和/或Rap，我们的初步数据表明，RasGRP3在发育中的血管对DAG/phorbol酯的反应中起着至关重要的作用，其机制类似于成人血管的通透性反应。因此，我们假设rasgrp3介导的Ras/Rap信号是粘附体连接调节的关键内皮控制点，在通过DAG的细胞信号传导占主导地位的情况下。我们通过外源性给药佛博尔酯的实验建立了这一理论，但我们假设这种情况在以DAG水平升高为特征的疾病状态（如糖尿病）中也会重现。我们提出，成人和发育中的胎儿糖尿病的血管并发症可以通过阻断RasGRP3活性来改善。这些假设将在三个目标中得到验证，这些目标将在细胞和分子水平上充分表征发育中的血管和内皮细胞对DAG/phorbol酯的依赖反应，并检查RasGRP3基因操作对胚胎和成人糖尿病内皮屏障功能和血管并发症的影响。

DESCRIPTION (provided by applicant): 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. RasGRP3 activates the small GTPases Ras and/or Rap, and our preliminary data point to a crucial role for RasGRP3 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 RasGRP3-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 RasGRP3 activity. These hypotheses will be tested in three aims that fully characterize the RasGRP3-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 RasGRP3 on endothelial barrier function and the vascular complications of diabetes in embryos and adults.