Ras/Rap Signaling and Endothelial Morphogenesis

描述（由申请方提供）：在发育期间和稳定血管中，内皮细胞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，其通过基于钙粘蛋白的相互作用连接单个细胞的肌动蛋白细胞骨架。虽然内皮粘附连接在血管发育中很重要，但令人惊讶的是，对它们的形成和调节知之甚少。在成熟的血管中，需要粘附连接来形成控制血液和组织部位之间的流体和大分子运动的屏障。第二信使甘油二酯（DAG）促进血管通透性并破坏内皮屏障功能，佛波醇酯模拟DAG活性。本提案的目的是确定我们最近发现的一种新型内皮DAG/佛波酯受体RasGRPS如何影响粘附连接的发育和对成人血管通透性的影响。RasGRP 3激活小GTP酶Ras和/或Rap，并且我们的初步数据指出RasGRP 3在发育中的血管对DAG/佛波醇酯的响应中起着至关重要的作用，其机制类似于成年血管的渗透性响应。因此，我们假设RasGRP 3介导的Ras/Rap信号传导是调节粘附连接的关键内皮控制点，在通过DAG的细胞信号传导占主导地位的情况下。我们通过外源性给予佛波醇酯实验性地建立了这一点，但我们认为这种情况在以DAG水平升高为特征的疾病状态（如糖尿病）中重演。我们认为阻断RasGRP 3活性可以改善成人和发育中胎儿糖尿病的血管并发症。这些假设将在三个目标，充分表征的RasGRP 3依赖性反应的发展中的血管和内皮细胞的DAG/佛波醇酯在细胞和分子水平上进行测试，并检查的后果的遗传操作的RasGRP 3内皮屏障功能和糖尿病的血管并发症在胚胎和成人。

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.