Ras/Rap Signaling and Endothelial Morphogenesis

描述(申请人提供)：内皮细胞在发育过程中和在稳定的血管中通过细胞-细胞相互作用相互通信。一个重要的结构是黏附连接，它通过基于钙粘附素的相互作用将单个细胞的肌动蛋白细胞骨架连接起来。尽管内皮细胞黏附连接在血管发育中很重要，但令人惊讶的是，人们对其形成和调节知之甚少。在成熟的血管中，粘附性连接需要形成一种屏障，控制血液和组织部位之间的液体和大分子的运动。第二信使二酰甘油(DAG)促进血管通透性，破坏内皮屏障功能，佛波酯模拟DAG活性。这项建议的目的是确定我们最近发现的一种新的内皮DAG/佛波酯受体RasGRPS如何影响成人血管的黏附连接和对血管通透性的影响。RasGRP3激活小GTP酶RAS和/或Rap，我们的初步数据表明，RasGRP3 IRF在发育中的血管对DAG/佛波酯的反应中起着至关重要的作用，其机制类似于成年血管的通透性反应。因此，我们假设RasGRP3介导的Ras/Rap信号是调控粘着连接的关键内皮控制点，在通过DAG的细胞信号占主导地位的情况下。我们通过外源性注射佛波酯来实验地建立这种情况，但我们假设这种情况在以DAG水平升高为特征的疾病状态中重现，如糖尿病。我们认为，阻断RasGRP3活性可以改善成人和发育中胎儿糖尿病的血管并发症。这些假说将在三个方面进行验证，在细胞和分子水平上完全表征发育中的血管和内皮细胞对DAG/佛波酯的依赖RasGRP3的反应，并检查RasGRP3基因操作对胚胎和成人糖尿病血管并发症和内皮屏障功能的影响。

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.