R-Ras signaling in vascular biology

描述（由申请人提供）：需要支持分析Ras家族小GTIPR-Ras和新型效应分子RLIP 76之间的相互作用;这种相互作用对R-Ras信号传导的影响; R-Ras/RLIP 76相互作用和信号传导调节血管细胞扩散、迁移、增殖和分化的机制;以及该信号节点在血管生成和损伤诱导的新生内膜增生导致动脉再狭窄中的作用。Ras GTP酶调节细胞增殖、分化、凋亡、粘附和迁移。特异性效应物与活化的Ras蛋白的结合传播信号级联，导致取决于Ras家族同种型的独特结果。R-Ras特别用于调节血管壁细胞的增殖、运动、增殖和分化，并在响应损伤的血管生成和血管再生期间调节这些过程。因此，R-Ras信号在血管细胞和血管功能中具有独特的多效性作用，但涉及的具体途径尚不清楚。我们在R-Ras效应子的蛋白质组学筛选中鉴定了RLIP 76，并发现RLIP 76调节R-Ras的一些独特细胞功能，包括细胞信号传导、扩散、迁移和体外血管生成。这是第一次鉴定具有这些不同细胞功能的R-Ras的特异性独特效应子。我们假设R-Ras/RLIP 76结合控制不同的信号传导途径来介导血管细胞功能和R-Ras在血管系统中的独特作用。为了验证这一假设，我们将绘制R-Ras和RLIP 76中的结合位点，并将鉴定将选择性破坏这种相互作用的突变。我们将使用这些突变体来确定阻断R-Ras/RLIP 76相互作用对血管壁细胞中R-Ras信号传导的影响。然后，我们将评估RLIP 76的推定支架功能，并将绘制血管细胞中RLIP 76/R-Ras信号传导复合物的蛋白质组分。接下来，我们将研究R-Ras/RLIP 76结合和表征的信号传导途径对R-Ras介导的血管细胞扩散、迁移、增殖和分化的贡献。最后，我们将研究R-Ras信号轴在体内的作用，即阻断血管生成，损伤诱导的新生内膜增生和动脉再狭窄。这些研究将表征控制细胞信号传导、迁移、增殖和血管功能的新型信号传导途径的调节和机制。因此，这些研究将为生理和病理生理功能的重要过程提供新的见解，并可能为心血管疾病的治疗提供新的治疗靶点。 公共卫生相关性：我们建议研究调节血管系统中两种主要病理反应中细胞生长和迁移的过程的分子和细胞机制：1）新血管形成（血管生成），其发生在损伤部位和肿瘤发展期间，其中肿瘤生长需要新血管，和2）动脉再狭窄，对动脉旁路引起的损伤的血管闭塞反应，支架插入和球囊扩张手术，每年影响美国多达40万人。我们已经确定了候选分子信号通路，我们将检查作为肿瘤血管生成和术后再狭窄的独特和选择性的代理商。因此，我们将在血管生成和动脉再狭窄的小鼠模型中使用生物化学、细胞生物学和生理学方法的组合来鉴定和表征用于治疗人类心血管疾病的新的治疗靶点。

DESCRIPTION (provided by applicant): Support is requested to analyze the interaction between the Ras family small GTPase R- Ras and a novel effector molecule, RLIP76; the effect of this interaction on R-Ras signaling; the mechanisms by which R-Ras/RLIP76 interactions and signaling regulate vascular cell spreading, migration, proliferation and differentiation; and the roles of this signaling node in angiogenesis and injury-induced neointimal hyperplasia leading to arterial restenosis. Ras GTPases regulate cell proliferation, differentiation, apoptosis, adhesion and migration. The binding of specific effectors to activated Ras proteins propagates signaling cascades resulting in unique outcomes depending on the Ras family isotype. R-Ras in particular functions to regulate adhesiveness, motility, proliferation and differentiation of cells of the vessel wall, and to modulate these processes during angiogenesis and vascular regeneration in response to injury. Thus R-Ras signaling has unique, pleiotropic effects in vascular cells and in vascular functions, but the specific pathways involved are unknown. We identified RLIP76 in a proteomic screen for R-Ras effectors and found that RLIP76 regulates some of the unique cellular functions of R-Ras including cell signaling, spreading, migration, and angiogenesis in vitro. This is the first identification of a specific, unique effector for R-Ras with these distinct cellular functions. We hypothesize that R-Ras/RLIP76 binding controls distinct signaling pathways to mediate vascular cell functions and the unique effects of R-Ras in the vasculature. To test this hypothesis we will map the binding sites in R-Ras and RLIP76 and will identify mutations which will selectively disrupt this interaction. We will use these mutants to determine the effect of blockade of R- Ras/RLIP76 interaction on R-Ras signaling in cells of the vessel wall. We will then evaluate a putative scaffolding function for RLIP76 and will map the protein components of the RLIP76/R- Ras signaling complex in vascular cells. Next we will investigate the contributions of R- Ras/RLIP76 binding and the characterized signaling pathways to R-Ras-mediated vascular cell spreading, migration, proliferation and differentiation. Lastly, we will investigate a role for this signaling axis in R-Ras functions in vivo, namely blockade of angiogenesis, and injury-induced neointimal hyperplasia and arterial restenosis. These studies will characterize the regulation and mechanisms of novel signaling pathways that control cell signaling, migration, proliferation and vascular function. Therefore these studies will provide new insights into processes essential in physiological and pathophysiological function and may indicate new therapeutic targets in the treatment of cardiovascular disease. PUBLIC HEALTH RELEVANCE: We propose to investigate the molecular and cellular mechanisms of a process regulating the growth and migration of cells in two major pathological responses in the vasculature: 1) new blood vessel formation (angiogenesis), which occurs at sites of injury and during tumor development, in which new blood vessels are required for tumor growth, and 2) arterial restenosis, a vessel-occluding response to injury resulting from arterial bypass, stent insertion and balloon dilatation surgeries, which affects as many as 400,000 people in the U.S. per year. We have identified candidate molecular signaling pathways which we will examine as unique and selective agents of tumor angiogenesis and post-surgical restenosis. Thus we will use a combination of biochemical, cell biological and physiological approaches in mouse models of angiogenesis and arterial restenosis to identify and characterize novel therapeutic targets for the treatment of human cardiovascular disease.