Kidney vascularization: semaphorin-mediated mechanisms.

描述（由申请方提供）：对肾器官发生过程中内皮细胞定向迁移的分子基础和发育中血管系统的模式知之甚少。我们发现VEGF是内皮细胞的化学引诱物，并指导内皮细胞向发育中的肾单位迁移。其他的引导信号也必然参与调节血管生长。这些引导线索的性质是未知的。两种VEGF共受体，神经纤毛蛋白1和2也是脑信号蛋白3A和3F的受体。脑信号蛋白是诱导轴突化学排斥的导向蛋白。Sema 3A通过与VEGF竞争神经纤毛蛋白结合来减少内皮细胞迁移。我们发现，sema 3A和3F在肾形态发生过程中表达，并定位于肾上皮细胞，与内皮细胞中的受体互补，表明脑信号蛋白对血管图案化很重要。本研究的目的是阐明内皮细胞定向迁移的机制，导致发育中的血管系统的空间组织和脑信号蛋白3A和3F在肾脏形态发生过程中的功能。我们推测sema 3A和3F是由肾上皮细胞产生的。动脉瘤细胞产生化学排斥信号，通过建立内皮细胞迁移的边界来调节VEGF的内皮细胞化学吸引。sema 3A、sema 3F和VEGF的配体与它们共有的受体结合的组合可能性可能导致血管形成的“路径”。我们还推测，信号蛋白的散射因子样特性可能是负责的，至少部分，肾上皮细胞的分支形态发生。为了验证我们的假设：1）我们将通过活细胞显微镜使用共培养、迁移试验和转基因小鼠中sema 3A和3F的细胞特异性过表达来研究sema 3A和3F引导因子对内皮细胞迁移的机制。2)我们将使用肾小管上皮细胞、器官培养物和转基因小鼠研究脑信号蛋白3A和3F在分支形态发生和小管发生中的功能。3)我们将研究FAK的作用和参与脑信号蛋白介导的指导和形态发生线索的下游信号机制。这一建议应提供新的和重要的信息，semaphorin诱导的定向迁移和推进我们的知识的分子机制，管理血管空间组织。了解细胞迁移的分子基础的指导线索，应使我们能够产生新的战略，诊断和治疗先天性肾脏异常，并在体外器官形成。

DESCRIPTION (provided by applicant): The molecular basis of directional migration of endothelial cells and the patterning of the developing vasculature during kidney organogenesis are poorly understood. We showed that VEGF is a chemoattractant for endothelial cells and directs the migration of endothelial cells towards developing nephrons. Other guidance cues are necessarily involved to modulate vascular growth. The nature of these guidance cues is unknown. Two VEGF co-receptors, neuropilins 1 and 2 are also receptors for semaphorins 3A and 3F. Semaphorins are guidance proteins that induce axon chemorepulsion. Sema 3A decreases endothelial cell migration by competing with VEGF for neuropilin binding. We showed that sema 3A and 3F are expressed during renal morphogenesis and localize to renal epithelial cells in a complementary fashion to their receptors located in endothelial cells, suggesting that semaphorins are important for vascular patterning. The objectives of this proposal are to elucidate the mechanisms of endothelial cells directional migration leading to the spatial organization of the developing vasculature and the function of semaphorins 3A and 3F during kidney morphogenesis. We hypothesize that sema 3A and 3F produced by renal epithe!ial cells generate chemorepulsive cues that modulate VEGF's endothelial cell chemoattraction by creating boundaries to endothelial cell migration. The combinatorial possibilities of ligand binding for sema 3 A, sema 3F and VEGF to their shared receptors may result in "paths" for vessel formation. We also hypothesize that the scatter factor-like properties of semaphorins may be responsible, at least in part, for branching morphogenesis of renal epithelia. To test our hypotheses: 1) we will study the mechanims of sema 3A and sema 3F guidance cues for endothelial cell migration by live cell microscopy using co-culture, migration assays, and cell-specific overexpression of sema 3A and 3F in transgenic mice. 2) We will examine the function of semaphorins 3A and 3F in branching morphogenesis and tubulogenesis using tubular epithelial cells, organ cultures and transgenic mice. 3) We will examine the role of FAK and the downstream signaling mechanisms involved in semaphorin-mediated guidance and morphogenetic cues. This proposal should provide novel and important information regarding semaphorin-induced directional migration and advance our knowledge of the molecular mechanisms governing vascular spatial organization. Understanding the molecular basis of guidance cues for cell migration should enable us to generate new strategies for diagnosis and treatment of congenital renal abnormalities and to develop organogenesis in vitro.