Signaling Mechanisms of VEGF Receptor in Vasculogenesis

性状：（申请人提供）：血管内皮生长因子-A VEGF-A或VEOF及其受体在控制血液中起着关键作用 血管生长和功能，如胚胎血管化和肿瘤 血管生成VEGF受体-i（Flt-1）和VEGF受体-2（Flk-1）均为 跨膜酪氨酸激酶，但它们的信号转导机制， 胚胎的发育过程尚不清楚。而fit-i基因的无效失活 导致严重的血管缺陷和早期胚胎死亡， 其激酶结构域的破坏不会导致血管缺陷。平冢 提出Fit-i仅仅是VEGF结合蛋白，其作用是限制 VEGF对FIk-1的可及性，而在fit-i无效突变体中， Flk-1信号传导增加导致血管缺陷。但是，null fit-1和fik-1的突变表型表明，fit-1和fik-1不起作用， 相反的角色。fit-1-/-无效突变体的主要缺陷是过度的 成血管细胞的承诺，但在flk-1无效突变，最初的承诺， 成血管细胞仍然存在。为了解决这些矛盾，我们建议 四个具体目标。特别地，Fik-1信号传导的水平将是 操纵，以查看Fik-1活性的上调是否会导致 fit-i-/-样突变表型，如果Flk-1活性下调， 在FLT-1-/-背景中将使FLT-1-/-表型最小化。Flk-1的调节 活性将通过几种方式实现：通过用FIT-I基因敲除小鼠进行繁殖， VEGF活性降低的小鼠，并通过敲入flk-1编码 组成型激活的FIk-1突变体和激酶减少的FIk-1突变体 活动对Fit-i激酶信号传导的要求将更彻底地 通过完全缺失编码以下的整个基因组序列来检查： fit-1激酶结构域。这些实验将为我们提供一个明确的答案。 我质疑Fit-i是一种主动信号分子还是一种被动VEGF 结合蛋白这些实验也可能为我们提供更广泛的 有机会解决VEGF受体介导的几个重要问题， 信号转导，特别是Flk-1信号转导在调节内皮细胞增殖中的作用 细胞增殖和/或存活。以上实验， 将有助于我们实现长期目标， VEGF介导的siarialino在血管发育中的机制。

DESCRIPTION: (provided by applicant): The vascular endothelial growth factor-A (VEGF-A, or VEOF) and its receptors play critical roles in controlling blood vessel growth and function, such as embryonic vascularization and tumor angiogenesis. VEGF receptor-i (FIt-i) and VEGF receptor-2 (Flk-1) are both transmembrane tyrosine kinases, but their signaling mechanisms in the context of developing embryos are unclear. While a null inactivation of the fit-i gene led to severe vascular defects and early embryonic lethality, a limited disruption of its kinase domain did not lead to vascular defects. Hiratsuka et al. proposed that Fit-i was a mere VEGF binding protein whose role was to limit VEGF accessibility to FIk-1, and that in the fit-i null mutants, it was an increase in Flk-1 signaling that led to the vascular defects. However, null mutant phenotypes of fit-i and fik-1 indicate that Fit-i and Fik-1 do not play opposite roles. The primary defect in fit-1-/- null mutants is the excessive hemangioblast commitment, but in flk-1 null mutants, the initial commitment to hemangioblasts still occurs. To address these inconsistencies, we have proposed four specific aims. In particular, the level of Fik-1 signaling will be manipulated to see if an up-regulation of the Fik-1 activity will result in a fit-i-/- -like mutant phenotype, and if a down-regulation of the Flk-1 activity in fit-1-/- background will minimize flt-1-/- phenotype. Modulation of Flk-1 activity will be achieved in several ways: by breeding fit-i knockout mice with mice of reduced VEGF activity and by knock-in of flk-1 cDNAs encoding a constitutively activated FIk-1 mutant and FIk-1 mutants with reduced kinase activities. The requirement for Fit-i kinase signaling will be more thoroughly examined by a complete deletion of the entire genomic sequence encoding for the Fit-i kinase domain. These experiments will provide a definitive answer to the question whether Fit-i is an active signaling molecule or a passive VEGF binding protein. These experiments may also provide us with extended opportunities to address several important issues of VEGF receptor mediated signaling, in particular the role of Flk-1 signaling in regulating endothelial cell proliferation and/or survival in vivo. Together, the above experiments will facilitate us to achieve our long term goal in understanding the mechanisms of VEGF-mediated siarialino in vascular development.