Signaling Mechanisms of VEGF Receptor in Vasculogenesis

描述：(申请人提供)：血管内皮生长因子-A 血管内皮生长因子(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，如胚胎血管形成和肿瘤 血管生成。血管内皮生长因子受体-I(FIT-I)和血管内皮生长因子受体-2(Flk-1)均为 跨膜酪氨酸激酶，但其信号机制的背景 目前尚不清楚胚胎的发育情况。而Fit-I基因的零失活 导致严重的血管缺陷和早期胚胎死亡，有限 其激酶结构域的破坏不会导致血管缺陷。平冢等人 艾尔提出Fit-I仅仅是一种血管内皮生长因子结合蛋白，其作用是限制 Fit-I缺失突变体中，Fit-I缺失突变体中的 导致血管缺陷的Flk-1信号增加。但是，为空 Fit-I和Fik-1的突变表型表明Fit-I和Fik-1不起作用 相反的角色。Fit-1-/-Null突变体的主要缺陷是过度 血管母细胞承诺，但在Flk-1缺失突变体中，最初的承诺是 血管母细胞仍然存在。为了解决这些不一致的问题，我们建议 四个具体目标。特别是，Fik-1信令的级别将是 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Fit-1-/-背景中将使Flt-1-/-表型最小化。Flk-1的调制 活动将通过几种方式实现：通过培育Fit-I基因敲除小鼠 血管内皮生长因子活性降低和Flk-1cDNAs敲入的小鼠 组分激活的Fik-1突变体和带有还原蛋白的Fik-1突变体 活动。对Fit-I激酶信号的要求将更加彻底 通过完全删除编码该基因的整个基因组序列进行检查 Fit-I激活域。这些实验将提供一个明确的答案 质疑FIT-I是主动信号分子还是被动的血管内皮生长因子 结合蛋白。这些实验也可能为我们提供更广泛的 解决血管内皮生长因子受体介导的几个重要问题的机会 信号转导，尤其是Flk-1信号转导在内皮调节中的作用 体内细胞增殖和/或存活。综上所述，上述实验 将有助于我们实现我们的长期目标，了解 血管内皮生长因子介导的唾液酸在血管发育中的作用机制。

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.