Role of Egfl7 in Vascular Development and Angiogenesis

描述(申请人提供)：血管生成和血管生成由一个复杂的生长因子及其同源受体系统控制。其中包括血管内皮生长因子/血管内皮生长因子受体和血管生成素/Tie信号通路，以及b-成纤维细胞生长因子、转化生长因子-β、肾上腺素及其受体。此外，还阐明了Notch信号在血管发育和动静脉命运调控中的重要性。虽然这些途径对血管发育的重要性已有文献记载，但其他关键因素可能仍未确定。利用“基因陷阱”的方法，我们最近发现了Egfl7，一个新的内皮限制基因，它编码一个分泌性蛋白，带有一个EMI结构域，两个EGF结构域，以及在Notch配体中发现的一个可能的DSL结构域。Egfl7在卵黄囊血岛中的新生血管及其祖细胞中特异表达。在成年人中，Egfl7在血管生成和动脉损伤过程中表达上调。我们的初步研究表明，EGFL7在体外与Notch 1和4结合，并介导几种已知的Notch效应器功能。在目前的提案中，我们将检验这样的假设，即EGFL7是Notch的新配体，EGFL7作为Notch激动剂发挥作用，并且EGFL7诱导的Notch信号在血管发育和血管生成过程中介导独特的和非冗余的过程。我们将在原代人内皮细胞中测试这些假说，并在ES细胞体外分化系统和小鼠中使用获得和功能丧失的方法。我们提出了以下目标：目标1：确定EGFL7在HUVEC和鸡绒毛膜尿囊膜模型中Notch信号转导中的作用。目的2：确定内皮细胞中Egfl7的过度表达是否导致血管发育缺陷。我们将通过产生Tie2-Egfl7转基因小鼠来强迫Egfl7在内皮细胞中表达，并通过产生VE-Caherin：TTA；tre-Egfl7转基因小鼠来诱导内皮细胞中Egfl7的表达。目的3：检测Egfl7功能是否在血管发育的早期阶段起关键作用。我们将在ES细胞和小鼠胚胎中培育出具有条件敲除等位基因和基于慢病毒的siRNA敲除的小鼠。

DESCRIPTION (provided by applicant): Vasculogenesis and angiogenesis are controlled by a complex system of growth factors and their cognate receptors. These include VEGF/VEGFR and angiopoietin/Tie signaling pathways, as well as b-FGF, TGF-beta, ephrins, and their receptors. In addition, the importance of Notch signaling for vascular development and arterial-venous fate specification has been elucidated. While the importance of these pathways for vascular development has been documented, it is likely that other critical factors remain unidentified. Using a "gene trap" approach, we recently identified Egfl7, a novel endothelial-restricted gene that encodes a secreted protein with an EMI domain, two EGF domains, and a putative DSL domain found in Notch ligands. Egfl7 is specifically expressed in the emerging vasculature and its progenitors in the yolk sac blood islands. In adults, Egfl7 is up-regulated during angiogenesis and arterial injury. Our preliminary studies indicate that EGFL7 binds to Notch 1 and 4 in vitro and mediates several of the known Notch effector functions. In the present proposal, we will test the hypothesis that EGFL7 is a novel ligand for Notch, that EGFL7 functions as a Notch agonist, and that EGFL7-induced Notch signaling mediates distinctive and non-redundant processes during vascular development and angiogenesis. We will test these hypotheses in primary human endothelial cells, and by using gain- and loss-of-function approaches in an ES cell in vitro differentiation system and in mice. We are proposing the following aims: Aim 1: Determine the role of EGFL7 in Notch signaling in HUVEC and in a chick chorioallantoic membrane model. Aim 2: Determine whether overexpression of Egfl7 in the endothelium leads to defects in vascular development. We will force expression of Egfl7 in endothelial cells by generating Tie2-Egfl7 transgenic mice, and induce Egfl7 expression in endothelial cells by generating VE-Cadherin:tTA;TRE-Egfl7 transgenic mice. Aim 3: Test whether Egfl7 function is crucial for early stages of vascular development. We will generate mice with a conditional knock-out allele and lentivirus-based siRNA knock-down in ES cells and mouse embryos.