Role of Egfl7 in Vascular Development and Angiogenesis

描述（由申请人提供）：血管生成和血管生成是由生长因子及其同源受体的复杂系统控制的。这些包括VEGF/VEGFR和血管生成素/Tie信号通路，以及b-FGF、tgf - β、ephrins及其受体。此外，Notch信号在血管发育和动静脉命运规范中的重要性也得到了阐明。虽然这些途径对血管发育的重要性已被证明，但其他关键因素可能仍未被确定。使用“基因陷阱”方法，我们最近发现了Egfl7，这是一种新的内皮限制基因，它编码一种分泌蛋白，该蛋白具有EMI结构域、两个EGF结构域和在Notch配体中发现的假定的DSL结构域。Egfl7在卵黄囊血岛的新生血管及其祖细胞中特异性表达。在成人中，Egfl7在血管生成和动脉损伤过程中上调。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效应功能。在本研究中，我们将验证以下假设：EGFL7是Notch的新型配体，EGFL7作为Notch激动剂发挥作用，以及EGFL7诱导的Notch信号在血管发育和血管生成过程中介导独特且非冗余的过程。我们将在原代人内皮细胞中测试这些假设，并在胚胎干细胞体外分化系统和小鼠中使用功能获得和功能丧失方法。我们提出以下目标：目的1：确定EGFL7在HUVEC和鸡绒毛膜尿囊膜模型中Notch信号传导中的作用。目的2：确定内皮细胞中过度表达Egfl7是否会导致血管发育缺陷。我们将通过产生Tie2-Egfl7转基因小鼠来强制内皮细胞表达Egfl7，并通过产生VE-Cadherin来诱导内皮细胞表达Egfl7:tTA；treegfl7转基因小鼠。目的3：测试Egfl7的功能是否在血管发育的早期阶段至关重要。我们将在胚胎干细胞和小鼠胚胎中产生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.