Priming of vascular tube morphogenesis: Novel role for VEGF and downstreamRhoA activation

在这个新的合作提案中，我们研究了我们关于VEGF作为一种免疫调节剂的能力的新发现。 在小鼠血管形成过程中， 通过检查血管组装，在体外的人EC中进行。Cleaver实验室已经证明， VEGFR 2或RhoA失活，在小鼠中成血管细胞出现时或之前，导致成血管细胞的完全丧失。 EC管形成，而EC管形成后RhoA的破坏导致EC显著增大 管，提示时间上不同的作用（tubulogenesis早期，抑制血管扩大后）。在 此外，Cleaver实验室的新工作表明，Cdc 42在早期或晚期的失活， 血管发育导致EC小管发生的显著缺陷。戴维斯实验室在体内也观察到了同样的现象 使用体外EC小管形成测定法测定表型。最近，戴维斯实验室定义了生长因子 在3D基质中EC小管发生和EC-周细胞管共组装的要求显示SCF，IL-3， SDF-1α、FGF-2和胰岛素（GF）在无血清条件下是这些过程所必需的。 重要的是，VEGF的添加对于这种定义的GF驱动的形态发生过程不是必需的，但它具有深远的意义。 在体内的影响，就像RhoA的影响一样。我们的合作已经形成了一个基本的模式- 转移观察表明VEGF作为上游引物通过RhoA活化来制备 EC/成血管细胞用于下游血管形态发生事件。事实上，内皮细胞的VEGF治疗 引发它们对这些促小管生成GF的反应;这些GF直接刺激EC尖端细胞，EC- 内衬管和周细胞补充到EC管。为了阐明VEGF引发信号，我们表明VEGF 促进RhoA活化，导致肌动蛋白应力纤维的形成，并增加局灶性粘连， FAK和桩蛋白的酪氨酸磷酸化，还激活蛋白激酶D（PKD）和Hsp 27。siRNA 抑制VEGFR 2、RhoA和PKD 2显著干扰VEGF诱导的引发。所有这些 新的见解定义了血管形成过程中的新步骤，EC启动，并提供了分子路线图 剖析VEGF如何通过RhoA激活作为引物来控制血管组装。 我们提出了三个具体的目标，以进一步研究这些新的见解的基本 VEGF诱导的和RhoA依赖的EC在体外和体内的启动过程，它们是： 目的1：检测VEGF依赖的EC信号传导和RhoA激活作为启动的中枢调节因子， in vivo. 目的2：鉴定和表征激活RhoA与VEGFR 2依赖性 信号传导，以便引发EC用于随后的管形态发生事件。 目的3：研究抑制VEGF引发和RhoA激活的基本EC机制， 包括Rasip 1和Arhgap 29的作用，它们是RhoA激活的抑制剂。

In this new collaborative proposal, we investigate our novel findings regarding the ability of VEGF to act as an upstream vascular morphogenic primer through activation of the small GTPase, RhoA, during mouse vascular development and in human ECs in vitro by examining blood vessel assembly. The Cleaver lab has shown that VEGFR2 or RhoA inactivation, at or before the appearance of angioblasts in mice, leads to complete loss of EC tubulogenesis, while disruption of RhoA later after EC tube formation leads to marked enlargement of EC tubes, suggestive of temporally distinct roles (tubulogenesis early, restraint of vessel enlargement later). In addition, new work from the Cleaver lab reveals that inactivation of Cdc42 at early or later time points during vascular development leads to marked defects in EC tubulogenesis. The Davis lab observes the same in vivo phenotypes using in vitro EC tubulogenesis assays. Recently, the Davis lab has defined growth factor requirements for EC tubulogenesis and EC-pericyte tube co-assembly in 3D matrices showing that SCF, IL-3, SDF-1α, FGF-2, and insulin (GFs) are necessary for these processes under serum-free defined conditions. Importantly, VEGF addition is not required for this defined GF-driven morphogenic process, yet it has profound effects in vivo, just like the influence of RhoA. Our collaborative work has led to a fundamental and paradigm- shifting observation demonstrating that VEGF acts as an upstream primer through RhoA activation to prepare ECs/angioblasts for downstream vascular morphogenic events. In fact, VEGF treatment of ECs specifically primes their responses to these pro-tubulogenic GFs; which directly stimulate an increase in EC tip cells, EC- lined tubes and pericyte recruitment to EC tubes. To elucidate VEGF priming signals, we show that VEGF promotes RhoA activation leading to formation of actin stress fibers with increased focal adhesions and tyrosine phosphorylation of FAK and paxillin, and also activates protein kinase D (PKD) and Hsp27. siRNA suppression of VEGFR2, RhoA, and PKD2 markedly interferes with VEGF-induced priming. Together, these new insights define a novel step during blood vessel formation, EC priming, and provide a molecular road map to dissect how VEGF acts as a primer through RhoA activation to control blood vessel assembly. We propose three specific aims to further investigate these novel insights into the fundamental process of VEGF-induced and RhoA-dependent EC priming in vitro and in vivo and they are; Aim1: To test VEGF-dependent EC signaling and RhoA activation as central regulators of priming, in vitro and in vivo. Aim2: To identify and characterize key RhoGEFs which activate RhoA in conjunction with VEGFR2-dependent signaling, in order to prime ECs for subsequent tube morphogenic events. Aim3: To investigate fundamental EC mechanisms that suppress VEGF priming and RhoA activation, including the role of Rasip1 and Arhgap29, which are inhibitors of RhoA activation.