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

在这项新的合作提案中，我们研究了我们关于血管内皮生长因子作为一种 小鼠血管过程中通过激活小GTP酶RhoA的上游血管形态发生引物 通过检测血管组装，在体外培养人内皮细胞。Cleaver实验室已经证明 在小鼠血管母细胞出现时或之前，VEGFR2或RhoA失活会导致完全丧失 EC小管形成后，RhoA的破坏导致EC明显增大 管，提示在时间上不同的作用(早期的管状发生，后来抑制血管的扩大)。在……里面 此外，Cleaver实验室的新工作表明，在早期或稍后的时间点， 血管发育导致内皮细胞小管发生明显缺陷。戴维斯实验室在活体内观察到了同样的情况 使用体外EC小管形成试验的表型。最近，戴维斯实验室定义了生长因子 3D基质中内皮细胞小管形成和周细胞管共组装的要求显示，SCF，IL-3， 在无血清限定的条件下，SDF-1α、成纤维细胞生长因子-2和胰岛素(GFS)是这些过程所必需的。 重要的是，这种明确的GF驱动的形态发生过程并不需要添加VEGF，但它具有深远的意义 体内效应，就像RhoA的影响一样。我们的协作工作导致了一个基本的和范例- 移位观察显示，血管内皮生长因子通过激活RhoA作为上游引物制备 ECS/血管母细胞对下游血管形态发生事件的影响。事实上，血管内皮生长因子对内皮细胞的特异性治疗 启动它们对这些促小管生成的GFS的反应；这些GFS直接刺激EC尖端细胞的增加，EC- 内衬管和周细胞向EC管的募集。为了阐明血管内皮生长因子的启动信号，我们证明了 促进RhoA激活，导致肌动蛋白应激纤维的形成，增加局灶性粘连和 蛋白激酶D(PKD)和热休克蛋白27(Hsp27)也被激活。小干扰RNA VEGFR2、RhoA和PKD2的抑制显著干扰了血管内皮生长因子诱导的启动。加在一起，这些 新的见解定义了血管形成过程中的一个新步骤，即EC启动，并提供了分子路线图 分析血管内皮生长因子是如何通过激活RhoA来调控血管组装的。 我们提出了三个具体目标来进一步研究这些对根本问题的新见解 血管内皮生长因子诱导和RhoA依赖的EC在体外和体内的启动过程，它们是； 目的：检测依赖血管内皮生长因子的EC信号转导和RhoA激活作为启动的中枢调节因子。 在活体内。 目的：鉴定和鉴定激活RhoA和VEGFR2依赖的关键RhoGEF 信号，以便为随后的管状形态发生事件准备内皮细胞。 目的：探讨EC抑制血管内皮生长因子(VEGF)启动和RhoA活化的基本机制。 包括Rasip1和Arhgap29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.