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

血管形态发生的启动：VEGF 和下游 RhoA 激活的新作用

基本信息

批准号：
9753627
负责人：
Ondine B Cleaver
金额：
$ 38.94万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9753627
关键词：
3-Dimensional Actins Address Angioblast Aorta Appearance Automobile Driving Basement membrane Biological Assay Blood Blood Vessels Blood capillaries Cardiovascular Diseases Cell Culture Techniques Cell Line Cells Cues Cyclic AMP Data Defect Deposition Development Diabetes Mellitus Disease Dissection Endothelial Cells Endothelium Event FGF2 gene Fibroblast Growth Factor Focal Adhesions Galium Genetic Models Growth Factor Human In Vitro Insulin Intercellular Junctions Interleukin-3 KDR gene Laboratories Malignant Neoplasms Maps Molecular Monomeric GTP-Binding Proteins Morphogenesis Mus PTK2 gene Pathway interactions Pericytes Phenotype Process Proteins Rho-associated kinase Role Secondary to Serum Signal Transduction Signaling Molecule Small Interfering RNA Stem Cell Factor Stress Fibers Suggestion Testing Time Tissues Tube Tyrosine Phosphorylation Vascular Endothelial Growth Factors Vascular Permeabilities Work blood vessel development cell type human model in vitro Model in vivo inhibitor/antagonist insight interest novel novel strategies paxillin progenitor protein kinase D protein kinase D2 recruit response restraint rho rhoA GTP-Binding Protein three-dimensional modeling

项目摘要

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.

在这个新的合作提案中，我们调查了有关VEGF充当能力的新颖发现小鼠血管中小的GTPase RhoA的激活，上游血管形态发生底漆通过检查血管组装，在体外开发和人类EC中。切肉刀实验室已经表明 VEGFR2或RHOA失活，在小鼠中或血管生成之前或之前，都会完全丧失 EC结节发生，而在EC管形成后稍后的RhoA破坏导致EC的显着膨胀试管，提示暂时不同的作用（早期的管结肠，稍后对血管增大的约束）。此外，切肉刀实验室的新作品表明，在早期或更晚的时间点灭活Cdc42 血管发育导致EC Tuberonesis明显缺陷。戴维斯实验室在体内观察使用体外EC小管发生测定的表型。最近，戴维斯实验室定义了生长因子在3D矩阵中共同组装EC结核病和EC-易期管的要求，表明SCF，IL-3， SDF-1α，FGF-2和胰岛素（GFS）对于这些过程在无血清定义条件下是必需的。重要的是，这种定义的GF驱动的形态学过程并不需要添加VEGF 在体内影响，就像Rhoa的影响一样。我们的协作工作导致了基本和范式 - 转移观察结果表明，VEGF通过RhoA激活充当上游底漆以制备 EC/血管细胞用于下游血管形态发生事件。实际上，vegf对EC的治疗特别素数对这些亲管gfs的反应；直接刺激EC细胞的增加，EC- 衬管和周细胞募集到EC管。为了阐明VEGF启动信号，我们证明了VEGF 促进RhoA激活，从而导致肌动蛋白应激纤维具有增加的局灶性粘合剂和 FAK和PAXILLIN的酪氨酸磷酸化，还激活蛋白激酶D（PKD）和HSP27。 sirna VEGFR2，RHOA和PKD2的抑制显着干扰了VEGF诱导的启动。在一起，这些新见解定义了在血管形成，EC启动过程中的新一步，并提供了分子路线图剖析VEGF如何通过RhoA激活作为底漆来控制血管组装。我们提出了三个特定的目标，以进一步研究这些新颖的见解 VEGF诱导的和RhoA依赖性的EC启动的过程在体外和体内； AIM1：测试依赖VEGF的EC信号传导和RhoA激活作为启动的中心调节剂，体外和体内。 AIM2：识别和表征关键的Rhogefs，与vegfr2依赖性一起激活Rhoa 信号传导，以使EC为随后的管形形态发生事件产生。 AIM3：研究抑制VEGF启动和RhoA激活的基本EC机制，包括RASIP1和ARHGAP29的作用，它们是RhoA激活的抑制剂。