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，我们研究了有关 VEGF 充当细胞因子的能力的新发现。在小鼠血管形成过程中，通过激活小 GTP 酶 RhoA，上游血管形态发生引物 Cleaver 实验室通过检查血管组装表明了这一点。 VEGFR2 或 RhoA 失活，在小鼠成血管细胞出现时或出现之前，导致完全丧失 EC 管发生，而 EC 管形成后 RhoA 的破坏导致 EC 显着增大管，提示在时间上不同的作用（早期的管形成，后来的血管扩张的抑制）。此外，Cleaver 实验室的新工作表明，Cdc42 在早期或后期的时间点失活血管发育导致 EC 管发生的明显缺陷戴维斯实验室在体内观察到同样的情况。最近，戴维斯实验室定义了生长因子。 3D 矩阵中 EC 管发生和 EC-周细胞管共组装的要求表明 SCF、IL-3、在无血清限定条件下，SDF-1α、FGF-2 和胰岛素 (GF) 对于这些过程是必需的。重要的是，这种定义的 GF 驱动的形态发生过程不需要添加 VEGF，但它具有深远的意义就像 RhoA 的影响一样，我们的合作工作产生了一个基本的范式—— 移动观察表明 VEGF 通过 RhoA 激活作为上游引物来制备 ECs/成血管细胞用于下游血管形态发生事件事实上，VEGF 专门针对 ECs 进行治疗。启动它们对这些促肾小管生长的 GF 的反应，直接刺激 EC 尖端细胞的增加，EC- 为了阐明 VEGF 启动信号，我们展示了 VEGF 的内衬管和周细胞募集。促进 RhoA 激活，从而形成肌动蛋白应力纤维，增加粘着斑和 FAK 和桩蛋白的酪氨酸磷酸化，还激活蛋白激酶 D (PKD) 和 Hsp27。 VEGFR2、RhoA 和 PKD2 的抑制显着干扰 VEGF 诱导的启动。新见解定义了血管形成、EC 启动过程中的一个新步骤，并提供了分子路线图剖析 VEGF 如何作为引物通过 RhoA 激活来控制血管组装。我们提出了三个具体目标，以进一步研究这些对基本原理的新颖见解 VEGF 诱导的和 RhoA 依赖性 EC 启动的体外和体内过程；目的 1：测试 VEGF 依赖性 EC 信号传导和 RhoA 激活作为启动、体外和体内。目标 2：识别和表征与 VEGFR2 依赖性结合激活 RhoA 的关键 RhoGEF 信号传导，以便为随后的管形态发生事件准备 EC。目标 3：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。