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-1?, 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.

Public Health Relevance

This work focuses on the ability of signaling molecules inside and outside the endothelial cell (EC) to support the formation of blood vessels that carry blood. We use ECs in cell culture for our studies, as well as genetic models of blood vessel development using mice. Understanding basic processes and identifying critical molecules and events driving blood vessel formation will lead to new approaches to alleviate blood vessel malfunctions in disease states such as cancer, cardiovascular disease, and diabetes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL136139-01
Application #
9262019
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Gao, Yunling
Project Start
2017-09-01
Project End
2021-06-30
Budget Start
2017-09-01
Budget End
2018-06-30
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Missouri-Columbia
Department
Pharmacology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Kim, Dae Joong; Norden, Pieter R; Salvador, Jocelynda et al. (2017) Src- and Fyn-dependent apical membrane trafficking events control endothelial lumen formation during vascular tube morphogenesis. PLoS One 12:e0184461