In this revised, collaborative renewal proposal, we investigate our novel findings regarding the ability of Rho, Ras and Rab GTPases to modulate cytoskeletal and membrane apical-basal polarization as well as vesicle trafficking to the apical membrane surface to control human endothelial cell (EC) tubulogenesis. Using state-of- the art in vitro and in vivo approaches, we have demonstrated a fundamental role for Cdc42 during this process with human ECs and during mouse vascular development and blood vessel growth. Inactivation of Cdc42 function leads to disruption of EC polarization in vivo and in vitro that results in a failure to properly form or organize EC lumen and tube networks. In addition, RhoA inactivation in vivo vs. in vitro does the opposite of Cdc42, where increased lumen formation occurs. Importantly, we have shown that Rasip1 and its binding partner, Arhgap29, function together to suppress RhoA signaling to allow EC tube formation to occur. To further investigate this process, we have developed a highly defined approach to elucidate when and where particular molecules and signaling pathways act. We can directly assess whether individual molecules or signals separately control intracellular vacuole formation, cytoskeletal polarization, vacuole trafficking along the tubulin cytoskeleton toward the apical surface, or vacuole fusion in the subapical region to create the apical membrane. We can perform these studies due to our ability to regulate the expression or activity of key molecules coupled with the ability to visualize intracellular vacuoles, the polarized cytoskeleton and the apical surface (in static or real-time video images) using EC apical labels such as GFP-caveolin1. Together, these results provide a molecular road map to elucidate how ECs change shape, polarize, reorient junctions, and move membranes to the apical surface; all with the ultimate goal of forming functional tubes that carry blood, a capacity essential for blood vessel formation, tissue viability, and tissue development. Here, we test the hypothesis that EC lumen formation depends on GTPase signaling cascades that promote the intracellular transport of membranes to the apical surface and polarized subcellular recruitment of critical effectors. We propose three specific aims to further investigate these novel insights into the fundamental process of EC tubulogenesis in vivo and in vitro and they are:
Aim #1. To elucidate the underlying molecules and mechanisms responsible for suppression of RhoA at the apical membrane in ECs;
Aim #2. To investigate Rab GTPase control of vacuole/vesicle formation, trafficking and fusion, leading to polarized EC apical membrane assembly and lumen formation;
Aim #3. To investigate the specific roles of key upstream guanine exchange factors (GEFs) vs. GTPase activating proteins (GAPs) regulating Cdc42, Rac, k-Ras and Rap1b during EC lumen formation.

Public Health Relevance

This work focuses on elucidating mechanisms that drive vascular progenitor cells to generate functional lumens, which can carry blood. Our collaborative team use cells that line blood vessels, termed endothelial cells (ECs), in cell culture as well as in mice, as model systems to study blood vessel formation. These studies will help identify new targets that may be targeted to help 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 #
5R01HL126518-06
Application #
10085257
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Ochocinska, Margaret J
Project Start
2014-12-01
Project End
2023-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
6
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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Norden, Pieter R; Kim, Dae Joong; Barry, David M et al. (2016) Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1. PLoS One 11:e0147758
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