In this new collaborative proposal, we investigate the ability of Rho GTPases to modulate both cytoskeletal and membrane apical-basal polarization to control endothelial cell (EC) tubulogenesis. Our previous studies demonstrated critical roles for Cdc42 and Rasip1 during this process. Using state-of-the art in vitro and in vivo approaches, we have demonstrated a fundamental role for Cdc42 during EC lumen formation using cultured human ECs and during mouse vascular development and blood vessel growth (previous studies and preliminary data shown here). 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 tube networks. In addition, together, we showed that Rasip1, a modulator of small GTPase function, is required for EC tubulogenesis, both in vivo and in vitro. We showed that Rasip1 is required to activate the GTPases Cdc42 and Rac1, as well as to suppress RhoA in ECs. Together, this work underlined the central importance of GTPase activity during blood vessel morphogenesis. We recently screened tubulogenic ECs and identified multiple modulators of GTPase activity as key regulators of this process. We have identified additional GTPases that work with Cdc42 to control vascular tubulogenesis, including Rac isoforms, k-Ras and Rap1. A key functional role of GTPases is to stimulate membrane trafficking from the basal to apical surface through vesicle transport along polarized microtubule tracks (which are enriched in acetylated tubulin and which are present in a subapical membrane domain to promote membrane fusion events at the apical surface). We observe strong enrichment of Rac, activated Src and Rasip1 at EC apical membranes and within vesicles that are being transported apically. We have also discovered novel regulators of tubulogenesis, including the GTPase effectors IQGAP1, MRCK?, and beta-Pix, and critical guanine exchange factors (GEFs) Dock6, Sos1 and FGD5, that activate Cdc42, Rac, and k-Ras during this process. In addition, we have found that Arhgap31 (inactivates Cdc42 and Rac) and Rasa1 (which inactivates Ras) are critical regulators, revealing that in order to properly control and balance EC tubulogenesis, negative regulators are necessary, while the Rasip1-associated Arhgap29, which inactivates Rho, is required for EC tubulogenesis, both in vivo and in vitro. Together, these results provide a molecular road map for how ECs change their shape, polarize and reorient junctions, all with the ultimate goal of forming functional tubes that carry blood, a capacity essential for blood vessel formation and tissue viability. Here, we propose to use genetic and in vitro approaches to dissect how complicated signaling pathways control specific cellular events to support blood vessel morphogenesis. The focus of this proposal is to elucidate the molecular and cellular mechanisms underlying how Cdc42 and other GTPases control cytoskeletal polarization and vesicular trafficking to drive EC morphogenesis and tubulogenesis. We propose three specific aims to examine the process of EC tubulogenesis in vivo and in vitro:
Specific Aim #1. To elucidate how the small Rho GTPase Cdc42 controls EC tubulogenesis via cytoskeletal polarization, apical membrane trafficking and EC junction formation to facilitate multicellular tube assembly and stability.
Specific Aim #2. To determine how Rac-, k-Ras-, and Rap1 interact with Cdc42 during EC tubulogenesis.
Specific Aim #3. To identify and functionally characterize critical GTPase GEFs and GAPs which positively and negatively regulate EC tubulogenesis.

Public Health Relevance

This work focuses on the ability of signaling molecules present within cells to support the formation of blood vessels, which carry blood. We are using cells that line blood vessels, endothelial cells (ECs) in cell culture for our studies, as well as models of blood vessel development using mice. Understanding of these basic processes will lead to new approaches to alleviating 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 #
5R01HL126518-02
Application #
8974855
Study Section
Special Emphasis Panel (ZRG1-CVRS-P (02))
Program Officer
Hanspal, Manjit
Project Start
2014-12-01
Project End
2018-11-30
Budget Start
2015-12-01
Budget End
2016-11-30
Support Year
2
Fiscal Year
2016
Total Cost
$378,505
Indirect Cost
$68,489
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
Liu, Xiaolei; Gu, Xiaowu; Ma, Wanshu et al. (2018) Rasip1 controls lymphatic vessel lumen maintenance by regulating endothelial cell junctions. Development 145:
Barry, David M; Koo, Yeon; Norden, Pieter R et al. (2016) Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II. Circ Res 119:810-26
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
Azizoglu, D Berfin; Cleaver, Ondine (2016) Blood vessel crosstalk during organogenesis-focus on pancreas and endothelial cells. Wiley Interdiscip Rev Dev Biol 5:598-617
Koo, Yeon; Barry, David M; Xu, Ke et al. (2016) Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth. Angiogenesis 19:173-90
Azizoglu, D Berfin; Chong, Diana C; Villasenor, Alethia et al. (2016) Vascular development in the vertebrate pancreas. Dev Biol 420:67-78
Davis, George E; Norden, Pieter R; Bowers, Stephanie L K (2015) Molecular control of capillary morphogenesis and maturation by recognition and remodeling of the extracellular matrix: functional roles of endothelial cells and pericytes in health and disease. Connect Tissue Res 56:392-402
Davis, George E; Cleaver, Ondine B (2014) Outside in: inversion of cell polarity controls epithelial lumen formation. Dev Cell 31:140-2