# Molecular regulation of Rho and Ras family GTPase activity controls vascular lumen formation.

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2021 · $391,458

## Abstract

SUMMARY
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.

## Key facts

- **NIH application ID:** 10085257
- **Project number:** 5R01HL126518-06
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Ondine B Cleaver
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $391,458
- **Award type:** 5
- **Project period:** 2014-12-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10085257

## Citation

> US National Institutes of Health, RePORTER application 10085257, Molecular regulation of Rho and Ras family GTPase activity controls vascular lumen formation. (5R01HL126518-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10085257. Licensed CC0.

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