Mechanisms of rhoa signaling in regulating vascular integrity and development

The National Heart, Lung, and Blood Institute awarded Western Washington University $460,658 on September 1, 2026, under the Cardiovascular Diseases Research program (CFDA 93.837) to characterize the role of RhoA signaling in regulating cranial vascular integrity and development.

The research will use zebrafish models with RhoA mutants and endothelial cell-specific gain-and-loss-of-function transgenic embryos exhibiting intracranial hemorrhage phenotypes to determine how RhoA and its effectors, including the Rho-associated protein kinases ROCK1/2, orchestrate endothelial cell activities that modulate vascular barrier function and blood vessel patterning. The work addresses the molecular and genetic mechanisms regulating cranial vascular integrity, with clinical relevance to hemorrhagic stroke, which caused 2.9 million deaths globally in 2019.

Performance period runs through May 31, 2029, with work conducted in Bellingham, Washington. This is a Project Grant, a standard research assistance mechanism. The award obligates the full $460,658 amount at present.

Abstract

Endothelial cells (ECS) form the innermost layer of all blood vessels and serve as a physical barrier to regulate vessel integrity and permeability. Disruption of the brain's EC barrier leads to hemorrhagic stroke, producing a spectrum of disease that includes neurologic dysfunction, disability, cognitive impairment, or death. Hemorrhagic stroke was the reported cause of death for 2.9 million people in 2019. IT is therefore of paramount clinical interest to define the molecular and genetic mechanisms that regulate cranial vascular integrity. Towards this long-term research goal, this proposal will characterize the role of the small, monomeric gtpase rhoa in regulating cranial vascular integrity and development. Rhoa acts as a master regulator in signal transduction cascades by activating effectors of cellular signaling, including the RHO-associated protein kinases ROCK1/2. Through its effectors, rhoa regulates several critical aspects of cellular biology including cytoskeletal dynamics, focal adhesion, and junctional remodeling - processes that modulate vascular integrity and development. Previous studies performed in vitro, in cell culture have ascribed both EC barrier-protective and EC barrier-disruptive functions to rhoa, making IT and its effectors attractive targets for vascular integrity studies and/or clinical therapies. However, the precise in vivo functions of rhoa in regulating blood vessel development and integrity are almost completely uncharacterized and, from a molecular perspective, IT is unclear how rhoa and its effectors orchestrate context-specific, highly coordinated, and contrasting EC activities. Aim 1 will use a zebrafish rhoa orthologue (RHOAA) mutant and EC-specific gain- and loss- of rhoa activity transgenic zebrafish embryos that exhibit dramatic intracranial hemorrhage phenotypes and vascular patterning defects to determine how rhoa regulates vascular integrity and development in vivo. Aim 2 will determine the in vivo functions of select rhoa-effectors (ROCK1, ROCK2, and additional effectors identified through preliminary analyses of human ECS) in regulating vascular integrity and development. Finally, aim 3 will use mass spectrometry-based proteomic analyses to identify proximal interacting proteins of rhoa, ROCK1, and ROCK2 in ECS. Combined, these aims are designed to expand our current knowledge of how rhoa signaling regulates vascular integrity and development. Undergraduate student researchers will participate in all aspects of this project, including experiment design, data collection, and dissemination of results. As rhoa/ROCK1/2 have been implicated in hemorrhagic stroke, cerebral ischemia, cerebral vasospasm, cerebral cavernous malformations, and malignant vascular tumors, this proposed work is expected to lead to the development of more specific and effective therapeutics for treating or preventing hemorrhagic stroke and other rhoa-associated vascular diseases. By identifying novel or uncharacterized regulators of EC morphogenesis, this proposal will also provide a foundation for future molecular and genetic studies of cranial vascular integrity and development.