EPHB4-RASA1 Inhibition of PIEZO1 Ras Activation Drives Lymphatic Valvulogenesis

Authors

Di Chen
Yipei Tang
Philip E Lapinski
David Wiggins
Eva M Sevick
Michael J Davis
Philip D King

Publication Date

11-8-2024

Journal

Circulation Research

DOI

10.1161/CIRCRESAHA.124.325383

PMID

39421925

PMCID

PMC11560524

PubMedCentral® Posted Date

11-8-2025

PubMedCentral® Full Text Version

Post-print

Abstract

BACKGROUND: EPHB4 (ephrin receptor B4) and the RASA1 (p120 Ras GTPase-activating protein) are necessary for the development of lymphatic vessel (LV) valves. However, precisely how EPHB4 and RASA1 regulate LV valve development is unknown. In this study, we examine the mechanisms by which EPHB4 and RASA1 regulate the development of LV valves.

METHODS: We used LV-specific inducible EPHB4-deficient mice and EPHB4 knockin mice that express a form of EPHB4 that is unable to bind RASA1 yet retains protein tyrosine kinase activity (EPHB4 2YP) to study the role of EPHB4 and RASA1 in LV valve development in the embryo and LV valve maintenance in adults. We also used human dermal lymphatic endothelial cells in vitro to study the role of EPHB4 and RASA1 as regulators of LV valve specification induced by oscillatory shear stress, considered the trigger for LV valve specification in vivo.

RESULTS: LV valve specification, continued valve development postspecification, and LV valve maintenance were blocked upon induced loss of EPHB4 in LV. LV valve specification and maintenance were also impaired in EPHB4 2YP mice. Defects in LV valve development were reversed by inhibition of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. In human dermal lymphatic endothelial cells, loss of expression of EPHB4 or its ephrin b2 ligand, loss of expression of RASA1, and inhibition of physical interaction between EPHB4 and RASA1 resulted in dysregulated oscillatory shear stress-induced Ras-MAPK activation and impaired expression of LV specification markers that could be rescued by Ras-MAPK pathway inhibition. The same results were observed when human dermal lymphatic endothelial cells were stimulated with the Yoda1 agonist of the PIEZO1 oscillatory shear stress sensor. Although Yoda1 increased the number of LV valves when administered to wild-type embryos, it did not increase LV valve number when administered to EPHB4 2YP embryos.

CONCLUSIONS: EPHB4 is necessary for LV valve specification, continued valve development postspecification, and valve maintenance. LV valve specification requires physical interaction between EPHB4 and RASA1 to limit activation of the Ras-MAPK pathway in lymphatic endothelial cells. Specifically, EPHB4-RASA1 physical interaction is necessary to dampen Ras-MAPK activation induced through the PIEZO1 oscillatory shear stress sensor. These findings reveal the mechanism by which EPHB4 and RASA1 regulate the development of LV valves.

Keywords

Receptor, EphB4, Animals, Humans, Mice, p120 GTPase Activating Protein, Lymphatic Vessels, Ion Channels, Endothelial Cells, Mice, Knockout, Cells, Cultured, Lymphangiogenesis, Mice, Inbred C57BL

Published Open-Access

yes

Recommended Citation

Chen, Di; Tang, Yipei; Lapinski, Philip E; et al., "EPHB4-RASA1 Inhibition of PIEZO1 Ras Activation Drives Lymphatic Valvulogenesis" (2024). Faculty, Staff and Student Publications. 284.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/284