RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity
Jun Kawasaki, Sandrine Aegerter, R. Dawn Fevurly, Akiko Mammoto, Tadanori Mammoto, Mustafa Sahin, John D. Mably, Steven J. Fishman, Joanne Chan
Jun Kawasaki, Sandrine Aegerter, R. Dawn Fevurly, Akiko Mammoto, Tadanori Mammoto, Mustafa Sahin, John D. Mably, Steven J. Fishman, Joanne Chan
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Research Article Vascular biology

RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity

Abstract

Vascular malformations are linked to mutations in RAS p21 protein activator 1 (RASA1, also known as p120RasGAP); however, due to the global expression of this gene, it is unclear how these mutations specifically affect the vasculature. Here, we tested the hypothesis that RASA1 performs a critical effector function downstream of the endothelial receptor EPHB4. In zebrafish models, we found that either RASA1 or EPHB4 deficiency induced strikingly similar abnormalities in blood vessel formation and function. Expression of WT EPHB4 receptor or engineered receptors with altered RASA1 binding revealed that the ability of EPHB4 to recruit RASA1 is required to restore blood flow in EPHB4-deficient animals. Analysis of EPHB4-deficient zebrafish tissue lysates revealed that mTORC1 is robustly overactivated, and pharmacological inhibition of mTORC1 in these animals rescued both vessel structure and function. Furthermore, overexpression of mTORC1 in endothelial cells exacerbated vascular phenotypes in animals with reduced EPHB4 or RASA1, suggesting a functional EPHB4/RASA1/mTORC1 signaling axis in endothelial cells. Tissue samples from patients with arteriovenous malformations displayed strong endothelial phospho-S6 staining, indicating increased mTORC1 activity. These results indicate that deregulation of EPHB4/RASA1/mTORC1 signaling in endothelial cells promotes vascular malformation and suggest that mTORC1 inhibitors, many of which are approved for the treatment of certain cancers, should be further explored as a potential strategy to treat patients with vascular malformations.

Authors

Jun Kawasaki, Sandrine Aegerter, R. Dawn Fevurly, Akiko Mammoto, Tadanori Mammoto, Mustafa Sahin, John D. Mably, Steven J. Fishman, Joanne Chan

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Figure 3

RASA1 participates in arteriovenous connections during vessel formation.

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RASA1 participates in arteriovenous connections during vessel formation....
(A–D) Arteriovenous connection assay was performed at 48 hpf. Balanced proportion of arterial (A, red) and venous (V, blue) ISVs in control embryo is shown (A). More venous ISVs were formed in rasa1a (750 μM; B) or ephb4a (500 μM, C) MO–injected embryos. Graph indicates the percentage increase in venous ISV over the balanced ratio (D; n = 20 per condition, mean ± SEM from 3 independent experiments; **P < 0.01. DA, dorsal aorta; PCV, posterior cardinal vein. (E–J) Synergistic experiment was performed using subthreshold, low doses of MOs. Representative images of control (E) and embryos injected with low dose ephb4a (150 μM; F) or rasa1a (250 μM; G) MO show no phenotype. Augmented caudal vascular defects with coinjection of low-dose ephb4a, rasa1a, or efnb2a (250 μM) MO are also shown (H–J). Red arrows indicate enlarged caudal vessels. Scale bars: 100 μm (A–C); 500 μm (E–J).