RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity
Jun Kawasaki, … , Steven J. Fishman, Joanne Chan
Jun Kawasaki, … , Steven J. Fishman, Joanne Chan
Published May 16, 2014
Citation Information: J Clin Invest. 2014;124(6):2774-2784. https://doi.org/10.1172/JCI67084.
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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 6

Overactive endothelial mTORC1 is consistently found in RASA1 mutation–positive patient tissues.

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Overactive endothelial mTORC1 is consistently found in RASA1 mutation–po...
Representative images of immunostaining with CD31 (PECAM-1, marker for endothelial cells), p-S6 (S235/236; marker for mTORC1 activation) or p-ERK1/2 (marker for increased RAF-MEK activation) from healthy tissue (A) or resected CM-AVMs from patients with RASA1 mutation (B and C). Black arrowheads indicate positive endothelial staining (overlapped staining with CD31); red arrowheads indicate no endothelial staining. (D–F) CM-AVM patient no. 1 (CM-AVM no. 1; B) had capillary stain on the back (D, enlarged in E) overlying a spinal cord AVM (F, sutures holding back open dura mater exposing AVM on spinal cord; photo, courtesy of Edward Smith, Boston Children’s Hospital). (G) CM-AVM patient no. 2 (CM-AVM no. 2; C) had CM-AVM on the lip. Scale bars: 50 μm (A–C).