Capillary malformation-arteriovenous malformation (CM-AVM) is a blood vascular anomaly caused by inherited loss-of-function mutations in RASA1 or EPHB4 genes, which encode p120 Ras GTPase-activating protein (p120 RasGAP/RASA1) and Ephrin receptor B4 (EPHB4). However, whether RASA1 and EPHB4 function in the same molecular signaling pathway to regulate the blood vasculature is uncertain. Here, we show that induced endothelial cell-specific (EC-specific) disruption of Ephb4 in mice resulted in accumulation of collagen IV in the EC ER, leading to EC apoptotic death and defective developmental, neonatal, and pathological angiogenesis, as reported previously in induced EC-specific RASA1-deficient mice. Moreover, defects in angiogenic responses in EPHB4-deficient mice could be rescued by drugs that inhibit signaling through the Ras pathway and drugs that promote collagen IV export from the ER. However, EPHB4-mutant mice that expressed a form of EPHB4 that is unable to physically engage RASA1 but retains protein tyrosine kinase activity showed normal angiogenic responses. These findings provide strong evidence that RASA1 and EPHB4 function in the same signaling pathway to protect against the development of CM-AVM independent of physical interaction and have important implications for possible means of treatment of this disease.

EPHB4 is a receptor protein tyrosine kinase that is required for the development of lymphatic vessel (LV) valves. We show here that EPHB4 is necessary for the specification of LV valves, their continued development after specification, and the maintenance of LV valves in adult mice. EPHB4 promotes LV valve development by inhibiting the activation of the Ras-MAPK pathway in LV endothelial cells (LEC). For LV specification, this role for EPHB4 depends on its ability to interact physically with the p120 Ras-GTPase-activating protein (RASA1) that acts as a negative regulator of Ras. Through physical interaction, EPHB4 and RASA1 dampen oscillatory shear stress (OSS)-induced Ras-MAPK activation in LEC, which is required for LV specification. We identify the Piezo1 OSS sensor as a focus of EPHB4-RASA1 regulation of OSS-induced Ras-MAPK signaling mediated through physical interaction. These findings contribute to an understanding of the mechanism by which EPHB4, RASA1 and Ras regulate lymphatic valvulogenesis.

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and most severe of congenital brain arteriovenous malformations, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-function de novo variants (2042.5-fold, p = 4.79 x 10-7). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (17.5-fold, p = 1.22 x 10-5), which cooperates with p120 RasGAP to regulate vascular development. Additional probands had damaging variants in ACVRL1, NOTCH1, ITGB1, and PTPN11. ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomic analysis defined developing endothelial cells as a likely spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant (Phe867Leu) exhibited disrupted developmental angiogenesis and impaired hierarchical development of arterial-capillary-venous networks, but only in the presence of a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have implications for patients and their families.