Angiogenesis depends upon EPHB4-mediated export of collagen IV from vascular endothelial cells.

Philip D King,Jiangping Wu,Taija Makinen,Thomas L Saunders,Elizabeth D Hughes,Di Chen,Magda N Hernandez Vasquez

doi:10.1172/jci.insight.156928

Abstract

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.

Highlights

Capillary malformation-arteriovenous malformation (CM-AVM) is an inherited autosomal dominant blood vascular disorder in humans that affects 1:10,000 to 1:100,000 individuals [1,2,3,4]
The same hemorrhagic and edematous phenotype was observed in TM-treated Ephb4fl/fl Ubert2cre embryos that carried distinct Ephb4fl alleles in which exon 1 was flanked by loxP sites (Supplemental Figure 2A, Table 1)
To determine if hemorrhage was associated with the apoptotic death of endothelial cell (EC), tissue sections from Ephb4fl/fl Ubert2cre embryos were stained with antibodies that detect the activated form of caspase 3

Summary

Introduction

Capillary malformation-arteriovenous malformation (CM-AVM) is an inherited autosomal dominant blood vascular disorder in humans that affects 1:10,000 to 1:100,000 individuals [1,2,3,4]. In approximately one third of patients, there are additional life-threatening fast flow blood vascular lesions that include AVM and arteriovenous fistulas. CM-AVM1, which accounts for approximately 50% of cases, is caused by mutations in the RASA1 gene that encodes the RASA1 protein known as p120 Ras GTPase-activating protein (p120 RasGAP) [1,2,3]. In growth factor receptor (GFR) signaling pathways, RASA1 interacts with the active GTP-bound form of the Ras small GTP-binding protein [9, 10]. This interaction increases the ability of Ras to hydrolyze bound GTP to GDP by several orders of magnitude resulting in the conversion of Ras to its inactive GDP-bound state. RASA1 acts as a negative regulator of GFR-induced Ras activation and downstream signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway that couple cell surface GFR-ligand recognition events to cellular outcomes [9, 10]

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Results

Conclusion