Afadin Facilitates Vascular Endothelial Growth Factor–Induced Network Formation and Migration of Vascular Endothelial Cells by Inactivating Rho-Associated Kinase Through ArhGAP29

Abstract

Objective— We previously reported that afadin, an actin filament-binding protein, regulated vascular endothelial growth factor–induced angiogenesis. However, the underlying molecular mechanisms are poorly understood. Here, we investigated the mechanisms of how Rho-associated kinase is activated in afadin-knockdown human umbilical vein endothelial cells (HUVECs) and how its activation is involved in defects of vascular endothelial growth factor–induced network formation and migration of the cells. Approach and Results— Knockdown of afadin or ArhGAP29, a GTPase-activating protein for RhoA, increased Rho-associated kinase activity and reduced the vascular endothelial growth factor–induced network formation and migration of cultured HUVECs, accompanied by the defective formation of membrane protrusions, such as lamellipodia and peripheral ruffles. Treatment of the afadin- or ArhGAP29-knockdown HUVECs with Rho-associated kinase inhibitors, Y-27632 or fasudil, partially restored the reduced network formation and migration as well as the defective formation of membrane protrusions. ArhGAP29 bound to afadin and was colocalized with afadin at the leading edge of migrating HUVECs. The defective formation of membrane protrusions in ArhGAP29-knockdown HUVECs was restored by expression of mutant ArhGAP29 that bound to afadin and contained a RhoGAP domain but not mutant ArhGAP29 that could bind to afadin and lacked the RhoGAP domain or mutant ArhGAP29 that could not bind to afadin and contained the RhoGAP domain. This suggested the requirement of both the interaction of afadin with ArhGAP29 and RhoGAP activity of ArhGAP29 for migration of HUVECs. Conclusions— Our results highlight a critical role of the afadin–ArhGAP29 axis for the regulation of Rho-associated kinase activity during vascular endothelial growth factor–induced network formation and migration of HUVECs.