Abstract 18516: Brag2, an Arf-GEF, Regulates Integrin-Dependent Endothelial Adhesion and is Involved in Developmental and Pathological Angiogenesis
β1-integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor (GEF) for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated silencing of Brag2 significantly blocked basal and VEGF-induced angiogenic sprouting (by 40 ± 7 %), tube formation and migration of HUVEC (49 % inhibition). Transfection with Brag2-siRNA differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and EC adhesion on β1-integrin-ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin-ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2 mediated recycling of αVβ3-integrins and endocytosis of β1-integrins and specifically of the active/matrix bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin-endocytosis. Furthermore, we found that Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin-endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Moreover, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.
- © 2013 by American Heart Association, Inc.