Abstract
β1-integrins are essential for angiogenesis. However, the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis are unclear. Brag2 is a guanine nucleotide exchange factor for the small GTPase ARF6, which is involved in the traffic of receptors and in the regulation of actin cytoskeleton. Here, we demonstrate that siRNA-mediated silencing of Brag2 reduced basal and VEGF-induced activation of Arf6 and significantly blocked basal and VEGF-induced angiogenic sprouting (by 40 ± 7 %), tube formation and 3-dimensional migration of EC. Transfection with Brag2 siRNA increased focal adhesions and enhanced adhesion of EC on β1-integrin ligands, such as fibronectin and collagen. In line with these results, silencing of Brag2 significantly increased the surface expression of the α5β1-integrin. Moreover, silencing of Brag2 significantly reduced the endocytosis of β1-integrins and specifically of the active (matrix bound) α5β1-integrin present in focal adhesions, suggesting that Brag2 may contribute to the disassembly of focal adhesions during migration via β1-integrin-endocytosis. Strikingly, in vivo silencing of the Brag2 orthologues in zebrafish embryos with morpholinos perturbed vascular development by leading to defects of the parachordal vessels, the dorsal longitudinal anastomotic vessel and the intersomitic vessels. Beyond developmental angiogenesis, in vivo silencing of Brag2 with intravitreal injection of shRNA significantly reduced pathological retinal neovascularization in the model of retinopathy of prematurity (32 % inhibition) and pathological choroidal neovascularization in a model of laser-induced choroidal neovascularization. These data reveal that the Arf6 GEF, Brag2 is essential for developmental and pathological angiogenesis probably by mediating EC migration and angiogenic sprouting through regulation of adhesion by promoting integrin internalization.
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