Abstract 14042: Brag2, an Arf6 Activator, Mediates Endocytosis of Beta1-Integrins in Endothelial Cells and Angiogenesis
The molecular mechanisms mediating angiogenesis are not completely understood. ADP-ribosylation factor 6 (ARF6) is a small GTPase activated by distinct guanine nucleotide exchange factors (GEF) and is involved in traffic and cytoskeletal organization in the cell periphery thereby regulating cell motility. Interestingly, the function of Arf6 is dependent on the upstream GEF activating it. Angiogenic factors such as VEGF have been shown to promote Arf6 activity. However, the regulation of Arf6 activity in endothelial cells (EC) is unclear. Recent evidence suggests that Brag2, a GEF for Arf6 GTPase, mediates receptor tyrosine kinase-induced Arf6 activity. In the present work we studied the role of Brag2 in angiogenesis. Human umbilical vein endothelial cells (HUVEC) express Brag2 as assessed by western blot. Silencing of Brag2 with siRNA significantly blocked angiogenic sprouting of HUVEC under basal conditions and after VEGF stimulation (by 40 ± 7 %) in a 3-dimensional spheroidal EC culture system and tube formation in a matrigel assay in comparison to scrambled siRNA-transfected EC. Moreover, silencing of Brag2 with siRNA significantly reduced the VEGF-induced 3-dimensional migration of EC (49 % inhibition), while not affecting proliferation. EC migration is dependent on adhesion to matrix proteins. Interestingly, knock down of Brag2 with siRNA significantly increased EC adhesion on the alpha5beta1-integrin ligand, fibronectin. In line with these results, silencing of Brag2 reduced the endocytosis of beta1-integrins and as consequence increased surface expression of the alpha5beta1-integrin in EC as assessed by FACS. In order to address the role of Brag2 in in vivo angiogenesis, we studied vessel formation in developing zebrafish embryos by employing the morpholino technology. Strikingly, in vivo silencing of the Brag2 homologue in zebrafish with a morpholino disturbed the formation of the parachordal vessels and led to defects in the dorsal longitudinal anastomotic vessel and in the intersomitic vessels. These data reveal for the first time that the Arf6 GEF, Brag2 is essential for in vivo angiogenesis probably by mediating EC migration and angiogenic sprouting through regulation of adhesion by promoting integrin internalization.
- © 2011 by American Heart Association, Inc.