βPix plays a dual role in cerebral vascular stability and angiogenesis, and interacts with integrin αvβ8

The growth of new blood vessels by angiogenesis and their stabilization by the recruitment of perivascular mural cells are thought to be two sequential, yet independent events. Here we identify molecular links between both processes through the beta Pix and integrin alpha(v)beta(8) proteins. Bubblehead (bbh) mutants with a genetic mutation in beta Pix show defective vascular stabilization. beta Pix is a guanine nucleotide exchange factor and scaffold protein that binds many proteins including Git1, which bridges beta Pix to integrins at focal adhesions. Here we show that the ability of beta Pix to stabilize vessels requires Git1 binding residues. Knockdown of Git1 leads to a hemorrhage phenotype similar to loss of integrin alpha(v), integrin beta(8) or beta Pix, suggesting that vascular stabilization through beta Pix involves interactions with integrins. Furthermore, double loss of function of beta Pix and integrin alpha(v) shows enhanced hemorrhage rates. Not only is vascular stability impaired in these embryos, but we also uncover a novel role of both beta Pix and integrin alpha(v)beta(8) in cerebral angiogenesis. Downregulation of either beta Pix or integrin alpha(v)beta(8) results in fewer and morphologically abnormal cerebral arteries penetrating the hindbrain. We show that this is coupled with a significant reduction in endothelial cell proliferation in bbh mutants or integrin alpha(v)beta(8) morphants. These data suggest that a complex involving beta Pix, GIT1 and integrin alpha(v)beta(8) may regulate vascular stability, cerebral angiogenesis and endothelial cell proliferation in the developing embryo. (C) 2011 Elsevier Inc. All rights reserved.