Ephrin-B2 induces migration of endothelial cells through the phosphatidylinositol-3 kinase pathway and promotes angiogenesis in adult vasculature

Objective - Ephrin- B2 plays a key role in vascular development. The purpose of this study was to elucidate the molecular mechanisms of ephrin- B2 signaling through the EphB receptor in endothelial cells and to determine whether ephrin- B2 contributes to in vivo angiogenesis in adult mice. Methods and Results - A chemotaxis assay on a polycarbonate membrane revealed that ephrin- B2/ Fc chimeric protein induced migration of human umbilical vein endothelial cells ( HUVECs) at a level 98% greater than control ( P < 0.01). To determine the signaling pathways activated in the HUVECs by Eph stimulation, phosphatidylinositol- 3 kinase ( PI3 kinase) activity was determined in an immune complex PI3 kinase assay. Serum- starved HUVECs were stimulated with ephrin- B2/ Fc and compared with unstimulated cells. PI3 kinase activity in stimulated cells was higher than that seen in unstimulated cells. In a chemotaxis assay, the PI3 kinase- specific inhibitor LY294002 blocked the migratory response of HUVECs induced by addition of ephrin- B2/ Fc. Finally, ephrin- B2/ Fc promoted angiogenesis in vivo in corneal neovascularization and Matrigel plug assays in adult mice, whereas LY294002 reduced angiogenesis in Matrigel that was induced by ephrin- B2/ Fc. Conclusions - Ephrin- B2/ Fc induces the migration of HUVECs through the PI3 kinase signaling pathway. Ephrin- B2/ Fc promotes in vivo angiogenesis in adult mice, suggesting that it contributes to adult angiogenesis.