EphB ligand, ephrinB2, suppresses the VEGF‐ and angiopoietin‐1‐induced Ras/mitogen‐activated protein kinase pathway in venous endothelial cells

ABSTRACT Interaction between ephrinB2 and EphB4 in endothelial cells at the arterial‐venous capillary interface is critical for proper embryonic capillary morphogenesis. However, the intracellular downstream signaling of ephrinB2‐EphB in vascular endothelial cells is unknown. This study examined the effect of ephrinB2induced activation of EphB kinases on vascular endothelial growth factor (VEGF)‐ and angiopoietin‐1 (Ang1)‐induced Ras/mitogen‐activated protein kinase (MAPK) signaling cascades in human umbilical vein endothelial cells (HUVECs). Reverse transcriptase‐polymer chain reaction results showed that HUVECs expressed three kinds of EphB kinases known to bind to ephrinB2: EphB2, EphB3, and EphB4. EphrinB2 not only increased the phosphorylation of EphB2 and EphB4 in a time‐dependent manner but also increased recruitment of p120‐Ras‐GTPase‐activating protein (p120‐RasGAP) to EphB2 and EphB4. Accordingly, ephrinB2 inhibited VEGF‐ and Ang1‐induced Ras‐MAPK activities, whereas ephrinB2 did not alter VEGF‐induced Flk phosphorylation or Ang1‐induced Tie2 phosphorylation. Furthermore, ephrinB2 suppressed VEGF‐ and Ang1‐induced proliferation and/or migration, which are mediated mainly through Ras/MAPK signaling cascades. From these results, we propose that ephrinB2‐EphB, signaling through Ras/MAPK cascade, may be critical for proper morphogenesis of capillary endothelium through the arrest of endothelial cell proliferation and migration at the arterial‐venous interface.