on September 18, 2003
Published online before print September 18, 2003, doi: 10.1161/01.ATV.0000096655.56262.56
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Submitted on August 13, 2003
Accepted on August 26, 2003
Ephrin-B2 Induces Migration of Endothelial Cells Through the Phosphatidylinositol-3 Kinase Pathway and Promotes Angiogenesis in Adult Vasculature
Hiromitsu Maekawa ;
From the Department of Cell Differentiation (H.M., Y.O., Y.I., Y.Y., T.S.), The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo; Department of Cardiovascular Medicine (H.M., R.N.), Graduate School of Medicine, The University of Tokyo, Tokyo; Department of Molecular Microbiology and Immunology (S.K.), Division of Endothelial Cell Biology, Nagasaki University Graduate School of Medicine, Nagasaki; and Department of Embryogenesis (H.M., H.K.) Division of Integrative Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
* To whom correspondence should be addressed. E-mail: sudato{at}sc.itc.keio.ac.jp.
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.
Key words: ephrin-B2 • angiogenesis • phosphatidylinositol-3 kinase • migration • endothelial cells
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