Ephrin-B2 Induces Migration of Endothelial Cells Through the Phosphatidylinositol-3 Kinase Pathway and Promotes Angiogenesis in Adult Vasculature

Abstract

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 (PϽ0.01).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. Key Words: ephrin-B2 Ⅲ angiogenesis Ⅲ phosphatidylinositol-3 kinase Ⅲ migration Ⅲ endothelial cells E ph receptor tyrosine kinases (RTKs) constitute the largest known family of RTKs identified, consisting of at least 14 receptors and 8 ephrin ligands. 1,2 Unlike most soluble RTK ligands, ephrins are membrane-attached cell surface molecules. 1 It has been shown that the ephrins and Ephs play essential roles in vascular development during embryogenesis. 3 Targeted mutation of ephrin-B2 in mice results in embryonic lethality at embryonic day 10.5 in homozygous nulls. 3 In these mice, vascular development is arrested at the primary plexus stage, and features of angiogenic remodeling are not observed. 3 Mouse embryos lacking EphB4 essentially phenocopy defects observed in ephrin-B2-null mice, suggesting reciprocal functions for the 2 molecules during vascular development in the embryo. 4 By contrast, little is known about the function of ephrin-B2 in the adult vasculature. Expression studies indicate that the endothelium of a subset of new vessels strongly expresses ephrin-B2 in adult angiogenesis as in tumor angiogenesis and in the female reproductive system, 5 suggesting that ephrin-B2 may play a role in adult angiogenesis. Here, by using in vivo model of angiogenesis in adult mice, we ask whether ephrin-B2 has an angiogenic effect. Conclusions-Ephrin-B2/FcSeveral proteins function in the Eph signaling pathway. Among these, phosphatidylinositol-3 (PI3) kinase, Grb2, Grb10, Nck, RasGAP, and Src are implicated in regulating cell morphology, attachment, and motility. 2,6,7 However, the molecular mechanism of angiogenesis induced by Eph activation has not been elucidated. In this study, we demonstrate that ephrin-B2 induces angiogenesis in vivo and that the PI3 kinase signaling pathway contributes to angiogenic ev...

Altered Gene Expression Related to Glomerulogenesis and Podocyte Structure in Early Diabetic Nephropathy of db/db Mice and Its Restoration by Pioglitazone

“…Although the pathophysiological significance of the kidney development-related genes we examined is not fully clarified in diabetic nephropathy, ephrin B2, HIF 1␣, Pod-1, and GLEPP1 participate in various stages and aspects of glomerulogenesis and are relevant to some types of glomerular injury, as previously suggested (26). Ephrin B2 is a transmembrane ligand of the ephrin B2 receptor (Eph) and its signaling pathway is required for vascular morphogenesis (36,37) and glomerular microvascular assembly (27). HIF-1␣ is critical for renal vasculogenesis and glomerulogenesis (30), and nuclear localization of HIF-1␣ increases in murine adriamycin nephrosis (38).…”

Receptor Tyrosine Kinase EphB4 Is a Survival Factor in Breast Cancer

“…9,12,14 -19 Reverse signaling via EphrinB2 stimulates migration and sprouting angiogenesis. 20 Growing evidence points to the expression of several Eph receptors in different cancer types. [21][22][23][24][25][26] The biological significance of this aberrant expression, however, is controversial.…”

Eph-B4 Mediates Vein Graft Adaptation By Regulation of eNOS

“…11,23 However, the regulation of successful vein graft adaptation to the arterial circulation, with just the "right amount" of wall thickening and dilation, without progression to neointimal hyperplasia and/or aneurysmal dilation, is not currently understood. 24 Downstream regulators of Eph-B4 signaling in the endothelial cell include Akt, ERK1/2 25,26 and caveolin-1. 8 Our finding that eNOS is phosphorylated with stimulation of Eph-B4 in adult venous endothelial cells is consistent with previous reports that have shown that activation of Eph-B4 enhances NO production in endothelial cells in vitro.…”