Eph-B4 Mediates Vein Graft Adaptation By Regulation of eNOS

Abstract

Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor ephrin type-B receptor 4 (Eph-B4), the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be via regulation of downstream eNOS activity. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type (WT) or eNOS-knockout (KO) mice. Mouse endothelial cells (EC) were stimulated with ephrin-B2/Fc without and with preclustering, without and with L-NAME (1 mM), and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. NO production was assessed using a NO-specific chemiluminescence analyzer. Cell migration was assessed using a transwell assay. Human and mouse vein grafts both showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS-KO mice showed less dilation and less wall thickening compared with WT vein grafts (n = 7; P < .05). Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n = 4; P < .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n = 4; P < .05). Ephrin-B2/Fc increased NO release from ECs (n = 3; P < .01) as well as a trend towards increased EC migration (n = 6; P = .11) in an eNOS-dependent fashion. eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation and mediates vein graft adaptation by regulation of eNOS activity.