title: Eph-B4 mediates vein graft adaptation by regulation of endothelial nitric oxide synthase.
abstract: OBJECTIVE
  Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor 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 through regulation of downstream eNOS activity.
  
  
  METHODS
  Mouse lung endothelial cells were stimulated with ephrin-B2/Fc, without and with preclustering, without and with the eNOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride or the Eph-B4 inhibitor NVP-BHG712, and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. Nitric oxide (NO) production was assessed using an NO-specific chemiluminescence analyzer. Cell migration was assessed using a Transwell assay. 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 or eNOS knockout mice.
  
  
  RESULTS
  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 (n = 3; P < .01) as well as increased endothelial cell migration (n = 6; P < .05) in an eNOS-dependent fashion. Both human and mouse vein grafts showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS knockout mice showed less dilation and less wall thickening compared with wild-type vein grafts (n = 7; P < .05).
  
  
  CONCLUSIONS
  eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation in vitro and may mediate vein graft adaptation by regulation of eNOS activity in vivo.
authors.affiliation: Department of Vascular Surgery, The Second Xiangya Hospital of Central South University, Changsha, China; Vascular Biology and Therapeutics Program and Department of Surgery, Yale University School of Medicine, New Haven, Conn.
authors.forename: Mo
authors.initials: M
authors.lastname: Wang
authors.affiliation: Vascular Biology and Therapeutics Program and Department of Surgery, Yale University School of Medicine, New Haven, Conn.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Collins
authors.affiliation: Vascular Biology and Therapeutics Program and Department of Surgery, Yale University School of Medicine, New Haven, Conn.
authors.forename: Trenton R
authors.initials: TR
authors.lastname: Foster
authors.affiliation: Vascular Biology and Therapeutics Program and Department of Surgery, Yale University School of Medicine, New Haven, Conn.
authors.forename: Hualong
authors.initials: H
authors.lastname: Bai
authors.affiliation: Vascular Biology and Therapeutics Program and Department of Surgery, Yale University School of Medicine, New Haven, Conn.
authors.forename: Takuya
authors.initials: T
authors.lastname: Hashimoto
authors.affiliation: Vascular Biology and Therapeutics Program and Department of Surgery, Yale University School of Medicine, New Haven, Conn.
authors.forename: Jeans M
authors.initials: JM
authors.lastname: Santana
authors.affiliation: Department of Vascular Surgery, The Second Xiangya Hospital of Central South University, Changsha, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Shu
authors.affiliation: Vascular Biology and Therapeutics Program and Department of Surgery, Yale University School of Medicine, New Haven, Conn; Department of Surgery, VA Connecticut Healthcare System, West Haven, Conn. Electronic address: alan.dardik@yale.edu.
authors.forename: Alan
authors.initials: A
authors.lastname: Dardik
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D036388:Ephrin-B2 / Q000494:pharmacology; D005838:Genotype; D006801:Humans; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D036224:Receptor, EphB4 / Q000378:metabolism*; D012501:Saphenous Vein / Q000201:enzymology / Q000473:pathology / Q000637:transplantation*; D015398:Signal Transduction; D013997:Time Factors; D066253:Vascular Remodeling; D014682:Vena Cava, Inferior / Q000187:drug effects / Q000201:enzymology / Q000473:pathology / Q000637:transplantation*
chemical_list: D004791:Enzyme Inhibitors; D036388:Ephrin-B2; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C086681:Ephb4 protein, mouse; D036224:Receptor, EphB4; D019331:NG-Nitroarginine Methyl Ester
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