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Eph B4 Receptor Signaling Mediates Endothelial Cell Migration and Proliferation via the Phosphatidylinositol 3-Kinase Pathway*

  • Jena J. Steinle
    Correspondence
    To whom correspondence should be addressed: Texas A & M University System Health Science Center, 702 SW HK Dodgen Loop, Medical Research Bldg., Rm. 202A, Temple, TX 76504. Tel.: 254-742-7144; Fax: 254-742-7145;
    Affiliations
    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, the Texas A & M University System Health Science Center, Temple, Texas 76504 and
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  • Cynthia J. Meininger
    Affiliations
    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, the Texas A & M University System Health Science Center, Temple, Texas 76504 and
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  • Reza Forough
    Affiliations
    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, the Texas A & M University System Health Science Center, Temple, Texas 76504 and
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  • Guoyao Wu
    Affiliations
    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, the Texas A & M University System Health Science Center, Temple, Texas 76504 and

    Department of Animal Science, Texas A & M University, College Station, Texas 77843
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  • Mack H. Wu
    Affiliations
    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, the Texas A & M University System Health Science Center, Temple, Texas 76504 and
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  • Harris J. Granger
    Affiliations
    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, the Texas A & M University System Health Science Center, Temple, Texas 76504 and
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  • Author Footnotes
    * This work was supported by NHLBI Grant 446221 from the National Institutes of Health (to H. J. G.) and Juvenile Diabetes Research Foundation (JDRF) Grants 2000-437 and 2002-228 (to C. J. M. and G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 13, 2002DOI:https://doi.org/10.1074/jbc.M207221200
      The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63%versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.
      Eph receptors are a family of 14 receptor tyrosine kinases, first noted in the nervous system (
      • Frisen J.
      • Holmberg J.
      • Barbacid M.
      ). They all possess an N-terminal globular domain, which folds into a compact β-sandwich and is necessary for ligand binding (
      • Frisen J.
      • Holmberg J.
      • Barbacid M.
      ). The ligands for the Eph receptors are arranged into two families based upon their attachment to the plasma membrane. Class A ephrins are attached to the outer leaflet of the plasma membrane via a glycosylphosphoinositide anchor. Class B ephrins are transmembrane proteins. Whereas ephrin ligands are insoluble in their natural state, soluble chimeric ephrin B2 ligands are commercially available to use as a tool to probe downstream signaling pathways.
      A role for ephrins in vascular growth and remodeling was first noted when gene knockout studies of Eph B4 or its ligand ephrin B2 resulted in embryonic lethality due to cardiovascular defects (
      • Adams R.H.
      • Klein R.
      ,
      • Wang H.U.
      • Chen Z.F.
      • Anderson D.J.
      ). These malformations involved disrupted angiogenesis of the yolk sac manifested as an apparent block in capillary plexus remodeling. There was limited ingrowth of capillaries into the neural tube, as well as defective interactions between endothelial and supporting cells in mutant mice (
      • Wang H.U.
      • Chen Z.F.
      • Anderson D.J.
      ). Additionally, work in Xenopus laevis using dominant negative Eph B4 receptors or missense expression of ephrin B1 or ephrin B2 demonstrated that interactions between Eph B4 and ephrin ligands are critical regulators of embryonic angiogenesis (
      • Helbling P.M.
      • Saulnier D.M.
      • Brandli A.W.
      ). After the initial studies on knockout mice andXenopus, much work has been done to determine cellular interactions of ephrins with one another (
      • Adams R.H.
      • Klein R.
      ,
      • Daniel T.O.
      • Stein E.
      • Cerretti D.P., St
      • John P.L.
      • Robert B.
      • Abrahamson D.R.
      ) and with other known signaling components (
      • Huynh-Do U.
      • Stein E.
      • Lane A.A.
      • Liu H.
      • Cerretti D.P.
      • Daniel T.O.
      ,
      • Kalo M.S.
      • Pasquale E.B.
      ). Activation of some Eph receptors promotes tube formation in renal endothelial cells but not in umbilical vein endothelial cells, suggesting that Eph receptor activation is specific for different types of endothelial cells (
      • Daniel T.O.
      • Stein E.
      • Cerretti D.P., St
      • John P.L.
      • Robert B.
      • Abrahamson D.R.
      ). Although much work has been done to determine phosphorylation sites for the Eph receptors (
      • Dodelet V.C.
      • Pasquale E.B.
      ), downstream signaling events resulting from receptor activation have received less attention.
      In this study, we first determined whether Eph receptors were present on human mesentery vascular endothelial cells. Once we concluded that these cells possess abundant Eph B4 receptors, we hypothesized that stimulation of Eph B4 receptors with ephrin B2 would result in activation of either the migration or proliferation phase of angiogenesis. Finally, we sought to examine the signaling pathways regulating these angiogenic events.

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