April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
EphrinB2-EphB4 Signals Control Lymphatic Valve Formation in Alkali Burn-Induced Corneal Lymphangiogenesis
Author Affiliations & Notes
  • Y. Fukushima
    Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  • F. Gomi
    Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  • S.-I. Nishikawa
    Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan
  • A. Uemura
    Division of Ophthalmology, Kobe University Graduate School of Medicine, Kobe, Japan
  • Footnotes
    Commercial Relationships  Y. Fukushima, None; F. Gomi, None; S.-I. Nishikawa, None; A. Uemura, None.
  • Footnotes
    Support  Santen Pharmaceutical
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 5707. doi:
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      Y. Fukushima, F. Gomi, S.-I. Nishikawa, A. Uemura; EphrinB2-EphB4 Signals Control Lymphatic Valve Formation in Alkali Burn-Induced Corneal Lymphangiogenesis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5707.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Reciprocal signals mediated by transmembrane ephrinB2 ligand and its cognate EphB4 receptor tyrosine kinase are requisite for morphogenetic remodeling of blood vessels and lymphatic vessels. While the ephrinB2-EphB4 signals regulate arterio-venous anastomoses in angiogenesis, their particular involvement in lymphangiogenesis remains elusive. In the present study, we examined roles of the ephrinB2-EphB4 signals in alkali burn-induced lymphangiogenesis in mouse corneas.

Methods: : Under anesthesia with intraperitoneal injections of sodium pentobarbital, corneal epithelia of 6-10 week-old wild-type, ephrinB2-tauLacZ, or EphB4-tauLacZ mice with C57BL/6 background were peeled with swabs soaked in 0.15N sodium hydroxide (NaOH). Seven days after the NaOH treatment, 4 µl of soluble mouse EphB4 proteins (4 mg/ml dissolved in phosphate buffered saline) were injected daily into subconjunctival spaces for 6 consecutive days. Whole-mount corneas were processed for immunohistochemistry with anti-PECAM-1, LYVE-1, integrin α9, and β-galactosidase antibodies.

Results: : In the corneal limbus of adult mice, we found periodic circumferential bands in static lymphatic vessels, in which endothelial cells (ECs) expressed PECAM-1 and integrin α9, but not LYVE-1. In these bands, ECs were invaginated within the lymphatic vessel lumens, forming valve-like structure. EphB4 was intensively expressed in the valvar ECs which were surrounded by ephrinB2-expressing lymphatic ECs. In newly formed lymphatic vessels, EphB4-expressing valves emerged around 7 days after the NaOH treatment. Pharmacological blockade of the ephrinB2-EphB4 signals perturbed lymphatic valve formation with beaded enlargement of lymphatic vessel walls.

Conclusions: : In adult corneas, EphB4 is constitutively expressed in valvar ECs of peripheral lymphatic vessels. In alkali burned-corneas, the ephrinB2-EphB4 signals control valve formation in lymphangiogenic vessels.

Keywords: neovascularization • cornea: basic science • inflammation 

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