May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Role of HB-EGF in Corneal Epithelial Wound Healing in vivo and in vitro
Author Affiliations & Notes
  • R. Yoshioka
    Ehime University, Toon, Japan
    Opthalmology,
  • A. Shiraishi
    Ehime University, Toon, Japan
    Opthalmology,
  • T. Kobayashi
    Ehime University, Toon, Japan
    Opthalmology,
  • S.-I. Morita
    Ehime University, Toon, Japan
    Opthalmology,
  • Y. Hayashi
    Ehime University, Toon, Japan
    Opthalmology,
  • S. Higashiyama
    Ehime University, Toon, Japan
    Biochemistry and Molecular Genetics,
  • Y. Ohashi
    Ehime University, Toon, Japan
    Opthalmology,
  • Footnotes
    Commercial Relationships  R. Yoshioka, None; A. Shiraishi, None; T. Kobayashi, None; S. Morita, None; Y. Hayashi, None; S. Higashiyama, None; Y. Ohashi, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2957. doi:https://doi.org/
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      R. Yoshioka, A. Shiraishi, T. Kobayashi, S.-I. Morita, Y. Hayashi, S. Higashiyama, Y. Ohashi; Role of HB-EGF in Corneal Epithelial Wound Healing in vivo and in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2957. doi: https://doi.org/.

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

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Abstract

Purpose: : Heparin binding EGF-like growth factor (HB-EGF) is a member of the EGF family that plays an important role in development and regeneration of various tissues. To investigate the function of HB-EGF in corneal epithelial wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter; HBlox/lox: K5-Cre (HB-/-). To determine the precise mechanisms of HB-EGF, we employed cultured corneal epithelial cells isolated from HB-/- (HB-/-CECs).

Methods: : Epithelial defects (2.0 mm) were created in the center of corneas of 6-8 weeks old HB-/-CECs and HBlox/lox, and the wounded area were monitored up to 5days. Corneal epithelial cells were isolated from HB-/-, and cultured under serum-free conditions. Third- or fourth-passage cells were used in this study. For in vitro wound healing assay, confluent HB-/-CECs were scraped with a 1ml pipette tip, and the extent of remaining acellular area were measured at 0, 12 and 24 hours after scraping in the presence or absence of 10ng/ml of HB-EGF. For cell attachment assay, HB-/-CECs were seeded to 96 well plate in 5×104cells/well, after 2 hour incubation in the presence or absence of HB-EGF, the number of cells adhered to the dishes were counted.

Results: : In vivo corneal wound was healed at 48hr in HBlox/lox, but it takes 86.7hr in HB-/-. Addition, the re-detachment of regenerated epithelium was frequently observed in HB-/-. In vitro wound healing, remaining acellular area at 0, 12, 24 hours after wounding was 100%, 77%, 60% in the absence of HB-EGF, 100%, 23%, 0% in the presence of HB-EGF, respectively. For cell attachment assay, 9% of the cells were attached in the absence of HB-EGF, while 16% of the cells were attached in the presense of HB-EGF.

Conclusions: : Corneal epithelial wound healing was impaired in HB-EGF deficient mouse corneas in vivo and in vitro.

Keywords: cornea: epithelium • wound healing • growth factors/growth factor receptors 
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