June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
HO-2 knockdown delays wound healing in Human Corneal Epithelial (HCE) cells by altering the signaling of EGFR and FAK mediated pathway
Author Affiliations & Notes
  • Adna Halilovic
    Pharmacology, New York Medical College, Valhalla, NY
  • Daohong Lin
    Pharmacology, New York Medical College, Valhalla, NY
  • Gregory Joseph
    Pharmacology, New York Medical College, Valhalla, NY
  • Brian Shkolnik
    Pharmacology, New York Medical College, Valhalla, NY
  • Michal Schwartzman
    Pharmacology, New York Medical College, Valhalla, NY
  • Footnotes
    Commercial Relationships Adna Halilovic, None; Daohong Lin, None; Gregory Joseph, None; Brian Shkolnik, None; Michal Schwartzman, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2572. doi:
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      Adna Halilovic, Daohong Lin, Gregory Joseph, Brian Shkolnik, Michal Schwartzman; HO-2 knockdown delays wound healing in Human Corneal Epithelial (HCE) cells by altering the signaling of EGFR and FAK mediated pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2572.

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

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Abstract

Purpose: Heme oxygenase (HO) represents an intrinsic cytoprotective and anti-inflammatory system. Inhibition of HO activity significantly impairs wound healing in human corneal epithelial (HCE) cells. We have shown that HO-2 knockdown in vitro attenuates corneal wound healing and that HO-2 null mice display an aberrant corneal wound healing following injury. In this study, we investigated the mechanisms that may contribute to HO-2 cytoprotective role in the corneal epithelium.

Methods: HCE cells were stably transfected with lentivirus HO-2 shRNA or non-target shRNA plasmids. Modified Boyden Chamber assay was used to study the migration of HO-2 knockdown cells. Flow cytometry cell cycle analysis was done using propidium iodide staining. Western blot was performed to evaluate the expression of proteins involved in migration and proliferation in HO-2 shRNA and non-target shRNA cells.

Results: HO-2 knockdown, using virus-mediated shRNA, impaired HCE wound healing by 35% and 25% at 24h and 48h after injury, respectively. Furthermore, HO-2 knockdown inhibited basal and EGF(10ng/mL)-stimulated HCE migration by 50% and 40%, respectively and was associated with a 35% decrease in p-Akt and 45% decrease in p-EGFR levels. Flow cytometric analysis showed that EGF-stimulated cell cycle progression was attenuated in HO-2 knockdown cells. Immunofluorescence of HO-2shRNA and control transfected cells revealed a cytoplasmic distribution of p-FAK. Upon injury, control cells displayed typical p-FAK as focal adhesion points in the membrane, whereas in HO-2 knockdown cells p-FAK had no focal distribution.

Conclusions: Knockdown of HO-2 results in attenuated corneal epithelial wound healing. Deficiency of HO-2 impairs wound healing by a mechanism that involves alterations in ERK, Akt and FAK signaling pathways, all of which have been shown to play an important role in growth factor-mediated cell migration and proliferation following injury.

Keywords: 482 cornea: epithelium • 765 wound healing • 714 signal transduction  
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