April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Knockdown of HO-2 but Not HO-1 Impairs Corneal Wound Healing
Author Affiliations & Notes
  • A. Halilovic
    Pharmacology,
    New York Medical College, Valhalla, New York
  • K. A. Patil
    Pharmacology,
    New York Medical College, Valhalla, New York
  • K. Castellano
    Pharmacology,
    New York Medical College, Valhalla, New York
  • L. Bellner
    Pharmacology,
    New York Medical College, Valhalla, New York
  • G. Cullaro
    Pharmacology,
    New York Medical College, Valhalla, New York
  • S. Inaganti
    Pharmacology,
    New York Medical College, Valhalla, New York
  • M. W. Dunn
    Pharmacology,
    Ophthalmology,
    New York Medical College, Valhalla, New York
  • M. L. Schwartzman
    Pharmacology,
    Ophthalmology,
    New York Medical College, Valhalla, New York
  • Footnotes
    Commercial Relationships  A. Halilovic, None; K.A. Patil, None; K. Castellano, None; L. Bellner, None; G. Cullaro, None; S. Inaganti, None; M.W. Dunn, None; M.L. Schwartzman, None.
  • Footnotes
    Support  NIH grant EY06513
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 380. doi:
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      A. Halilovic, K. A. Patil, K. Castellano, L. Bellner, G. Cullaro, S. Inaganti, M. W. Dunn, M. L. Schwartzman; Knockdown of HO-2 but Not HO-1 Impairs Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2010;51(13):380.

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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. We have shown that HO-1 induction accelerates corneal wound healing whereas HO-2 deletion results in impaired corneal wound healing with unresolved inflammation and chronic inflammatory complications (ulceration, perforation and neovascularization). This study was undertaken to examine the contribution of HO-1 and HO-2 to corneal wound healing in an in vitro epithelial scratch injury model.

Methods: : A scratch wound model was established using immortalized human corneal epithelial cells (HCE) treated with and without silencing inhibitory RNAs (siRNAs) against the human HO-1 and HO-2. Non-specific and GFP-conjugated siRNAs were used as controls. Wound healing at 12 and 24 h after injury was assessed by image analysis. Levels of HO-1, HO-2 and laminin 5 mRNA and protein were assessed by real time PCR and Western blot. Cell viability was measured by the MTT assay.

Results: : : Real time PCR of HO-1 and HO-2 mRNA and Western blot analysis indicated a specific knockdown of HO-1 and HO-2 gene expression by the corresponding siRNAs. Wound closure in HO-2 siRNA-treated cells was significantly impaired; only 50% of the scratch wound closed at 6 and 12 h after injury and 70% at 24 h after injury. In contrast, wound closure in HO-1 siRNA-treated cells was not significantly different than untreated cells or cells treated with non-specific siRNA. Interestingly, expression of laminin 5 was reduced by 40% in HO-2- but not in HO-1- knockdown cells.

Conclusions: : These results indicate that HO-2, which is highly expressed in the healthy corneal epithelium, is the critical HO component of the wound healing process in the cornea; its suppression inhibited laminin 5 expression, reduced cell migration and impairs healing, while HO-1 suppression had no effect. The mechanisms underlying HO-2 effects may reside in the reported cytoprotective properties of the HO products, biliverdin and carbon monoxide, that support the epithelial cell apparatus of the healing process.

Keywords: cornea: epithelium • wound healing • inflammation 
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