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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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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.
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.
: 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.
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.
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