Heme Oxygenase Is Essential for Corneal Epithelial Wound Healing

Abstract

Purpose: : Heme oxygenase (HO) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit the expression of inflammatory cytokines and proteins via its products, biliverdin/bilirubin and carbon monoxide (CO). Lack of HO activity results in impaired wound healing and the associated unresolved inflammation and chronic inflammatory complications include ulceration, perforation and neovascularization. This study was undertaken to investigate the effects of biliverdin and carbon dioxide releasing molecules (CORM) on epithelial scratch wound healing in vitro.

Methods: : A scratch wound model was established using immortalized human corneal epithelial cells (HCE). HCE cells were treated with chromium mesoporphyrin (CrMP, 10µM), an inhibitor of HO activity, with and without CORM-A1 (100µM) or biliverdin (10 µM) and further subjected to scratch injury. Hydroxyurea (0.5µM) was used to distinguish between cell proliferation and migration. Pictures were taken immediately after wounding and 12 and 24 hrs post injury. Images were analyzed by Axiovision software.

Results: : Injury to the cells resulted in an 80% wound closure within 24 hrs. Treatment of injured cells with biliverdin or CORM-A1 accelerated wound closure by 10% at 24 hrs. Addition of CrMP prior to injury markedly delayed wound closure by 30% at 12 hrs and 50% at 24 hrs post injury. However, addition of biliverdin and/or CORM-A1 to CrMP-treated cells reversed the effect of HO inhibition by enhancing cell migration and accelerating cell proliferation. Western blot analysis indicated that injury induced an increase in HO-1 expression and activity as well as an increase in p38 MAPK phosphorylation.

Conclusions: : These results clearly suggest that HO activity is essential for corneal epithelial wound healing. Inhibition of its activity impairs wound healing while amplification of its activity restores healing. The mechanisms by which HO contributes to cell migration and proliferation in response to injury may reside in the properties of CO and biliverdin, i.e., anti-oxidative, anti-inflammatory and cytoprotective properties.

Keywords: wound healing • inflammation • proliferation