Targeted suppression of HO-2 gene expression impairs the innate anti-inflammatory and repair responses of the cornea to injury

[摘要] Purpose: Heme oxygenase (HO)-2 ishighly expressed in the corneal epithelium and is a component of theheme oxygenase system that represents an intrinsic cytoprotective andanti-inflammatory system based on its ability to modulate leukocytemigration and to inhibit expression of inflammatory cytokines andproteins via its products biliverdin/bilirubin and carbon monoxide(CO). We have shown that in HO-2 null mice epithelial injuryleads to unresolved corneal inflammation and chronic inflammatorycomplications including ulceration, perforation and neovascularization.In this study, we explore whether a localized corneal suppression of HO-2is sufficient for disrupting the innate anti-inflammatory and repaircapability of the cornea. Methods: Silencing hairpin RNA (shRNA)against HO-2 was administered subconjunctivally (100 ng/eye) aswell as topically (100 ng/eye) starting one day before cornealepithelial debridement and once daily, thereafter. The cornealepithelium was removed using an Alger Brush in anesthetized mice.Re-epithelialization was assessed by fluorescein staining using adissecting microscope and image analysis. Inflammatory response wasquantified by myeloperoxidase activity. Levels of mRNA were measured byRT–PCR. Results: Local injection of HO-2-specificshRNAledtoa50%reduction in corneal HO-2 mRNA.Administration of HO-2-specific shRNA delayed cornealre-epithelialization when compared with the control shRNA-treated groupby 14%, 20%, and 12% at days 3, 4, and 7 after injury, respectively(n=18–24). The observed delay in the wound repair process in HO-2shRNA treated mice was accompanied by a threefold and 3.5 fold increasein the neovascular response at days 4 and 7 after injury. Further,local knockdown of HO-2 lead to an aberrant chronicinflammatory response, as shown by presence of high numbers ofinflammatory cells still present in the cornea at day 7 after injury;1.04±0.45×106 in HO-2 knockdown mice versus0.14±0.03×106 inflammatory cells in control mice. Matrixmetalloproteinase-2 (MMP-2) but not MMP-9 increasedfollowing injury and remained elevated in the injured corneas of the HO-2shRNA-treated eyes. Conclusions: Corneal knockdown of HO-2via local administration of HO-2-specific shRNA leads todelayed re-epithelialization, increased neovascularization and anaberrant inflammatory response similar to what is observed in the HO-2null mouse. The elevated MMP-2 expression may contribute to theincrease in neovascularization in corneas in which HO-2expression is suppressed.

[学科分类] 生物化学/生物物理