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

Abstract

Purpose: Heme oxygenase (HO)-2 is highly expressed in the corneal epithelium and is a component of the heme oxygenase system that represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins via its products biliverdin/bilirubin and carbon monoxide (CO). We have shown that in HO-2 null mice epithelial injury leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. In this study, we explore whether a localized corneal suppression of HO-2 is sufficient for disrupting the innate anti-inflammatory and repair capability of the cornea.

Methods: Silencing hairpin RNA (shRNA) against HO-2 was administered subconjunctivally (100 ng/eye) as well as topically (100 ng/eye) starting one day before corneal epithelial debridement and once daily, thereafter. The corneal epithelium was removed using an Alger Brush in anesthetized mice. Re-epithelialization was assessed by fluorescein staining using a dissecting microscope and image analysis. Inflammatory response was quantified by myeloperoxidase activity. Levels of mRNA were measured by RT-PCR.

Results: Local injection of HO-2-specific shRNA led to a 50% reduction in corneal HO-2 mRNA. Administration of HO-2-specific shRNA delayed corneal re-epithelialization when compared with the control shRNA-treated group by 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-2 shRNA treated mice was accompanied by a threefold and 3.5 fold increase in the neovascular response at days 4 and 7 after injury. Further, local knockdown of HO-2 lead to an aberrant chronic inflammatory response, as shown by presence of high numbers of inflammatory cells still present in the cornea at day 7 after injury; 1.04±0.45×10(6) in HO-2 knockdown mice versus 0.14±0.03×10(6) inflammatory cells in control mice. Matrix metalloproteinase-2 (MMP-2) but not MMP-9 increased following injury and remained elevated in the injured corneas of the HO-2 shRNA-treated eyes.

Conclusions: Corneal knockdown of HO-2 via local administration of HO-2-specific shRNA leads to delayed re-epithelialization, increased neovascularization and an aberrant inflammatory response similar to what is observed in the HO-2 null mouse. The elevated MMP-2 expression may contribute to the increase in neovascularization in corneas in which HO-2 expression is suppressed.

Figure 1

Effect of local HO-2 knockdown on HO-2 and HO-1 mRNA levels. Corneal A: HO-2, and B: HO-1 mRNA levels after control- and HO-2-shRNA-treatment. (**p<0.01 from control-shRNA-treated mice, n=2–4).

Figure 2

Effect of local HO-2 knockdown on corneal wound healing. A: Fluorescein-stained corneas after injury in control- and HO-2-shRNA-treated mice. B: Wound closure as percent from day 0 (*p<0.05 from non-treated, and control shRNA treated mice, n=3–24).

Figure 3

Effect of local HO-2 knockdown on the injury induced corneal neovascularization. A: Photographs showing neovascularization in control- and HO-2 shRNA-treated mice at days 4, and 7 after epithelial debridement. B: Neovascularization expressed as total length of penetrating vessels (***p<0.001 from control-shRNA-treated mice, n=10–21).

Figure 4

Effect of local HO-2 knockdown on the injury induced inflammatory response. Corneal MPO activity in control- and HO-2-shRNA-treated mice at days 2, 3, 4, and 7 after epithelial debridement (*p<0.05 from control-shRNA-treated mice, n=6–12).

Figure 5

Effect of local HO-2 knockdown on injury induced HO-1 mRNA expression and on HO-2 mRNA expression. Corneal HO-1 mRNA levels in A: control shRNA and B: HO-2-shRNA-treated corneas 1, 2, 3, 4, and 7 days post epithelial debridement (*p<0.05; **p<0.01 from corresponding uninjured corneas, n=5–7). Corneal HO-2 mRNA levels in C: control shRNA and D: HO-2-shRHA-treated corneas in uninjured corneas, and 1, 2, 3, 4, and 7 days post epithelial debridement (*p<0.05; **p<0.01; ***p<0.001 from control shRNA-treated uninjured corneas, n=5–10).

Figure 6

Effect of local HO-2 knockdown on injury induced MMP-2 and MMP-9 mRNA expression. Corneal mRNA levels of A: MMP-2 and B: MMP-9 in uninjured corneas and in control- and HO-2-shRNA-treated corneas 2, 4, and 7 days post epithelial debridement (*p<0.05 from uninjured, n=2–5).