Abstract
Purpose: :
Corneal diabetes leads to significant alterations of epithelial adhesive proteins and delayed wound healing. Diabetic corneas overexpress some proteinases, especially MMP-10 (M10) and cathepsin F (CF). The purpose was to improve wound healing and normalize marker expression in organ-cultured human diabetic corneas by silencing proteinase expression using adenovirus-driven shRNA (Ad-sh).
Methods: :
Sixteen pairs of age-matched autopsy human diabetic corneas (4 per group) were organ-cultured. Ad-sh viruses (Capital Biosciences) were added to cultures for 48 hours to silence MMP-10 and cathepsin F gene expression. Ad-sh were to either single target, both targets together, or both proteinases in combination (Combo) with Ad expressing c-met gene (Ad-cmet). Fellow control corneas received Ad-vector only. Quantitative RT-PCR confirmed shRNA silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Various diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections.
Results: :
Proteinase silencing significantly (p<0.02 except for Ad-shM10) reduced epithelial wound healing time (23% for Ad-shM10, 31% for Ad-shCF, and 36% for M10+CF). Combo treatment caused complete normalization of wound healing time (55% decrease vs. vector). Staining patterns of diabetic markers (α3β1 integrin and nidogen-1) were close to normal upon shRNA treatment. Staining for activated epidermal growth factor receptor (p-EGFR) and its signaling target p-Akt (reduced when M10 and CF were overexpressed) increased upon proteinase silencing. Addition of Ad-cmet also restored staining for p-p38. ShRNA treatments (especially combined with c-met overexpression) also increased diabetes-reduced staining for putative limbal stem cell markers, including ΔNp63α, keratins 15 and 17.
Conclusions: :
ShRNA silencing of proteinases overexpressed in diabetic corneas proved to be efficient in enhancing corneal epithelial marker staining and wound healing. Combination therapy using proteinase gene silencing and c-met overexpression normalized most studied parameters including the expression of putative limbal stem cell markers. Specific corneal gene therapy has a potential to become a viable option for treatment of diabetic keratopathy.
Keywords: cornea: epithelium • gene transfer/gene therapy • diabetes