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Alexander V. Ljubimov, Irina Epifantseva, Mehrnoosh Saghizadeh; Proteinase Gene Silencing Restores Wound Healing, Signaling, And Stem Cell Marker Expression In Human Organ-cultured Diabetic Corneas. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1838.
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© ARVO (1962-2015); The Authors (2016-present)
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).
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.
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.
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.
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