PURPOSE: Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression. METHODS: Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections. RESULTS: Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α₃β₁ integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain. CONCLUSIONS: Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
PURPOSE:Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression. METHODS: Sixteen pairs of organ-cultured autopsy humandiabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections. RESULTS:Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α₃β₁ integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain. CONCLUSIONS: Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
Authors: M Saghizadeh; D J Brown; R Castellon; M Chwa; G H Huang; J Y Ljubimova; S Rosenberg; K S Spirin; R B Stolitenko; W Adachi; S Kinoshita; G Murphy; L J Windsor; M C Kenney; A V Ljubimov Journal: Am J Pathol Date: 2001-02 Impact factor: 4.307
Authors: Sriniwas Sriram; Jennifer A Tran; Xiaoqing Guo; Audrey E K Hutcheon; Andrius Kazlauskas; James D Zieske Journal: Exp Eye Res Date: 2017-06-06 Impact factor: 3.467