title: Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing.
abstract: OBJECTIVE
  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.
authors.affiliation: Eye Program, Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Saghizadeh
authors.forename: Irina
authors.initials: I
authors.lastname: Epifantseva
authors.forename: David M
authors.initials: DM
authors.lastname: Hemmati
authors.forename: Chantelle A
authors.initials: CA
authors.lastname: Ghiam
authors.forename: William J
authors.initials: WJ
authors.lastname: Brunken
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Ljubimov
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D056646:Cathepsin F / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D003920:Diabetes Mellitus / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019573:Epithelium, Corneal / Q000378:metabolism; D005260:Female; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D053506:Matrix Metalloproteinase 10 / Q000096:biosynthesis / Q000235:genetics*; D008875:Middle Aged; D009924:Organ Culture Techniques; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014945:Wound Healing / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; D056646:Cathepsin F; D053506:Matrix Metalloproteinase 10
keywords: Akt; EGFR; MMP-10; c-met; cathepsin F; diabetic cornea; gene therapy; keratin; limbal stem cell; organ culture; p-38; wound healing