title: Normalization of wound healing and stem cell marker patterns in organ-cultured human diabetic corneas by gene therapy of limbal cells.
abstract: Overexpression of c-met and suppression of matrix metalloproteinase-10 (MMP-10) and cathepsin F genes was previously shown to normalize wound healing, epithelial and stem cell marker patterns in organ-cultured human diabetic corneas. We now examined if gene therapy of limbal cells only would produce similar effects. Eight pairs of organ-cultured autopsy human diabetic corneas were used. One cornea of each pair was treated for 48 h with adenoviruses (Ad) harboring full-length c-met mRNA or a mixture (combo) of Ad with c-met and shRNA to MMP-10 and cathepsin F genes. Medium was kept at the limbal level to avoid transduction of central corneal epithelium. Fellow corneas received control Ad with EGFP gene. After additional 5 (c-met) or 10 days (combo) incubation, central corneal epithelial debridement with n-heptanol was performed, and wound healing times were determined microscopically. Corneal cryostat sections were immunostained for diabetic and putative limbal stem cell markers, α3β1 integrin, nidogen-1, fibronectin, laminin γ3 chain, ΔNp63α, keratins 14, 15, and 17, as well as for activated signaling intermediates, phosphorylated EGFR, Akt, and p38. Limbal c-met overexpression significantly accelerated healing of 8.5-mm epithelial wounds over EGFP controls (6.3 days vs. 9.5 days, p < 0.02). Combo treatment produced a similar result (6.75 days vs. 13.5 days, p < 0.03). Increased immunostaining vs. EGFP controls for most markers and signaling intermediates accompanied c-met gene or combo transduction. Gene therapy of limbal epithelial stem cell compartment has a beneficial effect on the diabetic corneal wound healing and on diabetic and stem cell marker expression, and shows potential for alleviating symptoms of diabetic keratopathy.
authors.affiliation: Eye Program, Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Department of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA; University of California Los Angeles, Los Angeles, CA, USA.
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Saghizadeh
authors.affiliation: University of California Los Angeles, Los Angeles, CA, USA.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Dib
authors.affiliation: Center for Vision Research, Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, NY, USA; Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Brunken
authors.affiliation: Eye Program, Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Department of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA; University of California Los Angeles, Los Angeles, CA, USA. Electronic address: ljubimov@cshs.org.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Ljubimov
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000378:metabolism*; D003316:Corneal Diseases / Q000209:etiology / Q000473:pathology / Q000628:therapy*; D003920:Diabetes Mellitus / Q000473:pathology / Q000628:therapy*; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology*
chemical_list: D015415:Biomarkers
keywords: C-met; Cathepsin F; Cornea; Diabetes; Limbal gene therapy; MMP-10; Signaling pathways; Stem cell marker