NDLI: Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing.

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Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Epifantseva, Irina Hemmati, David M. Brunken, William J. Ljubimov, Alexander V. Saghizadeh, Mehrnoosh Ghiam, Chantelle A.
Description	Author Affiliation: Saghizadeh M ( Eye Program, Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.)
Abstract	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 ( 3ß1 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, ΔNp63 , ABCG2, keratins 15 and 17, and laminin Î³3 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.
ISSN	01460404
e-ISSN	15525783
DOI	10.1167/iovs.13-13233
Journal	Investigative Opthalmology & Visual Science
Issue Number	13
Volume Number	54
Language	English
Publisher	Association for Research in Vision and Ophthalmology
Publisher Date	2013-12-17
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Cathepsin F Genetics Corneal Diseases Diabetes Mellitus Gene Silencing Matrix Metalloproteinase 10 Stem Cells Metabolism Wound Healing Biosynthesis Cells, Cultured Pathology Epithelium, Corneal Genetic Therapy Immunohistochemistry Organ Culture Techniques Rna, Small Interfering Real-time Polymerase Chain Reaction Signal Transduction Cytology Comparative Study Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Discipline Ophthalmology
Content Type	Text
Resource Type	Article
Subject	Ophthalmology Sensory Systems Cellular and Molecular Neuroscience

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