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M. Saghizadeh, A. A. Kramerov, M. G. Castro, A. V. Ljubimov; Diabetic Corneal Gene Therapy With Adenoviral Vectors: Overexpression of Proteinases and C-Met in Human Organ-Cultured Corneas. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4307.
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© ARVO (1962-2015); The Authors (2016-present)
Poor wound healing of diabetic corneas may be due to elevated expression of proteinases, matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF), and/or impaired signaling of specific growth factors such as hepatocyte growth factor (HGF). The purpose was to examine the effects of overexpressing MMP-10 and CTSF in normal human organ cultured corneas, and of the HGF receptor, c-met proto-oncogene, in diabetic corneas using recombinant adenoviral (RAd) vectors.
Human corneas were obtained from the National Disease Research Interchange. Organ-cultured diabetic corneas were transduced with RAd expressing c-met gene (RAd-cmet) under cytomegalovirus (CMV) promoter. 350 or 500 µl RAd-cmet at 2.5x108 plaque forming units (pfu) per 1 ml were added to culture medium for 72 hr. Organ-cultured normal corneas were transduced with RAd expressing CTSF (RAd-CTSF) or MMP-10 (RAd-MMP-10) genes under CMV promoter. 200 or 300 µl RAd-CTSF or RAd-MMP-10 at 4x108 pfu/ml were added to culture medium for 72 hr. The other cornea of each pair was treated for 72 h with control RAd expressing β-galactosidase gene (RAd-gal; 32 or 48 µl RAd at 2.5x109 pfu/ml) in culture medium. After additional 4 days incubation, corneas were processed for immunohistochemistry or tested for healing of 5-mm epithelial circular wounds produced by n-heptanol application.
By immunofluorescence microscopy, corneas transduced with RAd-CTSF, RAd-MMP-10, and RAd-cmet had increased epithelial staining for the respective proteins compared to fellow corneas transduced with RAd-gal. The staining was more pronounce in basal cells for MMP-10 and CTSF. Stroma or endothelium did not show appreciable changes. Significantly faster healing was seen in diabetic corneas after c-met transduction. There was a tendency to slower wound healing of normal corneas after proteinase overexpression.
RAd seems to be a good vector to transduce the intact cornea with epithelial overexpression of target proteins identified by gene microarray analysis. The results support the role of MMP-10, CTSF and c-met in the migration of epithelial cells. Gene therapy with adenoviral vectors could be potentially useful for correcting diabetic corneal abnormalities.
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