Abstract
Purpose:
Gene therapy with overexpression of c-met and downregulation of matrix metalloproteinase-10 (MMP-10) and cathepsin F genes was shown to restore abnormal wound healing, as well as diabetic and stem cell marker expression in organ-cultured human diabetic corneas. The purpose of this study was to determine if these effects would be observed when gene therapy was applied to limbal cells only.
Methods:
Eight pairs of autopsy human corneas with long duration of diabetes (over 10 years) were organ-cultured. One cornea of each pair was treated for 48 hours with adenoviral constructs harboring full-length c-met gene or a mixture of adenoviruses with c-met gene and shRNA to MMP-10 and cathepsin F (combo treatment) genes. Fellow corneas of each pair received control adenovirus with EGFP gene. Medium was kept at the limbal level and lack of viral transduction in the central epithelium was verified by the absence of EGFP fluorescence. After 5 (c-met) or 10 days (combo) of incubation with adenoviruses, the corneal epithelium was debrided using 8.5-mm n-heptanol-soaked paper disks. Wound healing times were monitored. After completion of healing, corneas were embedded in OCT and checked by immunostaining for the expression of diabetic and putative limbal stem cell markers α3β1 integrin, ΔNp63α, ABCG2, keratins 15, 17, and 19, and N-cadherin.
Results:
Corneas with c-met overexpressed in the limbal epithelium achieved complete healing of 8.5-mm epithelial wounds significantly faster than EGFP controls (6.3 days vs. 9.5 days, p<0.02). Combo treatment similarly accelerated healing (6.75 days vs. 13.5 days, p<0.03). In some corneas, radially oriented cords of EGFP-positive cells extending towards the corneal center were evident during healing. Immunostaining showed increased expression of most putative limbal cell markers following c-met gene or combo transduction as compared to fellow EGFP-transduced corneas.
Conclusions:
The results suggest that specific gene therapy of corneal epithelial stem cell compartment has a normalizing beneficial effect on the diabetic corneal wound healing and stem cell marker expression. They corroborate the concept that limbal cells participate in and are important for corneal epithelial wound healing.
Keywords: 482 cornea: epithelium •
538 gene transfer/gene therapy •
721 stem cells