March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Proteinase Gene Silencing Restores Wound Healing, Signaling, And Stem Cell Marker Expression In Human Organ-cultured Diabetic Corneas
Author Affiliations & Notes
  • Alexander V. Ljubimov
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California
    Medicine, UCLA School of Medicine, Los Angeles, California
  • Irina Epifantseva
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California
  • Mehrnoosh Saghizadeh
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California
  • Footnotes
    Commercial Relationships  Alexander V. Ljubimov, None; Irina Epifantseva, None; Mehrnoosh Saghizadeh, None
  • Footnotes
    Support  EY13431, Cedars-Sinai Regenerative Medicine Institute and Department of Surgery
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1838. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Alexander V. Ljubimov, Irina Epifantseva, Mehrnoosh Saghizadeh; Proteinase Gene Silencing Restores Wound Healing, Signaling, And Stem Cell Marker Expression In Human Organ-cultured Diabetic Corneas. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1838.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: : Corneal diabetes leads to significant alterations of epithelial adhesive proteins and delayed wound healing. Diabetic corneas overexpress some proteinases, especially MMP-10 (M10) and cathepsin F (CF). The purpose was to improve wound healing and normalize marker expression in organ-cultured human diabetic corneas by silencing proteinase expression using adenovirus-driven shRNA (Ad-sh).

Methods: : Sixteen pairs of age-matched autopsy human diabetic corneas (4 per group) were organ-cultured. Ad-sh viruses (Capital Biosciences) were added to cultures for 48 hours to silence MMP-10 and cathepsin F gene expression. Ad-sh were to either single target, both targets together, or both proteinases in combination (Combo) with Ad expressing c-met gene (Ad-cmet). Fellow control corneas received Ad-vector only. Quantitative RT-PCR confirmed shRNA silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Various diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections.

Results: : Proteinase silencing significantly (p<0.02 except for Ad-shM10) reduced epithelial wound healing time (23% for Ad-shM10, 31% for Ad-shCF, and 36% for M10+CF). Combo treatment caused complete normalization of wound healing time (55% decrease vs. vector). Staining patterns of diabetic markers (α3β1 integrin and nidogen-1) were close to normal upon shRNA treatment. Staining for activated epidermal growth factor receptor (p-EGFR) and its signaling target p-Akt (reduced when M10 and CF were overexpressed) increased upon proteinase silencing. Addition of Ad-cmet also restored staining for p-p38. ShRNA treatments (especially combined with c-met overexpression) also increased diabetes-reduced staining for putative limbal stem cell markers, including ΔNp63α, keratins 15 and 17.

Conclusions: : ShRNA silencing of proteinases overexpressed in diabetic corneas proved to be efficient in enhancing corneal epithelial marker staining and wound healing. Combination therapy using proteinase gene silencing and c-met overexpression normalized most studied parameters including the expression of putative limbal stem cell markers. Specific corneal gene therapy has a potential to become a viable option for treatment of diabetic keratopathy.

Keywords: cornea: epithelium • gene transfer/gene therapy • diabetes 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×