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

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 (α₃β₁ 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, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 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.

Keywords: Akt; EGFR; MMP-10; c-met; cathepsin F; diabetic cornea; gene therapy; keratin; limbal stem cell; organ culture; p-38; wound healing.

Figure 1

Recombinant adenovirus-shM10 and rAV-shCF transduction into organ-cultured human diabetic corneas leads to a decrease in the expression of their respective target genes and proteins, M10, and CF. (A) Quantitative real-time RT-PCR of epithelial cell layers. (B) Immunofluorescent staining of corneal sections; exposure times were the same for each control and treated cornea. *P < 0.003. Scale bar: 30 μm. e, epithelium; s, stroma.

Figure 2

Various gene therapy treatments in organ-cultured diabetic corneas reduce epithelial wound healing time. M10 gene silencing slightly accelerates healing, whereas CF gene silencing results in significant acceleration. Wound healing time is further decreased when both proteinase genes are knocked down. Complete normalization of wound healing time is achieved with combined proteinase gene silencing by shRNA and c-met overexpression. Four pairs of corneas in each group were used, except for c-met (seven pairs were used). Data for c-met overexpression are from Saghizadeh et al. *P < 0.03.

Figure 3

Combined proteinase silencing (shM10 + shCF) in organ-cultured diabetic corneas normalizes the patterns of select diabetic markers, epithelial α₃β₁ integrin, and basement membrane nidogen-1, in the central cornea (top row) and limbus (bottom row). Arrow indicates interrupted limbal basement membrane staining in rAV-EGFP-treated cornea. Immunofluorescent staining of corneal sections. Scale bar: 30 μm.

Figure 4

Increased expression of limbal markers upon combined treatments in organ-cultured diabetic corneas. Top left: ΔNp63α. Both shM10 + shCF, and shM10 + shCF + c-met overexpression (Combo) treatments increase staining intensity and the number of positive basal epithelial cells. The same is true for ABCG2 (top right). Bottom left: laminin γ3 chain. Treatments markedly increase staining intensity and continuity. Bottom right: keratin 17. Both treatments increase staining intensity. Immunofluorescent staining of limbal corneal sections. Scale bar: 30 μm.

Figure 5

Combined proteinase silencing (shM10 and shCF) leads to increased expression (top rows) of activated EGFR (p-EGFR) and Akt (p-Akt). The same effect is seen when proteinase silencing is combined with c-met overexpression (Combo, bottom rows). Immunofluorescent staining of central corneal sections. Scale bar: 30 μm.

Figure 6

Proteinase silencing does not cause p38 MAP kinase activation (p-p38, top two rows). However, when this silencing is combined with c-met overexpression (Combo, bottom row), the expression of p-p38 increases. This result is in accordance with our previous data linking normalizing c-met effects on diabetic corneal wound healing with p38 activation. Immunofluorescent staining of central corneal sections. Scale bar: 40 μm.