Abstract
Purpose: :
Recently, we reported that histone deacetylase inhibitors such as trichostatin-A (TSA) and vorinostat inhibit fibrosis in the rabbit cornea in vivo. In our subsequent studies, we detected a simultaneous increase of TGIFs, the negative regulator of TGFβ, during TSA-mediated corneal fibrosis inhibition. It is yet unknown whether TGIFs increase a key step in TSA-mediated corneal fibrosis inhibition? We tested the hypothesis that anti-fibrotic effects of TSA are due to the TGIFs up-regulation using RNA knockdown approach.
Methods: :
Human corneal fibroblasts (HCF) were generated from donor corneas. Cultures were treated with a vehicle, TGFβ1 (1ng/ml), TSA (500nM) or TGFβ1+TSA. To test the role of TGIF, HCF were transfected with TGIF-specific siRNA (50 pmol) using lipofectamine and then exposed to TGFβ1+TSA. Fibrosis parameters were quantified by analyzing the mRNA and protein levels of alpha smooth muscle actin (SMA) with real-time PCR and immunobloting. TGIF levels in cultures were measured with real-time PCR and immunoprecipitation.
Results: :
TGFβ treatment of HCF caused robust fibrosis as indicated by a significant increase in mRNA and protein levels of SMA (4-7 fold). TGFβ also caused low transient increase in TGIFs mRNA (1.2-1.5 fold) presumably due to its feedback mechanism. TSA treatment significantly inhibited TGFβ-induced fibrosis as shown by a significant decrease in SMA mRNA and protein expression (60-75%±9; p<0.05-0.01) and coupled with a concurrent increase in TGIF expression (2.5-3.0 fold p<0.01) both in the presence and absence of TGFβ. siRNA transfection of HCF significantly inhibited TSA-induced TGIF increase (0.8-1.5 fold p<0.05) and reversed TSA-mediated inhibition of fibrosis as shown by notable increase in mRNA and protein levels of SMA.
Conclusions: :
TSA-mediated increase in TGIFs is a critical step for attenuating corneal fibrosis. Our data also suggests that TGIFs could be a novel target to inhibit corneal fibrosis.
Keywords: cornea: stroma and keratocytes • cornea: basic science • wound healing