Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity

Objective To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Procedure Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGF beta 1 to induce myofibroblast formation. The cultures were treated with either 5 mu M or 10 mu M SAHA for 72 h in the presence of TGF beta 1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying alpha-smooth muscle actin (alpha SMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays. Results Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGF beta 1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in alpha SMA mRNA and 86-88% (P < 0.001) decrease in alpha SMA+immunocytochemical staining. SAHA treatment also resulted in 4.5-to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 mu M dose; 0.5-fold decrease with 10 mu M dose). No change in fibroblast viability was observed with a 5 mu M SAHA dose, whereas a 10 rho M dose resulted in a moderate 17% decrease in cell viability. Conclusions Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGF beta-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.