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 ( MMP s) 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 β1 to induce myofibroblast formation. The cultures were treated with either 5 μ m or 10 μ m SAHA for 72 h in the presence of TGF β1. Real‐time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying α‐smooth muscle actin (α SMA ), a marker of myofibroblast formation and fibrosis. Real‐time PCR was used to determine the effects of SAHA on MMP 2 and MMP 9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays. Results Suberoylanilide hydroxamic acid ( SAHA ) significantly attenuated TGF β1‐induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3‐ to 3.5‐fold ( P  < 0.001) decrease in α SMA m RNA and 86–88% ( P  < 0.001) decrease in α SMA + immunocytochemical staining. SAHA treatment also resulted in 4.5‐ to 5.5‐fold ( P  < 0.01) decrease in MMP 9 expression. A dose‐dependent bimodal effect of SAHA on MMP 2 expression was noted (3.5‐fold increase with 5 μ m dose; 0.5‐fold decrease with 10 μ m dose). No change in fibroblast viability was observed with a 5 μ m SAHA dose, whereas a 10 μ m dose resulted in a moderate 17% decrease in cell viability. Conclusions Suberoylanilide hydroxamic acid ( SAHA ) can effectively inhibit TGF β‐induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro .