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

Abstract

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&#x3b2;1 to induce myofibroblast formation. The cultures were treated with either 5 &#x3bc;m or 10 &#x3bc;m SAHA for 72 h in the presence of TGF&#x3b2;1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying &#x3b1;-smooth muscle actin (&#x3b1;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&#x3b2;1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in &#x3b1;SMA mRNA and 86-88% (P < 0.001) decrease in &#x3b1;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 &#x3bc;m dose; 0.5-fold decrease with 10 &#x3bc;m dose). No change in fibroblast viability was observed with a 5 &#x3bc;m SAHA dose, whereas a 10 &#x3bc;m dose resulted in a moderate 17% decrease in cell viability. CONCLUSIONS: Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGF&#x3b2;-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.