Trichostatin A Blocks TGFβ-Induced Accumulation of Extracellular Matrix and Myofibroblast in Human Corneal Fibroblasts

Abstract

Purpose: : TGFβ is known to cause corneal fibrosis. Targeted suppression of TGFβ-induced profibrogenic genes is a novel approach to prevent corneal fibrosis. Trichostatin A (TSA) is a potent histone deacetylase inhibitor that has been shown to suppress TGFβ-induced collagen synthesis and fibrogenesis in the skin and kidneys. The aims of this study were to define noncytotoxic TSA doses for the HSF and examine the antifibrogenic effects of TSA on TGFβ-driven fibrosis in the cornea in vitro.

Methods: : Human corneal fibroblasts (HSF; 1-3 passages) were used. Toxicity of TSA and the effects of TSA on proliferation were investigated with trypan blue exclusion, MTT and MultiTox-Fluor assays. To study the effects of TSA on corneal wound healing, cultures were incubated with TSA (50-500nM) in presence/absence of TGFβ1 (1ng/ml) under serum-free conditions. Quantification of collagen Type IV, fibronectin and α-smooth muscle actin (SMA) mRNA was determined by real-time PCR using iQ SYBR Green Supermix and protein by immunoblotting and/or immunocytochemistry using appropriate antibodies. The cDNAs and total protein lysates were prepared using methods reported earlier. Statistical analysis was performed using ANOVA and Bonferonni-Dunn adjustments.

Results: : The effects of TSA on the HSF were both dose- and time-dependent. The tested concentrations of TSA 250nM or less were non-cytotoxic to HSF and did not affect normal HSF morphology and proliferation. As expected, TGFβ1 (1ng/ml) increased mRNA (1.3- to 4.1-fold) and protein (1.1- to 3.4-fold) expression of collagen Type IV, fibronectin and SMA in the HSF. Cultures treated with TSA showed a significant decrease in TGFβ1-stimulated production of collagen Type IV, fibronectin and SMA mRNA and protein levels at 100nM (43-77% ±3.7, p <0.1) and 250nM (69-93% ±5.2, p <0.01) concentrations.

Conclusions: : TSA significantly inhibited TGFβ1-induced accumulation of extracellular matrix and myofibroblasts in human cornea in vitro. The results suggest that TSA could be used to block cytokine-driven induction of profibrogenic events leading to corneal scarring in vivo.

Keywords: cornea: stroma and keratocytes • wound healing • growth factors/growth factor receptors