Histone deacetylase inhibitor attenuates TGF-β2 induced human conjunctival fibroblast activation

Abstract

Purpose: Excessive scar tissue formation of conjunctiva is a common cause of failure in glaucoma filtration surgery. Fibroblasts can be activated by a variety of inflammatory cytokines and differentiate into myofibroblasts, characterized by aSMA expression and extracellular matrix production, and this activation is known to play a pivotal role in fibrogenesis. Recent studies highlighted the role of antifibrotic activity of histone deacetylase (HDAC) inhibitors. The present study was aimed to evaluate the effects of HDAC inhibitor on TGF-ß2 induced human conjunctival fibroblast activation.

Methods: Human conjunctival fibroblasts were exposed to 5 ng/ml TGF-β2 to induce myofibroblast differentiation. The cultures were pretreated with suberoylanilide hydroxamic acid (SAHA 1.0-5.0 µM) for 1 hour and subsequently incubated with 5 ng/ml TGF-β2 for 48 hours. The effects of SAHA on human conjunctival fibroblasts were analyzed by Western blot analysis and immunocytochemistry. Cell viability and cytotoxicity were assessed using WST-8 assay and Hoechst 33342/propidium iodide (PI) dual staining, respectively.

Results: The expression of a-SMA in TGF-β2 treated human conjunctival fibroblasts was increased in both Western blot analysis (p=0.0009) and immunocytochemistry, and SAHA significantly reduced a-SMA expression (p=0.0011). TGF-β2 increased the proliferation of human conjunctival fibroblasts (p<0.0001), and the effect was significantly attenuated by SAHA (p<0.0001). SAHA did not cause any cellular toxicity at the tested doses.

Conclusions: HDAC inhibitor SAHA attenuated human conjunctival fibroblast differentiation into myofibroblast. Our results suggest that HDAC inhibitors might have a therapeutic potential in the prevention of excess scarring after glaucoma filtration surgery.