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V. Lopez, J. C. K. Tovey, M. R. Waggoner, J. W. Cowden, A. Sharma, R. R. Mohan; Vorinostat Effectively Reduces Tgfβ-Mediated Myofibroblast Formation in the Cornea. Invest. Ophthalmol. Vis. Sci. 2010;51(13):367.
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© ARVO (1962-2015); The Authors (2016-present)
Recently we demonstrated that Trichostatin-A, a histone deacetylase inhibitor, effectively reduces corneal scarring in rabbit eyes in vivo with no serious side effects. However, it is still not approved for treating patients. Vorinostat is a potent histone deacetylase inhibitor, and is currently in clinical use for treating cutaneous T-cell lymphoma in patients. The aim of this study was to examine the toxicity and efficacy of Vorinostat to control corneal haze.
An in vitro model of corneal scarring was used. Human corneal fibroblast (HSF) cultures generated from donor human corneas were grown in presence or absence of TGF-β (1nM) under serum free conditions with or without Vorinostat (5 or 25µM). Trypan blue assay and phase-contrast microscopy were used to evaluate Vorinostat cytotoxicity to HSF. The anti-fibrotic effects of Vorinostat were determined by evaluating expression of alpha-smooth muscle actin, fibronectin, F-actin, and Ki-67 with real-time PCR, western blotting, and immunocytochemistry. The iQ SYBR Green Supermix was used for real-time PCR and appropriate antibodies were used for immunoblotting. Total RNA, cDNAs, and protein lysates were prepared using standard methods. ANOVA and Bonferonni-Dunn adjustments were applied for statistical analysis.
Vorinostat showed dose-dependent anti-fibrotic effects on the HSF. The two tested Vorinostat concentrations (5µM and 25µM) did not cause toxicity or phenotypic changes to HSF. As expected, TGFβ1 (1ng/ml) showed significant increase in RNA (1.3 to 4.1-fold) and protein (1.1 to 3.4-fold) expression of SMA, fibronectin, F-actin, and Ki-67 in the HSF. Cultures treated with Vorinostat showed a significant decrease in TGFβ1-stimulated production of SMA, fibronectin, and F-actin (p <0.05 or p<0.01). Another noteworthy observation was that Vorinostat did not alter proliferation as detected with Ki-67 immunolabeling and DAPI nuclear staining. Five µM Vorinostat reduced TGFβ1-driven expression of SMA, F-actin, and fibronectin by 70-90% (± 4.0, p<0.1). The 25µM dose of Vorinostat was found more potent and showed 80-95% (± 5.0, p<0.01) reduction in the TGFβ1-induced expression of these proteins.
Vorinostat has potential application in treating corneal haze in vivo. Importantly, Vorinostat exhibits a dose-dependent effect without significant toxicity and jeopardizing corneal fibroblasts proliferation. Ongoing rabbit studies will elaborate Vorinostat’s clinical utility to prevent corneal haze in patients.
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