Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: cornea: stroma and keratocytes • cornea: basic science • wound healing