ABSTRACT
PURPOSE: Epithelial mesenchymal transition (EMT) plays a central role in the development of fibrotic complications of the lens. The current study is designed to check whether EMT of lens epithelial cells (LECs) is regulated by epigenetic modifications and to evaluate the effect of Trichostatin-A (TSA) on the transforming growth factor-ß (TGF-ß)-induced EMT. METHODS: Fetal human LECs (FHL124) were treated with TGF-ß2 in the presence or absence of TSA. Levels of mRNA, protein, as well as localization of α-smooth muscle actin (αSMA) were studied along with migration of LECs. Acetylation of histone H4 was analyzed and chromatin immunoprecipitation (ChIP) was carried out to study the level of acetylated histone H4 at the promoter of αSMA gene (ACTA2). Student's t-test was used for statistical analysis. RESULTS: TGF-ß2 treatment resulted in myofibroblast-like changes and increased migratory capacity of FHL124. Protein and mRNA expression of αSMA increased, and immunofluorescence revealed presence of extensive stress fibers. TSA treatment preserved epithelial morphology, retarded cell migration, and abrogated an increase in αSMA levels. TSA led to the accumulation of acetylated histone H4 that was reduced on TGF-ß2 treatment. However, increased level of histone H4 acetylation was found at the ACTA2 promoter region during TGF-ß treatment. CONCLUSIONS: The increased level of αSMA, a hallmark of EMT in LECs, is associated with increased level of histone H4 acetylation at its promoter region, and TSA helps in suppressing EMT by epigenetically reducing this level. TSA thus shows promising potential in management of fibrotic conditions of the lens.
Subject(s)
Actins/metabolism , Epithelial Cells/cytology , Epithelial-Mesenchymal Transition , Histones/metabolism , Promoter Regions, Genetic/physiology , Acetylation , Blotting, Western , Cell Movement/drug effects , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Fetus , Fluorescent Antibody Technique, Indirect , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Lens, Crystalline/cytology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Transforming Growth Factor beta2/antagonists & inhibitors , Transforming Growth Factor beta2/pharmacologyABSTRACT
We report a case of severe pigmented keratitis with poor prognosis, caused by Cladorrhinum bulbillosum. Antifungal treatment with topical natamycin and fluconazole eye drops and oral tablet fluconazole failed to heal the ulcer and resulted in perforation. The causative fungus, C. bulbillosum, was identified on the basis of its typical microscopic features and 98% sequence homology to ex-type isolate CBS 304.90 (accession no. FM955448). The results of an in vitro antifungal susceptibility test indicated that the isolate was susceptible to natamycin, amphotericin B, fluconazole and itraconazole. The present case is the third case of keratitis and the second case of human keratitis. Compromised immunity due to liver cirrhosis could lead to a failed prognosis even when the fungal isolate is highly susceptible to antifungal treatment.
