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K. N. Engin, S. Erdem Kuruca, G. Özcan Arcan, B. Çetin, E. Arcan, U. Serbes, K. Akgün Dar, S. Karadenizli, A. Kapucu; Evaluation of Apoptotic Effects of Single and Combined Use of Current Antifibrotic Agents on Human Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):469.
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© ARVO (1962-2015); The Authors (2016-present)
Mitomycin-C, 5-FU and Paclitaxel are current antifibrotics used to prevent fibrocellular scar formation in ophthalmic surgery but, their side effects on endothelium limits their use. Aside from its antioxidant effects, -tocopherol is known to be a neuromediator not only prevents tenon’s fibroblast proliferation and fibrocellular scar formation, but effects similar pathways with those of other antifibrotics as well. In this study, apoptotic effects of -tocopherol and other antifibrotics -in concordant doses with their in vivo uses, on human endothelial cells were examined in vitro in various combinations and durations.
Effects of antifibrotic agents in ECV304 Human Umblical Vein Endothelial Cell Cultures were evaluated in single 5 minnutes applications and in combination with 10 days of -tocopherol addition every 2 days. Paclitaxel, Mitomycin, 5-FU ve -tocopherol were used as antifibrotic agents. Apoptotic indexes (AI) were evaluated with photographs taken from the living cells via inverted microscope and apoptotic indexes was found with DAPI staining in fluorescence microscope. Apoptotic cells were demonstrated immunohistochemically in preperates stained with peroxidase labeled Caspace3 monoclonal antibodies and bcl-2 ve bax gene expressions were evaluated with Reverse Transcriptase-PCR. Mitotic indexes (MI) were evaluated to determine the proliferative trend of the cells.
Mitomycin and especially Paclitaxel groups demonstrated a significant AI increase, which were found to be augmented by -tocopherol addition. Either bcl-2/bax ratio decrease, or Caspase activity were found only in Mitomycin group, and again, further augmentation was achieved with -tocopherol addition. Significant difference were found in neither 5-FU nor 5-FU+-tocopherol groups. High proliferation was observed in -tocopherol group.
Combination with -tocopherol did not reduce the apoptotic effects of antifibrotics on human endothelial cells. On the other side, its additive effect with Paclitaxel and Mitomycin-C can be further investigated. Single and combined uses of 5-FU and -tocopherol have been found to be safe for, their long and short term effects were not statistically different from controls.
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