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F. Valamanesh, M. Berdugo, C. Goumeaux, A. Nagbou, A. Torriglia, F. Behar-Cohen; In Vitro Toxicity of Triamcinolone Acetonide on Bovine Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4454.
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© ARVO (1962-2015); The Authors (2016-present)
Corticosteroids are known for their antiangiogentic activities, however whether they induce direct toxicity on vascular endothelial cells remains unknown. We therefore have evaluated the potential toxicity of triamcinolone acetonide (TA) on bovine retinal endothelial cells in culture and characterized the type of induced cell death.
Subconfluent endothelial cells were treated with 0.1mg/ml or 1 mg/ml TA in 0.1% ethanol. Control cells were either untreated or exposed to 0.1% ethanol. Cell viability was evaluated at 24 hours, 72 hours and 5 days using MTT and lactate dehydrogenase (LDH) assays. Apoptosis was evaluated by TUNEL assay, annexin-binding and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, Microtubule-associated protein-light chain 3 (MAP-LC3) and LEI/L-DNase II.
TA induced a dose-dependant cell viability reduction on MTT assay, already observed at 24 hours. LDH test revealed that TA induced mostly necrosis at 1 mg/ml (25%). At 0.1mg/ml, apoptotic changes (annexin-binding) increased with the duration of TA exposure, but no nuclearization of Cytochrome C nor AIF were observed at any time point and no capsase 3 activation was detected. On the other hand, L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. No autophagic signs (MAP-LC3 positive vesicles) were detected.
Theseobservations demonstrate that TA antiangiogenic effects may be related to a direct toxic effect on endothelial cells and that the mechanism of cell death depends on the dose of TA. Higher dose induces mostly necrosis while lower dose induces a caspase-independent apoptosis.
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