Vitamins inhibit oxidant-induced apoptosis of corneal endothelial cells.
OBJECTIVE
To determine the effects of vitamins A, C, and E supplementation on lipid peroxidation and apoptosis in corneal endothelial cells.


METHODS
Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. The concentration of antioxidative vitamins (ascorbic acid, tocopherol, and retinoic acid) in the cells and supernatant was determined using reversed-phase high-performance liquid chromatography. Apoptosis was assessed by quantification of caspase-3-like activity, using annexin-V/propidium iodide stains for flow cytometry. Lipid peroxidation was assessed using the malondialdehyde method. Supplementation of antioxidative vitamins was tested in the setting of apoptosis.


RESULTS
Increasing levels of free iron led to a rapid loss of antioxidative vitamins in the supernatant and corneal endothelial cells. This was correlated with rising levels of malondialdehyde and increased apoptosis. Supplementation with ascorbic acid or alpha-tocopherol alone was not sufficient to prevent lipid peroxidation in the cells, whereas a combination of vitamins C and E was able to do so. In contrast, supplementation with vitamin A alone significantly reduced oxidative stress and apoptosis.


CONCLUSIONS
We present an in vitro model to test the direct influence of vitamin supplementation on corneal endothelial cells with regard to lipid peroxidation and apoptosis. We show that supplementation with antioxidative vitamins of corneal endothelial cells significantly prevents the generation of free-radical injury, lipid peroxidation, and consequent apoptosis.
Serbecic
Nermin
N
Department of Ophthalmology, SMZ-Ost, Donauspital, Vienna, Austria.
Beutelspacher
Sven Christoph
SC
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004338:Drug Combinations; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005296:Ferrous Compounds / Q000633:toxicity; D005434:Flow Cytometry; D015227:Lipid Peroxidation / Q000187:drug effects*; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000633:toxicity; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D014801:Vitamin A / Q000494:pharmacology*; D014810:Vitamin E / Q000494:pharmacology*
D017304:Annexin A5; D000975:Antioxidants; D004338:Drug Combinations; D005296:Ferrous Compounds; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D014801:Vitamin A; D014810:Vitamin E; C020748:ferrous sulfate; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D001205:Ascorbic Acid