Reoxygenation injury in a cultured corneal epithelial cell line protected by the uptake of lactoferrin

Shigeto Shimmura, Masaru Shimoyama, Maki Hojo, Kumiko Urayama, Kazuo Tsubota

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26 Citations (Scopus)


PURPOSE. To investigate whether reoxygenation after extended hypoxia causes cellular damage in cultured corneal epithelial cells and to demonstrate the protective effects of lactoferrin. METHODS. Immortalized human corneal epitheal cells (T-HCECs) were cultured to confluence in 96- well culture plates, subjected to stringent hypoxia (1% O2, 5% CO2, 94% N2 at 37°C) for 24 hours, and returned to normoxic conditions (5% CO2, 95% air at 37°C). Cell viability was observed by 1 μM propidium iodide staining 0, 2, 4, and 6 hours after reoxygenation. Inhibition studies were performed after 2 hours' reoxygenation, using 2mM iron chelator desferrioxamine and 0.2 mg/ml lactoferrin. Confocal immunocytochemistry for human lactoferrin and western blot analysis for lactoferrin-induced ferritin were performed in cultured T-HCECs to demonstrate the internalization of lactoferrin after application. Results. After 2 hours, reoxygenation of T-HCECs after hypoxia produced an increase in cell death that was significantly greater than that observed in normoxic control cells or in cells subjected to hypoxia for the same time span without reoxygenation. The addition of desferrioxamine and lactoferrin at the time of reoxygenation significantly attenuated cellular damage. Confocal immunocytochemistry revealed that lactoferrin is taken into the cytoplasm of T-HCECs as early as 30 minutes after application. This was also demonstrated in western blot analysis by the upregulation of intracellular ferritin at 18 hours by the addition of iron-bound lactoferrin but not by iron-free lactoferrin. CONCLUSION. Reoxygenation is responsible for increased cellular damage after extensive hypoxia, which is attenuated by chelators of free iron in the cytosol, including the major tear protein lactoferrin.

Original languageEnglish
Pages (from-to)1346-1351
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Issue number8
Publication statusPublished - 07-1998
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience


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