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N. C. Joyce, C. C. Zhu, D. L. Harris; Oxidative Stress Reduces Corneal Endothelial Proliferation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3962. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Determine whether oxidative stress affects the proliferative capacity of human corneal endothelial cells (HCEC).
Human corneas from young (<30 yo) and older donors (>50 yo) were obtained from NDRI, Philadelphia, PA. HCEC were cultured to Passage 2 according to published protocols. To test the effect of oxidative stress on induction of oxidative DNA damage, cells from young donors were treated for 4 hrs at 37°C with 0, 25, 50, 100, 200, or 500µM H2O2 and post-incubated for 24 hrs in medium containing 8% FBS without vitamin C or H2O2 to permit cell recovery. Cells were then fixed and prepared for ICC of 8-OHdG to evaluate oxidative DNA damage. Untreated HCEC from older donors acted as positive controls. The effect of H2O2 on viability was tested in the same cultures using a Live/Dead assay. To determine the effect of mild oxidative stress on proliferative capacity, Passage 2 HCEC were seeded at low density in 8% FBS without vitamin C. On Day 0, basal cell numbers were determined using a WST-8 cell proliferation assay kit. Remaining cells were treated for 1hr at 37°C with 0, 25, 50, or 100µM H2O2. Cells were then washed and permitted to recover in normal culture medium for 4 days. On Day 4, cell numbers were again determined. Cells were re-treated with H2O2 as previously followed by washing and incubation. Cell numbers were then determined on Days 6, 8, and 11. Results were compared to numbers from untreated HCEC from older donors.
Increasing concentrations of H2O2 increased nuclear oxidative DNA damage as indicated by staining for 8-OHdG, a marker for oxidative DNA damage. Viability was retained at all concentrations of H2O2 except 500µM. HCEC from young donors treated with 25µM H2O2 displayed growth characteristics similar to that of untreated controls with cell numbers plateauing by Day 6. Cells treated with 50µM or 100µM H2O2 exhibited reduced growth kinetics and fewer cells were present at the plateau phase. These same reduced growth kinetics were observed in untreated HCEC from older donors.
Treatment of HCEC from young donors with increasing concentrations of H2O2 causes oxidative DNA damage that closely resembles the damage observed in untreated HCEC cultured from older donors. Viability was only affected at higher levels of H2O2. The reduced proliferative capacity induced by 50µM or 100µM H2O2 treatment of HCEC from young donors is extremely similar to that exhibited by untreated HCEC from older donors, providing evidence that mild oxidative stress is capable of reducing proliferative capacity in HCEC and suggesting that oxidative stress contributes to the age-related reduction in proliferative capacity observed in HCEC.
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