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C.C. Luna, P.B. Liton, A. Hong, D.L. Epstein, P. Gonzalez; Extension of Life Span and Delay of Stress–Induced Premature Senescence of Trabecular Meshwork (TM) Cells Incubated at Low Oxygen Tension . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5161.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have observed in the past an increase in the number of cells with a senescent–associated phenotype in the TM of donors with primary open angle glaucoma (POAG). We have also shown that incubation of TM cells at increased oxygen concentrations can induce stress–induced premature senescence (SIPS). Since the physiologic concentration of oxygen in vivo is known to be much lower than that normally used in standard cell culture conditions, we investigated whether incubation at low oxygen concentration closer to the physiologic levels present in vivo can extend the proliferative capacity of TM cells and delay SIPS. Methods: Human and porcine trabecular meshwork cells were incubated for 15 days at either standard atmospheric oxygen concentration (20%) or at low oxygen (5%) concentration. Replicative potential was measured by the number of doubling cell populations necessary to reach permanent mitotic arrest in exponentially growing cultures. SIPS was evaluated in confluent cultures by measuring autofluorescence, senescence associated beta–galactosidase activity (sa–beta–gal), proliferative capacity, cell viability, and proteasome activity. Results: Human TM cell cultures incubated at 20% oxygen reached growth arrest characteristic of Hayflick 's limit at passage 10, while those maintained at 5% oxygen were still actively growing after passage 18. Confluent cultures of both human and porcine TM cells incubated at 20% oxygen compared to those incubated at 5% showed marked changes in phenotype and parameters associated with cellular senescence including: higher levels of autofluorescence (200%) and sa–beta–gal (200%), and a decrease in proteasome activity (52%) and proliferative capacity (45%) as well as a small reduction in cell viability (7%). Conclusions: The observed delay in replicative senescence and SIPS at low oxygen concentrations indicates that standard culture conditions at atmospheric oxygen are suboptimal for the propagation of TM cells in vitro. Chronic oxidative stress accelerates the onset of TM cellular senescence and might contribute to the observed accumulation of senescent cells in the TM of POAG donors.
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