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C.C. Luna, P.B. Liton, D.L. Epstein, P. Gonzalez; Effects of Chronic Oxidative Stress on Gene Expression Changes Induced by Cyclic Mechanical Stress in Human Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1854.
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© ARVO (1962-2015); The Authors (2016-present)
To analyze changes in gene expression induced by cyclic mechanical stress (CMS) in human trabecular meshwork (HTM) cells and evaluate whether chronic exposure to oxidative stress affects the ability of HTM cells to respond to mechanical stress.
Confluent HTM cell cultures were incubated 15 days in parallel at either 5% O2 (control), or 20% O2 (oxidative stress). Oxidative stress induced senescence was evaluated by sa–ß–gal and autofluorescence. Three cultures incubated at 20% oxygen and three at 5% oxygen were submitted to CMS and compared to six cultures incubated under the same conditions but not stressed. Changes in gene expression were evaluated by gene array analysis after 6 hours of CMS. The results from the arrays were validated by RT–PCR. Protein levels of secreted interleukin–6 (IL–6) and interleukin–8 (IL–8) were measured by enzyme–linked inmmunosorbent assay (ELISA).
Out of 37,000 gene transcripts, 10.4% showed significant changes (more than 1.5 fold) after CMS in HTM cells incubated at 5% oxygen. CMS induced extensive upregulation of genes involved in functional categories such as stress/defense, mitochondrial function, and extracellular matrix (ECM) regulators/modifiers; and downregulation of genes involved in cell adhesion, ECM proteins, and transcription regulation. Cells incubated at 5% oxygen had 65% more upregulated genes than those incubated at 20% oxygen. Genes such as IL–6, IL–8, thrombospondin, and prostaglandin synthases, among others, showed significant differences in their levels of induction at 5% and 20% oxygen. After CMS, protein levels of IL–8 and IL–6 increased in cells incubated at 5% oxygen, whereas IL–8 increased and IL–6 decreased in cells incubated at 20% oxygen.
Cyclic mechanical stress induces extensive gene expression changes in HTM cells that could potentially influence the levels of outflow resistance. Alterations in the ability to respond to cyclic mechanical stress in HTM cells incubated at elevated oxygen concentration suggest potential mechanisms by which chronic oxidative stress might contribute to the pathological increase in outflow resistance with age and in glaucoma.
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