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Elizabeth R Gaillard, Jennifer C. Tournear; Investigating the Cell Death Mechanisms of ARPE-19 Cells using Modified ARPE-Derived ECM to Model Aging and Disease. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1739.
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© 2017 Association for Research in Vision and Ophthalmology.
RPE cell death is a symptom of age related macular degeneration (AMD), but it is unclear what mechanisms of cell death are involved in this process. This study aims to modify ARPE-derived extra cellular matrix (ECM) in order to investigate the ARPE-19 cell death mechanism associated with various stresses modeling both age-related modifications and inflammation.
A series of modifications were performed on ECM derived from ARPE-19 cells to model aging and inflammation. These modifications included non-enzymatic glycation, A2E, blue light irradiated A2E and non-enzymatic nitration. These modifications were done on ECM coating 24-well plates. Post modification, healthy ARPE-19 cells were seeded onto the ECM and allowed to attach for 30 min. Unattached cells are removed and fixed for further study. Attached cells were allowed to grow prior to viability analysis. After 24 hours of growth, both unattached and attached cells were stained with Annexin-V and PI then subjected to analysis using flow cytometry to investigate apoptosis versus necrosis as mechanisms of cell death.
Cell viability is observed to decrease under all stresses when compared to unmodified ECM. Cells grown on blue light irradiated A2E modified ECM showed not only a loss in viability, but also a change in proliferation and cell morphology. There was evidence of both apoptosis and necrosis as mechanisms of cell death. When analyzing both the unattached and attached simultaneously, a further decrease in cell viability was observed suggesting that attachment to modified ECM is impaired.
These data suggest that more than one mechanism of cell death is involved under aging and disease conditions. Both necrotic and apoptotic cells were observed supporting the idea that cells have difficulty in both cell attachment and proliferation. This study provides insight into the mechanisms of RPE cell death in AMD and can help to further understand the pathogenesis of the disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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