Purpose
Atrophy of retinal pigment epithelial (RPE) cells and photoreceptors in the macula is characteristic of geographic atrophy (GA), an advanced state of dry age-related macular degeneration (AMD). RPE cells are subjected to chronic oxidative stress. Currently, the mechanism of oxidative stress-induced RPE cell death is controversial, with most pointing to a dominant role for apoptosis. However, apoptosis does not induce inflammatory response, which is not consistent with chronic inflammation observed in dry AMD. We set out to clarify the mechanism of oxidative stress-induced RPE cell death using cultured RPE cells. We test the hypothesis that necrosis is a major mechanism for oxidative stress-induced RPE cell death. Moreover, we have screened a collection of 2,000 FDA-approved compounds for molecules that inhibit oxidative stress-induced RPE necrosis.
Methods
ARPE-19 cells were treated with different concentration of H2O2 and harvested at different time points followed by cell death marker staining, as well as RNA, DNA and protein analyses. Compound screening was performed using MTT assay.
Results
Morphology and molecular hallmarks of dying ARPE-19 cells show strong evidence of necrotic cell death induced by oxidative stress. We observed partial chromosomal condensation (pyknosis) and permeability of cell membrane by propidium iodine (PI). ARPE-19 cells treated with H2O2 did not exhibit apoptotic DNA fragmentation, consistent with our finding that ARPE-19 cells have very low level of DFF45 protein, inhibitory subunit of DFF40 main apoptotic endonuclease. RPE cell death is accompanied by increased levels of PGAM5 and RIP3 both critical for undergoing programmed necrosis. Consistently, necrostatin-1, but not caspase inhibitor zVAD, rescued oxidative stress-induced RPE cell death. Additionally our results showed that autophagic cell death is not a result of H2O2 treatment in RPE cells. Furthermore, we screened FDA-approved compounds and identified candidates that successfully rescued RPE cells from oxidative stress-induced cell death.
Conclusions
We provide unequivocal evidence that necrosis is a major mechanism for oxidative stress-induced RPE cell death. We also identified natural compounds that can rescue oxidative stress-induced RPE cell death. Taken together, these findings provide novel insight into the mechanism of RPE cell death, and will be instrumental for developing novel therapeutics for dry AMD, especially GA.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium