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Gopalan Gnanaguru, Ariel Choi, Dhanesh Amarnani, Patricia A D'Amore; CD36-mediated uptake of oxidized LDL induces retinal pigment epithelial cell death through NLRP3 inflammasome activation. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6556.
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© ARVO (1962-2015); The Authors (2016-present)
Accumulation of lipids that become oxidized has been suggested to contribute to the pathogenesis of AMD. We have previously shown that oxidized LDL (ox-LDL), but not native LDL, are cytotoxic to human retinal pigment epithelial (RPE) cells. We therefore have examined the mechanism by which ox-LDL leads to RPE cell death.
Primary human fetal RPE (hf-RPE) cells were treated with 500 µg/ml of LDL or ox-LDL, and ARPE-19 cells were treated with 100 µg/ml of LDL or ox-LDL. CD36, NLRP3, and caspase-1 expressions were analyzed by RT-PCR, and western blot. Fluorescent probe assay was performed to confirm caspase-1 activation. The role of CD36 in ox-LDL uptake was studied using DiI-labeled-ox-LDL uptake and CD36 function blocking antisera. Lysosomal integrity was examined by labeling with Lysotracker DND-99. The role of inflammasome activation was assessed by measuring RPE cell death (LDH release) in the presence or absence of the NLRP3 inflammasome inhibitor isoliquiritigenin (10 µM/ml).
Treatment of hf-RPE and ARPE-19 cells with ox-LDL, but not with LDL, led to a significant increase (more than 2-fold, P<0.01) in CD36 mRNA at 24 hr and CD36 protein at 48-72 hr. Incubation of RPE cells for 30 min with 40 µg/ml of CD36-IgA antibody significantly (P<0.01) reduced DiI-labeled-ox-LDL uptake by 75% when compared to 40 µg/ml of control-IgA treated cells. Blocking of CD36 receptor for 48 hr reduced ox-LDL induced RPE cell death by 40%. Further analysis show that DND-99-positive lysosomes were reduced in RPE cells incubated with ox-LDL for 48 hr. The treatment of RPE cells with ox-LDL but not LDL induced NLRP3 mRNA levels over 6 fold and activated caspase-1. Incubation of ARPE-19 cells with isoliquiritigenin in the presence of ox-LDL reduced cell death by 95%.
Uptake of ox-LDL by RPE cells induces NLRP3 inflammasome activation, which can lead to cell death and/or the release or pro-inflammatory cytokines, and may contribute to the initiation and/or progression of AMD. Anyone of a number of steps along this pathway may represent novel targets for the prevention or attenuation of AMD.
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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