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Gopalan Gnanaguru, Ariel Choi, Dhanesh Amarnani, Wen Tseng, Leo A Kim, Patricia A D'Amore; Oxidized lipoproteins induce RPE cell death through NLRP3-inflammasome activation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2367.
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Oxidized phospholipids/lipoproteins have been shown to accumulate with age and in age-related macular degeneration. We hypothesize that the uptake of oxidized lipoproteins by RPE cells leads to lysosomal destabilization and cell death via NLRP3-inflammasome.
Primary human fetal RPE (hf-RPE) cells were treated with LDL or oxidized LDL (ox-LDL) at different doses (300, 400, and 500 µg/ml). Cell death was assessed by measurement of LDH release. Transepithelial resistance was assayed as a measure of RPE barrier function. Lysosomal integrity was revealed by immunostaining for lysosomal associated membrane protein-1 (LAMP-1). RNA and cell lysates were prepared from control, LDL (500 µg/ml), and ox-LDL (500 µg/ml)-treated cells and the expression of NLRP3, PYCARD, and caspase-1 examined by RT-PCR and western blot. IL-1b release was quantified by ELISA.
The results show that the treatment of RPE with 500 µg/ml of ox-LDL for 48 hr altered RPE cytoskeleton as evidenced by abnormal actin cytoskeleton organization. Further analysis of RPE barrier properties showed a significant decreased (P< 0.01) in transepithelial resistance following ox-LDL treatment for 48 hr. Analysis of media conditioned by RPE treated with ox-LDL for 48 hr revealed a dose-dependent increase in LDH release (over 50% LDH release at 500 µg/ml), a reflection of cell death. ox-LDL co-localized with LAMP-1, indicating its presence in the lysosomes. ox-LDL treatment for 24 hr led to a 4-fold increase in NLRP3 mRNA level and a 2-fold increase in PYCARD mRNA. Pro-caspase-1 and cleaved active caspase-1 protein levels were upregulated by treatment with ox-LDL for 48 hr. IL-1ß secretion was elevated were following 48 hr of exposure to ox-LDL.
Our results indicate that ox-LDL accumulation in the lysosome of hfRPE activates NLRP3 mediated cell death. Thus, normalization of lipid levels and/or inhibition of NLRP3 activation may attenuate/prevent RPE degeneration.
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