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Invest Ophthalmol Vis Sci. 2016 Sep 1;57(11):4704-12. doi: 10.1167/iovs.15-18663.

Oxidized Lipoprotein Uptake Through the CD36 Receptor Activates the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells.

Author information

1
Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
2
Program in Liberal Medical Education, Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States.
3
Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston, Massachusetts, United States 2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States 4Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States.

Abstract

PURPOSE:

Accumulation of oxidized phospholipids/lipoproteins with age is suggested to contribute to the pathogenesis of AMD. We investigated the effect of oxidized LDL (ox-LDL) on human RPE cells.

METHODS:

Primary human fetal RPE (hf-RPE) and ARPE-19 cells were treated with different doses of LDL or ox-LDL. Assessment of cell death was measured by lactate dehydrogenase release into the conditioned media. Barrier function of RPE was assayed by measuring transepithelial resistance. Lysosomal accumulation of ox-LDL was determined by immunostaining. Expression of CD36 was determined by RT-PCR; protein blot and function was examined by receptor blocking. NLRP3 inflammasome activation was assessed by RT-PCR, protein blot, caspase-1 fluorescent probe assay, and inhibitor assays.

RESULTS:

Treatment with ox-LDL, but not LDL, for 48 hours caused significant increase in hf-RPE and ARPE-19 (P < 0.001) cell death. Oxidized LDL treatment of hf-RPE cells resulted in a significant decrease in transepithelial resistance (P < 0.001 at 24 hours and P < 0.01 at 48 hours) relative to LDL-treated and control cells. Internalized ox-LDL was targeted to RPE lysosomes. Uptake of ox-LDL but not LDL significantly increased CD36 protein and mRNA levels by more than 2-fold. Reverse transcription PCR, protein blot, and caspase-1 fluorescent probe assay revealed that ox-LDL treatment induced NLRP3 inflammasome when compared with LDL treatment and control. Inhibition of NLRP3 activation using 10 μM isoliquiritigenin significantly (P < 0.001) inhibited ox-LDL induced cytotoxicity.

CONCLUSIONS:

These data are consistent with the concept that ox-LDL play a role in the pathogenesis of AMD by NLRP3 inflammasome activation. Suppression of NLRP3 inflammasome activation could attenuate RPE degeneration and AMD progression.

PMID:
27607416
PMCID:
PMC5024668
DOI:
10.1167/iovs.15-18663
[Indexed for MEDLINE]
Free PMC Article

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