Abstract
Purpose :
Retinal drusen are a risk factor in age-related macular degeneration. Drusen contain b-amyloid 1-42 (Aβ42) which contributes to retinal pigment epithelial (RPE) degeneration. In order to prevent the toxic effects of Aβ42 on RPE, the toll-like receptor 4 inhibitor curcumin was tested in vitro using RPE cells.
Methods :
RPE cells (ARPE-19; American Tissue Culture, CRL-2302) were grown to 90% confluency in DMEM with 10% fetal bovine serum (FBS), trypsinized, suspended at a concentration of 5.6x104/mL in DMEM supplemented with 0.1% FBS, and incubated for 2h at 37°C and 5% CO2. Cells were then treated with graded amounts of Aβ42 (ThermoFisher; 2fg to 200ng) for 2 hours. To test the protective effects of curcumin (Sigma), RPEs were treated with graded amounts of curcumin (0.03, 0.3, or 3uM) for 30 min prior to treatment with Aβ42. After treatment, calcein-AM (CAM) and ethidium homodimer-1 (EthD1) (ThermoFisher) were added to each sample. Cells were analyzed by flow cytometry using a Cyan ADP flow cytometer and Summit 4.3 software. The percent of live and dead RPE cells was counted; live cells were defined as CAM+/EthD1- and dead cells were defined as CAM-/EthD1+. In order to document Aβ42 internalization, Fluor-647 labeled Aβ42 (Aβ-647) was purchased from Anaspec. RPE cells were challenged with graded amount of labeled Aβ42 (0.2ng-200ng) for 2 hours in Millipore chamber slide and visualized by confocal microscopy.
Results :
RPE cells demonstrated a dose-dependent response to graded amounts of Aβ42 (2fg-200ng). The IC50 of Aβ42 on RPE cells was 0.41μM. Curcumin prevented Aβ42 induced toxicity and death in RPE cells: 200ng treatment killed 76.85% of cells (n=4, p=0.0005), which was reduced to 27.62% when cells were pretreated with 3uM curcumin (n=3, P=0.04). Aβ-647 challenged RPE cells demonstrated internalization into discrete cytoplasmic vacuole like positive staining. Notably, Aβ-647 challenged RPE reached a maximum that was not dose dependent. In addition, Aβ-647 internalization was partially blocked by curcumin pre-treatment.
Conclusions :
Aβ42 is indeed toxic to RPE cells in vitro with an IC50 of 0.41μM. The toll like receptor 4 inhibitor, curcumin, prevented Aβ42-mediated RPE death. Local RPE concentrations of Aβ42 may be higher in vivo than in vitro, and the degree of Aβ42 toxicity will be a balance of production of Aβ42, its aggregation, and its clearance by ABCB1 and other transporters.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.