Abstract
Purpose :
Accumulation in the Retinal Pigment Epithelial (RPE) cells of A2E, a pyridinium bis-retinoid, has the potential to cause RPE cell death and may contribute to the RPE cell atrophy and blue-light (BL) toxicity observed in AMD. The endocannabinoid receptor system is present in human RPE cells. It has been shown that level of anandamide, the major endocannabinoid, is significantly increased in retinal tissue from AMD donors. This study aimed to investigate the effects and the exosomes role in autophagy deficient RPE cells on the in vitro AMD-A2E-BL model, in the presence or absence of anandamide and Rimonoband, (a specific antagonist of CB1).
Methods :
By using A2E-loaded RPE cells and BL in-vitro, we can mimic the oxidative stress taking place in AMD. Signalling pathway and the exosomes role were assessed in the presence and absence of anandamide and Rimonoband, by Western-blot analysis, Tunable Resistive Pulse Sensing (TRPS), XTT proliferation assay, ELISA (IL-8, MCP-1) and confocal microscope.
Results :
Following damage to the autophagy mechanism, we found an increase in cell proliferation in various autophagy deficient RPE cells, expressing sh-RNA against G-proteins involved in autophagy process (Sh-53; Sh-74; Sh-53+74). Treatment with BL led to growth inhibition, and the autophagy deficient RPE cells found to be more effected (p<0.005). A2E and BL significantly (p<0.05) reduced the proliferation, compared to cells treated with BL alone. Anandamide successfully and significantly changed the amount of the pro-inflammatory chemokines, IL-8 and MCP-1, and increased the proliferation in autophagy deficient RPE cells. In addition, it was found that the exosomes' role changed in presence of A2E-BL treated cells vs. control.
Conclusions :
This research demonstrates that A2E-BL induces growth inhibition of autophagy deficient RPE cells and has negative affect on the proliferation of the cells. Moreover, these results demonstrate that anandamide via the CB1 receptor may attenuate AMD generated by the accumulation of A2E. This study is expected to select candidate therapeutic compounds that may contribute to delaying or arresting A2E-related toxicity to the RPE cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.