Unoprostone protects against cell death, reactive oxygen species, and apoptosis in a model retinitis pigmentosa system using expression of wild type and mutant rhodopsin (LB832)

Abstract

Objective. This study was to determine if unoprostone protects against deleterious effects evident in retinitis pigmentosa (RP). With the P23H mutation in rhodopsin, it is a dominant negative disease, causing the unfolded protein response (UPR) and ER stress. This leads to reactive oxygen species (ROS) production, apoptosis and ultimately cell death.Methods. Flp‐In T‐Rex‐293 cells were transfected with wild type (WT) or P23H mutant (MUT) rhodopsin in pcDNA5/FRT/TO with pOG44 under control of a tetracycline (tet) promoter. Cell growth, ROS and caspase were measured in non, mock, WT and MUT transfected cells with 1 µg/ml tet ± 100 nM unoprostone. ROS was measured with (5&6)‐chloromethyl‐2’,7’‐dichlorodihydrofluorescein diacetate,acetyl ester and caspase was detected using FITC‐VAD‐FMK.Results. There were no significant differences between non‐, mock‐ and WT (WT) cells ± unoprostone. At 120 h (n=3) the number of WT cells increased from 54.6 ± 1.2 to 111.7 ± 3.1, while the number of MUT cells decreased from 56.7 ± 3.6 to 19.0 ± 1.3. Unoprostone increased significantly (P<0.005) the number of MUT cells to 32.0 ± 0.5. ROS (FU, n=6) increased from 332.9 ± 0.7 in WT cells to 2816.8 ± 6.4 in MUT cells and this was significantly decreased (P<0.0005) to 1331.0 ± 3.0 with unoprostone. Caspase (FU, n=6) at 120 h increased from 365.7 ± 5.3 in WT cells to 895.5 ± 10.2 in MUT cells and this was significantly reduced (P<0.0005) to 523.2 ± 11.3 with unoprostone.Conclusions. The data shows that unoprostone can slow the pathological events (ER stress, ROS production, and apoptosis) and cell death seen in RP with UPR. Increased ROS generation and apoptosis are also seen in age related macular degeneration (AMD), suggesting unoprostone should also be investigated in dry AMD.Grant Funding Source: Supported by Sucampo AG