Apoptotic effects on human RPE cells through NaIO₃

Abstract

Purpose : Damage to the retinal pigment epithelium (RPE), a hallmark of AMD, leads to production of oxidative stress and hypoxia which in turn ends in retinal damage. Therefore, the influence of oxidative stress and hypoxia on a human RPE cell line (ARPE-19) was investigated in this study.

Methods : 300 µM NaIO₃ or CoCl₂ was applied to ARPE-19 cells (3h) to induce oxidative stress or hypoxia. Untreated ARPE-19 cells served as controls. After 4 days of cultivation, RT-qPCRs were performed (n=5/group). Cell samples were analyzed for specific genes relevant to autophagy (MTOR, SQSTM1, BNIP3) or apoptosis (FASLG, CASP3, CASP9). In addition, different concentrations of NaIO₃ (0.5 mM, 1 mM, 5 mM and 10 mM) were compared and analyzed by RT-qPCRs (n=10/group). Immunohistochemical stainings were made to visualize apoptotic effects (e.g. cleaved caspase 3) and a cell damage assay was performed (BrdU).

Results : 300 µM NaIO₃ or CoCl₂ led to increased CASP3 mRNA expression (rel. exp.: 1.44; p=0.018). In addition, a significantly increased FASLG expression was identified in the NaIO₃ group (rel. exp.: 4.17; p=0.026). No significant differences were found in regard to autophagy markers. The concentration series with NaIO₃ showed apoptotic as well as autophagic effects. An increased FASLG (rel. exp.: 3.77; p=0.008), MTOR (rel. exp.: 3.20; p=0.007), CASP9 (p=0.048), HIF1α (p=0.046), and NFkB expression (p=0.016) was noted with 0.5 mM NaIO₃. Additionally, 5 mM NaIO₃ led to significantly more FASLG (rel. exp.: 3.12; p=0.037) and NFkB (rel. exp.: 1.70; p=0.031). A higher expression of SQSTM1 (rel. exp.: 2.12; p=0.021) was observed for 10 mM NaIO₃. The cleaved caspase 3 staining visualized the apoptotic effect mediated by NaIO₃.

Conclusions : The RT-qPCR analyses and stainings suggest that both forms of damage have primarily an apoptotic effect on ARPE-19 cells, but higher concentrations of the stressors also induce autophagic effects. Further investigation at protein level should provide more insight into the effects of the stressors which is important for establishing novel therapeutic approaches.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.