Cell Viability and Transcriptome Changes Associated with Hydrogen Peroxide and Risuteganib Exposure in Human Retinal Cells In Vitro

Abstract

Purpose : Emerging research identifies elevated oxidative stress as central to the pathogenesis of many degenerative retinal diseases, such as dry age-related macular degeneration and retinitis pigmentosa. Treatment options for these diseases are limited, resulting in visual loss affecting millions of patients worldwide. In the present study, we investigated the cytotoxicity of hydrogen peroxide (H₂O₂) and protection by an investigational drug, risuteganib (RSG), in cultured human RPE (ARPE-19) and Müller (MIO-M1) cells.

Methods : ARPE-19 (n=3) and MIO-M1 (n=8-9) cells were treated according to the following five regimes: (1) untreated control for 36 h, (2) RSG treatment for 36 h, (3) untreated for 24 h, then H₂O₂ for 12 h, (4) RSG pre-treatment for 24 h, then H₂O₂ for 12 h, and (5) RSG for 24 h, then RSG and H₂O₂ co-treatment for 12 h. 400 µM RSG and 100 µM H₂O₂ were used. After exposure, cells were incubated for 48 h in fresh media before cell viability was measured by Trypan Blue dye exclusion assay. Statistical analysis was by Student’s t-test. Control, H₂O₂, and [RSG pre-treatment + H₂O₂] samples (n=6) were analyzed by RNA-seq for whole transcriptome gene expression. Differentially expressed genes were determined using edgeR and over-represented biological processes/pathways using goseq.

Results : H₂O₂ treatment significantly reduced cell viability by 27.7% (p=0.023) and 20.5% (p<0.0001) in ARPE-19 and MIO-M1 cells, respectively. Cell viability was significantly rescued by RSG pre-treatment (30.3%, p=0.015; 7.7%, p=0.0454) and RSG co-treatment (26.3%, p=0.027; 10.4%, p=0.0046) in ARPE-19 and MIO-M1 cells, respectively. RNA-seq showed H₂O₂ exposure regulated genes in the angiogenesis, immune system, cell adhesion/migration, cell proliferation/death, and metabolic processes. Biological pathway analysis showed integrin cell surface interaction was significantly regulated by H₂O₂. RSG effectively reversed the harmful effects of H₂O₂ treatment across many of the biological processes and pathways.

Conclusions : H₂O₂ induced significant cytotoxicity that was mitigated by RSG. Expression data showed H₂O₂ regulated genes across many disease-relevant processes and pathways, whereas RSG treatment lessened these effects. Our results suggest RSG may be effective in reducing oxidative stress-induced toxicity in retinal cells with relevance to human diseases.

This is a 2021 ARVO Annual Meeting abstract.