Effects of aflibercept, estradiol and brimonidine on human Retinal Pigment Epithelial cells (ARPE-19) and Müller cells (MIO-M1) exposed to cobalt chloride (CoCl₂) in vitro.

Abstract

Purpose : Cobalt chloride (CoCl₂) mimics hypoxic conditions and can affect cell viability. Tartrate brimonidine is a neuroprotective drug; estradiol modulates inflammatory response and can ameliorate retinal damage; aflibercept is an anti-VEGF drug. The present study evaluates and compares the protective effects of these drugs on ARPE-19 and MIO-M1 cells in vitro for the purpose to discover new therapies that can modulate the inflammatory/oxidative stress systems and reduce the damage caused by retinal diseases.

Methods : Human ARPE-19 and MIO-M1 cells were cultured separately for 24 hours in 96-well plates. Some cultures were pretreated for 6 hours with aflibercept (1x and 2x), estradiol (20nM and 40nM), and brimonidine (1x and 2x), whereas others were served as controls. Cells were then stressed with CoCl₂in different concentrations: 350 µM for ARPE-19 cells and 500 µM for MIO-M1 cells for 48 hours. Cultures were analyzed for cell viability (CV, MTT assay), reactive oxygen species (ROS/H2DCFDA assay), and mitochondrial membrane potential (MitoMP, JC-1 assay). The conditions were: untreated, solution-control, and the aflibercept, estradiol and brimonidine in different concentrations.

Results : The CoCl₂-treated ARPE-19 and MIO-M1 cells showed increased CV with aflibercept (2x for ARPE-19, p=0.0041; 1x and 2x for MIO-M1, p<0.0001 and p=0.020 respectively); CoCl₂-treated MIO-M1 cells showed higher MitoMP levels with 2x aflibercept (p=0.007). Cell viability increased with estradiol 20nM in ARPE-19 (p=0.008) and MIO-M1 (p<0.0001) CoCl₂-treated cells; Estradiol 40nM increased CV (p<0.0001) and MitoMP (p=0.015) in CoCl₂-treated MIO-M1 cells. CoCl2-treated ARPE-19 cells showed increased MitoMP in 1x and 2x brimonidine, p=0.0096 and p=0.0438, respectively. CoCl₂-treated MIO-M1 cells had increased CV with brimonidine (1x, p<0.0001 and 2x, p=0.004) and higher MitoMP levels (1x, p=0.029).

Conclusions : Each CoCl₂-treated cell line responded differently to the aflibercept, estradiol and brimonidine, some being rescued while other not affected. This suggests that combinations of drugs acting on different pathways may be needed to cyto-protect cells in retinal diseases. Our findings may be helpful to identify novel pathways and drugs to protect against retinal diseases, especially in AMD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.