EFFECTS OF ANTI-VEGF DRUGS ON CULTURED HUMAN RETINAL MÜLLER CELLS

Abstract

Purpose : Treatment with intravitreal anti-VEGF (vascular endothelial growth factor) injection is the primary treatment for key retinal vascular diseases. Müller cells are the predominant glial cells in the retina, providing structural and metabolic support to retinal neurons, and secretion/regulation of VEGF. This study was performed to investigate the change in Reactive Oxygen (ROS) production and mitochondrial membrane potential (ΔΨm), measures of cellular stress and injury, in human retinal Müller cells treated with anti-VEGF agents.

Methods : Immortalized human retinal Müller cells (MIO-M1, UCL Institute of Ophthalmology, UK) were plated in 24 well plates and treated for 24 hours with either ranibizumab, bevacizumab, aflibercept or ziv-aflibercept in 1x and 2x clinical concentrations. For ranibizumab, bevacizumab and aflibercept, 1x concentration was defined as the clinical dose (0.05ml) in 4ml of vitreous. For ziv-aflibercept, 1x was the clinical equivalent dose to aflibercept (2.0mg in 0.05ml) in 4ml of vitreous. The JC-1 detection kit was used to assess the mitochondrial membrane potential (ΔΨm). ROS level was assessed using the fluorescent dye 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCF-DA). The fluorescent signals were measured using the Biotek Synergy HT plate reader and the results were normalized to untreated 100%.

Results : No changes in ΔΨm were found in 1x and 2x concentrations of ranibizumab. Significant reduction of ΔΨm was observed in bevacizumab 2x dose (85.74±4.49, p=0.03), aflibercept 1x and 2x (85.28±2.87, p=0.0008 and 85.02±3.80, p=0.0031 respectively), and ziv-aflibercept 1x and 2x (85.39±3.47, p=0.0016 and 86.01 ± 4.62, p=0.0116 respectively). (Figure 1). ROS production was found significantly increased in both concentrations of all the drugs: ranibizumab 1x and 2x, 136.90±4.67 and 139.83±6.53, p<0.0001; bevacizumab 1x, 124±5.41 (p=0.0017) and 2x, 133.92±8.10 (p=0.0011); aflibercept 1x and 2x, 129.62±3.08 and 130.27±4.58 (both p<0.0001); ziv-aflibercept 1x, 140.27±9.92 (p=0.001) and 2x, 144.28±12.02 (p=0.0004).(Figure 2).

Conclusions : Cultured human retinal Müller cells showed signs of intracellular stress in the form of decreased ΔΨm and increased ROS production when treated with bevacizumab, aflibercept and ziv-aflibercept. Cells treated with ranibizumab showed increased ROS production but no changes in ΔΨm.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.