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Noor-Ul-Ain Shekoh, Jacklyn Nguyen, Paula Fukuhara, Cristina M Kenney, Baruch D Kuppermann; Effects of Bevacizumab on ARPE-19 gene expression. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4451.
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In age-related macular degeneration (AMD), retinal pigment epithelial cells (RPE) are dysfunctional and drusen formation occurs. Hypoxia results in upregulation of VEGFA leading to choroidal neovascularization. We simulated AMD conditions by treating immortalized RPE cells (ARPE-19) with amyloid beta (a component of drusen) and cobalt chloride which creates hypoxia. We then treated the ARPE-19 cells with the anti-VEGF agent Bevacizumab (Bv) and assessed gene expression in the angiogenic, oxidant and apoptosis pathways.
ARPE-19 cells were seeded in 6 well plates at a density of 0.5 million cells/well until 80% confluent. The cells were then treated with 250 µM cobalt chloride (CoCl2) and 25 µM amyloid beta (Aβ) separately and in combination (3 samples each). All the samples were then treated with 2x the clincal dose of Bevacizumab (equivalent to 0.05 ml of drug distributed in 4 ml of vitreous volume) for 24 hours. 3 cell samples were kept as controls and treated with 2x Bevacizumab only. Cells were lysed, RNA extracted and qRT-PCR was performed with primers to measure expression levels of SOD2 , GPX, BAX, Caspase-3, BCL2L13, HIF1A and VEGFA (markers for oxidation, apoptosis, and angiogenesis respectively) . Data were analyzed by the ΔΔCt Method and Fold change was calculated.
There were no appreciable changes in gene expression for HIF1A and BCL2L13. GPX gene expression was significantly decreased to 0.8 (p=0.0001), 0.67 (p=0.0002) and 0.72 (p=0.0001) folds for Bv+CoCl2+βA, Bv+βA & Bv+CoCl2, respectively. The gene expression for Caspase-3 was significantly increased to 0.3 (p=0.046), 0.5 (p=0.006) and 0.2 (p=0.041) folds for Bv+CoCl2+βA, Bv+βA & Bv+CoCl2, respectively, gene expression for VEGFA was significantly increased to 1.03 (p=0.01), 1.3 (p=0.0009) and 1.35 (p=0.0002) folds for Bv+CoCl2+βA, Bv+βA & Bv+CoCl2, respectively and SOD2 was significantly decreased to 0.3 (p=0.01) folds in Bv+CoCl2 samples. BAX gene expression was significantly increased to 0.3 (p=0.04) folds in Bv+CoCl2+βA samples.
The gene expression for HIF1A and BCL2L13 remained unaltered in all combinations. There was uniformly decreased gene expression for GPX in all combinations. The gene expression for Caspase-3 and VEGFA and SOD2 was variably altered. There was mildly increased gene expression for BAX in Bv+CoCl2+βA samples. These results suggest that Bv has significant effects on RPE gene expression.
This is a 2020 ARVO Annual Meeting abstract.
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