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Mohamed Mohamed, Jon Lucas Norman, Javier Cáceres-del-Carpio, Rodrigo Costa, Abdul Sami Memon, M. Tarek Moustafa, Shari R Atilano, Marilyn Chwa, Cristina M Kenney, Baruch D Kuppermann; Effects of anti-angiogenic drugs on expression patterns of epigenetic acetylation pathway genes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5028.
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© ARVO (1962-2015); The Authors (2016-present)
Epigenetic changes can affect cellular transcriptional activities and have been associated with retinal diseases. This study examines the effects of anti-angiogenic drugs on the transcription of acetylation genes in immortalized human RPE cells (ARPE-19) in vitro, to determine if epigenetic pathways are modulated by anti-angiogenic treatment.
ARPE-19 cells were treated with ranibizumab, bevacizumab, or aflibercept in 1X and 2X concentrations of the clinical intravitreal dose. Untreated cells were used as a control. RNA was isolated and reverse transcribed into copy DNA (cDNA). Quantitative polymerase chain reaction (Q-PCR) was performed in triplicates using primers for acetylation genes: HAT1 (histone acetyltransferase 1), and HDACs (histone deacetylases) 1, 6, and 11. HPRT1 (hypoxanthine phosphorbosyltransferase 1) and HMBS (hydroxymethylbilane synthase) were used as housekeeping genes. ΔΔCt (differences in cycle thresholds) was obtained and folds calculated using the formula 2ΔΔCt. Unpaired t test was used for statistical analysis.
Bevacizumab-treated cells expressed 0.73-fold of HDAC1 (p=0.0001), 1.3-fold of HDAC6 (p=0.004), and 1.52-fold of HDAC11 (p=0.009) at 1X concentration, and 1.39-fold of HDAC11 (p=0.013) at 2X concentration. Aflibercept-treated cells showed 0.74-fold of HDAC1 (p=0.0002), 1.11-fold of HDAC6 (p=0.037), and 1.27-fold of HDAC11 (p=0.02) at 1X concentration, and 1.48-fold of HDAC11 (p=0.023) at 2X concentration. Ranibizumab-treated cells expressed 1.26-fold of HDAC6 (p=0.018) at 1X concentration, and HDAC11 (1.32-fold, p=0.006) at 2X concentration. Untreated samples were assigned a value of 1. No statistically significant difference in HAT1 expression was found in any group.
Our results suggest that anti-angiogenic drugs can alter expression levels of acetylation genes in ARPE-19 cells. This is significant because the degree of histone acetylation regulates chromatin structure and function, which can impact transcriptional activities for numerous genes involved in age-related macular degeneration (AMD) pathogenesis. Our findings also suggest anti-VEGF drugs may have broader mechanisms of action than just binding vascular endothelial growth factor (VEGF) and blocking receptor interactions for angiogenesis inhibition.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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