Age-related macular degeneration (AMD) is a complex disease with both genetic and environmental factors implicated in its development and progression. Twin studies have shown that 46% to 71% of AMD severity can be explained by genetic factors, while environmental variations are responsible for 19% to 37%.1 Furthermore, environmental factors such as smoking, body mass index, serum C-reactive protein, and dietary intake were found to modify AMD genetic susceptibility.2–5
Epigenetics is the study of covalent modifications of the genome that can change gene structure and function without altering its sequence. These mechanisms include DNA methylation, histone acetylation/deacetylation, chromatin remodeling and noncoding RNA-mediated gene silencing. Epigenetic mechanisms can explain the impact of gene-environment interactions on different disease phenotypes.6
The introduction of antiangiogenic agents has revolutionized the management of wet AMD.7 Despite good results obtained by these drugs, 10% to 15% of patients continue to lose vision.8–12 This variability in patient response to therapy has been attributed to several clinical, behavioral, and genetic factors, as well as to possible gene-gene or gene-environmental interactions.13–16
This study examines the effects of antiangiogenic drugs on the transcription of acetylation genes in immortalized human retinal pigment epithelium cells (ARPE-19) in vitro, to determine if epigenetic pathways are modulated by antiangiogenic treatments.
Materials and Methods
Immortalized ARPE-19 cells were cultured until confluent in 175 cm2-flasks containing DMEM/F-12 culture medium (Dulbecco's Modification of Eagle's Medium; Mediatech, Manassas, VA), 10% dialyzed fetal bovine serum, supplementary antibiotics, and glucose. The cells were then plated into six-well plates for 24 hours. Culture media were removed and replaced with the same media containing aflibercept (Eylea; Regeneron, Tarrytown, NY), ranibizumab (Lucentis; Genentech, South San Francisco, CA), or bevacizumab (Avastin; Genentech, South San Francisco, CA) at one (1×) or two times (2×) the concentrations of the clinical intravitreal dose (12.5 μl/ml and 25 μl/ml, respectively). Cells were treated with antivascular endothelial growth factor (VEGF) drugs for 24 hours, and then pelleted for RNA isolation using a PureLink RNA Mini Kit (Ambion; Thermo Fisher Scientific, Waltham, MA). For real-time quantitative reverse transcription polymerase chain reaction (qRTPCR) analyses, 100 ng of individual RNA samples were reverse transcribed into complementary DNA using SuperScript VILO Master Mix (Thermo Fisher Scientific, Waltham, MA).
To identify genes associated with epigenetic pathways, we used the TaqMan Array 96-well plate (Thermo Fisher Scientific, Waltham, MA). This plate contains 28 genes that were specifically related to DNA methylation and transcription repression associated genes along with four candidate endogenous control genes. TaqMan Assay and complementary DNA samples from ARPE-19 cell cultures treated with aflibercept or bevacizumab at 1× concentration were added to each well, and qRT-PCR was performed using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA). Untreated samples were used as control.
After identification of possible target genes with the TaqMan Array, the transcription levels were verified using qRT-PCR in triplicate. We used primers for genes associated with epigenetic acetylation pathways: histone acetyltransferase 1 (HAT1) and histone deacetylases 1, 6 and 11 (HDAC1, HDAC6, and HDAC11). The qRT-PCR was performed on individual samples using a QuantiFast SYBR Green PCR Kit (Qiagen, Germantown, MD) on a StepOnePlus Real-Time PCR system. For the various target genes, housekeeping genes that had comparable amplification efficiencies to the genes of interest were chosen to maximize the accuracy of our differences in cycle threshold (ΔΔCt) values. The housekeeper genes were either hypoxanthine phosphoribosyltransferase 1 (HPRT1) or hydroxymethylbilane synthase (HMBS). Untreated samples were used as control. ΔΔCts were obtained, and folds were calculated using the formula 2^ΔΔCt.
Statistical analyses of the data were performed by unpaired t test using GraphPad Prism, Version 5 (GraphPad Software, La Jolla, CA). A P value less than .05 was considered statistically significant. Untreated samples (controls) were normalized to a value of 100% for comparison to treated samples.
Aflibercept-treated (1×) ARPE-19 cells exhibited changes in expression of all markers of histone deacetylation: HAT1 (1.18-fold, P = .02), HDAC1 (0.74-fold, P < .001), HDAC6 (1.11-fold, P = .04), and HDAC11 (1.27-fold, P = .02). At 2× concentration of aflibercept, only HAT1 (1.21-fold, P = .01) and HDAC11 (1.48-fold, P = .02) were significantly altered.
Bevacizumab-treated (1×) ARPE-19 cells also had significant change in the expression of all four genes compared with untreated cells: HAT1 (1.26-fold, P = .003), HDAC1 (0.73-fold, P < .001), HDAC6 (1.3-fold, P = .004), and HDAC11 (1.52-fold, P = .009). In cultures treated with 2× bevacizumab, expression of only HAT1 (1.97-fold, P = .01) and HDAC11 (1.39-fold, P = .01) were significantly affected compared with controls.
Compared with untreated APRE-19 cells, 1× ranibizumab-treated cells expressed higher levels of HAT1 (1.55-fold, P = .007) and HDAC6 (1.26-fold, P = .02), but none of the other epigenetic acetylation pathway markers were changed. In ARPE-19 cells treated with 2× ranibizumab, HAT1 (4-fold, P = .02) and HDAC11 (1.32-fold, P = .006) were the only epigenetic markers expressed differently compared with untreated cells (Table, Figure).
Fold Expression of Histone Acetylation Genes in Untreated and Anti-VEGF treated ARPE-19 cultures
Expression profiles for histone acetylation genes in untreated and anti-vascular endothelial growth factor treated human retinal pigment epithelium cell (ARPE-19) cultures.
*P < .05; **P < .01; ***P < .001
CT = difference in cycle thresholds; A1X = aflibercept at one time the concentration of clinical intravitreal dose; A2X = aflibercept at two times the concentration of clinical intravitreal dose; B1X = bevacizumab at one time the concentration of clinical intravitreal dose; B2X = bevacizumab at two times the concentration of intravitreal clinical dose; HAT = histone acetyltransferase; HDAC = histone deacetylase; R1X = ranibizumab at one time the concentration of clinical intravitreal dose; R2X = ranibizumab at two times the concentration of clinical intravitreal dose; UNT = untreated.
VEGF plays a key role in the pathogenesis of neovascularization and has been the main target for wet AMD therapy.17 Many studies have attempted to explain the resistance of a subset of patients to anti-VEGF therapy, but to date, pharmacogenetic studies have yielded inconclusive results. Several studies reported a significant association between specific single nucleotide polymorphisms, or SNPs, in genes known to be associated with AMD risk and anti-VEGF treatment outcomes.13,14,18–25 Conversely, other studies, including analyses of the major CATT and IVAN trials, were not able to prove this association.26–30 This inconsistency may be the result of different study designs, or due to the modification of genetic susceptibility by environmental factors.
Epigenetic factors have been implicated in the pathogenesis of AMD by selective transcription of genes involved in angiogenesis, inflammation, and oxidative stress pathways.31,32 These findings led us to the hypothesis that anti-VEGF drugs might interact with the epigenome of retinal cells, leading to differential responses to therapy in different patients. We used immortalized ARPE-19 cells to investigate this potential effect in vitro. Our results show that anti-VEGF drugs can alter the expression profiles of genes regulating the histone acetylation status in ARPE-19 cell line. We demonstrated this effect with both the clinically used concentration (1×) and a twofold concentration (2×).
Histone acetylation and deacetylation affect DNA transcription by two mechanisms: controlling the accessibility of DNA for transcription factors by altering chromatin folding properties, and modifying lysine residues to enhance specific binding sites for recruitment of repressors and activators of gene activity.6 HDAC inhibitors are potent angiogenesis suppressors in vivo and in vitro. Treatment of ARPE-19 cells with HDAC inhibitors increased the expression of clusterin protein; a major regulator of the complement pathway that acts to prevent cytolysis. Furthermore, HDAC inhibitors can significantly reduce ischemic retinal injury.33–35 Based on these findings, regulation of acetylation in the retina might have an important role in neuroprotection.
Our findings suggest the so-called drug resistance or tachyphylaxis may be in part due to the treatment interaction with different epigenome profiles of different patients. Greater understanding of this event could possibly help us in the future to tailor therapies for patients based on an epigenetic analysis, hopefully by means of a simple blood test.
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Fold Expression of Histone Acetylation Genes in Untreated and Anti-VEGF treated ARPE-19 culturesa
|Gene||Fold||P Value||Fold||P Value||Fold||P Value||Fold||P value||Fold||P Value||Fold||P Value|
|HDAC1||0.74||< .001||0.9||.25||0.73||< .001||0.97||.73||0.91||.54||1.34||.07|