Ascorbate Suppresses VEGF Expression in Retinal Pigment Epithelial Cells

Investigative Ophthalmology & Visual Science
David W SantGaofeng Wang

Abstract

To investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells. Dot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo-/- mice, which, like humans, cannot synthesize ascorbate de novo. Treatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome-3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the g...Continue Reading

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Citations

Dec 26, 2018·Oxidative Medicine and Cellular Longevity·Weiwei DuWei He
Mar 27, 2019·Therapeutic Advances in Ophthalmology·Nicholas Owen, Mariya Moosajee
Dec 29, 2020·Macromolecular Bioscience·Xiaoying ZhaQifeng Jiang
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Methods Mentioned

BETA
Dot-Blot
immunoprecipitation
hMeDIP-seq
Assay
ELISA
protein assay
RNA-seq

Software Mentioned

DESeq2
ABI 7900 HT
ImageJ
QuantStudio
UCSC genome browser
DeepTools
MACS2
STAR
IDR
edgeR

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