Ascorbate suppresses VEGF expression in retinal pigment epithelial cells

David W. Sant, Vladimir Camarena, Sushmita Mustafi, Yiwen Li, Zachary Wilkes, Derek van Booven, Rong Wen, Gaofeng Wang

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

PURPOSE. To investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells. METHODS. 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. RESULTS. 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 gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo-/-mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor. CONCLUSIONS. Ascorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD).

Original languageEnglish (US)
Pages (from-to)3608-3618
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number8
DOIs
StatePublished - Jul 1 2018

Keywords

  • 5-hydroxymethylcytosine
  • Age-related macular degeneration
  • Ascorbate
  • Retinal pigment epithelium
  • VEGF

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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