TY - JOUR
T1 - Ascorbate suppresses VEGF expression in retinal pigment epithelial cells
AU - Sant, David W.
AU - Camarena, Vladimir
AU - Mustafi, Sushmita
AU - Li, Yiwen
AU - Wilkes, Zachary
AU - van Booven, Derek
AU - Wen, Rong
AU - Wang, Gaofeng
N1 - Funding Information:
The authors thank Sheldon Miller at the National Eye Institute for providing hfRPE cells and Kevin Dickson, Christopher Gustafson, Blake Hampton, and Lena Dennison for their technical assistance in this research. Supported by National Institutes of Health Grant R01NS089525 and BrightFocus Grant M2017081 (to GW).
Funding Information:
The authors thank Sheldon Miller at the National Eye Institute for providing hfRPE cells and Kevin Dickson, Christopher Gustafson, Blake Hampton, and Lena Dennison for their technical assistance in this research. Supported by National Institutes of Health Grant R01NS089525 and BrightFocus Grant M2017081 (to GW). Disclosure: D.W. Sant, None; V. Camarena, None; S. Mustafi, None; Y. Li, None; Z. Wilkes, None; D. Van Booven, None; R. Wen, None; G. Wang, None
Publisher Copyright:
© 2018 The Authors.
PY - 2018/7
Y1 - 2018/7
N2 - 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).
AB - 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).
KW - 5-hydroxymethylcytosine
KW - Age-related macular degeneration
KW - Ascorbate
KW - Retinal pigment epithelium
KW - VEGF
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U2 - 10.1167/iovs.18-24101
DO - 10.1167/iovs.18-24101
M3 - Article
C2 - 30025088
AN - SCOPUS:85050339656
VL - 59
SP - 3608
EP - 3618
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 8
ER -