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

1 Citation (Scopus)

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

Fingerprint

Retinal Pigments
Vascular Endothelial Growth Factor A
Epithelial Cells
Mustelidae
Vitreous Body
DNA
Enzyme-Linked Immunosorbent Assay
Genes
RNA Sequence Analysis
Hypoxia-Inducible Factor 1
Choroid
Macular Degeneration
Gene Expression Profiling
DNA Sequence Analysis
Immunoprecipitation
Cultured Cells
Down-Regulation
Genome
Diet
Polymerase Chain Reaction

Keywords

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

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Ascorbate suppresses VEGF expression in retinal pigment epithelial cells. / Sant, David W.; Camarena, Vladimir; Mustafi, Sushmita; Li, Yiwen; Wilkes, Zachary; van Booven, Derek; Wen, Rong; Wang, Gaofeng.

In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 8, 01.07.2018, p. 3608-3618.

Research output: Contribution to journalArticle

Sant, David W. ; Camarena, Vladimir ; Mustafi, Sushmita ; Li, Yiwen ; Wilkes, Zachary ; van Booven, Derek ; Wen, Rong ; Wang, Gaofeng. / Ascorbate suppresses VEGF expression in retinal pigment epithelial cells. In: Investigative Ophthalmology and Visual Science. 2018 ; Vol. 59, No. 8. pp. 3608-3618.
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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).",
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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

PY - 2018/7/1

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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).

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KW - Ascorbate

KW - Retinal pigment epithelium

KW - VEGF

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