Becnel, Marianne

Bills, Kyle B

Conway, Matthew

Kay, Walker

Kim, Alexander

Kriak, John A

Reynolds, Jonathon

Sant, David W

Turner, Elizabeth L

English

eCommons at Roseman University

Vitamin C contributes to epigenetic regulation of genes related to diabetic retinopathy in retinal endothelial cellsElizabeth L. Turner*, Jonathon C. Reynolds*, Walker Kay, Marianne J. Becnel, Matthew Conway, Alexander Kim, John A. Kriak, Kyle Bills DC, PhD, David Sant PhD1Department of Biomedical Sciences, Noorda College of Osteopathic Medicine, Provo, UT   * Authors Contributed EquallyGlucose acts as a competitive inhibitor for vitamin C transport across the blood retinal barrier, and individuals with diabetes are known to have significantly lower vitamin C levels in the eyes than the general population. BackgroundMethodsPrimary, human retinal endothelial cells grown in 15.7 mM glucose were treated either with or without 50 μM vitamin C. RNA was extracted and RNA-seq was performed to determine transcriptional changes. Pathway analysis was performed using EnrichR, GOrilla, and Gene Set Enrichment Analysis (GSEA).ResultsVitamin C is an important cofactor for DNA methylation. A lack of vitamin C in the eyes of diabetics may contribute to dysregulation of the transcriptome and contribute to diabetic retinopathy. Figure 2. copied from Young et al. 2015 Annu Rev Nutr. As a cofactor, ascorbate participates in the cascade oxidation of 5mC, to 5hmC catalyzed by TET dioxygenases.Table 1. Table of pathways significantly downregulated in expression after treatment with vitamin c. All p-values are adjusted with the Banjamini-Hocherberg method. Figure 3. (A) Confocal imaging of 5hmC in primary retinal endothelial cells with or without 50 µM vitamin C or glutathione (GSH) for 5 days shows that global levels of 5hmC increase with vitamin C treatment, but not GSH. (B) Mean Fluorescent Intensity measurements of 5hmC staining of retinal endothelial cells treated with or without vitamin C or glutathione show that 5hmC levels increase dramatically with vitamin C. * P < 10-15, *** P < 10-20000.20.40.60.811.2Control VitC Glutathione***n.s.Figure 1. Diagram representing the competitive inhibition of glucose for vitamin C’s entry into the epithelial cells, therefore inhibiting entry into the retina. ConclusionLocal vitamin C deficiencies in the eyes of diabetics affect transcription in the retinal endothelial cells which may contribute to dysregulation of signaling related to the breakdown of the blood-retinal barrier in diabetic retinopathy.A BFigure 7. QuickGO flow-chart representing Gene Ontology pathway interactions involving terms related to leukocyte tethering or rolling. Red markings indicate pathways called as differential by GOrilla.  Figure 5. GSEA Traces from selected differential pathways.50 µM VitC0 µM VitCFigure 4. Heatmap showing the relative expression of differential genes. Red represents upregulated expression and blue represents downregulated expression. 437 genes were upregulated, and 308 genes were downregulated after treatment with vitamin C. Figure 6. qPCR confirmation of reduction in SELPgene. ANOVA p=0.0269, *p<0.05, **p<0.0150 µM VitC50 µM GSHControl

Vitamin C contributes to epigenetic regulation of genes related to diabetic retinopathy in retinal endothelial cells

https://ecommons.roseman.edu/cgi/viewcontent.cgi?article=1293&context=researchsymposium

Vitamin C contributes to epigenetic regulation of genes related to diabetic retinopathy in retinal endothelial cells

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