The palliative effects of folic acid on retinal microvessels in diabetic retinopathy via regulating the metabolism of DNA methylation and hydroxymethylation

Abstract

Diabetic retinopathy (DR) is one of the severe microvascular complications of diabetes. The protective effects of FA on retinal vascular endothelial cells against high glucose levels involve in multiple aspects in DR; however, the underlying mechanism is not fully elucidated. In present study, we investigated the transcriptome as well as genome-wide DNA methylation and hydroxymethylation signature in human retinal microvascular endothelial ACBRI 181 cells cultured within high glucose (HG) medium supplemented with or without FA by RNA-seq, MeDIP-seq, and hMeDIP-seq. Total 3308 differential expressed genes (DEGs) were involved in multiple biological processes and molecular functions containing angiogenesis, inflammation, S-adenosyl methionine metabolism, and hypoxia response. Moreover, the global DNA methylation and hydroxymethylation in ACBRI 181 cells with FA treatment were both compromised compared to HG. Combined with transcriptome data, four subclusters of DEGs with hyper- or hypomethylated promoters were further verified. Unexpectedly, promoters of these 487 genes all displayed a pattern of increased DNA hydroxymethylation. Furthermore, hyperglycemia rat model was established and administered with FA. The DNA methylation and hydroxymethylation changes of selected target genes COL1A1, ITGA7, MMP-14, and VEGFB confirmed by MeDIP-qPCR were consistent with the results in human ACBRI 181 cells. Finally, the presence of activated DNMT1 and TET2 induced by FA was determined in ACBRI 181 cells and hyperglycemia rat. Taken together, this research provided a resource of expression and epigenetic profiles in retinal microvascular endothelial cell, emphasizing a pharmacological mechanism of FA on DNA methylation and hydroxymethylation regulation in retinal microvessel cells of DR.

Keywords: DNA hydroxymethylation; DNA methylation; Diabetic retinopathy; folic acid.

Figure 1.

Expression profile of ACBRI 181 cells affected by HG and FA. (a) Venn diagram view of DEGs compared among NC, HG and FA (log₂ FC >1 or <-1, p < 0.01). (b) Biological process of GO and (c) KEGG analysis of the involved enriched functions. (d) Gene browser views of transcription of ALDH3A1, COL1A1, FGF23, ITGA7, MMP14, THBS1, and VEGFB. DEG: differential expressed genes; NC: negative control; HG: high glucose/hyperglycemia; FA: folic acid; FC: fold change; GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes

Figure 2.

Genome-wide DNA methylation and hydroxymethylation atlas in retinal epithelium. (a) The distribution of DMR (red) and DHMR (green) regions in genomic contexts in ACBRI 181 cells of NC, HG and FA in vitro (log₂ FC > 1 or < −1, FDR < 10⁻⁴). (b) The subclusters of DEGs with DMR and DHMR. (c) Pearson correlation of gene expression with DNA methylation and DNA hydroxymethylation. The coefficient value of each gene is calculated and averaged. (d) Isolation of primary rat retinal endothelial cells by positive CD31 and negative CD45. (e) DNA methylation and (f) hydroxymethylation at promoters of the target genes and (g) their transcription including ALDH3A1, COL1A1, FGF23, ITGA7, MMP14, THBS1, and VEGFB in rat retinal endothelial cells. qPCR was performed three individual experiments. ‘*’ and ‘^’ represent p value less than 0.05 by comparison between NC and HG, respectively. DMR: differential methylated regions; DHMR: differential hydroxymethylated regions; FPKM: fragments per kilobase of exon model per million mapped fragments; NC: negative control; HG: high glucose/hyperglycemia; FA: folic acid

Figure 3.

The role of DNMTs and TETs in retinal epithelium. (a) The enzyme activity of DNMTs and TETs in ACBRI 181 cells affected by HG and FA. (b) The enzyme activity of DNMTs and TETs in rat retinal endothelial cells affected by HG and FA. (c) ChIP-qPCR assay of DNMTs on promoter of MMP14. (d) ChIP-qPCR assay of TETs on promoter of MMP14. ChIP-qPCR was performed three individual experiments. ‘*’ and ‘^’ represent p value less than 0.05 by comparison between NC and HG, respectively