TET2-mediated ECM1 hypomethylation promotes the neovascularization in active proliferative diabetic retinopathy

Abstract

Background: Studies have shown that tet methylcytosine dioxygenase 2 (TET2) is highly expressed in diabetic retinopathy (DR), which reduces the DNA methylation of downstream gene promoters and activates the transcription. Abnormally expressed TET2 and downstream genes in a high-glucose environment are associated with retinal capillary leakage and neovascularization. Here, we investigated the downstream genes of TET2 and its potential association with neovascularization in proliferative diabetic retinopathy (PDR).

Methods: GSE60436, GSE57362, and GSE158333 datasets were analyzed to identify TET2-related hypomethylated and upregulated genes in PDR. Gene expression and promoter methylation of these genes under high glucose treatment were verified. Moreover, TET2 knockdown was used to assess its impact on tube formation and migration in human retinal microvascular endothelial cells (HRMECs), as well as its influence on downstream genes.

Results: Our analysis identified three key genes (PARVB, PTPRE, ECM1) that were closely associated with TET2 regulation. High glucose-treated HRMECs exhibited increased expression of TET2 and ECM1 while decreasing the promoter methylation level of ECM1. Subsequently, TET2 knockdown led to decreased migration ability and tube formation function of HRMECs. We further found a decreased expression of PARVB, PTPRE, and ECM1, accompanied by an increase in the promoter methylation of ECM1.

Conclusions: Our findings indicate the involvement of dysregulated TET2 expression in neovascularization by regulating the promoter methylation and transcription of downstream genes (notably ECM1), eventually leading to PDR. The TET2-induced hypomethylation of downstream gene promoters represents a potential therapeutic target and offers a novel perspective on the mechanism underlying neovascularization in PDR.

Keywords: DNA methylation; ECM1; Epigenetics; Neovascularization; Proliferative diabetic retinopathy (PDR); TET2.

Fig. 1

Identification of DMGs. A Heat map of the top 100 significant DMGs in the GSE57362 dataset. B Volcano plot of the DMGs in the GSE57362 dataset. C Volcano plot of the DEGs obtained from the GSE60436 dataset in the active FVM compared with the normal retina. D Volcano plot of the DEGs obtained from the GSE60436 dataset comparing the active and inactive FVM. E Venn diagram of the intersection of hypomethylated genes, upregulated genes in active FVM, and downregulated genes in inactive FVM. DMGs, differentially methylated genes; DEGs, differentially expressed genes; PDR, proliferative diabetic retinopathy; FVM, fibrovascular membranes

Fig. 2

Enrichment analysis of 62 hypomethylated upregulated genes and identification of TET2 downstream target genes. A KEGG enrichment analysis of 62 genes. B GO analysis of 62 genes. C Volcano plot of the DEGs in the GSE158333 dataset. D Venn diagram of all gene sets. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; DEGs, differentially expressed genes; FVM, fibrovascular membranes

Fig. 3

Validation of TET2-targeted hypomethylated upregulated genes. A Expression of TET2 and the three key genes. B Protein levels of three key genes in NC and HG groups. C Quantitative analysis of promoter methylation levels of the three key genes. D Agarose gel electrophoresis results of MS-PCR products of the key genes. MS-PCR, methylation-specific PCR; M, methylation bands; U, unmethylated bands; M/U, quantifying the ratio of the intensity of the methylated band to the unmethylated band; NC, normal control; HG, high glucose

Fig. 4

Verification of expression and promoter methylation of the three key genes. A Migration assay of HRMECs. B Tube formation assay of HRMECs. C Quantitative analysis of promoter methylation level of the three key genes. D Protein levels of three key genes after TET2 knockdown. E Quantitative analysis of promoter methylation levels of the three key genes. F Agarose gel electrophoresis results of MS-PCR products of the key genes. MS-PCR, methylation-specific PCR; M, methylation bands; U, unmethylated bands; M/U, quantifying the ratio of the intensity of the methylated band to the unmethylated band; NC, normal control; HG, high glucose

Fig. 5

Possible pathological mechanisms of retinal neovascularization and FVM proliferation in patients with PDR. TET2 overexpression in retinal endothelial cells is increased in the diabetic environment, prompting TET2 to act on the CpG island, reducing the methylation level of the ECM1 gene promoter. The gene of ECM1 opens and allows transcription, thereby increasing the expression level. Through a series of subsequent pathways, ECM1 promotes retinal neovascularization and FVM proliferation, ultimately leading to the development of PDR