Epigenetic Regulation of Placenta-Specific 8 Contributes to Altered Function of Endothelial Colony-Forming Cells Exposed to Intrauterine Gestational Diabetes Mellitus

Abstract

Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation.

Figure 1

Intrauterine exposure to GDM induces altered mRNA expression in neonatal ECFCs. Thirty-eight genes in GDM-exposed ECFCs exhibited either increased or decreased expression by at least 50% compared with controls (P < 0.01). A hierarchical clustering analysis of the 38 genes is illustrated.

Figure 2

PLAC8 is increased in GDM ECFCs, while NOS3 and ALX1 are decreased. A: qRT-PCR was performed to validate the results of the microarray analysis. Results were normalized to hypoxanthine phosphoribosyltransferase and to the mean control expression for each gene (n = 6 control and 12 GDM, *P < 0.05 by unpaired t test with Welch correction). B: Western blot analysis showed that PLAC8 was increased in most GDM ECFCs compared with controls. ALX1 and NOS3 were decreased in several GDM samples compared with controls. Vinculin is the loading control. C: Maternal plasma glucose levels in the glucose tolerance screen correlate with PLAC8 mRNA levels in neonatal ECFCs (r = 0.83 and P = 0.0001 by Pearson correlation).

Figure 3

Several CpG sites in the PLAC8 promoter and 1st intron are differentially hypomethylated in GDM-exposed ECFCs. A: The schematic shows the promoter and intron 1 of PLAC8, where there are two transcriptional start sites, E1A and E1B. Below the schematic is a graph illustrating the CpG frequency over each 1,000 bp region. The first start site E1A is denoted as “0” on the graph. PCR primers for bisulfite sequencing were generated to amplify CpG-rich regions at 1,357–1,617, 4,457–4,909, and 5,791–6,072, as shown by hash marks on the schematic. B: qRT-PCR identified PLAC8 mRNA variants present in control and GDM-exposed ECFCs. Two primer sets differentiating E1A or E1B were used to quantitate PLAC8 isoforms. Data were normalized to hypoxanthine phosphoribosyltransferase. n = 4 control, n = 7 GDM ECFC samples; *P < 0.001 by two-way ANOVA, followed by Šidák multiple comparisons. C and D: Bisulfite sequencing was performed on regions amplified by the primer sets shown in A and in Supplementary Table 1. C (1,357–1,617 region) and D (5,791–6,072 region) illustrate bisulfite sequencing data from representative control and GDM-exposed ECFC samples. ●, methylated CpGs; ○, unmethylated CpGs. Individual rows denote data from a single clone. The CpG site numbers are listed along the bottom. E: A correlation between CpG methylation frequency and PLAC8 mRNA expression is shown for 18 ECFC samples (n = 6 control and n = 12 GDM) by Pearson analysis. CpG methylation at site 1,557 was measured in 8–12 clones for each ECFC sample and is expressed as percent methylation. RNA expression was measured by qRT-PCR on parallel samples.

Figure 4

Depletion of PLAC8 reduces proliferation and increases senescence. PLAC8-specific siRNA was used to deplete PLAC8 from GDM-exposed ECFCs, and functional assays were performed. Transfection of an siControl was used as the control. A: Western blotting confirms efficient PLAC8 protein knockdown. The Western blot shows that the control siRNA did not affect protein levels. Vinculin is the loading control. B and C: Cell cycle analysis was conducted using flow cytometry. B: Dot plots from a representative experiment are shown. C: Quantitation of cells in S phase shows increased proliferation with PLAC8 depletion (n = 12 using five different GDM-exposed ECFC samples; *P < 0.05 by paired t test). D: Representative image of siControl- and siPLAC8-transfected GDM ECFCs stained for senescence-associated β-galactosidase (blue cells). E: Quantitation of senescent cells demonstrates increased senescence with PLAC8 depletion (n = 5 experiments using two different GDM-exposed ECFC samples; *P < 0.05 by paired t test).