Developmental programming in human umbilical cord vein endothelial cells following fetal growth restriction

Abstract

Background: Fetal growth restriction (FGR) is associated with an increased susceptibility for various noncommunicable diseases in adulthood, including cardiovascular and renal disease. During FGR, reduced uteroplacental blood flow, oxygen and nutrient supply to the fetus are hypothesized to detrimentally influence cardiovascular and renal programming. This study examined whether developmental programming profiles, especially related to the cardiovascular and renal system, differ in human umbilical vein endothelial cells (HUVECs) collected from pregnancies complicated by placental insufficiency-induced FGR compared to normal growth pregnancies. Our approach, involving transcriptomic profiling by RNA-sequencing and gene set enrichment analysis focused on cardiovascular and renal gene sets and targeted DNA methylation assays, contributes to the identification of targets underlying long-term cardiovascular and renal diseases.

Results: Gene set enrichment analysis showed several downregulated gene sets, most of them involved in immune or inflammatory pathways or cell cycle pathways. seven of the 22 significantly upregulated gene sets related to kidney development and four gene sets involved with cardiovascular health and function were downregulated in FGR (n = 11) versus control (n = 8). Transcriptomic profiling by RNA-sequencing revealed downregulated expression of LGALS1, FPR3 and NRM and upregulation of lincRNA RP5-855F14.1 in FGR compared to controls. DNA methylation was similar for LGALS1 between study groups, but relative hypomethylation of FPR3 and hypermethylation of NRM were present in FGR, especially in male offspring. Absolute differences in methylation were, however, small.

Conclusion: This study showed upregulation of gene sets related to renal development in HUVECs collected from pregnancies complicated by FGR compared to control donors. The differentially expressed gene sets related to cardiovascular function and health might be in line with the downregulated expression of NRM and upregulated expression of lincRNA RP5-855F14.1 in FGR samples; NRM is involved in cardiac remodeling, and lincRNAs are correlated with cardiovascular diseases. Future studies should elucidate whether the downregulated LGALS1 and FPR3 expressions in FGR are angiogenesis-modulating regulators leading to placental insufficiency-induced FGR or whether the expression of these genes can be used as a biomarker for increased cardiovascular risk. Altered DNA methylation might partly underlie FPR3 and NRM differential gene expression differences in a sex-dependent manner.

Keywords: DNA methylation; Developmental programming; Epigenetics; FPR3; Fetal growth restriction; Gene set enrichment analysis; Human umbilical cord vein endothelial cells; LGALS1; NRM; RNA-sequencing; Sex differences.

Fig. 1

Gene expression values of the genes that significantly differed between fetal growth restriction and control. TMM normalized gene expression of lectin galactoside-binding soluble 1 (LGALS1), formyl peptide receptor 3 (FPR3), nuclear envelope membrane protein (NRM) and RP5-855F14.1 in human umbilical vein endothelial cells collected from pregnancies complicated by fetal growth restriction (FGR) compared to control (CON). CPM, count per million. Data shown as mean ± SD

Fig. 2

DNA methylation at individual CpG positions for LGALS1. a The examined CpG positions in relation to the transcription start site (TSS); b DNA methylation at CpG1; c DNA methylation at CpG2; d DNA methylation at CpG3; e DNA methylation at CpG4 in fetal growth restriction (FGR) (n = 11) vs. control (n = 8). Data shown as Mean ± SD. Tested with two-way ANOVA with Bonferroni multiple comparison. LGALS1, lectin galactoside-binding soluble 1

Fig. 3

DNA methylation at individual CpG positions for NRM. a The examined CpG positions in relation to the transcription start site (TSS); b DNA methylation at CpG1; c DNA methylation at CpG2; d DNA methylation at CpG3; e DNA methylation at CpG4; f DNA methylation at CpG5; g DNA methylation at CpG6 in fetal growth restriction (FGR) (n = 11) vs. control (n = 8). Data shown as mean ± SD. Tested with two-way ANOVA with Bonferroni multiple comparison. NRM, nurim nuclear envelope membrane protein