Transgenerational inheritance of promoter methylation changes in extrauterine growth restriction-induced pulmonary arterial pressure disorders

Abstract

Background: This study aimed to investigate the influence of extrauterine growth restriction (EUGR) on pulmonary arterial pressure (PAP) and the transgenerational inheritance of promoter methylation changes in pulmonary vascular endothelial cells (PVECs) of 2 consecutive generations under EUGR stress.

Methods: After modeling, PAP values of F1 and F2 pups were investigated at 9-week-old. The methyl-DNA immune precipitation chip was used to analyze DNA methylation profiling. Differential enrichment peaks (DEPs) and regions of interest (ROIs) were identified, based on which Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and reactome pathway enrichments were analyzed.

Results: The F1 male rats in the EUGR group had significantly increased PAP levels compared to the control group; however, this increase was not observed in female rats. Interestingly, in F2 female rats, the EUGR group had decreased PAP. In the X chromosome of the F1 males, there were 16 differential ROI genes in the F1 generation, while in F2 females, there were 86 differential ROI genes. Similarly, there were 105 DEPs in the F1 generation and 38 DEPs in the F2 generation. In combination with the 5 common ROIs and 14 common DEPs, 18 genes were regarded as the key candidate genes associated with hereditable PAP variation in the EUGR model. Enrichment analysis showed that synaptic and neurotransmitter relative pathways might be involved in the process of EUGR-induced PAH development. Among common DEPs, Smad1 and Serpine1 were also found in 102 PAH-associated genes in the MalaCards database.

Conclusions: Together, there is a transgenerational inheritance of promoter methylation changes in the X chromosome in EUGR-induced PAP disorders, which involves the participation of synaptic and neurotransmitter relative pathways. Also, attenuated methylation of Smad1 and Serpine1 in the promoter region may be a partial driver of PAH in later life.

Keywords: DNA methylation; epigenetics; extrauterine growth restriction (EUGR); pulmonary arterial pressure (PAP); transgenerational inheritance.

Figure 1

The flow chart of investigation on 2 generations and the phenotype changes. The F1 male rats but not female rats in the EUGR group have significantly increased PAP versus the control group. In F2 female rats, the EUGR group has decreased PAP. *P<0.05; **P<0.01. EUGR, extrauterine growth restriction; PAP, pulmonary arterial pressure.

Figure 2

Major findings of DNA methylation changes in the X chromosome of PVECs. (A) In the X chromosome of F1 males, there are 16 differential ROI genes in the F1 generation (EUGR vs. Control), including 9 hypermethylation ROIs and 7 hypomethylation ROIs; (B) in the X chromosome of F2 females, there are 86 differential ROI genes, including 60 hypermethylation ROIs and 7 hypomethylation ROIs. (C) Between the 16-ROI set of F1 generation and the 86-ROI set of F2 generation, there are 5 common ROIs. (D) there were 105 DEPs in F1 generation and 38 DEPs in F2 generation, between which there are 14 common DEPs. (E) In combination with the 5 common DOIs and 14 common DEPs, they had a common gene Ptges3l1, and these 18 genes were regarded as the key candidate genes associated with hereditable PAP variation in the EUGR model. PVECs, pulmonary vascular endothelial cells; ROI, region of interest; EUGR, extrauterine growth restriction; DEPs, differential enrichment peaks; PAP, pulmonary arterial pressure.

Figure 3

Enrichment analysis of 18 candidate genes (limited in the X-chromosome). (A) The covered genes in 3 categories: BP terms (red), CC terms (blue), and MF terms (green). No significantly enriched GO terms are found; (B) enriched KEGG pathways: Basal transcription factors, Arachidonic acid metabolism, Steroid hormone biosynthesis, Herpes simplex infection, and Metabolic pathways; (C) enriched reactome pathways: Phase 1-inactivation of fast Na⁺ channels, Aryl hydrocarbon receptor signaling, The activation of arylsulfatases, HSF1 activation, Serotonin Neurotransmitter Release Cycle, Dopamine Neurotransmitter Release Cycle, Attenuation phase, Receptor-type tyrosine-protein phosphatases, Ub-specific processing proteases, and Neuronal System. BP, biological process; CC, cellular component; MF, molecular function; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

Enrichment analysis based on all common ROIs between 2 generations, (not confined to the X chromosome). There are 114 common ROIs (genes) in all chromosomes. (A) The GO summary for 3 categories; (B) all enriched GO terms, KEGG pathways, and reactome pathways (only one enriched KEGG pathway: RNA polymerase, and no reactome pathway). ROIs, regions of interest; GOI, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5

Enrichment analysis based on all common DEPs between 2 generations, (not confined to the X chromosome). There were 485 common DEPs (genes) in all chromosomes. (A) GO summary for 3 categories and the included genes; (B) all enriched GO terms, KEGG pathways, and reactome pathways. All the enriched terms are the BP category, such as regulation of synaptic vesicle transport, regulation of synaptic vesicle exocytosis, regulation of neurotransmitter secretion, regulation of calcium ion-dependent exocytosis, regulation of synaptic vesicle cycle, synaptic vesicle exocytosis, calcium ion regulated exocytosis, and synaptic vesicle transport establishment of synaptic vesicle localization. No other GO terms, KEGG pathways or reactome pathways are enriched. DEPs, differential enrichment peaks; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

A possible mechanism of the EUGR-induced PAH development. There are 102 PAH-associated genes acquired from the MalaCards database and 485 common DEPs (between F1 and F2) in all chromosomes. Two common factors are found: Smad1 and Serpine1. Based on this finding, a potential mechanism of the EUGR-induced PAH development is that: under the EUGR stress, PVECs have impaired methylation of Smad1 and Serpine1, and an increased expression of SMAD1 and SERPINE1 partially triggers PAH in the later life. PAH, pulmonary arterial hypertension; EUGR, extrauterine growth restriction; DEPs, differential enrichment peaks; PVEC, pulmonary vascular endothelial cells.