Prenatal inflammation exposure-programmed hypertension exhibits multi-generational inheritance via disrupting DNA methylome

Abstract

The multi-generation heredity trait of hypertension in human has been reported, but the molecular mechanisms underlying multi-generational inheritance of hypertension remain obscure. Recent evidence shows that prenatal inflammatory exposure (PIE) results in increased incidence of cardiovascular diseases, including hypertension. In this study we investigated whether and how PIE contributed to multi-generational inheritance of hypertension in rats. PIE was induced in pregnant rats by intraperitoneal injection of LPS or Poly (I:C) either once on gestational day 10.5 (transient stimulation, T) or three times on gestational day 8.5, 10.5, and 12.5 (persistent stimulation, P). Male offspring was chosen to study the paternal inheritance. We showed that PIE, irrespectively induced by LPS or Poly (I:C) stimulation during pregnancy, resulted in multi-generational inheritance of significantly increased blood pressure in rat descendants, and that prenatal LPS exposure led to vascular remodeling and vasoconstrictor dysfunction in both thoracic aorta and superior mesenteric artery of adult F2 offspring. Furthermore, we revealed that PIE resulted in global alteration of DNA methylome in thoracic aorta of F2 offspring. Specifically, PIE led to the DNA hypomethylation of G beta gamma (Gβγ) signaling genes in both the F1 sperm and the F2 thoracic aorta, and activation of PI3K/Akt signaling was implicated in the pathologic changes and dysregulated vascular tone of aortic tissue in F2 LPS-P offspring. Our data demonstrate that PIE reprogrammed DNA methylome of cells from the germline/mature gametes contributes to the development of hypertension in F2 PIE offspring. This study broadens the current knowledge regarding the multi-generation effect of the cumulative early life environmental factors on the development of hypertension.

Keywords: DNA methylation; Gβγ; LPS; PI3K/Akt signaling; hypertension; multi-generational inheritance; poly (I:C); prenatal inflammation exposure.

Fig. 1. Elevated systolic blood pressure (SBP) in adult F2–F4 descendants from F1 fathers experiencing prenatal inflammation exposure (PIE).

a Schematic diagram for experimental design. F1 generation: dams were randomized on embryonic day (E) 8.5 to control, LPS-P, LPS-T, Poly (I:C)-P, or Poly (I:C)-T groups. Dams of LPS-P or Poly (I:C)-P were intraperitoneally injected with LPS (0.79 mg/kg) or Poly (I:C) potassium salt (4 mg/kg based on the weight of Poly (I:C) itself) on E8.5, 10.5 and 12.5 (P indicates persistent), while dams of LPS-T or Poly (I:C)-T were intraperitoneally injected with LPS or Poly (I:C) on E10.5 (T indicates transient). F2–F4 generation: external control virgin females were mated with F1–F3 male offspring from each group to produce: F2 or F3 or F4 control, LPS-P, LPS-T, Poly (I:C)-P, or Poly (I:C)-T offspring, respectively. Postnatal litters were equalized to eight pubs and fed ad libitum for each generation. b, c The SBP of F2 offspring from the age of 8–28 weeks by tail-cuff method (b and c) for whose paternal line experiencing prenatal LPS and Poly (I:C) stimulation, respectively. Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01 denote the statistical comparison between control and F2 LPS-P (b) or F2 Poly (I:C)-P (c) groups at the same time point, while ^#P < 0.05 and ^##P < 0.01 denote the statistical comparison between control and F2 LPS-T (B) or F2 Poly (I:C)-T (c) group at the same time point (unpaired Student’s t test for the same time point). d, e A histogram representing the distribution of SBP frequency in F2 offspring for each group at 16 weeks is shown (d and e for whose paternal line experiencing prenatal LPS and Poly (I:C) exposure, respectively). f, g The SBP in F3 (f) and F4 (g) descendants from F1 control and LPS-P offspring. h, i The distribution of SBP frequency in F3 (h) and F4 (i) descendants from F1 control and LPS-P offspring. Data are expressed as mean ± SEM. *P < 0.05 and **P < 0 .01, versus the same generation control offspring at the same time point (f–g, unpaired Student’s t test for the same time point). The numbers of animals used in each group were eight from eight different litters.

Fig. 2. Prenatal LPS exposure leads to vascular remodeling in both the thoracic aorta and superior mesenteric artery (SMA) of adult F2 offspring.

a, b Histology of thoracic aortas (a) and SMA (b) from 16-week old F2 offspring by haematoxylin-eosin (H&E) staining. Representative microphotographs with different magnification for each group are shown. Vessel wall nearby the arrow direction represents endothelium. The values of vascular wall thickness, cross-sectional area, wall:lumen ratio, and VSMC width were quantified by using NIS-Elements BR software. Data are presented as means ± SEM. n = 6 from 3 litters for each group. Each dot represents one rat. **P < 0 .01 denotes the statistical comparison between two marked groups (unpaired Student’s t test).

Fig. 3. Prenatal LPS exposure leads to vasoconstrictor dysfunction in both the thoracic aorta and superior mesenteric artery (SMA) of adult F2 offspring.

a–d Vascular reactivity of both the thoracic aorta (a and c) and SMA (b and d) rings from 16-week-old F2 offspring. PE-induced cumulative concentration–response curves (a, b) and ACh-induced relaxation–response curves (c, d) were measured in endothelia-intact thoracic aorta and SMA rings from 16-week-old F2 offspring. Concentration–response curves are expressed as the percentage of contractions caused by KCl (60 mM), and relaxation–response curves are expressed as the percentage of the maximal PE-induced contractions. Data are presented as means ± SEM. n = 8 from 4 litters for each group for a–d. **P < 0 .01 denotes the statistical comparison between two marked groups (two-way ANOVA followed Sidak’s multiple comparisons test for a–d). The pD₂ (–log EC₅₀) values were calculated for each group and shown on the top left of each panel.

Fig. 4. Characteristic of genomic differentially methylated regions (DMRs) in the thoracic aorta of F2 LPS-P offspring.

a, b Significantly enriched Gene Ontology (GO) terms in the biologic process (BP) associated with hypomethylated DMRs (a) and hypermethylated DMRs (b) identified in aortic MeDIP-Seq data of F2 LPS-P offspring relative to control offspring. Each circle represents one gene (left) and the description of each GO item are shown (right). c, d Canonical pathway enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) terms from David of hypomethylated DMRs (c) and hypermethylated DMRs (d). The color shade and size of each circle reflect –log (P value) and the number of genes enriched in each KEGG pathway, respectively. n = 4 from 4 separate litters for each group for a–d. e, f DMRs functional analysis by Ingenuity pathway analysis (IPA). Top five enriched IPA canonical pathways (e) and the most significantly enriched IPA interaction networks (f) of hypomethylated DMRs identified in aortic MeDIP-Seq data of F2 LPS-P offspring relative to control offspring. The color shade of bar reflects –log (P value) and the horizontal axe represents the number of genes enriched in each canonical pathway, respectively. Red indicates the enriched canonical pathways; blue indicates the nearest genes associated with DMRs identified in aortic MeDIP-Seq data of F2 LPS-P offspring relative to control offspring (f).

Fig. 5. Hypomethylated DMRs within the promoter region along with downregulated mRNA expression of genes clustered in Gβγ signaling exists in both the F2 thoracic aorta and F1 sperm of PIE LPS-P offspring.

a Validation of the mRNA expression by qPCR in genes clustered in Gβγ signaling pathway in the thoracic aorta of F2 LPS-P offspring relative to control offspring. Each dot represents one rat. b, c Validation of DMRs in genes clustered in Gβγ signaling pathway identified in aortic MeDIP-Seq data by bisulfite sequencing PCR (BSP) in both the thoracic aorta of F2 offspring (b) and in the sperm of F1 offspring (c). BSP results were expressed as a percentage of DNA methylation in each sequence, detailed in Supplementary Figs. S4 and S5 for a and b, respectively.

Fig. 6. Activation of PI3K-Akt signaling is implicated in the enhanced vasoconstrictor response in F2 LPS-P offspring.

a Illustration of Gβγ Signaling pathway. Genes marked with red border own hypomethylated DMRs within its DNA sequence identified in aortic MeDIP-Seq data of F2 LPS-P offspring relative to control offspring. Genes marked with purple border belong to PI3K-Akt signaling. b Protein levels of phosphorylated (p)-Akt^ser473, p-S6^Ser235/236, Akt, and S6 in thoracic aortas of F2 offspring at the age of 16 weeks were determined by immunoblotting. β-actin was taken as internal control. Representative plots of two from eight offspring in each group (left) and statistical data of relative densitometry, normalized by β-actin (right), are shown. Data are presented as means ± SEM. *P < 0.05 and **P < 0 .01 denote the statistical comparison between the two marked groups (unpaired Student’s t test). c, d Effect of PI3K inhibitor LY294002 on the vascular reactivity of aortic rings from 16-week-old F2 offspring. PE-induced cumulative concentration–response curves (c) and Ach-induced relaxation–response curves (d) were measured in endothelia-intact aortic rings after 30 min treatment with LY294002 (20 μM) or vehicle control, as described in Fig. 3. Data are presented as means ± SEM. n = 8 from 4 litters for each group. * P < 0.05 and ** P < 0 .01 denote the statistical comparison between the two marked groups (two-way ANOVA followed Sidak’s multiple comparisons test for c and d). The pD2 (–log EC₅₀) values were calculated for each group and shown on the bottom of each panel.