Epigenetic and senescence markers indicate an accelerated ageing-like state in women with preeclamptic pregnancies

Abstract

Background: Preeclampsia is a pregnancy-specific hypertensive disorder characterized by proteinuria and/or multisystem involvement. Disease-specific therapy has yet to be developed due to the lack of understanding of underlying mechanism(s). We postulate that accelerated ageing in general, and particularly cellular senescence, play a role in its pathophysiology.

Methods: We compared women with preeclampsia vs. normotensive pregnancies with respect to epigenetic markers of ageing and markers of senescence in tissues/organs affected by preeclampsia (blood, urine, adipose tissue, and kidney).

Findings: We demonstrate that preeclamptic compared to normotensive pregnant women: (i) undergo accelerated epigenetic ageing during pregnancy, as demonstrated by an "epigenetic clock"; (ii) exhibit higher levels/expression of senescence-associated secretory phenotype factors in blood and adipose tissue; (iii) display increased expression of p16^INK4A in adipose tissue and renal sections, and (iv) demonstrate decreased levels of urinary α-Klotho (an anti-ageing protein) at the time of delivery. Finally, we provide data indicating that pre-treatment with dasatinib, a senolytic agent, rescues the angiogenic potential of mesenchymal stem cells (MSC) obtained from preeclamptic pregnancies, and promotes angiogenesis, even under pro-inflammatory conditions.

Interpretation: Taken together, our results identify senescence as one of the mechanisms underpinning the pathophysiology of preeclampsia. Therapeutic strategies that target senescent cells may offer novel mechanism-based treatments for preeclampsia.

Funding: This work was supported by NIH grants, R01 HL136348, R37 AG013925, P01 AG062413, R01 DK11916, generous gifts from the Connor Fund, Robert J. and Theresa W. Ryan and from The George G. Beasley family, the Noaber Foundation, and the Henry and Emma Meyer Professorship in Molecular Genetics.

Keywords: ageing; epigenetic clock; preeclampsia; senescence.

Fig. 1

Epigenetic ageing is accelerated during PE compared to normotensive pregnancies. (a) The epigenetic age of women with PE (n = 11) increased with progression of pregnancy from the first to the third trimester: first trimester, 30 ± 8 years; second trimester, 31 ± 9 years; and delivery, 32 ± 9 years. Epigenetic ages for women with normotensive pregnancies (n = 33) did not change over the course of their pregnancies (32 ± 6 years for all time points). There was a statistically significant difference in epigenetic ages change during the course of pregnancies in PE vs. normotensive pregnancies (P = 0·047, ANOVA for repeated measures) (b) When comparing the estimated epigenetic ages to the actual chronological ages (ΔE/CA), there was an age increase in PE vs. normotensive women at delivery, with a 2·4-year difference. Horvath's regression model was applied to the DNA methylation dataset to estimate DNA methylation (epigenetic) age, for women with PE and normotensive pregnancies.

Fig. 2

Higher senescence burden occurs in pregnant women with PE compared to normotensive pregnant women. (a) Women with PE (n = 11) have higher plasma p16^INK4A concentrations (0·66 [0·06–2·23]) than normotensive women (n = 13) (0·38 [0·38–0·38]), P = 0·026,) (Mann-Whitney U test) (b) Plasma p21^CIP1 concentrations were not different between the groups (normotensive: 113·5 [35·33–173·50], PE: 210·40 [57·37–616·10]; P = 0·167 (Mann-Whitney U test), NT = 13, PE = 11) (c) Urinary α-Klotho is lower in women with PE compared to those with normotensive pregnancies (94·66 [38·36–210·50] vs. 308·90 [157·90–799·10], respectively; P = 0·029, Mann-Whitney U test). Dots represent the individual values (mean value of the duplicate); lines represent the median with interquartile range. NT-normotensive, PE-preeclamptic pregnancies; *- P < 0·05, NS-not significant.

Fig. 3

Senescence-associated secretory phenotype (SASP) components are increased in adipose tissue of pregnant women with PE compared to normotensive pregnant women. (a) After protein isolation from the adipose tissue from PE (n = 8) compared to normotensive (n = 8) pregnancies, SASP component analysis was performed and indicated upregulation of both MCP-1 (4·76 ± 1·40 vs. 3·17 ± 1·44, respectively, P = 0·023, Student's t test) and (b) TNF-α (0·17 ± 0·04 vs. 0·12 ± 0·03, respectively, P = 0·015, Student's t test). Dots represent the individual values (mean value of the duplicate); lines represent the mean with SD. NT-normotensive, PE-preeclamptic pregnancies *-p < 0·05.

Fig. 4

Pregnant women with PE demonstrate stronger p16^INK4A nuclei staining of renal and adipose tissue sections, compared to normotensive pregnant women. (a) Abundant p16^INK4A staining in the kidney and adipose tissue sections of PE women, but not in NT pregnancies. (b-c) p16^INK4A expression was significantly increased in adipose tissue and renal sections in PE pregnancies compared to normotensive pregnancies: 1·04 [0·38–2·81 vs. 0·55 [ 0·00–0·61], P = 0·043 (Mann-Whitney U test) and 7·39 [3·72–13·93] vs. 0·03 [0·00–0·33], P = 0·034, Mann-Whitney U test), respectively. Individual values with median and interquartile ranges are presented in the graph. Red and black arrows indicate positive (brown) staining for p16^INK4A in the kidney and adipose tissue, respectively. NT-normotensive, PE-preeclamptic pregnancies, *- P < 0·05 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 5

Under pro-inflammatory conditions, senolytics improve the angiogenic potential of MSC isolated from PE pregnant women. (a) HUVECs pre-treated with the pro-inflammatory factor, TNF-α (50 ng/ml), demonstrate impaired angiogenesis compared to non-treated endothelial cells (P = 0·048, ANOVA for repeated measures); (b) The TNFα-treated endothelial cells were co-cultured with MSC isolated either from PE or NT pregnancies. The PE-MSC group showed a lower angiogenic potential compared to its normotensive counterpart (P = 0·024, ANOVA for repeated measures). (c) After PE-MSC were treated with senolytic agent, dasatinib, angiogenesis improved, reaching the angiogenic potential of NT-MSC (P > 0·05, ANOVA for repeated measures). There was a significant difference in total network growth per mm² in wells incubated with vehicle PE-MSC (treated with 1% DMSO) vs. dasatinib-treated PE-MSC (P = 0·016, ANOVA for repeated measures). Endothelial cells co-cultured with vehicle NT-MSC differed significantly from vehicle PE-MSC (P = 0·018, ANOVA for repeated measures). Lines represent total network growth per mm² measured as an average of sextuplicate with standard error mean. Analyzes were performed on the samples of 3 PE-MSC and 3 NT-MSC. Images were captured every 6 h and the data shown graphically are those obtained at the 24 h time points for each condition. HUVEC-human umbilical vein endothelial cells; MSC- mesenchymal stem cells; NT-normotensive, PE-preeclamptic women, **- P < 0·05