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Hypermethylation of delta-like homolog 1/maternally expressed gene 3 loci in human umbilical veins

insights into offspring vascular dysfunction born after preeclampsia

Yu, Yi-Chenb,*; Jiang, Yinga,c,g,*; Yang, Meng-Menga,c; He, Sai-Nana; Xi, Xiae,g; Xu, Ye-Taof,g; Hu, Wen-Shengd; Luo, Qionga,c

doi: 10.1097/HJH.0000000000001942

Background: Increasing epidemiological studies have confirmed the association between maternal preeclampsia and elevated blood pressure in their offspring. Though case–control or cohort studies have demonstrated long-term outcomes for the offspring of preeclampsia, it is still a question that how these changes were caused by genetic reasons or by preeclampsia itself.

Objective: In our study, we explored the potential epigenetic regulation of delta-like homolog 1–maternally expressed gene 3 (DLK1–MEG3) region in human umbilical vein endothelial cells (HUVECs), and its connection with endothelium-derived factors.

Study design: We recruited 58 singletons born with spontaneous conception (control group) and 67 singletons whose mother with preeclampsia (preeclampsia group), and detected the infants’ blood pressure and growth development index. To explore the potential mechanism, we did real-time PCR to test DLK1MEG3 imprinted genes and endothelium-derived factors. ELISA confirmed the protein secretion changes between two groups. In addition to confirm epigenetic alteration in preeclampsia HUVEC, we performed pyro-sequencing to detect methylation status of two different methylation regions: intergenic differential methylation region (IG-DMR) and MEG3 DMR which control the expression of DLK1 and MEG3. Furthermore, Person correlation was used to make sure the association of methylation alteration of IG-DMR and endothelium-derived factors.

Results: In our study, we found that DBP was significantly lower in preeclampsia offspring who born over 34 weeks compared with normal offspring (53.59 ± 1.38 vs. 59.9 ± 1.40 mmHg, P < 0.01), which leads to higher pulse pressure difference. Quantitative real-time PCR showed that imprinted gene DLK1 level significantly increased and MEG3 level decreased in HUVEC of preeclampsia group compared with control group, accompanying with lower expression of endothelial nitric oxide synthase and vascular endothelial growth factor (VEGF), higher expression of endothelin-1 (ET1), which are close related with vascular endothelial function. Meanwhile, ELISA assay of ET1, nitrite, VEGF were consistent with real-time results. Furthermore, abnormal expression of DLK1–MEG3 expression was caused by hypermethylation status of IG-DMR, And methylation status of IG-DMR highly correlated with ET1 concentration and nitrate concentration, these might be one of the mechanisms for impaired endothelial function (coefficient = 0.5806, P = 0.0115; coefficient = −0.4883, P = 0.0398).

Conclusion: Our results demonstrated that altered expression of imprinted genes DLK1 and MEG3 were caused by hypermethylation of IG-DMR in HUVEC of preeclampsia group, accompanied by lower secretion of nitrite, VEGF, and higher secretion of ET1. It might be one potential mechanism for higher risk of cardiovascular disease in preeclampsia offspring later in life.

aDepartment of Obstetrics, Women's Hospital

bDepartment of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University

cKey Laboratory of Reproductive Genetics (Ministry of Education)

dDepartment of Obstetrics, Hangzhou Maternal and Child Health Care Hospital, Hangzhou

eDepartment of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Shenzhen Hospital, Shenzhen

fDepartment of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

gDepartment of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut, USA

Correspondence to Qiong Luo, Department of Obstetrics, Women's Hospital, School of Medicine, Zhejiang University, No. 1, Xueshi Road, Shangcheng District, Hangzhou, China. Tel: +86 571 87061501; fax: +86 571 87061878; e-mail:

Abbreviations: CAD, coronary artery disease; CVD, cardiovascular disease; DLK1, delta-like homolog 1; DMR, differential methylated region; eNOS, endothelial nitric oxidase synthase; ET1, endothelin-1; HUVECs, human umbilical vein endothelial cell; IUGR, intrauterine growth restriction; LBW, low birth weight; MEG3, maternally expressed gene 3; VEGF, vascular endothelial growth factor

Received 8 May, 2018

Accepted 14 August, 2018

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