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Extrauterine growth restriction on pulmonary vascular endothelial dysfunction in adult male rats: the role of epigenetic mechanisms

Zhang, Liyana; Tang, Lilia; Wei, Jiakaia; Lao, Linjianga; Gu, Weizhongb; Hu, Qiongyaoa; Lv, Yinga; Fu, Linchena; Du, Lizhonga

doi: 10.1097/HJH.0000000000000309
ORIGINAL PAPERS: Genetics/epigenetics

Objective: Early postnatal life is considered as a critical time window for the determination of long-term metabolic states and organ functions. Extrauterine growth restriction (EUGR) causes the development of adult-onset chronic diseases, including pulmonary hypertension. However, the effects of nutritional disadvantages during the early postnatal period on pulmonary vascular consequences in later life are not fully understood. Our study was designed to test whether epigenetics dysregulation mediates the cellular memory of this early postnatal event.

Methods and results: To test this hypothesis, we isolated pulmonary vascular endothelial cells by magnetic-activated cell sorting from EUGR and control rats. A postnatal insult, nutritional restriction-induced EUGR caused development of an increased pulmonary artery pressure at 9 weeks of age in male Sprague–Dawley rats. Methyl-DNA immune precipitation chip, genome-scale mapping studies to search for differentially methylated loci between control and EUGR rats, revealed significant difference in cytosine methylation between EUGR and control rats. EUGR changes the cytosine methylation at approximately 500 loci in male rats at 9 weeks of age, preceding the development of pulmonary hypertension and these represent the candidate loci for mediating the pathogenesis of pulmonary vascular disease that occurs later in life. Gene ontology analysis on differentially methylated genes showed that hypermethylated genes in EUGR are vascular development-associated genes and hypomethylated genes in EUGR are late-differentiation-associated and signal transduction genes. We validated candidate dysregulated loci with the quantitative assays of cytosine methylation and gene expressions.

Conclusion: These results demonstrate that epigenetics dysregulation is a strong mechanism for propagating the cellular memory of early postnatal events, causing changes in the expression of genes and long-term susceptibility to pulmonary hypertension, and further providing a new insight into the prevention and treatment of EUGR-related pulmonary hypertension.

aDepartment of Neonatology

bDepartment of Pathology, The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, P.R. China

Correspondence to Lizhong Du, Professor, The Children's Hospital, Zhejiang University School of Medicine, Hangzhou 310003, P.R. China. Tel: +86 571 87068247; fax: +86 571 87033296; e-mail:

Abbreviations: ChIP, chromatin immunoprecipitation; DOHaD, developmental origins of health and disease; eNOS, endothelial nitric oxide synthase; EUGR, extrauterine growth restriction; GEO, gene expression omnibus; GO, gene ontology; IGF-1, insulin-like growth factor-1; IUGR, intrauterine growth restriction; MACS, magnetic-activated cell sorting; MeDIP-chip, methyl-DNA immune precipitation chip; PH, pulmonary hypertension; PPARγ, peroxisome proliferator-activated receptor gamma; PPHN, persistent pulmonary hypertension of the newborn; PVECs, pulmonary vascular endothelial cells; ROI, region of interest; TSS, transcription start site

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Received February 5, 2014

Received in revised form June 11, 2014

Accepted June 11, 2014

© 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins