| Part I:Epigenetics of Notchl regulation in pulmonary microvascular rarefaction following extrauterine growth restrictionObjective:Increased risk of adult-onset cardiovascular disease may originate from nutritional disturbances during perinatal period. In the search for mechanisms underlying the disadvantageous vascular phenotype presented in adult, growing data support the hypothesis that physiological adaptations in response to early nutritional deprivation may induce permanent alteration in the development of structures and function in cardiovascular system, In a EUGR rat model, we reported an elevated pulmonary arterial pressure (PAP) in adult and genome-wide epigenetic modification in pulmonary vascular endothelial cells (PVECs). However whether EUGR has an early impact on pulmonary vasculature, and hence results in pulmonary vascular dysfunction in later life is presently unknown.Methods:A rat model was used to investigate the physiological and structural effect of EUGR on early pulmonary vasculature by evaluating right ventricular systolic pressure (RVSP) and pulmonary vascular density of male rat. Epigentic modifications of Notch1gene in PVECs were evaluated.Results:1. EUGR decreased pulmonary vascular density with no significant impact on right ventricular systolic pressure at3weeks. Mean pulmonary pressure was increased at9weeks with no significant impact on pulmonary vascular density.2. Decreased transcription of Notchl was observed both at3and9weeks, in association with decreased downstream target gene, Hes-1at3weeks.3. Chromatin immunoprecipitation and bisulfite sequencing were performed to analyze the epigenetic modifications of the Notchl gene promoter in PVECs. EUGR caused a significantly increased H3K27me3in the proximal Notchl gene promoter, and increased methylation of single CpG sites in the distal Notchl gene promoter, both at3and9weeks.Conclusions:EUGR results in decreased pulmonary vascular growth in association with decreased Notchl in PVECs. This may be mediated by increased CpG methylation and H3K27me3in the Notchl gene promoter region. Part Ⅱ:Effect of Paternal Extrauterine Growth Restriction on female Offspring:Altered epigenetic programmingObjective:Environmental perturbations during early life are known to affect one’s risk of cardio-metabolic disease many years later. Furthermore, that risk can be inherited by future generations. It has been suggested that DNA methylation may play an important role in the transgenerational effect of developmental programming models. In a EUGR rat model, we reported an elevated pulmonary arterial pressure (PAP) in adult and genome-wide epigenetic modification in pulmonary vascular endothelial cells (PVECs). However whether EUGR has an transgenerational effect on pulmonary vasculature and potential mechanisms of transmission is presently unknown.Methods:1. Transgenerational model:Control and EUGR females from the FO generation were mated at age9weeks with nonsibling control or EUGR males. After confirmation of pregnancy, females were caged individually to produce the four experimental F1generation groups:C♂-C♀(both parents are controls); E♂-C♀(EUGR sire, control dam); E♂-E♀(EUGR sire, EUGR dam); C♂-E♀(control sire, EUGR dam). All females were fed ad libitum throughout pregnancy and suckling period.2. Rats were weighted at birth,3and9weeks. Assessment of pulmonary arterial pressure were performed in9-wk-old rats from both generation.3. Genomic DNA of pulmonary vascular endothelial cells (PVECs) from both generation were extracted. Genome-wide features of DNA methylation were analysed by bioinformatics, including GO enrichment of differential methylated genes and searching motif of overlap different methylated regions in FO and F1.Results:1. Weight:No significant differences were found among four groups of offspring. 2. Mean pulmonary arterial pressure (mPAP):An improved phenotype of significantly decreased pulmonary arterial pressure was observed in E♂-C♀females versus C♂-C♀group. Male offspring has normal pulmonary arterial pressure compared with control group.3. The amount of different methylated genes was significantly discreased in F1compared to F0, possiblely due to loss of differential methylation genes involved in regulation of blood pressure during parternal transmission.Conclusion:EUGR induces a paternal transgenerational effect on female offsprings, which have improved pulmonary vascular function. Loss of differential methylation genes involved in regulation of blood pressure may mediate the transgenerational effect. |
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