Gestational age-related patterns of AMOT methylation are revealed in preterm infant endothelial progenitors

OBJECTIVE Preterm birth is associated with altered angiogenesis and with increased risk of cardiovascular dysfunction and hypertension at adulthood. We previously demonstrated that in preterm newborns circulating cord blood endothelial progenitor cells (ECFC), responsible for angio/vasculogenesis, are reduced in number and display altered angiogenic properties. Altered angiogenic function was associated with a decreased expression of pro-angiogenic genes, among which the AMOT gene which is a strong positive regulator of angiogenesis. Such dysregulation may be related to epigenetic factors. In this study we analyse the methylation profiling of the AMOT gene during development, through a comparative analysis of the cord blood ECFC of preterm newborns and their term counterpart. METHODS We used both cloning-sequencing and pyrosequencing experiments to perform a comparative analysis of the DNA methylation profile of the promoter CpG island of AMOT gene in the cord blood ECFC of 16 preterm newborns (28-35 weeks gestational age-GA) and 15 term newborns (>37 weeks GA). RESULTS Twenty nine clones (obtained from 2 term newborns) and forty clones (obtained from 3 preterm newborns) were sequenced. The AMOT gene methylation rate was significantly higher in preterm compared to term newborns (4.5% versus 2.5% respectively: χ2 = 3.84; P = 1.8 10-02). Bisulfite pyrosequencing identified four CpG dinucleotides with significantly higher methylation levels in preterm newborns. This CpG-targeted methylation significantly decreased with increasing gestational age. CONCLUSIONS These findings highlight importance of pro-angiogenic AMOT gene methylation in ECFC, suggesting that epigenetic mechanisms may control the regulation of angiogenesis during development. Therefore they pave the way to specific short term and long term complications of preterm birth by altered angiogenesis.