DNA methylation of nos3 promoter in IUGR-guinea pig fetuses does not predict endothelial function and DNA methylation later in life

摘要

Background: Intrauterine growth restriction is associated with endothelial dysfunction and cardiovascular diseases in adult subjects. It has been proposed that changes in the DNA methylation pattern of key endothelial genes, such as the endothelial nitric oxide synthase gene (nos3), which could take place as early a fetal development, would precede the founding of an altered endothelial function. To address this issue, we studied the levels of eNOS expression, nos3 promoter DNA methylation pattern and ex vivo endothelial function in fetal and adult arteries from IUGR guinea pigs. Methods: Pregnant guinea pigs were submitted at mid-gestation to a surgery for implanting ameroid constrictors at both uterine arteries (IUGR), or a sham intervention (Control). In half of the sows, pregnancy was interrupted at term (~ 60 days) and fetuses were extracted by C-section, dissected and weight, whilst the other half was allowed to breed and the offspring were followed up to adulthood (10 months). Fetal and adult femoral arteries were isolated to assessed vascular function by wire-myography. Primary cultures of endothelial cells were obtained from fetal and adults aorta. In these cells levels of eNOS mRNA were quantified by qPCR and the DNA methylation of 12 CpG sites in nos3 promoter was determined by pyrosequencing. Results: In term fetuses, IUGR was associated with an increased expression of eNOS mRNA as well as a decrease in the DNA methylation status in CpG -170 (relative to the transcription start site) compared to controls fetuses. In contrast, endothelial-dependent relaxation in IUGR femoral arteries was comparable to controls, but with a reduced sensitivity. Endothelial-independent relaxation to NO was similarly reduced in sensitivity but not in the maximal effect in IUGR femoral arteries. In adult guinea pigs from IUGR pregnancies, eNOS mRNA levels were substantially decreased compared to control adults, and this change was associated with an increased DNA methylation level in CpG -170. Similarly, there was a reduced endothelial dependent relaxation, as well as a reduced sensitivity to NO, in adult IUGR femoral arteries compared to controls. Conclusion: This data suggests that changes in nos3 promoter DNA methylation observed in IUGR term fetuses would represent the exposure to an adverse intrauterine environment, however, this would not be associated with endothelial dysfunction and DNA methylation pattern on nos3 in the adult life. Conversely, the adult cardiovascular dysfunction associated to IUGR does not necessary is preceded by early changes in DNA methylation in endothelial cells.