N‐Acetylcysteine, a glutathione precursor, reverts vascular dysfunction and endothelial epigenetic programming in intrauterine growth restricted guinea pigs

Abstract

In humans, intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial programming in umbilical vessels. We aimed to determine the effects of maternal antioxidant treatment with N-acetylcysteine (NAC) on fetal endothelial function and endothelial nitric oxide synthase (eNOS) programming in IUGR guinea pigs. IUGR was induced by implanting ameroid constrictors on uterine arteries of pregnant guinea pigs at mid gestation, half of the sows receiving NAC in the drinking water (from day 34 until term). Fetal biometry and placental vascular resistance were followed by ultrasound throughout gestation. At term, umbilical arteries and fetal aortae were isolated to assess endothelial function by wire-myography. Primary cultures of endothelial cells (ECs) from fetal aorta, femoral and umbilical arteries were used to determine eNOS mRNA levels by quantitative PCR and analyse DNA methylation in the Nos3 promoter by pyrosequencing. Doppler ultrasound measurements showed that NAC reduced placental vascular resistance in IUGR (P < 0.05) and recovered fetal weight (P < 0.05), increasing fetal-to-placental ratio at term (∼40%) (P < 0.001). In IUGR, NAC treatment restored eNOS-dependent relaxation in aorta and umbilical arteries (P < 0.05), normalizing eNOS mRNA levels in EC fetal and umbilical arteries (P < 0.05). IUGR-derived ECs had a decreased DNA methylation (∼30%) at CpG -170 (from the transcription start site) and this epigenetic signature was absent in NAC-treated fetuses (P < 0.001). These data show that IUGR-ECs have common molecular markers of eNOS programming in umbilical and systemic arteries and this effect is prevented by maternal treatment with antioxidants.

Adult vascular dysfunction in foetal growth‐restricted guinea‐pigs is associated with a neonate‐adult switching in Nos3 DNA methylation

“…Additionally, an impaired foetal growth in humans is associated with vascular remodelling in the aorta at birth, as well as an increased aortic stiffness at childhood . Recent reports show that growth‐restricted guinea‐pig foetuses have an impaired NOS‐dependent relaxation in systemic and umbilical arteries which occurs in parallel with pro‐constrictive vascular remodelling in the aorta and femoral arteries, but not in the carotid artery . In this study, FGR adult guinea‐pigs showed an increased in vivo femoral vascular resistance with no diurnal variability, but a normal resistance in the carotid artery.…”

“…Specifically, altered expression of eNOS in FGR‐derived endothelium results from diverse epigenetic modifications in the NOS3 gene promoter, an effect that can be reprogrammed to a “normal type” by knocking‐down DNMT1 . Additionally, we recently reported that FGR results in an altered eNOS expression and Nos3 gene promoter DNA methylation in a guinea‐pig model of FGR, which is comparable between foetal systemic and umbilical arteries . Notably, maternal antioxidant treatment with N‐acetylcysteine (NAC) restored foetal growth by enhancing placental efficiency and reverting the functional and epigenetic programming of eNOS in arterial endothelium .…”

“…Additionally, we recently reported that FGR results in an altered eNOS expression and Nos3 gene promoter DNA methylation in a guinea‐pig model of FGR, which is comparable between foetal systemic and umbilical arteries . Notably, maternal antioxidant treatment with N‐acetylcysteine (NAC) restored foetal growth by enhancing placental efficiency and reverting the functional and epigenetic programming of eNOS in arterial endothelium . However, there are no studies addressing whether this epigenetic alteration in Nos3 or other endothelial‐related genes is maintained until adulthood as an early origin of the endothelial dysfunction.…”

Epigenetic Programming of Cardiovascular Disease by Perinatal Hypoxia and Fetal Growth Restriction

“…We recently addressed the role of oxidative stress in FGR by treating pregnant guinea pigs with N-acetyl cysteine, a glutathione precursor, during the second half of gestation. Our results show that maternal treatment with NAC restores fetal growth by increasing placental efficiency and reverses endothelial dysfunction in FGR guinea pigs [38]. Similarly, in ovo melatonin administration to chronic hypoxic chick embryos reduces the levels of oxidative stress markers (i.e., lipid peroxidation and protein nitration), by increasing the expression of glutathione peroxidase (GPx), an antioxidant enzyme [28].…”

“…Notably, the altered expression of eNOS was reversed by silencing DNMT1 expression in FGR EC, which restored the DNA methylation pattern at NOS3 promoter, as well as the regulation of eNOS expression induced by hypoxia [12]. Furthermore, using a guinea pig model of FGR, we compared the eNOS expression and DNA methylation pattern at Nos3 promoter to clarify whether these epigenetic changes occurring in umbilical EC would represent changes that take place in systemic arteries (i.e., aorta and femoral) [38]. We found comparable changes in eNOS expression which were associated with specific changes in DNA methylation of Nos3 promoter in the different FGR EC studied, suggesting the presence of a common programming of endothelial dysfunction in the umbilical-placental and systemic circulation.…”