Abstract

To test the hypothesis that chronic fetal hypoxia leading to oxidative stress directly induces epigenetic programming of eNOS expression in the fetal vasculature and that antioxidant therapy will have direct protective effects. The hypothesis will be tested by isolating the effects on epigenetic mechanisms affecting eNOS expression in vessels harvested from chick embryos incubated under normoxic or hypoxic conditions with and without N-acetylcysteine treatment. Methods Chick embryos will be incubated at 14% O2 from day 1 of incubation. This is an established model of FGR and 14% O2 approximates oxygen levels equivalent to those induced by human pregnancy at 3000 meters above sea level or those yielding hypoxic fetuses from sea level pregnancies affected by FGR (see ref. 12 for review). In human clinical practice, FGR as a result of suspected chronic fetal hypoxia may be only reliably diagnosed at around 25 weeks of gestation. Therefore, there is interest in identifying a translational antioxidant therapy that may rescue fetal endothelial dysfunction triggered by chronic fetal hypoxia once a diagnosis has been feasible. Therefore, treatment of chick embryos with antioxidants will start at day 13 of incubation, which is equivalent to ca. 25 weeks of gestation in human pregnancy. Altogether, this highlights the clinical relevance of the proposed animal model and the request for seed corn funding. Towards the end of the incubation period, at day 19, plasma samples from the chorioallantoic artery will be obtained to determine markers of oxidative stress. Additionally, the embryos will be killed and following biometry, the entire length of the aorta and hind limb vessels will be isolated for further analysis of the expression of eNOS and the methylation status of Nos3 gene promoter. In another cohort of embryos, NO-dependent and NO-independent mechanisms promoting endothelial relaxation will be determined in second order resistance femoral arteries via in vitro wire myography, as previously described9. Therefore, the project will link NO-dependent vascular dysfunction with epigenetic mechanisms regulating vascular eNOS expression in control and growth restricted chick embryos with or without treatment with an antioxidant. Projections of this proposal The longer term objective is to obtain sufficient training exposure and expertise to be able to set up this chick embryo model of FGR in Chile. Combined with our local expertise in mammalian models of FGR, such as the guinea pig, and with our clinical involvement in human pregnancy complicated by FGR, this will provide us with a layered integrative understanding of the different maternal, placental and fetal factors affecting FGR with the aim of designing potential candidate therapy translatable to the human clinical situation, such as gene delivery-based interventions.