Key points: Maternal hypertension is associated with increased rates of pregnancy pathologies, including fetal growth restriction, due at least in part to reductions in nitric oxide (NO) bioavailability and associated vascular dysfunction. Dietary nitrate supplementation, from beetroot juice (BRJ), has been shown to increase NO bioavailability and improve cardiovascular function in both preclinical and clinical studies. This study is the first to investigate effects of dietary nitrate supplementation in a pregnant animal model. Importantly, the effects of nitrate‐containing BRJ were compared with both ‘placebo’ (nitrate‐depleted) BRJ as well as water to control for potential nitrate‐independent effects. Our data show novel, nitrate‐independent effects of BRJ to lower blood pressure and improve vascular function in endothelial nitric oxide synthase knockout (eNOS^−/−) mice. These findings suggest potential beneficial effects of BRJ supplementation in pregnancy, and emphasize the importance of accounting for nitrate‐independent effects of BRJ in study design and interpretation. Maternal hypertension is associated with adverse pregnancy outcomes, including fetal growth restriction (FGR), due in part to reductions in nitric oxide (NO) bioavailability. We hypothesized that maternal dietary nitrate administration would increase NO bioavailability to reduce systolic blood pressure (SBP), improve vascular function and increase fetal growth in pregnant endothelial NO synthase knockout (eNOS^−/−) mice, which exhibit hypertension, endothelial dysfunction and FGR. Pregnant wildtype (WT) and eNOS^−/− mice were supplemented with nitrate‐containing beetroot juice (BRJ+) from gestational day (GD) 12.5. Control mice received an equivalent dose of nitrate‐depleted BRJ (BRJ−) or normal drinking water. At GD17.5, maternal SBP was measured; at GD18.5, maternal nitrate/nitrite concentrations, uterine artery (UtA) blood flow and endothelial function were assessed, and pregnancy outcomes were determined. Plasma nitrate concentrations were increased in both WT and eNOS^−/− mice supplemented with BRJ+ (P < 0.001), whereas nitrite concentrations were increased only in eNOS^−/− mice (P < 0.001). BRJ− did not alter nitrate/nitrite concentrations. SBP was lowered and UtA endothelial function was enhanced in eNOS^−/− mice supplemented with either BRJ+ or BRJ−, indicating nitrate‐independent effects of BRJ. Improvements in endothelial function in eNOS^−/− mice were abrogated in the presence of 25 mm KCl, implicating enhanced EDH signalling in BRJ− treated animals. At GD18.5, eNOS^−/− fetuses were significantly smaller than WT animals (P < 0.001), but BRJ supplementation did not affect fetal weight. BRJ may be a beneficial intervention in pregnancies associated with hypertension, endothelial dysfunction and reduced NO bioavailability. Our data showing biological effects of non‐nitrate components of BRJ have implications for both interpretation of previous findings and in the design of future clinical trials.

Adequate perfusion of the placental vasculature is essential to meet the metabolic demands of fetal growth and development. Lacking neural control, local tissue metabolites, circulating and physical factors contribute significantly to blood flow regulation. Nitric oxide (NO) is a key regulator of fetoplacental vascular tone. Nitrite, previously considered an inert end-product of NO oxidation, has been shown to provide an important source of NO. Reduction of nitrite to NO may be particularly relevant in tissue when the oxygen-dependent NO synthase (NOS) activity is compromised, e.g. in hypoxia. The contribution of this pathway in the placenta is currently unknown. We hypothesised that nitrite vasodilates human placental blood vessels, with enhanced efficacy under hypoxia. Placentas were collected from uncomplicated pregnancies and the vasorelaxant effect of nitrite (10-6-5x10-3 M) was assessed using wire myography on isolated pre-constricted chorionic plate arteries (CPAs) and veins (CPVs) under normoxic (pO2 ∼5%) and hypoxic (pO2 ∼1%) conditions. The dependency on the NO-sGC-cGMP pathway and known nitrite reductase (NiR) activities was also investigated. Nitrite caused concentration-dependent vasorelaxation in both arteries and veins, and this effect was enhanced by hypoxia, significantly in CPVs (P < 0.01) and with a trend in CPAs (P = 0.054). Pre-incubation with NO scavengers (cPTIO and oxyhemoglobin) attenuated (P < 0.01 and P < 0.0001, respectively), and the sGC inhibitor ODQ completely abolished nitrite-mediated vasorelaxation, confirming the involvement of NO and sGC. Inhibition of potential NiR enzymes xanthine oxidoreductase, mitochondrial aldehyde dehydrogenase and mitochondrial bc1 complex did not attenuate vasorelaxation. This data suggests that nitrite may provide an important reservoir of NO bioactivity within the placenta to enhance blood flow when fetoplacental oxygenation is impaired, as occurring in pregnancy diseases such as pre-eclampsia and fetal growth restriction.

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: (1) BKca channels (Paxillin, 10⁻⁵ M); (2) L-type calcium channels (Verapamil, 10⁻⁵ M); (3) Ryanodine receptor, RyR (Ryanodine, 10⁻⁵ M); (4) inositol 1,4,5-triphosphate receptor, IP3R, (2-Aminoethyl diphenylborinate, 2-APB, 3 × 10⁻³ M); (5) phospholipase C, PLC, (U73122, 10⁻⁵ M), and (6) GPCR-G_αi signaling, (Pertussis Toxin, 10⁻⁵ M). EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.

Increasing levels of estrogens across gestation are partly responsible for the physiological adaptations of the maternal vasculature to pregnancy. The G protein-coupled estrogen receptor (GPER) mediates acute vasorelaxing effects in the uterine vasculature, which may contribute to the regulation of uteroplacental blood flow. The aim of this study was to investigate whether GPER expression and vasorelaxation may occur following pregnancy. Elucidation of the functional signalling involved was also investigated. Radial uterine and third-order mesenteric arteries were isolated from non-pregnant (NP) and pregnant rats (P). GPER mRNA levels were determined and—concentration–response curve to the GPER-specific agonist, G1 (10⁻¹⁰–10⁻⁶ M), was assessed in arteries pre-constricted with phenylephrine. In uterine arteries, GPER mRNA expression was significantly increased and vasorelaxation to G1 was significantly enhanced in P compared with NP rats. Meanwhile, in mesenteric arteries, there was a similar order of magnitude in NP and P rats. Inhibition of L-type calcium channels and extracellular signal-regulated kinases 1/2 significantly reduced vasorelaxation triggered by G1 in uterine arteries. Increased GPER expression and GPER-mediated vasorelaxation are associated with the advancement of gestation in uterine arteries. The modulation of GPER is exclusive to uterine arteries, thus suggesting a physiological contribution of GPER toward the regulation of uteroplacental blood flow during pregnancy.