Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS^−/− mice: importance of nitrate‐independent effects

Abstract

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. Abstract 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.

Grape Seed Extract Polyphenols Improve Resistance Artery Function in Pregnant eNOS–/– Mice

“…### P < 0.001 eNOS −/− vs. WT; *P < 0.05 GSEP vs. H 2 O. N = 6-11 dams per group. the current study are consistent with our own data (Tropea et al, 2020) and those reported by others (Hefler et al, 2001;Poudel et al, 2013). Administration of a different polyphenol, resveratrol (3,5,4 -trihydroxystilbene), was reported to have no effect on SBP in eNOS −/− mice (Poudel et al, 2013).…”

“…Administration of a different polyphenol, resveratrol (3,5,4 -trihydroxystilbene), was reported to have no effect on SBP in eNOS −/− mice (Poudel et al, 2013). In contrast, our recent data demonstrated that supplementation with beetroot juice significantly lowered SBP in eNOS −/− mice, effects which we believe are potentially attributable to bioactive phytochemicals contained within the juice, including polyphenols and flavonoids (Tropea et al, 2020), in common with the active constituents of GSEP. The phenolic content of the extract used in the current study has been reported to be 94% and mostly made of polymers of catechin (Edirisinghe et al, 2008;Sivaprakasapillai et al, 2009), shown to provide a higher antioxidant ability compared to other commonly used antioxidants (Bagchi et al, 1997(Bagchi et al, , 1998.…”

“…In a recently published work (Finn-Sell et al, 2018), maternal supplementation with pomegranate juice had detrimental effects on fetal outcomes of both WT and eNOS −/− mice; whereas, treatment with resveratrol showed a trend toward an increase in fetal weight in eNOS −/− mice (Poudel et al, 2013) and the antioxidant drug Tempol (4-Hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) improved fetal growth in eNOS −/− mice (Stanley et al, 2012). More recently, maternal supplementation with either melatonin (Renshall et al, 2018), or with beetroot juice (Tropea et al, 2020), had no effects on eNOS −/− fetuses. In agreement with the latter data, our results showed that GSEP did not improve fetal growth in fetuses from eNOS −/− dams.…”

“…It appears that therapeutic interventions mostly fail to improve fetal outcomes in eNOS −/− mice. The growth potential of this homozygous knockout mouse as a model of FGR may be genetically limited (Tropea et al, 2020), thus representing a key limitation for studies in which intervention aim at ameliorating the FGR phenotype.…”

Saying NO to fetal growth restriction: Implications for beetroot juice and melatonin

“…The study reported in The Journal of Physiology by Tropea et al (2020) examined the effects of BRJ on blood pressure (BP) and endothelial function in pregnant endothelial nitric oxide synthase knockout mice (eNOS −/− ). eNOS is one of three NOS isoforms and the eNOS −/− mouse model is well established with respect to assessing maternal hypertension as related to fetal growth restriction (FGR) and vascular dysfunction (Tropea et al 2020). During pregnancy, NO maintains the low-resistance/high-flow environment of the utero-placental vascular system.…”