Investigating the association between nitrate dosing and nitrite generation by the human oral microbiota in continuous culture.

The generation of nitrite by the oral microbiota is believed to contribute to healthy cardiovascular function, with oral nitrate reduction to nitrite associated with systemic blood pressure regulation. There is the potential to manipulate the composition or activities of the oral microbiota to a higher nitrate-reducing state through nitrate supplementation. The current study examined microbial community composition and enzymatic responses to nitrate supplementation in sessile oral microbiota grown in continuous culture. Nitrate reductase (NaR) activity and nitrite concentrations were not significantly different to tongue-derived inocula in model biofilms. These were generally dominated by Streptococcus spp., initially, and a single nitrate supplementation resulted in the increased relative abundance of the nitrate-reducing genera Veillonella, Neisseria, and Proteus spp. Nitrite concentrations increased concomitantly and continued to increase throughout oral microbiota development. Continuous nitrate supplementation, over a 7-day period, was similarly associated with an elevated abundance of nitrate-reducing taxa and increased nitrite concentration in the perfusate. In experiments in which the models were established in continuous low or high nitrate environments, there was an initial elevation in nitrate reductase, and nitrite concentrations reached a relatively constant concentration over time similar to the acute nitrate challenge with a similar expansion of Veillonella and Neisseria. In summary, we have investigated nitrate metabolism in continuous culture oral biofilms, showing that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of putatively NaR-producing taxa.IMPORTANCEClinical evidence suggests that blood pressure regulation can be promoted by nitrite generated through the reduction of supplemental dietary nitrate by the oral microbiota. We have utilized oral microbiota models to investigate the mechanisms responsible, demonstrating that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of nitrate-reducing taxa.

Chronic developmental hypoxia alters mitochondrial oxidative capacity and reactive oxygen species production in the fetal rat heart in a sex-dependent manner.

Insufficient oxygen supply (hypoxia) during fetal development leads to cardiac remodeling and a predisposition to cardiovascular disease in later life. Previous work has shown hypoxia causes oxidative stress in the fetal heart and alters the activity and expression of mitochondrial proteins in a sex-dependent manner. However, the functional effects of these modifications on mitochondrial respiration remain unknown. Furthermore, while maternal antioxidant treatments are emerging as a promising new strategy to protect the hypoxic fetus, whether these treatments convey similar protection to cardiac mitochondria in the male or female fetus has not been investigated. Therefore, using an established rat model, we measured the sex-dependent effects of gestational hypoxia and maternal melatonin treatment on fetal cardiac mitochondrial respiration, reactive oxygen species (ROS) production, and lipid peroxidation. Pregnant Wistar rats were subjected to normoxia or hypoxia (13% oxygen) during gestational days (GDs) 6-20 (term ~22 days) with or without melatonin treatment (5 µg/ml in maternal drinking water). On GD 20, mitochondrial aerobic respiration and H2 O2 production were measured in fetal heart tissue, together with lipid peroxidation and citrate synthase (CS) activity. Gestational hypoxia reduced maternal body weight gain (p < .01) and increased placental weight (p < .05) but had no effect on fetal weight or litter size. Cardiac mitochondria from male but not female fetuses of hypoxic pregnancy had reduced respiratory capacity at Complex II (CII) (p < .05), and an increase in H2 O2 production/O2 consumption (p < .05) without any changes in lipid peroxidation. CS activity was also unchanged in both sexes. Despite maternal melatonin treatment increasing maternal and fetal plasma melatonin concentration (p < .001), melatonin treatment had no effect on any of the mitochondrial parameters investigated. To conclude, we show that gestational hypoxia leads to ROS generation from the mitochondrial electron transport chain and affects fetal cardiac mitochondrial respiration in a sex-dependent manner. We also show that maternal melatonin treatment had no effect on these relationships, which has implications for the development of future therapies for hypoxic pregnancies.

Enhanced Nitrite-Mediated Relaxation of Placental Blood Vessels Exposed to Hypoxia Is Preserved in Pregnancies Complicated by Fetal Growth Restriction.

Nitric oxide (NO) is essential in the control of fetoplacental vascular tone, maintaining a high flow-low resistance circulation that favors oxygen and nutrient delivery to the fetus. Reduced fetoplacental blood flow is associated with pregnancy complications and is one of the major causes of fetal growth restriction (FGR). The reduction of dietary nitrate to nitrite and subsequently NO may provide an alternative source of NO in vivo. We have previously shown that nitrite induces vasorelaxation in placental blood vessels from normal pregnancies, and that this effect is enhanced under conditions of hypoxia. Herein, we aimed to determine whether nitrite could also act as a vasodilator in FGR. Using wire myography, vasorelaxant effects of nitrite were assessed on pre-constricted chorionic plate arteries (CPAs) and veins (CPVs) from normal and FGR pregnancies under normoxic and hypoxic conditions. Responses to the NO donor, sodium nitroprusside (SNP), were assessed in parallel. Nitrate and nitrite concentrations were measured in fetal plasma. Hypoxia significantly enhanced vasorelaxation to nitrite in FGR CPAs (p < 0.001), and in both normal (p < 0.001) and FGR (p < 0.01) CPVs. Vasorelaxation to SNP was also potentiated by hypoxia in both normal (p < 0.0001) and FGR (p < 0.01) CPVs. However, compared to vessels from normal pregnancies, CPVs from FGR pregnancies showed significantly lower reactivity to SNP (p < 0.01). Fetal plasma concentrations of nitrate and nitrite were not different between normal and FGR pregnancies. Together, these data show that nitrite-mediated vasorelaxation is preserved in FGR, suggesting that interventions targeting this pathway have the potential to improve fetoplacental blood flow in FGR pregnancies.

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

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.

Nitrite mediated vasorelaxation in human chorionic plate vessels is enhanced by hypoxia and dependent on the NO-sGC-cGMP pathway.

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.

Effects of dietary nitrate supplementation, from beetroot juice, on blood pressure in hypertensive pregnant women: A randomised, double-blind, placebo-controlled feasibility trial.

Chronic hypertension in pregnancy is associated with significant adverse pregnancy outcomes, increasing the risk of pre-eclampsia, fetal growth restriction and preterm birth. Dietary nitrate, abundant in green leafy vegetables and beetroot, is reduced in vivo to nitrite and subsequently nitric oxide, and has been demonstrated to lower blood pressure, improve vascular compliance and enhance blood flow in non-pregnant humans and animals. The primary aims of this study were to determine the acceptability and efficacy of dietary nitrate supplementation, in the form of beetroot juice, to lower blood pressure in hypertensive pregnant women. In this double-blind, placebo-controlled feasibility trial, 40 pregnant women received either daily nitrate supplementation (70 mL beetroot juice, n = 20) or placebo (70 mL nitrate-depleted beetroot juice, n = 20) for 8 days. Blood pressure, cardiovascular function and uteroplacental blood flow was assessed at baseline and following acute (3 h) and prolonged (8 days) supplementation. Plasma and salivary samples were collected for analysis of nitrate and nitrite concentrations and acceptability of this dietary intervention was assessed based on questionnaire feedback. Dietary nitrate significantly increased plasma and salivary nitrate/nitrite concentrations compared with placebo juice (p < 0.001), with marked variation between women. Compared with placebo, there was no overall reduction in blood pressure in the nitrate-treated group; however there was a highly significant correlation between changes in plasma nitrite concentrations and changes in diastolic blood pressure in the nitrate-treated arm only (r = -0.6481; p = 0.0042). Beetroot juice supplementation was an acceptable dietary intervention to 97% of women. This trial confirms acceptability and potential efficacy of dietary nitrate supplementation in pregnant women. Conversion of nitrate to nitrite critically involves oral bacterial nitrate reductase activities. We speculate that differences in efficacy of nitrate supplementation relate to differences in the oral microbiome, which will be investigated in future studies.

From Pre-Clinical Studies to Clinical Trials: Generation of Novel Therapies for Pregnancy Complications.

Complications of pregnancy represent a significant disease burden, with both immediate and lasting consequences for mother and baby. Two key pregnancy complications, fetal growth restriction (FGR) and preeclampsia (PE), together affect around 10%-15% of all pregnancies worldwide. Despite this high incidence, there are currently no therapies available to treat these pregnancy disorders. Early delivery remains the only intervention to reduce the risk of severe maternal complications and/or stillbirth of the baby; however early delivery itself is associated with increased risk of neonatal mortality and morbidity. As such, there is a pressing need to develop new and effective treatments that can prevent or treat FGR and PE. Animal models have been essential in identifying and screening potential new therapies in this field. In this review, we address recent progress that has been made in developing therapeutic strategies for pregnancy disorders, some of which are now entering clinical trials.

RISING STARS: Bumps and bacteria: influence of the maternal microbiota on pregnancy outcomes.

During pregnancy, all major physiological systems undergo remarkable changes, driven largely by alterations in the maternal hormonal milieu. In healthy pregnancies, maternal cardiovascular and metabolic adaptation to pregnancy occurs to support fetal growth and maternal well-being. Impaired maternal adaptation to pregnancy is associated with a range of pregnancy complications, including gestational diabetes and preeclampsia. There is growing recognition of the importance of different maternal microbiota, including in the gut, vagina and oral cavity, in supporting normal maternal adaptations to pregnancy as well as evidence for microbial disturbances associating with pregnancy pathologies. Here, we aim to summarise emerging evidence demonstrating that differences in maternal microbiota associate with pregnancy outcomes and discuss potential therapeutic approaches under development that might restore an 'optimal' microbiome. In particular, we highlight recent work by ourselves and others exploring the role of the oral microbiome in pregnancy, given established links between poor oral health (e.g. periodontitis) and adverse pregnancy outcomes. Our research has focussed on specific nitrate-reducing oral bacteria which play a role in the generation of nitric oxide (NO) and other bioactive nitrogen oxides associated with cardiovascular health and maternal cardiovascular adaption to pregnancy. Ongoing research aims to define whether altered microbial profiles have clinical utility in the prediction of pregnancy pathologies, and whether interventions designed to optimise specific maternal microbiota could help prevent future complications.

CHronic hypERtension and L-citRulline studY (CHERRY): an Early-Phase Randomised Controlled Trial in Pregnancy.

Oral supplementation with L-citrulline, which is sequentially converted to L-arginine then nitric oxide, improves vascular biomarkers and reduces blood pressure in non-pregnant, hypertensive human cohorts and pregnant mice with a pre-eclampsia-like syndrome. This early-phase randomised feasibility trial assessed the acceptability of L-citrulline supplementation to pregnant women with chronic hypertension and its effects on maternal BP and other vascular outcomes. Pregnant women with chronic hypertension were randomised at 12-16 weeks to receive 3-g L-citrulline twice daily (n = 24) or placebo (n = 12) for 8 weeks. Pregnant women reported high acceptability of oral L-citrulline. Treatment increased maternal plasma levels of citrulline, arginine and the arginine:asymmetric dimethylarginine ratio, particularly in women reporting good compliance. L-citrulline had no effect on diastolic BP (L-citrulline: - 1.82 95% CI (- 5.86, 2.22) vs placebo: - 5.00 95% CI (- 12.76, 2.76)), uterine artery Doppler or angiogenic biomarkers. Although there was no effect on BP, retrospectively, this study was underpowered to detect BP changes < 9 mmHg, limiting the conclusions about biological effects. The increase in arginine:asymmetric dimethylarginine ratio was less than in non-pregnant populations, which likely reflects altered pharmacokinetics of pregnancy, and further pharmacokinetic assessment of L-citrulline in pregnancy is advised.Trial Registration EudraCT 2015-005792-25 (2017-12-22) and ISRCTN12695929 (2018-09-20).

Reconciling the nutritional and glucocorticoid hypotheses of fetal programming.

Fetal growth restriction associates with increased risk of adult cardiometabolic and neuropsychiatric disorders. Both maternal malnutrition [notably a low-protein (LP) diet] and stress/glucocorticoid exposure reduce fetal growth and cause persisting abnormalities (programming) in adult offspring. Deficiency of placental 11ß-hydroxysteroid dehydrogenase-2 (11ß-HSD2), which inactivates glucocorticoids, is reduced by an LP diet and has been proposed as a unifying mechanism. Here, we explored the importance of glucocorticoids and placental 11ß-HSD2 in dietary programming. Pregnant mice were fed a control or isocaloric LP diet throughout gestation. The LP diet first elevated fetal glucocorticoid levels, then reduced placental growth, and finally decreased fetal weight near term by 17%. Whereas the LP diet reduced placental 11ß-HSD2 activity near term by ∼25%, consistent with previous reports, activity was increased between 20 and 40% at earlier ages, implying that glucocorticoid overexposure in LP fetuses occurs via 11ß-HSD2-independent mechanisms. Consistent with this, heterozygous 11ß-HSD2(+/-) crosses showed that although both LP and 11ß-HSD2 deficiency reduced fetal growth, LP indeed acted independently of 11ß-HSD2. Instead, the LP diet induced the fetal hypothalamic-pituitary-adrenal axis per se. Thus, maternal malnutrition and placental 11ß-HSD2 deficiency act via distinct processes to retard fetal growth, both involving fetoplacental overexposure to glucocorticoids but from distinct sources.

Should the non-canonical pathway of nitric oxide generation be targeted in hypertensive pregnancies?

Hypertension in pregnancy is prevalent, affecting around 10% of pregnancies worldwide, and significantly increases the risk of adverse outcomes for both mothers and their babies. Current treatment strategies for pregnant women with hypertension are limited, and new approaches for the management of hypertension in pregnancy are urgently needed. Substantial evidence from non-pregnant subjects has demonstrated the potential for dietary nitrate supplementation to increase nitric oxide (NO) bioavailability and lower blood pressure, following bioactivation via the non-canonical NO pathway. Emerging data suggest this approach may also be of benefit in pregnant women, although studies are limited. This review aims to summarise the current evidence from preclinical and clinical studies of nitrate supplementation in pregnancy, drawing on data from non-pregnant populations where appropriate and highlighting key gaps in knowledge that remain to be addressed in future trials.

Fetoplacental vascular effects of maternal adrenergic antihypertensive and cardioprotective medications in pregnancy.

Maternal cardiovascular diseases, including hypertension and cardiac conditions, are associated with poor fetal outcomes. A range of adrenergic antihypertensive and cardioprotective medications are often prescribed to pregnant women to reduce major maternal complications during pregnancy. Although these treatments are not considered teratogenic, they may have detrimental effects on fetal growth and development, as they cross the fetoplacental barrier, and may contribute to placental vascular dysregulation. Medication risk assessment sheets do not include specific advice to clinicians and women regarding the safety of these therapies for use in pregnancy and the potential off-target effects of adrenergic medications on fetal growth have not been rigorously conducted. Little is known of their effects on the fetoplacental vasculature. There is also a dearth of knowledge on adrenergic receptor activation and signalling within the endothelium and vascular smooth muscle cells of the human placenta, a vital organ in the maintenance of adequate blood flow to satisfy fetal growth and development. The fetoplacental circulation, absent of sympathetic innervation, and unique in its reliance on endocrine, paracrine and autocrine influence in the regulation of vascular tone, appears vulnerable to dysregulation by adrenergic antihypertensive and cardioprotective medications compared with the adult peripheral circulation. This semi-systematic review focuses on fetoplacental vascular expression of adrenergic receptors, associated cell signalling mechanisms and predictive consequences of receptor activation/deactivation by antihypertensive and cardioprotective medications.

Altered Oral Nitrate Reduction and Bacterial Profiles in Hypertensive Women Predict Blood Pressure Lowering Following Acute Dietary Nitrate Supplementation.

BACKGROUND: The efficacy of dietary nitrate supplementation to lower blood pressure (BP) in pregnant women is highly variable. We aimed to investigate whether differences in oral microbiota profiles and oral nitrate-reducing capacity may explain interindividual differences in BP lowering following nitrate supplementation. METHODS: Participants recruited for this study were both pregnant and nonpregnant women, with or without hypertension (n=55). Following an overnight fast, plasma, saliva, and tongue scraping samples were collected for measurement of nitrate/nitrite concentrations, oral NaR (nitrate reductase) activity, and microbiota profiling using 16S rRNA gene sequencing. Baseline BP was measured, followed by the administration of a single dose of dietary nitrate (400 mg nitrate in 70 mL beetroot juice). Post-nitrate intervention, plasma and salivary nitrate/nitrite concentrations and BP were determined 2.5 hours later. RESULTS: Women with hypertension had significantly lower salivary nitrite concentrations (P=0.006) and reduced abundance of the nitrate-reducing taxa Veillonella(P=0.007) compared with normotensive women. Oral NaR activity was not significantly different in pregnant versus nonpregnant women (P=0.991) but tended to be lower in hypertensive compared with normotensive women (P=0.099). Oral NaR activity was associated with both baseline diastolic BP (P=0.050) and change in diastolic BP following acute nitrate intake (P=0.01, adjusted for baseline BP). CONCLUSIONS: The abundance and activity of oral nitrate-reducing bacteria impact both baseline BP as well as the ability of dietary nitrate supplementation to lower BP. Strategies to increase oral nitrate-reducing capacity could lower BP and enhance the efficacy of dietary nitrate supplementation, in pregnancy as well as in nonpregnant adults. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03930693.

Leptin receptors.

The hormone leptin, secreted predominantly from adipose tissue, plays a crucial role in the regulation of numerous neuroendocrine functions, from energy homeostasis to reproduction. Genetic deficiency as a consequence of leptin or leptin receptor mutations, although rare in humans, leads to early onset of chronic hyperphagia and massive obesity. In most human obesity, however, leptin levels are chronically elevated. Under these conditions of persistent hyperleptinaemia, and particularly when obesity is associated with a high-fat diet, leptin resistance develops, and signalling through the leptin receptor is curtailed, fuelling further weight gain. Here, we review the role of leptin receptors in the regulation of feeding and obesity development. Leptin receptors are found in each of the major components of the CNS "feeding" circuitry-the brainstem, hypothalamus and distributed reward centres. Through these receptors, leptin exerts influences on signalling and integration within these circuits to alter feeding behaviours. Although some progress is now being made with peptide analogues, the leptin receptor has not proved to be amenable to small molecule pharmacological intervention to date. Where clinical benefit from recombinant leptin administration has been achieved, this has been under circumstances of complete endogenous leptin deficiency or relative hypoleptinaemia such as in lipodystrophy.

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

Hypertension during pregnancy is a leading cause of maternal and fetal morbidity and mortality worldwide, increasing the risk of complications including preeclampsia, intracerebral hemorrhage and fetal growth restriction. Increased oxidative stress is known to contribute to poor vascular function; however, trials of antioxidant supplementation have raised concerns about fetal outcomes, including risk of low birthweight. Grape seed extract polyphenols (GSEP) have been suggested to promote cardiovascular protection, at least in part through antioxidant actions. We tested the hypothesis that administration of GSEP during pregnancy would reduce oxidative stress and improve resistance artery function with no detrimental effects on fetal growth, in an established model of maternal hypertension associated with vascular dysfunction, the endothelial NO synthase knockout (eNOS-/-) mouse. Pregnant C57BL/6J (WT) and eNOS-/- mice received either GSEP (200 mg/kg/day) or drinking water, between gestational (GD) day 10.5 and GD18.5. At GD17.5, maternal systolic blood pressure (SBP) was measured; at GD18.5, maternal malondialdehyde (MDA) concentrations, vascular function of aortic, mesenteric, uterine and posterior cerebral arteries was assessed, and fetal outcome evaluated. GSEP reduced maternal SBP (P < 0.01) and plasma MDA concentrations (P < 0.01) in eNOS-/- mice. Whilst there was no effect of GSEP on vascular reactivity of aortas, GSEP improved endothelial-dependent relaxation in mesenteric and uterine arteries of eNOS-/- mice (P < 0.05 and P < 0.001, respectively) and normalized lumen diameters of pressurized posterior cerebral arteries in eNOS-/- mice (P < 0.001). Supplementation with GSEP had no effect in WT mice and did not affect fetal outcomes in either genotype. Our data suggest that GSEP improve resistance artery function, potentially through antioxidant actions, and provide a basis to further investigate these beneficial effects including in the prevention of intracerebral hemorrhage. Maternal supplementation with GSEP may be a safe intervention to improve outcomes in pregnancies associated with hypertension and vascular dysfunction.

Pomegranate Juice Supplementation Alters Utero-Placental Vascular Function and Fetal Growth in the eNOS-/- Mouse Model of Fetal Growth Restriction.

The eNOS-/- mouse provides a well-characterized model of fetal growth restriction (FGR) with altered uterine and umbilical artery function and reduced utero- and feto-placental blood flow. Pomegranate juice (PJ), which is rich in antioxidants and bioactive polyphenols, has been posited as a beneficial dietary supplement to promote cardiovascular health. We hypothesized that maternal supplementation with PJ will improve uterine and umbilical artery function and thereby enhance fetal growth in the eNOS-/- mouse model of FGR. Wild type (WT, C57Bl/6J) and eNOS-/- mice were supplemented from E12.5-18.5 with either PJ in their drinking water or water alone. At E18.5 uterine (UtA) and umbilical (UmbA) arteries were isolated for study of vascular function, fetuses and placentas were weighed and fetal biometric measurements taken. PJ supplementation significantly increased UtA basal tone (both genotypes) and enhanced phenylephrine-induced contraction in eNOS-/- but not WT mice. Conversely PJ significantly reduced UtA relaxation in response to both acetylcholine (Ach) and sodium nitroprusside (SNP), endothelium dependent and independent vasodilators respectively from WT but not eNOS-/- mice. UmbA sensitivity to U46619-mediated contraction was increased by PJ supplementation in WT mice; PJ enhanced contraction and relaxation of UmbA to Ach and SNP respectively in both genotypes. Contrary to our hypothesis, the changes in artery function induced by PJ were not associated with an increase in fetal weight. However, PJ supplementation reduced litter size and fetal abdominal and head circumference in both genotypes. Collectively the data do not support maternal PJ supplementation as a safe or effective treatment for FGR.

Melatonin Increases Fetal Weight in Wild-Type Mice but Not in Mouse Models of Fetal Growth Restriction.

Fetal growth restriction (FGR) presents with an increased risk of stillbirth and childhood and adulthood morbidity. Melatonin, a neurohormone and antioxidant, has been suggested as having therapeutic benefit in FGR. We tested the hypothesis that melatonin would increase fetal growth in two mouse models of FGR which together represent a spectrum of the placental phenotypes in this complication: namely the endothelial nitric oxide synthase knockout mouse (eNOS-/-) which presents with abnormal uteroplacental blood flow, and the placental specific Igf2 knockout mouse (P0+/-) which demonstrates aberrant placental morphology akin to human FGR. Melatonin (5 µg/ml) was administered via drinking water from embryonic day (E)12.5 in C57Bl/6J wild-type (WT), eNOS-/-, and P0+/- mice. Melatonin supplementation significantly increased fetal weight in WT, but not eNOS-/- or P0+/- mice at E18.5. Melatonin did, however, significantly increase abdominal circumference in P0+/- mice. Melatonin had no effect on placental weight in any group. Uterine arteries from eNOS-/- mice demonstrated aberrant function compared with WT but melatonin treatment did not affect uterine artery vascular reactivity in either of these genotypes. Umbilical arteries from melatonin treated P0+/- mice demonstrated increased relaxation in response to the nitric oxide donor SNP compared with control. The increased fetal weight in WT mice and abdominal circumference in P0+/-, together with the lack of any effect in eNOS-/-, suggest that the presence of eNOS is required for the growth promoting effects of melatonin. This study supports further work on the possibility of melatonin as a treatment for FGR.

Postnatal remodeling of dendritic structure and spine density in gonadotropin-releasing hormone neurons.

The GnRH neurons represent the output cells of the neuronal network controlling gonadal function. Their activation initiates the onset of puberty, but the underlying mechanisms remain unclear. Using a GnRH-green fluorescent protein mouse model, we have been able to fill individual GnRH neurons with biocytin in the acute brain slice preparation to examine their morphological characteristics across puberty. GnRH neurons in prepubertal male mice [postnatal d 10-15 (PND10-15)] exhibited half as many dendritic and somal spines as adult male mice (>PND60; P < 0.05) but, surprisingly, a much more complex dendritic tree with 5-fold greater branch points (P < 0.05). Experiments examining somal and proximal dendritic spine numbers in vivo, in perfusion-fixed tissue from GnRH-green fluorescent protein mice, revealed the same pattern of approximately twice as many spines on adult GnRH neurons compared with PND10 male mice (P < 0.01). In contrast to the spine density alterations, reflecting changing excitatory input, confocal immunofluorescence studies revealed no differences in the numbers of vesicular gamma-aminobutyric acid transporter-immunoreactive elements adjacent to GnRH soma or proximal dendrites in prepubertal and adult male mice. Experiments evaluating dendritic tree structure in vivo (PND3, -10, and -35 and adult) revealed that GnRH neurons located in the rostral preoptic area, but not the medial septum, exhibited a more complex branching pattern at PND10, but that this was adult-like by PND35. These studies demonstrate unexpected dendritic tree remodeling in the GnRH neurons and provide evidence for an increase in direct excitatory inputs to GnRH neurons across the time of puberty.

The atrial natriuretic peptide (ANP) knockout mouse does not exhibit the phenotypic features of pre-eclampsia or demonstrate fetal growth restriction.

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP(+/+)), heterozygous (ANP(+/-)) and knockout (ANP(-/-)) mice. Maternal blood pressure did not differ between genotypes (E12.5, E17.5), and fetal weight (E18.5) was unaffected. Placental weight was greater in ANP(-/-) versus ANP(+/+) mice. Therefore, in our hands, the ANP model does not express phenotypic features of PE with FGR.

Metabolic pitfalls of CNS Cre-based technology.

Nestin-Cre mice have a significant metabolic phenotype that is hard to discern from current literature. Indeed, the Cre-lox system has numerous problems that can affect physiological parameters, and these are missed when the correct control strains are not used. Despite the increasing use of the Cre-lox system, these issues were not visible to the scientific community previously and may have affected published work. This makes it important to highlight the issues and raise awareness of the pitfalls of the Cre-lox system. Therefore, this perspective will discuss the impact of CNS and peripheral "off-target" Cre recombination on metabolic systems and describe the development of new approaches to obviate the difficulties.