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The aim of this study was to determine the iron (Fe) concentration profile within the lumen of the S2 renal proximal convoluted tubule (PCT) and to resolve whether this nephron segment transported Fe. To do this, we performed in vivo renal micropuncture on Wistar rats, collected PCT tubular fluid from superficial nephrons, and measured Fe concentration. The Fe concentration profile along the S2 PCT suggested significant Fe reabsorption. Proximal tubules were also microperfused in vivo with physiological solutions containing Fe and Zn, Cu, Mn, or Cd. PCTs perfused with 12μmol.l−1 55FeCl3 reabsorbed 105.2±12.7 fmol.mm−1.min−1 Fe, 435±52pmol.mm-1.min−1 Na, and 2.7±0.2nl.mm−1.min−1 water (mean ± SEM; n=19). Addition of ascorbate (1mmol.l−1) to the perfusate did not significantly alter Fe, Na, or water reabsorption. Supplementing the control perfusate with 60μmol.l−1 FeSO4 significantly decreased 55Fe uptake. Recalculating for the altered molar activity following addition of unlabeled Fe revealed a three-fold increase in Fe flux. Addition to the perfusate 12μmol.l−1 CuSO4, MnSO4, CdSO4, or ZnSO4 did not affect Fe, Na, or water flux. In conclusion, (1) in vivo, S2 PCTs of rat reabsorb Fe and (2) Fe is reabsorbed along the PCT via a pathway that is insensitive to Cu, Mn, Cd, or Zn. Together, these data demonstrate for the first time the hitherto speculated process of renal Fe filtration and subsequent tubular Fe reabsorption in a living mammal.

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 &lt, 0.001), and in both normal (p &lt, 0.001) and FGR (p &lt, 0.01) CPVs. Vasorelaxation to SNP was also potentiated by hypoxia in both normal (p &lt, 0.0001) and FGR (p &lt, 0.01) CPVs. However, compared to vessels from normal pregnancies, CPVs from FGR pregnancies showed significantly lower reactivity to SNP (p &lt, 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.

Supplemental Digital Content is available in the text., Rationale: Activation of the kynurenine pathway of tryptophan catabolism by infection and inflammation contributes to the development of systemic hypotension. Commercially-available kynurenine has direct vasorelaxant effects on arteries from several species and reduces systemic blood pressure when administered to normotensive or hypertensive rats. Objectives: To determine whether kynurenine promotes relaxation of human resistance arteries from normotensive and hypertensive pregnant women and to identify the vascular mechanism of its effects. Methods and Results: In isolated omental and myometrial resistance arteries from normotensive pregnant women, kynurenine (1 mmol/L) significantly reduced U46619-induced constriction (omentum N=14, P=2.4×10−3; myometrium N=21–25, P=2.6×10−4) and relaxed preconstricted arteries (N=53, P=1.0×10−11; N=20, P=8.8×10−3). Vasorelaxation persisted following endothelium removal (N=7, P=1.6×10−4) but was completely prevented by inhibition of large-conductance Ca2+-activated K+ channels (BKCa) channels with iberiotoxin (N=9, P=5.7×10−4) or paxilline (N=10, P=2.1×10−17). Accordingly, in isolated vascular smooth muscle cells from omental arteries, kynurenine increased the BKCa current (n=5–8, P=0.022) and the amplitude of spontaneous transient outward currents (n=6, P=0.031) but did not affect spontaneous transient outward current frequency. Kynurenine also increased Ca2+ spark frequency of pressurized omental arteries (n=8, P=0.031). Vasorelaxant effects of kynurenine persisted following inhibition of ryanodine receptors (N=7, P=0.48) but were moderately reduced by inhibition of adenylate cyclase (N=9, P=0.024). In arteries from women with preeclampsia, kynurenine similarly attenuated vasoconstriction (N=15, P=1.3×10−5) and induced BKCa-mediated vasodilation (N=16, P=2.0×10−4). Vasorelaxation in response to kynurenine and a specific BKCa activator, NS11021, was absent in fetal-derived placental resistance arteries in normal pregnancy and preeclampsia. Conclusions: Kynurenine dilates systemic arteries from multiple territories via BKCa activation. Notably, the vasorelaxatory capacity of kynurenine is preserved in preeclampsia, suggesting this approach may have translational potential for the treatment of hypertension in pregnancy. The data warrant further investigation of the potential to exploit this endogenous vasorelaxant as a new treatment for hypertensive pathologies.

Fetal growth restriction (FGR) affects around 5% of pregnancies and is associated with significant short- and long-term adverse outcomes. A number of factors can increase the risk of FGR, one of which is poor maternal diet. In terms of pathology, both clinically and in many experimental models of FGR, impaired uteroplacental vascular function is implicated, leading to a reduction in the delivery of oxygen and nutrients to the developing fetus. Whilst mechanisms underpinning impaired uteroplacental vascular function are not fully understood, interventions aimed at enhancing nitric oxide (NO) bioavailability remain a key area of interest in obstetric research. In addition to endogenous NO production from the amino acid l-arginine, via nitric oxide synthase (NOS) enzymes, research in recent years has established that significant NO can be derived from dietary nitrate, via the 'alternative NO pathway'. Dietary nitrate, abundant in green leafy vegetables and beetroot, can increase NO bioactivity, conferring beneficial effects on cardiovascular function and blood flow. Given the beneficial effects of dietary nitrate supplementation to date in non-pregnant humans and animals, current investigations aim to assess the therapeutic potential of this approach in pregnancy to enhance NO bioactivity, improve uteroplacental vascular function and increase fetal growth.

The endothelium is an established modulator of vascular tone; however, the recent discovery of the anti-contractile nature of perivascular adipose tissue (PVAT) suggests that the fat, which surrounds many blood vessels, can also modulate vascular tone. Both the endothelium and PVAT secrete vasoactive substances, which regulate vascular function. Many of these factors are common to both the endothelium and PVAT; therefore, this review will highlight the potential shared mechanisms in the modulation of vascular tone. Endothelial dysfunction is a hallmark of many vascular diseases, including hypertension and obesity. Moreover, PVAT dysfunction is now being reported in several cardio-metabolic disorders. Thus, this review will also discuss the mechanistic insights into endothelial and PVAT dysfunction in order to evaluate whether PVAT modulation of vascular contractility is similar to that of the endothelium in health and disease. Linked Articles This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue – Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc

Rationale: Maternal obesity pre-programmes offspring to develop obesity and associated cardiovascular disease. Perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the vasculature, which is reduced in hypertension and obesity. Objective: The objective of this study was to determine whether maternal obesity pre-programmes offspring to develop PVAT dysfunction in later life. Methods: Female Sprague–Dawley rats were fed a diet containing 10% (control) or 45% fat (high fat diet, HFD) for 12 weeks prior to mating and during pregnancy and lactation. Male offspring were killed at 12 or 24 weeks of age and tension in PVAT-intact or -denuded mesenteric artery segments was measured isometrically. Concentration–response curves were constructed to U46619 and norepinephrine. Results: Only 24-week-old HFD offspring were hypertensive (PM l-NMMA attenuated the anti-contractile effect of PVAT and increased contractility of PVAT-denuded arteries (PPPM A769662) was anti-contractile in PVAT-denuded (PPPP Conclusions: The diminished anti-contractile effects of PVAT in offspring of HFD dams are primarily due to release of a PVAT-derived contractile factor and reduced NO bioavailability.

INTRODUCTION: Potassium (K(+)) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries. METHODS: Human placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively. RESULTS: Kv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K(+) depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 - Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs. DISCUSSION: Kv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance.

The human fetoplacental vasculature is a low-resistance circulation with deoxygenated arterial relative to venous blood. The placenta lacks neuronal innervation suggesting that local physical (e.g., oxygenation; flow rate), paracrine (e.g., endothelial cell nitric oxide), and circulating (e.g., angiotensin II) factors will contribute to blood flow regulation in small fetoplacental vessels. Oxygenation (specifically hypoxia) has received particular attention. At the macro-level, hypoxic challenge increases vascular resistance, but the data's physiological relevance remains questionable. K(+) channels are a diverse family of proteins known to play important roles in the normal physiological functions of endothelial and smooth muscle cells of a variety of vascular beds. K(+) channels are categorized by their predicted transmembrane structure or gating properties. A small number of perfused placental cotyledon and isolated blood vessels studies have assessed K(+) channel activity. Specific activator/inhibitor application suggests functional voltage-gated channels, whereas toxin inhibitor studies have documented KCa channel activity. Pharmacological KATP channel activation significantly dilates preconstricted placental arteries and veins. There is a paucity of cell subtype-specific expression studies of placental K(+) channels. This review focuses on the roles of K(+) channels and oxygenation in controlling reactivity of small fetoplacental blood vessels.

Introduction There are no effective therapies for pre-eclampsia (PE), which remains a leading cause of considerable materno-fetal morbidity and mortality. Statins, widely utilized in cardiovascular disease, represent a candidate therapy for PE. Determination of statins’ ability to ameliorate the observed endothelial dysfunction in PE is lacking. Objective To determine the effect of short-term pitavastatin and pravastatin exposure on chorionic plate arteries (CPAs) and omental arteries (OAs) from normal and PE pregnancies. Methods CPAs and OAs from normal pregnancy (NP; N = 43 placentas, N = 20 biopsies) and PE (N = 18 placentas, N = 5 biopsies) pregnancies were mounted on a wire myograph. Contraction was assessed with KPSS (120 mM) and thromboxane-mimetic U46619 (0.1 nM–2 μM). Arteries were incubated for 2 h with 1 μM pitavastatin or pravastatin; time-controls in parallel. U46619 dose–response curves were repeated or NO-donor SNP (1 nM–100 μM) or endothelium-dependent bradykinin (0.1–1000 nM/L) following U46619 pre-constriction. All data are mean ± SEM. Results Neither statin significantly altered vascular reactivity in NP CPAs. CPAs show blunted SNP-induced relaxation in PE vs. NP (38 ± 10% vs. 28 ± 12% respectively; p = 0.038; Two-way ANOVA). Additionally, 1μM pitavastatin attenuated PE CPAs vasoconstriction compared to control (Emax, 152 ± 30% and 165 ± 33% respectively; p = 0.013; Two-way ANOVA) but vasodilation was unaffected. In NP OAs, 1μM pravastatin reduced vasoconstriction compared to time-control (111 ± 20% and 123 ± 23% respectively; p = 0.044; Two-way ANOVA) but did not affect vasodilation. Preliminary PE OA data suggests neither statin had a significant effect on vasoconstriction or vasodilatation (P > 0.05; Two-way ANOVA; N = 5). Discussion Data suggests statins are unlikely to be deleterious to placental vascular function in NP. Pitavastatin (PE CPAs) and pravastatin (NP OAs) have the ability to blunt agonist-induced vasoconstriction. Future work will focus on whether pitavastatin and pravastatin improve endothelial function in OAs from women with PE.

ObjectivesTo characterise Chorionic Plate Artery (CPA) function in maternal obesity, and investigate whether leptin exposure reproduces the obese CPA phenotype in normal-BMI women.Study designCPA responses to the thromboxane-A2 mimetic U46619 (pre/post leptin incubation), to the nitric oxide donor sodium nitroprusside (SNP) and the occurrence of tone oscillations (pre/post leptin incubation) were assessed in 46 term placentas from women of normal (18.5–24.9) or obese (>30) Body Mass Index (BMI).Outcome measuresArea Under the dose response Curve (AUC), maximum response (Vmax), sensitivity (EC50) to U46619 (pre/post leptin) and SNP; average vessel tone, oscillation amplitude and frequency (pre/post leptin).ResultsU46619 vasoconstriction was similar between BMI categories (p > 0.05), however vasodilatation to SNP was reduced in obesity (AUC p = 0.02, Vmaxp = 0.04) compared to normal-BMI women. Leptin incubation altered responses to U46619 in both normal-BMI (EC50 at 100 ng/ml leptin; p < 0.05) and obese women (AUC at 50 ng/ml; p < 0.05) but vasomotion was unaffected (p > 0.05).ConclusionsMaternal obesity is associated with altered placental vascular function which may adversely affect placental oxygen and nutrient transport, placing the fetus at risk. Leptin incubation altered CPA vascular function but did not reproduce the obese phenotype.

HIGHLIGHTS Short-term incubation with insulin increases the L-arginine transport in HUVECs. Short-term incubation with insulin increases the NO synthesis in HUVECs. Insulin induces relaxation in human placental vascular bed. Insulin attenuates the constriction induced by hydrogen peroxide in human placenta. The relaxation induced by insulin is dependent on BKCa channels activity in human placenta. Insulin induces relaxation in umbilical veins, increasing the expression of human amino acid transporter 1 (hCAT-1) and nitric oxide synthesis (NO) in human umbilical vein endothelial cells (HUVECs). Short-term effects of insulin on vasculature have been reported in healthy subjects and cell cultures; however, its mechanisms remain unknown. The aim of this study was to characterize the effect of acute incubation with insulin on the regulation of vascular tone of placental vasculature. HUVECs and chorionic vein rings were isolated from normal pregnancies. The effect of insulin on NO synthesis, L-arginine transport, and hCAT-1 abundance was measured in HUVECs. Isometric tension induced by U46619 (thromboxane A2 analog) or hydrogen peroxide (H2O2) were measured in vessels previously incubated 30 min with insulin and/or the following pharmacological inhibitors: tetraethylammonium (KCa channels), iberiotoxin (BKCa channels), genistein (tyrosine kinases), and wortmannin (phosphatidylinositol 3-kinase). Insulin increases L-arginine transport and NO synthesis in HUVECs. In the placenta, this hormone caused relaxation of the chorionic vein, and reduced perfusion pressure in placental cotyledons. In vessels pre-incubated with insulin, the constriction evoked by H2O2 and U46619 was attenuated and the effect on H2O2-induced constriction was blocked with tetraethylammonium and iberiotoxin, but not with genistein, or wortmannin. Insulin rapidly dilates the placental vasculature through a mechanism involving activity of BKCa channels and L-arginine/NO pathway in endothelial cells. This phenomenon is related to quick increases of hCAT-1 abundance and higher capacity of endothelial cells to take up L-arginine and generate NO.

Fetal growth restriction (FGR) is the inability of a fetus to reach its genetically predetermined growth potential. In the absence of a genetic anomaly or maternal undernutrition, FGR is attributable to “placental insufficiency”: inappropriate maternal/fetal blood flow, reduced nutrient transport or morphological abnormalities of the placenta (e.g., altered barrier thickness). It is not known whether these diverse factors act singly, or in combination, having additive effects that may lead to greater FGR severity. We suggest that multiplicity of such dysfunction might underlie the diverse FGR phenotypes seen in humans. Pregnant endothelial nitric oxide synthase knockout (eNOS−/−) dams exhibit dysregulated vascular adaptations to pregnancy, and eNOS−/− fetuses of such dams display FGR. We investigated the hypothesis that both altered vascular function and placental nutrient transport contribute to the FGR phenotype. eNOS−/− dams were hypertensive prior to and during pregnancy and at embryonic day (E) 18.5 were proteinuric. Isolated uterine artery constriction was significantly increased, and endothelium-dependent relaxation significantly reduced, compared with wild-type (WT) mice. eNOS−/− fetal weight and abdominal circumference were significantly reduced compared with WT. Unidirectional maternofetal 14C-methylaminoisobutyric acid (MeAIB) clearance and sodium-dependent 14C-MeAIB uptake into mouse placental vesicles were both significantly lower in eNOS−/− fetuses, indicating diminished placental nutrient transport. eNOS−/− mouse placentas demonstrated increased hypoxia at E17.5, with elevated superoxide compared with WT. We propose that aberrant uterine artery reactivity in eNOS−/− mice promotes placental hypoxia with free radical formation, reducing placental nutrient transport capacity and fetal growth. We further postulate that this mouse model demonstrates “uteroplacental hypoxia,” providing a new framework for understanding the etiology of FGR in human pregnancy.

Introduction The important immunoregulatory role of Kynurenine (Kyn) pathway tryptophan metabolism in the placenta is well established. Activity of this pathway is reduced in pre-eclampsia. Recently Kyn was shown to relax blood vessels and reduce blood pressure in animal models [1] . In a small study of non-pregnant humans (n = 5), Kyn relaxed omental arteries; this was attenuated by Linopirdine (Lin; 10 μM), an inhibitor of type 7 voltage-gated potassium (Kv7) channels [2] . Objectives To determine if Kyn contributes to regulation of vascular tone in systemic resistance arteries in pregnancy. Materials and methods Omental resistance arteries ( Results Treatment with 1, 3 or 6 mM Kyn dose-dependently attenuated arterial constriction to U46619 (N = 4–8, p = 0.003); greatest effect was observed at an intermediate U46619 dose of 10–7 M (change in constriction: control −0.19 ± 0.08 vs Kyn 6 mM −0.61 ± 0.13). Kyn induced relaxation of pre-constricted omental arteries (N = 7, p = 0.032; 0.70 ± 0.11 vs. 0.35 ± 0.08). Treatment with Lin 10 μM had no effect on Kyn-induced relaxation (N = 5, p = ns; 0.22 ± 0.12 vs. 0.17 ± 0.03). Conclusion This is the first study to demonstrate that Kyn has vasorelaxant effects on systemic resistance arteries in pregnancy. This supports our hypothesis that restoring Kyn pathway activity in pre-eclampsia could be a novel therapeutic target to attenuate maternal features of the disease. Kyn-induced relaxation was not inhibited by Lin 10 μM as previously reported outwith pregnancy, suggesting incomplete inhibition of Kv7 channels or an alternate compensatory mechanism.

OBJECTIVES: Deletion of the placental-specific P0 transcript of the insulin-like growth factor gene (Igf2) reduces placental growth from early pregnancy onwards. In Igf2 P0 knockout fetuses (P0), maternofetal flux of (14)C-methylaminoisobutyric acid ((14)C-MeAIB) mediated by system A amino acid transporter activity is increased at embryonic day 16 (E16), but this stimulation is not sustained, and by E19, fetal growth restriction (FGR) ensues. Here, we investigated whether upregulated (14)C-MeAIB transfer does occur concomitantly with a change in System A amino acid transporter activity and whether altered uteroplacental vascular function contributes to the FGR. We tested the hypothesis that FGR in P0 mice is attributable to altered nutrient transport rather than aberrant uteroplacental vascular function. METHODS: Plasma membrane vesicles were isolated from placentas of P0 and wild-type (WT) fetuses at E16 and E19. System A amino acid transporter activity was measured as sodium-dependent (14)C-MeAIB uptake over 60s. Wire myography was performed on uterine artery branches supplying P0 or WT implantation sites and agonist-induced constriction and dilation measured. RESULTS: Sodium-dependent uptake of (14)C-MeAIB (at 60s) was significantly (P�

Aim: The human fetoplacental vasculature has been suggested to be a low resistance/high flow system, but the mechanisms by which this state is achieved are unclear. Methods: This study assessed the effects of intraluminal flow and local oxygenation on isolated human placental chorionic plate arteries and veins at term using pressure myography. Results: Chorionic plate arteries and veins exhibit myogenic tone. A small but significant arterial vasodilatation was observed following exposure to hypoxia; chorionic plate veins contracted to a similar hypoxic stimulus. Under physiological conditions of pressure and oxygenation, increased luminal flow induced contraction in both chorionic plate arteries and veins. [Correction added after online publication 15th November 2011: ‘induced vasodilatation’ has been changed to ‘induced contraction’] Conclusions: Human fetoplacental vascular tone can be manipulated by local physical factors.

Despite their fundamental importance for normal cellular function, potassium (K) channels have been poorly studied in placental vascular tissues. This lack of experimental focus may relate to the fact that, as yet, no pregnancy complications have been directly attributable to a specific "channelopathy". K channel activity is central to normal cellular function. Vascular smooth muscle and endothelial cells within the fetoplacental circulation would be expected to be heavily influenced by the behaviour of K channels, as has been well-documented in other vascular beds. In this review, we summarise current understanding of K channel expression and activity in fetoplacental vasculature in normal and complicated pregnancies.

The obesity epidemic, including childhood obesity, is rapidly gaining strength as one of the most significant challenges to the health of the global community in the 21st Century. The proportion of women who are obese at the beginning of pregnancy is also increasing. These women and their babies are at high risk of pregnancy complications, and of programming for metabolic disease in adult life. In particular, maternal obesity is associated with aberrant fetal growth, encompassing both growth restricted and large for gestational age, or macrosomic fetuses. This article considers the potential effect of obesity and adipose tissue on placental nutrient exchange mechanisms in relation to aberrant fetal growth. The review emphasizes the dearth of work on this topic to date despite its importance to current and future healthcare of the population.

Objective. To determine if the phosphodiesterase type 5 inhibitor, sildenafil citrate improves endothelial-dependent relaxation of small arteries from women with preeclampsia. Methods. Myometrial and omental biopsies were taken from women participating in a randomized placebo-controlled trial using sildenafil citrate in women with preeclampsia. Vasoconstriction and endothelial-dependent relaxation of small arteries was measured utilizing wire myography. Results. Vasoconstriction and endothelial-dependent relaxation of myometrial and omental arteries were not altered in women taking sildenafil. Conclusion. Acute effects may have been lost as sildenafil administration occurred many hours prior to myography. Plasma sildenafil levels may have been lower than required for vascular response.

Control of vascular resistance and blood flow in the fetoplacental circulation is incompletely understood. Reactive oxygen species (ROS), physiological and pathophysiological regulators of vascular tone, are elevated in preeclampsia (PE), a disease of pregnancy characterized by increased fetoplacental vascular resistance. We tested the hypothesis that ROS modulate vascular reactivity in placental chorionic plate arteries. Wire myography was used to examine (1) the effects of acute exposure to ROS on arterial function in normal pregnancy and (2) the effects of maternal antioxidant supplementation on arterial reactivity in women at high risk for PE participating in the Vitamins in Pre-eclampsia (VIP) trial. ROS generated by xanthine plus xanthine oxidase enhanced basal tension, vasoconstriction in response to the thromboxane mimetic U46619, and relaxation in response to sodium nitroprusside. Hydrogen peroxide and peroxynitrite increased basal tone and relaxed preconstricted arteries (U44619), respectively. In women at risk for PE, chorionic plate artery constriction in response to U46619 was greater in the women receiving placebo compared to the women supplemented with the antioxidant vitamins C and E. ROS may regulate fetoplacental vascular resistance and blood flow in the short term, and chronic exposure to raised ROS could contribute to elevated fetoplacental vascular resistance in PE and fetal growth restriction (FGR).

Agonist-induced tone oscillations (rhythmic contractions and relaxations) occur in vascular beds to allow acute regulation of volume flow and thus the delivery of oxygen and nutrients to the tissue. Mechanisms responsible for the control of human placental vasomotor tone and blood flow are poorly characterized. This study aimed to characterise thromboxane-induced tone oscillations in human placental and myometrial arteries. Chorionic plate and myometrial arteries obtained from biopsies at term were mounted for isometric tension measurement. Tone oscillations were observed in chorionic arteries only when exposed to sub-maximal (

Decreased activity of "oxygen-sensitive" potassium (K) channels reduces hypoxia-induced contraction of pulmonary vascular tissue. "Oxygen-sensitive" channels are present in the fetoplacental vasculature, where vascular resistance is thought to increase in fetal growth restriction (FGR). We assessed placental gene expression of K(V)2.1, K(V)9.3 and K(IR)6.1, three "oxygen-sensitive" K channels in normal pregnancy and FGR.Biopsies were obtained from 12 normal pregnancies and 12 pregnancies complicated by FGR. Chorionic plate arteries and veins were dissected from the biopsies and a sample of placental tissue comprising chorionic plate and villus tissue was also collected. All samples were processed and analyzed using "real time" quantitative RT-PCR.K(V)2.1 and K(V)9.3 gene expression, in veins and placental homogenate, respectively, were increased in FGR. K(IR)6.1 gene expression was comparable in normal pregnancy and FGR samples.The gene expression of two voltage-gated K channels thought to be important for altered vascular reactivity following changes in oxygenation is increased in FGR.

Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that has been implicated in cardiovascular disease. The objective of the present study was to determine the vasoactive effects and underlying mechanisms of S1P on adult human maternal arteries. The isometric tensions of the omental and myometrial arteries isolated from normal pregnant women at term were assessed in response to incremental doses of S1P in the presence or absence of the nitric oxide (NO) synthase inhibitor N(G)nitro-L-arginine methyl ester (L-NAME). The putative involvement of Rho-associated kinases (ROCKs) in intact arteries and in those permeabilized with a-toxin, to study agonist-dependent calcium-sensitization, was assessed with the inhibitor Y27632. Real-time RT-PCR established the presence of mRNA encoding the S1P receptors (S1P 1 to 3 ), previously known as endothelial differentiation gene receptors (EDG1, 3 and 5), in both artery types. S1P induced a dose-dependent increase in the isometric tension of all the arteries. Y27632 reduced constriction due to S1P in intact arteries and reduced S1P-induced sensitization of contraction to submaximal activating Ca 2þ in permeabilized arteries. L-NAME also modulated S1P vasoactive responses in a tissue-specific manner. Two subgroups of omental arteries were identified, one of which utilizes the NO pathway. In myometrial arteries, S1P evoked oscillatory constrictions, whereas pretreatment with L-NAME resulted in only tonic constrictions of unaltered peak magnitude. The prominent vasoactive actions of S1P in the maternal arteries of pregnant women are modulated by inhibitors of ROCKs and NO bioavailability. The subtle tissue-specific functional differences in the modulation of S1P actions by NO have important implications for vascular tone regulation by this bioactive circulatory metabolite during pregnancy. nitric oxide, Rho-associated kinase, vasoconstriction

Poorly controlled diabetes in pregnancy, characterized by hypo- and hyperglycemia, is associated with adverse outcomes. We hypothesized that aberrant glucose levels affect vascular function in pregnancy. The effects of glucose concentration on constriction and endothelium-dependent relaxation in uterine arteries of normal C57BL/6 mice were examined. Ex-vivo arteries from 18 pregnant and 14 non-pregnant mice were mounted on a wire myograph, constricted with phenylephrine and relaxed with incremental doses of acetylcholine, in physiological saline solution containing 5 mmol/L glucose. Arteries were then exposed to solutions with 2, 5, 8 or 12 mmol/L glucose for 30 min and constriction/relaxation repeated. On altering glucose concentrations to 2, 8 or 12 mmol/L, maximal constriction was increased in arteries from pregnant but not from non-pregnant mice (paired t-test, p

Background Adenosine triphosphate (ATP)-sensitive potassium channels (KATP) are important modulators of vascular tone. Preliminary data from our laboratory suggests that KATP channels are expressed in the fetoplacental vasculature where addition of pinacidil, a specific KATP opener, promotes relaxation. We aimed to assess the effects of KRN2391 and KRN4884 on the fetoplacental vasculature, which are putative KATP channel openers. Materials and methods Functional activity of KATP channels was assessed in chorionic plate arteries and veins using wire myography. Cromakalim-, KRN2391- and KRN4884-induced relaxations were assessed in the presence and absence of agonist-induced pretone. Cromakalim, an established KATP channel opener, acted as control. Results KRN2391 evoked significantly greater relaxation of chorionic plate arteries and veins than either KRN4884 or cromakalim. KRN2391-induced relaxation of precontracted arteries and veins was reduced in the presence of inhibitiors of the nitric oxide pathway (L-NNA or LY83583). With KRN4884, there was no contribution of nitric oxide to the induced relaxation. Conclusions We conclude that KATP channels play an important role in controlling placental vascular tone. KRN2391 induces relaxation of human placental blood vessels by activation of KATP channels and via activation of nitric oxide-dependent pathways.

OBJECTIVE: Currently, no investigations reliably identify placental dysfunction in late pregnancy. To facilitate the development of such investigations we aimed to identify placental features that differ between normal and adverse outcome in late pregnancy in a group of pregnancies with reduced fetal movement. METHODS: Following third trimester presentation with reduced fetal movement (N = 100), placental structure ex vivo was measured. Placental function was then assessed in terms of (i) chorionic plate artery agonist responses and length-tension characteristics using wire myography and (ii) production and release of placentally derived hormones (by quantitative polymerase chain reaction and enzyme linked immunosorbant assay of villous tissue and explant conditioned culture medium). RESULTS: Placentas from pregnancies ending in adverse outcome (N = 23) were ~25% smaller in weight, volume, length, width and disc area (all p

Background: The regulation of vascular tone in the uterine circulation is a key determinant of appropriate uteroplacental blood perfusion and successful pregnancy outcome. Estrogens, which increase in the maternal circulation throughout pregnancy, can exert acute vasodilatory actions. Recently a third estrogen receptor named GPER (G protein-coupled estrogen receptor) was identified and, although several studies have shown vasodilatory effects in several vascular beds, nothing is known about its role in the uterine vasculature.Aim: The aim of this study was to determine the function of GPER in uterine arteries mainly during pregnancy. Uterine arteries were isolated from nonpregnant and pregnant rats.Methods: Vessels were contracted with phenylephrine and then incubated with incremental doses (10-12-10-5 M) of the selective GPER agonist G1.Results: G1 induced a dose-dependent vasodilation which was: 1) significantly increased in pregnancy, 2) endothelium-dependent, 3) primarily mediated by NO/cGMP pathway and 4) unaffected by BKca channel inhibition.Conclusion: This is the first study to show the potential importance of GPER signaling in reducing uterine vascular tone during pregnancy. GPER may therefore play a previously unrecognized role in the regulation of uteroplacental blood flow and normal fetus growth.

During pregnancy, trophoblasts migrate from the placenta into uterine spiral arteries, transforming them into wide channels that lack vasoconstrictive properties. In pathological pregnancies, this process is incomplete. To define the fundamental events involved in spiral artery remodeling, we have studied the effect of trophoblasts on vascular smooth muscle cells (SMCs). Here we demonstrate for the first time that apoptosis of SMCs can be initiated by invading trophoblasts. When trophoblasts isolated from normal placenta (primary trophoblasts) or conditioned medium was perfused into spiral or umbilical artery segments, apoptosis of SMCs resulted. Culture of human aortic SMCs (HASMCs) with primary trophoblasts, primary trophoblast-conditioned medium, or a trophoblast-derived cell line (SGHPL-4) also significantly increased SMC apoptosis. Fas is expressed by spiral artery SMCs, and a Fas-activating antibody triggered HASMC apoptosis. Furthermore, a Fas ligand (FasL)-blocking antibody significantly inhibited HASMC apoptosis induced by primary trophoblasts, SGHPL-4, or trophoblast-conditioned medium. Depleting primary trophoblast-conditioned medium of FasL also abrogated SMC apoptosis in vessels in situ. These results suggest that apoptosis triggered by the release of soluble FasL from invading trophoblasts contributes to the loss of smooth muscle from the walls of spiral arteries during pregnancy. Copyright © American Society for Investigative Pathology.

A successful pregnancy is dependent on liberal placental perfusion via the maternal and fetal circulations. Doppler waveform analyses of umbilical arteries suggest increased resistance to flow in the fetoplacental circulation of pregnancies complicated by intrauterine growth restriction (IUGR). Neither the site nor the mediators responsible for this altered vascular reactivity are known, to date. In placentas in normal pregnancy, reduced oxygenation promotes contraction of the in vitro-perfused placental cotyledon and modulates agonist-induced contraction of chorionic plate arteries and veins. Placental oxygenation has also been suggested to be reduced in IUGR. We tested the hypothesis that oxygen tension could directly modify placental chorionic plate vessel vasoreactivity in IUGR. Small arteries and veins from the chorionic plate were dissected from biopsies from placentas of pregnancies complicated by IUGR and were studied using parallel wire myography. Vasoconstriction at 20%, 7%, and 2% oxygen was assessed utilizing the thromboxane mimetic U46619. Experiments were also performed in the presence of 4-aminopyridine (4AP), a blocker of voltage-gated potassium channels. Increased oxygenation reduced venous vasoconstriction but did not modify arterial vasoconstriction. 4AP increased basal tone in arteries and veins. We suggest that venoconstriction in response to hypoxia may provide a mechanism for increased fetoplacental vascular resistance associated with IUGR.

Fetoplacental blood vessel constriction in response to reduced oxygenation has been demonstrated in placenta perfused in vitro. In pulmonary vessels, hypoxic vasoconstriction involves Ca 2+ influx into smooth muscle through membrane ion channels including voltage-gated Ca 2+ channels (VGCCs). We hypothesised that VGCCs are involved in agonist-induced constriction of fetoplacental resistance vessels and that their contribution is modulated by oxygen. Chorionic plate small arteries were studied using wire myography. Arteries were normalised at high (0.9 of L 13.3 kPa ) or low (0.9 of L 5.1 kPa ) stretch and experiments performed at 156, 38 or 15 mmHg oxygen. At low stretch, U46619 (thromboxane-mimetic) or KCl (smooth muscle depolarisation) constriction was greater at 38 than 156 or 15 mmHg oxygen. An L-type VGCC blocker nifedipine, inhibited KCl constriction by >85% but was less effective in U46619 constrictions (43–67%). At high stretch, nifedipine inhibition of KCl- and U46619-induced constriction was less at 15 than 38 or 156 mmHg oxygen. Oxygen did not affect constriction to U46619 or nifedipine-induced relaxation when vessels were normalised at high stretch. In conclusion, oxygen modulates chorionic plate arterial constriction at low stretch but regulation is lost at high stretch. U46619 constriction is underlain by VGCCs and nifedipine-insensitive processes; their relative contribution is influenced by oxygen.

Objective Endothelial dysfunction is central to the pathogenesis of pre-eclampsia (PE). This study aimed to determine if maternal characteristics, such as age, parity, BMI, smoking status and obstetric history, which affect the risk of developing pre-eclampsia, influenced endothelial function in myometrial arteries taken during an uncomplicated pregnancy. Study design As part of ongoing studies investigating endothelial function in normal and compromised pregnancies, myometrial vessels were isolated from biopsies taken at elective Caesarean section. A cohort of 119 women was identified and information regarding past pregnancy outcomes and medical history was obtained. Wire myography was used to compare endothelial-dependent relaxation in response to bradykinin between different patient groups. Results Maternal age, parity and a history of miscarriage did not affect endothelial-dependent relaxation of myometrial small arteries. Attenuated endothelial-dependent relaxation was seen in vessels taken from women with elevated BMI and enhanced relaxation was seen in women who had smoked during pregnancy. Vessels isolated from women with a history of past pregnancy complications did not show any significant difference in endothelial-dependant relaxation compared to women with uncomplicated histories. Conclusion Maternal factors may influence endothelial function in the absence of pregnancy complications. Endothelial-dependent relaxation of myometrial arteries, isolated from multiparous women with an uncomplicated index pregnancy, is comparable between women with and without a history of pregnancy complications.

Fetoplacental blood vessels constrict in response to endothelin (ET-1) or reduced oxygen tension in the placental cotyledon perfused in vitro. In nonplacental resistance arteries, hypoxia and ET-1 induce constriction by promoting Ca2+ influx into smooth muscle through membrane ion channels, which include voltage-gated Ca2+ channels (VGCCs). We hypothesized that VGCCs are involved in ET-1-induced constriction of fetoplacental resistance vessels and that their contribution to constriction is enhanced at low oxygen tension.Chorionic plate small arteries from term placentas were studied using parallel wire myography. Arteries were normalized at 0.9 of L(5.1 kPa) ("low stretch" approximately 25 mm Hg; approximating physiologic vascular pressure) or 0.9 of L(13.3 kPa) ("high stretch" approximately 42 mm Hg) and experiments performed at oxygen tensions of 156, 38, and 15 mm Hg.When chorionic plate arteries were normalized at low stretch, oxygen tension did not affect constriction to ET-1. Nifedipine (10(-4) M), a blocker of L-type VGCCs, inhibited ET-1 (EC80)-induced constriction to a similar extent at each oxygen tension (52% to 64% inhibition). In contrast, when arteries were normalized at high stretch, constriction to ET-1 was greater at 38 than at 156 or 15 mm Hg oxygen and nifedipine inhibition of ET-1-induced constriction was greater at 38 and 15 mm Hg than at 156 mm Hg oxygen.VGCCs and nifedipine-insensitive processes underlie the contractile response of chorionic plate arteries to ET-1 and their relative contribution to vasoconstriction is modulated by oxygen tension when vessels are normalized at high stretch. However, contrary to our hypothesis, the response of chorionic plate arteries to ET-1 is not modulated by oxygen when vessels are normalized at physiologic pressure.

Blood flow control within the fetoplacental circulation is poorly understood despite the essential role of the placenta in pregnancy. Our study compared an in vitro assessment of placental chorionic plate small artery function with in vivo measures of umbilical artery blood flow. Umbilical artery Doppler waveform analysis was performed on women (N=8) with uncomplicated pregnancies within 24 hours of delivery. Small arteries (n=25) were dissected from the placental chorionic plate from biopsies at term and studied using parallel wire myography. Chorionic plate arteries developed maintained constrictions to the thromboxane-mimetic U46619. Nitric oxide (NO) donation with the endothelial-independent agent sodium nitroprusside elicited significant relaxation compared to paired control vessels. This vasodilatation correlated significantly with both pulsatility and resistance indices measured in the umbilical artery. We conclude that Doppler indices in the umbilical artery are inversely proportional to the maximal relaxation achieved by chorionic plate small arteries in response to a NO donor.

We tested the hypothesis that crossing two mouse models of fetal growth restriction (FGR) of differing phenotype would induce more severe FGR than either model alone. Female endothelial nitric oxide synthase knockout mice (eNOS−/−) were mated with placental-specific Igf2 knockout males (P0). Resultant fetuses were no more growth restricted than those with P0 deletion alone. However, P0 deletion attenuated the reduced placental system A amino acid transporter activity previously observed in eNOS−/− mice. Manipulating maternal and fetal genotypes provides a means to compare maternal and fetal regulation of fetal growth.

Iron handling and gene expression of the divalent metal transporter, DMT1, in the kidney of the anemic Belgrade (b) rat.BackgroundWe have previously shown that the rat kidney reabsorbs metabolically significant amounts of iron and that it expresses the divalent metal transporter 1, DMT1. The Belgrade (b) rat carries a mutation in DMT1 gene, which causes hypochromic, microcytic anemia due to impaired intestinal iron absorption and transport of iron out of the transferrin cycle endosome. In the duodenum of b/b rats, expression of DMT1 mRNA and protein is increased, suggesting a feedback regulation by iron stores. The aim of this study was to investigate iron handling and DMT1 expression in the kidneys of Belgrade rats.MethodsAnimals were maintained for 3 weeks on a synthetic diet containing 185mg/kg iron (FeSO4), after which functional and molecular parameters were analyzed in male heterozygous (+/b) and homozygous (b/b) rats (N = 4 to 6 for each group).ResultsSerum iron concentration was significantly higher in b/b compared to +/b rats while urinary iron excretion rates were unchanged in b/b compared to +/b rats. Northern analysis using a rat DMT1 probe showed comparable mRNA levels between +/b and b/b animals. Western analysis and immunofluorescence microscopy performed using a polyclonal antibody against rat DMT1 showed that DMT1-specific immunoreactivity was almost absent in the kidneys of b/b rats compared to that seen in +/b animals.ConclusionOur results indicate that the G185R mutation of DMT1 causes protein instability in the kidneys of b/b rats. Given that +/b and b/b rats excrete comparable amounts of iron, the lack of DMT1 protein is compensated by an alternative, yet to be identified, mechanism.

The control of the blood flow within the fetoplacental circulation is poorly understood despite the essential role of the placenta in pregnancy. Our aim was to assess the vasoactive responses of veins from the placental chorionic plate. Biopsies were obtained from term placentae from uncomplicated pregnancies. Small veins from the chorionic plate were dissected free from surrounding tissue and studied using parallel wire myography. Human placental chorionic plate veins developed maintained constrictions to the thromboxane-mimetic U46619. Endothelium-dependent agonists did not promote venous relaxation. However, NO donation with the endothelial-independent agent, sodium nitroprusside, elicited significant relaxation. Venous constriction to U46619 and relaxation to sodium nitroprusside were modified by adjustment of media oxygen tension and normalization parameters. Human placental chorionic plate veins respond to vasoactive agents and may play a role in the control of the blood flow in the fetoplacental circulation.

The proliferation of nurstHed Initiatives arising from nurse specialist/practitioner posts In urology Is reflected In areas such as the management of bladder cancer, erectile dysfunction, stoma care, and prostate disease. The establishment of the role of urology specialist nurse In one North Oxfordshlre hospital led to the development of a nurstHed prostate assessment clinic for male patients with lower urinary tract symptoms arising from benign prostatic hyperplasia. A description of how training was conducted, and the subsequent reappraisal of competency, Is given In relation to physical examination and history-taking skills necessary for the development of this Initiative.

Despite the essential role of the placenta in pregnancy, the control of the blood flow within the fetoplacental circulation is poorly understood. A handful of myography studies have directly assessed the role of vasoactive agonists in fetoplacental vasculature contractility but have used a range of steady-state conditions. Our aim, therefore, was to determine the optimal vessel diameter and oxygen tension to assess vascular function in small arteries isolated from the chorionic plate of normal term placentae. Biopsies were obtained from term placentae from uncomplicated pregnancies. Small arteries were dissected from the chorionic plate, mounted onto a wire myograph in HCO3(-) -buffered physiological salt solution at 37 degrees C and equilibrated for 20 min. Two methods for normalization of the optimal length/diameter for contractility of chorionic plate small arteries were assessed. Both classical normalization (CN) and length-tension curve (LTC) methods produced similar data. These data were agonist-independent. Data for CN and LTC were unaffected but maximal force generation (for U46619) was decreased in reduced oxygen tensions. Using conditions for optimal tension production in chorionic plate small arteries the thromboxane-mimetic U46619 produced the greatest and most reproducible constrictive effect. Relaxations were only achieved with endothelial-independent agonists (sodium nitroprusside and papaverine).

We have demonstrated that the kidney plays an important role in iron balance and that metabolically significant reabsorption of this ion occurs in the loop of Henle and the collecting ducts [Wareing M, Ferguson CJ, Green R, Riccardi D, and Smith CP. J Physiol (Lond) 524: 581–586, 2000]. To test the possibility that the divalent metal transporter DMT1 (Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Nature 388: 482–488, 1997) could represent the apical route for iron entry in the kidney, we raised and affinity-purified an anti-DMT-1 polyclonal antibody and determined DMT-1 distribution in rat kidney by Western analysis, immunofluorescence, and confocal microscopy. The strongest DMT1-specific (i.e., peptide-protectable) immunoreactivity was found in the collecting ducts, in both principal and intercalated cells. Thick ascending limbs of Henle's loop and, more intensely, distal convoluted tubules exhibited apical immunostaining. Considerable intracellular DMT-1 immunoreactivity was seen throughout the nephron, particularly in S3 segments. The described distribution of DMT-1 protein is in agreement with our previous identification of nephron sites of iron reabsorption, suggesting that DMT-1 provides the molecular mechanism for apical iron entry in the distal nephron but not in the proximal tubule. Basolateral iron exit may be facilitated by a different system.

A luminal microperfusion technique was used to examine the K+ permeability of surface proximal convoluted tubules (PCT) in the kidney of anesthetized rats. Transtubular potassium concentration ([K+]) gradients were varied by altering the concentration of KCl in luminal perfusates, to which 32 mmol/l of the impermeant solute raffinose was also added to prevent net fluid reabsorption. The arithmetic mean transtubular [K+] gradient was highly predictive of net potassium flux, yielding an apparent K+ permeability of 31.9 ± 1.7 × 10−5 cm/s in the absence of fluid reabsorption. When compared using identical calculation techniques, we found this was not significantly different from the permeability derived in a previous study when fluid reabsorption was present [J. D. Kibble, M. Wareing, R. W. Wilson, and R. Green. Am. J. Physiol. 268 ( Renal Fluid Electrolyte Physiol. 27): F778–F783, 1995]. We conclude that fluid reabsorption does not affect the apparent permeability of the proximal tubule to potassium. The apparent permeability to86Rb, measured following its addition to luminal perfusates, was not significantly different from the value obtained for K+, suggesting that rubidium is a useful marker for net potassium movements in the PCT of the rat.

Fetal growth restriction (FGR) is a serious pregnancy complication, resulting in significant perinatal morbidity and mortality. Increased vascular resistance in the fetoplacental circulation is a hallmark of FGR and is associated with enhanced vasoconstriction of the resistance arteries in the placenta, the chorionic plate arteries (CPAs). Although the cause is unknown, FGR is associated with excess exposure to glucocorticoids (GCs), key mediators of vascular resistance in the systemic circulation. We hypothesized that GCs alter CPA reactivity, thereby contributing to the altered blood flow dynamics seen in FGR. We aimed to examine the acute and chronic effects of GCs on CPA reactivity and the operational mechanisms. Glucocorticoid receptors were highly expressed by CPA. 11β-Hydroxysteroid isoenzyme type 2 was detected within the endothelium, whereas 11β-hydroxysteroid isoenzyme type 1 was absent. Acute GC treatment significantly attenuated U46619-induced constriction. This effect was reversed by cotreatment with mifepristone or an endothelial NOS inhibitor. In contrast, chronic GC treatment potentiated U46619 constriction in a dose-dependent manner, which was partially abolished by mifepristone cotreatment. Similar effects were observed using a novel nonsteroidal glucocorticoid receptor-specific agonist. Chronic treatment with GCs altered the expression of several vasoactive factors, including thromboxane and bradykinin receptors, prokineticin-1, cyclooxygenase-2, and endothelial NOS. In summary, acute and chronic GC treatment exerts contrasting effects on CPA vasoreactivity. These opposing effects are consistent with temporal actions in other vascular beds and reflect activation of distinct nongenomic and genomic pathways. Chronic exposure to elevated GCs may contribute to the raised vascular resistance observed in the fetoplacental circulation in FGR.

Introduction Maternal obesity programmes offspring to develop obesity and associated cardiovascular disease. Perivascular adipose tissue (PVAT) exerts an anti-contractile effect in healthy blood vessels; an effect lost in male offspring of obese dams. However, the mechanism by which maternal obesity programmes PVAT dysfunction in offspring remains unknown. Methods Six week old female Sprague-Dawley rats were fed a control (10% fat) or 45% fat obesogenic diet (HFD) for 12 weeks before mating; during pregnancy and lactation. At weaning, female offspring were provided with the control diet until sacrifice at either 12 (12 wo) or 24 (24 wo) weeks of age. PVAT-denuded mesenteric arteries from pups, with or without exogenous PVAT, were mounted on a wire myograph and concentration-response curves were constructed to thromboxane A2 receptor agonist U46619 (10 nM-3 µM) ± 10 µM A769662, an activator of AMP-activated protein kinase (AMPK) ± glucosamine (an O-GlcNAcylator). Western blotting was used to asses protein expression in PVAT stimulated with or without glucosamine. Results Body weight, insulin levels and blood pressure were increased in HFD dams and their 12wo and 24 wo offspring compared to age-matched controls. Without PVAT, vessel contractions to U46619 were reduced in 12 wo offspring of HFD dams, effects mimicked in control arteries by pre-incubation with 10 mM glucosamine. PVAT from control, but not from HFD offspring, exerted an anti-contractile effect on the corresponding PVAT-denuded arteries at both ages. Pre-incubation of control PVAT with glucosamine diminished the anti-contractile effect at both ages. PVAT from HFD offspring pre-incubated with glucosamine had no effect on PVAT-denuded vessels but simultaneous AMPK activation within PVAT partially restored anti-contractile capability at both ages. Protein O-GlcNAcylation expression was increased in HFD PVAT and control PVAT incubated with glucosamine, whereas AMPK expression was decreased. Conclusions The enhanced protein O-GlcNAcylation and decreased AMPK expression in HFD PVAT may underlie the reduced anti-contractile effect of PVAT in female offspring of obese dams. Nevertheless, simultaneous AMPK activation within HFD PVAT partially restored the anti-contractile effects of PVAT.

The potency of adult-derived circulating progenitor endothelial colony forming cells (ECFCs) is drastically surpassed by their fetal counterparts. Human pregnancy is associated with robust intensification of blood flow and vascular expansion in the uterus, crucial for placental perfusion and fetal supply. Here, we investigate whether fetal ECFCs transmigrate to maternal bloodstream and home to locations of maternal vasculogenesis, primarily the pregnant uterus. In the first instance, endothelial-like cells, originating from mouse fetuses expressing paternal eGFP, were identified within uterine endothelia. Subsequently, LacZ or enhanced green fluorescent protein (eGFP)-labeled human fetal ECFCs, transplanted into immunodeficient (NOD/SCID) fetuses on D15.5 pregnancy, showed similar integration into the mouse uterus by term. Mature endothelial controls (human umbilical vein endothelial cells), similarly introduced, were unequivocally absent. In humans, SRY was detected in 6 of 12 myometrial microvessels obtained from women delivering male babies. The copy number was calculated at 175 [IQR 149–471] fetal cells per millimeter square endothelium, constituting 12.5% of maternal vessel lumina. Cross-sections of similar human vessels, hybridized for Y-chromosome, positively identified endothelial-associated fetal cells. It appears that through ECFC donation, fetuses assist maternal uterine vascular expansion in pregnancy, potentiating placental perfusion and consequently their own fetal supply. In addition to fetal growth, this cellular mechanism holds implications for materno-fetal immune interactions and long-term maternal vascular health.

Human placental expression of K(V)9.3, a voltage-gated K channel linked to tissue oxygenation responses, has been suggested at the messenger RNA level but tissue localisation has not been described. We aimed to: (1) produce an antibody to human K(V)9.3 and (2) assess channel expression and distribution in human placental tissue. We determined human placental protein expression and localisation using an antibody to K(V)9.3. Antibody specificity was confirmed by Western blotting. Staining was observed in syncytiotrophoblast microvillous membrane, endothelial cells (in intermediate, stem villi and chorionic plate blood vessels) and vascular smooth muscle cells (large diameter vessels only) by immunohistochemistry. Expression was unchanged in tissue from women with small-for-gestational age babies. It was concluded that K(V)9.3 is localised to human placental vascular tissues and syncytiotrophoblast.

Statins can have beneficial cholesterol-independent effects on vascular contractility, which may involve increases in the bioavailability of NO (nitric oxide) as a result of phosphorylation of eNOS (endothelial NO synthase). Although this has been attributed to phosphorylation of Akt (also known as protein kinase B), studies in cultured cells have shown that statins can phosphorylate AMPK (AMP-activated protein kinase); it is unknown whether this has functional effects in intact arteries. Thus we investigated the acute effects of simvastatin on resistance arterial contractile function, evaluating the involvement of NO, Akt and AMPK. Isolated rat mesenteric resistance arteries were mounted on a wire myograph. The effects of incubation (1 and 2 h) with simvastatin (0.1 or 1 μM) on contractile responses were examined in the presence and absence of L-NNA (N-nitro-L-arginine; 10 μM) or mevalonate (1 mM). Effects on eNOS, phospho-eNOS (Ser1177), and total and phospho-Akt and -AMPK protein expression were investigated using Western blotting. The effect of AMPK inhibition (compound C, 10 μM) on eNOS phosphorylation and contractile responses were also studied. Simvastatin (1 μM, 2 h) significantly reduced constriction to U46619 and phenylephrine and enhanced dilations to ACh (acetylcholine) in depolarized, but not in U46619-pre-constricted arteries. These effects were completely and partially prevented by L-NNA and mevalonate respectively. Simvastatin increased eNOS and AMPKα phosphorylation, but had no effect on Akt protein expression and phosphorylation after 2 h incubation. Compound C prevented the effects of simvastatin on eNOS phosphorylation and contractility. Thus simvastain can acutely modulate resistance arterial contractile function via mechanisms that involve the AMPK/phospho-eNOS (Ser1177)/NO-dependent pathway.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.

This study directly examined endothelial function of myometrial arteries in the uterus of women with gestational diabetes mellitus (GDM), healthy pregnant and non-pregnant women. It also examined whether endothelial function was affected by the changes in glucose concentration (from 2 to 12mmol/L) that is seen in poorly controlled diabetes.Ex vivo myometrial arteries from the uterus of women with GDM (N=16) as well as healthy pregnant (N=16) and non-pregnant women (N=15) were mounted on a wire myograph. The effect of changing glucose concentrations from 5mmol/L to 2, 8 and 12mmol/L glucose for 30min was studied.Myometrial arteries from women with GDM had significantly impaired endothelium-dependent relaxation compared to those from both healthy pregnant and non-pregnant women (two-way ANOVA, p0.001). Changes in glucose concentration had no effect on constriction or endothelium-dependent relaxation in any group studied (two-way ANOVA, p0.05).Fluctuations of glucose concentrations between 2 and 12mmol/L do not affect endothelial function. Endothelial dysfunction is present in the myometrial arteries of women with GDM, an inherent defect which may be responsible for some of the complications of GDM.

Adequate blood flow provision through alterations in maternal vascular function is essential during pregnancy for optimal fetal development. Abnormal uterine vasculature adaptation, resulting in aberrant blood flow to the placenta, has been implicated as a possible cause of fetal growth restriction (FGR). Our study aimed to develop strategies to evaluate murine vascular function in pregnancy using wire myography. Main uterine artery loop and branch vessels isolated from near-term pregnant mice showed significant contraction to phenylephrine (PE). Endothelial-dependent relaxation was noted with acetylcholine (ACH). U46619 elicited significant contraction of umbilical arteries and veins, but relaxation was only demonstrable with the nitric oxide (NO) donor sodium nitroprusside (SNP). In conclusion, our data suggest that murine uteroplacental and fetoplacental arteries show distinct responses to vasoactive agents. Furthermore, this study indicates that wire myography represents a robust technique for the assessment of murine uteroplacental and fetoplacental vascular function, which will aid evaluation of mouse genetic models of FGR.

BACKGROUND: During pregnancy, extravillous trophoblast invades the uterine wall and enters the spiral arteries. Remodelling ensues, with loss of vascular smooth muscle cells (SMCs) to create high flow, low resistance vessels. Pregnancies complicated by pre-eclampsia are characterized by incomplete arterial remodelling. Endovascular trophoblast is not easily accessible for studies to establish the pathogenesis of pre-eclampsia, so we have developed a model appropriate to carry out mechanistic studies of vessel wall transformation. METHODS AND RESULTS: Segments of human spiral artery were perfused with the choriocarcinoma cell line, BeWo; cells invaded the vessel wall and induced apoptosis of vascular SMC. Perfusion of vessels with BeWo-conditioned medium also induced SMC apoptosis, indicating the presence of a soluble apoptotic factor. BeWo express Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment of BeWo-conditioned medium with antibodies against FasL inhibited vascular SMC apoptosis in vitro. Antibodies that blocked TRAIL receptor function had no effect. Extracellular matrix degradation is also a prerequisite for vascular remodelling; BeWo express matrix metalloproteinase-12 (MMP-12) and BeWo-conditioned medium increased MMP-12 expression in spiral artery SMC. CONCLUSIONS: BeWo induce arterial remodelling via FasL- and MMP-12-dependent mechanisms. BeWo-derived factors up-regulate protease expression in spiral artery SMC to facilitate matrix breakdown. © The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Objective: To determine whether delivery mode influences placental chorionic plate arterial and venous vascular reactivity. Methods: Normal term placentas were obtained after vaginal delivery or cesarean section. Chorionic plate arterial and venous function was assessed by wire myography. Results: Sodium nitroprusside-induced arterial relaxation increased post cesarean section at 20% oxygen. Decreased U46619-induced venous contraction was observed in vaginal deliveries at 7% oxygen. Sodium nitroprusside-induced relaxation increased in vaginal delivery at 20% oxygen. Conclusion: Delivery mode does not alter chorionic plate arterial vascular reactivity under physiological conditions, however venous reactivity was modified, a factor for consideration when interpreting functional experimental data.