Untargeted analysis of plasma samples from pre-eclamptic women reveals polar and apolar changes in the metabolome.

INTRODUCTION: Pre-eclampsia is a hypertensive gestational disorder that affects approximately 5% of all pregnancies. OBJECTIVES: As the pathophysiological processes of pre-eclampsia are still uncertain, the present case-control study explored underlying metabolic processes characterising this disease. METHODS: Maternal peripheral plasma samples were collected from pre-eclamptic (n = 32) and healthy pregnant women (n = 35) in the third trimester. After extraction, high-resolution mass spectrometry-based untargeted metabolomics was used to profile polar and apolar metabolites and the resulting data were analysed via uni- and multivariate statistical approaches. RESULTS: The study demonstrated that the metabolome undergoes substantial changes in pre-eclamptic women. Amongst the most discriminative metabolites were hydroxyhexacosanoic acid, diacylglycerols, glycerophosphoinositols, nicotinamide adenine dinucleotide metabolites, bile acids and products of amino acid metabolism. CONCLUSIONS: The putatively identified compounds provide sources for novel hypotheses to help understanding of the underlying biochemical pathology of pre-eclampsia.

A role for two-pore potassium (K2P) channels in endometrial epithelial function.

The human endometrial epithelium is pivotal to menstrual cycle progression, implantation and early pregnancy. Endometrial function is directly regulated by local factors that include pH, oxygen tension and ion concentrations to generate an environment conducive to fertilization. A superfamily of potassium channels characterized by two-pore domains (K2P) and encoded by KCNK genes is implicated in the control of the cell resting membrane potential through the generation of leak currents and modulation by various physicochemical stimuli. The aims of the study were to determine the expression and function of K2P channel subtypes in proliferative and secretory phase endometrium obtained from normo-ovulatory women and in an endometrial cancer cell line. Using immunochemical methods, real-time qRT-PCR proliferation assays and electrophysiology. Our results demonstrate mRNA for several K2P channel subtypes in human endometrium with molecular expression of TREK-1 shown to be higher in proliferative than secretory phase endometrium (P < 0.001). The K2P channel blockers methanandamide, lidocaine, zinc and curcumin had antiproliferative effects (P < 0.01) in an endometrial epithelial cancer cell line indicating a role for TASK and TREK-1 channels in proliferation. Tetraethylammonium- and 4-aminopyridine-insensitive outwards currents were inhibited at all voltages by reducing extracellular pH from 7.4 to 6.6. Higher expression of TREK-1 expression in proliferative phase endometrium may, in part, underlie linked to increased cell division. The effects of pH and a lack of effect of non-specific channel blockers of voltage-gated potassium channels imply a role for K2P channels in the regulation of human endometrial function.

Inhibition of tissue transglutaminase 2 attenuates contractility of pregnant human myometrium.

Premature delivery remains a serious risk factor in pregnancy, with currently licensed tocolytics unable to offer significant improvement in neonatal outcome. Further understanding of the regulators of uterine contractility is required to enable the development of novel and more effective tocolytic therapies. The transglutaminase family is a class of calcium-dependent, transamidating enzymes, of which tissue transglutaminase 2 is a multifunctional enzyme with roles in cell survival, migration, adhesion, and contractility. The aim of the present study was to investigate the role of this enzyme in regulating the contractility of pregnant human myometrium. Tissue strips from biopsy samples obtained at elective cesarean section were either allowed to contract spontaneously or induced to contract with oxytocin, phenylephrine, or bradykinin. Activity integrals, used to measure contractile activity, were taken following cumulative additions of the reversible, polyamine transglutaminase inhibitors cystamine and mono-dansylcadaverine and the irreversible, site-specific transglutaminase inhibitors N-benzyloxycarbonyl-l-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine and 1,3-dimethyl-2[(oxopropyl)thio]imidazolium. The ability of cystamine and mono-dansylcadaverine to affect oxytocin-mediated calcium mobilization within primary cultured myometrial cells was also measured utilizing a calcium indicator. All inhibitors attenuated myometrial contractions in a concentration-dependent manner independent of the method of contraction stimulus. Similarly cultured myometrial cells preincubated with cystamine and mono-dansylcadaverine displayed an altered calcium response to oxytocin stimulation. Our findings demonstrate a potential role for tissue transglutaminase 2 in regulating uterine contractility in pregnant human myometrium that may be associated with the calcium signaling cascade required for contraction.

Detection of EETs and HETE-generating cytochrome P-450 enzymes and the effects of their metabolites on myometrial and vascular function.

Cytochrome P-450 (CYP450) enzymes of the CYP2 and -4 family in humans metabolize arachidonic acid to generate bioactive epoxyeicosatrienenoic acids (EETs) and hydroxyeicosatetrenoic acids (HETEs). We report significantly higher levels of CYP 2J2 protein expression following the onset of labor (n = 6, P < 0.05), implying increased EET-generating capacity within the uterus. Myometrial relaxation to 8,9-EET and 5,6-EET was observed, with the latter being inhibited by preincubation with 1 muM paxilline and is supported by whole cell recordings showing a modest effect of 5,6-EET on myometrial outward-current density (n = 4, P < 0.05). Only 5,6-EET of the EETs tested affected vascular reactivity (n = 6). Both 12- and 20-HETE (n = 5-6) caused vasoconstriction of partially depolarized blood vessels, with glibenclamide (n = 5) enhancing the effect of 12-HETE alone. Our findings signify a role for CYP2C9/19, -2J2, and -4A11/22 in late pregnancy, possibly related to the synthesis of lipid metabolites and downstream effects on vascular remodeling in the term pregnant uterus. The presence of CYP4A11/22 and their resultant procontractile metabolites could argue either a role in the control and initiation of labor and/or modification of the vascular delivery system to influence blood flow to the laboring uterus. The differential effects of the EETs and HETEs in the pregnant human uterus identify the CYP pathway as a novel modulator of myometrial and vascular physiology during late pregnancy.

Pharmacological profile of vascular activity of human stem villous arteries.

INTRODUCTION: The function of the placental vasculature differs considerably from other systemic vascular beds of the human body. A detailed understanding of the normal placental vascular physiology is the foundation to understand perturbed conditions potentially leading to placental dysfunction. METHODS: Behaviour of human stem villous arteries isolated from placentae at term pregnancy was assessed using wire myography. Effects of a selection of known vasoconstrictors and vasodilators of the systemic vasculature were assessed. The morphology of stem villous arteries was examined using IHC and TEM. RESULTS: Contractile effects in stem villous arteries were caused by U46619, 5-HT, angiotensin II and endothelin-1 (p ≤ 0.05), whereas noradrenaline and AVP failed to result in a contraction. Dilating effects were seen for histamine, riluzole, nifedipine, papaverine, SNP and SQ29548 (p ≤ 0.05) but not for acetylcholine, bradykinin and substance P. DISCUSSION: Stem villous arteries behave differently to vessels of the systemic vasculature and results indicate that the placenta is cut off from the systemic maternal vascular regulation. Particularly, endothelium-dependent processes were attenuated in the placental vasculature, creating a need to determine the role of the endothelium in the placenta in future studies.

Caspase-1-mediated cytokine release from gestational tissues, placental, and cord blood.

Distinguishing between fetal and maternal inflammatory responses is necessary for understanding the immune interplay either side of the placenta. Fetal immunity reaches maturity during extrauterine life and while basic inflammatory responses afford a certain degree of protection, fetuses are vulnerable to infection. With the discovery of inflammasomes-intracellular scaffolds that facilitate the elaboration of reactions resulting in the release of mature interleukin-1ß (IL-1ß)-it is necessary to consider how inflammatory stimuli are processed. The purinergic P2X7 receptor located on haematopoietic cells is a key intermediary in signal transduction initiated at Toll-like receptors (TLR) terminating in release of the mature IL-1ß product. We demonstrate herein that IL-1ß release from fetal membranes and mononuclear cells isolated from cord, placental, and maternal blood, obtained at term, is P2X7- and caspase-1 dependent. The P2X7-dependent release of the cytokine, which was highest from choriodecidua, was attenuated by progesterone (P4), prolactin and an NFkB inhibitor. The NLRP3 inflammasome appears necessary for the processing of IL-1ß in gestational tissues and leukocytes.

Functional characteristics of chorionic plate placental arteries from normal pregnant women and women with pre-eclampsia.

OBJECTIVES: We aimed to compare placental small artery function from women with pre-eclampsia and normal pregnancy. In particular, we wished to test the hypothesis that these arteries respond differently to an endothelium-dependent vasodilator, to the presence of nitric oxide, and to the presence of cyclic monophosphate nucleotides. METHODS: A small vessel wire myograph was used to study placental arteries (200 to 550 microm). Contractile function was assessed with vasopressin. Relaxation was assessed with the endothelium-dependent vasodilator, bradykinin, and the endothelium-independent vasodilators sodium nitroprusside (a nitric oxide donor) and papaverine (a phosphodiesterase inhibitor). RESULTS: The constrictor response to vasopressin did not differ between patient groups (p=0.79; repeated measures ANOVA). For both normal pregnancy and pre-eclampsia, the response of pre-constricted arteries to the endothelium-dependent vasodilator, bradykinin, was minimal. Vasorelaxation to sodium nitroprusside and papaverine was attenuated in pre-eclampsia compared to normal pregnancy (p=0.03 and p<0.001, respectively; repeated measures ANOVA). CONCLUSIONS: In pre-eclampsia, placental arteries exhibit an attenuated vasodilatory response to nitric oxide.

Measurement of vasoactive metabolites (hydroxyeicosatetraenoic and epoxyeicosatrienoic acids) in uterine tissues of normal and compromised human pregnancy.

BACKGROUND: Preeclampsia and intrauterine growth restriction define two disorders of a multifactorial etiology that compromise maternal and fetal well being as well as cardiovascular health in later life. Many of the overt symptoms of preeclampsia are attributable to the systemic endothelial dysfunction observed in the uteroplacental and systemic circulation, leading to a generalized vasoconstriction, hypertension and inadequate placental perfusion. Mounting evidence implicates nonprostanoid eicosanoids, epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) in the control of vascular function and dysfunction. OBJECTIVE: To determine whether levels of EETs and HETEs are altered in preeclampsia and intrauterine growth restriction compared with normal term pregnancy. METHODS: An analytical liquid chromatography-tandem mass spectrometry profiling method was utilized in order to analyze differential levels of EETs and HETEs in intrauterine tissues of term nonlaboring, laboring and preeclamptic women as well as women with a growth-restricted pregnancy. RESULTS: Placentae of preeclamptic women contained significantly (P < 0.05) larger amounts of 5-HETE, 12-HETE and 15-HETE known to possess either vasoconstrictive or proinflammatory actions. Laboring tissues were characterized by significantly higher (P < 0.05) EET levels in the amnion compared with the other clinical groups. EET and HETE levels in preeclampsia and intrauterine growth restriction were positively correlated (P < 0.05), whereas in normal and laboring pregnancies, EETs and HETEs were negatively correlated. CONCLUSION: Increased production of 5-HETE, 12-HETE and 15-HETE metabolites in preeclamptic placentae indicates an important role for this family of eicosanoids in the cause of this disease.

Interleukin-1 beta secretion from cord blood mononuclear cells in vitro involves P2X 7 receptor activation.

The purinergic receptor P2X(7) is activated by extracellular adenosine 5'-triphosphate (ATP) and promotes the efficient release of interleukin-1 beta. The authors examine protein and molecular expression of the P2X(7) receptor and its ability to stimulate interleukin-1 beta release in cord blood mononuclear cells (CBMCs) from placentae of term nonlaboring and laboring women. They show both mRNA and protein (78 kDa) expression for the P2X( 7) receptor in CBMCs of parturient and nonparturient women. Costimulation of CBMCs in vitro with bacterial endotoxin and ATP resulted in significantly (P < .05) enhanced interleukin-1 beta release in laboring (54.17 +/- 24.78 pg/mL(-1); n = 8) compared with nonlaboring (13.60 +/- 3.20 pg/mL(-1); n = 7) deliveries. Release of interleukin-1 beta in both groups was blocked by preincubation with oxidized ATP, a P2X(7) receptor antagonist. The authors provide evidence for a novel inflammatory pathway in the release of interleukin-1 beta, which may be linked to the onset of labor.

Quantitative profiling of epoxyeicosatrienoic, hydroxyeicosatetraenoic, and dihydroxyeicosatetraenoic acids in human intrauterine tissues using liquid chromatography/electrospray ionization tandem mass spectrometry.

A reversed-phase liquid chromatography negative ion electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated to quantify a range of physiologically relevant eicosanoids, including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs); 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs), and 5,6-, 8,15-, and 12,20-dihydroxyeicosatetraenoic acids (DiHETEs) in human intrauterine tissues. A solid-phase extraction method was employed to extract the eicosanoids, and gradient LC separation was performed on a Kromasil C(18) column. Mass spectrometric detection was performed by multiple reaction monitoring over a 31-min run time. The calibration curves were linear over the range of 4-400pmol/g tissue, and the intra- and interday precision and accuracy were within a coefficient of variation of 2.0 to 27.4% and 4.6 to 17.9%, respectively. The lower limit of quantitation was 1.0pmol/g tissue. The method was applied successfully to the characterization and quantitation of eicosanoids in the different compartments of human intrauterine tissues. Our results demonstrate significantly greater amounts of HETEs than of either the EETs or DiHETEs (P<0.001), irrespective of tissue type. Specifically, the metabolite 12-HETE was significantly more abundant (P<0.001) than all other HETEs. Of the EET metabolites, 5,6-EET predominated (P<0.001). A significant negative correlation between EETs and HETEs for all tissues (rho=-0.390, P<0.001) was identified, implying a biological feedback mechanism between these two arachidonate metabolite classes.

Progesterone enhances the tocolytic effect of ritodrine in isolated pregnant human myometrium.

OBJECTIVE: To evaluate the effect of natural progesterone on the relaxant effect of ritodrine on pregnant human oxytocin-induced myometrial contractility. STUDY DESIGN: Isometric tension recordings were performed under physiologic conditions on isolated myometrial strips taken from low-risk term pregnant women undergoing elective cesarean section. Cumulative effects of natural progesterone (10 (-11) to 10 (-5) mol/L) on oxytocin-induced myometrial contractility were evaluated. Contractile activity following ritodrine exposure was also investigated in myometrium pretreated with natural progesterone. RESULTS: Natural progesterone alone exerted a concentration-dependent relaxant effect on myometrial contractions. The concentration-response curve for ritodrine from natural progesterone pretreated myometrium was shifted to the left with a significant reduction ( P < .01) of 50% of the maximal response, contraction amplitude ( P < .05), and frequency ( P < .05). However, there was no significant difference in the mean maximal inhibition achieved ( P = .95). CONCLUSION: Natural progesterone increased the relaxant effect of ritodrine by reducing 50% of the maximal response, amplitude, and frequency of myometrial contraction, most likely through nongenomic actions. These results suggest that natural progesterone may be beneficial for preventing preterm birth in a low-risk population.

Hydrogen peroxide and superoxide anion modulate pregnant human myometrial contractility.

Reactive oxygen species (ROS) have the propensity to cause macromolecular damage with consequent modification of cellular function. We investigated the effects of two particular oxidants, superoxide (O2(-)) anions and hydrogen peroxide (H2O2), on oxytocin-induced myometrial contractility using biopsies from women undergoing Caesarean section at term gestation. Isometric tension recordings were performed and concentration-response curves derived after addition of test agents. A maximal reduction in myometrial contractility to 27.2 +/- 4.5% of control was observed followed application of H2O2. The enzyme scavenger catalase (CAT) reduced the inhibitory effect of H2O2 but had little effect at 10-fold lower concentrations. Addition of dialysed xanthine oxidase +/- hypoxanthine significantly inhibited contractility to 23.8.0 +/- 4.2% compared with control. Pre-incubation with superoxide dismutase and CAT diminished this effect. The non-specific potassium channel blocker, tetraethylammonium chloride (1 mM), had no effect on myometrial contractility. We conclude that human myometrium is susceptible to the effects of ROS, which may be produced by reperfusion-ischaemic episodes during labour. Our findings could, in part, explain the weak or prolonged depression of contractions characteristic of myometrial dysfunction culminating in difficult labours.

Vasoactive effects of neurokinin B on human blood vessels.

OBJECTIVES: Preeclampsia (PE) is a multisystem disease unique to human pregnancy. Abnormal placentation results in placental hypoperfusion leading to the secretion of a factor(s) by the placenta. Our aim was to investigate whether neurokinin B (NKB) is the circulating factor associated with PE. STUDY DESIGN: Vascular effects of NKB were assessed in blood vessels dissected from myometrial and omental biopsy specimens obtained at caesarean section from normal pregnant women (n = 26) or in mesenteric blood vessels obtained from nonpregnant female Wistar rats (n = 4). RESULTS: Incubation with NKB did not alter endothelial-dependent relaxation of omental or myometrial arteries. NKB produced a dose-dependent relaxation in preconstricted omental arteries and veins. NKB did not affect vasoactive responsiveness of rat mesenteric arteries. CONCLUSION: We conclude from these observations that NKB is not the circulating factor associated with increased vascular resistance in PE.

Myometrial and placental artery reactivity alone cannot explain reduced placental perfusion in pre-eclampsia and intrauterine growth restriction.

OBJECTIVES: (1) To investigate a possible association between myometrial and placental artery vasoreactivity and perfusion at the basal and chorionic plates, respectively. (2) To confirm that myometrial arteries from women with pre-eclampsia and intrauterine growth restriction exhibit an attenuated endothelium-dependent vasodilatory response. METHODS: Women with normal pregnancy, pre-eclampsia and intrauterine growth restriction had a magnetic resonance scan to assess placental perfusion using a technique called intravoxel incoherent motion. At delivery, myometrial and chorionic plate placental arteries were assessed on a wire myograph. Vessels were pre-constricted with the thromboxane mimetic U46619 and dilated with incremental doses of bradykinin. RESULTS: Pre-constricted myometrial arteries from women with pre-eclampsia or intrauterine growth restriction exhibited an attenuated vasodilatory response to bradykinin, compared with normal pregnancy (P < 0.0001). Pre-constricted placental arteries exhibited a minimal vasodilatory response in all three groups of women (P = 0.10). Maximal constrictor and vasodilatory responses of myometrial arteries were not associated with the perfusing fraction at the basal plate. Maximal constrictor and vasodilatory responses of chorionic plate placental arteries were not associated with the perfusing fraction at the chorionic plate. CONCLUSION: We confirm that myometrial arteries from women with pre-eclampsia or intrauterine growth restriction exhibit an attenuated endothelium-dependent vasodilatory response. Apart from vasoreactivity of small arteries, other factors may be involved in the control of placental perfusion.

Effects of a phosphodiesterase-5 (PDE5) inhibitor on endothelium-dependent relaxation of myometrial small arteries.

OBJECTIVE: In preeclampsia, endothelium-dependent function is markedly aberrant. Myometrial resistance arteries from women with preeclampsia show a minimal, wholly nitric oxide-mediated, bradykinin-induced relaxation. Our aim was to test that phosphodiesterase 5 (PDE5) inhibition could improve endothelium-dependent function in preeclampsia. Study design Small arteries dissected from myometrial biopsies obtained at cesarean section from normal pregnant women (N=22) or women with preeclampsia (N=24) were mounted on wire or pressure myographs. Vessels were constricted (arginine vasopressin or U46619) and relaxed (bradykinin) before and after incubation with a PDE5 inhibitor, UK-343664. RESULTS: Endothelium-dependent vasodilatation was decreased in vessels from women with preeclampsia. 100 nmol/L UK-343664 did not affect normal pregnant but significantly improved relaxation of the vessels from women with preeclampsia. CONCLUSION: A PDE5 inhibitor enhances endothelial function of myometrial vessels from women with preeclampsia, such that the behavior of these arteries approximates to those from normal women. These agents offer a potential therapeutic strategy for the management of preeclampsia.