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The uterine vasculature undergoes marked changes during pregnancy in order to provide the necessary increase in blood flow to support growth and nutrition of the uterus, placenta, and developing fetus. Pregnancy-associated uterine vascular transformations are orchestrated by a complex array of endocrine and cellular mechanisms to bring about structural modifications at the maternal–fetal interface, which collectively lead to development of the uteroplacental circulation. Understanding intrinsic uterine vascular remodeling in pregnancy is essential for understanding the physiologic and pathophysiologic regulation of maternal uterine blood flow. Aberrations of uterine vascular remodeling are potentially involved in the etiology of several pregnancy disorders, for example, preeclampsia, fetal growth restriction, and preterm labor; therefore, it is essential for subspecialist clinicians and investigators interested in reproductive physiology to fully understand the establishment of uteroplacental circulation. The foundational literature in this area is extensive; thus, a succinct review is likely to be a useful resource. Herein, we present and discuss a historical perspective on uterine vascular anatomy, maternal vascular growth associated with decidualization, trophoblast invasion, intervillous circulation, aberrations in uterine vascular modeling, and the clinical implications of improper development of the uteroplacental circulation. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To quantitate alcohol-induced alterations in the maternal uterine endothelial proteome utilizing iTRAQ-based mass spectrometry. Study design: Uterine artery endothelial cells from third trimester pregnant ewes were FAC sorted, validated and treated without or with binge-like alcohol. Lysates were trypsin digested, iTRAQ-labeled, and analyzed using nano LC MS/MS. Results: Alcohol significantly upregulated 14 and downregulated 17 proteins (P <0.05) including those related to cell structure, transcription/translation regulation, histones, Ca2+/NO, and redox balance. Gene Ontology and ArrayTrack analyses revealed alterations to protein processing, binding, and nutrient metabolism pathways. Further, alcohol altered proteins previously correlated with fetal alcohol spectrum disorders (FASD) and those that regulate epigenetic, transcriptional, and translational processes. Conclusions: Alcohol differentially alters the proteome in the maternal uterine compartment at the level of the endothelium. iTRAQ mass spectrometry provides a robust high throughput platform to comprehend the multi-mechanistic actions of alcohol and develop appropriate biomarkers and ameliorative measures for FASD. [Copyright &y& Elsevier]

Maternal alcohol consumption during pregnancy is a significant field of scientific exploration primarily because of its negative effects on the developing fetus, which is specifically defined as fetal alcohol spectrum disorders. Though the effects on the mother are less explored compared with those on the fetus, alcohol produces multiple effects on the maternal vascular system. Alcohol has major effects on systemic hemodynamic variables, endocrine axes, and paracrine factors regulating vascular resistance, as well as vascular reactivity. Alcohol is also reported to have significant effects on the reproductive vasculature including alterations in blood flow, vessel remodeling, and angiogenesis. Data presented in this review will illustrate the importance of the maternal vasculature in the pathogenesis of fetal alcohol spectrum disorders and that more studies are warranted in this field. [ABSTRACT FROM AUTHOR]

Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesisrelated mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis- related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓placental growth factor), adhesion molecules (↓angiopoietin-like-3; ↓collagen- 18A1;↓endoglin), proteases/matrix proteins/inhibitors (↓alanyl aminopeptidase; ↓collagen-4A3; ↓heparanase; ↓plasminogen, ↑plasminogen activator urokinase; ↓platelet factor-4; ↓plexin domain containing-1; ↓tissue inhibitor of metalloproteinases-3), transcription/ signaling molecules (↓heart and neural crest derivatives-2; ↓DNA-binding protein inhibitor;↓NOTCH-4;↓ribosomal protein- L13a1; ↓ribosomal protein large-P1), cytokines/chemokines (↓interleukin- 1B), and miscellaneous growth factors (↓leptin; ↓plateletderived growth factor-α); ↓transforming growth factor (TGF-α ↑TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional questions about FASD from both mechanistic as well as ameliorative perspectives. [ABSTRACT FROM AUTHOR]

Abstract: Little is known about alcohol effects on the utero-placental compartment during pregnancy. For the first time, we utilized 2-D DIGE quantitative proteomics to evaluate the role of the uterus in Fetal Alcohol Spectrum Disorders (FASD) pathogenesis. Uterine artery endothelial cells were isolated from pregnant ewes, FAC sorted, validated, and maintained in culture. To mimic maternal binge drinking patterns, cells were cultured in the absence or presence of alcohol (300mg/dl) in a compensating sealed humidified chamber system equilibrated with aqueous alcohol for 3h on 3 consecutive days for two weeks. CyDye switch combined with 2-D DIGE followed by MALDI-TOF and tandem MS/MS were utilized. Validation was performed using Western immunoblot analysis. Chronic binge-like alcohol significantly (P <0.05) decreased 30 proteins and increased 19 others. Gene-enrichment and functional annotation cluster analysis revealed significant enrichment (P <0.05) in three categories: glutathione S transferase, thioredoxin, and vesicle transport-related. Furthermore, alcohol differentially altered proteins with certain isoforms being downregulated while others were upregulated. In summary, binge alcohol has specific effects on the maternal uterine proteome, especially those related to oxidative stress. The current study also demonstrates a great need to utilize proteomic approaches for diagnostic, mechanistic and therapeutic aspects of FASD. [Copyright &y& Elsevier]

Oestrogen dramatically increases uterine blood flow (UBF) in ovariectomized (Ovx) ewes. Both the follicular phase and pregnancy are normal physiological states with elevated levels of circulating oestrogen. ICI 182 780 is a pure steroidal oestrogen receptor (ER) antagonist that blocks oestrogenic actions in oestrogen-responsive tissue. We hypothesized that an ER-mediated mechanism is responsible for in vivo rises in UBF in physiological states of high oestrogen. The purpose of the study was to examine the effect of an ER antagonist on exogenous and endogenous oestradiol-17β (E2β)-mediated elevations in UBF. Sheep were surgically instrumented with bilateral uterine artery blood flow transducers, and uterine and femoral artery catheters. Ovx animals ( n= 8) were infused with vehicle (35% ethanol) or ICI 182 780 (0.1–3.0μg min−1) into one uterine artery for 10 min before and 50 min after E2β was given (1μg kg−1i.v. bolus) and UBF was recorded for an additional hour. Intact, cycling sheep were synchronized to the follicular phase using progesterone, prostaglandin F2α(PGF2α) and pregnant mare serum gonadotrophin (PMSG). When peri-ovulatory rises in UBF reached near peak levels, ICI 182 780 (1 or 2μg (ml uterine blood flow)−1) was infused unilaterally ( n= 4 sheep). Ewes in the last stages of pregnancy (late pregnant ewes) were also given ICI 182 780 (0.23–2.0μg (ml uterine blood flow)−1; 60 min infusion) into one uterine artery ( n= 8 sheep). In Ovx sheep, local infusion of ICI 182 780 did not alter systemic cardiovascular parameters, such as mean arterial blood pressure or heart rate; however, it maximally decreased ipsilateral, but not contralateral, UBF vasodilatory responses to exogenous E2β by∼55–60% ( P< 0.01). In two models of elevated endogenous E2β, local ICI 182 780 infusion inhibited the elevated UBF seen in follicular phase and late pregnant ewes in a time-dependent manner by∼60% and 37%, respectively; ipsilateral≫ contralateral effects ( P< 0.01). In late pregnant sheep ICI 182 780 also mildly and acutely (for 5–30 min) elevated mean arterial pressure and heart rate ( P< 0.05). We conclude that exogenous E2β-induced increases in UBF in the Ovx animal and endogenous E2β-mediated elevations of UBF during the follicular phase and late pregnancy are partially mediated by ER-dependent mechanisms. [ABSTRACT FROM AUTHOR]

Pregnancy and the follicular phase of the ovarian cycle show elevation of uterine blood flow and associated increases in uterine artery endothelium (UAE) endothelial nitric oxide (NO) synthase (eNOS) expression. Nonetheless, a role for increased NO production during pregnancy and the follicular phase has only been inferred by indirect measures. The recent development of a uterine artery endothelial cell model further suggests that pregnancy is associated with reprogramming of cell signaling, such that eNOS may become more Ca2+ sensitive and be subject to regulation by Ca2+-independent kinases. This study describes for the first time the direct and simultaneous monitoring of NO production and intracellular free Ca2+ concentration ([Ca2+]i) in freshly isolated UAE from pregnant, follicular, and luteal sheep. The pharmacological agonists ionomycin (calcium ionophore) and thapsigargin (TG; endoplasmic reticulum Ca2+ pump inhibitor) were used to maximally elevate [Ca2+]i and fully activate eNOS as a measure of eNOS expression. NO production stimulated by ionomycin (5 μM) and TG (10 μM) were 1.95- and 2.05-fold, respectively, in pregnant-UAE and 1.34- and 1.37-fold in follicular-UAE compared with luteal-UAE. In contrast, the physiological agonist ATP (100 μM) stimulated a 3.43-fold increase in NO in pregnant-UAE and a 1.90-fold increase in follicular-UAE compared with luteal-UAE, suggesting that pregnancy and follicular phase enhance eNOS activation beyond changes in expression in vivo. 2-aminoethoxydiphenyl borate (APB; an inositol 1,4,5-trisphosphate receptor blocker) totally prevented the ATP-induced [Ca2+]i response but only partially inhibited NO production. Thus pregnancy-enhanced eNOS activation in UAE is mediated through [Ca2+]i-insensitive pathways as well as through a greater eNOS sensitivity to [Ca2+]i. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that pregnancy and the follicular phase are associated with increases in endothelial nitric oxide synthase (eNOS) mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide levels. Effect of ovariectomy, ovarian cycle, and pregnancy on eNOS expression.

Presents a study which tested that pregnancy and follicular phase are associated with increases in endothelial nitric oxide synthase (eNOS) messenger RNA and the expression of eNOS protein in nitric oxide levels. Methods; Results; Discussion.

Focuses on prolonged estrogen replacement therapy, presenting research on the systemic and uterine blood flow distribution during prolonged infusion of 17beta-estradiol. Description of research methods utilized; Analysis of statistical data compiled; Results of this research.

Presents a study that determined which reproductive and nonreproductive vascular beds exhibit alterations in vascular resistance and blood flow during prolonged (17beta-estradiol) (E2beta) infusion. Prolonged estrogen replacement therapy; Methodology; Cardiovascular effects of acute versus prolonged E2beta infusion; Experimental results.

Tests the hypothesis that increases in nitric oxide (NO) production during pregnancy are associated with elevations in protein expression of the constitutive isoform, endothelial cell NOS, in uterine but systemic arteries. Quantification of pregnancy-induced changes in endothelial ecNOS expression; Control adult heart rate and fetal growth measurements.

Part II. Presents a study on endothelial vasodilator production by uterine and systemic arteries and effects of pregnancy on nitric oxide synthase (NOS) expression. Immunohistochemical localization of ecNOS; Quantification of pregnancy-induced changes in endothelial ecNOS expression; Vascular smooth muscle; Vascular relaxation.

Examines whether nitric oxide is involved in crucial physiological adaptations of mammalian pregnancy. Vasodilation of the maternal systemic circulation; Increased uterine and fetoplacental blood flow; Uterus before parturition.

Studies endothelial vasodilator production by uterine and systemic arteries. Adenylate cyclase; Pressor responses; Uterine blood flow; Incubation procedures; Statistical analysis.

Studies the possible reproduction of endocrine and hemodynamic alterations in nonpregnant animals through prolonged treatment with estradiol-17 beta. Mean arterial pressure; Systemic vascular resistance; Systemic vasodilation; Attenuated pressor responses to infused angiotensin II (ANG II).

Examines the effect of nitric oxide (NO) during pregnancy. Maternal systemic vasodilation; Regulation of uterine and fetoplacental blood flow; Reduction of vascular reactivity during normal pregnancy; Variability of the relative contribution of nitric oxide depending on vascular bed and vessel size; Contribution of NO to low fetoplacental nitric oxide synthase in preeclampsia.

Nitric oxide (NO) production is essential to facilitate rises in uterine blood flow (UBF) during pregnancy. It has been proposed that the metabolites of E2β, 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 2-methoxyestradiol (2-ME2), and 4-methoxyestradiol (4-ME2) play a role in mediating vasodilation and rises in UBF during pregnancy. We previously showed that the E2β metabolites stimulate prostacyclin production in pregnancy-derived ovine uterine artery endothelial cells (P-UAECs); however, it is unknown whether the E2β metabolites also induce NO production. Herein, UAECs derived from nonpregnant and pregnant ewes were used to test the hypothesis that E2β metabolites stimulate NO production in a pregnancy-specific manner. Specific estrogen receptor (ER) and adrenergic receptor (AR) antagonists were used to determine the roles of ERs or ARs in E2β metabolite-induced NO production. E2β and its metabolites increased total nitric oxide metabolites (NOx) levels (NO2 + NO3) in P-UAECs, but not in NP-UAECs. Pretreatment with combined 1 µmol/L 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α antagonist) and 1 µmol/L 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-β antagonist) inhibited the rises in NOx levels stimulated by E2β and 2-ME2, but had no effect on 2-OHE2-, 4-OHE2-, or 4-ME2-stimulated rises in NOx levels. Pretreatment with yohimbine (α2-AR antagonist) and propranolol (β2,3-AR antagonist) inhibited the rises in NOx levels stimulated by 2-OHE2, but not by E2β, 4-OHE2, 2-ME2, or 4-ME2. These data demonstrate that E2β metabolites stimulate NO synthesis via ERs or ARs in UAECs in a pregnancy-specific manner, suggesting that these metabolites contribute to rises in vasodilation and UBF during pregnancy. [ABSTRACT FROM AUTHOR]

Examines the molecular and cellular signaling in the perinatal cardiovascular system. Improvement of the awareness of the importance of local systems related to the endothelium and its control of the perinatal cardiovascular system; Analysis of the various forms of stress into altered cardiovascular development in the fetus and neonate; Identification of the risk factors in small-for-gestational-age babies.

Intrauterine growth restriction ( IUGR) is interconnected with developmental programming of lifelong pathophysiology. IUGR is seen in human multifetal pregnancies, with stepwise rises in fetal numbers interfering with placental nutrient delivery. It remains unknown whether fetal blood analyses would reflect fetal nutrition, liver, and excretory function in the last trimester of human or ovine IUGR. In an ovine model, we hypothesized that fetal plasma biochemical values would reflect progressive placental, fetal liver, and fetal kidney dysfunction as the number of fetuses per gestation rose. To determine fetal plasma biochemical values in singleton, twin, triplet, and quadruplet/quintuplet ovine gestation, we investigated morphometric measures and comprehensive metabolic panels with nutritional measures, liver enzymes, and placental and fetal kidney excretory measures at gestational day ( GD) 130 (90% gestation). As anticipated, placental dysfunction was supported by a stepwise fall in fetal weight, fetal plasma glucose, and triglyceride levels as fetal number per ewe rose. Fetal glucose and triglycerides were directly related to fetal weight. Plasma creatinine, reflecting fetal renal excretory function, and plasma cholesterol, reflecting placental excretory function, were inversely correlated with fetal weight. Progressive biochemical disturbances and growth restriction accompanied the rise in fetal number. Understanding the compensatory and adaptive responses of growth-restricted fetuses at the biochemical level may help explain how metabolic pathways in growth restriction can be predetermined at birth. This physiological understanding is important for clinical care and generating interventional strategies to prevent altered developmental programming in multifetal gestation. [ABSTRACT FROM AUTHOR]

Introduces a series of articles that discuss recent research into the interplay between the fetal and maternal cardiovascular and endocrine systems and developmental programming.

In pregnancy, the uterine vasculature undergoes dramatic vasodilatory adaptations. Previously, vascular endothelial growth factor (VEGF) has been shown to stimulate endothelial nitric oxide synthase (eNOS) in uterine artery endothelial cells (UAECs) derived from pregnant ewes to a greater extent than those from non-pregnant ewes in a manner not fully explained by changes in the phosphorylation of eNOS. In this study, we used Fura-2 Ca2+ imaging and arginine-to-citrulline conversion eNOS activity assays to assess the importance of VEGF-stimulated Ca2+ responses in pregnancy-related changes in NO production in UAEC. In this study, we show that pregnancy-induced changes in VEGF-stimulated Ca2+ responses could account in part for the greater capacity of VEGF to stimulate eNOS in UAECs from pregnant versus non-pregnant animals. VEGF-stimulated Ca2+ responses in UAECs from pregnant and non-pregnant animals were mediated through VEGF receptor 2 and were detected in roughly 15% of all cells. There were no pregnancyspecific differences in area under the curve or peak height. UAECs from pregnant animals were more consistent in the time to response initiation, had a larger component of extracellular Ca2+ entry, and were more sensitive to a submaximal dose of VEGF. In UAECs from pregnant and non-pregnant animals Ca2+ responses and eNOS activation were sensitive to the phospholipase C/inositol 1,4,5-trisphosphate pathway inhibitors 2-aminoethoxydiphenylborane and U73122. Thus, changes in VEGF-stimulated [Ca2 + ]i are necessary for eNOS activation in UAECs, and pregnancy-induced changes in Ca2+ responses could also in part explain the pregnancy-specific adaptive increase in eNOS activity in UAECs. [ABSTRACT FROM AUTHOR]

Complex regulatory processes alter the activity of endothelial nitric oxide synthase ( eNOS) leading to nitric oxide (NO) production by endothelial cells under various physiological states. These complex processes require specific subcellular eNOS partitioning between plasma membrane caveolar domains and non-caveolar compartments., Translocation of eNOS from the plasma membrane to intracellular compartments is important for eNOS activation and subsequent NO biosynthesis. We present data reviewing and interpreting information regarding: (i) the coupling of endothelial plasma membrane receptor systems in the caveolar structure relative to eNOS trafficking; (ii) how eNOS trafficking relates to specific protein-protein interactions for inactivation and activation of eNOS; and (iii) how these complex mechanisms confer specific subcellular location relative to eNOS multisite phosphorylation and signalling., Dysfunction in the regulation of eNOS activation may contribute to several disease states, in particular gestational endothelial abnormalities (pre-eclampsia, gestational diabetes etc.), that have life-long deleterious health consequences that predispose the offspring to develop hypertensive disease, Type 2 diabetes and adiposity. [ABSTRACT FROM AUTHOR]

Background: Pregnancy-induced utero-placental growth, angiogenic remodeling, and enhanced vasodilation are all partly regulated by estradiol-17β-mediated activation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. However, very little is known about the effects of alcohol on these maternal utero-placental vascular adaptations during pregnancy and its potential role in the pathogenesis of fetal alcohol spectrum disorders (FASDs). In this study, we hypothesized that in vitro chronic binge-like alcohol will decrease uterine arterial endothelial eNOS expression and alter its multisite phosphorylation activity state via disruption of AKT signaling. To study the direct effects of alcohol on uterine vascular adaptations, we further investigated the effects of alcohol on estradiol-17β-induced uterine angiogenesis in vitro. Methods: Uterine artery endothelial cells were isolated from pregnant ewes (gestational day 120 to 130; term = 147), fluorescence-activated cell sorted, validated, and maintained in culture to passage 4. To mimic maternal binge drinking patterns, cells were cultured in the absence or presence of a lower (LD) or higher dose (HD) of alcohol in a compensating sealed humidified chamber system equilibrated with aqueous alcohol for 3 hours on 3 consecutive days. Immunoblotting was performed to assess expression of NO system-associated proteins and eNOS multi-site phosphorylation. Following this treatment paradigm, control and binge alcohol-treated cells were passaged, grown for 2 days, and then treated with increasing concentrations of estradiol-17β (0.1, 1, 10, 100 nM) in the absence or presence of LD or HD alcohol to evaluate estradiol-17β-induced angiogenesis index using BrdU proliferation assay. Results: LD and HD binge-like alcohol decreased uterine arterial eNOS expression ( p = 0.009). eNOS multisite phosphorylation activation state was altered: P635eNOS was decreased ( p = 0.017), P1177eNOS was not altered, and P495eNOS exhibited an inverse U-shaped dose-dependent relationship with alcohol. LD and HD alcohol decreased the major eNOS-associated protein cav-1 ( p < 0.001). However, the commonly implicated AKT pathway did not correlate with eNOS posttranslational modifications. Assessment of uterine vascular adaptation via angiogenesis demonstrated that alcohol abrogated the dose-dependent proliferative effects of estradiol-17β and thus blunted angiogenesis. Conclusions: Thus, the maternal uterine vasculature during pregnancy may be vulnerable to chronic binge-like alcohol. Altered eNOS multisite phosphorylation also suggests that alcohol produces specific effects at the level of posttranslational modifications critical for pregnancy-induced uterine vascular adaptations. Finally, the alcohol and estradiol-17β data suggest a negative impact of alcohol on estrogen actions on the uterine vasculature. [ABSTRACT FROM AUTHOR]

Pregnancy is a time of greatly increased uterine blood flow to meet the needs of the growing fetus. Increased uterine blood flow is also observed in the follicular phase of the ovarian cycle. Simultaneous fura-2 and 4,5-diaminofluoresceine (DAF-2) imaging reveals that cells of the uterine artery endothelium (UA Endo) from follicular phase ewes produce marginally more nitric oxide (NO) in response to ATP than those from luteal phase. However, this is paralleled by changes in NO in response to ionomycin, suggesting this is solely due to higher levels of endothelial nitric oxide synthase (eNOS) protein in the follicular phase. In contrast, UA Endo from pregnant ewes (P-UA Endo) produces substantially more NO (4.62-fold initial maximum rate, 2.56-fold overall NO production) in response to ATP, beyond that attributed to eNOS levels alone (2.07-fold initial maximum rate, 1.93-fold overall with ionomycin). The ATP-stimulated intracellular free calcium concentration ([Ca2+]i) response in individual cells of P-UA Endo comprises an initial peak followed by transient [Ca2+]i bursts that are limited in the luteal phase, not altered in the follicular phase, but are sustained in pregnancy and observed in more cells. Thus pregnancy adaptation of UA Endo NO output occurs beyond the level of eNOS expression and likely through associated [Ca2+]i cell signaling changes. Preeclampsia is a condition of a lack of UA Endo adaptation and poor NO production/vasodilation and is associated with elevated placental VEGF165. While treatment of luteal NP-UA Endo and P-UA Endo with VEGF165 acutely stimulates a very modest [Ca2+]i and NO response, subsequent stimulation of the same vessel with ATP results in a blunted [Ca2+]i and an associated NO response, with P-UA Endo reverting to the response of luteal NP-UA Endo. This demonstrates the importance of adaptation of cell signaling over eNOS expression in pregnancy adaptation of uterine endothelial function and further implicates VEGF in the pathophysiology of preeclampsia. [ABSTRACT FROM AUTHOR]

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI[sub 2]) may be as much a consequence of cellular reprogramruing at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI[sub 2] biosynthetic pathway (cPLA[sub 2], COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that chronic high-altitude hypoxia during pregnancy is associated with the upregulation of endothelial nitric oxide synthase (eNOS) gene expression and messenger ribonucleic acid (mRNA) in ovine uterine arteries. Increase of eNOS and mRNA levels; Effect of chronic hypoxia on basal endothelial activity; Modulating effect of endothelial NO in vascular activity.

Obesity and diabetes increase hypertensive disorders of pregnancy (HDP) risk, thus preventive interventions are heavily studied. How pregestational prediabetes and related interventions impact HDP risk is less characterized. Therefore, we searched and reviewed the literature to assess the impact on HDP risk of prediabetes and varied interventions. We identified 297 citations related to pregnancy, prediabetes, and early pregnancy interventions. We also reviewed the references and citations of included articles. We included five studies assessing HDP outcomes in women with first trimester hemoglobin A1c in the prediabetes range (5.7–6.4%). One prospective observational study demonstrated first trimester hemoglobin A1c (5.9–6.4%) is associated with increased HDP risk, while another prospective observational study and one retrospective observational study had similar trends without statistical significance. A small and underpowered randomized controlled trial demonstrated initiating gestational diabetes mellitus treatment (i.e., diet, monitoring, ± insulin) in response to first trimester hemoglobin A1c (5.7–6.4%) did not statistically reduce HDP compared with standard care. One retrospective observational study suggested metformin, when started early, may reduce HDP risk in patients with prediabetes. Pregestational prediabetes appears to increase HDP risk. Interventions (i.e., metformin, diet/glucose monitoring, and/or exercise) to reduce HDP risk require additional study with long-term follow-up. [ABSTRACT FROM AUTHOR]

Determines whether the lack of a critical cardiac output or flow-dependent oxygen consumption during states of low cardiac output in late gestation can be reproduced in nonpregnant sheep treated with estrogen. Oxygen transport characteristics of adult nonpregnant sheep during reduction of cardiac output; Increase in systemic tissue blood flow and oxygen consumption.

Studies how the acute administration of 17beta-estradiol leads to increase cardiac output, oxygen delivery, oxygen consumption and increased cardiac output in the nonpregnant sheep. Discussion on critical cardiac output; Materials and methods; Discussion of results.

All patients were intended to take statins throughout ACCORD-Lipid. Methods ACCORD-Lipid compared fenofibrate versus placebo in statin-treated patients with type 2 diabetes.[5] ACCORD-Lipid principally used simvastatin. [Extracted from the article]

Abstract Enhancement of vasodilation of uterine arteries during pregnancy occurs through increased connexin (Cx)43 gap junction (GJ) communication supporting more frequent and sustained Ca2+ 'bursts'. Such adaptation is lacking in subjects with preeclampsia (PE). Here we show TNF-alpha, commonly increased in PE subjects, inhibits Cx43 function and Ca2+ bursts in pregnancy-derived ovine uterine artery endothelial cells (P-UAEC) via Src and MEK/ERK phosphorylation of Cx43, and this can be reversed by PP2 or U0126. Of relevance to humans: (1) the nutraceutical Src antagonist t10, c12 CLA also recovers Ca2+ bursting in P-UAEC. (2) TNF-alpha can reduce and PP2 rescue Ca2+ bursting and NO output in human umbilical vein endothelium (HUV Endo) preparations. (3) Treatment of HUV Endo from PE subjects with PP2 alone can rescue bursting and NO output. We conclude TNF-alpha acts via Src more than MEK/ERK to inhibit GJ Cx43 function in PE subjects, and CLA may offer a potential therapy. Highlights • Pregnancy is associated with enhanced Ca2+ signaling in UA endothelial cells, mediated by enhanced Cx43 connectivity. • We now show TNF-alpha inhibits Cx43 function and so periodic Ca2+ bursts in pregnancy-derived UA endothelial cells. • TNF acts via Src to inhibit Cx43 by causing phosphorylation directly at positions 265 and indirectly at position 279/282. • TNF action is reversed by the Src inhibitor PP2 or the nutraceutical Src antagonist t10,c12 CLA or MEK/ERK inhibitor U0126. • In umbilical vessels from preeclamptic subjects, Src inhibitors promotes rescue of otherwise impaired Ca2+ and NO responses. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a common cause of maternal morbidity, characterized by impaired trophoblast invasion and spiral artery transformation resulting in progressive uteroplacental hypoxia. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation, and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to PE. Here we show that LIN28B is expressed ~1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in primary human trophoblast cultures. The expression of LIN28B mRNA and protein levels are significantly reduced in gestational age-matched preeclamptic vs. normal placentas, whereas LIN28A expression is not different. First trimester human placental sections displayed stronger LIN28B immunoreactivity in extravillous (invasive) cytotrophoblasts and syncytial sprouts vs. villous trophoblasts. LIN28B overexpression increased HTR8 cell proliferation, migration, and invasion, whereas LIN28B knockdown in JEG3 cells reduced cell proliferation. Moreover, LIN28B knockdown in JEG3 cells suppressed syncytin 1 (SYN-1), apelin receptor early endogenous ligand (ELABELA), and the chromosome 19 microRNA cluster, and increased mRNA expression of ITGß4 and TNF-a. Incubation of BeWo and JEG3 cells under hypoxia significantly decreased expression of LIN28B and LIN28A, SYN-1, and ELABELA, whereas TNF-a is increased. These results provide the first evidence that LIN28B is the predominant paralog in human placenta and that decreased LIN28B may play a role in PE by reducing trophoblast invasion and syncytialization, and by promoting inflammation. [ABSTRACT FROM AUTHOR]

Statins are widely prescribed, yet statin muscle pain limits their use, leading to increased cardiovascular risk. No validated therapy for statin muscle pain exists. The goal of the study was to assess whether metformin was associated with reduced muscle pain. A secondary analysis of data from the ACCORD trial was performed. An ACCORD sub‐study assessed patients for muscle cramps and leg/calve pain while walking, typical non‐severe statin muscle pain symptoms. We compared muscle pain between patients using a statin (n = 445) or both a statin and metformin (n = 869) at baseline. Overall patient characteristics were balanced between groups. Unadjusted analysis showed fewer reports of muscle cramps (35%) and leg/calve pain while walking (40%) with statins and metformin compared to statin only (muscle cramps, 42%; leg/calve pain while walking, 47%). Multivariable regression demonstrated a 22% odds reduction for muscle cramps (P = 0.049) and a 29% odds reduction for leg/calve pain while walking (P = 0.01). Metformin appears to reduce the risk of non‐severe statin muscle pain and additional research is needed to confirm the findings and assess metformin's impact on statin adherence and related cardiovascular outcomes. [ABSTRACT FROM AUTHOR]

Key points The catechol metabolites of 17β-oestradiol (E2β), 2-hydroxyoestradiol (2-OHE2) and 4-hydroxyoestradiol (4-OHE2), stimulate proliferation of pregnancy-derived ovine uterine artery endothelial cells (P-UAECs) through β-adrenoceptors (β-ARs) and independently of the classic oestrogen receptors (ERs)., Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2-OHE2- and 4-OHE2-induced P-UAEC proliferation, as well as proliferation induced by the parent hormone E2β and other β-AR signalling hormones (i.e. catecholamines)., Conversely, although 2-OHE2 and 4-OHE2 rapidly activate phosphatidylinositol 3-kinase (PI3K), its activation is not involved in catecholoestradiol-induced P-UAEC proliferation., We also show for the first time the signalling mechanisms involved in catecholoestradiol-induced P-UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2β- and catecholamine-induced proliferation., The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy., Abstract Previously we demonstrated that the biologically active metabolites of 17β-oestradiol, 2-hydroxyoestradiol (2-OHE2) and 4-hydroxyoestradiol (4-OHE2), stimulate pregnancy-specific proliferation of uterine artery endothelial cells derived from pregnant (P-UAECs), but not non-pregnant ewes. However, unlike 17β-oestradiol, which induces proliferation via oestrogen receptor-β (ER-β), the catecholoestradiols mediate P-UAEC proliferation via β-adrenoceptors (β-AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P-UAECs. P-UAECs were treated with 2-OHE2 and 4-OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24 h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)-AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist-induced P-UAEC proliferation. 2-OHE2 and 4-OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol-induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β-oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P-UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol-, 17β-oestradiol- and catecholamine-induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy. [ABSTRACT FROM AUTHOR]

Gestational iron deficiency (ID) can alter developmental programming through impaired nephron endowment, leading to adult hypertension, but nephrogenesis is unstudied. Iron status and renal development during dietary-induced gestational ID (<6 mg Fe kg-1 diet from Gestational Day 2 to Postnatal Day (PND) 7) were compared with control rats (198 mg Fe kg-1 diet). On PND2-PND10, PND15, PND30 and PND45, blood and tissue iron status were assessed. Nephrogenic zone maturation (PND2-PND10), radial glomerular counts (RGCs), glomerular size density and total planar surface area (PND15 and PND30) were also assessed. Blood pressure (BP) was measured in offspring. ID rats were smaller, exhibiting lower erythrocyte and tissue iron than control rats (PND2-PND10), but these parameters returned to control values by PND30-PND45. Relative kidney iron (µg g-1 wet weight) at PND2-PND10 was directly related to transport iron measures. In ID rats, the maturation of the active nephrogenic zone was later than control. RGCs, glomerular size, glomerular density, and glomerular planar surface area were lower than control at PND15, but returned to control by PND30. After weaning, the kidney weight/rat weight ratio (mg g-1) was heavier in ID than control rats. BP readings at PND45 were lower in ID than control rats. Altered kidney maturation and renal adaptations may contribute to glomerular size, early hyperfiltration and long-term renal function. [ABSTRACT FROM AUTHOR]

Placental angiogenesis is dramatically increased during pregnancy in association with the elevated placental blood flows to support the rapidly growing fetus. 2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) is an environmental toxicant and a ligand of aryl hydrocarbon receptor (AhR). Herein, we investigated the effects of TCDD on proliferation, migration, and viability of fetoplacental endothelial cells in response to a complete growth medium which contained serum and growth supplement using human umbilical cord vein (HUVECs) and artery (HUAECs) cells as models. We found that TCDD dose- and time-dependently inhibited ( p < 0.05) proliferation of HUVECs and HUAECs. Treatment with TCDD at 10 nM for 6 days inhibited ( p < 0.05) migration (by ∼30%) of HUAECs, but not HUVECs. TCDD at 10 nM also decreased ( p < 0.05) viability of HUVECs and HUAECs. Interestingly, specific AhR siRNA blocked ( p < 0.05) the TCDD-inhibited cellular responses in HUAECs, but not HUVECs. Nonetheless, TCDD at 10 nM neither affected the cell cycle progression, nor did it induce cell apoptosis in HUVECs and HUAECs. In addition, TCDD at 10 nM also did not alter activation of ERK1/2 and AKT1 in HUVECs and HUAECs. Collectively, TCDD suppresses proliferation and/or migration (two key steps of angiogenesis) of HUVECs and HUAECs independent and dependent of AhR, respectively. These data suggest that TCDD inhibited growth of HUVECs and HUAECs via decreasing cell viability. Thus, TCDD may inhibit fetoplacental angiogenesis, leading to negative pregnancy outcomes. [ABSTRACT FROM AUTHOR]

Normal pregnancy requires increased uterine endothelial cell driven vasodilation that is related to increases in sustained Ca2+ signaling via increased connexin 43 (Cx43) gap junction function. Preeclampsia, a hypertensive disorder of pregnancy associated with endothelial dysfunction, is also linked with down regulation of Ca2+ driven vasodilator production and increased levels of vascular endothelial growth factor (VEGF). Cx43 function can be acutely down-regulated by phosphorylation of multiple inhibitory residues and VEGF is known to promote phosphorylation of Cx43. Herein, we show that VEGF-165 promotes Cx43 phosphorylation at Ser-279/282 and Tyr-265 residues and blocks pregnancy-adapted Ca2+ signaling in ovine uterine artery endothelial cells (UAEC). Pharmacological Src and ERK kinase pathway inhibitors (PP2 and U0126) reverse these phosphorylations and rescue Ca2+ signaling. We also report a nutraceutical Src inhibitor, t10,c12 conjugated linoleic acid (10,12 CLA), rescues Ca2+ signaling in UAEC and therefore may have therapeutic potential for preeclampsia. [ABSTRACT FROM AUTHOR]

Estradiol 17β (E2β) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2β, 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME2), and 4-methoxyestradiol (4ME2) synthesized sequentially by cytochrome P450, family 1, subfamily A (CYP1A1) and B (CYP1B1), polypeptide 1, and catechol-O-methyltransferase (COMT) on ovarian cancer. Herein, we examined the expression of CYP1A1, CYP1B1, COMT, and estrogen receptor α (ERα) and β (ERβ) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2β, 2OHE2, 4OHE2, 2ME2, and 4ME2 in cell proliferation, and their interactive effects with AA on ovarian cells. We found the expression of CYP1A1, CYP1B1, COMT, ERα, and ERβ in most cell lines tested. Treating cells with physiological concentrations of E2β and its metabolites promoted (13%-42% of the control) IOSE-385 and OVCAR-3 proliferation. The ER blockade inhibited IOSE-385 (∼76%) and OVCAR-3 (∼87%) proliferative response to E2β but not to its metabolites. The ERα blockade inhibited (∼85%) E2β-stimulated OVCAR-3 proliferation, whereas ERβ blockade attenuated (∼83%) E2β-stimulated IOSE-385 proliferation. The AA at ≥250 μmol/L completely inhibited serum-stimulated cell proliferation in all cell lines tested; however, such inhibition in IOSE-385, OVCAR-3, and OVCA-432 was partially (∼10%-20%) countered by E2β and its metabolites. Thus, our findings indicate that E2β and its metabolites promote cell proliferation and antagonize the AA-suppressed cell proliferation in a subset of ovarian cancer cells, suggesting that blocking the actions of E2β and its metabolites may enhance AA’s antiovarian cancer activity. [ABSTRACT FROM PUBLISHER]

Abstract: Objectives: Uterine vascular resistance (UVR) is the ratio of systemic mean arterial pressure to mean uterine blood flow and is sensitive to changes in small arteries and arterioles. However, it provides little or no insight into changes in large, conduit arteries. Fluctuations in estrogen (E2) and progesterone (P4) levels during the ovarian cycle are thought to cause uterine resistance artery vasodilation; the effects on large arteries are unknown. Herein, our objective was to use the uterine vascular impedance, which is sensitive to changes in small and large arteries, to determine the effects of the ovarian cycle and pregnancy on the entire uterine vasculature. Study design: Uterine vascular perfusion pressure and flow rate were recorded simultaneously in anesthetized sheep in the nonpregnant (NP) luteal (NP-L, n =6) and follicular (NP-F, n =7) phases and in late pregnancy (CP, n =10). Impedance and metrics of impedance (input impedance Z 0, index of wave reflection R W, characteristic impedance Z C) were calculated. E2 and P4 levels were measured from jugular vein blood samples. Finally, from pressure-diameter tests post-mortem, large uterine artery circumferential elastic modulus (E Circ) was measured. Significant differences were evaluated by two-way ANOVA or Student''s t-test. Results: As expected, E2:P4 was higher in the NP-F group compared to the NP-L group (p <0.05). Also as expected, UVR and Z 0 decreased in the follicular phase compared to the luteal (p <0.05), but R W, Z C, and E Circ were unaltered. Pregnancy not only substantially decreased UVR (and Z 0) (p <0.00001) but also decreased Z C (p <0.001), R W (p <0.0001), E Circ (p <0.01), and pulse wave velocity (p <0.0001). Conclusions: The E2:P4 ratio mediates resistance artery vasodilatation in nonpregnant states, but has no effect on conduit artery size or stiffness. In contrast, pregnancy causes dramatic vasodilation and remodeling, including substantial reductions in conduit artery stiffness and increases in conduit artery size, which affect pulsatile uterine hemodynamics. [Copyright &y& Elsevier]

Covalent adduction of a NO moiety to cysteines ( S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10-30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro- L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two-dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, nine were novel SNO-proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation. J. Cell. Physiol. 227: 146-159, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The steroid hormone estrogen and its classical estrogen receptors (ERs), ER-α and ER-β, have been shown to be partly responsible for the short- and long-term uterine endothelial adaptations during pregnancy. The ER-subtype molecular and structural differences coupled with the differential effects of estrogen in target cells and tissues suggest a substantial functional heterogeneity of the ERs in estrogen signaling. In this review we discuss (1) the role of estrogen and ERs in cardiovascular adaptations during pregnancy, (2) in vivo and in vitro expression of ERs in uterine artery endothelium during the ovarian cycle and pregnancy, contrasting reproductive and nonreproductive arterial endothelia, (3) the structural basis for functional diversity of the ERs and estrogen subtype selectivity, (4) the role of estrogen and ERs on genomic responses of uterine artery endothelial cells, and (5) the role of estrogen and ERs on nongenomic responses in uterine artery endothelia. These topics integrate current knowledge of this very rapidly expanding scientific field with diverse interpretations and hypotheses regarding the estrogenic effects that are mediated by either or both ERs and their relationship with vasodilatory and angiogenic vascular adaptations required for modulating the dramatic physiological rises in uteroplacental perfusion observed during normal pregnancy. [ABSTRACT FROM AUTHOR]

The estrogen metabolite 2-methoxyestradiol (2-ME2) is one of the most potent antiangiogenic and proapoptotic endogenous steroids. Herein, we investigate the effects of 2-ME2 on angiogenesis of cultured primary ovine uterine artery endothelial cells (UAECs) from nonpregnant follicular (F-UAECs), nonpregnant luteal (L-UAECs), and pregnant ewes (P-UAECs). Uterine artery endothelial cells were treated with vehicle control, 10−8 mol/L 17β-estradiol (17βE2), or 10−9 to 10−6 mol/L 2-ME2. Angiogenesis, apotosis, and cell morphology were assessed by capillary tube formation, flowcytometry, and immunohistochemistry. 17βE2 stimulated while 10−6 mol/L 2-ME2 inhibited capillary tube formation in F-UAECs (P < .05). The inhibitory effects of 2-ME2 on angiogenesis were minimal in L-UAECs and were absent in P-UAECs when compared to controls. 10−6 mol/L 2-ME2 increased apoptosis and inhibited microtubular structure equally in pregnant and nonpregnant UAECs when compared to control or 17βE2 treatments. Thus, 2-ME2 inhibit capillary tube formation in F-UAECs while L-UAECs and P-UAECs are relatively unresponsive to the inhibitory effects of 2ME2 indicating that the pregnancy phenotypic state of the UAECs may modulate the action of 2-ME2 on capillary angiogenesis. [ABSTRACT FROM PUBLISHER]

Abstract: Currently, no single marker is sensitive and specific enough to be considered a reliable biomarker for prenatal alcohol exposure. To identify a proteomic signature profile for maternal alcohol consumption, we carried out high-throughput proteomics on maternal endothelial caveolae exposed to moderate binge-like alcohol conditions. In these specialized lipid-ordered microdomains that contain a rich assembly of proteins, we demonstrate that moderate binge-like alcohol resulted in a distinctive maternal caveolar proteomic signature with important proteins being dramatically decreased/knocked out in the alcoholic profile. These proteins span from histones and basic structural proteins like α tubulin to proteins involved in trafficking, deubiquitination, cell signaling, and cell–cell adhesion. The profile also suggests an important role for the mother and the uteroplacental compartment in the pathogenesis of fetal alcohol spectrum disorders (FASD). These data demonstrate that the caveolar proteomic signature created by alcohol shows a promising direction for early detection of FASD. [Copyright &y& Elsevier]

The follicutar phase of the ovine ovarian cycle demonstrates parallel increases in ovarian estrogens and uterine blood flow (UBF). Although estrogen and nitric oxide contribute to the rise in UBF, the signaling pathway remains unclear. We examined the relationship between the rise in UBF during the ovarian cycle of nonpregnant sheep and changes in the uterine vascular cGMP-dependent pathway and large-conductance Ca2+-activated K+ channels (BKCa). Nonpregnant ewes (n = 19) were synchronized to either follicular or luteal phase using a vaginal progesterone-releasing device (CIDR), followed by intramuscular PGF2α, CIDR removal, and treatment with pregnant mare serum gonadotropin. UBF was measured with flow probes before tissue collection, and second-generation uterine artery segments were collected from nine follicular and seven luteal phase ewes. The poreforming α-and regulatory β-subunits that constitute the BKCa, soluble guanylyl cyclase (sUC), and cGMP-dependent protein kinase G (cPKG) isoforms (cPKG1α and cPKG1β) were measured by Western analysis and cGMP levels by RIA. BKCa subunits were localized by immunohistochemistry. UBF rose >3-fold (P < 0.04) in follicular phase ewes, paralleling a 2.3-fold rise in smooth muscle cGMP and 32% increase in cPKG1α (P < 0.05). sGC, cPKG1β, and the BKCa α-subunit were unchanged. Notably, expression of β1- and β2-regulatory subunits rose 51 and 79% (P ⩽ 0.05), respectively. Increases in endogenous ovarian estrogens in follicular-phase ewes result in increases in UBF associated with upregulation of the cGMP- and cPKG-dependent pathway and increased vascular BKCa β/α-subunit stoichiometry, suggesting enhanced BKCa activation contributes to the follicular phase rise in UBF. [ABSTRACT FROM AUTHOR]

Objective. Determine the effect of endogenous estrogen versus estrogen replacement therapy (ERT) on bladder blood flow (BBF) and estrogen receptors (ERs). Methods. BBF was determined with radiolabeled microspheres in luteal, follicular, pregnant, oophorectomized (Ovx) sheep, and Ovx sheep with ERT. Estrogen receptors (ERα, ERβ) were quantified using Western blot analysis. Results. Compared to luteal and follicular ewes, BBF was reduced in pregnancy and following oophorectomy. Estrogen replacement therapy in Ovx sheep restored BBF to luteal levels. Estrogen receptor α predominated, whereas ERβ was not detectable. Estrogen receptor-α levels were unaffected by the ovarian cycle and increased in pregnancy, as well as in Ovx sheep with and without chronic ERT. Conclusion. The combination of diminished BBF and elevated ERα levels in both pregnant and Ovx sheep suggests an inverse relationship between BBF and ERα in the bladder. Although chronic ERT in Ovx sheep restored BBF, it did not restore ERα back to luteal levels. [ABSTRACT FROM AUTHOR]

Objective: Ethanol exposure during pregnancy may result in fetal alcohol syndrome (FAS). The mechanism by which this occurs is unknown. Recent studies in several organ systems, including the placenta, suggest that oxidative stress is involved. In this study we investigated the presence and levels of three oxidative stress markers in placental villous tissue exposed to ethanol. Methods: Villous tissues from normal placentas were perfused with Dulbeco’s modified Eagle’s medium (DMEM) with HEPES buffer, sodium bicarbonate, and glucose at pH 7.4. After stabilization, 100 mM ethanol was added to the perfusate. After 2 hours of perfusion, the tissue was removed, fixed and stained for nitrotyrosine, 4-hydroxy-2-nonenal (4HNE) and 8-hydroxyguanosine (8-OHDG). Staining within the trophoblasts was quantified with densitometry. Results: Nitrotyrosine and 4HNE immunostaining was seen in the trophoblasts. 4HNE was also seen in the stroma. In contrast, 8-OHDG was seen only in the stroma and endothelial cells in the fetal circulation. Ethanol exposure significantly increased nitrotyrosine levels in the trophoblasts beyond levels in the control tissue. Nitrotyrosine and 8-OHDG levels were also increased in stroma. Conclusion: Within the placental villi, markers of oxidative stress are present in the trophoblasts and stroma after a short period of ethanol exposure. There is an increase in oxidative stress, primarily involving the nitric oxide pathway, in the trophoblasts as well as DNA damage in the stroma. Lipid peroxidation is not acutely changed in our 2-hour exposure window. [Copyright &y& Elsevier]

Nitric oxide (NO) production has been shown to increase uterine blood flow and be elevated in ewes carrying multiple fetuses during late gestation. Vascular endothelial growth factor (VEGF) has been reported to increase eNOS expression and NO production in endothelial cell cultures. As angiogenesis and vasodilatation of the uterine and placental vascular beds are important at all stages of pregnancy, it is important to understand how VEGF and NO change throughout gestation in circulation. Therefore the objectives of the current study were to evaluate the systemic levels of VEGF and NO metabolite (NOx) throughout ovine gestation and to determine if there was an effect of sheep carrying singletons versus multiple fetuses. NOx and VEGF concentrations were analysed in systemic blood from pregnant ewes starting on day 27 of pregnancy and at multiple intermittent intervals throughout pregnancy until term. Blood samples from non-pregnant and postpartum ewes were also analysed. NOx concentrations in maternal blood expressed a biphasic pattern with NOx concentrations increasing ( P< 0.05) over non-pregnant values on days 40–69 of gestation, returning to non-pregnant concentrations from days 70–100, and again increasing ( P< 0.05) until term. Postpartum NOx concentrations were similar to non-pregnant values. While ewes carrying multiple fetuses had increased ( P< 0.05) concentrations of NOx on days 60–69, there were no differences in NOx concentrations in ewes carrying singletons or multiples from day 70–99 of gestation. Starting on day 100 and continuing throughout the duration of pregnancy, ewes carrying multiple fetuses had increased ( P< 0.05) concentrations of NOx compared to ewes carrying singletons. Concentrations of VEGF showed a different pattern from NOx with VEGF decreasing ( P< 0.05) from day 20–69 of pregnancy compared to non-pregnant ewes. Concentrations of VEGF returned to non-pregnant levels by day 70 and remained constant throughout the duration of pregnancy. On days 20–39, ewes carrying singleton fetuses had an increased VEGF concentration ( P< 0.05), whereas ewes carrying multiple fetuses demonstrated elevated VEGF concentrations from day 90–109 of gestation. Concentrations from non-pregnant and postpartum ewes did not differ ( P> 0.1). While there was no effect of fetal number on circulating VEGF concentrations, circulating levels of NOx were substantially increased ( P< 0.05) in ewes carrying multiple fetuses, compared to ewes carrying singletons. The pattern of the rise in NOx in circulating plasma was not directly associated with changes in VEGF regardless of the number of fetuses present. However, circulating concentrations of NOx and VEGF appear to, respectively, follow patterns of uterine blood flow and angiogenesis of the uterus. An understanding of these circulatory patterns may have important implications for fetal size, birth weight and fetal/developmental origins of adult disease. [ABSTRACT FROM AUTHOR]

Pregnancy and the follicular phase are physiological states of elevated oestrogen levels and rises in uterine blood flow (UBF). The dramatic increase in utero-placental blood flow during gestation is required for normal fetal growth and development. Oestrogen exerts its vasodilatory effect by binding to its specific oestrogen receptors (ER) in target cells, resulting in increased expression and activity of endothelial nitric oxide synthase (eNOS) to relax vascular smooth muscle (VSM). However, the regulation of endothelial versus VSM ERα and ERβ expression in uterine arteries (UAs) during the ovarian cycle, pregnancy and with exogenous hormone replacement therapy (HRT) are currently unknown. ER mRNA and protein localization was determined by in situ hybridization (ISH) using35S-labelled riboprobes and immunohistochemistry (IHC), respectively. UA endothelial (UAendo), UA VSM, omental artery endothelium (OA endo), and OA VSM proteins were isolated and ERα and ERβ protein expression was determined by Western analysis. We observed by ISH and IHC that ERα and ERβ mRNA and protein were localized in both UAendo and UA VSM. Immunoblot data demonstrated ovarian hormone specific regulation of ERα and ERβ protein in UAendo and UA VSM. Compared to luteal phase sheep, both ERα and ERβ levels in UAendo were elevated in follicular phase sheep. Whereas ERβ was elevated by pregnancy in UAendo and UA VSM, ERα was not appreciably altered. eNOS was increased in UAendo from follicular and pregnant sheep. Ovariectomized ewes (OVEX) had substantially reduced UAendo ERβ, but not UAendo ERα or OAendo ERα and ERβ. In contrast, OVEX increased UA VSM ERα and ERβ and decreased OA VSM ERα and ERβ. Treatment with oestradiol-17β (E2β), but not progesterone or their combination, increased UAendo ERα levels. The reduced ERβ in UAendo from OVEX ewes was reversed by E2β and progesterone treatment. While ERα and eNOS were not elevated in any other reproductive or non-reproductive endothelia tested, ERβ was augmented by pregnancy in uterine, mammary, placenta, and coronary artery endothelia. ERα and ERβ mRNA and protein are expressed in UA endothelium with expression levels depending on the endocrine status of the animal, indicating UA endothelium is a target for oestrogen action in vivo, and that the two receptors appear to be differentially regulated in a spatial and temporal fashion with regard to the reproductive status or HRT. [ABSTRACT FROM AUTHOR]

During normal pregnancy, elevated angiotensin II (Ang II) concentrations in the maternal and fetal circulations are associated with dramatic increases in placental angiogenesis and blood flow. Much is known about a local renin–angiotensin system within the uteroplacental vasculature. However, the roles of Ang II in regulating fetoplacental vascular functions are less well defined. In the fetal placenta, the overall in vivo vasoconstrictor responses of the blood vessels to Ang II infusion is thought to be less than that in its maternal counterpart, even though infused Ang II induces vasoconstriction. Recent data from our laboratories suggest that Ang II stimulates cell proliferation and increases endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) in ovine fetoplacental artery endothelial cells. These data imply that elevations of the known vasoconstrictor Ang II in the fetal circulation may indeed play a role in the marked increases in fetoplacental angiogenesis and that Ang II-elevated endothelial NO production may partly attenuate Ang II-induced vasoconstriction on vascular smooth muscle. Together with both of these processes, the high levels of Ang II in the fetal circulation may serve to modulate overall fetoplacental vascular resistance. In this article, we review currently available data on the expression of Ang II receptors in the ovine fetal placenta with particular emphasis on the effects of Ang II on ovine fetoplacental endothelium. The potential cellular mechanisms underlying the regulation of Ang II on endothelial growth and vasodilator production are discussed. [ABSTRACT FROM AUTHOR]