Placental eNOS Research Articles

Physalis angulata leaf extract ameliorates L-NG-nitroarginine methyl ester (L-NAME)-induced preeclampsia symptoms in rats through improved endothelial progenitor cells and endothelial cells due to reduced antiangiogenic factor and oxidative stress

Background: This study aimed to determine the ameliorative effects of Physalis angulata leaf extract on L-NG-nitroarginine methyl ester (L-NAME)-induced preeclampsia symptoms in rats. Methods: Phytochemical analysis of the extract was performed with liquid chromatography-high resolution mass spectrometry (LC-HRMS). Pregnant Wistar rats were randomly divided into five groups (n=6). Preeclampsia rats were injected with L-NAME on gestation days 9 to 18 (G9–G18), while sham rats were injected with the same vehicle volume. Three groups of preeclampsia rats were orally supplemented with the extract at doses of 500, 1500, and 2500 mg/kg body weight/day on G12–G18. The tail-cuff method measured blood pressures. Spectrophotometry determined urine protein levels. Serum soluble fms-like tyrosine kinase (sFlt)-1 levels were evaluated using an enzyme-linked immunosorbent assay (ELISA). Serum and placental malondialdehyde (MDA) levels, superoxide dismutase (SOD) activities, and nitric oxide (NO) levels were measured by colorimetry. Immunohistochemistry was used to determine tail artery endothelial nitric oxide synthase (eNOS), placental eNOS, and placental hypoxia-inducible factor (HIF)-1α expressions. Circulating angiogenic cells (CACs) and endothelial colony-forming cells (ECFCs) were counted using flow cytometry. Results: L-NAME injection increased blood pressures, 24-h total urine protein level, serum sFlt-1 level, serum and placental MDA levels, percentages of CACs and ECFCs, and placental HIF-1α expression. It also decreased serum and placental SOD activities, serum NO level, tail artery and placental eNOS expressions compared to the sham group. Physalis angulata leaf extract administration lowered blood pressures, urine protein level, sFlt-1 level, MDA levels, percentages of CACs and ECFCs, and placental HIF-1α expression. The extract increased SOD activities, NO level, tail artery and placental eNOS expressions compared to the preeclampsia group. Conclusions: Physalis angulata leaf extract reduces antiangiogenic factor and oxidative stress. It also enhances eNOS/NO signaling. Thus, it improves EPC and endothelial cell function and reverses L-NAME-induced hypertension and proteinuria in preeclampsia rats.

Expression of Endothelin-1 and Endothelial Nitric Oxide Synthase in Normal and Preeclamptic Placentae.

To investigate the expression of endothelin-1 (ET-1) and endothelial nitric oxide (NO) synthase (eNOS) in normal and preeclamptic (PE) placentae. The present cross-sectional analytical study was performed in normal and PE primigravidae (n = 10 in each group) who were admitted to the North Okkalapa General and Teaching Hospital from February 2019 to February 2020. Serum samples were collected immediately before delivery, and placental tissues were collected immediately after emergency or elective cesarean section. The expression of placental eNOS was measured by western blot, and the levels of ET-1 in placental tissue homogenates and in the serum were measured by enzyme-linked immunosorbent assay (ELISA). The PE group had significantly higher serum levels of ET-1 (median: 116.56 pg/mL; IQR: 89.14-159.62 pg/mL) than the normal group (median: 60.02 pg/mL; IQR: 50.89-94.37 pg/mL) (p < 0.05). However, statistically significant differences were not observed in the levels of ET-1 in placental tissue homogenates between normal and PE placentae (median: 0.007 pg/µg of total protein; IQR: 0.002-0.0123 pg/µg of total protein; and median: 0.005 pg/µg of total protein; IQR: 0.003-0.016 pg/µg of total protein respectively). The median and IQR values of relative placental eNOS expression were significantly higher in the PE group than in the normal group (p < 0.05). The serum levels of ET-1 level were not significantly correlated with placental ET-1 expression, and neither there was a significant correlation between placental ET-1 and eNOS expression in any of the groups. The serum levels of ET-1 were significantly higher in PE pregnant women compared with normal pregnant women, while the ET-1 levels of placental tissue homogenates were not significantly different. Serum ET-1 rather than placental ET-1 might play a major role in the pathogenesis of PE.

Modification of endothelial nitric oxide synthase by 4-oxo-2(E)-nonenal(ONE) in preeclamptic placentas

Preeclampsia (PE) is a leading cause of pregnancy complications, affecting 3–7% of pregnant women worldwide. The pathophysiology of preeclampsia involves a redox imbalance, oxidative stress and a reduced nitric oxide (NO) bioavailability. The molecular and cellular mechanisms leading to the dysfunction of the placental endothelial NO synthase (eNOS) are not clarified. This study was designed to investigate whether aldehydes generated by lipid peroxidation products (LPP), may contribute to placental eNOS dysfunction in PE. The analysis of placentas from PE-affected patients and normal pregnancies, showed a significant increase in protein carbonyl content, indicative of oxidative stress-induced protein modification, as shown by the accumulation of acrolein, 4-hydroxynonenal (HNE), and 4-oxo-2(E)-nonenal (ONE) adducts in PE placentas. In contrast, the levels of these LPP-adducts were low in placentas from normal pregnancies. Immunofluorescence and confocal experiments pointed out a colocalization of eNOS with ONE-Lys adducts, whereas eNOS was not modified in normal placentas. LC-MS/MS analysis of recombinant eNOS preincubated with ONE, allowed to identify several ONE-modified Lys-containing peptides, confirming that eNOS may undergo post-translational modification by LPP. The preincubation of HTR-8/SVneo human trophoblasts (HTR8) with ONE, resulted in ONE-Lys modification of eNOS and a reduced generation of NO. ONE inhibited the migration of HTR8 trophoblasts in the wound closure model, and this was partly restored by the NO donor, NOC-18, which confirmed the important role of NO in the invasive potential of trophoblasts. In conclusion, placental eNOS is modified by ONE in PE placentas, which emphasizes the sensitivity of this protein to oxidative stress in the disturbed redox environment of preeclamptic pregnancies.

AEROBIC EXERCISE AND AMELIORATED ENDOTHELIAL FUNCTION IN EXPERIMENTAL PREECLAMPSIA IN RAT MODEL p

Background: Preeclampsia is a life threatening state. Aerobic exercise has a beneficial effect on vascular endothelium function. Adropin is a newly discovered peptide that can increase by exercise and decreased in endothelial dysfunction. Objectives: Detecting the effect of practicing exercise on preeclampsia pathophysiology, and role of adropin. Patients and Methods: Twenty four adult female albino wistar rats were divided into 3 equal groups: group I: Sham-operated Time-mated pregnant rats (Sham), group II: Reduced uterine perfusion pressure pregnant rats (RUPP); placental ischemia was induced at gestation day (GD) 14 to mimic preeclampsia, and group III: exercise treated reduced uterine perfusion pressure pregnant rats (Ex-RUPP); swimming started from GD1 till the end of pregnancy period, 1h/day, 6 days/week added to preeclampsia induction. Maternal arterial blood pressure, micro-albuminuria, serum urea, creatinine and calculated glomerular filtration rate (GFR), plasma insulin, glucose and calculated HOMA-IR and placental eNOS, adropin, soluble fms–like tyrosine kinase 1 (sFlt-1), malondialdehyde (MDA), total antioxidant capacity (TAC), and TNF-α were measured. Placental and fetal tissues were weighed and placenta was histo-pathologically assessed. Results: Aerobic exercise during pregnancy caused significant reductions in all blood pressure values at GD17 and 19, serum urea, creatinine, micro-albuminuria, placental anti-angiogenic sflt-1, MDA, TNF-α, plasma glucose, insulin and HOMA-IR. In addition, exercise significantly increased the calculated creatinine clearance, placental eNOS and placental adropin, placental weight, fetal weight and number. Apoptotic trophoblasts and neutrophil aggregates were absent. Adropin was significantly positive correlated with eNOS together with a significant negative correlation to mean arterial blood pressure at GD19, sflt-1, MDA, insulin and HOMA-IR. Conclusion: Aerobic exercise, through increasing adropin level, was able to restore the angiogenic balance, and to decrease the oxidative stress, and inflammatory and insulin resistance states, thus improving endothelial function. This was reflected in improvement in the renal functions, blood pressure reduction with better maternal and fetal outcome.

Adhesive Capsulitis and Its Predictors in Saudi Diabetic Patients

Background: Preeclampsia is a life threatening state. Aerobic exercise has a beneficial effect on vascular endothelium function. Adropin is a newly discovered peptide that can increase by exercise and decreased in endothelial dysfunction. Objectives: Detecting the effect of practicing exercise on preeclampsia pathophysiology, and role of adropin. Patients and Methods: Twenty four adult female albino wistar rats were divided into 3 equal groups: group I: Sham-operated Time-mated pregnant rats (Sham), group II: Reduced uterine perfusion pressure pregnant rats (RUPP); placental ischemia was induced at gestation day (GD) 14 to mimic preeclampsia, and group III: exercise treated reduced uterine perfusion pressure pregnant rats (Ex-RUPP); swimming started from GD1 till the end of pregnancy period, 1h/day, 6 days/week added to preeclampsia induction. Maternal arterial blood pressure, micro-albuminuria, serum urea, creatinine and calculated glomerular filtration rate (GFR), plasma insulin, glucose and calculated HOMA-IR and placental eNOS, adropin, soluble fms–like tyrosine kinase 1 (sFlt-1), malondialdehyde (MDA), total antioxidant capacity (TAC), and TNF-α were measured. Placental and fetal tissues were weighed and placenta was histo-pathologically assessed. Results: Aerobic exercise during pregnancy caused significant reductions in all blood pressure values at GD17 and 19, serum urea, creatinine, micro-albuminuria, placental anti-angiogenic sflt-1, MDA, TNF-α, plasma glucose, insulin and HOMA-IR. In addition, exercise significantly increased the calculated creatinine clearance, placental eNOS and placental adropin, placental weight, fetal weight and number. Apoptotic trophoblasts and neutrophil aggregates were absent. Adropin was significantly positive correlated with eNOS together with a significant negative correlation to mean arterial blood pressure at GD19, sflt-1, MDA, insulin and HOMA-IR. Conclusion: Aerobic exercise, through increasing adropin level, was able to restore the angiogenic balance, and to decrease the oxidative stress, and inflammatory and insulin resistance states, thus improving endothelial function. This was reflected in improvement in the renal functions, blood pressure reduction with better maternal and fetal outcome.

Placental expression of endothelial and inducible nitric oxide synthase and NO metabolism in gestational hypertension: a case–control study

Objective: Hypertension is one of the most common medical disorders in pregnancy and a role of nitric oxide (NO) metabolism has been described. Thus, the present work aimed at determining placental gene expression of eNOS and iNOS, to measure NO and ONOO− levels in patients with gestational hypertension (GH). Methods: Fifteen patients with GH and 15 healthy pregnant controls were enrolled in the study. Placental tissue was taken immediately after delivery and was stored at −80 °C until analysis. A piece of frozen tissue was homogenized in the appropriate buffer. Total RNA was extracted and was reverse transcribed to obtain complementary DNA that was used for real-time PCR for iNOS and eNOS expression, whereas NO and ONOO− production were measured by commercially available kits. Results: Placental eNOS and iNOS mRNA levels were significantly reduced in GH when compared to controls. NO and ONOO− production were both significantly higher in GH than controls. Conclusions: The reduced eNOS and iNOS gene expression in women with GH reinforces the hypothesis that the mechanisms involving NO pathways, may promote oxidative damage, by contributing to the reduced blood flow and increased resistance in the feto–maternal circulation and suggests the use of NO modulators as useful tools in GH management.

PP073. Gestational hypertension: A study on placental expression of endothelial and inducible nitric oxide synthase and no metabolism

Hypertension is one of the most common medical disorders in pregnancy and a major cause of maternal and perinatal morbidity and death. In primates adequate development of the embryo, and later of the fetus, depends on a successful hemomonochorial placentation. Nitric oxide (NO) a low molecular weight mediator, induces vasodilatation, inhibits platelet aggregation, and prevents the adhesion of platelets to endothelial cells. Till date, no data are available regarding gestational hypertension (GH) placenta and no metabolism and related enzyme expression and activity. The present study aimed to evaluate eNOS and iNOS expression in the placentas of both normal and GH patients, by means of Real-Time quantitative PCR, measure placental nitric oxide and peroxynitrite levels in the same group of subjects, and correlate such findings with HELLP group already published. Fifteen patients with gestational hypertension and thirty healthy pregnant controls comparable for maternal and gestational age were enrolled in the study. Placental tissue was taken immediately after delivery. eNOS and iNOS mRNA levels were evaluated Real-Time quantitative PCR, whereas nitric oxide and peroxynitrite production was measured by a commercially available kit. Placental eNOS and iNOS mRNA levels were significantly reduced in GH (2,02-fold reduction and 2,33-fold reduction, respectively) when compared to controls. Conversely, NO and ONOO(-) production were significantly higher in GH group compared to control group (31.56±4.15nmol NO/mg prot vs. 23.98±5.14nmol NO/mg prot and 68.49±8.57 arbitrary fluorescence units vs 17.31±2.25 arbitrary fluorescence units; p<0, 05). Such results were compared to HELLP group obtained in an already published study. As from results herein reported, we can hypothesize that complex mechanisms involving NO pathways cause a placental vasculature damage. However, it is not easy to understand if these changes could be interpreted as causes or consequences of this pathologic state.

67: Single nucleotide polymorphisms (SNPs) in endothelial nitric oxide synthase (eNOS) gene and unexplained stillbirth

nitric oxide synthase (eNOS) gene and unexplained stillbirth Francesca Ferrari, Fabio Facchinetti, Huaizhi Yin, George Saade, Monica Longo Obstetrics and Gynaecology, University of Modena and Reggio Emilia, Mother-Infant department, Modena, Italy, The University of Texas Medical Branch, Obstetrics & Gynecology, Galveston, TX OBJECTIVE: Altered utero-placental perfusion has been implicated in the pathogenesis of unexplained stillbirth (SB). We previously reported that genetic polymorphisms of VEGF affecting placental vasculogenesis are associated with unexplained SB. Nitric oxide (NO) plays a critical role in regulation of uterine perfusion, and eNOS is the rate limiting enzyme in the NO production cascade. Previous studies suggest that eNOS gene SNPs may be associated with preeclampsia and idiopathic recurrent pregnancy loss. Our objective was to evaluate the presence and distribution of eNOS gene SNPs between unexplained SB and controls in a well-characterized cohort. STUDY DESIGN: Placentas were obtained from 50 unexplained SB and 46 livebirth controls. Classification of “unexplained” stillbirth was by Wigglesworth and Aberdeen criteria and included both appropriate (AGA-SB) and small for gestational age (SGA-SB, 10th%) SB. Placental DNA was extracted (Qiagen) and evaluated for 4 eNOS SNPs (rs1007311 A/G, rs891512 A/G, rs1800779 A/G and rs1800783 A/T) by real time PCR using specific taqman probes. The genotype and allelic distributions were compared between the groups using Chisquare (significance: p 0.05). RESULTS: All SNPs were in Hardy-Weinberg equilibrium. None of the SNPs were associated with SB overall. However, significant different genotype distribution emerged for eNOS-SNP rs1800783 A/T when comparing the subgroups of SGA-SB and AGA-SB to controls (p 0.004). Allele-A carriers of rs1800783 were more frequent in AGA-SB compared to both controls (p 0.003) and SGA-SB (p 0.001)(Fig. 1). CONCLUSION: This is the first study to report on placental eNOS polymorphisms and unexplained SB. Importantly we found that Allele A of rs1800783 eNOS SNP might play a role in the occurrence of unexplained stillbirth in the setting of adequate growth. Our results warrant further population-based analysis. 68 The influence of a mediolateral episiotomy during an operative vaginal delivery on the risk for Obstetric Anal Sphincter Injuries (OASIS) Joey de Vogel, Anneke van Beek, Dirk Gietelink, Marijana Vujkovic, Jan Willem de Leeuw, Dimitri Papatsonis Amphia hospital, Department of Obstetrics and Gynecology, Breda, Netherlands, Erasmus Medical Center, Department of Obstetrics and Gynecology, Rotterdam, Netherlands, Ikazia Hospital, Department of Obstetrics and Gynecology, Rotterdam, Netherlands OBJECTIVE: Operative vaginal delivery is associated with an increased risk of Obstetric Anal Sphincter Injuries (OASIS). It’s unclear if structural use of a mediolateral episiotomy at operative vaginal delivery will decrease this risk. The aim of our study was to evaluate the incidence of OASIS in women with operative vaginal delivery, and to assess if a mediolateral episiotomy is preventive for OASIS in these deliveries. STUDY DESIGN: A retrospective cohort study was performed using the obstetric-database of the Amphia Hospital, The Netherlands of deliveries from 2001 up to 2009. All patients with live born infants delivered by an operative vaginal delivery at a gestational age 34 weeks were included. Exclusion criteria were: multiple gestations, breech deliveries and the use of a median episiotomy. Continuous variables were compared using the Students t test or Mann-Whitney U test, the 2 test for categorical variables. Continuous variables were summarized as means with standard deviations, or medians with interquartile ranges. A logistic regression model was used for the risk assessment for OASIS when a mediolateral episiotomy was performed. Treatment effect was presented as adjusted odds ratio with 95% confidence interval. RESULTS: There were 2970 operative vaginal deliveries in the study period meeting the inclusion and exclusion criteria (Figure 1). The prevalence of OASIS was 5.7%. The absolute risk was 3.3% in the MLE group, compared to 15.5% in the MLEgroup (OR 0.19; 95% CI: 0.14-0.26). The risk estimation for OASIS remained similar (adjusted OR 0.20; 95% CI: 0.12-0.34), after logistic regression analysis with correction for gestational age, parity, birth weight, maternal age, use of epidural analgesia, indication for operative vaginal delivery, cephalic fetal position, and duration of the second stage. CONCLUSION: The use of a mediolateral episiotomy in operative vaginal delivery is associated with a fivefold decreased risk for developing OASIS. Therefore we advocate the structural use of a mediolateral episiotomy during an operative vaginal delivery in order to prevent OASIS. www.AJOG.org Intrapartum Fetal/Clinical Obstetrics Oral Concurrent Session 6

ENOS, NO, and the Activation of ERK and AKT Signaling at Mid-Gestation and Near-Term in an Ovine Model of Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) is a disease responsible for neonatal morbidity and mortality and perinatal death affecting 8% of all pregnancies. In sheep, IUGR that mimics the human IUGR disease closely can be brought on by environmental hyperthermia. Endothelial nitric oxidase synthase (eNOS) and nitric oxide (NO) are important in the regulation of blood flow in the fetal-placental circulation and are modulated by several factors including hypoxia. eNOS activity is also regulated by the phosphorylation of ERK1/2 and AKT proteins in various tissues. In a hyperthermic (HT) ovine model of IUGR with systemic hypertension and increased blood flow resistance, our objective was to determine the relationship between p-ERK, p-AKT, eNOS, and NO concentrations in the placenta, uterine, and umbilical vessels at mid-gestation and near-term.Eight pregnant ewes were exposed to hyperthermic conditions for either 55 or 80 days to induce IUGR. Sheep necropsies were performed at mid-gestation and near-term for collection of placentomes, umbilical vessels, and the uterine artery. Tissues were assessed for eNOS mRNA and protein, and p-ERK and p-AKT protein. Blood was collected for NO determination at the time of necropsy. Placental insufficiency and IUGR (PI-IUGR) pregnancies demonstrated: 1) reduced placental weight at mid-gestation and reduced placental and fetal weight near-term, 2) no changes in eNOS protein concentration in the uterine artery and umbilical vessels, but an increase in NO in umbilical vein blood at both time points, 3) no significant changes in signal transduction makers (ERK/AKT) in placental tissue at mid-gestation but a significant increase near-term in cotyledon tissues, and 4) an increase in p-AKT in the uterine vessels at term. The near-term findings of increased placental p-ERK and p-AKT proteins and umbilical vein NO concentration suggest one mechanism responsible for the increase in placental eNOS previously described in this PI-IUGR model characterized by fetal systemic hypertension and abnormal umbilical artery Doppler velocimetry.

Uterine artery notch is associated with increased placental endothelial nitric oxide synthase and heat shock protein

Objective. This study was undertaken to evaluate whether uterine artery notch is associated with placental expression of endothelial nitric oxide synthase (eNOS), Heat shock protein 70 (Hsp70), and Lectin-like oxidized low-density lipoprotein receptor (Lox) in pregnant women.Study design. Uterine artery Doppler was performed in 30 pregnant women divided into two groups with or without the presence of uterine artery notches. After delivery, placental expression of eNOS, Hsp70, and Lox were evaluated with western blot analysis.Results. Higher levels of placental eNOS (p = 0.02) and Hsp70 (p = 0.001) were expressed in the group with uterine artery notch compared with the group without uterine artery notch. There was no significant difference for placental Lox expression between group with uterine artery notch and without uterine artery notch (p = 0.17).Conclusion. Our results suggest that uterine artery notch is associated with increased placental expression of eNOS and Hsp in pregnant women.

Ontogeny of endothelial nitric oxide synthase mRNA in an ovine model of fetal and placental growth restriction

To determine: 1) placental eNOS mRNA concentration across gestation in normal ovine pregnancy and in an ovine model of intrauterine growth restriction (IUGR), and 2) placental eNOS protein concentration in early ovine pregnancy. A total of 24 sheep were studied with 12 ewes placed in hyperthermic (HT) conditions to induce IUGR and 12 were kept in control conditions. HT and control animals underwent euthanasia at 3 developmental time points (55, 95, & 130 days gestational age; dGA) in ovine placental & fetal development. Compared to controls, HT pregnancies showed 1) no differences in fetal weights at 55 dGA and 95dGA with significant reductions at 130 dGA, 2) significantly smaller placentae at 95 and 130 dGA with a trend for a reduction at 55 dGA, 3) significant decreases in cotyledon eNOS mRNA at 95 and 130 dGA, 4) a significant increase in caruncle eNOS mRNA expression at 130 dGA, 5) significant increase in eNOS protein in the caruncle, but not in the cotyledon at 55 dGA. Placental eNOS concentration is transcriptionally regulated at mid-gestation, while additional post-transcriptional regulation is also involved during early and late gestation in this model of placental and fetal growth restriction.

Increase in placental eNOS expression in an ovine model of fetal growth restriction

GROWTH RESTRICTION JUAN ARROYO, RUSSELL ANTHONY, THOMAS PARKER, GATES ROE, SANTOSH PANDIPATI, HENRY GALAN, University of Colorado Health Sciences Center, Obstetrics and Gynecology, Denver, Colorado, University of Colorado Health Sciences Center, Dept. of Pediatrics, Denver, Colorado OBJECTIVE: Endothelial nitric oxidase synthase (eNOS) and NO modulate fetal-placental blood flow and are regulated, in part, by hypoxia and shear stress. In an established hyperthermic (HT) ovine model of placental and fetal growth restriction (FGR) with numerous features seen in human FGR, we hypothesize that eNOS protein levels would be increased in the placental tissues of 130 day gestation (dGA) animals. STUDY DESIGN: HT ewes (n=4) were exposed to increased ambient temp for 80 days (starting at 35 dGA) and compared to control animals (n=4). At 125 dGA, fetal arterial catheters were placed for blood gas and systemic pressure measurements. Umbilical artery Doppler velocimetry studies were performed just prior to necropsy at 130 dGA. Placentomes were separated into caruncle and cotyledon compartments for western blot analysis (25 mg protein) with an eNOS mAb. eNOS expression was normalized to b-actin. Immunohistochemistry was used to localize eNOS. Data are expressed as meanGSEM and compared with t-tests with p!0.05 as significant. RESULTS: Compared to controls, 1) HT fetuses and placentae were smaller (p!0.001). 2) HT FGR fetuses showed higher S/D ratios (3.8G0.2 v 3.0G0.3; p!0.009) and systemic blood pressures (44.3G1.7 v 41.1G1.5mmHg; p!0.03). 3) FGR caruncle and cotyledon tissues showed a 1.5-fold increase in eNOS protein concentration. 4) HT fetuses had lower O2 saturation (52.2G7.0% v 33.1G11.0% p!0.008) and paO2 (18.9G1.5 mmHg v 13.9G1.9 mmHg; p!0.002). eNOS was localized to vascular endothelium and binucleated cells.

Reduced l-arginine Level and Decreased Placental eNOS Activity in Preeclampsia

Nitric oxide is produced enzymatically by the nitric oxide synthase (NOS), which converts L-arginine in the presence of oxygen to L-citrulline and NO. Moreover, it has been reported that asymmetric dimethylarginine (ADMA) acts as is an endogenous inhibitor of endothelial NOS (eNOS) by competing with the enzyme for L-arginine. In this study, we measured L-arginine and ADMA in normal and preeclamptic women, and also investigated the association between the Glu298Asp eNOS gene polymorphism and preeclampsia. Finally, we assessed eNOS expression levels in the placentas of both normal and preeclamptic patients, using Western blot and immunohistochemistry. L-arginine levels were found to be significantly lower in the preeclamptic women than in the normal pregnant women (p=0.02) but there were no significant differences in ADMA levels between the normal and preeclamptic women. We also determined there to be no association between the Glu298Asp eNOS gene and preeclampsia. With regard to placental eNOS expression, we detected a lower degree of eNOS expression in the preeclamptic syncytiotrophoblasts than in the normal syncytiotrophoblasts. We suggest that reduced L-arginine levels, rather than increased ADMA levels, contribute to the development of preeclampsia, and also that decreased placental eNOS expression constitutes a characteristic finding in preeclamptic placentas.

Expression of endothelial nitric oxide synthase traffic inducer in the placentas of women with pre-eclampsia

Objective To investigate the expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) in the placenta of women with pre-eclampsia (PE) and discuss its role in the pathogenesis of PE. Methods Western blot analysis was used to detect the expression of NOSTRIN and endothelial nitric oxide synthase (eNOS). The activity of eNOS in placental tissue was assayed by spectrophotometry. Serum and placental NO 2/NO 3, the stable metabolic end product of NO, was measured using nitrate reductase. Results Western blot analysis showed that the protein level of NOSTRIN was significantly higher in women with PE than in the control group ( P<0.01). However, no significant difference between groups was observed in the expression of placental eNOS ( P>0.05). The activity of eNOS was significantly decreased in women with PE (13.727±3.58 U/mg) compared with the control group (21.69±3.84 U/mg) ( P<0.01). The placental levels of NO 2/NO 3 were significantly lower in women with PE (27.53±8.51 μmol/mg) than in the control group (44.38±9.59 μmol/mg) ( P<0.01). The levels of serum NO 2/NO 3 were significantly lower in women with PE (48.56±8.49 μmol/L) than in the control group (65.37±9.61 μmol/L) ( P<0.01). A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in the placental tissues of women with PE ( r=−0.57, P<0.01). Conclusion The level of NOSTRIN expression in the placental tissues of women with PE is increased and is negatively correlated with the activity of eNOS—which may play an important role in the pathogenesis of PE.

Effect of betamethasone administration to the pregnant baboon at 0.75 gestation on placental eNOS distribution and activity

Betamethasone is frequently administered to pregnant women at risk of premature labor to accelerate fetal lung maturation. Maternally administered betamethasone produces pronounced changes in the fetal peripheral vasculature, raises fetal blood pressure and produces fetal growth restriction. Endothelial nitric oxide synthase (eNOS) plays an important role in regulating vascular tone. We hypothesized that effects of betamethasone on the fetal vasculature include decreased eNOS activity. We determined a significant depression of total placental eNOS protein measured by ELISA (betamethasone treated vs control, 0.48±0.28 vs 1.57±0.45, p<0.05) and immunohistochemistry in both syncytiotrophoblast and vascular endothelium. Following betamethasone exposure, eNOS mRNA and enzyme activity showed decreasing trends which were not statistically significant. eNOS protein was higher in the placentas of both control and betamethasone treated baboons in the presence of a female fetus compared with a male fetus. The same effect of the sex of the fetus was observed in the betamethasone group for eNOS mRNA. In conclusion, maternally administered betamethasone produces a consistent decrease in several indices of placental eNOS function that may play a role in the altered cardiovascular dynamics and fetal growth retardation produced by betamethasone administration in late pregnancy.

Effect of betamethasone administration to the pregnant baboon at 0.75 gestation on placental eNOS distribution and activity

Betamethasone is frequently administered to pregnant women at risk of premature labor to accelerate fetal lung maturation. Maternally administered betamethasone produces pronounced changes in the fetal peripheral vasculature, raises fetal blood pressure and produces fetal growth restriction. Endothelial nitric oxide synthase (eNOS) plays an important role in regulating vascular tone. We hypothesized that effects of betamethasone on the fetal vasculature include decreased eNOS activity. We determined a significant depression of total placental eNOS protein measured by ELISA (betamethasone treated vs control, 0.48±0.28 vs 1.57±0.45, p <0.05) and immunohistochemistry in both syncytiotrophoblast and vascular endothelium. Following betamethasone exposure, eNOS mRNA and enzyme activity showed decreasing trends which were not statistically significant. eNOS protein was higher in the placentas of both control and betamethasone treated baboons in the presence of a female fetus compared with a male fetus. The same effect of the sex of the fetus was observed in the betamethasone group for eNOS mRNA. In conclusion, maternally administered betamethasone produces a consistent decrease in several indices of placental eNOS function that may play a role in the altered cardiovascular dynamics and fetal growth retardation produced by betamethasone administration in late pregnancy.

Chemical stabilization of tetrahydrobiopterin by L-ascorbic acid: contribution to placental endothelial nitric oxide synthase activity.

The aim of this study was to characterize the mechanism of the chemical interaction between L-ascorbic acid (ASC) and tetrahydrobiopterin (BH(4)) in vitro and to examine its effect on the activity of endothelial nitric oxide synthase (eNOS) in first trimester human placentae. At room temperature, in Tris-HCl buffer (pH 7.4), both ASC and BH(4) were readily oxidized by dissolved O(2) or H(2)O(2). BH(4) was more sensitive to auto-oxidation, while ASC was more susceptible to oxidation by H(2)O(2). Addition of 36 micromol/l BH(4) to 143 micromol/l ASC increased the initial rate of ASC oxidation 3.2-fold in a catalase-sensitive manner, indicating that enhanced ASC oxidation is partly due to the formation of H(2)O(2). In the presence of catalase, BH(4) still stimulated 1.9-fold the initial rate of ASC oxidation, suggesting that another auto-oxidation product of BH(4), most probably quininoid-BH(2) (qBH(2)), could also stimulate ASC oxidation while itself being reduced back to BH(4). ASC prevented the auto-oxidation of BH(4) in a concentration-dependent fashion, with 3 mmol/l ASC providing an almost complete stabilization of 25 micromol/l BH(4). Importantly, basal eNOS activity in placental microsomes was stimulated 2.5-fold by 0.5 micromol/l BH(4), and 0.5 mmol/l ASC enhanced the BH(4)-stimulation 1.4-fold, with a smaller effect on basal eNOS activity. Taken together, the findings support the notion that the stabilizing action of ASC on BH(4) is related to the ASC-mediated reductive reversal of the auto-oxidation process of BH(4). Moreover, we demonstrated that concentrations of ASC present in the placenta as a common vitamin C supply are sufficient to protect cellular free BH(4) and may contribute to the stimulation of placental eNOS activity.