Preeclampsia Plasma Research Articles

OS076. The effect of lectin-like oxidized low density lipoprotein receptor monoclonal antibody (LOX-1 MAb) on the expression of eNOS in preeclamptic HUVECs model

Preeclampsia is a major cause of fetal and maternal morbidity and mortality and it still occurs in 7-10% of all pregnancies worldwide. The pathophysiology remains unclear and so it was named "a disease of theories". Clinical and biochemical evidences assumed that disruption of normal endothelial cells may be the primary cause in the pathogenesis of preeclampsia. Factors that cause endothelial dysfunction are not certainly known, but the evidence suggests that factors related to the placenta. In a previous study conducted, HUVECs were found to significantly increase the uptaking of Ox-LDL by endothelial cells in response to the exposure to preeclampsia plasma when compared with non-pregnant women plasma and plasma of pregnant women. Ox-LDL binds to LOX-1 in vascular cells, including endothelial cells and smooth muscle cells, and is thought to cause endothelial dysfunction in patients with preeclampsia. One sign of endothelial dysfunction is the decline in biological activity of endothelial derived NO. NO biological half-time in vivo is less than 1 second. Nitric oxide is produced by the five-electron oxidation of L-arginine which is catalyzed by the enzyme eNOS. This research will observes the expression of eNOS in HUVECs PE model which were exposed to 5, 10, 20μg/ml LOX-1 MAb (lectin-like oxidized LDL receptor monoclonal antibody). To know the effect of LOX-1 MAb on the expression of eNOS in Preeclamptic HUVECs Model. The study used HUVECs PE model which is HUVECs culture that was exposed with pre-eclamptic serum and it shown endothelial dysfunction. HUVECs culture was taken from the endothel of umbilical cord of newborn. HUVECs culture were divided into four groups: (1) HUVECs + Preeclamptic serum (HUVECs PE model), (2) HUVECs PE Model + LOX-1 MAb 5μg/ml, (3) HUVECs PE Model + LOX-1 MAb 10;μg/ml, 4) HUVECs PE Model + LOX-1 MAb 20μg/ml. We performed immunocytochemistry study using primary antibody (anti eNOS) while eNOS expression was observed in each group. The data were analyzed using ANOVA, Tukey's test, Correlation and Regression test. eNOS expression in group 1 was significantly different with group 2, 3 and 4 (p<0.05). Group 2 was significantly different with group 1, 3 and 4 (p<0.05). In group 4 and 5 there were no significant difference each other, but both were significantly different with group 1 and 2 (p<0.05). About 72% of the expression of eNOS in HUVECs PE model were influenced by exposure LOX-1 MAb and the other 28% were influenced by other factors. In the observation of HUVECs PE model, we found that the higher dosage of LOX-1 MAb given on the model, the eNOS expression will increase proportionally. Exposure of LOX-1 MAb will significantly increase eNOS expression on HUVECs PE model. We concluded that the higher dosage of LOX-1 MAb given on the HUVECs PE model, the eNOS expression will increase proportionally.

Amino acid profile of plasma and cerebrospinal fluid in preeclampsia

ObjectivesTo determine patterns in amino acids (arginine, GABA, glutamate and glutamine) and the diamine (agmatine) in plasma and cerebrospinal fluid (CSF) of mild and severe preeclampsia compared to control patients, using capillary zone electrophoresis to generate methods for refining diagnosis and prognosis and shed light on the pathophysiological mechanisms of preeclampsia. Study designThis is an observational case-control study in pregnant women that attended the emergency ward of the University Hospital, Mérida, Venezuela, during the period April, 2009–April 2010. Main outcome measuresMolar concentration of amino acids and diamine in plasma and CSF in control, mild and severe preeclampsia patients. ResultsAn increase in glutamate plasma levels was observed in mild preeclampsia and even higher in severe patients, while a biphasic response occurred in the CSF samples with a significant increment in mild preeclampsia patients and a decrease in severe preeclampsia patients. GABA significantly decreased both in plasma and CSF in mild preeclampsia with a tendency to return to normal levels in severe preeclampsia patients. Arginine CSF and plasma levels decreased in mild preeclampsia patients and even more in severe preeclampsia while agmatine significantly increased in plasma levels with no changes in CSF. ConclusionsThe results are discussed in terms of molecules that could be used as biomarkers of the severity of the disease and the possible involvement of these substances in the pathophysiology of preeclampsia.

Effects of Preeclamptic Plasma on Potassium Currents of Human Umbilical Vein Endothelial Cells

Endothelial cell (EC) dysfunction in preeclampsia (PE) may be mediated by humoral factors secreted by placenta, thereby affecting the EC vasoactive compound production. Possible targets of these factors include potassium channels, which are important in EC membrane potential control, calcium influx, and vasoactive compound release. Alterations in potassium channel function may thus contribute to the pathogenesis of PE. The present study compared the effects of 10% plasma from PE, normal pregnant (NP), or nonpregnant women (NS) on potassium currents of human umbilical vein ECs (HUVECs), using whole-cell patch clamp technique, with HUVECs in conventional culture medium (10% fetal bovine serum) as controls. Cells of all groups were similar in morphology and whole-cell capacitance. The fraction of cells with inward rectifier potassium channel (IRK) current in PE plasma (41.2%) was significantly lower than those in NP and NS plasmas (76.9% and 59.1%, respectively), although the IRK current density was similar among groups. The outward current components included the calcium-sensitive potassium channels (K(Ca)) and were partially blocked by 100 nmol/L apamin and 200 nmol/L iberiotoxin. The fraction with outward current in PE plasma (100%) was significantly higher than those in NP and NS plasmas (76.9% and 81.8%). The findings indicate inhibition of IRK expression by PE plasma in HUVEC culture, while K(Ca) expression may be facilitated probably as a compensatory response to diminished IRK. These data suggest that potassium channels may be a target of the pathogenic factor/factors in the plasma of patients with PE and may play roles in the pathogenesis of this condition.

Cardiovascular Biomarker Midregional Proatrial Natriuretic Peptide During and After Preeclamptic Pregnancies

Preeclampsia is associated with increased risk of cardiovascular disease. Midregional proatrial natriuretic peptide (MR-proANP), a precursor of the atrial natriuretic peptide, is a biomarker for cardiovascular disease. We obtained plasma from 184 pregnant women in gestational weeks 24 to 42 (normotensive pregnancies: n=77, preeclampsia: n=107), from 25 of these women at 5 to 8 years after index pregnancy (normotensive pregnancies: n=11, preeclampsia: n=14), and from 49 normotensive, nonpregnant women and analyzed them by immunoassay for MR-proANP. To investigate potential sources, placental and decidual atrial natriuretic peptide mRNA expression levels were analyzed by quantitative real-time PCR in 21 normotensive and 23 preeclamptic pregnancies, as well as in human heart and kidney samples. For further confirmation, we measured circulating MR-proANP and performed expression studies in a transgenic rat model for preeclampsia. MR-proANP was significantly elevated in maternal plasma in preeclampsia compared with normotensive pregnancies (135 versus 56 pmol/L; P<0.001). However, 5 to 8 years after pregnancy, there was no difference (formerly preeclamptic women versus formerly normotensive in pregnancy: 53 versus 49 pmol/L; P=0.5). Our preeclamptic rat model confirmed the acute MR-proANP differences between preeclamptic and normotensive pregnancies (10.9±1.9 versus 4.3±0.3 pmol/L; P=0.05). Atrial natriuretic peptide expression was high in the heart but negligible in the uteroplacental unit in both normotensive humans and rats, whereas expression in maternal and fetal hearts in the preeclamptic rats was significantly increased, compared with controls. MR-proANP is a serviceable biomarker in preeclampsia, both in humans and a rat model, probably reflecting cardiovascular hemodynamic stress.

Digifab and immunoneutralization of cardiotonic steroids in preeclampsia

Background. Elevated levels of Na + /K + -ATPase(NKA)-inhibitory cardiotonic steroids (CTS) including marinobufagenin (MBG) contribute to pathogenesis of preeclampsia (PE) via induction of vasoconstriction and of vascular stiffness. In PE, Digibind (affi nity-purifi ed digoxin antibody) exhibits benefi cial effects primarily due to immunoneutralization of CTS including MBG. Because Digibind is not available and the only other «clinically-usable» digoxin antibody is DigiFab (BTG International Ltd), we compared DigiFab and Digibind with respect to their ability to interact with CTS in PE plasma and to ex-vivo reverse PE-induced NKA inhibition. Objective. We compared effects of Digibind and Digifab on erythrocyte NKA from patients with PE and normotensive pregnant subjects. Using competitive fl uoroimmunoassays based on Eu-labeled DigiFab and Digibind we compared profi le of elution of digoxin-immunoreactive material following fractionation of PE plasma on reverse-phase HPLC column. Design and methods. 7 patients with PE (28 ± 2 years; gestational age, 39,0 ± 0,5 weeks, urinary protein excretion, 2,12 ± 0,46 g/24 hours, blood pressure 157 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant subjects (26 ± 1 years; gestational age, 37 ± 1 weeks, blood pressure 112 ± 2/75 ± 3 mm Hg) were studied. Results. PE was associated with substantial inhibition of erythrocyte NKA (1,47 ± 0,17 vs. 2,65 ± 0,16 umol Pi/mL/hr in control group, p < 0,001). At concentration 10 ug/mL (which mimics dose of Digibind administered in PE) both Digibind and DigiFab partially, but signifi cantly restored NKA activity (2,1 ± 0,2 and 2,05 ± 0,3 umol Pi/mL/hr, respectively). In competitive immunoassay Digibind and Digifab exhibited comparable cross-reactivity with MBG and endogenous ouabain. Following HPLC fractionation of plasma, both Digibind and Digifab detected PE-associated increase in CTS material in fractions 16–18; 176 vs. 75 pmoles for Digibind, control and preeclampsia, respectively; 221 vs. 70 pmoles for Digifab, control and PE, respectively. 4G4 anti-MBG monoclonal antibody detected preeclampsia-induced CTS increase (1056 vs. 421 pmoles) mainly in fraction 16. Conclusions. We conclude that: (i) Digifab and Digibind exhibit comparable cross-immunoreactivity with CTS; (ii) in patients with PE plasma levels of digoxin-like immunoreactivity are elevated proportionally when measured by both Digifab and Digibind and both antibodies reverse preeclampsia-induced NKA inhibition, and (iii) following HPLC fractionation of PE plasma, Digifab and Digibind interact with endogenous bufadienolides including MBG, rather than endogenous ouabain.

Differential expression and the anti-apoptotic effect of human placental neurotrophins and their receptors

Neurotrophin (NT) is important in the survival, maintenance and differentiation of neuronal tissue, and functions in follicle maturation, tumor growth, angiogenesis and immunomodulation; however, the expression of NT and its receptors (NTR) in human placenta and their influence on fetal growth are unclear. Here we investigated the correlation of NT and NTR in human placenta with uterine environment and fetal growth. TrkB, a NTR, mRNA was expressed on decidual and villous tissue and increased with gestational age, localizing in the trophoblast layer and endothelium by immunohistochemistry. Villous TrkB mRNA was significantly increased in preeclampsia (PE) than in controls and was higher in the normotensive small for gestational age (SGA) placenta, although it was not significant. It was also significantly increased in the small twin of discordant twin pregnancies. Brain-derived neurotrophic factor (BDNF), the main ligand of TrkB, was expressed in membranous chorion and villous tissue and was significantly higher in maternal plasma in normotensive SGA and PE than in controls. TrkB mRNA expression was up-regulated on cultured villous tissue explants and on JEG-3, a choriocarcinoma cell line, by H 2O 2 treatment. BDNF decreased apoptotic cells in H 2O 2-treated JEG-3, indicating that BDNF/TrkB signaling had anti-apoptotic effects against oxidative stress in JEG-3, suggesting a protective role of BDNF/TrkB in human villous tissue under unfavorable conditions in utero.

Is There Evidence of Separate Inflammatory or Metabolic Forms of Preeclampsia?

Objectives. To examine whether high insulin resistance versus high inflammation identifies subtypes of preeclampsia. Methods. A cytokine panel, glucose and insulin were measured in 37 preeclampsia plasma samples. Wilcoxon rank sum assessed median concentration of HOMAIR by pro-inflammatory:anti-inflammatory ratio. Regression stratifying by BMI and preterm birth was conducted. Results. There was no difference in median HOMAIR by the pro-inflammatory:anti-inflammatory ratio (p = 0.16). No subsets scatterplot clusters emerged. A positive correlation between HOMAlog and the ratio was significant (p = 0.04). Conclusions. No dichotomous subsets of preeclampsia by inflammation versus insulin resistance were detected. Contrary to our hypothesis, insulin resistance was higher as inflammation increased in preeclampsia.

VEGF in the Muscular Layer of Placental Blood Vessels: Immuno- Expression in Preeclampsia and Intrauterine Growth Restrictrion and its Association with the Antioxidant Status

The pathophysiology of preeclampsia (PE), a disorder occurring in 5% of all pregnancies, remains largely unknown, but early placental hypoxia and oxidative stress are known to be involved in the mechanism of the syndrome. Maternal plasma and placental tissue samples were collected from PE, intrauterine growth restriction (IUGR), and normotensive pregnant patients. The immunohistochemical expression of vascular endothelial growth factor (VEGF), malondialdehyde (MDA) production and the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase GSH-Px) were determined in the placental tissue. F2-isoprostane concentration and the ferric reducing ability of plasma (FRAP) were determined in maternal plasma. We found that the PE and IUGR groups showed a higher expression of VEGF in the muscular layer of fetal chorionic vessels. In addition, increased plasma F2 isoprostane levels and a significant reduction of FRAP in the plasma of PE women, as well as a lower activity of SOD in PE placentas and a higher activity of GSH-Px in IUGR placentas were found. Additionally, lower PlGF and higher sFlt1 levels were observed in the maternal plasma of PE and IUGR than control. We concluded that in a hypoxic environment, the placenta expresses VEGF in the muscular layer of fetal vessels. The development of PE could be related to the increased expression of VEGF, with decreased placental SOD activity and a decrease of both plasma F2-isoprostane and FRAP levels. In turn, the development of IUGR could be related to the association of decreased plasma FRAP levels and increased placental GSH-Px activity.

Quantitative detection of the hypermethylated RASSF1A gene in maternal plasma of pre-eclampsia

To investigate the level of hypermethylated ras association domain family 1A (RASSF1A) gene in maternal plasma of pre-eclampsia and its clinical value. Sixty pre-eclampsia women including 30 mild and 30 severe cases were selected, 60 women with normal pregnancy were studied as control. Free DNA from plasma samples was extracted, fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the concentrations of RASSF1A gene before and after methylation-sensitive restriction digestion. Meanwhile, beta-actin gene was detected as a control to confirm complete enzyme digestion. The median concentration of hypermethylated RASSF1A gene was 3.31-fold higher in samples from pre-eclamptic pregnancies than that in controls. There was significant difference between the mild and severe pre-eclamptic subjects (P<0.05), with the median concentrations of 1659 copies/mL and 2036.50 copies/mL, respectively. Hypermethylated RASSF1A gene in pre-eclampsia plasma was significantly increased and the concentrations were related to the severity of pre-eclampsia.

A proof‐of‐principle gel‐free proteomics strategy for the identification of predictive biomarkers for the onset of pre‐eclampsia

Objective Progress in the prevention and treatment of women at risk of pre‐eclampsia (PE) still remains hindered by the lack of clinical screening tools that can accurately predict which mothers are at risk. The identification and validation of predictive biomarkers is therefore seen as a critical milestone towards improved healthcare provision and the clinical testing of new therapeutic strategies. Gel‐free proteomic technologies offer the capability of analysing hundreds of plasma proteins simultaneously, but as yet these methods have not been applied to pregnancy complications. To assess the feasibility of such an approach to plasma biomarker research in pregnancy we have applied the technique to samples from women with PE to gestation‐matched controls.Sample Pooled plasma samples taken at time of disease from women with PE (n = 23) and gestation‐matched controls (n = 23).Methods Proteomics strategy for relative quantification of proteins using mass spectrometry.Results We identified several differences, including elevated levels of endoglin, PAPP‐A and PSG1 in PE plasma. Increased levels of endoglin were validated using immunoassay analysis of individual plasma samples.Conclusions Although at a relatively early stage, this mass spectrometry‐based approach shows promise as a tool to identify global protein changes in plasma. The application of these methods to pre‐disease samples is the next step in the identification of clinically useful biomarkers.

204. Proteomic analysis of the effluent from perfused placental cotyledons identifies proteins associated with pre-eclampsia

Pre-eclampsia (PE) is proposed to involve maternal systemic endothelial cell dysfunction which may be caused by excessive shedding of syncytiotrophoblast microparticles (STBM) from the placenta into the maternal circulation. We have examined STBM harvested by perfusion of placental cotyledons from PE and gestation-matched control (GMC) pregnancies. Following proteomic analysis, the proteins Calreticulin, Chloride intracellular channel 3 (CLIC3), Valosin-containing protein (VCP), Protein disulfide-isomerase A3 (ERp57) and Protein kinase C inhibitor protein 1 (14–3-3) were identified as having increased expression in PE samples. This result suggests that there may be increased rate of release of these proteins from the placenta into the maternal circulation. The aims of this work are to measure the expression of these proteins in placental extracts and maternal plasma, and to compare expression between PE and GMC using western blots and/or ELISA with antibodies specific to each protein. Placental expression was confirmed for all five proteins, however, CLIC3 was the only protein significantly increased in the placenta in PE (17.858µg+1.62, n = 27, t-test P &lt; 0.05) compared with GMC (10.478µg+0.76, n = 25). western blot determined there was a significant increase in calreticulin expression in plasma from PE women compared with GMC (as shown by Gu et al. 2008). Likewise, VCP was also increased in PE plasma (1810+280 density/0.25µl, n = 10) compared with GMC (884+260, n = 9). In contrast, the protein 14–3-3 was decreased in PE plasma (4 out of 27 PE plasma samples tested expressed 14–3-3) compared with GMC (10 out of 27 control plasma samples expressed 14–3-3). One of the western blots determined that there was a significant decrease in 14–3-3 expression in plasma from PE women (199.24+186 density/2.5µl, n = 10, un-paired t-test P &lt; 0.05) compared with GMC (2174.91 ± 723.29, n = 11). Neither ERp57 nor CLIC3 was detected in maternal plasma. Those proteins found to be significantly different in PE compared with GMC may be involved in the pathophysiology of PE. These results demonstrate that proteomic analysis of the effluent from perfused placental cotyledons is a reliable screening method to successfully identify proteins that have altered expression with PE. (1) Gu et al. Mole. Human Repro. April 16 2008

Homocysteine, Cysteine, and Related Metabolites in Maternal and Fetal Plasma in Preeclampsia

Homocysteine is associated with endothelial dysfunction and cardiovascular disease, and elevated concentrations of homocysteine have been found in preeclampsia. The purpose of this study was to investigate maternal and fetal concentrations of total homocysteine and related metabolites (including cysteine, choline, and betaine), and possible associations with infant birth weight. Women with preeclampsia (n=47) and controls (n=51), who underwent cesarean section, were included. Maternal plasma, umbilical vein, and artery plasma were analyzed. Median concentrations of homocysteine, cysteine, choline, and betaine were significantly higher in women with preeclampsia than controls, both in maternal and fetal plasma. There were no differences in folate and vitamin B12 concentrations between the groups, neither for maternal nor fetal samples. Maternal homocysteine concentration was a negative predictor for birth weight only in the preeclampsia group. Elevated homocysteine and cysteine concentration in maternal circulation in preeclampsia is reflected in the fetal circulation. The clinical significance of elevated homocysteine and cysteine concentrations in maternal and fetal compartments in preeclampsia remain to be explored, both regarding fetal growth and development of disease later in life.

PENGARUH PEMBERIAN KOMBINASI NAC DENGAN VITAMIN C DAN E TERHADAP STRES OKSIDATIF PADA HUVECs DIPAPAR PLASMA EKLAMPSIA

The blood plasma of preeclampsia contains oxidative stress factors. The oxidative stress is predicted asthe cause of dis-function and destruction of endothelial cell which will influence the severity of Preeclampsia and Eclampsia complications. Previous research showed that the treatment of antioxidant on preeclamsia was still controvertion. The  objectives of this research is to investigate the influence of the combination of NAC and vitamin C or vitamin E or bothof them on the level of H2O2, MDA, NO and GSH/GSSG ratio of endothelial cell culture exposed with Eclampsia plasma.The method wasan experimental study using HUVECs exposed with to 2%plasma Eclampsia as an Eclampsia model invitro. TheHUVECs were than treated with NAC 2µM only (group 1), NAC2µM + vitamin C 100 µM (group 2) , NAC 2 µM + vitamin E 100 µM (group 3), and NAC 2 µM + vitamin C 100 µM + vitamin E 100 µM (group 4) for 24 hours. The parameter measured in this study were H2O2, MDA, NO and GSH/GSSG ratio. Result showed that H2O2, MDA, and NO concentration are unsignificantly different among treatment groups although there was a decreasing on group treated with combination of NAC, vitamin C and Vitamin E.

Cell-Free mRNA Concentrations of Plasminogen Activator Inhibitor-1 and Tissue-Type Plasminogen Activator Are Increased in the Plasma of Pregnant Women with Preeclampsia

Detection of placental mRNA in maternal plasma has been reported in high-risk pregnancies. We attempted to investigate the concentrations of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) mRNA in maternal plasma in preeclampsia. Peripheral blood samples were obtained from healthy pregnant women before and after delivery and also from women with or without preeclampsia. Plasma was isolated from these samples, and RNA was extracted. Plasma PAI-1 and tPA mRNA concentrations were then measured by use of reverse transcription PCR assays. The concentrations were converted into multiples of the median (MoM) of the controls adjusted for gestational age. Data were stratified and analyzed according to the clinical severity of preeclampsia and quantitative distribution of blood pressure and proteinuria. The median (minimum-maximum) PAI-1 mRNA MoM values for women with preeclampsia and controls were 2.48 (0.82-8.53) and 1.00 (0.41-2.33), respectively, whereas the median (minimum-maximum) tPA mRNA MoM values were 3.33 (1.01-10.58) and 1.00 (0.95-1.20), respectively. The concentrations of both PAI-1 and tPA mRNA were significantly increased in cases of preeclampsia, compared with controls (P <0.0001). The MoM values of both mRNA species were directly correlated with the severity of preeclampsia and were greatest among a subgroup of hemolysis, increased liver enzymes, and low platelets pregnancies. Maternal plasma PAI-1 and tPA mRNAs are significantly increased in patients with preeclampsia and are positively correlated with the severity of preeclampsia.

Evidence for multiple circulating factors in preeclampsia

The purpose of this study was to enrich vasoactive factors that are present in the plasma of women with preeclampsia by the application of sequential fractionation and determination of the activity of each fraction in a bioassay. Pooled plasma from women with preeclampsia (n = 23) and matched control subjects (n = 23) was subjected to fractionation with ultrafiltration, targeted immunodepletion, or size exclusion chromatography. Myometrial arteries that were isolated from healthy cesarean section biopsy specimens (n = 28) were incubated with plasma fractions (2%, volume/volume), and their endothelial function was assessed by wire myography. Incubation of arteries with preeclampsia plasma or immunodepleted preeclampsia plasma had a deleterious effect on endothelial-dependent relaxation. Bioactivity of the plasma factors was absent in all fractions after either plasma ultrafiltration or separation with the use of size exclusion chromatography; however, activity was restored after recombination of these fractions. This study provides the first conclusive evidence that multiple synergistic factors, with a combined vasoactive effect, are present in the plasma of women with preeclampsia.

Reduced Cellular Glutathione Reductase Activity and Increased Adhesion Molecule Expression in Endothelial Cells Cultured With Maternal Plasma From Women With Preeclampsia

The purpose of the current study was to determine whether maternal circulating components could regulate oxidative status of glutathione redox cycle and adhesion molecule expression in endothelial cells (ECs). Maternal plasma was extracted from venous blood obtained from normal term pregnant women and from women with preeclampsia (PE). Normal and PE pregnancies were defined as American College of Obstetricians and Gynecologists criteria. Confluent ECs were incubated with EC growth medium (EGM) containing 20% plasma from women with normal (n = 8) and PE (n = 8) pregnancies for 4 hours. ECs incubated with EGM only were used as control. EC oxidative status was assessed by measuring cellular glutathione reductase (GR) and glutathione peroxidase (GPx) activities. Adhesion molecule expressions for intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), P-selectin, and E-selectin were determined by colorimetric assays detected on EC surface by UV spectrophotometer at OD 450 nm. Data are presented as mean +/- SE and analyzed by analysis of variance (ANOVA). A P value < .05 was set as statistically significant. Cellular GR activity was reduced approximately 35% in ECs treated with normal plasma and 70% in ECs treated with PE plasma compared to that in untreated control cells: 0.072 +/- 0.014 (P < .05), 0.039 +/- 0.006 (P < .01), versus 0.117 +/- 0.010 U/mg cellular protein, respectively. In contrast, GPx activity was slightly increased in ECs treated with normal plasma and significantly increased in ECs treated with PE plasma compared to that in untreated control cells: 0.059 +/- 0.005, 0.075 +/- 0.012 (P < .05) versus 0.044 +/- 0.002 U/mg cellular protein, respectively. P-selectin, E-selectin, and VCAM expressions were elevated in cells treated with normal plasma but significantly increased in cells treated with PE plasma compared to those of untreated controls: P-selectin--0.18 +/- 0.03, 0.35 +/- 0.04 versus 0.04 +/- 0.01 OD 450 nm, P < .01; E--selectin-0.06 +/- 0.02, 0.10 +/- 0.02 (P < .05) versus 0.03 +/- 0.01 OD 450 nm; VCAM--0.12 +/- 0.02, 0.16 +/- 0.03 (P < .01) versus 0.08 +/- 0.02 OD 450 nm, respectively. There was no difference for ICAM expression in cells treated with normal or PE plasma compared to controls. These data suggest that endothelial pro- and anti-oxidative status could be directly affected by circulating components during pregnancy. Reduced cellular GR activity and increased GPx activity accompany increased inflammatory reactions in ECs responding to circulating "toxic" factors in preeclampsia.

Evidence supporting that the excess of the sVEGFR-1 concentration in maternal plasma in preeclampsia has a uterine origin

Background. Preeclampsia has been considered an anti-angiogenic state. Two factors have been implicated in the genesis of this state: soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) and placental growth factor (PlGF). Indeed, the concentrations of PlGF, an angiogenic factor, are lower in preeclampsia than in normal pregnancy, while the opposite is the case for the anti-angiogenic factor, sVEGFR-1. The source of the excess sVEGFR-1 has not yet been determined. Since the placenta could be a source of sVEGFR-1, we conducted a study to determine whether there is a gradient in the plasma concentration of sVEGFR-1 and PlGF between the uterine vein and the antecubital vein in both patients with preeclampsia and normal pregnant women.Methods. A cross-sectional study was performed to determine the plasma concentrations of sVEGFR-1 and PlGF in the uterine and antecubital vein of patients with preeclampsia (n = 9) and normal pregnant women at term (n = 9). Plasma samples were collected from antecubital and uterine veins at the time of cesarean section. The concentrations of sVEGFR-1 and PlGF were determined using specific enzyme-linked immunoassays. The differences of plasma concentrations of sVEGFR-1 and PlGF between uterine and antecubital veins in both groups were compared by paired t-tests.Results. Patients with preeclampsia had a significantly higher mean plasma concentration of sVEGFR-1 in the uterine vein than in the antecubital vein (uterine vein: mean 13,675 ± 5,684 pg/ml vs. antecubital vein: mean 10,234 ± 4,700 pg/ml; paired t-tests, p = 0.04). In contrast, among normal pregnant women at term, there was no significant difference in plasma concentrations of sVEGFR-1 between the uterine and antecubital veins (uterine vein: mean 1,918 ± 665 pg/ml vs. antecubital vein: mean 1,750 ± 475 pg/ml; paired t-tests, p = 0.1). The mean plasma concentration of sVEGFR-1, either in the antecubital or uterine vein, was significantly higher in preeclampsia than in normal pregnancy (unpaired t-tests; both p < 0.001). There was no significant difference in the mean plasma concentration of PlGF between the uterine and the antecubital veins in both the preeclamptic (uterine vein, mean ± SD: 129 ± 106 pg/ml vs. antecubital vein, mean ± SD: 82 ± 43 pg/ml; paired t-tests, p = 0.2) and normal pregnancy groups (uterine vein, mean ± SD: 331 ± 254 pg/ml vs. antecubital vein, mean ± SD: 319 ± 259 pg/ml; paired t-tests, p = 0.4). The mean plasma concentration of PlGF, either in the uterine or antecubital vein, was lower in preeclampsia than in normal pregnancy (unpaired t-tests; p = 0.008 and 0.02 respectively).Conclusions. Plasma concentration of sVEGFR-1 was higher in the uterine vein than in the antecubital vein in women with preeclampsia. This provides evidence supporting the concept that the uterus is a potential source of the excess circulating sVEGFR-1 concentration in preeclamptic women.

S -Nitrosoalbumin–Mediated Relaxation Is Enhanced by Ascorbate and Copper

S-nitrosoalbumin (SNO-Alb) is a major reservoir of releasable nitric oxide (NO) in plasma. In preeclampsia, a pregnancy-specific disorder associated with endothelial dysfunction, we previously found significant elevations in plasma SNO-Alb concentrations and decreased plasma ascorbate (Asc) levels. This increased SNO-Alb may result from low-plasma Asc if Asc, along with transition metals (eg, copper [Cu]) are necessary for release of NO from S-nitrosothiols. We propose that vasodilator effects of SNO-Alb, mediated by release of NO, are fully realized only when Asc/Cu availability is sufficient. Relaxation responses to SNO-Alb or the control reduced human serum albumin (SH-Alb), and responses to pooled plasma from normal or preeclamptic pregnancies were examined in isolated mouse arteries. Arteries preconstricted with phenylephrine were exposed to SNO-Alb or SH-Alb at physiologically relevant concentrations. When free Cu was added in excess (10 mumol/L), NO release was not dependent on Asc. However, when Cu was added at lower (physiological) levels, NO release was dependent on Asc. The addition of Asc and Cu to SNO-Alb stimulated vasodilatory responses in isolated arteries >90%, whereas no change in the SH-Alb (5%) response was observed. Preeclampsia plasma with higher levels of SNO-Alb caused arteries to relax 44.1+/-4.7%, whereas normal pregnancy plasma caused 11.9+/-4.2% relaxation (P=0.007). These data indicate that SNO-Alb alone or in plasma can act as a potent vasodilator, and that sufficient Asc/Cu promotes this action. We suggest that the higher circulating levels of SNO-Alb, in women with preeclampsia, reflect a deficiency in Asc/Cu-mediated release of NO from SNO-Alb.

Role of Circulating S -Nitrosothiols in Control of Blood Pressure

The biological effects of nitric oxide (NO) are in large part mediated by S -nitrosylation of peptides and proteins to produce bioactive S -nitrosothiols (SNOs).1–3 The observation of abnormal SNO levels in numerous pathophysiological states2 suggests that dysregulation of SNO homeostasis may contribute to disease pathogenesis. For example, the hypotension of human sepsis is accompanied by increases in circulating levels of vasodilatory SNOs.3 Although such altered SNO levels may simply mirror NO production (eg, induction of inducible NO synthase in sepsis), they may also reflect changes specific to SNO biosynthesis and metabolism. Indeed, mice lacking a SNO-metabolizing enzyme are profoundly hypotensive under anesthesia.3 Thus, blood pressure is evidently regulated by both synthesis and turnover of SNOs. In this issue of Hypertension , Gandley et al4 extend this paradigm by proposing that a defect in SNO turnover contributes to the hypertension of preeclampsia. In the blood, S- nitrosoalbumin (SNO-albumin) and S -nitrosohemoglobin (SNO-Hb) constitute the major conduits for circulating NO bioactivity. Although both SNOs may influence blood pressure, they operate within distinct signaling circuits. SNO-Hb can be viewed as a principal regulator of SNO homeostasis, adaptively modulating NO chemistry to control NO bioactivity. SNO-Hb is formed by transfer of NO from heme-iron to Cysβ93 thiol on T to R structural transition (oxygenation) of the hemoglobin tetramer.5 SNO-Hb associates with the red blood cell (RBC) membrane via an interaction with the cytoplasmic domain of anion-exchanger 1 protein (CDAE1, Band 3); on deoxygenation (R→T) transfer of the NO group from SNO-Hb to a cysteine thiol within CDAE1 supports export of RBC vasodilatory activity.6 SNO-Hb thus serves as an O2 sensor and O2-dependent transducer of NO bioactivity. In contrast, it appears that rather than transducing a specific signal, albumin operates as a buffer to …

Plasma from women with severe pre-eclampsia increases microvascular permeability in an animal model in vivo.

Pre-eclampsia results in oedema, hypertension and proteinuria, and is associated with increased vascular permeability. A number of studies have pointed to the existence of a circulating macromolecule that induces this endothelial dysfunction. To test whether this circulating factor could increase vascular permeability, we have measured the effect of dialysed human plasma from pregnant women with mild or severe pre-eclampsia (pre-eclamptic toxaemia). Plasma was collected from patients with mild or severe pre-eclampsia and from normotensive women. Plasma was dialysed against frog Ringer's solution using a 12-14 kDa molecular-mass cut-off dialysis tubing. pi c (colloid osmotic pressure) was measured with a modified Hansen oncometer. Lp (hydraulic conductivity) and sigma (oncotic reflection coefficient) were measured in individually perfused frog mesenteric microvessels using the Landis-Michel technique during perfusion with dialysed plasma. Perfusion of vessels with normal plasma or plasma from patients with mild pre-eclampsia did not alter either Lp or sigma. However, plasma from patients with severe pre-eclampsia resulted in a 3.8+/-0.3-fold increase in Lp and a reduction in sigma from 0.96+/-0.03 to 0.80+/-0.11. There was a significant correlation between the change in sigma and the change in Lp, suggesting that the increase in permeability was due to an increase in pore size in these vessels. A circulating macromolecule in human plasma in severe pre-eclampsia is therefore able to increase vascular permeability in an animal model. The nature of the circulating macromolecule is not known, except that it is, or is bound to, a molecule greater than 12 kDa.