Syncytiotrophoblast Microvillous Membrane Research Articles

Altered expression of nutrient transporters in syncytiotrophoblast membranes in preeclampsia placentae

IntroductionExpression of nutrient transporters in the placenta affects fetal growth. This study reports the protein expression of nutrient transporters in the syncytial membranes [microvillous membrane (MVM) and basal membrane (BM)] of normotensive control and preeclampsia placentae. MethodsPlacentae were collected from fourteen normotensive control women and fourteen women with preeclampsia. The syncytiotrophoblast MVM and BM membranes were isolated. The protein expression of glucose transporter (GLUT1), vitamin B12 transporter (CD320) and fatty acid transporters (FATP2, FATP4) was assessed in both the membranes. ResultsComparison between membranes demonstrates similar CD320 protein expression in normotensive group whereas, in preeclampsia placentae it was higher in the BM as compared to MVM (p < 0.05). FATP2&4 protein expression was higher in the BM as compared to their respective MVM fraction in both the groups (p < 0.01 for both).Comparison between groups demonstrates higher GLUT1 expression in the MVM (p < 0.05) and BM (p < 0.05) whereas lower CD320 expression in the MVM (p < 0.05) of preeclampsia placentae as compared to their respective membranes in normotensive control. Furthermore, GLUT1 protein expression was positively associated and CD320 protein expression was negatively associated with maternal body mass index (BMI) (p < 0.05 for both). No difference was observed in the FATP2&4 protein expression. However, FATP4 protein expression was negatively associated with maternal blood pressure (p < 0.05 for MVM; p = 0.060 for BM) and birth weight (p < 0.05 for both membranes). DiscussionThe current study for the first time demonstrates differential expression of various transporters in the syncytiotrophoblast membranes of the preeclampsia placentae which may influence fetal growth.

Maternal glucagon-like peptide-1 is positively associated with fetal growth in pregnancies complicated with obesity.

Pregnant women with obesity are more likely to deliver infants who are large for gestational age (LGA). LGA is associated with increased perinatal morbidity and risk of developing metabolic disease later in life. However, the mechanisms underpinning fetal overgrowth remain to be fully established. Here, we identified maternal, placental, and fetal factors that are associated with fetal overgrowth in pregnant women with obesity. Maternal and umbilical cord plasma and placentas were collected from women with obesity delivering infants who were LGA (n=30) or appropriate for gestational age (AGA, n=21) at term. Maternal and umbilical cord plasma analytes were measured using multiplex sandwich assay and ELISA. Insulin/mechanistic target of rapamycin (mTOR) signaling activity was determined in placental homogenates. Amino acid transporter activity was measured in isolated syncytiotrophoblast microvillous membrane (MVM) and basal membrane (BM). Glucagon-like peptide-1 receptor (GLP-1R) protein expression and signaling were measured in cultured primary human trophoblast (PHT) cells. Maternal plasma glucagon-like peptide-1 (GLP-1) was higher in LGA pregnancies and positively correlated to birthweight. Umbilical cord plasma insulin, C-peptide, and GLP-1 were increased in obese-large for gestational age (OB-LGA) infants. LGA placentas were larger but showed no change in insulin/mTOR signaling or amino acid transport activity. GLP-1R protein was expressed in the MVM isolated from human placenta. GLP-1R activation stimulated protein kinase alpha (PKA), extracellular signal-regulated kinase-1 and-2 (ERK1/2), and mTOR pathways in PHT cells. Our results suggest elevated maternal GLP-1 may drive fetal overgrowth in obese pregnant women. We speculate that maternal GLP-1 acts as a novel regulator of fetal growth by promoting placental growth and function.

Effect of high altitude on human placental amino acid transport.

Women residing at high altitudes deliver infants of lower birth weight than at sea level. Birth weight correlates with placental system A-mediated amino acid transport capacity, and severe environmental hypoxia reduces system A activity in isolated trophoblast and the mouse placenta. However, the effect of high altitude on human placental amino acid transport remains unknown. We hypothesized that microvillous membrane (MVM) system A and system L amino acid transporter activity is lower in placentas of women living at high altitude compared with low-altitude controls. Placentas were collected at term from healthy pregnant women residing at high altitude (HA; >2,500 m; n = 14) or low altitude (LA; <1,700 m; n = 14) following planned, unlabored cesarean section. Birth weight, but not placenta weight, was 13% lower in HA pregnancies (2.88 ± 0.11 kg) compared with LA (3.30 ± 0.07 kg, P < 0.01). MVM erythropoietin receptor abundance, determined by immunoblot, was greater in HA than in LA placentas, consistent with lower placental oxygen levels at HA. However, there was no effect of altitude on MVM system A or L activity, determined by Na+-dependent [14C]methylaminoisobutyric acid uptake and [3H]leucine uptake, respectively. MVM abundance of glucose transporters (GLUTs) 1 and 4 and basal membrane GLUT4 were also similar in LA and HA placentas. Low birth weights in the neonates of women residing at high altitude are not a consequence of reduced placental amino acid transport capacity. These observations are in general agreement with studies of IUGR babies at low altitude, in which MVM system A activity is downregulated only in growth-restricted babies with significant compromise.NEW & NOTEWORTHY Babies born at high altitude are smaller than at sea level. Birth weight is dependent on growth in utero and, in turn, placental nutrient transport. We determined amino acid transport capacity in placentas collected from women resident at low and high altitude. Altitude did not affect system A amino acid transport across the syncytiotrophoblast microvillous membrane, suggesting that impaired placental amino acid transport does not contribute to reduced birth weight in this high-altitude population.

Proteomic identification of potential targets in the syncytiotrophoblast microvillous membrane for placental-specific therapeutic delivery.

A famous conjecture of Artin states that there are infinitely many prime numbers for which a fixed integer g is a primitive root, provided g≠−1 and g is not a perfect square. Thanks to work of Hooley, we know that this conjecture is true, conditional on the truth of the Generalized Riemann Hypothesis. Using a combination of Hooley's analysis and the techniques of Maynard–Tao used to prove the existence of bounded gaps between primes, Pollack has shown that (conditional on GRH) there are bounded gaps between primes with a prescribed primitive root. In the present article, we provide an unconditional proof of the analogue of Pollack's work in the function field case; namely, that given a monic polynomial g(t) which is not an vth power for any prime v dividing q−1, there are bounded gaps between monic irreducible polynomials P(t) in Fq[t] for which g(t) is a primitive root (which is to say that g(t) generates the group of units modulo P(t)). In particular, we obtain bounded gaps between primitive polynomials, corresponding to the choice g(t)=t.

FT Raman spectroscopy in the evaluation of biomarkers of normal and pathological placenta tissue

The basic precondition of proper intrauterine growth is appropriate supply of nutrients transported through placenta. Placenta capacity in the scope of transportation is dependent on transport systems and the structure of the basement membrane and syncytiotrophoblast microvillous membrane. The present pilot study demonstrates preliminary results of the analysis of placenta structure in the course of selected pathologies by FT Raman spectroscopy analysis. The observed changes of the molecular structure in the so-called average spectra, independent of methodical processing, may be an indicator of the efficiency of transportation controlled by syncytiotrophoblast. In particular, an increase in the intensity of dispersion and transfer within the frequency of 3425–3300 cm−1 demonstrate the dynamics of the interaction in the scope of hydrogen bonds in healthy tissues. Changes in the molecular structure within the frequency of 950–750 cm−1 and conformational changes within disulphide bonds differentiate the healthy tissue from the pathological one. Changes in the molecular structure observed in the FTR spectra are a spectroscopic image of placenta functions in the course of various pathologies. They also document a complex goal of our research that is finding spectroscopic biomarkers of regular and pathological placental tissue.

Apelin is a novel regulator of human trophoblast amino acid transport.

Apelin is an insulin-sensitizing hormone increased in abundance with obesity. Apelin and its receptor, APJ, are expressed in the human placenta, but whether apelin regulates placental function in normal body mass index (BMI) and obese pregnant women remains unknown. We hypothesized that apelin stimulates amino acid transport in cultured primary human trophoblast (PHT) cells and that maternal circulating apelin levels are elevated in obese pregnant women delivering large babies. Treating PHT cells with physiological concentrations of the pyroglutamated form [Pyr1]apelin-13 (0.1-10.0 ng/ml) for 24 h dose-dependently increased System A amino acid transport (P < 0.05) but did not affect System L transport activity. Mechanistic target of rapamycin (mTOR), extracellular signal-regulated kinase-1/2 (ERK1/2), and AMP-activated protein kinase-α (AMPKα) signaling were unaffected by apelin (P > 0.05). Plasma apelin was not different in obese women (BMI 35.8 ± 0.7, n = 21) with large babies compared with normal-BMI women (23.1 ± 0.5, n = 16) delivering normal birth weight infants. Apelin was highly expressed in placental villous tissue (20-fold higher vs. adipose), and APJ was present in syncytiotrophoblast microvillous membrane, but neither differed in abundance between normal-BMI and obese women. Phosphorylation (Thr172) of placental AMPKα strongly correlated with microvillous membrane APJ expression (P < 0.01, R = 0.63) but negatively correlated with placental apelin abundance (P < 0.01, R = -0.62). Neither placental APJ nor apelin abundance correlated with maternal BMI, plasma insulin, birth weight, or mTOR or ERK1/2 signaling (P > 0.05). Hence, apelin stimulates trophoblast amino acid uptake, establishing a novel mechanism regulating placental function. We found no evidence that apelin constitutes an endocrine link between maternal obesity and fetal overgrowth.

Placental amino acid transporter function adapts to maternal and fetal nutrient conditions

s / Placenta 35 (2014) A1eA23 A10 O-026. PLACENTAL AMINO ACID TRANSPORTER FUNCTION ADAPTS TO MATERNAL AND FETAL NUTRIENT CONDITIONS Eiji Shibata David, J. Askew, Yukiyo Aiko, Chiharu Tomonaga, Satoshi Aramaki, Hirohide Inagaki, Toru Hachisuga. Department of Obstetrics and Gynecology, University of Occupational and Environmental Health, Japan Background: Studies suggest that placental nutrient uptake activity is responsive to both maternal and fetal nutrient demands. We hypothesize that under conditions of limited nutrient availability to the fetus, as often present in preeclampsia (PE), IUGR, and insufficient weight-gain during pregnancy, a general adaptive response aimed to increase amino acid transport activity may be observed in the placenta. Method: A total of 40 placentas from full-term (n1⁄410) and pre-term (n1⁄410) normal pregnancies, IUGR (n1⁄410), and PE (n1⁄410) associated pregnancies were looked at by immunohistochemistry followed by relative qualitative scoring to compare expression levels and localization of System L, ASCT2, and mTOR proteins. Result: Syncytiotrophoblast (ST) in placenta of pregnancies complicated by IUGR or PE showed significant increases in the levels of 4F2hc and LAT1 compared to both full-term control and pre-term pregnancies seperately. Elevated mTOR protein was uniquely higher in IUGR placentas compared to full-term controls. Total cellular ASCT2 transporter protein levels were similar in all groups, however, levels of ASCT2 protein localized to the ST microvillous membrane (MVM) were significantly lower in IUGR compared to both full-term and pre-term pregnancies. Additionally, ASCT2 and mTOR protein levels were positively associated with maternal prepregnancy BMI. Conclusion: First, in conditions of limited nutrient availability, such as PE or IUGR, there is an overall increase in the level of System L and mTOR protein expression in the ST, suggestive of an adaptive response. Second, a decrease in ASCT2 protein at the ST MVM suggests a post-translational event that may decrease AAT activity in IUGR placentas. Third, a physiological link between transporter expression and pre-pregnancy BMI is suggested based upon a positive association observed with ASCT2 and mTOR expression values. O-027. ENDOMETRIAL PERIVASCULAR CELLS ESTABLISH UNIQUE CYTOKINE GRADIENTS IN DECIDUAL PROCESS Keisuke Murakami , Keiji Kuroda , Shintaro Makino , Atsuo Itakura , Satoru Takeda , Jan J. Brosens . Department of Obstetrics and Gynaecology, Juntendo University Faculty of Medicine, Japan; Division of Reproductive Health, Clinical Science Research Laboratories, Warwick Medical School, University of Warwick, Japan Aim: The endometrial perivascular microenvironment is rich in mesenchymal stem-like cells that express type 1 integral membrane protein Sushi domain containing 2 (SUSD2) but the role of these cells in the decidual transformation of this tissue in pregnancy is unknown. We used an antibody directed against SUSD2 to isolate perivascular (SUSD2+) and non-perivascular (SUSD2-) fibroblasts from mid-luteal biopsies. Method: SUSD2+ and SUSD2cells were purified from mid-luteal endometrial biopsies by magnetic bead separation and each cell population were expanded in vitro, then, subjected to RNA-seq analysis or differentiation experiments induced by cAMP andMPA to assess secretome profile. Result: On average, positive cells comprised 6.8% of endometrial stromal cells and 6-fold enriched in clonogenic cells. RNA sequencing revealed that cultures derived from SUSD2+ progenitor cells remain phenotypically distinct by the enrichment of novel and established endometrial perivascular signature genes including AOC3, NOTCH3, ELN, MYH11, DES, ACTA2, MCAM, and CSPG4. Decidualization transformed perivascular SUSD2+ -derived cells into the dominant source of a range of chemokines and cytokines, including CCL7, LIF and LTA. Conclusion: Our findings confirmed that SUSD2+ -derived cells remain the expression of perivascular signature genes even after in-vitro culture. Decidual response is spatially organized at the feto-maternal interface with differentiating perivascular cells establishing distinct cytokine and chemokine gradients that could have the key role in decidual process. O-028. LOCALIZATION OF TRPV5 (TRANSIENT RECEPTOR POTENTIAL VANILLOID RECEPTOR 5) PROTEIN IN HUMAN PLACENTAL TROPHOBLASTS Yoko Aoyama, David J. Askew, Yukiyo Aiko, Chiharu Tomonaga, Satoshi Aramaki, Hirohide Inagaki, Toru Hachisuga, Eiji Shibata. Department of Obstetrics and Gynecology, University of Occupational and Environmental

Down‐regulation of placental mTOR, insulin/IGF‐I signaling, and nutrient transporters in response to maternal nutrient restriction in the baboon

The mechanisms by which maternal nutrient restriction (MNR) causes reduced fetal growth are poorly understood. We hypothesized that MNR inhibits placental mechanistic target of rapamycin (mTOR) and insulin/IGF-I signaling, down-regulates placental nutrient transporters, and decreases fetal amino acid levels. Pregnant baboons were fed control (ad libitum, n=11) or an MNR diet (70% of controls, n=11) from gestational day (GD) 30. Placenta and umbilical blood were collected at GD 165. Western blot was used to determine the phosphorylation of proteins in the mTOR, insulin/IGF-I, ERK1/2, and GSK-3 signaling pathways in placental homogenates and expression of glucose transporter 1 (GLUT-1), taurine transporter (TAUT), sodium-dependent neutral amino acid transporter (SNAT), and large neutral amino acid transporter (LAT) isoforms in syncytiotrophoblast microvillous membranes (MVMs). MNR reduced fetal weights by 13%, lowered fetal plasma concentrations of essential amino acids, and decreased the phosphorylation of placental S6K, S6 ribosomal protein, 4E-BP1, IRS-1, Akt, ERK-1/2, and GSK-3. MVM protein expression of GLUT-1, TAUT, SNAT-2 and LAT-1/2 was reduced in MNR. This is the first study in primates exploring placental responses to maternal undernutrition. Inhibition of placental mTOR and insulin/IGF-I signaling resulting in down-regulation of placental nutrient transporters may link maternal undernutrition to restricted fetal growth.

Electron and Scanning Microscopic Observations on the Syncytiotrophoblast Microvillous Membrane Contribution to Preeclampsia in Early Placental Rats

Pre-eclampsia is a pregnancy-specific syndrome characterized by new-onset hypertension and proteinuria, occurring usually after 20 weeks’ gestation. The current study was carried out on 60 female Wistar rats. Group I: included virgin non-pregnant rats. Group II: included pregnant rats that were received saline solution (0.5 ml/100 g body weight) subcutaneously daily starting from day 7 to day 14 of gestation and served as control group. Group III: included pregnant rats that were treated with bestatin dissolved in saline in a dose of (40.0 μg/ml)/100 g body weight subcutaneously and daily starting from the same day of gestation and for the same duration as mentioned for group II, to make an animal model of preeclampsia. Hence several possible mechanisms of the activation in pre-eclampsia can be considered, all dependent on the syncytiotrophoblast microvillous surface membrane which is the placental surface in contact with maternal blood. Electron microscopic study revealed regions of syncytiotrophoblast in process of degenerative change were found. These regions were more abundant in pre-eclamptic specimens. Increase irregular deeply indented nuclei of the syncytiotrophoblast layer. Regions of syncytiotrophoblast revealed dilated cisterns of rough endoplasmic reticulum. The objectives of this study were to develop an approach to definitively identify and distinguish the syncytiotrophoblast ultrastructural changes to unambiguously determine the relative susceptibility to constitutive and placental oxidative stressinduced preeclampsia.

Expression of an electrically silent voltage-gated potassium channel in the human placenta

Human placental expression of KV9.3, a voltage-gated K channel linked to tissue oxygenation responses, has been suggested at the messenger RNA level but tissue localisation has not been described. We aimed to: (1) produce an antibody to human KV9.3 and (2) assess channel expression and distribution in human placental tissue. We determined human placental protein expression and localisation using an antibody to KV9.3. Antibody specificity was confirmed by Western blotting. Staining was observed in syncytiotrophoblast microvillous membrane, endothelial cells (in intermediate, stem villi and chorionic plate blood vessels) and vascular smooth muscle cells (large diameter vessels only) by immunohistochemistry. Expression was unchanged in tissue from women with small-for-gestational age babies. It was concluded that KV9.3 is localised to human placental vascular tissues and syncytiotrophoblast.

Differential expression of angiotensin II type 1 and type 2 receptors at the maternal–fetal interface: potential roles in early placental development

Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal-fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal-fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

Placental protein 13 (galectin-13) has decreased placental expression but increased shedding and maternal serum concentrations in patients presenting with preterm pre-eclampsia and HELLP syndrome

Placental protein 13 (PP13) is a galectin expressed by the syncytiotrophoblast. Women who subsequently develop preterm pre-eclampsia have low first trimester maternal serum PP13 concentrations. This study revealed that third trimester maternal serum PP13 concentration increased with gestational age in normal pregnancies (p < 0.0001), and it was significantly higher in women presenting with preterm pre-eclampsia (p = 0.02) and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome (p = 0.01) than in preterm controls. Conversely, placental PP13 mRNA (p = 0.03) and protein, as well as cytoplasmic PP13 staining of the syncytiotrophoblast (p < 0.05) was decreased in these pathological pregnancies compared to controls. No differences in placental expression and serum concentrations of PP13 were found at term between patients with pre-eclampsia and control women. In contrast, the immunoreactivity of the syncytiotrophoblast microvillous membrane was stronger in both term and preterm pre-eclampsia and HELLP syndrome than in controls. Moreover, large syncytial cytoplasm protrusions, membrane blebs and shed microparticles strongly stained for PP13 in pre-eclampsia and HELLP syndrome. In conclusion, parallel to its decreased placental expression, an augmented membrane shedding of PP13 contributes to the increased third trimester maternal serum PP13 concentrations in women with preterm pre-eclampsia and HELLP syndrome.

Activity and expression of Na+/H+ exchanger isoforms in the syncytiotrophoblast of the human placenta

The purpose of this study was to compare Na+/H+ exchanger (NHE) activity in the microvillous (MVM) and basal (BM) plasma membrane of the human placental syncytiotrophoblast and to determine the relative contribution of various NHE isoforms to this activity. Uptake of 22Na into isolated MVM vesicles in the presence of a H+ gradient, at initial rate, was four- to fivefold higher than that by BM vesicles (214+/-28 vs. 49+/-9 pmol/mg protein per 30 s, respectively, means+/-SEM, n=8, 6, P<0.001). The 22Na uptake by MVM, but not by BM, was reduced in the absence of a H+ gradient and in the presence of 500 microM amiloride. To determine the contribution of NHE1, NHE2 and NHE3 isoforms to NHE activity in MVM, we investigated the effect of amiloride analogues which show isoform selectivity. HOE 694, an analogue selective for NHE1 at low concentrations, inhibited 22Na uptake with an EC50 of 0.13+/-0.05 microM (n=6), whereas S3226, an analogue selective for NHE3 at low concentrations had an EC50 of 3.01+/-0.85 microM (n=5). To investigate this further, we measured recovery of syncytiotrophoblast intracellular pH (pHi) from an acid load using a H+-selective, fluorescent dye (BCECF) loaded into isolated intact placental fragments. This recovery was blocked in the absence of Na+ and the presence of amiloride (500 microM) and concentrations of HOE 694 and S3226 were comparable to those used in vesicle experiments. Overall these data show that under the conditions used NHE activity in the term placental syncytiotrophoblast is absent from BM. NHE activity in the MVM is attributable predominantly to NHE1.

Non-Gastric H +/K +ATPase is Present in the Microvillous Membrane of the Human Placental Syncytiotrophoblast

In humans, the non-gastric H +/K +ATPase (ATP1AL1) has previously been shown to be expressed in the epithelia of skin, kidney and colon. In this study we tested the hypothesis that the non-gastric H +/K +ATPase is localized to the syncytiotrophoblast, the transporting epithelium of the human placenta. Microvillous (MVM) and basal plasma membranes (BM) of the syncytiotrophoblast were isolated from term placenta and membrane proteins were separated using SDS–PAGE. The ATP1AL1 protein was identified as a 114 kD band in both MVM and BM by Western blot, however, the protein was more abundant in the MVM. Using immunocytochemistry H +/K +ATPase protein was localized in MVM but not BM. We constructed primers specific for ATP1AL1 and performed RT–PCR on RNA isolated from human placenta and human kidney. A product of the expected size could be detected in both tissues after 30 cycles of amplification. The sequence identity of this 517 nucleotide product was confirmed by sequencing and found to be identical to the human non-gastric H +/K +ATPase. The activity of this proton pump appears to be low in normal healthy placental at term, however, it is speculated that MVM non-gastric H +/K +ATPase may be important in pathological states. In conclusion, non-gastric H +/K +ATPase is present in the microvillous plasma membrane of the transporting epithelia of the human placenta.

Neutrophils are stimulated by syncytiotrophoblast microvillous membranes to generate superoxide radicals in women with preeclampsia

Objective This study was undertaken to determine whether syncytiotrophoblast microvillous membranes (STBMs) stimulate maternal neutrophils to produce superoxide radicals in women with preeclampsia. Study design Serum levels of tissue polypeptide antigen (TPA), which is a marker for STBM, were measured in 25 nulliparous women (10 with mild preeclampsia, 6 with severe preeclampsia, and 9 controls). Superoxide production by maternal neutrophils from cases and controls and by donor neutrophils cocultured with the STBMs from cases and controls was measured spectrophotometrically by reduction of ferricytochrome C. Results Maternal TPA levels were significantly greater among cases than controls ( P = .005). Superoxide production by maternal neutrophils and donor neutrophils cultured with STBM from cases of preeclampsia was greater than controls ( P values .006 and .019, respectively), and dose-response relationships were observed. Superoxide production by maternal leukocytes was correlated with superoxide induction by STBMs in culture ( P = .007). Conclusion STBMs in maternal blood induce neutrophils to generate superoxide radicals that may cause endothelial dysfunction in women with preeclampsia.

Matrix Metalloprotease-9, Placental Syncytiotrophoblast and the Endothelial Dysfunction of Pre-eclampsia

The maternal syndrome of pre-eclampsia is caused by generalized maternal endothelial cell dysfunction, arising directly or indirectly from factors of placental origin. Syncytiotrophoblast membrane microvesicular particles are shed from the placental surface into maternal blood in increased amounts in pre-eclampsia and, in vitro, both inhibit endothelial cell proliferation and cause marked changes in the morphology of the cultured cell monolayers. Because there is evidence that proteolytic activation and degradation of the underlying matrix can cause the same morphological changes, we tested the hypothesis that proteases intrinsic to syncytiotrophoblast microvillous membranes (STBM) are the cause of the in vitro endothelial changes.Purified STBM were analysed by zymography and western blotting. Although we could confirm the presence of urokinase plasminogen activator (uPA) in STBM we could demonstrate no intrinsic activity presumably because of its association with the plasminogen activator inhibitor-2 (PAI-2) which is also a component of STBM. We detected gelatinase activity and showed that it was due to the matrix metalloproteinase-9 (MMP-9). Its presence was confirmed in this location by immunohistocytochemistry.Protease inhibitors caused a small reversal of the effects of STBM on morphology and no effect on inhibition of proliferation. We conclude that the effect of STBM on endothelial cells is unlikely to be caused by intrinsic proteases.

Functional transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane to Xenopus oocytes.

The materno-fetal transfer of metabolites and nutrients requires the operation of specific transport mechanisms through syncytiotrophoblast membranes. Electrophysiological studies on these cells are scarce and, because of their syncytial nature, whole-cell current recordings have not been carried out. We have now studied whether or not ion channels from the human syncytiotrophoblast microvillous (hSM) membrane can be transplanted to Xenopus oocytes. Sixty-two percent of hSM-injected oocytes displayed lower resting potential and higher membrane conductance than uninjected cells. The increased membrane conductance was due to the incorporation of Cl(-) channels, because neither replacing Na(+) in the bathing solution by N-methyl- D-glucamine or K(+), nor withdrawing Ca(2+) had any significant effect on the currents elicited by voltage pulses. In contrast, substitution of Cl(-) by different anions markedly affected the membrane conductance, giving an anion selectivity sequence of I(-)>Br(-)>Cl(-)>methanosulfonate congruent with gluconate. In addition, disulfonic stilbenes and gluconate, but not anthracene-9-carboxylic acid, blocked the transplanted channels. These properties are compatible with those of placental Cl(-) "maxi" channels. It is concluded that functional Cl(-) channels from the hSM become effectively incorporated into the Xenopus oocyte membrane, where their function can be studied in detail.

Glucose Transport and System A Activity in Syncytiotrophoblast Microvillous and Basal Plasma Membranes in Intrauterine Growth Restriction

The mechanisms underlying the reduced fetal plasma concentrations of amino acids and glucose associated with intrauterine growth restriction (IUGR) remain to be fully established. The activity of the amino acid transporter system A has been shown to be reduced in the syncytiotrophoblast microvillous membrane (MVM) in IUGR, however the impact of these changes on transplacental transport is difficult to assess without information on system A activity in the basal plasma membrane (BM). In this study we measured system A activity and mediated D-glucose uptake using radiolabelled substrates and rapid filtration techniques, and glucose transporter isoform 1 (GLUT 1) protein expression using Western blots in MVM and BM isolated from human placentas. In term IUGR ( n=11) MVM system A activity was unaltered compared to controls ( n=9). In contrast, system A activity in MVM was reduced by 50 per cent ( P< 0.05) in preterm IUGR ( n=8, gestational age 28–36 weeks) as compared to controls ( n=8, gestational age 28–35 weeks). BM system A activity was unaltered in both IUGR groups. Similarly, MVM and BM GLUT 1 expression and mediated D-glucose uptake was not affected by IUGR. In all preterm IUGR pregnancies signs of severe fetal compromise were present whereas term IUGR fetuses were less affected. These data support the view that MVM system A activity is related to the severity of compromise in IUGR. The markedly reduced system A activity in MVM in preterm IUGR together with the unaltered activity in BM is consistent with a decreased transplacental transport of neutral amino acids in this pregnancy complication. The hypoglycemia present in utero in some IUGR fetuses is not caused by a decreased glucose transport capacity across the syncytiotrophoblast plasma membranes.

Downregulation of Placental Syncytin Expression and Abnormal Protein Localization in Pre-eclampsia

Development of placentation and successful pregnancy depend on co-ordinated interactions between the maternal decidua and myometrium, and the invasive properties of the fetal trophoblast. Syncytin, a protein encoded by the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W, is highly expressed in placental tissue. Previously, we have shown that the major site of syncytin expression is the placental syncytiotrophoblast, a fused multinuclear syncytium originating from cytotrophoblast cells. Here we present the first evidence that in pre-eclampsia, syncytin gene expression levels are dramatically reduced. Additionally, immunohistochemical examination of normal placentae and placentae from women with pre-eclampsia reveals that the syncytin protein in placental tissue from women with pre-eclampsia is localized improperly to the apical syncytiotrophoblast microvillous membrane as opposed to its normal location on the basal syncytiotrophoblast cytoplasmic membrane. Our previous results suggest that syncytin may mediate placental cytotrophoblast fusion in vivo and may play an important role in human placental morphogenesis. The present study suggests that altered expression of the syncytin gene, and altered cellular location of its protein product, may contribute to the aetiology of pre-eclampsia.

Endothelial function in myometrial resistance arteries of normal pregnant women perfused with syncytiotrophoblast microvillous membranes.

To investigate the effects of syncytiotrophoblast microvillous membranes (STBM) in concentrations, found in vivo in women with pre-eclampsia, on endothelial function in isolated resistance arteries. Department of Obstetrics and Gynaecology, Huddinge University Hospital, Stockholm. Twenty-nine myometrial resistance arteries isolated from biopsies of healthy term pregnant women, obtained during caesarean section. The myometrial arteries were mounted in a pressure arteriograph and perfused intraluminally for three hours with STBM (20 to 2000ng/mL) or with erythrocyte membranes or physiological salt solution as controls, all substituted with 0.5% bovine serum albumin. Bradykinin concentration-response curves were performed before and after perfusion. The bradykinin concentrationresponse curves were fitted to the Hill equation and maximal dilation and the pEC50 values were determined from these fits. Differences within groups were analysed with a paired Student's t test. Electron microscopic evaluation of the endothelium was performed. Neither STBM nor erythrocyte membrane perfusion affected maximal dilation or the pEC50 values of the bradykinin concentration-response curves at any concentration. Examination by electron microscopy showed no obvious damage to the endothelium after perfusion with STBM or erythrocyte membranes. Perfusion with STBM in concentrations up to 100 times those reported in pre-eclampsia has no significant effect on bradykinin-mediated dilation in isolated myometrial arteries.