Microvillous Membrane Research Articles

Localization of the placental BCRP/ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity

IntroductionThe breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. MethodsBCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 μM, 48 h). Results and discussionBCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts.

Placental Up-Regulation of Leptin and ARMS2 is Associated with Growth Discordance in Monochorionic Diamniotic Twin Pregnancies

Fetal growth discordance is a relatively common complication of monochorionic diamniotic (MCDA) twin pregnancies and is caused by a combination of maternal and placental factors. The aim of the study was to survey placental gene expression patterns and identify genes associated with growth discordance. Clinical samples comprised eight growth-discordant MCDA twin placentas (31+3-34+4 weeks gestational age) and six growth-concordant twin placentas (31+2-37 weeks gestational age). Gene expression libraries were constructed from placental biopsy samples and analyzed by RNA-sequencing. The distribution and relative abundance of mRNA transcripts expressed in the smaller and larger placentas from growth-discordant and concordant MCDA twins was remarkably similar. However, leptin (LEP) and age-related maculopathy susceptibility 2 (ARMS2) mRNA levels were exclusively up-regulated in all of the eight smaller growth-discordant twin placentas. Quantitative real-time PCR of independent biopsy samples confirmed the levels of differential mRNA expression for both genes. Immunohistochemical analysis of tissue sections from matching twin placentas showed increased leptin expression in 5-10% of blood vessel cells of the smaller placenta and marginally higher levels of ARMS2 expression in the microvillous membrane of the smaller placenta. Based on these findings, we speculate that up-regulation of leptin and ARMS2 forms part of an important survival mechanism to compensate for placental growth discordance. Since, leptin and ARMS2 are both expressed as soluble proteins, they may have clinical potential as measurable biomarkers for predicting the onset of growth discordance in MCDA twin pregnancies.

Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat.

System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and L-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and L-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [14C]α-methylaminoisobutyric acid ([14C]MeAIB) uptake, while 5mM L-arginine specifically and completely inhibited [3H]glycine uptake via human SNAT4, as well as [14C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [14C]MeAIB was almost completely inhibited by 20mM unlabeled MeAIB. L-Arginine (5mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and L-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

The influence of placental metabolism on fatty acid transfer to the fetus

The factors determining fatty acid transfer across the placenta are not fully understood. This study used a combined experimental and computational modeling approach to explore placental transfer of nonesterified fatty acids and identify the rate-determining processes. Isolated perfused human placenta was used to study the uptake and transfer of 13C-fatty acids and the release of endogenous fatty acids. Only 6.2 ± 0.8% of the maternal 13C-fatty acids taken up by the placenta was delivered to the fetal circulation. Of the unlabeled fatty acids released from endogenous lipid pools, 78 ± 5% was recovered in the maternal circulation and 22 ± 5% in the fetal circulation. Computational modeling indicated that fatty acid metabolism was necessary to explain the discrepancy between uptake and delivery of 13C-fatty acids. Without metabolism, the model overpredicts the fetal delivery of 13C-fatty acids 15-fold. Metabolic rate was predicted to be the main determinant of uptake from the maternal circulation. The microvillous membrane had a greater fatty acid transport capacity than the basal membrane. This study suggests that incorporation of fatty acids into placental lipid pools may modulate their transfer to the fetus. Future work needs to focus on the factors regulating fatty acid incorporation into lipid pools.

Down-regulation of the placental BCRP/ABCG2 transporter in response to hypoxia signaling

IntroductionThe BCRP/ABCG2 efflux transporter protects the developing fetus by limiting the transplacental transfer of drugs and chemicals and prevents the apoptosis of trophoblasts. The purpose of this study was to determine whether hypoxia-related signaling alters placental BCRP expression and function in vitro and in human pregnancies. MethodsHuman BeWo choriocarcinoma cells were treated with the hypoxia mimetic, cobalt chloride (CoCl2), or 3% oxygen for 24–48 h. Activation of HIF-1α signaling and regulation of BCRP was assessed using qPCR, ELISA, western blotting and a fluorescent substrate transport assay. In addition, healthy term placentas from high altitude pregnancies with chronic hypoxia were assessed for BCRP expression. ResultsCoCl2 and 3% oxygen increased HIF-1α protein signaling and decreased the mRNA and protein expression of BCRP by 30–75% in BeWo cells. Reduced BCRP expression corresponded with impaired efflux activity during hypoxia as evidenced by accumulation of the substrate Hoechst 33342. A number of transcription factors known to regulate BCRP, including AHR, NRF2 and PPARγ, were also coordinately down-regulated by 3% oxygen in BeWo cells. Moreover, women who gave birth at a high altitude (3100 m) exhibited signs of chronic placental hypoxia, including enhanced protein expression of the HIF-1α target GLUT1, and had reduced BCRP levels in microvillous membranes compared to women at a moderate altitude (1600 m). DiscussionThis study provides novel insight into the regulation of the placental BCRP transporter by hypoxia, which may be important for exposure of the fetus to chemicals during early development and in hypoxia-related pregnancy disorders.

Diagnostic and Research Aspects of Small Intestinal Disaccharidases in Coeliac Disease.

Disaccharidases (DS) are brush border enzymes embedded in the microvillous membrane of small intestinal enterocytes. In untreated coeliac disease (CD), a general decrease of DS activities is seen. This manuscript reviews different aspects of DS activities in CD: their utility in the diagnosis and their application to in vitro toxicity testing. The latter has never been established in CD research. However, with the recent advances in small intestinal organoid techniques, DS might be employed as a biomarker for in vitro studies. This includes establishment of self-renewing epithelial cells raised from tissue, which express differentiation markers, including the brush border enzymes. Determining duodenal DS activities may provide additional information during the diagnostic workup of CD: (i) quantify the severity of the observed histological lesions, (ii) provide predictive values for the grade of mucosal villous atrophy, and (iii) aid diagnosing CD where minor histological changes are seen. DS can also provide additional information to assess the response to a gluten-free diet as marked increase of their activities occurs four weeks after commencing it. Various endogenous and exogenous factors affecting DS might also be relevant when considering investigating the role of DS in other conditions including noncoeliac gluten sensitivity and DS deficiencies.

Microvilli Morphology Can Affect Efflux Active P-Glycoprotein in Confluent MDCKII -hMDR1-NKI and Caco-2 Cell Monolayers.

From fits of drug transport kinetics across confluent MDCKII-hMDR1-NKI and Caco-2 cell monolayers we estimated the levels of efflux active P-glycoprotein (P-gp) in these two cell lines (companion paper). In the present work, we compared the efflux active P-gp number to the total P-gp level, using liquid chromatography-tandem mass spectrometry, and showed that in Caco-2 cells total P-gp is about 10-fold greater than efflux active P-gp, whereas in MDCKII-hMDR1-NKI cells these values are within twofold. We further visualized the microvilli in MDCKII-hMDR1-NKI and Caco-2 cells using three-dimensional structured illumination super-resolution microscopy and found that the microvilli in Caco-2 cells are taller and more densely packed than those in MDCK-hMDR1-NKI cells. We hypothesized over 10 years ago that only P-gp at the tips of the microvilli contribute significantly to efflux activity, whereas the remaining P-gp are involved in a futile cycle of efflux of amphipathic drugs from the microvillus membrane, followed by their reabsorption into the same or nearby microvillous membranes. The difference between the levels of total and efflux active P-gp in Caco-2 cells can be explained by the more densely packed microvilli in Caco-2 cells, which would lead to a substantial fraction of P-gp not contributing to final release of drug into the apical chamber. Our results suggest that the effect of microvilli morphology differences between in vitro and in vivo systems must be considered when scaling transporter activity for efflux transporters of amphipathic compounds, for example, P-gp.

Protein expression of fatty acid transporter 2 is polarized to the trophoblast basal plasma membrane and increased in placentas from overweight/obese women

BackgroundObese and overweight women are more likely to deliver a large infant or an infant with increased adiposity, however the underlying mechanisms are not well established. We tested the hypothesis that placental capacity to transport fatty acid is increased in overweight/obese women. MethodsPregnant women with body mass index (BMI) ranging from 18.4 to 54.3 kg/m2 and with uncomplicated term pregnancies were recruited for collection of blood samples and placental tissue. Maternal and fetal levels of non-esterified fatty acids (NEFAs) were measured in plasma. The expression and localization of CD36/fatty acid translocase (FAT), fatty acid transport protein (FATP)2, and FATP4 was determined in fixed placental tissue and in isolated syncytiotrophoblast plasma membranes from normal and high BMI mothers. ResultsMaternal and fetal plasma NEFA levels did not correlate (n = 42). FATP2 and FATP4 expressions were higher in the basal plasma membrane (BPM) compared to the microvillous membrane (P < 0.001; n = 7) per unit membrane protein. BPM expression of FATP2 correlated with maternal BMI (P < 0.01; n = 30); there was no association between CD36/FAT or FATP4 expression and maternal BMI. ConclusionThe polarization of FATPs to the BPM will facilitate fatty acid transfer across the placenta. In overweight/obese pregnancies, the increased FATP2 expression could contribute to increased fatty acid delivery to the fetus and while we have no direct data we speculate that this could lead accelerated fetal growth or increased fat deposition.

Altered expression of human endogenous retroviruses syncytin-1, syncytin-2 and their receptors in human normal and gestational diabetic placenta.

Syncytins belong to the Human Endogenous Retrovirus family. The syncytin-1 receptor, SLC1A5, and syncytin-2 receptor, MFSD2, interact with their respective syncytin proteins to induce syncytiotrophoblast formation. However, there is no information about syncytins in gestational diabetic placenta. Therefore, we studied the expression and localization of syncytins and their receptors during normal placental development and in gestational diabetic placenta. Immunohistochemistry and Western-blot methods were performed with antibodies against syncytin-1, syncytin-2, SLC1A5 and MFSD2 in human first trimester placental tissues, normal term and gestational diabetic placentas. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts were determined by qRT-PCR in normal and diabetic term placentas. Cytoplasmic syncytin-1, syncytin-2, SLC1A5 and MFSD2 immunoreactions were observed in the trophoblastic layers in all placental samples. Some of the stromal cells showed strong cytoplasmic punctate staining. There were significantly weak syncytin-2 and MFSD2 immunoreaction intensities in diabetic placentas by ImageJ analysis, in parallel with decreased syncytin-2 and MFSD2 proteins in diabetic placentas by Western-blot. Protein expression of SLC1A5 increased dramatically in early pregnancy compared to term placenta. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts showed similar relative expression pattern by qRT-PCR. Syncytins were localized not only in cytotrophoblast cells and the basement membrane of the syncytiotrophoblast but also in the apical microvillous membrane and cytoplasm of syncytiotrophoblast, and some of the stromal cells and endothelium. Decreased syncytin-2 and MFSD2 proteins in gestational diabetic placentas might cause abnormal syncytiotrophoblast formation and possibly be involved in the pathology. Therefore, our study highlights an important potential relationship between syncytins and gestational diabetic placenta.

Cryptosporidiosis in Animals and Man: 2. Diagnosis

Different diagnostic methods including clinical examination, detection of endogenous developmental stages by histopathology and electron microscopy, detection of oocysts by concentration and purification of Cryptosporidium oocysts and staining techniques, immunological assays and molecular identification techniques are described. Clinical examination revealed that diarrhoea begins in infected neonates at 3-5 days post-infection and last for 4-17 days. High mortality due to cryptosporidiosis has been reported, even in the absence of other enteropathogens. Cryptosporidium infections can be diagnosed by histological examination of autopsy or biopsy material from the intestinal mucosa. Endogenous developmental stages can be recognized associated with villous atrophy, villous fusion and changes in the surface epithelium. Scanning and transmission electron microscopy have confirmed the intracellular but extra cytoplasmic location within parasitophorus vacuoles formed by a continuous covering of microvillous membranes. Many specialized staining procedures have been described to stain the wall and/or contents of mature oocysts. The stain of choice for many diagnostic laboratories has been acid-fast staining and safranine-methylene blue. Oocysts stained bright red whereas yeast, bacteria and other fecal debris only take up the counter stain (green or blue). More recently, several immunolabelling techniques using polyclonal or monoclonal antibodies have been developed to detect oocysts.

291: Gestational weight gain in twin pregnancies is not associated with discordance in birthweight or small for gestational age

291 Gestational weight gain in twin pregnancies is not associated with discordance in birthweight or small for gestational age Bahram Salmanian, Kathleen Antony, Diana Racusin, Kjersti Aagaard, Amirhoushang Shamshirsaz, Winston Campbell, Anthony Odibo, Alireza Shamshirsaz Baylor College of Medicine, Houston, TX, Washington University in St. Louis, St. Louis, MO, University of Connecticut, Storrs, CT, George Washington University, Washington, DC OBJECTIVE: Twins account for 3% of live births in the United States, and have a disproportionate burden of small-for-gestational age (SGA) and prematurity. Gestational weight gain guidelines (GWG) for women with twins are to gain significantly more weight than gravidae with singletons. The benefit of this increased GWG on birthweight has not been clearly demonstrated. We sought to apply published and original GWG guidelines for twins to assess association with growth discordance and SGA. STUDY DESIGN: This is a secondary analysis of a cohort of 600 gravidae carrying twins (1991-2011). GWG was categorized as insufficient, at goal, or excess using three different GWG guidelines: IOM guideline corrected for gestational age (GA), a validated gestational-week specific guideline (Guideline A), and a gestationalage specific guideline that we developed consisting of the IOM singleton GWG recommendation plus the contribution of an additional fetal-placental unit, including the estimated amniotic fluid weight, placental weight, and the “ideal” fetal weight (Guideline BK). The ideal fetal weight in this formula was calculated from coefficients for significant physiological variables affecting fetal growth, which were derived using backward stepwise multiple regression adjusting for chorionicity. The association between SGA <10th percentile defined using the twin chart versus traditional definitions of SGA were compared. Statistical analyses including adjusted OR, 95% confidence interval (CI), were performed. RESULTS: Using any of the three GWG models, GWG was not associated with growth discordance or SGA. CONCLUSION: GWG that is below the recommended IOM guidelines, below validated published guidelines, or our derived formula is not associated with growth discordance or SGA. The benefits of advising gravidae to gain significantly more weight than singletons do not appear to bear out in terms of twin growth. 292 Placental urate transport is GLUT9-mediated and unidirectional Benjamin Luscher, Camilla Marini, Xiao Huang, Christiane Albrecht, Matthias Hediger, Bruno Stieger, Daniel Surbek, Marc Baumann University Hospital of Bern, University of Bern, Departments of Obstetrics and Gynecology, Department of Clinical Research, Bern, Switzerland, University of Bern, Institute for Biochemestry and Molecular Medicine, Graduate School for Cellular and Biomedical Sciences (GCB), Bern, Switzerland, University of Bern, Institute for Biochemestry and Molecular Medicine, Bern, Switzerland, University Hospital of Zurich, Department of Clinical Pharmacology and Toxicology, Zurich, Switzerland OBJECTIVE: Pre-eclampsia is associated with increased maternal urate serum levels. Hyperuricemia might lead to long-term maternal cardiovascular risk and to fetal programming. The regulation of the placental urate transport system is not yet understood, but is likely to be predominantly regulated by GLUT-9 (glucose transporter 9), besides the multitransporter hOAT4 (human organic anion transporter). The aim of this study was to investigate the placental urate transport system and to characterize its transporter GLUT9. STUDY DESIGN: A transepithelial transport (Transwell ) model to assess urate transport activity was used. Electrophysiological techniques and radioactive ligand up-take assay were used to measure transport activity of GLUT9 overexpressed in Xenopus oocytes, and its activity under various conditions. We further analysed the placental GLUT9 expression using novel self-raised antibodies against human isoforms GLUT9a and -b. RESULTS: In the transwell system, urate is transported across the BeWo choriocarcinoma cell monolayer with 530 pmol/min. In the Xenopus oocytes system chloride replacing chloride with iodine resulted in a complete loss of urate transport. In radioactive up-take experiments iodine had no effect on urate transport. Both GLUT9 isoforms are present in the microvillous membrane (MVM) of the syncytiotrophoblast cells, but not in the basal membrane (BM). CONCLUSION: Our results for the first time show that urate transport in the placenta is unidirectional in the feto-maternal direction, because GLUT9 isoforms are expressed exclusively in the microvillous (apical) membrane of the trophoblast and because hOAT4 is known be localized on the basal membrane and to transport urate unidirectionally into trophoblast cells. Furthermore, GLUT9 uric acid transport activity is iodine-regulated by changing the mode of up-take from an electrogenic to an electroneutral transport. This may allow regulating urate transport by the change of an active to a passive transport. This study was funded by the Swiss National Fund NCCR grant “TransCure”

Computational modelling of amino acid exchange and facilitated transport in placental membrane vesicles.

Placental amino acid transport is required for fetal development and impaired transport has been associated with poor fetal growth. It is well known that placental amino acid transport is mediated by a broad array of specific membrane transporters with overlapping substrate specificity. However, it is not fully understood how these transporters function, both individually and as an integrated system. We propose that mathematical modelling could help in further elucidating the underlying mechanisms of how these transporters mediate placental amino acid transport.The aim of this work is to model the sodium independent transport of serine, which has been assumed to follow an obligatory exchange mechanism. However, previous amino acid uptake experiments in human placental microvillous plasma membrane vesicles have persistently produced results that are seemingly incompatible with such a mechanism; i.e. transport has been observed under zero-trans conditions, in the absence of internal substrates inside the vesicles to drive exchange. This observation raises two alternative hypotheses; (i) either exchange is not fully obligatory, or (ii) exchange is indeed obligatory, but an unforeseen initial concentration of amino acid substrate is present within the vesicle which could drive exchange.To investigate these possibilities, a mathematical model for tracer uptake was developed based on carrier mediated transport, which can represent either facilitated diffusion or obligatory exchange (also referred to as uniport and antiport mechanisms, respectively). In vitro measurements of serine uptake by placental microvillous membrane vesicles were carried out and the model applied to interpret the results based on the measured apparent Michaelis–Menten parameters Km and Vmax. In addition, based on model predictions, a new time series experiment was implemented to distinguish the hypothesised transporter mechanisms. Analysis of the results indicated the presence of a facilitated transport component, while based on the model no evidence for substantial levels of endogenous amino acids within the vesicle was found.

Na+, K+-ATPase and Ca2+-ATPase activities in basal and microvillous syncytiotrophoblast membranes from preeclamptic human term placenta

Objective: The aim of this study is to evaluate the effect of preeclampsia on the level of lipid peroxidation, activity and expression of both plasma membrane Ca2+- and Na+, K+-ATPases in syncytiotrophoblast. Methods: The level of lipid peroxidation was estimated by measuring TBARS. ATPase activities were quantified by a colorimetric method measuring the amount of inorganic phosphate during the assay. Expression of Ca2+- and Na+, K+-ATPases in syncytiotrophoblast plasma membranes and term placenta tissue sections was investigated using Western blot and immunohistochemistry, respectively. Results: Our results show a higher level of lipid peroxidation of syncytiotrophoblast plasma membranes from preeclamptic, as compared to uncomplicated pregnant women. Preeclampsia also significantly reduced the activity of Ca2+- and Na+, K+-ATPases; however, expression of both ATPases was unaffected. Conclusion: Our findings suggest that the reduction of Ca2+- and Na+, K+-ATPase activities during preeclampsia could be at least partially due to an increased level of lipid peroxidation of the syncytiotrophoblast plasma membranes.

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

Activation by intravesicular amino acids of system L-mediated serine uptake into human placental microvillous plasma membrane vesicles

s / Placenta 35 (2014) A1eA112 A99 Results: IF dams consumed 29±1% less food and gained less weight (P Ala>BCH>Pro>Gly>MeAIB>Met. Conclusion: The identity of amino acids present on the internal face of MVM dictates serine uptake capacity across MVM. The broad internal amino acid selectivity by system L in MVM indicates that both the composition and concentration of freely-exchangeable amino acids within syncytiotrophoblast is likely to regulate system L exchange capacity in MVM. Collectively these observations provide further experimental support for previous model predictions regarding serine uptake by system L exchange transporters. P2.123-N. STATUS ANALYSIS OF TUMOUR ASSOCIATED FACTORS IN HUMAN PLACENTAE AND STEM-LIKE CELLS DERIVED FROM PLACENTAL CELL LINES Rham Balahmar, Shiva Sivasubramaniam Nottingham Trent university,

Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I).

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.

A theoretical model of glucose transport suggests symmetric GLUT1 characteristics at placental membranes.

The process of glucose transport via the placenta is not fully deciphered. Here, we apply a theoretical model to compute glucose fluxes via the terminal villi of the human placenta for various sets of parameter values and conclude on characteristics of transport across the two bordering membranes. Based on available measured data, the spatial geometry of the terminal villi is being simulated. Within this region, glucose concentrations and fluxes are computed by a numerical scheme that solves the diffusion equation with boundary conditions that account for transporter mediated diffusion at the membranes. Feasible parameter values (ones that induce physiological glucose fluxes) are determined for four optional symmetry characteristics of the membranes. Confronting computed results with clinical knowledge reveals the most plausible scenario-symmetric activity of the transporter at the microvillous membrane. Thus, sensitivity analysis of the computed results enables deduction about micro-scale mechanisms at the bordering membranes based on macro-scale knowledge.

Differential levels of amino acid transporters System L and ASCT2, and the mTOR protein in placenta of preeclampsia and IUGR.

BackgroundSufficient amino acid transport activity (AAT) is indispensable for appropriate fetal growth. 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, intrauterine growth restriction (IUGR), and insufficient weight-gain during pregnancy, a general adaptive response aimed to increase amino acid transport activity may be observed in the placenta.MethodA total of 40 placentas from full-term (n = 10) and pre-term (average gestational period = 34.8 weeks, n = 10) normal pregnancies, IUGR (n = 10), and preeclampsia (n = 10) 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.ResultMicrovillous syncytiotrophoblast (ST) in placenta of pregnancies complicated by IUGR or preeclampsia (PE) showed significant increases in the levels of System L amino acid transport proteins 4F2hc and LAT1 compared to both full-term control and pre-term (early gestation control) pregnancies seperately (p < 0.05). Elevated mTOR protein was uniquely higher in IUGR placentas compared to full-term controls (P = 0.0026). 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 (P = 0.0006, 0.03, respectively). Additionally, ASCT2 and mTOR protein levels were positively associated with maternal pre-pregnancy BMI (P = 0.046, 0.048, respectively).ConclusionThere are three important findings based upon the present study. 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.

Expression and localization of the omega-3 fatty acid receptor GPR120 in human term placenta

Fatty acids can function as signaling molecules, acting through receptors in the cytosol or on the cell surface. G-Protein Receptor (GPR)120 is a membrane-bound receptor mediating anti-inflammatory and insulin-sensitizing effects of the omega-3 fatty acid docohexaenoic acid (DHA). GPR120 dysfunction is associated with obesity in humans. Cellular localization of GPR120 and the influence of maternal obesity on GPR120 protein expression in the placenta are unknown. Herein we demonstrate that GPR120 is predominantly expressed in the microvillous membrane (MVM) of human placenta and that the expression level of this receptor in MVM is not altered by maternal body mass index (BMI).

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.