Materno-fetal nutrient transfer across primary human trophoblast layer.

MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionallyon cultured primary trophoblasts, or bidirectionally on the Transwell® system using BeWo cells treated with forskolin. As forskolin can induce various gene alterations (e.g. cAMP response element genes), we aimed to establish a physiological primary trophoblast model for materno-fetal nutrient exchange studies without forskolin application. Methods: Human term cytotrophoblasts were isolated by enzymatic digestion and Percoll® gradient separation. The purity of the primary cells was assessed by flow cytometry using the trophoblast-specific marker cytokeratin-7. After screening different coating matrices, we optimized the growth conditions for the primary cytotrophoblasts on Transwell/ inserts. The morphology of 5 days cultured trophoblasts was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally transport studies were performed on the polarized trophoblasts in the Transwell® system. Results: During 5 days culture, the trophoblasts (>90% purity) developed a modest trans-epithelial electrical resistance (TEER) and a sizedependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ~400-70’000D). SEM analyses confirmed a confluent trophoblast layer with numerous microvilli at day six, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein ZO-1, and the membrane proteins ABCA1 and Na+/K+-ATPase. Vectorial glucose and cholesterol transport studies confirmed functionality of the cultured trophoblast barrier. Conclusion: Evidence from cell morphology, biophysical parameters and cell marker expressions indicate the successful and reproducible establishment of a primary trophoblast monolayer model suitable for transport studies. Application of this model to pathological trophoblasts will help to better understand the mechanism underlying gestational diseases, and to define the consequences of placental pathology on materno-fetal nutrient transport.

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.

IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue.

Importance of cholesterol and cholesterol transporters in the placental trophoblast during pregnancy

Membrane transporters are essential during pregnancy, being a core component of the exchange of nutrients, gases, and metabolic products between the mother and the developing fetus. Important compounds to be transported include vitamins and minerals, amino acids, glucose, as well as cholesterol. Cholesterol transport across the plasma membrane is mediated mainly by members of the ATP-binding cassette (ABC) transporter family. Cholesterol is present in every cell of the body, where it helps maintain the integrity of cell membranes and also plays an important role in cell signaling events. Cholesterol also acts as a precursor for the biosynthesis of steroids that include sex hormones, glucocorticoids, mineralcorticoids, as well as bile acids and oxysterols. Cholesterol transport is therefore crucial for a host of different physiological processes. The following chapter addresses the involvement and importance of ABC transporters in these different processes. The critical role that ABC transporters Play for a successful pregnancy outcome is highlighted by pathological processes that result malfunction of cholesterol transport during pregnancy. Avenues of future research are also described, which may help to further delineate the function and mechanism of action of ABC transporters.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Rapid fusion and syncytium formation of heterologous cells upon expression of the FGFRL1 receptor

The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.

Regulation of placental growth by aldosterone and cortisol

During pregnancy, trophoblasts grow to adapt the feto-maternal unit to fetal requirements. Aldosterone and cortisol levels increase, the latter being inactivated by a healthy placenta. By contrast, preeclamptic placental growth is reduced while aldosterone levels are low and placental cortisol tissue levels are high due to improper deactivation. Aldosterone acts as a growth factor in many tissues, whereas cortisol inhibits growth. We hypothesized that in preeclampsia low aldosterone and enhanced cortisol availability might mutually affect placental growth and function. Proliferation of cultured human trophoblasts was time- and dose-dependently increased with aldosterone (P < 0.04 to P < 0.0001) and inhibited by spironolactone and glucocorticoids (P < 0.01). Mineralo- and glucocorticoid receptor expression and activation upon agonist stimulation was verified by visualization of nuclear translocation of the receptors. Functional aldosterone deficiency simulated in pregnant mice by spironolactone treatment (15 μg/g body weight/day) led to a reduced fetal umbilical blood flow (P < 0.05). In rat (P < 0.05; R(2) = 0.2055) and human (X(2) = 3.85; P = 0.0249) pregnancy, placental size was positively related to plasma aldosterone. Autocrine production of these steroid hormones was excluded functionally and via the absence of specific enzymatic transcripts for CYP11B2 and CYP11B1. In conclusion, activation of mineralocorticoid receptors by maternal aldosterone appears to be required for trophoblast growth and a normal feto-placental function. Thus, low aldosterone levels and enhanced cortisol availability may be one explanation for the reduced placental size in preeclampsia and related disorders.

Does Aldosterone participate in placental angiogenesis via PLGF?

Introduction Angiogenic signals are a vital signal of placental integrity. Aldosterone has recently been shown to enhance placental growth factor (PlGF) expression in the peripheral vasculature [1] and to promote trophoblast growth [2]. The plgf gene possesses a functional mineralocorticoid receptor responsive element in the promoter region. Objectives Thus, we hypothesized that aldosterone adapts placental angiogenesis to trophoblast growth by secreting PlGF. Methods The human choriocarcinoma cell line BeWo and first and third trimester human primary trophoblasts cells were subjected to several syncytialization signals. Upon visual confirmation, the cultured cells were subjected to either control conditions, the known stimulator forskolin, and increasing amounts of aldosterone (10−9 to 10−6 M) with and without the competitive aldosterone receptor blocker spironolactone. After 6 and 24 h of incubation, RNA and protein were extracted. PlGF transcripts were quantified by Taqman PCR normalized to several housekeeping genes. Protein expression was quantified by ELISA. Results PlGF mRNA expression increased 3-fold with forskolin in BeWo cells. In this cell line, aldosterone could slightly stimulate PlGF production. In non-syncytialized primary human first trimester trophoblasts, aldosterone did not exert a specific effect. In contrast, the term primary human trophoblasts did respond with a 2.5-fold increase after incubation with aldosterone (10−7 M) in the presence of forskolin to allow forming a syncytial layer. PlGF protein was already slightly upregulated following 6 h of incubation with aldosterone. Conclusion We concluded that aldosterone does regulate PlGF expression in specified conditions during pregnancy. Inappropriately low aldosterone levels such as in preeclampsia might such not only compromise plasma volume and trophoblast growth but also placental vascularization and systemic PlGF availability. These observations merit further investigation.

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.

11beta-Hydroxysteroid dehydrogenase type 2 in pregnancy and preeclampsia

Cortisol availability is controlled by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which inactivates cortisol in cortisone, unable to bind to the glucocorticoid receptor. The 11beta-HSD2 enzyme activity limits either intracellular cortisol concentrations or within the uteroplacental compartment the transfer of cortisol into the fetal circulation. Mechanisms, by which 11beta-HSD2 activity is controlled, include transcriptional control, posttranscriptional modifications of 11beta-HSD2 transcript half-life, epigenetic regulation via methylation of genomic DNA and direct inhibition of enzymatic activity. The 11beta-HSD2 expression and activity is reduced in preeclampsia and the enzyme activity correlates with factors associated with increased vasoconstriction, such as an increased angiotensin II receptor subtype 1 expression, and notably fetal growth. Numerous signals such as proinflammatory cytokines known to be present and/or elevated in preeclampsia regulate 11beta-HSD2 activity. Shallow trophoblast invasion with the resulting hypoxemia seems to critically reduce available 11beta-HSD2 activity. A positive feedback exists as activated glucocorticoid receptors do enhance 11beta-HSD2 mRNA transcription and mRNA stability. No data are currently available on pregnancy and either epigenetic or direct effects on the activity of the translated enzyme.

Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts

The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

Hypoxic treatment of human dual placental perfusion induces a preeclampsia-like inflammatory response.

Preeclampsia is a human pregnancy-specific disorder characterized by a placental pro-inflammatory response in combination with an imbalance of angiogenic factors and clinical symptoms, including hypertension and proteinuria. Insufficient uteroplacental oxygenation in preeclampsia due to impaired trophoblast invasion during placentation is believed to be responsible for many of the molecular events leading to the clinical manifestations of this disease. We investigated the use of hypoxic treatment of the dual placental perfusion system as a model for preeclampsia. A modified perfusion technique allowed us to achieve a mean soluble oxygen tension within the intervillous space (IVS) of 5-7% for normoxia and <3% for hypoxia (as a model for preeclampsia). We assayed for the levels of different inflammatory cytokines, oxidative stress markers, as well as other factors, such as endothelin (ET)-1 that are known to be implicated as part of the inflammatory response in preeclampsia. Our results show a significant increase under hypoxia in the levels of different inflammatory cytokines, including IL-6 (P=0.002), IL-8 (P<0.0001), TNF-α (P=0.032) and IFN-γ (P=0.009) at 360 min in maternal venous samples (n=6). There was also a significant increase in ET-1 levels under hypoxia both on the maternal side at 30 min (P=0.003) and fetal side at 360 min (P=0.036) (n=6). Other markers of oxidative stress, including malondialdehyde and 8-iso-protaglandin F2α (P=0.009) also show increased levels. Overall, these findings indicate that exposure of ex vivo dually perfused placental tissue to hypoxia provides a useful model for mimicking the inflammatory response characteristic of preeclampsia. This would therefore provide a powerful tool for studying and further delineating the molecular mechanisms involved in the underlying pathophysiology of preeclampsia.

Thrombosis during pregnancy: Risks, prevention, and treatment for mother and fetus--harvesting the power of omic technology, biomarkers and in vitro or in vivo models to facilitate the treatment of thrombosis

Maternal thromboembolism and a spectrum of placenta-mediated complications including the pre-eclampsia syndromes, fetal growth restriction, fetal loss, and abruption manifest a shared etiopathogenesis and predisposing risk factors. Furthermore, these maternal and fetal complications are often linked to subsequent maternal health consequences that comprise the metabolic syndrome, namely, thromboembolism, chronic hypertension, and type II diabetes. Traditionally, several lines of evidence have linked vasoconstriction, excessive thrombosis and inflammation, and impaired trophoblast invasion at the uteroplacental interface as hallmark features of the placental complications. "Omic" technologies and biomarker development have been largely based upon advances in vascular biology, improved understanding of the molecular basis and biochemical pathways responsible for the clinically relevant diseases, and increasingly robust large cohort and/or registry based studies. Advances in understanding of innate and adaptive immunity appear to play an important role in several pregnancy complications. Strategies aimed at improving prediction of these pregnancy complications are often incorporating hemodynamic blood flow data using non-invasive imaging technologies of the utero-placental and maternal circulations early in pregnancy. Some evidence suggests that a multiple marker approach will yield the best performing prediction tools, which may then in turn offer the possibility of early intervention to prevent or ameliorate these pregnancy complications. Prediction of maternal cardiovascular and non-cardiovascular consequences following pregnancy represents an important area of future research, which may have significant public health consequences not only for cardiovascular disease, but also for a variety of other disorders, such as autoimmune and neurodegenerative diseases.