Primary Human Trophoblasts Research Articles

Placental expression of the angiogenic placental growth factor is stimulated by both aldosterone and simulated starvation

Aldosterone is an important factor supporting placental growth and fetal development. Recently, expression of placental growth factor (PlGF) has been observed in response to aldosterone exposure in different models of atherosclerosis. Thus, we hypothesized that aldosterone up-regulates growth-adaptive angiogenesis in pregnancy, via increased placental PlGF expression.We followed normotensive pregnant women (n = 24) throughout pregnancy and confirmed these results in a second independent first trimester cohort (n = 36). Urinary tetrahydroaldosterone was measured by gas chromatography-mass spectrometry and corrected for creatinine. Circulating PlGF concentrations were determined by ELISA. Additionally, cultured cell lines, adrenocortical H295R and choriocarcinoma BeWo cells, as well as primary human third trimester trophoblasts were tested in vitro. PlGF serum concentrations positively correlated with urinary tetrahydroaldosterone corrected for creatinine in these two independent cohorts. This observation was not due to PlGF, which did not induce aldosterone production in cultured H295R cells. On the other hand, PlGF expression was specifically enhanced by aldosterone in the presence of forskolin (p < 0.01) in trophoblasts. A pronounced stimulation of PlGF expression was observed with reduced glucose concentrations simulating starvation (p < 0.001).In conclusion, aldosterone stimulates placental PlGF production, enhancing its availability during human pregnancy, a response amplified by reduced glucose supply. Given the crucial role of PlGF in maintaining a healthy pregnancy, these data support a key role of aldosterone for a healthy pregnancy outcome.

Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters.

The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by Cold vapor-atomic fluorescence spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. We found the amino acid transporter subunits L-type amino acid transporter (LAT)1 and rBAT (related to b(0,+) type amino acid transporter) as well as the efflux transporter multidrug resistance associated protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-test P<0.05). The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is effluxed via MRP1 localized to the basal side of the STB. The findings can well explain why mercury is transported primarily towards the fetal side.

Fibulin-5 is upregulated in decidualized human endometrial stromal cells and promotes primary human extravillous trophoblast outgrowth

Interactions between the highly invasive trophoblasts and the maternal uterine decidual extracellular matrix (ECM) are crucial in the determination of a successful pregnancy. Fibulin-5 (FBLN5) is a member of the fibulin family that alters cell adhesive and invasive properties and is expressed in human villous cytotrophoblasts.We aimed to determine the expression and immunolocalization of FBLN5 in human first trimester decidua and examine the effect of FBLN5 in trophoblast invasion in vitro using a first trimester placental villous outgrowth assay. We demonstrated that FBLN5 mRNA expression is upregulated in response to cAMP-mediated decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported for the first time, FBLN5 protein production in first trimester decidual cells and also co-localization to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of exogenous FBLN5 on placental villous outgrowth in vitro and demonstrated that FBLN5 promotes EVT migration/invasion. This is the first study to identify FBLN5 in decidualized human endometrial stromal cells, first trimester decidua and EVT and determine a functional role for FBLN5 in human EVTs, suggesting that decidual and or EVT-derived FBLN5 regulates EVT invasion and placentation in women.

Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR.

TNF-α stimulates System A amino acid transport in primary human trophoblast cells mediated by p38 MAPK signaling.

Maternal obesity and gestational diabetes mellitus (GDM) increase the risk of delivering infants that are large for gestational age with greater adiposity, who are prone to the development of metabolic disease in childhood and beyond. These maternal conditions are also associated with increased levels of the proinflammatory cytokine TNF-α in maternal tissues and the placenta. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. TNF-α was previously shown to stimulate System A amino acid transport in primary human trophoblasts (PHTs), however the molecular mechanisms remain unknown. In this study, we tested the hypothesis that TNF-α regulates amino acid uptake in cultured PHTs by a mitogen-activated protein kinase (MAPK)-dependent mechanism. Treatment of PHTs with TNF-α significantly increased System A amino acid transport, as well as Erk and p38 MAPK signaling. Pharmacological antagonism of p38, but not Erk MAPK activity, inhibited TNF-α stimulated System A activity. Silencing of p38 MAPK using siRNA transfections prevented TNF-α stimulated System A transport in PHTs. TNF-α significantly increased the protein expression of System A transporters SNAT1 and SNAT2, but did not affect their mRNA expression. The effects of TNF-α on SNAT1 and SNAT2 protein expression were reversed by p38 MAPK siRNA silencing. In conclusion, TNF-α regulates System A activity through increased SNAT1 and SNAT2 transporter protein expression in PHTs. These findings suggest that p38 MAPK may represent a critical mechanistic link between elevated proinflammatory cytokines and increased placental amino acid transport in obese and GDM pregnancies associated with fetal overgrowth.

The expression and post-transcriptional regulation of FSTL1 transcripts in placental trophoblasts

IntroductionFollistatin-like-1 (FSTL1) is a widely expressed secreted protein with diverse but poorly understood functions. Originally described as a pro-inflammatory molecule, it has recently been reported to play a role in signaling pathways that regulate development and homeostasis. Distinctively, FSTL1 harbors within its 3′-UTR the sequence encoding microRNA-198 (miR-198), shown to be inversely regulated relative to FSTL1 expression and to exhibit opposite actions on cellular processes such as cell migration. We sought to investigate the expression of FSTL1 and to assess its interplay with miR-198 in human trophoblasts. MethodsWe used a combination of northern blot analyses, quantitative PCR, small RNA sequencing, western blot and immunohistochemistry to characterize FSTL1 and miR-198 expression in placental trophoblasts. We also used reporter assays to examine the post-transcriptional regulation of FSTL1 and assess its putative regulation by miR-198. ResultsWe detected the expression of FSTL1 transcript in both the human extravillous trophoblast line HTR-8/SVneo and in primary term human villous trophoblasts. We also found that the expression of FSTL1 was largely restricted to extravillous trophoblasts. Hypoxia enhanced the expression of FSTL1 protein in cultured primary villous trophoblasts. Interestingly, we did not detect any evidence for expression or function of mature miR-198 in human trophoblasts. DiscussionOur data indicate that placental FSTL1 is expressed particularly in extravillous trophoblasts. We also found no evidence for placental expression of miR-198, or for its regulation of FSTL1, implying that the post-transcriptional regulation of FSTL1 by miR-198 is tissue specific.

Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo.

The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts.

What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. X.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing else to disclose. None.

Expression and functional characterisation of System L amino acid transporters in the human term placenta.

BackgroundSystem L transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) mediate the uptake of large, neutral amino acids in the human placenta. Many System L substrates are essential amino acids, thus representing crucial nutrients for the growing fetus. Both LAT isoforms are expressed in the human placenta, but the relative contribution of LAT1 and LAT2 to placental System L transport and their subcellular localisation are not well established. Moreover, the influence of maternal body mass index (BMI) on placental System L amino acid transport is poorly understood. Therefore the aims of this study were to determine: i) the relative contribution of the LAT isoforms to System L transport activity in primary human trophoblast (PHT) cells isolated from term placenta; ii) the subcellular localisation of LAT transporters in human placenta; and iii) placental expression and activity of System L transporters in response to maternal overweight/obesity.MethodsSystem L mediated leucine uptake was measured in PHT cells after treatment with si-RNA targeting LAT1 and/or LAT2. The localisation of LAT isoforms was studied in isolated microvillous plasma membranes (MVM) and basal membranes (BM) by Western blot analysis. Results were confirmed by immunohistochemistry in sections of human term placenta. Expression and activity System L transporters was measured in isolated MVM from women with varying pre-pregnancy BMI.ResultsBoth LAT1 and LAT2 isoforms contribute to System L transport activity in primary trophoblast cells from human term placenta. LAT1 and LAT2 transporters are highly expressed in the MVM of the syncytiotrophoblast layer at term. LAT2 is also localised in the basal membrane and in endothelial cells lining the fetal capillaries. Measurements in isolated MVM vesicles indicate that System L transporter expression and activity is not influenced by maternal BMI.ConclusionsLAT1 and LAT2 are present and functional in the syncytiotrophoblast MVM, whereas LAT2 is also expressed in the BM and in the fetal capillary endothelium. In contrast to placental System A and beta amino acid transporters, MVM System L activity is unaffected by maternal overweight/obesity.

N-myc downstream-regulated gene 1 (NDRG1) mediates pomegranate juice protection from apoptosis in hypoxic BeWo cells but not in primary human trophoblasts

IntroductionN-Myc downstream-regulated gene 1 (NDRG1) expression is increased in placentas of human pregnancies with intrauterine growth restriction and in hypoxic cultured primary trophoblasts. We previously showed that elevated NDRG1 decreases trophoblast apoptosis induced by hypoxia. Separately, we found that pomegranate juice (PJ) decreases cell death induced by hypoxia in trophoblasts. Here, we test the hypothesis that PJ protects trophoblasts from hypoxia-induced apoptosis by modulating NDRG1 expression. MethodsQuantitative rtPCR was used to investigate the effects of PJ treatment on mRNA levels of 22 candidate genes involved in apoptosis, oxidative stress, and differentiation in trophoblasts. Western blotting and immunofluorescence were used to analyze NDRG1 protein levels. siRNA-mediated NDRG1 knockdown was used to investigate the role of NDRG1 in response to PJ in hypoxic BeWo choriocarcinoma cells and hypoxic cultured primary human trophoblasts. ResultsThe mRNA levels of eight genes were altered, with NDRG1 showing the largest response to PJ and thus, we pursued the role of NDRG1 here. PJ significantly increased NDRG1 protein expression in primary trophoblasts and in BeWo cells. Knockdown of NDRG1 in hypoxic BeWo cells in the presence of PJ yielded increased apoptosis. In contrast, knockdown of NDRG1 in hypoxic primary trophoblasts in the presence of PJ did not increase apoptosis. DiscussionWe conclude that the PJ-mediated decrease in cell death in hypoxia is partially mediated by NDRG1 in BeWo cells but not in primary trophoblasts. The disparate effects of NDRG1 between BeWo cells and primary trophoblasts indicate caution is required when extrapolating from results obtained with cell lines to primary trophoblasts.

ΔNp63 Regulates HTRA4 Expression During Extravillous Trophoblast Differentiation

During early placentation, trophoblast cell columns help anchor the developing embryo into the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB), distally these cells differentiate into invasive extravillous trophoblast (EVT). In the absence of normal EVT differentiation, pregnancies can terminate early or be complicated by fetal growth restriction. We previously reported that ΔNp63, a p53 family member, is highly expressed in CTB but is lost when these cells further differentiate into EVT. We now further explore the downstream targets of p63 during EVT differentiation using both primary CTB and the JEG3 human trophoblast. Using gene microarrays, we identified HTRA4 as one such p63 target: HTRA4 is a member of the high temperature requirement A family of serine proteases, and have been shown to regulate trophoblast fusogenic activity and invasion. p63‐overexpressing JEG3 cells showed decreased expression of HTRA4, and decreased cell migration; conversely, down‐regulation of p63 increased HTRA4 expression and restored cell migration. In primary human trophoblast, isolated from early placental tissues, we found that HTRA4 is expressed at low levels in p63‐high EGFR+/HLAG‐ CTBs, and at high levels in p63‐low EGFR‐/HLAG+ EVTs. Taken together, these data indicate that EVT differentiation is accompanied by loss of p63 and gain of HTRA4. Our data reveal a potentially novel p63‐HTRA4 axis, involved in trophoblast migration and invasion.

Sofalcone upregulates the nuclear factor (erythroid-derived 2)-like 2/heme oxygenase-1 pathway, reduces soluble fms-like tyrosine kinase-1, and quenches endothelial dysfunction: potential therapeutic for preeclampsia.

Preeclampsia is a severe complication of pregnancy, characterized by hypertension, oxidative stress, and severe endothelial dysfunction. Antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin, play key pathophysiological roles in preeclampsia. Heme oxygenase-1 (HO-1) is a cytoprotective, antioxidant enzyme reported to be downregulated in preeclampsia. Studies propose that inducing HO-1 may also decrease sFlt-1 production. Sofalcone, a gastric antiulcer agent in clinical use, is known to induce HO-1 in gastric epithelium. We aimed to investigate whether sofalcone induces HO-1 and reduces sFlt-1 release from primary human placental and endothelial cells and blocks endothelial dysfunction in vitro. We isolated human trophoblasts and endothelial cells (human umbilical vein endothelial cells) and also used uterine microvascular cells. We investigated the effects of sofalcone on (1) HO-1 production, (2) activation of the nuclear factor (erythroid-derived 2)-like 2 pathway, (3) sFlt-1 and soluble endoglin release, (4) tumor necrosis factor α-induced monocyte adhesion and vascular cell adhesion molecule upregulation, and (5) endothelial tubule formation. Sofalcone potently increased HO-1 mRNA and protein in both primary trophoblasts and human umbilical vein endothelial cells. Furthermore, sofalcone treatment caused nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 and transactivation of other nuclear factor (erythroid-derived 2)-like 2 responsive genes (NQO1, TXN, and GCLC). Importantly, sofalcone significantly decreased the secretion of sFlt-1 from primary human trophoblasts. Sofalcone potently suppressed endothelial dysfunction in 2 in vitro models, blocking tumor necrosis factor α-induced monocyte adhesion and vascular cell adhesion molecule 1 expression in human umbilical vein endothelial cells. These results indicate that in primary human tissues, sofalcone can potently activate antioxidant nuclear factor (erythroid-derived 2)-like 2/HO-1 pathway, decrease sFlt-1 production, and ameliorate endothelial dysfunction. We propose that sofalcone is a novel therapeutic candidate for preeclampsia.

NOD1 and NOD2 control the invasiveness of trophoblast cells via the MAPK/p38 signaling pathway in human first-trimester pregnancy

IntroductionNucleotide binding and oligomerization leucine-rich repeats (NLR) are recognized as members of pattern-recognition receptors that play important roles in host innate immune defenses. In this study, we have investigated the expression and regulation of nucleotide-binding oligomerization domains 1 and 2 (NOD1 and NOD2) on the invasiveness of primary human trophoblasts during the first trimester of pregnancy. MethodsThe expression of NOD1 and NOD2 by human first trimester villi from recurrent spontaneous abortion (RSA) patients and normal pregnancy was analyzed by immunochemistry, western blot and real-time RT-PCR, respectively. The effects of ligands on the regulation of their receptors and invasiveness of trophoblasts were examined. The ELISA was used to measure the secretion of matrix matallopeptidase-9 (MMP9) and matrix matallopeptidase-2 (MMP2) by trophoblasts. We also investigated the signaling pathways involved in invasion of trophoblasts. ResultsThe higher NOD1 and NOD2 were observed in villi from patients who experienced RSA compared with those who experienced a normal pregnancy. The ligands can up-regulate the expression of their receptors. After activation, NOD1 and NOD2 inhibited the invasion of trophoblast cells by decreasing MMP9 but not MMP2. After the activation of NOD1 and NOD2, the mitogen-activated protein kinase (MAPK)/p38 pathway was activated time dependently. Blocking this pathway with a specific inhibitor (SB203580) attenuated NOD1-and NOD2-regulated trophoblast invasions. DiscussionThese results suggest that abnormal elevated NOD1 and NOD2 expression in villi relate to pregnancy outcomes. Further, activation of NOD1 and NOD2 could have a bearing on our understanding of the invasive ability of trophoblasts.

Effects of serum from patients with early-onset pre-eclampsia, HELLP syndrome, and antiphospholipid syndrome on fatty acid oxidation in trophoblast cells.

The role of metabolic disorders of long-chain fatty acid oxidation in the development of pre-eclampsia (PE), hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome and antiphospholipid syndrome (APS) is unclear. The aim of this study was to research the effects of three serum on fatty acid oxidation in trophoblast cells. Primary human trophoblast cells and HTR8/SVneo cells were treated with serum from patients with early-onset severe PE (E-PE), E-PE with HELLP (PE-HELLP), APS, and normal pregnant women as controls (NC). Cells treated with free fatty acids (FFAs) of various lengths were used as controls. Triglyceride (TG) and FFA levels of the E-PE and APS groups were significantly higher than the PE-HELLP and NC groups (P < 0.05). Trophoblast cells treated with serum from the E-PE and APS groups showed obvious morphological changes and a large amount of lipid droplet deposition. Cells in the E-PE group had more severe damage of mitochondria ultrastructure, presenting with cell morphological changes, lipid droplet deposition, and mitochondria damage similar to the long-chain FFA group. FFA levels in the PE-HELLP group increased significantly compared with the NC group (P < 0.05), while TG levels did not change significantly (P > 0.05). Trophoblast cells treated with serum from the PE-HELLP group showed cellular morphology and mitochondria changes similar to the E-PE group, but had relatively less lipid droplet deposition. Serum from patients with E-PE, PE-HELLP, and APS with elevated levels of FFA had different effects on trophoblast cells, including cell morphology, lipid droplets deposition, and mitochondrial ultrastructure, suggesting disorders of lipid metabolism and fatty acid oxidation of various degrees and types. Serum from patients with E-PE led to damage similar to that of long-chain FFA in cell morphology, lipid droplet deposition and mitochondrial ultrastructure, indicating the correlation between disorders of long-chain fatty acid oxidation and the development of the disease.

Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways.

Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies.

Strong hypoxia reduces leptin synthesis in purified primary human trophoblasts

IntroductionOxygen availability severely affects placental function. During placental hypoxia, stabilization of hypoxia inducible factors (HIFs) affects transcription, and leptin gene expression concomitantly increases in vivo and in vitro. However, a causal relationship is uncertain. MethodsWe investigated the effect of oxygen availability on HIF-1 alpha (HIF1A) and leptin regulation in primary human trophoblasts isolated from six normal term placentae cultured at 0.1%, 1%, 3%, and 8% oxygen for 6 h, 24 h and 48 h. Gene expressions of leptin (LEP), leptin receptors (LEPR), HIF1A, insulin receptor (INSR) and further genes relevant in hypoxia (VEGFA, EPO, NOS2) or apoptosis (BCL2, BAX, Tp53) were examined. Leptin, HIF1A, INSR, phospho-AKT/AKT (insulin receptor signaling), caspase 3 and cleaved caspase 3 (apoptosis) proteins were measured. ResultsA hypoxic reaction with stabilization of HIF1A protein as well as up-regulation of HIF1A and VEGFA gene expressions, but without any hint for apoptosis, was present at 0.1% and 1% oxygen. However, leptin protein concentration (cell supernatants) peaked at 8% oxygen (normoxia) and was significantly reduced at 0.1% oxygen. There was no significant correlation between leptin and HIF1A, neither on the gene nor on the protein level. DiscussionElevated leptin gene expression in hypoxic placentas may not originate from trophoblasts, but from other placental cells, or from interaction of trophoblasts with other cells. Not only fetal hyperleptinemia, but also fetal hypoleptinemia under hypoxic conditions is conceivable. Strategies to prevent leptin dysregulation during pregnancy should be elucidated to protect the offspring from fetal programming of leptin resistance and adiposity in later life.

Primary Human Trophoblasts Expressing Four Essential Receptors of Hepatitis C Virus Entry but Resistance to Infection in Vitro

Background: Vertical transmission (VT) is generally considered to be the leading route of pediatric acquisition of Hepatitis C Virus (HCV). However, the mechanism of HCV VT has never been satisfactorily illustrated. Moreover, CD81, scavenger receptor class B type I (SR-B1), claudin 1 (CLDN1) and occludin (OCLN) were four necessary receptors for HCV cell infection. The expression of the essential receptors may explain the reason of HCV VT. Objective: To investigate the probable mechanism of HCV VT. Methods: We isolated trophoblasts from human term placental samples with approval from the Institutional Review Board at the Tongji Hospital of Tongji University and cultured the cells in vitro. Flow cytometry and Western blotting were used to detect the expression of the essential receptors for HCV infection on trophoblasts. Then we generated HCV pseudoparticle (HCVpp) and cell culture-produced HCV (HCVcc) to infect HEK-293T cells and trophoblasts,.At 72 h post-infection, HCVpp and HCVcc were determined in the infected cells. Results: Trophoblasts expressed CD81, SR-B1, CLDN1 and OCLN, which were essential for HCV cell entry. However, trophoblasts were not infected by HCVpp or HCVcc. Conclusion: Primary human trophoblasts possess the receptors necessary for HCV infection but resistant to infection in vitro.

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Objectives Preclinical studies suggest pravastatin is a candidate therapeutic for preeclampsia, leading to clinical trials. Surprisingly, the amount of functional interrogation of pravastatin (indeed all statins) on primary gestational tissues published thus far is very limited. The objective of this study was to determine the effect of statin administration to primary gestational tissues in vitro. Methods Pravastatin, simvastatin and rosuvastatin (HMG Co-A reductase inhibitors) were administered to primary endothelial cells (HUVECs), primary trophoblasts and preterm preeclamptic placental explants and the effect on sFlt1 and sEng secretion assessed. To identify the mechanism involved, farnesyl pyrophosphate, a HMG Co-A reductase pathway activator was added and sFlt1 and sENG was measured. Endothelial dysfunction was induced by addition of TNF α or trophoblast conditioned media to HUVECs, and the effect of statins on VCAM and monocyte adhesion determined. Finally HUVEC migration in the presence of sFlt1 ± statins was measured using the xCELLigence system (measures in real time). Results Statins significantly reduced sFlt1 release, reducing mRNA expression of the human placental specific variant, sFlt1-e15a and up-regulating heme-oxygenase-1. Pravastatin also significantly reduced sFlt1 release from preeclamptic placental explants. Concerning however, all three statins significantly increased secretion of sEng in a dose dependent manner. Addition of farnesyl pyrophosphate blocked statin-induced sFlt-1 reduction and sEng increase from HUVEC cells, indicating these changes are mediated via inhibition of HMG Co-A reductase. Primary trophoblast conditioned media and TNF α significantly upregulated markers of endothelial dysfunction, VCAM and Endothelin-1, which were quenched by adding statins. Statins also decreased monocyte adhesion to primary HUVECs, enhanced HUVEC tube forming and restored sFlt1 impaired HUVEC migration towards VEGF. Conclusions We characterized the effect of three statins on primary human endothelial cells and trophoblast cells. Our data confirms statins reduce sFlt1 and improve endothelial dysfunction. However, of potential concern is that statins increase sEng release. Disclosures F.C. Brownfoot: None. T.J. Kaitu’u-Lino: None. N. Hannan: None. R. Hastie: None. P. Cannon: None. K. Onda: None. S. Tong: None.

Neuregulin-1-mediated ErbB2-ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation.

ABSTRACTDuring placentation, foetal trophoblasts invade deeply into maternal tissue to establish a foeto–maternal circulation. We have previously shown that extravillous trophoblast (EVT) lineage cells express ErbB2 and ErbB3, of which the potential as an oncogenic unit is well established. However, a physiological function of this receptor combination in humans remains a puzzling question. Here, we demonstrate neuregulin 1 (NRG1) expression and secretion by human decidual stromal cells. Stimulation of human primary trophoblasts with exogenous NRG1 induced phosphorylation of ErbB2, ErbB3 and related downstream effectors. Co-immunoprecipitation experiments confirmed the formation of ErbB2–ErbB3 dimers upon ligand engagement. Along this line, receptor knockdown and ErbB3 neutralization strongly diminished NRG1-dependent activation of the signalling complex. Functional studies revealed that NRG1 promotes EVT formation in placental explant cultures. Although, in the presence of NRG1, basal and camptothecin-induced trophoblast apoptosis was significantly repressed, this effect was abolished upon ErbB3 inhibition. Notably, camptothecin provoked a strong reduction of trophoblast cell column size, whereas NRG1-treated explants were refractory to the compound. Taken together, our findings newly identify a physiological function of the NRG1–ErbB2–ErbB3 axis in trophoblast survival during human placental development.

Reply to “Letter to the editor: ‘Fatty acids and placental transport: insight or in vitro artifact?’”

reply: In our recent paper (2) we reported on the effects of fatty acids on trophoblast cellular signaling and amino acid transport activity in vitro. As with any other cell culture experiments, we are aware that this is an artificial environment on multiple levels. No matter how well designed the experiment, cell cultures in and of themselves cannot completely reproduce in vivo physiological conditions. However, despite their inherent limitations, cell culture experiments do provide an important tool for studying cell physiology. In vivo, cells are exposed to a multitude of effectors at variable concentrations and we concur with Dr. Keelan (1) that it is important to study effectors in combination. However, a problem arises with respect to which effectors should be included. For example, when studying effects of fatty acids on trophoblast cell function, should all effectors known to affect mTOR signaling and amino acid transport be included in the cell culture media for the experiment to be considered valid? By analogy, in studies of insulin effects on cell function, do all known effectors impinging on the insulin signaling pathway (adiponectin, leptin, and cytokines, just to mention a few) have to be included for the experiment to be physiologically relevant? If so, Dr. Keelan's argument invalidates most of the published studies in cell physiology. We believe that a reductionist approach in which single effector molecules are studied remains valid and we disagree with the argument that fatty acids should only be studied in combination. We reported that, in the presence of oleic acid, the effects of docosahexaenoic acid (DHA) alone were no longer predominant. Dr. Keelan suggests that this finding should be interpreted that DHA responses were an in vitro artifact. However, such an interpretation fails to appreciate that DHA counteracted the observed effects of oleic acid alone. Clearly, both DHA and oleic acid have the capacity to influence trophoblast signaling and function, both in isolation and when combined. In our paper we demonstrate that DHA reduces the activity of the amino transporters System A and System L in cultured primary human trophoblast cells. In a separate study following pregnant women supplemented with DHA, we found that placental DHA levels measured at term inversely correlated with System A and System L amino acid transport capacity (3). Such data from clinical samples strongly support the assertion that the reported in vitro effects are physiologically relevant and not an experimental artifact. As already discussed in our paper, we believe that these results merit further exploration of the effects of fatty acids on trophoblast function, both singularly and in combination.