EVT Cell Research Articles

ADAR1 could be a potential diagnostic target for intrauterine infection patients

Intrauterine infection (IUI) is mainly an ascending infection in which vaginal and cervical pathogens ascend to the uterus and can affect the fetus. Until now, there is still no effective diagnostic biomarker for IUI, such as chorioamnionitis (CAM) and funisitis (FUN). Deoxyribonucleic acid (DNA)/Ribonucleic acid (RNA) editing molecules such as apolipoprotein-B mRNA-editing complex (APOBEC) 3 families and Adenosine deaminase family acting on RNA (ADAR)1 were examined in chorioamniotic membranes and umbilical cord of 83 patient samples. Furthermore, Ureaplasma parvum induced ADAR1 was investigated in human HTR-8/SVneo EVT cell line. ADAR1 had a significantly higher area under the curve (AUC) (0.721 and 0.745) than other APOBEC3s or cytokines in CAM and FUN patients. In vitro, ureaplasma parvum was demonstrated to activate ADAR1 (p = 0.025) and reduce RIG-I, IRF3, IFN-\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\:{\\upalpha\\:}$$\\end{document}, and IFN-β expression in EVT cell line (p = 0.005, p = 0.010, p < 0.001, and p = 0.018, respectively). High expression of ADAR1 was strongly associated with CAM and FUN patients (multivariate analyses; p = 0.035 and p = 0.002). ADAR1 could be a potential diagnostic target for IUI, such as CAM and FUN patients.

Cytochrome c oxidase IV isoform 1 (COX4-1) regulates the proliferation, migration and invasion of trophoblast cells via modulating mitochondrial function

IntroductionSpontaneous miscarriage is a common complication of early pregnancy. Previous studies have shown that mitochondrial function plays an important role in establishment of a successful pregnancy. Cytochrome c oxidase subunit 4 isoform 1 (COX4I1), a component of electron transport chain complex Ⅳ, is required for coupling the rate of ATP production to energetic requirements. However, there is very limited research on its role in trophoblast biology and how its dysfunction may contribute to spontaneous miscarriage. MethodsPlacental villi (7–10 weeks gestational age) collected from either induced termination of pregnancy or after spontaneous miscarriage were examined for expression of COX4I1. COX4I1 was knocked down by siRNA transfection of primary isolates of EVT cells. Real-time cell analysis (RTCA) and 5-Ethynyl-2′-deoxyuridine (EdU) were used to detect changes in proliferation ability after COX4I1 knockdown of EVT cells. Migration and invasion indices were determined by RTCA. Mitochondrial morphology was observed via MitoTracker staining. Oxidative phosphorylation, ATP production, and glycolysis in COX4I1-deficient cells and controls were assessed by a cellular energy metabolism analyzer (Seahorse). ResultsIn placental villous tissue, COX4I1 expression was significantly decreased in the spontaneous miscarriage group. Knockdown of COX4I1 inhibited EVT cell proliferation, increased the migration and invasion ability and mitochondrial fusion of EVT cells. Mitochondrial respiration and glycolysis were impaired in COX4I1-deficient EVT cells. Knockdown of MMP1 could rescue the increased migration and invasion induced by COX4I1 silencing. DiscussionLow expression of COX4I1 leads to mitochondrial dysfunction in EVT, resulting in altered trophoblast function, and ultimately to pregnancy loss.

Extracellular vesicles in COVID-19 convalescence can regulate T cell metabolism and function

Long-term Tcell dysregulation has been reported following COVID-19 disease. Prolonged Tcell activation is associated with disease severity and may be implicated in producing long-covid symptoms. Here, we assess the role of extracellular vesicles (EV) in regulating Tcell function over several weeks post COVID-19 disease. We find that alterations in cellular origin and protein content of EV in COVID-19 convalescence are linked to initial disease severity. We demonstrate that convalescent donor-derived EV can alter the function and metabolic rewiring of CD4 and CD8 Tcells. Of note, EV following mild, but not severe disease, show distinctly immune-suppressive properties, reducing Tcell effector cytokine production and glucose metabolism. Mechanistically our data indicate the involvement of EV-surface ICAM-1 in facilitating EV-T cell interaction. Our data demonstrate that circulatory EV are phenotypically and functionally altered several weeks following acute infection, suggesting a role for EV as long-term immune modulators.

Snail mediates GDF-8-stimulated human extravillous trophoblast cell invasion by upregulating MMP2 expression

BackgroundExtravillous trophoblast (EVT) cell invasion is a tightly regulated process that requires for a normal pregnancy. The epithelial-mesenchymal transition (EMT) has been implicated in EVT cell invasion. Growth differentiation factor-8 (GDF-8), a member of the transforming growth factor-beta (TGF-β) superfamily, is expressed in the human placenta and promotes EVT cell invasion by upregulating the expression of matrix metalloproteinase 2 (MMP2). However, the underlying molecular mechanism of GDF-8-induced MMP2 expression remains undetermined. Therefore, the present study aims to examine the role of Snail and Slug, the EMT-related transcriptional regulators, in GDF-8-stimulated MMP2 expression and cell invasion in HTR-8/SVneo human EVT cell line and primary cultures of human EVT cells.MethodsHTR-8/SVneo and primary cultures of human EVT cells were used to examine the effect of GDF-8 on MMP2 expression and explore the underlying mechanism. For gene silencing and overexpression, the HTR-8/SVneo cell line was used to make the experiments more technically feasible. The cell invasiveness was measured by Matrigel-coated transwell invasion assay.ResultsGDF-8 stimulated MMP2 expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effect of GDF-8 on MMP2 expression was blocked by the inhibitor of TGF-β type-I receptors, SB431542. Treatment with GDF-8 upregulated Snail and Slug expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effects of GDF-8 on Snail and Slug expression were blocked by pretreatment of SB431542 and siRNA-mediated knockdown of SMAD4. Interestingly, using the siRNA knockdown approach, our results showed that Snail but not Slug was required for the GDF-8-induced MMP2 expression and cell invasion in HTR-8/SVneo cells. The reduction of MMP2 expression in the placentas with preeclampsia (PE) was also observed.ConclusionsThese findings discover the physiological function of GDF-8 in the human placenta and provide important insights into the regulation of MMP2 expression in human EVT cells.1EyNAiCz9UDZ9srU6xtfDxVideo

SERPINA5 may promote the development of preeclampsia by disruption of the uPA/uPAR pathway

Preeclampsia (PE) is the leading cause of maternal and fetal morbidity or mortality but lacks reliable methods for early diagnosis. In a previous study, serum SERPINA5 levels were higher in women with PE before the clinical manifestation of the disease. This study aimed to evaluate the efficacy of SERPINA5 in predicting PE and investigate its role in trophoblast cell biology. A multicenter, 2-stage observational case-control study was performed to develop and validate an early predictive PE model based on SERPINA5, maternal characteristics, and inflammatory factors. To further understand the relationship between SERPINA5 and PE, SERPINA5 was overexpressed or knocked down in extravillous trophoblast cells (EVT) and a pregnant rat model. After development and initial validation, a model that combined SERPINA5 and inflammatory factors had a high predictive ability for PE before 20 weeks gestation with an AUC of 0.90 (95% CI 0.83-0.96). It also demonstrated that SERPINA5 inhibited primary EVT cell invasion by disrupting the urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor (uPA/uPAR) pathway, in turn, is involved in the development of PE. In vivo experiments also proved that overexpression of SERPINA5 induced a PE-like syndrome (hypertension and proteinuria) in pregnant rats. Therefore, serum SERPINA5 is a promising early biomarker of PE, suggesting that it may be involved in placental development through its action on the uPA/uPAR system prior to the clinical manifestation of PE.

GDF-8 stimulates trophoblast cell invasion by inducing ALK5-SMAD2/3-mediated MMP2 expression.

Matrix metalloproteinases (MMPs) play a pivotal role in the regulation of cell invasion. Placental trophoblast cell invasion is a precisely regulated event. Dysregulation of MMPs has been linked to various placental diseases. Growth differentiation factor-8 (GDF-8), also known as myostatin, is a member of the transforming growth factor-beta (TGF-β) superfamily. GDF-8 and its putative receptors are expressed in human extravillous cytotrophoblast cells (EVTs). Although the pro-invasive effect of GDF-8 in human EVT cells has been recently reported, the underlying molecular mechanism remains largely unknown. In this study, we investigate the effects of GDF-8 on the expression of the two most important MMPs, MMP2 and MMP9, in the HTR-8/SVneo human EVT cell line. Our results show that GDF-8 significantly upregulates the expression of MMP2. The expression of MMP9 is not affected by GDF-8. Using a siRNA-mediated knockdown approach, we reveal that the stimulatory effect of GDF-8 on MMP2 expression is mediated by the ALK5-SMAD2/3 signaling pathway. Additionally, the knockdown of MMP2 attenuates the GDF-8-induced cell invasiveness. These findings deepen our understanding of the biological roles of GDF-8 in the regulation of human trophoblast cell invasion.

ASCL2 reciprocally controls key trophoblast lineage decisions during hemochorial placenta development

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast cell invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in mice identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development. We hypothesized that ASCL2 is a critical and conserved regulator of invasive trophoblast cell lineage development. In contrast to the mouse, the rat possesses deep intrauterine trophoblast cell invasion and spiral artery remodeling similar to human placentation. In this study, we investigated invasive/extravillous trophoblast (EVT) cell differentiation using human trophoblast stem (TS) cells and a loss-of-function mutant Ascl2 rat model. ASCL2 transcripts are expressed in the EVT column and junctional zone, which represent tissue sources of invasive trophoblast progenitor cells within human and rat placentation sites, respectively. Differentiation of human TS cells into EVT cells resulted in significant up-regulation of ASCL2 and several other transcripts indicative of EVT cell differentiation. Disruption of ASCL2 impaired EVT cell differentiation, as indicated by cell morphology and transcript profiles. RNA sequencing analysis of ASCL2-deficient trophoblast cells identified both down-regulation of EVT cell-associated transcripts and up-regulation of syncytiotrophoblast-associated transcripts, indicative of dual activating and repressing functions. ASCL2 deficiency in the rat impacted placental morphogenesis, resulting in junctional zone dysgenesis and failed intrauterine trophoblast cell invasion. ASCL2 acts as a critical and conserved regulator of invasive trophoblast cell lineage development and a modulator of the syncytiotrophoblast lineage.

Let-7 inhibits the migration and invasion of extravillous trophoblast cell via targeting MDM4

AimsExtravillous trophoblast (EVT) cells migration and invasion are important causes to preeclampsia (PE). Studies have shown that let-7 was involved in inhibiting proliferation and invasion of several cancer cells, however, its effect on EVT cells migration and invasion has hardly been reported. This study aimed to explore the relation between let-7 and EVT cell migration and invasion. MethodsMicroRNA (miRNA) and genes expression levels were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot (WB). Cell proliferation, migration and invasion were detected by cell counting kit-8 (CCK-8) assay, wound-healing assay and transwell assay. The binding site between let-7 and murine double minute 4 (MDM4) was identified using TargetScan. The targeting relation between let-7 and MDM4 was verified using dual luciferase reporter assay. ResultsThe results revealed that in placental tissues of PE patients, let-7, matrix metalloproteinase-2 (MMP-2) and MMP-9 were lowly expressed and tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2 were highly expressed. Let-7 silencing promoted the proliferation, migration, invasion of HTR8/SVneo cells and the expression levels of MMP-2 and MMP-9, however, it inhibited TIMP-1 and TIMP-2 expression levels, while overexpression of let-7 produced the opposite results. Furthermore, MDM4 is a target gene of let-7. Rescue experiments suggested that MDM4 siRNA partially reversed the effects of let-7 silencing on cells. ConclusionsLet-7 silencing promoted proliferation, migration and invasion in EVT cells through the up-regulation of MDM4. Our study provided new insights into the molecular mechanism of PE.

S100P enhances the motility and invasion of human trophoblast cell lines

S100P has been shown to be a marker for carcinogenesis where its expression in solid tumours correlates with metastasis and a poor patient prognosis. This protein’s role in any physiological process is, however, unknown. Here we first show that S100P is expressed both in trophoblasts in vivo as well as in some corresponding cell lines in culture. We demonstrate that S100P is predominantly expressed during the early stage of placental formation with its highest expression levels occurring during the first trimester of gestation, particularly in the invading columns and anchoring villi. Using gain or loss of function studies through overexpression or knockdown of S100P expression respectively, our work shows that S100P stimulates both cell motility and cellular invasion in different trophoblastic and first trimester EVT cell lines. Interestingly, cell invasion was seen to be more dramatically affected than cell migration. Our results suggest that S100P may be acting as an important regulator of trophoblast invasion during placentation. This finding sheds new light on a hitherto uncharacterized molecular mechanism which may, in turn, lead to the identification of novel targets that may explain why significant numbers of confirmed human pregnancies suffer complications through poor placental implantation.

The blocking of aquaporin-3 (AQP3) impairs extravillous trophoblast cell migration

Several aquaporins (AQPs) are expressed in extravillous (EVT) and villous trophoblast cells. Among them, AQP3 is the most abundant AQP expressed in chorionic villi samples from first trimester, followed by AQP1 and AQP9. Although AQP3 expression persists in term placentas, it is significantly decreased in placentas from preeclamptic pregnancies. AQP3 is involved in the migration of different cell types, however its role in human placenta is still unknown.Here, we evaluated the role of AQP3 in the migration of EVT cells during early gestation.Our results showed that Swan 71 cells expressed AQP1, AQP3 and AQP9 but only the blocking of AQP3 by CuSO4 or the silencing of its expression by siRNA significantly attenuates EVT cell migration.Our work provides evidence that AQP3 is required for EVT cell migration and suggests that an altered expression of placental AQP3 may produce failures in placentation such as in preeclampsia.

Oleic acid stimulation of motility of human extravillous trophoblast cells is mediated by stearoyl-CoA desaturase-1 activity.

Do fatty acids regulate development and motility of human extravillous trophoblast cells (EVTs)? Oleic acid is a promising lipid molecule that has beneficial effects on motility and development of human EVTs. Fatty acid uptake into trophoblast cells is important for maintaining cellular events during pregnancy, but the molecular mechanisms of action of various fatty acids, including trans fatty acids, saturated fatty acids and monounsaturated fatty acids, in EVT cell lines are not clear. Effects of oleic acid, elaidic acid, palmitic acid and stearic acid on HTR8/SVneo cells were assessed in diverse assays in a dose- and time-dependent manner. Effects of fatty acids on cell proliferation, migration, invasion and apoptosis (Annexin V expression, propidium iodide staining, TUNEL and invasion assays) of HTR8/SVneo cells were determined. Signal transduction pathways in HTR8/SVneo cells in response to fatty acids were determined by Western blot analyses. Regulation of fatty acids on oxidative conditions in EVTs were determined and validated by measurement of production of cellular reactive oxygen species, intracellular concentrations of free Ca2+and lipid peroxidation assays. In present study, we confirmed different effects of oleic acid and elaidic acid on migration, invasion, proliferation and apoptosis of the EVT cell line, HTR8/SVneo. We also investigated stearoyl-CoA desaturase-1 (SCD1) to determine if its activity contributed to oleic acid-induced migration of HTR8/SVneo cells. Next, we analyzed cell signaling molecules mediated by oleic acid and elaidic acid treatment, including MAPK and PI3K/AKT pathways in HTR8/SVneo cells. We further established whether selective inhibition of signaling molecules altered the ability of fatty acids to cause changes in migration and proliferation of HTR8/SVneo cells. Last, we examined the regulatory effects of oleic acid and SCD1 on oxidative stress in HTR8/SVneo cells. N/A. The lack of in vivo animal studies is a major limitation of this research. Effectiveness of oleic acid to stimulate migration of human EVT cells requires further investigation. Our results suggest that oleic acid can play an important role in promoting invasion of human EVT cell lines while both trans fatty acids and saturated fatty acids are not conducive to normal placentation. This may have implications for the prevention of pre-eclampsia and intrauterine growth restriction. This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810) awarded to G.S. and (No. HI17C0929) awarded to W.L. There are no conflicts of interest.

Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration

Background: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration.Methods: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software).Results: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 108 ± 2.5 × 108 particles/106 cells) compared to JEG-3 (2.86 × 108 ± 0.78 × 108 particles/106 cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (−exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes.Conclusion: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls.

Leptin-Promoted Human Extravillous Trophoblast Invasion Is MMP14 Dependent and Requires the Cross Talk Between Notch1 and PI3K/Akt Signaling1

The overexpression of leptin is a crucial feature for the maintenance of pregnancy. The effects of leptin on trophoblast invasion are important to its reproductive function, but the underlying mechanisms remain poorly understood. MMP14 is a member of matrix metalloproteinase (MMP) family that is closely involved in the invasion process. Here, we characterized the importance of MMP14 in the proinvasion effect of leptin on EVT cells and elucidated its molecular mechanisms. Transwell assay revealed that leptin promoted invasion of the immortalized EVT cell line HTR-8/SVneo in a dose- and time-related fashion. Further studies suggested that leptin enhanced HTR-8/SVneo cell invasion by up-regulating MMP14 expression and that knockdown of MMP14 by small interference RNA (siRNA) blocked the proinvasion effect of leptin. Notably, leptin promoted the expression of Notch1 receptor and activated its signaling in HTR-8/SVneo cells, and blocking this pathway by siRNA inhibited both leptin-enhanced MMP14 expression and invasiveness of HTR-8/SVneo cells. Such effects of Notch1 signaling were related with the activation of the PI3K/Akt pathway, which was significantly activated after leptin stimulation and was interfered by Notch1 signaling perturbation. Taken together, our observations suggest that leptin is an effective regulator of MMP14 expression, which consequently plays critical roles in invasion of EVT cells. The promoting effects of leptin on MMP14 require the cross talk between Notch1 and PI3K/Akt signaling pathways.

Extravillous trophoblast cell invasion is promoted by the CD44–hyaluronic acid interaction

IntroductionExtravillous trophoblast (EVT) cell invasion plays a crucial role in establishment of successful pregnancy. CD44, a cell-surface receptor for hyaluronic acid (HA), plays a key role in HA-mediated remodeling and degradation that triggers cancer cell invasion. However, few studies have reported on the expression or functions of CD44 in human EVT cells. We hypothesized that CD44-HA interaction was involved in invasion by EVT cells. MethodsTo test our hypothesis, we conducted in situ examinations of CD44 and HA expression in the human first-trimester placenta. We also assessed the methylation status of CD44 promoter and exon 1 regions in EVT cells. Finally, we conducted transwell cell invasion assays using EVT cell lines and EVT cells isolated from first-trimester human villous explant cultures. Results and discussionEVT cells, but not villous trophoblast cells, in the first-trimester placenta expressed CD44. HA was strongly expressed in adventitia surrounding the spiral uterine arterial walls of the decidua. The extent of demethylation of CD44 promoter and exon 1 CpG islands was increased in EVT cells compared to those of first-trimester chorionic villi (including villous trophoblast cells), suggesting that CD44 expression was, at least in part, associated with methylation status. Data from transwell cell invasion assay with siRNA knockdown of CD44 revealed that CD44 expression significantly promoted invasion by EVT cells in an HA-dependent manner. ConclusionsThe discovery of a CD44-HA interaction between EVT cells and the extracellular matrix contributes to our understanding of the mechanism underlying invasion by EVT cells.

The placental expression of Human Leukocyte Antigen HLA-F and HLA-E in early normal placentation

The placental expression of Human Leukocyte Antigen HLA-F and HLA-E in early normal placentation

The involvement of eukaryotic translation initiation factor 4E in extravillous trophoblast cell function

Extravillous trophoblast cells (EVT) are major players in placental implantation. They differentiate in the villous cell column, invade to the uterus and remodel the uterine spiral arteries. Trophoblast and tumor cells have similar invasion mechanisms, share similar biochemical mediators (e.g. c-myc, MMP9) and growth-factors (e.g. VEGF). The mRNA of these proteins has extremely structured 5-UTR and their translation is highly dependent on eukaryotic-translation-initiation-factor-4E (eIF4E). Cancer cells have elevated eIF4E and are more vulnerable to its silencing than normal cells. We speculated that like cancer, trophoblast function is highly eIF4E dependent. ObjectiveAnalyze eIF4E involvement in EVT differentiation and function. Study designEIF4E levels were assessed in first-trimester human placentae and in placental explants before and after EVT differentiation. The effect of eIF4E knockdown (siRNA, ribavirin) on the phenotype of placental explant and EVT cell lines (HTR-8/SVNEO) was evaluated. Tested parameters included eIF4E and its target levels, migration, invasion, cell death, cell cycle and cell count. ResultsHigh eIF4E levels were found in cytotrophoblast and especially EVT cells during their differentiation in the villi, compared to other placental cell types. EIF4E silencing increased cell death and cell cycle arrest in placental explants and HTR-8/SVNEO cells. Although it induced EVT outgrowth in the placental explants, it reduced HTR-8/SVNEO motility, reflecting the importance of using ex vivo models that include an intact placental microenvironment in its original architecture. ConclusionsOur results suggest that eIF4E prevents final EVT differentiation and supports placental cell proliferation and survival. A balance between cell proliferation and differentiation is crucial for placental development and implantation.

Effects of interleukin-6 on extravillous trophoblast invasion in early human pregnancy

Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors, including growth factors and cytokines, but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesized that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8(+) T cells, CD10(+) decidual stromal cells, CD14 macrophages, CD56(+) uterine natural killer cells, cytotrophoblast and EVT cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 were immunolocalized to EVT in placental bed biopsies from 8 to 20 weeks gestation and IL-6Rα expression was confirmed by western blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalized EVT cell line HTR-8/SVneo in a Matrigel(®) invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES) by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.

Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10−8 M), medroxyprogesterone acetate (10−7 M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states.

P30 Endothelin expression in ischemia/reperfusion-induced fetal growth restriction in the rat

Extravillous trophoblast cells (EVT) are major players in placental implantation. They differentiate in the villous cell column, invade to the uterus and remodel the uterine spiral arteries. Trophoblast and tumor cells have similar invasion mechanisms, share similar biochemical mediators (e.g. c-myc, MMP9) and growth-factors (e.g. VEGF). The mRNA of these proteins has extremely structured 5-UTR and their translation is highly dependent on eukaryotic-translation-initiation-factor-4E (eIF4E). Cancer cells have elevated eIF4E and are more vulnerable to its silencing than normal cells. We speculated that like cancer, trophoblast function is highly eIF4E dependent.Analyze eIF4E involvement in EVT differentiation and function.EIF4E levels were assessed in first-trimester human placentae and in placental explants before and after EVT differentiation. The effect of eIF4E knockdown (siRNA, ribavirin) on the phenotype of placental explant and EVT cell lines (HTR-8/SVNEO) was evaluated. Tested parameters included eIF4E and its target levels, migration, invasion, cell death, cell cycle and cell count.High eIF4E levels were found in cytotrophoblast and especially EVT cells during their differentiation in the villi, compared to other placental cell types. EIF4E silencing increased cell death and cell cycle arrest in placental explants and HTR-8/SVNEO cells. Although it induced EVT outgrowth in the placental explants, it reduced HTR-8/SVNEO motility, reflecting the importance of using ex vivo models that include an intact placental microenvironment in its original architecture.Our results suggest that eIF4E prevents final EVT differentiation and supports placental cell proliferation and survival. A balance between cell proliferation and differentiation is crucial for placental development and implantation.

Apoptosis of Extravillous Trophoblast Cells Limits the Trophoblast Invasion in Uterine but not in Tubal Pregnancy During FirstTrimester

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56 +NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8±0.7 per cent) is reduced compared to first trimester (5.6±0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4±2.9) to second (12.4±1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9±0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.