Trimester Extravillous Trophoblast Cell Line Research Articles

Cell-free fetal DNA impairs trophoblast migration in a TLR9-dependent manner and is reversed by hydroxychloroquine

Growing evidence suggests a relationship between elevated circulating cell-free fetal DNA (cffDNA) and preeclampsia. Hypomethylation of CpG motifs is a hallmark of cffDNA, allowing it to activate Toll-like receptor 9 (TLR9). Since TLR9 is expressed by the trophoblast, we hypothesize that placental-derived cffDNA accumulates at the maternal-fetal interface and limits trophoblast migration. We investigated this and if available therapeutics would have an impact. A human first trimester extravillous trophoblast cell line (Sw.71) was cultured in serum-free media after which cffDNA was isolated. Trophoblast cells were treated with media, cffDNA (375ng/ml), or as a positive control ODN 2216 (1M), a synthetic TLR9 agonist. For some experiments, cells were pretreated with A151 (5M), an antagonist of the DNA sensors TLR9, AIM2, and cGAS, prior to exposure to cffDNA or ODN 2216. Trophoblast cells were also treated with cffDNA or ODN 2216 together with aspirin (ASA, 10g/ml), aspirin-triggered lipoxin (ATL, 100nM), or hydroxychloroquine (HCQ, 1g/ml). After 48hrs, cell viability and cell migration were measured using the CellTiter assay and a two-chamber colorimetric assay. cffDNA and ODN 2216 significantly reduced trophoblast migration by 36.9 4.3% and 47.2 3.2%, respectively (p< 0.05), without affecting cell viability. Reduced cell migration in response to cffDNA or ODN 2216 was significantly reversed by A151 (p< 0.05). cffDNA inhibition of migration was significantly reversed by HCQ, but not ASA or ATL. ODN 2216 inhibition of migration was reversed by HCQ, ASA and ATL (p< 0.05). Our findings suggest that cffDNA exerts a local effect on placental function by limiting trophoblast migration through activation of DNA sensors. HCQ, which can inhibit TLR9 and cGAS, reverses this, while ASA, which can inhibit cGAS, does not. Thus, cffDNA inhibits trophoblast migration via TLR9. Further research can aid our understanding of the pathogenesis of preeclampsia to improve perinatal outcomes.

Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion.

Among several interleukin (IL)-6 family members, only IL-6 and IL-11 require a gp130 protein homodimer for intracellular signaling due to lack of intracellular signaling domain in the IL-6 receptor (IL-6R) and IL-11R. We previously reported enhanced decidual IL-6 and IL-11 levels at the maternal-fetal interface with significantly higher peri-membranous IL-6 immunostaining in adjacent interstitial trophoblasts in preeclampsia (PE) vs. gestational age (GA)-matched controls. This led us to hypothesize that competitive binding of these cytokines to the gp130 impairs extravillous trophoblast (EVT) differentiation, proliferation and/or invasion. Using global microarray analysis, the current study identified inhibition of interferon-stimulated gene 15 (ISG15) as the only gene affected by both IL-6 plus IL-11 vs. control or IL-6 or IL-11 treatment of primary human cytotrophoblast cultures. ISG15 immunostaining was specific to EVTs among other trophoblast types in the first and third trimester placental specimens, and significantly lower ISG15 levels were observed in EVT from PE vs. GA-matched control placentae (p = 0.006). Induction of primary trophoblastic stem cell cultures toward EVT linage increased ISG15 mRNA levels by 7.8-fold (p = 0.004). ISG15 silencing in HTR8/SVneo cultures, a first trimester EVT cell line, inhibited invasion, proliferation, expression of ITGB1 (a cell migration receptor) and filamentous actin while increasing expression of ITGB4 (a receptor for hemi-desmosomal adhesion). Moreover, ISG15 silencing further enhanced levels of IL-1β-induced pro-inflammatory cytokines (CXCL8, IL-6 and CCL2) in HTR8/SVneo cells. Collectively, these results indicate that ISG15 acts as a critical regulator of EVT morphology and function and that diminished ISG15 expression is associated with PE, potentially mediating reduced interstitial trophoblast invasion and enhancing local inflammation at the maternal-fetal interface. Thus, agents inducing ISG15 expression may provide a novel therapeutic approach in PE.

Caffeic acid protects human trophoblast HTR-8/SVneo cells from H2O2-induced oxidative stress and genotoxicity

Caffeic acid is highlighted as one of the major phenolic compounds present in foods with known antioxidant activity. This phenolic is among commonly consumed substances in everyday diet of pregnant women. However, there is not enough information on its effects during pregnancy, especially the most vulnerable early stage. Extravillous trophoblast cells are specific cells of the placenta that come in direct contact with maternal uterine tissue.Through this study we investigated the cytoprotective effects of caffeic acid on H2O2-induced oxidative damage in first trimester extravillous trophoblast cell line HTR-8/SVneo. Investigated concentrations (1–100 μM) of caffeic acid showed neither cytotoxic nor genotoxic effects on HTR-8/SVneo cells. The treatment with caffeic acid 100 μM significantly increased the percentage of cells in G2/M phase of the cell cycle, compared to non-treated cells. Pretreatment with caffeic acid (10 and 100 μM) attenuated oxidative DNA damage significantly, reduced cytotoxicity, protein and lipid peroxidation, and restored antioxidant capacity in trophoblast cells following H2O2 exposure. This beneficial outcome is probably mediated by the augmentation of GSH and effective ROS scavenging by caffeic acid. These promising results require further investigations to reveal the additional mechanisms/pathways and confirmation through studies in vivo.

Brominated diphenyl ether-47 differentially regulates cellular migration and invasion in a human first trimester trophoblast cell line

Polybrominated diphenyl ethers (PBDEs) are flame retardant compounds detected in human placenta and linked to adverse pregnancy outcomes. Impaired trophoblast migration and invasion during early pregnancy have been implicated as potential mechanisms of pregnancy disorders. The present study investigated the effect of BDE-47, a prevalent PBDE congener, on cell migration, invasion, and matrix metalloproteinase (MMP) expression in a human first trimester extravillous trophoblast cell line, HTR-8/SVneo. BDE-47 stimulated cell migration in HTR-SV/neo cells while decreasing invasion of cells into Matrigel. In addition, BDE-47 led to differential expression of MMP-1, -2, -3, and -9 at protein and mRNA levels. In summary, BDE-47 differentially regulated cellular migration and invasion with divergent changes in MMP expression in trophoblasts. Because proper regulation of trophoblast migration and invasion is critical for placental development and function, further research is warranted to determine if exposure to PBDEs disrupts trophoblast functions with increased risk for adverse pregnancy outcomes.

Nicotine Suppresses the Invasiveness of Human Trophoblasts by Downregulation of CXCL12 Expression through the Alpha-7 Subunit of the Nicotinic Acetylcholine Receptor

Smoke exposure during pregnancy has detrimental effects upon numerous fetal and neonatal outcomes. Nicotine (the main component of tobacco) has been suggested to affect placental development. During placental development, efficient invasion by trophoblasts is required for establishment of the fetus–maternal circulation. In this study we explored the regulation of trophoblast invasion by nicotine. An immortalized first trimester extravillous trophoblast cell line (HTR-8/SVneo cells) was used for all the experiments, which were treated by nicotine, methyllycaconitine, and C-X-C motif chemokine ligand 12 (CXCL12). Total RNA and protein were used to study the expressions of nicotinic acetylcholine receptors (nAChRs), and transwell assay was used to study invasiveness. Changes of RNA expression due to nicotine treatment were detected by RNA sequence. Level of CXCL12 mRNA was verified by quantitative PCR. We showed that HTR-8/SVneo expressed subunits α2–4, α7, α9, β1, and β2 of nAChRs. Nicotine downregulated CXCL12 expression and inhibited trophoblast invasion. Methyllycaconitine, as an antagonist of the α7 homopolymer, blocked the inhibitory effect of nicotine. CXCL12 could rescue the nicotine-induced inhibitory effect on invasion of HTR-8/SVneo cells. These results suggest that the α7 subunit of the nAChR has important roles in modulating trophoblast invasion through CXCL12.

H2S Improves the Rate of Migration in Trophoblast Cells

IntroductionThe migration of trophoblast cells during the first trimester of pregnancy is crucial to placentation process. Defective migration of trophoblastic cells may affect the remodelling (pseduovasculogenesis) of spiral arteries resulting in Hypoxia/Reoxygenation (H/R) episodes. These repeated H/R events can leads to oxidative stress and release of anti‐angiogenic, anti‐inflammatory and pro‐oxidant factors in maternal circulation causing endothelial dysfunction resulting in adverse pregnancy outcomes like preeclampsia and fetal growth restriction. The anti‐inflammatory, pro‐angiogenic and pro‐migratory effect of H2S on endothelial cells have been documented in literature. So we hypothesized that H2S or its mimics (NaHS) may also affect the migration capacity of trophoblast cells during placentation.MethodsThe immortalized first trimester extravillous trophoblast cell line (HTR‐8/SVneo) were given various treatments like 50μM NaHS, Hypoxia/Reoxygenation [H/R (one and two cycles)] and Hypoxia/Reoxygenation supplemented with 50μM NaHS. The cell migration was assessed using wound scratch assay following these various exposures. The percentage wound area covered was assessed in all groups. The difference in wound covered area was statistically analysed. [ten different zone in triplicate (i.e. thirty) were used for calculating the wound areas]ResultsThe percentage of wound covered area (rate of migration) increased in cells which received 50μM NaHS [control (32.8%), NaHS (67.5%)] whereas it reduced to 8.7% in Hypoxia/Reoxygenation one cycle exposed cells and more so (−18.9%) in the cells which received two cycles of Hypoxia/Reoxygenation. However the cells which received Hypoxia/Reoxygenation (one cycle and two cycles) and showed reduced migration, improved their rate of migration when supplemented with 50μM NaHS. [one cycle H/R (8.7%), one cycle H/R+NaHS (39.8) and two cycles H/R (−18.9%), two cycles H/R+NaHS (29.5%)]. The difference in wound covered area among various groups was statistically significant [(control vs NaHS; p&lt;0.001, control vs one cycle H/R; p&lt;0.001, control vs two cycle H/R; p&lt;0.001, one cycle H/R vs one cycle H/R+NaHS; p &lt;0.001, two cycle H/R vs two cycles H/R+NaHS; p&lt;0.001 (one‐way ANOVA with Bonferroni correction)].ConclusionThese results imply that H2S may promote the migration of trophoblastic cells, which might then lead to adequate conversion of spiral arteries into wider diameter low resistance vessels having good placental perfusion. In future the role of H2S on cellular and molecular factors responsible for cell migration needs to explored.IESC/T‐467/23.12.2014Support or Funding InformationF.8‐397/A‐397/2015/RsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of DJ-1 Downregulation on the Functions of the First Trimester Extravillous Trophoblasts.

DJ-1 ( PARK7) has been reported to be causative gene of Parkinson disease and also an oncogene. A loss in DJ-1 function can lead to cell death in neurodegenerative disease, or a gain of it can cause unregulated cell survival in cancer, respectively. DJ-1 protein is known to be expressed mainly in trophoblastic cells in the placenta with increased expression in the first trimester compared to later in term. However, its role in trophoblast regulation remains unknown. This study aimed to investigate the effect of DJ-1 regulation on a first trimester extravillous trophoblast cell line, HTR-8/SVneo. The effect of DJ-1 downregulation induced by small-interfering RNA on cell apoptosis, migration, and the pathway to regulate the cell function was assessed. Data of this study showed that DJ-1 downregulation increased apoptosis and reduced migration by regulating matrix metalloproteinase 2 and matrix metalloproteinase 9 in HTR-8/SVneo cells under both ambient and oxidative stress. Changes in cell function were demonstrated to be at least partly dependent on the AKT/S6 kinase beta-1 (S6K1) pathway. In summary, DJ-1 might play a protective role in maintaining trophoblastic cell functions through the AKT/S6K1-based pathway.

IGF2-derived miR-483-3p contributes to macrosomia through regulating trophoblast proliferation by targeting RB1CC1

What is the role of insulin-like growth factor 2 (IGF2)-derived miR-483-3p in macrosomia? IGF2-derived intronic miR-483-3p is overexpressed in macrosomia placentas, and miR-483-3p prompts HTR-8/SVneo extravillous trophoblast cell line proliferation through down-regulation of its target RB1 inducible coiled-coil 1 (RB1CC1). Macrosomia is a common pregnancy-associated disease and causes a number of adverse maternal and perinatal outcomes. The development of macrosomia is reportedly attributable to over proliferation of the placental cells. MicroRNAs (miRNAs) play an important role in the development of fetal and placenta by regulating their target genes. Here, we investigated the role of IGF2-derived intronic miR-483-3p in macrosomia. The expression of IGF2, miR-483-3p and its target gene in placental tissues from 30 pregnant women who had macrosomia was compared to those of 30 gestation-matched healthy pregnant controls. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR-8/SVneo cell was used. Placenta tissues were collected from pregnant women who had macrosomia without diabetes or other complications (n = 30) and healthy pregnant controls (n = 30). HTR-8/SVneo cells were transfected with specific miRNA mimics or inhibitors. MiRNA and mRNA isolated from placenta tissues or cells were measured by quantitative real-time PCR. Protein was measured by western blot. Cell proliferation was assayed using a colorimetric proliferation assay method. Cell cycle and apoptosis were analyzed by flow cytometry. The putative targets of miR-483-3p were predicted using the TargetScan, miRanda, miRDB and DIANA algorithms. Dual luciferase reporter assay was used to measure the relationship of miR-483-3p and RB1CC1. IGF2-derived miR-483-3p was overexpressed in macrosomia placentas. miR-483-3p promoted proliferation in HTR-8/SVneo cells and had a positive relationship with its host gene IGF2. Subsequently, RB1CC1 was confirmed as a direct target of miR-483-3p, which may be an important mediator of cell growth regulation for miR-483-3p. N/A. The level of IGF2 and its intronic miR-483-3p in the serum of these participants was not investigated. Further studies are required to understand the mechanisms underlying the cause of the increase of IGF2 and miR-483-3p in macrosomia. These findings give a new insight into the role of intronic miRNA and its host gene in the development of macrosomia. Furthermore, it may offer a new target for prognostic and therapeutic intervention for macrosomia. This work was supported by awards from National Natural Science Foundation of China (Nos. 81401213, 81673217, 81703260), Jiangsu Provincial Medical Youth Talent (No. QNRC2016110), Jiangsu Overseas Visiting Scholar Program for University Prominent Young & Middle-aged Teachers and Presidents, the Priority Academic Program for the Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine), the Education Department of Jiangsu Province (No. 16KJB330010), the Science and Technology Department of Jiangsu Province (No. BK20160227), the China Postdoctoral Science Foundation funded project (No. 2016M601892). The authors declare no competing financial interests.

Downregulated N-acetylglucosaminyltransferase III is involved in attenuating trophoblast migration and invasion under hypoxia–reoxygenation condition

Objective: Many studies have confirmed that N-acetylglucosaminyltransferase III (GnT-III) is correlated with tumor invasion and metastasis. However, the expression of GnT-III and its role in normal pregnancy and preeclampsia (PE) has not been reached. So the primary objective of this study is to determine GnT-III expression in normal pregnancy and whether its expression is vulnerable to oxidative stress in the trophoblast cells.Methods: Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used for the detection of GnT-III expression. Human first trimester extravillous trophoblast cell line (HTR8/SVneo) exposed to hypoxia/reoxygenation (H/R) condition was employed as an oxidative stress model in vitro to investigate the expression of GnT-III.Results: GnT-III was strongly expressed in cytotrophoblast (CTBs), syncytiotrophoblast (STBs) and the trophoblast columns (TCs) of human placental villi, and decidual cells in the maternal decidua. The expression of GnT-III was decreased in PE placentas compared with the normal control placentas. In addition, GnT-III was found to have decreased expression in H/R-exposed HTR8/SVneo cells, and the invasive and migratory abilities of HTR8/SVneo cells were attenuated, too.Conclusions: These findings suggest that GnT-III is an important regulator at the maternal-fetal interface during early pregnancy. Excessive oxidative stress can decrease GnT-III expression in trophoblast and the decreased expression of GnT-III may be involved in the development of preeclampsia.

PIM kinases 1, 2 and 3 in intracellular LIF signaling, proliferation and apoptosis in trophoblastic cells

Proviral insertion in murine (PIM) lymphoma proteins are mainly regulated by the Janus Kinase/Signal Transducer Activator of Transcription (JAK/STAT) signaling pathway, which can be activated by members of the Interleukin-6 (IL-6) family, including Leukemia Inhibitory Factor (LIF). Aim of the study was to compare PIM1, PIM2 and PIM3 expression and potential cellular functions in human first and third trimester trophoblast cells, the immortalized first trimester extravillous trophoblast cell line HTR8/SVneo and the choriocarcinoma cell line JEG-3. Expression was analyzed by qPCR and immunochemical staining. Functions were evaluated by PIM inhibition followed by analysis of kinetics of cell viability as assessed by MTS assay, proliferation by BrdU assay, and apoptosis by Western blotting for BAD, BCL-XL, (cleaved) PARP, CASP3 and c-MYC. Apoptosis and necrosis were tested by flow cytometry (annexin V/propidium iodide staining). All analyzed PIM kinases are expressed in primary trophoblast cells and both cell lines and are regulated upon stimulation with LIF. Inhibition of PIM kinases significantly reduces viability and proliferation and induces apoptosis. Simultaneously, phosphorylation of c-MYC was reduced. These results demonstrate the involvement of PIM kinases in LIF-induced regulation in different trophoblastic cell lines which may indicate similar functions in primary cells.

Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model

BackgroundFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by maternal antibodies against paternal human platelet antigens (HPAs) on fetal platelets. Antibodies against HPA-1a are accountable for the majority of FNAIT cases. We have previously shown that high levels of maternal anti-HPA-1a antibodies are associated with clinically significant reduced birth weight in newborn boys. Chronic inflammatory placental lesions are associated with increased risk of reduced birth weight and have previously been reported in connection with FNAIT pregnancies. The HPA-1a epitope is located on integrin β3 that is associated with integrin αIIb (the fibrinogen receptor) on platelets and megakaryocytes. Integrin β3 is also associated with integrin αV forming the αVβ3 integrin heterodimer, the vitronectin receptor, which is expressed on various cell types, including trophoblast cells. It is therefore thinkable that maternal anti-HPA-1a antibodies present during early pregnancy may affect placenta function through binding to the HPA-1a antigen epitope on invasive throphoblasts. The aim of the study was to examine whether interaction of a human anti-HPA-1a monoclonal antibody (mAb) with HPA-1a on trophoblast cells affect adhesion, migration and invasion of extravillous trophoblast cells.MethodsAn in vitro model with human anti-HPA-1a mAb, clone 26.4, and the first trimester extravillous trophoblast cell line HTR8/SVneo was employed. The xCELLigence system was utilized to assess the possible effect of anti-HPA-1a mAb on adhesion and migration of HTR8/SVneo cells. Specially designed chambers precoated with Matrigel were used to assess the effect on the invasive capacity of cells.ResultsWe found that human anti-HPA-1a mAb 26.4 partially inhibits adhesion and migratory capacity of HTR8/SVneo cells.ConclusionsOur findings suggest that anti-HPA-1a antibodies may affect trophoblast functions crucial for normal placental development. Future studies including primary throphoblast cells and polyclonal anti-HPA-1a antibodies are needed to confirm these results.

Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was measured by ELISA. aPL significantly increased trophoblast cellular and exosome expression of miR-146a-5p, miR-146a-3p, miR-155 and miR-210. aPL-induced up-regulation of trophoblast miR-146a-5p, miR-146a-3p and miR-210, but not miR-155, was inhibited by the TLR4 antagonist, LPS-RS. While inhibition or overexpression of miR-146a-5p had no effect on aPL-induced trophoblast IL-8 secretion, miR-146a-3p inhibition significantly reduced this response. aPL-induced trophoblast IL-8 secretion was inhibited by the presence of the TLR8-DN. In the absence of aPL, transfection of trophoblast cells with a miR-146a-3p mimic significantly increased IL-8 secretion and this was inhibited by the presence of the TLR8-DN. Patients with aPL and adverse pregnancy outcomes (APOs) expressed significantly higher levels of circulating miR-146a-3p compared with healthy pregnant controls with no pregnancy complications (P < 0.05). While the enrichment of miR-146a-3p in trophoblast-derived exosomes support the role of this miR acting in a paracrine or endocrine manner through exosome delivery, this has not been demonstrated. However, miR-146a-3p may also exert its pro-inflammatory effect intracellularly within the same trophoblast cell targeted by aPL. These findings provide a novel mechanism of trophoblast inflammation through miRs activating RNA-sensing receptors. Furthermore, circulating exosomal-associated miR-146a-3p in APS patients may serve clinically as a biomarker for related APOs. This study was supported in part by grants from the American Heart Association (#10GRNT3640032 to V.M.A.), the March of Dimes Foundation (Gene Discovery and Translational Research Grant #6-FY12-255 to V.M.A.), NICHD, NIH (R01HD049446 to V.M.A.), the Gina M. Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America (to S.M.G.), and the Yale University School of Medicine Medical Student Fellowship (to S.M.G.). The authors declare no competing financial interests. N/A.

ENaC mediates human extravillous trophblast cell line (HTR8/SVneo) invasion by regulating levels of matrix metalloproteinase 2 (MMP2)

IntroductionPlacenta dysfunction is thought to be the major etiological factor related to preeclampsia. The epithelial sodium channel (ENaC) has been localized in the apical plasma membrane of epithelia, mediating the active reabsorption of sodium in kidney, and be involved in the regulation of blood pressure. In previous studies, we found that the reduced expression of ENaC on placenta in preeclampsia patients. The aim of this study was to determine the role of MMP2 in the ENaC-induced trophoblast cell invasion ability, which is closely related to the occurrence of preeclampsia. MethodsHere we checked whether pregnancy related hormones human chorionic gonadotropin (HCG), prolactin and aldosterone could affect ENaC expression in the first trimester extravillous trophoblast cell line (HTR8/SVneo) by RT-PCR and Western blot. Cell invasion was studied by matrigel invasion assay. Tube formation assay was used to investigate the interaction between trophoblast cells and endothelial cells. The effects of ENaC on MMP2 were further determined by RT-PCR, western blot and gelatin zymography. ResultsWe demonstrated that HCG, prolactin and aldosterone could up-regulate the expression of αENaC in protein levels. Trophoblast cell invasion ability is stimulated when αENaC was up-regulated by aldosterone, and inhibited when ENaC was down-regulated by amiloride and αENaC specific RNAi (SiENA/ENaC). The interaction between HTR8/SVneo cells and HUVEC cells was enhanced when treated with aldosterone and weakened when treated with amiloride and SiRNA/ENaC. Amiloride and SiRNA/ENaC could inhibit MMP2 expression and activity. DisscutionAldosterone induced ENaC activity is important for trophoblast cells invasion. The results also indicate that ENaC could mediate trophoblast cells invasion ability through regulating expression and activity of matrix metalloproteinase-2 (MMP2).

Antiphospholipid antibodies effect over trophoblast cell function and pharmacological modulation

Event Abstract Back to Event Antiphospholipid antibodies effect over trophoblast cell function and pharmacological modulation Angela M. Alvarez1, Yonhatann Ramírez1, Sebastián Sanmartín2, Udo Markert3 and Angela Cadavid1* 1 University of Antioquia, Colombia 2 University of Valparaíso, Chile 3 University Hospital Jena, Germany Since antiphospholipid antibodies (aPL) are involved in impaired placental development in women with antiphospholipid syndrome (APS), the aim of this study was to determine the effect of aPL on the first trimester trophoblast cell line HTR-8/SVneo function and on the activation of the signal transducer and activator of the transcription 3 (STAT-3) pathway. IgGs purified from women with aPL and clinical manifestations of pregnancy morbidity and/or vascular thrombosis (PM and PM/VT groups), women with pregnancy morbidity without aPL (PM/aPL-), and also healthy women as controls (NHS) were used. First, the aPL binding to trophoblast cells was assessed by immunohistochemistry. Then, trophoblast viability, proliferation and invasion were evaluated. The effects of aspirin, enoxaparin and aspirin-triggered lipoxins (ATL) on antiphospholipid antibody-decreased trophoblast invasion were also evaluated. IgG from women with APS bound strongly to the first trimester extravillous trophoblast cell line HTR-8/SVneo. Reduced cell viability and STAT-3 activation were induced by IgG from women of PM group. On the other hand, the mitochondrial membrane integrity was reduced by IgG from women of PM/VT group, which in contrast induced an increase in proliferating trophoblast cells. Both PM and PM/VT IgG induced a decrease in trophoblast invasion. Aspirin, enoxaparin, and ATL restored the aPL-decreased invasion. Addition of the ATL receptor antagonist Boc-2, reversed the ATL-restored invasion. In conclusion, IgG from women with PM or PM/VT induced different effects on trophoblast cells. Trophoblast invasion was clearly altered but, interestingly, it was restored with enoxaparin, aspirin and, as shown for the first time with ATL. Additionally ATL has a receptor-mediated effect in trophoblast cells as demonstrated by addition of Boc-2. Acknowledgements CODI- Universidad de Antioquia Grant 91515; Sustainability Strategy Grupo Reproducción 2014-2015; Scientific International Cooperation: Colciencias Grant 91567 and CONICYT Grant PCCI 130017; AMA is fellow from Colciencias. Keywords: Antiphospholipid Syndrome, Antiphospholipid antibodies, Trophoblasts, STAT-3, trophoblast invasion Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Autoimmunity Citation: Alvarez AM, Ramírez Y, Sanmartín S, Markert U and Cadavid A (2015). Antiphospholipid antibodies effect over trophoblast cell function and pharmacological modulation. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00228 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 May 2015; Published Online: 14 Sep 2015. * Correspondence: MD, PhD. Angela Cadavid, University of Antioquia, Medellín, Colombia, angela.cadavid@udea.edu.co Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Angela M Alvarez Yonhatann Ramírez Sebastián Sanmartín Udo Markert Angela Cadavid Google Angela M Alvarez Yonhatann Ramírez Sebastián Sanmartín Udo Markert Angela Cadavid Google Scholar Angela M Alvarez Yonhatann Ramírez Sebastián Sanmartín Udo Markert Angela Cadavid PubMed Angela M Alvarez Yonhatann Ramírez Sebastián Sanmartín Udo Markert Angela Cadavid Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20μM BDE-47 for 24h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20μM BDE-47 for 24h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation.

MRNA induces RANTES production in trophoblast cells via TLR3 only when delivered intracellularly using lipid membrane encapsulation

Background Trophoblasts express Toll-like receptor 3 (TLR3). The artificial TLR3 ligand, PolyI:C, induces an inflammatory response in trophoblasts but an endogenous ligand has not been identified. Notably, inflammatory disorders of pregnancy are associated with increased circulating placenta-derived mRNA. Endogenous degraded, uncapped mRNA is recognized by TLR3 in other cell lines. Objective We tested the hypothesis that plasma-derived mRNA induces an inflammatory response in a trophoblast cell line via TLR3. Methods Experiments were performed in the human first trimester extravillous trophoblast cell line HTR-8/SV neo. Plasma-derived mRNA was amplified using modified template switching and final in vitro transcription. We compared free mRNA (which favors cell surface interaction) to liposomally encapsulated mRNA (which favors intracellular mRNA delivery). We tested for the specific requirement of TLR3 signaling using siRNA. We tested for involvement of the canonical signaling pathway downstream of TLR3 by measuring NF-κB and IFN regulatory factor transcriptional activity using firefly-luciferase constructs. Results Free mRNA did not induce RANTES production. In contrast, liposomal mRNA resulted in marked induction of RANTES production (non-stimulated control 3.4 ± 0.6 pg/mL, liposomal mRNA 169.7 ± 26.2 pg/mL, p < 0.001), and this RANTES production was abolished by siRNA for TLR3. Downstream of TLR3, liposomal mRNA-induced dose-response NF-κB and IFN regulatory factor transcriptional activity, and IFN beta production. Conclusion Plasma-derived 5′ uncapped mRNA delivered intracellularly signals to induce NF-κB activation and increase RANTES production via TLR3.

Tumour Necrosis Factor Alpha Stimulates the Production of Monocyte Chemoattractants by Extravillous Trophoblast Cells via Differential Activation of MAPK Pathways

The decidual microenvironment is characterized by a unique population of leukocytes composed primarily of CD56bright NK cells and macrophages. The latter are situated near trophoblast cells at the fetal–maternal interface and there is evidence that trophoblast cells are capable of recruiting macrophages to this site. This study sought to determine the role of tumour necrosis factor alpha (TNF) in the trophoblast-mediated recruitment of monocyte-derived macrophages to the fetal–maternal interface. The human first trimester extravillous trophoblast cell line HTR-8/SVneo was shown to express TNFR1 and to secrete the monocyte-attracting chemokines CCL2 and CCL5 after exposure to TNF in a dose-dependent manner. TNF-mediated stimulation of CCL2 secretion was completely inhibited by incubating the trophoblast cells with the p38-MAPK inhibitor SB203580, whereas CCL5 secretion was inhibited by treating the trophoblast cells with inhibitors specific for JNK (SP600125) and ERK kinase (U0126). Media conditioned by TNF-treated trophoblast cells significantly enhanced the ability of the monocyte cell line THP-1 to invade through Matrigel, and this effect was inhibited using antibodies specific for CCL2 and CCL5. These results support a role for TNF at the fetal–maternal interface as a regulator of macrophage recruitment by trophoblast cells.

Role of EG‐VEGF in human placentation: Physiological and pathological implications

Pre-eclampsia (PE), the major cause of maternal morbidity and mortality, is thought to be caused by shallow invasion of the maternal decidua by extravillous trophoblasts (EVT). Data suggest that a fine balance between the expressions of pro- and anti-invasive factors might regulate EVT invasiveness. Recently, we showed that the expression of the new growth factor endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is high in early pregnancy but falls after 11 weeks, suggesting an essential role for this factor in early pregnancy. Using human villous explants and HTR-8/SVneo, a first trimester extravillous trophoblast cell line, we showed differential expression of EG-VEGF receptors, PKR1 and PKR2, in the placenta and demonstrated that EG-VEGF inhibits EVT migration, invasion and tube-like organisation. EG-VEGF inhibitory effect on invasion was supported by a decrease in matrix metalloproteinase (MMP)-2 and MMP-9 production. Interference with PKR2 expression, using specific siRNAs, reversed the EG-VEGF-induced inhibitory effects. Furthermore, we determined EG-VEGF circulating levels in normal and PE patients. Our results showed that EG-VEGF levels were highest during the first trimester of pregnancy and decreased thereafter to non-pregnant levels. More important, EG-VEGF levels were significantly elevated in PE patients compared with age-matched controls. These findings identify EG-VEGF as a novel paracrine regulator of trophoblast invasion. We speculate that a failure to correctly down-regulate placental expression of EG-VEGF at the end of the first trimester of pregnancy might lead to PE.

Role of Hypoxia-Inducible Transcription Factors 1α and 2α in the Regulation of Plasminogen Activator Inhibitor-1 Expression in a Human Trophoblast Cell Line

The plasminogen activator inhibitors (PAIs) play critical roles in regulating hemostatic and invasive functions of trophoblasts through suppression of plasmin-dependent fibrinolysis and extracellular matrix degradation. The expression of PAI-1 is increased under hypoxic conditions, although the mechanism remains incompletely understood. In the current study we used HTR-8/SVneo cells, a first trimester extravillous trophoblast cell line, and siRNA technology to examine the role of hypoxia-inducible transcription factors (HIFs)-1α and -2α in the regulation of PAI-1 expression. Using serum-containing and serum-free media culture media it was initially noted that levels of PAI-1, but not PAI-2 protein, were markedly induced by hypoxic (2–3% oxygen) treatment. Under hypoxic conditions, Western blotting revealed that the presence of siRNAs to HIF-1α and HIF-2α suppressed expression of their respective proteins, whereas treatment with non-targeting and cyclophilin B siRNAs did not. Importantly, incubation with siRNA to HIF-1α or HIF-2α alone reduced PAI-1 protein levels to a similar extent, with the combined treatment inducing a more profound effect. The presence of HIF siRNAs reduced levels of PAI-1 mRNA as measured by quantitative real-time PCR, indicating that HIF-1α and HIF-2 α regulate PAI-1 expression at a transcriptional level. These results indicate that both HIF-1α and HIF-2α play important and similar roles in hypoxia-mediated stimulation of PAI-1 expression in HTR-8/SVneo cells. Our findings provide insight into the physiological regulation of trophoblast PAI-1 expression in early pregnancy when placental oxygen levels are low, as well as a mechanism for over-expression of placental PAI-1 noted in pregnancies with preeclampsia.

Induction of prostaglandin E2 production by leukemia inhibitory factor promotes migration of first trimester extravillous trophoblast cell line, HTR-8/SVneo

The invasion of first trimester extravillous trophoblast (EVT) to decidua is an important event in placentation. Leukemia inhibitory factor (LIF) is an essential factor for mouse implantation, and it is reported that LIF may be involved in human first trimester EVT invasion. Prostaglandin E2 (PGE2) is also known as a critical factor for first trimester EVT invasion. In this study, we investigated the role of LIF in PGE2 production and EVT invasion using a human first trimester EVT cell line, HTR-8/SVneo. Co-stimulation with LIF and IL-1beta induced higher amounts of PGE2 production and further migration of HTR-8/SVneo cells compared with that by stimulation with LIF or IL-1beta alone. Enhanced PGE2 production was most probably due to the enhanced expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). PGE2 produced by HTR-8/SVneo cells promoted the migration of HTR-8/SVneo cells. A COX-2 inhibitor suppressed PGE2 production and the migration of HTR-8/SVneo cells. Agonists to PGE2 receptors, EP1, EP2 and EP4, promoted the migration of HTR-8/SVneo cells. Moreover, stimulation with LIF up-regulated EP1, EP2 and EP4 expression in HTR-8/SVneo cells. It is suggested that LIF participates in placentation through EVT invasion by up-regulating PGE2 production and PGE2 receptor expression in first trimester EVT.