SVneo Cell Line Research Articles

Rosiglitazone blocks first trimester in-vitro placental injury caused by NF-\u03baB-mediated inflammation

Increased inflammation and abnormal placentation are common features of a wide spectrum of pregnancy-related disorders such as intra uterine growth restriction, preeclampsia and preterm birth. The inflammatory response of the human placenta has been mostly investigated in relation to cytokine release, but the direct molecular consequences on trophoblast differentiation have not been investigated. This study measured the general effects of LPS on both extravillous and villous trophoblast physiology, and the involvement of the transcription factors PPARγ and NF-κB, specifically using 1st trimester explants and HTR-8/ SVneo cell line models. While both proteins are known for their roles in inflammatory pathways, PPARγ has been identified as an important molecule in trophoblast differentiation, suggesting its potential role in mediating a crosstalk between inflammation and trophoblast differentiation. Here, LPS (1 µg/ml) exposure of first trimester placental villous explants resulted in secretion of inflammatory cytokines, induction of apoptosis and reduction in trophoblast cell proliferation. Additionally, LPS significantly reduced expression of the trophoblast differentiation proteins GCM1 and β-hCG, and increased invasion of the extravillous trophoblast. Activation of PPARγ by Rosiglitazone (10 µM) reversed the LPS-mediated effects on inflammatory cytokine release, trophoblast apoptosis and proliferation compared to controls. Lastly, markers of trophoblast differentiation and invasion reverted to control levels upon activation of PPARγ and concomitant inhibition of NF-κB (either by Rosiglitazone or NF-κB specific inhibitor), revealing a new role for NF-κB in trophoblast invasion. This study reveals a novel PPARγ - NF-κB axis that coordinates inflammatory and differentiation pathways in the human placenta. The ability to reverse trophoblast-associated inflammation with Rosiglitazone offers promise that the PPARγ – NF-κB pathway could one day provide a therapeutic target for placental dysfunction associated with both inflammation and abnormal trophoblast differentiation.

EIF5A1 promotes trophoblast migration and invasion via ARAF-mediated activation of the integrin/ERK signaling pathway

Trophoblast dysfunction is one mechanism implicated in the etiology of recurrent miscarriage (RM). Regulation of trophoblast function, however, is complex and the mechanisms contributing to dysregulation remain to be elucidated. Herein, we found EIF5A1 expression levels to be significantly decreased in cytotrophoblasts in RM villous tissues compared with healthy controls. Using the HTR-8/SVneo cell line as a model system, we found that overexpression of EIF5A1 promotes trophoblast proliferation, migration and invasion in vitro. Knockdown of EIF5A1 or inhibiting its hypusination with N1-guanyl-1,7-diaminoheptane (GC7) suppresses these activities. Similarly, mutating EIF5A1 to EIF5A1K50A to prevent hypusination abolishes its effects on proliferation, migration and invasion. Furthermore, upregulation of EIF5A1 increases the outgrowth of trophoblasts in a villous explant culture model, whereas knockdown has the opposite effect. Suppression of EIF5A1 hypusination also inhibits the outgrowth of trophoblasts in explants. Mechanistically, ARAF mediates the regulation of trophoblast migration and invasion by EIF5A1. Hypusinated EIF5A1 regulates the integrin/ERK signaling pathway via controlling the translation of ARAF. ARAF level is also downregulated in trophoblasts of RM villous tissues and expression of ARAF is positively correlated with EIF5A1. Together, our results suggest that EIF5A1 may be a regulator of trophoblast function at the maternal–fetal interface and low levels of EIF5A1 and ARAF may be associated with RM.

MiR-616-3p modulates cell proliferation and migration through targeting tissue factor pathway inhibitor 2 in preeclampsia.

Despite improvements in diagnosis and treatment, preeclampsia (PE) continues to pose a significant risk of maternal and foetal morbidity and mortality if not addressed promptly. An increasing number of studies have suggested that tissue factor pathway inhibitor 2 (TFPI2) acts as a suppressor gene, possibly inhibiting multiple serine proteases affecting cell proliferation and migration. It plays an essential role in the occurrence and development of PE, but the pathogenesis remains unclear. In our research, we performed western blotting, immunohistochemistry and qPCR assays to investigate TFPI2 and miR-616-3p expression in preeclamptic placental tissues. Cell assays were performed in HTR-8/SVneo and JEG3 cell lines. Cell proliferation and migration events were investigated by MTT, EdU and transwell assays. In conjunction with bioinformatics analysis, luciferase reporter assays were performed to elucidate the mechanism by which miR-616-3p binds to TFPI2 mRNA. We established that TFPI2 protein levels were significantly upregulated in PE placental tissues. In addition, we found that miR-616-3p binds specifically to the 3'-UTR region of TFPI2 mRNA. Furthermore, miR-616-3p knockdown or TFPI2 overexpression substantially impaired cell growth and migration, whereas miR-616-3p upregulation or TFPI2 knockdown stimulated cell proliferation and migration. This miR-616-3p/TFPI2 axis was also found to affect the epithelial-mesenchymal transition process in PE. Our results demonstrated that TFPI2 plays a vital role in the progression of PE and might provide a prospective therapeutic strategy to mitigate the severity of the disorder.

Immunoglobulins from sera of antiphospholipid syndrome patients are internalized in the HTR-8/SVneo cell line and cytotrophoblast in culture

Women with antiphospholipid syndrome (APS) experience pregnancy complications mostly due to impaired trophoblast cell functions. Antiphospholipid antibodies (aPL) affect extravillous trophoblast in vivo and in culture, but the mechanisms are still poorly understood. Previously, syncytiotrophoblast was shown to bind and internalize aPL, which was not replicated for extravillous cytotrophoblast in short term culture. Here, aPL binding and time dependent internalization was demonstrated with exposure to aPL in the extravillous cell line HTR-8/SVneo and isolated first trimester of pregnancy cytotrophoblast (CT) using immunocytochemistry and flow cytometry. Intracellular aPL were detectable from 2 h of culture, reaching 30.7 ± 3.1% (p < 0.001) positive cells in CT and 24.8 ± 7% (p < 0.01) in HTR-8/SVneo cells at 24 h and 33 ± 4.2% (p < 0.01) at 48 h. The data presented show that extravillous trophoblast cells internalize aPL in a time-dependent manner significantly more than control immunoglobulins after 24 h of exposure.

Downregulated lncRNA HOXA11-AS Affects Trophoblast Cell Proliferation and Migration by Regulating RND3 and HOXA7 Expression in PE

The long noncoding RNA HOXA11-AS displays abnormal expression in numerous human diseases. However, its function and biological mechanisms remain unclear in preeclampsia (PE). In this study, we report that HOXA11-AS is significantly downregulated in preeclamptic placental tissues and could contribute to the occurrence and development of PE. Silencing of HOXA11-AS expression could significantly suppress trophoblast cell growth and migration, whereas HOXA11-AS overexpression facilitated cell growth in the HTR-8/SVneo, JEG3, and JAR cell lines. RNA-seq analysis also indicated that HOXA11-AS silencing preferentially regulated numerous genes associated with cell proliferation and cell migration. Mechanistic analyses showed that HOXA11-AS could recruit Ezh2 and Lsd1 protein and regulate RND3 mRNA expression in the nucleus. In the cytoplasm, HOXA11-AS modulates HOXA7 expression by sponged miR-15b-5p, affecting trophoblast cell proliferation. Together, these data confirm that aberrant expression of HOXA11-AS is involved in the occurrence and development of PE and may act as a prospective diagnosis and therapeutic target in PE.

Involvement of mTOR, JNK and PI3K in the negative effect of ethanol and metformin on the human first-trimester extravillous trophoblast HTR-8/SVneo cell line

Our aim was to investigate the effect of two xenobiotics to which pregnant woman may be exposed, the drug of abuse ethanol (EtOH) (and its metabolite acetaldehyde (ACA)) and the therapeutic agent metformin (METF), on placentation-related processes in an extravillous trophoblastic (EVTs) cell line (HTR-8/SVneo cells). EtOH, ACA and METF (24 h) significantly reduced cell proliferation rates, culture growth, viability and migratory capacity of HTR-8/SVneo cells. Moreover, both EtOH (100 μM) and METF (1 mM) increased the apoptosis index and inhibited 3H-deoxy-D-glucose (3H-DG) and 3H-folic acid (3H-FA) uptake. mTOR, JNK and PI3K intracellular signaling pathways were involved in the effect of EtOH upon 3H-FA uptake and in the effect of METF upon cell viability, and mTOR and JNK in the effect of EtOH upon cell viability and 3H-DG uptake.We show that EtOH and METF have a detrimental effect in placentation-related processes of HTR-8/SVneo cells. Moreover, mTOR, JNK and PI3K appear to mediate some of these negative effects.

Down-Regulation of Neuropathy Target Esterase in Preeclampsia Placenta Inhibits Human Trophoblast Cell Invasion via Modulating MMP-9 Levels

Background/Aims: Neuropathy target esterase (NTE, also known as neurotoxic esterase) is proven to deacylate phosphatidylcholine (PC) to glycerophosphocholine as a phospholipase B. Recently; studies showed that artificial phosphatidylserine/PC microvesicles can induce preeclampsia (PE)-like changes in pregnant mice. However, it is unclear whether NTE plays a key role in the pathology of PE, a pregnancy-related disease, which was characterized by deficient trophoblast invasion and reduced trophoblast-mediated remodeling of spiral arteries. The aim of this study was to investigate the expression pattern of NTE in the placenta from women with PE and normal pregnancy, and the molecular mechanism of NTE involved in the development of PE. Methods: NTE expression levels in placentas from 20 pregnant women with PE and 20 healthy pregnant women were detected using quantitative PCR and immunohistochemistry staining. The effect of NTE on trophoblast migration and invasion and the underlying mechanisms were examined in HTR-8/SVneo cell lines by transfection method. Results: NTE mRNA and protein expression levels were significantly decreased in preeclamptic placentas than normal control. Over-expression of NTE in HTR-8/SVneo cells significantly promoted trophoblast cells migration and invasion and was associated with increased MMP-9 levels. Conversely, shRNA-mediated down-regulation of NTE markedly inhibited the cell migration and invasion. In addition, silencing NTE reduced the MMP-9 activity and phosphorylated Erk1/2 and AKT levels. Conclusions: Our results suggest that the decreased NTE may contribute to the development of PE through impairing trophoblast invasion by down-regulating MMP-9 via the Erk1/2 and AKT signaling pathway.

Decidual ACVR2A regulates extravillous trophoblast functions of adhesion, proliferation, migration and invasion in vitro

Decidual stromal cells form the largest proportion of maternal cells at the maternal-fetal interface. Our aim was to investigate the role of the pre-eclampsia associated decidual activin receptor, ACVR2A, in regulating trophoblast functions at this interface. St-T1b and HTR-8/SVneo cell lines were used to model decidual stromal and trophoblast cells respectively. St-T1b conditioned medium inhibited HTR-8/SVneo adhesion, proliferation, migration and invasion; all effects that were attenuated by decidual ACVR2A siRNA transfection. These findings suggest that altered decidual ACVR2A expression perturbs the maternal-fetal crosstalk involved in regulating trophoblast function at the interface, which may affect placentation and lead to pre-eclampsia.

BMAL1 facilitates trophoblast migration and invasion via SP1-DNMT1/DAB2IP pathway in recurrent spontaneous abortion.

The underlying mechanism about rhythms and epigenetics leading to aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA) remains unknown. Brain and muscle ARNT-like protein 1 (BMAL1) is considered as a crucial role in fertility, and polymorphism of BMAL1 gene has been reported to be associated with risk of miscarriage. However, the functional role of BMAL1 in RSA is not fully understood. Previous study shows the descended expression of DNA 5′-cytosine-methyltransferases 1 (DNMT1) in the villous of early pregnancy loss. Thus, understanding of the regulation of DNMT1 expression may be of significance for the elucidation of the process of RSA. Using HTR-8/SVneo and JEG-3 cell lines, we certified the induction of specificity protein 1 (SP1) to DNMT1 and DAB2 interaction protein (DAB2IP), respectively, both of which further activated matrix metallo-proteinase 2/9 (MMP2/9), bringing out changes in trophoblast migration and invasion. Notably, BMAL1 functioned as a positive upstream factor of SP1 only in HTR-8/SVneo cells but not in JEG-3 cells, inducing SP1-DNMT1/DAB2IP pathway and facilitating migration and invasion of trophoblasts. In addition, progesterone might restore the down-regulation of BMAL1 and downstream pathway in a dose-dependent manner. Last but not least, the decreased abundance of BMAL1 was correlated positively with that of SP1, DNMT1, DAB2IP, MMP2 and MMP9 in human villous specimens of RSA. Our results demonstrate that the induction of BMAL1 to SP1 contributes to the expression of DNMT1 and DAB2IP, respectively, activating trophoblast migration and invasion. The deregulation of the BMAL1-mediated pathway in RSA can be rescued by progesterone.

Lysophosphatidic acid-triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro.

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary-like structures, migration, and proliferation. The HTR-8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR-8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein-coupled receptors, LPA1 and LPA3 , were expressed in HTR-8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA3 . In addition, cyclooxygenase-2 and inducible nitric oxide synthase pathways participated in LPA-stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase-2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration-dependent manner. Finally, we observed that cyclooxygenase-2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA-triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal-fetal interface.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6β4 and α1β1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression. LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6β4-α1β1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling. N/A. The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available. These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders. This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

HTR-8/SVneo cell line contains a mixed population of cells

IntroductionThe placenta, a transient organ in humans, is essential for pregnancy maintenance and fetal development. Trophoblast and stromal cells are the main cell types present in human placenta. Trophoblast cells are derivatives of the trophectoderm layer and fulfill the endocrine, exchange, invasion and implantation processes of the placenta, whereas stromal cells are of extraembryonic mesenchymal origin and are important for villous formation and maintenance. Different cell lines were developed to study trophoblast functions including BeWo, JEG-3 and JAR from chorioncarcinoma while HTR-8/SVneo was developed using first trimester extravillous trophoblast infected with simian virus 40 large T antigen (SV40). These cell lines are largely used to study trophoblast functions including cell fusion, migration and invasion. Therefore, the purity of each cell lines is crucial in order to be able to use them as a model recapitulating trophoblast cells. MethodsHTR-8/SVneo, BeWo, JEG-3 and JAR were analyzed for epithelial and mesenchymal markers using immunofluorescence, real time PCR and Western blot. ResultsOur results showed that HTR-8/SVneo cell line contains two populations of cells suggesting the presence of trophoblast and stromal/mesenchymal cells. While all cells in BeWo, JEG-3 and Jar are positive for the trophoblast/epithelial marker CK7, HTR-8/SVneo cells contained few clusters of CK7 positive cells. Interestingly, vimentin expression was detected in a subset of HTR-8/SVneo cells and was completely absent from all other tested placental cell lines. DiscussionOur results unveil the presence of a heterogeneous population of trophoblast and stromal cells within HTR-8/SVneo cell line. This mixed population of cells should be taken into consideration when using this cell line to study trophoblast functions.

Increased Heme Oxygenase-1 and Nuclear Factor Erythroid 2-Related Factor-2 in the Placenta Have a Cooperative Action on Preeclampsia

Background: Previous studies have shown that oxidative stress is an important factor in preeclampsia (PE). Heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor-2 (Nrf2) are protective proteins that are involved in combating oxidative stress in the body. Nrf2 is also an essential upstream transcription factor regulating HO-1. This study was aimed at exploring the physiological roles of HO-1 and Nrf2 in PE. Methods: Serum and placenta were collected from 30 patients who presented with severe PE and 30 healthy pregnant females. HO-1 and Nrf2 levels in placenta were measured. Following stimulation of the HTR-8/SVneo cell line with tert-butylhydroquinone (tBHQ), an Nrf2 activator, nuclear Nrf2 protein and HO-1 mRNA levels were determined. Results: Compared with the healthy pregnancy group, HO-1 protein and mRNA levels were increased in placental samples obtained from the severe PE group (p < 0.01, p < 0.05). Similar increases were also observed for Nrf2 protein levels (p < 0.01). Nuclear Nrf2 protein and HO-1 mRNA levels were both increased in the HTR-8/SVneo cell line following stimulation with tBHQ (p < 0.05). Conclusion: Patients with severe PE may be protected against oxidative injury following an elevation in HO-1 and Nrf2 levels. Nrf2 is likely to have a synergistic effect on HO-1 in PE.

MiR-346 and miR-582-3p-regulated EG-VEGF expression and trophoblast invasion via matrix metalloproteinases 2 and 9.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an important regulator for embryo implantation and placental development, and is clinically associated with several obstetric disorders related to insufficient or inappropriate trophoblast invasion, such as recurrent abortion, preeclampsia, and intrauterine fetal growth restriction. This study was performed to identify the microRNAs targeting EG-VEGF, and evaluate the regulatory effect on trophoblast biology. miR-346 and miR-582-3p were initially identified via bioinformatic tools, and their specific binding sites on the EG-VEGF 3'UTR were further confirmed using dual luciferase and a co-transfection assays. miR-346 and miR-582-3p were demonstrated not only to suppress EG-VEGF expression, but also inhibit trophoblast invasion and migration in the JAR and HTR-8/SVneo cell lines. We further evaluated the effect of microRNAs in HTR-8/SVneo cells coexpressing EG-VEGF and miR-346 or miR-582-3p on matrix metalloproteinase (MMP 2 and MMP 9) and the tissue inhibitors of metalloproteinase (TIMP 1 and TIMP 2) using RT-PCR, western blotting and gelatin zymography. TIMP 1 and TIMP 2 were not affected by the two microRNAs, whereas the expressions and activities of MMP 2 and MMP 9 were significantly downregulated, which in turn inhibited the invasion ability of trophoblasts. In conclusion, miR-346 and miR-582-3p regulate EG-VEGF-induced trophoblast invasion through repressing MMP 2 and MMP 9, and may become novel diagnostic biomarkers or therapeutic targets for EG-VEGF-related obstetric disorders. © 2016 BioFactors, 43(2):210-219, 2017.

MiR-15b-AGO2 play a critical role in HTR8/SVneo invasion and in a model of angiogenesis defects related to inflammation

IntroductionmicroRNAs (miRs) have been shown to play critical roles in the regulation of trophoblast and endothelial cell functions, and one significant finding concerning the miR-15/16 family is that most members of this family are highly expressed in endothelial cells and contribute to functions, such as tube formation. The interaction between trophoblast and endothelial cell play an important role in normal placentation process. Therefore, the aims of this study were to investigate the expression of miR-15b in human placenta and to uncover the potential role of miR-15b as well as its target functional loop in trophoblast and endothelial cells. Whether inflammation could modulate the expression of miR-15b and its down-stream target was further investigated. Additionally, the potential link between miR-15b deregulation and preeclampsia was also explored in the placenta of patients diagnosed with preeclampsia. MethodsThe expression of miR-15b was studied in the placental tissue of a normal pregnancy using in situ hybridization, and the effects of miR-15b on proliferation, invasion, and angiogenesis were further explored in vitro using HTR-8/SVneo and HUVEC cell line models. A Lipopolysaccharides (LPS) treatment model in HTR-8/SVneo cell was utilized to explore the mechanism of how LPS treatment could lead to the activation of miR-15b expression. Western blot was used to detect the expression of proteins related to miR-15b mediated pathway in preeclamptic placentas. ResultsmiR-15b inhibits trophoblast cell invasion and endothelial cell tube formation by suppressing the expression of Argonaute 2 (AGO2), a major miRNA effecter protein. AGO2 is specifically localized to human placenta cytotrophoblast and endothelial cells, and it plays important roles in trophoblast cell invasion and endothelial cell tube formation. LPS treatment may lead to the overexpression of miR-15b and down-regulation of AGO2, which may be involved in shallow trophoblast cell invasion associated with the pathogenesis of preeclampsia. Chromatin immunoprecipitation assay indicates that increased occupancy of AGO2 to miR-15b promoter is responsible for the increased expression of miR-15b under the condition of LPS treatment. Furthermore, preeclamptic placentas have decreased expression of AGO2, but increased expression of miR-15b and TLR-4 compared to normal controls. DiscussionThis is the first report about the function of AGO2 in human trophoblast and endothelial cells in the placenta. The data indicates that the aberrant expression of miR-15b contributes to abnormal placentation by targeting AGO2 mRNA. This study provides insight into the potential role of the miR-15b and AGO2 functional loop in the placentation process.

Expression of the ciliary neurotrophic factor and its receptor α in human placenta of first and third trimester of gestation

The ciliary neurotrophic factor (CNTF) is a member of the IL-6 family of cytokines along with cardiotrophin-1, IL-11, leukemia inhibitory factor, oncostatin-M and IL-6 itself. These cytokines play an important role in the regulation of cellular processes such as gene activation and cell proliferation and differentiation. CNTF is a pleiotropic cytokine which effects are mediated via CNTF receptor α (CNTFRα). CNTF increases differentiation and/or survival in neuronal cells but it also has different effects on other cell types such as muscle cells, bone cells, adipocytes, retinal cells and pancreatic β-cells (1, 2). In addition, recent studies demonstrate that CNTF plays an important role in weight control since exogenously administration of CNTF has an anorectic effect in mice (3,4). Although many studies proved that CNTF plays different roles in many cell types, its role in the development of human placenta has never been investigated. In this study we investigated the expression of CNTF and CNTFRα in human trophoblast by, immunohistochemistry, immunocytochemistry and Western Blot analysis using normal first and third trimester human placentas and HTR-8/SVneo cell lines. Interestingly, using immunohistochemistry CNTF and CNTFRα were expressed in the cytotrophoblast and syncytiotrophoblast in the first and third trimester of gestation respectively. Moreover, the immunofluorescence analyses by confocal microscopy showed that CNTF is expressed in the cytoplasm and nuclei whereas CNTFRα is mainly expressed in the cell membrane and cytoplasm of HTR-8/SVneo cell line. In this study we demonstrated that CNTF and CNTFRα are normally expressed in human placenta and they may play an important role during placental development.

Expression of GRIM-19 in missed abortion and possible pathogenesis

To study the expression of a gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19) in the villi of patients with missed abortion and possible pathogenesis. Case-control study. University hospital. Villous samples were collected from a total of 50 women with missed abortion and 50 women with normal pregnancies. Villous samples from the missed-abortion group were collected by curettage or manual vacuum aspiration. The control villous samples were obtained by vacuum aspiration. Protein and messenger RNA levels of GRIM-19 in villous samples were measured by Western blot and real-time polymerase chain reaction, and the location of GRIM-19 in trophoblasts was detected by immunohistochemistry. Apoptotic cells and microvessel density in villous sample were assayed by TUNEL and immunostaining. Mitochondrial membrane potential and apoptotic changes of the HTR-8/SVneo cell line in response to down-regulation of GRIM-19 were evaluated by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide and annexin V/propidium iodide. Vascular endothelial growth factor production was measured by ELISA. GRIM-19 protein and messenger RNA levels in the villi from women with missed abortion were found to be significantly lower than in women who had normal pregnancies. Many more apoptotic cells and lower microvessel density were detected in villifrom patients with missed abortion. Knockdown of GRIM-19 in HTR-8/SVneo cells gave rise to a collapse of mitochondrial membrane potential and increase in apoptosis. Simultaneously, the vascular endothelial growth factor secretion in the HTR-8/SVneo cells culture medium decreased as GRIM-19 became down-regulated. The GRIM-19 deficiency in the villus may be associated with missed abortion via increasing apoptosis and affecting angiogenesis.

Effects of transforming growth factor-β1 on the proliferation and invasion of the HTR-8/SVneo cell line.

Transforming growth factor-β1 (TGF-β1) is involved in the regulation of trophoblast cell proliferation and invasion. However, the mechanism underlying this process remains unknown, which is predominantly due to the difficulty in obtaining and maintaining primary trophoblast cells in culture over a long period of time. The HTR-8/SVneo cell line is an immortalized trophoblast cell line, which has been reported to exhibit a number of similar characteristics to those of parental trophoblast cells. Therefore, the cell line has been a useful tool for the investigation of placental function and tumor progression. In the present study, the HTR-8/SVneo cell line was used as a model to investigate the TGF-β1/SMAD signaling pathway in the proliferation and invasion of trophoblast cells. The proliferation and invasion ability of HTR-8/SVneo cells was determined using the MTT and Transwell assays, respectively. In addition, reverse transcription polymerase chain reactions were performed to detect the mRNA expression of a panel of known downstream mediators of TGF-β1, including TGF-β receptor I (TβRI), SMAD4, SMAD3, SMAD7 and tissue inhibitor of metalloproteinases-1 (TIMP-1). The results indicated that TGF-β1 promotes the proliferation and invasion of the HTR-8/SVneo cell line at passage 90. Furthermore, the expression of TβRI, SMAD3 and SMAD4 were reduced following treatment with TGF-β1, while the expression of SMAD7 was increased and the expression of TIMP-1 remained unchanged following TGF-β1 treatment. These observations indicated that the effects of TGF-β1 on the proliferation and invasion of the HTR-8/SVneo cell line at passage 90 were different from those of parental trophoblasts, which is in contrast to the results of previous studies. It was concluded that the HTR-8/SVneo cell lines, which have been grown for over 90 passages, do not accurately represent parental trophoblast cells in studies of the TGF-β/SMAD signaling pathway.

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.

CDX2 Enhances HTR-8/SVneo Trophoblast Cell Invasion by Altering the Expression of Matrix Metalloproteinases

Background/Aims: The invasion of trophoblast cells into the maternal uterine decidua is critical for normal placentation, establishment of pregnancy and maintenance of fetal growth in humans. Several growth factors and cytokines have been implicated in trophoblast invasion, but the underlying regulatory mechanisms of invasion are not fully understood. Our earlier studies have found that caudal-related homeobox transcription factor 2 (CDX2) is hypomethylated in human pre-eclampsia placental tissues. However, whether CDX2 is involved in trophoblast invasion was unclear. Methods and Results: In this study, we investigated CDX2 function using a human HTR-8/SVneo cell line that overexpressed CDX2. Cell invasion assays demonstrated that CDX2 enhanced trophoblast cell invasiveness. Meanwhile, MTT assays revealed that CDX2 did not affect cell proliferation. Western blot analysis and quantitative real-time PCR demonstrated that the expression level of matrix metalloproteinase-9 (MMP-9) was significantly increased, whereas the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) was markedly suppressed in the CDX2-overexpressing trophoblast cells. The phosphoinositide-3-kinase (PI3K)/Akt signaling pathway is involved in proliferation, migration, metastasis and invasion. Our study showed that inhibition of PI3K/Akt signaling led to decreased expression of CDX2. Conclusion: We concluded that CDX2 is likely regulated by the PI3K/Akt signaling pathway during trophoblast cell invasion. Our findings may reveal new insights into the regulatory mechanisms of trophoblast cell invasion and may be an important contributor to the pathogenesis of pregnancy-related diseases.