Human Trophoblast Cell Line HTR-8 Research Articles

Procyanidin A1 acts as an antioxidant stressor in gestational diabetes

Purpose: To evaluate the mechanism and effect of Procyanidin A1 (PCA1) in gestational diabetes mellitus (GDM).Methods: Human trophoblast cell line HTR-8/Svneo was treated with 25 mM glucose for 24 h, and the effect of PCA1 on HTR-8/SVneo cell viability and proliferation was determined by CCK-8 assay and EdU proliferation kit. The expression of BAX, Bcl-2, cleaved Caspase-3 and cleaved caspase-9 was determined by western blotting. Cell apoptosis was assessed by Annexin V-FITC/ PI staining. Cellular generation of reactive oxygen species (ROS) was determined by ROS assay kit while superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were determined by the corresponding assay kits. The effect of PCA1 on Nrf2/HO-1 pathway was evaluated by determining the expression of Keap1, Nfr2, and HO-1.Results: Procyanidin A1 (PCA1) increased the viability of high glucose (HG) -induced HTR-8/SVneo cells and promoted cell proliferation. Furthermore, PCA1 significantly inhibited HG-induced BAX, cleaved caspase-3, and cleaved caspase-9 expression, resulting in further reduction in HG-induced cell apoptosis. High glucose (HG) induced a significant increase in intracellular ROS levels, and this HGinduced oxidative stress was inhibited by PCA1. Furthermore, PCA1 activated Nrf2/HO-1 pathway and this was responsible for its proliferative, anti-apoptosis and anti-oxidative effects.Conclusion: Procyanidin A1 promotes proliferation, and inhibits apoptosis and oxidative stress induced by high glucose in trophoblast cells by activating Nrf2/HO-1 signaling pathway. Therefore, it is a potential drug for the treatment of GDM.

CoCl2-mimicked hypoxia induces the assembly of stress granules in trophoblast cells via eIF2α phosphorylation-dependent and -independent pathways.

Hypoxia has been implicated in preeclampsia (PE) pathophysiology. Stress granules (SGs) are present in the placenta of patients with PE. However, the pathways that contribute to SG aggregation in PE remain poorly understood. The objective of the current study is to investigate this issue. We first established an in vitro hypoxia model using human trophoblast cell line HTR-8/SVneo treated with cobalt chloride (CoCl2). CCK8 assay and wound healing assay were conducted to assess the viability and migration of HTR-8/SVneo cells after exposure to CoCl2-mimicked hypoxia. SG component expression in HTR-8/SVneo cells treated with CoCl2 alone, or in combination with indicated siRNAs was evaluated by reverse transcription quantitative PCR (RT-qPCR), western blot and immunofluorescence staining. Our results found CoCl2-mimicked hypoxia inhibits the proliferation and migration of HTR-8/SVneo cells. The treatment of CoCl2 can induce SG assembly in HTR-8/Svneo cells. Mechanistically, both heme-regulated inhibitors (HRI) mediated eukaryotic translation initiation factor (eIF)2α phosphorylation pathway and 4E binding protein 1 (4EBP1) pathway are involved in SG formation under the stress of CoCl2-mimicked hypoxia. Hypoxia-induced SGs in trophoblast cells might contribute to the etiology of PE.

Modified Shoutai Pill inhibited ferroptosis to alleviate recurrent pregnancy loss

Ethnopharmacological relevanceModified Shoutai Pill, also called Jianwei Shoutai Pill (JSP), is a traditional Chinese medicine prescription that has been used as an effective agent for the treatment of miscarriage. Aim of the studyTo explore the potential molecular mechanism of JSP against recurrent pregnancy loss (RPL). Materials and methodsIn vivo, CBA/J mated DBA/2 mice were used to conduct RPL model, while CBA/J mated BALB/c mice were seen as the control group. Mice were orally administered with JSP, Fer-1 (a ferroptosis inhibitor) or distilled water from day 0.5–12.5 of gestation (GD 0.5–12.5). Pregnancy outcomes were analyzed and ferroptosis related indexes of the whole implantation sites were measured on GD 12.5. In vitro, human trophoblast cell line HTR-8/SVneo was cultured and treated with RAS-selective lethal small molecule 3 (RSL3) (a ferroptosis agonist) or different concentrations of JSP. Then, ferroptosis related indexes were tested to analyze whether JSP could inhibit ferroptosis in HTR-8/SVneo cells. ResultsIn vivo consequences demonstrated that JSP or Fer-1 alleviated pregnancy outcomes including lower resorption rate and abortion rate. In addition, excessive iron accumulation and MDA level were inhibited, while GSH and GPX content were raised under JSP or Fer-1 exposure. Also, JSP or Fer-1 enhanced protein expressions of GPX4 and SLC7A11 which suppress ferroptosis, and lightened protein expression of ACSL4 which boosts ferroptosis. In vitro, JSP rescued HTR-8/SVneo cell death and migration ability that were injured by RSL3. Furthermore, JSP inhibited RSL3-induced intracellular reactive oxygen species (ROS), lipid ROS and iron deposition. ConclusionsCollectively, our findings illustrated that the mechanism of JSP in treating RPL might be related to inhibiting ferroptosis, which provided a novel insight into the application of JSP in RPL intervention.

TMBIM4 Deficiency Facilitates NLRP3 Inflammasome Activation-Induced Pyroptosis of Trophoblasts: A Potential Pathogenesis of Preeclampsia

Impaired invasion of EVTs results in inadequate remodelling of arteries and poor placentation, leading to PE. TMBIM4 was found to promote the migration and invasion of human osteosarcoma U2-OS and breast cancer MCF7 cell lines. However, the effect of TMBIM4 on trophoblast biological behaviour and its relevance to PE pathophysiology remain unclear. In this study, we confirmed that TMBIM4 was highly expressed in cytotrophoblasts, syncytiotrophoblasts, and EVTs of the human placenta during early pregnancy. By comparing the expression levels of TMBIM4 in the placenta of women with normal-term pregnancy and PE, TMBIM4 was found to be significantly decreased in PE. Thereafter, we determined the expression of TMBIM4 in the LPS-treated first-trimester human trophoblast cell line HTR-8/SVneo (mimicking a PE-like cell model), and determined the effect of TMBIM4 on trophoblast function and its underlying mechanism. LPS treatment reduced the expression of TMBIM4 and induced NLRP3 inflammasome activity in HTR-8/SVneo cells. KO of TMBIM4 in the HTR-8/SVneo cell line impaired cell viability, migration, and invasion, which was more severe in the LPS/ATP-treated TMBIM4-KO cell line. Moreover, TMBIM4 deficiency enhanced NLRP3 inflammasome activity and promoted subsequent pyroptosis, with or without LPS/ATP treatment. The negative relationship between TMBIM4 expression and NLRP3 inflammatory activity was verified in PE placentas. Inhibiting the NLRP3 inflammasome with MCC950 in HTR-8/SVneo cells alleviated LPS/ATP-induced pyroptosis and damaged cell function in the TMBIM4-KO cell line. Overall, this study revealed a new PE-associated protein, TMBIM4, and its biological significance in trophoblast pyroptosis mediated by the NLRP3 inflammasome. TMBIM4 may serve as a potential target for the treatment of placental inflammation-associated PE.

LDHA deficiency inhibits trophoblast proliferation via the PI3K/AKT/FOXO1/CyclinD1 signaling pathway in unexplained recurrent spontaneous abortion.

Dysregulated trophoblast proliferation, invasion, and apoptosis may cause several pregnancy-associated complications, such as unexplained recurrent spontaneous abortion (URSA). Recent studies have shown that metabolic abnormalities, including glycolysis inhibition, may dysregulate trophoblast function, leading to URSA. However, the underlying mechanisms remain unclear. Herein, we found that lactate dehydrogenase A (LDHA), a key enzyme in glycolysis, was significantly reduced in the placental villus of URSA patients. The human trophoblast cell line HTR-8/SVneo was used to investigate the possible LDHA-mediated regulation of trophoblast function. LDHA knockdown in HTR-8/SVneo cells induced G0/G1 phase arrest and increased apoptosis, whereas LDHA overexpression reversed these effects. Next, RNA sequencing combined with Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that the PI3K/AKT signaling pathway is potentially affected by downstream genes of LDHA. Especially, we found that LDHA knockdown decreased the phosphorylation levels of PI3K, AKT, and FOXO1, resulting in a significant downregulation of CyclinD1. In addition, treatment with an AKT inhibitor or FOXO1 inhibitor also verified that the PI3K/AKT/FOXO1 signaling pathway influenced the gene expression of CyclinD1 in trophoblast. Moreover, p-AKT expression correlated positively with LDHA expression in syncytiotrophoblasts and extravillous trophoblasts in first-trimester villus. Collectively, this study revealed a new regulatory pathway for LDHA/PI3K/AKT/FOXO1/CyclinD1 in the trophoblast cell cycle and proliferation.

Decorin-induced, preeclampsia-associated microRNA-512-3p restrains extravillous trophoblast functions by targeting USF2/PPP3R1 axis.

Decorin (DCN) is a leucine-rich proteoglycan produced by chorionic villus mesenchymal cells anddecidual cells during human pregnancy. Studies from our laboratory demonstrated that decidua-derived DCN restrains multiple trophoblast functions including proliferation, migration, invasion andendovascular differentiation, mediated by DCN-binding to multiple tyrosine kinase receptors; expressed by the trophoblast. Furthermore, DCN was shown to be selectively over-produced by thedecidua in preeclampsia (PE) subjects and elevated in the second trimester maternal plasma in PE, before the appearance of clinical signs, presenting as a predictive biomarker for PE. Micro (mi)RNAs are single-stranded non-coding RNAs (17–25 nucleotides) that typically downregulate target genes by repressing translation or facilitating degradation of mRNAs. The human; placenta expresses many miRNAs, some of which are exclusively expressed by the trophoblast. Many; of these miRNAs are dysregulated in PE-associated placentas and some appear in the maternal blood as PE biomarkers. However, little is known about their contribution to the pathogenesis of PE, a multi-factorial disease associated with a hypo-invasive placenta. The objective of the present study was to examine whether exposure of extravillous trophoblast (EVT) to DCN affects expression of specific miRNAs, and to test the role of these miRNAs in altering EVT functions. We identified miR-512-3p, as one of the DCN-induced miRNAs, also upregulated in PE placentas. It was shown to be elevated in ectopic DCN-over-expressing or exogenous DCN-treated first trimester human trophoblast cell line HTR-8/SVneo. Use of miRNA-mimics and inhibitors revealed that miR-512-3p compromised trophoblast migration, invasion and VEGF-dependent endovascular differentiation. Finally, Protein Phosphatase 3 Regulatory Subunit B, Alpha (PPP3R1), a known target of miR-512-3p, was paradoxically elevated in miR-512-3p-overexpressing trophoblast and PE-associated placentas. Using Enrichr, a tool that consists of both a validated user-submitted gene list and a search engine for transcription factors, we found that PPP3R1 elevation resulted from the miRNA binding to and targeting Upstream Transcription Factor 2 (USF2) which targeted PPP3R1. These findings reveal a novel aspect of pathogenesis of PE and biomarker potentials of this miRNA in PE.

Effects of triclosan exposure on placental extravillous trophoblast motility, relevant IGF2/H19 signaling and DNA methylation-related enzymes of HTR-8/SVneo cell line

Triclosan (TCS) is an antimicrobial agent widely used in personal care products and a potential endocrine disruptor chemical (EDC). TCS can pass through the placental barrier. Any influence of EDCs on epigenetic changes of placenta and embryo may bring profound impact on later health. This study aimed to investigate the effects of TCS exposure on cell proliferation and migration, and the expression of imprinted genes IGF2/H19 and DNA methylation-related enzymes in human placental extravillous trophoblast cell line HTR-8/SVneo. After exposure to TCS levels of 0 (DMSO Control), 10−11, 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 3 × 10−5, 6 × 10−5, 10−4 M and incubated for up to 36 h, cell proliferation and migration were examined by CCK-8, EdU incorporation assay and wound healing assay; the mRNA levels of IGF2, H19, DNA methyltransferases (Dnmt3a, Dnmt3b and Dnmt1), ten-eleven translocation enzymes (Tet1, Tet2 and Tet3) and IGF2 Receptor (IGF2R) were analyzed by qRT-PCR. The protein levels of IGF2 were measured by Western blot and ELISA. The cell viability turned to decline at TCS treatment of 3 × 10−5 M and above (all p < 0.05, compared to the DMSO Control). The cell migration decreased at TCS 10−5 and 3 × 10−5 M treatment (p < 0.05), but consistently unchanged at low dose of TCS from 10−9 to 10−7 M (p > 0.05). TCS treatments below cytotoxicity doses (< 10−5 M) did not significantly alter the mRNA levels of IGF2, Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2 and Tet3, compared to DMSO Control treatment (all p > 0.05). The transcription level of H19 was up-regulated by TCS at 3 × 10−5 M. TCS at 10−7 and 6 × 10−5 M increased the protein level of IGF2 in cell supernatant. Our data suggest that high TCS exposure may suppress HTR-8/SVneo cells viability and migration, increase H19 gene expressions and IGF2 protein secretion. The exact mechanism of TCS action in human trophoblast needs further studies.

MicroRNA-3935 promotes human trophoblast cell epithelial-mesenchymal transition through tumor necrosis factor receptor-associated factor 6/regulator of G protein signaling 2 axis

BackgroundInsufficient migration and invasion during trophoblast epithelial-mesenchymal transition (EMT) results in the occurrence and development of preeclampsia (PE), and our previous study has screened 52 miRNAs, whose expression levels are altered in the placental samples from PE patients, compared with the normal group. Among those, miR-3935 is one of the miRNAs being most significantly down-regulated, indicating its involvement in PE. However, the exact effect and molecular mechanisms remain unknown.MethodsIn the present study, we investigate the roles and underlying mechanisms of miR-3935 in trophoblast EMT by use of the human extra-villous trophoblast cell line HTR-8/SVneo as well as human placental tissues and maternal blood samples obtained from 15 women with normal pregnancies and 15 women with PE. Experimental methods include transfection, quantitative reverse transcription-PCR (qRT-PCR), western blot, immunofluorescence staining, dual-luciferase assays, in vitro invasion and migration assays, RNA-Seq analysis, bisulfite sequencing and immunohistochemistry staining.ResultsMiR-3935 expression is significantly decreased in both placentas and peripheral blood specimens of PE, and functionally, miR-3935 promotes EMT of trophoblast cells. Mechanistically, TRAF6 is identified to be a direct target of miR-3935 and TRAF6 exerts its negative effect on EMT of trophoblast cells by inhibition of RGS2, which down-regulates the methylation status of promoter of CDH1 gene that encodes E-Cadherin protein through induction of ALKBH1, resulting in increase of E-Cadherin and subsequently insufficient trophoblast EMT.ConclusionsTogether these results uncover a hitherto uncharacterized role of miR-3935/TRAF6/RGS2 axis in the function of human trophoblasts, which may pinpoint the molecular pathogenesis of PE and may be a prognostic biomarker and therapeutic target for such obstetrical diseases as PE.

Lactobacillus crispatus promotes invasion of the HTR-8/SVneo trophoblast cell line

IntroductionRecent studies have shown that the endometrium possesses unique microbiomes, including Lactobacillus. However, the roles of these microbes are currently unknown, especially in placentation and the early stage of pregnancy. MethodsThe immortalized human first-trimester trophoblast cell line HTR-8/SVneo was cultured in the presence or absence of Lactobacillus crispatus. Invasive and migrative activities were directly evaluated using an optical microscope and a time-lapse imaging system. Protein levels of the invasion-related protein matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 were evaluated using ELISA. ResultsMatrigel invasion of HTR-8/SVneo cells was significantly increased by L. crispatus, though migration was not affected. The culture supernatant of L. crispatus also promoted invasion. Additionally, levels of the active forms of MMP-1 and MMP-2 in the cell culture medium were upregulated by L. crispatus treatment, but that of MMP-9 was not changed. DiscussionL. crispatus promotes trophoblast invasion with an increase in MMP-1 and MMP-2 activation. Our results might explain why Lactobacillus dominance in the endometrium seems beneficial for implantation. Nevertheless, further research is required to determine whether the promotion of trophoblast invasion by L. cripatus is favorable for successful placentation at the early stage of pregnancy.

Assembly of Cytoplasmic Stress Granules in Placentas in Women with Preeclampsia.

Inflammation is a well-recognized factor associated with preeclampsia (PE). Stress granules (SGs) have been shown to play an important role in regulating inflammation and immune responses. However, whether SGs are involved in the pathogenesis of PE has not been studied. Here, we evaluated the expression of SG components in placenta of pregnancies with PE. Placental samples or serum were collected from PE patients (n = 31) or healthy age-matched pregnancy (n = 17). mRNA expressions of SG-associated genes in placenta from PE or normal pregnancies were detected by real-time quantitative PCR, and protein expressions of HuR and G3BP were detected using western blot. Immunofluorescence staining was performed to evaluate SG components expression in placentas or 10% serum treated HTR-8/Svneo cells using antibodies against HuR and G3BP. Our study showed higher levels of elavl1, lsm2, lsm4, and ago1 mRNA expression and SG marker proteins expression in placental homogenates of PE patients. HuR/G3BP-positive SG structure was further observed in placental villi of PE by immunofluorescence assay. Besides, serum from PE patients could induce SG aggregation in human trophoblast cell line HTR-8/Svneo cells, suggesting the involvement of SGs in the development of PE.

Galectin-14 Promotes Trophoblast Migration and Invasion by Upregulating the Expression of MMP-9 and N-Cadherin.

Galectin-14 is specifically expressed in placental trophoblasts, and its expression is reduced in trophoblasts retrieved from the cervix of women destined to develop early pregnancy loss. However, the roles of galectin-14 in regulating trophoblasts and in the pathogenesis of pregnancy complication have never been investigated. In the current research, we aimed to investigate the roles of galectin-14 in the regulation of trophoblasts. Tissues of the placenta and villi were collected. Primary trophoblasts and human trophoblast cell line HTR-8/SVneo were used. Western blotting and RT-PCR were used to quantify gene expression. The siRNA-mediated galectin-14 knockdown and lentivirus-mediated overexpression were performed to manipulate the gene expression in trophoblasts. Transwell migration and invasion assays were used to evaluate cell migration and invasion capacity. Gelatin zymography was used to determine the gelatinase activity. Galectin-14 was significantly decreased in the villi of early pregnancy loss and the placenta of preeclampsia. Knockdown of galectin-14 in primary trophoblasts inhibited cell migration and invasion, downregulated the expression of matrix metalloproteinase (MMP)-9 and N-cadherin, the activity of MMP-9, and decreased the phosphorylation of Akt. Meanwhile, the overexpression of galectin-14 in HTR-8/SVneo promoted cell migration and invasion, upregulated the expression of MMP-9 and N-cadherin, the activity of MMP-9, and increased the phosphorylation of Akt. Increased Akt phosphorylation promoted cell migration and invasion and upregulated the expression and activity of MMP-9, while decreased Akt phosphorylation inhibited cell migration and invasion and downregulated the expression and activity of MMP-9. Thus, galectin-14 promotes trophoblast migration and invasion by enhancing the expression of MMP-9 and N-cadherin through Akt phosphorylation. The dysregulation of galectin-14 is involved in the pathogenesis of early pregnancy loss and preeclampsia.

Up-regulation of LncRNA NEAT1 induces apoptosis of human placental trophoblasts

The trophoblast apoptosis induced by placental oxidative stress is a contributor to the pathological development of preeclampsia (PE), whereas the molecular mechanism remains unclear. In this study, we explored the role and mechanism of Long non-coding RNA (LncRNA) NEAT1 in trophoblasts apoptosis. In the placenta tissues of PE patients and H2O2-treated human trophoblast cell line HTR-8/SVneo, the expressions of LncRNA NEAT1, p53, and estrogen receptor α (ESRα) were increased whereas miR-18a-5p expression was decreased. ESRα expression was up-regulated by LncRNA NEAT1 overexpression and down-regulated by miR-18a-5p overexpression in HTR-8/SVneo cells. LncRNA NEAT1 could release ESRα expression through sponging miR-18a-5p and the transcription of LncRNA NEAT1 was promoted by p53. miR-18a-5p overexpression suppressed H2O2-induced cell apoptosis in HTR-8/SVneo cells, while the inhibitory effect of miR-18a-5p overexpression on cell apoptosis was abrogated by LncRNA NEAT1 overexpression. In summary, LncRNA NEAT1 transcription was induced by p53 under oxidative stress condition, the high expression of LncRNA NEAT1 subsequently increased ESRα expression by sponging miR-18a-5p, thus inducing trophoblasts apoptosis.

1336-P: Trimethylamine N-Oxide Inhibits the Formation of Neutrophil Extracellular Traps to Regulate Placental Development in Gestational Diabetes Mellitus

Objective: Trimethylamine N-oxide (TMAO) is associated with risk of gestational diabetes mellitus (GDM), and TMAO levels in peripheral/cord blood of GDM mothers increased. Diabetes primes neutrophils to produce neutrophil extracellular traps (NETs), but it has not been reported in GDM. We determined the relationship between NETs and GDM, and explored potential role of TMAO. Methods: Maternal peripheral blood was collected during OGTT tests, and cord blood and placenta were collected during delivery. Plasma concentrations of cell-free double-stranded DNA (dsDNA) were compared between GDM and control mothers and association analysis was performed. Expression of myeloperoxidase (MPO) in placental tissues of GDM and control mothers was examined. Neutrophils from the blood of healthy donors were used to observe effects of TMAO on NETs formation. The effect of TMAO on human placental extravillous trophoblast cell line HTR-8/Svneo was examined. Results: Compared with controls, plasma concentrations of dsDNA in peripheral blood and cord blood were significantly increased in GDM mothers. Association analysis found that concentrations of dsDNA in peripheral and cord blood plasma were positively correlated with BMI and blood glucose levels, but not blood pressure. In addition, the placental thickness was positively correlated with dsDNA levels in cord blood plasma. MPO expression in the placental tissues of GDM mothers was also significantly higher than that of control mothers. TMAO-treated neutrophils could resist the formation of NETs stimulated by PMA or LPS, and TMAO promotes the proliferation, migration, invasion and angiogenesis of the cells. Conclusion: The formation of NETs was increased in GDM, and was positively correlated with BMI and blood glucose. TMAO could inhibit NETs formation and promote invasion and angiogenesis of human placental trophoblast cells, which may be involved in the development of the placenta. Disclosure X. Lin: None. X. He: None. X. Xiao: None.

Long Non-coding RNA Gas5 Is Associated With Preeclampsia and Regulates Biological Behaviors of Trophoblast via MicroRNA-21.

Preeclampsia is a lethal pregnancy specific hypertensive disorder involving multisystem. Despite extensive studies to investigate the causes of preeclampsia, the pathogenesis still remains largely unknown. Long non-coding RNAs (lncRNAs) are a diverse class of non-translated RNAs which play a crucial part in various biological phenomena. Although lncRNA Growth Arrest-Specific 5 (GAS5) aberrantly expressed in multiple cancer tissues and is implicated in multiple biological processes of tumor cells, little is known about its role in preeclampsia. In this study, 40 patients with preeclampsia and 32 gestational age matched normotension pregnant women were recruited. Using quantitative real-time polymerase chain reaction (qRT-PCR), we found higher expression of GAS5 in placenta of preclamsia affected women. The level of GAS5 existed strongly in correlation with Thrombin Time indicating coagulation function and other clinical parameters by Pearson correlation analysis. Then we constructed the GAS5 lentivirus expression vectors and transfected into human trophoblast cell lines HTR-8/SVneo and JEG-3. Using in vitro cell culture studies, we found an inhibited effect of GAS5 on proliferative ability, migratory ability and invasive ability however; no effect on apoptosis was detected. Further mechanistic analysis found that GAS5 modulated microRNA-21 (miR-21) in an opposite variation tendency by qRT-PCR and rescue experiment. In addition, inhibition of GAS5 promoted the activation of PI3K/AKT signaling pathway and its downstream proteins covering MMP-9 and TP53 as evident from our qRT-PCR and western blot analyses. Thus, we suggested that GAS5 might involve in pregnancy with preeclampsia by influencing the biological functions of trophoblast cells through the regulation of miR-21 and activation of PI3K/AKT signaling pathway and its downstream targets, which may contribute to reveal the nature of preeclampsia.

1037: Betamethasone reverses vasopressin induced inhibition of trophoblast migration

Elevated arginine vasopressin (AVP) is predictive of human preeclampsia (PreE) and causes a PreE phenotype in mice. Betamethasone (BMTZ), a corticosteroid administered to prevent preterm birth complications, inhibits AVP and stimulates serum/glucocorticoid regulated kinase 1 (SGK1), a serine/threonine kinase. In the AVP mouse model of PreE, early administration of BMTZ prevents preeclampsia. The objective of this study was to determine if BMTZ acts through SGK1 to inhibit the effects of AVP on trophoblast cell migration. Trophoblast migration was assessed using a human extravillous trophoblast cell line HTR-8/SVneo. Cells were plated into ibidi 2-well silicone inserts and allowed to adhere for 24 hours before removal of the insert creating a 500mm gap for migration analysis and treatment. Cells were treated with saline (n=7), AVP (n=7), AVP + BMTZ (n=3), or AVP + BMTZ + SGK1 inhibitor (n=3). Images were taken at 0 and 24 hours post-treatment. All wells were set up in duplicate and averaged for n=1. The area of the gap was measured and the percent closure for cell migration was calculated. Two-tailed Student t test or one way ANOVA multiple comparisons were performed with alpha=0.05. AVP treatment of trophoblast cells inhibited migration at 24 hours (Saline: 38% vs AVP: 25%, n=7 p< 0.05). Treatment of trophoblast cells with BMTZ prevented AVP induced inhibition of trophoblast migration (AVP: 25% n=7 vs AVP + BMTZ: 40% n=3, p< 0.05) with migration rates comparable to saline-treated cells (saline: 38% n=7 vs AVP + BMTZ: 40% n=3, p=0.99). The effect of BMTZ on AVP-induced inhibition of trophoblast cell migration was not mediated via SGK1 (AVP + BMTZ: 40% vs AVP + BMTZ + SGK1 inhibitor: 49%, n=3, p=0.51). These data demonstrate that AVP may play a role in placental dysfunction via inhibition of trophoblast migration. While BMTZ is sufficient to prevent the impaired trophoblast migration via AVP, these effects are mediated through a SGK1 independent mechanism.

CRY2 suppresses trophoblast migration and invasion in recurrent spontaneous abortion.

Disruption of circadian rhythms is associated with aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA). This study aims to explore the functional role and the mechanisms of cryptochrome 2 (CRY2), a fundamental component of the circadian clock, in regulating trophoblast migration and invasion. Human extravillous trophoblast cell line HTR-8/SVneo was used as a cell model. Cell migration and invasion were examined using wound healing assay and Transwell assay, respectively. The mRNA and protein levels were determined using quantitative real-time polymerase chain reaction and western blot, respectively. Luciferase reporter assay and chromatin immunoprecipitation assay were performed to explore the interaction between c-Myc to the brain and muscle ARNT-like protein 1 (BMAL1) promoter. CRY2 was highly expressed in human villous specimens of RSA. Furthermore, CRY2 overexpression impaired migration and invasion in HTR-8/SVneo cells, whereas CRY2 knockdown yielded the opposite results. Mechanistically, c-Myc bound to the BMAL1 promoter and induced BMAL1 transcription, both of which further activated matrix metalloproteinase 2/9 (MMP2/9) and facilitated migration and invasion in HTR-8/SVneo cells. CRY2 inhibited c-Myc-BMAL1 pathway and impaired migration and invasion of HTR-8/SVneo cells. Collectively, these findings demonstrate that CRY2 suppresses trophoblast migration and invasion via inhibiting c-Myc-BMAL1-MMP2/9 pathway.

Overexpression of Collapsin Response Mediator Protein 1 Inhibits Human Trophoblast Cells Proliferation, Migration, and Invasion.

Collapsin response mediator protein 1 (CRMP-1) is widely expressed in the nervous system and has tumor suppressive effects. Our previous studies have demonstrated that CRMP-1 was expressed in the trophoblasts of the whole stage of pregnancy with significantly increasing expression in the placenta of early-onset preeclampsia. Preeclampsia, especially early onset, is strongly associated with the dysfunction of trophoblast including proliferation, apoptosis, migration, and invasion. In this study, we found an inhibitory effect of CRMP-1 on proliferation, migration, invasion, and an enhanced effect on apoptosis in human trophoblast cell lines HTR-8/SVneo and JEG-3 by MTT assay, colony formation assay, cell viability assay, caspase 3/7 activity assay, scratch wound assay, and Matrigel Transwell assay. Overexpression of CRMP-1 in trophoblast cells led to downregulate expression of matrix metalloproteinase 2 and 9. The expression of CRMP-1 was detected by real-time quantitative polymerase chain reaction and Western blot analysis. Thus, we suggested that CRMP-1 might have implications for the pathogenesis of preeclampsia by regulating the biological behavior of trophoblast cells.

IL-33 promotes antiviral immunity and protects against congenital Zika disease in mice

Abstract Zika virus (ZIKV) infection during pregnancy can cause congenital Zika syndrome and other birth defects. The alarmin IL-33 can evoke strong antiviral immune responses during viral infection. However, whether IL-33 is involved in immune responses during ZIKV infection is not clear. In this study, we infected three-week-old wildtype (WT) and IL-33 knockout (KO) mice with ZIKV and analyzed the ZIKV-specific cytotoxic T lymphocyte (CTL) responses at 6 days post infection. We found that IL-33 deficiency resulted in decreased ZIKV-specific CTL responses as evidenced by lower numbers of IFN-g+TNF-a+ CD8+ T effectors. To determine if IL-33 contributes to anti-ZIKV immunity during pregnancy, we infected WT pregnant mice with ZIKV and found increased IL-33 in the placenta at a mid-gestational period (embryo E13.5) compared with that of uninfected ones. Likewise, elevated IL-33 expression was also observed in human trophoblast cell lines JEG-3 and HTR-8 upon ZIKV infection. To determine if IL-33 plays a protective role in congenital Zika disease, we infected pregnant WT and IL-33 KO mice by i.p. injection of ZIKV at E6.5 and found higher percentages of fetal demise and absorption in IL-33 KO mice compared with that in WT mice (16.7 % vs. 3.3 %) from necropsy on E13.5. In addition, the fetuses from IL-33 KO mice displayed smaller sizes than those of WT fetuses (55 mm2 vs. 48 mm2). Moreover, IL-33 deficient mice had lower numbers of virus-specific CD8+ T cells in the placenta, uterine and decidua. Importantly, IL-33 deficiency resulted in higher viral loads in both the placenta and fetus. In summary, our study highlights IL-33 as a key immune regulator in inducing anti-ZIKV CTL responses and protecting against congenital Zika disease.

Lithocholic acid activates mTOR signaling inducing endoplasmic reticulum stress in placenta during intrahepatic cholestasis of pregnancy

AimsIntrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder, which increases risks of adverse fetal outcomes. However, the pathophysiology is not fully understood. Here, we explored the roles of mTOR signaling and ER stress in placenta during ICP. Materials and methodsPlacental tissues were collected from normal and ICP pregnancies. mTOR signaling and endoplasmic reticulum stress were detected by immunohistochemistry in the placenta. The human placenta trophoblast cell line HTR-8/SVneo was used in vitro experiment. Key findingsICP placenta displayed histological abnormalities with fewer trophoblasts. Moreover, the expression of Bip and the phosphorylation of pS6(S235/236) or pAkt(S473) were higher comparing with normal placenta. In in vitro studies, the bile acids specifically to lithocholic acid rather than taurocholic acid or ursodeoxycholic acid, drastically increased the phosphorylation of pS6K1(T389), pS6(S235/236), or pAkt(S473), whereas the mTOR inhibitor can prohibit the upregulation. Similarly, the expressions of IRE1α and BiP increased sharply under lithocholic acid (20 μM) administration, while the same inhibitor can also decrease the expression. Additionally, transmission electron microscopy showed enlarged endoplasmic reticulum lumen under the lithocholic acid treatment. Furthermore, the cell viability reduced sharply under treatment with different dose of lithocholic acid. The mTOR inhibitor can reverse the decrease of cell viability to some extent. SignificanceBile acid can activate mTOR signaling which resulted in endoplasmic reticulum stress, leading to trophocyte viability decrease. mTOR pathway activation may be associated with the pathophysiology of ICP.

Upregulation of Angiogenic Factors via Protein Kinase C and Hypoxia-induced Factor-1α Pathways under High-glucose Conditions in the Placenta.

Abnormal glucose metabolism during pregnancy is an established risk factor for preeclampsia (PE). Disruption of the balance between placental angiogenic factors is linked to PE pathophysiology. We examined whether hypoxia-induced factor-1α (HIF-1α) and protein kinase Cβ (PKCβ) are involved in the regulation of placental angiogenic factors under high-glucose conditions in vitro. The human choriocarcinoma cell lines BeWo and JEG-3, and the human trophoblast cell line HTR-8/SVneo were cultured with 10 and 25 mmol/L glucose [control glucose group (CG) and high-glucose group (HG), respectively]. We examined the changes in HIF-1α, soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) expression in the CG and HG by real-time PCR and ELISA. PKC activation was also measured by ELISA. The expressions of HIF-1α, sFlt-1, PlGF, and VEGF were significantly higher in the HG than in the CG. PKC activity was significantly increased in the HG. High glucose affected the expression of angiogenic factors in choriocarcinoma cells via the PKCβ and HIF-1α pathways, suggesting their involvement in PE pathogenesis.