Human Trophoblast Cell Line Research Articles

The diagnostic value and regulatory mechanism of miR-200a targeting ZEB1 in pregnancy-induced hypertension

ABSTRACT Objective To investigate the diagnostic value and regulatory mechanism of miR-200a targeting ZEB1 in pregnancy-induced hypertension (PIH). Methods The expression of miR-200a and ZEB1 was detected in the placenta of PIH patients, and then the human trophoblastic cell line JEG-3 was transfected and divided into different groups: control group, NC group, ZEB1 siRNA group, miR-200a inhibitor group and miR-200a inhibitor group + ZEB1 siRNA group. After transfection, cell proliferation and migration/invasion were evaluated byMTT and Transwell assays, respectively, whereas apoptosis was assessed byflow cytometry. MiR-200a was measured by qRT-PCR, while ZEB1 was detectedby Western blotting. Results The expression of miR-200a was gradually increased in the placenta of patients with hypertension and mild or severe preeclampsia, while the mRNA and protein levels of ZEB1 were downregulated. A dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-200a andZEB1.Compared to the control, the miR-200a inhibitor caused a strongdecrease in miR-200a andan upregulation of ZEB1, with a significant enhancement ofcell proliferation, migration and invasion and a decrease in apoptosis. However, no significant alteration was observedin the miR-200a level after administration of ZEB1 siRNA, while ZEB1 was downregulated, with significant suppression of growth. Conclusion MiR-200a was upregulated in PIH patients, andinhibition of miR-200a may improve disease progression, as it could facilitatetrophoblastproliferation, migration and invasionandinhibitapoptosisby targeting ZEB1.

SIRT3 deficiency is resistant to autophagy-dependent ferroptosis by inhibiting the AMPK/mTOR pathway and promoting GPX4 levels.

Ferroptosis, an autophagy-dependent cell death, is characterized by lipid peroxidation and iron accumulation, closely associated with pathogenesis of gestational diabetes mellitus (GDM). Sirtuin 3 (SIRT3) has positive regulation on phosphorylation of activated protein kinase (AMPK), related to maintenance of cellular redox homeostasis. However, whether SIRT3 can confer autophagy by activating the AMPK-mTOR pathway and consequently promote induction of ferroptosis is unknown. We used human trophoblastic cell line HTR8/SVneo and porcine trophoblastic cell line pTr2 to deterimine the mechanism of SIRT3 on autophagy and ferroptosis. The expression of SIRT3 protein was significantly elevated in trophoblastic cells exposed to high concentrations of glucose and ferroptosis-inducing compounds. Increased SIRT3 expression contributed to classical ferroptotic events and autophagy activation, whereas SIRT3 silencing led to resistance against both ferroptosis and autophagy. In addition, autophagy inhibition impaired SIRT3-enhanced ferroptosis. On the contrary, autophagy induction had a synergistic effect with SIRT3. Based on mechanistic investigations, SIRT3 depletion inhibited activation of the AMPK-mTOR pathway and enhanced glutathione peroxidase 4 (GPX4) level, thereby suppressing autophagy and ferroptosis. Furthermore, depletion of AMPK blocked induction of ferroptosis in trophoblasts. We concluded that upregulated SIRT3-enhanced autophagy activation by promoting AMPK-mTOR pathway and decreasing GPX4 level to induce ferroptosis in trophoblastic cells. SIRT3 deficiency was resistant to high glucose- and erastin-induced autophagy-dependent ferroptosis and is, therefore, a potential therapeutic approach for treating GDM.

MicroRNA-26b-3p inhibits human trophoblast cell proliferation, invasion and resistance to apoptosis via targeting SHBG

Aberrant increased apoptosis and dysfunction in migration and invasion of trophoblast cell play key role in preeclampsia. However, the mechanism remains unclear. Our study investigated the roles of microRNA-26b-3p (miR-26b-3p) in trophoblast cell growth by targeting sex hormone binding globulin (SHBG). Human trophoblast cell line HTR8-SVneo was used in this study. Gain- and loss-of-functions of miR-26b-3p and SHBG were performed, and then the cell viability was detected with CCK-8 and EdU assays. Cell apoptosis was detected using flow cytometry, and the cell invasion and migration were evaluated with transwell assays. The expression level of SHBG, caspase-3 and Bcl-2 after miR-26b-3p transfection was measured with RT-PCR and western blot analysis. Matching the sequence of miR-26b-3p and SHBG was analyzed through bioinformatic prediction and confirmed with dual luciferase reporter assay. It is found that overexpression of miR-26b-3p inhibited HTR8-SVneo cell proliferation, invasion and migration, while increased cell apoptosis and induced cell cycle arrest at the G0/G1 phase. Elvated level of miR-26b-3p decreased the expression of SHBG. Bioinformatic prediction and dual luciferase assay identified the relation of miR-26b-3p and HTR8-SVneo. Downregulation of SHBG exhibited similar effects on HTR8-SVneo cell growth with overexpression of miR-26b-3p. Conclusion: MiR-26b-3p targets SHBG and decreases SHBG expression. It inhibits human trophoblast cell proliferation, invasion and resistance to death of trophoblast cell. This study provided a mechanism for preeclampsia. Sex hormone binding globulin: SHBG; miR-26b-3p: microRNA-26b-3p; CCK-8: Cell-Counting Kit-8; RT-PCR: Real time polymerase chain reaction; NC: miR-26b-3p mimic negative control or SHBG negative control siRNA; inhibitor NC: negative control inhibitor; TBST: Tris-buffered saline with Tween.

Cys-peptide mediates the protective role in preeclampsia-like rat and cell models

ObjectiveThe present study was designed to investigate whether the novel peptide cysteine-based peptide (Cys-peptide) had protective effects on preeclamptic animal and cell models. MethodsWe investigated effects of Cys-peptide on (1) preeclamptic symptoms (e.g. hypertension, proteinuria, fetal growth restriction (FGR)) in preeclampia-like rat models induced by lipopolysaccharides (LPS), (2) TNFα-induced cytotoxicity of human umbilical vascular endothelial cells (HUVECs) and HTR-8 cells (an immortalised human trophoblast cell line), (3) endothelial dysfunction and injured angiogenesis, (4) migration and invasion of trophoblast cells induced by TNFα. ResultsCys-peptide ameliorated LPS-induced hypertension, proteinuria and FGR and other PE symptoms in preeclampia-like rat models. In addition, Cys-peptide attenuated TNFα-induced cytotoxicity by decreasing soluble fms-like tyrosine kinase-1 (sFlt-1), endothelin-1 (ET-1) and tissue plasminogen activator (tPA) mRNA expression in both cells. Furthermore, Cys-peptide restored endothelial dysfunction and rescued angiogenesis caused by TNFα in vitro. Importantly, Cys-peptide could reverse insufficient ability to invade and migrate of trophoblast cells. ConclusionsThese results suggest Cys-peptide can play beneficial roles in preeclampsia-like rat and cell models. Therefore, we propose that Cys-peptide is probably a novel therapeutic candidate for PE.

A comparative study of key physiological stem cell parameters between three human trophoblast cell lines

Human trophoblast stem cells (TSCs) play a key role in the placenta. These cells are proliferative, undifferentiated, and can differentiate into mature trophoblast cell types. However, primary human TSCs are difficult to obtain. In our previous study, we established TSCs from human induced pluripotent stem cells (TShiPSC). Here, we aimed to characterize the identity of these TShiPSC cells by comparing them with BeWo choriocarcinoma cells and primary TSCs (CT cells). Compared with BeWo cells, CT and TShiPSC cells showed high secretion of human chorionic gonadotrophin (hCG) and syncytiotrophoblast differentiation ability. Global gene microarray analysis results showed that CT and TShiPSC cells, unlike BeWo cells, could be classified in the same group. Compared with BeWo cells, CT and TShiPSC cells showed high expression levels of TSC-specific genes and low expression of cancer adhesion and invasion genes. Analysis of placental barrier integrity showed that TShiPSC cells could form a good barrier. Prospective studies using TShiPSC cells hold great promise for elucidating the pathogenesis of infertility due to trophoblast defects.

Regulation of stanniocalcin-1 secretion by BeWo cells and first trimester human placental tissue from normal pregnancies and those at increased risk of developing preeclampsia.

Stanniocalcin‐1 (STC‐1) is a multi‐functional glycosylated peptide present in the plasma of healthy women postpartum and increased further in pregnancies complicated by preeclampsia. Although the STC‐1 gene is expressed by the placenta what regulates its secretion and from which cells at the feto‐maternal interface is unknown. Here, we demonstrate for the first time that the syncytiotrophoblast and cytotrophoblast are a major site of STC‐1 protein expression in first trimester placental tissue. Further, in response to low oxygen, first trimester chorionic villous tissue from pregnancies at increased risk of developing preeclampsia secreted significantly more STC‐1 than normal tissue under the same conditions. Using the human trophoblast cell line BeWo we have shown that low oxygen increased the secretion of STC‐1 but it required co‐stimulation with the Adenosine‐3', 5'‐cyclic monophosphate (cAMP) analogue, 8‐Bromo adenosine‐3', 5'‐cyclic monophosphate cAMP (8 Br‐cAMP) to reach significance. Inhibition of Hypoxia inducible factor 2α (HIF‐2α) and the Phosphatidylinositol‐3 kinase (PI3‐Kinase)/AKT/Serum and glucocorticoid‐induced kinase‐1(SGK‐1) pathway resulted in significant inhibition of STC‐1 secretion. As both low oxygen and cAMP are known to play a central role in placental function, their regulation of STC‐1 points to a potentially important role in the maintenance of a normal healthy pregnancy and we would hypothesize that it may act to protect against prolonged placental hypoxia seen in preeclampsia.

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.

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.

Exosomal encapsulation of miR-125a-5p inhibited trophoblast cell migration and proliferation by regulating the expression of VEGFA in preeclampsia

This study is aimed to examine the association between umbilical cord blood (UCB) derived exosomal microRNA (miRNA) with preeclampsia (PE) and to further explore the mechanism of a key differential gene (hsa-miR-125a-5p) in preeclampsia. Umbilical cord blood exosomal miRNA(exo-miRNA) from normal pregnant women and pregnant women with preeclampsia was processed via miRNA sequencing. Quantitative real-time polymerase chain reaction (QRT-PCR) was performed to assess the expression of miR-125a-5p in normal and PE placental tissues and peripheral blood derived exosomes in the third trimester. Human trophoblast cell line HTR8/SVneo was assigned as the negative control and miR-125a-5p mimics. QRT-PCR and Western blot were performed to identify the expressions of miR-125a-5p and vascular endothelial growth factor A (VEGFA). CCK8, flow cytometry, wound-healing and Transwell assays were used to analyze the effect of miR-125a-5p on HTR8/SVneo cell migration, proliferation, and cycle distribution. Tube formation was performed to estimate the angiogenesis ability of miR-125a-5p on HUVECs. In conclusion, miR-125a-5p expression in PE placental tissues was higher than in normal subjects, while the expression of VEGFA was lower in PE placental tissues. We then compared the miR-125a-5p mimics group with the negative control group and found that in the mimics group, the cell migration, proliferation and angiogenesis abilities were decreased, and more cells were arrested in the S stage. Our study systematically profiled the UCB exo-miRNA in normal and PE pregnant women and demonstrated that dysregulation of miR-125a-5p might affect HTR8/SVneo cell proliferation and migration and inhibit angiogenesis by regulating VEGFA, indicating that miR-125a-5p is involved in the progression of PE.

H19 regulates angiogenic capacity of extravillous trophoblasts by H19/miR-106a-5p/VEGFA axis.

To investigate the role and underlying mechanism of H19 in regulating angiogenic capacity of extravillous trophoblasts. Gain and loss of function experiments were performed using a human first-trimester extravillous trophoblast (EVT) cell line, HTR-8/SVneo cells. H19 was overexpressed or knocked down in HTR-8 cells by transfecting plasmid harboring whole-length H19 sequence (pH19) or siRNA specially targeting H19, respectively (siH19). Cell migration and tube-formation assay were assessed in the indicated groups. Gene expression was detected by RT-qPCR, Western blot, and ELISA assay. Overexpression of H19 in EVT cells increased cell migration and tube formation, while downregulation of H19 in EVT cells decreased cell migration and tube formation. Furthermore, we found that H19 played its role by VEGFA. In addition, we demonstrated the H19/miR-106a-5p/VEGFA regulatory axis in EVT. Experiments of the clinical specimen showed that H19 was very abundantly expressed in human first-trimester trophoblasts, and we found that the expression of H19 and VEGFA were significantly downregulated in the villous tissues from idiopathic recurrent miscarriage (RM) patients; moreover, the expression of H19 and VEGFA was positively correlated. H19/miR-106a-5p/VEGFA axis plays a role in regulating the angiogenic capacity of EVT, which might contribute to idiopathic RM.

ADATMS-7 regulates the focal adhesion kinase signaling and promotes invasiveness of trophoblasts in early pregnancy

IntroductionADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was recently identified to be associated with cell migration and invasion. However, its function on trophoblasts remains unknown. In this study, we are aimed to investigate the role of ADAMTS-7 on trophoblasts in human first trimester gestation. MethodsThe expression of ADAMTS-7 in trophoblasts and HTR8/SVneo cells is examined by immunohistochemistry and quantitative real-time PCR. BrdU incorporation and Annexin V/PI staining are utilized to measure the effect of ADAMTS-7 on the proliferation and apoptosis of HTR8/SVneo cells, respectively. In addition, we detect the role of ADAMTS-7 on the invasion ability of HTR8/SVneo cells using matrigel invasion assays. The activation of focal adhesion kinase (FAK) and integrinβ1 induced by ADAMTS-7 were determined by Western blot. ResultsADAMTS-7 and its substrate cartilage oligomeric matrix protein (COMP) were expressed in both primary human trophoblasts and human trophoblast cell lines. TGF-β1 induced a continuous and significant decrease of ADAMTS-7. Inversely, IL-1β up-regulated the ADAMTS-7 level in a dosage dependent manner. In addition, knockdown of ADAMTS-7 inhibited the growth and invasion of HTR8/SVneo cells. To the contrary, ADAMTS-7 overexpression promoted the growth and invasion of HTR8/SVneo cells. ADAMTS-7 knockdown led to a decreased level of FAK Tyr-397 phosphorylation. DiscussionOur results suggest that ADAMTS-7 may regulate trophoblasts invasion through focal adhesion kinase (FAK) signaling.

Resveratrol induces SIRT1-Dependent autophagy to prevent H2O2-Induced oxidative stress and apoptosis in HTR8/SVneo cells

IntroductionPre-eclampsia (PE) is a serious complication of pregnancy, and the likely pathogenic basis of early onset PE are placental dysfunction and increased oxidative stress. Resveratrol (RES) is a potent antioxidant which has shown beneficial effects in many diseases. The aim of this study was to investigate the protective effects of RES against oxidative stress-induced damage in trophoblasts, and elucidate the potential mechanisms. MethodsWe established an in vitro model of oxidative stress by exposing the human first-trimester extravillous trophoblast cell line HTR8/SVneo to H2O2. The level of oxidative stress was reflected by ROS, MDA and SOD. The viability of cells was determined by the MTS assay. Apoptosis was detected using Annexin V-FITC staining and flow cytometry. Levels of SIRT1(sirtuin 1) and autophagy-related proteins (LC3, Beclin-1, p62) were detected by western blot. Autophagosomes were observed by transmission electron microscopy (TEM). ResultsPre-treatment with RES significantly ameliorated H2O2-induced cytotoxicity, morphological damage, oxidative stress and apoptosis. Mechanistically, RES restored the levels of SIRT1 and autophagy-related proteins including LC3-II, Beclin-1 and p62 that were dysregulated by H2O2. Blocking autophagy by 3-methyladenine (3-MA) completely abolished the protective effects of RES, as did knocking down SIRT1. ConclusionRES may protect human trophoblasts against H2O2-induced oxidative stress by activating SIRT1-dependent autophagy, and therefore has therapeutic potential in PE.

Trophoblast toxicity of the neonicotinoid insecticide acetamiprid and an acetamiprid-based formulation

The neonicotinoid (Neo) insecticide family is a relatively new class of pesticides of growing use. There is an increasing concern that human exposure to environmental pollutants in utero may be associated with diseases in adulthood. A functional placenta and trophoblasts are a requisite for a healthy pregnancy. The aim of this study was to investigate whether the Neo Acetamiprid (Ace) and one of its commercial formulations (Ace CF) display toxic features to a human first trimester trophoblast cell line. HTR-8/SVneo cells were cultured in the presence of Ace or Ace CF (0.1–100 μM) for 4 and 24 h, and changes in cell viability, reactive oxygen species, antioxidant system and macromolecule damage levels were evaluated.Ace and Ace CF are cytotoxic for HTR-8/SVneo trophoblasts. Cell viability loss and oxidative imbalance were triggered by Ace and Ace CF treatments. Impact in the antioxidant enzymes catalase, superoxide dismutase and gluthatione S-transferase activities were observed after 24 h exposure to Ace CF. Moreover, Ace CF caused oxidative damage in proteins, lipids and DNA, whereas Ace only damaged proteins. To test oxidative stress as a toxicity mechanism, cells were pre-incubated with the antioxidant N-acetyl-l-cysteine (NAC), prior Neo treatment. NAC protected trophoblasts from cell death and prevented oxidative damage.Results demonstrate that Ace (as active principle or CF) is cytotoxic for human trophoblasts, and oxidative stress is a toxicity mechanism. Ace CF exhibited a more toxic effect than the active principle, in an identical exposure scenario.

Allopurinol inhibits excess glucose-induced trophoblast IL-1β and ROS production.

Pre-gestational diabetes is a risk factor for preeclampsia, a condition associated with inflammatory markers, a dysregulated angiogenic profile, and impaired placentation. Using an in vitro model, we previously reported that hyperglycemic levels of glucose induced a pro-inflammatory (IL-1β, IL-8, RANTES, GRO-α), anti-angiogenic (sFlt-1) and anti-migratory profile in a human trophoblast cell line. The IL-1β response to excess glucose was mediated by uric acid-induced activation of the NLRP3 inflammasome. Allopurinol is a xanthine oxidase inhibitor that inhibits uric acid and reactive oxygen species (ROS) production. Thus, we sought to test the effects of allopurinol on the IL-1β and other inflammatory, angiogenic and migratory responses that are triggered in the trophoblast by excess glucose. Under excess glucose conditions, allopurinol significantly inhibited trophoblast secretion of inflammatory IL-1β; caspase-1 activity; IL-8; RANTES; and GRO-α. Allopurinol also significantly inhibited excess glucose-induced trophoblast secretion of anti-angiogenic sFlt-1. The presence of IL1Ra significantly inhibited excess glucose-induced trophoblast IL-8 and GRO-α secretion but had no effect on RANTES or sFlt-1. Conversely, DPI, a ROS inhibitor, significantly inhibited excess glucose-induced trophoblast GRO-α and sFlt-1 secretion, but had no effect on IL-8 or RANTES. Together, our findings indicate that the xanthine oxidase inhibitor allopurinol inhibited excess glucose-induced trophoblast IL-1β secretion. Additionally, through its inhibition of both IL-1β and ROS production by the trophoblast, allopurinol reduced the additional pro-inflammatory and anti-angiogenic responses to excess glucose. Thus, allopurinol may be a candidate medication to prevent placental dysfunction and adverse pregnancy outcomes, such as preeclampsia, in pregnant women with diabetes.

HHV-6A Infection of Endometrial Epithelial Cells Affects miRNA Expression and Trophoblast Cell Attachment

We recently reported that human herpesvirus 6 (HHV-6) infection is frequently present in endometrial tissue of women with unexplained infertility, and that virus infection induces a profound remodulation of miRNA expression in human cells of different origin. Since specific miRNA patterns have been associated with specific pregnancy outcomes, we aimed to analyze the impact of HHV-6A infection on miRNAs expression and trophoblast receptivity in human endometrial cells. To this purpose, a human endometrial cell line (HEC-1A) was infected with HHV-6A and analyzed for alterations in the expression of miRNAs and for permissiveness to the attachment of a human choriocarcinoma trophoblast cell line (JEG-3). The results showed that HHV-6A infection of endometrial cells up-modulates miR22 (26-fold), miR15 (19.5-fold), and miR196-5p (12.1 fold), that are correlated with implant failure, and down-modulates miR18 (11.4 fold), miR101-3p (4.6 fold), miR181-5p (4.9 fold), miR92 (3.3 fold), and miR1207-5p (3.9 fold), characterized by a low expression in preeclampsia. Moreover, HHV-6A-infected endometrial cells infected resulted less permissive to the attachment of trophoblast cells. In conclusion, collected data suggest that HHV-6A infection could modify miRNA expression pattern and control of trophoblast cell adhesion of endometrial cells, undermining a correct trophoblast cell attachment on endometrial cells.

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.

1063: Endometrial and myometrial cell CXCL12 expression in response to mechanical injury: implications for placenta accreta.

In view of increasing incidence of placenta accreta spectrum disorders, there is an urge to better understand the molecular mechanisms of abnormal trophoblast invasion. CXCL12 and its receptor, CXCR4, have been implicated in many physiologic and pathologic processes including cell migration and trafficking, tissue healing in the setting of mechanical, ischemic and inflammatory damage and cancer metastasis. CXCL12 functions as a chemokine, attracting cells expressing CXCR4. Prior studies demonstrate that CXCL12 is produced by endometrium while CXCR4 is present on trophoblast cells. In this study, we sought to assess if tissue injury would affect the expression of CXCL12 in endometrial and myometrial cells as well as CXCR4 expression in trophoblast cells using an in vitro model. Cultured primary human endometrial stromal and myometrial cells were subjected to a scratch injury. Cells were collected 4 hours after injury and CXCL12 mRNA levels in injured and non-injured cells were assessed using RT-qPCR. CXCR4 mRNA levels were assessed in 2 human first-trimester trophoblast cell lines and in primary cytotrophoblast from term placentas with RT-qPCR. Baseline levels of CXCL12 mRNA were higher in myometrial than endometrial stromal cell (2-ΔCt; 0.076 vs 0.011 respectively, p< 0.01). Scratch injury resulted in significant increase in CXCL12 mRNA levels in endometrial stromal (2-ΔCt; 0.048 from 0.011, p=0.02) but not in myometrial cells (2-ΔCt; 0.076 vs 0.069, p >0.05). CXCR4 mRNA was expressed at high levels in cytotrophoblast from term placentas, but not in the established trophoblast cell lines. Injury results in enhanced expression of CXCL12 in cultured endometrial stromal cells. CXCL12 secretion will attract the CXCR4 expressing trophoblasts. These results suggest that activation of this axis as part of the normal healing process is likely a factor modulating the increased invasiveness of the trophoblast in the setting of uterine injury. Modulation of CXCL12 expression or use of CXCR4 antagonists may prevent excessive placental invasion associated with placenta accreta.

Metabolic disparities of different oxidative stress‑inducing conditions in HTR8/SVneo cells.

Placental oxidative stress is present throughout the duration of pregnancy, but it is when oxidative stress exceeds the normal physiological level that complications can occur. Trophoblast cell lines are commonly utilized for oxidative stress research due to their distinct uniform cell population and easy-to-apply interventions. However, conflicting results are often reported when different oxidative stress cell models are used. In this study, the aim was to characterize the intracellular and extracellular metabolite profiles of different oxidative stress cell models commonly used in the research of pregnancy complications. HTR8/SVneo human trophoblast cell lines were treated with five different oxidative stress-inducing conditions: Hypoxia (1% oxygen); hypoxia and reoxygenation; cobalt chloride (CoCl2; 300 µmol/l); sodium nitroprusside (SNP; 2.5 mmol/l); and the serum of women with preeclampsia (10% v/v). Intracellular metabolites were extracted from cells and extracellular metabolites were collected from spent media for metabolomic analysis via gas chromatography-mass spectrometry. The results demonstrated that there were distinct differences in the intracellular and extracellular metabolome between the different cell models. Meanwhile, treatments with exogenous drugs, such as CoCl2 and SNP, resulted in more similar metabolite profiles. These disparities between the different oxidative stress cell models will have implications for the applications of these results, and highlight the need for the standardization of oxidative stress cell models in obstetric research.

Suppressyn localization and dynamic expression patterns in primary human tissues support a physiologic role in human placentation

We previously identified suppressyn (SUPYN), a placental protein that negatively regulates the cell fusion essential for trophoblast syncytialization via binding to the trophoblast receptor for syncytin-1, ASCT2, and hypothesized that SUPYN may thereby regulate cell-cell fusion in the placenta. Here, we redefine in vivo SUPYN localization using specific monoclonal antibodies in a rare early placental sample, showing SUPYN localization in villous and extravillous trophoblast subtypes, the decidua and even in placental debris in the maternal vasculature. In human trophoblast cell lines, we show SUPYN alters ASCT2 glycosylation within the secretory pathway and that this binding is associated with inhibition of cell fusion. Using newly-optimized trophoblast isolation protocols that allow tracking of ex vivo cell fusion, we present transcription and translation dynamics of fusion-related proteins over 96 hours in culture and the effects of changes in ambient oxygen levels on these processes. We report converse syncytin-1 and SUPYN transcriptional and translational responses to surrounding oxygen concentrations that suggest both are important in the effects of hypoxia and hyperoxia on placental syncytialization. Our results suggest that SUPYN’s anti-fusogenic properties may be exerted at several sites in the maternal body and its dysregulation may be associated with diseases of abnormal placentation.

Aspirin enhances trophoblast invasion and represses soluble fms-like tyrosine kinase 1 production: a putative mechanism for preventing preeclampsia.

Recent studies suggested that prophylactic aspirin prior to 16 weeks of gestation in high-risk patients may reduce the risk of developing preeclampsia; however, the exact mechanism of aspirin's effect on the pathophysiology of preeclampsia is not clear. This study was designed to investigate the effect of aspirin on trophoblast cell function and its effect on soluble fms-like tyrosine kinase 1 (sFlt-1) production to elucidate the preventive mechanisms for preeclampsia. We used two human trophoblastic cell lines (HTR-8/SVneo and JAR) and freshly isolated cytotrophoblasts from normal and preeclamptic placenta at term to determine the effect of aspirin on trophoblast cell function. Trophoblasts were pretreated with aspirin, and then cell functions and sFlt-1 expression were assessed. Our results showed that aspirin promoted trophoblast invasion not only in HTR-8/SVneo and JAR cells, but also in isolated cytotrophoblasts. sFlt-1 production was repressed by aspirin in a dose-dependent manner. By adding Flt-1 recombinant protein, the trophoblast invasion ability was inhibited in HTR-8/SVneo cells, which was reversed by Flt-1 small interfering ribonucleic acid knockdown. In addition, metalloproteinase 2/9 expression and activity were activated by aspirin but inhibited by sFlt-1. Aspirin also downregulated Akt phosphorylation, and trophoblast invasiveness was facilitated under Akt inhibitor treatment. Aspirin enhances cell invasiveness and inhibits sFlt-1 production in trophoblasts. Moreover, sFlt-1 itself also inhibits trophoblast invasion. Our novel findings suggest that the preeclampsia prevention effect of aspirin may be exerted through these two mechanisms.