Human Extravillous Trophoblast Research Articles

Expression of GALNT2 in human extravillous trophoblasts and its suppressive role in trophoblast invasion

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on β1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of β1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on β1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of β1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.

Human Cytomegalovirus Infection Dysregulates the Canonical Wnt/β-catenin Signaling Pathway

Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected in utero. During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/β-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/β-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of β-catenin and expression of β-catenin target genes in these cells. HCMV infection leads to β-catenin protein accumulation in a discrete juxtanuclear region. Levels of β-catenin in membrane-associated and cytosolic pools, as well as nuclear β-catenin, are reduced after infection; while transcription of the β-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/β-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.

Treatment of Lipoxin A4 and its analogue on low-dose endotoxin induced preeclampsia in rat and possible mechanisms

Preeclampsia (PE) is known to represent an exaggerated maternal inflammatory response to pregnancy. Lipoxin A4 (LXA4), considered as an endogenous stop signal in inflammation, has been extensively studied pre clinically for its inflammatory pro-resolving effects. Thus, in the current study, we tested the effect of BML-111 (synthetic analogue of LXA4) on experimental PE rats induced by low-dose endotoxin (LPS) and of LXA4 on human extravillous trophoblast cell line (TEV-1). In vivo experiment results showed that systolic blood pressure, 24h-urinary albumin excretion, serum TNF-α and IL-8 levels and morphologic damage of placenta and kidney caused by LPS were all effectively alleviated by BML-111. LXA4 also inhibited LPS-triggered apoptosis, activation of NF-κB, TNF-α and IL-8 mRNA and protein expression in TEV-1 cells. At the same time, BML-111 protected the cells from LPS-reduced proliferation. The current study demonstrated for the first time that LXA4 could alleviate the symptoms of PE in endotoxin exposed rats.

Oxidative Stress Produced by Xanthine Oxidase Induces Apoptosis in Human Extravillous Trophoblast Cells

Oxidative stress has been recognized as an important factor in the pathophysiology of preeclampsia. It has been reported that the expression of xanthine oxidase (XO) in the cytotrophoblast and plasma hydrogen peroxide (H2O2) level are significantly higher in preeclamptics than in control women. The aim of this study was to clarify the biological influence of reactive oxygen species (ROS) produced by XO on extravillous trophoblast (EVT) cells. TCL1 cells, a human immortalized EVT cell line, were incubated with xanthine and XO (X/XO). We then measured the cell number, urate level of the culture media and the apoptotic cell ratio. Similar experiments were performed with additional administration of allopurinol, catalase, L-NAME or D-NAME, and with administration of H2O2 in substitution for X/XO. We assessed the effects of H2O2 on invasion ability, tube-like formation and protein expression of HIF1A and ITGAV of TCL1. Finally, the apoptotic cell ratio using primary cultured trophoblasts was measured following exposure to H2O2. X/XO decreased the relative cell number and increased the urate level and apoptotic cell ratio significantly. Elevation of the urate level and apoptotic cell ratio was attenuated by allopurinol and catalase, respectively. L-NAME and D-NAME had no influence on these effects. H2O2 also decreased the relative cell number. Pretreatment with H2O2 significantly inhibited the invasion ability, tube-like formation and HIF1A and ITGAV of TCL1. H2O2 also induced apoptosis in primary cultured trophoblasts. In conclusion, ROS produced by XO induced apoptosis and affected EVT function including invasion and differentiation.

Role of Integrin Switch and Transforming Growth Factor Beta 3 in Hypoxia-Induced Invasion Inhibition of Human Extravillous Trophoblast Cells1

The physiological hypoxic condition favors the angiogenesis in the placenta. However, it remains unclear how hypoxia regulates the invasion of human extravillous trophoblast cells. In the present study, we first showed that alpha5 integrin expression increased and alpha1 integrin expression decreased in human extravillous trophoblast cells cultured in 1% oxygen as compared with control cells cultured in 8% oxygen. Further data showed that the neutralizing antibody against alpha5 integrin increased the invasion of human extravillous trophoblast cells and the neutralizing antibody against alpha1 integrin inhibited the invasion of human extravillous trophoblast cells. Human extravillous trophoblast cells cultured in 1% oxygen showed reduced invasive capacity, which can be effectively blocked by alpha5 integrin neutralizing antibody. Moreover, human extravillous trophoblast cells exposed to 1% oxygen demonstrated increased expression of transforming growth factor-beta3 (TGFB3), and recombinant human TGFB3 inhibited the invasion of human extravillous trophoblast cells in a dose-dependent manner. The neutralizing antibodies against alpha5 integrin and TGFB3 markedly abrogated hypoxia-induced invasion inhibition in human extravillous trophoblast cells. These data indicate that hypoxia may inhibit the invasion of human extravillous trophoblast cells through inducing the integrin switch from alpha1 integrin to alpha5 integrin and promoting TGFB3 expression.

MicroRNA-155 inhibits proliferation and migration of human extravillous trophoblast derived HTR-8/SVneo cells via down-regulating cyclin D1

MiR-155 is known to participate in various cellular processes by targeting gene expression. We previously revealed a link between miR-155 and perturbation of trophoblast invasion and differentiation. This study aimed to investigate the target molecule(s) of miR-155 on the influence on the proliferation and migration of trophoblast cells. Bioinformatics analysis showed that, at the 3′ untranslated region (UTR) of cyclin D1, six bases are complementary to the seed region of miR-155. Luciferase assays and cyclin D1 3′UTR transfection assays validated that cyclin D1 3′UTR was the target of miR-155 in HTR-8/SVneo cells. Overexpression of miR-155 in HTR-8/SVneo cells reduced the level of cyclin D1 protein, decreased cell proliferation and invasion, and increased cell number at the G1 stage. Furthermore, the increased expression of miR-155 also regulated the protein levels of kinase inhibitory protein p27 and phosphorylated cytoskeletal protein filamin A. In conclusion, we found that cyclin D1 may be a target of miR-155 in HTR-8/SVneo cells, and demonstrated a negative regulatory role of miR-155 involved in cyclin D1/p27 pathway in proliferation and migration of the cells.

Leptin induces tube formation in first-trimester extravillous trophoblast cells

To study the roles of leptin on tube formation (as a measure of cellular angiogenesis) and expression of associated genes in first-trimester human extravillous trophoblast cells. The effects of leptin on tube formation and fatty acid uptake in first trimester extravillous placental trophoblast cells, HTR8/SVneo, were investigated. We also investigated the effects of leptin on the expression of genes involved in angiogenesis and lipid metabolism in these cells. Leptin at 25 ng/ml maximally stimulated tube formation in the first trimester placental trophoblast cells, HTR8/SVneo, by increasing tube length as well as numbers (10,100 ± 150 pixels) compared with those of control cells (2900 ± 50 pixels) p>0.05. Leptin-induced tube formation was not inhibited by the selective inhibitor of VEGF, indicating that its action was independent of VEGF. Leptin, however, significantly increased the expression of genes those are involved in angiogenesis pathways such as PECAM1, JAG1, CDH5, IL8, NRP1, SPHK1, S1PR1, CXCL 1 and 6, FGF1, EFNA3 and AKT1, as determined by PCR array. Leptin did not, however, stimulate expression of the primary angiogenic factors known in placenta such as VEGF or ANGPTL4, as determined by both qRTPCR and PCR array assays. Leptin increased 7-fold expression of FABP4, which is known to be involved in VEGF-mediated angiogenesis in endothelial cells. In addition, leptin treatment resulted a 48% increase in the uptake of docosahexaenoic acid, 22:6n-3 (DHA) which also stimulates tube formation in these cells. Leptin may play an important role in early placentation by stimulating several genes involved in angiogenic signalling pathway and fatty acid metabolism.

Expression of CD82 in Human Trophoblast and Its Role in Trophoblast Invasion

BackgroundWell-controlled trophoblast invasion at maternal-fetal interface is a critical event for the normal development of placenta. CD82 is a member of transmembrane 4 superfamily, which showed important role in inhibiting tumor cell invasion and migration. We surmised that CD82 are participates in trophoblast differentiation during placenta development.Methodology/Principal FindingsCD82 was found to be strongly expressed in human first trimester placental villous and extravillous trophoblast cells as well as in trophoblast cell lines. To investigate whether CD82 plays a role in trophoblast invasion and migration, we further utilized human villous explants culture model on matrigel and invasion/migration assay of trophoblast cell line HTR8/SVneo. CD82 siRNA significantly promoted outgrowth of villous explants in vitro (P<0.01), as well as invasion and migration of HTR8/SVneo cells (P<0.05), whereas the trophoblast proliferation was not affected. The enhanced effect of CD82 siRNA on invasion and migration of trophoblast cells was found associated with increased gelatinolytic activities of matrix metalloproteinase MMP9 while over-expression of CD82 markedly decreased trphoblast cell invasion and migration as well as MMP9 activities.Conclusions/SignificanceThese findings suggest that CD82 is an important negative regulator at maternal-fetal interface during early pregnancy, inhibiting human trophoblast invasion and migration.

Intracellular crosstalks between signal transducer and activator of transcription (STAT) and extracellular-regulated kinase (ERK) 1/2 regulating trophoblastic cells

Trophoblast proliferation and invasion are controlled by cytokines and growth factors present at the implantation site. Members of the Interleukin-6 (IL-6) family of cytokines trigger their effects through activation of intracellular cascades including the Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) pathway. Functions of several STAT molecules in trophoblast cells have been described, but the role of STAT1 remained unclear. Here, potential functions of STAT1 and its activation by Oncostatin M (OSM) have been investigated in an in vitro model.STAT1 expression and phosphorylation were analyzed in human term placenta tissue by immunohistochemistry. HTR-8/SVneo cells (immortalized human extravillous trophoblast cells) were stimulated with OSM, IL-6, IL-11, Leukemia Inhibitory Factor (LIF) and Granulocyte Macrophage Colony-Stimulating Factor. Expression and phosphorylation of STAT1 were analyzed by Western blotting and immunocytochemistry. Fludarabine and STAT1 siRNA were employed for STAT1 depletion. STAT1 transcriptional activity was evaluated by DNA-binding capacity assay. Cell viability and invasion were assessed by MTS and Matrigel assays, respectively.STAT1 was expressed in villous and extravillous trophoblast cells. Low phosphorylation was detectable exclusively in extravillous trophoblast cells. Only OSM and LIF induced phosphorylation of STAT1 in the in vitro model. Challenge with OSM increased cell invasion but not proliferation. Inhibition of STAT1 by fludarabine treatment or STAT1 siRNA transfection reduced cell viability and invasiveness in presence and absence of OSM.These results indicate the potential involvement of STAT1 in the regulation of trophoblast behavior. Furthermore, STAT 1 functions are more efficiently inhibited by blocking its expression than its phosphorylation.

The effect of lipopolysaccharide on MMP-2 and MMP-9 in HTR-8/SVneo cells

Brominated diphenyl ether (BDE)-47 is a prevalent flame retardant chemical found in human tissues and is linked to adverse pregnancy outcomes in humans. Because dysregulation of the prostaglandin pathway is implicated in adverse pregnancy outcomes, the present study investigates BDE-47 induction of prostaglandin synthesis in a human extravillous trophoblast cell line, HTR-8/SVneo, examining the hypothesis that BDE-47 increases generation of reactive oxygen species (ROS) to stimulate the prostaglandin response. Treatment with 20 μM BDE-47 significantly increased mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2) at 4, 12 and 24 h, and 24-h treatment significantly increased cyclooxygenase (COX)-2 cellular protein expression and prostaglandin E2 (PGE2) concentration in culture medium. The BDE-47-stimulated PGE2 release was inhibited by the COX inhibitors indomethacin and NS398, implicating COX activity. Exposure to 20 μM BDE-47 significantly increased ROS generation as measured by carboxydichlorofluorescein fluorescence, and this response was blocked by cotreatment with the peroxyl radical scavenger (±)-α-tocopherol. (±)-α-Tocopherol cotreatment suppressed BDE-47-stimulated increases of PGE2 release without significant effects on COX-2 mRNA and protein expression, implicating a role for ROS in post-translational regulation of COX activity. Because prostaglandins regulate trophoblast functions necessary for placentation and pregnancy, further investigation is warranted of BDE-47 impacts on trophoblast responses.

Laeverin/aminopeptidase Q induces trophoblast invasion during human early placentation

In primate placenta, extravillous trophoblast (EVT) invades maternal tissue in temporally- and spatially-regulated fashions. We previously identified a novel placenta-specific cell-surface aminopeptidase, laeverin/aminopeptidase Q, which is expressed on EVT-lineage cells in the fetal membrane. Laeverin possesses a peptide-binding site that is evolutionally unique to primates, suggesting possible involvement of laeverin in a primate-specific phenomenon during placentation. Thus, this study was designed to elucidate the molecular characteristics and physiological roles of laeverin in human EVT. Placental tissues of various developmental stages were subjected to immunostaining and western blotting. Effects of siRNA and a soluble form of recombinant laeverin on EVT cells isolated from primary villous explant cultures were examined using Matrigel invasion assays and cell proliferation assays. Laeverin was specifically immunolocalized to HLA-G-positive EVT in placentas from early and term pregnancy. In primary villous explant cultures, laeverin expression was induced on the cell surface of the outgrowing EVT. In western blotting, laeverin protein was detected as two distinct bands at 130 and 160 kDa along with a broad band ranging from 200 to 270 kDa. De-glycosylation treatment showed that these native laeverin isotypes are N-linked glycoproteins sharing a common 115-kDa core protein. In invasion assays, the reduction of laeverin expression by siRNA suppressed migration of the isolated EVT, while the soluble form of recombinant laeverin enhanced its migration. Laeverin is a specific cell-surface marker for human EVT and plays a regulatory role in EVT migration.

Pro-inflammatory cytokine-stimulated first trimester decidual cells enhance macrophage-induced apoptosis of extravillous trophoblasts

ObjectiveAs human blastocyst-derived extravillous trophoblasts (EVTs) invade the early decidua, they are positioned to interact with immune cells and resident decidual cells, and remodel spiral arteries into high capacity vessels that increase blood flow to the developing fetal-placental unit. Shallow EVT invasion elicits incomplete vascular transformation and reduces uteroplacental blood flow that presages adverse pregnancy outcomes. Excess macrophages in the decidua induce EVT apoptosis via tumor necrosis factor-alpha (TNF-α) secretion. Our previous observation that pro-inflammatory cytokines enhance neutrophil and macrophage activator granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in first trimester decidual cells is now extended to include: 1) the specific macrophage activator M-CSF; 2) macrophage activation and subsequent enhancement of EVT apoptosis by both GM-CSF and M-CSF. Study designQuantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay assessed M-CSF expression in first trimester decidual cells incubated with interleukin-1 beta (IL-1β) or TNF-α. Peripheral monocyte-derived macrophages pre-incubated with conditioned media from decidual cell cultures were co-cultured with a first trimester EVT cell line, HTR-8/SVneo cells. Macrophage activation was examined and EVT apoptosis evaluated by DNA fragmentation, caspase activation and cell membrane asymmetry. ResultsIL-1β or TNF-α significantly enhanced M-CSF expression in first trimester decidual cells. The conditioned media from these cultures activates macrophages, which promote caspase 3/7-dependent EVT apoptosis with antibodies against GM-CSF or M-CSF blocking this effect. ConclusionsPro-inflammatory cytokines increases synthesis of M-CSF in first trimester decidual cells. Both GM-CSF and M-CSF activate macrophages, which initiate caspase-dependent EVT apoptosis.

IFPA Award in Placentology Lecture: Molecular regulation of human trophoblast invasion

Invasion of extravillous trophoblast cell types into maternal uterine tissues is essential for successful human placental development and progression of pregnancy. Whereas endovascular trophoblasts migrate into the maternal spiral arteries, interstitial trophoblasts invade the decidual stroma, colonize the vessels from outside and communicate with diverse uterine cell types such as decidual stromal cells, macrophages and uterine NK cells. For example, interstitial trophoblasts expressing polymorphic human leukocyte antigen-C interact with uterine NK cells through binding to their killer immunoglobulin-like receptors which likely plays a role in trophoblast invasion and reproductive success of pregnancy. Both extravillous trophoblast subtypes are critically involved in the vascular transformation of the spiral arteries into dilated conduits ensuring appropriate blood flow into the intervillous space. Failures in this remodeling process are thought to be associated with severe forms of fetal growth restriction, preeclampsia and other pregnancy complications warranting studies on the molecular regulation of extravillous trophoblast differentiation. Moreover, interstitial trophoblast-derived hormones may regulate diverse biological functions in the decidua. In particular, human chorionic gonadotrophin has been shown to promote angiogenesis and to suppress apoptosis of endometrial stromal cells. In return, decidual cells produce a plethora of soluble factors controlling trophoblast invasion in a time- and distance-dependent manner. However, the underlying mechanisms have not been fully elucidated. Here, we will summarize autocrine as well as paracrine factors regulating invasion of extravillous trophoblasts and discuss critical signaling cascades involved. In addition, we will focus on key regulatory transcription factors controlling cell column proliferation and differentiation of the human extravillous trophoblast.

Endostatin Suppresses IGF-II-Mediated Signaling and Invasion of Human Extravillous Trophoblasts

Endostatin, a biological active fragment of the extracellular matrix protein collagen XVIII, is known to interfere with cellular motility in the context of pathological angiogenesis. However, the physiological role of endostatin remains largely elusive. Recent evidence suggested that the inhibitor is produced in human decidual cells of early pregnancy, indicating that endostatin could be involved in diverse reproductive processes, such as implantation and/or placental differentiation. To gain more insights into the role of endostatin, we here analyzed its effects on trophoblast motility, proliferation, and signaling using purified primary trophoblasts, first-trimester villous explant cultures, and trophoblastic SGHPL-5 cells. In vitro Transwell assays demonstrated that purified endostatin inhibited both basal and IGF-II-induced migration and invasion as well as outgrowth from villous explant cultures. In contrast, basal and IGF-II-stimulated proliferation was unaffected upon addition of the inhibitor. Analyses of IGF-II-associated downstream signaling events showed that endostatin interfered with activation of various signaling kinases such as ERK1/2, protein kinase B (Akt)/mammalian target of rapamycin/p70 S6 kinase, and focal adhesion kinase. Furthermore, virus-mediated, stable gene silencing of Akt1 in SGHPL-5 cells using a micro-RNA-adapted short hairpin RNA-expressing plasmid revealed that endostatin-mediated inhibition of IGF-II-induced Akt phosphorylation was critically dependent on the expression of the particular isoform. In conclusion, the data suggest that endostatin could be a physiological inhibitor of IGF-II-dependent trophoblast cell motility by suppressing focal adhesion kinase/Akt/mammalian target of rapamycin/p70 S6 kinase signaling.

The Loss of Endoglin Promotes the Invasion of Extravillous Trophoblasts

Endoglin is a coreceptor for TGF-β, which is expressed in syncytiotrophoblasts. The soluble form of endoglin (sEng) has been observed to increase in the serum of preeclamptic patients. Several studies have shown that endoglin is involved in cancer invasion. However, the role of endoglin in extravillous trophoblasts (EVT), which have an invasive phenotype, remains unknown. The present study was designed to investigate the expression and role of endoglin in human EVT. We found that endoglin was mainly expressed on cytotrophoblasts within the cell column during the first trimester and its expression decreased in the EVT by immunohistochemistry and immunocytochemistry. The expression of endoglin significantly increased after treatment with TGF-β1 and TGF-β3 in the human EVT cell line, HTR-8/SVneo, as detected by semiquantitative RT-PCR. To investigate the role of endoglin in EVT, the stable knockdown of endoglin was performed by lentiviral short hairpin RNA transfection into the HTR-8/SVneo cells. Although proliferation was not affected, the motility and invasiveness of the HTR-8/SVneo cells significantly increased by the knockdown of endoglin. Both the mRNA expression and secretion of urokinase-type plasminogen activator significantly increased in endoglin knockdown cells. The secretion of sEng was very low in HTR-8/SVneo, and the treatment of endoglin knockdown cells with 10 ng/ml sEng had no effect on their invasiveness. Therefore, the suppression of sEng was not involved in the increased invasiveness of endoglin knockdown cells. These results suggested that EVT increased their invasive function as a result of decreasing expression of transmembrane endoglin.

Interleukin (IL)11 mediates protein secretion and modification in human extravillous trophoblasts

Human trophoblast invasion and differentiation are essential for a successful pregnancy outcome. Dysregulation of these processes can lead to placental pathologies such as pre-eclampsia. The molecular mechanisms; however, are poorly understood. Interleukin (IL)11--a cytokine that regulates endometrial epithelial cell adhesion, trophoblast motility and invasion during implantation--may be involved in some of these processes. The effect of IL11 on protein expression was investigated in trophoblastic HTR8/SVneo cells and primary extravillous trophoblasts (EVTs) purified from first- trimester placentas. Two-dimension (2D)-differential in-gel electrophoresis analyses revealed that 731 spots were significantly differentially regulated by IL11 in HTR8/SVneo cells: seven spots were analyzed by liquid chromatography-tandem mass spectrometry and 14 unique proteins identified. Protein disulfide isomerase family A, member 3 (PDIA3; endoplasmic reticulum p57) and glucose-regulated protein 78 (GRP78) were further validated to be regulated by IL11 in HTR8/SVneo and primary EVT. One dimension western blot analysis confirmed that PDIA3 was down-regulated in EVT. 2D western blot analysis revealed that GRP78 was post-translationally modified following IL11 treatment. Moreover, IL11 stimulated the secretion of GRP78 in EVT. Data suggest that IL11, possibly via signal transducers and activators of transcription 3 signaling pathway, regulates PDIA3 protein expression and modification/secretion of GRP78. This is the first study to identify PDIA3 and GRP78 as IL11 targets in invasive trophoblasts and identifies a possible mechanism by which IL11 regulates trophoblast function.

Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy

During early pregnancy, extravillous trophoblast (EVT) cells are exposed to very low pO2 values. In this study, we investigated the proteolytic functions and invasiveness of human primary EVT cells under hypoxic conditions to show the early placental pathophysiology.Placental samples (from 5 to 10 weeks gestation) were obtained at termination of pregnancy. Cytotrophoblast cells were separated by Percoll® gradient method and cultured on Matrigel® to obtain an invasive phenotype (similar to EVT). The invasion capacity (Matrigel-coated invasion assay), migration of the cells (wound healing assay), activity and expression of matrix metalloproteinase (MMP)-2 and tissue inhibitor for MMP (TIMP)-2 (gelatin gel zymography, ELISA, and quantitative RT-PCR), and expression of membrane-type (MT)1-MMP (western blot) were investigated. All cultures (except for quantitative RT-PCR) were performed under 20% oxygen, 5% oxygen, and 5% oxygen with 3 repetitions of 0.1% oxygen hypoxic stimulation for 1 h.Invasion and MMP2 activity of the cells were significantly increased in 20% and decreased in 0.1% oxygen. There was no significant difference in cell migration among the oxygen environments. Concentrations of MMP2 in the supernatant and expression of MT1-MMP were increased in both the 0.1% and 20% oxygen environments. The MMP2 mRNA level was increased after 1-h stimulation with 0.1% oxygen. The TIMP2 concentration was increased only in 20% oxygen, but the mRNA level was decreased in 0.1% oxygen.These results suggested that hypoxia might inhibit the invasive capacity and MMP2 activation of EVT cells in the early first trimester of pregnancy. Decrease in TIMP2 production may reduce the MMP2/TIMP2/MT1-MMP complex and lead to this unique behavior of EVT cells under hypoxic conditions.

Decorin Is a Novel VEGFR-2-Binding Antagonist for the Human Extravillous Trophoblast

Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that (125)I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K(d)) of 566 pM, and DCN displaced this binding with an inhibition constant (K(i)) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function.

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

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

Characterization and role of NUMB in the human extravillous trophopblast

NUMB is a multifunctional protein involved in asymmetric cell differentiation, proliferation and maintenance. Four mammalian NUMB isoforms have been identified, which utilize the phosphotyrosine binding (PTB) domain and the proline rich region (PRR) domain to regulate cell growth and differentiation in the developing nervous system. The observation that a decrease in spongiotrophoblast number and thickness of placentae of null ( Numb −/− ) mouse embryos, which died at E10.5, suggests NUMB may play a role in placental development. In this study, we demonstrated for the first time, that NUMB isoforms 1, 2, 3, and 4 are present in the human placenta and the human extravillous trophoblast (EVT) cell line HTR8/SVneo. We report three novel isoforms, NUMB 7, 8, and 9, identified by cloning of RT-PCR products and sequencing. Corresponding sequences of novel isoforms were submitted to genebank (accession numbers for each new isoform: NUMB 7- EU265736, NUMB 8- EU265737 and NUMB 9- EU265738). Western blot analysis confirmed the presence of all NUMB isofoms in human placental samples in all trimesters and in EVT cells. NUMB immunosignals were extensively localized in human extravillous trophoblasts and decidual cells at the maternal–fetal interface. NUMB 8 appeared to be the predominant isoform in placental villi. Furthermore, cell migration studies revealed NUMB isoform 1 to be involved in EVT cell migration and NUMB isoforms 2 and 4 to induce EVT apoptosis.