First-trimester Extravillous Trophoblast Cell Research Articles

Effect of epigenetic modification of maspin on extravillous trophoblastic function

This study investigated the effect of epigenetic modification of maspin on extravillous trophoblastic function. The mRNA expression of maspin in placentae from normotensive and preeclamptic pregnant women was detected by RT-PCR. TEV-1 cells, a human first-trimester extravillous trophoblast cell line, were cultured and treated with CoCl(2) (300 μmol/L) to induce chemical hypoxia and with 5-aza (500 nmol/L) to induce demethylation. The mRNA expression of maspin in TEV-1 cells subjected to different treatments was determined by RT-PCR, and the proliferative and migratory abilities of TEV-1 cells were assessed by cell counting kit-8 (CCK-8) and Transwell assays. Our results showed that the maspin mRNA expression level in placentae from preeclamptic women was much higher than that from normotensive women. CoCl(2) or 5-aza could up-regulate the mRNA expression of maspin and significantly suppress the proliferation and migration of TEV-1 cells. It was concluded that the epigenetic modification in promoter region of maspin contributes to incomplete trophoblast invasion, which offers a novel approach for predicting and treating placental dysfunction.

Effects of increased fetuin-A in human trophoblast cells and associated pregnancy outcomes

The purpose of this study was to determine whether fetuin-A affects trophoblast viability and invasion, whether growth factors that bind receptors that activate tyrosine kinase are impaired by fetuin-A, and whether elevated maternal serum fetuin-A levels are associated with adverse pregnancy outcomes. We studied viability and invasion in first-trimester extravillous trophoblast cells that were exposed to fetuin-A, insulin-like growth factor, and placental growth factor. Insulin receptor substrates expression was assessed. We compared serum fetuin-A levels in 111 preeclampsia cases and 95 controls. Fetuin-A reduced extravillous trophoblast cell viability and invasion in the presence or absence of growth factors. Fetuin-A reduced insulin receptor substrate-1 and tyrosine phosphorylated insulin receptor substrate-1 expression in extravillous trophoblast cells that had been treated with insulin-like growth factor. Elevated serum fetuin-A levels were more prevalent in preeclampsia cases than control subjects, even after we controlled for diabetes mellitus, hypertension, and obesity. Fetuin-A may decrease trophoblast viability and invasion caused by the inhibition of receptor tyrosine kinase activity. Elevated serum levels of fetuin-A may be associated with preeclampsia.

Decreased Cyr61 under hypoxia induces extravillous trophoblasts apoptosis and preeclampsia

During placental development, oxygen environment is not only critical for trophoblasts migration and invasion, but also fundamental for appropriate placental perfusion. Cysteine-rich 61 (Cyr61, CCN1) was expressed in the extravillous trophoblasts (EVTs) and decreased in preeclampsia. Its regulatory properties in human first-trimester extravillous trophoblast cell line (TEV-1 cells) upon a low oxygen tension were investigated. The present study examined functional changes involved in adaptation to hypoxia of the TEV-1 cells, using cobalt chloride (CoCl(2)) as hypoxic mimic. It was found that hypoxia inhibited growth of TEV-1 cells and induced the increase of cell apoptosis (P<0.05). The Cyr61 expression in human EVTs was transcriptionally induced by CoCl(2). Inappropriate EVTs apoptosis has been implicated in the failure of trophoblasts to fully invade and modify the uterine environment and Cyr61 down-regulation, potentially leading to preeclampsia.

Inhibition of HLA-G Expression Via RNAi Abolishes Resistance of Extravillous Trophoblast Cell Line TEV-1 to NK Lysis

Remodelling of uterine spiral arteries occurs in the first trimester of pregnancy and involves an expanded and activated population of maternal natural killer (NK) cells in the decidua and extravillous trophoblast cells. Invasive trophoblasts encounter maternal NK cells during their invasion into the uterine tissue, posing the problem of susceptibility to NK lysis. Studies in vitro and in vivo suggested that the expression of HLA-G by invasive extravillous trophoblasts might provide invulnerability to NK cells, while there is still lack of direct evidence of HLA-G knockdown effect on trophoblast/NK interaction. A study was conducted to investigate the effects of down-regulated HLA-G on extravillous trophoblasts. The short hairpin RNA (shRNA) vector targeting HLA-G was constructed and transfected into the human first-trimester extravillous trophoblast cell line TEV-1. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) revealed that in HLA-G shRNA transfected cells, the expression of HLA-G was significantly decreased. HLA-G expression was also visualised by confocal imaging. The HLA phenotype of TEV-1 cells and inhibitory receptors expression in NK cells were analysed by flow cytometry. A comparison between HLA-G knockdown and non-knockdown cells showed a significant difference in the HLA expression profile without altering HLA-C and HLA-E. Both primary NK cells and NK-92 cell line exhibited potent cytotoxicity against HLA-G knockdown cells via standard 4-h 51Cr release assays. Expression of ILT2, ILT4 and KIR2DL4 in NK cells was unchanged after 4h of co-culture, while KIR2DL4 expression increased after 48h. We conclude that HLA-G contributes to trophoblast/NK interaction, acting as a key regulator of NK cytolysis in this human extravillous trophoblast cell model. In addition, TEV-1 cells share common HLA phenotype characters with extravillous trophoblast cells, and thus might be used as a good cell model. HLA-C expression in trophoblasts is not correlated with HLA-G translation and HLA-C alone was sufficient to boost HLA-E surface expression. In addition, RNA interference could be employed as a feasible and effective method to study HLA-G functions.

Establishment and Characterization of a Human First-Trimester Extravillous Trophoblast Cell Line (TEV-1)

Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGFbeta1). The responsive gene to TGFbeta1 treatment was confirmed by quantitative real-time PCR. The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGFbeta1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies.

Control of proliferation, migration, and invasiveness of human extravillous trophoblast by decorin, a decidual product.

Extravillous trophoblast (EVT) cells of the human placenta progressively lose their proliferative activity in situ as EVT cell columns migrate into and invade the decidua. It remains unclear whether this is due to a terminal differentiation of EVT cells along the invasive pathway with concomitant loss of proliferative ability, or a negative regulation by decidua-derived factors, or both mechanisms. Our earlier studies provided evidence for a negative regulation by a decidua-derived factor, transforming growth factor (TGF)-beta, which inhibited proliferation, migration, and invasiveness of first-trimester EVT cells in vitro. We further discovered that decidua also produces decorin, a proteoglycan that binds TGF-beta (and in some cases, inactivates TGF-beta), which is colocalized with TGF-beta in the decidual extracellular matrix. The present study used in vitro-propagated EVT cell lines to examine whether EVT cells retain their capacity for proliferation after the process of invasion; and whether decorin exerts any effect on EVT cell proliferation, migration, or invasiveness in a TGF-beta-dependent or TGF-beta-independent manner. We also examined whether trophoblastic cancer (choriocarcinoma) JAR and JEG-3 cells responded to decorin in a similar manner. Proliferation was measured using a colorimetric (MTT) cellularity assay and immunolabeling for the Ki-67 proliferation marker. Migration and invasiveness were measured in transwells by the ability of cells to cross 8-microm pores of polycarbonate membranes in the absence or presence of an additional matrigel barrier. These experiments revealed three points. First, EVT cells retained limited but significant proliferative ability in vitro after invading matrigel. Second, that decorin alone blocked EVT cell proliferation in a dose-dependent manner. This effect remained unaffected in an additional presence of TGF-beta, which exerted antiproliferative effects on its own. The antiproliferative effect of decorin was explained by an up-regulation of the p21 protein. Third, that decorin alone or TGF-beta alone exerted antimigratory and anti-invasive effects on EVT cells, but the addition of TGF-beta to decorin did not alter decorin action. And fourth, that choriocarcinoma cells were resistant to antiproliferative, antimigratory, and anti-invasive effects of decorin. These results suggest 1) that the invasive function of EVT cells is not associated with a terminal differentiation into a noncycling state; 2) that proliferation, migration, and invasiveness of EVT cells within the decidua are independently controlled by two decidual products, TGF-beta and decorin (decorin in the decidual extracellular matrix may serve as a storage mechanism for TGF-beta in an inactive state and may be activated by EVT cell proteolytic mechanisms, thus preventing overinvasion); and 3) that choriocarcinoma cells are refractory to negative regulation by both decidua-derived factors.

Regulation of human trophoblast migration and invasiveness.

The human placenta is an invasive structure in which highly proliferative, migratory, and invasive extravillous trophoblast (EVT) cells migrate and invade the uterus and its vasculature. Using in vitro propagated normal first-trimester EVT cells and immortalized EVT cells, which share all of the phenotypic and functional characteristics of the normal EVT cells, it has been shown that migration/invasion of human EVT cells is stringently regulated by many growth factors, their binding proteins, extracellular matrix (ECM) components, and some adhesion molecules in an autocrine/paracrine manner at the fetal-maternal interface in human pregnancy. Transforming growth factor beta (TGF-beta), decorin (a proteoglycan in the ECM), and melanoma cell adhesion molecule (Mel-CAM) inhibit, and insulin-like growth factor II (IGF-II), IGF-binding protein 1 (IGFBP-1), and endothelin 1 (ET-1) stimulate EVT cell migration/invasion. Inhibition of EVT cell migration by TGF-beta has been suggested to be due to upregulation of integrins, which make the cells more adhesive to the ECM. Its antiinvasive action is due to an upregulation of tissue inhibitor of matrix metalloprotease 1 (TIMP-1) and plasminogen activator inhibitor (PAI-1) and a downregulation of urokinase-type plasminogen activator (uPA). Molecular mechanisms of inhibition of migration/invasion of EVT cells by decorin and Mel-CAM remain to be identified. IGF-II action has been shown to be mediated by IGF type I receptors (IGF-RII) independently of IGF type I receptors (IGF-RI) and IGFBPs. This action of IGF-II appears to involve inhibitory G proteins and phosphorylation of mitogen-activated protein kinase (MAPK) (extracellular signal-regulated protein kinases 1 and 2 (ERK-1 and ERK-2)). IGFBP-1 stimulation of EVT cell migration appears to occur by binding its Arg-Gly-Asp (RGD) domain to alpha5beta1 integrin, leading to phosphorylation of focal adhesion kinase (FAK) and MAPK (ERK-1 and ERK-2). These studies may improve our understanding of diseases related to abnormal placentation, viz. hypoinvasiveness in preeclampsia and hyperinvasiveness in trophoblastic neoplasms.

Collagen phagocytosis by human extravillous trophoblast: potential role in trophoblastic invasion.

To study collagen phagocytosis by human extravillous trophoblast. First-trimester extravillous trophoblastic cell lines and primary trophoblast cell preparations were cultured in vitro with collagen-coated fluorescent latex beads and fluorescent-labeled collagen. Confocal microscopy was used to demonstrate internalization of collagen and beads. The effect of cytochalasin B, temperature, metabolic inhibitors, and cytokines was studied by culturing trophoblast cells with tritiated collagen. Acridine orange was used to stain for lysosomal compartments, and histochemical methods were used to demonstrate acid phosphatase in trophoblast cells. Both cell lines and primary culture cells internalize collagen and beads. Confocal microscopy unequivocally localized the phagocytosed material to the intracellular compartment. Inhibition by cytochalasin B and culture at 4C of uptake of [3H] collagen suggested that the process was phagocytosis. Cytokines and growth factors did not affect phagocytosis. Lysosomal compartments and acid phosphatase appear to colocalize. The continuous remodeling and turnover of collagen which occur in a wide variety of tissues under both physiologic and pathologic conditions are thought to be mediated by two pathways: one external (involving release of proteolytic enzymes), and the other internal (involving phagocytosis). Similar remodeling events are likely to occur during trophoblast invasion. Although current views emphasize the importance of the extracellular pathway, we postulate, on the basis of our findings, that both pathways are used, with the internal pathway probably being dominant. We hypothesize that the proteolytic enzymes (extracellular pathway) disrupt collagen matrices, thereby facilitating phagocytosis. It is teleologically sound to conceive of a dominant intracellular pathway, as it allows for more precise control of the process of invasion and is economical, as the products of collagen degradation can be used as energy sources or building blocks.

Autocrine-Paracrine Regulation of Human Trophoblast Invasiveness by Insulin-like Growth Factor (IGF)-II and IGF-Binding Protein (IGFBP)-1

Trophoblast growth and invasion of the uterus are tightly regulated by locally produced factors. Since insulin-like growth factor (IGF)-II is produced by the invasive human extravillous trophoblast (EVT) cells and IGF-binding protein (IGFBP)-1 by the decidual cellsin situthat are in proximity to each other, we examined the possible influence of these molecules on proliferation, migration, and invasiveness of first-trimester EVT cells in culture. These EVT cell functions were respectively measured by3H-TdR uptake,in vitromigration, and invasion assays. Secretion of invasion-associated enzymes was assessed by zymography, and IGF-binding moieties on the EVT cell were examined by affinity cross-linking. Proliferation of serum-starved EVT cells was stimulated by addition of serum but unaffected by a wide range of IGF-I, IGF-II, and IGFBP-1 concentrations. IGF-II and IGFBP-1 or their combination stimulated EVT cell invasiveness and migration, when assays were conducted in serum-reduced media. Affinity cross-linking studies failed to detect the type 1 IGF receptor, although several IGF-II-specific and IGF-II-preferring binding molecules including type 2 IGF receptor were identified on the EVT cell surface. IGF-II enhancement of invasion was unaffected in the presence of IGF-1 receptor-blocking antibody and IGF-1 failed to influence EVT cell invasion, indicating that type 1 IGF receptor was not responsible for the IGF-II effects. Secretion of gelatinases or plasminogen activators was unaltered by IGF-II or IGFBP-1. We conclude that trophoblast-derived IGF-II and decidua-derived IGFBP-1 provide autocrine/paracrine enhancement of trophoblast invasiveness largely by stimulating migration, an essential step in invasion.

Vascular endothelial growth factor stimulates proliferation but not migration or invasiveness in human extravillous trophoblast.

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that promotes angiogenesis and vascular hyperpermeability and interacts with two receptors, fms-like tyrosine kinase (Flt-1) and kinase domain-containing region (KDR). In situ localization in the pregnant human uterus revealed that VEGF mRNA is expressed primarily by the maternal decidua, whereas the receptor Flt-1 is expressed primarily by chorionic vascular endothelium and trophoblast cells-in particular, the extravillous trophoblast (EVT). We examined whether the mRNA and protein of VEGF and its receptors are expressed by invasive human first-trimester EVT cells propagated in culture and whether VEGF influences EVT cell proliferation, migration, and invasiveness. Proliferation was assessed by the uptake of [3H]thymidine. Invasion and migration across transwells were assessed by the degree of cellular transgression of a Millipore membrane coated, respectively, with and without Matrigel. Results of immunocytochemical and reverse transcription-polymerase chain reaction analysis revealed that both protein and mRNA of VEGF, Flt-1, and KDR were expressed by cultured normal EVT cells as well as their premalignant derivative produced by SV-40 Tag-immortalization, and BeWo choriocarcinoma cells. Under serum-free conditions, exogenous VEGF121 (the non-heparin-binding isoform) stimulated proliferation of all three cell lines in a concentration-dependent manner. The effects were abolished with a VEGF-neutralizing antibody. The same stimulatory effects on EVT cells were also seen with exogenous VEGF165 (a heparin-binding isoform), only after a cleaving of the heparin-binding domain with plasmin or a blocking of heparin binding sites with excess soluble heparan sulphate proteoglycans (HSPGs), suggesting a regulatory role of HSPGs. However, VEGF121 and VEGF165 (with and without the HSPG pretreatment) had no effect on normal EVT cell migration or invasiveness. Thus, VEGF may provide a dual role in angiogenesis and EVT cell proliferation during normal placental development.