Involvement of PPARγ in Human Trophoblast Invasion

The peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor superfamily that controls the expression of a large array of genes in a ligand-dependent manner. In the human placenta, PPARγ is specifically expressed in the villous cytotrophoblast and syncytiotrophoblast as well as in the extravillous cytotrophoblastic cells (EVCT) along their invasive pathway. The present study used two cellular models, primary cultures of trophoblastic cells differentiated in vitro in extravillous trophoblastic cells and a cell line (HIPEC65), which was established from a primary culture of EVCT transformed by T-SV40. We observed that natural (15d-PGJ₂) or synthetic ligands of PPARγ (rosiglitazone) inhibit cell invasion in a concentration-dependent manner, with no effect on cell proliferation. This is associated with a modulation of the expression of trophoblastic genes described to be directly involved in the control of EVCT invasiveness, such as GH-V (−20%), TGFβ2 (−30%), PAPP-A (−60%) and IL1β (+300%.). In order to identify PPARγ potential ligands at the fetomaternal interface, we purified LDL (low density lipoprotein) from human sera and oxidized them in vitro in the presence of copper. OxLDL inhibit in vitro extravillous trophoblast cell invasion, whereas native LDL have no effect. In situ OxLDL and their LOX-1 receptor, as well as PPARγ are immunodetected in trophoblasts at the maternofetal interface.     

Low Molecular Mass Polypeptide-2 in Human Trophoblast: Over-Expression in Hydatidiform Moles and Possible Role in Trophoblast Cell Invasion

Embryo implantation involves invasion of placental extravillous trophoblast cell (EVTs) into the uterus. Hyperactive EVT invasion occurs in hydatidiform moles and choriocarcinomas. We have previously demonstrated that the 20S proteasome is involved in mouse embryo implantation and its action is mediated via regulating the expression and activities of matrix metalloproteinase (MMP)-2 and MMP-9 in the EVTs. Our objective was to investigate whether low molecular mass polypeptide-2 (LMP2), a beta subunit of the 20S proteasome, is involved in the regulation of human trophoblast invasion. Normal human placentas or placentas from hydatidiform mole patients were collected and the expression of LMP2 in different cell types including trophoblastic column (TC), cytotrophoblast cells (CTB) and syncytiotrophoblast (STB) under different pathological states were studied by immunohistochemical analysis. Furthermore, the effect of LMP2 or proteasome on cell invasion was measured by using RNAi and inhibitors in a Matrigel invasion assay system in HTR-8/SVneo cells, a human invasive extravillous trophoblast cell line. Changes in the invasion-related molecules including MMP-2 and MMP-9 were also examined by using real time PCR and gelatin zymography. We demonstrated that the expression of LMP2 in TC of partial hydatidiform mole and complete hydatidiform mole, is higher than that in TC of normal human placentas. Besides, LMP2 knockdown significantly attenuated IL-1β-induced cell invasion in vitro, a response readily induced by proteasome inhibitors. In summary, over-expression of the 20S proteasome β-subunit LMP2 in trophoblast cells of hydatidiform moles may contribute to its highly invasive phenotype.     

Effects of human chorionic gonadotropin on trophoblast invasion

Recent studies have shown that different populations of trophoblastic cells of first-trimester human placenta express human chorionic gonadotropin/luteinizing hormone (hCG/LH) receptors. Among these trophoblastic cells, extravillous cytotrophoblasts (CTBs) not only express hCG/LH receptors but also present an invasive phenotype. CTB invasion is instrumental in blastocyst implantation and later in placentation and is tightly regulated in both time and space. This article describes some of the regulators of CTB invasion and summarizes and discusses the potential autocrine/paracrine role of hCG in trophoblast invasion.     

Expression pattern of the cell cycle promoter cyclin e in benign extravillous trophoblast and gestational trophoblastic lesions: correlation with expression of Ki-67.

In this study, a specific monoclonal antibody was used to immunohistochemically investigate correlated expression of the cell cycle promoter cyclin E and the proliferation marker Ki-67 in benign extravillous trophoblast and gestational trophoblastic lesions. Our data show that cyclin E is expressed in the normal extravillous trophoblast, with strongest levels of expression in the cell columns of anchoring villi. Differences could be observed in expression of Ki-67 in both normal extravillous trophoblast and gestational trophoblastic lesions. In the extravillous trophoblast of the cell columns, expression of cyclin E started more distal compared with Ki-67 and was maintained (with less intensity) into the deeper layers of interstitial trophoblast. In the benign trophoblastic lesions (exaggerated placental site [EPS] and placental site nodule [PSN]) and in the trophoblast proliferations on the surface of hydropic villi of hydatidiform moles (HM), the percentage of cells expressing cyclin E was higher than of those expressing Ki-67. The same observation could be made for a case of placental site trophoblastic tumor (PSTT). In contrast, choriocarcinomas (N=8), which are definitely malignant tumors, showed an opposite pattern, with a much higher percentage of strongly Ki-67-positive cells compared with cyclin E-positive cells. We conclude that cyclin E is expressed in benign extravillous trophoblast and gestational trophoblastic lesions, where a ratio cyclin E/Ki-67<1 characterizes choriocarcinomas, whereas PSTT and the benign lesions (HM, EPS, PSN) show expression of cyclin E in a higher percentage of cells than Ki-67 (cyclin E/Ki-67 ratio >1).     

Oxygen tension directs the differentiation pathway of human cytotrophoblast cells

During placental development, human cytotrophoblast cells can differentiate to either villous syncytiotrophoblast cells or invasive extravillous trophoblast cells. We hypothesize that oxygen tension plays a critical role in determining the pathway of cytotrophoblast differentiation. A highly purified preparation of cytotrophoblast cells from human third trimester placenta was cultured for 5 days in either 20% or 1% oxygen tension. The cells incubated at 20% oxygen formed a syncytium as determined by immunohistochemistry using an anti-desmosomal protein antibody that identifies cell membranes. In addition, the mRNA was markedly induced for syncytin, a glycoprotein shown to be essential for syncytiotrophoblast formation, and for human placental lactogen (hPL), which is a specific marker for syncytiotrophoblast cells. In contrast, the cell incubated at 1% oxygen tension did not fuse by morphologic analysis and did not express syncytin or hPL mRNA. However, these cells expressed abundant amounts of HLA-G, a specific marker for extravillous trophoblast cells, which was not seen in cells incubated at 20% oxygen tension. These results suggest that low oxygen tension directs differentiation along the extravillous trophoblast cell pathway while greater oxygen tension directs differentiation along the villous trophoblast cell pathway.     

Villous culture of first trimester human placenta--model to study extravillous trophoblast (EVT) differentiation.

During implantation and subsequent placentation the human extravillous trophoblast (EVT) cells invade the endometrium and maternal vasculature within the uterus. The origin of the EVT and signals triggering its differentiation, migration and invasion are poorly understood. First and second trimester human chorionic villi explants were used as a source of EVT and a variety of substrates which resemble extracellular matrix (ECM) in vivo have been tested to induce EVT differentiation and migration. The obtained results demonstrate that villous explants from both 5-7 and 8-10 weeks of gestation give rise to EVT cells in vitro if maintained on the surface of Matrigel or decidual extract supplemented collagen gel. Fetal calf serum (FCS) supplemented media was essential for EVT differentiation and villous trophoblast viability. Immunostaining of both EVT cells and cells from the cytotrophoblastic column with monoclonal antibody Ki67 (cell proliferation marker) indicate that EVT cells differentiate in vitro by proliferation from the tip of anchoring villi. These mononucleated, round-shaped, migrating cells are HLA-A,B,C class I antigen (W6/32) antibody and low molecular weight cytokeratin positive, and do not immunostain with PAI-1 (plasminogen activator inhibitor) and HPL antibodies. Differentiation of EVT was restricted to first trimester villous tissue; explants from second trimester placentae did not give rise to EVT. Tissue viability as monitored by glucose utilization, lactate, progesterone and hCG production rates correlated with EVT differentiation. The production rates for hCG demonstrated significant variation among individual placentae and was maintained constant for 10 days consistently only in explants cultured on decidual extract supplemented collagen matrix. The described villous tissue culture system may be, therefore, a unique in vitro model to study proliferation and differentiation of EVT from cytotrophoblastic columns, the regulation of EVT proliferation and differentiation, the role of ECM in the induction of the migration and the interaction of extravillous and villous trophoblast at the level of the cytotrophoblastic column.     

Osteopontin expression in gestational trophoblastic diseases: correlation with expression of the adhesion molecule, CEACAM1.

The human placenta is a complex tissue with multiple endocrine and nutritional functions and a unique capacity for rapid proliferation but tightly controlled invasion, differentiating it from malignant tumors. Osteopontin (OPN) is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. OPN also could be implicated in regulating implantation and placentation by promoting cellular migration and invasion in a placenta-specific fashion. We could demonstrate the expression pattern of OPN in the normal human placenta in which it is localized in the extravillous (intermediate) trophoblast and the villous cytotrophoblast. CEACAM1 is an adhesion molecule, which we have recently found to be expressed at the maternal-fetal interface of the normal placenta with a localization to the extravillous (invasive) trophoblast and in gestational trophoblastic disease (GTD) and also to be potentially implicated in trophoblast invasion and tumorigenesis. Both OPN and CEACAM1 have been shown to interact with integrin beta3. The purpose of this study was to investigate the expression pattern of OPN in GTD and to correlate it with the expression of CEACAM1. To analyze the expression of OPN, we performed immunohistochemistry on a total of 27 cases of GTD, including 21 hydatidiform moles and 6 choriocarcinomas, which had previously been characterized with respect to their CEACAM1 expression. Hydatidiform moles showed a positivity for OPN in villous cytotrophoblast and in the trophoblast proliferations on the villous surface. The strongest OPN expression could be observed in the choriocarcinomas with a heterogenous OPN expression pattern. CEACAM1 had shown similar results and was found to be expressed in choriocarcinoma. The expression pattern of osteopontin in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD (possibly as a functional complex with CEACAM1 and integrin beta3) and might be useful as an additional diagnostic marker for such lesions.     

The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function

Introduction

Trophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT).

Methods and results

Immunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast.

Discussion and conclusion

This is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4.     

Expression of endothelial nitric oxide synthase by extravillous trophoblast cells in the human placenta

Previous reports have documented the expression of endothelial nitric oxide synthase (eNOS) expression by the syncytiotrophoblast layer of the villus in the human placenta. In contrast, the underlying villous cytotrophoblast cells do not express the enzyme. Because extravillous cytotrophoblasts have not been as extensively investigated, our objective was to test whether these cells express eNOS. Using both a mouse monoclonal and a rabbit polyclonal antibody, we demonstrated immunoreactive eNOS in trophoblast cell columns emanating from anchoring villi in second trimester placentae. Cytokeratin positive trophoblast cells lying beneath remnant anchoring villi, lining decidual blood vessels and scattered throughout the basal plate of normal term and pre-eclamptic placentae also expressed immunoreactive eNOS. By Western analysis, the monoclonal and polyclonal antibodies were shown to be absolutely and relatively specific for eNOS, respectively. The finding of immunoreactive eNOS expression by extravillous trophoblast cells was substantiated by in situ hybridization. Using riboprobes generated from a bovine eNOS cDNA, we demonstrated specific hybridization in the endothelium of blood vessels in the umbilical cord, thus validating the in situ hybridization methodology, as well as specific hybridization in the extravillous trophoblast cells of the basal plate in normal term placenta. In conclusion, several different populations of extravillous trophoblast cells in the basal plate of the human placenta express eNOS.     

The Role of E-cadherin in the Motility and Invasion of Implantation Site Intermediate Trophoblast

During early pregnancy, intermediate (extravillous) trophoblast infiltrates the basal plate and invades the spiral arteries, a physiological process required to establish the maternal-fetal circulation. Immunostaining studies have shown that differentiation of trophoblast into this invasive subpopulation is associated with down-regulation of E-cadherin expression. To study the function of E-cadherin in trophoblast in vitro, we restored E-cadherin expression in an E-cadherin negative human implantation site intermediate trophoblastic cell line, IST-1, using a recombinant adenovirus, E-cad/Ad5 which constitutively expresses E-cadherin. In contrast to the control IST-1 cells which were individual and pleomorphic in shape, E-cad/Ad5 transduced cells were cohesive, uniform, and round. The motility and invasiveness of E-cad/Ad5 transduced IST-1 cells, as compared with the control cells, was significantly reduced. These effects were contact-dependent and were attenuated by a function-perturbing anti-E-cadherin antibody. In conclusion, our results indicate that expression of E-cadherin in IST-1 cells results in a contact-mediated inhibition of motility and invasion and suggest an important role for E-cadherin down-regulation in the intermediate trophoblast during implantation.     

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

OBJECTIVES: To study collagen phagocytosis by human extravillous trophoblast. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance

Antigen expression by villous and extravillous human trophoblast populations at discrete anatomical sites has been reviewed. The various different antigenic phenotypes have been highlighted using a panel of monoclonal antibodies reactive with characteristic trophoblast membrane antigens, a trophoblast-leucocyte common antigen, class I MHC antigens, epithelial cell cytokeratin and epithelial membrane markers. This approach has allowed three separate fetal trophoblast populations to be identified within term amniochorionic membranes, and also has facilitated further definition of trophoblast populations in maternal uterine tissues. Furthermore, antigenic alterations have been noted in the maternal uterine gland epithelium in pregnancy leading to the expression of a trophoblastic phenotype, thereby suggesting a mechanism of extrinsic regulation of gene expression in these tissues. The possible involvement in the immunoregulatory control of maternal responses in pregnancy of MHC-linked gene products expressed by trophoblast has been discussed.     

Expression,Regulation and Functional Characterization of Matrix Metalloproteinase-3 of Human Trophoblast

MMP-3 has been detected in human placenta and reduced expression of the enzyme was observed in invasive trophoblasts of patients with severe preeclampsia. However, detailed expression pattern, regulation and biological properties of the placental protease have not been elucidated so far. RT-PCR analyses, Western blotting and enzyme activity assays revealed that pro- and active form of MMP-3 were predominantly expressed in purified first trimester villous trophoblasts, in invasive cytotrophoblasts of differentiating explant cultures and in trophoblastic SGHPL-4 cells. Accordingly, immunofluorescene of first trimester placental tissues detected MMP-3 mainly in villous and extravillous cytotrophoblasts. IL-1β, an inducer of MMP-3 in decidual cells, increased secretion and activity of the protease in trophoblast supernatants in a dose- and time-dependent manner. IL-1β-stimulated production of the enzyme was suppressed in the presence of inhibitors of MAPK and AKT signalling. Similar to recombinant MMP-3, MMP-3 in supernatants of IL-1β-stimulated decidual stromal or SGHPL-4 cells degraded IGFBP-1 in vitro resulting in the appearance of cleavage products at approximately 25, 22, 17, 14 and 11 kD. However, cleavage assays using recombinant MMP-2 suggested that the gelatinase may contribute to IGFBP-1 degradation in trophoblast supernatants. Despite its effects on MMP-3 expression IL-1β failed to significantly alter invasion of SGHPL-4 cells through Matrigel-coated transwells. In conclusion, the data suggest that invasive trophoblast cell models secrete bioactive MMP-3. Inducible expression of the protease involves MAPK and AKT signalling. In addition to the decidua, MMP-3 of trophoblasts may contribute to the regulation of the IGF system by degrading IGFBP-1.