Experimental modulation of cell-cell adhesion,invasiveness and differentiation in trophoblast cells

The establishment of pregnancy in the human decisively depends on the competence of the early trophoblast to interact during implantation with (1). the uterine epithelium and subsequently (2). with the endometrial stroma and blood vessels. In the interaction with uterine epithelium cell-to-cell adhesion appears to be a critical element, involving initially (and astonishingly) apical cell poles of both epithelia. The subsequent invasion of the stroma includes both adhesive interactions with and degradation of extracellular matrix. How these different processes are regulated in detail remains largely unknown. While the invasiveness of the trophoblast is known to be regulated in local and temporal terms it has remained unclear so far whether trophoblast adhesiveness to cells and/or matrix is subject to a coupled regulation or whether both properties involve different, maybe sequentially effective, control mechanisms. It is also not known how the regulation of these activities is related to the differentiation pathways leading to the formation of noninvasive villous trophoblast serving endocrine as well as nutritive functions. This communication reviews experiments using normal cytotrophoblast cells isolated from first trimester or term placentae as well as malignant trophoblast (choriocarcinoma) cells treated with a panel of compounds known to modulate cell differentiation [retinoic acid, methotrexate, dibutyryl-cAMP, phorbol-(12-myristoyl-13-acetyl)-diester]. Parameters indicative of trophoblast differentiation [in particular chorionic gonadotrophin (hCG) secretion] as well as adhesion to uterine epithelial cells and invasion into extracellular matrix in vitro were monitored. While expression of differentiation parameters was increased by all drug treatments, adhesion to uterine epithelial cells in vitro was reduced. Modulation of invasiveness, however, followed a different pattern: while it was reduced in normal trophoblast cells it was even increased in choriocarcinoma cells with various substances. The response of cells with respect to production of extracellular matrix proteins or matrix-degrading proteinases showed a complex pattern that again lacked a stringent correlation with hCG production and adhesion, and in addition also with invasive behavior. These results suggest that adhesiveness of trophoblast to uterine epithelial cells and invasiveness into the uterine stroma (extracellular matrix) are subject to different control mechanisms. They support the view that trophoblast-endometrium interactions involve a cascade of various adhesion and migration processes whose cellular and molecular basis is complex but accessible to experimental investigation using a variety of available in vitro systems.     

Cytogenetics of trophoblasts from complete hydatidiform moles

The risk of developing choriocarcinoma following a complete hydatidiform mole (CHM) is 2000-4000 times greater than the risk following a normal pregnancy. To understand more fully the increased susceptibility of the molar trophoblast to malignant transformation, we separated the trophoblastic cells from the stromal cells in 14 complete moles and cultured them for cytogenetic analysis. The numerical and structural abnormalities found were compared with those found in the trophoblasts from normal pregnancy and malignant choriocarcinoma cell lines. The percentage of polyploid cells was 2.8 times greater in molar trophoblasts than in normal trophoblasts. Although we found no consistent chromosomal abnormality in the molar trophoblasts, these cells were significantly more vulnerable to chromosomal breakage than the molar fibroblasts, normal trophoblasts, normal fibroblasts, and maternal decidual cells. Out of a total of 103 breakpoints observed in 338 cells, 42 coincided with known fragile sites, 18 with the location of protooncogenes, 27 with breakpoints reported in other neoplasia, and 18 with breakpoints found in four choriocarcinoma cell lines. The chromosomes in choriocarcinoma cell lines have hypotetraploid mode and many structural rearrangements. Our results suggest that the genetic instability found in the molar trophoblasts may be responsible for progressive karyotypic changes and greater susceptibility to malignant transformation.     

hCMV induced IL-6 release in trophoblast and trophoblast like cells.

BACKGROUND: Human cytomegalovirus (hCMV) infection of the trophoblasts is a crucial event in virus transmission from mother to child, being one responsible factor for intrauterine infection of the unborn. Differences of virus replication in trophoblasts depending on time point of pregnancy and degree of differentiation of trophoblasts might influence this transmission. Furthermore, immunological reactions of the trophoblasts to hCMV infection might be important defence mechanisms too. OBJECTIVES: hCMV replication and interleukin-6 release in trophoblasts and trophoblast like cells (choriocarcinoma cells) was investigated. STUDY DESIGN: Trophoblasts from term and 1st trimester placentas were isolated and infected with hCMV. hCMV production and release to the supernatant as well as interleukin-6 release and interleukin-6 mRNA production by these infected cells was measured. Choriocarcinoma cell lines (JEG-3, JAR) were treated the same. Non-infected trophoblasts were used as controls. RESULTS: In 1st trimester trophoblast, term trophoblasts and JEG-3 permissive hCMV replication was observed, although with different kinetics and efficiency. In JAR no complete virus replication was seen. High levels of interleukin-6 were measured in the supernatants of all hCMV infected cells immediately after infection. IL-6 mRNA upregulation was seen 48 h after infection in those cell types replication of hCMV occurred (1st trimester trophoblasts, term trophoblasts, JEG-3). At that time-point hCMV immediate early proteins appeared. In JARs no virus production and no IL-6 mRNA upregulation was seen, and IL-6 levels in the supernatant of these hCMV infected cells declined significantly until day 6 after infection compared to mock infected cells. CONCLUSION: These observations show that hCMV replication is influenced by the degree of trophoblast differentiation. Interleukin-6 is upregulated by hCMV infection, but is independent of complete virus replication.     

Platelet-endothelial cell adhesion molecule-1 is expressed by a subpopulation of human trophoblasts: a possible mechanism for trophoblast-endothelial interaction during haemochorial placentation

The terminal event in the establishment of the haemochorial placenta in the human is the invasion of trophoblasts into the maternal vessels, a process in which trophoblasts interact directly with the vascular endothelium and degrade the vascular basement membrane and the tunica elastica of the vessels. To further understand this heterotypic cellular interaction, we investigated the expression by human trophoblasts of the vascular cell adhesion molecule platelet- endothelial cell adhesion molecule-1 (PECAM-1) as a possible mediator of the adhesive interaction between trophoblasts and endothelium. In vitro, human trophoblasts were found to express PECAM-1 mRNA and protein. Indirect immunofluorescence indicated a diffuse staining pattern, which was most intense in a subpopulation of trophoblast cells. Co-incubation of trophoblasts with endothelial cells showed interaction between these two cell types with strong expression of PECAM-1 at points of trophoblast-endothelial cell contact, suggesting that this cell adhesion molecule participates in this heterotypic cell interaction. Immunohistochemical localization of PECAM-1 in chorionic villi and first trimester implantation sites showed that, in vivo, only extravillous interstitial and endovascular trophoblasts were positive. In first trimester placentae, villous trophoblast and extravillous trophoblast in other locations than around or within the decidual vessels did not express this molecule. In term placentae, villous trophoblast did not express these adhesion molecules except for two specimens examined. This study demonstrates that PECAM-1 is expressed by a subset of human trophoblasts in vitro and in vivo. Its tissue localization suggests that PECAM-1 is important in mediating the adhesive interaction between trophoblasts and maternal vascular endothelium during the process of haemochorial placentation. Regulation of PECAM-1 expression by human trophoblasts may play a critical role in normal and abnormal vascular invasion during implantation and placentation.      

人绒毛膜促性腺激素对JEG-3细胞表达VEGF的影响

目的: 揭示人绒毛膜促性腺激素(hCG)对滋养层细胞表达血管内皮生长因子(VEGF)的影响。方法: 以滋养层细胞来源的JEG-3细胞系为研究对象,采用半定量逆转录多聚酶链反应(RT-PCR)方法检测VEGFmRNA的表达。结果: 分别用(0、50、500、5000、25000)U/LhCG处理48h后,JEG-3细胞中VEGF的表达被诱导增高,且包括VEGF₁₂₁、VEGF₁₆₅、VEGF₁₈₉在内的VEGF亚型均受到诱导。另外,观察VEGF在25000U/LhCG处理的JEG-3细胞中表达的时间变化,结果发现VEGF在JEG-3细胞中的表达在经过短暂的诱导后略降低。结论: hCG可能通过调节VEGF在滋养层细胞或滋养层细胞来源的绒癌细胞中的表达,进而影响细胞的增殖、迁移或浸润转移。     

Evaluation of Cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry

Villous trophoblast cells (TC) obtained from first trimester and term human placentae after trypsin/Percoll gradient isolation were immunodepleted of contaminant cells. The level of purity was assessed by the intracellular expression of the pan trophoblast marker cytokeratin-7 (CK7) and comparisons were made with the GB25 trophoblast-specific (cytotrophoblast+syncytiotrophoblast) cell surface marker. The presence of contaminating cells was traced with intracellular vimentin, or cell surface CD2, CD36, and CD163 markers and evaluated by flow cytometric analysis. The pattern of CK7 expression by trophoblast cells was also analyzed by immunofluorescence microscopy. Most batches of TC from first trimester or term placentae (92+/-3% and 96+/-2%, respectively) showed a high percentage of CK7 expressing cells, with less than 2% contaminating vimentin positive cells. In some batches of TC with a lower percentage (65+/-4%) of CK7-expressing cells, no vimentin was found, but a low percentage of CD36-expressing cells was evidenced, with no presence of CD2, and/or CD163-expressing cells. The intracellular CK7 signal correlated significantly with that of GB25 (p<0.05) cell surface expression in TC of term placentae. The choriocarcinoma BeWo and Jar cell lines also showed high levels (>92%) of CK7-expressing cells. Conversely, the control U87 astrocytoma cell line showed a high percentage (>90%) of vimentin but no CK7-expressing cells. These results provide evidence that the mutually exclusive pattern of intracellular CK7/vimentin expression of human TC can be used for evaluation by flow cytometry of the purity of primary human trophoblast cells.     

人滋养层细胞侵袭力分子调节机制研究

胎盘是妊娠过程中形成的暂时性的特异器官,它在母体和胎儿间起着重要的桥梁作用。滋养层细胞具有类似于肿瘤细胞迁移和浸润的能力,作为胎盘组织的主要组成细胞之一,在胚胎植入、胎盘的形成和发育等许多生理过程中发挥着重要的作用,但同时也是多种毒素和病原微生物入侵时的靶细胞。滋养细胞侵入过度将导致绒毛膜癌等疾病的发生,浸润不足则可能造成流产、子痫前期等妊娠期疾病。目前,诸多研究表明在胎盘形成过程中,有许多分子和信号通路参与对其滋养层细胞的调控,但滋养层细胞迁移与浸润的具体机制尚不完全明确。本文对人滋养层细胞侵袭力分子调节机制进行了系统论述,旨在为研究病理性妊娠的发生、发展过程提供参考。     

Efficacy of a recombinant chimeric anti-hCG antibody to prevent human cytotrophoblasts fusion and block progesterone synthesis

PROBLEM: A recombinant chimeric antibody against hCG (cPIPP) has been engineered and expressed at high yield in plants. The purpose of this work was to enquire whether this antibody is competent to neutralize the bioactivity of hCG on human trophoblasts. METHODS: Cytotrophoblast cells, isolated from term placentae were maintained in culture for 3 days in presence or absence of humanized chimeric anti-hCG antibodies. Progesterone secreted was quantitated by ELISA. Fusion and cyto-architecture of the cells was studied by light and electron microscopy. Modulation of E-cadherin was investigated using RT-PCR and immunocytochemistry. RESULTS: Recombinant chimeric anti-hCG antibody blocked the synthesis of progesterone by trophoblasts. No fusion of cytotrophoblasts to form syncytium took place. E-cadherin, a vital cell adhesion molecule involved in cell-to-cell interaction did not show differentiation related decline in its expression in presence of the antibody. CONCLUSION: Recombinant chimeric anti-hCG antibody (cPIPP) was effective to neutralize hCG induced bioactivities in the human derived trophoblast cells.     

Expression of Epstein-Barr Virus-Induced Gene 3, an Interleukin-12 p40-Related Molecule, throughout Human Pregnancy : Involvement of Syncytiotrophoblasts and Extravillous Trophoblasts

In human pregnancy, trophoblasts are the only cells of fetal origin in direct contact with the maternal immune system: syncytiotrophoblasts are in contact with maternal blood, whereas extravillous trophoblasts are in contact with numerous maternal uterine natural killer (NK) cells. Therefore, trophoblasts are thought to play a key role in maternal tolerance to the semiallogeneic fetus, in part through cytokine production and NK cell interaction. Epstein-Barr virus-induced gene 3 (EBI3) encodes a soluble hematopoietin receptor related to the p40 subunit of interleukin-12. Previous studies indicated that EBI3 is expressed in the spleen and tonsils, and at high levels in full-term placenta. To investigate further EBI3 expression throughout human pregnancy, we generated monoclonal antibodies specific for EBI3 and developed an EBI3 enzyme-linked immunosorbent assay. Immunohistochemical experiments with EBI3 monoclonal antibody on first-, second-, and third-trimester placental tissues demonstrated that EBI3 was expressed throughout pregnancy by syncytiotrophoblasts and extravillous trophoblasts (cytotrophoblast cell columns, interstitial trophoblasts, multinucleated giant cells, and trophoblasts of the chorion laeve). EBI3 expression was also induced during in vitro differentiation of trophoblast cell lines. In addition, large amounts of secreted EBI3 were detected in explant cultures from first-trimester and term placentae. Consistent with these data, EBI3 levels were strongly up-regulated in sera from pregnant women and gradually increased with gestational age. These data, together with the finding that EBI3 peptide is presented by HLA-G, suggest that EBI3 is an important immunomodulator in the fetal-maternal relationship, possibly involved in NK cell regulation.     

Osteopontin facilitates invasion in human trophoblastic cells via promoting matrix metalloproteinase-9 in vitro

Successful implantation of embryo and placentation depend on proper trophoblast proliferation and differentiated into specialized invasive trophoblast. However, little is known about the regulatory factors and mechanisms in trophoblast proliferation and differentiation. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. It has been identified that OPN is highly expressed in invasive trophoblasts in human placenta. In this study, we demonstrated that OPN is constitutively expressed in highly invasive phenotype of human choriocarcinoma cell lines of JAR and JEG-3 cells, and OPN could promote trophoblast proliferation and invasion, partly through promoting MMP-9 secretion. Inhibition of OPN will compromise the abilities of proliferation and invasion in JAR and JEG-3 cell lines. Our data showed that the expression of OPN in trophoblast may participate in placentation, OPN expression defects may be involved in gestational trophoblastic diseases.     

CXCL12 controls over-invasion of trophoblasts via upregulating CD82 expression in DSCs at maternal-fetal interface of human early pregnancy in a paracrine manner

Tetraspanin CD82 has been identified as a potential contributor to controlling trophoblast invasiveness in human first-trimester pregnancy. However, it is unclear how the regulation of CD82 expression at maternal-fetal interface. The present study is to investigate the effect of the trophoblast-derived CXCL12 on CD82 expression in decidual stromal cells (DSCs) that in turn controls trophoblast cell invasiveness. In-cell Western was used to evaluate the expression of CD82 in DSCs. A co-culture model was established to investigate the reciprocal interaction between trophoblasts and DSCs via CXCL12/CXCR4 and CD82 expression. We found that both anti-CXCL12 and anti-CXCR4 neutralizing antibody can eliminate increase of CD82 expression in DSCs induced by the trophoblasts supernatant. Moreover, the invasiveness of trophoblasts pre-treated with anti-CXCR4 neutralizing antibody was significantly decreased. Interestingly, when DSCs were pre-treated with anti-CXCR4 neutralizing antibody, the trophoblasts invasiveness in the co-culture was enhanced, and thus anti-CXCR4 neutralizing antibody can reverse the decrease of trophoblasts invasiveness induced by CD82. The trophoblast cell-derived CXCL12 does not only increase the invasiveness in an autocrine manner, but also control the over-invasion of trophoblasts through promoting CD82 expression in DSCs in a paracrine manner, which maintains a physiological balance of human trophoblasts invasiveness via the cross-talk between trophoblasts and DSCs.     

Extracellular pH modulates the secretion of fibronectin isoforms by human trophoblast

Differentiation of human trophoblast from the proliferative to the invasive phenotype takes place in a hypoxic and thus likely an acidic microenvironment. During differentiation, the secretion pattern of fibronectin isoforms changes. Therefore, we analysed the relation between extracellular pH, secretion of fibronectin splice variants and invasiveness. By means of immunohistochemistry and biochemistry, cellular non-oncofetal fibronectins were found in placental stroma and around extravillous trophoblast, whereas oncofetal isoforms only marked the extracellular matrix of extravillous trophoblast. In vitro, mesenchymal cells produced non-oncofetal fibronectins only, whereas choriocarcinoma cell lines, extravillous trophoblast and choriocarcinoma/trophoblast hybrid cells secreted both non-oncofetal and oncofetal isoforms. When the pH of the culture medium was either lowered or increased (between 6.0 and 8.0), the trophoblast hybrids, but not choriocarcinoma and mesenchymal cells, responded with increased secretion of fibronectins and a shift towards oncofetal isoforms. These changes were preserved after pH normalisation. Histochemical determination of local tissue acidity revealed that the site of the lowest detectable tissue pK coincided with the starting point of invasion, the proximal part of trophoblastic cell columns. Therefore, it is concluded that the local pH plays an important role as regulator of differentiation of human trophoblast as reflected by the synthesis of oncofetal fibronectins by the invasive phenotype of extravillous trophoblast.     

Production of interferons in human placental trophoblast subpopulations and their possible roles in pregnancy.

The human cytotrophoblasts are the first fetal cells to arise during embryogenesis and are the progenitor cells to villous (noninvasive), syncytiotrophoblast (noninvasive), "intermediate" extravillous (invasive), and "anchoring" extravillous (invasive) trophoblast subpopulations. These trophoblast subpopulations were isolated from first- and third-trimester placentae and were stimulated with Sendai virus, granulocyte-macrophage colony-stimulating factors (GM-CSF), and platelet-derived growth factor (PDGF) to produce interferons (IFNs). GM-CSF and PDGF induced very low levels of IFN in first-trimester extravillous and villous trophoblast subpopulations. Highly proliferating and invasive intermediate extravillous trophoblast cultures produced five- to eightfold more IFNs than villous trophoblast cultures and two- to fivefold more IFN than the syncytiotrophoblast cultures when stimulated with Sendai virus. Syncytiotrophoblast cultures produced higher levels of IFNs (up to twofold) than villous trophoblast cultures when stimulated with the same virus. Pretreatment of first-trimester extravillous and villous trophoblast cultures with GM-CSF and PDGF followed by infection with Sendai virus resulted in greater IFN production than when the cultures were stimulated with virus alone. The levels of IFN produced were dependent on the type of trophoblast, the type of inducer, and the stage of differentiation of the trophoblasts. The purified trophoblast IFNs have potent antiviral activities when assayed on human amniotic WISH cells, and they inhibited proliferation of normal trophoblasts and trophoblast-derived malignant cells in vitro without any toxicity. Furthermore, the trophoblast IFNs activated NK cell activity and suppressed mitogen-stimulated lymphocyte proliferation at concentrations of between 10 and 1,000 IU/ml. The possible functions of the trophoblast IFNs during pregnancy are discussed with respect to human placental and fetal protection and development.     

Expression of placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in human normal and malignant invasive trophoblastic cells

We recently identified two novel aminopeptidases, placental leucine aminopeptidase (P-LAP) and adipocyte-derived leucine aminopeptidase (A-LAP). Enzymatically, P-LAP degrades oxytocin, vasopressin, and angiotensin III, while A-LAP degrades angiotensin II and kallidin. In this study we investigated the expression and localization of P-LAP and A-LAP in human trophoblastic cells in the normal placenta (n = 26), gestational choriocarcinoma (n = 8), and placental site trophoblastic tumor (n = 3). On immunoblot analysis both P-LAP and A-LAP proteins were detected in normal placenta and five choriocarcinoma tissues, as well as in two choriocarcinoma cell lines. Immunohistochemical staining of normal placental tissues demonstrated that P-LAP was not only localized in villous syncytiotrophoblasts but also highly expressed in extravillous trophoblasts (EVTs) invading the decidua or maternal spiral arteries. The expression level of P-LAP on these invasive EVTs reached a maximum during the late first to second trimesters of pregnancy, and it decreased in the third trimester. Similarly, A-LAP was strongly expressed in EVTs invading the decidua or spiral arteries in the second trimester of pregnancy, while it was weakly or moderately expressed in villous cytotrophoblasts or EVTs located in the cell columns. These two aminopeptidases were more strongly expressed in all eight choriocarcinomas and three placental site trophoblastic tumors and mainly localized to the intermediate-type trophoblastic tumor cells invading the uterine myometrium or stromal vessels. In summary P-LAP and A-LAP were predominantly expressed in the invasive phenotype of EVTs during placentation, as well as in the invasive tumor cells of trophoblastic neoplasms. These results suggest the involvement of these aminopeptidases in invasiveness of both normal and malignant intermediate-type trophoblasts possibly through degradation of specific peptide substrates.