Invasion Of Extravillous Trophoblast Cells Research Articles

Extracellular matrix components of the placental extravillous trophoblast: immunocytochemistry and ultrastructural distribution.

Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, "i". Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen "i" was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with "i" in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus.

TGFβ regulation of trophoblast function

Placenta growth factor (PIGF) is a homodimeric glycoprotein, 46–50 kDa in size, belonging to the vascular endothelial growth factor (VEGF) sub-family. It exists as two isoforms, PIGF-1 and -2, the latter having a heparin-binding domain. Like VEGF, it is a potent angiogenic factor; however, PIGF homodimers interact with the VEGF receptor Flt-1 (fms-like tyrosine kinase), but not with the kinase domain-containing region (KDR). Since PIGF is made by the human placenta and extravillous trophoblast (EVT) cells of the human placenta express Flt-1 in situ, these cells may be responsive to PIGF. Therefore, this study examined whether first trimester EVT cells propagated in vitro expressed the mRNA or the protein of Flt-1 and PIGF, and whether exogenous PIGF-1 had any effect on EVT cell proliferation, migration or invasiveness. Immunocytochemical and RT-PCR analyses revealed that both normal and SV40 Tag-immortalized EVT cells expressed the protein and mRNA for Flt-1, but not for P1GF-1 or -2. Exogenous PIGF-1 stimulated proliferation (measured by 3H-thymidine uptake) of normal EVT cells in a concentration-dependent manner, but only in the presence of excess heparan sulphate proteoglycans (HSPGs). These results raise two possibilities: that exogenous PIGF-1 (in spite of having a low affinity for heparin) was sequestered away from its receptor because of binding to heparan sulphate proteoglycans on the EVT cell surface or the ECM, or that HSPGs could modify the interaction between Flt-1 and PIGF. PIGF-1, in the presence or absence of HSPGs, however, had no effect on EVT migration or invasiveness, when measured with a transwell invasion (in the presence of Matrigel™) or migration (in the absence of Matrigel™) assay. These findings place PIGF amongst a large group of growth factors that promote EVT cell proliferation without influencing their migratory or invasive behaviours, and suggest that PIGF-Flt-1 interactions may be regulated by HSPGs in situ.

Functional Role of Cell Surface Integrins on Human Trophoblast Cell Migration: Regulation by TGF-β, IGF-II, and IGFBP-1

Trophoblast invasion of the human uterus is stringently controlled by the microenvironment. Invasive extravillous trophoblast cells in situ as well as in culture express a selective repertoire of cell surface integrins. Since migration is a necessary step in the invasion cascade, we tested whether certain integrins or invasion-regulating molecules, i.e., TGF-β, IGF-II, and IGFBP-1 produced at the fetomaternal interface had a functional role on trophoblast migration. Flow cytometric analysis of integrin expression and the use of an in vitro cell migration assay revealed that exogenous TGF-β upregulates integrin expression and reduces migratory ability of the invasive trophoblast, whereas IGF-II has no effect on integrin expression but stimulates migration. Trophoblast migration was inhibited in the presence of α5 and β1 integrin blocking antibodies, indicating its dependence on the expression of these subunits. Furthermore, IGFBP-1, which contains an RGD sequence recognizing certain integrins, stimulated migration, an effect that was blocked by pretreatment with anti-α5 or -β1 blocking Abs. These studies demonstrate that the migration of first trimester invasive trophoblast in vitro (1) requires the expression of α5 and β1 integrin subunits, (2) is inhibited by TGF-β, possibly due to increased cell adhesiveness to the extracellular matrix, (3) is stimulated by IGF-II by an as yet undetermined mechanism, and (4) is stimulated by IGFBP-1, likely by interaction with the RGD binding site of the α5β1 integrin. The invasion-regulating effects of TGF-β, IGF-II, and IGFBP-1 may thus, at least in part, be due to their migration-regulating effects on the invasive trophoblast.

Trophoblastic localization and production of immunoreactive endothelin-1 in human placenta

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.

Proliferation and differentiation of human first trimester cytotrophoblast in vitro

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.

Extracellular matrix molecules in the morphogenesis of the human placenta

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.

Isolation and characterization of basic fibroblast growth factor from first trimester trophoblast

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.