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Schools of Surgical and Reproductive Sciences,3
Clinical and Laboratory Sciences,4
Medical Education Development,5 University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cytokines, implantation, placenta, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transforming growth factor betas (TGFBs) are members of a large superfamily of cytokines, including activins, inhibins, and bone morphogenic proteins [6]. The TGF family is composed of three related 25-kDa homodimeric proteins: TGFB1, 2, and 3. The TGFBs exert their biological effects by binding to cell surface receptors, designated types I, II, and III, that signal through the SMAD family of proteins [7]. Several studies have suggested that the different TGFB isoforms may regulate trophoblast invasion [8–10]. We recently localized the three TGFB isoforms in the placenta and placental bed during early gestation [11]. There was extracellular immunoreactivity for TGFB1 in cytotrophoblast columns and islands, around decidual stromal cells, and associated with fibrinoid surrounding both decidual and myometrial portions of transformed spiral arteries, although extravillous trophoblast cells were themselves negative. Similar immunoreactivity was observed for TGFB2 with additional variable intracellular staining in cytotrophoblast islands and decidual stromal cells. While TGFB3 mRNA and protein were detected in placental homogenates by RT-PCR and ELISA, respectively, TGFB3 was not detected using immunohistochemistry in placenta or placental bed. There was no indication of temporal regulation of TGFB1, 2, and 3 [11].
Although the TGFB isoforms, and in particular TGFB1, have been widely studied in association with cancer progression and metastases, there is limited information on their role in trophoblast invasion. Graham et al. [8] demonstrated that TGFB (isotype not specified) could inhibit the invasive capacity of primary isolates of first-trimester extravillous trophoblast cells. In addition, Caniggia et al. [10] reported a decrease in the outgrowth of extravillous trophoblast cells from placental villous explants in the presence of TGFB3. The reduced invasion observed in the presence of TGFB1 by Graham et al. [8] was associated with an increase in tissue inhibitor of matrix metalloprotease (TIMP) 1 levels. TGFB1 has also been shown to reduce cell growth both by decreasing the proliferative rate and by inducing apoptosis [12]. Based on these results, the aim of this study was to determine the role of TGFB1, 2, and 3 in invasion of extravillous trophoblast cells and explore a potential mechanism for biological activity. We therefore hypothesized that the different TGFB isoforms would 1) inhibit extravillous trophoblast invasion in vitro, 2) increase the apoptotic index and/or decrease the proliferative index of trophoblast cells, and 3) decrease levels of active proteases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Placental samples were obtained from women undergoing elective surgical termination of pregnancy at the Royal Victoria Infirmary, Newcastle upon Tyne, UK. The study was approved by the Joint Ethics Committee of Newcastle and North Tyneside Health Authority and the University of Newcastle upon Tyne, and all women gave informed written consent. All samples were obtained at 8–10 wk gestational age, as determined by ultrasound measurement of crown rump length before pregnancy termination. Following uterine evacuation, the placental tissue was immediately suspended in sterile saline and transported to the laboratory, where it was washed two to three times in sterile phosphate-buffered saline to remove excess blood. Chorionic villous tips were dissected, minced to approximately 0.5 mm3, and resuspended in culture medium (DMEM:F12 containing 10% heat-inactivated fetal bovine serum [FBS], penicillin/streptomycin, and amphoteracin B [all from Sigma, Poole, UK]) such that 15 µl of the explant suspension contained approximately 10 mg tissue.
Invasion Assay
Tissue culture well inserts containing a membrane with a pore size of 8 µm (24-well format; Becton Dickinson, Franklin Lakes, NJ) were coated with 10 µl growth factor-reduced Matrigel (Becton Dickinson) as per the manufacturer's instructions. All tissue culture was performed in a standard 37°C 5% CO2 in air incubator. Fifteen microliters of the prepared explants were placed on top of the Matrigel, and 200 µl culture medium were added to the lower chamber and then incubated overnight to allow adherence of the explant to the Matrigel. The next morning (Day 1), a further 600 µl medium were added to the lower chamber and 200 µl to the upper chamber of the insert. At the end of the incubation period, the Matrigel and explant were removed, and the upper side of the membrane was cleaned with a cotton wool bud. For assessment of the number of invaded cells, the filters were stained with hematoxylin and eosin and mounted on glass microscope slides with supermount and DPX. Each slide was blinded, and the total number of cells that had invaded onto the underside of the filter was counted manually by one investigator (H.A.O.) at 100x magnification. In addition, conditioned medium was collected from each chamber and stored at –80°C until required for analysis.
Validation of Invasion Assay
Preliminary experiments were performed to determine the time course of invasion and the phenotype of invaded cells. Explants from four placentas (each in duplicate) were incubated as described and the filters harvested and counted on Day 3, 4, 5, 6, and 7. To assess the phenotype of the invaded cells, filters, were fixed in 100% ethanol for immunohistochemistry (n = 3).
To verify the identity of the invading cells as extravillous trophoblast cells, the undersides of the filters were immunostained for cytokeratin 7 (reactive with villous and extravillous trophoblast) and with a panel of antibodies reactive predominantly with villous (ITGA6 [integrin alpha 6], ITGB3 [integrin beta 3], epidermal growth factor receptor [EGFR]) and extravillous (ITGA1 [integrin alpha 1], HLA-G, ERBB2) trophoblast.
Immunohistochemistry was performed using an avidin-biotin peroxidase method (Vectastain Elite; Vector Laboratories, Peterborough, UK). Details of the primary antibodies are given in Table 1. The reaction was developed with Fast diaminobenzidine (DAB) (Sigma) tablets. Washes between each step were performed in TBS (0.15 M Tris-buffered 0.05 M saline, pH 7.6). Sections were counterstained with Mayer hematoxylin (BDH, Poole, UK) and mounted in DPX synthetic resin (Raymond Lamb, London, UK). Omission of primary antibody or substitution with nonimmune mouse serum for the primary antibody were both included as controls.
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Effect of TGFB Family of Cytokines on Trophoblast Invasion
Two approaches were undertaken to determine the effect of the TGFB family on trophoblast invasion. First, exogenous TGFB1, 2, and 3 were added to determine the direct effects of these cytokines on trophoblast invasion. Second, isotype-specific neutralizing antibodies and cytokines + neutralizing antibodies were used to inhibit the activity of any endogenous TGFB. Exogenous cytokines (TGFB1, 2, 3: 0.05, 0.5, and 5 ng/ml; n = 5), neutralizing antibodies to TGFB1, 2, and 3 (mouse anti-human TGFB1, 10 µg/ml, n = 9; goat anti-human TGFB2, 1 µg/ml, n = 9; goat anti-human TGFB3, 10 µg/ml, n = 10), or cytokine and neutralizing antibody (TGFB1, 5 ng/ml + mouse anti-human TGFB1, 10 µg/ml, n = 7; TGFB2, 5 ng/ml + goat anti-human TGFB2, 1 µg/ml, n = 7; TGFB3, 5 ng/ml + goat anti-human TGFB3, 10 µg/ml, n = 7) were added in the appropriate concentration to the medium and Matrigel. Controls for the neutralizing antibody experiments included the use of anti-hLAP (goat anti-human latency associated peptide-TGFB1, 1 µg/ml, n = 9), nonimmune goat IgG (10 µg/ml, n = 11), or nonimmune mouse IgG (10 µg/ml, n = 5). Neutralizing antibody concentrations were chosen to give a minimum of 50% neutralization based on manufacturers' specifications. All cytokines and antibodies were purchased from R&D Systems (Abingdon, UK). Each experiment was performed in triplicate. To remove intersubject variability, results are expressed as an invasion index determined by normalizing data from the test filters to their respective control filters.
Immunohistochemistry
To determine whether TGFB1, 2, and 3 altered trophoblast apoptosis and proliferation, at the end of invasion experiments the explants on the Matrigel-coated upper surface of the filters were fixed in 10% neutral-buffered formalin for 24 h and processed into paraffin wax (n = 4). Serial sections (3 µm) were immunostained for M30 (neoepitope of cytokeratin 18 exposed after caspase-mediated cleavage) and MKI67 (proliferation marker). Sections were also stained for cytokeratin 7 and HLA-G to distinguish cytotrophoblast cell columns and extravillous (HLA-G positive) from villous (HLA-G negative) trophoblast. Immunohistochemistry was performed as described previously. The sections were scored semiquantitatively for the approximate percentage of immunopositive cells [13] (0 = no immunopositive cells, 1 = <10% immunopositive cells, 2 = 11–44% immunopositive cells, 3 = 45–80% immunopositive cells, 4 = >80% immunopositive cells) by one investigator (G.E.L.) who was blinded to the identity of the sample. Villous and extravillous trophoblast, identified by HLA-G staining, were scored separately.
Substrate Gel Zymography
To determine the levels of secreted proteases and their inhibitors, zymographic analysis was performed as described previously [14]. Briefly, conditioned medium samples were collected following culture as described previously, and 20 µg of total protein were resolved in a 12% SDS-PAGE containing either 2 mg/ml gelatin (for MMP2 and MMP9) or 2 mg/ml casein and 0.025 units/ml plasminogen (for uPA, also known as PLAU) (American Diagnostica Inc., Greenwich, CT). The gels were then washed in 2.5% Triton X-100 to remove the SDS and incubated overnight at 37°C in a solution of 50 mM Tris and 5 mM CaCl2 to allow the enzymes to digest the substrate. Reverse zymography was performed to determine the levels of secreted TIMPs. 50 µg total protein was resolved in a 15% SDS-PAGE containing 2 mg/ml gelatin and a mixture of MMP9 and MMP2 (kind gift from Dr. Dylan Edwards, University of East Anglia, Norwich, UK). After electrophoresis, the gels were incubated overnight in 2.5% Triton X-100, 50 mM Tris, and 5 mM CaCl2 at room temperature on a shaking table, washed, and incubated for a further 16–18 h in 50 mM Tris and 5 mM CaCl2 at 37°C. After incubation, the gels were stained with Coomassie Brilliant Blue R250, destained, preserved, and dried. The dried gels were then scanned, and densitometry was performed (UnScan-It, Silk Scientific Co., Orem, UT). To remove intersubject variability, all results are normalized to their respective controls (n = 7).
Statistical Analysis
Data are presented as means ± SEM. Statistical analyses were performed using the StatView statistical software package (Abacus Concepts Inc., Berkley, CA). Statistical significance was determined using one-way ANOVA followed by Fisher post hoc analysis unless otherwise stated. Student t-test was used when only two sets of data were compared. All statistical tests were two sided, and differences were considered statistically significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the time course experiments are shown in Figure 1. Cell counts increased progressively after Day 2, and by Day 6 113 (±40) cells had invaded through the filter. It was arbitrarily decided to count invaded cell numbers for all future experiments on Day 6, as this provided an adequate but not excessive number of cells for quantification.
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Phenotype of Invaded Cells
Filters stained with a panel of antibodies directed against villous trophoblast cells ITGA6, ITGB3, and EGFR showed little or no immunoreactivity (Fig. 2, A–C). All cells on filters labeled with markers of extravillous trophoblast (ITGA1, HLA-G, ERBB2) showed a high level of immunostaining for all antibodies tested (Fig. 2, D–F). As expected, all cells on filters immunostained for cytokeratin 7 (Fig. 2G) were immunopositive, and there was no immunoreactivity in the absence of primary antibody (negative control; Figure 2H). This staining pattern is consistent with an extravillous trophoblast cell phenotype.
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Effect of TGFB1, 2, and 3 on Extravillous Trophoblast Cell Invasion
TGFB1 produced a dose-dependent decrease in the mean invasion index of extravillous trophoblast cells: 0.05 ng/ml, 31%, P < 0.005; 0.5 ng/ml, 25%, P < 0.02; 5 ng/ml, 55%, P < 0.0001 (Fig. 3). There was also an inhibition of invasion in response to TGFB2 and TGFB3 but only at the highest concentration tested (5 ng/ml): TGFB2, 36%, P < 0.03; TGFB3, 29%, P < 0.03 (Fig. 3).
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The efficacy of the neutralizing antibodies was tested in the presence of their respective exogenous cytokine (5 ng/ ml). In all cases the isoform-specific neutralizing antibody prevented the cytokine-mediated inhibition of trophoblast invasion: TGFB1, P < 0.02; TGFB2, P < 0.03; TGFB3, P < 0.02 (Fig. 4A). Addition of the neutralizing antibodies to TGFB1, 2, and 3 to the Matrigel and medium in the absence of exogenous cytokine led to an increase in the mean invasion index of extravillous trophoblast cells compared to controls: TGFB1, 148%, P < 0.04; TGFB2, 141%, P < 0.05; TGFB3, 234%, P < 0.003 (Fig. 4B). There was no difference in invasion in the presence of the human latency-associated protein of TGFB1 compared to untreated controls. Additional negative controls included inclusion of either nonimmune goat IgG or nonimmune mouse IgG, which also did not alter the invasive capacity of the explants compared to untreated controls (Fig. 4B).
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Effect of TGFB1, 2, and 3 on Placental Explant Apoptosis and Proliferation
Representative immunostaining for M30, MKI67, and HLA-G is shown in Figure 5. In general there was an equal distribution of M30-positive (Fig. 5B) cells in both villous and extravillous trophoblast cells, as distinguished by HLA-G immunoreactivity (Fig. 5A). There was no difference in the proportion of M30-positive villous or extravillous trophoblast cells in explants that had been incubated in the presence of TGFB1, 2, and 3 (villous: control 2.8 ± 0.7; TGFB1 2.5 ± 0.5; TGFB2 1.5 ± 0.3; TGFB3 1.4 ± 0.4; extravillous: control 2.8 ± 0.7; TGFB1 2.5 ± 0.8; TGFB2 1 ± 0.4; TGFB3 1.1 ± 0.8). In addition, there was no difference in the proportion of M30-positive villous trophoblast cells in explants that had been cultured in the presence of neutralizing antibodies to TGFB1, 2, and 3 (control 2.7 ± 0.2; anti-TGFB1 2.8 ± 0.2; anti-TGFB2 2.3 ± 0.2; anti-TGFB3 2.3 ± 0.3). By contrast, there was a significant increase in the proportion of M30-positive extravillous trophoblast cells in explants cultured in an anti-TGFB1 neutralizing antibody but not in neutralizing antibodies to the other isoforms (control 1.5 ± 0.3; anti-TGFB1 2.8 ± 0.3 P < 0.01; anti-TGFB2 1.3 ± 0.2; TGFB3 1.5 ± 0.3).
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No MKI67-positive cells were observed in extravillous trophoblast cells, although a high proportion of MKI67-positive cells were observed in cytotrophoblast columns proximal to the HLA-G-positive cytotrophoblast in distal columns, suggesting that trophoblast cells in the columns were highly proliferative (Fig. 5C). There was no difference in the proportion of cells that were positive for MKI67 in the villous trophoblast cells of the explants incubated in different concentrations of TGFB1, TGFB2, or TGFB3 compared to controls (control 2.5 ± 0.5; TGFB1 1.9 ± 0.4; TGFB2 2.3 ± 0.1; TGFB3 2.4 ± 0.1). In addition, there was no difference in the proportion of MKI67-positive villous trophoblast cells in explants cultured in neutralizing antibodies to the three cytokines (control 1.2 ± 0.2; anti-TGFB1 1.6 ± 0.3; anti-TGFB2 2.0 ± 0.4; anti-TGFB3 1.8 ± 0.3).
Substrate Gel Zymography for Secreted Proteases
There was a decrease in the levels of secreted MMP9 in response to TGFB1 (5 ng/ml, P < 0.03), TGFB2 (5 ng/ml, P < 0.03), and TGFB3 (5 ng/ml, P < 0.003) compared to controls (Fig. 6). In contrast, there was no difference in the levels of secreted MMP2 in response to any of the cytokines tested (Fig. 6). In addition, there was no difference in the levels of secreted TIMP1 and TIMP3 in response to TGFB1 (TIMP1 1.02 ± 0.009, TIMP3 1.03 ± 0.02), TGFB2 (TIMP1 1.08 ± 0.05, TIMP3 1.13 ± 0.08) or TGFB3 (TIMP1 1.07 ± 0.03, TIMP3 1.15 ± 0.09) compared to controls. There was also a decrease in the levels of secreted uPA in response to TGFB1 (P < 0.008), TGFB2 (P < 0.05), and TGFB3 (P < 0.02) compared to controls (Fig. 7). Taken together these results suggest that there is an overall reduction in the level of proteases in the presence of all three TGFB isoforms.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The dose-dependent decrease in trophoblast invasion in the presence of TGFB1 is consistent with previous reports [8, 15]. At the highest dose of TGFB1 tested (5 ng/ml), this effect could be completely blocked by the presence of an anti-TGFB1 neutralizing antibody. Furthermore, invasion was increased in the presence of neutralizing antibody alone. There are two possible explanations for this result. First, TGFB1 could originate from trophoblast to limit its own invasive capacity. Second, although growth factor-reduced Matrigel was used in the present study, TGFB1 is still present (manufacturers' specifications 1.7 ng/ml); this could be the source of the TGFB1 that is neutralized by the anti-TGFB1 antibody. The neutralizing antibody to hLAP (TGFB1) did not affect the invasive capacity of the trophoblast, suggesting that the TGFB1 present in the assay system was already in an active form and was not bound to hLAP. The present finding agrees with Graham et al. [8, 15], who demonstrated that TGFB1 (10 ng/ml) inhibited invasion of isolated first-trimester extravillous trophoblast cells (HTR-8) and their immortalized line (HTR-8/SVneo) in a similar assay. The invasive potential of the choriocarcinoma cell lines JAR and JEG-3 was not affected by this cytokine. In contrast, in an alternative invasion assay, where cells are grown on beads and then embedded in a fibrin clot, Tse et al. [16] demonstrated that TGFB1 (10 ng/ ml) increased the length of cellular processes of a first-trimester extravillous trophoblast cell line (SGHPL-4), suggesting an increase in invasion. These differences may reflect differences in extracellular matrix substrate and cell type used. The assay in the current study uses placental explants and Matrigel (reconstituted basement membrane), which we consider to be more physiological than transformed cell lines and single components of the extracellular matrix. Caniggia et al. [10], using villous explants plated on Matrigel, studied the effects of TGFB1 on trophoblast outgrowth (migration) and observed no effect of antibodies and oligonuceotides to TGFB1. Cell migration is only one component of the invasive pathway that also requires attachment to the extracellular matrix and its degradation.
The effect of TGFB2 and 3 on trophoblast in in vitro invasion assays has not previously been reported. In the present study, TGFB2 and 3 inhibited the invasion of extravillous trophoblast cells from placental explants at 5 ng/ ml, though no significant effect was seen at lower concentrations. These effects were blocked by addition of the respective neutralizing antibodies. In common with TGFB1, invasion was also increased in response to neutralizing antibodies. Again this could reflect autocrine control of invasive capacity or be a reflection of TGFB2 or 3 in the Matrigel. The presence of TGFB2 or 3 in Matrigel is not documented by the manufacturers, although by ELISA we failed to detect TGFB2 or 3 in Matrigel (data not shown). Using antibodies and oligonucleotides to TGFB2, Caniggia et al. [10] observed no effect on outgrowth from placental villous explants. In contrast, inhibition of TGFB3 increased outgrowth from the distal end of the villous tip and increased migration into Matrigel, leading to the conclusion that TGFB3 inhibits growth and differentiation of trophoblast cell columns.
Several studies have suggested that the TGFB family of cytokines have a growth inhibitory effect on some tumors by growth arrest and increased apoptosis [8, 17]. We therefore hypothesized that TGFB-mediated inhibition of invasion was due to reduced trophoblast proliferation. MKI67 staining was frequent in villous and proximal column trophoblast but was not seen on HLA-G-positive extravillous trophoblast, consistent with our previous observations [18] and those of Caniggia et al. [19]. In contrast, Nishimura et al. [20] reported MKI67 staining throughout HLA-G-positive explant outgrowths. However, their explants were cultured in 3% oxygen, which is known to increase villous and proximal column trophoblast proliferation [19, 21]. More important, we found no difference in the number of MKI67-positive proliferative cells in the presence or absence of TGFB or neutralizing antibodies to TGFB. This contrasts with Graham et al. [17], who reported a decrease in the uptake of 3[H]-thymidine by a primary trophoblast cell line, HTR-8, in response to TGFB1. The differences in results may reflect the different methodologies employed; while both MKI67 and 3[H]-thymidine are indicators of proliferative activity, they are known to identify different populations of cells, at least in breast cancer [22].
There are no previous studies of TGFB-mediated apoptosis in trophoblast. As TGFB is generally regarded as a death-inducing agent [12, 23], we hypothesized that reduced trophoblast invasion may be mediated, at least in part, by increased apoptosis. Contrary to our hypothesis, we found no increase in the number of M30-positive cells in the explants. While this observation needs confirmation, it is interesting to note that TGFB-mediated apoptosis is known to be context dependent; in mesenchymal cells, TGFB1 has recently been shown to activate the prosurvival phospatidylinositol 3-kinase/AKT pathway [24]. Further, cell death effects are dependent on the presence or absence of other growth factors. While neither TGFB nor TNF [25] alone appears to induce trophoblast apoptosis, they may act synergistically within the placental bed to induce cell death, thereby limiting invasion [26].
Cellular invasion requires proteolytic degradation of extracellular matrix molecules. Extravillous trophoblast cells use two main pathways: the MMPs and the uPA system. The MMPs are a family of more than 23 zinc-binding enzymes that include the collagenases (MMP1 and MMP4), the stromelysins (MMP3 and MMP10), and the gelatinases (MMP2 and MMP9) [27, 28]. Trophoblast cells are a major source of MMP2 and MMP9 [29]. The activity of the MMPs is regulated primarily by TIMP1, 2, and 3. In the present study, secreted levels of MMP9 were decreased in the presence of TGFB1, although levels of MMP2 and TIMP1 and 3 were not altered. This is consistent with the reported effect of TGFB1 on primary isolates of cytotrophoblast cells [30]. In contrast, Graham et al. [8] reported that TGFB1 increased the levels of TIMP1 and MMP2 mRNA in HTR-8 cells, although there was no difference in MMP2 activity when measured by zymography. We also found that secreted levels of MMP9 were reduced in the presence of TGFB2 and 3. The effect of these cytokines on cellular protease and protease inhibitor activity has not been previously examined in trophoblast cells, although several studies in models of tumor invasion confirm a general increase in protease inhibitor and decrease in protease levels [31–33].
The main components of the uPA system include the serine proteinase uPA, the uPA receptor (uPAR also known as PLAUR), and the plasminogen activator inhibitors 1 and 2 (PAI1 and 2, also known as SERPINE1 and 2) [34]. The uPA is secreted as a single chain pro-enzyme (pro-uPA) that, on binding to the uPAR, is converted into a two-chain, high-molecular-weight (50 kDa), active enzyme [35]. Free uPA or uPA bound to uPAR can convert plasminogen into plasmin, which in turn degrades extracellular matrix proteins or activates other invasion-associated enzymes including the MMPs [36]. In addition to its proteolytic function, uPA has been demonstrated to be a migration-stimulatory molecule for the trophoblast cell line HTR-8/SVneo [37]. Inhibition of both free uPA and uPAR-bound uPA is mediated mostly by PAI1 [38]. In the present study, secreted levels of uPA were decreased in the presence of TGFB1. This is in agreement with Graham et al. [8], who reported that there was a reduction in the levels of secreted uPA by HTR-8 cells in response to TGFB1. The same group went on to demonstrate an increase in PAI1 and a decrease in uPA levels in the cell line HTR-8/SVneo in response to TGFB1 [39]. Binding of PAI1 to the uPA/uPAR complex results in the entire complex being internalized and uPAR returned to the cell surface and PAI1 and uPA degraded [38]. Thus, it is expected that an increase in PAI1 would not only inhibit uPA activity but also decrease the levels of secreted uPA, as seen in the present study. TGFB2 and 3 were also found to reduce secreted levels of uPA. It is likely therefore that the TGFB family of cytokines elicits their anti-invasive effects by altering the amount of active protease available for ECM digestion and stimulating cellular migration.
This study reports a modification of the Matrigel invasion assay using placental villous explants as the source of invading cells. Previous studies [10, 39] of placental villous explant cultures on Matrigel have examined surface outgrowth of extravillous trophoblast cells, thus assessing migration rather than invasion. Extravillous trophoblast cell columns form on contact with Matrigel [40], invade through the Matrigel, and attach to the underlying filter. The invaded cells from the underside of the filters were characterized immunohistochemically as extravillous trophoblast cells. This approach therefore provides a useful model of extravillous trophoblast invasion in which all the important cell types are present without the need for isolation of one cell type or the use of transformed cell lines.
In conclusion, there is a dose-dependent decrease in extravillous trophoblast invasion in the presence of TGFB1. We have previously demonstrated that the decidua is a source of this cytokine during early pregnancy and therefore acts in a paracrine manner [11]. In addition, we have also demonstrated an inhibition in the level of invasion in response to TGFB2 and 3, but only at the highest concentration tested. In contrast to TGFB3, extravillous trophoblast cells and decidua are both immunopositive for TGFB2, and there may therefore be both an autocrine and a paracrine role for this cytokine [11]. The effects of the TGFB family on trophoblast invasion appear to be mediated not by altered proliferation/apoptosis but rather by a reduction in protease activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gendie Lash, School of Surgical and Reproductive Sciences, 3rd Floor, William Leech Building, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom. FAX: 44 191 222 5066; g.e.lash{at}ncl.ac.uk
Received: 27 January 2005.
First decision: 10 February 2005.
Accepted: 20 April 2005.
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