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Biology of Reproduction 67, 1628-1637 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Effect of Matrigel on Human Extravillous Trophoblasts Differentiation: Modulation of Protease Pattern Gene Expression1

Anne Tarradea, Frederic Goffind, Carine Munautd, René Lai-Kuenb, Viviane Tricottetb, Jean-Michel Foidartd, Michel Vidaudc, Francis Frankenned, and Danièle Evain-Brion2,a

a INSERM 427, Laboratoire de Microscopie Electronique, b Laboratoire de Génétique Moléculaire (MV), c Faculté des Sciences Pharmaceutiques et Biologiques, 75006 Paris, France d Laboratoire de Biologie des Tumeurs, Domaine du Sart-Tilman, B-4000 Liège, Belgique


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human placenta is characterized by extensive trophoblast invasion of the uterus. Indeed, extravillous cytotrophoblast cells invade the decidua and the upper third of uterine spiral arteries in the myometrium. This invasion is reflected in situ by the expression of specific markers. In order to study this invasion process, we have established an in vitro culture model of human extravillous trophoblast isolated from first trimester chorionic villi. The aim of this study was to investigate the effect of a composite matrix, the Matrigel required for the culture of this homogenous population of extravillous trophoblasts (EVCT), on their in vitro differentiation. The effect of Matrigel was studied on different markers characterized by immunocytochemistry and by real-time polymerase chain reaction assay of transcripts. In addition, the expression of 12 different matrix metalloproteases and their inhibitors were investigated. We show that human extravillous cytotrophoblasts acquire an invasive phenotype on Matrigel associated with a specific pattern of protease gene expression. This in vitro model will be of interest to study the cellular mechanisms involved in abnormal trophoblast invasion observed in poor placentation and preeclampsia.

developmental biology, growth factors, placenta, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human placenta is characterized by extensive trophoblast invasion of the uterus, leading to direct contact between trophoblasts and maternal blood in a process known as hemomonochorial placentation [1]. The placenta is also responsible for the production of a variety of specific hormones [2].

The key cell of the human placenta is the cytotrophoblast. After nidation, this cell differentiates along two different pathways, toward villous cytotrophoblasts (VCTs) and extravillous cytotrophoblasts (EVCTs) [3, 4]. Villous cytotrophoblasts fuse to form a multinucleated syncytiotrophoblast, which covers the chorionic villi and bathes in maternal blood. The syncytiotrophoblast has a key role in exchanges between the fetus and mother and produces large amounts of steroid [5] and protein hormones such as hCG [6], human placental lactogen (hPL), [7], and placental growth hormone [8]. Invasive EVCTs arise from the proliferative zone of the proximal column of the anchoring villi. EVCT migrate through the endometrium, interact with decidual cells and immunocompetent cells, and differentiate into multinucleated placental bed giant cells. In addition, they can invade the maternal spiral arteries, mediate the destruction of the arterial wall, and replace the endothelium, forming endovascular trophoblasts [4, 9]. This process of migration and invasion is influenced by extracellular matrix proteins. Indeed, the amount, composition, and orientation of extracellular matrix deposed by the trophoblast of cell columns or deeper in the junctional zone by decidual cells vary to a large extent [9, 10]. Around the invasive trophoblasts, the matrix shows a mosaic pattern, including different composants such as collagen IV, laminin, and heparan sulfate. Therefore, EVCTs modulate their receptors to the composants of the matrix, i.e., the integrins, during their migration. Indeed, integrin {alpha}6ß4 is expressed by proliferative cells in the columns and is downregulated during invasion [11, 12]. {alpha}5ß1 expression is then upregulated, and {alpha}vß1 and {alpha}1ß1 expression appears on invasive cells in arterial walls [4, 9, 10, 12]. The transition between the proliferative and invasive phenotype is characterized by downregulation of molecules associated with mitosis and upregulation of cell cycle inhibitors [13]. It is also associated with downregulation of epidermal growth receptor (EGF-R) expression and upregulation of the protooncogene c-erbB2 [14, 15] and other adhesion molecules such as connexin [16] and cadherin [17]. Invasive EVCTs also express human leukocyte antigen (HLA)-C, HLA-E, and HLA-G antigens involved in trophoblast immunotolerance [18].

During the invasion process, EVCTs express matrix metalloproteinases (MMPs), which are zinc-dependent proteolytic enzymes that cleave all the constituents of the extracellular matrix.

At least 21 MMPs have thus far been identified [19]. According to their structure and substrate, members of the MMP family can be classified into four groups: gelatinases (MMP-2 and MMP-9), collagenases (MMP-1, -8, and -13), stromelysins (MMP-3, -7, -10, -11, and -12), and MMPs containing a transmembrane domain near their carboxyl terminus, which localizes the enzymes on the plasma membrane (MMP-14, -15, -16, and -17). MMP activity is tightly regulated by several physiological inhibitors, known as tissue inhibitors of metalloproteinase (TIMP) 1 to 4 [20]. The serine proteinase urokinase (uPA), as well as PAI-1, one of its physiological inhibitors [21], are also known to be expressed by EVCT.

Human placenta cells, both in situ and in vitro, produce uPA [22, 23] and several MMPs: gelatinases A and B [24–26], collagenase [26–28], stromelysin [29] and MT-MMP [30, 31]. To control the activities of proteinases, human trophoblast cells produce inhibitors of MMPs: TIMP 1, 2, and 3 [32] and PAI-1 and -2 [23, 33].

There is a large body of evidence that MMPs and uPA, as well as their inhibitors (TIMPs, PAI-1) and receptors (uPAR), play a key role in cellular invasion [34–36]. This involves not only a trail-blazing action but also the release and activation of many growth factors and cytokines bound to the extracellular matrix (ECM) [37] and the shedding of active extracellular domains of proteins anchored to the outer cell membrane [35]. These proteases are probably involved in tissue remodeling and in the paracrine and chemotactic processes associated with EVCT invasion through decidual tissue.

The aim of this in vitro study was to investigate protease gene expression during the differentiation of a homogeneous population of extravillous trophoblasts isolated from first-trimester human chorionic villi and cultured on Matrigel, a composite extracellular matrix required for their in vitro culture [38–40]. The cultured cells were characterized by means of immunocytochemistry, and the pattern of gene expression was assessed with real-time polymerase chain reaction (PCR).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues

Placental tissues from first-trimester (7–12 wk) elective terminations were obtained from the Department of Obstetrics and Gynecology at Broussais and Saint-Vincent-de-Paul Hospitals. The tissue was washed in Ca2+-, Mg2+-free Hanks balanced salt solution supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin. Chorionic villi were dissected, rinsed, and minced for cell isolation.

Isolation and Purification of Trophoblasts Differentiating into EVCT

This procedure was adapted from a previous technique [38] based on the intensity of the enzymatic digestion of the anchoring chorionic villi. Trophoblast cells from the column are directly accessible to enzymatic digestion and are released from the tissue in aggregates, while the isolation of VCTs requires more intensive enzymatic digestion of the chorionic villi [40]. Briefly, chorionic villi were incubated in Hanks solution containing 0.125% trypsin (Difco Laboratories, Detroit, MI), 4.2 mM MgSO4, 25 mM Hepes, and 50 Kunitz units/ml DNase type IV (Sigma, Saint-Quentin Fallavier, France) for 35 min at 37°C without agitation.

After tissue sedimentation, the supernatant was filtered (100-µm pore size). Hanks solution was added to the tissue and sedimented twice. Trypsin digestion was stopped with 5% fetal calf serum (FCS). Cells were centrifuged at 300 x g for 10 min, diluted to a concentration of 5–6 x 105 cells/2 ml, and then plated on Matrigel-coated (5–6 mg/ml; Collaborative Biomedical Products, Le pont de Claix, France) 35-mm Falcon culture dishes.

Cells were maintained in Dulbecco modified Eagle medium (DMEM; Gibco, Grand Island, NY), supplemented with 10% FCS (Biological Industries, Beth Haemek, Israel), 2 mM glutamine, 25 mM Hepes, 100 IU/ml penicillin, and 100 µg/ml streptomycin and incubated with 5% CO2 at 37°C. After 3 h, cells were washed three times to eliminate debris and were incubated with complete medium.

Immunocytochemistry

After 90 min and 48 h of culture on dishes coated with Matrigel, EVCTs were fixed in 4% PFA for 20 min at room temperature, washed in PBS, and permeabilized for 4 min in 0.3% Triton X-100-PBS. Cells were incubated with 7% donkey serum in PBS or 7% goat serum in PBS for 30 min to reduce nonspecific binding. Primary antibodies diluted in PBS containing 1% BSA were added overnight at 4°C (Table 1). Cells were washed in PBS-0.1% Tween, then incubated with anti-rabbit IgG, donkey biotinylated species-specific F(ab')2 fragment (Amersham, Les Ulis, France) at 1:200, anti-mouse IgG, goat biotinylated species-specific F(ab')2 fragment (Amersham, Les Ulis, France), or anti-rat IgG, goat biotinylated species-specific F(ab')2 fragment (Amersham) for 1 h and washed in PBS-0.1% Tween. Staining was revealed with streptavidin-fluorescein (Amersham) diluted 1:400 in the dark for 1 h. Slides were mounted in mounting medium (Vector Laboratories, Burlingame, CA) and examined and photographed with an Olympus BX60 epifluorescence microscope. Controls omitted the primary antibody or used nonspecific mouse IgG of the same isotype.


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  		TABLE 1. Antibodies used for immunocytochemistry

Quantification of Specific Transcripts by Real-Time Reverse Transcription Polymerase Chain Reaction

Total RNA was extracted from cultured EVCT using Qiagen RNeasy Mini Kit (Courtabeuf, France). Complementary DNA synthesis and PCR amplification were performed as described previously [40, 41]. All PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA) and the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The nucleotide sequences of the amplification primers are listed in Table 2. Each sample was analyzed in duplicate and a calibration curve was run in parallel in each analysis. To control for sample-to-sample differences in RNA concentration and quality, transcripts were normalized to human PPIA (cyclophilin A). Three culture dishes were pooled for each determination. Three different primary cultures were analyzed at the indicated time in culture. The results are expressed as the mean ± SEM of the three different experiments.


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  		TABLE 2. Probes and primers used in real-time PCR

Scanning Electron Microscopy

Cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 h at room temperature. The cultures were then dehydrated with increasing concentrations of acetone and dried with a critical-point drying apparatus (Balzers Union, Zurich, Switzerland) using acetone and liquid CO2. The dried specimens were coated with a 30-nm layer of gold in a vacuum evaporator.

Invasion Assays

To assess the invasive potential of cytotrophoblasts, cultured EVCTs were plated on Transwell inserts (6.5 mm; Costar, Cambridge, MA) containing polycarbonate filters with 8-µm pores, as previously described [39]. The upper side was coated with 10 µl of diluted Matrigel (5–6 mg/ml), and 2.5 x 105 cells were plated in 200 µl of DMEM supplemented with 10% FCS, 2 mM glutamine, 25 mM Hepes, 100 IU/ml penicillin, and 100 µg/ml streptomycin; 600 µl of the same medium was added to the well. Transwells were incubated with 5% CO2 at 37°C.

After 24, 48, and 72 h of culture, the Transwell inserts were washed three times with PBS and cells were fixed for 1 h in 4% paraformaldehyde at 4°C. Samples were rinsed and fixed for 10 min at -20°C in methanol. Cells were incubated with 7% goat serum in PBS for 30 min to reduce nonspecific binding. Cytokeratin 07 antibody (1:200; Dako, Trappes, France) diluted in PBS containing 1% BSA was added for 3 h at room temperature. Cells were washed in PBS-Tween 0.1% and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h, then washed in PBS-Tween 0.1%. Filters were dissected with a scalpel, and the upper face of the filter was placed in contact with a superfrost slide, mounted in mounting medium (Vector Laboratories), and examined and photographed with an Olympus BX60 epifluorescence microscope.

Semiquantitative Reverse Transcription Polymerase Chain Reaction

MMP-1, -2, -3, -8, -9, -11, -12, -14 (MT1-MMP), -15 (MT2-MMP), and -16 (MT3-MMP); TIMP-1, -2, and -3, uPA (urokinase); and PAI-1 (plasminogen activator inhibitor 1) expression was evaluated as follows.

The reverse transcription polymerase chain reaction (RT-PCR) amplifications were performed in an automated instrument (Gene Amp PCR system 9600) using the Gene Amp Thermostable rTth Reverse Transcriptase RNA PCR kit (Perkin-Elmer). In order to monitor the efficiency of the RT-PCR assay, synthetic RNAs were constructed and used as internal standards (see below). Preliminary experiments were carried out to determine the conditions in which the amount of DNA obtained after amplification was directly proportional to the amount of RNA submitted to amplification. For quantitative RT-PCR measurements, 0.1–10 ng of total RNA and a determined copy number of multistandard synthetic RNA (mssRNA) internal control were used per 25 µl reaction mixture (final volume). The RT step (70°C for 15 min) was followed by a 2-min incubation step at 95°C (to denature RNA-DNA heteroduplexes) and then by a 30-cycle PCR amplification. The PCR conditions were 94°C for 15 sec, 68°C for 20 sec, and 72°C for 10 sec. The sequences of the specific oligonucleotide primers and the size of the respective amplification products are given in Table 3. The amplified products were separated by polyacrylamide gel electrophoresis, stained (Gelstar, BioProducts), and quantified (Wluor-S Multi Imager, Bio-Rad). In all assays, the actual concentration of the specific mRNA calculated as copy number equivalent to mssRNAs was expressed per unit of 28S rRNA measured on the same solution of RNA.


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  		TABLE 3. Primers used in semiquantitative RT-PCR

Construction and Use of the mssRNAs

Synthetic RNAs were created in order to monitor, in each sample, possible variations in the efficiency of the RT-PCR amplification. These synthetic RNAs display two main characteristics. First, they can be amplified by primers used for RT-PCR amplification of the endogenous mRNA of interest. Second, the size of their amplification product is different from the size of the RT-PCR product amplified from the mRNA, enabling their discrimination after electrophoresis. Generation of the synthetic standard RNA for MMP-1, -2, -3, -8, -9, -11, and -14 mRNA and 28S rRNA was previously described [42, 43]. MMP-12, -15, and -16; TIMP-1, -2, and -3; PAI-1; and uPA mRNA were monitored with a second mssRNA. For the construction of this multistandard RNA, a double-stranded synthetic DNA (Fig. 1) was synthesised. A sequence-verified construct was then linearized, purified, and used as a template for RNA synthesis (SP6 polymerase, SP6/T7 transciption kit; Roche Molecular Biochemicals). The resulting mssRNA was then purified and quantified. RT-PCR amplification of this mssRNA using the different pairs of primers generating amplification products larger (245–271 base pairs [bp]) than those obtained from the cellular RNA (155–210 bp; see Table 1), enabling their discrimination by electrophoresis.



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  		FIG. 1. A) Sequence of the synthetic DNA used as template for the synthesis of the mssRNA internal control. B) Arrangement of the forward and reverse primer pairs in the synthetic standard DNA insert (open boxes) and SP6 polymerase initiation site (black box) in the pSPT18 plasmid

The validation of the RT-PCR quantification using mssRNA has been previously described [43].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Trophoblast Growth Arrest on Matrigel

As previously shown [40], the isolation procedure used here allowed us to isolate a homogenous population of EVCTs, as characterized by specific marker expression. As illustrated in Figure 2 and quantified in Table 4, after plating on Matrigel for 90 min, all these mononucleated cells express cytokeratin 07 and TGFß2, both of which are markers of EVCT (Fig. 2, middle and lower panels, left side). The cells remained cytokeratin 07 and TGFß2 positive at 48 h of culture (Fig. 2, middle and lower panels, right side; Table 4).



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  		FIG. 2. Upper panel) Immunostaining of Ki-67, a nuclear antigen present during all active phases of the cell cycle. The extravillous trophoblasts show strong nuclear staining (76% of cells) after 90 min on Matrigel (left panel); the Ki-67 signal decreased after 48 h of culture, when only 19% of cells were positive (right panel). Immunostaining of cytokeratin 7 (middle panel) and TGFß2 (lower panel) in isolated human EVCT cultured for 90 min (left panel) or 48 h on Matrigel (right panel). All the extravillous trophoblasts expressed these two specific markers


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  		TABLE 4. Transcript levels and immunodetection of specific markers of extravillous trophoblasts after 90 min and 48 h of culture on Matrigel.a

Observed 90 min after plating on Matrigel, 76% of the cells were proliferative, as shown by immunostaining for Ki-67 (a nuclear antigen present during all the active phases of the cell cycle, namely G1, S, G2, and mitosis; Fig. 2, upper panel, left side) and cyclin B1 (data not shown). After 48 h of culture on Matrigel, only 19% of cells were Ki-67 positive (Fig. 2, upper panel, right side), and cyclin B1 was no longer expressed (data not shown).

Trophoblast Invasion of Matrigel-Coated Transwells

Cells layered on Matrigel-coated Transwells were observed by scanning electron microscopy (Fig. 3A, right panel) and light microscopy after immunostaining with an antibody against cytokeratin 07 (Fig. 3A, left panel). Many cell projections were visible on the lower side of the filter after 48 h (Fig. 3A, right panel), and their numbers were maximal after 48–72 h (Fig. 3B).



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  		FIG. 3. A) Extravillous trophoblasts isolated from first-trimester placentas were cultured on Matrigel-coated Transwell filters. After 48 h, the cells were fixed and stained with anti-cytokeratin 07 antibody. The filters were removed from the inserts and the top of the filter was placed in contact with a slide. Transwells were examined with an epifluorescence microscope. After 48 h, fluorescent (cytokeratin-positive) cell projections (cp) appeared in the pores (left panel). After 48 h, cells were fixed for scanning electron microscopy. The filters were removed from the inserts and the bottom of the filter was observed. After 48 h (right panel), cell projections appeared in the pores. B) Time course of cytotrophoblast invasion. Cytotrophoblasts were cultured for 1.5, 24, 48, or 72 h on diluted-Matrigel-coated Transwell filters. Invasion was quantified by counting the number of cytokeratin cell projections that penetrated the filter pores in 20 fields. The figure represents one of four experiments. The results are means ± SEM of three filters

Effect of Matrigel on Specific EVCT Marker Expression

Cells were immunostained with antibodies against EGF-R (A), c-erbB2 (B), integrin subunits {alpha}6 (C) and {alpha}5 (D), hPL (E), and HLA-G, and positive cells were counted (Table 4). They were observed 90 min after plating on Matrigel (Fig. 4) and after 48 h of culture on Matrigel (Fig. 5). After 48 h on Matrigel, none of the cells were positive in immunocytochemistry for EGF-R, while all were positive for c-erbB2 and hPL. The {alpha}6 integrin subunit was detected in 58% of cells 90 min after plating on Matrigel and in 0% of cells after 48 h of culture on Matrigel. The {alpha}5 subunit was detected in 42% of cells 90 min after plating on Matrigel and in 99% of cells after 48 h of culture on Matrigel. CK7, c-erbB2, HLA-G, TGFß2, and hPL transcript levels, as measured by real-time quantitative PCR, were not significantly affected by culture on Matrigel (Table 4), whereas EGF-R transcript levels fell significantly.



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  		FIG. 4. Immunostaining of EGF-R (top left), c-erbB2 (top right), {alpha}6 (middle left), and {alpha}5 (middle right) integrin subunit and hPL (bottom left) in isolated human EVCT cultured for 90 min compared with the IgG isotype control (bottom right)



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  		FIG. 5. Immunostaining of EGF-R (top left), c-erbB2 (top right), {alpha}6 (middle left), and {alpha}5 (middle right) integrin subunit and hPL (bottom left) in isolated human EVCT cultured for 48 h on Matrigel compared with the IgG isotype control (bottom right)

Effect of Matrigel on Protease and Protease Inhibitor Expression

The protease and protease inhibitor expression profile was determined in EVCT by semiquantitative PCR before and after 48 h of culture on Matrigel. Figure 6 illustrates a Southern blot of representative PCR products. The relative expression of the different MMPs and protease inhibitors in trophoblast cultures are shown in Figures 7 and 8 and Table 5. MMP-7 and -11 and stromelysin 3 were not expressed at either time point. MMP1, -3, -8, and -9 were weakly expressed (less than a thousand copies per ng of mRNA) (Fig. 7A). In contrast, MMP-2 and -12, MT1-MMP, and MT2-MMP were strongly expressed (Fig. 7B). TIMP-1 and -2 were strongly expressed, and TIMP-3 was weakly expressed (Fig. 8). Culture on Matrigel for 48 h increased MMP-12, MT2-MMP, TIMP-2, and TIMP-3 expression and reduced MMP-2 and TIMP-1 expression. MT1-MMP expression was not affected.



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  		FIG. 6. Southern blots of representative RT-PCR products obtained from EVCT cells before and after a 48-h culture on Matrigel. Endogenous target mRNA and mssRNA internal controls are coamplified by the same primers, but their products differ in size. Arrows indicate RT-PCR products from the mssRNA or the endogenous mRNA. The number of mssRNA copies submitted to RT-PCR is indicated



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  		FIG. 7. MMP gene expression by extravillous trophoblasts assayed by RT-PCR before culture and after 48 h of culture on Matrigel. A) MMP-1, -3, -7, -8, -9, and -11 expression. MMP-7 and -11 were not expressed, while MMP-1, -3, -8, and -9 were weakly expressed. Matrigel had no affect on the expression of these MMPs. B) MMP-2, -12, MT1-MMP, and MT2-MMP were strongly expressed. MMP-12 and MT2-MMP transcripts increased 20-fold and 14-fold (* indicates P < 0.05), respectively, whereas MMP-2 transcripts fell fivefold. MT1-MMP expression was unaffected



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  		FIG. 8. TIMPs gene expression in extravillous trophoblasts, as assayed by semiquantitative RT-PCR, before culture and after 48 h of culture on Matrigel. TIMP-1, -2, and -3 were strongly expressed; TIMP-2 and TIMP-3 expression increased after 48 h of culture on Matrigel (1.6-fold and 22-fold respectively, P < 0.05). TIMP-1 expression decreased 1.5-fold


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  		TABLE 5. uPA and PAI-1 transcript levels in EVCT.a

As shown in Table V, uPA expression was initially low and increased markedly during cell culture (P < 0.05). The PAI-1 inhibitor was strongly expressed and increased by almost twofold at 48 h of culture on Matrigel.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human trophoblast differentiation along the invasive pathway has been well characterized in situ in the anchoring villi by means of immunohistochemistry [9]. The cells switch in situ from a proliferative state in the proximal part of the column to an invasive, nonproliferative phenotype at the distal part of the column [13]. This is associated with changes in cell-to-cell contacts [44–46], integrin expression [10–12], receptor [14, 15], and soluble factor secretion [9, 47].

In this study, we used a recently described method to prepare human EVCTs [40]. We first confirmed, by means of immunocytochemistry and real-time PCR, the purity of the cell preparation and the presence of specific markers of EVCTs [40]. As previously shown [25, 38, 40], EVCTs do not attach to the plastic dishes and require a matrix to survive in culture. One of the matrices used to culture these cells in vitro is Matrigel, which is a composite matrix rich in laminin and collagen and therefore presents similarities with the matrix present around the invasive trophoblast in the decidua.

Freshly isolated cells were Ki-67 positive, as tested after short exposure to Matrigel. (Immunocytochemical analysis of freshly isolated unattached cells collected after cytospinning was unsuccessful.) The strong expression of Ki-67 by cytotrophoblasts after 90 min of culture on Matrigel is indicative of DNA synthesis [48]. Ki-67 is a marker of proliferative cells in first-trimester chorionic villi, such as villous CT and cells from the tip of the column [13, 47]. This rules out contamination by syncytiotrophoblast fragments, as recently observed with a completely different method of VCT isolation [49]. In addition, 100% of cells expressed TGFß2, a marker specific for syncytiotrophoblast and EVCTs but not for VCTs [45]. The population of Ki-67-positive and TGFß2-positive cells thus corresponds to cytotrophoblasts of the proximal anchoring villi, which are termed proliferative EVCTs or intermediate trophoblasts by certain authors [47, 50]. The same cells also expressed EGF-R and integrin subunit {alpha}6, which also characterizes the proximal layer of the trophoblast column in situ [14, 15].

After 48 h of culture on Matrigel, these cells expressed surface c-erbB2, hPL, and HLA-G and were EGF-receptor negative. Interestingly, transcript levels of c-erbB2, hPL, and HLA-G were not modulated by 48 h of culture on Matrigel, while their immunodetection was clearly modified. Mühlauser et al. [15], using in situ hybridization, found c-erbB2 cRNA in the cell columns, whereas the gene product was not expressed by proliferative trophoblasts; a similar expression profile has also been described for HLA-G [51, 52]. These results point to posttranscriptional differences between proliferative and invasive cytotrophoblasts.

A major characteristic of invasive cells is metalloprotease secretion. The distribution of MMPs and TIMPs along the invasive pathway of EVCTs (from the tip of anchoring villi to the invaded vessels) has been established by means of immunohistochemistry and in situ hybridization [32, 52–54]. Several groups have published conflicting data on MMP expression by cultured trophoblast cells. These studies used different cell types and cell isolation-purification procedures and focused on different stages of pregnancy. The present study, based on quantitative RT-PCR, establishes the pattern of MMP and TIMP expression in a well-defined population of purified EVCT, together with the modifications induced by adherence and culture on Matrigel. MMP2, MT1-MMP, MMP9, uPA, PAI-1, TIMP2, and TIMP3 but not MT11 have been shown to be expressed in situ in cytotrophoblasts from the column [29, 31, 34, 54–56]. A similar pattern was obtained here with isolated EVCT: MMP2, MT1-MMP, uPA, and PAI-1 were significantly expressed, whereas MMP9 was barely detected and MMP11 was undetectable. The three MMP inhibitors were also expressed, TIMP1 and TIMP2 strongly and TIMP3 weakly. Once again, this expression pattern matches that of proliferating, columnar ECVTs. MMP9 expression of EVCT has been associated with the invasive behavior of these cells [54, 57] and anti-MMP9 antibodies have been shown to block EVCT invasion [26]. It is therefore surprising that MMP9 is only weakly expressed in our model of invasive EVCTs. But this model, as any in vitro model, is far from reproducing the complexity of the in vivo cellular environment, especially at the cytokine and growth-factor levels. Because the MMP9 expression is known to be regulated by a large array of factors, as expected by the large variety of responsive elements present on the promoter region of its gene, the low expression levels observed here could simply reflect a difference in the expression stimulator/inhibitor ratio as compared with the in vivo situation.

The strong expression of MMP2, MT1-MMP, TIMP2, uPA, and PAI-1 is consistent with the invasive phenotype of EVCT, as these proteases and inhibitors have been linked, alone or in combination, with cellular invasion in numerous models [35]. In addition, our data obtained with isolated cells strongly suggest that MMP1, MMP3, and MMP8 are expressed at low or very low levels and that MMP12 and MT2-MMP are expressed at high levels. Immunohistochemical data obtained by others [58] have shown that MMP3 is also very weakly expressed by EVCT in vivo.

Matrigel is a reconstituted basal membrane that, in some respects, is similar to the decidual ECM in that it is rich in laminin and relatively poor in collagen IV. In addition, some growth factors and cytokines are still bound to the matrix and can be mobilized by extracellular proteinases [59]. The most noteworthy modification induced by cultivating isolated EVCTs on Matrigel for 48 h was a marked increase in TIMP2 and MMP12 expression. These changes could point to a less invasive phenotype, as suggested by an increased TIMP2 to MT1-MMP and MMP2 ratio, but would endow the cells with the potential to cross vessel walls by massive elastase expression (MMP12). This new phenotype is similar to that which EVCT should acquire to penetrate the vessel wall after migrating through the decidual matrix, providing additional evidence that trophoblast phenotype and behavior are largely controlled by its environment.

Human EVCTs acquire an invasive phenotype on Matrigel associated with a specific pattern of protease gene expression. This in vitro model will be of interest in the study of the cellular mechanisms involved in abnormal trophoblast invasion observed in poor placentation and preeclampsia.


   ACKNOWLEDGMENTS
 
We thank the department of Obstetrics and Gynecology of Broussais and Saint-Vincent-de-Paul Hospitals, Paris, for donating placental tissues. We also thank Marie-Rose Pignon for her excellent technical assistance.


   FOOTNOTES
 
1 This work was supported by a grant from INSERM-CFB and by FNRS (F.G.). Back

2 Correspondence: Danièle Evain-Brion, INSERM U 427, Faculté des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75006 Paris, France. FAX: 33 1 44 07 39 92; evain{at}pharmacie.univ-paris5.fr Back

Received: 28 November 2001.

First decision: 20 December 2001.

Accepted: 5 June 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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