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Inhibits Trophoblast Migration through Elevation of Plasminogen Activator Inhibitor-1 in First-Trimester Villous Explant Cultures
Department of Obstetrics and Gynecology (S.B., J.P., J.H., P.H., M.K.), University of Vienna, A-1090 Vienna, Austria; and School of Biological Sciences (J.D.A.), University of Manchester, M13 0JH Manchester, United Kingdom
Address all correspondence and requests for reprints to: Martin Knöfler, Department of Obstetrics and Gynecology, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna. E-mail: martin.knoefler{at}akh-wien.ac.at.
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Abstract |
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could impair trophoblast invasion. Using first-trimester placental explant cultures, we have demonstrated that the cytokine inhibits in vitro migration of extravillous trophoblasts (EVT) on collagen I, and invasion through Matrigel. To elucidate the underlying mechanism, proliferation and differentiation of EVT in vitro were analyzed by immunohistochemistry of serial sections, Western blotting, zymography, ELISA, and RT-PCR from RNA pools of mechanically separated cell populations. At 24 h of cultivation in the presence or absence of TNF, anchorage and proliferation of trophoblasts had occurred to generate cell columns containing viable, post-mitotic, differentiated EVT [positive for integrins 1 and 5, matrix metalloproteinase (MMP)-2, and human leukocyte antigen-G1; negative for proliferating cellular nuclear antigen, cytokeratin 18 neoepitope, and in 5-Bromo-2-deoxy-uridine labeling]. At 72 h, control cells had broken away from the column to migrate through the extracellular matrix; whereas, in contrast, TNF-treated EVT remained as contiguous cell columns, despite increased MMP-9 expression. Thus, in vitro MMP9 activity appears not to be essential for trophoblast migration. Expression of plasminogen activator inhibitor (PAI)-1 was elevated in TNF-treated EVT, and adding antibodies that inhibit PAI-1 activity restored migration, whereas tissue-inhibitor-of-metalloproteinases-1-blocking antibodies were ineffective. Induction of PAI-1 by TNF could be related to restricted trophoblast invasion in preeclampsia. |
Introduction |
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One leading hypothesis to account for preeclampsia is maladaptation of the maternal immune system (7), with enhanced expression of proinflammatory cytokines linked to failed trophoblast invasion. Several lines of evidence support the idea that TNF could be one such mediator. Higher staining intensities were detected in decidual stroma of hypertensive placentas (8), and increased levels of TNF and its soluble carrier protein, TNF receptor (TNFR)I, were measured in preeclamptic sera (9, 10). Hypoxic conditions, which might occur in preeclampsia under reduced perfusion, enhanced TNF secretion from villous tissue in vitro (11). There are possible associations among elevated TNF, preterm labor, and recurrent abortion (12, 13). Besides its adverse effects, a role in normal placental hormone production, trophoblast turnover, and matrix metalloproteinase (MMP) expression has been suggested (14, 15, 16), so that the role of this cytokine must be evaluated in the context of its pleitrophic properties (17). Here, we investigated the influence of TNF on the complex differentiation program of the anchoring villus using explants of first-trimester placenta. EVT generated by these organ cultures express differentiation-specific markers in a spatiotemporally regulated manner that parallels closely EVT differentiation in vivo (18). Using this in vitro system, we demonstrate that TNF inhibits EVT migration through increases in plasminogen activator inhibitor (PAI)-1 expression, which could suggest a link between elevated levels of TNF and PAI-1 in preeclampsia (19) and failed trophoblast invasion.
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Materials and Methods |
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Collagen type I (rat tail) and Matrigel were obtained from Becton Dickinson (Bedford, MA). DMEM, Ham’s F-12, and fetal calf serum were from Life Technologies, Inc. (Paisley, UK). Sodium bicarbonate, RNALater, and Tri-Reagent were from Sigma (St. Louis, MO). 5-Bromo-2-deoxy-uridine (BrdU) Labeling and Detection Kit I was purchased from Roche (Mannheim, Germany), recombinant human TNF was obtained from Eubio (Vienna, Austria), and zymogram gels were from Invitrogen (Carlsbad, CA). Primary antibodies used for immunohistochemistry are listed in Table 1
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First-trimester placentae were obtained with the permission of the local ethical committee, at elective termination of selected pregnancies performed by evacuation between the 8th and 10th weeks. Tissues were collected and washed with ice-cold PBS, stored in cold explant culture medium (see recipe below), and processed within 2 h.
Explant culture
Preparation of first-trimester placental explants was performed as described elsewhere (20). Briefly, small pieces of tissue (2–3 mm) from the periphery were dissected under the microscope and incubated for 1 h in serum-free culture medium (DMEM:Ham’s F12 with 50 µg/ml gentamycin) in a humidified chamber at 37 C, 5% CO2. For preparation of extracellular matrix (ECM)-coated plates, 1 ml collagen I was mixed with 10x DMEM (1:10) and 7.5% sodium bicarbonate (1:5). Matrigel was diluted in explant medium to a final concentration of 8 mg/ml. For each explant, one drop of ECM protein solution (85 µl) was placed in the center of a 12-well culture dish (Costar, Corning, NY). In experiments with TNF, 1 or 10 ng/ml of the recombinant, human cytokine was added to the ECM solution. After formation of gels (30 min at 37 C), the dissected tissue pieces were carefully put on the top of each gel drop, covered with 50 µl medium, and incubated for 4 h to allow anchorage. Subsequently, explants were supplemented with 1ml medium in the absence or presence of 1 or 10 ng/ml TNF. For each condition, five to eight explants were analyzed, and experiments were repeated five times with different placentae. After an additional 24 h and 72 h, supernatants were collected, frozen in liquid nitrogen, and stored at -80 C. The explants were washed twice in PBS and either fixed for immunohistochemistry or proceeded for RNA or protein isolation. For blocking studies, inhibitory antibodies were added to the gel matrix at final concentrations of 5 µg/ml antihuman TNF (R&D Systems, Minneapolis, MN), 5 µg/ml antihuman tissue inhibitor of metalloproteinases (TIMP)-1 (R&D Systems), or 10 µg/ml [antihuman PAI-1, clone MA-56A7C10 (21)], respectively, and cytokine was added after the 4-h attachment period.
Immunohistochemistry
After 30 min of fixation at 4 C in 2% paraformaldehyde, explants were washed two times (each 10 min) in PBS (0.18% Na2HPO4; 0.03% KH2PO4; 0.8% NaCl, pH 7.4) and soaked with 0.5 M sucrose/PBS for at least 1 h at room temperature. Then the samples were covered with OCT Compound (Sakura, Zoetermonde, Netherlands), frozen in liquid nitrogen, and kept at -80 C. Four-micrometer serial cryostat sections were prepared, postfixed with 1% paraformaldehyde (10 min, 4 C), washed (three times, 5 min, in PBS), and treated with 0.1% Triton X-100/PBS for another 5 min. After three washes in PBS (each 5 min) and 30 min of incubation in blocking solution (NEN Life Science Products, Boston, MA), slides were incubated overnight with primary antibody, washed three times in PBS (each 5 min), followed by 5 µg/ml fluorescein isothiocyanate-conjugated goat antimouse or 5 µg/ml goat antirabbit antibodies (1 h; Molecular Probes, Eugene, OR). As a negative control, the primary antibody was replaced by buffer or isotype IgG. Finally, all sections were counterstained with 1 µg/ml DAPI (4'6-diamidine-2'-phenylindole dihydrochloride) from Roche and covered with flouromount G (Soubio, Birmingham, AL).
BrdU labeling
For detection of proliferation in the organ cultures, BrdU Labeling and Detection Kit I (Roche) was used according to the manufacturer’s instructions. After attachment to the gel matrix, villous explants were incubated 24 h in the presence of 10 µM BrdU. Subsequently, tissues were snap-frozen in liquid nitrogen, and sections were cut and fixed in Ethanol-Fixans (15 mM glycine/70% ethanol, 20 min, -20 C). BrdU was detected with primary monoclonal anti-BrdU and secondary antimouse Ig fluorescein antibodies.
RNA extraction and RT-PCR
RNA pools were prepared after mechanical separation of villi and EVT from approximately 200 first-trimester placental explants cultivated on Matrigel. After 72 h of incubation, cultures were placed on ice, washed twice with cold PBS, and covered with RNALater (Qiagen, Hilden, Germany). Separation was done using small forceps and scissors under the stereomicroscope by cutting the explants at the attachment site. Villi were separated from tissue containing cell column and distal EVT with minimal cross-contamination. Pooled tissues were frozen in liquid nitrogen and homogenized with a microdismembrator (Braun Biotech International, Melsungen, Germany). Next, the disrupted tissue was covered with Tri-reagent, and total RNA was isolated according to the manufacturer’s instructions. Four micrograms of each RNA, quantitated with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) were used for synthesis of first-strand cDNA using 10 U/µl Superscript (Life Technologies, Inc.). Semiquantitative PCR amplification (45 sec, 96 C, 1 min annealing temperature, 80 sec 72 C) was performed with PCR Reagent System (Life Technologies, Inc.) in a RoboCycler Gradient 96 (Stratagene, Amsterdam, Netherlands) using 0.5 U Taq polymerase. Cycle numbers were optimized within the linear range of individual PCR reactions. Oligonucleotide primers, annealing temperatures, and cycle numbers are listed in Table 2. In all experiments, a possible DNA contamination was checked by negative control RT-PCR in which reverse transcriptase was omitted in the reverse transcription step. The PCR products were analyzed on 1.5% agarose gels containing ethidium bromide and photographed under UV radiation. All PCR fragments were sequence-verified on a 16-capillary sequencer by using the nonradioactive ABI PRISM Terminator Cycle Sequencing Ready Reaction Kit as specified by the supplier (Applied Biosystems, Foster City, CA).
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MMP-2 and MMP-9 secretion was evaluated by substrate gel zymography of supernatants from 72-h explants. Protein concentration was determined by Bio-Rad Assay Reagent according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). Conditioned medium containing 250 ng protein was mixed with the same volume of 2x sample buffer [0.125 M Tris-HCl, pH6.8; 20% glycerol; 4% sodium dodecyl sulfate (SDS); 0,005% bromophenol blue], incubated for 10 min at room temperature and loaded on nonreducing 10% polyacrylamide gels containing 0.1% gelatin (Novex Invitrogen, Carlsbad, CA). Proteins were separated by electrophoresis at a constant voltage of 125 V/gel for 90 min as described by the supplier. Gels were washed with Novex Renaturing Buffer (2.5% Triton X-100) with gentle agitation for 30 min and incubated overnight at 37 C with Novex Developing Buffer (0.05 M Tris-HCl; 0.2 M NaCl; 5 mM CaCl2; and 0.02% Brij 35, pH 7.2). Gels were stained with 0.5% Coomassie brilliant blue R-250 and destained overnight in 10% acetic acid/30% methanol.
Western blot analysis
Thirty micrograms of protein of the supernatant were mixed with 5x SDS-sample buffer (100 mM Tris-HCl, pH 6.8; 5% SDS; 25% glycerol; 0.01% bromophenol blue), reduced with 1% ß-mercaptoethanol, heated for 3 min at 95 C, and separated on a 10% SDS-polyacrylamide gels. Equal loading of protein was monitored by Ponceau S staining of membranes. Proteins were then transferred onto 0.45 µm nitrocellulose (Schleicher & Schuell, Dassel, Germany) in a buffer containing 25 mM Tris-HCl (pH 8.3), 0,5% SDS, 192 mM glycine, 20% ethanol and blocked with 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0,3%Tween-20 [Tris-buffered saline with Tween 20 (TBST)] containing 3% nonfat dry milk for at least 1 h at room temperature. The blots were then incubated overnight at 4 C with either 1 µg/ml mouse monoclonal TIMP-3 (Clone 136–13H4, Chemicon, Temecula, CA) or 2 µg/ml mouse monoclonal PAI-1 (Clone 6, Oncogene, Boston, MA) antibodies in TBST containing 0.5% nonfat dry milk. After five washing steps in TBST (5 min each), blots were incubated 1 h at room temperature with a peroxidase-linked antimouse IgG (NA 931, Amersham Pharmacia Biotech, Buckinghamshire, UK, 1:80.000). Signals were developed using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
Densitometry
Bands obtained in Western blot analyses were densitometrically scanned using the documentation system Epi Chemi II darkroom (UVP, CA). Signals were quantitated using the program Labworks (UVP).
ELISA
Total human free and MMP-bound TIMP-1 was measured in supernatants with a TIMP-1 ELISA according to the manufacturer’s instructions (Amersham Pharmacia Biotech). Within- and between-assay precision levels were 9.3% and 12.4%, respectively; sensitivity was 1.25 ng/ml. Soluble TNFRI was determined by using Quantikine Human sTNF RI Elisa (R&D Systems). The detection limit was less than 3.0 pg/ml; inter- and intraassay CVs were 8.8% and 6.9%, respectively. The total amount of urokinase plasminogen activator (uPA) was quantitated using CaoChrom uPA Antigen ELISA as described by the supplier (Hyphen BioMed, Andresy, France).
Statistical analysis
Statistical analyses were performed with Sigma Stat Statistical Software (Jandl Corp., Chicago, IL) using ANOVA or Student’s paired t test. A P-value <0.05 was considered statistically significant. Errors are given ± SEM.
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Results |
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inhibits trophoblast migration and invasion
Depending on the type of ECM substrate, EVT emerging from first-trimester placental explant cultures either migrated on top of the gels (collagen I) or invaded underneath the anchorage site (Matrigel), as previously described (18). Upon seeding on collagen I, proliferation of EVT, attachment, flattening at the distal parts of the tips, outgrowth, and spreading of EVT at the ECM surface were observed within 72 h (Fig. 1). In the presence of 10 ng/ml TNF, anchorage and proliferation to form cell columns took place, but migration of EVT away from the site of origin was not observed. Explants cultured on Matrigel at 72 h showed cytokeratin 7-positive EVT detached from the proliferating cell column and migrating deeper into the surrounding gel. However, this type of distal invasion could not be observed in 284 out of 315 TNF-treated explant cultures isolated from 21 different placentas. Although anchorage and column formation were not affected, EVT did not move away from the proximal areas of the explant in the presence of TNF, forming instead a shell-like structure underneath the Matrigel surface. This build-up of cells suggested that EVT generated in the initial (24 h) proliferative burst were inhibited from migrating. The effect of cytokine was abolished upon treatment with TNF-blocking antibodies (not shown).
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). Human leukocyte antigen (HLA)-G and integrin 5 are expressed in EVT but not in villous trophoblast, and RT-PCR demonstrated that a clean separation could be achieved, with negligible levels of expression in the villous tissue pool. Semiquantitative RT-PCR revealed that mRNA levels of integrins 1, 5, 6, and HLA-G1 in EVT might not be affected by TNF treatment.
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on EVT differentiation in proximal and distal columns and in migrating cells (Fig. 2B
). EVT-specific expression of HLA-G1 and integrin 1 and suppression of integrin 6 in the invasive cells were confirmed. Interestingly, 1 was absent from proximal parts of the column but present more distally, suggesting that the in vitro system closely mimics the in vivo differentiation process (22). The nonmigrating EVTs seen in the presence of TNF also expressed integrin 1 and HLA-G1, but now in close proximity to the villus, suggesting that their initial differentiation program was not disturbed.
TNF does not affect cell cycle progression or activation of EVT-specific proteins
TNFRI mRNA expression was detectable in villi and EVT of both controls and TNF-treated cultures (Fig. 2A). Increased production of soluble TNFRI was observed in supernatants of TNF-treated explants (Fig. 3A). Inducible secretion of the extracellular receptor domain was inhibited upon addition of TNF-blocking antibodies. In control tissue sections, TNFRI protein was detectable in cytotrophoblasts of the villus, the cell column, and in EVT, but not in the syncytium (Fig. 3B). TNFRII was weakly expressed only in villous mesenchymal cells, suggesting that the effect of the cytokine on EVT was mediated through TNFRI.
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-mediated cell death (Fig. 3B). However, M30-positive trophoblasts could be detected in areas of the proximal cell column. Counting of all nonvillous trophoblasts of the explants revealed that 9.6% ±1.2 nuclei stained positive in controls, whereas 13.8% ±1.2 nuclei produced the epitope under TNF (P = 0.06), indicating a trend toward increased apoptosis in cells in proximal columns.
To study the influence of TNF on trophoblast growth, explants were analyzed for proliferating cellular nuclear antigen (PCNA) and S-phase progression by BrdU labeling during the first 24 h after attachment to Matrigel (Fig. 4). In controls, nuclear expression of PCNA and incorporation of BrdU were observed in the cell column, whereas no signals could be observed in cytotrophoblasts attached to the Matrigel surface. Interestingly, 5ß1 and 1 integrin, HLA-G1, MMP-2 (not shown), and the cyclin-dependent kinase inhibitor p57KIP2 increased in distal cytotrophoblasts of the cell column, suggesting activation of EVT-specific proteins before the onset of invasion. Similarly, growth-arrested cytotrophoblasts expressing EVT marker proteins could be detected in the presence of TNF. A total of 6.0 ± 0.7% and 6.8 ± 0.3% EVT reacted with M30 antibodies in the absence or presence of cytokine, respectively (P = 0.34); 82 ± 9.3% and 78 ± 8.7% nuclei were BrdU labeled in controls and TNF-treated cell columns, respectively (P = 0.6), suggesting that the cytokine did not influence cell cycle progression.
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-mediated inhibition
To investigate the role of proteolysis in TNF-mediated inhibition of migration, MMP-2, MMP-9, and TIMPs-1, -2, and -3 were examined (Fig. 5). RT-PCR revealed considerable expression of MMP-9 both in villi and EVT of TNF-supplemented explants (Fig. 5A). Using gelatin zymography (Fig. 5B), the pro-form of MMP-9 (92 kDa) was detectable in supernatants of cultures only in the presence of TNF (9 of 10 cultures analyzed). Active MMP-9 enzyme was not detected at 72 h in two different pools of control supernatants (48 and 200 first-trimester explants) nor in individual explants (8–10 weeks; n = 11). Thus, at least in vitro, MMP-9 activity is not a prerequisite for EVT differentiation and invasion.
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). Production of soluble TIMP-1 (Fig. 5C) and TIMP-3 (Fig. 5D) did not change upon addition of TNF, whereas TIMP-2 was undetectable by ELISA in supernatants of controls and cytokine-treated organ cultures (not shown).
TNF restricts trophoblast invasion through elevation of PAI-1
PAI-1 (but not PAI-2) is detectable in EVT in vivo (24, 25). Components of the uPA/PAI system were analyzed in explant culture by RT-PCR (Fig. 6A). Expression of transcripts encoding tissue-type plasminogen activator (tPA), PAI-1, uPA, and its receptor (uPAR) was detectable in RNA pools of villi and EVT. Interestingly, a rise of PAI-1 protein was detectable in supernatants of TNF-treated first-trimester explants (Fig. 6B). In comparison with untreated cultures (100%), increases to 270% and 340%, respectively, were observed in two different pools of TNF-supplemented explants. The increase of PAI-1 in nonmigrating trophoblasts was also observed by immunohistochemistry, whereas protein expression was below the detection limit in EVT of controls (Fig. 6C). Both uPA and uPAR were found to be expressed in the matrix-anchored trophoblasts, regardless of the presence of the cytokine. Accordingly, levels of soluble uPA of supernatants did not change upon TNF treatment (Fig. 6D).
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depend on PAI-1, blocking studies using inhibitory antibodies were performed (Fig. 7). Despite the presence of TNF, the antibody MA-56A7C10, which blocks the active conformation of PAI-1 (21), restored migration (13 of 16 cultures on Matrigel; Fig. 7A). TIMP-1-neutralizing antibodies, which increased outgrowth/migration in control explants (four of five cultures), could not relieve the adverse effects of TNF (eight of eight cultures on Matrigel). Similarly, TNF--treated explants recovered the migratory phenotype upon addition of the PAI-1-blocking antibody in explants cultivated on collagen I (five of six; Fig. 7B), whereas TIMP-1-inhibiting antibodies were ineffective (not shown).
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Discussion |
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and its receptors in the placenta and the reproductive tract suggests multiple roles throughout pregnancy (17). Depending on gestational age, trophoblast, other placental and decidual cell types produce TNF, TNF receptor I and II, and their soluble forms (8, 26, 27, 28, 29, 30, 31). Attempts to elucidate the effect of TNF on trophoblast invasiveness using a variety of cell culture models have led to contradictory conclusions. TNF failed to regulate invasion of isolated first-trimester villous trophoblast through Matrigel, whereas reduction of cell adhesion/motility was observed using the trophoblastic cell line HTR-8/Svneo (32, 33). On the other hand, a stimulatory role of the cytokine was suggested because it was shown to increase total gelatinolytic and MMP-9 activity of isolated cytotrophoblasts (14). Stimulation of choriocarcinoma cells with TNF increased their proliferative capacity, whereas purified cytotrophoblasts of first and third trimester do not alter growth but initiate an apoptotic cascade (16, 30, 34). Thus, depending on the quality of individual trophoblast preparations and the degree of transformation of the trophoblastic cell line, the cytokine may exert distinct effects, making it difficult to draw conclusions on its role in EVT formation and differentiation.
To overcome these problems, we used human villous explant cultures of first-trimester placentae, allowing investigation of the effects of TNF on anchoring villus morphogenesis in a system that preserves the spatial relationships between differentiating trophoblast and adjacent stroma. Treatment with TNF affected neither attachment nor the proliferation and cell column formation that occurs during the early culture period (24 h). Instead, a considerable number of HLA-G1/1 integrin-positive cytotrophoblasts accumulated at the site of anchorage. These cells were post-mitotic but failed to detach or invade into the ECM. Thus, the TNF effect cannot be explained by elevated proliferation with reduced differentiation. Although different early and final stages of the apoptotic cascade were not evaluated in this study, we have investigated the cytokeratin 18 neoepitope as a marker of apoptotic trophoblasts (23). Based on this, some programmed cell death was detectable in proximal cells of the cytotrophoblast columns, and this increased upon addition of TNF. In contrast, levels of apoptosis in differentiated EVT were very low and not increased by TNF treatment. Thus, it appears that neither increased apoptosis nor elevated proliferation can be responsible for the effect of TNF. We conclude that the cytokine specifically inhibits in vitro trophoblast migration/invasion, which could mirror a putative in vivo situation upon increase of TNF in the placental bed.
TNF likely acts through TNFRI, which was found to be expressed in cytotrophoblasts of the villus, the cell column, and distal EVT in explants, whereas TNFRII was absent from all trophoblast. These results are in agreement with observations made in vivo (34). Soluble TNF-RI was previously shown to be secreted from isolated first- and term-trimester trophoblasts (27, 34), and TNF increased production in explant culture supernatants. Inducible release of soluble receptor might provide a mechanism for attenuating the adverse effects of TNF under some circumstances such as exaggerated production of the cytokine during preterm labor or chorioamnionitis (35, 36).
MMP-2 and -9 have been suggested to be important for trophoblast invasion (37, 38, 39). In agreement with previous data (14), induction of MMP-9 by TNF may occur in both villous trophoblasts and EVT. Levels of soluble TIMP-1 and -3, which inhibit MMP-2 and -9 (40), were not altered. Interestingly, antibody-mediated inhibition of TIMP-1 function increased EVT migration in control explants. However, the antimigratory effect of TNF- was not inhibited by blocking TIMP, suggesting that the signaling pathway restraining migration is independent of gelatinase activity. MMP-9 mRNA seemed to be low in villi of controls and could not be observed in untreated EVT. Accordingly, MMP-9 activity could not be detected in supernatants of unstimulated cultures. That data suggest that MMP-9 may not be necessary for EVT invasion/migration in vitro; and other MMPs, such as MMP-2, might play a predominant role in the process. This assumption would be in agreement with a recent report showing that MMP-9 is mainly expressed in villous cytotrophoblasts cells but few EVT of early placental tissue, whereas in situ gelatinase activity correlates with sites (cell column, EVT) of abundant MMP-2 expression (41).
PAI-1 was undetectable in distal EVT of controls by immunohistochemistry but could be easily recognized in the nonmigratory cytotrophoblasts of TNF-treated explants, and in the corresponding culture supernatants, suggesting a role in the antimigratory effect. Indeed, antibody-mediated inhibition of TNF-induced PAI-1 restored migration of EVT. TNF did not affect soluble uPA levels. TNF-dependent induction of PAI-1 seems to be a property shared with endothelial cells, which show reduced migration upon treatment with the cytokine (42, 43). Elevated PAI-1 may also protect nonmigratory cytotrophoblasts from TNF-induced apoptosis, which has been previously noticed in the promyelocytic leukemia cell line HL-60 (44). Thus, the present data indicate that the effect of TNF on EVT migration could be mediated via the uPA/PAI-1 system, which has been shown to be important in cell migration in other cell types (45).
PAI-1 may block trophoblast migration through multiple mechanisms. It can directly block uPA receptor (uPAR)-bound uPA and thus inhibit ECM degradation initiated by cell-associated uPA (46). In addition, PAI-1 can prevent cell migration by inhibiting the interaction between vitronectin and integrin vß3 or binding of vitronectin to uPAR (47, 48). Both uPA and integrin vß3 have been suggested to play a role in trophoblast invasiveness (49, 50).
Finally, our results could suggest a link between elevated TNF levels, enhanced PAI-1 expression, and impaired trophoblast invasion in preeclampsia. Increased levels of PAI-1 were detected in plasma and syncytium of preeclamptic women, whereas elevated TNF concentrations were detected in preeclamptic sera, villi, and some decidual stromal cells (8, 9, 19). Our data show that TNF increases soluble PAI-1 produced by explant cultures, which according to immunohistochemistry and RT-PCR is likely derived from both syncytio- and cytotrophoblast. We suspect that defects in the vascular connection of mother and fetus may result in local hypoxia and production of TNF (11), which could lead to reduced trophoblast migration through a PAI-1-coupled mechanism. Because decreased invasiveness likely provokes local hypoxia and inflammation, more TNF might be generated at the maternal-decidual interface, which could further restrict invasion. This negative feedback cycle could perpetuate conditions favoring preeclampsia.
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Acknowledgments |
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Footnotes |
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Abbreviations: BrdU, 5-Bromo-2-deoxy-uridine; DAPI, 4'6-diamidine-2'-phenylindole dihydrochloride; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; EVT, extravillous trophoblasts; HLA, human leukocyte antigen; MMP, matrix metalloproteinase; PAI, plasminogen activator inhibitor; PCNA, proliferating cellular nuclear antigen; SDS, sodium dodecyl sulfate; TBST, Tris-buffered saline with Tween 20; TIMP, tissue inhibitor of metalloproteinases; TNFR, TNF receptor; tPA, tissue-type plasminogen activator; uPA, urokinase plasminogen activator; uPAR, uPA receptor.
Received August 4, 2003.
Accepted October 31, 2003.
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D. Iacob, J. Cai, M. Tsonis, A. Babwah, C. Chakraborty, R. N. Bhattacharjee, and P. K. Lala Decorin-Mediated Inhibition of Proliferation and Migration of the Human Trophoblast via Different Tyrosine Kinase Receptors Endocrinology, December 1, 2008; 149(12): 6187 - 6197. [Abstract] [Full Text] [PDF]
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C. J. Lockwood, C. Oner, Y. H. Uz, U. A. Kayisli, S. J. Huang, L. F. Buchwalder, W. Murk, E. F. Funai, and F. Schatz Matrix Metalloproteinase 9 (MMP9) Expression in Preeclamptic Decidua and MMP9 Induction by Tumor Necrosis Factor Alpha and Interleukin 1 Beta in Human First Trimester Decidual Cells Biol Reprod, June 1, 2008; 78(6): 1064 - 1072. [Abstract] [Full Text] [PDF]
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 C. J. Lockwood, C.-F. Yen, M. Basar, U. A. Kayisli, M. Martel, I. Buhimschi, C. Buhimschi, S. J. Huang, G. Krikun, and F. Schatz Preeclampsia-Related Inflammatory Cytokines Regulate Interleukin-6 Expression in Human Decidual Cells Am. J. Pathol., June 1, 2008; 172(6): 1571 - 1579. [Abstract] [Full Text] [PDF]
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J. Prast, L. Saleh, H. Husslein, S. Sonderegger, H. Helmer, and M. Knofler Human Chorionic Gonadotropin Stimulates Trophoblast Invasion through Extracellularly Regulated Kinase and AKT Signaling Endocrinology, March 1, 2008; 149(3): 979 - 987. [Abstract] [Full Text] [PDF]
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 S. J. Rogerson, V. Mwapasa, and S. R. Meshnick Malaria in Pregnancy: Linking Immunity and Pathogenesis to Prevention Am J Trop Med Hyg, December 1, 2007; 77(6_Suppl): 14 - 22. [Abstract] [Full Text] [PDF]
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H. Horita, E. Kuroda, T. Hachisuga, M. Kashimura, and U. Yamashita Induction of prostaglandin E2 production by leukemia inhibitory factor promotes migration of first trimester extravillous trophoblast cell line, HTR-8/SVneo Hum. Reprod., July 1, 2007; 22(7): 1801 - 1809. [Abstract] [Full Text] [PDF]
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G. St. J. Whitley, P. R. Dash, L.-J. Ayling, F. Prefumo, B. Thilaganathan, and J. E. Cartwright Increased Apoptosis in First Trimester Extravillous Trophoblasts from Pregnancies at Higher Risk of Developing Preeclampsia Am. J. Pathol., June 1, 2007; 170(6): 1903 - 1909. [Abstract] [Full Text] [PDF]
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C. J. Lockwood, P. Toti, F. Arcuri, E. Norwitz, E. F. Funai, S.-T. J. Huang, L. F. Buchwalder, G. Krikun, and F. Schatz Thrombin Regulates Soluble fms-Like Tyrosine Kinase-1 (sFlt-1) Expression in First Trimester Decidua: Implications for Preeclampsia Am. J. Pathol., April 1, 2007; 170(4): 1398 - 1405. [Abstract] [Full Text] [PDF]
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 Y. Purwosunu, A. Sekizawa, K. Koide, A. Farina, N. Wibowo, G. H. Wiknjosastro, S. Okazaki, H. Chiba, and T. Okai Cell-Free mRNA Concentrations of Plasminogen Activator Inhibitor-1 and Tissue-Type Plasminogen Activator Are Increased in the Plasma of Pregnant Women with Preeclampsia Clin. Chem., March 1, 2007; 53(3): 399 - 404. [Abstract] [Full Text] [PDF]
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S. J. Renaud, S. K. Macdonald-Goodfellow, and C. H. Graham Coordinated Regulation of Human Trophoblast Invasiveness by Macrophages and Interleukin 10 Biol Reprod, March 1, 2007; 76(3): 448 - 454. [Abstract] [Full Text] [PDF]
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 Y. Hu, J. P. Dutz, C. D. MacCalman, P. Yong, R. Tan, and P. von Dadelszen Decidual NK Cells Alter In Vitro First Trimester Extravillous Cytotrophoblast Migration: A Role for IFN-{gamma} J. Immunol., December 15, 2006; 177(12): 8522 - 8530. [Abstract] [Full Text] [PDF]
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 C. Leisser, L. Saleh, S. Haider, H. Husslein, S. Sonderegger, and M. Knofler Tumour necrosis factor-{alpha} impairs chorionic gonadotrophin {beta}-subunit expression and cell fusion of human villous cytotrophoblast Mol. Hum. Reprod., October 1, 2006; 12(10): 601 - 609. [Abstract] [Full Text] [PDF]
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J. Pollheimer, T. Loregger, S. Sonderegger, L. Saleh, S. Bauer, M. Bilban, K. Czerwenka, P. Husslein, and M. Knofler Activation of the Canonical Wingless/T-Cell Factor Signaling Pathway Promotes Invasive Differentiation of Human Trophoblast Am. J. Pathol., April 1, 2006; 168(4): 1134 - 1147. [Abstract] [Full Text] [PDF]
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C. J. Lockwood, P. Matta, G. Krikun, L. A. Koopman, R. Masch, P. Toti, F. Arcuri, S.-T. J. Huang, E. F. Funai, and F. Schatz Regulation of Monocyte Chemoattractant Protein-1 Expression by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} in First Trimester Human Decidual Cells: Implications for Preeclampsia Am. J. Pathol., February 1, 2006; 168(2): 445 - 452. [Abstract] [Full Text] [PDF]
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 G. Buchwalter, C. Gross, and B. Wasylyk The Ternary Complex Factor Net Regulates Cell Migration through Inhibition of PAI-1 Expression Mol. Cell. Biol., December 15, 2005; 25(24): 10853 - 10862. [Abstract] [Full Text] [PDF]
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I. Kenis, S. Tartakover-Matalon, N. Cherepnin, L. Drucker, A. Fishman, M. Pomeranz, and M. Lishner Simvastatin has deleterious effects on human first trimester placental explants Hum. Reprod., October 1, 2005; 20(10): 2866 - 2872. [Abstract] [Full Text] [PDF]
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X. Zhang, K. E. Green, C. Yallampalli, and Y. L. Dong Adrenomedullin Enhances Invasion by Trophoblast Cell Lines Biol Reprod, October 1, 2005; 73(4): 619 - 626. [Abstract] [Full Text] [PDF]
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 C. Nicola, A. V. Timoshenko, S. J. Dixon, P. K. Lala, and C. Chakraborty EP1 Receptor-Mediated Migration of the First Trimester Human Extravillous Trophoblast: The Role of Intracellular Calcium and Calpain J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4736 - 4746. [Abstract] [Full Text] [PDF]
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L. Belkacemi, G. E. Lash, S. K. Macdonald-Goodfellow, J. D. Caldwell, and C. H. Graham Inhibition of Human Trophoblast Invasiveness by High Glucose Concentrations J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4846 - 4851. [Abstract] [Full Text] [PDF]
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S. J. Renaud, L.-M. Postovit, S. K. Macdonald-Goodfellow, G. T. McDonald, J. D. Caldwell, and C. H. Graham Activated Macrophages Inhibit Human Cytotrophoblast Invasiveness In Vitro Biol Reprod, August 1, 2005; 73(2): 237 - 243. [Abstract] [Full Text] [PDF]
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G. E. Lash, H. A. Otun, B. A. Innes, J. N. Bulmer, R. F. Searle, and S. C. Robson Inhibition of Trophoblast Cell Invasion by TGFB1, 2, and 3 Is Associated with a Decrease in Active Proteases Biol Reprod, August 1, 2005; 73(2): 374 - 381. [Abstract] [Full Text] [PDF]
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T. Saarela, M. Hiltunen, S. Helisalmi, S. Heinonen, and M. Laakso Tumour necrosis factor-{alpha} gene haplotype is associated with pre-eclampsia Mol. Hum. Reprod., June 1, 2005; 11(6): 437 - 440. [Abstract] [Full Text] [PDF]
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