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BOR - Papers in Press, published online ahead of print March 17, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026716
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BIOLOGY OF REPRODUCTION 71, 244–252 (2004)
DOI: 10.1095/biolreprod.103.026716
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Interaction of Cocultured Decidual Endothelial Cells and Cytotrophoblasts in Preeclampsia1

S. Campbell3, J. Rowe3, C.J. Jackson4, and E.D.M. Gallery2,3

Perinatal Research Group,3 Kolling Institute of Medical Research Sutton Arthritis Research Laboratory,4 Institute of Bone and Joint Research, The University of Sydney at Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Disturbed cell-cell communication between trophoblasts and the maternal endothelium may be responsible for the deficient endovascular invasion seen in preeclampsia. In vitro studies have been hampered by lack of suitable models to directly examine interactions between these cell types. Using a bilayer coculture model, we examined the effect of decidual endothelial cells on matrix metalloproteinase secretion and the migration of cytotrophoblasts from preeclamptic pregnancies. Cells were incubated on semipermeable membranes in 20% or 2% O2 with or without the tumor promoter phorbol 12-myristate 13-acetate, which activates matrix metalloproteinase-2 and -9 in endothelial cells. Cytotrophoblasts from preeclamptic pregnancies secreted significantly less matrix metalloproteinase-2 and -9 than their normal counterparts. Although decidual endothelial cells downregulated cytotrophoblast migration in normal pregnancy, this was not observed in cocultures with cytotrophoblasts from preeclamptic pregnancies. In addition, cytotrophoblasts from preeclamptic pregnancies altered phorbol myristate acetate-induced activation of endothelial matrix metalloproteinases. Hypoxia increased cytotrophoblast migration when cells were incubated alone but not in coculture with decidual endothelial cells due to increased adhesion between the two cell types. These results suggest dysfunctional interactive regulation of migration and matrix metalloproteinase secretion in preeclampsia that could result in abnormal endovascular trophoblast invasion of the maternal vasculature.

decidua, placenta, pregnancy, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Migration and invasion of trophoblasts into the uterine wall and maternal vasculature are pivotal to the success of placentation. Interstitial trophoblasts invade the uterine tissue, anchoring the placenta to the uterus, and preparing the decidual spiral arteries for endovascular migration [1, 2]. Extravillous trophoblasts migrate to the maternal spiral arteries, traverse the endothelium, and then migrate along its luminal surface, partially replacing it and the musculoelastic tissue that normally maintains vessel integrity. This process converts the uterine spiral arteries into low-resistance, high-capacitance vessels allowing sufficient blood flow across the materno-fetal interface to meet the demands of the growing fetus.

Cellular invasion of the extracellular matrix is a complex, multistep process involving the concerted action of adhesive, degradative, and migratory pathways [3]. Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes capable of degrading almost all components of the extracellular matrix [4]. Because basement membranes are the major structural hindrance for invading cells, the two gelatinases, MMP-2 and MMP-9 (which cleave type IV collagen, the main component of basement membranes), are therefore regarded as key enzymes in the invasion process. MMP expression is induced in a variety of cell types by a number of stimuli, including growth factors such as vascular endothelial growth factor (VEGF) [5], chemical agents such as the phorbol esters [6], physical stress such as hypoxia [7], and cell-matrix and cell-cell interactions [8, 9].

Increasing evidence suggests that early embryonic development takes place in a relatively hypoxic environment and that low oxygen tension has both a physiological role in organogenesis, and an important regulatory role in angiogenesis [10], trophoblast invasion and differentiation [11, 12]. Although the exact mechanisms by which oxygen modulates these events are unknown, MMP and tissue inhibitors of MMP (TIMP) production may be involved.

Defects in the process of placentation precede several disorders of pregnancy, including preeclampsia. This multifactorial, multisystem disorder classically manifests in the third trimester, with clinical features reflecting widespread maternal vascular dysfunction, probably due to endothelial cell activation and damage [13]. The critical defect, however, occurs much earlier in pregnancy. Histological examination of the placental bed has revealed that the ability of trophoblasts to enter and transform the spiral arteries appears to be impaired in preeclampsia. Many in situ and in vitro studies have shown functional defects in trophoblasts isolated from preeclamptic pregnancies [14–17]. However, trophoblast invasion and degradation of the extracellular matrix are governed not only by intrinsic trophoblast cell programming but also by interaction with the maternal cellular environment [18]. Therefore, disturbed cell-cell communication between trophoblasts and the maternal endothelium may be responsible for deficient endovascular invasion.

Surprisingly, there have been few reports specifically examining interactions between trophoblasts and vascular cells of the spiral arteries. Most studies have been limited to immunohistochemical analysis of placental and uterine tissues [19–21] or placental explant models [22]. To examine interactions between pure populations of decidual endothelial cells and cytotrophoblasts, we developed an in vitro bilayer coculture model [23] and reported an interactive regulation of cytotrophoblast migration and MMP-9 secretion in normal pregnancy [24]. In the study reported here, we investigated the hypothesis that in cocultures involving cytotrophoblasts from preeclamptic pregnancies, there would be downregulation of MMP production or activation and that this would be reflected in reduced cytotrophoblast migration.


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

Subjects were normally pregnant and preeclamptic women who were delivered by cesarean section before the onset of labor. The study was approved by the Human Research Ethics Committee of Royal North Shore Hospital, and all subjects gave written informed consent. A woman was considered normal if her blood pressure never rose above 130/80 mm Hg during the index pregnancy and the cesarean section was performed for obstetric indications. Preeclampsia was defined as follows [25]: de novo hypertension (blood pressure > 140/90 mm Hg), proteinuria (>300 mg/ day or 2+ dipstick), and hyperuricemia (serum uric acid > 0.35 mmol/L) occurring after 20-wk amenorrhoea.

Decidual Endothelial Cell Preparation

Endothelial cells were isolated from decidual biopsies (0.5–1.0 g) collected at cesarean section as described previously [26]. Briefly, decidual tissue was enzyme digested at 37°C in 0.08% pronase/0.12% trypsin in Hank balanced salt solution. Cells were manually squeezed from the digested tissue, then purified by positive selection using Dynabeads M-450 (Dynal, Oslo, Norway) coated with the lectin ulex europaeus 1 (Sigma, St. Louis, MO). This procedure resulted in a population of endothelial cells of >95% purity as identified by immunohistochemistry for Factor VIII-related antigen (DAKO, Carpinteria, CA). Cells were maintained in 40% pooled human pregnancy serum in Medium 199 (Sigma) with added heparin (100 µg/ml; Sigma) and endothelial cell growth supplement (a partially purified bovine brain extract prepared in our laboratory, ~50 µg/ ml) in a humidified 95% air and 5% carbon dioxide atmosphere. Experimental incubations were conducted at passage 2.

Cytotrophoblast Preparation

Cytotrophoblasts were isolated from placental villous tissue as described previously [24]. Briefly, cells were harvested by washing and mincing followed by an overnight enzyme digestion at 4°C in 0.25% trypsin (Sigma), 0.02% DNase 1 (Boeringer Mannheim, Mannheim, Germany), 0.02% sodium EDTA (Sigma) in Hank balanced salt solution [27]. Cells were fractionated by density gradient centrifugation at 800 x g for 30 min at 4°C on a preformed continuous gradient of isotonic Percoll (Amersham Pharmacia Biotech, Buckinghamshire, England). Cells sedimenting at densities between 1.048 and 1.062 g/ml were collected, and nontrophoblastic cells removed by immunoadsorption with immobilized monoclonal anti-HLA-I and -II antibody-coated Dynabeads M-450 (Dynal). Cells not bound to these antibodies were washed and seeded immediately for experimental incubation. This procedure resulted in a population of cytotrophoblasts of >95% viability as measured by trypan blue staining and >95% purity as assessed by immunocytochemistry of cytospins prepared from cell suspensions immediately following isolation, using a Shandon Cytospin 3 (Shandon Scientific, Cheshire, England). Cytotrophoblasts were identified by the presence of cytokeratin-7 (clone OV-TL 12/30; epithelial marker) and absence of Factor VIII-related antigen (endothelial cell marker), Leukocyte Common Antigen (leukocyte marker), CD-63 (macrophage marker), and vimentin (mesenchymal cell marker). All antibodies were purchased from DAKO, and immunocytochemistry was performed according to the manufacturer's instructions.

The secretion of progesterone by all trophoblastic cells isolated was measured as an indicator of their functional integrity [24]. Levels of progesterone were measured in conditioned media collected after a 20-h incubation (1 x 106 cells/ml in 10% bovine calf serum in Medium 199) by a routine diagnostic autoanalytic method (Immulite 2000; Diagnostic Products Corporation, Los Angeles, CA).

Coculture Experiments

Bilayer coculture experiments were conducted as described previously [23, 24]. Briefly, Millicell-PCF culture plate inserts (Millipore, Bedford, MA) of 12-µm pore size in 24-well plates (Nunc, Roskilde, Denmark) were coated on each side with bovine collagen Type 1 (Collaborative Biomedical Products, Bedford, MA). Well bases were coated with 0.1% gelatin (BDH Ltd., Poole, England). After adherence of decidual endothelial cells (7 x 104 cells/insert) to the lower surface of the insert membrane, the insert was inverted and cells were washed and quiesced in serum-free Medium 199 for 1 h. Incubations began with the addition of freshly isolated cytotrophoblasts (1.6 x 105 cells/insert) to the upper side of the insert. The total incubation volume was 0.5 ml/well.

Each coculture experiment used cytotrophoblasts (CTB) and decidual endothelial cells (DEC) obtained from individual subjects. Cytotrophoblasts from preeclamptic women (PECTB) were incubated with decidual endothelial cells from normal women (PECTB with NDEC, n = 6 cell preparations of each cell type) and with decidual endothelial cells from preeclamptic women (PECTB with PEDEC, n = 6 cell preparations of each cell type). Incubations with PECTB were compared with incubations with normal cells (NCTB with NDEC, n = 6 cell preparations of each). Control incubations with cytotrophoblasts alone and decidual endothelial cells alone on their respective sides of the inserts were also conducted on the same 24-well plate treated identically to the coculture situation.

Incubations were conducted in duplicate for 20 h in 10% charcoal-stripped bovine calf serum (Sigma) in Medium 199, in the absence or presence of 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma). Prior to the experiment, gelatinases present in the bovine calf serum were removed by chromatography through a gelatin-Sepharose affinity column (Pharmacia Biotech, Uppsala, Sweden) [28]. At the end of incubation, conditioned media from both sides of the membrane were pooled, centrifuged at 176 x g for 5 min, and stored at –70°C until analysis.

In three experiments, additional cells (alone and in coculture) on the same 24-well plate were treated with recombinant human (rh) VEGF165 (R&D Systems, Minneapolis, MN). The rhVEGF165 (50 ng/ml) was added alone or pretreated with plasmin (4 U/ml; Sigma) to cleave the VEGF165 heparin-binding domain [29]. For 3 h before the beginning of the experimental incubations, VEGF (1 µg/ml) was predigested at 37°C with an equal volume of plasmin (8 U/ml). A control condition with carrier protein and plasmin was treated identically.

In two experiments using cells from normal pregnancies and three experiments using cells from preeclamptic pregnancies (incubated alone and in coculture), an identical 24-well plate was incubated under hypoxic conditions (2% O2 {cong} pO2 14 mm Hg) in a Forma Water-Jacketed Tri-gas Precision Incubator, as described previously [30].

Analysis of Supernatants

MMPs were examined as previously described [24]. Activities of the gelatinases (MMP-2 and MMP-9) were analyzed by zymography. Interstitial collagenase (MMP-1) was detected by Western immunoblot analysis. The gels and blots were scanned into a Macintosh computer and the intensity of the bands was semiquantitated using NIH Image (National Institute of Health, Bethesda, MD). TIMP-1 was measured in conditioned media using a commercially available Biotrak ELISA kit (Amersham Pharmacia Biotech, Buckinghamshire, England).

Assessment of Cytotrophoblast Migration

At the end of incubation, the upper membrane surface was manually scraped off, and cells on the lower membrane surface and well base were fixed in buffered 4% paraformaldehyde and immunostained with monoclonal antibody to cytokeratin (clone LP34) (1:200 dilution; DAKO) with hematoxylin counterstaining as described previously [24]. The primary antibody was detected with a secondary antibody and streptavidin-biotin complex, linked to peroxidase (DAKO) and the chromagen 3,3'-diaminobenzidine tetrahydrochloride (DAKO). An irrelevant primary mouse monoclonal antibody was used as a negative control in all cases. Cytokeratin-stained trophoblast cell bodies and processes that had migrated through the membrane pores and adhered to the lower surface of the membrane, or had migrated completely through the decidual endothelial cell monolayer and settled onto the base of the wells were counted under a light microscope. Total cytotrophoblast migration was calculated as the sum of these two counts.

Statistical Methods

To allow comparison of MMPs on different gels, an aliquot of the same positive control containing Medium 199 with 0.5% bovine calf serum was loaded onto all gels. MMP activity data were normalized by expression as percent of positive loading control. For clarity, MMP data are illustrated in figures as means ± SEM (standard error of the mean). Cytotrophoblast migration is shown in figures as individual data points or as mean ± SEM. Unpaired Mann-Whitney tests were used for comparisons of NCTB versus PECTB alone and in coculture with DEC. Within-group changes in response to different experimental conditions and coculture were analyzed by the Wilcoxon rank sum test. These nonparametric tests make no assumptions about the normality of the data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Progesterone Secretion by Cytotrophoblasts from Preeclamptic Pregnancies

All cytotrophoblasts from preeclamptic pregnancies secreted progesterone (82.58 ± 11.23 nmol/106 cells, mean ± SEM) and levels were not significantly different from those of normal pregnancies (82.56 ± 57.36 nmol/106 cells, mean ± SEM). As for normal cytotrophoblasts, the level of progesterone secretion by cells from preeclamptic pregnancies was not correlated with technical aspects of the isolation procedure such as the length of time between delivery of the placenta and collection of cotyledons or the length of overnight enzymatic digestion of placental tissue. Gestation at delivery was significantly lower (P < 0.001) in the preeclamptic (media n = 218, range = 82 days; n = 12) than in the normal group (median = 274, range = 8 days; n = 6). There was no correlation between progesterone secretion and gestation at delivery of the placentas in either the normal (r = 0.303, n = 6) or preeclamptic group (r = 0.511, n = 12).

Gelatinase Secretion under Basal Conditions

Under basal conditions, latent but not active forms of MMP-9 and MMP-2 were visible on gelatin zymograms. As shown in Figure 1, a and b, under basal conditions, levels of latent MMP-9 and MMP-2 were significantly lower when cytotrophoblasts from preeclamptic pregnancies (n = 12) were incubated alone compared with cytotrophoblasts from normal pregnancies (n = 6) (P < 0.01 and P < 0.05, respectively). As reported previously [24], an interactive decrease of secreted levels of MMP-9 was shown in cocultures of normal cytotrophoblasts and decidual endothelial cells. To determine whether this also occurred with cytotrophoblasts from preeclamptic pregnancies, they were cocultured with decidual endothelial cells from normal or preeclamptic women, under basal conditions and in response to PMA, and results compared with cocultures of cells from normal women (NCTB/NDEC). Results, expressed as a percentage of the sum of levels secreted by the two cell types when they were incubated alone, are shown in Figure 1c (MMP-9) and Figure 1d (MMP-2). Levels of secreted MMP-9 were significantly lower (P < 0.05) when cytotrophoblasts from preeclamptic pregnancies were cocultured with decidual endothelial cells from either normal (n = 6) or preeclamptic women (n = 6) compared with the total MMP-9 secretion by the two cell types incubated alone (Fig. 1c). This decrease was similar to that seen in basal cocultures using normal cytotrophoblasts (Fig. 1c). Under basal conditions, levels of latent MMP-2, as analyzed by zymography, were not significantly different when cytotrophoblasts from preeclamptic pregnancies were cocultured with decidual endothelial cells from either normal or preeclamptic women compared with the total amount of MMP-2 secreted by the two cell types incubated alone (Fig. 1d). This lack of effect was also seen in basal cocultures using normal cytotrophoblasts (Fig. 1d).



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  		FIG. 1. Levels of secreted MMP-9 and MMP-2 in normal pregnancy (N) and in preeclampsia (PE). a) Levels of latent MMP-9 and b) latent MMP-2 secreted by cytotrophoblasts from normal and preeclamptic pregnancies incubated alone (n = 6 normal and n = 6 preeclamptic cytotrophoblast cell preparations, each from independent patient samples). Results (illustrated as mean ± SEM) are expressed as a percentage of secretion by normal cytotrophoblasts under basal conditions (##, P < 0.01 levels of latent MMP-9 in cytotrophoblasts from preeclamptic versus normal pregnancies; #, P < 0.05 levels of latent MMP-2 in cytotrophoblasts from preeclamptic versus normal pregnancies; **, P < 0.01 levels of latent MMP-9 from preeclamptic cytotrophoblasts in the presence of PMA, compared with the same cells under basal conditions). c) Levels of latent MMP-9 and d) latent MMP-2 in cocultures of normal cytotrophoblasts with normal decidual endothelial cells (NCTB/NDEC, n = 6 cell preparations of each cell type) and in cocultures of preeclamptic cytotrophoblasts with normal or preeclamptic decidual endothelial cells (PECTB/ NDEC, n = 6 cell preparations of each cell type; and PECTB/PEDEC, n = 6 cell preparations of each cell type). All cell preparations were from independent patient samples. Results (illustrated as mean ± SEM) are expressed as a percentage of the sum of the secretion by the same cells (normal or preeclamptic) incubated alone, either under basal conditions or in the presence of PMA (*, P < 0.05 latent MMP-9 and MMP-2 secreted in coculture versus sum of cells alone; #, P < 0.05 levels of latent MMP-2 in coculture under basal conditions versus in the presence of PMA; {phi}, P < 0.05 levels of latent MMP-2 secreted by cocultures of preeclamptic cells [PECTB/PEDEC] versus normal cells [NCTB/NDEC], both in the presence of PMA)

MMP-1 Secretion in Preeclampsia

Levels of MMP-1 secreted by decidual endothelial cells from preeclamptic women (n = 6) were very low in contrast with the easily measurable levels from their normal counterparts (n = 6), as previously reported [6]. By Western blotting, levels of MMP-1 from normal decidual endothelial cells were unaffected by coculture with cytotrophoblasts from normal (n = 6) or preeclamptic (n = 6) pregnancies (data not shown).

TIMP-1 Secretion in Preeclampsia

Levels of TIMP-1 secreted by normal cytotrophoblasts, quantitated by ELISA, were much lower than that of normal decidual endothelial cells (12.42 ± 2.73 ng/106 cells versus 542.74 ± 134.08 ng/106 cells, mean ± SEM). Even at these low levels, it was apparent that levels of TIMP-1 were significantly lower (P < 0.05) from cytotrophoblasts from preeclamptic pregnancies (n = 6) than from their normal counterparts (n = 6), while levels of TIMP-1 were similar from decidual endothelial cells from normal and preeclamptic women (Fig. 2). Although levels of TIMP-1 were lower from cytotrophoblasts from preeclamptic than from normal pregnancies, there was no difference in levels of TIMP-1 in coculture (NCTB/NDEC, n = 6, and PECTB/ PEDEC, n = 6) compared with the sum of TIMP-1 secreted by the two cell types incubated alone (Fig. 2).



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  		FIG. 2. Levels of secreted TIMP-1 from cytotrophoblasts and decidual endothelial cells from normal and preeclamptic pregnancies. Cytotrophoblasts and decidual endothelial cells from normal pregnancies (n = 6 NCTB and n = 6 NDEC cell preparations) were incubated alone and in coculture; cytotrophoblasts and decidual endothelial cells from preeclamptic pregnancies (n = 6 PECTB and n = 6 PEDEC cell preparations) were incubated alone and in coculture. All cell preparations were from independent patient samples. Results are illustrated as mean ± SEM (#, P < 0.05 TIMP-1 secretion by normal versus preeclamptic cytotrophoblasts)

Effect of PMA on Gelatinase Secretion

PMA was used as a positive control in the coculture system because it has been shown to stimulate MMP production and activate gelatinases in decidual endothelial cells [6, 24]. However, in these experiments, PMA had little overall effect on MMP secretion by cytotrophoblasts. When cells were incubated alone, it stimulated latent MMP-9 secretion by cytotrophoblasts from preeclamptic (P < 0.01) but not normal pregnancies (Fig. 1a). Coculture incubations under basal conditions showed an overall reduction of MMP-9 levels for each combination (P < 0.05) but not of MMP-2 levels (Fig. 1, c and d). In the presence of PMA, there was a similar proportional inhibition of secreted levels of MMP-9 in coculture (Fig. 1c). There was a small but significant relative inhibition of secreted levels of MMP-2 in coculture in the presence of PMA in incubations including cytotrophoblasts from preeclamptic pregnancies (Fig. 1d). This was not seen when normal cytotrophoblasts were cocultured with normal decidual endothelial cells.

Although PMA activated MMP-9 and MMP-2 in decidual endothelial cells from both normal and preeclamptic women, it did not activate either enzyme in cytotrophoblasts from either normal or preeclamptic pregnancies, as shown by zymography in Figure 1, a and b. However, cytotrophoblasts from preeclamptic pregnancies cocultured with decidual endothelial cells from either normal or preeclamptic women in the presence of PMA displayed higher levels of active MMP-9 (P < 0.05) than coculture using normal cytotrophoblasts. The introduction of abnormal cytotrophoblasts (from preeclamptic pregnancies) to decidual endothelial cell cultures also resulted in marked variability of decidual endothelial cell levels of active MMP-2, with final active MMP-2 levels ranging from 50% to over 600% of those from preeclamptic cells incubated alone. This difference between the effects in coculture of cytotrophoblasts from preeclamptic pregnancies (highly variable response) and normal pregnancies (little variation in response) is illustrated in Figure 3.



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  		FIG. 3. Levels of active MMP-2 in response to PMA. Cocultures of cells from normal pregnancies (NCTB/NDEC, n = 6 cell preparations of each cell type) were compared with those from preeclamptic pregnancies (PECTB/NDEC, n = 6 cell preparations of each cell type; and PECTB/ PEDEC, n = 6 cell preparations of each cell type). All cell preparations were from independent patient samples. Results (shown as individual data points) are expressed as percentage change from levels secreted by cells incubated alone in the presence of PMA. Highly variable levels of active MMP-2 by decidual endothelial cells in response to coculture were seen with cytotrophoblasts from preeclamptic but not normal pregnancies

Effect of VEGF on MMP and TIMP-1 Secretion in Normal Pregnancy

MMP secretion was examined in response to the physiological growth factor VEGF. Although VEGF (1 µg/ml) had no statistically significant effect on levels of MMP secreted by decidual endothelial cells, it increased the levels of MMP-2 (measured by zymography) and MMP-1 (measured by Western blotting) in all three cell preparations examined (Fig. 4, a and b). VEGF had no effect on MMPs secreted by CTB (data not shown). VEGF increased (P < 0.05) levels of secreted TIMP-1 (measured by ELISA) by decidual endothelial cells (n = 3), as shown in Figure 4c, but had no effect on TIMP-1 secreted by cytotrophoblasts. Predigestion of VEGF with plasmin did not alter these responses.



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  		FIG. 4. The effect of VEGF on decidual endothelial cell secretion of (a) the gelatinases MMP-9 and MMP-2, (b) MMP-1, and (c) TIMP-1. Results (illustrated as mean ± SEM) are shown for three decidual endothelial cell preparations, each from independent patient samples (*, P < 0.05 decidual endothelial cells under basal conditions versus in response to VEGF)

Cytotrophoblast Migration

The migration of cytotrophoblasts from preeclamptic pregnancies through a collagen-coated matrix and an endothelial monolayer was assessed and compared with that of cells from normal pregnancies. There was no correlation between basal cytotrophoblast migration and the gestation at delivery of the placentas in either normal (r = 0.276, n = 6) or preeclamptic (r = 0.219, n = 12) pregnancies.

As shown in Figure 5, total migration (the number of cytokeratin-positive cells on the underside of the insert and on the base of the well) was not significantly different in cytotrophoblasts from preeclamptic (n = 12) versus normal (n = 12) pregnancies due to outliers in the preeclamptic group, but the median number of migrated cytotrophoblasts from preeclamptic pregnancies was lower than normal cytotrophoblasts under all conditions examined. PMA had no effect on the migration of cytotrophoblasts from preeclamptic pregnancies, in contrast with the migration of normal cells, which was decreased (P < 0.01). Coculture with either NDEC or PEDEC gave similar results and have therefore been shown as a single group in Figure 5. Although coculture with decidual endothelial cells resulted in a significant reduction (P < 0.05) in the migration of normal cytotrophoblasts, it had no effect on the migration of cytotrophoblasts from preeclamptic pregnancies (Fig. 5).



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  		FIG. 5. Cytotrophoblast migration in the absence and presence of PMA. Cytotrophoblasts from normal (n = 12 cell preparations) and from preeclamptic pregnancies (n = 12 cell preparations) were cultured alone or with decidual endothelial cells. All cell preparations were from independent patient samples. Decidual endothelial cells are shown in the figure as a single group because coculture of cytotrophoblasts with either normal or preeclamptic decidual endothelial cells were not different. Results are shown as individual data points with the median as a bar (**, P < 0.01 normal cytotrophoblasts under basal conditions versus in response to PMA; *, P < 0.05 normal cytotrophoblasts alone versus coculture under basal conditions)

Effect of Hypoxia

The pO2 of medium incubated in 2% O2 was measured to determine the level of hypoxia achieved. A pO2 of ~17 mm Hg was reached in media within 15 min of incubation in 93% N2, 5% CO2, 2% O2 and was maintained throughout a 24-h incubation period. Hypoxia (2% O2) had no effect on the production of MMP-9, MMP-2, or TIMP-1 by either normal or preeclamptic cytotrophoblasts or decidual endothelial cells incubated alone or in coculture (data not shown).

Figure 6 shows the effect of hypoxia on cytotrophoblast migration. Group data are shown for all cytotrophoblast populations because cytotrophoblasts from preeclamptic and normal pregnancies displayed similar responses to hypoxia, and, as can be seen in Figure 5, there was no significant difference in the migration of cytotrophoblasts from preeclamptic or normal pregnancies under standard culture conditions. Cytotrophoblasts (n = 5) cultured alone for 20 h in 2% O2 showed a significant increase in migration (P < 0.05) compared with cells in 20% O2 (Fig. 6a). This effect of hypoxia was lost when cytotrophoblasts were cocultured with decidual endothelial cells (n = 5). However, as shown in Figure 6b, there was a difference in the final disposition of the cytotrophoblasts in hypoxic cocultures. At the end of the 20-h incubation period in 2% O2, a greater proportion of these cells had remained adherent to the lower surface of the membrane than was seen in cocultures in 20% O2 (P < 0.05), i.e., more cytotrophoblasts adhered to the endothelial cell monolayer after a 20-h incubation period than migrated all the way through to the base of the well.



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  		FIG. 6. The effect of hypoxia on cytotrophoblast migration. a) Total migration by cytotrophoblasts (n = 5 cell preparations from independent patient samples) incubated alone, with PMA, in coculture with decidual endothelial cells, or both, under standard culture conditions (20% O2) or under hypoxic conditions (2% O2). Cytotrophoblasts from normal and preeclamptic pregnancies were grouped together for analysis. Results are illustrated as mean ± SEM (#, P < 0.05 total migration by cytotrophoblasts incubated alone ± PMA in 20% O2 versus 2% O2; {phi}, P < 0.05 total migration by cytotrophoblasts in 2% O2 incubated alone under basal conditions versus in coculture ± PMA; *, P < 0.05 total migration by cytotrophoblasts in 20% O2 incubated alone under basal conditions compared with those in the presence of PMA ± decidual endothelial cells). b) The percentage of cytotrophoblasts on the lower surface of the membrane after incubation in hypoxic conditions. Cytotrophoblasts (n = 5 cell preparations from independent patient samples) were incubated alone, with PMA, in coculture with decidual endothelial cells, or both, under standard culture conditions (20% O2) or under hypoxic conditions (2% O2). Cytotrophoblasts adherent to the lower surface of the membrane were calculated as a percentage of total cytotrophoblasts that had migrated. Results are illustrated as mean ± SEM (#, P < 0.05 percentage of cytotrophoblasts on the lower surface of the insert in coculture under basal conditions in 20% O2 versus 2% O2)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Successful placentation is the consequence of controlled trophoblast interactions with the maternal cellular environment. It is therefore likely that shallow trophoblast invasion and insufficient remodeling of the maternal vasculature, as seen in preeclampsia, is not due solely to the intrinsic properties of the invading cells but also to disturbed cell-cell communication between the placental and maternal cells. This is the first report examining this interaction at a cellular level.

Using cells from 12 preeclamptic women under control conditions, we found that levels of secreted MMP-9 and MMP-2, as measured by zymography, were significantly lower from cytotrophoblasts from preeclamptic compared with normal pregnancies. This work confirms and extends a previous report [17], where third trimester cytotrophoblasts from three preeclamptic pregnancies were found to express reduced levels of MMP-9 protein and mRNA. In contrast, another report [31] showed no significant difference in the level of latent MMP-9 secreted by third-trimester trophoblasts from 10 preeclamptic pregnancies compared with normal cells. This discrepancy may be due to either the different cytotrophoblast isolation techniques used or to different inclusion criteria for the patient groups. The latter study isolated trophoblasts from the maternal aspect of the placenta, whereas we discarded this portion of the placenta, and furthermore, used HLA class I- and class II-coated magnetic beads to remove contaminating cells and to ensure a homogeneous population of villous cytotrophoblasts, free of maternal cell contamination. The latter study included subjects who had experienced labor and vaginal delivery, and of the subjects who delivered by cesarean section, it was not stated whether they labored before surgery. In addition, the latter study included subjects with 1+ dipstick proteinuria. In the study reported here, women who had been in labor (whether delivered by cesarean section or vaginally) were excluded from the study because labor affects decidual expression of MMPs and TIMP-1 [32]. To ensure that all women within the preeclamptic group were unequivocally preeclamptic, only subjects with 2+ or more proteinuria by dipstick or >300 mg/24 h were included due to the high incidence of false negatives reported for 1+ dipstick measurements [33].

In addition to a reduction in levels of secreted gelatinases, levels of secreted TIMP-1 were also significantly lower from cytotrophoblasts from preeclamptic pregnancies, as measured by Western blotting, than from their normal counterparts. Cytotrophoblast progesterone secretion was measured as an indicator of their functional integrity and was not different between the two patient groups. To our knowledge, this is the first report of reduced levels of TIMP-1 by cytotrophoblasts in preeclampsia. Levels of TIMP-1 from cytotrophoblasts were very low compared with those of decidual endothelial cells, corrected for cell number. As reported previously [24], levels of secreted TIMP-1 were not different between decidual endothelial cells from normal and preeclamptic women.

Using a function-perturbing antibody, MMP-9 has been shown to be essential for cytotrophoblast invasion in vitro [16]. However, the role of MMP-2 has not been investigated in this way. Both MMP-9 and MMP-2 degrade collagen type IV, the main component of basement membranes, and are likely to be important for endovascular trophoblasts to traverse the maternal endothelium and gain access to the maternal circulation. Although histological examination of placental bed biopsies from preeclamptic women shows abundant interstitial migration, endovascular invasion fails to proceed beyond the superficial portion of the spiral arteries and fewer vessels are breached [21]. The reduced levels of secreted gelatinases by cytotrophoblasts from preeclamptic pregnancies in vitro may at least partly explain the abnormally shallow endovascular invasion seen in vivo. To study this, we examined cytotrophoblast migration through a collagen-coated semipermeable membrane. Although total cytotrophoblast migration tended to be lower in all experimental conditions when cells were from preeclamptic pregnancies, there was no significant difference between the two patient groups due to intersubject variability. To our knowledge, there is only one other report comparing the motility of cultured cytotrophoblasts in preeclampsia. Third-trimester cytotrophoblasts from four preeclamptic pregnancies were compared with second- and third-trimester cytotrophoblasts from four normal pregnancies (although the mode of delivery was not stated) and it was found that invasion through Matrigel-coated membranes after 72 h of culture was lower in the preeclamptic compared with the normal group [17]. Reduced trophoblast outgrowths have also been reported from placental explants obtained from three preeclamptic women and grown on Matrigel for 5 days [34]. In light of the present study, it would be of interest to know if impaired outgrowth from preeclamptic explants were apparent after just 1 day in culture.

Coculture of cytotrophoblasts from both normal and preeclamptic pregnancies with decidual endothelial cells resulted in a specific reduction of levels of latent MMP-9, but not MMP-2 or MMP-1. This occurred with cytotrophoblasts from both normal and preeclamptic pregnancies and indicates an interactive regulation of MMP-9 secretion between the maternal and placental cells. Because zymography measures steady-state protein levels, it is also possible that the decrease in MMP-9 levels may be due to increased degradation of MMP-9 in coculture or increased sequestering of MMP-9 onto the extracellular matrix in coculture. Findings for migration and levels of secreted MMP-9 were concordant in normal cytotrophoblasts (i.e., there was inhibition of migration), but not for cytotrophoblasts from preeclamptic pregnancies (i.e., there was no inhibition of migration). This altered regulation of cytotrophoblast migration by the maternal endothelium suggests dysfunctional control mechanisms in cytotrophoblasts from preeclamptic pregnancies and, in particular, the possibility that other processes such as adhesion properties or other proteases are involved.

Despite a small increase in latent MMP levels from cytotrophoblasts from preeclamptic pregnancies in response to PMA, levels remained lower than in normal cytotrophoblasts. The differential effect of PMA on levels of secreted MMP was reflected in its effect on cytotrophoblast migration, as PMA caused a reduction in migration in cells from normal but not preeclamptic pregnancies. The combination of PMA and coculture had no extra effect on MMP levels from normal cells. However, there was a specific suppression of levels of both MMP-9 and MMP-2 in this situation when cytotrophoblasts from preeclamptic pregnancies were involved. PMA induced activation of both MMP-2 and MMP-9 in decidual endothelial cells, but not cytotrophoblasts. Levels of active MMP-2 from decidual endothelial cells were variable when they were cocultured with cytotrophoblasts from preeclamptic pregnancies. Such variability was not observed in cocultures using cytotrophoblasts from normal pregnancies and was particularly evident when both the cytotrophoblasts and decidual endothelial cells were from preeclamptic women. This indicates dysfunctional maternal-fetal interactive regulation of MMP-2 activation in preeclampsia. Levels of active MMP-9 from decidual endothelial cells were also affected by coculture with cytotrophoblasts from preeclamptic but not normal pregnancies. Interestingly, this increase corresponded with higher migration of preeclamptic (but not normal) cytotrophoblasts, but this did not reach statistical significance (P = 0.060). The results indicate that normal regulatory mechanisms between maternal and placental cells are disturbed in preeclampsia.

Because we previously showed no stimulation by the tumor promoter PMA on MMP production and/or activation by cytotrophoblasts from normal pregnancies, an alternative physiologically relevant putative stimuli was examined. Because VEGF has been shown to increase trophoblast motility [35], exogenous VEGF was added to some incubations, but no effect on cytotrophoblast migration or levels of secreted MMP were found, even after cleavage of the heparin-binding domain. The response of endothelial cells to VEGF was different from that of trophoblasts. It caused a modest increase in levels of latent MMP-2 and MMP-1 from decidual endothelial cells in accordance with previous reports using endothelial cells from different vascular beds [5] and resulted in a significant increase in levels of TIMP-1.

There is a growing body of work indicating a role for hypoxia in early placental development and associated abnormalities, and it is clear from in vitro studies that oxygen tension plays a regulatory role in normal trophoblast differentiation along the invasive pathway [36]. It has been suggested that one mechanism underlying deficient endovascular invasion in preeclampsia could be a defect in the ability of the trophoblasts to respond to changes in oxygen tension during early placental development [37]. However, it is the second wave of endovascular invasion that is defective in pregnancies that later develop preeclampsia, suggesting an interactive disturbance between invasive trophoblasts and host maternal cells, perhaps involving an abnormal response to the local rise in oxygen tension occurring near decidual vessels at that stage in pregnancy. An abnormal response to increasing oxygen tension may inhibit trophoblast endovascular invasion and thus result in local placental hypoxia and relative ischemia due to intermittent constriction of these vessels, which have retained their muscle coat. Indeed, the morphologic changes seen histologically in placentas from preeclamptic pregnancies have been reproduced in vitro by exposing third-trimester placental explants to hypoxic conditions [38]. In addition, preeclampsia is associated with increased expression of VEGF, which is upregulated by hypoxia [39, 40]. Despite evidence for a pathophysiological role for hypoxia in the progression of preeclampsia, a causative role for hypoxia remains unclear.

In our experiments where cytotrophoblasts were incubated alone, 20 h of exposure to hypoxia resulted in an increase in cytotrophoblast migration. This hypoxia-induced increase was observed whether cells were from normal or preeclamptic pregnancies and suggests that there is no intrinsic defect in the ability of preeclamptic trophoblasts to respond to changes in oxygen tension. These results are in keeping with a previous report [41] of hypoxic stimulation of normal first-trimester trophoblast invasiveness. In contrast, it has also been shown that first- and second-trimester trophoblast invasion through Matrigel-coated membranes was greatly reduced in response to hypoxia [11], partly attributed to an inability to switch their integrin repertoire completely. In the current experiments, when cytotrophoblasts were cocultured with decidual endothelial cells, this hypoxia-enhanced cytotrophoblast migration was no longer evident. One explanation is paracrine inhibition of hypoxia-induced cytotrophoblast migration by decidual endothelial cells. Whether such a regulator is produced by decidual endothelial cells in response to hypoxia per se or in response to factors released by hypoxic cytotrophoblasts is unknown. VEGF has been shown to be upregulated in cultured placental cells in response to hypoxia [42], whereas its antagonistic soluble receptor, VEGFR1, is upregulated in cultured endothelial cells in response to both hypoxia and VEGF [43, 44]. Our findings of increased trophoblast migration in response to hypoxia could represent an autocrine mechanism involving VEGF induction, whereas the decrease seen in coculture may involve a paracrine pathway involving VEGFR1. Although our experiments showed no effect of exogenous VEGF on cytotrophoblast migration, we cannot exclude the possibility that the cytotrophoblast response to hypoxia may be dose- or isoform-specific.

Coculture under hypoxic conditions caused a higher proportion of the migrated cytotrophoblasts to adhere to the lower surface of the membrane (in contact with the endothelium) rather than the base of the well. When cocultures were performed under standard conditions or when cytotrophoblasts were cultured alone (under standard or hypoxic conditions), the reverse was seen. This suggests that hypoxia enhances adhesive interactions between cytotrophoblasts and decidual endothelial cells, and although the physiological relevance of this interaction is not understood, hypoxic induction of cellular adhesion receptors [12, 45] may be involved, and this may also explain other histological abnormalities seen in preeclampsia, such as macrophage cuffing of decidual vessels [46]. Interestingly, hypoxic stimulation of integrin expression in microvascular endothelial cells is thought to occur partially through an autocrine-paracrine action of VEGF [45]

Although hypoxia increased the number of migratory cells in our experiments, it had no effect on levels of MMP-1, -2, -9, or TIMP-1 from cytotrophoblasts. Likewise, it had no effect on levels of secreted MMPs from decidual endothelial cells, although it has been shown to upregulate MMP-2 expression in endothelial cells from several other sources [47]. In agreement, a previous report [7] found that hypoxia had no effect on MMP-9 or MMP-2 in a first-trimester cell line, but it decreased secretion of TIMP-1. This indicates that hypoxia may cause a shift in balance between MMPs and their inhibitors, favoring increased MMP activity. This hypothesis is not supported in the study reported here, as levels of secreted TIMP-1 were unaffected by hypoxia. The regulation of trophoblast migration in response to hypoxia may involve other MMPs or TIMPs or another mechanism altogether.

In conclusion, growing evidence suggests that preeclampsia is the result of an interactive disturbance between maternal and placental cells within the utero-placental unit and not just simply an intrinsic abnormality in trophoblast cell programming. The capacity to examine direct interactions between maternal decidual endothelial cells and invading placental cytotrophoblasts affords a unique insight into their physiological regulation in normal pregnancy and its disturbance in preeclampsia. In this study, the use of primary extravillous trophoblasts was not possible because there is no reliable early marker for preeclampsia and first- and early second-trimester tissue is not readily available from ongoing pregnancies where the pregnancy outcome (be it normal or preeclamptic) is later known. There have, however, been numerous reports of the use of villous trophoblasts for invasion studies [16, 17, 48], and we have previously shown that cultured third-trimester villous cytotrophoblasts share characteristics expressed by their extravillous counterparts, namely migratory behavior and gelatinase secretion [24]. With the caveats inherent in extrapolating this in vitro study to the in vivo physiological situation, we have demonstrated significant functional abnormalities in cytotrophoblasts from preeclamptic pregnancies and disturbed cell-cell communication between cytotrophoblasts and decidual endothelial cells in preeclampsia. In addition, decidual endothelial cells were shown to limit hypoxia-mediated migratory behavior of cytotrophoblasts.


   ACKNOWLEDGMENTS
 
The authors are grateful to Sister Mei Ling Chang and the consultant obstetricians and their registrars at Royal North Shore Hospital and North Shore Private Hospital for assistance in sample collection. Thanks are also due to Fiona Elliott for technical assistance, and PaLMs, Royal North Shore Hospital, St. Leonards, Australia, for technical assistance with routine procedures, particularly progesterone measurements.


   FOOTNOTES
 
1 Supported by a Project Grant and Postgraduate Biomedical Research Scholarship from the National Health and Medical Research Council of Australia and a Royal North Shore Hospital Centenary Foundation Postgraduate Scholarship. Back

2 Correspondence: E.D.M. Gallery, Department of Renal Medicine, The University of Sydney at Royal North Shore Hospital, St. Leonards, NSW 2065, Australia. FAX: 612 9436 3719; eileeng{at}med.usyd.edu.au Back

Received: 23 December 2003.

First decision: 19 January 2004.

Accepted: 4 March 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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