Hum. Reprod. Advance Access originally published online on September 27, 2007
Human Reproduction 2007 22(11):2834-2841; doi:10.1093/humrep/dem303
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BeWo cells stimulate smooth muscle cell apoptosis and elastin breakdown in a model of spiral artery transformation
1 Maternal and Fetal Health Research Centre, Maternal and Fetal Health Research Group, University of Manchester, St. Mary's Hospital, Manchester M13 0JH, UK 2 Centre for Developmental and Endocrine Signalling, Division of Basic Medical Sciences, St. George's, University of London, Cranmer Terrace, London SW17 0RE, UK
3 Correspondence address. Tel: +44-161-276-6487; Fax: +44-161-276-6134; E-mail: john.aplin{at}manchester.ac.uk
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BACKGROUND: During pregnancy, extravillous trophoblast invades the uterine wall and enters the spiral arteries. Remodelling ensues, with loss of vascular smooth muscle cells (SMCs) to create high flow, low resistance vessels. Pregnancies complicated by pre-eclampsia are characterized by incomplete arterial remodelling. Endovascular trophoblast is not easily accessible for studies to establish the pathogenesis of pre-eclampsia, so we have developed a model appropriate to carry out mechanistic studies of vessel wall transformation.
METHODS AND RESULTS: Segments of human spiral artery were perfused with the choriocarcinoma cell line, BeWo; cells invaded the vessel wall and induced apoptosis of vascular SMC. Perfusion of vessels with BeWo-conditioned medium also induced SMC apoptosis, indicating the presence of a soluble apoptotic factor. BeWo express Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment of BeWo-conditioned medium with antibodies against FasL inhibited vascular SMC apoptosis in vitro. Antibodies that blocked TRAIL receptor function had no effect. Extracellular matrix degradation is also a prerequisite for vascular remodelling; BeWo express matrix metalloproteinase-12 (MMP-12) and BeWo-conditioned medium increased MMP-12 expression in spiral artery SMC.
CONCLUSIONS: BeWo induce arterial remodelling via FasL- and MMP-12-dependent mechanisms. BeWo-derived factors up-regulate protease expression in spiral artery SMC to facilitate matrix breakdown.
Key words: apoptosis/Fas ligand/matrix metalloproteinase/trophoblast invasion/vascular remodelling
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Introduction |
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Maintenance of a successful pregnancy is dependent on the ability of the trophoblast to invade the uterus and remodel the spiral arteries. The process of remodelling, known as physiological change, converts these vessels into high flow, low resistance conduits that lack maternal vasomotor control (Pijnenborg et al., 1983
; 2006; Kam et al., 1999; Benirschke and Kaufmann, 2000). This transformation is necessary to meet the increasing demands of the developing fetus for blood and oxygen.
Early in pregnancy, extravillous trophoblast detaches from cell columns (Aplin, 1991), and invades the uterine wall via one of two routes. Interstitial trophoblast invades the uterine stroma as far as the first third of the myometrium, while endovascular trophoblast enters the spiral arteries and migrates to a similar depth, in a retrograde manner. The combined actions of these two cell populations lead to spiral artery transformation (Pijnenborg et al., 1983; Kam et al., 1999). Vessel remodelling involves a partial loss of endothelium, and replacement of the internal elastic lamina and musculo-elastic media with an amorphous fibrinoid material in which trophoblast is embedded (Brosens et al., 1967; Pijnenborg et al., 1983). Diseases of pregnancy such as pre-eclampsia, fetal growth restriction and second trimester miscarriage are all associated with impaired arterial remodelling. Shallow trophoblast invasion, decreased numbers of invasive trophoblasts (Khong et al., 1986; Naicker et al., 2003) and the absence of endovascular trophoblast from the myometrial segments of spiral arteries have all been observed in pre-eclampsia (Kadyrov et al., 2003). Spiral artery remodelling is also reduced or absent in hypertensive diseases of pregnancy (Pijnenborg et al., 1991), in the maternal uterus of small-for-gestational-age infants (Khong et al., 1986) and in late sporadic miscarriage (Ball et al., 2006).
We and others have previously shown that first trimester cytotrophoblasts induce apoptosis of vascular cells during arterial remodelling (Cartwright et al., 2002; Ashton et al., 2005; Crocker et al., 2005; Red-Horse et al., 2006). When introduced into the lumen of isolated human spiral artery segments, these cells colonize the vessel wall and induce endothelial and smooth muscle cell (SMC) death. To do this, trophoblasts utilize members of the tumour necrosis factor (TNF) family of apoptosis-inducing ligands. First trimester cytotrophoblasts release soluble Fas ligand (FasL) (Abrahams et al., 2004; Harris et al., 2006), which effects apoptosis following ligation of the Fas receptor on arterial endothelial cells and SMC (Ashton et al., 2005). They also express TNF-related apoptosis inducing ligand (TRAIL), a membrane-bound ligand which binds to TRAIL Receptor-1 (TRAIL-R1) and TRAIL Receptor-2 (TRAIL-R2) present on arterial SMC (Keogh et al., 2007). Along with trophoblast-derived proteases and cytokines present in the uterine environment, these ligands regulate the complex process of arterial transformation.
The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as a model of trophoblast (Church and Aplin, 1998; Egawa et al., 2002; Li et al., 2003; Ishimatsu et al., 2005). As the availability of systems to study physiological change is limited, we have examined whether BeWo can remodel spiral artery segments in a manner similar to endovascular trophoblasts. We have also studied the mechanisms they employ to induce apoptosis of vascular SMC.
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Reagents
Reagents were purchased from the following sources: mouse anti-human Fas (CD95) monoclonal antibody, mouse anti-human matrix metalloproteinase-2 (MMP-12) monoclonal antibody (catalytic domain), mouse anti-human MMP-12 monoclonal antibody (hemopexin domain), recombinant human MMP-12 western blotting standard, rabbit anti-human active caspase-3 polyclonal antibody (R&D Systems, Minneapolis, MN, USA); mouse anti-human FasL monoclonal antibody (clone NOK-1), mouse anti-human FasL monoclonal antibody (clone NOK-2) (BD Pharmingen, San Diego, CA, USA); mouse IgG2a
(clone UPC-10), rabbit anti-human cleaved poly-ADP-ribose polymerase (PARP, p85 fragment) polyclonal antibody (Promega Corporation (Madison, WI, USA); mouse anti-human cytokeratin-7 monoclonal antibody (clone OV-TL 12/30), rabbit anti-mouse biotinylated antibody, swine anti-rabbit biotinylated antibody, streptavidin-fluorescein isothiocyanate (FITC), FITC-conjugated rabbit anti-mouse IgG, horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG, FITC-conjugated swine anti-rabbit IgG, DakoCytomation A/S (Glostrup, Denmark); mouse anti-human CD34 monoclonal antibody, Serotec (Oxford, UK); mouse anti-human TRAIL monoclonal antibody, mouse anti-human TRAIL receptor-1 monoclonal antibody, mouse anti-human TRAIL receptor-2 monoclonal antibody, Alexis Biochemicals (distributed by Axxora, Nottingham, UK); in situ cell death detection kit (TUNEL), Roche (Lewes, UK); annexin V-FITC apoptosis detection kit, BD Pharmingen (Oxford, UK); Vectashield mounting medium, Vector Laboratories Inc. (Burlingame, CA, USA); OCT embedding medium, Raymond A Lamb (London, UK); CellTracker CM-Dil, Molecular Probes (distributed by Invitrogen Ltd, Paisley, UK); tissue culture medium, Cambrex (Wokingham, UK); fetal bovine serum (FBS), Gibco (distributed by Invitrogen Ltd); Matrigel, BD Discovery Labware (Bedford, MA, USA); collagen I, collagen IV, elastin, fibronectin, laminin, Sigma-Aldrich Inc. (Saint Louis, MO, USA); RainbowTM molecular weight markers, HybondTM-P PVDF membrane, ECL Plus western blotting detection reagents, Amersham Biosciences UK Ltd. (Chalfont St.Giles, UK); Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich and were of AnalR grade.
Tissue
Informed consent was obtained for all myometrial and placental tissue used in this study, and local ethical committee approval was in place. Normal first trimester placenta (8–12 weeks gestation) was obtained at elective termination of pregnancy (surgical or medical). Umbilical cords were obtained from normal term placentae within 30 min of caesarean section or vaginal delivery. Term decidual/myometrial biopsies taken from non-placental bed tissue were obtained from women with normal pregnancies at elective caesarean section.
Vessel explant model
Dissection and perfusion of spiral arteries were performed as previously described (Cartwright et al., 2002; Cartwright and Wareing, 2006). Briefly, unmodified spiral arteries were dissected from term decidual/myometrial biopsies under sterile conditions and mounted on glass cannulae in a pressure myography perfusion chamber (Living Systems Instrumentation, Burlington, VT, USA). Arteries were denuded of endothelium by passing a column of air through the vessel and then perfused with the appropriate medium or cells, as indicated. The ends of each vessel were tied and the arteries were incubated for up to 72 h in 1:1 DMEM:Ham's F12 culture medium supplemented with 10% FBS, glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml).
Umbilical artery segments were dissected under sterile conditions from umbilical cords and cannulated with a needle and syringe. Sterile phosphate-buffered saline (PBS) was forced through the segments to remove the endothelium. Arteries were then perfused with the appropriate medium or cells, as indicated. The ends of each vessel were tied and the arteries were incubated for up to 96 h in 1:1 DMEM:Ham's F12 culture medium supplemented with 10% FBS, glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml).
Cell culture and labelling
BeWo cells were cultured in 1:1 DMEM:Ham's F12 culture medium supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml). The human aortic SMC (HASMC) cell line was cultured as previously described (Harris et al., 2006; Keogh et al., 2007) and maintained in Kaighn's modification of Ham F12 medium supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml). All cells were incubated with 95% air and 5% carbon dioxide at 37°C in a humidified incubator.
Co-culture assay
Proliferating HASMC were labelled with CellTracker (1 µg/ml) in serum-free medium for 5 min at 37°C, followed by 15 min at 4°C. HASMC were washed twice in PBS and transferred back to serum-containing medium, before BeWo cells were added to the SMC, at a ratio of approximately 1:1. Cells were maintained in a 1:1 ratio of SMC medium:BeWo medium. After 48 h, adherent and non-adherent cells were collected. Cells were centrifuged, the supernatant discarded and the pellets were analysed for phosphatidylserine externalization (to detect SMC apoptosis; described below). The number of apoptotic SMC [positive for both CellTracker (red) and annexin V (green)] was quantified by flow cytometry.
For experiments requiring the addition of blocking antibodies, SMC were labelled with CellTracker as described above and transferred back into serum-containing medium. BeWo were added and allowed 2 h to adhere. After this time, cultures were washed twice in PBS and maintained in serum-free medium (1:1 ratio of SMC medium:BeWo medium). TRAIL receptor blocking antibodies (final concentration 5 µg/ml) were also added at this time. After 48 h, the number of SMC externalizing phosphatidylserine in each co-culture was quantified by flow cytometry, as described below.
Conditioned medium and blocking antibody treatments
BeWo-conditioned culture medium was produced in the presence or absence of serum by culturing BeWo cells in a T75 flask with 15 ml of medium for 48 h. Conditioned medium was diluted 1:1 (v/v) with unconditioned medium before use. For flow cytometry experiments using the FasL blocking antibody NOK-2, serum-free unconditioned medium (control) or BeWo-conditioned medium was incubated with NOK-2 (10 µg/ml) or an isotype-matched control antibody IgG2a (10 µg/ml) for 1 h at room temperature. HASMC were then incubated with the depleted medium for 2 h, also in the absence of serum.
Immunohistochemistry
After culture, vessels were fixed with 2% (v/v) paraformaldehyde in PBS for 30 min and then incubated with 0.5 M sucrose in PBS for at least 1 h. The tissue was placed in OCT embedding medium, frozen and stored at –80°C. Transverse sections (10 µm) of frozen vessels were prepared using a cryostat, transferred to poly-L-lysine-coated slides and stored at –80°C. For immunostaining of vessel sections, slides were warmed to room temperature and fixed with 4% (v/v) paraformaldehyde for 20 min. For immunostaining of BeWo, cells were cultured on glass cover slips and fixed with 4% (v/v) paraformaldehyde for 20 min. Following a washing step in PBS, both cover slips and tissue sections were permeabilized with 0.1% (v/v) Triton-X in PBS for 5 min, washed in PBS and allowed to air dry before addition of the primary antibody. Primary and secondary antibodies were diluted in PBS and applied to the tissue or cover slip for 1 h, during which the slides/cover slips were placed in a humidified chamber at room temperature. Samples were protected from light once the secondary antibody was applied. Following each antibody incubation, the slides or cover slips were washed thrice in PBS. Antibody dilutions used were as follows: active caspase-3 (0.25 µg/ml), CD34 (1:10), cleaved PARP (1:100), cytokeratin-7 (1:40), FasL (NOK-1; 1:50), MMP-12 (1:20), TRAIL (1:20), FITC-conjugated rabbit anti-mouse and swine anti-rabbit secondary antibodies (1:40), biotinylated mouse-anti rabbit (1:200) and biotinylated rabbit-anti swine secondary antibodies (1:500) and streptavidin-FITC (1:100). Sections and cover slips were mounted using Vectashield mounting medium containing propidium iodide and stored at 4°C in the dark until viewed. Slides were analysed at room temperature using either an Olympus IX70 inverted fluorescence microscope or a Biorad Radiance 2100 confocal microscope with a 10 or a 40 x oil immersion objective lens and LaserSharp 2000 image analysis software.
A modified protocol was used to stain BeWo cells for FasL or TRAIL. After incubation with primary antibody and subsequent PBS washes, cells were incubated with a biotinylated secondary antibody for 1 h. Following further washes, streptavidin–FITC was applied for 1 h, cells were washed thrice in PBS and mounted as previously described.
TUNEL staining
Vessel sections were fixed using 4% (v/v) paraformaldehyde in PBS for 20 min at room temperature, washed in PBS for 20 min and allowed to air dry. Slides were then incubated with permeabilization solution [0.1% (v/v) Triton in 0.1% (w/v) sodium citrate in H2O] for 8 min and washed in PBS. Slides were allowed to air dry before incubation with 16 µl of TUNEL reagent per tissue section. The working TUNEL reagent solution was prepared according to the manufacturer's instructions, although the TUNEL enzyme provided was diluted 1:5 with PBS to reduce background fluorescence. Slides were incubated in a humidified chamber for 1 h in the dark, then washed in PBS. Slides were mounted using Vectashield mounting medium containing propidium iodide and stored at 4°C in the dark. Quantification of the number of TUNEL-positive cells per vessel section was performed blind, using an Olympus IX70 inverted fluorescence microscope. TUNEL-positive cells present in the two layers of cells closest to the lumen were excluded to omit residual endothelial cells or adherent trophoblast from the counts. A minimum of six sections were stained and counted per vessel.
Annexin V assay
Phosphatidylserine externalization was quantified by flow cytometry, using a commercially available annexin V-FITC apoptosis detection kit following the manufacturer's guidelines. Following treatment, HASMC were washed twice in PBS, trypsinized and collected. The culture medium was also retained and pooled with the adherent cells. Cells were centrifuged, the supernatant discarded and the cell pellets resuspended in kit binding buffer. The cells were centrifuged again, the supernatant discarded and the pellet re-suspended in kit buffer (100 µl/pellet) containing annexin V solution (5 µl/pellet) and propidium iodide (2.5 µg/ml). Samples were incubated in the dark for 10 min and the percent of annexin V-positive cells was analysed using a Coulter Epics Elite flow cytometer.
TRAIL expression
Proliferating BeWo cell monolayers were washed twice in PBS, trypsinized and collected in serum-containing medium. Cells were centrifuged, the supernatant discarded and the pellets washed thrice in PBS containing BSA (0.5%) and NaN3 (0.1%). Cells were incubated on ice with a mouse anti-human TRAIL antibody (25 µg/ml) or a control IgG2 (25 µg/ml) for 1 h. Cells were then washed thrice in PBS/BSA/NaN3 and incubated with a rabbit-anti mouse FITC-conjugated secondary antibody (1:40 dilution) for 1 h in the dark. Cells were again washed thrice in PBS/BSA/NaN3 and the percent of fluorescent cells was quantified using flow cytometry.
Western blotting
HASMC were washed with PBS, incubated on ice in lysis buffer [1 x PBS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin], scraped from dishes, centrifuged and the lysates retained for analysis. Equal amounts of protein were separated by SDS–PAGE on 10% gels and transferred to PVDF membranes. Membranes were blocked [Tris-buffered saline (TBS), 0.1% (v/v) Tween, 5% (w/v) milk powder], and then probed with mouse anti-human MMP-12 antibody (1:200) prepared in TBS–Tween [3% (w/v) BSA], followed by an HRP-conjugated secondary antibody (1:1000). Following washing, proteins were detected by enhanced chemiluminescence.
Statistics
Data were compared using either a repeated measures ANOVA or paired t-test (parametric) or a Kruskal–Wallis test (non-parametric). Appropriate post-hoc tests were applied and all statistical analyses performed using GraphPad Prism software, version 4 (GraphPad Software, San Diego, CA, USA). Significance was taken as P < 0.05. Data are presented as the mean ± SEM from at least three independent experiments.
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Using an ex vivo model of endovascular trophoblast invasion, we have studied the ability of BeWo to invade segments of human spiral artery and induce apoptosis of medial SMC. To facilitate rapid access to the SMC layers, vessel segments were denuded of endothelium prior to perfusion. Removal of the arterial endothelium was confirmed by CD34 staining (data not shown). Forty-eight hours after introduction into the lumen of spiral artery segments, cytokeratin-7 positive cells could clearly be observed in the vessel wall (Fig. 1A), although the extent of invasion was quite variable, with some regions more highly colonized than others. Areas of SMC apoptosis were less heterogeneous; subpopulations of TUNEL-positive cells were observed throughout the arterial segments following perfusion with either BeWo cells or their conditioned medium (Fig. 1C and D). In contrast, control vessels exhibited minimal SMC apoptosis (Fig. 1B). Immunohistochemistry using antibodies against active caspase-3 and cleaved PARP confirmed that SMC death was occurring by apoptosis (data not shown).
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3 independent experiments, with each experiment performed on vessels from a different biopsyAlthough the number of TUNEL-positive SMC observed within spiral arteries was increased after the introduction of cells, this did not reach statistical significance (Fig. 2A). However, BeWo-conditioned medium induced SMC apoptosis significantly above control levels at 24, 48 and 72 h (P < 0.05). Denuded segments of umbilical artery, which contain many more layers of SMC, were also perfused with BeWo cells (Fig. 2B). This resulted in a significant increase in the number of TUNEL-positive SMC after 48 h (P < 0.05). Conditioned medium also increased apoptosis of umbilical artery SMC after 24 h (P < 0.05), 48 h (P < 0.05) and 96 h (P < 0.01) in culture. These data suggest that pro-apoptotic mediators are secreted by BeWo cells. FasL and TRAIL are utilized by first trimester cytotrophoblasts to induce SMC apoptosis during spiral artery remodelling (Harris et al., 2006
; Keogh et al., 2007). A majority of BeWo cells were shown to express surface FasL (Fig. 3A), 68.4 ± 3.5% (mean ± SEM, n = 3; flow cytometry). In contrast, surface TRAIL expression was only detected on 15.4 ± 5.2% (n = 4; flow cytometry) of cells. Confocal microscopy confirmed these findings and revealed that TRAIL is present in vesicles within the cytoplasm of some, but not all, cells (Fig. 3B).
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To further study the soluble pro-apoptotic factors released by BeWo cells, a HASMC cell line was cultured in BeWo-conditioned medium (Fig. 4A), and apoptosis was quantified using phosphatidylserine externalization measured by flow cytometry. SMC apoptosis was significantly increased after treatment with conditioned medium, with
30% of SMC externalizing phosphatidylserine after 2, 4 and 24 h (P < 0.001) in culture. To test for soluble FasL, serum-free conditioned medium was pretreated with FasL-blocking antibody or control IgG. Conditioned medium containing control IgG increased the number of apoptotic SMC significantly above levels observed in control cultures (P < 0.01; Fig. 4B). When the conditioned medium was pretreated with a FasL-blocking antibody, levels of SMC apoptosis were significantly decreased (P < 0.01).
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To investigate whether TRAIL, which exists predominantly as a membrane-bound ligand, induces SMC apoptosis, a dual culture system was employed in which fluorescently labelled SMC make direct contact with BeWo. After 48 h,
25% of SMC were apoptotic, in contrast to 5% in monoculture (Fig. 5A). This experiment was repeated in the presence of antibodies that blocked TRAIL-R1 and TRAIL-R2. As previously observed, the presence of BeWo cells significantly increased the percent of SMC externalizing phosphatidylserine (P < 0.01; Fig. 5B), although due to the absence of serum, the baseline level of apoptosis was increased. The addition of TRAIL receptor blocking antibodies had no effect on the level of SMC apoptosis observed in the co-cultures.
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The specialized extracellular matrix (ECM) environment of the vessel wall may affect target cell susceptibility to apoptosis. SMC were cultured in the absence of serum on culture plates which were either uncoated or coated with collagen I, collagen IV, elastin, Matrigel, fibronectin or laminin. Cells were stimulated with a Fas-activating antibody and after 48 h, phosphatidylserine externalization was quantified. Approximately 30% of SMC grown on uncoated plates were apoptotic; this figure rose to 55% when SMC were challenged with a Fas-activating antibody (Fig. 6). Surprisingly, none of the matrix components tested offered any protection against Fas-induced apoptosis.
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To successfully remodel the spiral arteries, trophoblasts must degrade ECM components, including elastin fibres within the medial SMC layers. BeWo express the elastolytic enzyme MMP-2 (Di Simone et al., 2006
; Mandl et al., 2006), but express little or no MMP-9 (Mandl et al., 2006; Morgan et al., 1998). In this study, we assessed expression of the elastase MMP-12 (macrophage metalloelastase) by BeWo using immunohistochemistry and western blotting. BeWo predominantly expressed the active 45 kDa form of MMP-12 (Fig. 7A), which exhibited a punctuate distribution within the cytoplasm (Fig. 7B).
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During the invasion process, trophoblast may promote protease expression in spiral artery SMC. To investigate this, we cultured HASMC in vitro with BeWo-conditioned medium for 24 h and measured the expression of MMP-12 by immunoblotting. Levels of active MMP-12 were not significantly increased following treatment with BeWo-conditioned medium (Fig. 8A), nor did induction of SMC apoptosis using a Fas-activating antibody or etoposide alter MMP-12 expression. However, expression of MMP-12 was increased in the medial SMC layer of spiral arteries perfused with BeWo-conditioned medium (Fig. 8C). Vessels perfused with control medium exhibited negligible staining (Fig. 8B).
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Discussion |
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At present, the complex processes which regulate vessel transformation are poorly understood, primarily due to the lack of suitable cellular and tissue models in which to study this phenomenon. It is possible to observe arterial remodelling in human decidual/myometrial biopsies, but histology only supplies a snapshot of the process. Most rodent systems are limited by the lower level of trophoblast invasion, the later stage of pregnancy at which it occurs (Adamson et al., 2002
) and the greater role in vascular transformation ascribable to uterine NK cells (Ashkar and Croy, 2001). Trophoblast invasion and arterial remodelling has also been studied by transplanting human first trimester placental villi into mammary fat pads or beneath the kidney capsules of Scid mice (Red-Horse et al., 2006). Data from these studies confirm a role for trophoblast-induced endothelial cell and SMC apoptosis in the process of vascular transformation. To tackle the problem from a different angle, our laboratory has developed an ex vivo model of endovascular trophoblast invasion and spiral artery remodelling where trophoblast is introduced into human spiral artery segments that can be maintained at high viability for several days (Cartwright et al., 2002; Cartwright and Wareing, 2006).
In this study, we have examined the ability of BeWo cells to invade and remodel segments of spiral artery. BeWo cells exhibit many of the characteristics of invasive trophoblast: morphologically they are mononucleate and they secrete small amounts of placental alkaline phosphatase and human chorionic gonadotrophin (Friedman and Skehan, 1979). Akin to endovascular trophoblast, BeWo adhered to the vessel wall and penetrated the smooth muscle layers beneath. However, regions of invasion were highly variable; several intramural cells could be observed in some vessel sections, yet adjacent sections contained none. This may explain why conditioned culture medium but not the cells themselves induced SMC apoptosis in perfused vessel segments, a result which indicates the release of soluble pro-apoptotic factors. It is possible that secretion occurs only after cells have attached to the vessel wall, generating a high local concentration of factor(s). Vessels perfused with conditioned medium from adherent cultures probably achieve a more uniform exposure to the apoptotic factors, resulting in an increased overall level of apoptosis.
In common with first trimester cytotrophoblasts (Harris et al., 2006), BeWo cells employ FasL to induce SMC apoptosis; however, unlike primary cytotrophoblast (Keogh et al., 2007), they do not appear to utilize TRAIL, perhaps because of its relatively low level of expression on the cell surface. The heterogeneity of BeWo cell invasion may reflect this fact, or alternatively, the absence of the endothelium from the arterial segments may have hindered the initial stages of adhesion. We find this possibility unlikely, however, as the extent of BeWo invasion in several vessels that contained remnants of endothelium was no different to the level of invasion observed in denuded vessels.
We also investigated whether components of the ECM have the ability to enhance cell survival, such that trophoblast-mediated breakdown of matrix components may promote SMC loss during vessel remodelling. This did not prove to be the case. Despite this, catabolism of ECM is still required to effect vessel transformation. Elastin fibres within the arterial media, and the internal elastic lamina present in myometrial vessels, must be degraded to facilitate a permanent increase in vessel diameter and to abolish vasomotor control. BeWo cells express high levels of the elastase MMP-2 (Di Simone et al., 2006; Mandl et al., 2006), but negligible MMP-9 (Mandl et al., 2006; Morgan et al., 1998). Here we show that they also express the 45 kDa active form of MMP-12, which has documented elastolytic activity (Shapiro et al., 1993).
In addition to trophoblast, vascular SMC may also play a role in elastin breakdown. Elastases including cathepsin S and K are up-regulated by vascular SMC in mouse models of atherosclerosis (Cheng et al., 2004), thus elastase expression may be increased in spiral artery SMC during the remodelling process. Here we show that SMC cultured in vitro express active MMP-12 and that the level of protein expression does not change following treatment with BeWo-conditioned culture medium. In contrast, spiral artery SMC in situ do not express MMP-12 until they are challenged with BeWo-conditioned medium. Cultured SMC may constitutively express MMP-12 because their phenotype is more similar to that of spiral artery SMC exposed to trophoblast-derived factors. Nevertheless, these data serve as a reminder that experiments performed using cell lines must be repeated using primary cells/tissue. Taking these findings into account, we propose that trophoblasts release soluble factors which increase protease expression and/or activity in vascular SMC. These proteases can then act locally to degrade matrix components within the vessel wall, allowing easier access to invading trophoblasts. Thus, trophoblast may facilitate their own invasion by inducing cooperative breakdown of elastin fibres by medial SMC.
Studies of the placental bed have shown that spiral artery remodelling is a gradual, multi-step process that occurs throughout the first and second trimesters of pregnancy (Benirschke and Kaufmann, 2000). Consequently, this phenomenon must be tightly regulated to prevent sudden loss of vessel integrity. It is likely that a number of mechanisms act concurrently to facilitate vessel transformation, and that the process involves factors derived from the invading trophoblast, the decidualized endometrium, resident leukocytes and the maternal endocrine system. There is evidence to suggest that changes in vascular structure, such as arterial dilation and disorganization of the smooth muscle layers, occur prior to the arrival of extravillous trophoblast (Craven et al., 1998). Changes such as replacement of the endothelium and SMC with trophoblast embedded in fibrinoid, only occur in the presence of trophoblast (Pijnenborg et al., 1983). Dedifferentiation of vascular SMC and breakdown of mural ECM by trophoblast- and SMC-derived proteases are other features of physiological change that require further investigation.
Our current findings demonstrate that following introduction of BeWo cells into arteries, SMC apoptosis occurs slowly and in a highly controlled manner. This is essential if vessel integrity is to be maintained and is representative of the changes observed in vivo. Although we demonstrate here that BeWo-derived FasL mediates SMC apoptosis, our results do not exclude the possibility that BeWo may also stimulate resident vascular SMC to produce apoptotic factors. Invading cells may promote the expression and/or release of FasL by neighbouring SMC, so that cell death is regulated in a paracrine manner. However, the present study has shown that BeWo cells can induce apoptosis of spiral artery SMC via release of soluble FasL, via a mechanism similar to that of primary trophoblast (Harris et al., 2006). Genetic manipulation of BeWo prior to vessel perfusion may be useful in delineating key molecules involved in trophoblast invasion. We therefore propose this as an experimental model system to allow further investigation of vessel transformation in vitro.
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Funding |
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The Wellcome Trust, UK (069939, L.K.H., R.J.K., P.N.B., J.E.C, G.S.W, J.D.A); British Heart Foundation Intermediate Fellowship, M.W.
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Submitted on May 4, 2007; resubmitted on July 2, 2007; accepted on August 28, 2007.
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