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5ß11
a Departments of Obstetrics and Gynaecology,
b Pathological Sciences, and School of Biological Sciences, University of Manchester, Manchester M13 0JH, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1ß1 and 5ß1 and produced an extracellular matrix containing oncofetal fibronectin, as in vivo. Function-blocking antibodies were used to investigate the role of the integrin-fibronectin interaction in anchoring villus development on collagen I. Antibodies to fibronectin and the integrin subunits 5 and ß1, added at 24 h, all changed the pattern of cytotrophoblast outgrowth. Anti-fibronectin caused cell rounding within the cytotrophoblast sheet and increased the population of single cells at its periphery. Anti-integrin 5 caused rounding and redistribution of cells within the outgrowth. In the presence of anti-integrin ß1, cell-collagen interactions within the sheet were destabilized, often leading to the appearance of an annulus of aggregated cells at the periphery. These results show that 1) mesenchymal villi retain the potential to form anchoring sites until at least the end of the first trimester, 2) adhesion to a permissive extracellular matrix stimulates cytotrophoblast proliferation and differentiation along the extravillous lineage, 3) integrin 5ß1-fibronectin interactions contribute significantly to anchorage of the placenta to uterine extracellular matrix. We suggest that as the developing placenta ramifies, new sites of anchorage form whenever peripheral villi contact decidua. This process is predicted to contribute to the stability of the placental-decidual interface.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most information on the cytotrophoblast phenotype has been gained from histological studies of pregnancy hysterectomies [2], while the invasive potential of normal cytotrophoblast has been confirmed using primary cultures [5]. However, currently available cell isolation protocols produce a mixture of different cytotrophoblast populations [6,7] that suffer from alterations in phenotype and the possible loss of critical properties in vitro [8]. Explant culture offers the potential to maintain the basic architecture of placental tissue at anchorage sites. We and others have previously shown that explants of placental villi from the first trimester of pregnancy can progress into the extravillous lineage in vitro [9–11]. Contact coculture of first-trimester mesenchymal villi with decidual tissue has been shown to support anchoring villus formation and cytotrophoblast migration in vitro [10–12], but the mechanism by which maternal tissue influences these morphogenetic events is currently uncertain. There are likely to be paracrine effects mediated by diffusible substances [12].
There is also the possibility that physical anchorage of trophoblast to a permissive extracellular matrix (ECM) could trigger progression into the extravillous lineage. Signals from the ECM, transduced by integrins, have been shown to mediate alterations in cell shape, adhesion, and migration, and also to play a role in the regulation of proliferation and differentiative transitions in other cell types [13]. Fibronectin (FN) is abundant at sites of anchoring villus formation in vivo; since cytotrophoblast produces the oncofetal isoform (oFN [14]), which is distinguishable by virtue of a unique glycopeptide epitope [15], it is clear that both maternal and trophoblast-derived FN are present in the ECM encountered by trophoblast in the extravillous pathway [16]. Cytotrophoblast undergoes a change of cell surface phenotype during its differentiation into the extravillous lineage [17–21], with loss of integrin 6ß4 and rapid up-regulation of other integrins including the FN receptor 5ß1. In the present study, we have undertaken to dissect the differentiative pathway of cytotrophoblast, seeking to distinguish steps that may occur as a result of interaction with the ECM from those that require other signals. On the basis of observations of the cell surface and ECM phenotype of extravillous cytotrophoblast, we focus on the role of integrin-FN interactions in these events.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagen type I (rat tail tendon), Matrigel, and agarose were obtained from Stratech Scientific Ltd. (Luton, UK). Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 were from Gibco (Life Technologies, Paisley, UK) or Sigma (Poole, UK). Fetal calf serum (FCS) was from Gibco; bromodeoxyuridine (BrdU) was from BCL (Lewes, UK).
Antibodies
Details of the primary antibodies used in this study are given in Table 1. Rabbit polyclonal anti-FN was raised to plasma FN and was previously shown to inhibit cell adhesion to FN monolayers [22]. Monoclonal antibody (mAb) X18A4 is specific to the oncofetal isoform of FN (oFN [23]). Monoclonal Ab 13 to integrin ß1 recognizes all heterodimeric (xß1) complexes involving this subunit and is a potent inhibitor of integrin ß1-mediated cell adhesion [22, 24, 25]. Monoclonal Ab B1E5 to integrin 5 is also function-inhibiting [22]. Several antibodies to members of the integrin v family are listed in Table 1 and were used for localization studies. For function-blocking experiments, mouse mAb LM609 to integrin vß3 was used. Monoclonal Ab C8F12 to v was obtained from C.H. Streuli (University of Manchester). Anti-proliferating cell nuclear antigen (PCNA) antibody was from Dako (High Wycombe, UK).
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Tissue Collection
Normal placenta was obtained at elective termination of pregnancy by evacuation in the first trimester. The tissue ranged from 8 to 12 wk gestation. It was collected into PBS with antibiotics for transport to the laboratory. For culture experiments, small pieces of tissue (~2–3 mm) containing several floating mesenchymal villi were dissected from the periphery. Specimens of this "zero-time" tissue were fixed and sectioned for comparative histology.
Culture On Gels
Gels were prepared by placing a drop (~85 µl) of the appropriate substrate in 12-well culture dishes. One percent agarose was prepared by melting a stock of 2% agarose in double-strength salt solution and mixing with double-strength DMEM (prediluted from 10-strength stock). Nine parts of concentrated collagen I (4.75–5.25 mg/ml) were mixed with 1 part of 10-strength medium, and the pH was adjusted to neutral. Matrigel was aliquoted at 4°C and warmed to 37°C to gel. Gels were produced at least 24 h before use and incubated in medium at 37°C to allow full contracture.
After the villous tissue was dissected, medium was drained from the gel droplets, and the tissue was arranged on top with villi in a radial orientation and gently covered with ~20 µl of culture medium. The cultures were incubated for 2 h to allow tissue to adhere to the gel, and then carefully flooded with 1 ml of culture medium (1:1 DMEM:F12, 10% FCS). Culture was continued in an atmosphere of 5% CO2 in air at 37°C for various time periods up to 7 days. Cultures were inspected and media changed daily. For identification of newly synthesized DNA, BrdU was added to the cultures at 100 µM for the periods specified in Results. In order to measure the effects on the explants of antibodies to integrins or FN, an initial period of 24-h anchorage to the gel was allowed. After this time, the culture medium was changed, and antibody was introduced at the concentration given in Table 1. Media were subsequently changed every 24 h with the introduction of fresh antibody. Antibody concentrations were based on maximal inhibition in cell adhesion assays carried out using BeWo cells [22]. Explant morphology naturally varies, but the antibody inhibition data are representative of explants of at least 6 placentas cultured in triplicate.
Tissue Processing and Microscopy
Tissue was fixed and processed for semithin, light, and transmission electron microscopy examination, and was also snap-frozen, cryosectioned, and processed for immunohistochemistry as previously described [11, 18]. For visualization of incorporated BrdU, sections were mounted on poly-L-lysine-coated slides and pretreated with 2 M HCl (1 h, room temperature), then neutralized by immersion in 0.1 M borate pH 8.5, washed in PBS, and incubated overnight in mouse anti-BrdU antibody (1/20; BCL) as previously described [18]. PCNA staining used antibody at 1/100 and the same standard immunoperoxidase method. Whole mounts and semithin sections were imaged using a Kontron KS400 system (Kontron Elektronik GmbH, Eching, Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagen I supported efficient and rapid (within 3 h) attachment of the explanted tissue (Table 2). Radial outgrowth from one to several cells deep was apparent in the plane of the gel surface (Figs. 1, 2, 3A, 3C; see also Fig. 9A), and all outgrowing cells were cytokeratin-positive (Fig. 3, C and D). Elongated bipolar cells were prominent at the edge of the outgrowths (Figs. 1D, 3A, 4A; see also 8B and 9A). Outgrowth occurred specifically from the tips of mesenchymal villi (Fig. 1) and was visible as early as 12–18 h of culture, although in some cases activity became apparent only on the second day in vitro. Not all tips produced outgrowths even when they appeared to be stably attached to the gel surface. However, multiple foci of outgrowth were usually observed from adjacent villi, and with increasing time these fused to form a continuous sheet of cytotrophoblast resembling a shell (Figs. 1B and 3A). Most of the outgrowth activity occurred between Days 1 and 4 (Fig. 2). The size of the outgrowth varied widely between cultures (Fig. 2). This variation was apparent in different cultures produced from the same or different placentas, and appeared to depend mainly on the size of the sites of origin where villous tips contacted the gel surface. This behavior could be observed in tissue obtained from all gestational ages under study (8–12 wk), and no alteration was observed in this range. Within the sheet, cells were multilayered and had prominent cytoplasmic glycogen masses, intermediate filament bundles, and desmosomal junctions (Fig. 4). Some cells penetrated a short distance into the collagen gel, but extensive infiltration or erosion of the collagen gel was not apparent (Figs. 3C, 4A, 4B; see also 9A). A population of detached single cells could be observed at the periphery of the cytotrophoblastic shell (Fig. 3A; see also 8B). These cells were mainly rounded and did not migrate further.
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The tissue interactions observed with the three gel matrices used for the establishment of first-trimester (8–12 wk) villous explants were quite distinct (Table 2). Attachment of tissue to agarose was poor. No morphological alterations were evident; the villous surfaces remained smooth throughout the experiment (up to 72 h), and there was no evidence from microscopic examination of either whole mounts or semithin sections that any new extravillous trophoblast was being produced. It was concluded that agarose did not support cytotrophoblast outgrowth, and these cultures were not studied further. With Matrigel, attachment was as rapid and efficient as on collagen I (Table 2). Examination of whole mounts at 24 h revealed only subtle changes at the gel-villus tip interface, such as the appearance of rounded surface projections of individual cytotrophoblast cells (Fig. 3B). Cross sections through contact areas showed that cytotrophoblast columns had formed at attachment sites, and invasive fronts of cells proceeded to advance from the surface deeper into the gel bed. The downward direction of migration accounted for the difficulty of observing this phenomenon in whole mounts. In cryosections, several cell columns (often merging) could be seen to develop below the tissue where groups of cells migrated into the gel (Fig. 3D). There was little or no single-cell infiltration of the gel. The cells in the columns were large, rounded, and cytokeratin-positive (Fig. 3D).
Cell Proliferation
Cytotrophoblast proliferation in cultures on matrices of agarose, Matrigel, and collagen I was studied by metabolic labeling with BrdU (Fig. 5). The results showed focal proliferation of cytotrophoblast of proximal columns in culture. Cells of the first few layers of the proximal column were BrdU-positive. In addition, staining was observed in a few villous cytotrophoblast cells at sites not involved in column formation, an observation that supports the continuing viability of the subsyncytial cytotrophoblast population in vitro. On agarose, there were no columns formed in culture, but stem cytotrophoblast cells also incorporated BrdU (not shown).
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The pattern of cell proliferation was also studied by immunolocalization of PCNA (not shown). Anti-PCNA stained predominantly cytotrophoblast cells of the proximal column, but staining was also found more distally among the differentiating cytotrophoblast. Thus considerably more cells stained for PCNA than labeled with BrdU.
In cultures on collagen I, BrdU incorporation was studied as a function of time (Fig. 6). Particular attention was paid to cytotrophoblast of the proximal column, which is the main source of invasive extravillous trophoblast both in vivo and in vitro. BrdU was added to cultures for 5 h on Day 1 (1–6 h and 19–24 h) (Fig. 6). The results showed that cytotrophoblast of the proximal column maintained its proliferative potential for the first 24 h. When BrdU was added on Day 2 (43–48 h), little labeling was observed, indicating that cell proliferation ceased by 48 h of culture (not shown). Dependency of proliferation on factors present in FCS was evaluated by comparison with cultures maintained in serum-free conditions. BrdU incorporation was similar in the two experiments (not shown), suggesting that the presence of serum is not of critical importance for the proliferative burst.
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Integrin and FN Expression by Cytotrophoblast
The above results indicated that cell-ECM interactions were critical in the stimulation of de novo column development in vitro. Cultures on gels of collagen I were selected for further studies of the role of integrins at the cell surface and ECM components in these events. Initially it was necessary to establish the patterns of expression of relevant receptors.
Cytotrophoblasts in the developing columns expressed integrins 1, 5, and ß1 uniformly after 5-day culture (Fig. 7, A, D, and E). At earlier time points, 1 was seen at more distal locations in the outgrowth, while 5 and ß1 were always uniformly positive. The distribution of these two subunits was pericellular within the columns and also strong at the cell-collagen interface. Integrin 2 was observed in cells in the column, but there was considerable variation in the level of expression (Fig. 7B). More proximal cells in the column sometimes appeared more strongly positive, and no particular accumulation of reactivity was apparent at the cell-collagen interface. Integrin 4 was weak or absent (Fig. 7C). Integrin 6 and ß4 subunits were present in regions of cytotrophoblast close to the villous tissue but were lost in more distal areas of outgrowths. Several antibodies were applied that recognize integrins of the v family: either v itself, the vß3 complex, or subunits ß3 or ß5. Staining in the developing outgrowths was weak or negative. These data are summarized in Table 3.
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Localization studies with a polyclonal anti-FN antibody showed widespread staining within the column outgrowth and both at the surface and within the subjacent collagen gel (not shown). To evaluate the contribution of trophoblast-derived fibronectin (oFN) to this matrix, immunolocalization was carried out with mAb X18A4 (Fig. 7F). This showed evidence for production in the outgrowing cells with intracellular staining, accumulation as streaks between cells, and increased staining at the surface of the collagen gel. Staining was also carried out with anti-laminin antibodies (not shown). Columns contained immunoreactive laminin in streaks and spots. However, there was no specific accumulation of laminin at the gel surface beneath outgrowths (not shown).
Integrin and FN Function
The results suggested that the FN-integrin 5ß1 interaction may be important as a mediator of cytotrophoblast adhesion and/or outgrowth. The involvement of FN, 5, and ß1 was evaluated using function-perturbing antibodies. Explants were first established for 24 h to allow initial anchorage to the collagen gel and the appearance of foci of trophoblast outgrowth, then exposed to antibody for further periods of up to 5 days. Data are illustrated in Figures 8 and 9 and summarized in Table 4. Control experiments showed development of sheet outgrowths with a uniform appearance in both whole mounts (Fig. 8A) and semithin sections (Fig. 9A). The outgrowths were 1–5 cells thick, with an increase in thickness towards the distal edge (Figs. 8B and 9A). Minimal penetration or infiltration of the gel occurred. Antibodies to integrin 5 (Fig. 8C, 8D, 9C) and integrin 5ß1 (Fig. 9B) disrupted the outgrowths, giving rise to rounding of the cells within the columns. This produced a "lacy" appearance in which gaps appeared in the cell sheet. These antibodies also reduced the incidence of elongated or detached cells at the periphery. Morphological changes were also evident in the presence of function-blocking anti-ß1 in the culture medium (Fig. 8E, 8F, 9D). Fewer elongated cells were apparent at the leading edge of the outgrowth. Condensation of cells (as seen in Fig. 8F) often began to be visible at the edges of outgrowths 1 or 2 days after the first exposure to antibody, and this could progress to the formation of a discrete annulus of cells at the periphery of the culture (Figs. 8E and 9D). Between the annulus and the villous tissue, cells aggregated into elongated radial cords (Fig. 8E). Some loss of thickness of the collagen gel was apparent in these areas, as suggested by the need to adjust the focus of the microscope downwards to visualize the network of cells present between the tissue and annulus. When the antibody was added at 48 h, after more column development was evident in the cultures, further spreading of cytotrophoblast across the gel surface was blocked (not shown).
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As with antibodies to integrin 5ß1, antibody to FN caused disruption to the structure of the outgrowths without completely inhibiting their formation (Fig. 8G, 8H, 9E). Cell rounding occurred within the outgrowing cytotrophoblast sheets. A few infiltrating cells were apparent within the collagen gel matrix (Fig. 9E). In addition, a substantially larger population of scattered, rounded cells became evident at the periphery of the explant (Fig. 8G and 8H).
Since antibodies to the ß1 integrins did not produce a complete inhibition of outgrowth, there was a need to examine the possibility that other integrins may be important. Integrins of the v family act as alternative receptors for FN. Addition of function-blocking antibodies to either v or the vß3 complex, in the presence or absence of antibodies to integrin ß1, had no consistently inhibitory effect on column outgrowth.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5ß1 and 1ß1 and loss of 6ß4. The development of new anchorage sites is specific to the tips of mesenchymal villi in culture; it is not observed in other regions of the villous tree, either at lateral sites in mesenchymal villi or in intermediate villi. This suggests the novel hypothesis that different parts of the villous tree have different developmental potential. Variations might be intrinsic to the cytotrophoblast or might arise as a result of paracrine signalling. Variations in signals reaching the cytotrophoblasts could arise as a result of differences in either the underlying mesenchyme or overlying syncytium in different locations in the villus.
The morphology of cells in the newly formed columns, with abundant intermediate filaments and cytoplasmic glycogen masses, is consistent with features seen in vivo [1]. However, further differentiation in vivo leads to a massive infiltration of single cytotrophoblasts and the formation of giant cells in the placental bed [2]. Neither of these latter phenomena was observed in explant culture; cytotrophoblasts showed cell surface characteristics expected of cells in distal anchoring columns but did not differentiate further to acquire the surface phenotype that has been reported in cells migrating in vivo within maternal blood vessels, such as expression of integrin 4ß1 or vß3 [26]. These findings indicate that cell-matrix interaction at the tip of the mesenchymal villus is sufficient to trigger the first steps of entry into the extravillous lineage but not to produce the further changes characteristic of the endovascular cytotrophoblast population. It is therefore likely that paracrine interactions with maternal or fetal stromal, vascular, or blood-borne substances are required to complete this transition [27]. Numerous factors have been shown to increase the migratory activity of cultured first-trimester cytotrophoblasts (reviewed in [12]). Others have an opposite effect: thus transforming growth factor ß increases production of oFN [28] and increases surface expression of integrins 1, 5, and v with a concomitant decrease in migratory activity [29]. Blocking the function of its receptor endoglin stimulates outgrowth from villous explant cultures on Matrigel [30]. Activin-A stimulates cytotrophoblast outgrowth from explants, and this is reversible by follistatin [31]. Under conditions of low oxygen tension, cytotrophoblasts remain for longer in the proliferative pool, and the up-regulation of integrin 1 is inhibited [32].
When tissue was explanted into agarose, viability was maintained, as indicated by the incorporation of BrdU into stem cytotrophoblasts beneath the villous syncytium, but no new extravillous trophoblast was observed to develop, and there was no evidence of adhesion and migration into the surrounding gel. In contrast, anchoring sites developed on collagen I and Matrigel. This indicates that simple physical contact is not sufficient to stimulate column formation and suggests the likelihood of a signalling process that involves interaction between a physiological ECM and cognate receptors on the villus. Since the responses to collagen I and Matrigel differ—the former promoting migration across the surface and the latter stimulating invasion by the cell column—it appears that the respective signalling pathways are distinct. The details of these early recognition and signalling events remain to be defined.
The results indicate that villous tissue from the peripheral regions of the developing placenta retains the potential to generate new anchoring sites at least until the end of the first trimester. Thus as the placenta grows and ramifies, it may continue to develop new anchorage sites, reinforcing attachment to the uterine wall. Examination of the morphology of the developing placenta indicates that indeed anchoring sites increase rapidly in number as the placenta grows during the first trimester [33].
Integrins of the ß1 family including 1ß1 and 5ß1 are up-regulated, as seen in columns in vivo [20]. We have also observed 2ß1, which was not reported in previous studies [20, 21]. In principle, either 1ß1 or 2ß1 could act as a receptor for collagen I, mediating cytotrophoblast adhesion and migration over the gel [34, 35]. Integrin 1ß1 was initially restricted to distal edges of the cytotrophoblast sheet in culture but was later strongly expressed throughout outgrowths. However, 2ß1 did not appear uniformly on cells in the columns and was often not present in cells at the gel interface. In contrast, 5 and ß1 were always clearly concentrated at this location, as was FN, which is abundant at corresponding sites in vivo [16]. At least some of this FN was of trophoblastic origin as indicated by the presence of the oncofetal glycopeptide epitope [14, 23]. This suggests that FN acts as a bridging ligand between the collagen matrix and integrin 5ß1 at the cytotrophoblast surface, mediating anchorage and/or migratory activity. Other FN receptors, including those of the integrin v subfamily and 4ß1 were examined, but levels were very low and current evidence suggests that 5ß1 is the major FN receptor in this system.
Function-blocking antibodies to 5, ß1, and the 5ß1 complex all disrupted the nascent columns, causing alterations in cell patterning within the outgrowing sheet. These alterations are consistent with inhibitory effects on cell-matrix interaction. In addition, a modest increase in penetration of the gel by cells was observed. Anti-FN antibody also disrupted outgrowth architecture, though to a lesser extent than the anti-integrin antibodies, and, in addition, triggered the appearance of a significant population of rounded single cells near the periphery of the outgrowth. These are assumed to have detached from the distal edge of the outgrowth. They remained viable for at least 7 days but migrated no further. These observations are consistent with a role for cell-FN interaction in anchorage site integrity [36, 37].
Although the anti-integrin ß1 antibody used in this study inhibits all members of this integrin subfamily, cell-FN and cell-collagen interactions can also be mediated by v integrins, which have been reported on trophoblast [26, 29]. However, expression was low in the explant model, and when the respective function-blocking reagents were included along with anti-ß1, no additional inhibitory effect was observed on the outgrowth. It is important to note that experiments carried out using function-blocking antibodies did not probe tissue-gel attachment, but rather cytotrophoblast outgrowth, which requires prior attachment. Thus the experiment required that initial adhesion occurred before the reagent could be applied. After antibodies were added that disrupted FN-receptor interaction, outgrowth was reduced but not completely blocked. One possible explanation is that currently unrecognized receptor-ligand interactions were operating that do not involve the integrin ß1 subfamily. Another possibility is that antibody access to cell surface locations was incomplete.
The explant model of placental anchoring site development in vitro has revealed a novel aspect of placental function: the capacity to form new peripheral anchoring sites from floating mesenchymal villi that contact the decidual surface. Defects in this process might have important ramifications in pregnancy pathologies such as spontaneous abortion and pre-eclampsia. This model therefore represents a powerful tool for the investigation of a novel aspect of placental function. We have shown that early events in anchoring site morphogenesis are dependent on signals from the ECM and that cell-FN interactions are important in anchorage. We have also shown that these events are sensitive to perturbations in either ECM composition or integrin activity.
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ACKNOWLEDGMENTS |
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v; Arnoud Sonnenberg for anti-6; and Caroline Damsky for anti-5. |
FOOTNOTES |
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2 Correspondence: John D. Aplin, Research Floor, St. Mary's Hospital, Manchester M13 0JH, UK. FAX: 0161 276 6134; japln{at}mh1.mcc.ac.uk
3 Current address: Ljiljana Vi;aacovac, INEP, University of Belgrade, Zemun, Yugoslavia.
Accepted: November 5, 1998.
Received: June 29, 1998.
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-N-acetyl galactosaminylation at the threonine residue of a defined peptide sequence creates the oncofetal peptide epitope in human fibronectin. J Biol Chem 1989; 264:10472–10476.6ß4 in human trophoblast and its loss from extravillous cells. Placenta 1993; 14:203–215.[CrossRef][Medline]1-6 integrin subunits in human developing and term placenta. Lab Invest 1991; 65:347–356.[Medline]1ß1 and 2ß1 integrin-mediated cell adhesion to collagen V. J Cell Sci 1996; 109:1865–1874.[Abstract]2ß1 integrin-mediated cell adhesion. J Cell Sci 1998; 111:1127–1135.[Abstract]This article has been cited by other articles:
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S. Tartakover-Matalon, N. Cherepnin, M. Kuchuk, L. Drucker, I. Kenis, A. Fishman, M. Pomeranz, and M. Lishner Impaired migration of trophoblast cells caused by simvastatin is associated with decreased membrane IGF-I receptor, MMP2 activity and HSP27 expression Hum. Reprod., April 1, 2007; 22(4): 1161 - 1167. [Abstract] [Full Text] [PDF]
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L.J. Ayling, G.St.J. Whitley, J.D. Aplin, and J.E. Cartwright Dimethylarginine dimethylaminohydrolase (DDAH) regulates trophoblast invasion and motility through effects on nitric oxide Hum. Reprod., October 1, 2006; 21(10): 2530 - 2537. [Abstract] [Full Text] [PDF]
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P.A. Elustondo, G.E. Hannigan, I. Caniggia, and D.J. MacPhee Integrin-Linked Kinase (ILK) Is Highly Expressed in First Trimester Human Chorionic Villi and Regulates Migration of a Human Cytotrophoblast-Derived Cell Line Biol Reprod, May 1, 2006; 74(5): 959 - 968. [Abstract] [Full Text] [PDF]
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 F. Mondon, T.-M. Mignot, R. Rebourcet, H. Jammes, J.-L. Danan, F. Ferre, and D. Vaiman Profiling of oxygen-modulated gene expression in early human placenta by systematic sequencing of suppressive subtractive hybridization products Physiol Genomics, June 16, 2005; 22(1): 99 - 107. [Abstract] [Full Text] [PDF]
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 J. B. Laoag-Fernandez, H. Matsuo, H. Murakoshi, A. L. Hamada, B. K. Tsang, and T. Maruo 3,5,3'-Triiodothyronine Down-Regulates Fas and Fas Ligand Expression and Suppresses Caspase-3 and Poly (Adenosine 5'-Diphosphate-Ribose) Polymerase Cleavage and Apoptosis in Early Placental Extravillous Trophoblasts in Vitro J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4069 - 4077. [Abstract] [Full Text] [PDF]
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H. A. Coppock, A. White, J. D. Aplin, and M. Westwood Matrix Metalloprotease-3 and -9 Proteolyze Insulin-Like Growth Factor-Binding Protein-1 Biol Reprod, August 1, 2004; 71(2): 438 - 443. [Abstract] [Full Text] [PDF]
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M.-J. Lee, Y. Ma, L. LaChapelle, S. S. Kadner, and S. Guller Glucocorticoid Enhances Transforming Growth Factor-{beta} Effects on Extracellular Matrix Protein Expression in Human Placental Mesenchymal Cells Biol Reprod, May 1, 2004; 70(5): 1246 - 1252. [Abstract] [Full Text] [PDF]
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S. Bauer, J. Pollheimer, J. Hartmann, P. Husslein, J. D. Aplin, and M. Knofler Tumor Necrosis Factor-{alpha} Inhibits Trophoblast Migration through Elevation of Plasminogen Activator Inhibitor-1 in First-Trimester Villous Explant Cultures J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 812 - 822. [Abstract] [Full Text] [PDF]
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Y. Nishioka, T. Higuchi, Y. Sato, S. Yoshioka, K. Tatsumi, H. Fujiwara, and S. Fujii Human migrating extravillous trophoblasts express a cell surface peptidase, carboxypeptidase-M Mol. Hum. Reprod., December 1, 2003; 9(12): 799 - 806. [Abstract] [Full Text] [PDF]
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 L. Pavan, A. Tarrade, A. Hermouet, C. Delouis, M. Titeux, M. Vidaud, P. Therond, D. Evain-Brion, and T. Fournier Human invasive trophoblasts transformed with simian virus 40 provide a new tool to study the role of PPAR{gamma} in cell invasion process Carcinogenesis, August 1, 2003; 24(8): 1325 - 1336. [Abstract] [Full Text] [PDF]
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L. Qin, Y.-L. Wang, S.-X. Bai, S.-H. Ji, W. Qiu, S. Tang, and Y.-S. Piao Temporal and Spatial Expression of Integrins and Their Extracellular Matrix Ligands at the Maternal-Fetal Interface in the Rhesus Monkey During Pregnancy Biol Reprod, August 1, 2003; 69(2): 563 - 571. [Abstract] [Full Text] [PDF]
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T. Higuchi, H. Fujiwara, H. Egawa, Y. Sato, S. Yoshioka, K. Tatsumi, K. Itoh, M. Maeda, J. Fujita, and S. Fujii Cyclic AMP enhances the expression of an extravillous trophoblast marker, melanoma cell adhesion molecule, in choriocarcinoma cell JEG3 and human chorionic villous explant cultures Mol. Hum. Reprod., June 1, 2003; 9(6): 359 - 366. [Abstract] [Full Text] [PDF]
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V. Jokimaa, S. Oksjoki, H. Kujari, E. Vuorio, and L. Anttila Altered expression of genes involved in the production and degradation of endometrial extracellular matrix in patients with unexplained infertility and recurrent miscarriages Mol. Hum. Reprod., December 1, 2002; 8(12): 1111 - 1116. [Abstract] [Full Text] [PDF]
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Y. Sato, H. Fujiwara, T. Higuchi, S. Yoshioka, K. Tatsumi, M. Maeda, and S. Fujii Involvement of Dipeptidyl Peptidase IV in Extravillous Trophoblast Invasion and Differentiation J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4287 - 4296. [Abstract] [Full Text] [PDF]
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G. Xu, M.-J. Guimond, C. Chakraborty, and P. K. Lala Control of Proliferation, Migration, and Invasiveness of Human Extravillous Trophoblast by Decorin, a Decidual Product Biol Reprod, August 1, 2002; 67(2): 681 - 689. [Abstract] [Full Text] [PDF]
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M.O. Babawale, M.A. Mobberley, T.A. Ryder, M.G. Elder, and M.H.F. Sullivan Ultrastructure of the early human feto-maternal interface co-cultured in vitro Hum. Reprod., May 1, 2002; 17(5): 1351 - 1357. [Abstract] [Full Text] [PDF]
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F. Lyall, H. Simpson, J. Nicola Bulmer, A. Barber, and S. Courtenay Robson Transforming Growth Factor-{beta} Expression in Human Placenta and Placental Bed in Third Trimester Normal Pregnancy, Preeclampsia, and Fetal Growth Restriction Am. J. Pathol., November 1, 2001; 159(5): 1827 - 1838. [Abstract] [Full Text] [PDF]
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L. M. Gleeson, C. Chakraborty, T. McKinnon, and P. K. Lala Insulin-Like Growth Factor-Binding Protein 1 Stimulates Human Trophoblast Migration by Signaling through {{alpha}}5{beta}1 Integrin via Mitogen-Activated Protein Kinase Pathway J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2484 - 2493. [Abstract] [Full Text] [PDF]
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Y. Ma, C. J. Lockwood, A. L. Bunim, D. A. Giussani, P. W. Nathanielsz, and S. Guller Cell Type-Specific Regulation of Fetal Fibronectin Expression in Amnion: Conservation of Glucocorticoid Responsiveness in Human and Nonhuman Primates Biol Reprod, June 1, 2000; 62(6): 1812 - 1817. [Abstract] [Full Text]
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B. A. Kilburn, J. Wang, Z. M. Duniec-Dmuchkowski, R. E. Leach, R. Romero, and D. R. Armant Extracellular Matrix Composition and Hypoxia Regulate the Expression of HLA-G and Integrins in a Human Trophoblast Cell Line Biol Reprod, March 1, 2000; 62(3): 739 - 747. [Abstract] [Full Text]
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