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Program in Fetal Health and Development, Samuel Lunenfeld Research Institute, Mount Sinai Hospital; Departments of Obstetrics and Gynecology (I.C., C.V.T., J.W.K.R., S.J.L.), Pediatrics (I.C.), and Immunology (M.L.), University of Toronto, and the Division of Immunology and Cancer Research, Hospital for Sick Children (M.L.), Toronto, Ontario, Canada M5G 1X5
Address all correspondence and requests for reprints to: Dr. Isabella Caniggia, Mount Sinai Hospital, Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5. E-mail: caniggia{at}mshri.on.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6ß4 integrin, a laminin
receptor; and acquire 5ß1 integrin, a FN
receptor (7). Once the EVT invade the endometrium, they express the
1ß1 integrin, a collagen/laminin receptor.
Thus, specific changes in ECM proteins and their receptors are
associated with the acquisition of an invasive phenotype by the EVT
(4).
Growth factors and cytokines play a role in regulating trophoblast
differentiation along the invasive pathway in both an autocrine and a
paracrine fashion. Interleukin-1ß (8) and epidermal growth factor (9)
stimulate invasion of isolated first trimester trophoblast cells. In
contrast, transforming growth factor-ß (TGFß) inhibits the
differentiation of isolated first trimester trophoblast cells toward an
invasive phenotype (10). TGFß1 in vitro reduces first
trimester trophoblast proliferation, promotes their differentiation by
inducing syncytium formation, and inhibits their invasiveness by
inducing the expression of tissue inhibitor of metalloproteinases (11).
Within the human placenta, TGFß1 and TGFß2 have been localized by
immunostaining to both decidual and trophoblast cells at the
fetal-maternal interface throughout gestation (10). During the first
trimester, in situ hybridization revealed that TGFß1
messenger RNA (mRNA) was primarily localized to the
syncytiotrophoblast, with low levels in EVT, trophoblast columns, and
large decidual cells (12). TGFß2 can also inhibit first trimester
trophoblast migration in vitro and up-regulate the
expression of 5 and ß1 integrin subunits
(13).
TGFß binds to specific cell surface receptors, namely the TGFß type I receptor (R-I), TGFß type II receptor (R-II), betaglycan, and endoglin (14, 15). R-I and R-II are serine/threonine kinases, which in the presence of ligand, form a heteromeric complex that appears essential for signaling (15). Cross-linking experiments have demonstrated that isolated term trophoblast cells and placental membranes express betaglycan, R-I, and R-II (16).
Endoglin is a homodimeric glycoprotein that binds TGFß1 and TGFß3 with high affinity (14) and is primarily expressed on endothelial cells. Throughout placental development, endoglin is expressed at high levels on the syncytiotrophoblast and is transiently up-regulated on EVT within the columns (6, 17). Endoglin associates with R-II and R-I to form a functional receptor complex (18, 19). Endoglin when transfected into the U937 monocytic line was shown to modulate specific responses to TGFß1 (20), suggesting that it acts as a regulatory component of the receptor complex. Thus, endoglin expressed at the time of cell sprouting and column formation may play a critical role in the process of trophoblast migration and invasion.
Genbacev et al. (21) described a novel human villous explant culture system in which induction of EVT differentiation can be studied in intact villi. In contrast to isolated CTB, the explant system permits the dissection of the molecular events associated with the transition from polarized to nonpolarized cells that occurs in anchoring villi. As endoglin is expressed in cells undergoing this transition, we have used this model to investigate its role in the regulation of trophoblast differentiation along the invasive pathway. It has been suggested that TGFß from decidua, and to a minor extent from trophoblasts, regulates trophoblast invasion during first trimester (11). We, therefore, reasoned that interfering with its effects in the explant culture system might stimulate EVT migration and invasion. We thus analyzed the effects of antibody and antisense oligonucleotides to endoglin on the outgrowth, proliferation, and migration of trophoblasts, production of FN, and changes in integrin expression. We found that endoglin plays a key regulatory role in the process of trophoblast differentiation along the invasive pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments in the presence of 10% (vol/vol) FBS demonstrated that DMEM/F12 supported greater EVT sprouting and migration than DMEM. To study the effects of various agents on EVT differentiation, we then used a serum-free villous explant culture system. Villous explants of 5–8 weeks gestation were incubated overnight in DMEM/F12 or DMEM/F12 plus 10% (vol/vol) FBS to promote attachment of the distal villous tips to the Matrigel. After this incubation period, explants were washed with fresh medium and cultured in either serum-free DMEM/F12 or DMEM/F12 supplemented with varying concentrations of FBS (0.5% and 10%). In serum-free medium, the number of EVT per villous tip was 1.58 ± 0.08; it was 1.32 ± 0.17 in 0.5% FBS and 1.26 ± 0.02 in 10% FBS (mean ± SEM of three separate experiments, each performed in triplicate), suggesting that villous explant cultures were viable for at least 6 days in a serum-free medium. All subsequent experiments were performed with DMEM/F12 in the absence of serum.
The viability of the explant cultures was assessed by measuring hCG and progesterone production rates in the culture medium collected at the time of medium change every 48 h. Both hCG and progesterone concentrations were measured by RIAs (Coat-A-Count hCG immunoradiometric assay and progesterone, Diagnostics Products Corp., Los Angeles, CA). Results are expressed for progesterone as nanograms per 0.1 g wet wt tissue and for hCG as international units per 0.1 g wet weight tissue.
Antibodies
Murine monoclonal antibody (mAb) 44G4 specific for human
endoglin was produced as previously described (22). IgG purified from
ascites was used in all functional assays. Rat mAb 7D3 against
cytokeratin was a generous gift from Drs. S. Fisher and C. Damsky (San
Francisco, CA). Murine mAb TS2/7 against the 1 integrin
subunit was provided by Dr. M. Hemler (Boston, MA). Mouse mAb P1D6
against the 5 integrin subunit was obtained from
Chemicon (Temecula, CA); rat mAb GoH3 against the 6
integrin subunit was purchased from Serotec Canada (Toronto, Canada),
and the neutralizing rabbit polyclonal antibody to TGFß was obtained
from R&D (Minneapolis, MN). Purified mouse IgG from Coulter (Hialeah,
FL) and rat IgG from Sigma Chemical Co. (Toronto, Canada) were used as
negative controls. Human recombinant TGFß1, TGFß2, and TGFß3
isoforms were purchased from R&D (Minneapolis, MN).
Immunohistochemistry
Villous explants kept in culture for 6 days in the presence of
sense or antisense oligonucleotides to endoglin were dissected away
from the insert membrane with the supporting Matrigel. Explants and
placental tissue of 10 weeks gestation were fixed for 1 h at 4 C
in 4% (vol/vol) paraformaldehyde, cryoprotected by incubation in 10%
(vol/vol) glycerol for 30 min and 50% (vol/vol) OCT compound
(Tissue-Tek, Miles, Elkhart, IN) for 18 h, embedded in 100% OCT,
and frozen in liquid nitrogen. Ten-micron sections were cut with a
cryostat and mounted on poly-L-lysine-coated slides. To
verify the quality of the tissue and select the most representative
sections, every tenth one was stained with hematoxylin and eosin;
neighboring sections were selected and stained using the avidin-biotin
immunoperoxidase method. Endogenous peroxidase enzyme activity was
quenched with 3% (vol/vol) hydrogen peroxide in 0.01 M
Tris-HCl, pH 7.4, containing 0.15 M NaCl or methanol for 10
min. Nonspecific binding sites were blocked using 5% (vol/vol) normal
horse serum and 1% (wt/vol) BSA in Tris buffer for 40 min at 23 C. In
the case of murine monoclonal antibodies, a higher background was
observed, and it was necessary to preincubate the sections with 5%
(wt/vol) Texas red-conjugated goat antimouse IgG antibody for 1 h
at 23 C before incubation with primary antibody at 4 C for 1 h.
Optimal antibody concentrations were established in preliminary
experiments by titration and were used as follows: 44G4, 5 µg/ml;
rabbit anti-TGFß, 20 µg/ml; P1D6, 20 µg/ml; GoH3, 0.5 µg/ml;
TS2/7, 20 µg/ml; and 7D3, 10 µg/ml. The slides were washed three
times with Tris buffer, then incubated with a 200-fold dilution of
biotinylated goat antirabbit IgG or a 300-fold dilution of biotinylated
horse antimouse or antirat IgG for 1 h at 4 C. After washing three
times with Tris buffer, the slides were incubated with an avidin-biotin
complex for 1 h. Slides were washed again in Tris buffer and
developed in 0.075% (wt/vol) 3,3-diaminobenzidine in Tris buffer, pH
7.6, containing 0.002% (vol/vol) H2O2, giving
rise to a brown product. After light counterstaining with toluidine
blue, slides were dehydrated in an ascending ethanol series, cleared in
xylene, and mounted. In control experiments, primary antibodies were
replaced with nonimmune mouse or rat IgG or blocking solution [5%
(vol/vol) normal goat serum and 1% (wt/vol) BSA].
Effect of antibody to endoglin on EVT formation
Villous explants, prepared from placentas of 5–8 weeks
gestation, were incubated for 16 h in DMEM/F12. Explant cultures
were then washed with fresh serum-free medium and incubated in
serum-free DMEM/F12 medium containing increasing concentrations of mAb
44G4 IgG (0.1–10 µg/ml). DMEM/F12 medium with or without antibody
was replaced every 48 h. Antibody addition was thus performed on
days 1, 3, and 5 of culture. The morphological integrity of villous
explants and their EVT differentiation were monitored daily for up to 6
days.
Antisense oligonucleotides and their effects on EVT formation
Phosphorothioate oligonucleotides were synthesized on a DNA
synthesizer and purified by capillary electrophoresis. Oligonucleotides
of 16 bp targeted against sequences adjacent to the AUG initiation
codon of human endoglin (23) mRNA were synthesized. Previous studies
have demonstrated that antisense oligonucleotides targeted to sequences
adjacent to initiation codons are most efficient in inhibiting
translation (24). Furthermore, 16-mer oligonucleotides are short enough
to be taken up efficiently and provide sufficient specificity for
hybridization to the corresponding target mRNA (24). The sequences of
the antisense and sense endoglin oligonucleotides were
5'-GCGTGCCGCGGTCCAT-3' and 5'-ATGGACCGCGGCACGC-3', respectively. An
oligomer with the same composition as the antisense oligonucleotide,
but with a scrambled sequence, 5'-GCGGGCCTCGTTCCAG-3', was also
synthesized and used as a negative control. Oligonucleotides were
dissolved in water, and their concentrations were estimated by optical
density at OD260. Antisense or sense oligonucleotides
(5–10 µM) were added to the villous explants on days 1
and 3 of culture. EVT sprouting and migration from the distal end of
the villous tips were recorded daily for up to 6 days.
FN production
Villous explants of 5–8 weeks gestation were incubated
overnight in DMEM/F12. Explants were then washed and incubated in
DMEM/F12 containing either 10 µg/ml mAb 44G4 or nonimmune IgG and 10
µM antisense, scrambled, or sense endoglin
oligonucleotides. The medium with or without the various agents was
changed on day 3 and was replaced on day 5 by methionine-cysteine-free
low glucose DMEM containing 25 µCi/ml
[35S]methionine/cysteine with or without the same
antibodies or oligonucleotides. The cultures were metabolically labeled
for 18 h. Conditioned culture media were collected and diluted
with an equal amount of 25 mM Tris-HCl buffer (pH 7.4),
0.15 M NaCl, and 0.5% (vol/vol) Triton X-100, and FN was
isolated using gelatin-Sepharose as previously described (25). Briefly,
50 µl gelatin-Sepharose suspension were added to 500 µl medium, and
the samples were incubated overnight at 4 C. The gelatin-Sepharose
beads were centrifuged and washed three times in Tris/Triton X-100
buffer, and FN was eluted by boiling for 5 min in 1% (vol/vol) SDS and
electrophoresed on a 4–12% (wt/vol) polyacrylamide gradient gels.
Radiolabeled FN was revealed by autoradiography and quantitated using a
PhosphorImager (410A and ImageQuant software, Molecular Dynamics,
Sunnyvale, CA).
[3H]Thymidine incorporation into DNA
Villous explants of 5–8 weeks gestation, cultured for
48 h with and without antisense oligonucleotides to endoglin, were
incubated in the presence of 1 µCi [3H]thymidine/ml
medium. After 6 h of incubation, explants were washed with PBS,
fixed in 4% paraformaldehyde for 1 h, embedded in OCT, and
processed for cryostat sections as previously described. Ten-micron
sections were mounted on 3-amino-propyl-tryethoxysilane-precoated
slides and coated with NBT-2 emulsion (Eastman Kodak, Rochester, NY).
Slides were developed after 3 days using Kodak D-19 developer,
counterstained with eosin, and examined by brightfield microscopy.
Data analysis
All data are presented as the mean ± SEM of at
least three separate experiments performed in triplicate. Statistical
significance was determined by one-way ANOVA, followed by assessment of
differences using Student-Newman-Keuls test for nonpaired groups.
Significance was defined as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endoglin in vivo is up-regulated in first trimester
placenta trophoblasts as they break through the syncytium (6). We,
therefore, examined whether an antibody to endoglin (mAb 44G4) could
alter the early events of EVT differentiation along the invasive
pathway. Exposure of villous explants of 5–8 weeks gestation to 44G4
IgG was associated with an increase in EVT outgrowth from the distal
end of the villous tips and a higher number of cells migrating into the
surrounding matrix (Fig. 1B
). Stimulation
of EVT outgrowth and migration by 44G4 IgG was specific, as incubation
of explants with an equivalent amount of nonimmune murine IgG or medium
alone had no effect (Fig. 1A). Furthermore addition of 44D7 IgG (10
µg/ml; IgG1 isotype) reactive with CD98 antigen, a cell surface
molecule expressed at high levels on syncytiotrophoblast (26), had no
stimulatory effect (data not shown).
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). Control
explants, cultured in the presence of sense endoglin oligonucleotides,
exhibited no such effect (Fig. 1C).
Further experiments demonstrated that 24 h after the addition of
44G4 IgG (day 2 of culture), there was a significant increase in EVT
outgrowth and migration from 0.20 ± 0.03 in the control group to
2.03 ± 0.46 in the antibody-treated group (n = 4;
P < 0.005; Fig. 2A).
After 5 days of treatment (day 6), the number of EVT outgrowths
increased from 0.64 ± 0.09 in control IgG-treated explants to
3.2 ± 0.5 in the 44G4 IgG-treated explants (n = 10;
P < 0.05; Fig. 2A). Subsequent experiments
demonstrated that the stimulatory effect of 44G4 IgG was dose dependent
and was maximal at 1 µg/ml (Fig. 2B).
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). After 5 days of exposure, the number of EVT per villous
tip increased from 2.08 ± 0.47 in sense-treated explants to
8.46 ± 1.7 in antisense-treated cultures (Fig. 2C). The antisense
endoglin effect on trophoblast migration and invasion was specific, as
incubation of explants with an equivalent amount of either sense
endoglin (Fig. 2C) or scrambled antisense endoglin oligonucleotide (not
shown) had no effect. Antisense endoglin stimulated EVT outgrowth and
migration in a concentration-dependent manner, with maximal stimulation
observed at 10 µM (Fig. 2D).
Characterization of trophoblast differentiation along the invasive
pathway in villous explants cultures
Previous reports indicate that stem trophoblasts within the
villous core and at the proximal site of the column, where trophoblasts
start to migrate away from the stem villi, undergo proliferation (21),
whereas differentiated EVT do not. Therefore, we investigated whether
EVT outgrowth triggered by antisense endoglin treatment was due to cell
division or migration. [3H]Thymidine autoradiography of
explants exposed to antisense endoglin oligonucleotides showed villous
trophoblast proliferation within the villous tip at the proximal site
of the forming column, whereas neither differentiated EVT, which have
invaded the surrounding Matrigel, nor mesenchymal cells in the villous
core showed any DNA synthesis (Fig. 3).
This suggests that EVT within the column do not divide, and that
blockage of endoglin most likely induces cell migration from the
villous core.
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). The 6 integrin subunit
was found on polarized CTB within the villi and on the nonpolarized
trophoblasts in the proximal columns (Fig. 4A). The 5
integrin subunit was minimally expressed on polarized trophoblasts or
syncytium, but was present on EVT within the columns. EVT that had
migrated further away in the Matrigel were negative for the
5 integrin (Fig. 4B). All trophoblast cells, including
CTB within the villi, syncytiotrophoblast, and EVT stained positively
for cytokeratin, confirming the epithelial-like nature of the cells
forming the columns and migrating into the Matrigel (Fig. 4C). EVT that
had migrated into Matrigel were positive for the 1
integrin (Fig. 4D). A polyclonal antibody to TGFß showed staining of
the syncytiotrophoblast and stroma of the villi, suggesting that TGFß
was present in the culture system. Migrating EVT and the Matrigel
itself, known to contain TGFß, showed weak positive staining (Fig. 4E). No reactivity was observed in the explants stained with control
IgG (Fig. 4F).
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), high levels of endoglin were
observed in syncytiotrophoblast, and weak positive immunoreactivity was
also noticed in CTB. In endoglin antisense oligonucleotide-treated
villous explants, endoglin expression was low in syncytiotrophoblast
and absent in CTB (Fig. 5B). As little EVT outgrowth was observed under
basal culture conditions, the expression of endoglin in trophoblast
columns could only be studied in antisense-treated explants. The
staining of endoglin in EVT of explants treated with antisense endoglin
was weakly positive (Fig. 5E) compared with sections of the same
explant stained with control IgG (Fig. 5F). In addition, endoglin
expression in proximal columns of explants (Fig. 5E) was much reduced
compared with sections of 10 weeks gestation placenta stained under
similar conditions (Fig. 5H). When a subsequent section of this
placenta was stained for 5 integrin (Fig. 5G), the
transition zone in the proximal column was clearly visualized as
negative for 5, but positive for endoglin (Fig. 5H). The
5 integrin in explants treated with antisense endoglin
was also found to be highly expressed in EVT within proximal and distal
columns (Fig. 5D). These data suggest that antisense endoglin
treatment, which promotes EVT outgrowth and migration in explant
cultures, induces a decrease in endoglin expression at the level of the
transition zone, followed by an increase in expression of the
5 integrin FN receptor.
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shows a representative experiment in
which both 44G4 IgG and antisense endoglin oligonucleotides induced a
significantly greater production of FN than that observed in control
IgG or sense oligonucleotide-treated cultures. PhosphorImager analysis
of all data demonstrated 8- and 5-fold increases in FN synthesis (five
independent experiments performed in triplicate; P <
0.05) for 44G4 IgG and antisense endoglin-treated explants,
respectively, relative to control sense or DMEM/F12 alone (Fig. 7, A and B). FN production in villous
explants cultured in the presence of a scrambled antisense endoglin
oligonucleotide was similar to that in sense-treated explants or in
medium alone.
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shows a representative experiment
demonstrating that treatment of villous explants with TGFß1 and
TGFß3, but not TGFß2, decreased FN synthesis compared with that in
explants treated with medium alone. PhosphorImager analysis of three
different experiments showed that the TGFß1 and TGFß3 inhibitory
effect was statistically significant compared with that of control
DMEM/F12 alone (Fig. 8B).
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, A and B). Addition of
exogenous TGFß1 and TGFß3 to explant cultures incubated with
antisense oligonucleotides or antibody to endoglin, which binds both
isoforms, did not alter the stimulatory effect of antisense
oligonucleotide and antibody to endoglin on FN synthesis. In contrast,
addition of TGFß2, which does not interact with endoglin, overcame
the antibody and antisense oligonucleotide stimulatory effect on FN
synthesis. TGFß2, but not TGFß1 or TGFß3, inhibited the EVT
outgrowth and migration induced by the antisense endoglin treatment
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, Vicovac et al. (28) showed that first trimester floating villi cocultured with decidua parietalis retained the capacity to differentiate in vitro into anchoring villi. Similar observations were reported for villi cultured in Matrigel by Genbacev et al. (21). Our studies in the absence of serum support these findings and suggest that villous explants reproduce the events occurring during the formation of anchoring villi. Local breakdown of the syncytium by trophoblasts and their outgrowth were observed, generating columns of EVT that migrate and invade the surrounding matrix. Maintenance of hormone (progesterone and hCG) production by the explants suggests that these structures retain the viability and functional integrity found in other studies (21). The slightly lower progesterone production observed in our system is probably due to culture in serum-free medium and hence reflects a reduced precursor pool.
Trophoblast invasion in vivo is accompanied by changes in
the spatial distribution of ECM, such as loss of laminin and appearance
of a matrix enriched in FN (3, 4). During implantation and trophoblast
invasion, oncofetal FN has been found at the level of the placental
uterine junction in the matrix surrounding the anchoring villi (27) and
in soluble form in amniotic fluid, suggesting a role in regulating
uterine-placental adherence. Human trophoblasts from both first
trimester and term placenta synthesize oncofetal FN, which is deposited
in the ECM (29). Studies in vitro with isolated trophoblasts
indicate that FN synthesis and secretion are attributed mostly to the
5 integrin-positive cells (30).
Increasing evidence suggests that TGFß modulates FN production in various cell types, including trophoblasts. TGFß, exogenously added or derived from serum, as demonstrated with neutralizing antibodies, was reported to stimulate oncofetal FN production by trophoblast cells in vitro (29). Although the villous explants of 5–8 weeks gestation under basal conditions synthesized and released FN into the medium, we found that interfering with endoglin synthesis is associated with increased FN production. As antibody and antisense treatments trigger EVT outgrowth in the explant system, this increased FN production is probably due to an increased number of differentiating trophoblasts producing FN and/or increased FN expression. Thus, our data suggest that TGFß may be a negative regulator of trophoblast differentiation along the invasive pathway in the explant system and that endoglin appears to be required to mediate this effect. These data are seemingly in contradiction with the observation that TGFß1 does not affect the rate of isolated first trimester trophoblast invasion in vitro (9). These cells are known to acquire an invasive phenotype when cultured in vitro (31). In the explant system, earlier events in trophoblast differentiation along the invasive pathway are taking place, namely breaking through the syncytium of an intact villi before acquisition of the invasive phenotype. The observation that endoglin expression along this pathway is confined to this area of transition and the proximal columns suggests that regulation of this pathway by TGFß is at this level. As production of FN is mostly attributed to the EVT within the columns, this suggests that the trophoblast cells are differentiating into FN-producing cells once they are removed from the regulatory influence of endoglin.
The observation that blockage of endoglin accelerates trophoblast migration and invasion is also supported by the finding that villous trophoblast cells at the proximal site of the forming column undergo DNA synthesis, an event that is known to occur in the early phases of the normal program of trophoblast differentiation along the invasive pathway (21). Furthermore, we observed changes in integrin expression that mimic the EVT differentiation pathway observed in vivo in anchoring villi of first trimester placenta (4, 6) and in vitro in cocultures of first trimester villous and decidual tissue (28).
The observation that antisense oligonucleotides and antibodies to endoglin stimulated trophoblast migration and invasion suggest that this process is normally regulated by TGFß, a finding supported by our data showing that addition of 10 ng/ml TGFß1 and TGFß3, but not TGFß2, decreased FN synthesis in villous explants.
We found that antisense endoglin treatment of villous explants decreased the endoglin protein content in syncytiotrophoblast and CTB compared with that in control explants cultured in the presence of sense oligonucleotides. Together with the stimulatory effects observed by adding the 44G4 antibody directly to the culture system, these data suggest that the morphological and biochemical events induced by antisense oligonucleotides are due to a reduction in endoglin expression. In addition, we demonstrated that antisense treatment reduced endoglin expression on EVT within the proximal part of the column compared with that found in vivo at 10 weeks gestation in placenta, suggesting that the antisense treatment was also acting on cells of the proximal column. It is unlikely that the formation of EVT outgrowths is a nonspecific response to antisense treatment. First neither sense nor scrambled antisense endoglin oligonucleotides sequence alter villous morphology or FN production. Secondly, TGFß1 and TGFß3 antisense oligonucleotides induce EVT migration, whereas antisense TGFß2 do not (unpublished observations). Moreover, similar stimulatory effects on trophoblast migration and invasion were produced after incubation with an antibody reactive to endoglin. Support for the role of endoglin in controlling EVT differentiation was provided by the observation that TGFß1 and TGFß3 isoforms did not alter the stimulatory effects of antisense and antibody to endoglin on villous outgrowth and FN synthesis. However, TGFß2, which does not bind endoglin, could inhibit the effects of antisense and antibody to endoglin. In contrast, addition of TGFß2 alone to explants did not affect FN production. Thus, it is possible that removal of the primary inhibitory system may unmask other inhibitory pathways by which TGFß2 may act.
The mechanism(s) by which endoglin mediates TGFß effects needs to be elucidated. TGFß R-II and R-I, which are serine kinases, form a heteromeric receptor complex upon ligand binding that appears to be essential for TGFß signaling (15). It is plausible that in EVT, endoglin associates with these TGFß signaling receptors and regulates the inhibitory effect of TGFß on EVT differentiation.
During the first trimester of gestation, TGFß is colocalized with one of its natural inhibitors, decorin, in the ECM of decidual tissue, suggesting that this proteoglycan may aid TGFß storage or limit its activity within the decidual ECM (12). Our findings suggest that TGFß produced by the villi is a negative regulator of trophoblast differentiation along the invasive pathway. The expression of endoglin at the transitional zone from polarized to nonpolarized trophoblasts appears essential to the mediation of this negative regulation by TGFß1 and/or possibly TGFß3. Blocking endoglin expression in this transition phase triggers EVT outgrowth and migration and FN production. Thus, trophoblast invasion, characteristic of normal human placentation, is dependent on an intricate balance between positive and negative regulators. Our data suggest that endoglin is a critical negative regulator of this system. This raises the possibility that inappropriate expression or function of endoglin might contribute to the major complications of pregnancy, such as preeclampsia or choriocarcinoma, which are associated with abnormal trophoblast invasion and placenta development.
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Acknowledgments |
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Footnotes |
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2 Career scientist of the Ontario Ministry of Health.
3 Terry Fox Research Scientist of the National Cancer Institute of
Canada.
Received April 10, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 Y. Hu, J. P. Dutz, C. D. MacCalman, P. Yong, R. Tan, and P. von Dadelszen Decidual NK Cells Alter In Vitro First Trimester Extravillous Cytotrophoblast Migration: A Role for IFN-{gamma} J. Immunol., December 15, 2006; 177(12): 8522 - 8530. [Abstract] [Full Text] [PDF]
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), medium alone (
), or nonimmune mouse IgG1 (10 µg/ml;
). *, P < 0.05, antibody-treated
vs. controls nonimmune IgG1 or medium alone. B,
Dose-dependent effect of mAb 44G4 on EVT formation. The number of EVT
outgrowths per villous tip was measured at an antibody concentration
varying from 0.01–10 µg/ml. *, P < 0.05,
antibody-treated vs. untreated. C, Time-dependent effect
of antisense endoglin oligonucleotides on the number of EVT outgrowths
per villous tip. 
, Antisense endoglin oligonucleotides; 
, sense
endoglin oligonucleotides; 
