1 Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, 2100 Copenhagen,
Denmark
2 Department of Anatomy, University of California, San Francisco, CA 94143-0452,
USA
* Author for correspondence (e-mail: lund{at}inet.uni2.dk)
Accepted 3 June 2003
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SUMMARY |
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Key words: Invasion, Tissue remodeling, Plasminogen deficiency, Implantation, Matrix metalloproteinases
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INTRODUCTION |
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One proposed function of decidualization is to protect the mother from the
invasive nature of the embryo. Transplanting the mouse embryo outside of the
uterus results in extensive invasion and growth of the embryo
(Kirby, 1965). It is likely
that the decidua protects the uterus from inappropriate invasion of the embryo
through a physical barrier as well as by the production of inhibitors of
proteolytic enzymes, such as the tissue inhibitors of metalloproteinases
(TIMPs) and plasminogen activator inhibitor type 1 (PAI1) that are produced by
the decidual cells (Alexander et al.,
1996
; Das et al.,
1997
; Reponen et al., 1995;
Teesalu et al., 1996
).
From gestation days 7.5-12, fetal blood vessels form and invade the yolk
sac placenta to provide nutrient and gas exchange until the true
chorioallantoic placenta is formed, a process that begins at gestation day 9.
By day 10 of gestation, three zones of the placenta can be visualized: the
labyrinth layer, which is composed of embryonic trophoblast cell strands
containing maternal blood, the junctional zone spongiotrophoblast layer, and
the trophoblast giant cell zone that isolates the embryo from the mother
(Rinkenberger and Werb, 2000).
Embryonic vessel formation begins by day 10 in the labyrinth layer; these
vessels contain nucleated fetal erythrocytes, indicating that placental
circulation has begun (Adamson et al.,
2002
). It is in the labyrinth layer that the maternal and
embryonic circulations are in closest proximity, and where gas and nutrient
exchange will take place. Failure to implant or to establish a functional
placenta results in death and resorption of the embryo. Malformation of the
placenta results in intrauterine growth retardation and birth of runted
embryos with poor survival prospects
(Cross et al., 1994
;
Rinkenberger and Werb, 2000
;
Rossant and Cross, 2001
).
Trophoblast invasion depends on the finely tuned balance between activated
proteases and protease inhibitors, enabling the extracellular degradation and
phagocytosis of maternal cells and extracellular matrix. Based on both
expression and functional studies, at least three classes of proteases, matrix
metalloproteinases (MMPs), serine proteases and cysteine proteases, could be
involved in the extracellular matrix remodeling that facilitates trophoblast
invasion (Cross et al., 1994;
Alexander et al., 1996
;
Afonso et al., 1997
). The
expression of the serine protease urokinase-type plasminogen activator (uPA)
(Sappino et al., 1989
;
Teesalu et al., 1996
;
Teesalu et al., 1998
) and
several MMPs, including MMP2, MMP9, MMP11 and mouse ColA
(Alexander et al., 1996
;
Teesalu et al., 1999
;
Balbin et al., 2001
) in both
trophoblast giant cells and/or the decidua, indicates that they may function
in the tissue remodeling and cell invasion processes that take place during
implantation and placentation. However, the results of gene deletion mouse
models have indicated that although protease activity is crucial for
implantation success, there is considerable functional redundancy among the
various classes of proteases. Deleting the plasminogen/plasmin system does not
affect the viability of homozygous knockout animals
(Carmeliet et al., 1994
;
Bugge et al., 1995a
;
Bugge et al., 1995b
;
Bugge et al., 1996
;
Ploplis et al., 1995
;
Teesalu et al., 1999
). None of
the MMP-null mice have so far been shown to be infertile
(Itoh et al., 1998
;
Sternlicht and Werb, 1999
;
Teesalu et al., 1999
;
Vu and Werb, 2000
). Taken
together, these data suggest that the protease interactions are complex to
ensure that appropriate maternal and fetal connections are made during
implantation.
In the absence of plasminogen (PLG), skin wound healing, postlactational
mammary gland involution and tumor metastasis are delayed but ultimately do
occur (Rømer et al.,
1996; Bugge et al.,
1998
; Lund et al.,
1999
; Lund et al.,
2000
). In the case of wound healing, there is functional overlap
between PLG and one or more MMPs, such that a complete arrest of healing
requires both PLG deficiency and MMP inhibition
(Lund et al., 1999
). These
data led us to hypothesize that there may also be a functional overlap between
PLG and the MMPs during implantation and placentation. To test this
hypothesis, we have studied the effect of galardin (GM6001), a hydroxamate
broad-spectrum inhibitor of MMPs (Grobelny
et al., 1992
; Levy et al.,
1998
), on implantation and placentation in wild-type and
Plg-deficient mice.
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MATERIALS AND METHODS |
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Animals and tissue treatment
The mice used in these experiments were Plg gene targeted mice of
a mixed 129/Black Swiss background (Bugge
et al., 1995a) backcrossed to NIHS outbred mice for 8-12
generations. For breeding of experimental animals, heterozygous
Plg+/- mice were used as breeding pairs. In all
experiments the wild-type control mice were littermates to the
Plg-deficient mice. Stud males were littermates to the female
experimental mice. Genotyping of the Plg alleles was performed as
described (Bugge et al.,
1996
). To generate embryos of known genotype in mothers of the
same genotype, homozygous Plg-/- and
Plg+/+ females were mated to homozygous
Plg-/- and Plg+/+ males, respectively,
with proven fertility and checked for pregnancy by occurrence of vaginal plugs
in the morning [midnight=0 days post-coitum (dpc)]. Galardin was administered
intraperitoneally at 150 mg/kg body weight as a 30 mg/ml slurry in 4%
carboxymethylcellulose (CMC) in PBS. Injections began at 3.5 dpc and were
administered every 24 hours thereafter until 13.5 dpc. The mice used for
histological analysis were anesthetized by injection of 0.03 ml/10 g of a 1:1
mixture of Dormicum (Midazolam, 5 mg/ml) and Hypnorm (Fluanison, 5 mg/ml and
Fentanyl, 0.1 mg/ml). The mice were perfused intracardially with 10 ml
ice-cold PBS followed by 10 ml 4% paraformaldehyde (PFA). The uteri were
removed, the number of implantation and/or resorption sites was determined
macroscopically and tissues were fixed overnight in 4% PFA. The tissue was
dehydrated and embedded in paraffin wax. Animal care at the University of
Copenhagen and Rigshospitalet, Copenhagen, Denmark was in accordance with
national and institutional guidelines.
Antibodies for immunohistochemistry
Rabbit polyclonal antibodies (pAb) raised against the mouse receptor for
urokinase-type plasminogen activator (uPAR)
(Solberg et al., 2001) were
used at a concentration of 10 µg/ml. Rabbit pAb against fibrin/fibrinogen
were used at a dilution of 1:1000, as described previously
(Bugge et al., 1995a
;
Suh et al., 1995
). CD-34 rat
monoclonal antibody (mAb) (clone MEC 14.7)
(Baumhueter et al., 1993
) was
obtained from HyCult Biotechnology, Uden, Netherlands.
Immunohistochemistry
Paraffin wax-embedded sections were deparaffinized in xylene, hydrated
through graded ethanol to aqueous solution and digested with 0.03% trypsin for
10 minutes at 37°C, blocked in 5% swine serum in TBS with 0.25% BSA
(TBS-BSA) and incubated with the primary antibody in TBS-BSA. Then a
biotin-conjugated swine-anti-rabbit pAb (DAKO E431) was applied, followed by
AP-ABC (DAKO K 0376). Slides were developed with Fast Red (DAKO) and
counterstained with Mayers Hematoxylin. As negative controls, we used
pre/non-immune rabbit IgG. As a control for the specificity of the staining
obtained for uPAR we used analogous tissue from uPAR-deficient mice
(Bugge et al., 1995b).
In situ hybridization
35S-labeled RNA sense and antisense probes were generated by in
vitro transcription from subclones of the following mouse cDNA: MMP9, MMP14,
the spongiotrophoblast marker 4311, placental lactogen 1 (PL1) and PECAM, as
described (Lund et al., 1996;
Lund et al., 1999
;
Rømer et al., 1996
;
Baldwin et al., 1994
;
Colosi et al., 1987
; Lescisin
et al., 1988). Tissue sections were deparaffinized in xylene, hydrated through
graded ethanol/water dilutions and the in situ hybridization was carried out
as described (Lund et al.,
1996
). For each mRNA, expression was detected with two
non-overlapping probes with identical results. Sense probes were included in
all experiments and in all cases gave negative results.
Statistical analysis
Statistical evaluation of differences between groups of mice was done with
the Wilcoxon rank sum test.
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RESULTS |
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Expression of uPAR and MT-1-MMP during implantation and
placentation
The localization of protease activities is determined in part by the
location of their receptors. Two molecules that could localize plasmin and MMP
activity are the receptor for urokinase plasminogen activator and the
membrane-type MMP MT1-MMP or MMP14. We therefore next examined the expression
of uPAR by immunohistochemistry, using highly specific polyclonal antibodies
against mouse uPAR (Solberg et al.,
2001). We also examined MT1-MMP expression by in situ
hybridization. Localization studies were done on sections of implantation
sites at 7.5, 8.5, 10.5 and 12.5 dpc.
In 7.5 dpc implantation sites, uPAR staining was seen in the maternal vessels, as well as in the decidua cells, with the most intense uPAR immunoreactivity in the less differentiated of these cells towards the peripheral zone of the decidua (Fig. 1C). Weak to moderate uPAR staining was also seen in the trophoblast cells, identified by staining for cytokeratin (data not shown). No staining was seen with either pre-immune rabbit IgG (Fig. 1D) or with the polyclonal immune IgG on equivalent tissue obtained from uPAR-deficient mice (data not shown). The same uPAR staining pattern was seen at 8.5 dpc implantation sites (Fig. 1G,O). At 12.5 dpc implantation sites the remaining mesometrial decidua cells (Fig. 1K), as well as the giant trophoblast cells, identified by PL1 expression were positive for uPAR immunoreactivity (Fig. 1K, and data not shown). uPAR staining was also seen in the maternal vessels and in the spongiotrophoblast cells, identified by detection of mRNA for the spongiotrophoblast marker 4311 (Fig. 1K, and data not shown). uPAR staining was also detected in the maternal vessels, as well as in the embryonic vessels present in the labyrinth layer. The embryonic vessels were easily recognized by their content of nucleated red blood cells (data not shown).
|
Delayed decidualization and angiogenesis at 7.5 dpc in
Plg-deficient mice treated with galardin
We next examined the impact of galardin treatment on embryo implantation
sites in Plg-deficient and littermate wild-type mice. Plugged female
mice were either treated daily with 150 mg/kg of galardin or mock treated from
3.5 dpc until they were sacrificed at 7.5 dpc. In histological sections, all
implantation sites from mock-treated wild-type mice had an elongated egg
shape, whereas 33% of the implantation sites from galardin-treated wild-type
mice and Plg-deficient mice and 60% of the implantation sites from
galardin-treated Plg-deficient mice were rounded instead of egg
shaped (Table 2 and
Fig. 2A, parts a-d).
Morphometric analysis revealed that the decidual length was significantly
shorter in the mock-treated Plg-deficient mice than in the
mock-treated wild-type mice (P<0.001), whereas galardin-treatment
in both genotypes led to only moderately and non significantly shorter
decidual length (P>0.05 for both genotypes). The most pronounced
difference was however seen between the galardin-treated
Plg-deficient mice and the mock-treated wild-type mice
(P<0.0001) (Fig.
2B).
|
|
|
MMP-9 and plasminogen are both involved in angiogenesis
(Pepper, 2001). We observed
that 60% of the 7.5 dpc implantation sites from galardin-treated
Plg-deficient mice had a decreased decidual vascularization compared
with mock-treated Plg-deficient mice. The endothelial cells were
identified by their expression of PECAM mRNA by situ hybridization
(Fig. 3A, parts i-n,
Table 2).
Retarded embryonic growth at 8.5 dpc in Plg-deficient mice
treated with galardin
To study the effect of galardin and Plg-deficiency at later stages
of implantation, we treated mice of both genotypes daily with galardin or
vehicle alone from 3.5 dpc to 8.5 dpc, and then analyzed the implantation
sites. Only slight differences in the size and shape of the decidua were
observed in implantation sites from the four groups of mice
(Fig. 2, parts e-h). At 8.5
dpc, 50% of the embryos from Plg-deficient mice treated with galardin
were runted and resembled the mock-treated wild-type embryos at the 7.5 dpc
developmental stage (Table 2,
Fig. 2, parts e-h). Although
MMP-9 and PL-1 mRNA were detected in the trophoblast giant cells of
implantation sites from all four groups of mice
(Fig. 3B, parts a-d), the
expression pattern of MMP-9 in galardin-treated Plg-deficient mice
8.5 dpc was characteristic of the pattern of mock-treated wild-type mice 7.5
dpc (compare Fig. 3B, part d
and Fig. 3A, part a). The
vascularization of the decidua in 8.5 dpc galardin-treated
Plg-deficient mice, detected by PECAM expression
(Fig. 3B,n), was also like that
seen in 7.5 dpc mock-treated wild-type implantation sites, indicating that the
interaction of the embryos with the maternal vasculature was delayed in the
galardin-treated Plg-deficient mice and probably contributed to the
phenotype observed at 8.5 dpc.
|
|
|
In the galardin- and mock-treated wild-type placentas, as well as in the mock-treated Plg-deficient placentas, the fetal vessels were uniformly distributed throughout the labyrinth as shown by immunohistochemical staining of CD34 (Fig. 4N; not shown) and by in situ hybridization for PECAM mRNA (not shown). However, in the galardin-treated Plg-deficient placentas, fetal blood vessels were abnormal in a third of all placentas and not detected in the labyrinth layer by either CD34 staining (Fig. 4Q) or PECAM mRNA in situ hybridizations (not shown), indicating that Plg-deficiency and galardin treatment synergistically impair the fetal vessel formation. The defective vascularization of the labyrinth placenta may reduce feto-maternal exchange of gases and nutrients, causing growth retardation and, in the severely affected placentas, embryonic death. In the remaining placentas an apparent normal vessel system was observed as detected by gross histological analysis at day 12.5 dpc.
In the 12.5 dpc placentas from galardin-treated mice of both genotypes and from mock-treated Plg-deficient mice a large amount of fibrin was detected in the middle of the placenta surrounding the central artery. However, no increase in the amount of fibrin(ogen) was observed in the rest of the placental tissue (Fig. 4O,R). It is unlikely that impaired fibrin degradation is a primary cause of the phenotype of the placentas from galardin-treated Plg-deficient mice.
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DISCUSSION |
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These data raise the question of how the two enzyme systems interact in
vivo. In wild-type mice, uPA is expressed in maternal vessels in the decidua
close to the embryo and in trophoblast cells of the ectoplacental cone
(Teesalu et al., 1996). As
shown in the present study uPAR is expressed in the decidua cells, in the
maternal vessels and in trophoblast cells, whereas MMP-9 and mColA are also
expressed in the giant trophoblast cells
(Alexander et al., 1996
;
Balbin et al., 2001
). In
contrast to an earlier report based on immunohistochemical analysis of
cultured blastocysts, we found that MT1-MMP was expressed in maternal vessels
in the decidua, in cells surrounding the remnant of the uterine lumen and in
the undifferentiated decidua, but not in giant cells as described
(Tanaka et al., 1998
). MMP2,
MMP3 and MMP11 are all expressed in the undifferentiated decidua
(Alexander et al., 1996
;
Teesalu et al., 1999
). A delay
in the development of the decidua in response to protease inhibition by
galardin has been demonstrated previously
(Alexander et al., 1996
), and
could arise as a result of inhibition of several enzymes. Our results
demonstrate that although inhibition of either MMPs or loss of plasminogen
leads to an initial effect on decidualization at 7.5 dpc, none of the embryos
in these groups of mice show signs of growth retardation. Therefore,
deficiency of neither plasminogen nor MMPs alone impairs normal embryonic and
placental development, probably owing to a functional overlap between the two
classes of proteases (Lund et al.,
1999
). This redundancy may be extensive as mice deficient for both
uPA and MMP11 have normal embryonic and placental development and are fertile
(Teesalu et al., 1999
).
Likewise, mice deficient for MMP9 and PLG are viable and fertile (L.R.L.,
unpublished). However, this is not surprising given the role of galardin as a
broad-spectrum inhibitor (Grobelny et al.,
1992
; Levy et al.,
1998
). Furthermore, each protease might have multiple and distinct
functions during various stages of implantation and placental development, as
well as during embryogenesis. Future genetic experiments and/or access to
inhibitors specific for individual MMPs will be required to identify the MMPs
involved and elucidate their functional roles and interactions with
plasminogen (Johnsen et al.,
1998
).
The placentas from galardin-treated Plg-deficient mice exhibited
improper vascularization and development of the labyrinth layer. The width of
the labyrinth layer of placentas in galardin-treated Plg-deficient
mice is significantly less than in the other three groups of mice. The
development of the labyrinth layer is essential for embryo survival because
after gestation day 10.5, all exchange of gases and nutrients takes place
there. Failure to establish a proper feto-maternal circulatory system
eventually will result in death and resorption of the embryo
(Cross et al., 1994).
Histological analysis revealed that the entire labyrinth structure in
galardin-treated Plg-deficient mice was abnormal, both trophoblast
differentiation and villous morphogenesis appeared to be defective. As fetal
vessels grow into the branched villous as a secondary event, the observed lack
of vessels in the labyrinth may be a secondary effect
(Rossant and Cross, 2001
).
This phenotype is seen only in galardin-treated Plg-deficient mice;
thus, the result strongly suggests that both plasmin and MMP activities
directly or indirectly are involved in vascularization of the labyrinth, and
that there is a functional overlap between the two types of activities, so
that each of them alone is adequate for proper vascularization. Expression
studies, showing co-localization of mRNA for uPA and MMP9 10.5 dpc in giant
trophoblast cells at the fetomaternal interface
(Teesalu et al., 1998
;
Teesalu et al., 1999
) support
this hypothesis. However, the functional implication of co-expression of uPA
and MMP-9 can be definitively clarified only by the generation of mice
deficient for both uPA and MMP9. The giant trophoblast cells were also shown
to express uPAR by immunohistochemical staining, thus enabling a focalized
uPA-catalyzed proteolysis at the fetomaternal interface. The functional
importance of this is shown by in situ zymography data reported by Teesalu et
al. (Teesalu et al., 1998
),
demonstrating in situ uPA-dependent proteolytic activity.
The combined loss of plasminogen and MMP inhibition also affected the
normal development of the trophoblast giant cell. At 12.5 dpc the placentas
from galardin-treated Plg-deficient mice displayed abnormal spatial
distribution of trophoblast giant cells, which remained accumulated in the
middle of the placenta in a condensed cluster of PL1-positive cells; in
addition an abnormal multicellular layer of trophoblast giant cells was
observed towards the periphery of the placenta. This finding indicates a
potential defect in the migration or differentiation of these cells as a
result of MMP-inhibition and lack of plasminogen. However, to distinguish
clearly between a differentiation and/or migration defect future studies with
additional and more specific markers
(Adamson et al., 2002) will be
required. A defect in giant cell migration is in accordance with previous in
vitro studies of cultured blastocysts, where galardin treatment inhibited the
migration of the trophoblast cells
(Librach et al., 1991
;
Behrendtsen et al., 1992
).
The placentas from galardin-treated mice of both genotypes separated during
collection between the decidual layer and the spongiotrophoblast layer. This
could be due to dead or dying tissue at the interface or to accumulation of
maternal blood at the interface which is unable to enter into the labyrinth
layer. However, it is tempting to speculate that cell-cell or cell-matrix
adhesion of these layers was abnormal. Inhibition of MMPs or lack of
plasminogen leads to abnormal adhesion and migration of cells. Cleavage of
laminin-5 -chain by plasmin has been shown to be important for the
formation of hemidesmosomes of cells in vitro
(Goldfinger et al., 1998
).
Furthermore, cleavage of laminin-5
-chain by MMP2
(Giannelli et al., 1997
) and
MT1-MMP (Koshikawa et al.,
2000
) induces migration of cells cultured on laminin 5. The
mechanism for cell-cell and cell-matrix adhesion in placenta has not been
elucidated in the present study, but abnormal adhesion of the cell layers due
to malformation of hemidesmosomes may prevent adequate development of the
maternal-fetal interface, resulting in growth retardation or fetal death.
Even though the galardin treatment was stopped after 13.5 dpc the placental
insufficiency persisted and pups that reached parturition had high perinatal
mortality. Malformation of placentas is known to result in intrauterine growth
retardation and birth of runted embryos with poor survival prospects.
Moreover, MMPs and PLG are necessary for the normal development of the mammary
gland (Witty et al., 1995;
Thomasset et al., 1998
;
Lund et al., 2000
). Thus, the
observed lack of milk in pup stomachs indicates that the poor postnatal
survival rate of pups delivered by galardin-treated mice of both genotypes
might also be due to impaired lactation resulting from impaired mammary
development.
Mutation of almost every gene that regulates angiogenesis or vascular
development results in placental defects of varying severity
(Rossant and Cross, 2001;
Adamson et al., 2002
). Both
MMPs and plasminogen have a role in angiogenesis in a number of developmental
and pathological processes (Pepper,
2001
). Our data show that MMPs and the plasminogen system
synergize during the development of vascular connections between the mother
and fetus. Placental development is a highly sensitive indicator of the genes
involved in vascular development. The challenge remains to elucidate the
molecular targets of these proteases that direct each stage of trophoblast and
placental development.
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ACKNOWLEDGMENTS |
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