1 Department of Medicine, Baylor College of Medicine, Houston, TX 77030,
USA
2 Department of Anatomy, University of California, San Francisco, CA 94143-0452,
USA
* Author for correspondence (e-mail: farrahk{at}bcm.tmc.edu )
Accepted 1 November 2001
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Summary |
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Key words: Matrix metalloproteases, MMP-2, Branching morphogenesis, Lung development, Egf signaling
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Introduction |
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Morphogens expressed in the distal epithelium of the lung can signal
mesenchyme differentiation (Motoyama et
al., 1998; Park et al.,
2000
). Sonic hedgehog (Shh), which is widely expressed in the
distal epithelium of the lung, disrupts normal epithelial to mesenchymal ratio
by increasing proliferation of mesenchyme when expressed ectopically
(Bellusci et al., 1997a
). In
its absence, or that of Gli2 and Gli3, which encode
zinc-finger DNA binding proteins and act as transcription factors downstream
of Shh, the esophagus fails to separate from trachea
(Motoyama et al., 1998
;
Park et al., 2000
). These data
indicate that the mesenchyme, not the epithelium, is the most likely target of
Shh signaling.
Secreted growth factors also play critical roles as signaling molecules in
lung morphogenesis (Hogan,
1999; Warburton et al.,
2000
). The functional role of fibroblast growth factors (Fgfs) has
been revealed by targeted expression of a dominant-negative Fgf receptor
(Fgfr) in the primordial respiratory epithelium
(Peters et al., 1994
).
Abrogation of Fgf signaling results in the complete absence of epithelial
branching and differentiation in mouse lung
(Peters et al., 1994
).
Deletion of Fgfr2(IIIb) results in viable mice with severe defects of lung
(Arman et al., 1999
). Fgf10
expressed in the mesenchyme at the earliest stage of lung development is
required for migration and proliferation of primordial endoderm
(Bellusci et al., 1997b
;
Sekine et al., 1999
). In its
absence, embryos lack lung buds (Min et
al., 1998
).
Epidermal growth factor (Egf) family members have been implicated in the
development of the respiratory system in a diverse range of animal species
from Drosophila to mammals (Hogan
and Yingling, 1998; Kramer et
al., 1999
; Warburton et al.,
2000
). Moreover, defective signaling through the Egf receptor
(Egfr) has been demonstrated in low birth-weight human infants at high risk
for respiratory distress syndrome (Fondacci
et al., 1994
). Previous studies from this laboratory have
established a role for Egf in lung development and epithelial maturation
(Miettinen et al., 1995
). Mice
deficient in Egfr (Egfr-/-) die shortly after birth from
respiratory failure (Miettinen et al.,
1995
; Miettinen et al.,
1997
). Furthermore, deficient alveolization and septation in these
mice correlates with low expression of SP-C and thyroid-specific transcription
factor (TTF-1) (Miettinen et al.,
1997
). It remains unclear as to what is the molecular basis for
the defect in alveolization, and whether instructive signals originating from
the mesenchyme or the epithelium are lacking.
While epithelial-mesenchymal interactions during lung development require
secreted factors that allow branching morphogenesis in the lung, the nature of
these factors remains poorly understood. Egf regulates expression and
activation of ECM-degrading enzymes (van
der Zee et al., 1998). Indeed, the cleft palate seen in some
Egfr-/- mice may be due to decreased expression of MMPs
(Miettinen et al., 1999
).
These findings suggest a plausible role for MMPs as downstream effector
molecules of growth factor signaling in lung development. Thus, the
alveolization defects in Egfr-/- mice may directly result from
abnormal protease expression and/or function. In the present study we have
tested the hypothesis that epithelial-mesenchymal interactions during lung
development may be mediated through regulation of proteolysis. We sought to
determine the factors originating in the mesenchyme that are necessary for
prenatal epithelial branching, and postnatal completion of the alveolization
process. Here we identify MMP-2 (gelatinase A) and MT1-MMP (MMP 14) as key
proteases in lung development, and show that their activity occurs downstream
of Egfr signaling.
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Materials and Methods |
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Whole lungs from E12.5 Egfr+/- mice were dissected and embryos
were genotyped by Southern blot analysis as described previously
(Miettinen et al., 1995).
Wild-type and Egfr-/- fibroblasts were separated from endoderm
following a 30 minute treatment with 0.05% trypsin/EDTA (Gibco-BRL) and single
cell suspensions were washed once in the medium (1:1 DMEM/F-12, 10% FCS,
gentamycin 50 µg/ml) before seeding onto tissue culture-treated plates.
Cells were grown to confluence and were deprived of serum factors 24 hours
prior to stimulation with Fgf or Egf. All cells were used in experiments at
passages 2-5.
In vivo MMP inhibition
Timed-pregnant mice were injected intraperitoneally with a broad,
class-specific metalloproteinase inhibitor (GM6001)
(Galardy et al., 1994) at 100
µg/g, or with carrier reagent (PBS), daily for 3 days starting at E10.5.
Embryonic lungs were collected on E13.5, which corresponds to 1 day after
initiation of the pseudoglandular stage (E12.5-16.5) of lung development. This
concentration of GM6001 is shown to inhibit MMPs in animal models
(Zheng et al., 2000
) and at
higher concentrations we did not see additional effects (data not shown).
GM6001 is
N-[(2R)-2-(hydroxyamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan
methylamide] (Galardy et al.,
1994
).
Lung organ culture and in vitro MMP inhibition
Wild-type E10.5 and E11.5 whole lungs were isolated and placed on top of
polycarbonate membrane (VWR) floating on 1 ml of serum-free medium (1:1
DMEM/F-12, Transferrin 10 µg/ml, BSA 1 µg/ml, gentamycin 50 µg/ml) in
24-well culture plates. Lung organ cultures were treated with 20 µM of
GM6001, a MMP inhibitor (Galardy et al.,
1994) synthesized by AMS Scientific Inc., or control medium plus
1:1000 dilution of vehicle (DMSO), and placed in 5% CO2 at 37°C
for 4 days. In order to obtain quality images of the lung, explants were
removed from polycarbonate membranes, fixed in 4% paraformaldehyde (PFA),
photographed, and the total number of branches determined for each organ. This
concentration of GM6001 used here is the same as that used for tissue culture
inhibition in our laboratory (Alexander et
al., 2001
; Chin and Werb,
1997
); we observed no significant differences at higher
concentrations of GM6001 (data not shown).
RNA isolation and RNAse protection assay (RPA)
We extracted total cellular RNA from lung tissue using RNAzol B (TEL-TEST,
Inc.) according to the manufacturer's protocol. RPA was performed on 10 µg
of RNA using the In vitro Transcription Kit and the RPA Kit (Pharmingen)
according to the manufacturer's protocol. Briefly, a 1011 bp cDNA fragment
from the protein-coding region of the mouse MT1-MMP gene was cloned into
pT7Blue (Novagen) (Apte et al.,
1997). After linearization of the plasmid with BamHI, T7
RNA polymerase was used to synthesize the 1011 bp antisense probe that was
labeled with [
-32P]UTP. For synthesis of the MMP-2 antisense
RNA probe, pSP65 was linearized with EcoRI, and SP6 RNA polymerase
was used to generate and label the probe. A total of 1x106
cpm of radiolabeled probe was used to hybridize to 10 µg of total RNA
extracted from the lungs. After the incubation, the hybridization mixture was
treated with a mixture of RNAseA and RNAseT1. The protected hybridized RNA
fragments were recovered by ethanol precipitation, resolved on 5% acrylamide,
8 M urea gels, exposed to Kodak film overnight, and the bands quantified using
Quantity One software (Bio-Rad).
Histology
Mouse embryonic lungs were fixed in 4% paraformaldehyde (PFA) and embedded
in paraffin; sections were cut and standard histological techniques used for
histology (hematoxylin and eosin, H and E). The trachea and lungs from
3-week-old mice were infused with 4% PFA at 20 cm water pressure overnight at
4°C before paraffin embedding.
In situ zymography
DQTM gelatin, fluorescein conjugate (Molecular Probes) was
reconstituted according to the manufacturer's recommendations and diluted at
1:1 with 2% agar immediately before addition to cryostat sections of the lungs
of newborn mice, followed by incubation at ambient temperature for up to 3
hours. This modification resulted in better localization of the gelatinase
activity. Fluorescence intensity was monitored for 3 hours and lung sections
from wild-type and Egfr-/- mice were photographed at equal time
points. For inhibition of MMP activity, cryostat sections of the lung were
incubated with 10 mM of 1,10-phenanthroline or control buffer for 1 hour prior
to incubation with 1:1 dilution of DQTM gelatin and 2% agar.
Gelatin gel zymography
Whole lung homogenates were lysed in 120 mM Tris-HCl buffer pH 8.7, 0.1%
NP-40 and 5% glycerol. Insoluble aggregates and nuclei were removed by
centrifugation and protein in the supernatant was quantified by standard
protocols (Bradford). Samples (5 µg of protein) were added to
non-denaturing loading buffer and separated in 10% SDS-polyacrylamide gels
containing 0.02% gelatin. SDS was then removed by three 20 minute washes with
2.5% Triton X-100 before incubation for 24 hours at 37°C in the developing
buffer (50 mM Tris-HCl pH 8, 5 mM CaCl2, 0.02% NaN3).
Gels were then fixed and stained with 50% methanol and 10% acetic acid
containing 0.3% w/v Coomassie Blue. MMP-2 and MMP-9 (gelatinase B) activity
appeared as clear bands. Optical density of the clear bands was visualized
using the Chemi-Doc gel documentation system (Bio-Rad) and quantified using
Quantity One quantitation software (Bio-Rad). Results are shown as means and
standard error of the mean. Statistical analysis were performed using the
unpaired student's t test, with significant differences considered as
P<0.05.
In situ hybridization (ISH)
We used RNA probes prepared from plasmid vectors pSP65, pSP65 92b, pBSmo
Sl-2, TRMII, p1011as, pSK T-1 and pTIMP-2
(Alexander et al., 1996;
Apte et al., 1997
) for MMP-2,
gelatinase B, stromelysin-1, stromelysin-2, MT1-MMP, TIMP-1 and TIMP-2,
respectively. The labeling of the probes and in situ hybridization (ISH) on
prepared paraffin sections of lung tissue were carried out as previously
described (Alexander et al.,
1996
; Apte et al.,
1997
; Chin and Werb,
1997
). Briefly, the plasmids were linearized and
[35S]UTP (1000 Ci/nmol, Amersham)-labeled probes were transcribed
from the SP6 promoter using a transcription kit (Promega). The probes were
fractionated with Sephadex G-50 (Pharmacia), precipitated with ethanol, mixed
with hybridization mixture, and placed on the pretreated sections. The
sections were incubated overnight at 55°C, washed in high stringency
conditions, and dipped in autographic emulsion (Kodak NTB2). After exposure
for 4-8 days, the emulsion was developed and the sections were counterstained
with hematoxylin and mounted.
Statistics
All data are representative of at least three independent experiments with
four to five mice in each in vivo experiment and are expressed as
means±s.e.m. Significant differences (*),
P0.05, are expressed relative to sham-treated control using the
Student's t-test. In vitro studies are representative of at least
three independent experiments performed in triplicate, unless otherwise
stated.
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Results |
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Inhibition of MMPs severely retards branching morphogenesis in the developing lung. The lung forms primary buds on the second day of embryonic stage E10.5. If MMPs are involved, then inhibition of MMPs should affect branching morphogenesis at this period of development. We injected timed-pregnant mice intraperitoneally with a broad, class-specific metalloproteinase inhibitor (GM6001) at 100 µg/g, or with carrier reagent (PBS), daily for three days starting at E10.5. Embryonic lungs were collected on E13.5, which corresponds to one day after the initiation of the pseudoglandular stage (E12.5-16.5) of lung development. Normal developing lung at E13.5 was characterized by elaboration of multiple epithelial branches into the hypercellular mesenchyme, shown by an arrow pointing to a freshly forming branch in Fig. 3c. Inhibition of MMPs resulted in severe retardation of branching in the developing lung, compared with sham-treated animals (Fig. 3). There was a lack of tertiary and fourth order branching of the endoderm that is normally seen by E13 in the developing lung. The cells forming the invaginating epithelium were also poorly organized and had lost their tight association with the mesenchyme. Furthermore, the mesenchyme in the embryos treated with GM6001 appeared poorly organized and hypoplastic compared with the sham-treated embryos. Slowing of lung branching in response to MMP inhibition was dose-dependent, as GM6001 given at 10 µg/g or 1 µg/g resulted in milder or no apparent branching defects, respectively (data not shown).
|
We next examined whether blockage of MMPs specifically inhibits branching
in the developing lungs. Isolated embryonic lungs in culture are capable of
branching ex vivo for several days in the absence of serum factors
(Shannon et al., 1998).
Inhibition of metalloproteinases using soluble GM6001 in the absence of serum
resulted in retardation of branching by two-to threefold compared with the
control (Fig. 4A,B). In
addition, cells in the explant cultures treated with GM6001 were viable and
displayed normal branching when returned to control media (data not
shown).
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MMP-2 and MT1-MMP are expressed during lung development
We next sought to determine which MMPs are expressed in embryonic lung. We
examined expression of stromelysin-1 (MMP-3), stromelysin-2 (MMP-10),
gelatinase A (MMP-2), gelatinase B (MMP-9), MT1-MMP (MMP-14), and their
natural inhibitors, tissue inhibitor of metalloproteinases 1 (TIMP-1) and
TIMP-2 in three well characterized stages of embryonic lung development
(Warburton et al., 2000):
embryonic stage/primary budding (9.5-11.5 days post-coitum; E9.5-11.5),
pseudoglandular (E12.5-16.5), and canalicular (E17-18) stages by in situ
hybridization (ISH). Expression of stromelysin-1 (MMP-3), stromelysin-2
(MMP-10), gelatinase B (MMP-9) or TIMP-1 mRNA were not detected in the
epithelium or the mesenchyme at any stages
(Fig. 5, absence of white
grains; and data not shown). By contrast, we found abundant expression of mRNA
for gelatinase A (MMP-2), MT1-MMP (MMP-14) and TIMP-2 in the embryonic lung at
E11.5, E13.5, E18.5 (Fig. 5,
arrows pointing to white grains; and data not shown). MMP-2 expression was
confined to cells of mesenchymal origin (fibroblasts, cartilage, and
endothelium) and was completely absent from lung epithelium
(Fig. 6e,f and inset, absence
of white grains). By contrast, MT1-MMP and TIMP-2 were expressed in both the
epithelium and the mesenchyme, although the epithelium appeared to express
lower message levels than the mesenchyme
(Fig. 6a-d and insets).
Incubation with mouse sense probes for MMP-2, MT1-MMP and TIMP-2 performed
under parallel conditions showed no tissue binding (data not shown).
|
|
Egfr-/- lungs lack activated MMP-2 due to a deficiency in
MT1-MMP gene expression
We next looked at the expression pattern of MMP-2, and MT1-MMP mRNAs by in
situ hybridization. Interestingly, there was abundant expression of MMP-2 and
TIMP-2, but little to no expression of MT1-MMP in Egfr-/- mice
(Fig. 7; and data not shown).
ProMMP-2 is proteolytically cleaved by MT1-MMP to yield active MMP-2
(Holmbeck et al., 1999).
Because it is widely accepted that ISH is not a quantitative method for
assessing mRNA in the tissue, we chose to use an RNAse protection assay (RPA)
to quantify the mRNA level in 1- and 9-day-old lungs from wild-type and
Egfr-/- mice. The RPA confirmed the ISH data obtained from
embryonic lung showing that, while lungs from 1-day-old postnatal
Egfr-/- mice express MMP-2 mRNA at levels similar to their
littermate controls, much less MT1-MMP mRNA was detected in lungs of
Egfr-/- mice (Fig.
8a). There was also a decrease in MMP-2 mRNA in 9-day-old
Egfr-/- lungs compared with the wildtype. Egfr-/- mice
rarely live beyond the first day of life and the occasional mice that live to
a few weeks do not thrive, suffering from severe emaciation and dehydration.
Thus, not surprisingly, we found less MMP-2 mRNA from these 9-
(Fig. 8b) or 7-day-old (data
not shown) Egfr-/- mice compared with the littermate controls.
These data are consistent with our in situ data
(Fig. 7; and data not shown).
Thus, Egfr signaling is essential in lung organogenesis and for proper
activation of MMP-2 through a mechanism that involves the expression of
MT1-MMP.
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|
Egfr signaling directly regulates MT1-MMP gene expression in
embryonic lung fibroblasts
We next asked whether Egfr signaling directly and/or specifically regulates
MT1-MMP gene expression in the embryonic lung mesenchyme. E12 lung fibroblasts
from wild-type, but not Egfr-/- mice stimulated with Egf for 16
hours upregulated MT1-MMP mRNA tenfold
(Fig. 8c). Furthermore, Egf
regulation of MT1-MMP was specific, since Fgf-2 failed to increase MT1-MMP
message in the wild-type embryonic fibroblasts
(Fig. 8c). RPA studies
represent at least three independent experiments.
Newborn lungs from MMP-2-/- mice phenocopy abnormal lung
branching in Egfr-/- mice
Our data point to a role of Egfr signaling for proper activation and
expression of MMP-2 during lung development. To determine whether regulation
of MMP-2 function is necessary for lung morphogenesis, we examined lungs from
the mice lacking MMP-2 (Itoh et al.,
1997). Although these mice are born apparently normal
(Itoh et al., 1997
), upon
careful examination we observed that 15% of newborn MMP-2-/- mice
gasped for breath and in all mice the lungs had abnormally large alveolar
spaces, fewer septations and thinner interstitial tissue than in control
littermates (Fig. 9), which is
similar to, but slightly less severe than, the phenotype seen in
Egfr-/- lungs.
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Discussion |
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Tubular structures such as lung follow a highly stereotyped and detailed
process of invasion into the mesenchyme to form the final 3D structure of the
organ. Interestingly, exogenous addition of collagenase to organ cultures
inhibits lung branching (Ganser et al.,
1991). However, there is no evidence that collagenase is expressed
at this time in morphogenesis. Instead, our in vivo inhibition studies, in
which we treated pregnant mice with GM6001, point to a positive role for MMPs
in epithelial cell branching, because embryonic lungs failed to undergo
secondary and tertiary branching.
What is the functional role of ECM-degrading enzymes during branching
morphogenesis in vivo? We found that branch formation required functional
MMPs. The simplest hypothesis is that MMPs are required for degradation of an
ECM protein that is a necessary component for migration of epithelial cells
during invagination. During development lung ECM is in a constant state of
turnover. One role of MMP-2 could be to cleave ECM proteins such as elastin,
laminin-5 or collagen types IV or VII, producing bioactive fragments, which
would then induce migration of epithelial cells in vitro
(Karelina et al., 2000;
Koshikawa et al., 2000
;
Salo et al., 1999
).
MT1-MMP is the downstream target of Egfr signaling
In this study we provide strong evidence for the role of MMPs in branching
morphogenesis during embryonic lung development. Lung buds in mice are first
formed from invagination of a single epithelial endoderm tube into the
splanchnic mesoderm at E9.5 (Hogan,
1999; Warburton et al.,
2000
). The 3D growth of the primary lung buds may be controlled by
the Shh and its downstream transcriptional molecules Gli2 and Gli3
(Bellusci et al., 1997a
),
whereas the pattern of secondary branch formation is most likely controlled by
growth factors (Hogan, 1999
;
Warburton et al., 2000
) and
proteases as shown by our studies. Egfr and many of its ligands are abundantly
expressed in the developing mouse lung, suggesting an autocrine and/or
paracrine interaction in the developing mouse embryo
(Warburton et al., 1992
).
Embryonic lung synthesizes several Egfr ligands and responds to Egf,
Tgf-
and amphiregulin by precocious branching
(Schuger et al., 1996
;
Warburton et al., 1992
).
Most MMPs (e.g. MMP-2) are secreted as inactive zymogens. Therefore, their
activators (e.g. MT1-MMP) may ultimately control ECM degradation during
morphogenesis (Caterina et al.,
2000). Our data point to MT1-MMP and its activation of MMP-2 as a
major downstream target of signaling by this receptor tyrosine kinase that
results in branching morphogenesis in lung development. We provide direct
evidence for the role of MMP-2 during lung branching morphogenesis and show
that MT1-MMP, a potent activator for the same enzyme is upregulated by an Egfr
ligand in culture. Although expression of MMP-2 mRNA and protein was only
slightly decreased in mice with a null mutation in Egfr, its activation was
greatly compromised. However, MT1-MMP, which is required for cell surface
activation of MMP-2, is downregulated in the absence of Egfr signaling. Since
proMMP-2 can also be activated by other enzymes, it is not surprising that we
found a small amount of active MMP-2 in the whole lung homogenates.
MMP-2 null mice phenocopy the Egfr-/- lung phenotype
If the major target of signaling through the Egfr is activation of MMP-2,
then MMP-2-/- mice should have the same phenotype as
Egfr-/- mice. Mice with null mutations of MMP-2 and Egfr showed
strikingly similar abnormal distal airway branching and abnormal tissue
architecture by histology. This raises the question of what substrates of
MMP-2 are involved in airway development. MMP-2 cleaves many proteins in the
interstitial space, basement membrane and on cell surfaces
(Werb, 1997). Elastin and
collagen remodeling are specific to lung and are necessary for alveolar
development (Wright et al.,
1999
). Since active MMP-2 in the lung is a potent elastinolytic
enzyme (Shipley et al., 1996
),
its absence in both Egfr-/- and MMP-2-/- lungs is
expected to result in significant disturbance in alveolization postnatally.
Indeed, in support of this hypothesis, we found severely impaired
alveolization concomitant with abnormal elastin deposition in 15% of the
MMP-2-/- mice (F.K. and K.R., unpublished). Thus elastin is one of
the in vivo targets of MMP-2 in lung that is controlled by Egf signaling.
These data provide further evidence for the coordinated signaling through Egfr
and MMP-2 activation in lung branching morphogenesis.
However, does Egfr originally activate pathways other than MMP-2
activation? Although MMP-2-/- mice exhibited only mild respiratory
compromise, these mice may have a spectrum of lung diseases that may only be
manifest clinically after a pathological insult
(Shapiro, 2000). By contrast,
mice deficient in Egfr signaling die shortly after birth from respiratory
failure and lack epithelial cell maturity
(Miettinen et al., 1995
). This
suggests that there are additional downstream targets of Egfr signaling other
than MMPs. Thus, while some features in branching morphogenesis are shared
between these mice, lack of MMP-2 activation alone does not produce the lethal
respiratory failure in Egfr-/- mice. It is unlikely that this is
due to a direct role of MT1-MMP, because MT1-MMP null mice do not manifest
respiratory failure (Holmbeck et al.,
1999
). More likely, these affects are due to the functions of Egfr
signaling as a survival factor (Gibson et
al., 1999
; Sibilia et al.,
2000
).
Our data indicate that MMPs in vivo are downstream effectors of growth factors mediating growth and differentiation. However, the epithelial basement membrane and the ECM of the mesenchymal stroma must be degraded for proper branching and invagination of the epithelial cells to proceed. MMP-2 is expressed constitutively throughout the mesenchyme, although it may be regulated locally at the activation level by the Egfr through expression of MT1-MMP on both epithelial and mesenchyme. Although we suspect abnormal elastin deposition, we have not elucidated the full component of protein targets of these MMPs in the lung in vivo.
Membrane-bound MMP-2, in close association with its activator MT1-MMP,
plays an important role, not only in branching of the epithelium, but also in
the angiogenesis and cell invasion that are required for lung development
(Haas et al., 1998;
Haas et al., 1999
). Although
growth retardation in MT1-MMP-/- mice
(Holmbeck et al., 1999
) and
poor branching morphogenesis are seen in MMP-2-/- mice, they do not
exhibit a phenotype as severe as the in vivo inhibition of MMPs. This is
reminiscent of previous in vitro studies showing that inhibition of MMPs can
block endothelial tube formation and correlate MMP-2 activity with sprouting
of endothelial tubes from pre-existing microvessel segments
(Haas and Madri, 1999
).
Furthermore, microvascular endothelial cells upregulate expression of MMP-2
and MT1-MMP when grown on 3D type I collagen matrices
(Haas et al., 1998
). Whether
other members of the MMP family act during lung morphogenesis in the absence
of MMP-2 activation, or whether coordination of epithelial and vascular
development is essential, are subjects for further study.
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Acknowledgments |
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