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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2045-2053
Stromelysin-1 (MMP-3)-Independent Gelatinase Expression and
Activation in Mice
By
H.R. Lijnen,
J. Silence,
B. Van Hoef, and
D. Collen
From the Center for Molecular and Vascular Biology, University of
Leuven, Leuven, Belgium.
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ABSTRACT |
A potential physiological role of stromelysin-1 (MMP-3) in the
expression or activation of gelatinase A (MMP-2) or gelatinase B
(MMP-9) in the wall of injured arteries was studied with the use of
homozygous MMP-3-deficient (MMP-3 /) mice. One week
after perivascular electric injury of the carotid or femoral artery in
wild-type (MMP-3+/+) or MMP-3/ mice,
70 kD and 65 kD proMMP-2 levels were enhanced by twofold to fourfold,
with corresponding increases of 20- to 40-fold for active 61 kD and 58 kD MMP-2, and of 10- to 80-fold for 94 kD proMMP-9. Active MMP-2
species represented approximately one third of the total MMP-2
concentration for both MMP-3+/+ and
MMP-3/ mice. Active 83 kD MMP-9 was not detected in
noninjured carotid or femoral arteries, whereas one week after injury
its contribution to the total MMP-9 level was 11% to 18% for
MMP-3+/+ and MMP-3/ mice.
Immunostaining of arterial sections confirmed enhanced expression of
both MMP-2 and MMP-9 after vascular injury. Double immunostaining
showed colocalization of MMP-9 with macrophages in the adventitia,
whereas MMP-2 was also detected mainly in the adventitia but failed to
colocalize with smooth muscle cells. Cell culture experiments confirmed
comparable ratios of active versus latent MMP-2 in skin fibroblasts and
smooth muscle cells derived from MMP-3+/+ and
MMP-3/ mice. Addition of plasmin(ogen) did not
significantly affect activation of proMMP-2. In
MMP-3+/+ and MMP-3/ macrophages,
comparable levels of 94 kD proMMP-9 were detected, and
plasmin(ogen)-mediated conversion to 83 kD MMP-9 was obtained in both
genotypes. These data thus indicate that proMMP-2 activation may occur
via a plasmin- and MMP-3-independent mechanism, whereas plasmin can
directly activate proMMP-9 via a MMP-3-independent mechanism.
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INTRODUCTION |
MATRIX METALLOPROTEINASES (MMPs) are
involved in the accelerated breakdown of extracellular matrix
associated with normal tissue remodeling and with pathological
conditions, such as arthritis, tumor invasion, and
metastasis,1-4 and also play a role in smooth muscle cell
migration.5 MMPs are secreted as zymogens that are
extracellularly activated by organomercurial compounds, by several
proteinases (including plasmin, trypsin, chymotrypsin, kallikrein,
cathepsin G, or neutrophil elastase), by oxygen radicals, or by
association with the cell surface.1,4,6 In vitro, plasmin
activates proMMP-3 (stromelysin-1), proMMP-9 (gelatinase B), proMMP-10
(stromelysin-2), and proMMP-13 (collagenase-3), but its in vivo role in
modulating MMP activity is not clearly established.7-9
Several positive feedback mechanisms operate in MMP activation. Thus,
MMP-3 and MMP-10 can superactivate procollagenase, generating
collagenase with a 5- to 12-fold higher specific
activity.8,10 Besides proMMP-1 (interstitial collagenase)
MMP-3 can also activate proMMP-9 and, thus, appears to play a key role
in activation of the MMP family.11,12 ProMMP-3 has been
detected in cultures of medial and intimal smooth muscle cells and in
fibroblasts, and MMP-3 mRNA expression was reported in human
atherosclerotic plaque, mainly associated with
macrophages.13,14 Some controversy exists on the activation
mechanisms of proMMP-2 (gelatinase A) and proMMP-9. It was reported
that trypsin can activate proMMP-915,16 but is ineffective
in activating proMMP-2.17,18 Direct activation of proMMP-9
but not proMMP-2 by plasmin was also reported by some authors,9 whereas others found that plasmin is inefficient in activating both progelatinases.12,15,17-19 In addition,
plasminogen activator-dependent pathways have been proposed for the
activation of both proMMP-2 and proMMP-9 during cancer invasion and
metastases.20,21
Because MMP-3 can activate proMMP-9,11,12 it was
hypothesized that plasmin may be involved in proMMP-9 activation
indirectly through activation of proMMP-3.12 In the present
study, we have evaluated a potential physiological role of active MMP-3
in gelatinase activation by monitoring gelatinase A and B expression
and activation in mice deficient in MMP-3.
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MATERIALS AND METHODS |
Proteins and Reagents
MMP-3-deficient (MMP-3/) and wild-type
(MMP-3+/+) mice of the same genetic background (B10.RIII)
were a kind gift of Dr J. Mudget (Merck Research Lab, Rahway,
NJ).22 The mice were rederived by back crossing into a BL6
background until 50% BL6. Homozygosity of offspring was confirmed by
genotyping of tail tip DNA using Southern blotting (data not shown).
Mice were kept in microisolation cages on a 12-hour day-night cycle and
fed regular chow. The animals were anesthetized by intraperitoneal
injection of 60 mg/kg Nembutal (Abbott Laboratories, North Chicago,
IL), and all experiments were performed in accordance with the guiding
principles of the American Physiological Society and the International
Society on Thrombosis and Haemostasis.23 Mice were 8 to 14 weeks old with body weight (mean ± standard error of the mean
[SEM]) of 27 ± 1.0 g (n = 17) or 21 ± 0.4 g (n = 5) for male
or female MMP-3+/+ mice and 31 ± 0.9 g (n = 17) or 25 ± 0.4 g (n = 7) for male or female MMP-3/
mice.
Human fibrinogen, plasminogen, and rabbit polyclonal antisera against
murine t-PA and u-PA were obtained and characterized as
described.24,25 Statistical analysis was performed using Student's t-test.
Vascular Injury Model
Perivascular electric injury to the femoral or carotid artery of mice
was performed essentially as described elsewhere.26 Briefly, the arteries were exposed by blunt-end dissection and injured
by electric current (1.4 V during 2 seconds) at distances of 1 mm over
a total length of 2 or 3 mm. The vessel segments (control noninjured or
injured) were embedded in Tissue-Tek (Laborimpex, Brussels, Belgium), snap-frozen in precooled 2-methyl butane, and
stored at  80°C. Seven-micrometer-thick sections were made throughout the whole artery (about 700 sections per artery) and stained
with hematoxylin-eosin or with the appropriate antiserum or were used
for fibrin overlay as described below.
From separate experiments, control noninjured arteries and injured
femoral or carotid arteries were dissected free of tissue and frozen at
80°C. These arteries were pulverized under liquid nitrogen
and incubated for 1 hour at 4°C with 60 µL extraction buffer (10 mmol/L sodium phosphate buffer, pH 7.2, containing 150 mmol/L NaCl, 1%
Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium
deoxycholate, and 0.2% sodium azide). After extensive vortexing and
centrifugation at 13,000 revolutions per minute for 5 minutes, the
supernatants were used for determination of the protein concentration
(BCA protein assay; Pierce, Rockford, IL) and equivalent amounts of
total protein were applied to zymography on gelatin- or
casein-containing gels as described below.
Zymographic Analysis
For zymographic analysis of plasminogen activator activity, arterial
extracts were electrophoresed on a 12.5% acrylamide gel cast with 1%
nonfat dry milk and 5 µg/mL human plasminogen under nonreducing
conditions.27 For zymographic analysis of gelatinase activity, arterial extracts were electrophoresed on a 10% Tris-Glycine gel with 0.1% gelatin (Novex, SanverTECH, Bouchout,
Belgium).28 The amount of lysis of the
substrate gel (area × intensity) was quantitated using the
Quantimed 600 image analysis software (Leica, Cambridge, UK), and
expressed in arbitrary units of lysis obtained per mg total protein in
the extract.
In situ zymography with 7-µm arterial cryosections was performed by
fibrin overlay as described.29 Zymography was performed at
37°C for 1 to 2 hours, without or with addition to the gel of
antibodies against murine t-PA or u-PA (final concentration 40 µg/mL). To compare activities between different experiments, data
were expressed as a ratio of the lysis observed in injured sections
versus noninjured control sections measured on the same overlay. Data
are reported as mean ± SEM of four to six experiments (different
animals) with, in each experiment, two to four sections analyzed in
duplicate.
Histology and Immunocytochemistry
Seven-micrometer-thick arterial sections were stained with
hematoxylin-eosin or with the appropriate antiserum as described below.
Primary polyclonal antisera used were rabbit antimurine MMP-3
(homemade), rabbit antimurine MMP-9 (homemade), and sheep antihuman
MMP-2 (Biodesign, Kennebunk, ME). Primary monoclonal antibodies used
were rat antimouse macrophage-specific Mac-3 (clone M3/84; Pharmingen,
San Diego, CA), biotinylated mouse antihuman smooth muscle  -actin
(clone 1A4; Sigma, St Louis, MO), and biotinylated rat antimurine
panleukocyte antigen CD45 (clone 30 F11.1; Pharmingen).
Immunostaining for the MMPs was performed using appropriate
peroxidase-labeled secondary antibodies (Dakopatts, Copenhagen, Denmark); immunostaining for Mac-3 was done by using biotinylated rabbit antirat immunoglobulins (Dakopatts) and the Tyramide Signal Amplification kit (Dupont NEN, Brussels, Belgium), whereas
for -actin and CD45 biotinylated secondary antibodies were used in combination with the Vectastain system (ABC Elite kit, Vector Laboratories Inc, Burlingame, CA). Peroxidase activity was developed by
incubating sections in 0.05 mol/L Tris-HCl buffer, pH 7.0, containing
0.06% 3,3 -diaminobenzidine and 0.01%
H2O2 followed by counterstaining with Harris'
hematoxylin. Specificity of the staining was confirmed by omission of
the primary antibody or by replacing it with equivalent amounts of
isotype-matched nonimmune IgG or serum.
Colocalization of MMP-2 or MMP-9 with smooth muscle cells or
macrophages was investigated by using a double immunofluorescence approach by which MMP-stained cells appeared red, -actin or Mac-3 stained cells green, and double-labeled cells stained
yellow.30
Cell counts were performed in a blinded manner on transverse arterial
sections using a computer-assisted image analysis system (IP Plus 1.0;
CN Road, Zellik, Belgium). Medial and intimal cell nuclei were counted
at equally spaced positions across the artery.
Cell Culture Experiments
Mice were injected intraperitoneally with 0.5 mL of a 4%
thioglycollate solution,31 and 3 days later peritoneal
macrophages were harvested through a catheter after injection of 5 mL
of a 5% glucose solution. Macrophages were grown in RPMI containing phorbol 12-myristate 13-acetate (final concentration
107 mol/L), 2 mmol/L glutamine, 4.5 g/L glucose, 100 U/mL penicillin, and 0.1 mg/mL streptomycin and washed with serum-free
medium (RPMI containing 0.02% lactalbumin hydrolysate), and samples
were removed at different time points (0-72 hours).
To obtain smooth muscle cells, the aorta was cut into small fragments
(<1 mm3), which were incubated in plates coated with
collagen (collagen S, type I, at 30 µg/mL in phosphate-buffered
saline) in Dulbecco's modified Eagle's medium (DMEM) containing 1 × nonessential amino acids (NEAA), 10 ng/mL basic
fibroblast growth factor, 20% fetal calf serum, 2 mmol/L glutamine,
100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified
CO2-incubator at 37°C.
To obtain fibroblasts, skin dissected from the abdomen was cut in small
pieces and incubated in plates coated with collagen. The cells were
grown in DMEM without sodium pyruvate, containing 4.5 g/L glucose, 20%
fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, 0.1 mg/mL
streptomycin, 1 × NEAA and 10 ng/mL basic fibroblast growth
factor. At confluency, the cells were washed with serum-free medium,
and samples of the conditioned medium were removed at different time
intervals (0-72 hours). In some experiments, plasminogen (final
concentration 10 µg/mL) or plasmin (final concentration 2 µg/mL)
was added to the serum-free conditioned medium, and samples were
collected on aprotinin (final concentration 20 KIU/mL).
For immunoadsorption, serum-free conditioned medium (1-mL samples) was
mixed with insolubilized antibodies against murine MMP-3 or MMP-9 (100 µL IgG.Sepharose 4B suspension containing approximately 2 µg
IgG/µL) and incubated overnight at 4°C. After centrifugation and
extensive washing of the gel, bound protein was eluted with 50 µL
Tris, pH 6.8, containing 10% glycerol, 30 mg/mL SDS and 70 µg/mL
bromophenol blue.
Extracellular Matrix Degradation by Peritoneal Macrophages
Peritoneal macrophages, stimulated as described above, were plated on
3H-proline-labeled matrix from human umbilical vein
endothelial cells at a density of 106 cells per well, and
at timed intervals (0-48 hours) aliquots of the medium were removed to
determine released radiolabel.31
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RESULTS |
Vascular Injury Model
Histology and immunocytochemistry.
Hematoxylin-eosin staining of arterial sections taken 1 week after
injury showed the formation of a small neointima both in MMP-3+/+ and MMP-3/ mice, similar
to what was reported previously in this model.26,29,32 This
is illustrated in Fig 1 for
MMP-3/ mice, showing no intima in the control
artery (panel a) and a small neointima in the injured artery (panel b).
Intimal and medial areas determined at the center of the injury
(position 3 of the schematic representation of the injured artery in
Fig 1) were 0.0045 ± 0.0009 mm2 and 0.0082 ± 0.0009 mm2 (n = 11) in MMP-3+/+ mice, with
corresponding values of 0.0034 ± 0.0006 mm2 and 0.0065 ± 0.0003 mm2 (n = 22) in
MMP-3/ mice (mean ± SEM of n
determinations in total, using 4 arteries of MMP-3+/+ and 6 arteries of MMP-3/ mice), yielding
intima/media ratios of 0.63 ± 0.13 and 0.51 ± 0.08 for
MMP-3+/+ and MMP-3/ mice,
respectively (P = .41). At the borders of the injury (positions 2 and 4), the intimal and medial areas were 0.0037 ± 0.001 mm2 and 0.011 ± 0.001 mm2 (n = 6) in
MMP-3+/+ mice compared with 0.0041 ± 0.0009 mm2 and 0.0094 ± 0.001 mm2 (n = 10) in
MMP-3/ mice, yielding intima/media ratios of
0.36 ± 0.11 and 0.43 ± 0.09, respectively (P = .65).
Nuclear cell counts showed a comparable cell population in uninjured
(normal) sections as well as at the border and at the center of the
injury in the neointima of MMP-3+/+ and
MMP-3/ mice. In the media, cell counts were
also comparable in uninjured sections of MMP-3+/+ and
MMP-3/ arteries and somewhat lower (P = .22) at the borders of the injury in MMP-3/
arteries. In contrast, at the center of the injury the media was
virtually depleted of cells in both genotypes (data not shown).
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| Fig 1.
Light microscopic analysis (original magnification
×200) of sections of noninjured control arteries and of sections
taken from the center of injured arteries (position 3 in the inset) after 1 week in MMP-3/ mice. Staining is performed
with hematoxylin-eosin (panels a and b) or with antiserum against MMP-2
(panels c and d) or against MMP-9 (panels e and f). The inset shows a
longitudinal section through the artery, and the arrows indicate the
presumed migration of smooth muscle cells. Positions 1 and 5 correspond
to normal sections, positions 2 and 4 to the borders of the injury, and position 3 to the center of the injury (modified from Carmeliet et
al26). The arrows and arrowheads indicate the internal and external elastical lamina, respectively.
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The cell population 1 week after injury was heterogeneous as shown by
immunostaining for -actin (smooth muscle cells), CD45 (leukocytes),
or Mac-3 (macrophages; Fig 2). In both
genotypes, the neointima at the center of the injury contained mainly
CD45 and Mac-3 positive cells, but no -actin positive cells. At the borders of the injury, mainly CD45 positive cells and occasionally macrophages were detected in neointima and media of
MMP-3+/+ and MMP-3/ mice.
-Actin positive cells at the borders of the injury were detected in
the media but not in the neointima (data not shown). Mac-3 positive
cells were mainly detected in the adventitia, which also shows
significant background staining for CD45 (Fig 2). It cannot be excluded
that adherent leukocytes contribute to the observed CD45 staining.
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| Fig 2.
Identification of different cell types in the center of
the injury (position 3) 1 week after injury in MMP-3+/+
(upper panel) or MMP-3/ (lower panel) mice.
Immunostaining is performed with antiserum against -actin (panels a
and d), against CD45 (panels b and e), or against Mac-3 (panels c and
f). The arrows and arrowheads indicate the internal and external
elastical lamina respectively (original magnification ×200).
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Immunostaining for MMP-2 (Fig 1, panels c and d) and for MMP-9 (Fig 1,
panels e and f) showed enhanced expression of both gelatinases after
vascular injury in MMP-3+/+ and in
MMP-3/ mice. Double immunofluorescence
analysis by confocal laser microscopy using cocktails of an MMP-2- or
MMP-9-specific polyclonal antibody and a cell-type specific monoclonal
antibody (-actin or Mac-3) showed colocalization of MMP-9 with
macrophages, mainly in the adventitia (Fig
3). MMP-2 positive staining was also observed mainly in the adventitia
and did not colocalize with the few smooth muscle cells that were
present (Fig 3). Staining against MMP-3 was weakly positive in the
adventitia of injured arteries of MMP-3+/+ mice and was
negative in uninjured arteries as well as in injured sections of
MMP-3/ mice (not shown).
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| Fig 3.
Immunostaining and cellular localization of MMP-2 and
MMP-9 in arterial sections (position 3) 1 week after electric injury in
MMP-3+/+ mice. Single immunostaining was performed for
MMP-2 or MMP-9 (appearing red) and for -actin or Mac-3 (appearing
green). Cells in which MMPs and -actin or Mac-3 are colocalized
appear yellow. The elastic membranes are visualized by their
autofluorescence. The arrows and arrowheads indicate the internal and
external elastical lamina respectively (original magnification ×480).
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Gelatinase expression.
Zymography on gelatin-containing gels showed the presence of two
molecular forms of proMMP-2 (Mr 70 or
65 kD) and of active MMP-2 (Mr 61 or 58 kD), as
well as proMMP-9 (Mr 94 kD) and active MMP-9
(Mr 83 kD) in extracts of injured arteries from
MMP-3+/+ and MMP-3/ mice
(Fig 4A). The identity of both gelatinases
was confirmed by Western blotting (not shown).
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| Fig 4.
Zymographic analysis on (A) gelatin- or (B)
casein-containing gels of arterial extracts (5 µg total protein)
obtained from MMP-3+/+ or MMP-3/ mice
without (lane 1) or 1 week after (lane 2) vascular injury. Lanes 3 and
4 in (B) represent arterial extracts obtained from t-PA/ or u-PA/ mice,
respectively.
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Quantitative analysis showed significantly enhanced levels of both
latent and active forms of MMP-2 and MMP-9 1 week after injury in
MMP-3+/+ and MMP-3/ mice
(Table 1). A 65 kD proMMP-2 was the main
MMP-2 species, and 1 week after injury of the carotid or femoral artery
in MMP-3+/+ or MMP-3/ mice its
levels were enhanced approximately twofold relative to noninjured
control arteries. Similarly, the levels of 70 kD proMMP-2 were twofold
enhanced in injured carotid or femoral arteries of MMP-3+/+
mice, and threefold to fourfold in MMP-3/
mice. However, active MMP-2 levels were significantly more enhanced 1 week after injury: 58 kD MMP-2 levels in extracts of carotid or femoral
arteries were enhanced by a factor of 29 or 18 in MMP-3+/+
mice, with corresponding values of 45 and 29 in
MMP-3/ mice. MMP-2 levels of 61 kD were
undetectable in all control arteries and were enhanced 1 week after
vascular injury to similar levels in MMP-3+/+ and
MMP-3/ mice.
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Table 1.
Gelatinase Levels After Injury of Femoral or Carotid
Artery of MMP-3+/+ or MMP-3/ Mice as
Determined With Arterial Extracts on Gelatin Zymography
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Total MMP-2 levels (lysis observed with all MMP-2 species) 1 week after
injury were comparable in carotid and femoral arteries of both
MMP-3+/+ and MMP-3/ mice. The
contribution of active molecular forms of MMP-2 (61 kD plus 58 kD) in
the total MMP-2 level was also comparable in carotid and femoral
arteries of MMP-3+/+ mice (33% and 32%) and
MMP-3/ mice (36% and 28%).
ProMMP-9 levels 1 week after injury relative to noninjured control
arteries were enhanced by more than one order of magnitude in
MMP-3+/+ mice (10- or 80-fold in carotid or femoral artery)
as well as in MMP-3/ mice (40-fold in carotid
and femoral artery). Active 83 kD MMP-9 was not detectable in any of
the control arteries, whereas 1 week after injury comparable levels
were detected in MMP-3+/+ and
MMP-3/ mice (Table 1). Again, total MMP-9
levels (latent plus active) 1 week after injury were comparable, and
the contribution of active MMP-9 to the total level was relatively
constant in carotid and femoral arterial extracts of
MMP-3+/+ mice (11% or 13%) and
MMP-3/ mice (18% or 13%).
Plasminogen activator activity.
Zymography on casein-containing gels with extracts of carotid or
femoral arteries showed the presence of both t-PA
(Mr about 70 kD) and u-PA (Mr
about 50 kD) activity (Fig 4B). Quantitative analysis showed that t-PA
activity levels in noninjured carotid or femoral arteries were similar
in MMP-3+/+ and MMP-3/ mice, and
did not increase significantly within 1 week after vascular injury
(data not shown). u-PA activity levels were also similar in noninjured
arteries of MMP-3+/+ and MMP-3/
mice and increased significantly (P < .01 versus control) in the femoral arteries of MMP-3+/+ and
MMP-3/ mice 1 week after injury. No
significant differences were observed between MMP-3+/+ and
MMP-3/ mice (data not shown).
In situ zymography with arterial sections on fibrin overlays showed a
comparable fibrinolytic activity in the carotid and femoral arteries of
MMP-3+/+ and MMP-3/ mice. The
ratio of the fibrinolytic activity in the injured carotid artery versus
control sections taken from corresponding areas of noninjured arteries
was (mean ± SEM of n experiments) 1.4 ± 0.26 (n = 8) in
MMP-3+/+ mice and 1.6 ± 0.17 (n = 9) in
MMP-3/ mice, with corresponding values for
the femoral artery of 2.4 ± 0.46 (n = 8) and 2.7 ± 0.39 (n = 11), respectively. This assay detects primarily t-PA activity as shown
by our finding that lysis of the fibrin gel was reduced by  90% on
addition of anti-t-PA antibodies, but was not affected by addition of
anti-u-PA antibodies (data not shown).
Cell Culture Experiments
Skin fibroblasts.
Zymography on gelatin-containing gels of serum-free conditioned medium
of fibroblasts derived from MMP-3+/+ or
MMP-3/ mice showed the presence of 94 kD
proMMP-9, 70 kD and 65 kD proMMP-2, and 58 kD MMP-2, whereas 61 kD
MMP-2 and 83 kD MMP-9 were undetectable (Fig 5A). The contribution of the two
differently glycosylated proMMP-2 forms and of active 58 kD MMP-2 to
the total MMP-2 level was relatively constant in time and comparable
for MMP-3+/+ and MMP-3/ samples
(Table 2). Addition of plasminogen or
plasmin to the culture medium did not significantly affect the
distribution of active and latent forms of MMP-2 in
MMP-3+/+ or MMP-3/ samples (Fig
5A).
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| Fig 5.
(A) Zymographic analysis on gelatin-containing gels of
72-hour samples of serum-free conditioned medium of fibroblasts derived from MMP-3+/+ or MMP-3/ mice without
(lane 1) or with (lane 2) addition of plasmin. (B) Zymography on
casein-containing gels in the absence of plasminogen of serum-free
conditioned medium of fibroblasts derived from MMP-3+/+
(lane 1) or MMP-3/ (lane 2) mice, and of the eluates
of MMP-3+/+ samples without plasminogen (lane 3) or
with plasminogen (lane 4) after adsorption with anti-MMP-3 IgG.
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Table 2.
Contribution (%) of Different Molecular Forms of MMP-2
to the Total MMP-2 Level in Serum-Free Conditioned Medium of
Fibroblasts or Smooth Muscle Cells Derived From
MMP-3+/+ or MMP-3/ Mice
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The levels of proMMP-9 in MMP-3+/+ samples were comparable
with those in the MMP-3/ samples (not shown).
ProMMP-3 levels in MMP-3+/+ samples corresponded to 88 ± 27, 130 ± 26, or 180 ± 52 arbitrary units per
106 cells/mL at 24, 48, or 72 hours. The identity of MMP-3
was confirmed by immunoadsorption (Fig 5B). Zymography on
casein-containing gels cast in the absence of plasminogen after
immunoadsorption with anti-MMP-3 IgG indeed showed the presence of 56 kD proMMP-3 in MMP-3+/+ samples collected without
plasminogen and of both 56 kD proMMP-3 and a slightly lower
Mr (active) MMP-3 species in samples with plasminogen (Fig 5B, lane 4).
Vascular smooth muscle cells.
Zymography on gelatin-containing gels of serum-free conditioned medium
of smooth muscle cells derived from MMP-3+/+ or
MMP-3/ mice showed the presence of 70 kD and
65 kD proMMP-2 and 58 kD MMP-2. A 61 kD MMP-2 and 83 kD MMP-9 were
undetectable in all samples, whereas proMMP-9 was detected only in
MMP-3/ mice and proMMP-3 only in
MMP-3+/+ mice (not shown).
The contribution of the 70 kD and 65 kD proMMP-2 forms to the total
MMP-2 level at different time points was relatively constant and
comparable for MMP-3+/+ and
MMP-3/ samples. Active 58 kD MMP-2 levels
were somewhat but not significantly (P > .01) higher in
MMP-3/ compared with MMP-3+/+
samples (Table 2). Addition of plasmin(ogen) to the culture medium did
not significantly affect the distribution of active and latent forms of
MMP-2 (not shown).
ProMMP-9 was detected in low concentration in the
MMP-3/ samples (9 ± 2, 47 ± 6, or 160 ± 37 arbitrary units per 106 cells/mL at 24, 48, or 72 hours) but not in any MMP-3+/+ sample.
ProMMP-3 was detected only in MMP-3+/+ samples (9 ± 2, 37 ± 4, or 130 ± 32 arbitrary units per 106
cells/mL at 24, 48, or 72 hours). The identity of MMP-3 was confirmed by immunoadsorption of conditioned medium with a rabbit polyclonal antiserum raised against murine MMP-3 as described above. Furthermore, zymography on casein-containing gels in the absence of plasminogen, after immunoadsorption of MMP-3+/+ samples with anti-MMP-3
IgG showed the presence of both 56 kD proMMP-3 and a slightly lower
Mr species similarly as observed with fibroblasts
(not shown).
Macrophages.
Zymography on gelatin-containing gels of serum-free conditioned medium
of peritoneal macrophages derived from MMP-3+/+ or
MMP-3/ mice showed the presence only of
proMMP-9 in comparable amounts in MMP-3+/+ and
MMP-3/ samples. Addition of plasminogen to
the culture medium resulted in significant conversion of 94 kD proMMP-9
to 83 kD MMP-9 as well in MMP-3+/+ as in
MMP-3/ samples, as confirmed by gelatin
zymography after immunoadsorption with anti-MMP-9 IgG
(Fig 6). In addition, an MMP-9 species with Mr about 60 kD was generated (Fig 6, lane 3).
Immunoadsorption with anti-MMP-3 IgG and casein zymography in the
absence of plasminogen confirmed that plasmin-mediated conversion of 56 kD proMMP-3 to a lower Mr (active) MMP-3 had
occurred in the MMP-3+/+ samples (not shown).
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| Fig 6.
Zymographic analysis on gelatin-containing gels of
72-hour samples of serum-free conditioned medium of macrophages from
MMP-3+/+ or MMP-3/ mice obtained
without (lane 1) or with (lane 2) plasminogen and of the eluates of
plasminogen-containing samples after adsorption with anti-MMP-9 IgG
(lane 3).
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Aorta Segments
Zymography of arterial extracts on gelatin-containing gels showed the
presence of latent and active forms of MMP-2 and of latent MMP-9. The
levels of the different MMP-2 species and of 94 kD proMMP-9 were
comparable in MMP-3+/+ and MMP-3/
samples, whereas 83 kD MMP-9 could not be clearly identified, possibly
because of the low levels of total MMP-9 (data not shown). Immunostaining for MMP-2 or MMP-9 confirmed comparable expression of
both gelatinases in aortas of MMP-3+/+ and
MMP-3/ mice.
Extracellular Matrix Degradation by Peritoneal Macrophages
Invasion of macrophages into the peritoneal cavity measured 3 days
after injection of thioglycollate was lower in
MMP-3/ mice (5.5 ± 0.66, n = 21) than in
MMP-3+/+ mice (8.5 ± 0.94, n = 16; P = .011).
Lysis of a 3H-proline-labeled subendothelial matrix by
stimulated macrophages from both MMP-3+/+ and
MMP-3/ mice was significantly higher in the
presence than in the absence of plasminogen, and the time course of
lysis was comparable for MMP-3+/+ and
MMP-3/ macrophages (data not shown).
| |
DISCUSSION |
The gelatinase (type IV collagenases) class of human matrix
metalloproteinases comprises a 72 kD molecule (gelatinase A or MMP-2)
and a 92 kD species (gelatinase B or MMP-9); both MMPs degrade
denatured forms of collagen (gelatin) as well as several types of
native collagens. Gelatinases have been shown to be associated with
many connective tissue cells and with
monocytes/macrophages.5,9 Both gelatinases are secreted in
a latent form, and activation of proMMP-2 and proMMP-9 appears to occur
via different mechanisms. Serine proteinases such as trypsin and
plasmin were shown to activate proMMP-9 but not
proMMP-2.9,15-18 However, some investigators reported that
purified proMMP-9 is not efficiently activated by plasmin.12 Recently, it was shown that membrane type-1 MMP
converts 72 kD proMMP-2 to an intermediate 64 kD species, which can be activated by plasmin after conversion to a 62 kD
molecule.33
Stromelysin-1 (MMP-3), which can be generated from proMMP-3 by the
action of plasmin,9 activates proMMP-99,11,12
but not proMMP-2.9 Okada et al12 reported that
MMP-3 converts 92 kD proMMP-9 to an active species of 64 kD that lacks
both NH2- and COOH-terminal peptides and can cleave native
collagens including the 2 chain of type I collagen and collagen
types III, IV, and V. Ogata et al,11 on the other hand,
found that MMP-3 activates proMMP-9 by sequential cleavage at two sites
in the NH2-terminal region yielding an inactive
intermediate species of 86 kD and a fully active form of 82 kD.
Previously, an intermediate 83 kD species of MMP-9 was described as a
fully active form,21,34,35 possibly because the further
conversion was inhibited by TIMP-1 copurified with
proMMP-9.12,15 Taken together, these findings may indicate
that plasmin does not directly activate proMMP-9 but may, nevertheless,
be indirectly involved in its activation through activation of
proMMP-3.12 Thus, MMP-3 may play a key role in the
activation of proMMP-9. In the present study, we have evaluated a
potential physiological role of active MMP-3 (plasmin dependent or
independent) in gelatinase activation with the use of mice that are
genetically deficient in MMP-3.22
Enhanced expression of latent and active forms of MMP-2 and MMP-9 was
previously reported in vascular wall cells of balloon-injured rat
carotid arteries36 and in femoral and carotid arteries of mice after perivascular electric injury.37 Therefore, we
have applied a vascular injury model to MMP-3+/+ and
MMP-3/ mice to monitor the ratios of active
to latent gelatinases. In this model, 1 week after injury the media in
the center of the injury is virtually depleted of smooth muscle cells,
and a small neointima is formed at the borders of the injury associated
with migration of smooth muscle cells from the borders into the center of the injury.26 One week after vascular injury, neointima
formation and cell accumulation in the media and intima was very
similar in MMP-3+/+ and MMP-3/
mice. Immunostaining of arterial sections showed significantly enhanced
expression of MMP-2 and MMP-9 in MMP-3+/+ as well as in
MMP-3/ mice. Double immunostaining showed
colocalization of MMP-9 with macrophages mainly in the adventitia,
whereas MMP-2 was also detected mainly in the adventitia but failed to
colocalize with smooth muscle cells. Possibly MMP-2 is secreted by
fibroblasts or infiltrating inflammatory cells. However, it should be
kept in mind that at 1 week after injury proliferating/migrating smooth
muscle cells do not stain very well for -actin.26
Quantitative analysis of the different gelatinase species in arterial
extracts showed that the contribution of active MMP-2 to the total
MMP-2 levels was very similar before and after injury for
MMP-3+/+ and MMP-3/ mice,
indicating that MMP-2 expression and activation occurs independently of
MMP-3. Active 83 kD MMP-9 was not detectable in control uninjured
femoral or carotid arteries but was present at 1 week after injury in
comparable amounts in MMP-3+/+ and
MMP-3/ arteries, indicating that activation
of 94 kD proMMP-9 can occur in the absence of MMP-3. Analysis of
plasminogen activator activity (t-PA and u-PA mediated) in arterial
extracts did not show differences in plasminogen activating potential
between both genotypes.
These studies in the vascular injury model were complemented with cell
culture experiments. The contribution of active MMP-2 to the total
MMP-2 level in conditioned medium of fibroblasts or smooth muscle cells
was comparable for MMP-3+/+ and
MMP-3/ mice and was not significantly
affected by addition of plasmin(ogen). Addition of plasmin(ogen) to the
culture medium of macrophages resulted in comparable conversion of 94 kD proMMP-9 to 83 kD MMP-9 in MMP-3+/+ and
MMP-3/ samples. It was shown previously that
u-PA-mediated plasmin activates proMMP-9 (and also proMMP-3,
proMMP-12, and proMMP-13), contributing to media destruction and
aneurysm formation during atherosclerosis.38
Taken together, these data, obtained in an in vivo model as well as in
cell culture experiments, do not show impaired proMMP-9 activation in
the absence of MMP-3. Generation of active MMP-9 in contrast was most
evident in the presence of plasmin(ogen). ProMMP-9 activation, thus,
appears to be possible via plasmin-dependent and MMP-3-independent
mechanisms. The hypothesis that active MMP-3 is required for activation
of proMMP-9, and that the effect of plasmin on activation of proMMP-9
is mediated via activation of proMMP-3 to active MMP-3, thus, is
contradicted by our finding that plasmin can activate proMMP-9 also in
the absence of MMP-3 as schematically illustrated below.
[View Larger Version of this Image (9K GIF file)]
However, we have previously shown that
active MMP-9 can be detected in plasminogen-deficient
fibroblasts,39 possibly as a result of activation of
proMMP-9 by MMP-3 or other active MMPs.
The data obtained in this study with the use of
MMP-3/ mice thus do not exclude direct
activation of proMMP-9 by MMP-3 but indicate that MMP-3 or other MMPs
that critically depend on MMP-3 for their activation do not play a
physiological role in activation of proMMP-9.
| |
FOOTNOTES |
Submitted July 18, 1997;
accepted November 7, 1997.
Supported by grants from the Flemish Fund for Scientific Research (FWO,
contract G.0293.98) and from the IUAP (contract P4/34).
Address reprint requests to H.R. Lijnen, PhD, Center for Molecular and
Vascular Biology, KU Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Dr J. Mudget (Merck Research Laboratories, Rahway,
NJ) for kindly supplying us with breeding pairs of wild-type and
MMP-3-deficient mice and to Dr G. Murphy (Strangeways Research Laboratory, Cambridge, UK) for murine MMP-3 and MMP-9. Double immunostainings were kindly performed by Dr F. Lupu (Thrombosis Research Institute, London, UK). We are grateful to Dr P. Carmeliet and
Dr L. Moons (Center for Transgene Technology and Gene Therapy, Leuven,
Belgium) for helpful discussions. Skilful technical assistance by A. Dewulf, L. Frederix, G. Lemmens, I. Vanlinthout, and M. Verstreken is
gratefully acknowledged.
| |
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Abstract
Introduction
Abstract