Expression of matrix-degrading enzymes in pulmonary vascular
remodeling in the rat
Smita
Thakker-Varia1,
Carol A.
Tozzi1,2,
George J.
Poiani1,2,
Joanne P.
Babiarz1,
Linda
Tatem1,
Frank J.
Wilson1, and
David J.
Riley1
1 Departments of Medicine and
of Neuroscience and Cell Biology, University of Medicine and
Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway
08854-5635; and 2 New Jersey
Veterans Affairs Health Care System, Lyons, New Jersey 07939-9988
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ABSTRACT |
Exposure of rats
to hypoxia causes pulmonary arterial remodeling, which is partly
reversible after return to air. We hypothesized that degradation of
excess collagen in remodeled pulmonary arteries in the posthypoxic
period is mediated by endogenous matrix metalloproteinases (MMPs).
Total proteolytic, collagenolytic, and gelatinolytic activities, levels
of stromelysin-1 and tissue inhibitor of metalloprotease-1 (TIMP-1),
and immunolocalization of stromelysin-1 in main pulmonary arteries were
determined after exposure of rats to 10%
O2 for 10 days followed by
normoxia. We observed transient increases in total proteolytic,
collagenolytic, and gelatinolytic activities and expression of ~72-,
68-, and 60-kDa gelatinases by zymography within 3 days of cessation of
hypoxic exposure. The level of TIMP-1 increased as the stromelysin-1
level increased. Immunoreactive stromelysin-1 was localized
predominantly in the luminal region of normal and hypertensive
pulmonary arteries. These results are consistent with the notion that
endogenous MMPs may mediate the breakdown of excess collagen in
remodeled pulmonary arteries during the early posthypoxic period.
extracellular matrix proteins; blood vessels; peptide
peptidohydrolases; hypertension; collagen
 |
INTRODUCTION |
THE PULMONARY ARTERY undergoes major structural
reorganization during sustained elevation of pulmonary arterial
pressure. As the pulmonary arterial wall enlarges during remodeling,
vascular cells must migrate, requiring the breakdown of extracellular
matrix barriers. Increased activity of serine proteases in pulmonary arteries has been observed during early remodeling in
monocrotaline-induced pulmonary hypertension (40), suggesting serine
proteases are involved in early remodeling. After removal of the
hypertensive stimulus, the excess extracellular matrix proteins that
have accumulated during exposure to hypoxia decrease toward normal
levels (20). The decrement in content of highly insoluble collagen and
elastin molecules in a relatively brief time suggests that matrix
metalloproteinases (MMPs) play a role in the resorption of vascular
collagen.
MMPs are a family of degradative enzymes expressed during periods of
active remodeling, such as embryonic development, wound healing, and
involution of tissues (38). Members of this family are secreted in a
latent form and are cleaved by other proteases to
lower-molecular-weight active forms. In addition, latent MMPs can be
activated by nonproteolytic processes such as organomercurial compounds, plasmin, and reactive oxygen species. The counterregulatory tissue inhibitor of metalloproteinase (TIMP) modulates MMP activities, and the balance between MMP and TIMP is thought to determine overall turnover of matrix proteins.
There is considerable evidence that MMPs are important in the
maintenance of the integrity and stability of systemic blood vessels
(8). For example, mutations in TIMP-3 genes are thought to be the cause
of a rare genetic form of macular degeneration (Sorsby's fundus
dystrophy) in humans, and a stromelysin promoter variant is associated
with progression of atherosclerosis (8). In atherosclerosis, MMPs
colocalize with foamy macrophages within regions of atherosclerotic
plaques, and cytokines, such as tumor necrosis
factor-
, that induce production of MMPs may be
involved in initiating a cascade of events leading to plaque rupture
and vascular occlusion. Mechanical injury to the wall of blood vessels in animal models induces MMPs, and administration of an MMP inhibitor substantially reduces the rate of migration of the smooth muscle cells,
suggesting that MMPs play a role in restenosis. These observations suggest that inflammation plays a key role in modulating MMP expression in systemic vessels.
The aim of this study was to determine whether expression of
interstitial collagenase, stromelysin-1, and gelatinases was increased
during recovery from hypoxic pulmonary hypertension in the rat. We also
analyzed the level of TIMP-1 expression to determine whether an
imbalance between MMPs and their principal inhibitor occurs during
regression of pulmonary artery remodeling. In the rat hypoxic model of
pulmonary hypertension, little if any inflammation is demonstrable by
ultrastructural examination of blood vessels (18). This model,
therefore, provides an opportunity to study MMP expression in
remodeling in the absence of vascular injury or inflammatory disease.
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MATERIALS AND METHODS |
Animals. Outbred 6-wk-old male rats
(n = 224) and adult male
Hartley-strain guinea pigs (n = 4;
Charles River Breeding Laboratories) were used. Animals were retained
in the animal quarters for 1 wk and were fed standard animal chow and
water ad libitum. Guinea pigs were killed by a lethal intraperitoneal
injection of pentobarbital sodium, and dermal collagen was extracted
and used as a substrate for collagenase. Institutional guidelines for
animal use were followed.
Antibodies. Sheep anti-rabbit
stromelysin-1 polyclonal antibody was provided by M. Lark (Merck Sharpe
and Dohme Research Laboratories, Rahway, NJ), goat anti-mouse TIMP-1
polyclonal antibody by D. Denhardt (Rutgers University, Piscataway, NJ)
(7), and rabbit anti-human
5-subunit polyclonal antibody
by S. Johansson (Uppsala University, Uppsala, Sweden) (4).
Nucleic acid probes. The probes were a
1,300-bp EcoR I-BamH I fragment of rat
transin (the rat homologue of stromelysin-1) (pKSTREB) (17), a 700-bp
EcoR I fragment of rat stromelysin-2 (5), a 1,250-bp
EcoR I cDNA fragment of rat uterine smooth muscle
collagenase 54 (21), a 700-bp Hind III-EcoR I
fragment of mouse TIMP-1 cDNA (P TIMP-F) (10), and a 1,500-bp
EcoR I fragment of human
pro-
1(I) collagen cDNA (Hf677) (2).
Hypoxic exposure and hemodynamic
measurements. Rats were exposed to 10%
O2-90%
N2 (hypoxia) at ambient pressure
(15). Groups of animals were studied before exposure to hypoxia
(day
0), and after exposure to hypoxia
for 10 days (day
10) or to hypoxia for 10 days
followed by recovery in air for 1, 3, 7, or 14 days
(days 11,
13,
17, and
24, respectively). In addition, one
group (n = 3) was exposed to hypoxia
for 3 days and used to measure steady-state mRNA levels for
pro-
1(I) collagen as a positive control
for Northern blot analysis. Age-matched control animals breathed room
air. Hypoxic animals were fed standard rat chow and given water ad libitum, and the amount of food was weighed. Control animals were fed
the same amount as hypoxic animals to ensure similar final body
weights. Mean right ventricular pressure (RVP) was measured in
anesthetized rats injected intraperitoneally with 50 mg/kg of
pentobarbital sodium; the ratio of weights of cardiac ventricles [right ventricle/(left ventricle + septum), RV/(LV+S)] and
hematocrit were measured as previously described (15).
Light microscopy and thickness of pulmonary
arterioles. Photomicrography of hematoxylin and
eosin-stained tissue sections and measurements of pulmonary artery wall
thickness (vessels with external diameters 110-180
µm) were performed as previously described (20).
Preparation of tissues. The main
pulmonary artery, the entire left extrapulmonary artery, and the
proximal 3 mm of the right extrapulmonary artery were excised en bloc.
Individual tissues were used for all assays except Northern blot
analysis and SDS-PAGE analysis, for which tissues were pooled
(n = 5-6). For
immunohistochemistry, lungs were perfused via the trachea with a 1:1
solution of OCT (Miles Scientific) and PBS, frozen in liquid nitrogen,
and stored at
70°C.
Hydroxyproline and protein
measurements. Pulmonary artery segments were
homogenized and then hydrolyzed in 6 N HCl at 116°C for 48 h, and
total protein and hydroxyproline contents were assayed as previously
described (20).
Total protease activity. Degradation
of [14C]carboxymethyl transferrin by pulmonary artery
homogenates was used to assay for noncollagenolytic activity (35). The
substrate was prepared by reaction of reduced bovine transferrin with
[14C]acetic anhydride
(108 mCi/mM; Amersham). Pulmonary arteries were homogenized (model PT
10/35, Brinkman Instruments) in transferrin buffer (0.1 M
Tris · HCl-0.15 M NaCl, pH 7.5) and centrifuged at
9,000 g for 15 min at 4°C. The
pellet was resuspended at 10 mg/ml in transferrin buffer and stored at
70°C. Tissue homogenates (20 µl) were
incubated with 25 µl of
[14C]carboxymethyl
transferrin and 530 µl of buffer (0.1 M
Tris · HCl, 0.15 M NaCl, 10 mM
CaCl2, and 5 mM
MgCl2, pH 7.5). The mixture was
incubated at 37°C for 1 h, and 200 µl of 10% TCA
were added, followed by centrifugation at 10,000 g for 15 min at 4°C. Radioactivity in the supernatant was counted by liquid scintillation spectrometry (model LS 6000IC, Beckman Instruments). To determine the classes of
enzymes, homogenates from day
13 animals were incubated with EDTA
(100 µM), 1,10-phenanthroline (100 µM), phenylmethylsulfonyl fluoride (PMSF; 100 µM), and 3,4-dichloroisocoumarin (100 µM; Sigma) (3).
Collagenase activity. Type I collagen
extracted from dermis (12) was labeled with
[14C]acetic anhydride
(108 mCi/mM; Amersham) (11), digested with pepsin (25), and used as
substrate [specific activity, 6.7 × 104
counts · min-1
(cpm) · mg
1]. Tissues
were homogenized in 0.15 M NaCl and centrifuged at 6,000 g for 20 min at 4°C, and the
pellet was washed in 0.15 M NaCl. The pellet was resuspended at 10 mg/ml in a 0.04 M Tris buffer with 0.15 M NaCl, 0.01 M
CaCl2, 250 µg/ml
of streptomycin, and 200 U/ml of penicillin G, pH 7.5, frozen in liquid
nitrogen, and stored at
70°C. Tissue
homogenate (50 µl) was incubated with 50 µl of
[14C]collagen, 2 mM
4-aminophenylmercuric acetate (Sigma), and 350 µl of
buffer (0.04 M Tris, 0.15 M NaCl, 10 mM
CaCl2, and 5 mM MgCl2, pH 7.5) for 48 h at
37°C. The mixture was centrifuged at 10,000 g for 10 min, 0.04 M phosphotungstic
acid and 50 µl of 2 N HCl were added to the
supernatant, and radioactivity was counted.
Gelatinolytic activity. The substrate
was prepared by denaturing
[14C]collagen in
boiling water for 15 min (19). Tissue homogenate (50 µl), prepared as described in
Preparation of tissues, was incubated
with 50 µl of
[14C]gelatin and 350 µl of buffer (0.04 M Tris, 0.15 M NaCl, 10 mM CaCl2, and 5 mM
MgCl2, pH 7.5) for 48 h at
37°C. After precipitation with 12% (wt/vol) TCA, the
radioactivity in the supernatant was counted.
Gelatin zymography. Tissues were
homogenized in NP-40 lysis buffer (0.1 M Tris, 1% NP-40, and 0.15 M
NaCl, pH 8.0) containing 5 mM EDTA and 2 mM PMSF. Homogenates were
incubated for 1 h at 4°C, centrifuged at 27,000 g for 20 min, and stored at
70°C. Extracts were diluted with 2% SDS, 0.15% glycerol,
0.25 M Tris · HCl (pH 6.8), and 0.1% bromphenol blue
(19), and protein content was assayed (BCA protein assay kit, Pierce
Chemical). Samples were loaded without boiling onto a 12%
polyacrylamide gel copolymerized with 0.1% gelatin and separated by
electrophoresis at 4°C (13). The gels, incubated for 60 min in
2.5% Triton X-100 and then for 24 h at 37°C in 50 mM
Tris · HCl (pH 8.0) containing 5 mM
CaCl2 to allow for gelatin
digestion, were stained in 0.25% Coomassie blue containing 50%
methanol-10% acetic acid followed by destaining in 10% methanol-10%
acetic acid. Enzymatic activity in the gel was visualized as negative
staining, and enzyme sizes were referenced to molecular-weight markers.
Western blot analysis. Pulmonary
artery extracts, prepared as described for gelatin zymography, were
denatured in Laemmli's buffer (16) containing
-mercaptoethanol for
10-15 min and subjected to SDS-PAGE with a 12.5% polyacrylamide
gel. The protein was blotted onto polyvinylidene fluoride membranes
(Millipore) with a semidry transfer unit (Hoefer Scientific
Instruments) and buffer (48 mM Tris, 39 mM glycine, 0.375 mM SDS, and
20 mM methanol) for 60 min at 80 mA. Equal loading was based on
comparison with
5-integrin (4),
a protein that did not change during pulmonary hypertension. The
membranes were blocked for 1 h with a 4% solution of dry milk powder
in 0.2% Tween 20-PBS. The polyvinylidene fluoride membranes were
incubated overnight at 4°C with nonimmune sera or immune sera:
anti-rabbit stromelysin-1 or anti-mouse TIMP-1 antibodies. Membranes
were washed, incubated with
125I-protein A (0.1 µCi/ml) or
125I-protein G (0.1 µCi/ml; ICN Biomedicals) for 1 h, washed extensively, air-dried, and exposed to X-AR-5 photographic film (Kodak) for 24 h.
Molecular weights were compared with size markers. Density of bands was
scanned with an imaging detector (model GS-670, Bio-Rad Laboratories).
RNA analysis. Total RNA was extracted
by the guanidine isothiocyanate method, and Northern blot analysis was
performed (20) with 20 µg or more of total RNA.
Nitrocellulose filters (Schleicher & Schuell) were hybridized to nick
translated cDNA probes (24) labeled with
[32P]dCTP
(3,000 Ci/mM; ICN Biomedicals) to a specific activity of ~1 × 108 cpm/µg. As a
positive control, a probe for pro-
1(I)
collagen was used (2), which has been shown to increase at 3 days of hypoxia compared with control pulmonary arteries (20). Southern blot
analysis (27) was performed with the
32P-labeled cDNA probes mentioned
above.
RT-PCR. Total RNA (50 µg) was used to synthesize single-stranded cDNA using
750 units of Moloney mouse leukemia virus reverse transcriptase
(GIBCO) and 50-100 pM of
oligo(dT)12-18 (Pharmacia LKB
Biotechnology). Single-stranded cDNA was used for RT-PCR and primed
with two oligonucleotide primers,
5'-CCGTCCAGAAGATCGATGCA-3' and
5'-CCATCTACACAGAGACAGTT-3', complementary to the rat
transin cDNA (15), and 5'-ACCACCTTATACCAGCGTTA-3' and
5'-AAACAGGGAAACACTGTGCA-3', complementary to mouse TIMP-1
cDNA (10). Amplification was performed in a 100-µl
reaction mixture (50 mM NaCl, 10 mM KCl, 10 mM
Tris · HCl, 1.5 mM
MgCl2, 3 mM dithiothreitol,
gelatin, and 200 µM each nucleotide
5'-triphosphate, pH 8.8) containing 2.5 U of
Taq DNA polymerase (Perkin-Elmer). The
reaction was carried out with a DNA thermal cycler (model 480, Perkin-Elmer Cetus) for 30 cycles as follows: denaturation at 94°C,
1.5 min; annealing at 54°C, 1 min; extension at 75°C, 1.5 min;
and final extension, 10 min. The RT-PCR products were electrophoresed
on a 1.5% agarose gel, DNA fragments were blotted onto nitrocellulose,
and Southern blot analysis was performed.
Immunohistochemistry. Frozen sections
of peripheral lung tissue (6 µm thick) were cut at
17°C on a cryostat microtome (model 855, American Optical
Reichert Scientific Institute) and mounted on gelatinized slides that
were air-dried and stored desiccated at
70°C. Tissue
sections were warmed to room temperature, hydrated in a humidified
chamber for 2 h at 4°C, OCT was removed, and tissues were fixed in
75% ethanol for 5 min. After PBS rinses, 1% BSA in PBS with 0.05%
azide as a blocking agent was applied for 2 h at room temperature or
overnight at 4°C. The blocking agent was removed, and sheep
anti-rabbit stromelysin-1 (1:50 to 1:5,000 dilutions) was applied
overnight at 4°C. The primary antibodies were removed by
aspiration, and the slides were thoroughly washed with PBS. The
secondary antibody, rhodamine-conjugated rabbit anti-sheep IgG (1:1,000
dilution; Cappel) with PBS, was applied for 2 h at room temperature or
overnight at 4°C. Tissues were washed thoroughly in PBS, mounted in
gelvatol (Air Products and Chemicals) with
N-propyl galate, and photographed with
T-Max 400 film (Kodak) on a microscope (Labophot Fluorescence
Microscope, Nikon) equipped with an epifluorescence attachment.
To evaluate the specificity of the primary antibody, five experiments
were performed on pulmonary artery tissue sections obtained from
day
13 animals. First, rabbit
stromelysin-1 antibody (1:50) was incubated with rat stromelysin-1 in
PBS for 30 min at 37°C, held overnight at 4°C, and centrifuged
at 1,500 g for 10 min. Tissues
incubated with the supernatants containing the absorbed antibody showed
weak, nonspecific fluorescence, indicating that the antibody was
immunologically specific for rat stromelysin-1. Second, tissues were
stained with anti-mouse desmin, and immunoreactivity was observed only
in vascular smooth muscle cells, demonstrating specificity of unrelated
antibodies. Third, tissues were evaluated for autofluorescence before
their reaction with secondary antibody, and none was observed. Fourth,
fluorescence with the conjugated secondary antibody was examined.
Fifth, tissues were reacted with an appropriate nonimmune serum
followed by conjugated secondary antibody. Weak, nonspecific
fluorescence was observed in the latter two experiments.
Statistical analysis.
2 Analysis with Yates'
correction was used to assess nonparametric data (28). Data were
analyzed with one-way ANOVA (28) followed by Duncan's post hoc test
(9). A P value of <0.05 was
considered significant.
 |
RESULTS |
Animal, hemodynamic, and biochemical
results. Animal survival was 100% (33/33) in control
group and was 98% (188/191) in the hypoxic group
[
2 = 1.34, not
significant (NS)]. Body weights were not significantly different
between hypoxic animals and their age-matched controls on any day. Mean
RVP increased twofold on day
10 compared with day
0, decreased by
day
13 compared with
day
10, and was not different from that of
control levels on day
24 (Fig.
1A). The RV/(LV+S) increased by day
10, was decreased on
day
13 compared with
day 10, but was above control levels on
day
24 (Fig.
1B). Hematocrit increased during
hypoxia, decreased during recovery, but remained elevated on
day
24 (Fig.
1C). The medial walls of muscular
pulmonary arteries were thickened 2.5-fold on
day
10, decreased on
day
13 compared with
day
10, and were at the control level on
day
24 (Fig.
1D). Hydroxyproline and protein
contents of main pulmonary arteries were ~75 and 150%, respectively,
greater than control levels on day
10; both decreased on
day
13 compared with
day
10, and both were at control levels on
day
17 (Fig. 1,
E and
F).

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Fig. 1.
Measurements of pulmonary hypertension and vascular
remodeling. Rats were exposed to 10%
O2 (hypoxia) at ambient pressure
for 10 days or were exposed to hypoxia for 10 days and allowed to
recover in air for 1, 3, 7, or 14 days
(days 11,
13,
17, and
24, respectively).
A: mean right ventricular pressure
(RVP). B: ratio of weight of right
ventricle to that of left ventricle plus septum [RV/(LV+S)].
C: hematocrit.
D: medial wall thickness of muscular
pulmonary arteries. E: hydroxyproline
content. F: protein content. Data are
means ± SE; n = 4-6 animals
except for D where
n = 18 and 4-5 arteries/animal
were measured. * P < 0.05 compared with day 0.
P < 0.05 compared with day 10.
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Proteolytic activity. Degradation of
[14C]carboxymethyl
transferrin was at control level on
days
10 and
11, was elevated 3.5-fold on
day
13, and was at control level on
day
24 (Fig.
2A).
Protease activity on day
13 was inhibited ~64% by 100 µM EDTA and ~47% by 100 µM
1,10-phenanthroline, both MMP inhibitors (3), and ~15% by 100 µM PMSF and ~15% by 100 µM
3,4-dichloroisocoumarin, both serine protease inhibitors (3) (all
P < 0.05, n = 5-6 pulmonary arteries).
These results indicate that MMPs are the predominant proteases in
day
13 pulmonary arteries. Collagenolytic activity was at control levels on day
10, increased fivefold at day
13 (P < 0.05, n = 5-6 pulmonary
arteries), decreased on day 17 compared with
day
10, and was at control levels on
day
24 (Fig. 2B). Gelatinolytic activity was at
control levels on day
10 compared with
day
0, increased on
days
11,
13, and
17, and was at control level on
day
24 (Fig.
2C). Collagenolytic and
gelatinolytic activities were completely inhibited by 100 µM EDTA (data not shown). On zymographic analysis,
two bands of gelatinolytic activities were observed at ~68 kDa and
faintly at ~60 kDa on day
0 (Fig.
3). Three bands of ~72, 68, and 60 kDa
were observed on days
10,
11, 13, and
17, with the greatest density of bands
on day
11 (Fig. 3). The density of the bands
appeared to decrease on day
17 compared with
day
11.

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Fig. 2.
Proteolytic activity in pulmonary arteries. Conditions are as described
in Fig. 1. A: total proteolytic
activity. B: collagenolytic activity.
C: gelatinolytic activity.
Collagenolytic activity was not measured on
day 11. Data are means ± SE;
n = 5-6.
* P < 0.05 compared with
day 0.
P < 0.05 compared with
day 13.
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Fig. 3.
Gelatinase activity in pulmonary artery tissue. Homogenates of
pulmonary artery tissues (15 µg of protein) were
separated on an acrylamide gel copolymerized with gelatin and examined
for gelatinolytic activity. Equal loading was checked by measuring
protein levels before gel was loaded. Lanes indicate days of exposure
to hypoxia or recovery from hypoxia. Presence of ~72-, 68-, and
60-kDa band was faint on day 0 and was increased at 10 days of
hypoxia and 1, 3, and 7 days of recovery.
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Stromelysin and TIMP-1. An ~57-kDa
stromelysin-1 protein, expressed at all times, was increased on
day
13 compared with
day 10 and decreased by
day
24 (Fig.
4A).
Scanning densitometer readings indicated a twofold increase in
stromelysin-1 protein levels on day
13 compared with all other days
(P < 0.05;
n = 5 pulmonary arteries). A
constitutively expressed ~28.5-kDa TIMP-1 protein was faintly visible
on day
0. The signal appeared greater on
days 10,
13,
and 17 than on
day 0 and appeared to decrease by day
24 (Fig.
4B). Equal loading of samples was
demonstrated by unchanged levels of
5-integrin protein throughout
the hypoxic and recovery periods (Fig.
4C). Densitometry readings for
TIMP-1 showed a threefold increase in TIMP-1 levels on
day
13 compared with
day
10 (P < 0.05; n = 6 pulmonary arteries).
Densitometry readings were consistent for stromelysin-1
(n = 4 tissues) and TIMP-1
(n = 6 tissues). Hybridization signals for rat stromelysin-1 (transin), stromelysin-2, and TIMP-1 mRNAs were not detectable under low-stringency conditions by
Northern blot analysis (Fig. 5). This was
not an artifact because pro-
1(I)
collagen mRNA was detected on day
0 and increased on day 3 of hypoxia with the same method (Fig. 5). No stromelysin-1 or TIMP-1
products were detected by RT-PCR analysis after 30 cycles (not shown).

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Fig. 4.
Autoradiograms of immunoblots for antibodies to stromelysin-1 and
tissue inhibitor of metalloprotease (TIMP)-1. Homogenates of pulmonary
artery tissue (20 µg of protein) were examined during
hypoxia and recovery from hypoxia. A:
stromelysin-1. Left: ~57-kDa
stromelysin-1 protein (arrow). Right:
corresponding densitometer measurement of stromelysin-1 protein
indicating expression at all times, with increased expression on day
13. B: TIMP-1.
Left: ~28.5-kDa TIMP-1 protein
(arrow) was expressed at all times.
Right: corresponding densitometer
measurements of TIMP-1 protein indicating apparent increased expression
on days 10 and
13 and reduction toward control by
day 24.
C: 150-kDa
5-integrin protein control
showed no change during hypoxia and recovery. Immunoblots are
representative of n = 4-5 blots
for each protein, showing same results.
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Fig. 5.
Autoradiogram of Northern hybridizations to rat RNA. Total RNA (10 µg) was applied to filters and hybridized with
radiolabeled probes. Lane 1,
day 0 (control) and lane 2, 3-day hypoxic rats showing 5.3- and
4.7-kb bands for pro- 1(I) collagen
(arrows). Lane 3,
day 13 rat transin (rat homologue of
stromelysin-1). Lane 4,
day 13 rat stromelysin-2.
Lane 5, day 13 TIMP-1. No
hybridization signals are seen in
lanes 3-5.
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Immunohistochemistry. Immunoreactive
stromelysin-1 was detected predominantly in the luminal portion of the
vessel wall of muscular pulmonary arteries on
day 0 (Fig.
6A). The
intensity of the stain appeared slightly increased on
days
10,
11, and
13 compared with
days
0 and
17 (Fig. 6,
B-E).
An accompanying hematoxylin and eosin-stained section of a
day
17 muscular pulmonary artery is shown
(Fig. 6F) for orientation. In
separate control experiments, tissues were incubated with the
following: rabbit stromelysin-1 antibody absorbed with rat
stromelysin-1, PBS in place of the primary antibody followed by
incubation with the conjugated secondary antibody, and nonimmune serum
followed by the conjugated secondary antibody. For all control
experiments, weak, nonspecific fluorescence was observed on
days
0,
10, and
13 (Fig. 6,
G-I).

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Fig. 6.
Immunolocalization of stromelysin-1 in muscular pulmonary arterioles.
A: control;
B: 10-day hypoxia;
C:
day 11;
D:
day 13;
E:
day 17;
F: hematoxylin and eosin-stained
section as reference. Arrow, muscular pulmonary vessel.
G-I:
immunofluorescence of stromelysin-1 after incubation of tissues with
PBS in place of primary antibody followed by secondary antibody.
G:
day 0; H:
day 10;
I:
day 13. Pulmonary arteries were incubated
with PBS followed by rhodamine-conjugated rabbit anti-sheep IgG. Note
weak, nonspecific staining at these times. d, Day; av, alveolus; aw,
airway; bv, blood vessel. Bar in E, 20 µm.
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DISCUSSION |
We observed a rapid decrease in collagen content of main pulmonary
arteries of rats during the first several days after removal from a
hypoxic environment. Transient increases in total proteolytic, collagenolytic, and gelatinolytic activities and increased expression of stromelysin protein and the ~68-kDa gelatinase were noted 1-3 days after return to normoxia. A temporal correlation between the
expression of MMPs and rapid decrease in vascular collagen content
suggests an association between collagen resorption and MMP activity.
The relative proportion of collagen to noncollagen proteins remained
constant throughout remodeling and regression. This observation
suggests that the rates of turnover of cells and matrix proteins
occurred proportionately, consistent with "physiological"
resorption of collagen. The processes controlling removal of excess
collagen in remodeled arteries may be analogous to the involutional
loss of collagen in the postpartum uterus, which is mediated by marked
increases in levels of MMP activities (38).
We studied in greatest detail proteases expressed on
day
13, the time of peak total proteolytic
and collagenolytic activities as well as of stromelysin protein
expression. Total proteolytic activity on
day
13 was reduced 47-64% by MMP
inhibitors, and ~15% of total activity was blocked by serine
protease inhibitors, suggesting that MMPs are the predominant proteases
expressed on day
13. Interstitial collagenase was
observed to increase fivefold on day
13. In contrast, gelatinolytic
activity and expression of the ~72-, 68-, and 60-kDa gelatinases by
zymography peaked on day
11. The reason for the earlier
expression of gelatinase is not known, but we speculate that
degradation of basement membranes could be occurring before breakdown
of interstitial collagens. Gelatinase degrades basement membrane
components (collagen types IV, V, VII, and X and fibronectin) as well
as partially degraded collagen (38). The stimulus for basement membrane
degradation could be changes in tissue structure, perhaps caused by a
decrease in smooth muscle cell hypertrophy or cell death during
involutional loss of cell mass during regression of pulmonary
hypertension. As discussed below, stromal expression of MMPs may be a
consequence of changes in cell shape or stimulation by oxygen-derived
free radicals produced in blood vessels during the transition from
hypoxia to normoxia. Further work is needed to determine whether
breakdown of basement membranes or other structural components occurs
before degradation of interstitial collagens to explain the early
appearance of gelatinase.
Stromelysin-1, an MMP with broad substrate specificity (38), was
studied using molecular, biochemical, and immunohistochemical methods.
In homogenates prepared from main pulmonary arteries, we observed
constitutive expression of stromelysin-1 by immunoblot and a transient
twofold increased level above control on
day
13. The location of immunoreactive
stromelysin-1 appeared to be mainly on the luminal portion of the
muscular pulmonary arteries, but the resolution of the photomicrographs
was not sufficient to determine the cellular source(s) of
stromelysin-1. Procollagenase, in contrast, is predominantly localized
in the media and adventitia within connective tissue-type mast cells,
which are abundant in remodeled pulmonary arteries of rats (33). We
observed differences in the temporal expression of stromelysin-1 by
immunoblot, which showed an increase only on
day
13, and by immunohistochemistry, which
showed increased staining on days
10,
11, and
13. It is possible that central and
distal pulmonary arteries express stromelysin-1 differently in response
to a hypertensive stimulus. It is known that differences in the pattern
of structural remodeling in central and peripheral arteries occur,
which is probably attributed to the composition of extracellular matrix
proteins, populations of cells in the vessel walls, and differences in
cellular and molecular responses to the hypertensive stimulus (14, 31). It is possible, for example, that a subset of smooth muscle cells capable of expressing stromelysin-1 is present during active remodeling in small branches. Alternatively, different hemodynamic stresses might
signal the expression of stromelysin-1 in the two types of vessels. A
systematic study contrasting MMP expression in small and large arteries
is needed to determine the significance of these observations.
Stromelysin-1 mRNA was not detected in pulmonary artery tissue by
Northern blot analysis under low-stringency conditions. Stromelysin-1
RT-PCR products were not visualized after 30 cycles. These observations
suggest that posttranscriptional mechanisms are more likely to
contribute to high levels of stromelysin-1 protein observed during the
posthypoxic period. These posttranscriptional mechanisms may involve
increased translation of stromelysin-1 protein or stromelysin-1 derived
from cells migrating into the artery during remodeling.
We examined the changes in TIMP-1 protein in regression of pulmonary
artery remodeling. TIMP-1 was expressed at low levels in normotensive
pulmonary arteries but appeared to increase during the late
hypertensive (day
10) and recovery phases compared
with control pulmonary arteries. The level of TIMP-1 appeared to be greatest on day
13, the time of peak proteolytic
activities and greatest expression of MMPs. It is difficult to draw
conclusions about these changes in protein levels on TIMP-1-MMP balance
in tissue because of sequestration of enzyme, kinetics of
enzyme-inhibitor binding, and turnover rates of both components.
Several possible mechanisms may be responsible for activation of MMPs
in pulmonary arteries. First, the change in tissue oxygen tension
during the transition from hypoxia to normoxia may have activated
latent MMPs and induced proteolytic activity. Various reactive oxygen
species, including hypochlorous acid (HOCl), hydrogen peroxide
(H2O2),
and hydroxyl radical generated by hypoxanthine/xanthine oxidase (X/XO),
activate isolated latent procollagenase, and their activation
potentials are comparable to other nonproteolytic activators (26).
Neutrophil procollagenase and progelatinase are activated by HOCl (36),
an effect that is enhanced by addition of cathepsin G (6). Incubation
of human vascular smooth muscle cell culture medium containing latent
progelatinase with X/XO resulted in activation of progelatinase (22).
Noninflammatory cells, such as UMR-106 osteosarcoma cells (23) and
Walker 256 carcinosarcoma cells (29), secrete latent MMPs that are
activated by addition of HOCl and
H2O2,
respectively. It is possible that oxygen free radicals were generated
during the increase in tissue oxygen tension that occurred after the
rats were removed from the hypoxic environment, analogous to the
generation of oxygen free radicals during ischemia-reperfusion. It has been speculated that the mechanism of nonproteolytic activation of procollagenase involves a conformational change in the enzyme molecule that disrupts a cysteine-zinc atom interaction and frees the
zinc atom to participate in proteolytic reactions (30). It is possible
that oxygen free radical generation is an initiating event in induction
of MMP activity during the posthypoxic period.
A second possible mechanism modulating MMP expression may involve
changes in mechanical forces on cells after the reduction in blood
pressure in the posthypoxic period. Werb and colleagues (1, 34, 37)
observed that alterations in cell shape, actin cytoskeleton, and
cellular interactions with integrins can induce production of
stromelysin and interstitial collagenase. These results suggest that
expression of MMPs may be under the control of physical changes in
tissue architecture. In tissues, proteolysis is stimulated by removing
a distending force from the body of a gravid rat uterus (39).
Experiments from our laboratory (32) suggest that release of static
mechanical tension from isolated pulmonary arteries induces proteolysis
and expression of MMPs. In main pulmonary arteries, alterations of MMP
expression may be a consequence of physical forces when blood pressure
is reduced after return to normoxia.
In conclusion, our results show increased proteolysis in main pulmonary
arteries of rats that is restricted to the period of rapid reduction in
collagen content after reversal of hypoxic pulmonary hypertension. The
observation that temporally controlled proteolysis occurs in vessels in
the absence of major inflammatory changes suggests that noninflammatory
mechanisms may control endogenous MMP expression in pulmonary arteries.
Future experiments are needed to establish whether a causal
relationship exists between collagen resorption and MMP activity and
whether proteolysis in pulmonary arteries plays a role in regulation of
pulmonary hemodynamics.
 |
ACKNOWLEDGEMENTS |
We thank Selina Boykin and Marcella Spioch for secretarial
assistance, James Fox for technical assistance, and Drs. John J. Jeffrey (Albany Medical College, Albany, NY), Lynn M. Matrisian (Vanderbilt University School of Medicine, Nashville, TN), Shiu Yeh Yu
(University of Rochester School of Medicine and Dentistry, Rochester,
NY), David T. Denhardt (Rutgers University, Piscataway, NJ), Michael W. Lark (Merck Sharpe and Dohme Research Laboratories, Rahway, NJ), and
Staffan Johansson (Uppsala University, Uppsala, Sweden) for probes and
reagents.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-24264 and HL-07467; the Barbara Cornwall Wallace Respiratory
Research Laboratory; the American Lung Association of New Jersey; the
American Heart Association/New Jersey Affiliate; the University of
Medicine and Dentistry of New Jersey Cardiovascular Institute; and the
Medical Research Service of the Department of Veterans Affairs.
Address for reprint requests: S. Thakker-Varia, Div. of Pulmonary and
Critical Care Medicine, Dept. of Medicine, UMDNJ-Robert Wood Johnson
Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635.
Received 15 July 1997; accepted in final form 14 April 1998.
 |
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