1 Department of Medicine and Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba R3E 0V9; 2 Vancouver Hospital, University of British Columbia, Vancouver, British Columbia V6H 3Z6; 3 Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3A 1R9; 4 The Guy's King's College and St. Thomas' School of Medicine, King's College London, London SE1 9RT; and 5 Glaxo Wellcome, Stevenage SG1 2NY, United Kingdom
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ABSTRACT |
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In severe or
chronic asthma, there is an increase in airway smooth muscle cell
(ASMC) mass as well as an increase in connective tissue proteins in the
smooth muscle layer of airways. Transforming growth factor-
(TGF-
) exists in three isoforms in mammals and is a potent regulator
of connective tissue protein synthesis. Using immunohistochemistry, we
had previously demonstrated that ASMCs contain large quantities of
TGF-
1-3. In this study, we demonstrate that bovine ASMC-derived
TGF-
associates with the TGF-
latency binding protein-1 (LTBP-1)
expressed by the same cells. The TGF-
associated with LTBP-1
localizes TGF-
extracellularly. Furthermore, plasmin, a serine
protease, regulates the secretion of a biologically active form of
TGF-
by ASMCs as well as the release of extracellular TGF-
.
The biologically active TGF-
released by plasmin induces ASMCs to
synthesize collagen I in an autocrine manner. The autocrine induction
of collagen expression by ASMCs may contribute to the
irreversible fibrosis and remodeling seen in the airways of some asthmatics.
plasmin; bovine; transforming growth factor- latency binding
protein-1; fibrosis; remodeling
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INTRODUCTION |
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THE FUNCTIONAL ABNORMALITY in asthma is characterized by obstruction to flow during expiration (1). The obstruction to flow may occur from increased airway reactivity and contraction of the smooth muscle cells surrounding the airways (15). In addition, airway obstruction occurs as a consequence of mucus plugs consisting of an inflammatory exudate and desquamated surface airway epithelial cells in the lumen (1). The wall of the airways is thickened by inflammatory cells such as eosinophils, lymphocytes, and mast cells as well as by mucosal edema and vasodilation (1, 15). Although these changes in the airways may be reversible, there are also more permanent changes that occur, characterized by subepithelial fibrosis due to collagens I, III, and V and fibronectin (32). In addition, airway smooth muscle cell (ASMC) hyperplasia and hypertrophy occur, and there is an increase in connective tissue proteins such as collagen, elastin, laminin, hyaluronan, versican, tenascin, and fibronectin around the smooth muscle cells (5, 6, 32).
On the basis of animal models of injury, it has been observed that
there is recruitment and activation of inflammatory cells before
fibrotic changes (7, 16, 34). Activated inflammatory cells
and structural cells are induced to release a number of proinflammatory
and fibrotic cytokines, including transforming growth factor-
(TGF-
) (7, 16, 18, 34, 39). TGF-
, a multifunctional
protein, is one of the most potent regulators of inflammation and
connective tissue synthesis (2). TGF-
exists in three
isoforms in mammals: TGF-
1, TGF-
2, and TGF-
3 (2, 18,
27). TGF-
1 appears to be the most common isoform associated
with disorders characterized by inflammation and fibrosis (2) due to the observation that at sites of injury,
TGF-
1 is released in large quantities by platelets (10)
and inflammatory cells such as macrophages (16). TGF-
1
is a chemoattractant for inflammatory cells and fibroblasts as well as
a mitogen to immature fibroblasts (7, 16). In addition,
TGF-
induces fibroblasts to synthesize a variety of extracellular
matrix proteins such as collagens, elastin, proteoglycans, and
fibronectin (25). The role of TGF-
in airway fibrosis
and remodeling is currently unclear. Minshall et al. (26)
demonstrated that TGF-
1 mRNA and immunoreactivity were increased in
the submucosal eosinophils of asthmatics but not of normal controls. In
addition, Redington et al. (29) demonstrated an increase
in the quantity of TGF-
1 in the bronchoalveolar lavage fluid from
asthmatic patients compared with normal controls and that the levels of
TGF-
were further increased in asthmatics after exposure to
allergen. Together, these observations suggest that, in asthmatic
airways, there is an increase and release of TGF-
1 that may be
important in the pathogenesis of airway fibrosis and remodeling.
We had previously demonstrated that TGF-1-3 (18,
19) and TGF-
receptors are ubiquitously present in ASMCs
(20). The intracellular presence of TGF-
would only be
relevant if the ASMCs were able to secrete TGF-
, which, once
released, could function in an autocrine or paracrine fashion. TGF-
is synthesized as a 100-kDa pro-TGF-
(27), and before
secretion, the pro region, called the latency-associated peptide (LAP),
is cleaved but remains noncovalently associated with TGF-
(27). When TGF-
is secreted in association with LAP as
latent TGF-
(L-TGF-
), it cannot interact with its receptor and is
biologically inactive (27). Because TGF-
and its
receptors are so ubiquitously expressed, the most critical regulation
of TGF-
action is the generation of a biologically active form of
TGF-
by removal of the LAP (27). Additionally, in some
instances, L-TGF-
1 is associated with the high-molecular-weight latent TGF-
binding protein-1 (LTBP-1) (35, 38). LTBP-1
bound to L-TGF-
1 targets TGF-
1 to the extracellular matrix (ECM), a process that serves as a reservoir of TGF-
1 (38).
Plasmin is a serine protease and has been shown to release L-TGF-
1
from its association with LTBP-1 (38) and has been
demonstrated to activate L-TGF-
1 by removal of the LAP
(38). In this paper, we demonstrate that subconfluent
primary cultures of ASMCs spontaneously release large quantities of
biologically active TGF-
, which is regulated by plasmin. In
addition, the TGF-
secreted by ASMCs is complexed with LTBP-1
generated by the same cells, which is also released by plasmin.
Furthermore, the plasmin-mediated release of biologically active
TGF-
can induce ASMCs to synthesize procollagen I in an autocrine
manner. These findings demonstrate that ASMCs can generate biologically
active TGF-
, which may contribute to the pathogenesis of airway
fibrosis and remodeling.
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MATERIALS AND METHODS |
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Materials.
-Modified Eagle's medium (
MEM), fetal calf serum (FCS),
insulin-transferrin-selenium, and other cell culture ingredients were
from GIBCO BRL (Burlington, Ontario, Canada).
[3H]thymidine was purchased from ICN (Irvine, CA).
Anti-collagen type I (rabbit) was from Cedarlane Laboratories (Hornby,
Ontario, Canada). Anti-
-smooth muscle actin (mouse),
anti-fibronectin (mouse),
2-antiplasmin
(
2-AP), and the plasmin substrate
N-p-tosyl-Gly-Pro-Lys p-nitroanilide
were from Sigma (St. Louis, MO). Recombinant human TGF-
3, porcine
TGF-
1 and TGF-
2, and anti-LTBP-1 (mouse) were from R&D Systems
(London, Ontario, Canada). Anti-pan-TGF-
1-3 (rabbit) was from
Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture.
Bovine trachealis muscle was obtained from a local abattoir, B. J. Packers (Beausejour, Manitoba, Canada) or J. & L. Beef (Surrey, British
Columbia, Canada), and the ASMCs were isolated as previously described
(9). Briefly, after removal of the trachealis muscle, the
tissue was minced and the slurry was suspended in a digestion buffer
consisting of Hanks' balanced salt solution (GIBCO BRL), 600 U/ml
collagenase (GIBCO BRL), 10 U/ml elastase (Sigma), and 2 U/ml protease
(Sigma). The isolated cells were suspended in 10% FCS (GIBCO BRL) to
neutralize the proteases, filtered through a 70-µm nylon mesh
(Biodesign, Carmel, NY), and washed in MEM with 10% FCS and
antibiotic-antimycotic solution (GIBCO BRL). Cell numbers were
determined using a hemocytometer, and cells were cultured in 100-mm
dishes at 250-500,000 cells/dish. Experiments were performed when
the cells were either subconfluent (60-70%) or confluent. For
confluent experiments, the cells were grown in serum-free
MEM
containing antibiotics plus 2 ng/ml insulin, 1.34 ng/ml selenium, and
1.1 ng/ml transferrin. In some experiments, confluent monolayers were
cultured in the presence of TGF-
1, -2, or -3, anti-pan-TGF-
antibody, or plasmin in the absence or presence of anti-pan-TGF-
1-3 antibody, aprotinin, or
2-AP. In experiments
requiring in vitro wounding, the confluent ASMC layers were scratched
with a rubber policeman. In experiments with in vitro wounds,
monolayers with 24 wounds were cultured in the absence and presence of
2-AP, aprotinin, or anti-pan-TGF-
antibodies. In some
experiments, the subconfluent cells were cultured in the absence and
presence of the plasmin inhibitor
2-AP or aprotinin. The
smooth muscle identity of the cells was confirmed immunohistochemically
by their expression of smooth muscle
-actin (data not shown and Ref.
9).
Collection of conditioned medium.
At various times after incubation of cells, the overlying
cell-conditioned medium (CM) was removed, stored in sterile siliconized Eppendorf tubes in the presence of the protease inhibitors (leupeptin 1 mg/ml, Amersham; aprotinin 1 mg/ml and pepstatin 1 mg/ml, both from
Sigma), and frozen at 80°C until used. In those instances where CM
was used to detect plasmin activity, the CM was collected without the
addition of protease inhibitors (17). In some instances, the cells remaining after removal of CM were used for protein extraction and Western analysis.
CCL-64 mink lung epithelial growth inhibition assay for TGF-.
The CCL-64 growth inhibitor assay that was used to quantitate TGF-
has been extensively described by us (16-19).
Briefly, subconfluent cells maintained in TGF-
-free 0.2% bovine
calf plasma,
MEM, 10 mM HEPES (pH 7.4), penicillin (25 mg/ml), and
streptomycin (25 mg/ml) and cultured at 4.5 × 104
cells/0.5 ml in 24-well Costar plates (Flow Laboratories, Mississauga, Ontario, Canada) in neutral CM or CM that was acidified and
subsequently neutralized were added. After 22 h, the cells were
pulsed with 0.2 µCi of [3H]thymidine for 3 h, at
37°C and lysed with 0.5 ml of 1 N NaOH for 30 min at room
temperature, and [3H]thymidine incorporation was
quantitated using liquid scintillation counting techniques. A standard
curve using porcine TGF-
1 was included in each assay. In those
instances where the CM demonstrated quantities of TGF-
that were in
excess of the limits of detection by the CCL-64 assay, the sample was
diluted 1:4, 1:10, etc., before the TGF-
in the sample was quantitated.
TGF- immunoassays.
Quantikine TGF-
1 and TGF-
2 immunoassay kits (R&D Systems,
Minneapolis, MN) were used to identify the presence of TGF-
1 or
TGF-
2 in acidified CM from ASMCs according to the manufacturer's instructions.
Western blotting and immune detection. Whole cell protein extracts were obtained by removing cells into 1.5-ml Eppendorf tubes and lysing cells on ice with triple-detergent lysis buffer [50 mM Tris · HCl, pH 8.0, 0.15 M NaCl, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 5 mg/ml sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride]. Protein concentration was calculated using the Bradford assay (Bio-Rad, Hercules, CA). Protein extracts were run on SDS-PAGE according to the method of Laemmli (22) at 200 V for 45 min at room temperature. Molecular size was determined by running prestained molecular markers (Amersham, Buckinghamshire, UK). Gels were transferred to nitrocelullose membrane (Bio-Rad) using CAPS transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, and 20% (vol/vol) methanol] for 1 h at 120 V at 4°C. Blots were blocked for 1 h at room temperature in Tris-buffered saline containing 0.2% Tween 20 (TBS-T) and 5% skim milk (wt/vol). Blots were incubated overnight at 4°C with primary antibody (1 µg/ml) in 1% skim milk (wt/vol) TBS-T and incubated with the appropriate secondary antibody for 1 h at room temperature [1:2,000 in 1% skim milk (wt/vol) TBS-T]. Blots were washed three times for 10 min in TBS-T, and antibody detection was carried out using the ECL system (Amersham) according to the manufacturer's instructions. Relative absorbance was determined using the Quantity I imaging system (Bio-Rad).
Immunoprecipitations.
Bovine tracheal smooth muscle pieces were microdissected from whole
tracheae and stored at 80°C before protein extraction. Pieces (~1
g) were pulverized while frozen and homogenized in triple-detergent
lysis buffer on ice. Samples were further sonicated on ice, and
500-1,000 µg were used for immunoprecipitation. Samples were
precleared by incubation with 1 µg of rabbit IgG and 10 µl of
protein A/G agarose for 30 min at 4°C with rocking. After a brief
centrifugation at 12,000 rpm, the supernatant was removed to a fresh
tube and incubated with 1-2 µg of anti-TGF-
1-3 (Santa Cruz) for 1 h at 4°C with rocking. Protein A/G agarose (20 µl) was added and incubated for 1 h at 4°C with rocking. Immune
complexes were pelleted by centrifugation for 10 min at 12,000 rpm, and the pellets were washed three times with RIPA buffer [50 mM
Tris · HCl (pH 7.5), 150 mM NaCl, 1% (vol/vol) Nonidet P-40,
0.5% (wt/vol) sodium deoxycholate, and 0.1% (wt/vol)
SDS]. Pellets were resuspended in Laemmli sample buffer (nonreducing)
and boiled before being run on SDS-PAGE.
Plasmin assay.
Plasmin was quantitated by measuring the increase in absorbance at 405 nm due to cleavage of the plasmin-specific chromogenic substrate
N-p-tosyl-Gly-Pro-Lys p-nitroanilide
(17). The analysis of all experimental and standard curve
samples was performed in 96-well flat-bottom microtiter plates (Flow
Laboratories) using a Titertek Multiskan MCC/340 spectrophotometer
(Flow Laboratories). The standard curve was made by incubating 2 mM of
N-p-tosyl-Gly-Pro-Lys p-nitroanilide
in the presence of a range of concentrations of plasmin (Sigma; from
1 × 104 to 1 × 10
2 U/ml). To
measure plasmin generated by ASMCs, 100 µl of either ASMC-derived CM
or serum-free
MEM control medium were incubated with 100 µl of the
plasmin substrate. The samples were incubated for 5 h at 37°C.
Absorbance was measured, and the background absorbance from serum-free
MEM was subtracted from all experimental and standard curve samples.
The quantity of plasmin present was then derived from the values
obtained in the standard curve and presented as units of plasmin
activity per milliliter of medium.
Statistical analysis.
Statistical analysis of TGF- in CM of subconfluent and confluent
ASMCs was performed using the Graphpad InStat software program (San
Diego, CA) or ANOVA. The statistical analysis of the relative absorbance of the Western blots was done using the Mann-Whitney test
for independent samples.
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RESULTS |
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Release of TGF- from cultures of ASMCs.
To determine whether primary cultures of ASMCs release TGF-
, the
serum-free CM from subconfluent and confluent cultures was examined
using the CCL-64 bioassay (16-19). Neutral CM from
subconfluent ASMCs, representing TGF-
in an already active
form, contained large quantities of TGF-
activity (Fig.
1A). In addition,
total TGF-
, representing the combination of TGF-
in its
biologically active and latent forms, was also present in large
quantities (Fig. 1A). Under these conditions, 61.5% of the
TGF-
released by subconfluent ASMCs was in an active form. However,
ASMC cultures that had just reached confluence for 1 day contained less
active and total TGF-
compared with subconfluent cultures (Fig.
1A). After 15 days of confluence, markedly less active
TGF-
was detectable, and the total TGF-
detected in the CM was
also decreased (Fig. 1A). After 15 days of confluence, the
percent of active TGF-
was 2.2%. With the use of an immunoassay to
identify the isoforms of TGF-
present, CM from both subconfluent and
confluent ASMCs secreted TGF-
1 almost exclusively (Fig.
1B).
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Induction of collagen synthesis by ASMCs is regulated by TGF-.
Although many connective tissue proteins may be regulated by TGF-
(2, 4) the experiments in this study were designed to
detect procollagen I as an index of connective tissue synthesis. Subconfluent ASMCs constitutively express large quantities of procollagen
1(I) and
2(I), which, in the
presence of a pan-TGF-
1-3 neutralizing antibody, was markedly
decreased (Fig. 5A). These findings demonstrate that procollagen I synthesis by ASMCs is regulated
by TGF-
in an autocrine fashion. Furthermore, confluent compared
with subconfluent (Fig. 5A) monolayers of ASMCs
constitutively express small quantities of procollagen I. However,
confluent ASMCs can be induced to synthesize increased quantities of
procollagen I in a dose-dependent manner in the presence of purified
TGF-
1, TGF-
2, and TGF-
3 (Fig. 5B). It is of note
that the representative blot selected for Fig. 5B
demonstrates that the expression of collagen I in the presence of 0.5 ng/ml of TGF-
2 is decreased. However, when the relative absorbance
using densitometry was done on all the samples, there was a significant
induction of collagen I expression in the presence of 0.5 ng/ml of
TGF-
2.
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Association of TGF- with LTBP-1.
Despite the release of large quantities of TGF-
by subconfluent
ASMCs, the CM from the same cells at confluence contained no TGF-
(Fig. 1A). TGF-
in association with its LAP can be
complexed with LTBP-1 generated by fibroblasts (38). If
this were to occur in cultures of ASMCs, then the TGF-
in CM would
be diminished but localized on ASMCs by its association to LTBP-1.
LTBP-1 was expressed equally by subconfluent and confluent ASMCs (data
not shown). However, the CM of subconfluent cells compared with
confluent cells contained large quantities of LTBP-1 (Fig.
7A). It is then possible that
the TGF-
released by subconfluent ASMCs associates with the LTBP-1
present in the CM and that the LTBP-1/L-TGF-
complex then interacts
with the ASMCs or the ECM generated by the ASMCs. It has previously
been demonstrated that plasmin can release LTBP-1 from its association
with ECM (27, 38). Because confluent ASMCs cultured in the
presence of plasmin resulted in a dramatic increase in LTBP-1
immunoreactivity in the CM, these findings confirmed that LTBP-1 that
is extracellular to ASMCs was present and could be released by the
actions of plasmin (Fig. 7B). Further confirmation that
plasmin releases LTBP-1 from ASMCs was obtained when a marked reduction
of LTBP-1 in CM was observed in the presence of aprotinin (Fig.
7B). Because plasmin also resulted in increased TGF-
activity in the CM (Fig. 3A), these findings suggest that
the plasmin-mediated release of TGF-
may be due to its actions on
LTBP-1 and L-TGF-
associated with LTBP-1. To determine whether
TGF-
1 from ASMCs is associated with LTBP-1, protein extracts from
ASMCs (data not shown) or the trachealis muscle were immunoprecipitated
with anti-TGF-
1-3 antibodies, and the presence of LTBP-1 was
detected by using Western analysis (Fig. 7C). Lane
1, which contains A/G-associated proteins, did not demonstrate any
immunoreactivity with LTBP-1 antibody (Fig. 7C). However,
the proteins obtained by immunoprecipitation with TGF-
1-3
antibodies contained detectable LTBP-1 (lane 2, Fig. 4).
These findings then demonstrate that TGF-
is associated with LTBP-1
expressed by ASMCs obtained directly from the airways.
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DISCUSSION |
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Our findings demonstrate for the first time that the serine
protease plasmin is generated by ASMCs, where it functions to release a
biologically active form of not only TGF-1 but also TGF-
2 from
ASMCs. Furthermore, once released, the presence of TGF-
1 and -
2
induces the same cells to synthesize procollagen I in an autocrine
fashion. Last, it is of interest that LTBP-1 has consistently been
demonstrated to be associated with the extracellular matrix (37,
38). However, this is the first observation demonstrating that
not only can LTBP-1 be present in solution but there may also be a
potential role for LTBP-1 in the smooth muscle layer of airways. This
is also the first observation to demonstrate that wounding of confluent
ASMCs results in plasmin-mediated release of active TGF-
1, which
then leads to an autocrine induction of collagen I synthesis.
With rare exception, the TGF- isoforms are secreted by cells in a
biologically latent form (16, 17, 27). The most important mechanism in the regulation of TGF-
activity is dependent on the
conversion of L-TGF-
to its active form (27). Several
mechanisms of activation of L-TGF-
have been described
(27), but ASMC-derived L-TGF-
is activated by plasmin.
Plasmin is cleaved from the proenzyme plasminogen by the actions of two
plasminogen activators (PA), tissue-type PA (tPA) and urokinase-type PA
(uPA), which, in turn, are controlled by plasminogen activator
inhibitors (PAIs) (24). Currently, there are no reports
demonstrating the regulation of the plasmin/plasminogen system in
ASMCs, but there are a few reports describing the role of this system
in vascular smooth muscle cells (VSMCs). Although the mechanism is not
known, in models of VSMC injury, there is an increase in the expression
of tPA, uPA, and PAI-1, leading to pericellular activation of plasmin
(30). In this study, the expression of all the members of
the plasminogen/plasmin system in ASMCs has not been described, but it
has been demonstrated that plasmin activity is present in CM from
subconfluent cultures of ASMCs. The presence of plasmin in these
conditions is associated with large quantities of active TGF-
1.
Because aprotinin, an inhibitor of plasmin activity (24),
totally abrogates the generation of active TGF-
without affecting
the total TGF-
secreted by the same cells, these findings confirm
the importance of plasmin-mediated posttranslational activation of
L-TGF-
from ASMCs. The presence of plasmin when ASMCs are
subconfluent results in the release of primarily the TGF-
1 isoform.
However, when ASMCs are confluent, the addition of plasmin results in
the release of both TGF-
1 and TGF-
2. Previously, the actions of
plasmin have been described as being restricted to the release of
TGF-
1 (37, 38). The current findings suggest that in
addition to the release of TGF-
1, plasmin may be important in the
release of biologically active TGF-
2 that may be associated with the
ASMCs extracellularly. Demonstrating that plasmin is critical to the
activation of L-TGF-
1 from ASMC is complementary to the observation
that plasmin activates L-TGF-
1 from alveolar macrophages (17,
21), endothelial cells (27), and VSMCs (8,
23, 30). Collectively, these observations support plasmin as an
important physiological substance in the activation of L-TGF-
1 by
several different phenotypes of cells. Because the in vitro model is
composed of a single cell monolayer of ASMCs, the source of plasmin
must be the cells themselves. However, in vivo, additional sources of
plasmin could be inflammatory cells such as macrophages, mast cells,
eosinophils, neutrophils, and lymphocytes (5, 15, 37) that
are present in the walls of asthmatic airways. These inflammatory cells
generate proteases such as plasmin when they are activated in a
nonspecific manner or are stimulated to facilitate migration into
tissue (11, 36). It is then conceivable that at times of inflammation
in the airways, an increase in plasmin activity occurs and may lead to
the release of TGF-
.
Confluent monolayers of ASMCs in vitro may resemble some
characteristics of ASMCs in situ (12, 13). When ASMCs are
confluent, they do not release TGF-. In vitro wounding of confluent
monolayers or conditions of subconfluence could be considered analogous
to in vivo injury to the smooth muscle layer of the airways (12, 13). When the ASMC monolayers are disrupted, there is a release of TGF-
, plasmin, and procollagen I synthesis. In this context the
presence of plasmin releases both TGF-
1 and -
2, which, in turn,
induce collagen I synthesis. In vivo, an injury to the ASMCs could
occur during airway inflammation that may be due to a myriad of
inflammatory mediators such as proteases, oxygen radicals, and
cytokines (34). Injury in a localized region could
resemble the culture conditions of subconfluent cells or wounded
monolayers. If this were to occur in vivo, the release of biologically
active TGF-
in these localized areas could result in ASMC connective tissue synthesis. Such a cycle of injury and inflammation may be
repeated many times in patients with asthma (15, 34),
which could contribute to the remodeling of airways described earlier (6, 13, 15, 32).
At subconfluence, there are large amounts of active and latent TGF-
present in the CM. Yet the CM from the same cells when allowed to
become confluent contains markedly decreased quantities of active and
latent TGF-
. TGF-
is easily degraded and can become adherent to
plastic (31), which may account for loss of some activity
in the CM. However, it is also possible that the TGF-
1 released by
subconfluent ASMCs may be preserved. It is of interest that
plasmin-mediated release of active TGF-
results in the removal of
LAP from L-TGF-
(3), but the LAP can reassociate with
the active TGF-
, resulting in L-TGF-
(3). L-TGF-
1
can complex with the LTBP-1 (37, 38), which is present in
abundant quantities in the CM of subconfluent ASMCs. The association of
L-TGF-
with LTBP-1 could then interact with the ECM generated by the
ASMCs (35, 37). Confluent ASMCs do not secrete TGF-
,
but the addition of plasmin results in increased TGF-
activity and
LTBP-1 in the CM. TGF-
complexed with LTBP-1 is susceptible to
release by plasmin (35, 37), suggesting that the addition
of plasmin to confluent ASMCs releases the LTBP-1 from its interactions
with the ECM and TGF-
from its associations with LTBP-1. These
findings are highly significant because they suggest that, if TGF-
is released by ASMCs at some point, the same TGF-
complexed with
LTBP-1 can later be present on ASMCs or the ECM generated by ASMCs.
TGF- is one of the most potent inducers of connective tissue
synthesis (2, 40). Because connective tissue proteins, including collagens and fibronectin, have been shown to be increased in
the airways of asthmatics (6, 32), it was of interest to
observe that subconfluent monolayers and monolayers with wounds expressed increased quantities of collagen regulated by TGF-
in an
autocrine manner. Furthermore, confluent ASMCs that synthesize small
quantities of collagen can respond to TGF-
1-3 by induction of
collagen I synthesis. This would suggest that, in the event of increase
in TGF-
1, -
2, or -
3 levels in the airways, induction of
connective tissue synthesis is likely to occur. Physiologically, a
localized increase of TGF-
in the airways could occur by the actions
of plasmin or ASMC injury or at times of inflammation in the airways of
asthmatics when the sources of TGF-
1 could be eosinophils,
macrophages, and lymphocytes (15, 26, 29, 34). Although
TGF-
3 was detected in ASMCs by immunohistochemistry (18,
19), the release of TGF-
3 could not be adequately identified by an assay used previously to detect and characterize TGF-
isoforms (18, 21). It is possible that TGF-
3 is not released by
ASMCs under the conditions described, or, alternatively, TGF-
3 is
released but in quantities that cannot be detected by the current assay.
In conclusion, our findings demonstrate that for in vitro ASMCs that
resemble conditions of injury, there is plasmin-mediated release of
TGF-, which in an autocrine fashion results in collagen synthesis. These findings suggest that the TGF-
expressed by ASMCs may be important in the pathogenesis of airway connective tissue
synthesis and remodeling.
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ACKNOWLEDGEMENTS |
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We thank Carolin Hoette for helping prepare the manuscript and Dr. Kevin Craib for the statistical analysis.
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FOOTNOTES |
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*
Amanda Coutts and Gang Chen contributed equally to this
work.
Address for reprint requests and other correspondence: N. Khalil, Division of Respiratory Medicine, Univ. of British Columbia,
655 West 12th Ave., Vancouver, BC, Canada V5Z 4R4 (E-mail: nasreen.khalil{at}bccdc.hnet.bc.ca).
Funding for this work was provided by Glaxo Wellcome. A. Coutts was supported in part by a Manitoba Lung Association Fellowship. S. Hirst is the recipient of Wellcome Trust Research Career Development Fellowship 051435 and was a visiting assistant professor in the Department of Physiology, University of Manitoba, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 January 2000; accepted in final form 8 December 2000.
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