Several growth factors, including platelet-derived growth
factor (PDGF), have been implicated in the mechanism of lung and airway
remodeling. In the present study, we evaluated whether thrombin may
promote lung and airway remodeling by increasing PDGF production from
lung and airway epithelial cells. Conditioned medium (CM) was prepared
by treating epithelial cells with increasing concentrations of
thrombin; before use in the assays, CM was treated with hirudin until
complete inhibition of thrombin activity. CM from epithelial cells
stimulated the proliferation of lung fibroblasts and bronchial smooth
muscle cells. Anti-PDGF antibody significantly inhibited this CM
proliferative activity, implicating PDGF in this effect. Enzyme
immunoassay and RT-PCR demonstrated that thrombin induced the secretion
and expression of PDGF from bronchial and alveolar epithelial cells.
RT-PCR showed that epithelial cells express the thrombin receptors
protease-activated receptor (PAR)-1, PAR-3, and PAR-4. The PAR-1
agonist peptide was also found to induce PDGF secretion from epithelial
cells, suggesting that the cellular effect of thrombin occurs via a
PAR-1-mediated mechanism. Overall, this study showed for the first time
that thrombin may play an important role in the process of lung and
airway remodeling by stimulating the expression of PDGF via its
cellular receptor, PAR-1.
coagulation; lung fibrosis; bronchial asthma; platelet-derived
growth factor receptor
 |
INTRODUCTION |
STRUCTURAL REMODELING
of the lung tissue is believed to be the consequence of repeated cycles
of epithelial injury and repair process associated with chronic
pulmonary disease (3). Remodeling of the alveolar
compartment of the lung, such as that occurring in pulmonary fibrosis,
is characterized by interstitial and intra-alveolar deposition of
connective tissue and collapse and distortion of air spaces by scar
contraction (5). Tissue remodeling is also a severe
complication of airway disorders, particularly of bronchial asthma. It
is believed that airway remodeling leading to thickening of the
bronchial wall is responsible for the underlying irreversible airway
hyperreactivity observed in patients with chronic bronchial asthma
(23). The morphological changes of airway remodeling include epithelial sloughing, thickening of the epithelial basement membrane, edema, inflammatory cell infiltration of the submucosa, hyperplasia of mucus glands, accumulation of myofibroblasts, and thickening of medial smooth muscle layers (2,
7, 8). Several growth factors such as
platelet-derived growth factor (PDGF), basic fibroblast growth factor
(bFGF), transforming growth factor-
, epidermal growth factor (EGF),
and insulin-like growth factor, secreted by inflammatory or resident
structural cells, have been implicated in the mechanism of the
structural changes associated with lung and airway remodeling
(15, 16).
Thrombin is a serine protease that, besides its critical role in
thrombosis and hemostasis, has also been described as inducing cellular
and molecular events relevant to tissue remodeling. For example,
thrombin may induce platelet aggregation and platelet release of
thromboxane A2, serotonin, procoagulant, and growth factors. Thrombin may also stimulate chemotaxis of monocytes and lymphocytes, the motility and proliferation of fibroblasts and smooth
muscle cells, and the secretion of growth factors and proteases from
the vascular endothelium (9, 11,
22). These observations together with previous studies
(10, 18, 19) showing increased generation of thrombin in pulmonary diseases suggest that thrombin may
also play an important role in the pathogenesis of lung and airway
remodeling. We hypothesized that thrombin may also indirectly promote
lung and airway remodeling by increasing the production of growth
factors from alveolar and bronchial epithelial cells. To demonstrate
this possibility, in the present study, we evaluated the effect of
thrombin on the secretion of PDGF from alveolar and bronchial
epithelial cells and the effect of conditioned medium (CM) of these
cells stimulated with thrombin on the proliferation of lung fibroblasts
and bronchial smooth muscle cells. It is known that the cellular
effects of thrombin depend on stimulation of the protease-activated
receptor (PAR)-1, PAR-3, or PAR-4 (6). In the present
study, we also evaluated the expression of these receptors in alveolar
and bronchial epithelial cells.
 |
MATERIALS AND METHODS |
Reagents.
Dulbecco's modified Eagle's medium (DMEM), L-glutamine,
vitamin solution, sodium pyruvate, nonessential amino acids,
transferrin, bovine pituitary extract, TRIzol reagent, and Superscript
preamplification system were purchased from GIBCO BRL (Life
Technologies, Grand Island, NY). Fetal bovine serum (FBS) was from
BioWhittaker (Walkersville, MD); and penicillin, streptomycin, and
dexamethasone were from Nacalai Tesque (Kyoto, Japan). BSA, Ham's F-12
medium, epinephrine, gentamicin, amphotericin B, insulin,
triiodothyronine, cholera toxin, endothelial growth supplement,
L-leucine, L-lysine, L-methionine, HEPES, and nonspecific protease from Streptomyces griseus
were from Sigma (St. Louis, MO). Human EGF was from Higeta Shouyu
(Tokyo, Japan). Hydrocortisone and retinoic acid were from Wako (Osaka, Japan). bFGF, cDNA probes of PDGF-A and PDGF-B, goat anti-human PDGF
antibody, and Quantikine enzyme immunoassay kits for EGF, bFGF, and
PDGF-AB were from R&D Systems (Minneapolis, MN). All other chemicals
and reagents used were of the best quality commercially available.
Cell culture.
The human lung fibroblast cell line WI-38 was obtained from RIKEN Cell
Bank (Ibaraki, Japan). The human lung carcinoma-derived alveolar
epithelial cell line A549 and the human bronchial epithelial cell line
BEAS-2B were obtained from the American Type Culture Collection
(Manassas, VA). Normal human bronchial epithelial (NHBE) cells and
human bronchial smooth muscle cells were purchased from Clonetics
(Walkersville, MD) as primary culture cells. Both lung fibroblasts and
A549 cells were cultured in DMEM containing 10% heat-inactivated FBS,
50 µg/ml of penicillin, 50 µg/ml of streptomycin, 2 mM
L-glutamine, 2% vitamin solution, 110 µg/ml of sodium
pyruvate, and 0.1 mM nonessential amino acids. NHBE cells were cultured in CCMD161 medium (Clonetics) supplemented with 30 µg/ml of bovine pituitary extract, 0.5 µg/ml of BSA, 0.5 µg/ml of epinephrine, 50 µg/ml of gentamicin, 50 ng/ml of amphotericin B, 0.5 ng/ml of human
EGF, 0.5 µg/ml of hydrocortisone, 5 µg/ml of insulin, 7 ng/ml of
triiodothyronine, 10 µg/ml of transferrin, and 0.1 ng/ml of retinoic
acid. BEAS-2B cells were cultured in serum-free Ham's F-12 medium
supplemented with 5 µg/ml of insulin, 5 µg/ml of transferrin, 20 ng/ml of human EGF, 0.1 µM dexamethasone, 20 ng/ml of cholera toxin,
30 µg/ml of bovine pituitary extract, and 1 µM retinoic acid. Nasal
epithelial cells were isolated from nasal polyps of patients with
chronic sinusitis according to the method previously described
(27). Briefly, stroma from polyploid lesions was removed,
and the epithelial cells were dissociated by incubation with 0.1%
nonspecific protease from Streptomyces griseus in Ham's
F-12 medium at 4°C for 20 h. The cell suspension was then
filtered through a 60-µm Nitex mesh, centrifuged at 500 g
for 10 min, washed, and resuspended in DMEM-Ham's F-12 medium containing 0.5 mM L-leucine, 0.5 mM L-lysine,
0.1 mM L-methionine, 0.3 mM MgCl2, 0.4 mM
MgSO4, 1 mM CaCl2, 9 mg/l of phenol red, 14 mM
NaHCO3, 7 mM L-glutamine, 50 µg/ml of
penicillin, 50 µg/ml of streptomycin, 10 µg/ml of insulin, 0.1 µg/ml of hydrocortisone, 0.1 µg/ml of cholera toxin, 5 µg/ml of
transferrin, 25 ng/ml of human EGF, 8 µg/ml of endothelial growth
supplement, 50 µg/ml of bovine pituitary extract, and 30 mM HEPES.
The human bronchial smooth muscle cells were positive for smooth muscle
cell
-actin but negative for von Willebrand factor. The cells were
cultured with a culture kit (Clonetics) containing a modified MCDB131
medium supplemented with 5% FBS, 50 µg/ml of gentamicin, 50 ng/ml of amphotericin B, 0.5 ng/ml of human EGF, 5 µg/ml of insulin, and 2 ng/ml of human FGF. All cells were cultured in 10-cm dishes in an
atmosphere composed of 5% CO2 and 95% air. Confluent
cells were harvested by a brief exposure to 0.025% trypsin-0.02% EDTA in HEPES-buffered saline (50 mM HEPES and 150 mM NaCl, pH 7.4) and
passaged after 5~7 days.
Preparation of CM.
A549, NHBE, and BEAS-2B cells were grown to 80~90% confluence in
10-cm dishes, and human nasal epithelial cells were grown in 12-well
tissue culture plates. After incubation for 24 h in basal medium
without supplements, the cells were washed and incubated for 48 h
in the presence of varying concentrations of thrombin. The CM was
harvested, centrifuged at 1,200 g for 15 min, and stored at
80°C until used. To evaluate the participation of the thrombin receptor (PAR-1), after preincubation in FBS- and supplement-free medium for 24 h, NHBE cells were treated with the PAR-1 agonist peptide (SFLLRNP) or its negative control (YFLLRNP) for 48 h, and
then the CM was collected and used in the assays.
Proliferation assay.
CM was used in the proliferation assays after it was treated with
hirudin (250 antithrombin units) at 37°C for 30 min and after
confirmation of the absence of thrombin activity in CM with the
chromogenic substrate S2238 for thrombin (Chromogenix, Mölndal, Sweden) by amidolytic assay. The proliferative effect of CM on fibroblasts and bronchial smooth muscle cells was assessed by a
colorimetric method as previously described (21). Briefly, after the cells were cultured in 96-well plates up to 70~80% of confluence, their growth was arrested by culturing overnight in medium
without FBS and growth factors. After the cells were washed, 100 µl
of hirudin-treated CM were applied to each well and incubated for
48 h under an atmosphere of 5% CO2 and 95% air.
After being appropriately washed, the cells were fixed by treatment
with 10% Formalin for 30 min; the wells were then washed, and 1%
(wt/vol) methylene blue in 0.01 M borate buffer was added. After the
wells were washed with borate buffer, a 1:1 dilution of ethanol in 0.1 M HCl was added, and the absorbance at 650 nm was measured with a
microplate reader. This method has been previously validated with a
number of characterized growth factors (21). In addition, proliferative assays were also carried out with a cell-counting kit
purchased from Dojindo (Kumamoto, Japan); this kit contains a
tetrazolium compound, WST-1, a sodium salt of
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate, which is used for the colorimetric determination of cell number.
In separate experiments to evaluate whether the proliferative effect of
CM depends on the presence of PDGF, the CM prepared from cells
stimulated with the same concentration of thrombin (250 nM) was treated
with varying concentrations of anti-human PDGF antibody at 37°C for
30 min. The anti-PDGF antibody-treated CM was then used in
proliferation assays performed as described above.
Enzyme immunoassay.
Enzyme immunoassays were carried out to assess the concentration of
growth factors in CM. Commercial immunoassay kits (described in
Reagents) were used to measure the concentrations of
PDGF-AB, bFGF, and EGF.
RNA isolation and RT-PCR.
RT-PCR was carried out to evaluate the expression of thrombin receptors
(PAR-1, PAR-3, and PAR-4) and PDGF receptors (
and
) in alveolar
and airway epithelial cells, lung fibroblasts, and bronchial smooth
muscle cells. Total RNA was extracted from confluent cells by the
guanidine isothiocyanate procedure with the TRIzol Reagent. Two
micrograms of total RNA were reverse transcribed with oligo(dT) primer
and the Superscript preamplification system kit following the
manufacturer's instructions. The primers used in the PCR are listed in
Table 1. The PCR mixture contained PCR buffer (100 mM Tris·HCl, pH 8.3, and 200 mM KCl), 25 mM
MgCl2, 10 µM deoxyribonucleosides (dATP, dGTP, dCTP, and
dTTP), 10 µM each forward and reverse primer, an appropriate amount
of diethyl pyrocarbonate-treated distilled water, and 1 U of
Taq polymerase (Boehringer Mannheim, Mannheim, Germany). The
mixture was then divided into aliquots in PCR tubes containing 2 µl
of cDNA sample, and the amplification was performed with a PC-800
Programmable Temp control system cycler (Astec, Fukuoka, Japan). The
PCR products were then electrophoresed on a 2% agarose gel and stained
with 0.5 µg/ml of ethidium bromide. PCR of samples treated without RT
was also carried out to ensure the absence of contaminating DNA in
total RNA prepared from epithelial cells.
Southern hybridization.
Southern hybridization was carried out to assess the effect of thrombin
on gene expression of PDGF in airway epithelial cells. Preparation of
total RNA and PCR products was performed as described in RNA
isolation and RT-PCR. After the PCR products were resolved on a 2% agarose gel, the gel was incubated in a solution containing 1.5 M NaCl and 500 mM NaOH for 30 min at room temperature and then
incubated again in a solution containing 1 M ammonium acetate and 20 mM
NaOH at room temperature for 30 min. The cDNA was transferred for
24 h by capillary action onto a Hybond-N+ membrane (Amersham Pharmacia Biotech, Tokyo, Japan) and fixed to the membrane by ultraviolet irradiation with a Stratalinker (Stratagene, La Jolla, CA).
The membrane was then prehybridized in a solution containing 20 mM
Tris·HCl, pH 7.5, 750 mM NaCl, 2.5 mM EDTA, 1% SDS, 5×
Denhardt's solution, 0.5 µg/ml of denatured salmon sperm DNA, and
50% formamide and then hybridized with cDNA probes of human PDGF-A,
PDGF-B, or human glyceraldehyde-3-phosphate dehydrogenase (Cayman
Chemical) that were labeled with [
-32P]dCTP with a DNA
labeling kit (Takara Shuzo, Kyoto, Japan).
Northern hybridization.
Northern hybridization was carried out with a digoxigenin Northern
starter kit (Boehringer Mannheim). Ten micrograms of total RNA were
electrophoretically separated on 1.2% agarose gels and transferred to
positively charged Boehringer Mannheim nylon membranes by capillary
elution. The membranes were hybridized with digoxigenin-11-UTP-labeled RNA probes. Immunologic detection was then performed following the
manufacturer's instructions.
Statistical analysis.
All data are expressed as means ± SE unless otherwise specified.
The difference between three or more variables was calculated by
analysis of variance. Statistical analyses were carried out with the
StatView 4.1 package software for the Macintosh (Abacus Concepts,
Berkeley, CA).
 |
RESULTS |
Effect of CM on proliferation of lung fibroblasts and bronchial
smooth muscle cells.
To evaluate the role of thrombin in the secretion of PDGF from
bronchial and alveolar epithelial cells, the proliferative activity of
CM from these cells stimulated with varying concentrations of thrombin
on lung fibroblasts and bronchial smooth muscle cells was assessed
after complete inhibition of residual thrombin activity in the CM with
hirudin. In the methylene blue-based proliferation assays, CM from the
primary bronchial epithelial cells (NHBE cells) significantly
stimulated the proliferation of fibroblasts and bronchial smooth muscle
cells in a dose-dependent manner (Fig. 1A). Similarly, CM
from the alveolar epithelial cell line A549 significantly stimulated
the proliferation of fibroblasts and bronchial smooth muscle cells in a
dose-dependent fashion (Fig. 1B). These findings were in
good agreement with the results obtained with the cell-counting kit
containing the tetrazolium salt WST-1. For preparation of the control
sample, aliquots of culture medium were incubated in the presence of
the same concentrations of thrombin but in the absence of epithelial
cells; after treatment with hirudin, the control medium was also used
in the proliferative assays. These control media did not show
proliferative activity on lung fibroblasts (data not shown), further
ensuring that hirudin completely inhibited residual thrombin activity
in our assays.

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Fig. 1.
Effects of conditioned medium (CM) from normal human
bronchial epithelial (NHBE; A) and alveolar epithelial
(A549; B) cells treated with indicated concentrations of
thrombin for 48 h on proliferation of lung fibroblasts (open bars)
and bronchial smooth muscle cells (solid bars). Cell supernatant was
used in this assay as CM. Residual thrombin activity in CM was
inhibited by hirudin before the assay. Lung fibroblasts and bronchial
smooth muscle cells were incubated in each CM for 48 h. CM from
NHBE and A549 cells stimulated the proliferation of fibroblasts and
bronchial smooth muscle cells. Values are means ± SE, with 100%
being the no. of cells that proliferated in the presence of CM from
epithelial cells prepared with 0 nM thrombin. Assays were done in
triplicate in 5 separate experiments. * P < 0.05. ** P < 0.01. # P < 0.05. ## P < 0.03 [all compared with
control samples (0 nM thrombin)].
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Effect of anti-PDGF antibody on proliferative activity of CM from
airway epithelial cells.
To assess whether the proliferative activity of CM depends on the
secretion of PDGF from epithelial (NHBE) cells, the inhibitory effect
of anti-PDGF antibody that recognizes PDGF-AA, PDGF-BB, and PDGF-AB was
evaluated. Compared with control samples (0 µg/ml of anti-PDGF
antibody), the anti-PDGF antibody significantly inhibited the
proliferative activity of CM prepared with NHBE cells stimulated with
250 nM thrombin in a dose-dependent manner (Fig.
2). This inhibition of
fibroblast proliferation was significant above 10 µg/ml of the
anti-PDGF antibody.

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Fig. 2.
Effect of anti-platelet-derived growth factor (PDGF)
antibody on proliferative activity of CM. CM prepared from NHBE cells
stimulated with the same concentration of thrombin (250 nM) was treated
with indicated concentrations of anti-human PDGF antibody at 37°C for
30 min before the assay. Fibroblasts were incubated in each medium for
48 h. Values are means ± SE expressed as percentage over
basal medium that was taken as 100% of fibroblast proliferation.
Assays were done in triplicate in 5 separate experiments.
* P < 0.05. ** P < 0.005 [both
compared with control sample (0 µg/ml of anti-PDGF antibody)].
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Secretion of PDGF and other growth factors by epithelial cells
treated with thrombin.
To assess whether thrombin stimulation increases the secretion of PDGF
and other growth factors from epithelial cells, the concentration of
these growth factors in CM was measured. As shown in Fig.
3, thrombin significantly and
specifically stimulated the secretion of PDGF-AB but not that of bFGF
or EGF from NHBE cells. The increase in the concentration of PDGF-AB in
CM stimulated with thrombin was significant from a thrombin
concentration of 62.5 nM for BEAS-2B cells (Fig.
4A); in A549 cells
(Fig. 4B), thrombin stimulated the secretion of PDGF-AB at a
thrombin concentration of 62.5 nM and reached a plateau with 125 nM
thrombin. Thrombin also significantly increased the concentration of
PDGF-AB in CM prepared with primary cells from bronchial or nasal
epithelium; thrombin increased PDGF-AB concentration from a
concentration of 62.5 nM for NHBE cells (Fig.
5A) and 125 nM for
nasal epithelial cells (Fig. 5B). The secretion of PDGF-AB
from thrombin-treated BEAS-2B cells was also time dependent (Fig.
6).

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Fig. 3.
Concentrations of epidermal growth factor (EGF), basic
fibroblast growth factor (bFGF), and PDGF-AB in CM from NHBE cells
treated with 125 nM thrombin. The cells were grown in 12-well tissue
culture plates. They were incubated in the presence (125 nM; solid
bars; +) and absence (open bars; ) of thrombin for 48 h, and
then the concentration of each growth factor in CM was determined with
commercial enzyme immunoassay kits.
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Fig. 4.
Effect of various concentrations of thrombin on PDGF-AB
secretion from established epithelial cell lines. BEAS-2B
(A) and A549 (B) cells were grown in
10-cm-diameter dishes. The cells were incubated with indicated
concentrations of thrombin for 48 h, and then the concentration of
PDGF-AB in CM was measured. Values are means ± SE. Assays were
done in triplicate in 2 separate experiments. * P < 0.01. ** P < 0.001. # P < 0.0001 [all compared with
control samples (0 nM thrombin)].
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Fig. 5.
Effect of indicated concentrations of thrombin on PDGF
secretion from primary bronchial (NHBE; A) and nasal
epithelial (B) cells. NHBE cells were grown in
10-cm-diameter dishes, and human nasal epithelial cells were grown in
12-well tissue culture plates. The cells were incubated with for
48 h, and then the concentration of PDGF-AB in CM was determined.
Values are means ± SE. Assays were done in triplicate in 2 separate experiments. * P < 0.001;
** P < 0.001. # P < 0.005 [both compared with control samples (0 nM thrombin)].
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Fig. 6.
Time course of PDGF-AB secretion from thrombin-treated
epithelial cells (BEAS-2B). BEAS-2B cells were grown in 12-well tissue
culture plates and incubated in the presence (solid bars) and absence
(open bars) of thrombin (125 nM) for indicated times, and then the
concentration of PDGF-AB in CM was measured. Values are means ± SE. Assays were done in triplicate in 2 separate experiments.
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Effect of thrombin on PDGF gene expression in epithelial cells.
The effect of thrombin on the gene expression of PDGF-A and PDGF-B in
NHBE cells was evaluated by RT-PCR and Southern blot analysis. As shown
in Fig. 7, thrombin significantly
increased the expression of the PDGF-B gene but not that of the PDGF-A
gene in NHBE cells. Similarly, Northern blot analysis also showed that thrombin stimulates the expression of the PDGF-B gene at a
transcriptional level (data not shown).

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Fig. 7.
Effect of thrombin on gene expression of PDGF in
epithelial (NHBE) cells. Total RNA was prepared with NHBE cells treated
with and without thrombin (125 nM) for 48 h, and RT-PCR and
Southern hybridization (top) were performed as described in
MATERIALS AND METHODS. The intensities of the bands were
densitometrically quantified (bottom), and the results were
normalized to the internal standard band glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Thrombin increased the expression of PDGF-B gene
but not that of PDGF-A gene in NHBE cells.
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Effect of PAR-1 agonist peptide on PDGF secretion in epithelial
cells.
To evaluate the participation of the thrombin receptor on PDGF
secretion induced by thrombin, the effect of the PAR-1 agonist peptide
on the concentration of PDGF in CM from NHBE cells was assessed. Like
thrombin, the PAR-1 agonist peptide also induced PDGF-AB secretion from
NHBE cells in a dose-dependent manner (Fig. 8). This effect was
significant from a peptide concentration of 25 µM. The negative
control peptide did not induce PDGF-AB secretion (data not shown).
These results suggest that thrombin stimulation of PDGF-AB secretion
from airway epithelial cells is mediated by PAR-1.

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Fig. 8.
Concentration of PDGF-AB in CM from protease-activated
receptor (PAR)-1 agonist peptide-treated epithelial cells. NHBE cells
were grown in 12-well tissue culture plates and incubated with
indicated concentrations of PAR-1 agonist peptide for 48 h, and
then the concentration of PDGF-AB in CM was determined. Values are
means ± SE. Assays were done in triplicate in 2 separate
experiments. Control peptide did not affect PDGF secretion (data
not shown). * P < 0.05 compared with control
sample (0 µM PAR-1 agonist).
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Expression of PARs in epithelial cells.
To evaluate the type of thrombin receptors expressed by epithelial
cells, mRNAs of PAR-1, PAR-3, and PAR-4 were analyzed by RT-PCR. RT-PCR
showed that both alveolar (A549) and airway (NHBE) epithelial cells
expressed not only PAR-1 but also PAR-3 and PAR-4; however, the
bronchial epithelial cell line BEAS-2B did not express PAR-3 (Fig.
9).

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Fig. 9.
RT-PCR analysis of PAR-1, PAR-3, and PAR-4 in epithelial
cells. Total RNA was prepared with cells incubated in basal medium
without supplements for 24 h, and RT-PCR was performed as
described in MATERIALS AND METHODS. Both alveolar (A549)
and bronchial (NHBE) epithelial cells expressed not only PAR-1 (592 bp)
but also PAR-3 (513 bp) and PAR-4 (542 bp) thrombin receptors, but
BEAS-2B cells did not express PAR-3.
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Expression of PDGF receptors in epithelial cells, lung fibroblasts,
and bronchial smooth muscle cells.
Because it is known that the effect of PDGF depends on the presence of
its receptors,
and
, on target cells, the expression of PDGF
receptors was evaluated. RT-PCR showed that both PDGF receptors (
and
) are expressed by epithelial cells, lung fibroblasts, and
bronchial smooth muscle cells (Fig.
10).

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Fig. 10.
RT-PCR of PDGF receptors in NHBE cells, lung
fibroblasts, and bronchial smooth muscle cells (BSMC). Total RNA was
prepared with the respective cells incubated in basal medium without
supplements for 24 h, and RT-PCR was performed as described in
MATERIALS AND METHODS. Both PDGF- (501 bp) and PDGF-
(216 bp) receptors were expressed by NHBE cells, lung fibroblasts, and
BSMC.
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 |
DISCUSSION |
This study showed that CM from alveolar and bronchial epithelial
cells treated with thrombin significantly stimulates the proliferation
of lung fibroblasts and bronchial smooth muscle cells compared with
that in the medium of untreated cells. Immunoassays and inhibition
experiments revealed that the increased growth of these cells depends
on the enhanced production and secretion of PDGF-AB induced by thrombin
in epithelial cells.
It is well known that increased thrombin generation occurs in the lungs
of most patients with inflammatory diseases of the pulmonary parenchyma
and airways and that it plays a critical role in tissue remodeling
(10, 18, 19). After injury, a tissue factor-activated factor VII complex is formed on lung and airway
epithelial and/or endothelial cells, leading to activation of the
extrinsic pathway of coagulation, thrombin generation, fibrin
formation, and fibrosis (4, 12,
14). In the lung and airways, generated thrombin may
modulate the tissue repair response by altering vascular permeability,
by stimulating the secretion of proteases and the production or
degradation of components of the connective tissue, and by promoting
adhesion, spreading, and proliferation of fibroblasts and smooth muscle
cells (1). In addition to its direct effects, thrombin may
also play a role in lung tissue remodeling by stimulating the secretion
of growth factors from several cells. For example, it has been reported that thrombin may induce increased expression of PDGF from lung fibroblasts, vascular smooth muscle cells, endothelial cells, and
alveolar macrophages (13, 19,
20, 22, 26). In the present study, we hypothesized that thrombin may also participate in
tissue remodeling by stimulating the secretion of growth factors from
alveolar and bronchial epithelial cells.
The CM prepared by treating alveolar and bronchial epithelial cells
with different concentrations of thrombin was found to stimulate the
proliferation of lung fibroblasts and bronchial smooth muscle cells.
This CM effect did not depend on the direct proliferative effect of
thrombin because its activity in CM was blocked completely by hirudin
before use in the proliferative assays. Measurement of growth factors
in CM showed that thrombin upregulated the secretion of PDGF-AB from
bronchial epithelial cells in dose- and time-dependent manners; RT-PCR
analysis showed that this increased secretion of PDGF-AB depends on
increased mRNA expression of PDGF-B. It is worth noting that
upregulation of PDGF-AB was induced by thrombin not only in transformed
epithelial cell lines but also in primary bronchial and nasal
epithelial cells, suggesting that this effect is probably also observed
in vivo at sites of inflammation. Inhibition assays carried out with anti-PDGF antibody confirmed that PDGF was responsible for the proliferative activity of CM from epithelial cells. Overall, these findings suggest that stimulation of PDGF secretion from epithelial cells may constitute another mechanism of thrombin participation in
lung and airway remodeling. The biological relevance of PDGF in the
mechanism of tissue remodeling is illustrated by the important role it
plays in the pathogenesis of fibroproliferative processes such as wound
healing, pulmonary fibrosis, atherosclerosis, myelofibrosis, scleroderma, and inflammatory joint diseases. Properties that implicate
PDGF in the mechanism of tissue remodeling are its strong mitogenic and
chemotactic activities on fibroblasts and smooth muscle cells and its
ability to stimulate angiogenesis and the secretion of cytokines (e.g.,
transforming growth factor-
) that, in turn, may upregulate the
expression of extracellular matrix components (17,
24, 25). A previous study (24)
has shown that the PDGF-B chain is the polypeptide that is mainly
expressed in lung tissues from patients with lung fibrosis
(24); the demonstration in the present study that thrombin
upregulates the expression of PDGF-B but not that of PDGF-A reinforces
the important role that thrombin plays in the process of lung fibrosis.
The cellular effects of thrombin have been found to depend on
stimulation of the thrombin receptors PAR-1, PAR-3, and PAR-4 (6). After cellular activation by thrombin, a new
NH2-terminal peptide, which functions as a tethered ligand
for the receptor itself, is unmasked on PAR-1, PAR-3, or PAR-4. The
tethered ligand of PAR-1 has been shown to promote proliferation of
fibroblasts and vascular smooth muscle cells, cytokine secretion, and
deposition of collagen in the extracellular matrix. To evaluate whether
PAR-1 also mediates the effect of thrombin in primary airway epithelial cells (NHBE cells), the effect of the PAR-1 agonist peptide on PDGF-AB
secretion was assessed. The PAR-1 agonist peptide induced PDGF-AB
secretion from NHBE cells in a similar fashion as thrombin. RT-PCR
analysis showed that besides the PAR-1 gene, PAR-3 and PAR-4 genes are
also expressed by bronchial (NHBE and BEAS-2B) and alveolar (A549)
epithelial cells. These findings suggest that PARs may play an
important role in the mechanism of lung and airway remodeling by
mediating the mitogenic and secretory activity of thrombin in
epithelial cells.
PDGF is known to occur in the form of three dimers (PDGF-AA, PDGF-BB,
and PDGF-AB) and to bind to specific high-affinity receptor subunits
(
, 
, and 
) on the surface of responsive cells
(fibroblasts and smooth muscle cells). The effect of PDGF depends on
the type of PDGF receptor subunit available on the cell surface.
PDGF-BB can bind to any one of these receptor subunits; PDGF-AA can
bind only to an 
-receptor; and PDGF-AB can bind either to an

-receptor or to an 
-receptor (24). In the
present study, RT-PCR analysis showed that both the
- and
-subunits were equally expressed by lung fibroblasts and bronchial
smooth muscle cells. These findings explain the proliferative activity
of PDGF-rich CM on fibroblasts and bronchial smooth muscle cells
observed in the present study. In addition, airway epithelial cells
express both subunit receptors, suggesting that they may also play an
important role in epithelial regeneration in the lung and airways.
In conclusion, the results of this study showed for the first time that
1) thrombin regulates PDGF expression and secretion from
nasal, bronchial, and alveolar epithelial cells; 2) the
cellular effect of thrombin on PDGF secretion by epithelial cells is
mediated by its receptor PAR-1; 3) airway epithelial cells
express the thrombin receptors PAR-1, PAR-3, and PAR-4; and
4) the thrombin-induced secretion of PDGF is sufficient for
stimulating the proliferation of fibroblast and bronchial smooth muscle
cells. Overall, these findings suggest that thrombin may play an
important role in the process of lung and airway remodeling by
stimulating the secretion of PDGF via its PAR-1 receptor from alveolar
and bronchial epithelial cells.
Address for reprint requests and other correspondence: E. C. Gabazza, Third Dept. of Internal Medicine, Mie Univ. School of Medicine, Edobashi 2-174, Tsu-city, Mie 514-8507, Japan (E-mail: gabazza{at}clin.medic.mie-u.ac.jp).
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