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Regular Articles |
From the Centre for Cardiopulmonary Biochemistry and Respiratory
Medicine*
and the Haematology
Department,
Haemostasis Research Unit, Royal
Free and University College London Medical School, The Rayne Institute,
London, United Kingdom
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1(I) procollagen and CTGF mRNA levels (3.0 ±
0.4-fold and 6.3 ± 0.4-fold respectively,
(P < 0.01), and total inflammatory cell
number. UK-156406, administered at an anticoagulant
dose, attenuated lung collagen accumulation in response to
bleomycin by 35 ± 12% (P < 0.05),
inhibited 1(I) procollagen and CTGF mRNA levels by 50%
and 35%, respectively (P < 0.05),
but had no effect on inflammatory cell recruitment. This is the first
report showing that direct thrombin inhibition abrogates lung collagen
accumulation in bleomycin-induced pulmonary fibrosis.
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Introduction |
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A number of reports have examined the effects of modulating the coagulation cascade in ALI. For example, exogenous delivery of the highly specific direct thrombin inhibitor hirudin and AT III, have been shown to be protective in animal models of ALI.15-17 In addition, heparin, which inhibits serine proteases by potentiating the formation of AT-III/serine protease complexes, but also has anti-inflammatory properties, lead to improved gas exchange in an animal model of ALI.18 Finally, heparin has also been shown to attenuate bleomycin-induced pulmonary fibrosis in mice,19 although it was uncertain in this study whether heparin was delivered at an anticoagulant dose and whether the protective effects were because of the its direct anti-proliferative effects.
Thrombin plays a central role in blood coagulation by converting
soluble plasma fibrinogen into an insoluble fibrin clot and promoting
platelet aggregation and degranulation, but also mediates a number of
biological responses that may play important roles in subsequent
inflammatory and tissue repair responses. Thrombin activates
endothelial cells,20
acts as a chemoattractant for
inflammatory cells21-23
and fibroblasts,24
and stimulates fibroblast proliferation24-26
and
procollagen production.27
Most of the cellular effects of
thrombin are mediated by a unique family of ubiquitously expressed
cell-surface receptors called protease-activated receptors (PARs),
which are activated by limited proteolysis rather than direct ligand
binding.28
To date, four PARs have been characterized, of
which three, (PAR-1, -3, and -4), are activated by thrombin, although
PAR-1 has been shown to be the major receptor involved in mediating
thrombin’s mitogenic, profibrotic, and proinflammatory effects
in vitro.27,29,30
After the interaction of
thrombin with its receptors, most of its cellular effects are thought
to be mediated via the induction and release of a host of secondary
mediators.31
For example, there is good evidence that the
mitogenic effects of thrombin for lung fibroblasts in vitro
are mediated, at least in part via the autocrine production of PDGF-AA
and up-regulation of the PDGF- receptor.10
More
recently, we have shown that thrombin is also a potent inducer of
connective tissue growth factor (CTGF) production by human lung
fibroblasts via direct proteolytic activation of PAR-1.32
CTGF is a fibroblast mitogen, chemoattractant, and promoter of
procollagen and fibronectin production in
vitro.33
It has therefore been proposed that some of
the profibrotic effects of thrombin in vitro may also be
mediated, at least in part, by increased production of this growth
factor. CTGF is also induced in response to transforming growth
factor-ß1 and is thought to be responsible for
mediating some of its downstream fibrogenic effects.34
Further evidence that CTGF may be important in tissue repair and
fibrosis has been provided by studies showing that repeated
subcutaneous injection of CTGF into newborn mice leads to increased
connective tissue deposition,33
and that CTGF expression
is dramatically increased in a number of fibrotic and
fibroproliferative disorders, including pulmonary
fibrosis35
and in animal models of this
disease.36
Despite the evidence that thrombin may play an important role in the
pathogenesis of pulmonary fibrosis, there have been no previous reports
that have specifically addressed whether direct thrombin inhibition
affects collagen accumulation in this disorder. The aim of this study
was therefore to examine the effect of a potent and highly selective
direct thrombin inhibitor, UK-156406 (Pfizer Central Research,
Sandwich, Kent, UK) on thrombin-induced fibroblast responses
in vitro and in bleomycin-induced pulmonary fibrosis in
rats. Our data show that UK-156406 blocked the profibrotic effects of
thrombin in vitro when used at equimolar concentration to
the protease and attenuated lung collagen accumulation after
bleomycin-induced lung injury. We further show that the protective
effect of direct thrombin inhibition on lung collagen accumulation in
this model was preceded by significant reductions in both
1(I) procollagen and CTGF mRNA levels, but not
by changes in inflammatory cell recruitment. This is, to our knowledge,
the first report to show that direct thrombin inhibition is associated
with attenuation of both 1(I) procollagen
and CTGF mRNA levels, and ultimately an abrogation in lung collagen
accumulation in an animal model of pulmonary fibrosis.
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Materials and Methods |
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Purified human -thrombin (catalog no.T4393) was obtained from
Sigma Chemical Co. Ltd., (Poole, Dorset, UK). The direct thrombin
inhibitor, UK-156406 was a generous gift from Dr. Andrew Gray (Pfizer
Central Research, Sandwich, Kent, UK). The cDNA probe for human CTGF
was inserted into the EcoRI and NotI sites of
pBluescript and kindly provided by Dr. Raj Beri (AstraZeneca R&D
Charnwood, Loughborough, UK). The cDNA probe for FISP12 (the murine
orthologue of CTGF), encompassing nucleotides 1663 to 2930 was
generated from a plasmid (pBluescriptfisp12del) kindly provided by Dr.
Joseph A. Lasky (Tulane University, New Orleans, LA). The
pBluescriptfisp12del plasmid was subcloned from a plasmid (A12/pMexNeo
I) originally obtained from Dr. Rolf-Peter Ryseck (Bristol-Myers Squibb
Pharmaceutical Research Institute, Princeton, NJ). The cDNA probe for
1(I) procollagen (probe Hf677) was kindly
provided by Dr. M. L. Chu (Thomas Jefferson University,
Philadelphia, PA). Rat-specific PAR-1 antibodies were generated by
immunizing rabbits with the agonist sequence
SFFLRNPSENTFELVPL-NH2 and purified by affinity
chromatography37
and were a generous gift from Professor
Eleanor Mackie (University of Melbourne, Melbourne, Australia).
Fibroblast Culture
Human fetal lung fibroblasts (HFL-1; American Type Culture
Collection, Rockville, MD), were maintained in Dulbecco’s modified
Eagle’s medium (DMEM), supplemented with penicillin, streptomycin, and
10% (v/v) newborn calf serum (NCS), (Imperial Laboratories, Andover,
Hampshire, UK). Cells were routinely passaged, tested for mycoplasma
infection, and used for experiments between passages l5 to 20. In all
in vitro experiments, UK-156406 was preincubated with human
-thrombin (10 nmol/L to 1 µmol/L) in serum-free DMEM for 60
minutes at 37°C. To examine the effects of thrombin alone, the
protease was similarly preincubated in serum-free DMEM under the same
conditions.
Animal Model of Pulmonary Fibrosis
Male Lewis rats, aged 6 weeks and weighing 140 to 170 g, were
anesthetized by intramuscular injection of 0.75 to 1.0 ml/kg Hypnorm
(fentanyl citrate, 0.315 mg/ml, and fluanisone, 10 mg/ml; Janssen
Pharmaceutical, High Wycombe, UK). Bleomycin sulfate (Lundbeck, Luton,
UK) was administered by a single intratracheal injection (l.5 mg/kg
body weight in 0.3 ml of sterile saline) as described
previously.38
Control animals received 0.3 ml of saline
alone. In initial experiments, groups of six rats were killed by
pentobarbitone overdose after 6 days to allow assessment of thrombin
levels in BALF, as described previously.39
Separate groups
of two rats were sacrificed 1, 3, 6, and 14 days after bleomycin
instillation for immunohistochemical assessment of thrombin and PAR-1.
Lungs were fixed by intratracheal instillation of 4% paraformaldehyde,
the trachea ligated, and the inflated lungs and heart removed en
bloc. Tissues were fixed and transferred to 15% sucrose in
phosphate-buffered saline (PBS), before alcohol dehydration and
embedding in paraffin wax. For assessment of the effect of UK-156406 on
lung collagen accumulation after bleomycin-induced lung injury at 14
days, UK-156406 (0.5 mg/kg/hour in 0.9% sterile saline) was
administered continuously via osmotic minipumps (Alzet, Palo Alto, CA)
implanted subcutaneously, 24 hours before bleomycin instillation, to
groups of six animals. Drug control animals received minipumps
containing saline alone. An additional series of animals was killed 6
days after bleomycin or saline instillation for measurement of blood
coagulation parameters, total and differential cell counts in BALF, and
for Northern analysis of lung tissue CTGF and
1(I) procollagen mRNA levels. For
measurement of coagulation parameters, blood was collected from the
inferior vena cava of animals after laparotomy, and was immediately
mixed, 10:1 with a solution of 3.8% trisodium citrate (w/v). For
measurement of total lung collagen and CTGF and procollagen mRNA
levels, the vasculature was perfused with 5 ml of sterile saline
containing 100 U/ml heparin. The lungs were removed, weighed, and
immediately snap-frozen in liquid N2 after
removing the trachea and major airways.
Fibroblast Proliferation Assay
Fibroblast proliferation was assessed using a colorimetric assay based on the uptake and subsequent elution of the dye methylene blue as previously described.40 Briefly, cells were seeded at 5 x 103 cells/well into 96-well plates (Nunc, Life Technologies, Paisley, Scotland, UK) in DMEM and 5% NCS. Control medium, thrombin, or thrombin plus UK-156406 (10 nmol/L to 1 µmol/L) were added to cell cultures for 48 hours. Cells were rinsed with PBS, fixed with formol-saline, and stained with a solution of methylene blue for 30 minutes. Plates were rinsed with borate buffer, bound dye was eluted from the cell monolayer by addition of acidified alcohol, and the absorbance was measured at 650 nmol/L using a microplate spectrophotometer. In some experiments, changes in fibroblast cell number were confirmed by direct cell counting with a standard hemocytometer (British Drug House/Merck, UK).
Determination of Fibroblast Procollagen Production in Vitro and Total Lung Collagen in Vivo
Fibroblast procollagen production in vitro and total lung collagen in vivo were assessed by quantitating hydroxyproline by reverse-phase HPLC as previously described.38,41,42 Briefly, for in vitro studies, cells were seeded at 105 cells/ml in 2.4-cm diameter wells in DMEM and 5% NCS, grown to visual confluence and exposed to either control medium, thrombin, or thrombin plus with UK-156406 (10 nmol/L to 1 µmol/L) for 48 hours. At the end of the incubation period, proteins in the media and cell layer were ethanol precipitated and separated from free amino acids by filtration (0.45 µmol/L). Filters were hydrolyzed in HCl and hydrolysates prepared for quantitation of hydroxyproline by HPLC analysis (Beckman System Gold, Beckman, High Wycombe, UK), after derivatization with 7-chloro-4-nitrobenzofuran (Sigma) as previously described.42 For measurement of total lung collagen, powdered lung tissue was weighed and was similarly hydrolyzed in HCl and prepared for HPLC analysis after diluting hydrolysate aliquots (1:100). The total amount of collagen in each lung was calculated, assuming that lung collagen contains 12.2% w/w hydroxyproline43 and results were expressed as total lung collagen (mg).
Northern Analysis of CTGF and 1(I) Procollagen
mRNA Levels
For Northern analysis of CTGF mRNA levels in vitro,
fibroblasts were seeded at 2 x 105
cells/ml
in 6-cm diameter dishes in DMEM and 5% NCS as described
previously.32
Briefly, on reaching visual confluence,
cells were quiesced for 24 hours and exposed to control media,
thrombin, or thrombin plus with UK-156406, (10 nmol/L to 100 nmol/L) in
serum-free conditions. After 90 minutes, total RNA was isolated with
TRIzol reagent according to the manufacturer’s instructions (Gibco
BRL, Paisley, UK). For Northern analysis of CTGF and
1(I) procollagen mRNA levels in
vivo, total RNA was isolated with TRIzol reagent, from powdered
lung tissue, kept frozen. Total RNA from fibroblast cultures (5 µg),
and lung tissue (10 µg) were mixed with RNA loading buffer (Sigma),
and electrophoresed on a formaldehyde 1% (w/v) agarose gel. RNA
loading and integrity was visualized and quantitated by fluorescent
scanning of the gel (Fuji, FLA 3000) before transfer to nylon membranes
(Hybond N; Amersham International, High Wycombe, UK) and fixation by UV
crosslinking. Membranes were hybridized overnight in Denhardt’s-based
standard hybridization solution at 65°C in the presence of the
[32P]-dCTP-labeled cDNA probes for human CTGF
(fibroblast cultures), or 1(I) procollagen
and FISP12 (lung tissue). At the end of the hybridization, membranes
probed for CTGF and FISP12 were rinsed at low stringency [2x standard
saline citrate (SSC), 0.1% sodium dodecyl sulfate (SDS), for 5 minutes
at room temperature], once at medium stringency (0.5x SSC, 0.1% SDS,
for 25 minutes at 65°C) and once at high stringency (0.1x SSC, 0.1%
SDS, for 5 minutes at 65°C). Membranes probed for
1(I) procollagen were rinsed at low
stringency (2x SSC, 0.1% SDS, 5 minutes at room temperature, followed
by 20 minutes at 65°C) and high stringency (0.1x SSC, 0.1% SDS for
20 minutes at 65°C). All membranes were exposed to phosphorimage
storage screens (Fuji) for 2 to 4 hours and mRNA levels were
quantitated by phosphorimage analysis (Fuji FLA 3000).
Estimation of Active Thrombin Levels in Rat Bronchoalveolar Lavage
BALF was centrifuged at 2000 x g for 10 minutes at 4°C and thrombin levels in the supernatant were estimated using a previously described spectrophotometric assay.6 Briefly a 100-µl aliquot of the supernatant was mixed with 50 µl of 0.05 mol/L Tris, 0.1 mol/L NaCl (pH 7.35) buffer solution and 50 µl of the chromphore S2238 (1 mmol/L; H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochloride; Chromogenix, Quadratech, Surrey, UK) at 37°C. Absorbance was read on a spectrophotometer at 405 nmol/L at regular intervals up to 60 minutes and BALF thrombin levels were derived by extrapolation from a thrombin standard curve.
Immunohistochemical Localization of Thrombin and PAR-1
Lung tissue sections (5 µm) were cut and mounted on glass slides before dewaxing in xylene and rehydration in ethanol according to standard histological procedures. Tissue endogenous peroxidase activity was blocked by incubating sections with 3% hydrogen peroxide (Sigma) before washing in PBS and incubation with normal goat serum (DAKO, High Wycombe, UK) (1:40 dilution). Active thrombin in lung sections was localized as previously described.44 Briefly, sections were incubated with recombinant hirudin (1:50 dilution, Sigma), followed by a purified rabbit anti-hirudin antibody (1:1200 dilution; American Diagnostica, Greenwich, CT) for 1 hour each at 37°C. For immunohistochemical localization of PAR-1, sections were incubated with rabbit anti-rat PAR-1 primary antibodies37 for 1 hour at room temperature. All sections were washed in PBS and incubated with a biotinylated goat anti-rabbit secondary antibody (1:300 dilution, DAKO) for 60 minutes and similarly washed in PBS. Sections were incubated with a streptavidin/peroxidase complex (1:300 dilution, DAKO) for a further 60 minutes, followed by a solution of 600 µg/ml of 3,3'-diaminobenzidine (Sigma) and 0.03% hydrogen peroxide. Sections were washed, counterstained with hematoxylin, dehydrated, and mounted with DPX mountant (Merck, Poole, UK). Control sections were incubated with normal goat serum or an isotype-specific, nonimmune rabbit IgG primary antibody (DAKO) instead of primary antibodies.
Assessment of Blood Clotting Parameters in Rat Plasma
Plasma was prepared by centrifugation of citrated blood samples at 2000 x g for 15 minutes. The plasma mean activated partial thromboplastin time (APTT) and the mean prothrombin time (PT) were assessed using Actin FS and Thromboplastin IS (Dade Behring, Marburg, Germany), respectively. Parameters were measured in duplicate using a mechanical KC-4A coagulometer (Amelung, Lemgo, Germany).
BALF Total Cell Number and Differential Cell Counts
BALF total cell number was assessed as previously described.39 Briefly, BALF cells were pelleted by centrifugation at 300 x g for 10 minutes at 4°C. Cell pellets were re-suspended in DMEM (1 ml) and cells were counted with a standard hemocytometer. Cytospins were prepared by centrifuging 100-µl aliquots of the cell suspension for 3 minutes at 450 rpm (Cytospin 2; Shandon, Southern Products Ltd., Cheshire, UK). Slides were air-dried and stained with Diffquik Stain (Dade Behring, Dûdingen, Switzerland) and differential cell counts were performed by two investigators by counting a minimum of 500 cells per slide. Cells were identified as macrophages/monocytes, neutrophils, or lymphocytes and data were expressed as a percentage of the total number of lavageable cells.
Statistical Analysis
All data are presented as means ± SEM for six replicates, unless otherwise indicated. Statistical evaluation was performed using an unpaired Student’s t-test for single, and analysis of variance for multiple-group comparisons. A P value <0.05 was considered significant.
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To confirm the previous observation that BALF thrombin levels are
elevated after bleomycin-induced lung injury, we assessed these levels
in our model at a time point where they would be expected to be maximal
(6 days). We found that BALF thrombin levels were indeed significantly
increased from 0.30 ± 0.02 ng/ml in saline control animals to
2.97 ± 0.68 ng/ml in animals given bleomycin
(n = 6, P < 0.01). We also
performed experiments to determine the immunohistochemical localization
of active thrombin and the major thrombin receptor, PAR-1, within the
lung after bleomycin-induced lung injury at 1, 3, 6, and 14 days
(Figure 1)
. In saline-instilled rats, the
appearance of the lung under light microscopy appeared normal with only
weak positive staining for active thrombin that was associated with
resident macrophages (Figure 1, a and b)
. In contrast, in
bleomycin-instilled rats, there was evidence of a diffuse
inflammatory-cell influx at days 1 and 3, which was replaced by
interstitial fibrosis with multiple inflammatory foci by day 6, and
eventually mature regional fibrotic areas by day 14.38
In
the lungs of these animals there was intense and widespread staining
for active thrombin, which was predominately associated with
macrophages within inflammatory and fibroproliferative foci (Figure 1, c and d)
, and also fibroblast-like interstitial cells (Figure 1d
,
arrows). These changes were evident throughout the time course but
appeared maximal 6 days after bleomycin instillation. PAR-1 was
consistently expressed on the bronchial epithelium of lung sections
from both bleomycin- and saline-treated animals. However, in contrast
to the parenchyma that was negative in saline-treated animals (Figure 1, e and f)
, there was a dramatic increase in the expression of PAR-1,
which again was associated with macrophages in inflammatory and
fibroproliferative foci and was also maximal on day 6, in
bleomycin-treated animals (Figure 1, g and h)
.
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Before performing in vivo experiments with UK-156406,
we assessed the efficacy of this compound to block fibroblast responses
to thrombin in vitro. Figure 2
shows a representative experiment for the effect of UK-156406 on human
fetal lung fibroblast (HFL-1) proliferation (Figure 2a)
and procollagen
production (Figure 2b)
in response to an optimal concentration of
thrombin (10 nmol/L). Thrombin stimulated fibroblast proliferation and
procollagen production, assessed after 48 hours, by 72 ± 5% and
121 ± 6%, respectively, relative to media control cells (both
P < 0.01). These fibroblast responses to thrombin were
inhibited by UK-156406 in a dose-dependent manner from equimolar
concentrations onwards (P < 0.05), and were
completely blocked when UK-156406 was added in 10-fold excess
(P < 0.01). In addition, UK-156406 had no
effect on basal fibroblast proliferation or fibroblast procollagen
production at all concentrations examined up to 1 µmol/L.
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, respectively. The
characteristic increase in thrombin-stimulated CTGF mRNA levels at 90
minutes was decreased by 65% for fibroblasts exposed to thrombin in
the presence of equimolar concentrations of UK-156406
(P < 0.05), and by 88% when UK-156406 was
added in 10-fold excess (P < 0.01). In
addition, at these concentrations, UK-156406 had no effect on basal
CTGF mRNA levels. Effect of UK-156406 on Blood Clotting Parameters in Vivo
To ensure that UK-156406 was administered to animals at an
anticoagulant dose, the compound was delivered by continuous
subcutaneous infusion via an osmotic minipump and two ex
vivo assays of thrombin-dependent coagulation, the mean APTT and
PT, were measured when serum concentration of the drug would have
reached steady-state levels, based on available pharmacokinetic data
from Pfizer. Figure 3
shows that the mean
PT of UK-156406-treated animals (Figure 3a)
was prolonged from
24.5 ± 1.1 seconds to 31.4 ± 1.6 seconds
(P < 0.01), whereas the mean APTT (Figure 3b)
was increased from 17.0 ± 0.8 seconds to 24.5 ± 1.1 seconds
(P < 0.01) compared to animals receiving drug
vehicle alone. In addition, intratracheal bleomycin alone had no effect
on these clotting parameters and the prolongation of APTT and PT in
animals given UK-156406 was the same in all animals, whether they had
previously received intratracheal bleomycin or saline (data not shown).
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Figure 4
shows the effect of
UK-156406 on total lung collagen accumulation in response to bleomycin,
assessed by quantitating hydroxyproline in lung hydrolysates at 14
days. In bleomycin-instilled animals, total lung collagen was increased
by 124% compared with animals given intratracheal saline
(P < 0.01, n = 6). This
increase in lung collagen accumulation was reduced by 35%
(P < 0.05, n = 6) in
bleomycin-treated rats given UK-156406 compared with animals given
bleomycin and drug vehicle alone. In addition, UK-156406 had no effect
on basal lung collagen levels in animals given intratracheal saline.
The results shown are representative of three separate experiments
performed, in which statistically significant differences in total lung
collagen between drug-treated animals and those given bleomycin and
drug vehicle alone were always obtained.
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1(I)
Procollagen mRNA Levels in Vivo
To begin to examine the mechanism by which direct thrombin
inhibition attenuates bleomycin-induced pulmonary fibrosis, we assessed
whether the changes in lung collagen at the protein level were preceded
by a reduction in rat CTGF and 1(I)
procollagen mRNA levels. Figure 5
shows
the data expressed as fold increases in CTGF and
1(I) procollagen mRNA levels greater than
control levels assessed on day 6, for six animals per group, (Figure 5a)
, as well as representative Northern blots. In lung tissue from
bleomycin-treated animals, CTGF and 1(I)
procollagen mRNA levels were increased 6.3 ± 0.4-fold and
3.0 ± 0.4-fold, respectively (P < 0.01).
These mRNA levels were reduced by 35% for CTGF and by 50% for
1(I) procollagen in bleomycin-treated animals
given UK-156406, compared to bleomycin-treated animals given drug
vehicle alone (n = 6, P <
0.05). Finally, UK-156406 had no effect on CTGF and
1(I) procollagen mRNA levels in saline-treated
animals.
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Thrombin is known to exert potent effects on inflammatory cell
migration in vitro.21-23
To assess whether
direct thrombin inhibition affected inflammatory cell recruitment in
this model, we examined the effect of UK-156406 on total
inflammatory cell number and the relative proportions of inflammatory
cells in BALF, 6 days after bleomycin instillation (Table 1)
. As expected, total inflammatory cell
number in BALF was dramatically increased in bleomycin-treated animals
compared with saline controls (P < 0.01) and
there was a characteristic significant increase in the proportion of
both neutrophils and lymphocytes and a corresponding reduction in the
proportion of macrophages, compared to saline-treated controls
(P < 0.01). However, UK-156406 had no effect on
both total cell number and the relative proportion of inflammatory
cells in bleomycin or in saline-treated control animals.
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1(I) procollagen and CTGF mRNA levels, but not
inflammatory cell recruitment. Active Thrombin and PAR-1 Are Increased in Bleomycin-Induced Pulmonary Fibrosis
Thrombin levels in BALF had previously been reported to be increased in bleomycin-treated animals compared to saline controls,11,12 but the localization of thrombin in the lung after bleomycin injury had not been described. In this study we confirmed that BALF thrombin levels were elevated in bleomycin-treated animals. The specificity of this assay for thrombin was further confirmed by showing that thrombin activity in BALF could be abolished in the presence of the highly selective thrombin inhibitor, hirudin (data not shown). Immunohistochemical studies performed to localize thrombin in lung after bleomycin instillation revealed that thrombin was predominantly localized to macrophages in inflammatory and fibroproliferative foci and also fibroblast-like interstitial cells, and that immunoreactivity of thrombin was maximal on day 6. These findings are in accord with previous reports of peak thrombin levels11,12 and procoagulant activity associated with alveolar macrophages in BALF of bleomycin-treated rats.45
There are a number of potential mechanisms that may lead to increased expression of thrombin on macrophages after bleomycin injury. For example, the active protease may leak into the alveolar space from the vascular compartment as a result of chronic activation of the coagulation cascade, after the extensive and continued endothelial injury caused by bleomycin. However, macrophages have been shown to express both tissue factor, the primary cell-surface initiator of the extrinsic coagulation cascade in vitro46 as well as membrane assembly sites for generation of the prothrombinase complex,47 responsible for activating the intrinsic coagulation cascade. Macrophages are therefore thought to be able to accelerate membrane-dependent coagulation reactions and facilitate the local extravascular generation of thrombin, independent of classical blood coagulation. Indirect support that these effects may be important mechanisms occurring in the human lung has come from studies showing that thrombin is present on the surface of normal human alveolar macrophages44 and that expression of tissue factor on these cells is dramatically increased in patients with pulmonary fibrosis.8
As well as assessing the immunohistochemical localization of thrombin, we also examined expression of the major cellular receptor for thrombin, PAR-1. Although PAR-1 expression had never been described in the lung, PAR-1 mRNA had previously been detected in cultured alveolar macrophages.48 Furthermore, specific agonists of this receptor have been shown to cause bronchoconstrictor responses in experimental animals,49 suggesting that the lung does indeed express functional PAR-1 receptors. Our studies showed that PAR-1 was expressed on the bronchial epithelium in the lungs of all experimental animals, with no evidence of altered expression after bleomycin instillation. However, expression of PAR-1 in the lung parenchyma was dramatically different in bleomycin-treated animals compared to controls, where similar to thrombin, positive staining was predominately associated with macrophages in inflammatory and fibroproliferative foci and was also maximal 6 days after bleomycin instillation.
UK-156406 Blocks the Profibrotic Effects of Thrombin in Vitro
Before examining the effects of direct thrombin inhibition in the bleomycin model of pulmonary fibrosis, and as thrombin has a very high affinity for its receptor,50 we first determined whether UK-156406, could block thrombin-induced fibroblast responses in vitro. UK-156406 is a hydrophilic, small peptide-based reversible inhibitor of thrombin proteolytic activity. It is very potent with a dissociation constant (Ki) of 0.39 nmol/L and displays good selectivity over other serine proteases, including factor Xa, plasmin, and factor VIIa (data provided by Pfizer, patent no. WO9513274). We showed that UK-156406 inhibited thrombin-induced fibroblast proliferation, procollagen production, and CTGF mRNA levels in a dose-dependent manner from equimolar concentrations of the protease onwards. These effects were comparable to those obtained with hirudin in previous reports,27,32 so that this compound was deemed suitable for further evaluation in vivo.
Steady-State Levels of UK-156406 Prolong Clotting Parameters in Rat Plasma
UK-156406 has an in vivo half-life of 
1 hour.
Because thrombin is generated chronically after bleomycin-induced lung
injury, we decided to deliver the compound continuously via osmotic
minipumps, inserted into rats subcutaneously. Initial pilot studies
revealed that at higher doses (up to 1.5 mg/kg/hour), UK-156406 caused
local subcutaneous bleeding around the pump insertion site. In
addition, two ex vivo assays of thrombin-dependent
coagulation, the APTT and PT became unrecordably high (>180 seconds)
at this dose (data not shown). In final experiments, a dose of 0.5
mg/kg/hour proved optimal, as it caused significant prolongation of the
APTT and PT, without noticeable hemostatic complications. In addition,
at this dose, subcutaneous delivery of UK-156406 was as effective at
prolonging the APTT and PT as previously reported in in vivo
studies, in which hirudin was administered as a continuous intravenous
infusion,15,16
or twice daily by subcutaneous
injection.30
We further calculated that this dose of
UK-156406 would generate a plasma steady-state concentration
approaching 1 µmol/L and that the concentration of thrombin detected
in BALF after bleomycin instillation would be in the range of 50 to 100
nmol/L. Although this concentration is greater than that of thrombin
used in the in vitro experiments of this study (10 nmol/L),
fibroblast responses to thrombin in vitro are exerted over a
range of concentrations between 10 pmol/L to 200
nmol/L,27,32
with a plateau concentration for
thrombin-induced fibroblast proliferation and procollagen production of
10 nmol/L.27
In addition, the concentration of BALF
thrombin after bleomycin instillation is similar to the concentration
of thrombin detected in BALF from patients with pulmonary
fibrosis9
and also in blood clotting.51
Because microvascular damage after bleomycin injury is widespread and
associated with extensive vascular leak,52
and given the
small molecular weight of UK-156406 (611.7) it is theoretically
at least feasible that the concentration of UK-156406 within the
pulmonary interstitium may approach that of plasma, and therefore be
sufficiently high to inhibit thrombin-mediated responses in the lung.
UK-156406 Reduces Lung Collagen Accumulation in Bleomycin-Induced Pulmonary Fibrosis
Evaluation of the effect of UK-156406 on bleomycin-induced lung injury showed that direct thrombin inhibition attenuated lung collagen accumulation in this model. Given thrombin’s pluripotent effects, there are a number of potential mechanisms by which direct thrombin inhibition may have afforded protection after bleomycin instillation, including blocking thrombin’s procoagulant effects (ie, fibrin deposition). Intra-alveolar accumulation of fibrin has been extensively documented in bleomycin-induced pulmonary fibrosis and in human studies of fibrotic lung disease,2-4,53 and fibrin is thought to influence the fibrotic response by acting as a provisional matrix on which fibroblasts can proliferate and produce collagen in combination with fibronectin.54 There is also evidence that fibrin can act as a reservoir of fibrogenic growth factors and cytokines that are released during fibrinolysis.55 Fibrin has further been shown to protect active thrombin from inhibition from its physiological inhibitors so that it can remain available to exert its biological effects when bound to the provisional matrix.56 Support for a role of fibrin in bleomycin-induced pulmonary fibrosis has come from studies using genetically modified mice that either overexpress or are completely deficient in plasminogen-activator inhibitor-1 (PAI-1), an endogenous inhibitor of fibrinolysis. In mice overexpressing PAI-1 (favoring fibrin persistence), collagen deposition was increased after bleomycin instillation, whereas in PAI-1-deficient mice (favoring fibrin clearance), collagen levels were similar to wild-type controls.57 However, the role of fibrin in this model remains controversial as fibrinogen knockout mice are not protected from developing pulmonary fibrosis in response to bleomycin.58,59
Modulation of thrombin’s numerous cellular effects may provide another important mechanism by which UK-156406 may have reduced lung collagen accumulation in this model, particularly in view of the fact that we have been able to demonstrate extensive expression of PAR-1 in the lung after bleomycin injury. PAR-1 has been reported to be expressed by a number of different cell types in vitro, including fibroblasts,31 but in this study, PAR-1 seemed to be predominantly localized to both bronchial epithelial cells as well as macrophages in inflammatory and fibroproliferative foci. Although we were unable to demonstrate specific staining for PAR-1 on fibroblasts in the rat lung in vivo using conventional light microscopy, this may not be surprising given the elongated morphology of the interstitial fibroblast within the pulmonary parenchyma. However, we have able to show that primary rat lung fibroblasts isolated from male Lewis rats express PAR-1 at both the mRNA and protein level at the earliest passage number and that these cells further exhibit normal fibroblast responses to PAR-1 agonists and thrombin in vitro (data not shown).
In support of the hypothesis that UK-156046 may have exerted its
protective effects in this model by directly interfering with
thrombin-mediated fibroblast responses, we show that
thrombin-induced fibroblast proliferation, procollagen, and CTGF mRNA
levels were almost completely abolished in in vitro studies,
when the inhibitor was used at concentrations that are likely to be
attained at the dose used in this study in vivo. We also
show that the previously reported increases in both CTGF and
1(I) procollagen mRNA levels in response to
bleomycin-induced lung injury36,60
were dramatically
reduced in animals given UK-156406. This is, to our knowledge, the
first report that a reduction in CTGF mRNA levels correlates with
reduced 1(I) procollagen mRNA levels and
ultimately lung collagen accumulation in an animal model of tissue
fibrosis.
There is increasing in vitro evidence that thrombin exerts its profibrotic effects via the up-regulation of secondary mediators, including PDGF.10 We recently reported that thrombin also induces fibroblast CTGF production,32 but the role of CTGF in mediating the profibrotic effects of thrombin, as well as the contribution of CTGF to lung collagen accumulation in this model remain unclear. Although our in vivo experiments do not test causality directly, the coordinate down-regulation of both CTGF and procollagen gene expression by direct thrombin inhibition makes it tempting to speculate that the effects of thrombin on lung collagen deposition, may be mediated, at least in part, via a CTGF-dependent mechanism.
A final mechanism by which direct thrombin inhibition may influence the fibrotic response in this model may involve modulation of thrombin’s effects on inflammatory cell recruitment and activation. Thrombin is a potent chemoattractant for inflammatory cells61 and stimulates the release of a number of proinflammatory cytokines, including monocyte chemotactic factor-1, interleukin-6, and interleukin-8,62,63 predominantly via PAR-1-dependent mechanisms. Thrombin further influences inflammatory cell trafficking by inducing the expression of cell surface adhesion molecules, such as P-selectin and intercellular adhesion molecule-1.64 Our results showed that inflammatory cell profiles in BALF after bleomycin instillation were unaffected by UK-156406, suggesting that inflammatory cell recruitment was not affected by direct thrombin inhibition. However, as thrombin is also known to stimulate the production of a number of fibrogenic cytokines, including PDGF from alveolar macrophages in vitro,65 preventing thrombin-mediated PAR-1 activation of resident and recruited inflammatory cells may be another important mechanism by which direct thrombin inhibition may have attenuated lung collagen accumulation in this model. Future studies using PAR-1 knockout mice will be critical in further elucidating the contribution of the cellular versus the procoagulant effects of thrombin in promoting lung collagen deposition after bleomycin instillation. However, the recent finding that fibrinogen-null mice are not protected from bleomycin-induced lung injury58,59 combined with our data, point to the possibility that the cellular effects of thrombin may play a key role in the fibrotic response after bleomycin injury.
Therapeutic Implications of This Study
Activation of the coagulation cascade is a feature of a number of lung disorders associated with inflammation and excessive deposition of extracellular matrix proteins including idiopathic pulmonary fibrosis,2-4 pulmonary fibrosis associated with systemic sclerosis,9,10 ALI/acute respiratory distress syndrome5 and more recently, airway remodeling in asthma.66 Our findings suggest that modulation of the coagulation cascade, and more specifically the profibrotic effects of coagulation proteases, may therefore warrant further evaluation as potential therapeutic strategies for treatment of these disorders. PAR-1 antagonists, blocking antibodies, and antisense oligonucleotides that are currently being developed as potential anti-thrombotic agents67,68 may in the future provide an opportunity for selectively interfering with the profibrotic effects of thrombin, while avoiding potential hemostatic complications associated with direct proteolytic inhibition of coagulation proteases.
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Acknowledgements |
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Footnotes |
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Supported by the Medical Research Council, U.K (Clinical Training Fellowship to D.C.J.H), The Wellcome Trust (program grant 051154), and The Middlesex Hospital Special Trustees.
D. C. J. H. and N. R. G. contributed equally to this work.
Accepted for publication July 5, 2001.
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