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Introduction
Pulmonary fibrosis (PF) is a progressive and often fatal
human disease characterized by irreversible fibrosis
resulting from excessive repair of damaged
alveoli[1]. While early studies have suggested that fibrosis occurs in the
interstitium of the lungs, more recent studies have suggested that
the initial injury in alveolar epithelial cells (AEC) causes
intra-alveolar fibrosis and the collapse of alveolar capillary
units[2]. These latter observations suggest that PF may be an
ongoing form of acute lung injury occurring sequentially at
discrete sites in the lungs. The evolving theory about the
pathogenesis of PF is that severity of fibrogenic response in the
lungs is directly related to the severity of lung injury in the
early phase, including sequential AEC injury, capillary
endothelial cells injury, and infiltrations of inflammatory
cells[3]. The observations emphasize that effective therapies for
these disorders must be given early in the natural history of the
disease, prior to the development of extensive lung
destruction and fibrosis.
Bleomycin is an anticancer agent prescribed for various
cancers, including that of the lungs. However, this drug has
a dose-dependent pulmonary toxicity, including lung
fibro-sis, which limits its clinical
use[4]. The toxic effect of this agent has been utilized advantageously in a number of experimental approaches to induce PF in animal models.
Several groups of workers have reported that in the early stages
of bleomycin-induced lung damage, the lesions are
associated with biochemical and functional changes that resemble
those of human PF, including inflammatory cell infiltration,
increased collagen content, and reduced lung volume and
compliance[5,6]. It is useful to assess the effects of potential
therapeutic agents.
Angiotensin II, which is mainly generated by the
angiotensin converting enzyme (ACE) and chymase, is a peptide
that plays a crucial role in regulating blood pressure and
sodium homeostasis[7]. Recent studies have shown that
angiotensin II is also closely associated with tissue injury and
fibrogenesis in circulatory organs[8] and
lungs[9]. The vast majority of the actions of angiotensin II are thought to be
mediated via the angiotensin type I (AT1) receptor. The AT1
receptor has been localized to fibroblasts, vascular smooth
muscle cells, macrophages, and AEC in patients with PF
induced by chronic obstructive pulmonary
disease[10]. Human lung fibroblast proliferation and the synthesis of the
extracellular matrix in human lung fibroblasts increased after
stimulation by angiotensin II in
vivo[11]. The AT1 receptor mediates AEC apoptosis in response to angiotensin
II[12]. A high concentration of ACE has been observed in bronchoalveolar
lavage (BAL) fluid from patients with idiopathic pulmonary
fibrosis (IPF)[13]. In an animal model of radiation-induced PF,
concentrations of ACE and angiotensin II increased in lung
tissue homogenates[14]. The administration of an ACE
inhibitor or an AT1 receptor antagonist significantly
attenuated PF in animal models[15,16]. These studies suggest that
the process of PF is promoted by an activated local
renin-angiotensin system in the lungs via the AT1 receptor. Taken
together, the evidence from these studies suggest that
angiotensin II plays an important role in the promotion of PF,
and the AT1 receptor is a prominent receptor for the
transmission of its signaling. However, these studies focused on
fibrogenic progression, and the relationship between the AT1
receptor and lung injury in the early phase in
vivo remains unclear. The aims of this study are to clarify whether the
AT1 receptor located in the lungs is induced in the early
stage of PF induced by bleomycin, and whether early lung
injury could be reduced by valsartan, an antagonist of the
AT1 receptor.
Materials and methods
Animals Male Sprague-Dawley (SD) rats (200_220 g;
Shanghai Laboratory Animal Center, Chinese Academy of
Sciences, Shanghai, China) were maintained in a controlled
environment and provided with water and standard rodent
food. All rats were acclimatized to their new surroundings
for 1 week prior to the animal experiments, which were
performed at the Animal Department (Shanghai Institute of
Material Medica, Shanghai, China) and approved by the
Shanghai Animal Care and Use Committee.
Experimental protocols One hundred and ninety SD rats
were randomly divided into 7 groups (n=40 in A, B, C, D
groups, n=10 in E, F, G groups). The rats in group A and B
were injected intratracheally with 2 mL/kg saline, and then
given saline or 60
mg·kg-1·d-1 valsartan gavaged at 9:00 AM
for 21 d. Those in group C, D, E, F and G were given saline,
valsartan (60, 30, 15
mg·kg-1·d-1, Qianhong, Changzhou,
China), or 0.5
mg·kg-1·d-1 dexamethasone (Taihe, Tianjin,
China) gavage at 9:00 AM for 21 d after an intratracheal
injection of bleomycin sulfate (8 U/kg, 16 U/mL in saline, Sigma,
St Louis, MO, USA). The day of intratracheal injection with
bleomycin or saline was designated d 0. The rats were
eutha-nized by sodium barbital (80 mg/kg, intraperitoneal) on d 1, 3,
7 and 21 after bleomycin or saline administration. The lung
vasculature was perfused free of blood, and then the left
lungs were removed from the trachea and hilar nodes and
weighed. The left lungs of half of the animals were fixed in
4% phosphate buffered paraformaldehyde for
histopathological preparation. BAL fluid samples were collected from
the right lung of each animal (5 rats from each group) for
inflammatory cell count and protein concentration assay.
For all the animals, recovery of lavage fluid was about 80%
of the utilized lavage volume. Left lung tissues were frozen
in liquid nitrogen for measurements of hydroxyproline,
malondialdehyde, myeloperoxidase, ACE activity,
caspase-3 activity, mRNA expression, and protein expression profiles.
Histopathological evaluation Rat lung tissues were
processed for routine paraffin embedding, and serial sections (5
μm) were stained with hematoxylin and eosin (H&E) and a
modified Masson trichrome to assess the degree of
inflammation and fibrosis. Fibrosis and alveolitis were scored by
blinding using the previously described semiquantitative
criteria[17].
Hydroxyproline assay in lung tissues Frozen lung
tissues were homogenized by a Polytron tissue homogenizer in
saline containing 0.1 mol/L phenylmethylsulfomyl fluoride.
The homogenized sample was hydrolyzed in 6 mol/L HCl
and the hydroxyproline concentration was quantified by
chloramine-T in duplicate lung tissue samples as previously
described[18].
Malondialdehyde concentration, ACE activity, and
myeloperoxidase activity assay in lung tissues Lung tissue
samples were homogenized at a w/v ratio of 1:10 in cold
Tris-HCl buffered saline (pH 7.4, 10 mmol/L Tris-HCl, 0.1 mmol/L
EDTA-2Na, 10 mmol/L Saccharose, 0.8% sodium chloride
solution). One portion of each homogenate was centrifuged
at 3000×g for 10 min at 4 °C, and the supernatant was used
for the measurement of malondialdehyde concentration and
ACE activity. The malondialdehyde concentration was
determined by the thiobarbituric acid method as previously
described[19]. ACE activity was analyzed, as previously
described[20], by measuring the hydrolysis of
Hippuryl-L-histidyl-L-leucine ( Hip-His-Leu; Sigma, USA) substrate to
histidyl-leucine (His-Leu), which fluoresces at an excitation
wavelength of 365 nm and an emission wavelength of 495
nm. The reaction was stopped by 0.28 mol/L NaOH.
o-Phthalaldehyde solution (Sigma, USA) bound to the
His-Leu product, and the amount of tagged His-Leu was
determined by spectrofluorophotometry
(RF-5301 PC spectro-fluorophotometer; Shimadzu, Kyoto, Japan). Another
portion of each homogenate was used to analyze
myeloperoxidase activity with a microplate reader (Molecular Devices,
Sunnyvale, CA, USA) using a myeloperoxidase activity kit
(Jiancheng, Nanjing, China). Changes in absorbance at 450
nm were measured with the microplate reader.
Caspase-3 activity assay in lung tissues Caspase-3
activity was determined according to the method
of Stennicke and
Salvesen[21]. Lung tissue samples were suspended at a
w/v ratio of 1:10 in Tris-HCl buffered saline, and
homogenized 3 times at 4 °C. The homogenate was centrifuged at
12 000×g for 10 min at 4 °C, and pre-incubation was carried
out in a medium containing 150 µg protein of the cytosolic
sample and 1 mL caspase buffer {20
mmol/L Pipes, 100 mmol/L NaCl, 10 mmol/L dithiothreitol, 1 mmol/L EDTA, 0.1%
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate
(CHAPS), and 10% sucrose, pH 7.2} at 37 °C for 0.5 h,
followed by incubation with 10 µL fluorogenic substrate
Ac-DEVD-AMC (N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-
methyl-coumarin; Sigma, USA). Active caspase-3
cleaved the substrate and released the fluorogenic
AMC. AMC fluorescence was quantified by spectrofluorophotom-etry
using an excitation wavelength of 380 nm and an emission
wavelength of 460 nm. For the control, we added 10 µL of
Ac-DEVD-CHO (N-Acetyl-Asp-Glu-Val-Asp-al; Sigma,
USA), the specific inhibitor blocking cleavage of the
fluorogenic substrate, to the incubation medium.
Protein and cell count in BAL fluid The total cell
number in BAL fluid were examined immediately following lavage.
The remainder of each sample was centrifuged at
1200×g for 15 min at 4 °C. The supernatant fractions were used to
measure total protein concentration. The protein concentration
in BAL fluid was measured using the bicinchoninic acid
(BCA) method (Pierce Chemical, Rockfold, IL, USA) and
expressed as grams of protein per liter of BAL fluid (g/L).
Collagen I and collagen III gene expression by
RT-PCR Lung tissues were ground into a powder in liquid nitrogen,
and the gene expression of collagen I, collagen III, and
GAPDH were measured using RT-PCR. Total RNA was
extracted using the TRIzol Reagent (Invitrogen Life
Techno-logies, Paisley, UK) according to the manufacturer's
instructions. The yield and purity of the isolated RNA
solution were determined by
A260/A280 readings on a
spectro-photometer. Reverse transcription was performed on 2 µg of
RNA with random primers and Moloney murine leukemia
virus reverse transcriptase (Promega, Madison, WI,
USA).The PCR were carried out with the primers shown in Table 1.
Amplifications consisted of 10 min at 95 °C,
following 30 cycles of 60 s at 95 °C, 45 s at 49_62 °C, and 75 s at 72 °C.
The PCR products were analyzed by electrophoresis on an
agarose gel, stained with ethidium bromide, and photographed.
Western blot analysis of AT1 Supernatants of the lung
tissue homogenate were collected after 30 min of
centrifugation at 12 000 ×g, and protein concentration was measured
by BCA protein assay (Pierce Chemical, USA). An aliquot
of 20 µg supernatants were placed in sample buffer (4%
sodium dodecyl sulfate, 125 mmol/L Tris-HCl, pH 6.8,
1% β-mercaptoethanol, 50% (v/v) glycerol, and 0.01%
(w/v) bromophenol blue) and denatured at 100 °C for 10 min. The
denatured samples were separated on 8% sodium dodecyl
sulfate-polyacrylamide electrophoresis gels. The proteins
were transferred to nitrocellulose membranes (Amersham
Pharmacia Biotech, Bucks, UK) by electroblotting. The blots
were blocked for 1 h with 5% (w/v) fat-free milk powder in
phosphate buffered solution (PBS) (0.1% Tween-20), and
subsequently incubated with primary rabbit antibodies
against the AT1 receptor (Novus Biologicals, Littleton, CO,
USA) and mouse primary antibodies against GAPDH (Abcam,
Cambridge, UK) at 4 °C overnight. The blots were washed 3
times in PBS with 0.1% Tween-20. They were then incubated
for 1 h with anti-rabbit (Abcam, UK) and anti-mouse (Abcam,
UK) antibodies conjugated with horseradish peroxidase.
Immunoreactive bands were detected by chemiluminescence
(Amersham Pharmacia Biotech, UK) and exposure to Kodak
film (Eastman Kodak, USA).
In situ detection apoptosis Briefly, paraffin-embedded
tissues were sectioned (5 µm), and antigen retrieval was
performed using citrate buffer. In situ detection of DNA
fragmentation was made by TUNEL assay (Roche, Mannheim,
Germany) according to the manufacturer's instructions. The
slides were then observed under a fluorescence microscope
(Leica, Mannheim, Germany).
Statistical methods Results are given as mean±SD.
One-way ANOVA followed by the least significant difference
method was used to determine differences among
groups for all continuous parameters. The Mann-Whitney or Chi-square
tests were applied for non-continuous parameters. The
statistical significance level was set at P<0.05.
Results
Effect of valsartan on mortality, body weight and lung
weight in rats Table 2 shows that the mortality of rats in the
bleomycin group was higher than that in the saline group,
while that of the 3 valsartan groups and the dexamethasone
group was lower than that of the bleomycin group. The
mortality of the valsartan 30 and the 60
mg·kg-1·d-1 groups
was 0. Table 3 presents the results of body weight. Rats
only receiving bleomycin lost weight significantly after
bleomycin injection, but the rats receiving dexamethasone
(0.5 mg·kg-1·d-1) or valsartan (15, 30, 60
mg·kg-1·d-1) after
being treated with bleomycin gained body weight. The body
weight of the 3 bleomycin-valsartan-treated groups was more
than that of bleomycin-dexamethasone-treated group
on d 21, and the rats receiving 60
mg·kg-1·d-1 valsartan gained
more body weight than the 2 other valsartan groups. The
body weight of the control rats receiving valsartan and
saline was similar (Table 3). The left lung was weighed and
the relative weight (lung weight/body weight×100%) was
calculated for each animal. The increase in wet weight of the
left lung is one of the indices representing lung edema. As
shown in Table 3, the wet and relative weights of the left
lung increased significantly after bleomycin treatment. The
increased relative weight of the left lung induced by
bleo-mycin declined significantly following valsartan and
dexamethasone treatment, and the decline of the rats treated with
bleomycin and valsartan (30, 60
mg·kg-1·d-1) were obviously
more than those with only bleomycin (P<0.05). These
results indicated that this compound ameliorated lung edema,
and this negative result caused by bleomycin and valsartan
(60 mg·kg-1·d-1) was most apparent among the 3 doses of
valsartan and dexamethasone.
Effect of valsartan on the degree of lung
fibrosis Hydroxyproline content, an efficient index of collagen
deposition, and collagen I/III mRNA expression were
measured in the lung samples. As shown in Figure 1A, valsartan
(60, 30, 15
mg·kg-1·d-1) and dexamethasone depressed the
high content of hydroxyproline induced by bleomycin, and
the inhibitory degree of valsartan showed a
dose-dependent manner. On mRNA expression of collagen I/III, the
high expression in the bleomycin group on d 21 was
mitigated by 60
mg·kg-1·d-1 valsartan (Figure 1B).
To further elucidate the histopathological changes
associated with bleomycin-induced lung fibrosis and the
efficacy of valsartan, the sections were stained with H&E and
Masson trichrome. On d 21, marked histopathological
changes, such as large fibrous areas, collapsed alveolar
spaces, and traction bronchiectasis in the subpleural and
peribronchial regions, were seen in the bleomycin-treated
rats. Although fibrotic lesions were observed in the rats
receiving bleomycin and valsartan (60
mg·kg-1·d-1), the
extent of fibrosis was markedly less severe compared with
that of the bleomycin group and bleomycin-dexamethasone
group (Figure 1C). To confirm the effects of valsartan on the
histopathology of bleomycin-induced lung fibrosis, the
overall grades of fibrotic changes of the lungs were estimated by
numerical scoring by blinding. As shown in Table 4,
the 21-d treatment with 60
mg·kg-1·d-1 valsartan resulted in the
depression of fibrosis induced by bleomycin significantly
(P<0.01).
AT1 receptor in the early phase The level of AT1 receptor
expression in the bleomycin group was measured in lung
homogenates by Western blotting. In lung homogenates on
d 1, 3, and 7, an anti-AT1 receptor antibody recognized a
protein with an apparent molecular mass of 46 kDa. AT1 receptor
expression located in lung tissues increased gradually from
d 1 to 7 after bleomycin treatment (P<0.01; Figure 2).
Lung injury in the early phase Malondialdehyde
induction in the tissue samples and the increased protein
concentration in BAL fluid are 2 other important markers of lung
injury, with the first indicating the increased level of
production of oxygen-free radicals and the latter indicating alveolar
edema. Bleomycin-treated rats showed marked increases in
malondialdehyde (Figure 3) and protein concentration (Figure
4); these levels peaked on d 7 after bleomycin treatment. Rats
receiving valsartan (60
mg·kg-1·d-1) showed significantly
lower increases in these levels. A similar situation was found
with myeloperoxidase activity (Figure 4), alveolitis scores in
lung tissue (Table 5), and the number of total inflammatory
cells in BAL fluid (Table 6). The level of malondialdehyde
concentration, protein concentration, myeloperoxidase
activity and cell count were positively correlated with AT1
receptor expression in lung tissues in bleomycin-treated rats
in the early stage, and valsartan reduced all of these increases.
These data collectively indicate that the AT1 receptor
contributes to lung injury induced by bleomycin.
ACE activity in the early phase ACE activity is an index
of pulmonary endothelial cell metabolic function, and
decreased ACE activity has been proposed as an early
indicator of bleomycin lung
toxicity[22]. In the bleomycin group, the tendency of ACE activity declined gradually from d 1,
and was lowest on d 7 (Figure 5). Valsartan reduced the
decrease induced by bleomycin in the time course. These
results implied that angiotensin II augmented bleomycin lung
toxicity via the AT1 receptor.
Apoptosis in lung tissues in the early
phase In the bleomycin-treated rats, the first event noted was endothelial
damage of the lung vasculature, and apoptosis of AEC
accompanied. Apoptosis in lung tissues from d 1 to d 7 was
analyzed by in situ TUNEL assay. TUNEL-positive nuclei in
the alveolar septum increased gradually from d 1 and were
highest on d 7 in the bleomycin-treated rats (Figure 6A).
There were obviously less apoptosis nuclei in the lung
tissues of the valsartan-treated rats than the bleomycin-treated
rats, especially on d 3 and d 7.
In addition, apoptosis was detected as an increase in the
total activity of caspase-3 in the lung tissues, measured by
enzyme assay of lung homogenates (Figure 6B). As early as
the first day after instillation of bleomycin intratracheally,
lung caspase-3 activity increased by 100%, and peaked
on d 7, but the increase was prevented by the co-administration
of valsartan.
Discussion
Inhibitors of ACE or antagonists of the AT1 receptor have
been shown to have antifibrotic effects in the
heart[23], kidney[24] and
liver[25]. The first report of antifibrotic actions in
the lungs by the ACE inhibitor
captopril[26], published many years ago, was recently extended by the demonstration that
the AT1 antagonists, losartan,
L158809[16] and
candesartan[9] have even more potent antifibrotic potential in the lung than
ACE inhibitors. The present work extends those
observations by showing that another AT1 antagonist, valsartan,
also delays lung fibrosis induced by bleomycin in rats, and
one of the mechanisms by which AT1 antagonists act is
through the inhibition of lung injury in the early phase,
including inflammation, apoptosis and oxidative stress.
Bleomycin-induced lung injury is associated with the
recruitment of inflammatory cells into the injured lungs
during the first 7 d[27]. This is accompanied by a rapid increase
in pulmonary microvascular leak, which is manifested as an
increase in total protein in BAL
fluid[28]. Assessment of these early inflammatory responses in the
present study was
revealed by increases of total inflammatory
cell number in BAL fluid in bleomycin-treated
rats compared with saline-treated rats. Assessment
of myeloperoxidase activity and alveolitis scores within the
lungs revealed similar increases. Valsartan reduced the increases of total inflammatory
cell number, alveolitis scores and myeloperoxidase activity,
suggesting that the increase of the AT1 receptor may contribute
to these increased responses in bleomycin-treated rats.
Earlier work has shown that AEC undergoing apoptosis
in response to Fas ligand, tumor necrosis factor-α or
bleomycin begin secreting angiotensin II into the
extracellular space within hours of exposure, at least
in vitro[29_31]. Those studies also implied that the autocrine production of
angiotensin II and its binding to the AT1 receptor on AEC
were required for apoptosis in response to these agents. In
a previous study, the ACE inhibitor captopril or the caspase
inhibitor ZVAD-fmk had essentially equal ability, blocking
the appearance of apoptotic epithelial cells in rats exposed
to intratracheal bleomycin and preventing subsequent
collagen deposition, which suggests that the blockade of
fibrogenesis by captopril is indeed related to the inhibition
of apoptosis in vivo[11].
The present study, which shows that valsartan depressed the apoptosis from d 1 to d 7 and
extracellular matrix in later phage induced by bleomycin, was
consistent with the findings that valsartan is able to block
both apoptosis and collagen deposition in rats. To capillary
endothelial cells apoptosis, the action of angiotensin II were
prevented in circulation and kidney via AT1 receptor
inhibitors, although there is little paper studying in lung
update, [32]. AT1 receptor signaling augments endothelial
cells apoptosis in the process of oxidative stress-induced
vascular injury[33]. The apoptosis of endothelial and
epithelial cells in the lungs increases the permeability of the
air-blood barrier and enhances the infiltration of inflammatory
cells[34], which is one of mechanisms promoting lung fibrosis
in idiopathic PF patients and bleomycin-treated
rats[35,36]. ACE activity in located lung tissues, an index of pulmonary
endothelial cell metabolic function[37], decreased in the early
stage in the bleomycin-treated rats, which shows that
endothelial cells in lung tissues were injured.
On the other hand, recent findings suggest that
oxidative stress may play an important role in the pathogenesis of
tissue fibrosis affecting apoptosis of both structural and
inflammatory cells and altering the cytokine
microenvironment balance[38,39]. It has been demonstrated that the
presence of oxidative stress may lead to the damage, activation
and/or apoptosis of AEC either directly, through an
imbalanced intracellular redox equilibrium, or indirectly, by
activating redox-sensitive effector pathways such as
transcription factors and angiotensin-converting enzymes, increasing
the conversion of angiotensinogen into angiotensin II
which can be considered a mediator of oxidative
stress[40]. These responses
are correlated with high levels of malondialdehyde,
which indicate that the increased lipid peroxidation is
produced by oxygen-free
radicals[41]. Data from this study
revealed that bleomycin significantly increased
malondial-dehyde production in lung tissues. This effect could be a
secondary event, following a bleomycin-induced increase in
free radical generation and/or decrease in lipid peroxidation
protecting enzymes. Previous studies have reported that
bleomycin-induced lung toxicity is related to redox cycling
of an iron-bleomycin complex, which in turn catalyzes the
formation of reactive oxidative species with ultimate
progression of lipid peroxidation[42]. The results of this study
show that valsartan attenuates increases in lung tissue
malondialdehyde concentration induced by bleomycin,
suggesting that angiotensin II is involved in oxidative stress in
bleomycin-treated lung tissues via AT1.
The view that angiotensin II promotes
human lung fibroblasts proliferation via the activation of the AT1 receptor
in vitro was demonstrated by accumulating
evidence[43]. A hypothesis testified that
angiotensin II generation contributes directly to the fibroproliferative response to lung injury and
collagen synthesis in vivo[44], but little was done with regards
to the relationship of early lung injury and angiotensin
II in bleomycin-induced lung fibrosis. In the present study, we
found that in the early phase, bleomycin-induced apoptosis,
inflammation and oxidative stress of lung tissue were
significantly attenuated by the AT1 receptor antagonist, valsartan.
These results show that angiotensin II also contributes to
early lung injury, suggesting that a new mechanism of local
renin-angiotensin system involved in the pathology of lung
fibrosis. These studies are in agreement with the possibility
that AT1 receptor antagonists may hold potential for the
treatment of PF in humans.
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