Transforming growth factor-
1 upregulation is independent of
angiotensin in paraquat-induced lung fibrosis
Chung-Ming Chen
a
,
b
, Hsiu-Chu Chou
c
, Hsun-Hui Hsu
b
, Leng-Fang Wang
d
,∗
aGraduate Institute of Medical Sciences, Taipei Medical University, Taipei, TaiwanbDepartment of Pediatrics, Taipei Medical University Hospital, Taipei, Taiwan
cDepartment of Anatomy, College of Medicine, Taipei Medical University, No. 250, Wu Hsing Street, Taipei, Taiwan dDepartment of Biochemistry, College of Medicine, Taipei Medical University, No. 250, Wu Hsing Street, Taipei, Taiwan
Received 8 April 2005; received in revised form 18 July 2005; accepted 5 August 2005 Available online 22 September 2005
Abstract
Transforming growth factor-
1 (TGF-1) contributes to the fibrosis of injured organs. Angiotensin II (Ang II) is an inducer
of TGF-
1 in cells of the heart and kidneys, and the regulation of TGF-1 by Ang II has not yet been confirmed in lung tissue.
We evaluated the role of TGF-
1 and its relationship with Ang II in paraquat-induced lung fibrosis. Adult male Sprague–Dawley
rats were treated intraperitoneally with paraquat (20 mg/kg) or saline in the control group. On days 1, 3, 7, and 21 after paraquat
treatment, TGF-
1 and collagen gene expressions, TGF-1 protein, angiotensin-converting enzyme (ACE) activity, Ang II, and
hydroxyproline contents were measured in lung tissue. Lung TGF-
1 mRNA expression progressively increased and reached a
peak on day 7 after paraquat treatment. Increases in TGF-
1 mRNA expression and TGF-1 levels preceded the onset of increased
collagen I mRNA expression and hydroxyproline contents. c-myc mRNA expressions were inversely correlated with TGF-
1 protein
levels in paraquat-treated lungs. Lung ACE activity decreased after paraquat administration and the decrement was maximal on
day 7. Lung Ang II concentrations immediately decreased after paraquat administration and the values were not related to TGF-
1
levels. We conclude that TGF-
1 is upregulated and contribute to the paraquat-induced lung fibrosis and this effect is independent
of the renin–angiotensin system.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Paraquat; Angiotensin-converting enzyme; Angiotensin; Transforming growth factor; Hydroxyproline
1. Introduction
Paraquat dichloride (1,1
-dimethyl-4,4
-bipyridilium
dichloride; methyl viologen) is an effective and widely
used herbicide in most countries. The intentional and
accidental ingestion of commercial liquid formulations
of paraquat has caused a large number of human
fatali-ties. According to epidemiological data in the National
∗Corresponding author. Tel.: +886 2 27361661; fax: +886 2 27360399.
E-mail address: [email protected] (L.-F. Wang).
Poison Center in Taiwan during 1985 and 1997, paraquat
poisoning was the leading cause of poisoning-induced
death in Taiwan (
Satoh and Hosokawa, 2000
). Paraquat
produces toxicity in humans and the lungs are one of
the primary target organs (
Forman et al., 1982
). The
toxic effects of paraquat on the lungs result in
monary edema, hypoxia, respiratory failure, and
pul-monary fibrosis. Survivors of paraquat poisoning may
be left with a restrictive type of long-term pulmonary
dysfunction (
Yamashita et al., 2000
).
Transforming growth factor-
1 (TGF-1) is a key
growth factor that initiates tissue repair and its sustained
0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.08.004
production underlies the development of tissue fibrosis
(
Border and Ruoslahti, 1992
). In experimental models of
lung fibrosis, TGF-
1 is an important upstream effector
of collagen gene expression by a variety of approaches,
including the administration of the TGF-
1 gene and
TGF-
1 to the lung (
Gauldie et al., 1999; Kenyon et al.,
2003
). Angiotensin II (Ang II) produced from
prote-olytic processing of angiotensinogen is documented to
be an inducer of TGF-
1 expression in cells of the heart
and kidneys (
Campbell and Katwa, 1997; Klahr and
Morrissey, 1998; Kupfahl et al., 2000
). Although
TGF-1 has been reported to play a role in pulmonary fibrosis
induced with paraquat and hyperoxia (
Ruiz et al., 2003
),
its relationship with Ang II has not yet been confirmed
in lung tissue. The aims of this study were to evaluate
the role of TGF-
1 and to determine its relationship with
angiotensin in paraquat-induced lung fibrosis.
2. Materials and methods
2.1. Animals
This study was approved by the Institutional Animal Use
Committee at Taipei Medical University and was performed
using adult male Sprague–Dawley rats (with approximate body
weights of 230–250 g) maintained on a standard laboratory diet
and water ad libitum. Rats were treated intraperitoneally with
paraquat (20 mg/kg, Sigma Chemical, St. Louis, MO, USA)
or saline in the control group. On days 1, 3, 7, and 21 after
paraquat treatment, rats were anesthetized by intraperitoneal
pentobarbital (50 mg/kg), and the lungs were removed from the
chest and immediately frozen in liquid nitrogen for
determi-nation of TGF-
1 and collagen gene expressions, angiotensin
converting-enzyme (ACE) activity, the Ang II concentration,
and TGF-
1 contents. Another set of rats was used for
mea-surement of lung hydroxyproline as an estimate of collagen
content.
2.2. TGF-
1, collagen, and c-myc gene expressions by
reverse transcription-polymerase chain reaction
(RT-PCR)
Lung tissue was ground into a powder in liquid nitrogen,
and the gene expressions of TGF-
1, collagen I, collagen III,
c-myc were measured using RT-PCR. Total RNA was extracted
using the TRIzol Reagent (Invitrogen Life Technologies,
Pais-ley, UK) according to the manufacturer’s instructions. Yield
and purity of the isolated RNA solution were determined by
A260 and A280 readings on a spectrophotometer. Reverse
tran-scription was performed on 3
g of RNA with oligo-dT primers
and avian myeloblastosis virus reverse transcriptase (Roche,
Indianapolis, IN, USA). The PCR were carried out with the
primers shown in
Table 1
. The PCR products were analyzed
by electrophoresis on an agarose gel, stained with ethidium
bromide, and photographed. To determine the linear range of
the PCR, the intensity of the amplified products was plotted
against the cycle number. At least three samples (range 3–6)
on each day were analyzed in each group.
2.3. Measurements of ACE activity, Ang II, and TGF-
1
levels in lung tissue
Lung tissue was homogenized in lysis buffer and
cen-trifuged at speeds according to the manufacturer’s
instruc-tions. The supernatant solution was used for measurements
of ACE activity, Ang II, and TGF-
1 levels with
enzyme-linked immunosorbent assay kits from Buhlmann Labs AG,
Switzerland; SPI-BIO, Massy Cedes, France; and Biosource,
Camarillo, CA, USA, respectively. One unit of ACE
activ-Table 1
Oligonucleotide sequences of the primers used
Primer Sequence Product size (bp)
TGF-1
Sense 5-GCT CGC TTT GTA CAA CAG CA-3 280
Antisense 5-GAG TTC TAC GTG TTG CTC CA-3
Collagen I
Sense 5-GCT GCC TTT TCT GTT CCT TT-3 185
Antisense 5-GGA TTT GAA GGT GCT GGG TA-3
Collagen III
Sense 5-GCC ACC CTG AAC TCA AGA GT-3 446
Antisense 5-GCC ATC CTC TAG AAC TGT GT-3
c-myc
Sence 5-AGG AAC TAT GAC CTC GAC TAC G-3 293
Antisense 5-AGT AGC TCG GTC ATC ATC TCC AG-3
-Actin
Sense 5-TTG TAA CCA ACT GGG ACG ATA TGG-3 764
ity was defined as the amount of enzyme required to release
1
mol/min of hippuric acid. TGF-1 and Ang II were
expressed as
g/g of protein and ng/g of protein, respectively.
2.4. Hydroxyproline assay of lung tissue
The hydroxyproline contents of lung tissues were
deter-mined and the data were expressed as
g/g wet lung tissue
(
Reddy and Enwemeka, 1996
). Total lung tissues from
con-trol and paraquat-treated rats were frozen in liquid nitrogen
and lyophilized using a freeze-dry system (Labconco, Kansas
City, MO, USA). The lyophilized lung tissue was thoroughly
homogenized in distilled water using a polytron homogenizer.
Aliquots of standard hydroxyproline and lung tissue samples
were hydrolyzed and mixed with a buffered chloramine-T
reagent, and oxidation was allowed to proceed at room
tem-perature. The chromophore was developed with the addition
of Ehrlich’s aldehyde and was incubated. Absorbance of each
sample was read at 550 nm using a spectrophotometer and was
plotted against the concentration of standard hydroxyproline.
2.5. Histological evaluation
After sacrifice, right lung was isolated and inflation-fixed
in formalin at a pressure of 20 H
2O. Subsequently, the lobes of
the lung were separated and sectioned sagittally. The sagittal
sections were embedded in paraffin, and 5-
m-thick sections
were made and stained with hematoxylin and eosin. The
sec-tions were examined by light microscopy and assessed for the
presence of hemorrhage, intra-alveolar edema, and fibrosis.
2.6. Statistical analysis
Results are presented as the mean
± S.E.M. Comparisons
between control and paraquat-treated groups at each time point
were made using unpaired Student’s t-test. Differences were
considered significant at P < 0.05.
3. Results
Sixty rats received paraquat treatment in this
study. Three deaths occurred between 1 and 3 days
Fig. 1. TGF-1, collagen I, collagen III, and c-myc gene expressions in control and paraquat-treated rat lungs. (A) Lung TGF-1 mRNA expression progressively increased after paraquat treatment and the value had reached a peak and statistical significance on day 7. (B) Col-lagen type I mRNA expressions were comparable between the control and paraquat-treated lungs on days 1 and 3, and values had signifi-cantly increased on day 7 after paraquat treatment. (C) Collagen type III mRNA expressions had significantly increased on days 1 and 21 after paraquat treatment when compared with the control group. (D) c-myc mRNA expressions were increased in paraquat-treated lungs and the values were significantly higher on days 1, 7, and 21 when compared with the control group (*P < 0.05,**P < 0.01,***P < 0.001).
after paraquat treatment. Between days 3 and 21
no deaths occurred. Twenty-five rats were used for
hydroxyproline measurements. The rest were used for
measurements of TGF-
1 and collagen gene
expres-sions, ACE activity, Ang II concentration, and TGF-
1
content.
3.1. TGF-
β1, collagen, and c-myc gene expressions
in control and paraquat-treated rat lungs
Lung TGF-
1 mRNA expression progressively
increased after paraquat treatment and the value reached
a peak and statistical significance on day 7 when
com-pared with the control group (
Fig. 1
A). Collagen type I
mRNA expressions were comparable between the
con-trol and paraquat-treated lungs on days 1 and 3, and the
values had significantly increased on day 7 (
Fig. 1
B).
Collagen type III mRNA expressions had significantly
increased on days 1 and 21 after paraquat treatment
when compared with the control group (
Fig. 1
C). c-myc
mRNA expressions were increased in paraquat-treated
lungs and the values were significantly higher on days
1, 7, and 21 when compared with the control group
(
Fig. 1
D).
3.2. Effects of paraquat treatment on lung ACE
activity and Ang II concentration
Lung ACE activity gradually decreased after paraquat
administration and the values were significantly lower in
paraquat-treated group on days 3 and 7 when compared
with the control group (
Fig. 2
A). Lung Ang II
concen-trations immediately decreased after paraquat
adminis-tration and the values reached statistical significance on
days 1, 7, and 21 (
Fig. 2
B).
3.3. Effects of paraquat treatment on lung TGF-β1
and hydroxyproline contents
Paraquat-treated rats exhibited a progressive increase
in lung TGF-
1 levels and the values reached
statisti-cal significance on days 3 and 7 (
Fig. 3
A). The values
then decreased after day 7 and returned to the
con-trol level by day 21 after paraquat treatment.
Hydrox-yproline contents of the lung tissue were comparable
among control and paraquat-treated rats on days 1, 3,
and 7 and the value reached a maximum on day 21
(
Fig. 3
B).
3.4. Histology
The histological appearance of the lungs is illustrated
in
Fig. 4
. Examination of random fields under a light
microscopy revealed lung structure progressively
disor-ganized and inflammatory cellular infiltrate increased in
the interstitium and airspaces as paraquat-treated rats
aged. Alveolar hemorrhage and capillary stasis were
found mostly in rats on day 3 after paraquat treatment
(
Fig. 4
C).
Fig. 2. ACE activities and Ang II levels in lung tissues of control and paraquat-treated rats. (A) Lung ACE activity progressively decreased after paraquat treatment, and the activities were significantly lower in paraquat-treated groups on days 3 and 7 when compared with the control group. (B) Lung Ang II concentrations immediately decreased after paraquat administration and the values had reached statistical significance on days 1, 7, and 21 when compared with the control group (*P < 0.05,**P < 0.01).
4. Discussion
Acute respiratory distress syndrome (ARDS) is a
rather heterogenous disorder, and the clinical course is
divided into three phases: (1) an early exudative phase of
lung inflammation and edema; (2) a proliferative phase
with pneumocyte and fibroblast proliferation; and (3)
a final fibrotic phase with collagen deposition and
pul-monary fibrosis (
Meduri, 1996
). We previously reported
that an intraperitoneal paraquat injection (35 mg/kg) in
rats produced a picture resembling the initial
inflamma-tory phase of ARDS; rats exhibit increased wet lung
weight, inflammatory responses, and total protein
con-tent in bronchoalveolar lavage fluid (
Chen and Lua,
2000
). However, pulmonary fibrosis is a major
deter-minant of the prognosis associated with ARDS.
Many inflammatory cytokines, particularly TGF-
1,
are involved in the pathogenesis of ARDS (
Dhainaut
et al., 2003
). TGF-
1 is mitogenic and chemotactic for
fibroblasts, monocytes, and macrophages, and promotes
accumulation of the extracellular matrix (
Blobe et al.,
Fig. 3. TGF-1 and hydroxyproline contents in control and paraquat-treated rat lungs. (A) Lung TGF-1 levels progressively increased after paraquat treatment and values reached statistical significance on days 3 and 7. Values then decreased after day 7 and had returned to the control level by day 21 after paraquat treatment. (B) Hydroxyproline contents of lung tissues were significantly higher in paraquat-treated rats than control rats on day 21 (*P < 0.05,**P < 0.01).
2000
). TGF-
1 not only participates in the active early
phase of acute lung injury and contributes to the
develop-ment of pulmonary edema, but is also associated with the
late phase of acute lung injury and leads to pulmonary
fibrosis (
Dhainaut et al., 2003
). Collagen is the major
extracellular matrix component of the lungs and is vital
for maintaining the normal lung architecture. Types I and
III collagen are the most abundant collagen subtypes in
the lungs (
Kirk et al., 1984
). They are present in the
adventitia of pulmonary arteries, the interstitium of the
Fig. 4. Light micrographs of lung sections stained with hematoxylin and eosin from control (A) and paraquat-treated rats on days 1 (B), 3 (C), 7 (D), and 21 (E) after paraquat treatment (×400). The central component of the alveolar wall is the capillary (+) and its associ-ated connective tissue. On each side faces the alveolus, flat, squamous pneumocyte type I cell (arrowhead) is interposed between the capil-lary and air spaces. Pneumocyte type II cell (arrow) lines the alveolus which shows a round shaped nucleus and is surrounded by a noticeable amount of cytoplasm. Large alveolar macrophage ( ) were found in the alveolar wall or free in the alveolar space. Bar = 50m.
bronchial tee, the interlobular septa, the bronchial
lam-ina propria, and the alveolar interstitium. In this study,
we found that increased TGF-
1 mRNA expression
pre-ceded the increase in collagen I mRNA expression and
increased hydroxyproline content following the increase
in TGF-
1 levels of lung tissues. Collagen III mRNA
expression was significantly increased on day 1 after
paraquat treatment, but the result was not related to
TGF-1 mRNA expression. This finding implies the presence
of other pathways for collagen production and is
con-sistent with the observations of
Batra et al. (2003)
, who
found that TGF-
1 does not increase collagen III
synthe-sis in human lung fibroblasts. Previous studies also found
that N-terminal procollagen peptide-III is elevated in
tra-cheal aspirate, serum, and bronchoalveolar lavage fluid
from ARDS patients within 24 h of diagnosis (
Chesnutt
et al., 1997; Marshall et al., 2000
).
Transforming growth factors are multifunctional
growth factors that are also involved in lung
organo-genesis. c-myc, a member of the basic
helix-loop-helix leucine zipper family of transcription factors, is
important for lung differentiation (
Kim et al., 2003
).
In this study, we found that higher c-myc expressions
in paraquat-treated lungs and the values were inversely
correlated with TGF-
1 protein levels. These data are
consistent with the findings of
Kim et al. (2003)
and
indicate that lung differentiation was diminished
dur-ing the fibrotic stage of acute lung injury. Oxidative
agents generated from xanthine and xanthine oxidase
induces c-myc expression (
Shibanuma et al., 1988
) and
2-h paraquat treatment increases in vivo lung xanthine
oxidase activity (
Waintrub et al., 1990
). We speculate
that higher c-myc expressions in paraquat-treated lungs
than those in control lungs might be due to
paraquat-induced xanthine oxidase.
ACE is distributed along the luminal pulmonary
endothelial surface and hydrolyzes Ang I to Ang II.
ACE activity decreases at an early stage of acute lung
injury and can be used as a marker of underlying
pul-monary capillary endothelial dysfunction (
Orfanos et al.,
2000
). In this study, we used 20 mg/kg of paraquat and
found that ACE activity had decreased on days 3 and 7
after paraquat treatment. This finding is consistent with
the observations of
Roth et al. (1979)
and
Venkatesan
(2000)
, who found that lung ACE activity had
signifi-cantly decreased on days 1 and 4 after 50 and 25 mg/kg
paraquat treatment, respectively. These studies indicate
that the paraquat-mediated decrease in lung ACE
activ-ity is dose dependent. The reduction in ACE activactiv-ity was
secondary to necrosis of pulmonary capillary endothelial
cells (
Hollinger et al., 1980
). In this study, the alteration
in ACE activity was concordant with the histological
appearance that showed prominent pulmonary
hemor-rhage on day 3 after paraquat treatment.
Ang II is documented to be an inducer of
TGF-1 expression in the heart and kidneys (
Campbell and
Katwa, 1997; Klahr and Morrissey, 1998; Kupfahl et al.,
2000
). However, the role of Ang II in pulmonary
fibro-sis has not yet been clarified. In this study, we found that
paraquat treatment decreased lung Ang II levels before
the rise in TGF-
1 and hydroxyproline levels and the fall
in ACE activity. These data indicate that Ang II is not
an upstream activator of TGF-
1 in paraquat-induced
lung injury, and lower lung Ang II levels may result
from decreased angiotensinogen synthesis. These
find-ings are inconsistent with the observations of
Marshall
et al. (2004)
, who found increased lung Ang II
con-centrations in bleomycin-induced lung injury. Several
mechanisms may activate TGF-
1, including pathways
involving interleukin-13 (
Lee et al., 2001
), CD36 and
thrombospondin-1 (
Yehualaeshet et al., 1999
), as well
as reactive oxygen intermediates including the
super-oxide anion and hydrogen persuper-oxide (
Bellocq et al.,
1999
). Paraquat treatment increases lung xanthine
oxi-dase activity and generates superoxide anion and
hydro-gen peroxide (
Waintrub et al., 1990; Suntres, 2002
).
In addition to the lungs, the kidneys are another
tar-get organ for paraquat toxicity in rats. The
nephrotox-icity caused by paraquat is prominent and appears to
involve convoluted renal tubules and proximal
tubu-lar cells (
Murray and Gibson, 1972; Mølck and Friis,
1997
). We speculate that the discrepancy in lung Ang II
levels between paraquat and bleomycin studies might
be due to other mechanisms activating TGF-
1 and
the nephrotoxicity associated with paraquat treatment
that might alter the activity of the renin–angiotensin
system.
In conclusion, we found that increase in TGF-
1
mRNA expression and TGF-
1 levels preceded the onset
of increased collagen I mRNA expression and
hydrox-yproline content and decreased Ang II levels in lung
tissues with paraquat-induced lung injury. These results
confirm previous study that TGF-
1 plays an
impor-tant role in the fibroproliferative phase of
paraquat-induced lung injury and this effect is independent of the
renin–angiotensin system.
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