1 Section of Pulmonary
Diseases, Connective tissue growth factor (CTGF) is a
newly described 38-kDa peptide mitogen for fibroblasts and a promoter
of connective tissue deposition in the skin. The
CTGF gene promotor
contains a transforming growth factor-
transforming growth factor- CONNECTIVE TISSUE GROWTH FACTOR (CTGF) is a newly
described fibroblast mitogen and promoter of collagen deposition. CTGF
was first isolated from human umbilical artery endothelial cells (4) and subsequently was found to be expressed by human foreskin and dermal
fibroblasts (12) but not by other cell types such as epithelial cells
or leukocytes (15). It is a 38-kDa, cysteine-rich peptide (12) and
shares homology with several other peptides, including CEF-10 (24),
Cyr61 (16) and Fisp-12 (21). Transforming growth factor- There is evidence that CTGF may be involved in the pathogenesis of
dermal fibrosis. This is not unexpected, because TGF- A prior publication has shown that
fisp-12 mRNA, a murine gene homologous
to CTGF, is expressed in murine lung
(21). TGF- Rat CTGF cloning and sequencing. RNA
was isolated from rat lung fibroblasts (RLFs) with an acid-phenol
extraction reagent (Ultraspec). A reverse transcription-polymerase
chain reaction (RT-PCR) was performed on 0.5 µg of total
RLF RNA with an RT-PCR kit (Promega). Primers were designed
using sequences from GenBank and the MacMolly software program.
Identical 19-base regions of the human
CTGF and mouse
fisp-12
genes separated by 199 bases were identified. The homologous regions
chosen include bases 934-953 and 1152-1170 for human
CTGF and bases 2693-2712 and
2911-2929 for fisp-12. Additional
bases were added to these two sequences to provide restriction
endonuclease sites at the end of the PCR product. The primer
sequences were
5'-CC- After gel purification of BamH I and
Pst I endonuclease (Promega)
double-digestion products of the vector and the RT-PCR-derived CTGF
cDNA fragment, the rat CTGF fragment was ligated into the multiple
cloning region of the Bluescript SK+ vector (Stratagene). Colonies were
screened with a battery of restriction endonucleases. A selected colony
matched with the expected result of ligation of the CTGF cDNA fragment
into the Bluescript vector. A large plasmid preparation was isolated
from the favorable colony, and the plasmid was sequenced with the
Sequenase Kit, version 2 (USB).
Isolation of human, rat, and mouse lung
fibroblasts. RLFs were isolated from 8-wk-old male
Sprague-Dawley rats exactly as previously described (3). Human lung
fibroblasts (HLFs) were isolated from sections of grossly normal lung,
which were resected for anatomic considerations during the course of
curative surgical therapy for lung cancer. Primary HLFs were obtained
through mechanical and enzymatic dissociation using a modification of
the method used in obtaining RLF isolates. Briefly, a portion of the
resected lobe distant from the cancerous lung nodule was resected, and then parenchyma from peripheral lung tissue (within 2 cm of the pleura
to avoid large vessels and bronchi) was diced into 1-mm pieces and
rinsed several times with PBS to remove blood. The tissue was then
placed in Dulbecco's modified Eagle's medium (DMEM) with 0.05%
trypsin, 0.008% collagenase, and 0.005% DNase (Sigma) and stirred for
30 min at 37°C before being filtered through 100-µm nylon mesh.
Filtered cells were washed twice in DMEM with 10% fetal bovine serum
(FBS), Fungizone, penicillin (100 U/ml), and streptomycin (25 mg/ml).
Cells were seeded at 106
cells/175-cm2 flask and washed
twice to remove nonadherant cells after 1 day of incubation at 37°C
in 95% air-5% CO2. When cultures
were subconfluent, they were passed for staining characterization and
subcultured or cryopreserved in DMEM with 10% DMSO. Studies were
performed on passages 4-7.
Mouse lung fibroblasts (MLFs) were isolated from 6- to 8-wk-old female
C57BL/6 and BALB/c mice. Briefly, mice were rendered insensitive with
tribromoethanol (Aldrich) intraperitoneally and exsanguinated via the
abdominal aorta. The lungs and heart were exposed, and the lungs were
perfused free of blood with 20 ml of normal saline injected into the
right ventricular outflow tract. The trachea was cannulated, and 3 ml
of warmed dispase were rapidly injected into the lungs. Then 0.5 ml of
low-melting-point agarose, warmed to 37°C, was injected slowly into
the trachea, and the chest cavity was covered with ice for 2 min. The
lungs were then removed and placed in dispase at room temperature for
45 min. The lung parenchyma was gently teased from the large airways
with forceps and incubated in DMEM containing DNase for 5 min on a stir
plate. Large pieces of lung tissue were removed and discarded with a
sterile 100-µm filter. The cells in the filtrate were placed on ice
for 10 min and then pelleted at 130 g
for 8 min. The cells were resuspended in DMEM with glutamine,
streptomycin, penicillin, and 10% FBS and placed in a bacteriological
plate for 35 min at 37°C to allow unwanted macrophages to attach.
The unattached cells were aspirated and plated in tissue culture plates
in DMEM with 10% FBS, and the medium was changed every 3 days.
Second-passage HLFs and MLFs obtained via the described methods
appeared spindle shaped and morphologically like mesenchymal cells and
also proliferated in response to platelet-derived growth factor (PDGF).
Immunohistochemistry was performed on two separate isolations of HLFs
and MLFs obtained in this manner. The following primary antibodies were
employed: anti-vimentin (Sigma); anti- Bleomycin exposure. C57BL/6 and BALB/c
mice (25 g; obtained from Charles River) were rendered insensitive with
tribromoethanol intraperitoneally before sterile tracheal cut-down
surgery. Four units per kilogram of bleomycin (Bristol-Meyers) in a
volume of 0.05 ml of 0.9% NaCl were administered into the tracheal
lumen. Preliminary studies demonstrated that this dose would result in fibrosis with a low mortality. A similar volume of sterile 0.9% NaCl
was instilled into control mice. After administration of bleomycin, the
neck wound was closed with a clip, and the animals were allowed to
recover from anesthesia on a warming plate.
Lung tissue procurement. At designated
time points after exposure (8, 16, and 30 days), the mice were
anesthetized with an intraperitoneal injection of tribromoethanol.
After ligation of the abdominal aorta, the chest cavity was exposed.
The left lung was tied off at the main stem bronchus and removed. The
left lung was then immediately wrapped in foil and placed in liquid
nitrogen for 30 min before storage at Hydroxyproline assay. Total
hydroxyproline content of the left lung was measured as an assessment
of lung collagen content. A spectrophotometric assay was used to
quantify lung hydroxyproline (14). Briefly, left lungs were removed
from the Northern analysis: RNA isolation.
Murine lungs were removed from the Twenty micrograms of total rat lung RNA in denaturation buffer were
added to each well of a 1.2% formaldehyde-agarose gel and separated
overnight via electrophoresis. RNA was transferred from the gel to a
nylon membrane (Immobilon-N) by capillary action overnight.
Prehybridization solution and hybridization diluent consisted of
6× saline-sodium phosphate-EDTA buffer, 5×
Denhardt's solution, 1% SDS, and 20 µg/ml of salmon sperm DNA.
Prehybridization took place for 3-5 h at 62°C, and
hybridization took place overnight at 62°C. cDNA templates were
randomly primed with
32P-radiolabeled CTP and the
Ready-To-Go DNA labeling kit (Pharmacia). The rat CTGF plasmid was
cloned in our laboratory (as described in Rat CTGF
cloning and sequencing). 36B4 (a human
ribosomal phosphoprotein), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), or 18S rRNA, using a fragment from a murine 18S rRNA plasmid
(American Type Culture Collection), were used as loading controls.
Labeled probes were separated from unincorporated nucleotides with the
TE Midi SELECT-D, G-50 spin columns (5 Prime Statistical Analysis. All data are
reported as means ± SE. ANOVA was used for comparisons between
treated and control groups for hydroxyproline data. A nonparametric
Student's t-test was used for
comparison of densitometric values from Northern blots. P values < 0.05 were considered
significant.
The sequence of the rat cDNA fragment that was amplified with RT-PCR
and ligated into the Bluescript vector is shown in Fig. 1. The cloned fragment of rat CTGF is 96%
homologous with that of human CTGF and 90% with that of mouse Fisp-12.
The homology between the rat sequence and that of human CTGF and mouse
Fisp-12 clearly identifies the cloned RT-PCR fragment as rat CTGF and validates its use as a probe template for Northern analysis.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
1 (TGF-
1) response element.
Because TGF-
1 expression is upregulated in several models of
fibroproliferative lung disease, we asked whether CTGF is also
upregulated in a murine lung fibrosis model and whether CTGF could
mediate some of the fibrogenic effects associated with TGF-
1. A
portion of the rat CTGF gene was
cloned and used to show that primary isolates of both murine and human
lung fibroblasts express CTGF mRNA in vitro. There was a greater than
twofold increase in CTGF expression in both human and murine lung
fibroblasts 2, 4, and 24 h after the addition of TGF-
1 in vitro. A
bleomycin-sensitive mouse strain (C57BL/6) and a bleomycin-resistant
mouse strain (BALB/c) were given bleomycin, a known lung fibrogenic
agent. CTGF mRNA expression was upregulated in the sensitive, but not
in the resistant, mouse strain after administration of bleomycin. In
vivo differences in the CTGF expression between the two mouse strains
were not due to an inherent inability of BALB/c lung fibroblasts to
respond to TGF-
1 because fibroblasts from untreated BALB/c mouse
lung upregulated their CTGF message when treated with TGF-
1 in
vitro. These data demonstrate that CTGF is expressed in lung
fibroblasts and may play a role in the pathogenesis of lung fibrosis.
1; collagen; mice
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
1 (TGF-
1)
has been shown to upregulate CTGF expression in vitro (8).
1 is known to
be increased in fibroproliferative dermal diseases, including
scleroderma, and a positive correlation between CTGF expression and
skin sclerosis has been reported (10, 11). In vitro studies have
demonstrated that TGF-
1 is the only known inducer of CTGF mRNA
expression (12). Dermal fibroblast CTGF expression is upregulated in
response to TGF-
1 through a novel TGF-
1 response element in the
CTGF promotor (8, 15). With the use of
a rat skin wound model, it has been shown that upregulation of CTGF
mRNA expression follows increases in TGF-
1 mRNA expression (12),
suggesting that CTGF is also induced by the increased expression of
TGF-
1 in vivo. Because dermal fibroblasts synthesize CTGF and also proliferate in response to CTGF, others (7) have proposed
that CTGF may act as an autocrine mitogen induced by TGF-
1.
1 expression is upregulated in many models of fibrotic
lung disease, including murine models of bleomycin-induced lung
fibrosis (18, 19). We postulated that if CTGF expression plays an
important role in lung fibrogenesis, it would be increased during
development of the disease. To test this hypothesis, we exposed
bleomycin-sensitive (C57BL/6) and bleomycin-resistant (BALB/c) strains
of mice to bleomycin via intratracheal injection and probed for CTGF
mRNA expression at 8, 16, and 30 days after bleomycin administration. In addition, we showed that CTGF was upregulated in both human and
mouse lung fibroblasts after treatment with TGF-
1 in vitro.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-gAg-CTT-TCT-ggC-TgC-ACC-3' and
5'-
T-CTC-Cgt-ACA-TCT-TCC-Tg-3'
and were ordered from Oligos ETC. The underlined sequences represent
the BamH I and Pst I
restriction endonuclease sites, respectively.
-smooth muscle actin (clone
1A4, Sigma), anti-desmin (clone 33, Dakopatt), anti-cytokeratin (clone
CAM 5.2, Becton Dickinson), anti-macrophage (clone Mac378, Dakopatt),
and anti-CD32 (rat anti-mouse, which recognizes natural killer cells,
monocytes, macrophages, granulocytes, mast cells, and B lymphocytes;
PharMingen). Mouse IgG was also used as a control for the mouse
monoclonal primary antibodies. The secondary antibodies used for
immunohistochemical identification were biotinylated goat anti-rat and
goat anti-mouse (Jackson Immunologicals). One hundred percent of the
HLFs and MLFs stained positively for vimentin, 100% of the MLFs and
89% of the HLFs stained for
-smooth muscle actin, 22% of the MLFs
and 5% of the HLFs stained for desmin, less than 1% of the MLFs
stained for CD32, none of the HLFs were positive for anti-macrophage
staining, and neither the MLF nor the HLF isolates stained for keratin.
Therefore, we believe that the human and murine lung cells isolated
were predominantly myofibroblasts.
70°C for later
Northern analysis or hydroxyproline measurement. The right lung was
perfused with 10% neutral Formalin through a tracheal cannula at a
pressure of 15 cmH2O for 15 min.
The trachea was then clamped, and the right lung was removed from the
chest cavity and placed in fresh fixative overnight at 4°C before
processing in paraffin.
70°C freezer and homogenized in 5% trichloroacetic
acid (1:9 wt/vol). The homogenized samples were centrifuged for
10 min at 4,000 g, and the pellet was
washed twice with distilled water, then hydrolyzed for 16 h at
100°C in 6 N HCl. Hydrolysates were assayed colorimetrically at 561 nm with dimethylaminobenzaldehyde to quantify hydroxyproline. Hydroxyproline content is expressed as micrograms hydroxyproline per
left lung.
70°C freezer and
immediately dissociated in 2 ml of 4.2 M GTC with the Tissue Tearor
(Biospec Products). The samples were transferred to Corex tubes and
centrifuged at 10,000 g to remove
unwanted precipitate. The supernatant was gently layered over 1 ml of
5.7 M CsCl, and the samples were spun in a Beckman table top centrifuge
at 85,000 rpm for 5 h at 21°C. The supernatant was carefully
aspirated and discarded. The remaining pellet was suspended in 200 µl
of water. Thereafter, 8 µl of 5 M NaCl along with 500 µl of
ice-cold ethanol were added to precipitate the RNA. The RNA was washed
once with cold 80% ethanol. The pellet was resuspended in diethyl
pyrocarbonate-treated water and stored at
70°C
until use.
3 Prime, Boulder,
CO). Hybridized membranes were washed with 2× SSC with 0.5% SDS,
then 0.2× saline-sodium citrate buffer with with 0.5% SDS at 37 and 62°C. The hybridized autoradiographic signal was detected with Biomax Film (Kodak) and quantified on a densitometric scanner (Bio-Rad).
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Fig. 1.
Comparison of rat connective tissue growth factor (CTGF) cDNA sequence
with that of human CTGF and mouse Fisp-12. Mismatched bases are
shaded.
Primary isolates of HLFs constitutively expressed CTGF mRNA in vitro as
shown in Fig.
2A. After
exposure to recombinant TGF-1 (10 ng/ml), there was an upregulation
of CTGF expression at the earliest time point tested (2 h). We chose to
use 10 ng/ml of TGF-
1 because this concentration has been shown to
induce expression of CTGF in human foreskin fibroblast cultures (7).
The TGF-
1-induced increase in CTGF mRNA remained elevated while in
the presence of TGF-
1 for at least 24 h. These Northern analysis
results were duplicated with an HLF isolate from a second donor. The
combined results of three in vitro experiments from two HLF isolates
are shown in Fig. 2B. These data
demonstrate a significant upregulation of CTGF message in HLFs exposed
to TGF-
1 in vitro. We also performed Northern analysis for CTGF
expression in primary cultures of human epithelial cells and in A549
cells (a malignant human lung epithelial cell line) but found no CTGF
expression in the presence or absence of TGF-
1 (data not shown).
Figure 3 demonstrates that neither tumor
necrosis factor (TNF-
; 10 ng/ml), interleukin-1
(IL-1
; 1 ng/ml), nor PDGF-AB (10 ng/ml) upregulates CTGF expression
4 h after exposure, whereas TGF-
1 (10 ng/ml) does. Thus, of the cytokines tested to date, only TGF-
1 is capable of upregulating CTGF
mRNA expression.
|
|
Figure 4 demonstrates the difference in lung collagen accumulation, as assessed by hydroxyproline measurement, between groups of five C57BL/6 and BALB/c mice 8, 16, and 30 days after the administration of bleomycin or saline control. There was a statistically significant increase in lung collagen accumulation in the bleomycin-treated C57BL/6 mice compared with C57BL/6 mice treated with saline and BALB/c mice treated with bleomycin at 16 and 30 days. Furthermore, the hydroxyproline content in bleomycin-treated C57BL/6 mice continued to increase over time after exposure, whereas this did not occur in the BALB/c strain. There were no statistical differences in lung hydroxyproline content between the saline-treated C57BL/6 and BALB/c mice at 16 and 30 days after treatment (16-day C57BL/6, 48.4 ± 3.6 µg; 16-day BALB/c, 39.7 ± 2.4 µg; 30-day C57BL/6, 62.9 ± 4.4 µg; 30-day BALB/c, 61.2 ± 3.2 µg). Both strains of mice had a small, but statistically significant, increase in lung hydroxyproline content in bleomycin-treated mice compared with their strain-specific saline control mice at 8 days. Histology of lung tissue sections from these two strains at 16 and 30 days after bleomycin exposure correlated with the hydroxyproline data and showed subpleural fibrosis in the C57BL/6 mice and near normal architecture in the BALB/c mice (data not shown).
|
Mouse lung RNA was isolated from both the C57BL/6 and BALB/c strains 8, 16, and 30 days after the administration of bleomycin or an equal volume of saline as a negative control. As shown in Fig. 5, there was an upregulation in the CTGF mRNA expression (3.85 ± 2.03-fold saline control) in the C57BL/6 lungs at the earliest time point tested (8 days) that preceded histological evidence of collagen deposition. CTGF mRNA expression was statistically elevated in bleomycin-treated C57BL/6 lungs compared with C57BL/6 saline controls at 16 and 30 days after exposure (the bleomycin-treated and saline-treated ratios were 2.45 ± 0.19- and 2.46 ± 0.46-fold saline control, respectively). In contrast, CTGF mRNA expression was not upregulated in the BALB/c strain at any of the time points tested after bleomycin exposure. The 18S ribosomal RNA band was used as a loading control.
|
Because there was a difference in the expression of CTGF mRNA between
the two strains in vivo, we investigated whether or not there would be
a difference in CTGF mRNA expression exhibited in MLFs isolated from
the two strains in vitro after the addition of TGF-1
(10 ng/ml). Figure 6 shows that both
C57BL/6 and BALB/c mouse strains upregulate their CTGF mRNA expression
to a similar magnitude and over a similar time frame in response to
TGF-
1.
|
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DISCUSSION |
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The data presented in this paper represent the first studies designed
to determine whether human and murine lung fibroblasts express CTGF, as
reported previously for dermal fibroblasts (12). We found
that both human and mouse lung mesenchymal cells express CTGF message
constitutively in vitro (Figs. 2 and 5). Our finding that TGF-1
upregulates CTGF mRNA expression in murine and human lung fibroblasts
in vitro is similar to findings others have described with human dermal
fibroblasts. We found that other proinflammatory cytokines released
early in lung injury, e.g., TNF-
and IL-1
, did not upregulate
CTGF under the conditions we employed. The lack of effect of early
inflammatory cytokines (TNF-
and IL-1
) on CTGF expression has
not, to our knowledge, been shown previously using any source of
fibroblasts. Thus TGF-
1 remains the only known inducer of CTGF mRNA
expression. A novel TGF-
1 motif has been described in the
CTGF promotor (8). Once anti-CTGF
antibodies are commercially available, studies that demonstrate, using
Western analysis, that CTGF peptide expression is increased along with CTGF mRNA would be important.
Like TGF-1, CTGF is capable of inducing normal rat kidney (NRK)
fibroblast proliferation under serum-free conditions in vitro (15).
However, unlike TGF-
1, CTGF is not capable of inducing anchorage-independent growth of NRK fibroblasts (15). In those NRK
studies, anti-CTGF antibodies and antisense oligomers were used to show
that TGF-
1-induced anchorage-dependent growth is mediated by CTGF. A
recent preliminary study reported that CTGF is a mitogen for HLFs in
vitro (22). Because our data demonstrate that HLFs express CTGF mRNA,
CTGF has the potential to act as a mitogen in vivo through an autocrine
mechanism.
TGF-1 has been shown to be a mitogen for smooth muscle cells and
human foreskin fibroblasts in vitro through a PDGF-dependent pathway
(13, 25). At low concentrations, TGF-
1 stimulates proliferation
through induction of PDGF-A chain synthesis. At higher concentrations,
TGF-
1 may inhibit mesenchymal cell proliferation by decreasing
transcription of the PDGF-
receptor, to which PDGF-A chain binds
(2). Investigators have studied the mitogenic effect of TGF-
1 on 3T3
cells from mouse embryos with a homozygous deletion of the PDGF-
receptor (23). They found that PDGF-
receptor-deficient 3T3 cells
still proliferate in response to TGF-
1, although the magnitude of
the mitogenic response is reduced by about one-half. Other
investigators have shown that at least 70% of the mitogenic activity
of TGF-
1 for cells can be blocked with an anti-PDGF antibody (2). It
is possible that these studies may be reconciled by the observation
that a polyclonal anti-PDGF antibody is capable of recognizing CTGF on
Western analysis, although there is minimal homology between CTGF and
PDGF (11, 26). Alternatively, there may be inherent differences in
response to various mitogens between embryonic 3T3 cell clones and more
differentiated smooth muscle and fibroblast isolates. At present,
TGF-
1 appears to act as a mitogen through both a PDGF-A
chain-dependent and a PDGF-independent CTGF-mediated pathway.
CTGF may also be responsible for mediating the collagen deposition
generally attributed to TGF-1. The addition of CTGF to cultured
dermal fibroblasts results in the upregulation of fibronectin and type
I collagen transcription (7). Upregulation of CTGF mRNA preceded the
phase of collagen deposition in a rat skin wound model (12) as well as
in our model of bleomycin-induced lung fibrosis. To our knowledge,
there are no published data regarding the effect of blocking CTGF on
collagen deposition in vivo.
We sought to determine whether there is a difference in CTGF expression between the bleomycin-sensitive (C57BL/6) and bleomycin-resistant (BALB/c) mouse strains after bleomycin exposure. There were no differences in lung CTGF mRNA expression between the strains in unexposed animals. However, bleomycin-sensitive C57BL/6 mice showed an increase in whole lung CTGF message expression after bleomycin administration, whereas bleomycin-resistant BALB/c mice did not. The greater than twofold increase in CTGF expression observed in mice that developed fibrosis is likely to underestimate the induction of this gene by bleomycin, because the areas of lung fibrosis were focal and represented less than 25% of the lung parenchyma. These data are consistent with a role for CTGF in lung fibrogenesis.
There was no difference in the upregulation of CTGF mRNA expression
between cultured lung C57BL/6 and BALB/c fibroblasts treated with
TGF-1. Thus the lack of CTGF induction in the BALB/c mice in vivo
apparently is not due to an intrinsic inability of the BALB/c MLFs to
upregulate CTGF expression in response to TGF-
1. However, others
have described the upregulation of TGF-
1 mRNA expression in the
lungs of these two murine strains after bleomycin exposure (1, 18, 19).
TGF-
1 is upregulated in C57BL/6 mice 7-14 days after
intratracheal bleomycin administration (18, 19). Baecher-Allan and
Barth (1) have performed semiquantitative PCR on lung RNA from
bleomycin-treated mice and found that TGF-
1 expression is
upregulated in both C57BL/6 and BALB/c strains, although there was a
sevenfold increase in the C57BL/6 mice compared with a threefold
increase for the BALB/c mice.
It is important to recognize that TGF-1 must be activated before it
can bind to its receptor. TGF-
1 may be activated by acid hydrolysis
or proteolytic cleavage (20). Therefore, TGF-
1 mRNA expression alone
may not reflect the activity of TGF-
1 in lung injury models. We have
previously shown that there is a difference in lung inflammation
between C57BL/6 and BALB/c mice after bleomycin administration (17).
The observed differences in lung inflammation between the two strains
may be due to the lack of TNF-
upregulation in the BALB/c strain
compared with the C57BL/6 strain after treatment with bleomycin (17).
It is also possible that the lack of lung inflammation in the BALB/c
strain may result in decreased conversion of TGF-
1 to its active
form.
Earlier differences have also been described between mouse strains in
the sequence of events that occur between exposure to bleomycin and the
development of lung inflammation and eventual lung fibrosis. A greater
than 2.5-fold increase in bleomycin hydrolase activity, an enzyme that
reduces the efficiency of bleomycin-mediated oxygen radical formation
to less than 1%, is present in the lungs of BALB/c mice compared with
C57BL/6 mice (6). Also, a comparative lack of poly(ADP-ribose)
polymerase activity, as a reflection of DNA breakage, has been
described in BALB/c mice when contrasted to C57BL/6 mice after
bleomycin exposure (9). A possibility to further explain differences
between murine strains in response to bleomycin is that the BALB/c mice
do not suffer the same degree of DNA damage in response to bleomycin as
do C57BL/6 mice. Therefore, less TNF- is released, resulting in less
inflammation and TGF-
1 activation, which in turn leads to less CTGF
expression and less fibrosis.
The concept of cytokine networks has been put forward to explain the
pathogenesis of pulmonary fibrosis (5). The present study suggests that
CTGF is upregulated by TGF-1 and has the potential to mediate some
of the mitogenic and collagen deposition effects attributed to TGF-
1
in the lung. The upregulation of CTGF in the bleomycin-sensitive
strain, but not in the resistant strain, at time points preceding and
during the fibrotic phase of the lung injury adds credence to the
hypothesis that CTGF is playing a significant role in the pathogenesis
of bleomycin-induced lung fibrosis.
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ACKNOWLEDGEMENTS |
---|
The authors thank Mary Cheles of the Tulane Medical Center Anatomic Histopathology Laboratory for technical assistance.
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants K08-HL-03374 (to J. A. Lasky) and K08-HL-03569 (to L. Ortiz); National Institute of Environmental Health Sciences Grant R01-ES-06766 (to A. R. Brody); and American Lung Association Grant RG 183-N (to J. A. Lasky). We also acknowledge the continued support of the Tulane/Xavier Center for Bioenvironmental Research and the Tulane Cancer Center.
Address for reprint requests: J. A. Lasky, Tulane Univ. Medical Center, 1430 Tulane Ave., SL-9, New Orleans, LA 70112.
Received 13 August 1997; accepted in final form 13 April 1998.
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