1 Section of Pulmonary Diseases, Critical Care, and Environmental Medicine, Departments of 2 Pharmacology and 3 Pathology and the Lung Biology Program, Tulane University Medical Center, New Orleans, Louisiana 70112
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ABSTRACT |
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The present study was
undertaken to investigate the effects of treatment with the
angiotensin-converting enzyme (ACE) inhibitor enalapril in a mouse
model of pulmonary hypertension induced by bleomycin. Bleomycin-induced
lung injury in mice is mediated by enhanced tumor necrosis factor-
(TNF) expression in the lung, which determines the murine strain
sensitivity to bleomycin, and murine strains are sensitive (C57BL/6) or
resistant (BALB/c). Bleomycin induced significant pulmonary
hypertension in C57BL/6, but not in BALB/c, mice; average pulmonary
arterial pressure (PAP) was 26.4 ± 2.5 mmHg (P < 0.05) vs. 15.2 ± 3 mmHg, respectively. Bleomycin treatment
induced activation of nuclear factor (NF)-
B and activator protein
(AP)-1 and enhanced collagen and TNF mRNA expression in the lung of
C57BL/6 but not in BALB/c mice. Double TNF receptor-deficient mice (in
a C57BL/6 background) that do not activate NF-
B or AP-1 in response
to bleomycin did not develop bleomycin-induced pulmonary hypertension
(PAP 14 ± 3 mmHg). Treatment of C57BL/6 mice with enalapril
significantly (P < 0.05) inhibited the development of
pulmonary hypertension after bleomycin exposure. Enalapril treatment
inhibited NF-
B and AP-1 activation, the enhanced TNF and collagen
mRNA expression, and the deposition of collagen in bleomycin-exposed
C57BL/6 mice. These results suggest that ACE inhibitor treatment
decreases lung injury and the development of pulmonary hypertension in
bleomycin-treated mice.
pulmonary hypertension; tumor necrosis factor; nuclear factor-B; activator protein-1; angiotensin II
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INTRODUCTION |
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PULMONARY HYPERTENSION IS a prominent feature of interstitial lung diseases (ILD), including systemic sclerosis, granulomatous diseases, and idiopathic pulmonary fibrosis (IPF) (47). In ILD, the presence of pulmonary hypertension significantly alters survival (47). In these disorders, pulmonary vascular pathology characterized by concentric intimal fibrosis, medial hypertrophy, and adventitial fibrosis can be identified (47). Traditionally, it has been assumed that hypoxic vasoconstriction is responsible for the development of pulmonary hypertension associated with ILD (47). Growing evidence suggests that the upregulation of cytokines and growth factors in the lung contribute to the development of the pulmonary hypertension associated with these diseases (47).
Bleomycin induction of lung injury in mice is a well-established model
of ILD, resulting in pulmonary fibrosis (14, 43). Endotracheal instillation of bleomycin in mice is followed by upregulated expression of lung cytokines, development of lung inflammation, and accumulation of collagen in the lung (14, 43). Among the cytokines upregulated in the lung of
bleomycin-treated mice, tumor necrosis factor (TNF)- plays a
fundamental role in the pathogenesis of bleomycin-induced pulmonary
fibrosis (32, 36, 37). The upregulation of TNF expression
correlates with the murine strain sensitivity to bleomycin
(32). Inbred mice of the C57BL/6 strain, which are
sensitive to bleomycin, upregulate lung TNF expression, develop lung
inflammation, and accumulate collagen in their lung in response to
bleomycin exposure (32-34). In contrast, BALB/c mice,
a strain resistant to bleomycin, do not upregulate lung TNF expression
or develop pulmonary fibrosis in response to bleomycin
(32-34).
ANG II is the product of the proteolytic cleavage of ANG I by angiotensin-converting enzyme (ACE). Increased concentrations of ANG II are found in bleomycin-treated or irradiated animals preceding the development of fibrosis (20, 44). Elevated levels and activity of ACE have been found in serum and bronchoalveolar lavage fluid of patients with fibrotic lung diseases such as sarcoidosis, asbestosis, silicosis, and IPF (8, 19, 45) and in animal models of pulmonary hypertension (3, 24). The biological effects of enhanced ACE expression in the injured lung appear to be the result of a local rather than a global action (38, 41). This is suggested by data demonstrating isolated enhanced expression of the ACE gene and protein at the site of cell proliferation in the intima of pulmonary arteries of patients with pulmonary hypertension (38, 41).
In animal models of pulmonary hypertension, the inhibition of ACE
attenuates the fibroproliferative effects of chronic hypoxia and
partially reverses the vascular neointimal formation and smooth muscle
cell proliferation (27, 29). The mechanisms responsible for the ability of ACE inhibitors to decrease the fibroproliferative reaction observed in pulmonary hypertension are not completely understood. Recently, we have shown that bleomycin-induced lung injury
is characterized by nuclear factor (NF)-B activation in mouse lung
(30). This enhanced NF-
B activation appears to promote expression of inflammatory mediators and collagen deposition, and
interventions that inhibit NF-
B activation have been shown to
ameliorate bleomycin-induced lung injury (7, 9). Recent studies have shown that ACE inhibitors are capable of regulating the
activation of the NF-
B transcription factor in animal models of
atherosclerosis (15).
Therefore, we hypothesized that ACE inhibition would decrease
activation of transcription factors and inhibit TNF and collagen expression in the mouse lung, thus ameliorating bleomycin-induced lung
injury and pulmonary hypertension. In the present investigation, we
studied the effect of enalapril on the activation of the transcription factors NF-B and activator protein (AP)-1, the expression of TNF and
1(I)-procollagen mRNA, the induction of inflammation, and the accumulation of collagen in the lung of bleomycin-treated mice.
We have also measured pulmonary hemodynamics, by right heart catheterization, in intact spontaneously breathing mice.
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METHODS |
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Chemicals. Stock solutions (5 U/ml) of bleomycin (Blenoxan; kindly donated by Bristol-Meyer-Squibb Pharmaceuticals, Princeton, NJ) were prepared immediately before use with endotoxin-free water. All other chemicals were of the highest grade commercially available (specific vendors and their locations denoted).
Animals.
Animal protocols were approved by the Tulane University Committee on
the Use and Care of Animals. Specific pathogen-free female C57BL/6 and
BALB/c (Charles River Laboratories, Kingston, NY) mice weighing
20-25 g (6-10 wk old) were housed in pathogen-free cabinets
and provided with water ad libitum. Mice genetically deficient in both
p55 and p75 receptors [p55(/
)-p75(
/
)] were generated, on a
C57BL/6 genetic background, by gene targeting in embryonic stem cells
at Immunex (Seattle, WA) and have been described previously (33,
35).
Bleomycin exposure.
Animals were anesthetized with intraperitoneal tribromoethanol (250 mg/kg; Aldrich, Milwaukee, WI) and exposed to bleomycin as previously
described (33). Briefly, the trachea was exposed, and 4 U/kg bleomycin in 0.05 ml of 0.9% NaCl was slowly instilled in the
tracheal lumen. Control mice received the same volume of sterile
saline. After exposure, the skin incision was closed, and the animals
were allowed to recover on a warming plate. Hemodynamic evaluations
were performed 14 days after bleomycin exposure (33). After hemodynamic evaluation, the thorax was opened, and the descending aorta was severed. The left lungs were removed and stored at 80°C for subsequent analysis. The right lungs were perfused with formalin in
situ as described below.
Enalapril treatment. In preliminary studies, a dose of 5 mg/kg enalapril (Merck, West Point, PA) was found to attenuate 95% of the systemic and pulmonary vascular pressor response to ANG I (dose range 0.1-1 µg/kg iv). Enalapril was given daily, at a dose of 5 mg/kg as an intraperitoneal injection, for 14 days after bleomycin exposure. The first enalapril dose was given 1 h after the endotracheal administration of bleomycin.
Measurements of pulmonary vascular responses and cardiac output. The techniques for measurement of pulmonary hemodynamics have been described previously (4). After administration of bleomycin or vehicle (14 days), mice were anesthetized with thiopentobarbital (85-95 µg/g ip) and ketamine (3 µg/g ip) and placed on a thermoregulated surgical table. Body temperature was monitored and maintained at 37°C with a water-jacketed heating blanket. The trachea was cannulated (PE-90 tubing) to maintain a patent airway, and the animals breathed air enriched with 95% O2-5% CO2. A femoral artery was cannulated for the measurement of systemic arterial pressure. Systemic arterial pressure was measured with a Viggo-Spectramed transducer (Viggo Spectramed, Oxnard, CA) attached to a polygraph (model 7; Grass Instruments, Quincy, MA). Heart rate was monitored electronically from the systolic pressure pulses with a tachometer (model 7P44A; Grass).
Pulmonary arterial pressure (PAP) was measured in anesthetized mice with the use of a single-lumen catheter (Nu-Med, Hopkinton, NY). The catheter (145 mm in length, 0.25 mm OD) has a specially curved tip to facilitate passage through the right heart, main pulmonary artery, and the left or right pulmonary artery. Immediately after placement of the pulmonary catheter (30 min in average), pressure in the main pulmonary artery was measured with a pressure transducer (Schneider/Namic, Glenns Falls, NY), and mean PAP was derived electronically and recorded continuously. For the determination of pulmonary arterial wedge pressure, the catheter was advanced to the left or right pulmonary artery and wedged with continuous measurement of the pressure waveform. Cardiac output was measured by the thermodilution technique. A known volume (20 µl plus catheter dead space) of 0.9% NaCl solution at 23°C was injected in the right atrium, and changes in blood temperature were measured in the root of the aorta. A cardiac output computer (Cardiotherm 500; Columbus Instruments, Columbus, OH) equipped with a small animal interface was used. Thermodilution curves were recorded on a chart recorder (Western Graphtec, Irvine, CA), and pulmonary and systemic arterial pressures were monitored continuously. Catheter placement was verified at postmortem examination. Arterial blood gases and pH were monitored with a Corning 178 analyzer with a 50-µl blood sample withdrawn through a femoral artery catheter and were within the physiological range. In control-treated mice arterial PO2 (PaO2), arterial PCO2 (PaCO2), and pH averaged 450 Torr, 35 Torr, and 7.45 units, respectively. In bleomycin-treated mice PaO2, PaCO2, and pH averaged 124 Torr, 28 Torr, and 7.42 units, respectively.cDNA probes.
cDNA templates used for experiments are as follows: the 1.101-kb murine
TNF was obtained for American Type Culture Collection (Rockville, MD)
and has been described elsewhere (32, 33). The murine
1(I)-procollagen cDNA was a kind gift from Dr. Eero Vuorio (Turku University, Turku, Finland) and has been described previously (23). The murine 18S cDNA was used for loading
control, as previously described (32, 33).
Northern analysis.
After hemodynamic studies, the mice were killed by exanguination. Lung
tissue was isolated and immediately snap-frozen in liquid nitrogen.
Total RNA was extracted from the lung using a cesium chloride method
for Northern analysis (32, 33). RNA was separated by
electrophoresis (20 µg/lane) on a 1.2% formaldehyde-agarose gel,
transferred to an Immobilon-N transfer membrane (Millipore, Bedford,
MA), and hybridized overnight at 62°C with
[32P]dCTP-labeled (ICN, Irvine, CA) random-primed cDNA
probes as previously described (32-34). Membranes
were probed first for TNF, then stripped and reprobed for
1(I)-procollagen and, as a loading control, for 18S.
Blots were developed for 72 h using Biomax films and intensifying
screens (Kodak). To quantitate mRNA, membranes were exposed to a Fuji
PhosphorImager (Fujix BAS 1000; Fuji, Stamford, CT) plate
overnight and scanned. Quantitative analysis was determined with the
use of McBAS 2.5 software (Fuji USA). For each mRNA band, the results
were normalized to the internal control (18S) and expressed as a degree
of increase between the bands for the control and the bands for the
bleomycin- and bleomycin- and enalapril-treated animals.
Lung hydroxyproline content. Lung hydroxyproline concentration was determined spectrophotometrically according to the method of Kivirikko et al. (18). Briefly, left lungs from control-, enalapril-, bleomycin-, and bleomycin- and enalapril-treated mice were homogenized in 5% trichloroacetic acid (1:9 wt/vol) and centrifuged for 10 min at 4,000 g. The pellet was then washed two times with distilled water and hydrolyzed for 16 h at 100°C in 6 N HCl. Hydroxyproline in the hydrolyzate was assessed colorimetrically at 561 nm with p-dimethylaminobenzaldehyde. Hydroxyproline content was computed as micrograms hydroxyproline per whole left lung.
Lung morphology and evaluation of right ventricular hypertrophy. The heart was perfused with 0.9% NaCl to remove residual blood, and the right lung was fixed in situ for 2 h by intratracheal instillation of 10% neutral formalin (Sigma, St. Louis, MO) at a constant pressure of 30 cmH2O and was preserved in fixative for 24 h. Lung tissues were then sectioned sagittally and embedded in paraffin. Sections (4 µm thick) were generated and mounted on positively charged slides (Fisher Scientific, Pittsburgh, PA). Slides were stained with hematoxylin-eosin for light microscopic examination. Immediately after death, hearts were resected to evaluate right ventricular hypertrophy. The atria were removed up to the plane of the atrial-ventricular valves. The right ventricle (RV) free wall was then dissected free of the left ventricle (LV) and septum. The RV and LV plus septum were weighed and the RV-to-LV + septum ratio was calculated.
Electrophoretic mobility shift assay.
To generate nuclear extracts, lungs were minced on ice and homogenized
with a Dounce homogenizer in 2 ml buffer [0.32 M sucrose, 3 mM
magnesium chloride, 0.5 mM EGTA, 1 mM HEPES, 0.1% Triton X, 0.5 mM
phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, and 10 µg/ml leupeptin]. Homogenates were filtered and centrifuged at 4°C
at 700 g for 10 min. Pellets were resuspended in 200 µl lysis buffer [20 mM HEPES, 125 nM sodium chloride, 5 mM magnesium chloride, 12% vol/vol glycerol, 5 mM dithiothreitol (DTT), 0.5 mM
PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin], sonicated on
ice for 15 s, centrifuged at 14,000 rpm at 4°C, and frozen at
70°C until used.
Statistics. All values are expressed as means ± SE. Differences between murine strains were analyzed using ANOVA with Fisher's protected least-significant difference test for pair-wise comparison (Statview 4; Abacus Concept, Berkeley, CA). A P value <0.05 was considered significant.
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RESULTS |
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Enalapril treatment blocks the systemic and pulmonary vascular hemodynamic effects of ANG I. Enalapril (5 mg/kg ip) significantly decreased the mean arterial pressure in C57BL/6 mice. The mean arterial blood pressure of control- and enalapril-treated mice was 94 ± 9 and 83 ± 7 mmHg, respectively. Enalapril attenuated 95% of the systemic and pulmonary vascular pressor response to ANG I in C57BL/6 mice. Control-treated mice responded to ANG I (doses of 0.3 and 1 µg/kg iv) with significant increases in arterial blood pressure (+38.3 ± 8 and +48 ± 8 mmHg, respectively). This pressor response to ANG I was significantly decreased by enalapril treatment (+6 ± 4 and +7 ± 5 mmHg, respectively).
Enalapril treatment also inhibited the pressor effect of ANG I on the pulmonary vasculature in C57BL/6 mice. The mean pulmonary arterial blood pressure of control-treated mice was 12.7 ± 1 mmHg. Control-treated mice responded to ANG I (doses of 0.3 and 1 µg/kg iv) with significant increases in mean pulmonary pressure (+3.4 ± 0.6 and +4.3 ± 0.4 mmHg, respectively). These pressor responses were significantly (P < 0.001) inhibited by enalapril treatment (+0.8 ± 0.4 and +0.7 ± 0.6, respectively).Bleomycin induces pulmonary hypertension in C57BL/6, but not in
BALB/c, mice.
Bleomycin treatment resulted in the development of pulmonary
hypertension in a strain-specific manner (Figs.
1 and 2).
Compared with vehicle-treated C57BL/6 mice, bleomycin treatment
significantly increased right atrial pressure (RA) (4.2 ± 0.4 vs.
7.9 ± 0.8 mmHg), PAP (13.2 ± 1.9 vs. 26.4 ± 2.5 mmHg), pulmonary vascular resistance (PVR; 0.9 ± 0.08 vs. 2 ± 0.15 mmHg · ml1 · min
1),
and RV free wall weight (0.23 ± 0.02 vs. 0.39 ± 0.04) in
C57BL/6 mice (Figs. 1 and 2). In contrast, bleomycin exposure
did not significantly alter RA (4.2 ± 0.8 vs. 4.3 ± 0.9 mmHg), PAP (13.2 ± 2.1 vs. 15.2 ± 3 mmHg), PVR (0.90 ± 0.1 vs. 0.92 ± 0.11 mmHg · ml
1 · min
1), or RV
free wall weight (0.23 ± 0.02 vs. 0.21 ± 0.05) in BALB/c mice compared with vehicle-treated BALB/c mice (Fig. 2). Bleomycin did
not significantly alter cardiac output in either murine strain (Fig.
2).
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Bleomycin-induced pulmonary hypertension is a TNF receptor-mediated
event.
Because bleomycin-induced pulmonary hypertension develops only in the
C57BL/6 mouse strain, compared with the BALB/c strain, which is similar
to what has been previously reported for bleomycin-induced TNF
expression in the lung, we studied whether TNF interaction with its
receptors was an important determinant of the changes in pulmonary
hypertension. To test this, we exposed double [p55(/
)-p75(
/
)] TNF receptor-deficient mice (developed on a C57BL/6 genetic background) to bleomycin and measured the pulmonary pressure and vascular resistance in these mice. As seen in Figs. 1 and 2 and in contrast to
bleomycin-exposed C57BL/6 mice, bleomycin exposure did not significantly alter RA (4.1 ± 0.8 vs. 3.9 ± 0.9 mmHg), PAP
(13.4 ± 2.1 vs. 14 ± 3 mmHg), PVR (0.91 ± 0.1 vs.
0.89 ± 0.11 mmHg · ml
1 · min
1), or RV
free wall weight (0.23 ± 0.04 vs. 0.22 ± 0.03) in
p55(
/
)-p75(
/
) TNF receptor-deficient mice compared with
p55(
/
)-p75(
/
) TNF receptor-deficient mice exposed to vehicle
alone (Figs. 1 and 2).
TNF receptors mediate NF-B and AP-1 activation in response to
bleomycin.
An important effect of the interaction of TNF with its receptors is the
activation of transcription factors (such as NF-
B and AP-1) that
regulate the expression of TNF-sensitive genes such as collagen, matrix
metalloproteinases, and other cytokines involved in fibrosis such as
transforming growth factor (TGF)-
(31, 32, 50). Gel
shift electrophoresis of nuclear extracts obtained from the lungs of
bleomycin-treated mice 14 days after exposure demonstrated evidence of
NF-
B activation in the lungs from C57BL/6 mice. In contrast, no
evidence of NF-
B activation was found in nuclear extracts isolated
from the lungs of BALB/c or double [p55(
/
)-p75(
/
)] TNF
receptor-deficient mice exposed to bleomycin (Fig.
3). The enhanced NF-
B activation
observed in bleomycin-exposed C57BL/6 mice was found as early as 30 min after bleomycin exposure (data not shown). The specificity of the
observed NF-
B binding was demonstrated by the fact that the binding
was inhibited by addition of a nonlabeled NF-
B oligonucleotide. Also
the use of an antibody specific to the p50, but not the p65, subunit of
NF-
B caused a shift of the NF-
B complexes (Fig. 3).
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Enalapril ameliorates bleomycin-induced lung injury in C57BL/6
mice.
To evaluate the effects of persistent ACE inhibition on
bleomycin-induced lung injury and bleomycin-induced pulmonary
hypertension in bleomycin-sensitive (C57BL/6) mice, we studied the
effects of enalapril treatment on the bleomycin-induced loss of weight in C57BL/6 mice, as shown in Table 1.
Bleomycin treatment induced a significant (P < 0.05)
loss in body weight (measured 14 days after intratracheal exposure) in
C57BL/6 mice (4.1 ± 0.3 g). No significant weight loss was
found in control- or enalapril-treated mice exposed to bleomycin
(21.7 ± 0.3 and 20.4 ± 0.2 g, respectively).
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Enalapril inhibits bleomycin-induced pulmonary hypertension in
C57BL/6 mice.
As described above, enalapril treatment inhibited the bleomycin-induced
remodeling of the pulmonary circulation in C57BL/6 mice (Fig. 5). The
effects of enalapril treatment on the bleomycin induction of pulmonary
hypertension in C57BL/6 mice are shown in Table
2. Enalapril treat- ment significantly
(P < 0.05) attenuated the increases in RA, PAP, and
PVR observed in C57BL/6 mice in response to bleomycin. Enalapril
treatment did not alter the mean pulmonary artery wedge pressure or the
cardiac output in bleomycin-treated mice.
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Enalapril treatment inhibits TNF mRNA expression in the lungs of bleomycin-treated C57BL/6 mice. Because bleomycin-induced lung injury correlates with enhanced TNF expression in the mouse lung, we evaluated whether enalapril treatment decreased TNF expression in the lungs of bleomycin-treated C57BL/6 mice. Bleomycin exposure resulted in an enhanced TNF mRNA expression in the lungs of C57BL/6 mice 14 days after bleomycin exposure, as assessed by Northern analysis (Fig. 7A). Compared with control-treated animals (1.8 ± 0.2 AU), bleomycin-treated mice demonstrated a significant (P < 0.05) increase (2.9 ± 0.4 AU) in TNF mRNA expression in their lungs (Fig. 7C). This enhanced TNF expression was significantly inhibited (1.1 ± 0.3 AU) by enalapril treatment (Fig. 7C).
Enalapril inhibits NF-B and AP-1 activation in the lungs of
bleomycin-treated C57BL/6 mice.
We studied whether the inhibitory effect of enalapril on TNF and
collagen mRNA expression is associated with inhibition of the activity
of NF-
B and AP-1 transcription factors. As shown in Figs.
8 and 9,
bleomycin exposure resulted in enhanced NF-
B (Fig. 8A)
and AP-1 (Fig. 9A) activation in the lungs of C57BL/6 mice
14 days after exposure. In contrast, enalapril treatment significantly
inhibited NF-
B (Fig. 8B) and AP-1 (Fig. 9B)
activation in response to bleomycin.
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DISCUSSION |
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In this study, pulmonary hypertension is a prominent feature of ILD in humans (47). Here we demonstrate the development of pulmonary hypertension in a murine model of bleomycin-induced lung injury. Mice treated with bleomycin develop fibroproliferative responses characterized by enhanced expression of cytokines, infiltration of lung parenchyma by inflammatory cells, apoptosis of alveolar epithelial cells, fibroblast proliferation, upregulated collagen gene expression, and excess deposition of collagen in the lung (1, 10, 32, 36, 43). The sequences of events leading to bleomycin-induced pulmonary hypertension are less well understood. Traditionally, it has been thought that hypoxic vasoconstriction resulting from severe lung damage induces pulmonary vascular remodeling (47). However, a more plausible explanation is that endothelial and smooth muscle cell proliferation observed in the pulmonary circulation is induced as a result of the same inflammatory environment that is present in the lung parenchyma (47). Consistent with this hypothesis is our observation that the development of bleomycin-induced pulmonary hypertension correlates well with the murine strain sensitivity to bleomycin (32-34). C57BL/6 mice are sensitive, whereas BALB/c mice are resistant, to bleomycin-induced lung injury (32-34). In this study, we found that pulmonary hypertension develops in C57BL/6 mice exposed to bleomycin and that it is absent in BALB/c mice.
TNF plays a fundamental role in the pathogenesis of bleomycin-induced
lung injury, and antagonism of TNF with the use of anti-TNF antibodies
or soluble TNF receptors ameliorates bleomycin-induced lung injury
(32, 36, 37). We have previously reported that TNF
expression in the mouse lung is a major determinant of the difference
in murine strain sensitivity to bleomycin (32, 34). We
have reported that animals deficient in both TNF receptors [p55(/
)-p75(
/
)] are protected from bleomycin-induced lung injury (33). These double TNF receptor-deficient mice were
developed on a bleomycin-sensitive (C57BL/6) genetic background and
exhibit enhanced TNF expression in their lungs in response to bleomycin (33). In the present work, we report that TNF receptors
appear to also be important contributors to the development of
bleomycin-induced pulmonary hypertension, since double TNF
receptor-deficient mice are protected from bleomycin-induced pulmonary
hypertension (Fig. 3). These observations complement recent published
reports suggesting that TNF overexpression in the lung of rodents is
associated with lung inflammation and fibrosis and the spontaneous
development of pulmonary hypertension (6, 42).
An important finding of the present study is that treatment with the
ACE inhibitor enalapril significantly reduced bleomycin-induced lung
injury (Table 1 and Fig. 5) and prevented the development of
bleomycin-induced pulmonary hypertension (Table 2) in C57BL/6 mice. The
protection against bleomycin-induced lung injury and pulmonary
hypertension by enalapril was associated with reduced transcription
factor (NF-B and AP-1; Figs. 8 and 9) activation and expression of
inflammatory (TNF) and fibrogenic [
1(I)-collagen] genes (43). These effects of enalapril translated into
decreased lung inflammation, reduced collagen deposition, and decreased muscle thickness in the media of small vessels from bleomycin-treated mice (Fig. 5B). The selection of enalapril in the present
work was based on its long half-life, which allows for daily
administration. Whether similar results can be achieved by using other
ACE inhibitors or ANG II receptor antagonists may be determined in
future studies.
The mechanisms responsible for modulation of TNF and collagen gene
expression by enalapril in response to bleomycin appear to involve
attenuation of transcription factor activation in the lung. Prominent
among the transcription factors activated by bleomycin in the mouse
lung are NF-B and AP-1, which regulate the transcription of multiple
genes involved in the inflammatory and fibroproliferative responses
(2, 31, 50). Bleomycin has been proposed to activate NF-
B by promoting expression of inflammatory cytokines such as TNF
or via production of reactive oxygen species (7).
Consistent with this concept, our data demonstrate decreased NF-
B
and AP-1 activation in the lungs of bleomycin-resistant (BALB/c) or
double [p55(
/
)-p75(
/
)] TNF receptor-deficient mice.
Furthermore, our data also demonstrate that enalapril treatment reduced
TNF mRNA expression and NF-
B and AP-1 activation in the lungs of bleomycin-treated C57BL/6 mice. Therefore, these data suggest that
enalapril inhibition of TNF expression greatly reduced activation of
transcription factors and subsequently decreased the downstream effects
of TNF on fibrogenic genes (30-37). In addition to
enhancing TNF expression, bleomycin has been proposed to activate
NF-
B by increasing the production of reactive oxygen species
(7). The use of antioxidative agents inhibits
bleomycin-induced inhibitor factor
B (I
B) degradation, prevents
NF-
B activation, and decreases the expression of inflammatory
cytokines and the accumulation of collagen in response to bleomycin
(9, 12). The mechanisms leading to bleomycin-induced
activation of AP-1 are not well understood. Bleomycin has been shown to
activate the c-Jun NH2-terminal kinase, a member of the
mitogen-activated protein kinase (MAPK) family involved in AP-1
activation (40). However, the present work suggests that
most of the activation of AP-1 in the lungs of C57BL/6 mice can be
explained by the enhanced TNF expression observed in the lungs in
response to bleomycin. This is suggested by the fact that neither
BALB/c, which do not upregulate TNF (32, 33), nor double
[p55(
/
)-p75(
/
)] TNF receptor mice demonstrated enhanced AP-1
activation in response to bleomycin (Figs. 3 and 4).
The specific mechanisms by which enalapril attenuates NF-B and AP-1
activation are not completely understood (13). The most
accepted explanation to address the inhibitory effects of enalapril
treatment on the activation of NF-
B and AP-1 is that there may be
common elements between the signal transduction for ANG II and
signaling events leading to NF-
B and AP-1 activation. The ACE
inhibitor might in turn downregulate these elements. A potential target
is MAPK kinase (MAPKK), which has been shown to induce activation of
the I
B kinase kinase (IKK) kinases, leading to I
B phosphorylation
and NF-
B activation (16, 17, 26, 56).
Consistent with this hypothesis, we have demonstrated in preliminary
studies that endotracheal administration of bleomycin to mice is
followed by an enhanced activity of the IKK-
, but not IKK-
,
subunit of the IKK (30). Activation of the AT1
receptor is followed by receptor endocytosis and rapid tyrosine
phosphorylation of platelet-derived growth factor and epidermal growth
factor receptors that provide docking sites for the upstream activation of the Src family of tyrosine kinases and the formation of downstream complexes leading to Ras activation (13). This scaffolding
step facilitates activation of MAPK and can be inhibited by
AT1 receptor antagonists as described above (5,
25).
In addition to inhibiting the expression of inflammatory
mediators, enalapril may prevent the well-known effects of ANG II on
cell proliferation and apoptosis (21, 25, 46). In
general, activation of AT1 receptors mediates cell
proliferation (21, 25), whereas inhibition of cell growth
and induction of apoptosis follow activation of AT2
receptors (28, 46, 54). Therefore, it has been proposed
that the balance between the expression of these two receptors is an
important determinant of the response to ANG II (21). In
human lung fibroblasts, ANG II exerts a mitogenic response via
AT1 receptors, which are the only ANG II receptors present
on these cells (21). The mitogenic response of ANG II on
human lung fibroblasts appears to be mediated via an autocrine production of TGF- and can be inhibited by treatment of these cells
with the AT1 receptor antagonist losartan
(21). ANG II also induces DNA synthesis and cell growth in
human pulmonary artery smooth muscle cells (25). ANG II
mediates this mitogenic effect on smooth muscle cells by triggering
activation of MAPK, and pretreatment of these cells with the specific
MAPK inhibitor PD-98059 inhibits ANG II-induced DNA synthesis
(25). MAPK activation in response to ANG II is mediated
via AT1 receptors and can be readily inhibited by losartan
treatment (25). Our results demonstrating attenuated AP-1
activation in enalapril-treated C57BL/6 mice correlates well with these
observations, since activation of MAPKs constitutes the primary signal
leading to AP-1 activation (25).
In addition to the induction of cell proliferation, ANG II is also capable of inducing apoptosis (54). Excessive apoptosis has been closely linked to the pathogenesis of lung fibrosis and bleomycin-induced lung injury (10, 11, 34). Uhal and associates (49) reported that myofibroblasts isolated from fibrotic human lung secreted soluble mediators capable of inducing apoptosis in alveolar epithelial cells. These mediators were identified as angiotensinogen and its active peptide ANG II (52). In subsequent reports, these authors demonstrated that the ANG II-induced apoptosis in human and rat epithelial cells could be modulated by treatment with the ACE inhibitor captopril (48). Furthermore, these investigators reported that the capacity of TNF to induce apoptosis in epithelial cells depends on the ability of the target cells to generate ANG II de novo (51, 53). These observations are important, since we have indicated that TNF is required for the development of bleomycin-induced lung fibrosis in mice (1, 32, 33, 36). TNF induction of apoptosis depends on its interaction with TNF receptors, and double TNF receptor-deficient mice demonstrated significantly less apoptosis in alveolar macrophages than did C57BL/6 or BALB/c mice in response to bleomycin (34). In the present work, we found that double TNF receptor-deficient mice do not develop pulmonary hypertension in response to bleomycin. Therefore, it is possible that, by inhibiting TNF expression, enalapril modulates the induction of TNF-mediated apoptosis in the endothelium of pulmonary vessels. This effect may be important, since it has been reported that the ability of the endothelial cell to survive apoptosis confers a survival advantage that allows for the uncontrolled endothelial cell proliferation observed in pulmonary hypertension (47).
In summary, enalapril treatment protects mice from the
development of bleomycin-induced fibrosis and pulmonary hypertension. Enalapril treatment promotes lung homeostasis by inhibiting cell proliferation, decreasing collagen expression, and reducing
TNF-mediated inflammation. Enalapril inhibition of TNF and
1(I)-collagen expression appears to be mediated via
attenuation of NF-
B and AP-1 activation in the mouse lung. The
protective effects of enalapril may involve inhibition of kinases
located upstream of the IKK and MAPKK. Further studies are necessary to
identify these kinases and reactions. The present data also suggest a
need for further studies looking at the effects of ACE inhibitors in
the treatment of pulmonary hypertension and fibrotic lung diseases in humans.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Peschon for the generous gift of tumor necrosis factor receptor-deficient mice.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants HL-03569 (to L. A. Ortiz), HL-03374 (to J. A. Lasky), ES-08663 (to M. Friedman), and HL-6000 (to A. J. Hyman and P. J. Kadowitz).
Address for reprint requests and other correspondence: L. A. Ortiz, Section of Pulmonary Diseases, Critical Care, and Environmental Medicine, Dept. of Medicine SL9, Tulane Univ. Medical Center, New Orleans, LA 70112-2699 (E-mail: lortiz{at}tulane.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 12, 2002;10.1152/ajplung.00144.2001
Received 25 April 2001; accepted in final form 24 December 2001.
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