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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Idiopathic pulmonary fibrosis (IPF) is characterized by an alveolitis with epithelial and endothelial damage progressing to fibrosis. Numerous mediators have been implicated in this complex process. Studies in humans have shown that endothelin-1 (ET-1), a vasoconstrictor and mitogenic peptide, is a mediator in IPF. To determine the role of ET-1 and endothelin-converting enzyme (ECE)-1 and the effect of bosentan, an ET receptor antagonist, in an animal model of IPF, we studied three groups of rats (n = 6 each): Group 1, control, received saline; Group 2, fibrosis, received 1.5 U bleomycin intratracheally; Group 3, fibrosis-bosentan treated, received bleomycin and bosentan daily by gavage. After 28 d, right upper lobes were fixed for immunohistochemistry (IHC) and sections were stained with antisera to ET-1 and ECE-1 and graded semiquantitatively. Sections from left lungs were embedded in paraffin and stained for light microscopic morphometry to quantitate the fibrosis. By IHC, we found increased ET-1 immunoreactivity (ir) in airway epithelium and inflammatory cells, and ECE-1-ir in airway epithelium, type II pneumocytes and endothelial cells (p < 0.05). By morphometry, the volume fraction (Vv) of connective tissue (CT) increased and the Vv of air decreased in the fibrosis group compared with that in the control group. Bosentan reduced the Vv of CT and increased the Vv of air compared with that in the fibrosis group (p < 0.05). These results indicate that ET-1 is involved in the pathogenesis of pulmonary fibrosis in the rodent model and that blockage of its receptors reduces the fibrosis.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Idiopathic pulmonary fibrosis (IPF), also known as cryptogenic fibrosing alveolitis, is a generally fatal condition of unclear etiology (1, 2). Its principal pathologic characteristics include endothelial and epithelial damage, inflammation with
neutrophils, macrophages, and lymphocytes, followed by proliferation of type II pneumocytes and fibroblasts, and collagen
deposition. The pathogenesis of IPF is complex, involving mediators, cytokines and growth factors, produced by a variety of
cells within the lung (1). One potential mediator is endothelin-1
(ET-1). First isolated and purified from the culture medium of
porcine aortic endothelial cells by Yanagisawa and coworkers
(3), ET-1 is a 21-residue peptide that belongs to a family of potent vasoactive peptides, also including ET-2 and ET-3 (3, 4).
These three isoforms have a similar structure, produced from 
200-residue preproendothelins encoded by three different
genes found in the genomic DNA library of several species, including humans and rodents. Preproendothelins generate 38-residue biologically inactive intermediates, termed big ETs,
that are processed to mature ETs. Big ET-1 is cleaved to ET-1
by endothelin-converting enzyme-1 (ECE-1) that is a membrane-bound metalloprotease (5). ET-1 has numerous and diverse actions, including vasoconstriction (3, 4, 6), bronchoconstriction, and growth promotion (7). The effects of ETs are
mediated by two principal specific receptors, ET-A, which has
a substantially greater affinity for ET-1 than do ET-2 and ET-3 (10), and ET-B, with a similar affinity for ET-1, -2, and -3 (11).
ET-1 has been associated with pulmonary fibrosis in humans. In
a previous study, we demonstrated using immunohistochemistry (IHC) and in situ hybridization, an increased expression of
ET-1 in airway epithelium, and type II pneumocytes of patients
with IPF compared with control subjects and with patients with
nonspecific fibrosis (12). Our findings were confirmed by those
of Uguccioni and colleagues (13) who also described elevated
ET-1 plasma levels in patients with IPF compared with control
subjects. Thus, ET-1 appears to play an important role in the
pathogenesis of IPF in humans. There is little information, however, on the role of ET-1 in animal models of this disease. One model of IPF that has withstood the test of time is produced by bleomycin in mice and rats (14). Thus, we chose the
rat model of bleomycin-induced pulmonary fibrosis to investigate the role of ET-1 in this disease. Specifically, we examined
the expression of ET-1 and ECE-1 using IHC. We also hypothesized that if ET-1 is an important mediator in pulmonary
fibrosis, administration of an ET receptor antagonist should
reduce the amount of fibrosis in the bleomycin model; to do
this, we chose bosentan, the orally active ET-A and ET-B receptor antagonist (15). Our immunohistochemical findings suggest that ET-1 is indeed involved in the pathogenesis of pulmonary fibrosis in this model, and that the fibrosis can be reduced
significantly using bosentan.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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General Protocol
A total of 18 male Fisher rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 260 ± 5 g were studied. All animals were free of respiratory or other diseases, caged in pairs, and provided with a standard pelleted ration of chow and water ad libitum. We studied three groups of six animals each: Group 1, control, received saline; Group 2, fibrosis, received 1.5 U of bleomycin sulphate (Blenoxane®; Bristol-Myers Squibb, Princeton, NJ); Group 3, fibrosis-bosentan-treated, received bleomycin plus, concurrently, bosentan, the orally active ET-A and B receptor antagonist (F. Hoffmann-La Roche, Basel, Switzerland).
All animals were killed 28 d after saline or bleomycin administration. Lung tissues from the control and the fibrosis groups were examined for ET-1 and ECE-1 expression by IHC and those of all three groups evaluated for the extent of fibrosis by light microscopic morphometry.
Induction of Pulmonary Fibrosis and Administration of Bosentan
The rats were weighed, anesthetized intraperitoneally with sodium pentobarbital (35 mg/kg), and intubated with a size 16 catheter. Saline or bleomycin was injected through the endotracheal catheter using a 1-ml syringe. Control animals received 0.6 ml of sterile saline followed by 0.6 ml of air. Groups 2 and 3 received a single dose of 1.5 U bleomycin sulphate in 0.6 ml of saline, also followed by 0.6 ml air to distribute the drug equally in the lungs. For Group 3, bosentan was prepared fresh each week by suspension in 5% arabic gum, and 100 mg/kg were administered by gastric gavage daily for the 28-d period.
Fixation and Preparation of the Pulmonary Tissues
The animals were anesthetized intraperitoneally with pentobarbital (35 mg/kg), heparinized (1,000 units/kg), and exsanguinated via the femoral artery. The heart and lungs were removed en bloc, and the lungs were dissected free. For IHC, the right upper lobes of Groups 1 and 2 were separated from the right middle, lower, and cardiac lobes. They were fixed by instillation through the airways of 4% paraformaldehyde in phosphate-buffered saline (PBS) (0.01 M phosphate buffer at pH 7.2 with 0.15 M NaCl) for 3 h, then transferred to PBS containing 15% sucrose and 0.1% sodium azide, and stored at 4° C.
For light microscopic morphometry, the left lungs were fixed by instillation of 10% formalin through the bronchus using a 16-gauge catheter, overnight, at a constant pressure of 15 mm Hg. Then their volumes were measured, using the Archimedes principle, by immersing them in water, they were sliced in the sagittal direction from the hilus, and four slices from each animal were embedded in paraffin using standard histologic techniques, sectioned at 5 µm, and stained with hematoxylin-eosin.
Immunohistochemistry
IHC was performed on the lungs of Groups 1 and 2 using the avidin-biotin-peroxidase-complex (ABC) method (16) with the Vectastain Elite kit (Vector Laboratories, Burlingame, CA) using polyclonal ET-1 and ECE-1 antisera raised as described previously (17). Cryostat sections (10-µm thick) were cut from the right upper lobe, picked up on poly-L-lysine-coated slides, and dried at 37° C overnight. The sections were then washed in PBS three times for 5 min each and incubated in 10% normal goat serum for 30 min at room temperature followed by incubation with ET-1 or ECE-1 antisera overnight at 4° C. After three more washes in PBS, sections were incubated with biotinylated goat antirabbit IgG antiserum for 45 min, washed in PBS, and incubated with ABC for 45 min. Immunoreactive sites were developed by immersion of the sections in a solution containing 0.01% hydrogen peroxide and 0.025% 3,3'-diaminobenzidine. Sections were counterstained with Harris' hematoxylin, dehydrated, cleared in toluene, and mounted. For negative controls, sections were incubated with 10% normal goat serum instead of the primary antisera, or with the antisera preabsorbed with the respective antigens prior to addition to sections.
For the assessment of the sections, they were randomized, coded, and examined by light microscopy without knowledge of treatment groups, for the localization of ET-1 immunoreactivity (ir) and ECE-1-ir in the following cell types: airway epithelium, type II pneumocytes, vascular endothelium, and inflammatory cells (including macrophages, neutrophils, and lymphocytes). Immunostaining was graded semiquantitatively from 0 to 4, using a previous method (12): Grade 0, no staining; Grade 1, focal staining; Grades 2, 3, and 4, diffuse weak, moderate, and strong staining, respectively.
Light Microscopic Morphometry
This was done using a point-counting technique, slightly modified from the method used by Zwikler and colleagues (18). The slides were examined at a magnification ×250 using a Zeiss Photomicroscope II (Zeiss, Oberkochen, Germany) to which a projection screen was attached with a grid of 64 points. Slides from all three groups of rats were randomized, coded, and examined without knowledge of the treatment. For each slide, we systematically point-counted one field in 10, thus essentially sampling the entire lung tissue. Points were assigned to the following structures: (1) airways, (2) vessels (arteries and veins) > 20 µm diameter, and (3) parenchyma. Each structure was further subdivided into compartments (Table 1): for the airways, we point-counted lumen air, lumen inflammatory cells, wall tissue, and wall inflammatory cells; for the vessels, lumen, intima plus media, adventitial connective tissue, and adventitial inflammatory cells; for the parenchyma, alveolar air, alveolar inflammatory cells, connective tissue, and inflammatory cells in the connective tissue. For each compartment, we summed the points and expressed them as volume fraction (Vv) of the total. We obtained the absolute volume of each compartment by multiplying its Vv by the volume of the left lung. We compared the three groups with respect to the degree of fibrosis and to the effect of bosentan on the bleomycin-treated animals. From these basic data on individual compartments, we did secondary calculations to obtain: (1) total air = airway lumen air + alveolar air, (2) total connective tissue = airway wall tissue + vascular adventitial tissue + parenchymal connective tissue, and (3) total inflammatory cells = airway wall inflammatory cells + vascular adventitial inflammatory cells + parenchymal connective tissue inflammatory cells.
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Statistical Analysis
The results were expressed as means ± standard error, using the number of animals. Statistical analyses for the IHC were done by Student's unpaired t test and for the morphometry by one-way analysis of variance, using proprietary software (Systat, Evanston, IL). A value of p < 0.05 was considered significant.
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RESULTS |
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INTRODUCTION
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DISCUSSION
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The initial weights of the animals in the control group and in the fibrosis- and fibrosis-bosentan-treated groups were 281 ± 4, 236 ± 7, and 265 ± 5 g, respectively. The rats receiving bleomycin lost weight 1 to 2 wk after the instillation, but thereafter gained weight. By Day 28, the weights of the animals in the three groups were 310 ± 5, 245 ± 10, and 267 ± 7 g, respectively. The final left lung volumes for control, fibrosis-, and fibrosis-bosentan-treated groups were 3.53 ± 0.30, 2.28 ± 0.23, and 2.92 ± 0.35 ml, respectively; the volumes of the left lungs were significantly smaller in the fibrosis group than in the control group (p = 0.024).
Descriptive Light Microscopy
The lungs from the control animals that received only saline showed a normal architecture, with intact parenchyma, vessels, and airways (Figure 1A). No inflammation or fibrosis was observed in this group. The lungs in Groups 2 and 3 that received bleomycin were qualitatively, albeit not quantitatively (see MORPHOMETRY below), similar and showed multiple scattered foci of fibrosis (Figure 1B and C): in these areas, the architecture of the lung was distorted and replaced by simplified boxlike air spaces separated by wide bands of connective tissue, containing inflammatory cells (neutrophils, macrophages, and lymphocytes) and lined by hyperplastic type II pneumocytes (Figure 1D).
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To address the question of the effect of bosentan on normal lungs, we had previously assessed this in a model termed "postobstructive pulmonary vasculopathy," also in rats, produced by unilateral ligation of one pulmonary artery (19); in this model, the contralateral (nonligated) lung is normal, and we had one group of rats treated with bosentan, one not treated. The lungs from these rats had also been fixed by instillation of 10% formalin through the bronchus at a pressure of 15 mm Hg, and sagittal slices were embedded in paraffin and stained with hematoxylin-eosin; we did not, however, measure their lung volumes nor perform morphometry. Nevertheless, by light microscopy, we found no differences in the lungs of these groups, both being normal, ruling out any significant effect of the bosentan. This had been previously confirmed by Dr. M. Clozel (personal communication).
Immunohistochemistry
The semiquantitative grading of the IHC results are in Figures 2 and 3, representative photomicrographs are in Figure 4. In the fibrosis group, ET-1-ir was increased in airway epithelium (p = 0.30) and inflammatory cells (p < 0.001) compared with the control group (Figure 2); its expression was also increased in type II pneumocytes in the fibrosis group, but the increment was not statistically significant.
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ECE-1-ir (Figure 3) was increased in the airway epithelium (p = 0.006), in type II pneumocytes (p = 0.013), and in vascular endothelium (p = 0.035) of the fibrosis group compared with the control group. ECE-1-ir was unchanged in inflammatory cells.
No immunostaining was seen in the sections stained for the negative control experiments (Figure 4). In addition, although bosentan is an ET-1 receptor antagonist and would not be expected to affect the expression of the ligand or of the converting enzyme, we stained slides from four of the rats in the bosentan-treated group and found (data not shown) a small reduction in the staining for both ET-1 and ECE-1 that could readily be attributed to the reduction in the inflammation and fibrosis in the bosentan-treated group compared with that in the fibrosis group (see below). The effect of bosentan on the expression of ET-1 in normal lungs was determined by immunohistochemical staining slides of control lungs from the aforementioned "postobstructive pulmonary vasculopathy" model: we found minimal staining of the pulmonary tissue sections (results not shown), consistent with the fact that bosentan did not alter the expression of ET-1 in our lungs.
Morphometry
The values of the Vv of the individual pulmonary compartments are in Table 1. In the fibrosis group, there was a significant decrease in the Vv of parenchymal alveolar air and increase in the Vv of connective tissue of the airways, the vessels, and the parenchyma; the Vv of adventitial and parenchymal inflammatory cells were also significantly increased in this group. In Group 3, the addition of bosentan produced a significant decrease in the Vv of airway wall tissue, parenchymal connective tissue, and adventitial inflammatory cells, and a significant increase in the Vv of parenchymal alveolar air compared with the fibrosis Group 2. The secondarily derived Vv of total air, connective tissue, and inflammatory cells are plotted in Figure 5. The Vv of total connective tissue rose in the fibrosis group compared with the control group (p < 0.05), with a concomitant fall in the Vv of total air (p < 0.001). Although the Vv of total inflammatory cells was small, it was nevertheless increased in the fibrosis group compared with the control group (p = 0.005). In the fibrosis-bosentan-treated group, the Vv of total air increased and the Vv of total connective tissue decreased compared with the fibrosis group (p = 0.003 and p = 0.001, respectively), although they still remained significantly different from control. There was also a small reduction in the Vv of inflammatory cells.
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p < 0.05) compared
with the fibrosis group. The fibrosis-bosentan-treated group, however, still had a significantly lower Vv of air and higher Vv of connective tissue than the control group (
p < 0.05).
The values of the absolute volumes of the lung compartments are in Table 2. In the fibrosis group, the absolute volumes of alveolar air were significantly decreased, whereas those of the adventitial inflammatory cells were significantly increased (p < 0.05) compared with the control group. The total absolute volumes of air, connective tissue, and inflammatory cells are shown in Figure 6. The total volume of air significantly decreased and that of inflammatory cells significantly increased in the fibrosis group compared with that in the control group (p = 0.002 and 0.01, respectively).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The principal findings of the present study were that the expression of ET-1 was increased in airway epithelial and inflammatory cells, and that of ECE-1 was increased in airway epithelium, type II pneumocytes, and vascular endothelium of the rats with bleomycin-induced fibrosis compared with the control group, and that bosentan, the orally active ET-A and B receptor antagonist, significantly reduced the Vv of connective tissue and increased the Vv of air compared with the fibrosis group.
We had previously shown in adult human lungs of patients with IPF, using IHC and in situ hybridization, that ET-1 expression in epithelial cells of airways and type II pneumocytes was increased compared with lungs of control subjects and of patients with nonspecific fibrosis (12); the ET-1-ir was particularly prominent in those areas with active granulation tissue deposition. In addition, ET-1 was increased in the endothelium of those patients who had IPF plus pulmonary hypertension, pointing to disease-specific activation of cell types. The present study suggests that in the rodent model of bleomycin-induced lung fibrosis, ET is also important in mediating the fibrosis since ET-1-ir was increased in epithelial cells, as was ECE-1-ir, and that bosentan, the ET receptor antagonist, significantly reduced the fibrotic response.
The model of bleomycin-induced fibrosis in rodents and mice, used for more than 20 yr, closely mimics IPF and has been used by many investigators (14, 20, 21). The model is also directly relevant to the human since pulmonary fibrosis is an important side effect of bleomycin when used as a chemotherapeutic agent. The sequence of events, with initial endothelial and epithelial damage, alveolitis with infiltration by several varieties of inflammatory cells, including neutrophils, lymphocytes, and macrophages, followed by proliferation of type II pneumocytes and fibroblasts with collagen deposition is believed to mimic the sequence in humans, although in the latter, early events have been more elusive. In our study, the group of animals with fibrosis had a reduction in lung volume by about one-third, with a 25% reduction in the Vv of air and a doubling of the Vv of total connective tissue (Figure 5), consistent with findings in humans with moderately severe IPF (1, 12).
Semiquantitative grading of the immunohistochemical staining for ET-1 in the rats after 28 d of bleomycin showed that its expression was increased principally in airway epithelial cells and inflammatory cells as well as in type II pneumocytes, although in the latter it did not attain statistical significance; the grading results for ECE-1 revealed an increased staining in
airway epithelial and type II pneumocytes and in the endothelial cells. ET-1 and ECE-1, the enzyme responsible for its conversion from big ET-1, could have autocrine as well as paracrine
effects in lung fibrosis and be involved in both its exudative and
proliferative phases. In the exudative phase with endothelial and
epithelial damage, inflammatory cells, in particular neutrophils
and macrophages, play an important role in cytokine production,
including interleukin-1 (IL-1), IL-6, IL-8, tumor necrosis factor-
(TNF-), and macrophage chemotactic protein (22, 23); IL-1
and TNF- increase ET-1 expression in several cell types (6).
Endo and colleagues (24) showed that TNF-, transforming
growth factor-
(TGF-), and IL-8 stimulate ET-1 synthesis in
tracheal epithelial cell cultures of guinea pigs. More recently,
we showed that IL-1 and TNF- increase ET-1 and ECE-1 expression by normal human bronchial epithelial cells (25).
Thus, ET-1 released from inflammatory cells could have autocrine actions in the chemotaxis of other inflammatory cells and
paracrine actions on epithelial cells. These effects, as well as the
epithelial damage produced by the bleomycin (14), are likely to
be responsible for the upregulation of ET-1 and ECE-1 expression in the airway and alveolar epithelial cells. In the present
study, the lack of increased ET-1-ir staining in endothelial
cells is not surprising because, in these animals, we did not see
the arteriopathy observed previously in those patients with
IPF developing pulmonary hypertension and right heart failure (12). Interestingly, however, there was increased ECE-1-ir
in the endothelial cells of the pulmonary vessels in the fibrosis group: this was not unexpected since the expression of ECE-1
has been shown to precede that of ET-1 in the neointima of
rats after angioplasty (26). The absence of a significant difference in staining for ECE-1-ir in inflammatory cells, in the face
of a difference of ET-1-ir, is unclear; to our knowledge, there
are no studies to date that have addressed this point.
ET-1 has, in addition to vasoactive and proinflammatory
effects, important proliferative actions that are directly relevant to pulmonary fibrosis: it is a known mitogen with the ability to stimulate DNA synthesis in numerous cell types, including smooth muscle cells, fibroblasts, and endothelial cells,
although its role may be modulated depending on the types of
cell, culture, or environmental conditions (reviewed in 27). In
the epithelial and fibroblastic proliferative phase of pulmonary fibrosis, ET-1 can have both autocrine and paracrine effects, the former by epithelial-derived ET-1 stimulating the
proliferation of type II pneumocytes and of small airway epithelial cells (28). Paracrine action could occur from inflammatory cells to epithelial cells, or from both of these cell types to
fibroblasts; intercellular interactions between epithelial and fibroblastic cells have been shown to be particularly important
in pulmonary fibrosis (29). Endothelins have been shown to
act synergistically with other growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF),
TGF-, basic fibroblastic growth factor (b-FGF), and insulinlike
growth factor (IGF) in the migration and/or proliferation of
smooth muscle cells (6, 27). These and other cytokines (e.g.,
IL-1, TNF-) may originate from macrophages and other inflammatory cells, and from the epithelial cells, stimulating ET-1
synthesis and acting in concert to stimulate fibroblastic proliferation. One growth factor that interacts prominently with ET-1
is TGF-1: in the bleomycin model in rats, TGF- is expressed
early in macrophages, later in epithelial cells, and the influx of
alveolar macrophages into the lungs is prevented by corticosteroids (20, 21).
ET-1 induces proliferation by binding to the specific ET receptors at the cell surface, and via the activation of protein kinase C, stimulates the expression of the protooncogenes c-jun and c-fos in several cell types, including Swiss 3T3 fibroblasts (9, 27). In a recent study, Marini and colleagues (30) found that cultured pulmonary bronchial epithelial cells incubated with ET-1 increased fibronectin gene expression, mediated by the ET-A receptor, suggesting a role in inducing the subepithelial fibrosis observed in asthma; a similar process may be operative in IPF since fibronectin is a potent chemotactic agent for fibroblasts. In another recent study, Wang and colleagues (26) found an increased expression of ET-1 and ET-3, ECE-1, and ET-A and ET-B receptor mRNA, and ET-ir at various times after angioplasty-induced neointimal formation in the rat carotid artery; there was an early rise in ECE-1 mRNA (6 and 24 h after ballooning), and a later rise in ET-ir (14 d). These findings, together with our data, are consistent with the notion that ET-1 plays a role, along with other cytokines and mediators and growth factors, in both the inflammatory and proliferative role in the genesis of pulmonary fibrosis, at least in the rodent bleomycin-induced model.
Because the effects of ET-1 are mediated by specific receptors, it seemed logical to attempt to prevent or to abrogate the process with one of the newly available ET receptor antagonists. We chose bosentan because it blocks ET-A and ET-B receptors, both of which may be involved in proliferation, and because it is orally active (15). ET receptor antagonists have been studied in several animal models of human diseases and proved to be effective. Eddahibi and colleagues (31) and Chen and coworkers (32) demonstrated that oral bosentan largely protected rats against the development of chronic hypoxic pulmonary hypertension and the associated right ventricular hypertrophy. Furthermore, bosentan effectively reduced blood pressure in cyclosporine-induced hypertension in rats and marmosets (33), and it was found to lower systemic and pulmonary vascular pressures in patients with heart failure (34).
In the present study, the animals that were treated with bosentan had a significant albeit incomplete reduction in the fibrosis compared with those in Group 2 that did not receive the ET receptor antagonist. Indeed, both the Vv and absolute volumes of total air and the Vv of total connective tissue compartments, as well as left lung volumes, were significantly improved, although they did not reach normal levels; these findings suggest that the endothelin system is an important link in the pathogenetic chain of events leading to pulmonary fibrosis, and that bosentan or similar agents potentially could be valuable in the treatment of IPF. The fact that the absolute volume of connective tissue was unchanged is not surprising because the reduction in Vv of the connective tissue was offset by the partial return to normal of the left lower lobar volumes. Our data also suggest that bosentan had an effect on the inflammatory component of the fibrosis, although the small Vv occupied by these cells precluded demonstration of a statistically significant effect. Several other agents, including nitric oxide, prostacyclin, and atrial natriuretic peptides, act as ET antagonists and can counteract the proliferative response to ET-1 in various cell types through one or more pathways (27). Although ET-1 can act as a mitogen through both ET-A and ET-B receptors (26), under certain conditions, ET-B receptors may inhibit proliferation (35). This possibility, along with the fact that other mediators are involved in pulmonary fibrosis, may explain why we observed an incomplete prevention or abrogation of the fibrotic response in the bleomycin model. On could question whether the dose of bosentan was sufficient to inhibit the fibrosis and whether a higher dose might have reduced the fibrosis even more. Although such an effect cannot be entirely ruled out, the evidence from other models, in particular hypertension, suggests that the dose that we used was appropriate for blocking the ETA and ETB receptors (31, and M. Clozel, personal communication).
Although bosentan would not be expected to alter the expression of the ligand ET or of its converting enzyme, in the slides from the bosentan-treated group we found a small reduction in the staining for both ET-1 and ECE-1, readily attributable to the reduced inflammation and fibrosis in this group compared with the fibrosis group. In support of this notion, Lariviere and colleagues (36) found, in the rat model of DOCA-salt-induced hypertension, that bosentan had no significant effect on ir-ET-1 in segments of thoracic aorta; they also found that bosentan reduced blood pressure and increased plasma ET-1 levels, consistent with the receptor antagonist effects of bosentan, resulting in continued and increased presence of ET-1 in the bloodstream since it is prevented from binding to its receptors. In addition, we ascertained the effects of bosentan on normal lungs from our rat model or postobstructive pulmonary vasculopathy (19) treated with bosentan, finding no differences in the light microscopic morphology compared with the rats that did not receive bosentan. In their studies, Clozel (personal communication) also found that bosentan had no effect on normal lungs.
In summary, the results of the present study strongly support a role for endothelin, specifically ET-1, in the model of pulmonary fibrosis induced by bleomycin in the rat. These findings are in agreement with those in humans, either in IPF, or in the pulmonary fibrosis associated with disease such as scleroderma, where, in addition to the parenchymal fibrosis, there is a vasculopathy that may lead to pulmonary hypertension (37). Indeed, in our previous study (12), we found that the expression of ET-1 was prominent in the endothelial cells in addition to the epithelial cells of the subset of patients with IPF who had pulmonary hypertension. The finding that bosentan significantly abrogated the fibrosis suggests that it may be a useful therapeutic tool in humans with IPF, and perhaps even more so in those patients with superimposed pulmonary hypertension such as scleroderma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. René P. Michel, Department of Pathology, McGill University, 3775 University St., Room B15, Montréal, QC, H3A 2B4 Canada.
(Received in original form July 29, 1996 and in revised form February 19, 1997).
Drs. Michel and Giaid are the recipients of grants MT-7727 and MT-1257, respectively, from the Medical Research Council of Canada.Dr. Giaid is supported by the Heart and Stroke Foundation.
Acknowledgments: The writers thank Dr. M. Yanagisawa for his valuable help and Dr. F. Hu for expert technical assistance. Bleomycin was kindly provided by Bristol-Myers (Princeton, New Jersey), and bosentan was provided by F. Hoffmann-La Roche (Basel, Switzerland).
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References |
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ABSTRACT
INTRODUCTION
METHODS
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REFERENCES
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