Airway fibrosis in rats induced by vanadium
pentoxide
James C.
Bonner1,
Annette B.
Rice1,
Cindy R.
Moomaw2, and
Daniel L.
Morgan3
Laboratories of 1 Pulmonary
Pathobiology, 2 Experimental
Pathology, and 3 Toxicology,
National Institute of Environmental Health Sciences, National
Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
Vanadium pentoxide
(V2O5)
is a cause of occupational asthma and bronchitis. We previously
reported that intratracheal instillation of rats with
V2O5
causes fibrosis of the lung parenchyma (J. C. Bonner, P. M. Lindroos,
A. B. Rice, C. R. Moomaw, and D. L. Morgan. Am.
J. Physiol. Lung Cell. Mol. Physiol. 274:
L72-L80, 1998). In this report, we show that intratracheal
instillation of
V2O5 induces airway remodeling similar to that observed in individuals with
asthma. These changes include airway smooth muscle cell thickening, mucous cell metaplasia, and airway fibrosis. The transient appearance of peribronchiolar myofibroblasts, which were desmin and vimentin positive, coincided with a twofold increase in the thickness of the
airway smooth muscle layer at day 6 after instillation and preceded the development of airway fibrosis by
day 15. The number of nuclear profiles
within the smooth muscle layer also increased twofold after
V2O5
instillation, suggesting that hyperplasia accounted for thickening of
the smooth muscle layer. The majority of cells incorporating
bromodeoxyuridine at day 3 were
located in the connective tissue surrounding the airway smooth muscle
wall that was positive for vimentin and desmin. These data suggest that
myofibroblasts are the principal proliferating cell type that
contributes to the progression of airway fibrosis after
V2O5 injury.
asthma; myofibroblasts; smooth muscle cell; collagen; metals
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INTRODUCTION |
INCREASING ATTENTION has been focused on metals
as agents in air pollution particles that could cause adverse
respiratory effects. Inhalation of vanadium compounds may result in
occupational bronchial asthma and bronchitis in individuals working in
the petrochemical industry (21, 23), and vanadium has been reported to
stimulate constriction of bronchi isolated from humans and experimental
animals (9, 24). Vanadium-containing residual oil fly ash particles
collected from emissions of petrochemical plants have also been
reported to induce airway hyperresponsiveness in rats (13), and
residual oil fly ash-induced lung inflammation is dependent at least in
part on vanadium compounds (11, 19). Additionally, ambient air
pollution particles <10 µm in diameter collected from urban areas
contain vanadium, which may contribute to the toxic effects of these
particles on lung macrophages and myofibroblasts in vitro (4).
Although vanadium and vanadium-containing particles cause lung
inflammation and airway hyperreactivity, it is not known whether vanadium exposure results in airway remodeling similar to that observed
in individuals with chronic asthma. Asthma is a complex disorder
characterized by airway hyperresponsiveness and progressive inflammation. The inflammatory response in the airways of asthmatic patients may lead to fibroproliferative changes, such as an increase in
airway smooth muscle cell (SMC) mass (12,15), mucous cell metaplasia within the lining of the airway epithelium (6), and the development of
irreversible airway fibrosis (5, 6, 17, 27). An increase in airway wall
smooth muscle results in an enhanced contractile response and an
amplified narrowing of the airway lumen during an asthmatic attack
(17). The deposition of extracellular matrix proteins by connective
tissue cells accumulating and proliferating beneath the airway
epithelium (i.e., airway fibrosis) contributes to chronic, irreversible
narrowing of the airway lumen (5, 27).
The principal collagen-producing connective tissue cell type in the
fibrotic response is likely a myofibroblast phenotype (i.e.,
contractile interstitial cell) (1, 34). Myofibroblasts possess
characteristics of fibroblasts (e.g., positive for vimentin and
procollagen) and SMC (e.g., positive for desmin and smooth muscle
-actin) (34). It has been suggested that myofibroblasts contribute
to restrictive airway disease by depositing collagen and thereby
promoting airway fibrosis (5, 27). Myofibroblasts also have the
potential to differentiate into SMC (28), and ultrastructural studies
of airways from asthmatic patients suggest that myofibroblast-to-SMC
differentiation contributes to increased airway smooth muscle mass
observed in asthma (14).
The purpose of this study was to investigate the progression of airway
remodeling after a single intratracheal instillation of vanadium
pentoxide
(V2O5)
to determine whether this metal causes constrictive airway pathology
consistent with its asthma-like effects in humans and rodents. We
report that
V2O5
instillation causes airway smooth muscle thickening, mucous cell
metaplasia in the airway epithelial lining, and a marked increase in
the proliferation of peribronchiolar myofibroblasts. These
proliferative events precede the development of airway fibrosis.
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METHODS |
Intratracheal instillation.
V2O5
suspensions (pH 7.2) were vortexed thoroughly, then bath sonicated for
30 min at 25°C before instillation. Male Sprague-Dawley rats
(Charles River) weighing ~200 g were instilled intratracheally with
200 µl of sterile saline or 1 mg/kg (0.2 mg/rat)
V2O5
(Aldrich Chemical, Milwaukee, WI) suspended in 200 µl of saline, as
previously reported (3). Rats received a single injection of
bromodeoxyuridine (BrdU; 50 mg/kg ip) 1 h before they were killed. At
3, 6, and 15 days after instillation, the animals (3 saline-instilled
and 5 V2O5-instilled
per time point) were overdosed with an intraperitoneal injection of
pentobarbital sodium (Nembutal), and the lungs were removed en bloc.
The lungs were instilled with neutral buffered Formalin in PBS, pH 7.2, the trachea was tied off, and the lungs were immersed in Formalin
overnight. After fixation, the lung tissues were embedded in paraffin
and cut into 6-µm-thick sections. Sections were mounted and stained
for hematoxylin and eosin, Masson's trichrome for collagen, and alcian
blue-periodic acid-Schiff (PAS) for identification of mucin-containing
goblet cells. Serial sections were used for desmin, vimentin, and BrdU immunohistochemistry.
Immunohistochemistry.
Lung tissue was fixed overnight in 10% neutral buffered Formalin.
Immunohistochemistry was performed using the avidin-biotin peroxidase
method. Tissue sections (6 µm) were deparaffinized with xylene and
dehydrated with a series of graded alcohol solutions to automation
buffer (AB) consisting of 5% NaCl and 2% HCl (Biomeda, Foster City,
CA). Endogenous peroxidase was blocked in 3% (vol/vol) H2O2
for 15 min. After the sections were washed twice with AB, blocking was
performed with 5% normal goat serum for 20 min at 25°C. Without
being rinsed, the slides were incubated with a primary rabbit
anti-desmin antibody (Accurate Antibodies, Westbury, NY) at a dilution
of 1:200 for 30 min at 25°C, a primary mouse anti-vimentin antibody
(clone LN6, Accurate Antibodies) for 60 min at 25°C, or a 1:50
dilution of primary monoclonal mouse anti-BrdU antibody (Becton
Dickinson, Mountain View, CA) for 30 min at room temperature. For
detection of desmin, a rabbit Elite kit (Vector Laboratories, Burlingame, CA) was used as follows: sections were washed twice with
AB, then incubated for 30 min with a 1:400 dilution of biotinylated secondary goat anti-rabbit IgG. Slides were washed again and incubated with the Elite avidin-biotin complex (Vector Laboratories) for 30 min.
For detection of vimentin, a Biogenex kit (Biogenex, San Ramon, CA) was
used as follows: sections were washed once with AB, then incubated with
biotinylated secondary antibody for 20 min at 25°C. After the
sections were washed with AB, the kit label antibody was applied for 20 min at 25°C. For BrdU, sections were washed twice with AB, then
incubated for 30 min with the Elite avidin-biotin complex (Vector
Laboratories) for 30 min. For staining of all antigens (desmin,
vimentin, and BrdU), slides were washed 5 times with AB, and then the
antibody complex was visualized using a diaminobenzidine tablet (10 mg;
Sigma Chemical, St. Louis, MO) dissolved in 20 ml of AB containing 12 µl of 30%
H2O2
for 6 min in the dark. All slides were then rinsed in running tap water, counterstained with hematoxylin (Harelco, Gibbstown, NJ), dehydrated through a series of graded alcohols to xylene, and covered
with a coverslip with Permount (Fisher Scientific, Fair Lawn, NJ).
Morphometric analysis.
Morphometric evaluation was carried out on rats at 3, 6, and 15 days
after instillation with saline or
V2O5.
Five airways were measured per rat, and at least three saline- and
three
V2O5-instilled animals were evaluated at each time point. Bronchioles that presented a
closed circular or oval profile were selected. The thickness of the
brown-staining, desmin-positive airway smooth muscle layer or the
blue-staining, trichrome-positive peribronchiolar collagen layer was
measured on digitized microscopic images (magnification ×400) of
histological sections with the NIH Image Program (National Institutes
of Health, Bethesda, MD), as described previously (8). The ratio of
area to perimeter was used as an index of smooth muscle thickness or
airway collagen thickness, where the area is defined as the entire ring
of smooth muscle or collagen. The NIH Image Program allows for manual
outlining of the desmin-stained smooth muscle layer or the
trichrome-stained collagen layer and computes the area within the
outlined ring of tissue. The perimeter is the airway basement membrane
circumference. Thus we corrected for the variability in bronchiolar
diameter (i.e., perimeter). Airway smooth muscle thickness was also
verified by conventional morphometry, wherein the thickness of the
smooth muscle layer from the base of the columnar epithelium to the
proximal (inner) edge of the vimentin-positive adventitia was measured
using an eyepiece reticle. The smooth muscle wall thickness was
routinely evaluated at two points on opposite sides of the short axis
of the elliptical profiles, and measurements were made at locations where cell borders appeared sharp to minimize tangential sectioning. Similar verification was performed to determine thickness of the trichrome-positive layer (i.e., airway fibrosis), where measurements with an eyepiece reticle were made from the base of the columnar epithelium to the distal (outer) edge of the blue-staining collagen surrounding the airway. For analysis of time-course data, one-way ANOVA
was performed to determine an effect of exposure. If this analysis was
significant, two-sample t-tests were
performed on treatment effects at each time point.
 |
RESULTS |
V2O5
stimulates airway smooth muscle thickening.
Desmin was demonstrated as a specific SMC marker and clearly stained
SMC in normal bronchioles from saline-instilled rats but did not stain
the airway epithelium or lung cells residing within the lung parenchyma
(Fig.
1A).
V2O5
instillation caused a thickening of the desmin-positive bronchiolar SMC
layer by day 6 (Fig.
1B). Desmin-positive peribronchiolar
cells were also abundant 6 days after
V2O5
instillation and were identified as myofibroblasts (see below).
Proliferating airway SMC were detected by BrdU immunohistochemical staining at day 3, indicating that at
least some of the SMC thickening observed at day
6 was due to replicating SMC (Fig.
1B). However, these BrdU-positive
SMC were few in number and represented <15% of the total number of
BrdU-positive cells surrounding the airway at day
3 after instillation. The majority of BrdU-positive
cells were observed in the connective tissue layer surrounding the SMC band. Quantitative morphometry of the bronchiolar SMC layer with use of
the NIH Image Program showed a 2.2- to 2.5-fold increase in the
area-to-perimeter ratio (i.e., thickness) of the smooth muscle layer
that peaked 6 days after
V2O5
instillation and remained thickened at day
15 (Fig. 2). Measurements
made by eyepiece reticle were somewhat more variable and indicated 2- to 4.5-fold increases in smooth muscle thickness. To assess whether the
increase in smooth muscle mass was due to hypertrophy or hyperplasia,
we counted nuclear profiles on hematoxylin- and eosin-stained serial
sections of the same airways that we used for smooth muscle
area/perimeter measurements. The airway cross sections from
saline-instilled rats contained 28 ± 5 nuclear profiles compared
with 67 ± 17 nuclear profiles in airway cross sections from
V2O5-instilled
rats at day 6. Thus there was a
2.3-fold increase in airway SMC nuclear profiles. Because the
morphometry data indicated a 2.2- to 2.5-fold increase in smooth muscle
thickness, these data suggest that the increase in smooth muscle mass
after
V2O5
exposure is due primarily to SMC hyperplasia.

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Fig. 1.
Airway smooth muscle thickening after vanadium pentoxide
(V2O5)
instillation as visualized by desmin immunohistochemistry.
A: saline-instilled
(day 6) rat lung showing normal
bronchiolar wall architecture with desmin-positive smooth muscle layer
(brown stain, arrowheads) residing beneath ciliated epithelial cells.
B:
V2O5-instilled
(day 6) lung showing increased
desmin-positive airway smooth muscle (arrowheads) beneath an activated
mucous metaplastic epithelium. Inset:
bromodeoxyuridine (BrdU) staining at day
3 after
V2O5
treatment; a group of subepithelial cells in smooth muscle layer is
shown undergoing DNA synthesis. Original magnification, ×400.
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Fig. 2.
Quantitative measurement of airway smooth muscle cell thickness after
V2O5-induced
airway injury. Smooth muscle thickness in bronchiole cross sections was
evaluated from 3 animals per saline-instilled group or 5 animals per
V2O5-instilled
group at 3, 6, and 15 days after instillation. Smooth muscle cell layer
was defined as desmin positive, vimentin negative (see Fig. 3). Values
are means ± SE. Statistical deviation represents
variation among individual animals:
* P < 0.05. ** P < 0.01.
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Identification of peribronchiolar myofibroblasts during
V2O5-induced
airway remodeling.
Mesenchymal cells staining positively for desmin and vimentin were
abundant around bronchioles possessing thickened airway smooth muscle
at day 6 after instillation (Fig.
3). Airway SMC stained only for desmin and
not vimentin, whereas the airway epithelium was negative for both
markers. Because vimentin is a well-known fibroblast marker and desmin
is a marker of SMC, these results indicated that proliferating
mesenchymal cells surrounding
V2O5-injured airways were most likely myofibroblasts. To verify the identity of
desmin-positive, vimentin-positive myofibroblasts, serial sections of
the peribronchiolar region were viewed by high-magnification oil-immersion light microscopy. The majority of connective tissue cells
within this region stained positively for cytoplasmic desmin and
vimentin, which further indicated that these cells were mainly myofibroblasts (Fig. 4). Some inflammatory
cells (e.g., macrophages) that infiltrated this region were positive
for vimentin but not for desmin (data not shown). As mentioned above,
few SMC were BrdU positive (Fig. 1,
inset), and the majority of
BrdU-positive cells were observed in the connective tissue layer
surrounding the SMC band (Fig. 5).
Quantitation of BrdU-positive peribronchiolar cells within the airway
smooth muscle layer or underlying vimentin-positive layer of
approximately equal thickness showed that the majority of proliferating
cells were not SMC but connective tissue cells possessing a
myofibroblast phenotype (Figs. 5 and
6).

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Fig. 3.
Use of desmin and vimentin immunohistochemistry to differentiate airway
smooth muscle cells and peribronchiolar myofibroblasts after
V2O5
injury. Six days after instillation of
V2O5,
serial sections of Formalin-fixed, paraffin-embedded lung sections were
incubated with control IgG (A),
anti-vimentin antibody (B), or
anti-desmin antibody (C).
Arrowheads, airway smooth muscle layer.
A shows no positive brown staining
as expected for a control IgG antibody. In
B, vimentin-positive cells are
peribronchiolar, and no positive staining was observed in airway smooth
muscle layer or in airway epithelium.
C shows intense staining for desmin in
airway smooth muscle layer and also in peribronchiolar mesenchymal
cells, but not in airway epithelium. These data demonstrate presence of
desmin-positive, vimentin-positive myofibroblasts adjacent to airways.
Original magnification, ×200.
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Fig. 4.
Desmin (A) and vimentin
(B) immunohistochemistry in
peribronchiolar serial sections demonstrating cytoplasmic
colocalization in numerous connective tissue cells. High-magnification,
oil-immersion light microscopy of desmin-positive, vimentin-positive
region was performed to determine whether these antigens were expressed
in different cell populations or whether both antigens were expressed
by same connective tissue cell types. Majority of peribronchiolar
connective tissue cells surrounding airway smooth muscle layer
(arrowheads) contained desmin and vimentin immunoreactivity, indicating
that these cells were mainly a myofibroblast phenotype. Original
magnification ×1,200.
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Fig. 5.
BrdU immunohistochemistry showing proliferation of peribronchiolar
cells underlying bronchiolar epithelium (ep) at day
3 after
V2O5
instillation. Although some BrdU-positive cells were observed in
desmin-positive, vimentin-negative airway smooth muscle cell (smc)
layer (see Fig. 1), >85% of peribronchiolar BrdU-positive cells were
observed in connective tissue (ct) layer surrounding airway smooth
muscle band that was vimentin positive and desmin positive (see Fig.
3). Arrowheads, BrdU-positive nuclei. Lu, airway lumen. Original
magnification, ×800.
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Fig. 6.
Quantitation of subepithelial BrdU-positive cells in bronchioles from
V2O5-instilled
rats. BrdU counts were made within desmin-positive, vimentin-negative
airway smooth muscle layer and surrounding desmin-positive,
vimentin-positive connective tissue layer, where BrdU counts were made
within 5 µm of smooth muscle layer (see Fig. 5). Five bronchioles
were evaluated from 3 saline control rats and 5 V2O5-instilled
rats. Values are means ± SE and represent variation among
individual animals.
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Activation of the bronchiolar epithelium by
V2O5.
The bronchiolar epithelium could be important in driving the
proliferation of the airway SMC or peribronchiolar connective tissue
cells after airway injury (Figs. 1 and 5).
V2O5
instillation caused activation of the airway epithelium, defined as
mucous cell metaplasia (a serous cell-to-goblet cell phenotypic
change), where mucin in the goblets was detected by alcian blue-PAS
stain. BrdU-positive airway epithelial cells were rarely observed
(Figs. 1 and 5), which indicated that activation of the airway
epithelium did not involve hyperplasia. Saline-instilled control
airways possessed a predominance of ciliated epithelial cells and no
detectable goblet cells (Fig. 7). After
V2O5
injury, the alcian blue-PAS stain showed that 30-40% of the
airway epithelial cells had differentiated to goblet cells (Figs. 7 and
8). A quantitative assessment revealed that
numbers of goblet cells increased maximally by day
6 after V2O5
instillation and declined to nearly saline control levels by
day 15 (Fig. 8).

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Fig. 7.
Alcian blue-periodic acid-Schiff (PAS) staining demonstrating mucous
cell metaplasia in airway epithelium of
V2O5-instilled
rats. A: cross section of a bronchiole
from a control (saline-instilled) rat at day
6 after instillation showing a predominance of ciliated
epithelial cells lining airway lumen and a lack of mucus-producing
goblet cells. B: bronchiole from a
V2O5-instilled
rat at day 6 after instillation shows
numerous mucus-positive (purple-staining) goblet cells (arrowheads).
Quantitation of alcian blue-PAS-positive goblet cells is shown in Fig.
8. Original magnification, ×200.
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Fig. 8.
Quantitation of alcian blue-PAS-positive goblet cells during course of
airway fibrogenesis after
V2O5-induced
lung injury. Number of alcian blue-PAS-positive cells in each
bronchiole cross section were counted in 5 bronchiole cross sections
per lung from 3 animals per saline-instilled group or 5 animals per
V2O5-instilled
group at 3, 6, and 15 days after instillation. AB, automation buffer.
Values are means ± SE. Statistical deviation represents variation
among individual animals: * P < 0.05.
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Airway fibrosis during
V2O5-induced
airway injury.
The deposition of mature collagen around bronchioles was detected by
trichrome staining. In a previous study of
V2O5-induced lung fibrosis, we showed that total lung hydroxyproline increased fourfold by day 15 after instillation
(26), yet this quantitative measurement includes collagen deposited
within lesions in the lung parenchyma as well as around airways. To
measure changes in peribronchiolar collagen deposition, we used
morphometry to quantitate the thickness of the trichrome-positive
layer. The thickness of the subepithelial trichrome-positive layer
increased by 3.1- to 3.9-fold at day
15 after
V2O5
instillation, as determined by area/perimeter measurements with the NIH
Image Program (Figs. 9 and
10). More variable measurements were
obtained with eyepiece reticle measurements, and the magnitude-increase
values for collagen thickness among saline- and
V2O5-instilled
groups ranged from 2.5- to 7-fold depending on the specific site within
the airway wall that was measured (data not shown). This indicated that
area/perimeter measurements obtained from the computer-assisted NIH
Image Program were more reliable than eyepiece reticle measurements.
Significant increases in the thickness of the trichrome-positive layer
were not observed before day 15, and
no increases were observed in the saline-instilled control animals
(Fig. 10).

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Fig. 9.
Collagen deposition in subepithelial fibrotic lesions in
peribronchiolar region at day 15 after
V2O5
instillation. Saline-instilled (A)
or
V2O5-instilled
lung sections (B) were stained with
Masson's trichrome to detect mature collagen (blue stain, arrowheads).
C: high magnification of bronchiole
from a
V2O5-instilled
animal showing blue-staining collagen fibrils beneath airway
epithelium. Quantitative morphometry of trichrome-positive,
peribronchiolar collagen is shown in Fig. 10. Original magnification,
×200 (A and
B) and ×400
(C).
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Fig. 10.
Quantitative morphometry of trichrome-positive, peribronchiolar
collagen after airway injury caused by
V2O5
instillation. Five bronchiole cross sections per lung were evaluated
from 3 animals per saline-instilled group or 5 animals per
V2O5-instilled
group at 3, 6, and 15 days after instillation. Collagen layer was
defined as trichrome positive (see Fig. 9). Collagen deposition was
significantly increased only at day 15 after
V2O5 instillation. Values are means ± SE. Statistical deviation represents variation among individual
animals: * P < 0.05.
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DISCUSSION |
In this study, we have shown that the intratracheal instillation of
V2O5,
a transition metal associated with occupational asthma, induced
remodeling of the rat bronchiolar architecture and caused
fibroproliferative changes in the airway wall that are consistent with
the pathology of asthma. These features included mucous cell
metaplasia, airway smooth muscle thickening, proliferation of
peribronchiolar myofibroblasts, and airway fibrosis. Our definition of
airway fibrosis encompasses subepithelial fibrosis (i.e., basement membrane thickening) and peribronchiolar fibrosis surrounding the
smooth muscle layer. Increasing attention has been focused on the
significance of airway fibrosis in asthma, and it has been proposed
that airway myofibroblasts participate in the increased production of
collagen that leads to airway fibrosis (5, 13). To our knowledge, this
is the first report of metal-induced airway remodeling that is
consistent with the fibroproliferative pathology seen in asthma.
Myofibroblasts may contribute to vanadium-induced airway remodeling in
at least two ways. First, the contractile nature of myofibroblasts
might contribute to persistent narrowing of the airway lumen. Second,
the deposition of collagen by peribronchiolar myofibroblasts could
contribute to airway narrowing by forming scar tissue around the
airway. We observed a transient appearance of peribronchiolar
myofibroblasts that peaked at day 6 after
V2O5 instillation (Fig. 3) and then declined by day
15, leaving a thickened collagen sheath around the
airway (Fig. 9). In tissues other than lung, myofibroblasts appear
transiently within days of injury and decrease in number as healing
occurs (32). The loss of peribronchiolar myofibroblasts in our study
between days 6 and
15 could be due to removal of cells by
apoptosis or differentiation of myofibroblasts to
airway SMC.
In addition to their role in promoting airway fibrosis, it has been
suggested that myofibroblasts migrate and differentiate into SMC, and
this is one possible explanation for the increase in smooth muscle wall
mass seen in asthma (14). The more classic explanation for increased
smooth muscle mass involves SMC hyperplasia and hypertrophy (12, 15). A
hyperplastic growth response could arise when SMC are stimulated to
proliferate in response to mitogens released by the activated airway
epithelium, inflammatory cells such as macrophages, or the SMC
themselves. In the present study, the BrdU-labeling index in the airway
smooth muscle layer at any given time was low compared with the numbers
of BrdU-positive peribronchiolar cells in the surrounding connective
tissue layer. However, it is possible that we missed the peak of
proliferating SMC, since we did not investigate time points before
day 3 after instillation. Our
quantitation of nuclear profiles in the airway smooth muscle wall
showed a 2.3-fold increase in cell number, which was nearly identical
to the magnitude increase in area/perimeter measurements for SMC
thickness caused by
V2O5
instillation. This indicates that the SMC thickening that we observed
is due mainly to cell hyperplasia. However, this does not rule out the
possibility that some proliferating myofibroblasts adjacent to the
smooth muscle wall migrated and differentiated into SMC, thereby
increasing smooth muscle wall thickness. Our data suggest that SMC
hypertrophy plays only a minor role in the thickening of the airway wall.
A variety of growth factors and cytokines have been reported to
stimulate the proliferation or differentiation of myofibroblasts and
SMC. For example, platelet-derived growth factor (PDGF) isoforms and
transforming growth factor (TGF)-
are potent mitogens for mesenchymal cells (fibroblasts, myofibroblasts, and SMC) and are upregulated during fibroproliferative lung disease (16, 22). We
recently reported that tyrosine kinase inhibitors specific for PDGF or
epidermal growth factor receptors reduced pulmonary fibrosis in rats
instilled with
V2O5
(26). Basic fibroblast growth factor (FGF-2) is normally sequestered
within the basement membrane of airways (31) but, when released, is
mitogenic for human airway SMC and also upregulates the PDGF receptor
-subtype to render these cells more responsive to the mitogenic
effects of PDGF (2). Targeted expression of interleukin (IL)-11 to airways with the Clara cell 10-kDa promoter caused airway remodeling and subepithelial fibrosis that was characterized by increased collagen
and increased desmin and smooth muscle
-actin-containing cells,
including myofibroblasts and SMC (33). TGF-
1, a major inducer of
collagen deposition by myofibroblasts and fibroblasts, is upregulated
during the progression of lung fibrosis (18). Furthermore, TGF-
1
induces fibroblasts to differentiate to a smooth muscle
-actin-positive myofibroblast phenotype (10). Proinflammatory
cytokines such as IL-1
and tumor necrosis factor-
(TNF-
) are
also increased after lung injury, and neutralizing antibodies to
TNF-
have been reported to block pulmonary fibrosis (25). Thus it
appears that the fibrogenic response is orchestrated by a variety of
cytokines and growth factors that mediate myofibroblast growth and
collagen deposition.
It is likely that
V2O5
stimulates several cell types in the airways to produce cytokines. For
example, we found that
V2O5 was a strong inducer of mucous cell metaplasia in vivo (Fig. 4), and
activation of human airway epithelial cells in vitro by vanadium compounds has been reported to stimulate the secretion of IL-6, IL-8,
and TNF-
(7). In addition, we previously reported that V2O5
stimulates the secretion of IL-1
by rat alveolar macrophages (3).
Therefore, epithelial cells and macrophages could function as effector
cells in vanadium-induced airway fibrosis. Also, the mesenchymal target
cell types (i.e., SMC and myofibroblasts) could themselves act as a
source of cytokines and growth factors after V2O5
stimulation. The mechanisms through which vanadium compounds increase
cytokine production have not been clarified, but several studies have
reported that vanadium stimulates a variety of signaling events in
epithelial cells and fibroblasts, including tyrosine phosphorylation
(30), mitogen-activated protein kinase activation (29, 35), and
activation of nuclear factor-
B (20). One or more of these signaling
pathways could be linked to induction of cytokine gene expression.
In summary, we have shown that
V2O5
instillation causes airway remodeling similar to that observed in
individuals with asthma and chronic bronchitis. These changes include
airway SMC thickening, mucous cell metaplasia, and airway fibrosis. The
transient appearance of peribronchiolar myofibroblasts, which were
desmin and vimentin positive, coincided with an increase in airway
smooth muscle mass and preceded the development of airway fibrosis.
These data support the idea that myofibroblasts contribute to airway
fibrosis. Because V2O5
is a cause of occupational asthma, this model should be useful for
investigating the cellular and molecular mechanisms of airway SMC
thickening and airway fibrosis.
 |
ACKNOWLEDGEMENTS |
The authors thank Paul Nettesheim, Julie Foley, Robert Maronpot,
and Robert Langenbach for helpful discussions during the preparation of
the manuscript. The authors greatly appreciate the excellent technical
assistance of Herman Price.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. C. Bonner,
NIEHS, PO Box 12233, Research Triangle Park, NC 27709 (E-mail:
bonnerj{at}niehs.nih.gov).
Received 1 March 1999; accepted in final form 23 July 1999.
 |
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