INTRODUCTION

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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.

	METHODS

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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

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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.

View larger version (128K): [in this window] [in a new window]   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.

View larger version (16K): [in this window] [in a new window]   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.

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).

View larger version (110K): [in this window] [in a new window]   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.

View larger version (129K): [in this window] [in a new window]   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.

View larger version (115K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   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.

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).

View larger version (122K): [in this window] [in a new window]   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.

View larger version (13K): [in this window] [in a new window]   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.

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).

View larger version (76K): [in this window] [in a new window]   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).

View larger version (15K): [in this window] [in a new window]   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.

	DISCUSSION

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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.