Occupational exposure to vanadium pentoxide (V₂O₅) has been associated with an increased incidence of chronic obstructive airway disease and a reduction in lung function 1. V₂O₅ is the most common commercial form of vanadium and is the primary form found in industrial exposure situations 2. Occupational exposure to V₂O₅ occurs during the cleaning of oil-fired boilers and furnaces, during handling of catalysts in chemical plants, and during the refining, processing, and burning of vanadium-rich fossil fuels 3.

We previously reported that V₂O₅ causes airway disease in rats that is similar to the pathology of asthma and bronchitis in humans 4. These pathologic changes include mucous cell hyperplasia, increased airway smooth muscle mass, and peribronchiolar fibrosis. Lung fibroblasts are thought to play a major role in V₂O₅-induced airway remodeling in vivo, as these cells proliferate around airways following injury and deposit collagen which defines the airway fibrotic lesion 45.

Vanadium compounds exert cellular stress via inhibition of protein tyrosine phosphatases (PTPs) in cells 6 and through the generation of reactive oxygen species 78. In particular, vanadium compounds have been shown to stimulate release of H₂O₂ in several pulmonary cell types, including alveolar macrophages 9, human lung epithelial cells 10, and human lung fibroblasts 11. Vanadium-induced oxidative stress has been reported to increase the phosphorylation of MAP kinases through the epidermal growth factor receptor (EGFR) 12 and stimulate activation of multiple transcription factors including p53 13, AP-1 14, NF-κB 15 and STAT-1 8. These transcription factors play major roles in cell proliferation, apoptosis, differentiation, and the induction of pro-inflammatory mediators. These cellular responses, in turn, determine the overall pathologic outcomes (e.g., inflammation, fibrosis) that lead to the development of V₂O₅-induced bronchitis.

While much is known about signal transduction pathways that are activated by vanadium-induced oxidative stress, much less is know about genes that are regulated by these signaling pathways. In this study, we investigated V₂O₅-induced gene expression in cultured normal human lung fibroblasts using microarray analysis in order to gain a better understanding of the genes that mediate the pathogenesis of fibrosis.

Exposure of human lung fibroblasts to V₂O₅ resulted in significantly altered expression of over 1400 genes on the Affymetrix Human Genome U133A 2.0 Array. The majority of significantly changed genes were suppressed by V₂O₅ exposure over the 24 hr time course. Four major temporal patterns of gene expression were identified by hierarchical clustering analysis; progressively induced genes (Fig. 1A and 1B), genes that were induced in a biphasic manner (Fig. 1C), progressively suppressed genes (Fig. 1D) and early induced, late suppressed genes (Fig. 1E). Examples of genes from each of these temporal categories are shown in Fig. 2. The cellular localization and functions of selected genes from each of these categories is shown in Table 1.

An analysis of the biological processes (gene ontology categories) affected by V₂O₅ exposure in human lung fibroblasts was performed using the NIH DAVID program 21. This analysis revealed that certain GO categories were unique to V₂O₅-induced genes, including chemotaxis, inflammatory response, immune response, and cell-cell signaling (Table 2). GO categories that were unique to suppressed genes included ubiquitin cycle, cell cycle, DNA repair, nuclear transport, and programmed cell death. A few categories such as RNA processing were common to induced and suppressed genes.

While analysis of GO biological processes was useful in assessing the overall numbers of significantly changed genes in various functional categories, we selectively grouped genes that have been shown to play important roles in various aspects of tissue injury, repair, and remodeling. These categories included A) cytokines and chemokines, B) growth factors, C) STAT signaling, D) cell cycle regulation, E) oxidative stress, and F) TGF-β signaling (Fig. 3). The functions and cellular localization of representative genes from each of these categories is shown in Table 3. A number of cytokines and chemokines were induced over the time course, including IL8, IL-6, CCL8, CXCL9, and CXCL10, while IL15 was suppressed in a time-dependent manner (Fig. 3A). VEGF, HGF, and HBEGF were progressively induced, while FGF2 and FGF9 were suppressed (Fig. 3B). CTGF was induced early (4 hrs) and suppressed late. Members of the STAT signaling pathway were differentially regulated (Fig. 3C). IRF-1 was induced in a biphasic manner. SOCS3 was progressively induced over the time course, while SOCS1 and IFNGR were progressively suppressed. Genes encoding cell cycle regulation were mainly suppressed, including CDKN1B and CDKN1C, which function to inhibit cell cycle progression (Fig. 3D). Oxidative stress genes were differentially regulated. In particular, SOD2 and PIPOX, which function in peroxide generation, were progressively induced (Fig. 3E). OXR1 and OXSR1, which are protective against oxidative stress, were suppressed. Genes involved in TGF-β signaling and collagen deposition were suppressed, including TGFB2, SMAD1, SMURF1, COL1A1, COL1A2, and COL3A1 (Fig. 3F).

Taqman quantitative real time RT-PCR was used to validate a dozen selected genes that were induced or suppressed by V₂O₅ exposure. We chose to validate 3 genes from each of the following categories (growth factors, chemokines, transcription factors, oxidative stress) that appear to have important roles in inflammation, repair, or fibrosis. The results obtained with Taqman quantitative RT-PCR closely mirrored the patterns of temporal induction or suppression observed in the microarray experiment (Fig. 4).

Occupational exposure to vanadium oxides has been associated with an increased incidence of obstructive airway disease and a reduction in lung function 1. In the present study, we investigated the temporal expression of genes in normal human lung fibroblasts exposed V₂O₅. We previously reported that 10 μg/cm² V₂O₅, the same dose used in our microarray experiment, causes minimal cytotoxicity (<10%) to fibroblasts or epithelial cells over a 24 hr time period 1011. This concentration of V₂O₅ also causes several well-defined phenotypic changes in lung fibroblasts including a marked increase in H₂O₂ by fibroblasts 11, phosphorylation of the signal transducer and activator of transcription (STAT-1) 8, and increased expression of heparin-binding EGF-like growth factor, HBEGF 11. Our current study identified genes regulated by V₂O₅ that could play potentially important roles in oxidative stress, inflammation, growth, and apoptosis during V₂O₅-induced lung injury, remodeling and repair. Moreover, our investigation suggests that fibroblasts play an important role in orchestrating the responses of other pulmonary cell types, including neutrophils, airway epithelial cells, lymphocytes, and endothelial cells. The postulated roles of selected genes that were validated by RT-PCR in mediating V₂O₅-induced inflammation, repair, and fibrosis are illustrated in Fig. 5.

A variety of genes encoding cytokines and chemokines were induced or suppressed by V₂O₅. For example, V₂O₅ induced IL8 and IL6, which play important roles in acute inflammation. We validated the strong induction of IL8 mRNA by RT-PCR. Vanadium rich oil fly ash has been reported to increase IL8 and IL6 mRNA and protein expression in normal human airway epithelial cells 2526. Moreover, workers exposed to vanadium-rich fuel oil ash have increased IL8 protein in nasal fluid 27. Chemokines induced by V₂O₅ could play important roles in the immune response. Notably, V₂O₅ induced CXCL9 (Mig) and CXCL10 (inducible protein-10), both of which were validated by RT-PCR. CXCL9 and CXCL10 are STAT1-dependent chemokines that function in the recruitment of lymphocytes 28. We previously showed that V₂O₅ activates STAT1 in lung fibroblasts 8 and mice deficient in STAT1 are susceptible to pulmonary fibrosis 29. Moreover, we have observed intratracheal V₂O₅ exposure in rats causes lymphocytic accumulation surrounding airways and small blood vessels, as well causing proliferation of lymphocytes within the bronchus-associated lymphatic tissue adjacent to large airways 30. It is possible that STAT1-dependent induction of CXCL9 and CXCL10 could be a mechanism for lymphocyte accumulation around airways and blood vessels following lung injury by V₂O₅.

Polypeptide growth factors have a variety of functions in airway remodeling that occurs after metal-induced lung injury. Our genomic analysis identified several growth factors that were validated by RT-PCR. Each of these genes had a different temporal pattern of expression. First, vascular endothelial cell growth factor (VEGF) was progressively induced after V₂O₅ treatment. Li and coworkers showed that vanadium induces the expression of VEGF in a mouse epithelial cell line through the activation of ERK 31. VEGF promotes angiogenesis by stimulating the proliferation of vascular endothelial cells and fibroblasts 32. Our data suggest that fibroblasts could function to promote the formation new blood vessels in V₂O₅-induced airway fibrotic lesions by signaling endothelial cells via VEGF protein or it is possible that secreted VEGF could stimulate fibroblast replication. Second, HBEGF gene expression was increased in a biphasic manner. HBEGF functions both in fibroblast mitogenesis and in epithelial repair 1011. Third, connective tissue growth factor (CTGF) was increased transiently in human lung fibroblasts and then suppressed. We have also reported that V₂O₅ increases CTGF mRNA in the lungs of rats exposed by intratracheal instillation 30. The temporal differences in the expression of VEGF, HBEGF, and CTGF after V₂O₅ treatment remain unclear. We have reported that the early induction of HBEGF is due to peroxide dependent activation of MAP kinases 11. We have also observed that V₂O₅-induced CTGF expression requires MAP kinases (Ingram and Bonner, unpublished observation). The late induction of HBEGF and VEGF could be due to the delayed induction of a transcriptional regulator gene that is increased in response to V₂O₅-induced oxidative stress. One such transcriptional regulator that serves as a master switch for growth factor induction is the early growth response (EGR1) gene. EGR1 was significantly induced at 4 and 24 hr following V₂O₅ treatment in both microarray and RTPCR experiments. EGR1 is induced by a variety of factors including cellular stress and functions as a transcriptional regulator to increase the expression of growth factor genes such as VEGF 33.

Other growth response genes, including the growth arrest specific (GAS1) gene and Bcell translocation genes (BTG1 and BTG2), were progressively suppressed in a time dependent manner after V₂O₅ exposure. BTG1, BTG2, and GAS1 are all anti-mitogenic factors that mediate growth arrest of fibroblasts 343536. Cyclin-dependent kinase inhibitors, CDKN1B p27(Kip1) and CDKN1C p57(Kip2), were also progressively suppressed. These two kinase inhibitors mediate growth arrest and serve as tumor suppressors 3738. Overall, our data suggests that V₂O₅ stimulates the growth and survival of fibroblasts by suppressing genes encoding anti-mitogenic factors (GAS1, BTG2, CDKN1B, and CDKN1C). In particular, our RT-PCR results validated GAS1 suppression in V₂O₅-exposed fibroblasts. While the increased expression of growth factors (i.e., VEGF, HBEGF, CTGF) by fibroblasts exposed to V₂O₅ is likely important in promoting fibroblast growth and survival, the reduced expression of GAS1 by V₂O₅ could be equally important in promoting fibroblast replication and survival. Moreover, V₂O₅ progressively suppressed GAS1 over the entire time course of the experiment, indicating sustained loss of growth arrest control when growth factors such as VEGF, HBEGF, and CTGF were maximally induced.

We found that V₂O₅ induced or suppressed a number of genes that are involved in oxidative stress. Vanadium compounds have been reported to activate several transcription factors and induce the release of inflammatory mediators through the generation of H₂O₂ 13148. Also, we previously reported that human lung fibroblasts exposed to V₂O₅ release micromolar amounts of H₂O₂ in vitro 12 to 18 hrs after V₂O₅ exposure 11. Two genes encoding peroxide-generating enzymes, SOD2 and PIPOX, were validated by RT-PCR. SOD2 was progressively increased over the 24 hr time course of V₂O₅ exposure. SOD2 serves as a major protective anti-oxidant defense enzyme that converts superoxide anion to H₂O₂ 39. V₂O₅ undergoes redox chemistry to generate superoxide anion, so it is possible that SOD2 plays a role in reducing V₂O₅-induced lung injury. L-pipecolate oxidase (PIPOX), a peroxisomal oxidase, was also progressively induced by V₂O₅. PIPOX utilizes molecular oxygen as a substrate with H₂O₂ as a product 40. While V₂O₅ induces genes that generate peroxide (SOD2, PIPOX), we also validated suppression of the oxidative resistance gene (OXR1). Volkert and colleagues discovered the human OXR1 gene using a functional genomics approach in a search for genes that function in protection against oxidative damage 41. While OXR1 is protective against oxidative stress, the precise function of this gene is not well understood. Because OXR1 is protective against oxidative injury, suppression of this gene could contribute to V₂O₅-induced oxidative stress. Also, the temporal suppression of OXR1 occurs as PIPOX (a pro-oxidative stress gene) is temporally induced.

V₂O₅ causes airway fibrosis in rats in vivo, and it is well known that increased collagen production defines the fibrotic lesion 4. TGF-β is an essential mediator of collagen production by fibroblasts. Our results showed that TGFB2, along with its associated signaling intermediates SMAD1 and SMURF1, were all progressively suppressed by V₂O₅. Moreover, several major collagen genes (COL1A2, COL1A1, COL3A1) were suppressed as well. These data indicate that V₂O₅ does not directly stimulate fibroblasts to deposit collagen. Instead, it is likely that TGF-β or other factors signals produced by neighboring pulmonary cell types to increase collagen production. TGF-β mRNA is increased in the lungs of rats treated with V₂O₅. Therefore, during V₂O₅-induced fibrogenesis fibroblasts do not appear to be effectors of their own collagen deposition, but likely require other cell types (e.g., macrophages) as a source of TGF-β.

While we used lung fibroblasts in our study, it is highly relevant to consider the effect of V₂O₅ exposure on gene expression by other lung cell types, including epithelial cells. Li and colleagues used microarray analysis to investigate gene expression changes in human bronchial epithelial cells exposed to vanadium or zinc and identifed a small set of genes that could be used as biomarkers for discriminating vanadium from zinc 42. They also reported that IL8 and PTGS2 (COX-2) were induced several-fold by vanadium but not by zinc. IL8 and PTGS2 were also strongly induced in human lung fibroblasts by vanadium in our study. In fact, we previously reported that COX-2 null mice are susceptible to V₂O₅-induced lung fibrosis, which emphasized an important protective role for the PTGS2 gene during fibrogenesis 43.

A variety of genes were induced or suppressed in normal human lung fibroblasts by vanadium pentoxide (V₂O₅) that appear to have important functions in inflammation, fibrosis and repair. Our data suggest that both the induction of genes that mediate cell proliferation and chemotaxis (VEGF, CTGF, HBEGF), as well as suppression of genes involved in growth arrest and apoptosis (GAS1), is important to the lung fibrotic reaction to V₂O₅. The induction of interferon-inducible, STAT1-dependent chemokines (CXCL9 and CXCL10) could contribute to both suppression of fibroblast proliferation and lymphocyte accumulation. The strong induction of IL8 likely contributes to neutrophilic inflammation. An increase in peroxide-generating enzymes (PIPOX, SOD2) is consistent with H₂O₂ production by V₂O₅, while the reduced expression of protective oxidative response genes (e.g., OXR1) could further contribute to oxidative damage. Overall, our study reveals a wide variety of candidate genes that could mediate V₂O₅-induced airway remodeling after occupational and environmental exposures.

V₂O₅, vanadium pentoxide; STAT-1, signal transducer and activator of transcription; GAS1, growth arrest specific gene; VEGF, vascular endothelial cell growth factor; CTGF, connective tissue growth factor; CXCL10, Chemokine (C-X-C motif) ligand 10; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PTGS-2, prostaglandin synthase 2; OXR1, oxidative resistance gene; SOD2, superoxide dismutase-2; PIPOX, L-pipecolate oxidase.

The author(s) declare that they have no competing interests.

JLI and JCB designed the experiments, performed the data analysis, and drafted the manuscript. JLI, AAM, EAT, JBM, and DGW performed cell culture, RNA isolation, and validated changes in selected genes by Taqman quantitative real-time RT-PCR. LJP performed with microarray hybridizations. RST performed statistical analysis on the microarray data. All authors read and approved the final manuscript.