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Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis.

ABSTRACT: BACKGROUND: Exposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5 in vitro in order to identify candidate genes that could play a role in inflammation, fibrosis, and repair during the pathogenesis of V2O5-induced bronchitis. METHODS: Normal human lung fibroblasts were exposed to V2O5 in a time course experiment. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Selected genes that were significantly changed in the microarray experiment were validated by RT-PCR. RESULTS: V2O5 altered more than 1,400 genes, of which ~300 were induced while >1,100 genes were suppressed. Gene ontology categories (GO) categories unique to induced genes included inflammatory response and immune response, while GO categories unique to suppressed genes included ubiquitin cycle and cell cycle. A dozen genes were validated by RT-PCR, including growth factors (HBEGF, VEGF, CTGF), chemokines (IL8, CXCL9, CXCL10), oxidative stress response genes (SOD2, PIPOX, OXR1), and DNA-binding proteins (GAS1, STAT1). CONCLUSION: Our study identified a variety of genes that could play pivotal roles in inflammation, fibrosis and repair during V2O5-induced bronchitis. The induction of genes that mediate inflammation and immune responses, as well as suppression of genes involved in growth arrest appear to be important to the lung fibrotic reaction to V2O5.

SUBMITTER: Ingram JL

PROVIDER: S-EPMC1865536 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis.

Ingram Jennifer L JL Antao-Menezes Aurita A Turpin Elizabeth A EA Wallace Duncan G DG Mangum James B JB Pluta Linda J LJ Thomas Russell S RS Bonner James C JC

Respiratory research 20070425

<h4>Background</h4>Exposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5 in vitro in order to identify candidate genes that could play a role in inflammation, fibrosis, and repair during the pathogenesis of V2O5-induced bronchitis.<h4>Methods</h4>Normal human lung fibroblasts were exposed to V2O5 in a time course experiment. Gene expression was measured at various time points over a 24 ...[more]

PMID: 17459161

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Project description:Exposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5 in vitro in order to identify candidate genes that could play a role in airway remodeling associated with V2O5-induced bronchitis. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Expression data were preprocessed using RMA with a log2 transformation. Statistical analysis was performed in R using the affylmGUI package using a linear model with contrasts between untreated control and V2O5-exposed fibroblasts. Genes identified as statistically significant were filtered by selecting only those genes that exhibited a > 2-fold change. Quantitative real-time RT-PCR was utilized to confirm expression of selected genes. More than 2000 genes were significantly changed in response to V2O5 over the time course of our experiment. Genes altered by V2O5 were involved in biologic processes related to cell growth and differentiation, oxidative stress responses, immune regulation, and interferon signaling and apoptosis. In particular, V2O5 induced genes that encode growth factors involved in epithelial repair (HB-EGF) or angiogenesis (VEGF), peroxide generating enzymes (SOD2), pro-inflammatory enzymes (PGHS2), while suppressing genes involved in growth arrest (GAS1, STAT-1) and cell cycle inhibition (CDKN1B). Our study also identified a variety of novel genes that could be used as biomarkers of V2O5-induced bronchitis or could serve as candidate genes for disease progression. Experiment Overall Design: Experimental Design Experiment Overall Design: Normal adult human lung fibroblasts (ATCC 16 Lu) were purchased from American Type Culture Collection (Rockville, MD). Fibroblasts were seeded into 175 cm2 plastic culture flasks and grown to confluence in 10% fetal bovine serum (FBS)/Dulbeccoâs modified Eagleâs medium (DMEM), then trypsin-liberated, and seeded into 150 mm dishes. Confluent monolayers were rendered quiescent for 24 hrs in serum-free defined medium (SFDM) that consisted of Hamâs F-12 medium with 0.25% BSA with an insulin/transferrin/selenium supplement. Cells were treated with 10 Âµg/cm2 vanadium pentoxide, V2O5 (Aldrich Chemical, Milwaukee, WI) or SFDM and RNA was harvested from the fibroblast cultures at 1, 4, 8, 12 and 24 hrs post-treatment. RNA from an SFDM control was harvested at each of these time points to normalize the V2O5 treatment at the same corresponding time point. Three replicate chips were analyzed for SFDM and V2O5 treatment groups at each of the five time points tested. Experiment Overall Design: Microarray Hybridizations and Data Analysis Experiment Overall Design: Human lung fibroblast RNA was isolated using RNeasy columns (Qiagen, Valencia, CA). RNA quality was verified by spectrophotometry and gel electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Probe preparation and hybridization to the microarray was performed in the CIIT Gene Expression Core Facility using standard Affymetrix procedures. Double-stranded cDNA was synthesized from RNA using an oligo-dT24-T7. Biotinylated cRNA was synthesized from an aliquot of the cDNA template using the T7 RNA Transcript Labeling Kit (ENZO Diagnostics, Farmingdale NY). The labeled cRNA was then fragmented, hybridized to Affymetrix Human Genome U133A 2.0 arrays (Affymetrix, Santa Clara, CA), and stained using phycoerythrein-conjugated streptavidin (Molecular Probes, Eugene, OR). Experiment Overall Design: Statistical Analysis and Data Processing Experiment Overall Design: The microarray data were preprocessed using RMA with a log base 2 (log2) transformation. Statistical analysis of the data was performed in R using the affyImGUI package. To identify genes with significant changes in expression following V2O5 exposure, all treatment groups were analyzed using a linear model with contrasts between untreated fibroblasts and V2O5-exposed fibroblasts at each time point. Probability values were adjusted for multiple comparisons using a false discovery rate of 5% (FDR = 0.05). Genes identified as statistically significant were subject to an additional filter by selecting only those genes that exhibited a > 2-fold change from the untreated fibroblasts.

Project description:Both environmental exposure to vanadium pentoxide (V2O5, V+5 for its ionic counterparts) and fibroblast senescence are associated with pulmonary fibrosis, but whether V+5 causes fibroblast senescence remains unknown. We found in a dose-response study that 2-40 μM V+5 caused human lung fibroblasts (HLF) senescence with increased senescence-associated β-galactosidase activity and p16 expression, while cell death occurred at higher concentration (LC50, 82 μM V+5). Notably, measures of reactive oxygen species (ROS) production with fluorescence probes showed no association of ROS with V+5-dependent senescence. Preloading catalase (polyethylene-conjugated), a H2O2 scavenger, did not alleviate the cellular senescence induced by V+5. Analyses of the cellular glutathione (GSH) system showed that V+5 oxidized GSH, increased GSH biosynthesis, stimulated cellular GSH efflux and increased protein S-glutathionylation, and addition of N-acetyl cysteine inhibited V+5-elevated p16 expression, suggesting that thiol oxidation mediates V+5-caused senescence. Moreover, strong correlations between GSSG/GSH redox potential (Eh), protein S-glutathionylation, and cellular senescence (R2 > 0.99, p < 0.05) were present in V+5-treated cells. Studies with cell-free and enzyme-free solutions showed that V+5 directly oxidized GSH with formation of V+4 and GSSG in the absence of O2. Analyses of V+5 and V+4 in HLF and culture media showed that V+5 was reduced to V+4 in cells and that a stable V+4/V+5 ratio was rapidly achieved in extracellular media, indicating ongoing release of V+4 and reoxidation to V+5. Together, the results show that V+5-dependent fibroblast senescence is associated with a cellular/extracellular redox cycling mechanism involving the GSH system and occurring under conditions that do not cause cell death. These results establish a mechanism by which environmental vanadium from food, dietary supplements or drinking water, can cause or contribute to lung fibrosis in the absence of high-level occupational exposures and cytotoxic cell death.

Project description:BACKGROUND: Elevated levels of air pollution are associated with increased risk of lung cancer. Particulate matter (PM) contains transition metals that may potentiate neoplastic development through the induction of oxidative stress and inflammation, a lung cancer risk factor. Vanadium pentoxide (V2O5) is a component of PM derived from fuel combustion as well as a source of occupational exposure in humans. In the current investigation we examined the influence of genetic background on susceptibility to V2O5-induced inflammation and evaluated whether V2O5 functions as a tumor promoter using a 2-stage (initiation-promotion) model of pulmonary neoplasia in mice. RESULTS: A/J, BALB/cJ (BALB), and C57BL/6J (B6) mice were treated either with the initiator 3-methylcholanthrene (MCA; 10 microg/g; i.p.) or corn oil followed by 5 weekly aspirations of V2O5 or PBS and pulmonary tumors were enumerated 20 weeks following MCA treatment. Susceptibility to V2O5-induced pulmonary inflammation was assessed in bronchoalveolar lavage fluid (BALF), and chemokines, transcription factor activity, and MAPK signaling were quantified in lung homogenates. We found that treatment of animals with MCA followed by V2O5 promoted lung tumors in both A/J (10.3 +/- 0.9 tumors/mouse) and BALB (2.2 +/- 0.36) mice significantly above that observed with MCA/PBS or V2O5 alone (P < 0.05). No tumors were observed in the B6 mice in any of the experimental groups. Mice sensitive to tumor promotion by V2O5 were also found to be more susceptible to V2O5-induced pulmonary inflammation and hyperpermeability (A/J>BALB>B6). Differential strain responses in inflammation were positively associated with elevated levels of the chemokines KC and MCP-1, higher NFkappaB and c-Fos binding activity, as well as sustained ERK1/2 activation in lung tissue. CONCLUSIONS: In this study we demonstrate that V2O5, an occupational and environmentally relevant metal oxide, functions as an in vivo lung tumor promoter among different inbred strains of mice. Further, we identified a positive relationship between tumor promotion and susceptibility to V2O5-induced pulmonary inflammation. These findings suggest that repeated exposures to V2O5 containing particles may augment lung carcinogenesis in susceptible individuals through oxidative stress mediated pathways.

Dataset Information

Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis.

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Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis.

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