Vanadium Pentoxide Exposure Causes Strain-Dependent Changes in Mitochondrial DNA Heteroplasmy, Copy Number, and Lesions, but Not Nuclear DNA Lesions.

Interstitial lung diseases (ILDs) are lethal lung diseases characterized by pulmonary inflammation and progressive lung interstitial scarring. We previously developed a mouse model of ILD using vanadium pentoxide (V2O5) and identified several gene candidates on chromosome 4 associated with pulmonary fibrosis. While these data indicated a significant genetic contribution to ILD susceptibility, they did not include any potential associations and interactions with the mitochondrial genome that might influence disease risk. To conduct this pilot work, we selected the two divergent strains we previously categorized as V2O5-resistant C57BL6J (B6) and -responsive DBA/2J (D2) and compared their mitochondrial genome characteristics, including DNA variants, heteroplasmy, lesions, and copy numbers at 14- and 112-days post-exposure. While we did not find changes in the mitochondrial genome at 14 days post-exposure, at 112 days, we found that the responsive D2 strain exhibited significantly fewer mtDNA copies and more lesions than control animals. Alongside these findings, mtDNA heteroplasmy frequency decreased. These data suggest that mice previously shown to exhibit increased susceptibility to pulmonary fibrosis and inflammation sustain damage to the mitochondrial genome that is evident at 112 days post-V2O5 exposure.

Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5).

Interstitial lung diseases (ILDs) are characterized by injury, inflammation, and scarring of alveoli, leading to impaired function. The etiology of idiopathic forms of ILD is not understood, making them particularly difficult to study due to the lack of appropriate animal models. Consequently, few effective therapies have emerged. We developed an inbred mouse model of ILD using vanadium pentoxide (V2O5), the most common form of a transition metal found in cigarette smoke, fuel ash, mineral ores, and steel alloys. Pulmonary responses to V2O5, including dose-dependent increases in lung permeability, inflammation, collagen content, and dysfunction, were significantly greater in DBA/2J mice compared to C57BL/6J mice. Inflammatory and fibrotic responses persisted for 4 mo in DBA/2J mice, while limited responses in C57BL/6J mice resolved. We investigated the genetic basis for differential responses through genetic mapping of V2O5-induced lung collagen content in BXD recombinant inbred (RI) strains and identified significant linkage on chromosome 4 with candidate genes that associate with V2O5-induced collagen content across the RI strains. Results suggest that V2O5 may induce pulmonary fibrosis through mechanisms distinct from those in other models of pulmonary fibrosis. These findings should further advance our understanding of mechanisms involved in ILD and thereby aid in identification of new therapeutic targets.

Epigenetic considerations in medicine.

Epigenetic modifications are gene regulatory mechanisms that allow rapid adaptation to the environment. These mitotically stable and meiotically heritable changes are sensitive to environmental conditions especially during developmental periods, and they are essential to understanding how information in the DNA sequence is utilized. Recent research in this area has led to excitement and questions about medical applications of epigenetics.

Vanadium pentoxide (V(2)O(5)) induced mucin production by airway epithelium.

Exposure to environmental pollutants has been linked to various airway diseases and disease exacerbations. Almost all chronic airway diseases such as chronic obstructive pulmonary disease and asthma are caused by complicated interactions between gene and environment. One of the major hallmarks of those diseases is airway mucus overproduction (MO). Excessive mucus causes airway obstruction and significantly increases morbidity and mortality. Metals are major components of environmental particulate matters (PM). Among them, vanadium has been suggested to play an important role in PM-induced mucin production. Vanadium pentoxide (V(2)O(5)) is the most common commercial source of vanadium, and it has been associated with occupational chronic bronchitis and asthma, both of which are MO diseases. However, the underlying mechanism is not entirely clear. In this study, we used both in vitro and in vivo models to demonstrate the robust inductions of mucin production by V(2)O(5). Furthermore, the follow-up mechanistic study revealed a novel v-raf-1 murine leukemia viral oncogene homolog 1-IKK-NF-κB pathway that mediated V(2)O(5)-induced mucin production. Most interestingly, the reactive oxygen species and the classical mucin-inducing epidermal growth factor receptor (EGFR)-MAPK pathway appeared not to be involved in this process. Thus the V(2)O(5)-induced mucin production may represent a novel EGFR-MAPK-independent and environmental toxicant-associated MO model. Complete elucidation of the signaling pathway in this model will not only facilitate the development of the treatment for V(2)O(5)-associated occupational diseases but also advance our understanding on the EGFR-independent mucin production in other chronic airway diseases.

Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice.

BACKGROUND: Multi-walled carbon nanotubes (MWCNTs) are widely used in many disciplines due to their unique physical and chemical properties. Therefore, some concerns about the possible human health and environmental impacts of manufactured MWCNTs are rising. We hypothesized that instillation of MWCNTs impairs pulmonary function in C57BL/6 mice due to development of lung inflammation and fibrosis. METHODS: MWCNTs were administered to C57BL/6 mice by oropharyngeal aspiration (1, 2, and 4 mg/kg) and we assessed lung inflammation and fibrosis by inflammatory cell infiltration, collagen content, and histological assessment. Pulmonary function was assessed using a FlexiVent system and levels of Ccl3, Ccl11, Mmp13 and IL-33 were measured by RT-PCR and ELISA. RESULTS: Mice administered MWCNTs exhibited increased inflammatory cell infiltration, collagen deposition and granuloma formation in lung tissue, which correlated with impaired pulmonary function as assessed by increased resistance, tissue damping, and decreased lung compliance. Pulmonary exposure to MWCNTs induced an inflammatory signature marked by cytokine (IL-33), chemokine (Ccl3 and Ccl11), and protease production (Mmp13) that promoted the inflammatory and fibrotic changes observed within the lung. CONCLUSIONS: These results further highlight the potential adverse health effects that may occur following MWCNT exposure and therefore we suggest these materials may pose a significant risk leading to impaired lung function following environmental and occupational exposures.

Vanadium pentoxide induces pulmonary inflammation and tumor promotion in a strain-dependent manner.

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.

Airway Exposure to Modified Multi-walled Carbon Nanotubes Perturbs Cardiovascular Adenosinergic Signaling in Mice.

The broad list of commercial applications for multi-walled carbon nanotubes (MWCNT) can be further expanded with the addition of various surface chemistry modifications. For example, standard commercial grade MWCNT (C-grade) can be carboxylated (COOH) or nitrogen-doped (N-doped) to suite specific utilities. We previously reported dose-dependent expansions of cardiac ischemia/reperfusion (I/R) injury, 24 h after intratracheal instillation of C-grade, COOH, or N-doped MWCNT in mice. Here, we have tested the hypothesis that airway exposure to MWCNT perturbs cardiovascular adenosinergic signaling, which could contribute to exacerbation of cardiac I/R injury. 100 µL of Vehicle or identical suspension volumes containing 100 µg of C-grade, COOH, or N-doped MWCNT were instilled into the trachea of CD-1 ICR mice. 1 day later, we measured cyclic adenosine monophosphate (cAMP) concentrations in cardiac tissue and evaluated arterial adenosinergic smooth muscle signaling mechanisms related to nitric oxide synthase (NOS) and cyclooxygenase (COX) in isolated aortic tissue. We also verified cardiac I/R injury expansion and examined both lung histology and bronchoalveolar lavage fluid cellularity in MWCNT exposed mice. Myocardial cAMP concentrations were reduced (p < 0.05) in the C-grade group by 17.4% and N-doped group by 13.7% compared to the Vehicle group. Curve fits to aortic ring 2-Cl-Adenosine concentration responses were significantly greater in the MWCNT groups vs. the Vehicle group. Aortic constrictor responses were more pronounced with NOS inhibition and were abolished with COX inhibition. These findings indicate that addition of functional chemical moieties on the surface of MWCNT may alter the biological responses to exposure by influencing cardiovascular adenosinergic signaling and promoting cardiac injury.

Oxidative stress and antioxidants in the pathogenesis of pulmonary fibrosis: a potential role for Nrf2.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disorder in which excessive deposition of extracellular matrix leads to irreversible scarring of interstitial lung tissue. The etiology of IPF remains unknown, but growing evidence suggests that disequilibrium in oxidant/antioxidant balance contributes significantly. IPF is currently regarded as a fibroproliferative disorder triggered by repeated alveolar epithelial cell injury. Oxidative stress plays a role in many processes involved in alveolar epithelial cell injury and fibrogenesis. Here we review the role of oxidative stress in IPF, and other forms of pulmonary fibrosis, with particular attention to antioxidant defenses regulated by the redox-sensitive transcription factor nuclear factor, erythroid derived 2, like (Nrf2). Nrf2 binds specific antioxidant response elements (AREs) in the promoter of antioxidant enzyme and defense protein genes and regulates their expression in many tissue types. Nrf2 protects from several phenotypes in which enhanced oxidative burden contributes to disease pathogenesis, including cancer, acute lung injury, and pulmonary fibrosis. We suggest that promoter polymorphisms in human NRF2 may contribute to IPF susceptibility, although this hypothesis has not been tested. Pulmonary fibrosis is a highly complex disease and involves multiple genes and processes, and new therapies for cellular and molecular targets involved in pathogenic mechanisms are needed.

Vanadium-induced STAT-1 activation in lung myofibroblasts requires H2O2 and P38 MAP kinase.

Vanadium compounds present in air pollution particulate matter activate signal transduction pathways in pulmonary cell types leading to pathological outcomes including aberrant cell proliferation, apoptosis, and cytokine expression. Vanadium has been proposed to activate transcription factors via the generation of hydrogen peroxide (H2O2). We investigated the mechanisms through which vanadium pentoxide (V2O5), the major form of vanadium released from the industrial burning of fuel oil, activated the signal transducer and activator of transcription (STAT)-1. V2O5-induced STAT-1 activation was blocked by catalase and N-acetyl-L-cysteine (NAC), suggesting vanadium-induced generation of H2O2. Surprisingly, however, V2O5 did not increase H2O2 levels released by rat lung myofibroblasts into cell culture supernatants. Instead, these quiescent myofibroblasts spontaneously released micromolar concentrations of H2O2, and the addition of V2O5 reduced H2O2 levels in cell culture supernatants within minutes. V2O5 suppressed H2O2 for as long as 24 h. Differences in the temporal activation of STAT-1 and p38 MAPK were observed following V2O5 or H2O2 treatment, and STAT-1 activation by V2O5 or H2O2 was attenuated by an inhibitor of the EGF receptor tyrosine kinase (AG1478) or p38 MAPK (SB203580). The phosphorylation of p38 MAPK by V2O5 was inhibited by NAC and catalase, yet the EGF receptor inhibitor AG1478 had no effect on V2O5-induced p38 MAPK activation. Collectively, our findings support the novel hypothesis that H2O2 spontaneously generated by myofibroblasts fuels vanadium-induced activation of STAT-1. Moreover, p38 MAPK and EGF receptor activation are required for V2O5-induced STAT-1 activation.

Mast cells contribute to altered vascular reactivity and ischemia-reperfusion injury following cerium oxide nanoparticle instillation.

Cerium oxide (CeO2) represents an important nanomaterial with wide ranging applications. However, little is known regarding how CeO2 exposure may influence pulmonary or systemic inflammation. Furthermore, how mast cells would influence inflammatory responses to a nanoparticle exposure is unknown. We thus compared pulmonary and cardiovascular responses between C57BL/6 and B6.Cg-Kit(W-sh) mast cell deficient mice following CeO2 nanoparticle instillation. C57BL/6 mice instilled with CeO2 exhibited mild pulmonary inflammation. However, B6.Cg-Kit(W-sh) mice did not display a similar degree of inflammation following CeO2 instillation. Moreover, C57BL/6 mice instilled with CeO2 exhibited altered aortic vascular responses to adenosine and an increase in myocardial ischemia/reperfusion injury which was absent in B6.Cg-Kit(W-sh) mice. In vitro CeO2 exposure resulted in increased production of PGD2, TNF-α, IL-6 and osteopontin by cultured mast cells. These findings demonstrate that CeO2 nanoparticles activate mast cells contributing to pulmonary inflammation, impairment of vascular relaxation and exacerbation of myocardial ischemia/reperfusion injury.

Mouse models of bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is a component of many interstitial lung diseases, including idiopathic pulmonary fibrosis, a chronic, progressive disease for which there is currently no effective therapy. Bleomycin has been widely used in rodents to model pulmonary fibrosis for the study of mechanisms involved in fibrogenesis and for evaluation of potential therapies. Bleomycin induces DNA strand breaks, resulting in pulmonary inflammation, injury, and subsequent interstitial fibrosis. This unit describes methods for delivering bleomycin, either directly into the lung or systemically, to create models of pulmonary fibrosis in rodents. Also described is a rapid and easy procedure for measuring lung collagen content to quantify the severity of fibrosis.

Susceptibility of signal transducer and activator of transcription-1-deficient mice to pulmonary fibrogenesis.

The signal transducer and activator of transcription (Stat)-1 mediates growth arrest and apoptosis. We postulated that lung fibrosis characterized by excessive proliferation of lung fibroblasts would be enhanced in Stat1-deficient (Stat1-/-) mice. Two weeks after bleomycin aspiration (3 U/kg), Stat1-/- mice exhibited a more severe fibroproliferative response and significantly elevated total lung collagen compared to wild-type mice. Growth factors [epidermal growth factor (EGF) or platelet-derived growth factor (PDGF)] enhanced [3H]thymidine uptake in lung fibroblasts isolated from Stat1-/- mice compared to wild-type mice. Interferon (IFN)-gamma, which signals growth arrest via Stat1, inhibited EGF- or PDGF-stimulated mitogenesis in wild-type fibroblasts but enhanced [3H]thymidine uptake in Stat1-/- fibroblasts. Moreover, IFN-gamma treatment in the absence of growth factors induced a concentration-dependent increase in [3H]thymidine uptake in Stat1-/- but not wild-type fibroblasts. Mitogen-activated protein kinase (ERK-1/2) phosphorylation in response to PDGF or EGF did not differ among Stat1-/- and wild-type fibroblasts. However, Stat3 phosphorylation induced by PDGF, EGF, or IFN-gamma increased twofold in Stat1-/- fibroblasts compared to wild-type fibroblasts. Our findings indicate that Stat1-/- mice are more susceptible to bleomycin-induced lung fibrosis than wild-type mice due to 1) enhanced fibroblast proliferation in response to growth factors (EGF and PDGF), 2) stimulation of fibroblast growth by a Stat1-independent IFN-gamma signaling pathway, and 3) increased activation of Stat3.

Complement factor 3 mediates particulate matter-induced airway hyperresponsiveness.

Epidemiologic studies have suggested that exposure to airborne particulate matter (PM) can exacerbate allergic airway responses; however, the mechanism(s) are not well understood. We and others have recently shown that development of airway hyperresponsiveness (AHR) may be a complement-mediated process. In the present study, we examined the role of complement factor 3 (C3) in the development of PM-induced AHR and airway inflammation by comparing responses between C3-deficient (C3(-/-)) and wild-type mice. Mice were exposed to 0.5 mg of ambient particulate collected in urban Baltimore. Forty-eight hours later, airway responsiveness to intravenous acetylcholine was assessed and bronchoalveolar lavage was conducted. PM exposure of wild-type mice resulted in significant increases in AHR, whereas it did not significantly increase airway reactivity in C3(-/-) mice. Interestingly, PM induced similar inflammatory responses in both wild-type and C3(-/-) mice. Immunohistochemical staining demonstrated marked C3 deposition in the airway epithelium and connective tissue of wild-type mice after PM exposure. These results suggest that exposure to PM may induce AHR through activation of complement factor 3 in the airways.