Nicholas J. Kenyon

Simvastatin Inhibits Airway Hyperreactivity: Implications for the Mevalonate Pathway and Beyond

RATIONALE Statin use has been linked to improved lung health in asthma and chronic obstructive pulmonary disease. We hypothesize that statins inhibit allergic airway inflammation and reduce airway hyperreactivity via a mevalonate-dependent mechanism. OBJECTIVES To determine whether simvastatin attenuates airway inflammation and improves lung physiology by mevalonate pathway inhibition. METHODS BALB/c mice were sensitized to ovalbumin over 4 weeks and exposed to 1% ovalbumin aerosol over 2 weeks. Simvastatin (40 mg/kg) or simvastatin plus mevalonate (20 mg/kg) was injected intraperitoneally before each ovalbumin exposure. MEASUREMENTS AND MAIN RESULTS Simvastatin reduced total lung lavage leukocytes, eosinophils, and macrophages (P < 0.05) in the ovalbumin-exposed mice. Cotreatment with mevalonate, in addition to simvastatin, reversed the antiinflammatory effects seen with simvastatin alone (P < 0.05). Lung lavage IL-4, IL-13, and tumor necrosis factor-alpha levels were all reduced by treatment with simvastatin (P < 0.05). Simvastatin treatment before methacholine bronchial challenge increased lung compliance and reduced airway hyperreactivity (P = 0.0001). CONCLUSIONS Simvastatin attenuates allergic airway inflammation, inhibits key helper T cell type 1 and 2 chemokines, and improves lung physiology in a mouse model of asthma. The mevalonate pathway appears to modulate allergic airway inflammation, while the beneficial effects of simvastatin on lung compliance and airway hyperreactivity may be independent of the mevalonate pathway. Simvastatin and similar agents that modulate the mevalonate pathway may prove to be treatments for inflammatory airway diseases, such as asthma.

Vitamin A Deficiency Decreases and High Dietary Vitamin A Increases Disease Severity in the Mouse Model of Asthma

The Th1/Th2 paradigm has become an important issue in the pathogenesis of asthma, characterized by normal Th1 and elevated Th2 cytokine expression. Vitamin A deficiency (VAD) can produce a Th1 bias, whereas high-level dietary vitamin A can promote a Th2 bias. We used the OVA exposure mouse model to determine the contributions of vitamin A-deficient, control (4IU/g), and high-level vitamin A (250-IU/g) diets to the development of allergic airway inflammation and hyperresponsiveness. VAD reduced serum IgE and IgG1 responses, pulmonary eosinophilia, and the levels of IL-4 and IL-5 in bronchoalveolar lavage specimens, whereas the 250-IU/g diet increased serum IgE. Also, VAD blocked pulmonary hyperresponsiveness following methacholine challenge while the 250-IU/g diet exacerbated pulmonary hyperresponsiveness. In conclusion, VAD diminished and high-level dietary vitamin A enhanced the development of experimental asthma in this model system. These data suggest that excessive intake of vitamin A may increase the risk or severity of asthma in industrialized countries whereas vitamin A deficiency continues to increase mortality from infectious diseases in developing countries.

Airway fibrosis in a mouse model of airway inflammation.

BALB/c mice were sensitized to ovalbumin by systemic injection and then exposed for up to 8 weeks to ovalbumin aerosols in whole body chambers. A pattern of airway inflammation, mucous cell hypertrophy and hyperplasia, and airway remodeling with submucosal fibrosis was observed as lesions evolved over time. Larger conducting airways were removed from the lungs by microdissection. Airway fibrosis was quantified by direct assay for collagen content, which was significantly increased after 4 and 8 weeks of exposure to ovalbumin aerosol. Based upon PCR analysis of mRNA levels in the airways, most of the newly synthesized collagen was Type I. Relaxin, administered by continuous infusion over the second half of a 4-week exposure to ovalbumin, was able to inhibit the accumulation of collagen in the airways of exposed mice. Thus, stimulation of collagen degradation by an activator of collagen breakdown by matrix metalloproteinases appears to be an effective therapeutic strategy in prevention of airway fibrosis in this animal model. Whole body plethysmography of unrestrained mice indicated functional changes in airway reactivity in the lungs of exposed animals occurring in conjunction with the reported structural changes. This result indicates that the ovalbumin-exposed mouse may be a suitable model for examining structure-function relationships in the lungs of animals with a predictable time course of airway inflammation, remodeling, and fibrosis and for testing potential new drugs for treatment of asthma or chronic bronchitis at a mechanistic level.

Simvastatin Inhibits Goblet Cell Hyperplasia and Lung Arginase in a Mouse Model of Allergic Asthma: A Novel Treatment for Airway Remodeling?

Airway remodeling in asthma contributes to airway hyperreactivity, loss of lung function, and persistent symptoms. Current therapies do not adequately treat the structural airway changes associated with asthma. The statins are cholesterol-lowering drugs that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting step of cholesterol biosynthesis in the mevalonate (MA) pathway. These drugs have been associated with improved respiratory health, and ongoing clinical trials are testing their therapeutic potential in asthma. We hypothesized that simvastatin treatment of ovalbumin (OVA)-exposed mice would attenuate early features of airway remodeling by a mevalonate-dependent mechanism. BALB/c mice initially were sensitized to OVA and then exposed to 1% OVA aerosol for 2 weeks after sensitization for 6 exposures. Simvastatin (40 mg/kg) or simvastatin plus MA (20 mg/kg) were injected intraperitoneally before each OVA exposure. Treatment with simvastatin attenuated goblet cell hyperplasia, arginase-1 protein expression, and total arginase enzyme activity, but it did not alter airway hydroxyproline content or transforming growth factor-β1. Inhibition of goblet cell hyperplasia by simvastatin was mevalonate-dependent. No appreciable changes to airway smooth muscle cells were observed in any control or treatment groups. In conclusion, in an acute mouse model of allergic asthma, simvastatin inhibited early hallmarks of airway remodeling, which are indicators that can lead to airway thickening and fibrosis. Statins are potentially novel treatments for airway remodeling in asthma. Additional studies using subchronic or chronic allergen exposure models are needed to extend these initial findings.

Arginases I and II in lungs of ovalbumin-sensitized mice exposed to ovalbumin: Sources and consequences

Arginase gene expression in the lung has been linked to asthma both in clinical studies of human patients and in the well-studied mouse model of ovalbumin-induced airway inflammation. Arginase is thought to regulate NO levels in the lung by its ability to divert arginine, the substrate for nitric oxide synthases that produce citrulline and NO, into an alternative metabolic pathway producing ornithine and urea. In the present study arginase I and arginase II concentrations were measured in isolated microdissected airway preparations from sensitized Balb/c mice exposed to ovalbumin aerosol. We found that arginase II was constitutively expressed in the airways of normal mice, whereas arginase I was undetectable in normal airways, while its expression was increased in airways of mice exposed to ovalbumin. The expression of arginase I strongly correlated with the presence of lung inflammation, as quantified by differential cell counts in lung lavage, suggesting that most, or all, of the arginase I in lungs of mice exposed to ovalbumin is present in the inflammatory cells rather than in the airway epithelium. There was also a significant correlation between increased expression of arginase I in the isolated airways and decreased lung compliance. On the other hand, while we found arginase II expression to also be significantly increased in airways from mice exposed to ovalbumin as compared with normal airways, the relative increase was much less than that observed for arginase I, suggesting that there was a smaller contribution of inflammatory cells to the arginase II content of the airways in mice exposed to ovalbumin. There was no apparent correlation between the content of arginase in isolated airways and exhaled NO concentration in the expired air from mice exposed to ovalbumin. However, there was a correlation between exhaled NO concentration from mice exposed to ovalbumin and the lymphocyte content of the lung lavage. The concentration of arginine found in isolated airways from Balb/c mice exposed for 2 weeks to ovalbumin was about half of the value found in isolated microdissected airways from normal mice. Treatment of mice systemically with an arginase inhibitor significantly increased the amount of NO produced, as measured as the amount of nitrite+nitrate (NOx) in lung lavage supernatant prepared from mice exposed to ovalbumin. Our results are consistent with the hypothesis that the response of the lung to ovalbumin challenge includes an adaptive response in the large airways regulating the concentration of arginine within cells of the airway epithelium and subepithelial layer, by shunting of arginine into the metabolic pathway for increased synthesis of NO.

Geoepidemiology of COPD and idiopathic pulmonary fibrosis

Progress in improving patient outcomes and advancing therapeutics in chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) is hampered by phenotypic heterogeneity and variable responsiveness to clinical interventions that are not fully explained by currently held disease paradigms for COPD and IPF. Although these chronic lung diseases differ in their geoepidemiology and immunopathogenesis, emerging evidence suggest that organ-specific autoimmunity may underlie subphenotypes of COPD and IPF. In particular, the links to tobacco smoking, diet, gender, and environment are explored in this review. We also highlight potential mechanisms that could guide future investigations in both laboratory and clinical settings. A paradigm shift is needed in how we think about COPD and IPF, based on geoepidemiology and a broader understanding of disease pathogenesis that may ultimately lead to new therapies and improved patient outcomes.

Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin

Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, l-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses - inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration - were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nomega-hydroxy-nor-l-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2 knockout mice exposed to ovalbumin.

Continuous Droplet Removal upon Dropwise Condensation of Humid Air on a Hydrophobic Micropatterned Surface

Combination of two physical phenomena, capillary pressure gradient and wettability gradient, allows a simple two-step fabrication process that yields a reliable hydrophobic self-cleaning condenser surface. The surface is fabricated with specific microscopic topography and further treatment with a chemically inert low-surface-energy material. This process does not require growth of nanofeatures (nanotubes) or hydrophilic–hydrophobic patterning of the surface. Trapezoidal geometry of the microfeatures facilitates droplet transfer from the Wenzel to the Cassie state and reduces droplet critical diameter. The geometry of the micropatterns enhances local coalescence and directional movement for droplets with diameter much smaller than the radial length of the micropatterns. The hydrophobic self-cleaning micropatterned condenser surface prevents liquid film formation and promotes continuous dropwise condensation cycle. Upon dropwise condensation, droplets follow a designed wettability gradient created with micropatterns from the most hydrophobic to the least hydrophobic end of the surface. The surface has higher condensation efficiency, due to its directional self-cleaning property, than a plain hydrophobic surface. We explain the self-actuated droplet collection mechanism on the condenser surface and demonstrate experimentally the creation of an effective wettability gradient over a 6 mm radial distance. In spite of its fabrication simplicity, the fabricated surface demonstrates self-cleaning property, enhanced condensation performance, and reliability over time. Our work enables creation of a hydrophobic condenser surface with the directional self-cleaning property that can be used for collection of biological (chemical, environmental) aerosol samples or for condensation enhancement.

Design-of-experiment optimization of exhaled breath condensate analysis using a miniature differential mobility spectrometer (DMS).

Analytical instruments that can measure small amounts of chemicals in complicated biological samples are often useful as diagnostic tools. However, it can be challenging to optimize these sensors using actual clinical samples, given the heterogeneous background and composition of the test materials. Here we use gas chromatography-differential mobility spectrometry (GC/DMS) to analyze the chemical content of human exhaled breath condensate (EBC). Ultimately, this system can be used for non-invasive disease diagnostics. Many parameters can be adjusted within this instrument system, and we implemented a factorial design-of-experiments to systematically test several combinations of parameter settings while concurrently analyzing effects and interactions. We examined four parameters that affect sensitivity and detection for our instrument, requiring a 2(4) factorial design. We optimized sensor function using EBC samples spiked with acetone, a known clinical biomarker in breath. Two outputs were recorded for each experiment combination: number of chemicals detected, and the amplitude of acetone signal. Our goal is to find the best parameter combination that yields the highest acetone peak while also preserving the largest number of other chemical peaks in the spectra. By optimizing the system, we can conduct further clinical experiments with our sensor more efficiently and accurately.

Differential effects of simvastatin on IL-13-induced cytokine gene expression in primary mouse tracheal epithelial cells.

BackgroundAsthma causes significant morbidity worldwide in adults and children alike, and incurs large healthcare costs. The statin drugs, which treat hyperlipidemia and cardiovascular diseases, have pleiotropic effects beyond lowering cholesterol, including immunomodulatory, anti-inflammatory, and anti-fibrotic properties which may benefit lung health. Using an allergic mouse model of asthma, we previously demonstrated a benefit of statins in reducing peribronchiolar eosinophilic inflammation, airway hyperreactivity, goblet cell hyperplasia, and lung IL-4 and IL-13 production.ObjectivesIn this study, we evaluated whether simvastatin inhibits IL-13-induced pro-inflammatory gene expression of asthma-related cytokines in well-differentiated primary mouse tracheal epithelial (MTE) cell cultures. We hypothesized that simvastatin reduces the expression of IL-13-inducible genes in MTE cells.MethodsWe harvested tracheal epithelial cells from naïve BALB/c mice, grew them under air-liquid interface (ALI) cell culture conditions, then assessed IL-13-induced gene expression in MTE cells using a quantitative real-time PCR mouse gene array kit.ResultsWe found that simvastatin had differential effects on IL-13-mediated gene expression (inhibited eotaxin-1; MCP-1,-2,-3; and osteopontin (SPP1), while it induced caspase-1 and CCL20 (MIP-3α)) in MTE cells. For other asthma-relevant genes such as TNF, IL-4, IL-10, CCL12 (MCP-5), CCL5 (RANTES), and CCR3, there were no significant IL-13-inducible or statin effects on gene expression.ConclusionsSimvastatin modulates the gene expression of selected IL-13-inducible pro-inflammatory cytokines and chemokines in primary mouse tracheal epithelial cells. The airway epithelium may be a viable target tissue for the statin drugs. Further research is needed to assess the mechanisms of how statins modulate epithelial gene expression.