Airway inflammation and remodeling are major features of chronic obstructive pulmonary disease (COPD), whereas pulmonary hypertension is a common comorbidity associated with a poor disease prognosis. Recent studies in animal models have indicated that increased arginase activity contributes to features of asthma, including allergen-induced airway eosinophilia and mucus hypersecretion. Although cigarette smoke and lipopolysaccharide (LPS), major risk factors for COPD, may increase arginase expression, the role of arginase in COPD is unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling using an animal model of COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pretreated by inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or vehicle. Repeated LPS exposure increased lung arginase activity, resulting in increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios. Both ratios were reversed by ABH. ABH inhibited the LPS-induced increases in pulmonary IL-8, neutrophils, and goblet cells as well as airway fibrosis. Remarkably, LPS-induced right ventricular hypertrophy, indicative of pulmonary hypertension, was prevented by ABH. Strong correlations were found between arginase activity and inflammation, airway remodeling, and right ventricular hypertrophy. Increased arginase activity contributes to pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD, indicating therapeutic potential for arginase inhibitors in this disease.
Chronic obstructive pulmonary disease (COPD) is characterized by a progressive decline in lung function and airflow limitation that is not fully reversible. Chronic inflammation, characterized by increased numbers of neutrophils, macrophages, CD8+, and CD4+ T lymphocytes and B cells in the lung, could contribute to structural changes underlying the airflow limitation: emphysema and airway remodeling (Hogg and Timens, 2009). Airway remodeling in COPD is predominantly characterized by mucus cell hyperplasia and peribronchiolar fibrosis (Pauwels et al., 2001). In addition, pulmonary hypertension, a comorbidity present in a large proportion of COPD patients, may lead to right ventricular hypertrophy and pulmonary vascular remodeling (Wright et al., 1983; Peinado et al., 1999; MacNee, 2010).
Recent studies in animal models and in patients (Meurs et al., 2002; Zimmermann et al., 2003; Morris et al., 2004; Maarsingh et al., 2006, 2008a,b, 2009a,b; Lara et al., 2008; North et al., 2009; Vonk et al., 2010) have indicated a major role for increased arginase activity in the pathophysiology of asthma. Increased activity of arginase, which converts l-arginine to l-ornithine and urea, decreases the l-arginine bioavailability to constitutive and inducible isoforms of nitric-oxide synthase (NOS) in the airways. This results in decreased production of bronchodilatory NO as well as increased synthesis of proinflammatory and procontractile peroxynitrite, which contribute to the development of allergen-induced airway hyperresponsiveness (Meurs et al., 2002; Maarsingh et al., 2006, 2009a). Treatment with inhaled arginase inhibitors protected against allergen-induced airway obstruction, airway hyper-responsiveness, and airway inflammation in guinea pig (Maarsingh et al., 2008b) and mouse (North et al., 2009; Takahashi et al., 2010) models of acute allergic asthma. Using repeatedly allergen-challenged guinea pigs, we recently demonstrated that increased arginase activity also has a major role in airway remodeling in chronic asthma, as indicated by effective inhibition of these features by inhalation of the specific (Baggio et al., 1999) arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) (Maarsingh et al., 2011). In addition to changes in NO metabolism, arginase activity may increase l-ornithine downstream products such as polyamines and l-proline, thereby promoting cell proliferation and collagen synthesis, respectively (Maarsingh et al., 2009b).
Although several studies have revealed the important role of arginase, particularly arginase I, in the pathophysiology of asthma, little is known about its role in COPD (Maarsingh et al., 2008a). Although asthma and COPD share some common features, their pathogenesis is distinct. Therefore, studies designed to address the role of arginase in the pathophysiology of COPD are indicated.
Increased arginase activity was demonstrated in the late 1970s in sputum from patients with chronic bronchitis (Chachaj et al., 1978; Kochanski et al., 1980) and more recently in bronchoalveolar lavage fluid (Hodge et al., 2011) and platelets (Guzman-Grenfell et al., 2011) from patients with COPD. Interestingly, cigarette smoke has been shown to increase arginase I expression in rat lung (Gebel et al., 2006) and in airways of patients with mild asthma (Bergeron et al., 2007). High constitutive expression of arginase I has also been demonstrated in azurophilic granules from human neutrophils (Munder et al., 2005), which are known to be released in COPD (Tetley, 2005). Increased arginase activity and decreased NO synthesis have also been implicated in pulmonary arterial hypertension (Morris et al., 2003), a comorbidity of COPD. Although these studies have indicated that arginase may be increased in COPD, its role in pathophysiology of the disease is largely undetermined.
In the present study, we investigated the role of arginase in airway inflammation, remodeling, and pulmonary hypertension in our recently described guinea pig model of LPS-induced COPD (Pera et al., 2011).
Materials and Methods
Outbred, male, specified pathogen-free Dunkin Hartley guinea pigs (Harlan, Heathfield, United Kingdom) weighing 350–400 g were used. All protocols were approved by the University of Groningen Committee for Animal Experimentation.
Guinea pigs were challenged by intranasal instillation with either 200 μl of LPS (O55:B5; Sigma-Aldrich, St. Louis, MO) (5 mg/ml in saline) or 200 μl of saline twice weekly for 12 consecutive weeks (Pera et al., 2011). Thirty minutes before each instillation, animals inhaled a nebulized (whole-body exposure) dose of the arginase inhibitor ABH in phosphate-buffered saline (PBS) (25 mM nebulizer concentration, 15 minutes) or PBS (15 minutes), using a DeVilbiss nebulizer (Somerset, PA) (Meurs et al., 2006). Twenty-four hours after the last instillation, the guinea pigs were humanely euthanized by experimental concussion, followed by rapid exsanguination. Heart and lungs were immediately resected and kept in Krebs-Henseleit buffer or on ice, respectively, for further processing.
Arginase Activity Assay.
Arginase activity, expressed as picomoles urea produced per milligram protein per minute, was determined in lung homogenates by measuring the conversion of [14C]-l-arginine to [14C]urea at 37°C (Maarsingh et al., 2009a).
Amino Acid Quantification.
Frozen lung tissue was homogenized in Tris-HCl buffer (50 mM Tris-HCl, 150 mM NaCl; pH 7.5) and centrifuged (12,000 g; 20 minutes; 4°C) to remove insoluble material. In the supernatants, concentrations of the amino acids l-ornithine, l-arginine, and l-citrulline were determined using high-performance liquid chromatography followed by tandem mass spectrometry as described recently (Maarsingh et al., 2011). Average ratios of these amino acids were calculated as the mean of individual ratios from each animal.
Interleukin-8 (IL-8) was determined in lung homogenates using an enzyme-linked immunosorbent assay for guinea pig IL-8 according to manufacturer’s instructions (Cusabio Biotech, Wuhan, China).
Transverse frozen cross-sections (4 µm) of the middle right lung lobe were used for histologic and immunohistochemical analyses. Neutrophils were identified by staining sections for tissue nonspecific alkaline phosphatase activity (Westerhof et al., 2001). MUC5AC antibody (Neomarkers, Fremont, CA) was used to identify MUC5AC-expressing goblet cells (Bos et al., 2007). Sections were counterstained with hematoxylin. Airways within sections were digitally photographed (40–200× magnification) and classified as cartilaginous or noncartilaginous. Measurements were performed using ImageJ (U.S. National Institutes of Health, Bethesda, MD) or NIS (Nikon Instruments Europe, Amstelveen, The Netherlands) quantification software. Neutrophils in the airway adventitia and submucosa were expressed as the number of positively stained cells per millimeter basement membrane length (Pera et al., 2011). Parenchymal neutrophils were expressed as a percentage of total cell counts (Pera et al., 2011). MUC5AC-positive cells in the epithelium were expressed as number of cells per millimeter basement membrane length (Pera et al., 2011).
The upper right lung lobe was inflated and fixed with formalin at 25 cmH2O constant pressure for 24 hours, and embedded in paraffin. For evaluation of pulmonary vascular dimensions, sections (4 µm) were stained with Weigert’s elastin (resorcin/fuchsin) and Van Gieson stain (van Suylen et al., 1998). Pulmonary vessel dimensions were determined as described recently (Pera et al., 2011). For evaluation of airway wall collagen, sections were stained with Sirius red and counterstained with hematoxylin. The positively stained area in the airway wall, from the adventitial border to the basement membrane, of noncartilaginous airways was determined as described recently (Pera et al., 2011). The airway wall collagen area was normalized to the square of the basement membrane length.
To evaluate right ventricular hypertrophy, Fulton’s index, i.e., the ratio of the right ventricle weight and the sum of the septum and left ventricle weights, was determined.
Lungs were analyzed for hydroxyproline as an estimate of collagen content, using chloramine T and Erlich’s solution, as described previously (Pera et al., 2011).
Data are presented as mean ± S.E.M. Statistical differences between means were calculated using one-way analysis of variance (ANOVA), followed by a Bonferroni or Newman-Keuls multiple comparison test, as indicated in the legends. Correlations were determined using a Pearson correlation coefficient test. Differences were considered significant when P < 0.05.
LPS Increases Arginase Activity in the Lung.
Repeated LPS challenge in vivo increased arginase activity (2.2-fold) in lung homogenates ex vivo (Fig. 1A). In both LPS- and saline-challenged animals in vivo treatment with inhaled ABH did not significantly change the arginase activity measured ex vivo (Fig. 1A). In addition to ex vivo arginase enzyme activity, concentrations of the amino acids l-ornithine, l-arginine, and l-citrulline were determined in lung homogenates (Table 1) and were used to calculate amino acid ratios, which reflect arginase and NOS activities in the lung. Repeated LPS challenge increased the l-ornithine/l-arginine and l-ornithine/l-citrulline ratios in the lung (Fig. 1, B and C), indicating that the endogenous arginase activity is increased and that the balance between arginase and NOS activity is shifted toward arginase. ABH treatment did not affect the amino acid ratios in saline-challenged animals. However, in the LPS-challenged animals, ABH treatment reduced both the l-ornithine/ l-arginine ratio and the l-ornithine/ l-citrulline ratio to levels below those observed in the PBS-treated, saline-challenged animals (Fig. 1, B and C). As expected, a clear correlation (P = 0.030, R = 0.65) was found between arginase activity and the l-ornithine/ l-arginine ratio (Fig. 1D). Collectively, these data indicate that LPS instillation induces increased arginase activity in the lung, which is inhibited by ABH in vivo.
LPS Induces Neutrophilia and IL-8 Increase in the Lung.
Neutrophils are a major inflammatory cell type involved in COPD pathogenesis and are a rich source of arginase (Munder et al., 2005). Repeated LPS challenge increased neutrophil numbers in both cartilaginous (2.9-fold) and noncartilaginous (3.2-fold) airways as well as in the parenchyma (2.0-fold), as determined by histochemical analysis (Fig. 2, A–C). ABH treatment reduced the neutrophil numbers in these compartments by 83, 60, and 56%, respectively, but did not affect neutrophil numbers in saline-challenged animals (Figs. 2, A–C). To assess potential mechanisms involved in arginase-induced neutrophilia in the LPS-challenged animals, we determined levels of the neutrophil chemoattractant IL-8 in lung homogenates. Figure 2D indicates that the neutrophil infiltration by repeated LPS challenge is induced by a significant increase of IL-8 in the lung, which was fully inhibited by inhalation of ABH. As expected, a positive correlation between the IL-8 levels and the number of neutrophils in the lung was observed in all four experimental groups (P = 0.012, R = 0.49). Significant correlations between arginase activity and neutrophilia [in airways (P = 0.040, R = 0.63) and parenchyma (P = 0.036, R = 0.63)] and between arginase activity and IL-8 levels (P = 0.012, R = 0.75) were found in saline-treated animals after repeated saline or LPS challenge (Fig. 6, A–C). Taken together, these data suggest that LPS-induced arginase activity contributes to neutrophilia by increasing IL-8 levels.
LPS Increases MUC5AC Expression in the Airways.
Repeated in vivo LPS challenge significantly increased (2.2-fold) the number of MUC5AC-positive cells in the epithelium of cartilaginous airways, as determined by immunohistochemistry (Fig. 3), indicating mucus hypersecretion. ABH treatment fully inhibited the LPS-induced MUC5AC expression, whereas it had no effect in saline-challenged animals (Fig. 3).
To evaluate fibrotic changes, lungs were analyzed for hydroxyproline as an estimate of collagen content. Repeated LPS challenge induced a significant 1.7-fold increase in total lung hydroxyproline content (Fig. 4A). ABH treatment inhibited the LPS-induced increase in hydroxyproline by 75%, whereas it had no effect on the hydroxyproline content in saline-challenged animals (Fig. 4A).
To assess changes in collagen deposition in the airways, Sirius red staining was evaluated in the airway wall of noncartilaginous airways. Similar to the increase in hydroxyproline content, LPS induced a 1.9-fold increase in airway wall collagen content (Fig. 4B). ABH fully inhibited the LPS-induced collagen deposition in the airway wall, whereas it did not affect the collagen content in the airway wall of saline-challenged animals (Fig. 4B). The importance of arginase in fibrotic processes is further supported by the significant correlations between arginase activity and lung hydroxyproline content (P = 0.002, R = 0.82) as well as airway collagen content (P = 0.002, R = 0.76) (Fig. 6, D and E).
Right Ventricular Hypertrophy.
Repeated LPS challenge induced right ventricular hypertrophy as indicated by a significant 1.4-fold increase in Fulton’s index (Fig. 5). In support, a significant correlation (P = 0.002, R = 0.83) between arginase activity and the Fulton’s index was observed (Fig. 6F). ABH treatment fully inhibited the LPS-induced right ventricular hypertrophy, whereas ABH had no effect in saline-challenged animals (Fig. 5).
Pulmonary Vessel Wall Dimensions.
To evaluate pulmonary vessel wall dimensions, pulmonary artery medial area and pulmonary arteriole wall area were determined in formalin-fixed, paraffin-embedded guinea pig lung sections stained with Weigert’s elastin and Van Gieson stain. Neither repeated LPS challenge nor ABH treatment affected the medial area of pulmonary arteries or wall area of pulmonary arterioles (data not shown). In addition, there was no evidence of intimal proliferation in the pulmonary vessels of either classification (data not shown).
This is the first study to demonstrate the effectiveness of an arginase inhibitor in preventing pulmonary inflammation, airway remodeling, and pulmonary hypertension in an animal model of COPD. Thus, inhaled ABH protected against increase in lung IL-8, neutrophil infiltration, mucus hypersecretion, and airway fibrosis induced by repeated intranasal LPS challenge in guinea pigs. In addition, repeated LPS challenge induced right ventricular hypertrophy, a major feature of pulmonary hypertension, which was similarly inhibited by inhalation of ABH. The contribution of arginase to inflammatory and remodeling processes is apparent from the inhibitory effects of ABH on these parameters and is also supported by the strong correlations between lung arginase activity and the indices of pulmonary inflammation and tissue remodeling. To date, this is the only study to examine the contribution of arginase to the pathophysiology of COPD. Our findings suggest a major role for increased arginase activity in this disease.
Repeated LPS challenge in vivo increased arginase activity in guinea pig lung homogenates determined ex vivo. This presumably reflects increased arginase expression induced by the LPS challenge, because arginase is a constitutively active enzyme. Unfortunately, because of a lack of specific antibodies against guinea pig arginase it is not possible to determine arginase protein expression in a direct manner. However, transcriptional profiling studies using microarray analysis detected increased pulmonary arginase gene expression in mice after LPS challenge (Brass et al., 2008; Gungor et al., 2010). The lack of effect of ABH inhalation on the LPS-induced arginase activity ex vivo (ABH not being present in the assay) suggests that arginase does not regulate its own expression. The current finding is in contrast with observations from animal models of asthma where allergen-induced increase in arginase activity is prevented by arginase inhibitors (Maarsingh et al., 2008b, 2011).
Although the changes in absolute amino acid concentrations did not reach statistical significance, the increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios in the lung reflect the LPS-induced increase in ex vivo arginase activity and decreased NOS activity. Previous studies have indicated that the amino acid ratios, rather than absolute concentrations, are a superior indicator of arginase activity and amino acid bioavailability (Morris et al., 2003, 2004). The LPS-induced increase in l-ornithine/l-citrulline ratio indicates that the increased arginase activity competes with NOS for l-arginine. Treatment with inhaled ABH prevented the LPS-induced increase in l-ornithine/l-arginine ratio, indicating that endogenous arginase activity was indeed inhibited by ABH. Moreover, the reduction in the l-ornithine/l-citrulline ratio by ABH indicates restoration of NOS activity by the arginase inhibitor.
Interestingly, ABH treatment of the LPS-challenged animals resulted in attenuation of both ratios below the levels observed for saline-challenged animals. For the l-ornithine/l-citrulline ratio this suggests an LPS-induced increase in iNOS activity, resulting in increased l-citrulline production, as also reflected by the trend toward an increase of the l-citrulline concentration in the lungs of ABH-treated, LPS-challenged animals (Table 1). Indeed, increased expression of iNOS by LPS is well established (Förstermann and Kleinert, 1995) and could also account for the decreased l-ornithine/l-arginine ratio, because recycling of l-citrulline is an important source of l-arginine under inflammatory conditions (Xie and Gross, 1997; Morris, 2005). This is also supported by previous observations that argininosuccinate synthetase, an enzyme that plays a key role in the conversion of l-citrulline to l-arginine, is upregulated in the lung by LPS treatment in vivo (Nagasaki et al., 1996; Gungor et al., 2010). Taken together, our findings indicate that LPS induces increased arginase activity in the lung in vivo, which is inhibited by inhaled ABH, thereby favoring NOS activity and NO production. Anti-inflammatory and antifibrotic actions of NO (Dekkers et al., 2009) may contribute to the inhibition by ABH of LPS-induced pathology, as is also discussed below. However, the direct role of NO in these actions remains to be established.
The relevance of the increased arginase activity observed in our guinea pig model of COPD is supported by other studies. Increased arginase activity previously was found in bronchoalveolar lavage fluid (Hodge et al., 2011) and platelets (Guzman-Grenfell et al., 2011) of patients with COPD. In addition to LPS, arginase gene expression is also induced by cigarette smoke, as shown in a transcriptome analysis of rat lung (Gebel et al., 2006). Similarly, subcutaneous injection of cigarette smoke extract in rabbits resulted in increased arginase expression and activity in cavernous tissue (Imamura et al., 2007). Moreover, arginase immunostaining in the airways is further increased in patients with mild asthma who smoke compared with nonsmoking patients with asthma (Bergeron et al., 2007).
Pulmonary neutrophils are increased in COPD, and correlations between airway neutrophil numbers and COPD severity have been found (Tetley, 2005). In our model, ABH inhalation strongly inhibited LPS-induced neutrophilia, indicating that the increase of arginase activity importantly contributes to the inflammation. To investigate potential mechanisms underlying this anti-inflammatory effect of ABH, we determined concentrations of the major neutrophil-attracting chemokine IL-8 in whole-lung homogenates. LPS-induced neutrophilia was associated with increased IL-8 in the lung, whereas both neutrophil influx and IL-8 increase were inhibited by ABH. Our data suggest that increased arginase activity contributes to neutrophilia by increasing IL-8 levels in the lung.
A potential mechanism underlying arginase-enhanced IL-8 production and neutrophilia is by promoting nuclear factor-кB activity through decreasing NO synthesis. NO inhibits nuclear factor-кB by nitrosylation of this transcription factor (Ckless et al., 2007). In addition, increased arginase activity causes uncoupling of iNOS and subsequent production of the proinflammatory oxidant species peroxynitrite (Maarsingh et al., 2008a), which induces IL-8 expression in various cell types (Zouki et al., 2001; Sarir et al., 2010). Accordingly, breakdown of peroxynitrite reduces smoke-induced IL-8 levels in sheep lung (Lange et al., 2011).
Increased MUC5AC expression is observed in the airway epithelium of patients with COPD and can be induced by cigarette smoke and LPS, as well as by neutrophil elastase and peroxynitrite (Caramori et al., 2004). In the current study, ABH fully inhibited the LPS-induced MUC5AC expression in the guinea pig airway epithelium, indicating a role for increased arginase activity in mucus hypersecretion. The effect of ABH may be the result of the inhibition of IL-8 production and airway neutrophilia, which may both contribute to increased MUC5AC expression (Fischer and Voynow, 2000; Bautista et al., 2009). Moreover, ABH may reduce the LPS-induced MUC5AC expression by inhibiting peroxynitrite formation and restoring NO production (Ramnarine et al., 1996).
Previous studies suggested a role for arginase in fibrotic processes. Increased arginase expression was demonstrated in bleomycin-induced lung fibrosis in mice (Endo et al., 2003), and arginase activity was associated with lung collagen content in a model of allograft fibrosis in rats (Liu et al., 2005). In addition, repeated allergen challenge-induced lung hydroxyproline increase in guinea pigs was inhibited by inhaled ABH (Maarsingh et al., 2011). In the present study, we confirm the importance of increased arginase in the development of lung fibrosis (as assessed by measuring lung hydroxyproline content). It is noteworthy that our present study is the first to show that arginase inhibition prevents fibrosis in the airway wall, a major pathologic feature of COPD and a key determinant of airflow limitation in obstructive airways diseases (Matsuba and Thurlbeck, 1972).
Increased arginase activity may contribute to airway fibrosis via its role in inflammation but also through increased production of l-ornithine and its downstream product l-proline, a precursor of collagen (Wu and Morris, 1998). In accordance, transforming growth factor-β, a major profibrotic factor, has been shown to induce arginase activity in the rat lung and in isolated fibroblasts, whereas transforming growth factor-β-induced collagen synthesis was reduced by arginase inhibition in lung fibroblasts of rats and mice in vitro (Liu et al., 2005; Kitowska et al., 2008; Warnken et al., 2010). In addition, restoration of NO production and reduced peroxynitrite formation may also play a part in the antifibrotic effects of ABH (Wani et al., 2007; Naura et al., 2010).
Our data show that repeated LPS challenge also induces right ventricular hypertrophy, a feature of pulmonary hypertension, a known comorbidity of COPD (MacNee, 2010). The LPS-induced right ventricular hypertrophy was prevented by ABH. Therefore, our data suggest that repeated LPS challenge results in pulmonary hypertension via induction of arginase. Pulmonary hypertension and associated right ventricular hypertrophy may result from (a combination of) vascular remodeling and functional changes in the vessel wall, both leading to increased resistance in the pulmonary vasculature (MacNee, 2010). In our model, we did not observe changes in pulmonary vessel dimensions after repeated LPS instillation or by treatment with inhaled ABH, suggesting that increased resistance in the pulmonary vessels and subsequent right ventricular hypertrophy occur through exaggerated constriction of the vessels rather than remodeling of the vessel wall. In this respect, endothelial dysfunction caused by reduced endothelial NOS activity has been proposed as a potential mechanism (Morris et al., 2008). This is also supported by associations between pulmonary hypertension and reduced l-arginine and NO levels (Xu et al., 2004), whereas inhalation of NO and oral l-arginine therapy decrease pulmonary arterial pressure in primary or secondary pulmonary hypertension (Morris et al., 2003). Increased metabolism of l-arginine by enhanced arginase II activity in the endothelium has been shown to contribute to the reduced l-arginine and NO levels (Morris et al., 2003; Xu et al., 2004). In addition, hypoxia, which is a major contributor in COPD-related pulmonary hypertension, upregulates arginase in human lung microvascular endothelial cells (Krotova et al., 2010). Our data suggest that COPD-associated pulmonary hypertension can effectively be targeted by inhalation of arginase inhibitors. This indicates that increased arginase activity may contribute to pulmonary hypertension and right ventricular hypertrophy in COPD, possibly by inducing endothelial dysfunction in pulmonary vessels. Considering the severely detrimental impact of a pulmonary hypertension diagnosis on the prognosis for patients with COPD and the current lack of an effective pharmacological treatment, the potential for arginase inhibitors as a treatment of pulmonary hypertension is a finding of significant importance.
In conclusion, our study demonstrates that increased arginase activity plays a major role in pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD. This is the first study to provide evidence for a role of increased arginase activity in COPD and to identify arginase inhibitors as potential treatment of this disease.
Participated in research design: Pera, Zaagsma, Meurs, Maarsingh.
Conducted experiments: Pera, Zuidhof, Smit, Menzen, Klein, Flik, Maarsingh.
Contributed analytic tools: Klein, Flik.
Performed data analysis: Pera, Zuidhof, Smit, Maarsingh.
Wrote or contributed to the writing of the manuscript: Pera, Zaagsma, Meurs, Maarsingh.
- Received October 1, 2013.
- Accepted February 18, 2014.
This study was supported by Merck Sharp & Dohme, Oss, The Netherlands, and The Graduate School of Behavioral and Cognitive Neurosciences, University of Groningen, Groningen, The Netherlands.
- 2(S)-amino-6-boronohexanoic acid
- analysis of variance
- chronic obstructive pulmonary disease
- interleukin 8
- inducible nitric-oxide synthase
- nitric oxide
- nitric-oxide synthase
- phosphate-buffered saline
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics