Marieke Smit

Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD

Airway remodelling and emphysema are major structural abnormalities in chronic obstructive pulmonary disease (COPD). In addition, pulmonary vascular remodelling may occur and contribute to pulmonary hypertension, a comorbidity of COPD. Increased cholinergic activity in COPD contributes to airflow limitation and, possibly, to inflammation and airway remodelling. This study aimed to investigate the role of acetylcholine in pulmonary inflammation and remodelling using an animal model of COPD. To this aim, guinea pigs were instilled intranasally with lipopolysaccharide (LPS) twice weekly for 12 weeks and were treated, by inhalation, with the long-acting muscarinic receptor antagonist tiotropium. Repeated LPS exposure induced airway and parenchymal neutrophilia, and increased goblet cell numbers, lung hydroxyproline content, airway wall collagen and airspace size. Furthermore, LPS increased the number of muscularised microvessels in the adventitia of cartilaginous airways. Tiotropium abrogated the LPS-induced increase in neutrophils, goblet cells, collagen deposition and muscularised microvessels, but had no effect on emphysema. In conclusion, tiotropium inhibits remodelling of the airways as well as pulmonary inflammation in a guinea pig model of COPD, suggesting that endogenous acetylcholine plays a major role in the pathogenesis of this disease.

Bronchoconstriction Induces TGF-β Release and Airway Remodelling in Guinea Pig Lung Slices

Airway remodelling, including smooth muscle remodelling, is a primary cause of airflow limitation in asthma. Recent evidence links bronchoconstriction to airway remodelling in asthma. The mechanisms involved are poorly understood. A possible player is the multifunctional cytokine TGF-β, which plays an important role in airway remodelling. Guinea pig lung slices were used as an in vitro model to investigate mechanisms involved in bronchoconstriction-induced airway remodelling. To address this aim, mechanical effects of bronchoconstricting stimuli on contractile protein expression and TGF-β release were investigated. Lung slices were viable for at least 48 h. Both methacholine and TGF-β1 augmented the expression of contractile proteins (sm-α-actin, sm-myosin, calponin) after 48 h. Confocal fluorescence microscopy showed that increased sm-myosin expression was enhanced in the peripheral airways and the central airways. Mechanistic studies demonstrated that methacholine-induced bronchoconstriction mediated the release of biologically active TGF-β, which caused the increased contractile protein expression, as inhibition of actin polymerization (latrunculin A) or TGF-β receptor kinase (SB431542) prevented the methacholine effects, whereas other bronchoconstricting agents (histamine and KCl) mimicked the effects of methacholine. Collectively, bronchoconstriction promotes the release of TGF-β, which induces airway smooth muscle remodelling. This study shows that lung slices are a useful in vitro model to study mechanisms involved in airway remodelling.

Muscarinic receptor stimulation augments TGF-β1-induced contractile protein expression by airway smooth muscle cells.

Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-β(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M(2) and M(3) receptors were stimulated with methacholine (MCh), TGF-β(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-α-actin and calponin by TGF-β(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-α-actin stress fibers by TGF-β(1). MCh did not augment TGF-β(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-β(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3β and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-β(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD.

Arginase Inhibition Prevents Inflammation and Remodeling in a Guinea Pig Model of Chronic Obstructive Pulmonary Disease

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.

Bronchoprotection by olodaterol is synergistically enhanced by tiotropium in a guinea pig model of allergic asthma

The novel once-daily β2-agonist bronchodilator drug olodaterol has recently been shown to be effective in patients with allergic asthma for >24 hours. An increased cholinergic tone common to these patients may decrease the effectiveness of β2-agonists. This could provide a rationale for combination therapy with olodaterol and the long-acting anticholinergic tiotropium to aim for a once-daily treatment regimen. In guinea pigs, we evaluated the protective effects of olodaterol, alone and in combination with tiotropium, on airway responsiveness to histamine, which is partially mediated by a cholinergic reflex mechanism. In addition, using a guinea pig model of acute allergic asthma, we examined the cooperative effects of these bronchodilators on allergen-induced early (EAR) and late (LAR) asthmatic reactions, airway hyper-responsiveness (AHR) to histamine, and airway inflammation. It was demonstrated that the protective effect of olodaterol against histamine-induced bronchoconstriction was synergistically enhanced and prolonged in the presence of tiotropium. In addition, tiotropium synergistically augmented both the reversal of and the protection against the allergen-induced AHR after the EAR by olodaterol. Olodaterol and tiotropium were highly effective in inhibiting the magnitude of the allergen-induced EAR and LAR, and both reactions were fully inhibited by the combination of these drugs. It is remarkable that these effects were not associated with an effect on inflammatory cell infiltration in the airways. In conclusion, the results indicate that combination therapy with olodaterol and tiotropium may be highly effective in the treatment of allergen-induced asthmatic reactions and AHR.

Novel drug targets for asthma and COPD: Lessons learned from in vitro and in vivo models

Asthma and chronic obstructive pulmonary disease (COPD) are highly prevalent respiratory diseases characterized by airway inflammation, airway obstruction and airway hyperresponsiveness. Whilst current therapies, such as β-agonists and glucocorticoids, may be effective at reducing symptoms, they do not reduce disease progression. Thus, there is a need to identify new therapeutic targets. In this review, we summarize the potential of novel targets or tools, including anti-inflammatories, phosphodiesterase inhibitors, kinase inhibitors, transient receptor potential channels, vitamin D and protease inhibitors, for the treatment of asthma and COPD.

Cooperative signaling by TGF-β1 and WNT-11 drives sm-α-actin expression in smooth muscle via Rho kinase-actin-MRTF-A signaling

Airway smooth muscle (ASM) remodeling is a key feature in asthma and includes changes in smooth muscle-specific gene and protein expression. Despite this being a major contributor to asthma pathobiology, our understanding of the mechanisms governing ASM remodeling remains poor. Here, we studied the functional interaction between WNT-11 and TGF-β1 in ASM cells. We demonstrate that WNT-11 is preferentially expressed in contractile myocytes and is strongly upregulated following TGF-β1-induced myocyte maturation. Knock-down of WNT-11 attenuated TGF-β1-induced smooth muscle (sm)-α-actin expression in ASM cells. We demonstrate that TGF-β1-induced sm-α-actin expression is mediated by WNT-11 via RhoA activation and subsequent actin cytoskeletal remodeling, as pharmacological inhibition of either Rho kinase by Y27632 or actin remodeling by latrunculin A attenuated sm-α-actin induction. Moreover, we show that TGF-β1 regulates the nuclear expression of myocardin-related transcription factor-A (MRTF-A) in a Rho kinase-dependent fashion, which in turn mediates sm-α-actin expression. Finally, we demonstrate that TGF-β1-induced MRTF-A nuclear translocation is dependent on endogenous WNT-11. The present study thus demonstrates a WNT-11-dependent Rho kinase-actin-MRTF-A signaling axis that regulates the expression of sm-α-actin in ASM cells.

The novel compound Sul-121 inhibits airway inflammation and hyperresponsiveness in experimental models of chronic obstructive pulmonary disease.

COPD is characterized by persistent airflow limitation, neutrophilia and oxidative stress from endogenous and exogenous insults. Current COPD therapy involving anticholinergics, β2-adrenoceptor agonists and/or corticosteroids, do not specifically target oxidative stress, nor do they reduce chronic pulmonary inflammation and disease progression in all patients. Here, we explore the effects of Sul-121, a novel compound with anti-oxidative capacity, on hyperresponsiveness (AHR) and inflammation in experimental models of COPD. Using a guinea pig model of lipopolysaccharide (LPS)-induced neutrophilia, we demonstrated that Sul-121 inhalation dose-dependently prevented LPS-induced airway neutrophilia (up to ~60%) and AHR (up to ~90%). Non-cartilaginous airways neutrophilia was inversely correlated with blood H2S, and LPS-induced attenuation of blood H2S (~60%) was prevented by Sul-121. Concomitantly, Sul-121 prevented LPS-induced production of the oxidative stress marker, malondialdehyde by ~80%. In immortalized human airway smooth muscle (ASM) cells, Sul-121 dose-dependently prevented cigarette smoke extract-induced IL-8 release parallel with inhibition of nuclear translocation of the NF-κB subunit, p65 (each ~90%). Sul-121 also diminished cellular reactive oxygen species production in ASM cells, and inhibited nuclear translocation of the anti-oxidative response regulator, Nrf2. Our data show that Sul-121 effectively inhibits airway inflammation and AHR in experimental COPD models, prospectively through inhibition of oxidative stress.

Precision cut lung slices: A novel in vitro model to demonstrate that bronchoconstriction is a key player in airway remodelling

Objective Medication errors in ambulatory care are frequent. Because the process of prescribing and medication therapy is more complex than in hospital settings adequate methods to avoid medication errors are difficult to implement. In our study we investigated a campaign from a health insurance company that had the aim to increase medication safety within insurants with polypharmacy and multimorbidity. Method An expert team consisting of clinical pharmacologists and a pharmacist analysed the medication of insurants for risk prescriptions who had an additional telephonic health coaching. Attending physicians received a report and were offered to contact the experts for counselling. Results of medication analyses of 400 insurants were categorized in eight types of prescribing errors and feedback from the physicians was recorded. Differences in medication therapy after counselling were scanned. Main outcome measure The frequency of medication errors in eight categories and physicians’ feedback, further changes in medications of physicians responding. Results Insurants were 48% female and 52% male and took an average of 13.4 drugs regularly. 16.8% of the physicians contacted replied, 13.3% had a counselling conversation. 29.2% of the physicians replying gave a positive response to the campaign, 13.8 % a negative and for 56.9 % a neutral feedback was given. Out of a total of n=2524 errors 26.8% occurred in the category missing indication, 6.8% in PRISCUS medication, 33.3% in interactions, 15.3% in wrong dosage, 0.9% in contraindication, 2.8% in dosage adaption to renal function, 6.9% in double medication and 7.2% in gap of prescribing. Conclusion A large number of medication errors appear in ambulatory care and consequently adverse drug events and hospital admissions are more likely to happen. Medication check by pharmacotherapy experts and pharmacological consulting with a high response of physicians can be a key tool to reduce these errors. Cooperation of a health insurance company and clinical pharmacologists is an effective association but needs to be optimized for better acceptance and more rapid ability to react.