Cigarette smoke restricts the ability of mesenchymal cells to support lung epithelial organoid formation.

Adequate lung epithelial repair relies on supportive interactions within the epithelial niche, including interactions with WNT-responsive fibroblasts. In fibroblasts from patients with chronic obstructive pulmonary disease (COPD) or upon in vitro cigarette smoke exposure, Wnt/ß-catenin signalling is distorted, which may affect interactions between epithelial cells and fibroblasts resulting in inadequate lung repair. We hypothesized that cigarette smoke (CS), the main risk factor for COPD, interferes with Wnt/ß-catenin signalling in fibroblasts through induction of cellular stress responses, including oxidative- and endoplasmic reticulum (ER) stress, and thereby alters epithelial repair support potential. Therefore, we assessed the effect of CS-exposure and the ER stress inducer Thapsigargin (Tg) on Wnt/ß-catenin signalling activation in MRC-5 fibroblasts, and on their ability to support lung epithelial organoid formation. Exposure of MRC-5 cells for 15 min with 5 AU/mL CS extract (CSE), and subsequent 6 h incubation induced oxidative stress (HMOX1). Whereas stimulation with 100 nM Tg increased markers of both the integrated stress response (ISR - GADD34/PPP1R15A, CHOP) and the unfolded protein response (UPR - XBP1spl, GADD34/PPP1R15A, CHOP and HSPA5/BIP), CSE only induced GADD34/PPP1R15A expression. Strikingly, although treatment of MRC-5 cells with the Wnt activator CHIR99021 upregulated the Wnt/ß-catenin target gene AXIN2, this response was diminished upon CSE or Tg pre-exposure, which was confirmed using a Wnt-reporter. Furthermore, pre-exposure of MRC-5 cells to CSE or Tg, restricted their ability to support organoid formation upon co-culture with murine pulmonary EpCam+ cells in Matrigel at day 14. This restriction was alleviated by pre-treatment with CHIR99021. We conclude that exposure of MRC-5 cells to CSE increases oxidative stress, GADD34/PPP1R15A expression and impairs their ability to support organoid formation. This inhibitory effect may be restored by activating the Wnt/ß-catenin signalling pathway.

Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies.

Airway cholinergic nerves play a key role in airway physiology and disease. In asthma and other diseases of the respiratory tract, airway cholinergic neurons undergo plasticity and contribute to airway hyperresponsiveness and mucus secretion. We currently lack human in vitro models for airway cholinergic neurons. Here, we aimed to develop a human in vitro model for peripheral cholinergic neurons using human pluripotent stem cell (hPSC) technology. hPSCs were differentiated towards vagal neural crest precursors and subsequently directed towards functional airway cholinergic neurons using the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic neurons were characterized by ChAT and VAChT expression, and responded to chemical stimulation with changes in Ca2+ mobilization. To culture these cells, allowing axonal separation from the neuronal cell bodies, a two-compartment PDMS microfluidic chip was subsequently fabricated. The two compartments were connected via microchannels to enable axonal outgrowth. On-chip cell culture did not compromise phenotypical characteristics of the cells compared to standard culture plates. When the hPSC-derived peripheral cholinergic neurons were cultured in the chip, axonal outgrowth was visible, while the somal bodies of the neurons were confined to their compartment. Neurons formed contacts with airway smooth muscle cells cultured in the axonal compartment. The microfluidic chip developed in this study represents a human in vitro platform to model neuro-effector interactions in the airways that may be used for mechanistic studies into neuroplasticity in asthma and other lung diseases.

Responsivity and Reproducibility of Sputum Inflammatory Biomarkers During COPD Exacerbation and Stable Phases - A Pilot Study.

INTRODUCTION: There is a great interest to identify airway biomarkers to evaluate the potential and efficacy of anti-inflammatory therapeutic interventions. In this pilot study, we compared cytokine mRNA and protein levels of IL-6, IL-8, CCL2, CCL4, and TNF-α, as well as LTB-4 expression regarding their reproducibility and responsivity in induced sputum in COPD patients. METHODS: We recruited a cohort of 17 patients with a moderate COPD exacerbation, necessitating antibiotics and/or oral corticosteroids. Patients were followed for two consecutive stable phase visits. Cytokine mRNA and protein levels were measured in induced sputum samples. RESULTS: IL-6 and CCL4 protein levels decreased from exacerbation to stable phase, whereas their mRNA expression showed the same trend (not statistically significant). Coefficients of variation were overall lower (ie, more favorable for responsiveness) at protein levels compared to mRNA levels. No significant differences were observed in the reproducibility between cytokine mRNA expression and protein measurements. IL-6, IL-8, CCL2, and TNF-α gene expression levels yielded moderate to high intraclass correlation coefficients and/or Spearman correlation coefficients between both stable phase samples in contrast to their protein levels. CONCLUSION: Our findings suggest that several protein levels yield better responsivity with lower noise-to-signal ratios compared to their respective mRNA levels. In contrast, cytokine mRNA expression was more reproducible as it varied less in a stable state than proteins. Future studies are needed with a larger sample size to further evaluate the differences of responsivity and reproducibility between cytokine mRNA and protein measurements, not only during exacerbations.

The Stress of Lung Aging: Endoplasmic Reticulum and Senescence Tête-à-Tête.

Beyond the structural changes, features including the dysregulation of endoplasmic reticulum (ER) stress response and increased senescence characterize the lung aging. ER stress response and senescence have been reported to be induced by factors like cigarette smoke. Therefore, deciphering the mechanisms underlying ER and senescent pathways interaction has become a challenge. In this review we highlight the known and unknown regarding ER stress response and senescence and their cross talk in aged lung.

Pharmacological Screening Identifies SHK242 and SHK277 as Novel Arginase Inhibitors with Efficacy against Allergen-Induced Airway Narrowing In Vitro and In Vivo.

Arginase is a potential target for asthma treatment. However, there are currently no arginase inhibitors available for clinical use. Here, a novel class of arginase inhibitors was synthesized, and their efficacy was pharmacologically evaluated. The reference compound 2(S)-amino-6-boronohexanoic acid (ABH) and >200 novel arginase inhibitors were tested for their ability to inhibit recombinant human arginase 1 and 2 in vitro. The most promising compounds were separated as enantiomers. Enantiomer pairs SHK242 and SHK243, and SHK277 and SHK278 were tested for functional efficacy by measuring their effect on allergen-induced airway narrowing in lung slices of ovalbumin-sensitized guinea pigs ex vivo. A guinea pig model of acute allergic asthma was used to examine the effect of the most efficacious enantiopure arginase inhibitors on allergen-induced airway hyper-responsiveness (AHR), early and late asthmatic reactions (EAR and LAR), and airway inflammation in vivo. The novel compounds were efficacious in inhibiting arginase 1 and 2 in vitro. The enantiopure SHK242 and SHK277 fully inhibited arginase activity, with IC50 values of 3.4 and 10.5 µM for arginase 1 and 2.9 and 4.0 µM for arginase 2, respectively. Treatment of slices with ABH or novel compounds resulted in decreased ovalbumin-induced airway narrowing compared with control, explained by increased local nitric oxide production in the airway. In vivo, ABH, SHK242, and SHK277 protected against allergen-induced EAR and LAR but not against AHR or lung inflammation. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. SIGNIFICANCE STATEMENT: Arginase is a potential drug target for asthma treatment, but currently there are no arginase inhibitors available for clinical use. We have identified promising novel arginase inhibitors for the potential treatment of allergic asthma that were able to protect against allergen-induced early and late asthmatic reactions. Our new inhibitors show protective effects in reducing airway narrowing in response to allergens and reductions in the early and late asthmatic response.

Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD.

BACKGROUND: Goblet cell metaplasia, a common feature of chronic obstructive pulmonary disease (COPD), is associated with mucus hypersecretion which contributes to the morbidity and mortality among patients. Transcription factors SAM-pointed domain-containing Ets-like factor (SPDEF) and forkhead box protein A2 (FOXA2) regulate goblet cell differentiation. This study aimed to (1) investigate DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation and (2) compare this in airway epithelial cells from patients with COPD and controls during mucociliary differentiation. METHODS: To assess DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation, primary airway epithelial cells, isolated from trachea (non-COPD controls) and bronchial tissue (patients with COPD), were differentiated by culture at the air-liquid interface (ALI) in the presence of cytokine interleukin (IL)-13 to promote goblet cell differentiation. RESULTS: We found that SPDEF expression was induced during goblet cell differentiation, while FOXA2 expression was decreased. Importantly, CpG number 8 in the SPDEF promoter was hypermethylated upon differentiation, whereas DNA methylation of FOXA2 promoter was not changed. In the absence of IL-13, COPD-derived ALI-cultured cells displayed higher SPDEF expression than control-derived ALI cultures, whereas no difference was found for FOXA2 expression. This was accompanied with hypomethylation of CpG number 6 in the SPDEF promoter and also hypomethylation of CpG numbers 10 and 11 in the FOXA2 promoter. CONCLUSIONS: These findings suggest that aberrant DNA methylation of SPDEF and FOXA2 is one of the factors underlying mucus hypersecretion in COPD, opening new avenues for epigenetic-based inhibition of mucus hypersecretion.

The ß2-subtype of adrenoceptors mediates inhibition of pro-fibrotic events in human lung fibroblasts.

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by ß-adrenoceptor activation. We aimed, first, to characterise the expression pattern of ß-adrenoceptor subtypes in human lung fibroblasts and, second, to probe ß-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding ß(2)-adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for ß(1)- and ß(3)-adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed ß-agonist-induced G(s)-signalling. Proliferation of MRC-5 cells (determined by [(3)H]-thymidine incorporation) was significantly inhibited by ß-agonists including the ß(2)-selective agonist formoterol (-logIC(50), 10.2) and olodaterol (-logIC(50), 10.6). Formoterol's effect was insensitive to ß(1)-antagonism (GCP 20712, 3 µM), but sensitive to ß(2)-antagonism (ICI 118,551; apparent, pA (2), 9.6). Collagen synthesis in MRC-5 cells (determined by [(3)H]-proline incorporation) was inhibited by ß-agonists including formoterol (-logIC(50), 10.0) and olodaterol (-logIC(50), 10.3) in a ß(2)-blocker-sensitive manner. α-Smooth muscle actin, a marker of myo-fibroblast differentiation, was down-regulated at the mRNA and the protein level by about 50% following 24 and 48 h exposure to 1 nM formoterol, a maximally active concentration. In conclusion, human lung fibroblasts exclusively express ß(2)-adrenoceptors and these mediate inhibition of various markers of pro-fibrotic cellular activity. Under clinical conditions, anti-fibrotic actions may accompany the therapeutic effect of long-term ß(2)-agonist treatment of bronchial asthma and COPD.

Reduction of fibrogenesis by selective delivery of a Rho kinase inhibitor to hepatic stellate cells in mice.

One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] has been shown to reduce fibrosis in animal models. However, kinase expression is ubiquitous, so any inhibitor may affect many cell types. We hypothesize that cell-specific delivery of a kinase inhibitor will be beneficial. Therefore, we conjugated Y27632 to the carrier mannose-6-phosphate (M6P) human serum albumin (HSA), which is taken up specifically in activated HSC through the M6P/insulin-like growth factor II receptor. This conjugate decreased protein expression of phosphorylated myosin light chain 2 (pMLC2) and vinculin, downstream of Rho kinase, in activated primary HSC and decreased the migration and contraction of HSC. In an ex vivo model, free Y27632 decreased contractility of rat aortas, whereas the Y27-conjugate did not, showing that the Y27-conjugate does not affect nontarget tissue. In chronic CCl(4)-induced liver fibrosis, both free drug and conjugate reduced HSC activation; however, only the Y27-conjugate significantly reduced collagen deposition. Treatment with the Y27-conjugate, but not with free drug, reduced pMLC2 expression in livers 24 h after injection, demonstrating prolonged inhibition of the Rho kinase pathway. The Rho kinase inhibitor Y27632 can be specifically targeted to HSC using M6PHSA, decreasing its effects in nontarget tissues. The targeted drug effectively reduced fibrotic parameters in vivo via the inhibition of the Rho kinase pathway.

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.

Alternative mechanisms for tiotropium.

Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summarizes data from a variety of investigations that provide insights into possible mechanisms to account for the effects of tiotropium. The report summarizes the discussion on basic and clinical research in this field.

Muscarinic M3 receptor stimulation increases cigarette smoke-induced IL-8 secretion by human airway smooth muscle cells.

Acetylcholine is the primary parasympathetic neurotransmitter in the airways and is known to cause bronchoconstriction and mucus secretion. Recent findings suggest that acetylcholine also regulates aspects of remodelling and inflammation through its action on muscarinic receptors. In the present study, we aimed to determine the effects of muscarinic receptor stimulation on cytokine production by human airway smooth muscle cells (primary and immortalised cell lines). The muscarinic receptor agonists carbachol and methacholine both induced modest effects on basal interleukin (IL)-8 and -6 secretion, whereas the secretion of RANTES, eotaxin, vascular endothelial growth factor-A and monocyte chemoattractant protein-1 was not affected. Secretion of IL-8 and -6 was only observed in immortalised airway smooth muscle cells that express muscarinic M3 receptors. In these cells, methacholine also significantly augmented IL-8 secretion in combination with cigarette smoke extract in a synergistic manner, whereas synergistic effects on IL-6 secretion were not significant. Muscarinic M3 receptors were the primary subtype involved in augmenting cigarette smoke extract-induced IL-8 secretion, as only tiotropium bromide and muscarinic M3 receptor subtype selective antagonists abrogated the effects of methacholine. Collectively, these results indicate that muscarinic M3 receptor stimulation augments cigarette smoke extract-induced cytokine production by airway smooth muscle. This interaction could be of importance in patients with chronic obstructive pulmonary disease.

Airway hyperresponsiveness in asthma: lessons from in vitro model systems and animal models.

Airway hyperresponsiveness (AHR) is a hallmark clinical symptom of asthma. At least two components of AHR have been identified: 1) baseline AHR, which is persistent and presumably caused by airway remodelling due to chronic recurrent airway inflammation; and 2) acute and variable AHR, which is associated with an episodic increase in airway inflammation due to environmental factors such as allergen exposure. Despite intensive research, the mechanisms underlying acute and chronic AHR are poorly understood. Owing to the complex variety of interactive processes that may be involved, in vitro model systems and animal models are indispensable to the unravelling of these mechanisms at the cellular and molecular level. The present paper focuses on a number of translational studies addressing the emerging central role of the airway smooth muscle cell, as a multicompetent cell involved in acute airway constriction as well as structural changes in the airways, in the pathophysiology of airway hyperresponsiveness.

Inhibition of allergen-induced airway remodelling by tiotropium and budesonide: a comparison.

Chronic inflammation in asthma and chronic obstructive pulmonary disease drives pathological structural remodelling of the airways. Using tiotropium bromide, acetylcholine was recently identified as playing a major regulatory role in airway smooth muscle remodelling in a guinea pig model of ongoing allergic asthma. The aim of the present study was to investigate other aspects of airway remodelling and to compare the effectiveness of tiotropium to the glucocorticosteroid budesonide. Ovalbumin-sensitised guinea pigs were challenged for 12 weeks with aerosolised ovalbumin. The ovalbumin induced airway smooth muscle thickening, hypercontractility of tracheal smooth muscle, increased pulmonary contractile protein (smooth-muscle myosin) abundance, mucous gland hypertrophy, an increase in mucin 5 subtypes A and C (MUC5AC)-positive goblet cell numbers and eosinophilia. It was reported previously that treatment with tiotropium inhibits airway smooth muscle thickening and contractile protein expression, and prevents tracheal hypercontractility. This study demonstrates that tiotropium also fully prevented allergen-induced mucous gland hypertrophy, and partially reduced the increase in MUC5AC-positive goblet cell numbers and eosinophil infiltration. Treatment with budesonide also prevented airway smooth muscle thickening, contractile protein expression, tracheal hypercontractility and mucous gland hypertrophy, and partially reduced MUC5AC-positive goblet cell numbers and eosinophilia. This study demonstrates that tiotropium and budesonide are similarly effective in inhibiting several aspects of airway remodelling, providing further evidence that the beneficial effects of tiotropium bromide might exceed those of bronchodilation.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Insulin induces airway smooth muscle contraction.

BACKGROUND AND PURPOSE: Recently, the use of inhaled insulin formulations for the treatment of type I and type II diabetes has been approved in Europe and in the United States. For regular use, it is critical that airway function remains unimpaired in response to insulin exposure. EXPERIMENTAL APPROACH: We investigated the effects of insulin on airway smooth muscle (ASM) contraction and contractile prostaglandin (PG) production, using guinea-pig open-ring tracheal smooth muscle preparations. KEY RESULTS: It was found that insulin (1 nM-1 microM) induced a concentration-dependent contraction that was insensitive to epithelium removal. These sustained contractions were susceptible to inhibitors of cyclooxygenase (indomethacin, 3 microM), Rho-kinase (Y-27632, 1 microM) and p42/44 MAP kinase (PD-98059, 30 microM and U-0126, 3 microM), but not of PI-3-kinase (LY-294002,10 microM). In addition, insulin significantly increased PGF(2alpha)-production which was inhibited by indomethacin, but not Y-27632. Moreover, the FP-receptor antagonist AL-8810 (10 microM) and the EP(1)-receptor antagonist AH-6809 (10 microM) strongly reduced insulin-induced contractions, supporting a pivotal role for contractile prostaglandins. CONCLUSIONS AND IMPLICATIONS: Collectively, the results show that insulin induces guinea-pig ASM contraction presumably through the production of contractile prostaglandins, which in turn are dependent on Rho-kinase for their contractile effects. The data suggest that administration of insulin as an aerosol could result in some acute adverse effects on ASM function.

Airway smooth muscle phenotype and function: interactions with current asthma therapies.

Asthma incidence has climbed markedly in the past two decades despite an increased use of medications that suppress airway inflammation and repress contraction of smooth muscle that encircles the airways. Asthmatics exhibit episodes of airway inflammation that potentiates reversible airway smooth muscle spasm. A hallmark diagnostic symptom of asthma is airway hyperresponsiveness to inhaled non-allergic stimuli, such as methacholine, that directly induce airway smooth muscle contraction. Inhaled gluccocorticoids are used for first-line prevention of airway inflammation, and are frequently combined with inhaled beta2-adrenoceptor agonists that can effectively relax airway smooth muscle and restore airway conductance. Leukotriene receptor antagonists and anti-cholinergics can also be used in many patients to ensure optimal control of symptoms. With increasing disease duration irreversible airway restriction develops from inflammation-driven fibro-proliferative airway remodeling that includes increased deposition of extracellular matrix, the accumulation of airway smooth muscle, and increased numbers of myofibroblasts. Mature airway smooth muscle cells are phenotypically plastic, enabling them to subserve contractile, proliferative, migratory and secretory functional responses that contribute to airway remodeling and persistent hyperresponsiveness. This review assesses current understanding of acute and chronic effects of common anti-asthma medications on the diverse phenotype and functional characteristics of airway smooth muscle cells. Furthermore, we describe the significance of these effects in the treatment of asthma symptoms and pathogenesis.