The novel TRPA1 antagonist BI01305834 inhibits ovalbumin-induced bronchoconstriction in guinea pigs.

BACKGROUND: Asthma is a chronic respiratory disease in which the nervous system plays a central role. Sensory nerve activation, amongst others via Transient Receptor Potential Ankyrin 1 (TRPA1) channels, contributes to asthma characteristics including cough, bronchoconstriction, mucus secretion, airway hyperresponsiveness (AHR) and inflammation. In the current study, we evaluated the efficacy of the novel TRPA1 antagonist BI01305834 against AHR and inflammation in guinea-pig models of asthma. METHODS: First, a pilot study was performed in a guinea-pig model of allergic asthma to find the optimal dose of BI01305834. Next, the effect of BI01305834 on (1) AHR to inhaled histamine after the early and late asthmatic reaction (EAR and LAR), (2) magnitude of EAR and LAR and (3) airway inflammation was assessed. Precision-cut lung slices and trachea strips were used to investigate the bronchoprotective and bronchodilating-effect of BI01305834. Statistical evaluation of differences of in vivo data was performed using a Mann-Whitney U test or One-way nonparametric Kruskal-Wallis ANOVA, for ex vivo data One- or Two-way ANOVA was used, all with Dunnett's post-hoc test where appropriate. RESULTS: A dose of 1 mg/kg BI01305834 was selected based on AHR and exposure data in blood samples from the pilot study. In the subsequent study, 1 mg/kg BI01305834 inhibited AHR after the EAR, and the development of EAR and LAR elicited by ovalbumin in ovalbumin-sensitized guinea pigs. BI01305834 did not inhibit allergen-induced total and differential cells in the lavage fluid and interleukin-13 gene expression in lung homogenates. Furthermore, BI01305834 was able to inhibit allergen and histamine-induced airway narrowing in guinea-pig lung slices, without affecting histamine release, and reverse allergen-induced bronchoconstriction in guinea-pig trachea strips. CONCLUSIONS: TRPA1 inhibition protects against AHR and the EAR and LAR in vivo and allergen and histamine-induced airway narrowing ex vivo, and reverses allergen-induced bronchoconstriction independently of inflammation. This effect was partially dependent upon histamine, suggesting a neuronal and possible non-neuronal role for TRPA1 in allergen-induced bronchoconstriction.

The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices.

Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced remodeling. Because of the different mechanism of action of PDE4 inhibitors and anticholinergics, we hypothesized functional interactions of these two drug classes. Guinea pig precision-cut lung slices were preincubated with the PDE4 inhibitors CHF-6001 or roflumilast and/or the anticholinergics tiotropium or glycopyorrolate, followed by stimulation with methacholine (10 µM) or TGF-ß1 (2 ng/ml) for 48 h. The inhibitory effects on airway smooth muscle remodeling, airway contraction, and TGF-ß release were investigated. Methacholine-induced protein expression of smooth muscle-myosin was fully inhibited by CHF-6001 (0.3-100 nM), whereas roflumilast (1 µM) had smaller effects. Tiotropium and glycopyrrolate fully inhibited methacholine-induced airway remodeling (0.1-30 nM). The combination of CHF-6001 and tiotropium or glycopyrrolate, in concentrations partially effective by themselves, fully inhibited methacholine-induced remodeling in combination. CHF-6001 did not affect airway closure and had limited effects on TGF-ß1-induced remodeling, but rather, it inhibited methacholine-induced TGF-ß release. The PDE4 inhibitor CHF-6001, and to a lesser extent roflumilast, and the LAMAs tiotropium and glycopyrrolate inhibit bronchoconstriction-induced remodeling. The combination of CHF-6001 and anticholinergics was more effective than the individual compounds. This cooperativity might be explained by the distinct mechanisms of action inhibiting TGF-ß release and bronchoconstriction.

Combination therapy of tiotropium and ciclesonide attenuates airway inflammation and remodeling in a guinea pig model of chronic asthma.

BACKGROUND: The long-acting anticholinergic tiotropium has recently been registered for the treatment of asthma, and its use is associated with a reduction in exacerbation frequency. Anti-inflammatory and anti-remodeling effects of tiotropium have been demonstrated in in vitro and in vivo models. Because tiotropium treatment is used in combination with inhaled corticosteroids, potential additive effects between the two would be clinically relevant. Therefore, the aim of this study was to investigate additive effects between tiotropium and ciclesonide on airway inflammation and remodeling in guinea pig models of asthma. METHODS: Guinea pigs (n = 3-8/group) were sensitized and challenged with ovalbumin in an acute (single challenge) and a chronic model (12 weekly challenges) of allergic asthma. Animals were treated with vehicle, nebulized tiotropium (0.01-0.3 mM) and/or intranasally instilled ciclesonide (0.001-1 mg/kg) before each challenge. Bronchoalveolar lavage fluid and lungs were collected for analysis of airway inflammation and remodeling. RESULTS: Tiotropium and ciclesonide treatment, alone or in combination, did not inhibit airway inflammation in the acute asthma model. In a dose-finding study, low doses of tiotropium and ciclesonide inhibited airway eosinophilia and airway smooth muscle thickening in the chronic asthma model. Threshold doses of 0.01 mM tiotropium (nebulizer concentration) and 0.01 mg/kg ciclesonide were selected to investigate potential additive effects between both drugs. At these doses, tiotropium and ciclesonide did not inhibit airway eosinophilia or airway smooth muscle thickening when administered alone, but significantly inhibited these allergen-induced responses when administered in combination. CONCLUSIONS: Combined treatment with low doses of tiotropium and ciclesonide inhibits airway inflammation and remodeling in a guinea pig model of chronic asthma, suggesting that combined treatment with anticholinergics and corticosteroids may have anti-inflammatory and anti-remodeling activity in allergic airway diseases. Since tiotropium is registered as a therapy for asthma added on to corticosteroid treatment, these beneficial effects of the combination therapy may be clinically relevant.

L-thyroxine promotes a proliferative airway smooth muscle phenotype in the presence of TGF-ß1.

Hypothyroidism may reduce, whereas hyperthyroidism may aggravate, asthma symptoms. The mechanisms underlying this relationship are largely unknown. Since thyroid hormones have central roles in cell growth and differentiation, we hypothesized that airway remodeling, in particular increased airway smooth muscle (ASM) mass, may be involved. To address this hypothesis, we investigated the effects of triiodothyronine (T3) and l-thyroxine (T4) in the absence and presence of the profibrotic transforming growth factor (TGF)-ß1 on human ASM cell phenotype switching. T3 (1-100 nM) and T4 (1-100 nM) did not affect basal ASM proliferation. However, when combined with TGF-ß1 (2 ng/ml), T4 synergistically increased the proliferative response, whereas only a minor effect was observed for T3. In line with a switch from a contractile to a proliferative ASM phenotype, T4 reduced the TGF-ß1-induced contractile protein expression by ∼50%. Cotreatment with T3 reduced TGF-ß1-induced contractile protein expression by ∼25%. The synergistic increase in proliferation was almost fully inhibited by the integrin αvß3 antagonist tetrac (100 nM), whereas no significant effects of the thyroid receptor antagonist 1-850 (3 µM) were observed. Inhibition of MEK1/2, downstream of the integrin αvß3, also inhibited the T4- and TGF-ß1-induced proliferative responses. Collectively, the results indicate that T4, and to a lesser extent T3, promotes a proliferative ASM phenotype in the presence of TGF-ß1, which is predominantly mediated by the membrane-bound T4 receptor αvß3. These results indicate that thyroid hormones may enhance ASM remodeling in asthma, which could be of relevance for hyperthyroid patients with this disease.

Muscarinic M3 receptors on structural cells regulate cigarette smoke-induced neutrophilic airway inflammation in mice.

Anticholinergics, blocking the muscarinic M3 receptor, are effective bronchodilators for patients with chronic obstructive pulmonary disease. Recent evidence from M(3) receptor-deficient mice (M(3)R(-/-)) indicates that M3 receptors also regulate neutrophilic inflammation in response to cigarette smoke (CS). M(3) receptors are present on almost all cell types, and in this study we investigated the relative contribution of M(3) receptors on structural cells vs. inflammatory cells to CS-induced inflammation using bone marrow chimeric mice. Bone marrow chimeras (C56Bl/6 mice) were generated, and engraftment was confirmed after 10 wk. Thereafter, irradiated and nonirradiated control animals were exposed to CS or fresh air for four consecutive days. CS induced a significant increase in neutrophil numbers in nonirradiated and irradiated control animals (4- to 35-fold). Interestingly, wild-type animals receiving M(3)R(-/-) bone marrow showed a similar increase in neutrophil number (15-fold). In contrast, no increase in the number of neutrophils was observed in M3R(-/-) animals receiving wild-type bone marrow. The increase in keratinocyte-derived chemokine (KC) levels was similar in all smoke-exposed groups (2.5- to 5.0-fold). Microarray analysis revealed that fibrinogen-α and CD177, both involved in neutrophil migration, were downregulated in CS-exposed M(3)R(-/-) animals receiving wild-type bone marrow compared with CS-exposed wild-type animals, which was confirmed by RT-qPCR (1.6-2.5 fold). These findings indicate that the M(3) receptor on structural cells plays a proinflammatory role in CS-induced neutrophilic inflammation, whereas the M(3) receptor on inflammatory cells does not. This effect is probably not mediated via KC release, but may involve altered adhesion and transmigration of neutrophils via fibrinogen-α and CD177.

Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells.

BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct effects of endogenous non-neuronal acetylcholine on epithelial cell differentiation. METHODS: Human airway epithelial cells from healthy donors were cultured at an air-liquid interface (ALI). Cells were exposed to the muscarinic antagonist tiotropium (10 nM), interleukin (IL)-13 (1, 2 and 5 ng/mL), or a combination of IL-13 and tiotropium, during or after differentiation at the ALI. RESULTS: Human airway epithelial cells expressed all components of the non-neuronal cholinergic system, suggesting acetylcholine production. Tiotropium had no effects on epithelial cell differentiation after air exposure. Differentiation into goblet cells was barely induced after air exposure. Therefore, IL-13 (1 ng/mL) was used to induce goblet cell metaplasia. IL-13 induced MUC5AC-positive cells (5-fold) and goblet cells (14-fold), as assessed by histochemistry, and MUC5AC gene expression (105-fold). These effects were partly prevented by tiotropium (47-92%). Goblet cell metaplasia was induced by IL-13 in a dose-dependent manner, which was inhibited by tiotropium. In addition, tiotropium reversed goblet cell metaplasia induced by 2 weeks of IL-13 exposure. IL-13 decreased forkhead box protein A2 (FoxA2) expression (1.6-fold) and increased FoxA3 (3.6-fold) and SAM-pointed domain-containing ETS transcription factor (SPDEF) (5.2-fold) expression. Tiotropium prevented the effects on FoxA2 and FoxA3, but not on SPDEF. CONCLUSIONS: We demonstrate that tiotropium has no effects on epithelial cell differentiation after air exposure, but inhibits and reverses IL-13-induced goblet cell metaplasia, possibly via FoxA2 and FoxA3. This indicates that non-neuronal acetylcholine contributes to goblet cell differentiation by a direct effect on epithelial cells.

Noncanonical WNT-5A signaling regulates TGF-ß-induced extracellular matrix production by airway smooth muscle cells.

Transforming growth factor ß (TGF-ß), a key mediator of fibrotic responses, is increased in asthma and drives airway remodeling by inducing expression of extracellular matrix (ECM) proteins. We investigated the molecular mechanisms underlying TGF-ß-induced ECM expression by airway smooth muscle cells and demonstrate a novel link between TGF-ß and Wingless/integrase 1 (WNT) signaling in ECM deposition. Airway smooth muscle expresses abundant WNT ligands, with the noncanonical WNT-5A being the most profoundly expressed. Interestingly, WNT-5A shows ∼2-fold higher abundance in airway smooth muscle cells isolated from individuals with asthma than individuals without asthma. WNT-5A is markedly induced in response to TGF-ß (4-16-fold; EC50 0.3 ng/ml) and is required for collagen and fibronectin expression by airway smooth muscle. WNT-5A engages noncanonical WNT signaling pathways, as inhibition of Ca(2+) and c-Jun N-terminal kinase (JNK) signaling attenuated this TGF-ß response, whereas the canonical WNT antagonist Dickkopf 1 (DKK-1) did not. Accordingly, WNT-5A induced JNK phosphorylation and nuclear translocation of nuclear factor of activated T cells c1 (NFATc1). Furthermore, silencing of the WNT-5A receptors Frizzled 8 (FZD8) and RYK attenuated TGF-ß-induced ECM expression. Collectively, these findings demonstrate that noncanonical WNT-5A signaling is activated by and necessary for TGF-ß-induced ECM production by airway smooth muscle cells, which could have significance in asthma pathogenesis.

Glucocorticosteroids and ß2-adrenoceptor agonists synergize to inhibit airway smooth muscle remodeling.

Airway remodeling, including increased airway smooth muscle (ASM) mass and contractility, contributes to increased airway narrowing in asthma. Increased ASM mass may be caused by exposure to mitogens, including platelet-derived growth factor (PDGF) and collagen type I, which induce a proliferative, hypocontractile ASM phenotype. In contrast, prolonged exposure to insulin induces a hypercontractile phenotype. Glucocorticosteroids and ß2-adrenoceptor agonists synergize to increase glucocorticosteroid receptor translocation in ASM cells; however, the impact of this synergism on phenotype modulation is unknown. Using bovine tracheal smooth muscle, we investigated the effects of the glucocorticosteroids fluticasone (10 nM), budesonide (30 nM), and dexamethasone (0.1-1 µM) and the combination of low concentrations of fluticasone (3-100 pM) and fenoterol (10 nM) on ASM phenotype switching in response to PDGF (10 ng/ml), collagen type I (50 µg/ml), and insulin (1 µM). All glucocorticosteroids inhibited PDGF- and collagen I-induced proliferation and hypocontractility, with the effects of collagen I being less susceptible to glucocorticosteroid action. At 100-fold lower concentrations, fluticasone (100 pM) synergized with fenoterol to prevent PDGF- and collagen I-induced phenotype switching. This inhibition of ASM phenotype switching was associated with a normalization of the PDGF-induced decrease in the cell cycle inhibitors p21(WAF1/CIP1) and p57(KIP2). At this concentration, fluticasone also prevented the insulin-induced hypercontractile phenotype. At even lower concentrations, fluticasone (3 pM) synergized with fenoterol to inhibit this phenotype switch. Collectively, these findings indicate that glucocorticosteroids and ß2-agonists synergistically inhibit ASM phenotype switching, which may contribute to the increased effectiveness of combined treatment with glucocorticosteroids and ß2-agonists in asthma.

Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells.

Asthmatics have elevated levels of IL-17A compared to healthy controls. IL-17A is likely to contribute to reduced corticosteroid sensitivity of human airway epithelium. Here, we aimed to investigate the mechanistic underpinnings of this reduced sensitivity in more detail. Differentiated primary human airway epithelial cells (hAECs) were exposed to IL-17A in the absence or presence of dexamethasone. Cells were then collected for RNA sequencing analysis or used for barrier function experiments. Mucus was collected for volume measurement and basal medium for cytokine analysis. 2861 genes were differentially expressed by IL-17A (Padj < 0.05), of which the majority was not sensitive to dexamethasone (< 50% inhibition). IL-17A did inhibit canonical corticosteroid genes, such as HSD11B2 and FKBP5 (p < 0.05). Inflammatory and goblet cell metaplasia markers, cytokine secretion and mucus production were all induced by IL-17A, and these effects were not prevented by dexamethasone. Dexamethasone did reverse IL-17A-stimulated epithelial barrier disruption, and this was associated with gene expression changes related to cilia function and development. We conclude that IL-17A induces function-specific corticosteroid-insensitivity. Whereas inflammatory response genes and mucus production in primary hAECs in response to IL-17A were corticosteroid-insensitive, corticosteroids were able to reverse IL-17A-induced epithelial barrier disruption.

Diesel exhaust particles distort lung epithelial progenitors and their fibroblast niche.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by inflammation and impaired tissue regeneration, and is reported as the fourth leading cause of death worldwide by the Centers for Disease Control and Prevention (CDC). Environmental pollution and specifically motor vehicle emissions are known to play a role in the pathogenesis of COPD, but little is still known about the molecular mechanisms that are altered following diesel exhaust particles (DEP) exposure. Here we used lung organoids derived from co-culture of alveolar epithelial progenitors and fibroblasts to investigate the effect of DEP on the epithelial-mesenchymal signaling niche in the distal lung, which is essential for tissue repair. We found that DEP treatment impaired the number as well as the average diameter of both airway and alveolar type of lung organoids. Bulk RNA-sequencing of re-sorted epithelial cells and fibroblasts following organoid co-culture shows that the Nrf2 pathway, which regulates antioxidants' activity, was upregulated in both cell populations in response to DEP; and WNT/ß-catenin signaling, which is essential to promote epithelial repair, was downregulated in DEP-exposed epithelial cells. We show that pharmacological treatment with anti-oxidant agents such as N-acetyl cysteine (NAC) or Mitoquinone mesylate (MitoQ) reversed the effect of DEP on organoids growth. Additionally, a WNT/ß-catenin activator (CHIR99021) successfully restored WNT signaling and promoted organoid growth upon DEP exposure. We propose that targeting oxidative stress and specific signaling pathways affected by DEP in the distal lung may represent a strategy to restore tissue repair in COPD.

A transcriptomics-guided drug target discovery strategy identifies receptor ligands for lung regeneration.

Currently, there is no pharmacological treatment targeting defective tissue repair in chronic disease. Here, we used a transcriptomics-guided drug target discovery strategy using gene signatures of smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke, identifying druggable targets expressed in alveolar epithelial progenitors, of which we screened the function in lung organoids. We found several drug targets with regenerative potential, of which EP and IP prostanoid receptor ligands had the most profound therapeutic potential in restoring cigarette smoke-induced defects in alveolar epithelial progenitors in vitro and in vivo. Mechanistically, we found, using single-cell RNA sequencing analysis, that circadian clock and cell cycle/apoptosis signaling pathways were differentially expressed in alveolar epithelial progenitor cells in patients with COPD and in a relevant model of COPD, which was prevented by prostaglandin E2 or prostacyclin mimetics. We conclude that specific targeting of EP and IP receptors offers therapeutic potential for injury to repair in COPD.

The integrin-blocking peptide RGDS inhibits airway smooth muscle remodeling in a guinea pig model of allergic asthma.

RATIONALE: Airway remodeling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. The mechanisms driving these changes are, however, incompletely understood. Recently, an important role for extracellular matrix proteins in regulating ASM proliferation and contractility has been found, suggesting that matrix proteins and their integrins actively modulate airway remodeling. OBJECTIVES: To investigate the role of RGD (Arg-Gly-Asp)-binding integrins in airway remodeling in an animal model of allergic asthma. METHODS: Using a guinea pig model of allergic asthma, the effects of topical application of the integrin-blocking peptide RGDS (Arg-Gly-Asp-Ser) and its negative control GRADSP (Gly-Arg-Ala-Asp-Ser-Pro) were assessed on markers of ASM remodeling, fibrosis, and inflammation induced by repeated allergen challenge. In addition, effects of these peptides on human ASM proliferation and maturation were investigated in vitro. MEASUREMENTS AND MAIN RESULTS: RGDS attenuated allergen-induced ASM hyperplasia and hypercontractility as well as increased pulmonary expression of smooth muscle myosin heavy chain and the proliferative marker proliferating cell nuclear antigen (PCNA). No effects were observed for GRADSP. The RGDS effects were ASM selective, as allergen-induced eosinophil and neutrophil infiltration as well as fibrosis were unaffected. In cultured human ASM cells, we demonstrated that proliferation induced by collagen I, fibronectin, serum, and platelet-derived growth factor requires signaling via RGD-binding integrins, particularly of the alpha(5)beta(1) subtype. In addition, RGDS inhibited smooth muscle alpha-actin accumulation in serum-deprived ASM cells. CONCLUSIONS: This is the first study indicating that integrins modulate ASM remodeling in an animal model of allergic asthma, which can be inhibited by a small peptide containing the RGD motif.

The laminin ß1-competing peptide YIGSR induces a hypercontractile, hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma.

BACKGROUND: Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established. METHODS: Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro. RESULTS: Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline- and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR. CONCLUSION: These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide.

Arginase inhibition protects against allergen-induced airway obstruction, hyperresponsiveness, and inflammation.

RATIONALE: In a guinea pig model of allergic asthma, using perfused tracheal preparations ex vivo, we demonstrated that L-arginine limitation due to increased arginase activity underlies a deficiency of bronchodilating nitric oxide (NO) and airway hyperresponsiveness (AHR) after the allergen-induced early and late asthmatic reaction. OBJECTIVES: Using the same animal model, we investigated the acute effects of the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) and of L-arginine on AHR after the early and late reaction in vivo. In addition, we investigated the protection of allergen-induced asthmatic reactions, AHR, and airway inflammation by pretreatment with the drug. METHODS: Airway responsiveness to inhaled histamine was measured in permanently instrumented, freely moving guinea pigs sensitized to ovalbumin at 24 hours before allergen challenge and after the allergen-induced early and late asthmatic reactions by assessing histamine PC(100) (provocative concentration causing a 100% increase of pleural pressure) values. MEASUREMENTS AND MAIN RESULTS: Inhaled ABH acutely reversed AHR to histamine after the early reaction from 4.77 +/- 0.56-fold to 2.04 +/- 0.34-fold (P < 0.001), and a tendency to inhibition was observed after the late reaction (from 1.95 +/- 0.56-fold to 1.56 +/- 0.47-fold, P < 0.10). Quantitatively similar results were obtained with inhaled l-arginine. Remarkably, after pretreatment with ABH a 33-fold higher dose of allergen was needed to induce airway obstruction (P < 0.01). Consequently, ABH inhalation 0.5 hour before and 8 hours after allergen challenge protected against the allergen-induced early and late asthmatic reactions, AHR and inflammatory cell infiltration. CONCLUSIONS: Inhalation of ABH or l-arginine acutely reverses allergen-induced AHR after the early and late asthmatic reaction, presumably by attenuating arginase-induced substrate deficiency to NO synthase in the airways. Moreover, ABH considerably reduces the airway sensitivity to inhaled allergen and protects against allergen-induced bronchial obstructive reactions, AHR, and airway inflammation. This is the first in vivo study indicating that arginase inhibitors may have therapeutic potential in allergic asthma.

Mouse Lung Tissue Slice Culture.

Precision-cut lung slices (PCLS) represent an ex vivo model widely used in visualizing interactions between lung structure and function. The major advantage of this technique is that the presence, differentiation state, and localization of the more than 40 cell types that make up the lung are in accordance with the physiological situation found in lung tissue, including the right localization and patterning of extracellular matrix elements. Here we describe the methodology involved in preparing and culturing PCLS followed by detailed practical information about their possible applications.

Mesenchymal WNT-5A/5B Signaling Represses Lung Alveolar Epithelial Progenitors.

Chronic obstructive pulmonary disease (COPD) represents a worldwide concern with high morbidity and mortality, and is believed to be associated with accelerated ageing of the lung. Alveolar abnormalities leading to emphysema are a key characteristic of COPD. Pulmonary alveolar epithelial type 2 cells (AT2) produce surfactant and function as progenitors for type 1 cells. Increasing evidence shows elevated WNT-5A/B expression in ageing and in COPD that may contribute to the disease process. However, supportive roles for WNT-5A/B in lung regeneration were also reported in different studies. Thus, we explored the role of WNT-5A/B on alveolar epithelial progenitors (AEPs) in more detail. We established a Precision-Cut-Lung Slices (PCLS) model and a lung organoid model by co-culturing epithelial cells (EpCAM+/CD45-/CD31-) with fibroblasts in matrigel in vitro to study the impact of WNT-5A and WNT-5B. Our results show that WNT-5A and WNT-5B repress the growth of epithelial progenitors with WNT-5B preferentially restraining the growth and differentiation of alveolar epithelial progenitors. We provide evidence that both WNT-5A and WNT-5B negatively regulate the canonical WNT signaling pathway in alveolar epithelium. Taken together, these findings reveal the functional impact of WNT-5A/5B signaling on alveolar epithelial progenitors in the lung, which may contribute to defective alveolar repair in COPD.

Delivery system for budesonide based on lipid-DNA.

Budesonide is a hydrophobic glucocorticoid with high anti-inflammatory activity for the treatment of asthma, inflammatory bowel disease and rheumatoid arthritis. A micellar drug-delivery system based on lipid-DNA may provide a strategy to maximize its drug efficacy and reduce adverse effects. In this work, we report the use of lipid-DNAA (UU11mer), featuring two hydrophobic alkyl chains and forming micelles at a comparatively low critical micelle concentration, to render budesonide water-soluble with a high loading capacity (LC). The inhibition of interleukin-8 (IL-8) release shows that the new delivery system retains the inhibitory activity in cell-based assays. In conclusion, this research provides a novel approach to formulate and administer budesonide in a non-invasive manner, which dramatically improves its water-solubility while retaining its bioavailability.

Arginase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma.

BACKGROUND: Using guinea pig tracheal preparations, we have recently shown that endogenous arginase activity attenuates inhibitory nonadrenergic noncholinergic (iNANC) nerve-mediated airway smooth muscle relaxation by reducing nitric oxide (NO) production--due to competition with neuronal NO-synthase (nNOS) for the common substrate, L-arginine. Furthermore, in a guinea pig model of allergic asthma, airway arginase activity is markedly increased after the early asthmatic reaction (EAR), leading to deficiency of agonist-induced, epithelium-derived NO and subsequent airway hyperreactivity. In this study, we investigated whether increased arginase activity after the EAR affects iNANC nerve-derived NO production and airway smooth muscle relaxation. METHODS: Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5-16 Hz)-induced relaxation was measured in tracheal open-ring preparations precontracted to 30% with histamine in the presence of 1 microM atropine and 3 microM indomethacin. The contribution of NO to EFS-induced relaxation was assessed by the nonselective NOS inhibitor Nomega-nitro-L-arginine (L-NNA, 100 microM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine (nor-NOHA, 10 microM). Furthermore, the role of substrate availability to nNOS was measured in the presence of exogenous L-arginine (5.0 mM). RESULTS: At 6 h after ovalbumin-challenge (after the EAR), EFS-induced relaxation (ranging from 3.2 +/- 1.1% at 0.5 Hz to 58.5 +/- 2.2% at 16 Hz) was significantly decreased compared to unchallenged controls (7.1 +/- 0.8% to 75.8 +/- 0.7%; P < 0.05 all). In contrast to unchallenged controls, the NOS inhibitor L-NNA did not affect EFS-induced relaxation after allergen challenge, indicating that NO deficiency underlies the impaired relaxation. Remarkably, the specific arginase inhibitor nor-NOHA normalized the impaired relaxation to unchallenged control (P < 0.05 all), which effect was inhibited by L-NNA (P < 0.01 all). Moreover, the effect of nor-NOHA was mimicked by exogenous L-arginine. CONCLUSION: The results clearly demonstrate that increased arginase activity after the allergen-induced EAR contributes to a deficiency of iNANC nerve-derived NO and decreased airway smooth muscle relaxation, presumably via increased substrate competition with nNOS.

Inhalation of the Rho-kinase inhibitor Y-27632 reverses allergen-induced airway hyperresponsiveness after the early and late asthmatic reaction.

BACKGROUND: In guinea pigs, we have previously demonstrated that the contribution of Rho-kinase to airway responsiveness in vivo and ex vivo is enhanced after active sensitization with ovalbumin (OA). Using conscious, unrestrained OA-sensitized guina pigs, we now investigated the role of Rho-kinase in the development of airway hyperresponsiveness (AHR) after the allergen-induced early (EAR) and late asthmatic reaction (LAR) in vivo. METHODS: Histamine and PGF2alpha PC100-values (provocation concentrations causing 100% increase in pleural pressure) were assessed before OA-challenge (basal airway responsiveness) and after the OA-induced EAR (5 h after challenge) and LAR (23 h after challenge). Thirty minutes later, saline or the specific Rho-kinase inhibitor Y-27632 (5 mM, nebulizer concentration) were nebulized, after which PC100-values were reassessed. RESULTS: In contrast to saline, Y-27632 inhalation significantly decreased the basal responsiveness toward histamine and PGF2alpha before OA-challenge, as indicated by increased PC100 -values. Both after the allergen-induced EAR and LAR, AHR to histamine and PGF2alpha was present, which was reversed by Y-27632 inhalation. Moreover, there was an increased effectiveness of Y-27632 to reduce airway responsiveness to histamine and PGF2alpha after the EAR and LAR as compared to pre-challenge conditions. Saline inhalations did not affect histamine or PGF2alpha PC100-values at all. Interestingly, under all conditions Y-27632 was significantly more effective in reducing airway responsiveness to PGF2alpha as compared to histamine. Also, there was a clear tendency (P = 0.08) to a more pronounced degree of AHR after the EAR for PGF2alpha than for histamine. CONCLUSION: The results indicate that inhalation of the Rho-kinase inhibitor Y-27632 causes a considerable bronchoprotection to both histamine and PGF2alpha. Moreover, the results are indicative of a differential involvement of Rho-kinase in the agonist-induced airway obstruction in vivo. Increased Rho-kinase activity contributes to the allergen-induced AHR to histamine and PGF2alpha after both the EAR and the LAR, which is effectively reversed by inhalation of Y-27632. Therefore, Rho-kinase can be considered as a potential pharmacotherapeutical target in allergic asthma.

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, ß2-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.