Sophie Bos

Caveolae and Caveolins in the Respiratory System

Caveolae are flask-shaped invaginations of the plasma membrane that are present in most structural cells. They owe their characteristic Omega-shape to complexes of unique proteins, the caveolins, which indirectly tether cholesterol and sphingolipid-enriched membrane microdomains to the cytoskeleton. Caveolins possess a unique scaffolding domain that anchors receptors, ion channels, second messenger producing enzymes, and effector kinases, thereby sequestering them to caveolae, and modulating cellular signaling and vesicular transport. The lungs express numerous caveolae and high levels of caveolins; therefore they likely play an important role in lung physiology. Indeed, recent and ongoing studies indicate important roles for caveolae and caveolins in the airway epithelium, airway smooth muscle, airway fibroblasts, airway inflammatory cells and the pulmonary vasculature. We review the role of caveolae and caveolins in lung cells and discuss their involvement in cellular signaling associated with asthma, COPD, lung cancer, idiopathic pulmonary fibrosis and pulmonary vascular defects.

Muscarinic M₃ receptors contribute to allergen-induced airway remodeling in mice

Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the muscarinic M3 receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the muscarinic M3 receptor. The objective of this work was to study the contribution of the muscarinic M3 receptor to allergen-induced remodeling using muscarinic M3 receptor subtype-deficient (M3R(-/-)) mice. Wild-type (WT), M1R(-/-), and M2R(-/-) mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R(-/-) mice (30-100%), whereas M1R(-/-) and M2R(-/-) mice responded similarly to WT mice. In addition, airway smooth muscle and pulmonary vascular smooth muscle mass were 35-40% lower in saline-challenged M3R(-/-) mice compared with WT mice. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and T helper type 2 cytokine expression, was similar or even enhanced in M3R(-/-) mice. Our data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the muscarinic M3 receptor, and not via M1 or M2 receptors. No stimulatory role for muscarinic M3 receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response, and may involve bronchoconstriction.

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin‐1, but the functional role of caveolae and caveolin‐1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin‐1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)‐β1. As assessed by Western analysis and laser scanning cytometry, caveolin‐1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF‐β1 induced profound increases in the contractile phenotype markers sm‐α‐actin and calponin in cells that also accumulated abundant caveolin‐1; however, siRNA or shRNAi inhibition of caveolin‐1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF‐β1. The failure by TGF‐β1 to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor‐4E binding protein(4E‐BP)1 phosphorylation with caveolin‐1 knockdown, indicating that caveolin‐1 expression promotes TGF‐β1 signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin‐1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin‐1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin‐1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.

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.

HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice

Chronic obstructive pulmonary disease (COPD) constitutes a major health burden. Studying underlying molecular mechanisms could lead to new therapeutic targets. Macrophages are orchestrators of COPD, by releasing pro-inflammatory cytokines. This process relies on transcription factors such as NF-κB, among others. NF-κB is regulated by lysine acetylation; a post-translational modification installed by histone acetyltransferases and removed by histone deacetylases (HDACs). We hypothesized that small molecule HDAC inhibitors (HDACi) targeting class I HDACs members that can regulate NF-κB could attenuate inflammatory responses in COPD via modulation of the NF-κB signaling output. MS-275 is an isoform-selective inhibitor of HDAC1-3. In precision-cut lung slices and RAW264.7 macrophages, MS-275 upregulated the expression of both pro- and anti-inflammatory genes, implying mixed effects. Interestingly, anti-inflammatory IL10 expression was upregulated in these model systems. In the macrophages, this was associated with increased NF-κB activity, acetylation, nuclear translocation, and binding to the IL10 promoter. Importantly, in an in vivo model of cigarette smoke-exposed C57Bl/6 mice, MS-275 robustly attenuated inflammatory expression of KC and neutrophil influx in the lungs. This study highlights for the first time the potential of isoform-selective HDACi for the treatment of inflammatory lung diseases like COPD.

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.