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Cystic fibrosis (CF) is a genetic disease that causes multiple airway abnormalities. Two major respiratory consequences of CF are airway hyperresponsiveness (AHR) and airway remodeling. Airway smooth muscle (ASM) is hypothesized to be responsible for the airway dysfunction, since their thickening is involved in remodeling, and excessive contraction by the ASM may cause AHR. It is unclear whether the ASM is intrinsically altered to favor increased contractility or proliferation or if microenvironmental influences induce pathological behavior in vivo. In this study, we examined the contractile and proliferative properties of ASM cells isolated from healthy donor and CF transplant lungs. Assays of proliferation showed that CF ASM proliferates at a higher rate than healthy cells. Through calcium analysis, no differences in contractile activation in response to histamine were found. However, CF ASM cells lagged in their reuptake of calcium in the sarcoplasmic reticulum. The combination CFTR corrector and potentiator, VX-809/770, used to restore CFTR function in CF ASM, resulted in a reduction in proliferation and in a normalization of calcium reuptake kinetics. These results show that impaired CFTR function in ASM cells causes intrinsic changes in their proliferative and contractile properties.

Background Bronchial thermoplasty (BT) is a novel technique used in the treatment of subjects with severe refractory asthma. Radiofrequency is provided to airway walls during bronchoscopy in order to reduce airway remodeling. Several clinical studies have reported an improvement in subjects’ symptoms following BT. However, how BT affects the airway architectures and inflammatory mediators in the airways has not been yet fully elucidated. Methods Fourteen subjects with severe asthma were recruited in this study according to the criteria of ATS severe asthma definition. The study subjects undertook bronchial biopsy during the bronchoscopy procedure at baseline and 6 weeks after the initial BT treatment. The obtained samples were stained with antibodies for α-smooth muscle actin (α-SMA); protein gene product (PGP) 9.5, a specific nerve marker; von Willebrand factor (vWF), a marker for blood vessels; interleukin-17A (IL-17A) and transforming growth factor-β1 (TGF-β1). Results The expression of α-SMA and PGP9.5 were significantly reduced post-BT. There was no significant difference in the number of blood vessels between baseline and post-BT. In addition, BT did not affect the production of IL-17A and TGF-β1 in the airways. The changes in the expression of α-SMA and PGP9.5 had no significant correlation with the improvement of pulmonary function. Conclusion and Clinical Relevance: This study suggests that BT reduces airway smooth muscle mass and the airway innervation without affecting vasculature and the production of inflammatory mediators in the airways of subjects with severe asthma.

Cystic fibrosis (CF) is an autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Many patients with CF have asthma-like symptoms and airway hyperrespon...

The increased mass of airway smooth muscle (ASM) in the airways of asthmatic patients may contribute to the pathology of this disease by increasing the capacity for airway narrowing. Evidence for the airway epithelium as a participant in ASM remodeling is accruing. To investigate mechanisms by which airway epithelial cells induce ASM cell (ASMC) proliferation, we have employed a co-culture model to explore markers of ASMC proliferative phenotype. Co-culture with epithelial cells led to incorporation of bromodeoxyuridine into ASMCs, indicating augmented proliferation and an associated increase in mRNA of the pro-proliferative co-transcription factor Elk1. Although the mitogen heparin-binding epidermal growth factor (HB-EGF) was augmented in the co-culture supernatant, the ASMC epidermal growth factor receptor (EGFR), an effector of HB-EGF induced proliferation, did not mediate epithelial-induced proliferation. The co-culture increased the expression of ASMC mRNA for the pro-inflammatory cytokines IL-6 and IL-8 as well as the pro-proliferative microRNA miR-210. The transcriptional repressor Max-binding protein (Mnt), a putative target of miR-210, was transcriptionally repressed in co-cultured ASMCs. Together, these data indicate that the airway epithelium-induced proliferative phenotype of ASMCs is not driven by EGFR signaling, but rather may be dependent on miR210 targeting of tumor suppressor Mnt.

Enhanced recovery after surgery (ERAS) is an interprofessional program that can lead to hastened patient recovery and reduced time in hospital. Nursing staff play a key role in the implementation of enhanced recovery protocols. This issue focalizes the role of nurses in ERAS program for patients submitted to Thoracic Surgery, in particular for cases of major lung resection performed by a minimally invasive surgical approach (VATS, video assisted thoracic surgery).

Constriction of airways during asthmatic exacerbation is the result of airway smooth muscle (ASM) contraction. Although it is generally accepted that ASM is hypercontractile in asthma, this has not been unambiguously demonstrated. Whether airway hyperresponsiveness (AHR) is the result of increased ASM mass alone or also increased contractile force generation per unit of muscle directly determines the potential avenues for treatment.To assess whether ASM is hypercontractile we performed a series of mechanics measurements on isolated ASM from intrapulmonary airways and trachealis from human lungs. We analysed the ASM and whole airway proteomes to verify if proteomic shifts contribute to changes in ASM properties.We report an increase in isolated ASM contractile stress and stiffness specific to asthmatic human intrapulmonary bronchi, the site of increased airway resistance in asthma. Other contractile parameters were not altered. Principal component analysis (PCA) of unbiased mass spectrometry data showed clear clustering of asthmatic subjects with respect to ASM specific proteins. The whole airway proteome showed upregulation of structural proteins. We did not find any evidence for a difference in the regulation of myosin activity in the asthmatic ASM.In conclusion, we showed that ASM is indeed hyperreactive at the level of intrapulmonary airways in asthma. We identified several proteins that are upregulated in asthma that could contribute to hyperreactivity. Our data also suggest enhanced force transmission associated with enrichment of structural proteins in the whole airway. These findings may lead to novel directions for treatment development in asthma.

Purpose: Chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLN) are a new class of nanodevices to effectively deliver anti-cancer drugs to tumoral cells. This study investigated their antitumoral effects ‘per se’, using a mathematical model validated on experimental data. Methods: OLN were prepared and characterized either in vitro or in vivo. TUBO cells, established from a lobular carcinoma of a BALB-neuT mouse, were investigated following 48 h of incubation in the absence/presence of different concentrations of OLN. OLN internalization, cell viability, necrosis, apoptosis, cell cycle and reactive oxygen species (ROS) production were checked as described in the Method section. In vivo tumor growth was evaluated after subcutaneous transplant in BALB/c mice of TUBO cells either without treatment or after 24 h incubation with 10% v/v OLN. Results: OLN showed sizes of about 350 nm and a positive surface charge (45 mV). Dose-dependent TUBO cell death through ROS-triggered apoptosis following OLN internalization was detected. A mathematical model predicting the effects of OLN uptake was validated on both in vitro and in vivo results. Conclusions: Due to their intrinsic toxicity OLN might be considered an adjuvant tool suitable to deliver their therapeutic cargo intracellularly and may be proposed as promising combined delivery system.

Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

Pathogen persistence in the respiratory tract is an important preoccupation, and of particular relevance to infectious diseases such as tuberculosis. The equilibrium between elimination of pathogens and the magnitude of the host response is a sword of Damocles for susceptible patients. The alveolar macrophage is the first sentinel of the respiratory tree and constitutes the dominant immune cell in the steady state. This immune cell is a key player in the balance between defense against pathogens and tolerance toward innocuous stimuli. This review focuses on the role of alveolar macrophages in limiting lung tissue damage from potentially innocuous stimuli and from infections, processes that are relevant to appropriate tolerance of potential causes of lung disease. Notably, the different anti-inflammatory strategies employed by alveolar macrophages and lung tissue damage control are explored. These two properties, in addition to macrophage manipulation by pathogens, are discussed to explain how alveolar macrophages may drive pathogen persistence in the airways.

Background Inhaled oxidative toxicants present in ambient air cause airway epithelial injury, inflammation, and airway hyperresponsiveness. Effective adaptation to such environmental insults is essential for the preservation of pulmonary function, whereas failure or incomplete adaptation to oxidative injury can render the host susceptible to the development of airway disease. Objective We sought to explore the mechanisms of airway adaptation to oxidative injury. Methods For a model to study pulmonary adaptation to oxidative stress–induced lung injury, we exposed mice to repeated nose-only chlorine gas exposures. Outcome measures were evaluated 24 hours after the last chlorine exposure. Lung mechanics and airway responsiveness to methacholine were assessed by using the flexiVent. Inflammation and antioxidant responses were assessed in both bronchoalveolar lavage fluid and lung tissue. Using both loss or gain of function and genomic approaches, we further dissected the cellular and molecular mechanisms involved in pulmonary adaptation. Results Repeated exposures to oxidative stress resulted in pulmonary adaptation evidenced by abrogation of neutrophilic inflammation and airway hyperresponsiveness. This adaptation was independent of antioxidant mechanisms and regulatory T cells but dependent on residential alveolar macrophages (AMs). Interestingly, 5% of AMs expressed forkhead box P3, and depletion of these cells abolished adaptation. Results from transcriptomic profiling and loss and gain of function suggest that adaptation might be dependent on TGF-β and prostaglandin E2. Conclusion Pulmonary adaptation during oxidative stress–induced lung injury is mediated by a novel subset of forkhead box P3–positive AMs that limits inflammation, favoring airway adaptation and host fitness through TGF-β and prostaglandin E2.

Introduction: Lung adaptation from pulmonary insults is an essential requirement for host fitness and survival. Lung epithelial injury and inflammation trigger oxidative stress which further damages the tracheobronchial tree. Failure or incomplete adaptation to oxidative injury favours the development of severe airway disease. Objectives: Establish a new model to explore airway adaptation to oxidative injury in order to elucidate key mechanisms for preserving lung function. Methods: To establish a model for the study of adaptation to oxidative stress induced-lung injury, we exposed mice to repeated chlorine gas exposures. Outcome measures were assessed 24h after the last chlorine exposure. Lung function mechanics were explored using FlexiVent. Inflammation and anti-oxidant responses were assessed in both bronchoalveolar lavage and lung tissue. Knock-out mice, depletion, adoptive transfer and gene array approaches were used to further explore the mechanistic. Results: Here we report a striking respiratory airway adaptation in mice after repeated chlorine exposures. Mice rapidly adapt by controlling the neutrophilic inflammation and airway hyperresponsiveness. After excluding antioxidant mechanisms and regulatory T cells, we found that adaptation was mediated by resident alveolar macrophages, programmed with a potent pro-phagocytic capacity. Notably, 5% of alveolar macrophages expressed Foxp3 and depletion of these cells abolished adaptation. Conclusions: This new model of adaptation to oxidative stress-induced lung injury lead us to discover a subset of alveolar macrophages Foxp3 + , which may provide new cellular mechanism in resolving inflammation leading to airway adaptation and host fitness. CIHR MOP-126131.

Background Bronchial vascular remodelling may contribute to the severity of airway narrowing through mucosal congestion. Interleukin (IL)-17A is associated with the most severe asthmatic phenotype but whether it might contribute to vascular remodelling is uncertain. Objective To assess vascular remodelling in severe asthma and whether IL-17A directly or indirectly may cause endothelial cell activation and angiogenesis. Methods Bronchial vascularization was quantified in asthmatic subjects, COPD and healthy subjects together with the number of IL-17A+ cells as well as the concentration of angiogenic factors in the sputum. The effect of IL-17A on in vitro angiogenesis, cell migration and endothelial permeability was assessed directly on primary human lung microvascular endothelial cells (HMVEC-L) or indirectly with conditioned medium derived from normal bronchial epithelial cells (NHBEC), fibroblasts (NHBF) and airway smooth muscle cells (ASMC) after IL-17A stimulation. Results Severe asthmatics have increased vascularity compared to the other groups, which correlates positively with the concentrations of angiogenic factors in sputum. Interestingly, we demonstrated that increased bronchial vascularity correlates positively with the number of subepithelial IL-17A+ cells. However IL-17A had no direct effect on HMVEC-L function but it enhanced endothelial tube formation and cell migration through the production of angiogenic factors by NHBE and ASMC. Conclusions & clinical relevance Our results shed light on the role of IL-17A in vascular remodelling, most likely through stimulating the synthesis of other angiogenic factors. Knowledge of these pathways may aid in the identification of new therapeutic targets.

Rationale Chlorine gas (Cl2) is a potent oxidant and trigger of irritant induced asthma. We explored NF-E2–related factor 2 (Nrf2)-dependent mechanisms in the asthmatic response to Cl2, using Nrf2-deficient mice, buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and sulforaphane (SFN), a phytochemical regulator of Nrf2. Methods Airway inflammation and airway hyperresponsiveness (AHR) were assessed 24 and 48 h after a 5-min nose-only exposure to 100 ppm Cl2 of Nrf2-deficient and wild type Balb/C mice treated with BSO or SFN. Animals were anesthetized, paralyzed and mechanically ventilated (FlexiVent™) and challenged with aerosolized methacholine. Bronchoalveolar lavage (BAL) was performed and lung tissues were harvested for assessment of gene expression. Results Cl2 exposure induced a robust AHR and an intense neutrophilic inflammation that, although similar in Nrf2-deficient mice and wild-type mice at 24 h after Cl2 exposure, were significantly greater at 48 h post exposure in Nrf2-deficient mice. Lung GSH and mRNA for Nrf2-dependent phase II enzymes (NQO-1 and GPX2) were significantly lower in Nrf2-deficient than wild-type mice after Cl2 exposure. BSO reduced GSH levels and promoted Cl2-induced airway inflammation in wild-type mice, but not in Nrf2-deficient mice, whereas SFN suppressed Cl2-induced airway inflammation in wild-type but not in Nrf2-deficient mice. AHR was not affected by either BSO or SFN at 48 h post Cl2 exposure. Conclusions Nrf2-dependent phase II enzymes play a role in the resolution of airway inflammation and AHR after Cl2 exposure. Moderate deficiency of GSH affects the magnitude of acute inflammation but not AHR.

Anna Binda,1 Alice Panariti,1 Andrea Barbuti,2 Carmen Murano,1 Roberta Dal Magro,1 Massimo Masserini,1,3,4 Francesca Re,1,3,4 Ilaria Rivolta,1,3,4 1School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy; 2Department of Biosciences, ThePaceLab and Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA), University of Milan, Milano, Italy; 3Milan Center for Neuroscience (NeuroMI), University of Milano-Bicocca, Monza, Italy; 4Nanomedicine Center NANOMIB, University of Milano-Bicocca, Milano, Italy Purpose: Nanotechnologies turned out to be promising in the development of diagnostic and therapeutic approaches toward neurodegenerative disorders. However, only a very scant number of nanodevices until now proved to be effective on preclinical animal models. Although specific tests in vivo are available to assess the potential toxicity of these nanodevices on cognitive functions, those to evaluate their biosafety in vitro on neurons are still to be improved. Materials and methods: We utilized the patch-clamp technique on primary cultures of cortical neural cells isolated from neonatal rats, aiming to evaluate their electrical properties after the incubation with liposomes (mApoE-PA-LIPs), previously proved able to cross the blood–brain barrier and to be effective on mouse models of Alzheimer’s disease (AD), both in the absence and in the presence of β-amyloid peptide oligomers. Results: Data show a high degree of biocompatibility, evaluated by lactate dehydrogenase (LDH) release and MTT assay, and the lack of cellular internalization. After the incubation with mApoE-PA-LIPs, neuronal membranes show an increase in the input resistance (from 724.14±76 MΩ in untreated population to 886.06±86MΩ in the treated one), a reduction in the rheobase current (from 29.6±3 to 24.2±3pA in untreated and treated, respectively), and an increase of the firing frequency, consistent with an ultimate increase in intrinsic excitability. Data obtained after co-incubation of mApoE-PA-LIPs with β-amyloid peptide oligomers suggest a retention of liposome efficacy. Conclusion: These data suggest the ability of liposomes to modulate neuronal electrical properties and are compatible with the previously demonstrated amelioration of cognitive functions induced by treatment of AD mice with liposomes. We conclude that this electrophysiological approach could represent a useful tool for nanomedicine to evaluate the effect of nanoparticles on intrinsic neuronal excitability. Keywords: neurodegenerative disorders, nanomedicine, action potential, electrophysiology, patch clamp, β-amyloid peptide