Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis.

Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal 3D cocultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell cocultures. Gain- and loss-of-function experiments showed that BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1 null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide tools to understand the mechanisms of respiratory diseases at the single-cell level.

Regulation of MHC Class I Expression in Lung Epithelial Cells during Inflammation.

Lung infections are a perennial leading cause of death worldwide. The lung epithelium comprises three main cell types: alveolar type I (AT1), alveolar type II (AT2), and bronchiolar cells. Constitutively, these three cell types express extremely low amounts of surface MHC class I (MHC I) molecules, that is, <1% of levels found on medullary thymic epithelial cells (ECs). We report that inhalation of the TLR4 ligand LPS upregulates cell surface MHC I by ∼25-fold on the three subtypes of mouse lung ECs. This upregulation is dependent on Nlrc5, Stat1, and Stat2 and caused by a concerted production of the three IFN families. It is nevertheless hampered, particularly in AT1 cells, by the limited expression of genes instrumental in the peptide loading of MHC I molecules. Genes involved in production and response to cytokines and chemokines were selectively induced in AT1 cells. However, discrete gene subsets were selectively downregulated in AT2 or bronchiolar cells following LPS inhalation. Genes downregulated in AT2 cells were linked to cell differentiation and cell proliferation, and those repressed in bronchiolar cells were primarily involved in cilium function. Our study shows a delicate balance between the expression of transcripts maintaining lung epithelium integrity and transcripts involved in Ag presentation in primary lung ECs.

An update in club cell biology and its potential relevance to chronic obstructive pulmonary disease.

Club cells are found in human small airways where they play an important role in immune defense, xenobiotic metabolism, and repair after injury. Over the past few years, data from single-cell RNA sequencing (scRNA-seq) studies has generated new insights into club cell heterogeneity and function. In this review, we integrate findings from scRNA-seq experiments with earlier in vitro, in vivo, and microscopy studies and highlight the many ways club cells contribute to airway homeostasis. We then discuss evidence for loss of club cells or club cell products in the airways of patients with chronic obstructive pulmonary disease (COPD) and discuss potential mechanisms through which this might occur.

ABL kinase inhibition promotes lung regeneration through expansion of an SCGB1A1+ SPC+ cell population following bacterial pneumonia.

Current therapeutic interventions for the treatment of respiratory infections are hampered by the evolution of multidrug resistance in pathogens as well as the lack of effective cellular targets. Despite the identification of multiple region-specific lung progenitor cells, the identity of molecules that might be therapeutically targeted in response to infections to promote activation of progenitor cell types remains elusive. Here, we report that loss of Abl1 specifically in SCGB1A1-expressing cells leads to a significant increase in the proliferation and differentiation of bronchiolar epithelial cells, resulting in dramatic expansion of an SCGB1A1+ airway cell population that coexpresses SPC, a marker for type II alveolar cells that promotes alveolar regeneration following bacterial pneumonia. Furthermore, treatment with an Abl-specific allosteric inhibitor enhanced regeneration of the alveolar epithelium and promoted accelerated recovery of mice following pneumonia. These data reveal a potential actionable target that may be exploited for efficient recovery after pathogen-induced infections.

Pathology of Idiopathic Pulmonary Fibrosis Assessed by a Combination of Microcomputed Tomography, Histology, and Immunohistochemistry.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease with the histology of usual interstitial pneumonia (UIP). Although the pathologist's visual inspection is central in histologic assessments, three-dimensional microcomputed tomography (microCT) assessment may complement the pathologist's scoring. We examined associations between the histopathologic features of UIP and IPF in explanted lungs and quantitative microCT measurements, including alveolar surface density, total lung volume taken up by tissue (%), and terminal bronchiolar number. Sixty frozen samples from 10 air-inflated explanted lungs with severe IPF and 36 samples from 6 donor control lungs were scanned with microCT and processed for histologic analysis. An experienced pathologist scored three major UIP criteria (patchy fibrosis, honeycomb, and fibroblastic foci), five additional pathologic changes, and immunohistochemical staining for CD68-, CD4-, CD8-, and CD79a-positive cells, graded on a 0 to 3+ scale. The alveolar surface density and terminal bronchiolar number decreased and the tissue percentage increased in lungs with IPF compared with controls. In lungs with IPF, lower alveolar surface density and higher tissue percentage were correlated with greater scores of patchy fibrosis, fibroblastic foci, honeycomb, CD79a-positive cells, and lymphoid follicles. A decreased number of terminal bronchioles was correlated with honeycomb score but not with the other scores. The three-dimensional microCT measurements reflect the pathological UIP and IPF criteria and suggest that the reduction in the terminal bronchioles may be associated with honeycomb cyst formation.

Role of Secretoglobin+ (club cell) NFκB/RelA-TGFß signaling in aero-allergen-induced epithelial plasticity and subepithelial myofibroblast transdifferentiation.

Repetitive aeroallergen exposure is linked to sensitization and airway remodeling through incompletely understood mechanisms. In this study, we examine the dynamic mucosal response to cat dander extract (CDE), a ubiquitous aero-allergen linked to remodeling, sensitization and asthma. We find that daily exposure of CDE in naïve C57BL/6 mice activates innate neutrophilic inflammation followed by transition to a lymphocytic response associated with waves of mucosal transforming growth factor (TGF) isoform expression. In parallel, enhanced bronchiolar Smad3 expression and accumulation of phospho-SMAD3 was observed, indicating paracrine activation of canonical TGFßR signaling. CDE exposure similarly triggered epithelial cell plasticity, associated with expression of mesenchymal regulatory factors (Snai1 and Zeb1), reduction of epithelial markers (Cdh1) and activation of the NFκB/RelA transcriptional activator. To determine whether NFκB functionally mediates CDE-induced growth factor response, mice were stimulated with CDE in the absence or presence of a selective IKK inhibitor. IKK inhibition substantially reduced the level of CDE-induced TGFß1 expression, pSMAD3 accumulation, Snai1 and Zeb1 expression. Activation of epithelial plasticity was demonstrated by flow cytometry in whole lung homogenates, where CDE induces accumulation of SMA+Epcam+ population. Club cells are important sources of cytokine and growth factor production. To determine whether Club cell innate signaling through NFκB/RelA mediated CDE induced TGFß signaling, we depleted RelA in Secretoglobin (Scgb1a1)-expressing bronchiolar cells. Immunofluorescence-optical clearing light sheet microscopy showed a punctate distribution of Scgb1a1 progenitors throughout the small airway. We found that RelA depletion in Secretoglobin+ cells results in inhibition of the mucosal TGFß response, blockade of EMT and reduced subepithelial myofibroblast expansion. We conclude that the Secretoglobin-derived bronchiolar cell is central to coordinating the innate response required for mucosal TGFß1 response, EMT and myofibroblast expansion. These data have important mechanistic implications for how aero-allergens trigger mucosal injury response and remodeling in the small airway.

Serine Protease Imbalance in the Small Airways and Development of Centrilobular Emphysema in Chronic Obstructive Pulmonary Disease.

Epithelial dysfunction in the small airways may cause the development of emphysema in chronic obstructive pulmonary disease. C/EBPα (CCAAT/enhancer binding protein-α), a transcription factor, is required for lung maturation during development, and is also important for lung homeostasis after birth, including the maintenance of serine protease/antiprotease balance in the bronchiolar epithelium. This study aimed to show the roles of C/EBPα in the distal airway during chronic cigarette smoke exposure in mice and in the small airways in smokers. In a model of chronic smoke exposure using epithelial cell-specific C/EBPα-knockout mice, significant pathological phenotypes, such as higher protease activity, impaired ciliated cell regeneration, epithelial cell barrier dysfunction via reduced zonula occludens-1 (Zo-1), and decreased alveolar attachments, were found in C/EBPα-knockout mice compared with control mice. We found that Spink5 (serine protease inhibitor kazal-type 5) gene (encoding lymphoepithelial Kazal-type-related inhibitor [LEKTI], an anti-serine protease) expression in the small airways is a key regulator of protease activity in this model. Finally, we showed that daily antiprotease treatment counteracted the phenotypes of C/EBPα-knockout mice. In human studies, CEBPA (CCAAT/enhancer binding protein-α) gene expression in the lung was downregulated in patients with emphysema, and six smokers with centrilobular emphysema (CLE) showed a significant reduction in LEKTI in the small airways compared with 22 smokers without CLE. LEKTI downregulation in the small airways was associated with disease development during murine small airway injury and CLE in humans, suggesting that LEKTI might be a key factor linking small airway injury to the development of emphysema.

Distal-type bronchiolar adenoma of the lung expressing p16INK4a - morphologic, immunohistochemical, ultrastructural and genomic analysis - report of a case and review of the literature.

Bronchiolar adenoma (BA) of the lung is a rare benign neoplasm. Because of a chest abnormal shadow indicated by health checkup, a 77-year-old female nonsmoker underwent computed tomography, revealing an 8 mm ground glass nodule in the peripheral field of the right lower lobe. Wedge resection of the nodule was performed, with a frozen diagnosis of primary lung adenocarcinoma. The localized, 8 × 4 × 3 mm-sized, jelly-like mass microscopically revealed a lepidic-growing lesion composed of ciliated columnar cells, mucous cells and basal cells surrounded by mucin pool. Neither nuclear atypia nor mitotic activity was noted. Immunohistochemically, the ciliated, mucous and basal cells were positive for TTF-1 and p16INK4a . Mucous cells were positive for napsin A and focally expressed MUC5AC. MUC6 was negative. Basal cells were positive for CK5/6, p40, p63 and podoplanin. Human papillomavirus genome was undetectable by in situ hybridization. Ultrastructurally, the bronchiolar epithelial tubules consisted of two layers, the inner nonciliated microvillous cells and the outer basal-like cells, and some of the inner cells were filled with mucin granules in cytoplasm. Molecular analysis of the tumor failed to show driver mutations. The final diagnosis was distal-type BA. The postoperative course was uneventful for 6 months.

TiO2 Nanomaterials Non-Controlled Contamination Could Be Hazardous for Normal Cells Located in the Field of Radiotherapy.

Among nanomaterials (NMs), titanium dioxide (TiO2) is one of the most manufactured NMs and can be found in many consumers' products such as skin care products, textiles and food (as E171 additive). Moreover, due to its most attractive property, a photoactivation upon non-ionizing UVA radiation, TiO2 NMs is widely used as a decontaminating agent. Uncontrolled contaminations by TiO2 NMs during their production (professional exposure) or by using products (consumer exposure) are rather frequent. So far, TiO2 NMs cytotoxicity is still a matter of controversy depending on biological models, types of TiO2 NMs, suspension preparation and biological endpoints. TiO2 NMs photoactivation has been widely described for UV light radiation exposure, it could lead to reactive oxygen species production, known to be both cyto- and genotoxic on human cells. After higher photon energy exposition, such as X-rays used for radiotherapy and for medical imaging, TiO2 NMs photoactivation still occurs. Importantly, the question of its hazard in the case of body contamination of persons receiving radiotherapy was never addressed, knowing that healthy tissues surrounding the tumor are indeed exposed. The present work focuses on the analysis of human normal bronchiolar cell response after co-exposition TiO2 NMs (with different coatings) and ionizing radiation. Our results show a clear synergistic effect, in terms of cell viability, cell death and oxidative stress, between TiO2 NMS and radiation.

Concurrent absorption and secretion of airway surface liquids and bicarbonate secretion in human bronchioles.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm2) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( HCO3- ), which can be enhanced pharmacologically by cAMP as well as Ca2+-mediated agonists. Concurrent secretion and absorption of surface liquid along with HCO3- secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

Autophagy proteins are required for club cell structure and function in airways.

Epithelial cells that line lung airways produce and secrete proteins with important roles in barrier function and host defense. Secretion of airway goblet cells is controlled by autophagy proteins during inflammatory conditions, resulting in accumulation of mucin proteins. We hypothesized that autophagy proteins would also be important in the function of club cells, dominant secretory airway epithelial cells that are dysregulated in chronic lung disease. We found that in the absence of an inflammatory stimulus, mice with club cells deficient for the autophagy protein Atg5 had a markedly diminished expression of secreted host defense proteins secretoglobulin family 1A, member 1 (Scgb1a1) and surfactant proteins A1 and D (Sftpa1 and Sftpd), as well as abnormal club cell morphology. Adult mice with targeted loss of Atg5 also showed diminished levels of host defense proteins in regenerating cells following ablation with naphthalene. A mouse strain with global deficiency of Atg16-like 1 (Atg16l1), an Atg5 binding partner, had a similar loss of host defense proteins and abnormal club cell morphology. Cigarette smoke exposure reduced levels of Scgb1a1 in wild-type mice as expected. Smoke exposure was not required to trigger club cell abnormalities in mice bearing the human ATG16 variant Atg16l1T300A/T300A, which had low Scgb1a1 levels independent of this environmental stress. Evaluation of lung tissues from former smokers with severe chronic obstructive pulmonary disease showed evidence of reduced autophagy and SCGB1A1 expression in club cells. Thus, autophagy proteins are required for the function of club cells, independent of the cellular stress of cigarette smoke, with roles that appear to be distinct from those of other secretory cell types.

Systematic Analysis of Cell-Type Differences in the Epithelial Secretome Reveals Insights into the Pathogenesis of Respiratory Syncytial Virus-Induced Lower Respiratory Tract Infections.

Lower respiratory tract infections from respiratory syncytial virus (RSV) are due, in part, to secreted signals from lower airway cells that modify the immune response and trigger airway remodeling. To understand this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea versus small airway bronchiolar cells. A workflow was established using telomerase-immortalized human epithelial cells that revealed highly reproducible cell type-specific differences in secreted proteins and nanoparticles (exosomes). Approximately one third of secretome proteins are exosomal; the remainder are from lysosomal and vacuolar compartments. We applied this workflow to three independently derived primary human cultures from trachea versus bronchioles. A total of 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate. Fifteen proteins unique to RSV-infected primary human cultures from trachea were regulated by epithelial-specific ets homologous factor. A total of 106 proteins unique to RSV-infected human small airway epithelial cells was regulated by the transcription factor NF-κB. In this latter group, we validated the differential expression of CCL20/macrophage-inducible protein 3α, thymic stromal lymphopoietin, and CCL3-like 1 because of their roles in Th2 polarization. CCL20/macrophage-inducible protein 3α was the most active mucin-inducing factor in the RSV-infected human small airway epithelial cell secretome and was differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insights into the complexity of innate responses and regional differences in the epithelial secretome participating in RSV lower respiratory tract infection-induced airway remodeling.

RUMI is a novel negative prognostic marker and therapeutic target in non-small-cell lung cancer.

Recent comprehensive next-generation genome and transcriptome analyses in lung cancer patients, several clinical observations, and compelling evidence from mouse models of lung cancer have uncovered a critical role for Notch signaling in the initiation and progression of non-small-cell lung cancer (NSCLC). Notably, Rumi is a "protein O-glucosyltransferase" that regulates Notch signaling through O-glucosylation of Notch receptors, and is the only enzymatic regulator whose activity is required for both ligand-dependent and ligand-independent activation of Notch. We have conducted a detailed study on RUMI's involvement in NSCLC development and progression, and have further explored the therapeutic potential of its targeting in NSCLC. We have determined that Rumi is highly expressed in the alveolar and bronchiolar epithelia, including club cells and alveolar type II cells. Remarkably, RUMI maps to the region of chromosome 3q that corresponds to the major signature of neoplastic transformation in NSCLC, and is markedly amplified and overexpressed in NSCLC tumors. Notably, RUMI expression levels are predictive of poor prognosis and survival in NSCLC patients. Our data indicates that RUMI modulates Notch activity in NSCLC cells, and that its silencing dramatically decreases cell proliferation, migration, and survival. RUMI downregulation causes severe cell cycle S-phase arrest, increases genome instability, and induces late apoptotic-nonapoptotic cell death. Our studies demonstrate that RUMI is a novel negative prognostic factor with significant therapeutic potential in NSCLC, which embodies particular relevance especially when considering that, while current Notch inhibitory strategies target only ligand-dependent Notch activation, a large number of NSCLCs are driven by ligand-independent Notch activity.

Both Ways at Once: Keeping Small Airways Clean.

The small airways of the lungs are under constant assault from the pathogens and debris in the air that they must conduct to alveoli. Although hygiene is of paramount importance for respiratory health, the underlying principles of airway clearance have not been well integrated or established. Newly emerging concepts of simultaneous absorption and secretion of airway surface liquid (ASL) and the role of [Formula: see text] in the maturation of mucins have advanced from experimental evidence as well as observations from the congenital disease cystic fibrosis (CF) to present a novel model that integrates microanatomy with organ physiology to meet the constant challenge of cleaning small airways.

Syndecan 4 Mediates Nrf2-dependent Expansion of Bronchiolar Progenitors That Protect Against Lung Inflammation.

The use of lung progenitors for regenerative medicine appears promising, but their biology is not fully understood. Here, we found anti-inflammatory attributes in bronchiolar progenitors that were sorted as a multipotent subset of mouse club cells and found to express secretory leukocyte protease inhibitor (SLPI). Notably, the impaired expression of SLPI in mice increased the number of bronchiolar progenitors and decreased the lung inflammation. We determined a transcriptional profile for the bronchiolar progenitors of Slpi-deficient mice and identified syndecan 4, whose expression was markedly elevated as compared to that of wild-type mice. Systemic administration of recombinant syndecan 4 protein caused a substantial increase in the number of bronchiolar progenitors with concomitant attenuation of both airway and alveolar inflammation. The syndecan 4 administration also resulted in activation of the Keap1-Nrf2 antioxidant pathway in lung cells, which is critically involved in the therapeutic responses to the syndecan 4 treatment. Moreover, in 3D culture, the presence of syndecan 4 induced differentiated club cells to undergo Nrf2-dependent transition into bronchiolar progenitors. Our observations reveal that differentiative switches between bronchiolar progenitors and club cells are under the Nrf2-mediated control of SLPI and syndecan 4, suggesting the possibility of new therapeutic approaches in inflammatory lung diseases.

Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma.

Ubiquitous expression of a resident K-Ras(G12V) oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-Ras(G12V) expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-Ras(G12V) expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras(G12V) oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC(+) alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras(G12V) expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10(+) Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC(+) ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-Ras(G12V)-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC(+) ATII lesions. Finally, activation of K-Ras(G12V) during embryonic development under the control of a Sca1 promoter yielded CC10(+), but not SPC(+), hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC(+) ATII cells were able to yield malignant adenocarcinomas.

Postnatal development of the bronchiolar club cells of distal airways in the mouse lung: stereological and molecular biological studies.

Club (Clara) cells are nonciliated secretory epithelial cells present in bronchioles of distal pulmonary airways. So far, no information is available on the postnatal differentiation of club cells by a combination of molecular biological, biochemical, and stereological approaches in the murine lung. Therefore, the present study was designed to investigate the changes in the club cell secretory proteins (CC10, surfactant proteins A, B and D) and club cell abundance within the epithelium of bronchioles of distal airways during the postnatal development of the mouse lung. Perfusion-fixed murine lungs of three developmental stages (newborn, 15-day-old and adult) were used. Frozen, unfixed lungs were used for cryosectioning and subsequent laser-assisted microdissection of bronchiolar epithelial cells and RT-PCR analyses. High resolution analyses of the three-dimensional structures and composition of lung airways were obtained by scanning electron microscopy. Finally, using design-based stereology, the total and average club cell volume and the volume of secretory granules were quantified by light and transmission electron microscopy. Our results reveal that murine club cells are immature at birth and differentiate postnatally. Further, increase of the club cell volume and number of intracellular granules are closely correlated to the total lung volume enlargement. However, secretory granule density was only increased within the first 15 days of postnatal development. The differentiation is accompanied by a decrease in glycogen content, and a close positive relationship between CC10 expression and secretory granule abundance. Taken together, our data are consistent with the concept that the morphological and functional differentiation of club cells is a postnatal phenomenon.

Role of endogenous hydrogen sulfide in nerve-evoked relaxation of pig terminal bronchioles.

Hydrogen sulfide (H2S) is a gasotransmitter employed for intra- and inter-cellular communication in almost all organ systems. This study investigates the role of endogenous H2S in nerve-evoked relaxation of pig terminal bronchioles with 260 µm medium internal lumen diameter. High expression of the H2S synthesis enzyme cystathionine γ-lyase (CSE) in the bronchiolar muscle layer and strong CSE-immunoreactivity within nerve fibers distributed along smooth muscle bundles were observed. Further, endogenous H2S generated in bronchiolar membranes was reduced by CSE inhibition. In contrast, cystathionine ß-synthase expression, another H2S synthesis enzyme, however was not consistently detected in the bronchiolar smooth muscle layer. Electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked smooth muscle relaxation. Inhibition of CSE, nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and of ATP-dependent K+, transient receptor potential A1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) channels reduced the EFS relaxation but failed to modify the GYY4137 response. Raising extracellular K+ concentration inhibited the GYY4137 relaxation. Large conductance Ca2+-activated K+ channel blockade reduced both EFS and GYY4137 responses. GYY4137 inhibited the contractions induced by histamine and reduced to a lesser extent the histamine-induced increases in intracellular [Ca2+]. These results suggest that relaxation induced by EFS in the pig terminal bronchioles partly involves the H2S/CSE pathway. H2S response is produced via NO/sGC-independent mechanisms involving K+ channels and intracellular Ca2+ desensitization-dependent pathways. Thus, based on our current results H2S donors might be useful as bronchodilator agents for the treatment of lung diseases with persistent airflow limitation, such as asthma and chronic obstructive lung disease.

Interaction between corticosteroids and muscarinic antagonists in human airways.

BACKGROUND: To date there is emerging clinical evidence to add long-acting anti-muscarinic agents (LAMAs) with inhaled corticosteroid (ICSs) in asthma, but the pharmacological rationale that supports the use of such a combination has not yet been explained. The aim of this study was to pharmacologically investigate the interaction between the ICS beclomethasone and the LAMA glycopyrronium on the human airway smooth muscle (ASM) tone. METHODS: We investigated the rapid non-genomic bronchorelaxant effect of beclomethasone and glycopyrronium, administered alone and in combination, in human isolated bronchi and bronchioles. Experiments were carried out also in passively sensitized airways and the pharmacological analysis of drug interaction was performed by Bliss Independence method. RESULTS: The acute administration of beclomethasone and glycopyrronium induced a significant relaxation of passively sensitized ASM pre-contracted with histamine, by causing submaximal/maximal inhibition of the contractile tone in both medium bronchi and bronchioles. Beclomethasone was characterized by a rapid non-genomic and epithelium independent bronchorelaxant effect. In passively sensitized airways, this effect seemed to be dependent by the activation of a Gsα--cyclic adenosine monophosphate (cAMP)--protein kinase A cascade. While no synergistic interaction was detected in non-sensitized bronchi, the beclomethasone/glycopyrronium combination synergistically enhanced the relaxation of passively sensitized medium and small bronchi. The synergistic interaction between beclomethasone and glycopyrronium was associated with an increase of cAMP concentrations. CONCLUSIONS: Our study provides for the first time the pharmacological rationale for combining low doses of an ICS plus a LAMA.

Sex-related differences in long-term pulmonary outcomes of neonatal hyperoxia in mice.

AIM: Premature infants are often exposed to hyperoxia to maintain adequate oxygenation, which may lead to the development of bronchopulmonary dysplasia (BPD). Sex-specific differences exist in the development and severity of BPD. Only a few studies have examined the mechanisms underlying these sex-related differences. The aim of the present study is to examine the sex-related long-term effects of neonatal hyperoxia on the lungs of adult mice. MATERIALS AND METHODS: Newborn mice were exposed to 95% oxygen (hyperoxia) for 96 hours and were allowed to recover in room air to adulthood (8 weeks of age). Lung tissues were excised at 4 days, 14 days, or 8 weeks of age. Short-term effects of neonatal hyperoxia on the mouse lung and sex-related differences in pulmonary function, airway hyper-responsiveness, and lung structure in adult mice were assessed. RESULTS: Neonatal hyperoxia was found to have no differential effect on body weight, muscarinic acetylcholine receptor gene expression, or bronchiolar epithelial thickness in adult mice. Respiratory resistance was increased and sensitivity to methacholine was decreased in male adult mice following exposure to neonatal hyperoxia, whereas delayed alveolarization was observed in female adult mice following exposure to neonatal hyperoxia. CONCLUSIONS: The findings of the present study demonstrate that neonatal hyperoxia differentially affects pulmonary outcome in female and male adult mice.