Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma

BACKGROUND: Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation. METHODS: A total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils. RESULTS: Eosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p < 0.05) in vitro. The expression of Wnt-5a, TGF-β1, collagen, and fibronectin genes in ASMC was significantly higher after 24 h of co-culture with eosinophils from asthmatic subjects, while co-culture of ASMC with eosinophils from healthy subjects increased only TGF-β1 and fibronectin gene expression. ASMC proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p < 0.05). CONCLUSIONS: Eosinophils enhance Wnt-5a, TGF-β1, fibronectin, and collagen gene expression in ASMC and promote proliferation of these cells in asthma. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02648074

Tiotropium sustains the anti-inflammatory action of olodaterol via the cyclic AMP pathway

Mesenchymal cells (fibroblasts) of the airway wall respond to cholinergic stimulation by releasing pro-inflammatory and chemotactic cytokines and may thus contribute to chronic inflammation of the lung. Here, we studied the anti-inflammatory potential of olodaterol, a long acting beta2-adrenergic receptor agonist, and tiotropium, a long-acting muscarinic receptor antagonist, and whether they interact at the level of the cyclic AMP dependent signaling pathway. Pulmonary fibroblasts of asthmatic (n = 9) and non-asthmatic (n = 8) subjects were stimulated with the muscarinic receptor agonist carbachol and interleukin-1beta (IL-1 beta) in presence or absence of tiotropium or olodaterol alone, or their combination. We also measured cAMP levels and phosphorylation of the cAMP response element binding protein (CREB). As single components, carbachol, olodaterol and tiotropium did not affect IL-6 and IL-8 release. Carbachol concentration-dependently enhanced the production of IL-1beta-induced IL-6 and IL-8, which was blocked by the simultaneous addition of tiotropium. The combination of olodaterol plus tiotropium further reduced IL-6 and IL-8 release. Olodaterol induced cAMP and the phosphorylation of CREB, an effect counteracted by carbachol, but rescued by tiotropium. We conclude that olodaterol plus tiotropium cooperate to decrease the inflammatory response in pulmonary fibroblasts in vitro

L1 syndrome mutations impair neuronal L1 function at different levels by divergent mechanisms

Mutations in the human L1CAM gene cause neurodevelopmental disorders collectively referred to as L1 syndrome. Here, we investigated cellular pathomechanisms underlying two L1 syndrome mutations, R184Q and W1036L. We demonstrate that these mutations cause partial endoplasmic reticulum (ER) retention of L1, reduce L1 cell surface expression, but do not induce ER stress in neuronal NSC-34 cells. We provide evidence that surface trafficking of mutated L1 is affected by defective sorting to ER exit sites and attenuated ER export. However, in differentiated neuronal cultures and long-term cultured hippocampal slices, the L1-R184Q protein is restricted to cell bodies, whereas L1-W1036L also aberrantly localizes to dendrites. These trafficking defects preclude axonal targeting of L1, thereby affecting L1-mediated axon growth and arborization. Our results indicate that L1 syndrome mutations impair neuronal L1 function at different levels, firstly by attenuating ER export and secondly by interfering with polarized neuronal trafficking

The three A's in asthma - airway smooth muscle, airway remodeling & angiogenesis

Asthma affects more than 300 million people worldwide and its prevalence is still rising. Acute asthma attacks are characterized by severe symptoms such as breathlessness, wheezing, tightness of the chest, and coughing, which may lead to hospitalization or death. Besides the acute symptoms, asthma is characterized by persistent airway inflammation and airway wall remodeling. The term airway wall remodeling summarizes the structural changes in the airway wall: epithelial cell shedding, goblet cell hyperplasia, hyperplasia and hypertrophy of the airway smooth muscle (ASM) bundles, basement membrane thickening and increased vascular density. Airway wall remodeling starts early in the pathogenesis of asthma and today it is suggested that remodeling is a prerequisite for other asthma pathologies. The beneficial effect of bronchial thermoplasty in reducing asthma symptoms, together with the increased potential of ASM cells of asthmatics to produce inflammatory and angiogenic factors, indicate that the ASM cell is a major effector cell in the pathology of asthma. In the present review we discuss the ASM cell and its role in airway wall remodeling and angiogenesis

Bronchial Smooth Muscle Cells of Asthmatics Promote Angiogenesis through Elevated Secretion of CXC-Chemokines (ENA-78, GRO-alpha, and IL-8)

<p>Background: Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors.</p><p>Objective: To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors.</p><p>Methods: Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay.</p><p>Results: Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p <0.001). Levels of ENA-78, GRO-alpha and IL-8 were significantly elevated in CM of BSMC from asthma patients (p <0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC.</p><p>Conclusions: BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-alpha and IL-8) produced by BSMC. Implications: CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.</p>