Disease-specific expression and regulation of CCAAT/enhancer-binding proteins in asthma and chronic obstructive pulmonary disease
ABSTRACT CCAAT/enhancer-binding proteins (C/EBPs) control cell proliferation; lack of C/EBPalpha correlates with increased proliferation of bronchial smooth muscle cells (BSMCs) of asthmatic patients.
We sought to assess disease-specific expression of C/EBPalpha, beta, delta, and epsilon and the effects of budesonide (10(-8) mol/L) and formoterol (10(-8) mol/L).
Expression and function of C/EBPalpha, beta, delta, and epsilon BSMCs of control subjects (n = 9), asthmatic patients (n = 12), and patients with chronic obstructive pulmonary disease (COPD; n = 10) were determined.
The control group expressed C/EBPalpha, beta, delta, and epsilon, which were upregulated by serum (5%). Budesonide completely inhibited C/EBPalpha and beta expression; formoterol increased C/EBPalpha expression (2-fold). C/EBPdelta and epsilon expression were not affected by the drugs. The asthmatic group did not appropriately express C/EBPalpha. Basal levels of C/EBPbeta, delta, and epsilon were upregulated by serum (5%). Budesonide and formoterol increased C/EBPbeta levels (3.4-fold and 2.5-fold, respectively), leaving C/EBPalpha, delta, and epsilon levels unaffected. The COPD group normally expressed C/EBPalpha, beta, and epsilon, which were upregulated by serum treatment (5%). Basal levels of C/EBPdelta were downregulated by serum in 7 of 10 BSMC lines. Budesonide inhibited C/EBPalpha and beta expression, upregulated C/EBPdelta (3.2-fold), and had no effect on C/EBPepsilon. Formoterol upregulated C/EBPalpha expression (3-fold) but not the other C/EBPs. Protein analysis and electrophoretic mobility shift assay confirmed the disease-specific expression pattern of C/EBPalpha in asthmatic patients and C/EBPdelta in patients with COPD.
The expression and regulation of C/EBPs in BSMCs of asthmatic patients and patients with COPD seems disease specific. Budesonide and formoterol modulate C/EBP expression in a drug- and disease-specific pattern.
The data could provide a method to discriminate between asthma and COPD at an early disease stage.
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ABSTRACT: 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. To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors. 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. 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-α 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. BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC. 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.PLoS ONE 12/2013; 8(12):e81494. DOI:10.1371/journal.pone.0081494 · 3.53 Impact Factor
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ABSTRACT: For oxygen supply, airway wall cells depend on diffusion though the basement membrane, as well as on delivery by micro-vessels. In the asthmatic lung, local hypoxic conditions may occur due to increased thickness and altered composition of the basement membrane, as well as due to edema of the inflamed airway wall. In our study we investigated the effect of hypoxia on proliferation and pro-inflammatory and pro-angiogenic parameter production by human bronchial smooth muscle cells (BSMC). Furthermore, conditioned media of hypoxia-exposed BSMC was tested for its ability to induce sprout outgrowth from endothelial cells spheroids. BSMC were cultured in RPMI1640 (5% FCS) under normoxic (21% O2) and hypoxic (1% and 5% O2) conditions. Proliferation was determined by cell count and Western blot analysis for cyclin E and Proliferating Cell Nuclear Antigen (PCNA). Secretion of IL-6, IL-8, ENA-78 and VEGF-A was analyzed by ELISA. BSMC conditioned medium was tested for its angiogenic capacity by endothelial cell (EC)-spheroid in vitro angiogenesis assay. Proliferation of BSMC obtained from asthmatic and non-asthmatic patients was significantly reduced in the presence of 1% O2, whereas 5% O2 reduced proliferation of asthmatic BSMC only. Hypoxia induced HIF-1α expression in asthmatic and non-asthmatic BSMC, which coincided with significantly increased release of IL-6, IL-8 and VEGF-A, but not ENA-78. Finally, endothelial sprout outgrowth from EC spheroids was increased when exposed to hypoxia conditioned BSMC medium. Hypoxia had dualistic effects on proliferative and inflammatory responses of asthmatic and non-asthmatic BSMC. First, hypoxia reduced BSMC proliferation. Second, hypoxia induced a pro-inflammatory, pro-angiogenic response. BSMC and EC may thus be promising new targets to counteract and/or alleviate airway wall remodeling.PLoS ONE 02/2014; 9(2):e89875. DOI:10.1371/journal.pone.0089875 · 3.53 Impact Factor
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ABSTRACT: 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 β2-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-1β (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-1β-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 inflamamtory response in pulmonary fibroblasts in vitro.Pulmonary Pharmacology & Therapeutics 11/2013; 27(1). DOI:10.1016/j.pupt.2013.11.001 · 2.57 Impact Factor