• Javier Milara, Teresa Peiró, Adela Serrano, Ricardo Guijarro, Cristóbal Zaragozá, Herman Tenor, and Julio Cortijo.
• Clinical Research Unit (UIC), University General Hospital Consortium, Valencia, Spain; Department of Biotechnology, Universidad Politécnica de Valencia, Spain; Research Foundation of General Hospital of Valencia, Spain. Electronic address: xmilara@hotmail.com.
• Pulm Pharmacol Ther. 2014 Aug 1; 28 (2): 138-48.

BackgroundEpithelial to mesenchymal transition (EMT) is under discussion as a potential mechanism of small airway remodelling in COPD. In bronchial epithelium of COPD and smokers markers of EMT were described. In vitro, EMT may be reproduced by exposing well-differentiated human bronchial epithelial cells (WD-HBEC) to cigarette smoke extract (CSE). EMT may be mitigated by an increase in cellular cAMP.ObjectiveThis study explored the effects of roflumilast N-oxide, a PDE4 inhibitor on CSE-induced EMT in WD-HBEC and in primary bronchial epithelial cells from smokers and COPD in vitro.MethodsWD-HBEC from normal donors were stimulated with CSE (2.5%) for 72 h in presence of roflumilast N-oxide (2 nM or 1 μM) or vehicle. mRNA and protein of EMT markers αSMA, vimentin, collagen-1, E-cadherin, ZO-1, KRT5 as well as NOX4 were quantified by real-time quantitative PCR or protein array, respectively. Phosphorylated and total ERK1/2 and Smad3 were assessed by protein array. cAMP and TGFβ1 were measured by ELISA. Reactive oxygen species (ROS) were determined by DCF fluorescence, after 30 min CSE (2.5%). Apoptosis was measured with Annexin V/PI labelling. In some experiments, EMT markers were determined in monolayers of bronchial epithelial cells from smokers, COPD versus controls.ResultsRoflumilast N-oxide protected from CSE-induced EMT in WD-HBEC. The PDE4 inhibitor reversed both the increase in mesenchymal and the loss in epithelial EMT markers. Roflumilast N-oxide restored the loss in cellular cAMP following CSE, reduced ROS, NOX4 expression, the increase in TGFβ1 release, phospho ERK1/2 and Smad3. The PDE4 inhibitor partly protected from the increment in apoptosis with CSE. Finally the PDE4 inhibitor decreased mesenchymal yet increased epithelial phenotype markers in HBEC of COPD and smokers.ConclusionsRoflumilast N-oxide may mitigate epithelial-mesenchymal transition in bronchial epithelial cells in vitro.Copyright © 2014 Elsevier Ltd. All rights reserved.

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