Roflumilast N-oxide inhibits bronchial epithelial to mesenchymal transition induced by cigarette smoke in smokers with COPD

BACKGROUND: Epithelial 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. OBJECTIVE: This 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. METHODS: WD-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. RESULTS: Roflumilast 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. CONCLUSIONS: Roflumilast N-oxide may mitigate epithelial-mesenchymal transition in bronchial epithelial cells in vitro

Estudio farmacológico de la acción de un antimuscarínico de larga duración (LAMA) en la activación de fibroblastos pulmonares humanos

El remodelado de las vías aéreas es un proceso patológico observado en enfermedades de carácter obstructivo e inflamatorio como la Enfermedad Pulmonar Obstructiva Crónica (EPOC); En condiciones inflamatorias, los fibroblastos pulmonares residentes se activan y transforman en miofibroblastos, células con fenotipo contráctil, proliferativo y con actividad secretora de matriz extracelular, la acumulación de miofibroblastos contribuye a la progresión del remodelado pulmonar, y a la limitación del flujo aéreo propio de pacientes EPOC. Promoviendo la evolución de la enfermedad y un peor pronóstico. La presente tesis doctoral establece las posibles rutas de activación de dicha transición celular, empleando distintos mecanismos de activación como lo son: Carbacol (CCh: Activación colinérgica); factor de crecimiento transformante-β1 (TGFβ1: Activación colinérgica no neuronal) o extracto de humo de tabaco (EHT: Activación oxidativa), y estudia el efecto farmacológico de un antimuscarínico de larga duración (LAMA). Se concluye que el fármaco empleado es capaz de paliar la transición celular en cuestión y en consecuencia, su uso podría enlentecer los procesos fibróticos característicos del remodelado pulmonar observado en la EPOC