Effects of aclidinium on human lung fibroblast activation in vitro

Introduction: Muscarinic activation of human lung fibroblasts is associated with pathological remodelling in the airways of patients with asthma and chronic obstructive pulmonary disease (COPD). Aims: To investigate the in vitro effects of aclidinium bromide, a novel, long-acting muscarinic antagonist, on human lung fibroblast activation. Methods: Lung fibroblasts, isolated from human bronchus, were pre-incubated with aclidinium (10 -9 M-10 -7 M), the ERK 1/2 inhibitor PD98059 (10μM) or the cAMP analogue dbcAMP (1mM) for 30 min and then exposed to carbachol (10μM) for 48 h. Collagen type I and α-smooth muscle actin (αSMA) expression were measured by RT-PCR, Western blot (WB) and immunofluorescence. ERK 1/2 phosphorylation was measured by WB and intracellular cAMP levels by cAMP Biotrak enzyme immunoassay. Fibroblast proliferation was assessed using a BrdU kit, and fibroblast migration by wound closure assay. Results: Aclidinium, PD98059 and dbcAMP attenuated carbachol-induced increases in αSMA and collagen type I mRNA and protein levels. Aclidinium and dbcAMP prevented carbachol-induced increases in phospho-ERK 1/2. Carbachol (10μM) prevented isoprenaline (1μM)-induced cAMP upregulation, which was completely reversed by aclidinium 10 -7 M. Carbachol-dependent increases in lung fibroblast proliferation (2-fold) were reduced by aclidinium 10 -7 M (1.1-fold), PD98059 (1.3-fold) and dbcAMP (1.2-fold). Aclidinium 10 -7 M, PD98059 and dbcAMP reduced fibroblast wound closure by 30%, 28% and 40%, respectively. Conclusions: Aclidinium blocks carbachol-induced lung fibroblast proliferation probably by a direct effect at muscarinic receptors. Aclidinium may alleviate lung fibroblast activation in patients with asthma and COPD.