Inhibition of TGF-β induced lung fibroblast to myofibroblast conversion by phosphodiesterase inhibiting drugs and activators of soluble guanylyl cyclase

Abstract

Pulmonary fibroblast to myofibroblast conversion is a pathophysiological feature of idiopathic pulmonary fibrosis and COPD. This conversion is induced by transforming growth factor (TGF)-β derived from epithelial cells as well as activated macrophages that have infiltrated the lung. Preventing this conversion might be a favourable therapeutic approach. Within this study we examined the activity of different members of the phosphodiesterase (PDE) family in primary human lung fibroblasts and various lung fibroblast cell lines both before and after TGF-β induced differentiation to myofibroblasts as reflected by the expression of alpha-smooth muscle actin. We showed that the predominant PDE activities in lung fibroblasts are attributed to PDE5, PDE1 and to a smaller extent to PDE4. cyclic GMP (cGMP)-hydrolyzing activity declines by about half after differentiation to myofibroblasts in all pulmonary fibroblasts investigated, which is accompanied by a down-regulation of PDE5 protein. Lung fibroblast to myofibroblast differentiation is blocked by treatment with the PDE4 inhibitor piclamilast alone, depending on the TGF-β concentration applied, and in combination with prostaglandin E₂ (PGE₂) in a synergistic manner. Despite the high PDE5 activity the PDE5 inhibitor sildenafil by itself as well as in combination with brain natriuretic peptide or the nitric oxide-donor DETA-NONOate shows no inhibiting effects. However, combining sildenafil with the guanylyl cyclase (GC) activator BAY58-2667 and ODQ (which sensitizes GC for activation by BAY58-2667) suppressed TGF-β induced differentiation. In summary, our data indicate that drugs interfering with the cyclic AMP (cAMP)-as well as with the NO-cGMP-pathway offer the therapeutic opportunity to prevent the differentiation of pulmonary fibroblasts to myofibroblasts in lung fibrosis.

Introduction

Fibrosis, to a various extent, is a hallmark of different respiratory diseases like idiopathic pulmonary fibrosis, asthma and chronic obstructive pulmonary disease (COPD). In COPD there is a complex remodelling process in the peripheral lung, resulting in emphysema and fibrosis of the small airways. Patients with idiopathic pulmonary fibrosis suffer from massively interspersed interstitial fibrotic foci in the lung containing palisades of fibroblasts, myofibroblasts and huge amounts of extracellular matrix deriving from these cells (Pardo and Selman, 2002). During the different stages of fibrosis progression the phenoptype of lung fibroblasts changes from a migratory phenotype, to a proliferative and profibrotic one, which is called a myofibroblast. The myofibroblast is characterized, besides having a strong production of extracellular matrix (e.g. collagen), by the expression of alpha-smooth muscle actin, which is not present in fibroblasts and contributes to the altered mechanical characteristics of the lung (Adler et al., 1989). The myofibroblast somehow has a hybrid phenotype between a fibroblast and a smooth muscle cell. For a long time, a well-accepted theory stated that a common pathogenic mechanism underlies all fibrotic lung diseases. This was thought to include an initial inflammation accompanied by a strong accumulation of intra-alveolar macrophages providing e.g. large amounts of transforming growth factor (TGF)-β, inducing the conversion of fibroblasts to the contractile myofibroblasts. Therefore enhanced TGF-β concentrations have been detected in various fibrotic diseases including idiopathic pulmonary fibrosis (Khalil et al., 1991, Broekelmann et al., 1991), sarcoidosis (Salez et al., 1998) and cystic fibrosis (Wojnarowski et al., 1999). It has also been shown that TGF-β is expressed by macrophages of the terminal airway and alveoli (De Boer et al., 1998). However, it became evident that anti-inflammatory treatment with glucocorticoids did not improve the outcome of idiopathic pulmonary fibrosis (Mapel et al., 1996) and that fibrosis results from an epithelial injury followed by an abnormal wound healing process (for review see Selman et al., 2001). Under those conditions, especially in the late stage of the fibrotic lung disease, TGF-β was detected in bronchiolar epithelial cells, epithelial cells of the honeycomb cysts and particularly in activated hyperplastic type II pneumocytes. Thus, epithelial cells seem to be the main source of profibrotic TGF-β (Khalil et al., 1991, Khalil et al., 1996).

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of related proteins which can be subdivided into 11 isoenzymes based on their amino acid sequences, sensitivity to different activators and inhibitors as well as their ability to hydrolyze either preferentially cyclic AMP (cAMP), cyclic GMP (cGMP) or both (Francis et al., 2001). Inhibitors of PDE4, which specifically modulate intracellular cAMP concentrations, and inhibitors of cGMP-specific PDE5 are in clinical development for the treatment of respiratory diseases (Lee et al., 2005, Lipworth, 2005). Thus, the PDE4-selective inhibitor roflumilast is currently in phase III clinical trials for asthma and COPD, whereas the PDE5-selective sildenafil is approved for the treatment of pulmonary arterial hypertension and is in phase II trials for COPD. Up to now the therapeutic value of PDE family-specific inhibitors in the treatment of lung fibrosis by prevention of the differentiation of fibroblasts to myofibroblasts has not been evaluated in detail.

In this study we analyzed the activity of phosphodiesterases 1, 2, 3, 4 and 5 in three human lung fibroblast cell lines (HFL-1, GM06114 and IMR-90) as well as primary human lung fibroblasts before and after TGF-β1-induced fibroblast to myofibroblast conversion by measuring the enhanced expression of the myofibroblast marker alpha-smooth muscle actin. We addressed the question whether inhibition of phosphodiesterase 4 and 5 alone, inhibition of PDE4 in combination with prostaglandin E₂ (PGE₂), or inhibition of PDE5 in combination with guanylyl cyclase (GC) activators might interfere with the TGF-β1-induced differentiation of fibroblasts to myofibroblasts. We provide evidence that TGF-β1-induced alpha-smooth muscle actin expression in human lung fibroblasts can be suppressed by inhibition of PDE4 alone and in a synergistic fashion in combination with PGE₂. In contrast, inhibition of PDE5 by itself did not affect alpha-smooth muscle actin expression but when combined with the soluble GC (sGC) activator BAY58-2667 plus ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one; sensitizes the sGC for activation by BAY58-2667) or BAY41-2272, alpha-smooth muscle actin expression was affected.