Pulmonary, gastrointestinal and urogenital pharmacologyThe phosphodiesterase 4 inhibitor rolipram protects against cigarette smoke extract-induced apoptosis in human lung fibroblasts
Introduction
The pathogenesis of COPD remains unclear, but involves aberrant inflammatory responses of the lung caused by chronic cigarette smoke or particle exposure, which can be aggravated by infections (Shapiro, 1995). The complex pathology of this disease includes bronchitis associated with airway inflammation and mucous obstruction, as well as emphysema resulting from the loss of alveolar surface area for gas exchange. The mechanisms underlying alveolar destruction remain unclear, but include protease/anti-protease imbalance, oxidative stress, and apoptosis (Barnes et al., 2003, Saetta et al., 2001, Sakao et al., 2003, Shapiro, 1995).
The clinical management of COPD involves both pharmacological and non-pharmacological treatments. Long-acting bronchodilators such as long-acting β2 agonists and long-acting anti-muscarinic drugs are employed in the symptomatic management of COPD, yet have limited clinical efficacy (Penning-van Beest et al., 2011). Inhaled corticosteroids represent the main anti-inflammatory treatment used in COPD therapy despite a relatively modest clinical efficacy in this disease relative to asthma (Burge et al., 2000, Calverley et al., 2003).
Recently, phosphodiesterase-4 (PDE4) inhibitors have been shown to provide therapeutic benefit, particularly in patients with late stage COPD. Phosphodiesterases (PDEs) are cAMP- or cGMP-specific enzymes which have a ubiquitous distribution in most human cells. Eleven PDE isozymes have been identified to date (Boswell-Smith and Spina, 2007). Of these, PDE4, which hydrolyzes cAMP, is expressed in lung structural cells such as smooth muscle cells, airway epithelium, and inflammatory cells (i.e., neutrophils, lymphocytes, and macrophages) (Torphy, 1998). Phase III clinical studies have shown that the PDE4 inhibitor roflumilast significantly improved post-bronchodilator forced expiratory volume in 1 s (FEV1) and reduced exacerbation rate, and dyspnea severity in the clinical phenotype of chronic bronchitis (Calverley et al., 2009, M2-127 and M2-128 study groups, 2009). PDE4 inhibitors are known to mitigate mucociliary malfunction, lung fibrotic and emphysematous remodeling, and oxidative stress (Cortijo et al., 2009). In vivo studies have confirmed that the PDE4 inhibitor roflumilast can alleviate emphysema in mice exposed to chronic cigarette smoke (Cortijo et al., 2009).
Apoptosis may represent an important factor in the pathogenesis of COPD, which contributes to emphysematous lung destruction in response to cigarette smoke (Sakao et al., 2003). Lung tissues from patients with COPD displayed greater numbers of apoptotic cells than control lungs or lungs from smokers without COPD (Henson et al., 2006, Sakao et al., 2003). These findings suggest that inhibition of cigarette smoke-induced apoptosis may have a beneficial effect in COPD progression. However, the effects of PDE4 inhibitors on apoptosis of lung structural cells remain poorly understood in the context of COPD therapy. In the cigarette smoke-induced emphysema model, the PDE4 inhibitor GPD-1116 attenuated emphysema by inhibiting cigarette smoke-induced matrix metalloproteinase (MMP)-12 activity and protecting lung cells from apoptosis (Mori et al., 2008). We have previously shown that the xanthine derivatives (i.e., aminophylline or theophylline) protected against apoptosis of MRC-5 cells through the inactivation of caspases-3 and -8 (Kim et al., 2011). Therefore, the current study was designed to determine whether rolipram, a PDE4 inhibitor, could protect against apoptosis of lung fibroblasts in response to exposure to aqueous cigarette smoke extract (CSE), by directly inhibiting the extrinsic apoptotic pathway.
Section snippets
Preparation of CSE
Kentucky 1R3F research-reference filtered cigarettes (The Tobacco Research Institute, University of Kentucky, Lexington, KY) were smoked using a peristaltic pump (VWR, Radnor, PA). The filters were cut from the cigarettes prior to the experiments. Each cigarette was smoked in 6 min with a 17 mm butt remaining. Four cigarettes were smoked through 40 ml of cell growth medium, and this solution was referred to as 100% strength CSE. The solution was adjusted to a pH of 7.45 and used within 15 min of
Rolipram pre-treatment protects against CSE-induced MRC-5 cell death
We first investigated the effect of rolipram on CSE-induced MRC-5 cell viability. MRC-5 cells were exposed to 20% CSE for 12, 24 and 48 h, and cell viability was determined by the MTT assay. As shown in Fig. 1A, differences in cell viability were not evident at 12 h, whereas cell viability was decreased in a time-dependent manner from 24–48 h exposure. Next, MRC-5 cells were pre-treated for 1 h with rolipram at various concentrations (10–30 μM), and then exposed to 20% CSE for 24 h (a time point
Discussion
Among the pharmacotherapeutic treatments currently available for COPD, none can reverse or prevent disease progression. Inhaled bronchodilators (i.e., β2-adrenoreceptor agonists and muscarinic receptor antagonists) represent the current mainstays of COPD therapy, but primarily serve to relieve symptoms. The rationale for developing PDE4 inhibitors to treat COPD is based on the ability of these compounds to increase intracellular cAMP levels in immune, pro-inflammatory and structural cells, in
Conclusion
In summary, we report here for the first time that the PDE4 inhibitor rolipram protects lung fibroblasts from CSE induced-apoptosis via inhibiting caspase-3 and caspase-8 activation. The activation of Akt may play a contributory role in the anti-apoptotic role of rolipram. The limitations of this in vitro study include the use of CSE, which provides an approximation of cigarette smoke exposure, but may not recapitulate all the effects of mainstream cigarette smoke. These findings may provide
Acknowledgments
This work was supported by Research Grants funded by the Korea Association of Internal Medicine (KAIM) in 2011 and Gachon University Gilhospital in 2011 (to Jeong-Woong Park).
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2016, Pesticide Biochemistry and PhysiologyCitation Excerpt :Two mechanisms for increasing live cells and decreasing necrotic and apoptotic cells by PDEIs can be addressed. One is the inhibition of caspase-3 and caspase-8 which has been already confirmed [37] and the other one is the increase in cytosolic cAMP which activates the Akt-dependent antiapoptotic pathway through stimulation of protein kinase A [17,38]. One of the significant inflammatory factors is TNF-α which its antagonists have found a place in the treatment of diseases associated with inflammation [39].
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