Respiratory Research, Gilead Sciences, 199 East Blaine St, Seattle, WA 98102, USA.
Respir Res. 2012 Mar 27;13(1):28. doi: 10.1186/1465-9921-13-28.
Small airway narrowing is an important pathology which impacts lung function in chronic obstructive pulmonary disease (COPD). The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins. This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting β2 adrenergic agonist indacaterol, both approved therapeutics for COPD, on fibroblast functions that contribute to inflammation and airway fibrosis.
The effects of roflumilast and indacaterol treatment were characterized on transforming growth factor β1 (TGFβ1)-treated normal human lung fibroblasts (NHLF). NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion. Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed.
Evaluation of roflumilast (1-10 μM) showed no significant inhibition alone on TGFβ1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF. A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies. When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone. Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response element-binding protein (CREB), an effect that was protein kinase A-dependent. Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF.
These results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD.
小气道狭窄是一种重要的病理学改变，它会影响慢性阻塞性肺疾病（COPD）患者的肺功能。成纤维细胞和肌成纤维细胞的积累通过产生和释放细胞因子、促纤维化因子和细胞外基质蛋白等介质，导致炎症、重塑和纤维化。本研究探讨了磷酸二酯酶 4 抑制剂罗氟司特与长效β2 肾上腺素能激动剂茚达特罗联合应用（均为 COPD 的批准治疗药物）对促进炎症和气道纤维化的成纤维细胞功能的影响。
研究了罗氟司特和茚达特罗治疗对转化生长因子β1（TGFβ1）处理的正常人肺成纤维细胞（NHLF）的影响。评估了 NHLF 中成纤维细胞增殖介质内皮素-1（ET-1）和结缔组织生长因子（CTGF）的表达、肌成纤维细胞标志物α平滑肌肌动蛋白的表达以及纤维连接蛋白（FN）的分泌。使用肿瘤坏死因子-α（TNF-α）诱导 NHLF 分泌趋化因子 C-X-C 基元配体 10（CXCL10）、趋化因子 C-C 基元配体 5（CCL5）和粒细胞-巨噬细胞集落刺激因子（GM-CSF），并评估了药物抑制作用。
研究发现，1-10 μM 罗氟司特单独应用时，对 TGFβ1 诱导的 ET-1 和 CTGF mRNA 转录物、ET-1 和 FN 蛋白产生、α平滑肌表达或 TNF-α诱导的 CXCL10、CCL5 和 GM-CSF 分泌无明显抑制作用。当用罗氟司特治疗时，观察到 ET-1 和 CTGF 的浓度依赖性抑制作用，并选择亚最大浓度进行联合研究。当在罗氟司特中加入低浓度的茚达特罗（0.1 nM）时，观察到所有评估的炎症和纤维化介质均有显著抑制作用，这优于单独使用任何一种药物的抑制作用。罗氟司特加茚达特罗联合治疗可显著增加环磷酸腺苷反应元件结合蛋白（CREB）的磷酸化，这是一种蛋白激酶 A 依赖性的作用。还发现蛋白激酶 A 的抑制作用可逆转茚达特罗和罗氟司特对 CTGF 的抑制作用。
这些结果表明，在 LABA 中加入罗氟司特可抑制原代成纤维细胞/肌成纤维细胞功能，从治疗角度来看，这可能会影响 COPD 中小气道重塑和气道阻塞相关的肺成纤维细胞前炎症和促纤维化介质的释放。
