Pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-ß1.

BACKGROUND: Pirfenidone, an antifibrotic agent used for the treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-ß1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-ß1-induced human fibrotic lung fibroblasts and lung fibrosis has not been elucidated. METHODS: The effects of pirfenidone were evaluated in lung fibroblasts isolated from fibrotic human lung tissues after TGF-ß1 exposure. The ability of two new pharmacological targets of pirfenidone, collagen triple helix repeat containing protein 1(CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), to mediate contraction of collagen gels and migration toward fibronectin were assessed in vitro. RESULTS: Compared to control lung fibroblasts, pirfenidone significantly restored TGF-ß1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-ß1- and CTHRC1-induced fibroblast activity, upregulation of bone morphogenic protein-4(BMP-4)/Gremlin1, and downregulation of α-smooth muscle actin, fibronectin, and FHL2, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression, but did not downregulate CTHRC1. CONCLUSIONS: Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic targets for lung fibrosis.

Sensitive detection of viable circulating tumor cells using a novel conditionally telomerase-selective replicating adenovirus in non-small cell lung cancer patients.

Circulating tumor cells (CTCs) have a crucial role in the clinical outcome of cancer patients. Detection of non-small cell lung cancer (NSCLC) using an antibody against epithelial cell adhesion molecule (EpCAM) in captured CTCs has low sensitivity; the loss of epithelial markers leads to underestimation of CTCs with mesenchymal phenotype. We propose a new approach for detection of viable CTCs, including those with epithelial-mesenchymal transition status (EMT-CTCs), using the new telomerase-specific replication-selective adenovirus (OBP-1101), TelomeScan F35. Peripheral venous blood samples and clinicopathological data were collected from 123 NSCLC patients. The sensitivity of CTC detection was 69.1%, and for patients with stage I, II, III and IV, it was 59.6%, 40.0%, 85.7%, and 75.0%, respectively. Among the EMT-CTC samples, 46% were vimentin positive and 39.0% of non-EMT-CTC samples were EpCAM positive. Patients testing positive for EMT-CTCs at baseline had poor response to chemotherapy (P = 0.025) and decreased progression-free survival (EMT-CTC positive vs. negative: 193 ± 47 days vs. 388 ± 47. days, P = 0.040) in comparison to those testing negative. TelomeScan F35 is a highly sensitive CTC detection system and will be a useful screening tool for early diagnosis of NSCLC patients. Mesenchymal-phenotype CTCs are crucial indicators of chemotherapeutic efficacy in NSCLC patients.

Combination of glycopyrronium and indacaterol inhibits carbachol-induced ERK5 signal in fibrotic processes.

BACKGROUND: Airway fibrosis is one of the pathological features of chronic obstructive pulmonary disease (COPD), and recent studies revealed that acetylcholine plays an important role in the development of airway remodeling by stimulating proliferation and collagen synthesis of lung fibroblasts. This study was designed to examine the effects of a long-acting muscarinic receptor antagonist (LAMA) glycopyrronium and a long-acting ß2 adrenergic receptor agonist (LABA) indacaterol on acetylcholine-mediated fibrotic responses in lung fibroblasts. METHODS: After carbachol (CCh) or transforming growth factor-ß1 (TGF-ß1) exposure, the response to glycopyrronium and indacaterol was determined in vitro in fibroblasts isolated from mild-to-moderate COPD lung tissue. The ability of fibroblasts to mediate the contraction of collagen gels was assessed. The expression of α-smooth muscle actin (α-SMA) and the phosphorylation of extracellular-signal-regulated kinase 5 (ERK5) were determined by immunoblot. TGF-ß1 was quantified by ELISA and acetylcholine was quantified by liquid chromatography tandem-mass spectrometry. RESULTS: CCh stimulated fibroblast-mediated collagen gel contraction and α-SMA expression and TGF-ß1 release by fibroblasts. Blockade of autocrine TGF-ß1 attenuated CCh-mediated fibrotic responses, while TGF-ß1 did not stimulate acetylcholine release. Glycopyrronium plus indacaterol significantly attenuated CCh- and TGF-ß1-mediated fibrotic responses through inhibition of ERK5 phosphorylation. Notably, the magnitudes of CCh- and TGF-ß1-stimulated gel contraction, CCh-induced TGF-ß1 release, and ERK5 phosphorylation were greater in fibroblasts isolated from COPD subjects than in those from non-smokers. CONCLUSIONS: CCh induced TGF-ß1 self-sustaining signaling loops by potentiating ERK5 signaling and promoted myofibroblast activity. This autocrine signaling mechanism may be an attractive therapeutic target to block the fibrotic response, which was modulated by the combination of glycopyrronium and indacaterol.

Oncostatin M modulates fibroblast function via signal transducers and activators of transcription proteins-3.

Oncostatin M (OSM), an inflammatory cytokine of the interleukin-6 (IL-6) superfamily, plays a key role in various biological processes such as modulation of extracellular matrix (ECM), cell proliferation, cell survival, and induction of inflammation. It has been reported that OSM was increased in asthma and pulmonary fibrosis, and thus OSM may play a role in airway remodeling and the development of lung parenchymal fibrosis. Recruitment of lung fibroblasts to the sites of airway injury and subsequent differentiation into myofibroblasts is believed to contribute to excess ECM deposition. In the current study, we assessed the ability of OSM to modulate fibroblast collagen gel contraction, migration toward fibronectin, and expression of α-smooth muscle actin (α-SMA). We demonstrated that OSM augments gel contraction, chemotaxis, and α-SMA expression. OSM-augmented fibroblast chemotaxis was mediated by the signal transducer and activator of transcription (STAT3) and p38 mitogen-activated protein kinase, while augmentation on gel contraction and α-SMA expression was mediated by STAT3. Neither transforming growth factor-ß1 nor PGE2 was involved in mediating OSM effect on the cells. The Th2 cytokines IL-4 and IL-13, which also are believed to play an important role in promoting lung fibrosis and airway remodeling, act through STAT3, and we demonstrated the potential for additive effects of OSM with IL-4 and IL-13. The present study supports the concept that OSM may contribute to tissue remodeling, which may be additive with IL-4 or IL-13. Blockade of OSM or OSM-mediated STAT3 signaling could be a therapeutic target to regulate lung fibrotic mechanisms.