DEC1 is involved in circadian rhythm disruption-exacerbated pulmonary fibrosis.

BACKGROUND: The alveolar epithelial type II cell (AT2) and its senescence play a pivotal role in alveolar damage and pulmonary fibrosis. Cell circadian rhythm is strongly associated with cell senescence. Differentiated embryonic chondrocyte expressed gene 1 (DEC1) is a very important circadian clock gene. However, the role of DEC1 in AT2 senescence and pulmonary fibrosis was still unclear. RESULTS: In this study, a circadian disruption model of light intervention was used. It was found that circadian disruption exacerbated pulmonary fibrosis in mice. To understand the underlying mechanism, DEC1 levels were investigated. Results showed that DEC1 levels increased in lung tissues of IPF patients and in bleomycin-induced mouse fibrotic lungs. In vitro study revealed that bleomycin and TGF-ß1 increased the expressions of DEC1, collagen-I, and fibronectin in AT2 cells. Inhibition of DEC1 mitigated bleomycin-induced fibrotic changes in vitro and in vivo. After that, cell senescence was observed in bleomycin-treated AT2 cells and mouse models, but these were prevented by DEC1 inhibition. At last, p21 was confirmed having circadian rhythm followed DEC1 in normal conditions. But bleomycin disrupted the circadian rhythm and increased DEC1 which promoted p21 expression, increased p21 mediated AT2 senescence and pulmonary fibrosis. CONCLUSIONS: Taken together, circadian clock protein DEC1 mediated pulmonary fibrosis via p21 and cell senescence in alveolar epithelial type II cells.

PM2.5 exposure-induced senescence-associated secretory phenotype in airway smooth muscle cells contributes to airway remodeling.

Fine particulate matter (PM2.5) has been linked to increased severity and incidence of airway diseases, especially chronic obstructive pulmonary disease (COPD) and asthma. Airway remodeling is an important event in both COPD and asthma, and airway smooth muscle cells (ASMCs) are key cells which directly involved in airway remodeling. However, it was unclear how PM2.5 affected ASMCs. This study investigates the effects of PM2.5 on airway smooth muscle and its mechanism. We first showed that inhaled particulate matter was distributed in the airway smooth muscle bundle, combined with increased airway smooth muscle bundle and collagen deposition in vivo. Then, we demonstrated that PM2.5 induced up-regulation of collagen-I and alpha-smooth muscle actin (α-SMA) expression in rat and human ASMCs in vitro. Next, we found PM2.5 led to rat and human ASMCs senescence and exhibited senescence-associated secretory phenotype (SASP) by autophagy-induced GATA4/TRAF6/NF-κB signaling, which contributed to collagen-I and α-SMA synthesis as well as airway smooth muscle remodeling. Together, our results provided evidence that SASP induced by PM2.5 in airway smooth muscle cells prompted airway remodeling.

BMAL1/p53 mediating bronchial epithelial cell autophagy contributes to PM2.5-aggravated asthma.

BACKGROUND: Fine particulate matter (PM2.5) is associated with increased incidence and severity of asthma. PM2.5 exposure disrupts airway epithelial cells, which elicits and sustains PM2.5-induced airway inflammation and remodeling. However, the mechanisms underlying development and exacerbation of PM2.5-induced asthma were still poorly understood. The aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a major circadian clock transcriptional activator that is also extensively expressed in peripheral tissues and plays a crucial role in organ and tissue metabolism. RESULTS: In this study, we found PM2.5 aggravated airway remodeling in mouse chronic asthma, and exacerbated asthma manifestation in mouse acute asthma. Next, low BMAL1 expression was found to be crucial for airway remodeling in PM2.5-challenged asthmatic mice. Subsequently, we confirmed that BMAL1 could bind and promote ubiquitination of p53, which can regulate p53 degradation and block its increase under normal conditions. However, PM2.5-induced BMAL1 inhibition resulted in up-regulation of p53 protein in bronchial epithelial cells, then increased-p53 promoted autophagy. Autophagy in bronchial epithelial cells mediated collagen-I synthesis as well as airway remodeling in asthma. CONCLUSIONS: Taken together, our results suggest that BMAL1/p53-mediated bronchial epithelial cell autophagy contributes to PM2.5-aggravated asthma. This study highlights the functional importance of BMAL1-dependent p53 regulation during asthma, and provides a novel mechanistic insight into the therapeutic mechanisms of BMAL1. Video Abstract.

Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic disease with high mortality. Currently, pirfenidone and nintedanib are the only approved drugs for IPF by the U.S. Food and Drug Administration (FDA), but their efficacy is limited. The activation of multiple phosphotyrosine (pY) mediated signaling pathways underlying the pathological mechanism of IPF has been explored. A Src homology-2 (SH2) superbinder, which contains mutations of three amino acids (AAs) of natural SH2 domain has been shown to be able to block phosphotyrosine (pY) pathway. Therefore, we aimed to introduce SH2 superbinder into the treatment of IPF. Methods: We analyzed the database of IPF patients and examined pY levels in lung tissues from IPF patients. In primary lung fibroblasts obtained from IPF patient as well as bleomycin (BLM) treated mice, the cell proliferation, migration and differentiation associated with pY were investigated and the anti-fibrotic effect of SH2 superbinder was also tested. In vivo, we further verified the safety and effectiveness of SH2 superbinder in multiple BLM mice models. We also compared the anti-fibrotic effect and side-effect of SH2 superbinder and nintedanib in vivo. Results: The data showed that the cytokines and growth factors pathways which directly correlated to pY levels were significantly enriched in IPF. High pY levels were found to induce abnormal proliferation, migration and differentiation of lung fibroblasts. SH2 superbinder blocked pY-mediated signaling pathways and suppress pulmonary fibrosis by targeting high pY levels in fibroblasts. SH2 superbinder had better therapeutic effect and less side-effect compare to nintedanib in vivo. Conclusions: SH2 superbinder had significant anti-fibrotic effects both in vitro and in vivo, which could be used as a promising therapy for IPF.

Pleural mesothelial cell migration into lung parenchyma by calpain contributes to idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia. It is unknown why fibrosis in IPF distributes in the peripheral or named sub-pleural area. Migration of pleural mesothelial cells (PMC) should contribute to sub-pleural fibrosis. Calpain is known to be involved in cell migration, but the role of calpain in PMC migration has not been investigated. In this study, we found that PMCs migrated into lung parenchyma in patients with IPF. Then using Wt1tm1(EGFP/Cre)Wtp /J knock-in mice, we observed PMC migration into lung parenchyma in bleomycin-induced pleural fibrosis models, and calpain inhibitor attenuated pulmonary fibrosis with prevention of PMC migration. In vitro studies revealed that bleomycin and transforming growth factor-ß1 increased calpain activity in PMCs, and activated calpain-mediated focal adhesion (FA) turnover as well as cell migration, cell proliferation, and collagen-I synthesis. Furthermore, we determined that calpain cleaved FA kinase in both C-terminal and N-terminal regions, which mediated FA turnover. Lastly, the data revealed that activated calpain was also involved in phosphorylation of cofilin-1, and p-cofilin-1 induced PMC migration. Taken together, this study provides evidence that calpain mediates PMC migration into lung parenchyma to promote sub-pleural fibrosis in IPF.

Splicing factor SRSF6 mediates pleural fibrosis.

Pleural fibrosis is defined as an excessive deposition of extracellular matrix that results in destruction of the normal pleural tissue architecture and compromised function. Tuberculous pleurisy, asbestos injury, and rheumatoid pleurisy are main causes of pleural fibrosis. Pleural mesothelial cells (PMCs) play a key role in pleural fibrosis. However, detailed mechanisms are poorly understood. Serine/arginine-rich protein SRSF6 belongs to a family of highly conserved RNA-binding splicing-factor proteins. Based on its known functions, SRSF6 should be expected to play a role in fibrotic diseases. However, the role of SRSF6 in pleural fibrosis remains unknown. In this study, SRSF6 protein was found to be increased in cells of tuberculous pleural effusions (TBPE) from patients, and decellularized TBPE, bleomycin, and TGF-ß1 were confirmed to increase SRSF6 levels in PMCs. In vitro, SRSF6 mediated PMC proliferation and synthesis of the main fibrotic protein COL1A2. In vivo, SRSF6 inhibition prevented mouse experimental pleural fibrosis. Finally, activated SMAD2/3, increased SOX4, and depressed miRNA-506-3p were associated with SRSF6 upregulation in PMCs. These observations support a model in which SRSF6 induces pleural fibrosis through a cluster pathway, including SRSF6/WNT5A and SRSF6/SMAD1/5/9 signaling. In conclusion, we propose inhibition of the splicing factor SRSF6 as a strategy for treatment of pleural fibrosis.

VEGF/Src signaling mediated pleural barrier damage and increased permeability contributes to subpleural pulmonary fibrosis.

The distribution of fibrosis in idiopathic pulmonary fibrosis (IPF) is subpleural with basal predominance. Alveolar epithelial cell was considered as the key cell in the initial phase of IPF. However, the idea of activation and damage of alveolar epithelial cells is very difficult to explain why fibrosis distributes in the subpleural area. In this study, human pleural mesothelial cell (PMC) line and primary rat PMC was used as in vitro model. Intraperitoneal injection of bleomycin was used for making a pulmonary fibrosis model. The integrity of cultured monolayer PMCs was determined by transepithelial electric resistance (TEER). Pleural permeability was estimated by measuring paracellular transport of fluorescein isothiocyanate (FITC)-conjugated dextran. Changes in lung tissue of patients with IPF were analyzed by Masson's and immunofluorescence staining. We found bleomycin induced PMCs damage and increased PMCs permeability; increased PMCs permeability aggravated bleomycin-induced subpleural inflammation and pulmonary fibrosis. Moreover, bleomycin was found to activate VEGF/Src signaling which increased PMCs permeability. In vivo, inhibition of VEGF/Src signaling prevented bleomycin-induced subpleural pulmonary fibrosis. At last, activation of VEGF/Src signaling was confirmed in subpleural area in patients with IPF. Taken together, our findings indicate that VEGF/Src signaling mediated pleural barrier damage and increased permeability which contributes to subpleural pulmonary fibrosis.

Bleomycin induced apical-basal polarity loss in alveolar epithelial cell contributes to experimental pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrosing interstitial lung disease with limited therapeutic options and a median survival of 3 years after diagnosis. Dysregulated epithelial regeneration is key event involved in initiating and sustaining IPF. The type II alveolar epithelial cells (AECIIs) play a crucial role for epithelial regeneration and stabilisation of alveoli. Loss of cell apical-basal polarity contributes to fibrosis. AECII has apical-basal polarity, but it is poorly understood whether AECII apical-basal polarity loss is involved in fibrosis. Bleomycin is a traditional inducer of pulmonary fibrosis. Here firstly we observed that bleomycin induced apical-basal polarity loss in cultured AECIIs. Next, cell polarity proteins lethal (2) giant larvae 1 (Lgl1), PAR-3A, aPKC and PAR-6B were investigated. We found bleomycin induced increases of Lgl1 protein and decreases of PAR-3A protein, and bleomycin-induced PAR-3A depression was mediated by increased-Lgl1. Then Lgl1 siRNA was transfected into AECIIs. Lgl1 siRNA prevented apical-basal polarity loss in bleomycin-treated AECIIs. At last, Lgl1-conditional knockout mice were applied in making animal models. Bleomycin induced pulmonary fibrosis, but this was attenuated in Lgl1-conditional knockout mice. Together, these data indicated that bleomycin mediated AECII apical-basal polarity loss which contributed to experimental pulmonary fibrosis. Inhibition of Lgl1 should be a potential therapeutic strategy for the disease.

Crosstalk between pleural mesothelial cell and lung fibroblast contributes to pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive and fibrosing interstitial pneumonia of unknown cause. The main feature of IPF is a heterogeneous appearance with areas of sub-pleural fibrosis. However, the mechanism of sub-pleural fibrosis was poorly understood. In this study, our in vivo study revealed that pleural mesothelial cells (PMCs) migrated into lung parenchyma and localized alongside lung fibroblasts in sub-pleural area in mouse pulmonary fibrosis. Our in vitro study displayed that cultured-PMCs-medium induced lung fibroblasts transforming into myofibroblast, cultured-fibroblasts-medium promoted mesothelial-mesenchymal transition of PMCs. Furthermore, these changes in lung fibroblasts and PMCs were prevented by blocking TGF-ß1/Smad2/3 signaling with SB431542. TGF-ß1 neutralized antibody attenuated bleomycin-induced pulmonary fibrosis. Similar to TGF-ß1/Smad2/3 signaling, wnt/ß-catenin signaling was also activated in the process of PMCs crosstalk with lung fibroblasts. Moreover, inhibition of CD147 attenuated cultured-PMCs-medium induced collagen-I synthesis in lung fibroblasts. Blocking CD147 signaling also prevented bleomycin-induced pulmonary fibrosis. Our data indicated that crosstalk between PMC and lung fibroblast contributed to sub-pleural pulmonary fibrosis. TGF-ß1, Wnt/ß-catenin and CD147 signaling was involved in the underling mechanism.

miR-4739 mediates pleural fibrosis by targeting bone morphogenetic protein 7.

BACKGROUND: Pleural fibrosis is defined as excessive depositions of matrix components that result in pleural tissue architecture destruction and dysfunction. In severe cases, the progression of pleural fibrosis leads to lung entrapment, resulting in dyspnea and respiratory failure. However, the mechanism of pleural fibrosis is poorly understood. METHODS: miR-4739 levels were detected by miRNA array and real-time PCR. Real-time PCR, western blotting and immunofluorescence were used to identify the expression profile of indicators related to fibrosis. Target gene of miR-4739 and promoter activity assay was measured by using dual-luciferase reporter assay system. In vivo, pleural fibrosis was evaluated by Masson staining and miR-4739 level was detected by In situ hybridization histochemistry. FINDINGS: We found that bleomycin induced up-regulation of miR-4739 in pleural mesothelial cells (PMCs). Over-regulated miR-4739 mediated mesothelial-mesenchymal transition and increased collagen-I synthesis in PMCs. Investigation on the clinical specimens revealed that high levels of miR-4739 and low levels of bone morphogenetic protein 7 (BMP-7) associated with pleural fibrosis in patients. Then we next identified that miR-4739 targeted and down-regulated BMP-7 which further resulted in unbalance between Smad1/5/9 and Smad2/3 signaling. Lastly, in vivo studies revealed that miR-4739 over-expression induced pleural fibrosis, and exogenous BMP-7 prevented pleural fibrosis in mice. INTERPRETATION: Our data indicated that miR-4739 targets BMP-7 which mediates pleural fibrosis. The miR-4739/BMP-7 axis is a promising therapeutic target for the disease. FUND: The National Natural Science Foundation of China.

Cigarette smoking aggravates bleomycin-induced experimental pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death. The cause of IPF is poorly understood. Although several environmental and occupational factors are considered as risk factors in IPF, cigarette smoking seems to be the most strongly associated risk factor. Here firstly, we treated mice with cigarette (16 mg tar, 1.0 mg nicotine in each cigarette) smoking and tried to explore the role of cigarette smoking in pulmonary fibrosis. Mice were continuously subjected to smoke for about 1 h each day (12 cigarettes per day, 5 days per week) during 40 days. Bleomycin was administrated by intraperitoneal injection at a dose of 40 mg/kg on days 1, 5, 8, 11 and 15. We found bleomycin induced pulmonary fibrosis in mice, and cigarette smoking augmented bleomycin-induced fibrosis reflected by both in fibrotic area and percentages of collagen in the lungs. Then we prepared and employed cigarette smoke extract (CSE) in cell models and found that CSE could induce the activation of p-Smad2/3 and p-Akt, as well as collagen-I synthesis and cell proliferation in lung fibroblasts and pleural mesothelial cells (PMCs). TGF-ß1 signaling mediated CSE-induced PMCs migration. Moreover, in vitro studies revealed that CSE had superimposed effect on bleomycin-induced activation of TGF-ß-Smad2/3 and -Akt signaling. TGF-ß-Smad2/3 and -Akt signaling were further augmented by cigarette smoking in the lung of bleomycin-treated mice. Taken together, these findings represent the first evidence that cigarette smoking aggravated bleomycin-induced pulmonary fibrosis via TGF-ß1 signaling.

Lethal (2) giant larvae regulates pleural mesothelial cell polarity in pleural fibrosis.

Pleural fibrosis is barely reversible and the underlying mechanisms are poorly understood. Pleural mesothelial cells (PMCs) which have apical-basal polarity play a key role in pleural fibrosis. Loss of cell polarity is involved in the development of fibrotic diseases. Partition defective protein (PAR) complex is a key regulator of cell polarity. However, changes of PMC polarity and PAR complex in pleural fibrosis are still unknown. In this study, we observed that PMC polarity was lost in fibrotic pleura. Next we found increased Lethal (2) giant larvae (Lgl) bound with aPKC and PAR-6B competing against PAR-3A in PAR complex, which led to cell polarity loss. Then we demonstrated that Lgl1 siRNA prevented cell polarity loss in PMCs, and Lgl1 conditional knockout (ER-Cre+/-Lgl1flox/flox) attenuated pleural fibrosis in a mouse model. Our data indicated that Lgl1 regulates cell polarity of PMCs, inhibition of Lgl1 and maintenance of cell polarity in PMCs could be a potential therapeutic treatment approach for pleural fibrosis.

Inhibition of angiotensin II and calpain attenuates pleural fibrosis.

Pleural fibrosis is associated with various inflammatory processes such as tuberculous pleurisy and bacterial empyema. There is currently no ideal therapeutic to attenuate pleural fibrosis. Some pro-fibrogenic mediators induce fibrosis through inflammatory processes, suggesting that blockage of these mediators might prevent pleural fibrosis. The MeT-5A human pleural mesothelial cell line (PMC) was used in this study as an in vitro model of fibrosis; and intra-pleural injection of bleomycin with carbon particles was used as an in vivo mouse model of pleural fibrosis. Calpain knockout mice, calpain inhibitor (calpeptin), and angiotensin (Ang) II type 1 receptor (AT1R) antagonist (losartan) were evaluated in prevention of experimental pleural fibrosis. We found that bleomycin and carbon particles induced calpain activation in cultured PMCs. This in vitro response was associated with increased collagen-I synthesis, and was blocked by calpain inhibitor or AT1R antagonist. Calpain genetic or treatment with calpeptin or losartan prevented pleural fibrosis in a mouse model induced by bleomycin and carbon particles. Our findings indicate that Ang II signaling and calpain activation induce collagen-I synthesis and contribute to fibrotic alterations in pleural fibrosis. Inhibition of Ang II and calpain might therefore be a novel strategy in treatment of pleural fibrosis.

miR-18a-5p Inhibits Sub-pleural Pulmonary Fibrosis by Targeting TGF-ß Receptor II.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death within 3-5 years of diagnosis. Sub-pleural pulmonary fibrosis is a pathological hallmark of IPF. Bleomycin treatment of mice is a an established pulmonary fibrosis model. We recently showed that bleomycin-induced epithelial-mesenchymal transition (EMT) contributes to pleural mesothelial cell (PMC) migration and sub-pleural pulmonary fibrosis. MicroRNA (miRNA) expression has recently been implicated in the pathogenesis of IPF. However, changes in miRNA expression in PMCs and sub-pleural fibrosis have not been reported. Using cultured PMCs and a pulmonary fibrosis animal model, we found that miR-18a-5p was reduced in PMCs treated with bleomycin and that downregulation of miR-18a-5p contributed to EMT of PMCs. Furthermore, we determined that miR-18a-5p binds to the 3' UTR region of transforming growth factor ß receptor II (TGF-ßRII) mRNA, and this is associated with reduced TGF-ßRII expression and suppression of TGF-ß-Smad2/3 signaling. Overexpression of miR-18a-5p prevented bleomycin-induced EMT of PMC and inhibited bleomycin-induced sub-pleural fibrosis in mice. Taken together, our data indicate that downregulated miR-18a-5p mediates sub-pleural pulmonary fibrosis through upregulation of its target, TGF-ßRII, and that overexpression of miR-18a-5p might therefore provide a novel approach to the treatment of IPF.

Crosstalk between calpain activation and TGF-ß1 augments collagen-I synthesis in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown cause that typically leads to respiratory failure and death within 3-5years of diagnosis. TGF-ß1 is considered a major profibrotic factor. However, TGF-ß1 is necessary but not sufficient to the pathogenesis of fibrotic lesion of the lungs. Recent observations have revealed that calpain, a calcium dependent protease, plays a pivotal role in tissue remodeling and fibrosis. However, the mechanism of calpain mediating pulmonary fibrosis is not understood. Calpain conditional knockout (ER-Cre(+/-)capns1(flox/flox)) mice and primary human lung fibroblasts (HLFs) were used here to investigate the relationship between calpain and TGF-ß1. Calpain knockout mice were protected from fibrotic effects of bleomycin. Bleomycin induced increases in TGF-ß1 via calpain activation in HLFs. Moreover, TGF-ß1 also activated calpain. This crosstalk between calpain activation and TGF-ß1 triggered the downstream signaling pathway including TGF-ß1 Smad2/3 and non-Smad (Akt) pathways, as well as collagen-I synthesis. Taken together, our data indicate that the crosstalk between calpain activation and TGF-ß1 augments collagen-I synthesis in HLFs and in pulmonary fibrosis. Intervention in the crosstalk between calpain activation and TGF-ß1 is a novel potential strategy to prevent pulmonary fibrosis.

Bleomycin induced epithelial-mesenchymal transition (EMT) in pleural mesothelial cells.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis. Recent studies revealed that pleural mesothelial cells (PMCs) undergo epithelial-mesenchymal transition (EMT) and play a pivotal role in IPF. In animal model, bleomycin induces pulmonary fibrosis exhibiting subpleural fibrosis similar to what is seen in human IPF. It is not known yet whether bleomycin induces EMT in PMCs. In the present study, PMCs were cultured and treated with bleomycin. The protein levels of collagen-I, mesenchymal phenotypic markers (vimentin and α-smooth muscle actin), and epithelial phenotypic markers (cytokeratin-8 and E-cadherin) were measured by Western blot. PMC migration was evaluated using wound-healing assay of culture PMCs in vitro, and in vivo by monitoring the localization of PMC marker, calretinin, in the lung sections of bleomycin-induced lung fibrosis. The results showed that bleomycin induced increases in collagen-I synthesis in PMC. Bleomycin induced significant increases in mesenchymal phenotypic markers and decreases in epithelial phenotypic markers in PMC, and promoted PMC migration in vitro and in vivo. Moreover, TGF-ß1-Smad2/3 signaling pathway involved in the EMT of PMC was demonstrated. Taken together, our results indicate that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis.