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Background Chromodomain helicase DNA-binding protein 4 (CHD4) has been shown to contribute to DNA repair and cell cycle promotion; however, its roles in cancer initiation and progression remain largely unknown. This study aimed to demonstrate the role of CHD4 in the development of non-small cell lung cancer (NSCLC) and determine the potential mechanisms of action. Methods By using immunohistochemistry, the expression levels were evaluated in both cancer and non-cancerous tissues. Subsequently, CHD4 knockdown and overexpression strategies were employed to investigate the effects of CHD4 on cell proliferation, migration, along with the growth and formation of tumors in a xenografts mouse model. The protein expression levels of CHD4, PHF5A and ROCK/RhoA markers were determined by Western blot analysis. Results Compared with non-cancerous tissues, CHD4 was overexpressed in cancer tissues and CHD4 expression levels were closely related to clinical parameters of NSCLC patients. In H292 and PC-9 cell lines, CHD4 overexpression could promote the proliferative and migratory potential of NSCLC cells. Furthermore, down-regulation of CHD4 could reduce the proliferative and migratory ability in A549 and H1299 cell lines. Meanwhile, knockdown of CHD4 could decrease the tumorigenicity in nude mice. Finally, we demonstrated that one of the mechanisms underlying the promotive effect of CHD4 on NSCLC proliferation and migration may be through its interaction with PHD finger protein 5A (PHF5A) and subsequent activation of the RhoA/ROCK signaling pathway. Conclusions CHD4, which is highly expressed in cancer tissue, could be an independent prognostic factor for NSCLC patients. CHD4 plays an important role in regulating the proliferative and migratory abilities of NSCLC via likely the RhoA/ROCK pathway by regulating PHF5A.

Background Tyrosine kinase inhibitors (TKIs), such as gefitinib, are widely used as standard treatments for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. However, the subsequent inevitable drug resistance has become a major challenge in clinical treatment. The aim of this study was to investigate the role of tissue-type plasminogen activator (PLAT) in gefitinib resistance in NSCLC. Methods The function of PLAT was determined using gefitinib-resistant cells and a nude mouse model. The gene knockdown was achieved by Lentivirus based RNA silence technique. Expression of relevant genes and proteins, cell viability, proliferation, apoptosis, cell cycle, reactive oxygen species levels, mitochondrial membrane potential and differential gene expression was detected by RT-qPCR, western blot, cell counting kit-8 assay, EdU incorporation, flow cytometry, JC-1 dye assay and complementary DNA arrays. The effects of PLAT knockdown on tumorigenesis was analyzed in vivo. Results Gefitinib-resistant cells expressed higher levels of PLAT and that knockdown of PLAT in resistant cells restored gefitinib sensitivity. Tumor proliferation was limited in vivo following PLAT knockdown. Moreover, PLAT knockdown affected mitochondrial function, caused caspase activation and cell cycle arrest, and activated TNF-α signaling, leading to apoptosis of gefitinib-resistant PC9 cells. Conclusions Our results suggest that PLAT reduces apoptosis of NSCLC cells and knockdown of PLAT enhances anticancer effect of gefitinib by upregulating TNF-α signaling.

According to multiple studies, the objective response rate of PD‐1/PD‐L1 inhibitors in the second‐line treatment of unscreened non‐small cell lung cancer (NSCLC) is only approximately 20%. Predictive biomarkers of treatment efficacies are still under investigation. In selected NSCLC patients with PD‐L1 expression ≥ 50%, the response rate of pembrolizumab in first‐line treatment can reach 44.8%. Moreover, patients with a higher tumor mutation burden (TMB) tend to achieve a better response with nivolumab. Besides PD‐L1 expression and TMB, taking all these indicators into consideration would hypothetically maximize the clinical response in a specific subgroup of patients. Our study aims to accumulate large and complete samples and clinical data to verify the biomarkers and their cutoff values related to the efficacy of PD‐1/PD‐L1 inhibitors in Chinese NSCLC patients, and to construct a comprehensive predictive model by combining multi‐omics data with contemporary machine learning techniques. NSCLC patients administered treatment of anti‐PD‐1/PD‐L1 antibodies or a combination with other drugs have been enrolled. The estimated enrollment is 250 participants. A sophisticated predictive model of immunotherapy response in the Chinese population has not yet been developed. It is clinically and practically imperative to comprehensively evaluate the possible indicators of Chinese NSCLC patients through multiple test platforms, such as next generation sequencing, PCR, or immunohistochemistry. This study is registered in the Chinese Clinical Trial Registry (ChiCTR1900021395).

Background Increasing evidence indicates that gut microbiota plays an important role in cancer progression. However, the underlying mechanism remains largely unknown. Here, we report that broad-spectrum antibiotics (ABX) treatment leads to enhanced metastasis by the alteration of gut microbiome composition. Methods Cancer LLC and B16-F10 cell metastasis mouse models, and microarray/RNA sequencing analysis were used to reveal the regulatory functions of microbiota-mediated circular RNA (circRNA)/microRNA (miRNA) networks that may contribute to cancer metastasis. Results The specific pathogen-free (SPF) mice with ABX treatment demonstrated enhanced lung metastasis. Fecal microbiota transplantation (FMT) from SPF mice or Bifidobacterium into germ-free mice significantly suppressed lung metastasis. Mechanistically, gut microbiota impacts circRNA expression to regulate levels of corresponding miRNAs. Specifically, such modulations of gut microbiota inhibit mmu_circ_0000730 expression in an IL-11-dependent manner. Bioinformatics analysis combined with luciferase reporter assays revealed reciprocal repression between mmu_circ_0000730 and mmu-miR-466i-3p. We further showed that both mmu-miR-466i-3p and mmu-miR-466 f-3p suppresses a number of genes involved in epithelial-mesenchymal transition (EMT) and stemness of cancer stem cells such as SOX9. Conclusions These results provide evidence of a previously unrecognized regulatory role of non-coding RNAs in microbiota-mediated cancer metastasis, and thus, the microbiome may serve as a therapeutic target.

Background Epigenetic alterations are involved in most cancers, but its application in cancer diagnosis is still limited. More practical and intuitive methods to detect the aberrant expressions from clinical samples using highly sensitive biomarkers are needed. In this study, we developed a novel approach in identifying, visualizing, and quantifying the biallelic and multiallelic expressions of an imprinted gene panel associated with cancer status. We evaluated the normal and aberrant expressions measured using the imprinted gene panel to formulate diagnostic models which could accurately distinguish the imprinting differences of normal and benign cases from cancerous tissues for each of the ten cancer types. Results The Quantitative Chromogenic Imprinted Gene In Situ Hybridization (QCIGISH) method developed from a 1013-case study which provides a visual and quantitative analysis of non-coding RNA allelic expressions identified the guanine nucleotide-binding protein, alpha-stimulating complex locus (GNAS), growth factor receptor-bound protein (GRB10), and small nuclear ribonucleoprotein polypeptide N (SNRPN) out of five tested imprinted genes as efficient epigenetic biomarkers for the early-stage detection of ten cancer types. A binary algorithm developed for cancer diagnosis showed that elevated biallelic expression (BAE), multiallelic expression (MAE), and total expression (TE) measurements for the imprinted gene panel were associated with cell carcinogenesis, with the formulated diagnostic models achieving consistently high sensitivities (91–98%) and specificities (86–98%) across the different cancer types. Conclusions The QCIGISH method provides an innovative way to visually assess and quantitatively analyze individual cells for cancer potential extending from hyperplasia and dysplasia until carcinoma in situ and invasion, which effectively supplements standard clinical cytologic and histopathologic diagnosis for early cancer detection. In addition, the diagnostic models developed from the BAE, MAE, and TE measurements of the imprinted gene panel GNAS, GRB10, and SNRPN could provide important predictive information which are useful in early-stage cancer detection and personalized cancer management.

Background/Aims: Tyrosine kinase inhibitor gefitinib significantly improves the survival of patients with non-small-cell lung cancer (NSCLC) by inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase. However, patients eventually develop resistance to gefitinib through uncharacterized mechanisms. It is known that plasminogen activator urokinase receptor (PLAUR) plays an important role in cell proliferation, migration and apoptosis. However, the role of PLAUR, particularly exosomal PLAUR in gefitinib resistance in NSCLC has not been reported. The aim of this study is to determine the relationship between PLAUR and gefitinib resistance. Methods: In this study, a tethered cationic lipoplex nanoparticle (TCLN) biochip containing molecular beacons was used as probes to detect PLAUR mRNA in plasma exosomes from patients with gefitinib-sensitive and -resistant NSCLC. In vitro , Real-time PCR was used to examine the expression of PLAUR mRNA and Western blot was applied to examine the expression of related proteins. The gene knockdown was achieved by Lentivirus based RNA silence technique. The cell counting kit-8 assay and EdU incorporation were used to examine cell proliferation. The flow cytometry was applied to determine cell apoptosis and cell cycle, while the mitochondrial membrane potential was measured by JC-1 dye assay. Signaling pathway affected by PLAUR knockdown was identified by cDNA Microarray. The effect of PLAUR knockdown on tumorigenesis was analyzed in vivo . Results: We found that the exosomal PLAUR mRNA in the plasma of gefitinib-resistant NSCLC patients was significantly increased compared to that of gefitinib-sensitive NSCLC patients. The PLAUR mRNA and soluble PLAUR protein were also significantly increased in gefitinib-resistant human lung adenocarcinoma PC9R cells compared to gefitinib-sensitive PC9 cells. Silencing PLAUR in PC9R cells impaired mitochondrial membrane potential and increased cell apoptosis via EGFR/p-AKT/survivin signaling pathway. Furthermore, EGFR was upregulated in the geftinib-resistant PC9R cells, and knockdown of EGFR significantly increased cell apoptosis. Conclusions: Taken together, our results demonstrated that PLAUR induces geftinib-resistance through EGFR/p-AKT/survivin signaling pathway in gefitinib-resistant human lung adenocarcinoma cells. PLAUR could be a novel therapeutic target for gefitinib-resistant NSCLC patients.

BACKGROUND The authors built a model for lung cancer diagnosis previously based on the blood biomarkers progastrin-releasing peptide (ProGRP), carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), and cytokeratin 19 fragment (CYFRA21-1). In the current study, they examined whether modification of the model to include relevant clinical information, risk factors, and low-dose chest computed tomography screening would improve the performance of the biomarker panel in large cohorts of Chinese adults. METHODS The current study was a large-scale multicenter study (ClinicalTrials.gov identifier NCT01928836) performed in a Chinese population. A total of 715 participants were enrolled from 5 regional centers in Beijing, Henan, Nanjing, Shanghai, and Chongqing between October 2012 and February 2014. Serum biomarkers ProGRP, CEA, SCC, and CYFRA21-1 were analyzed on the ARCHITECT i2000SR. Relevant clinical information was collected and used to develop a patient risk model and a nodule risk model. RESULTS The resulting patient risk model had an area under the receiver operating characteristic (ROC) curve of 0.7037 in the training data set and 0.7190 in the validation data set. The resulting nodule risk model had an area under the ROC curve of 0.9151 in the training data set and 0.5836 in the validation data set. Moreover, the nodule risk model had a relatively higher area under the ROC curve (0.9151 vs 0.8360; P = 0.001) compared with the American College of Chest Physician model in patients with lung nodules. CONCLUSIONS Both the patient risk model and the nodule risk model, developed for the early diagnosis of lung cancer, demonstrated excellent discrimination, allowing for the stratification of patients with different levels of lung cancer risk. These new models are applicable in high-risk Chinese populations. Cancer 2018;124:262-70. © 2017 American Cancer Society.

// Fanglei Liu 1 , Chunxue Bai 1 and Zhongliang Guo 2 1 Department of Respiratory Medicine, Zhongshan Hospital Affiliated to Fudan University, Shanghai, China 2 Department of Respiratory Medicine, Dongfang Hospital Affiliated to Tongji University, Shanghai, China Correspondence to: Zhongliang Guo, email: guozhongliangshzs@163.com Keywords: small cell lung cancer, osteopontin, prognosis, proliferation, epithelial-mesenchymal transition Received: May 30, 2017 Accepted: June 26, 2017 Published: July 26, 2017 ABSTRACT Osteopontin (OPN) is known to be overexpressed in numerous carcinomas. Although abundant OPN has been reported to be correlated with poor survival in non-small cell lung cancer (NSCLC) but their clinical and prognostic significance in SCLC remains unknown. In this study, RNA-sequencing was used to obtain gene expression data in SCLC tissue samples and OPN expression levels were then investigated using qPCR, immunohistochemical and Western blot. We found OPN was one of the most upregulated genes. Besides, the correlation of OPN with tumor clinicopathological characteristics was evaluated and we found OPN was associated with advanced tumor stages. In addition, Kaplan-Meier survival analysis and Cox analyses revealed OPN expression was an independent predictor for overall survival (OS) (P= 0.013) and progression-free survival (PFS) (P=0.008). A high level of OPN was correlated with pT classification and pN classification (P

Mucus hypersecretion is a prominent mechanism in airway inflammation. Muc5ac is a major component of mucus and can be activated by reactive oxygen species (ROS). Peroxiredoxin 6 (Prdx6) highly expresses in airway epithelium and protects the airway from oxidative stress. In this study, we investigated the roles of Prdx6 in lipopolysaccharide (LPS)-induced mucin production in mice. We found that the levels of H2O2 and the Muc5ac mRNA were significantly increased in Prdx6 (-/-) mice compared to those in C57BL/6J mice after LPS instillation, which were markedly inhibited by epithelial growth factor receptor (EGFR) inhibitor Elrotinib. In vitro studies showed that mRNA levels of Prdx6 were decreased while H2O2 and Muc5ac were increased in a dose-dependent manner after LPS exposure, with significant increase in Prdx6 knockdown bronchial epithelial cells compared with those in normal epithelial cells. LPS-induced Muc5ac release was significantly inhibited by EGFR inhibitor, p38 inhibitor and JNK inhibitor, but not ERK1/2 inhibitor, indicating that the H2O2-EGFR-MAPK pathway is likely involved in the responses. This study indicated that Prdx6 decreased LPS-induced Muc5ac increase and played important roles in mucin hypersecretion after LPS exposure.

Lian Wu,1,2 Jing Zhang,3 Jie Ming Qu,4 Chun-xue Bai,3 Mervyn J Merrilees5 1Department of Community and Health Services, Unitec, 2Department of Pharmacology & Clinical Pharmacology, University of Auckland, Auckland, New Zealand; 3Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 4Department of Pulmonary Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 5Department of Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand Abstract: A reduced content of alveolar elastic fibers is a key feature of COPD lung. Despite continued elastogenic potential by alveolar fibroblasts in the lung affected by COPD, repair of elastic fibers does not take place, which is due to increased levels of the chondroitin sulfate proteoglycan versican that inhibits the assembly of tropoelastin into fibers. In this study, primary pulmonary fibroblast cell lines from COPD and non-COPD patients were treated with a small interfering RNA (siRNA) against versican to determine if knockdown of versican could restore the deposition of insoluble elastin. Versican siRNA treatment reduced versican expression and secretion by pulmonary fibroblasts from both COPD and non-COPD patients (P

In this study, we utilized AQP3-knockout mice as the in vivo model and AQP3-knockdown human bronchial epithelial cells (HBECs) as the in vitro model. Airway injury was experimentally induced by intra-tracheal injection of naphthalene. HE staining, transmission and scanning electron microscope were performed to evaluate self-healing capacity in vivo. Transwell and wound-healing assays were performed to evaluate epithelial cell migration in vitro. We found that both the airway epithelial cells of AQP3-knockout mice and AQP3-knockdown HBECs exhibited an obviously impaired self-healing capacity with defective epithelial cell migration through AQP3-facilitated glycerol transport. In addition, glycerol supplementation could largely correct defective injury healing and epithelial cell migration. For the first time, we found evidence for distinct defects in AQP3-deficient airway epithelial cell migration. Mechanistic analysis showed AQP3-facillitated glycerol transport plays a role in airway epithelial self-healing after injury.

Gefitinib, an epidermal growth factor receptor (EGFR)-specific drug, is effective for ~1 year, after which resistance is inevitable. Calpain 2 (CAPN2) is known to serve a role in the drug response and resistance in certain cancer therapies. However, the full function of CAPN2, particularly in non-small cell lung cancer, has not yet been elucidated. In the present study, CAPN2 expression in gefitinib-resistant lung adenocarcinoma cells was investigated. CAPN2 function in these cells was further evaluated using gene knockdown both in vitro and in vivo. The results demonstrated that CAPN2 was strongly associated with gefitinib-resistance, and CAPN2 mRNA and protein expression levels were significantly increased in gefitinib-resistant cell lines. Furthermore, CAPN2 knockdown inhibited gefitinib-resistant cell proliferation in vitro and in vivo. CAPN2 conferred gefitinib-resistance by inhibiting cell apoptosis and arresting the cell cycle. CAPN2 knockdown also induced caspase activation and mitochondrial dysfunction, and its function in gefitinib resistance appeared to be largely mediated by EGFR/protein kinase B/survivin signaling pathway activation. These results suggest that CAPN2 is responsible for EGFR-tyrosine kinase inhibitor resistance, and CAPN2 inhibition may be used to provide therapeutic benefits in the treatment of gefitinib resistance.

Background The aim of present study was to investigate the effects and mechanisms of peroxiredoxin (Prdx) 6 on cecal ligation and puncture (CLP) induced acute lung injury (ALI) in mice. Methods The cecal of male Prdx 6 knockout and wildtype C57BL/6J mice were ligated and perforated. Stool was extruded to ensure wound patency. Two hours, 4 h, 8 h and 16 h after stimulation, the morphology, wet/dry ratio, protein concentration in bronchial alveolar lavage fluid (BALF) were measured to evaluate lung injury. Myeloperoxidase (MPO) activity, hydrogen peroxide (H2O2), malondialdehyde (MDA), total superoxide dismutase (SOD), xanthine oxidase (XOD), CuZn-SOD, total anti-oxidative capability (TAOC), glutathione peroxidase (GSH-PX), catalase (CAT) in lungs were measured by assay kits. The mRNA expression of lung tumor necrosis factor (TNF-α), interleukin (IL)-1β, and matrix metalloproteinases (MMP) 2 and 9 were tested by real-time RT-PCR. The nuclear factor (NF)-κB activity was measured by TransAM kit. Results CLP-induced ALI was characterized by inflammation in morphology, increased wet/dry ratio, elevated protein concentration in BALF and higher level of MPO activity. The levels of H2O2, MDA, and XOD were significantly increased and SOD, CuZn-SOD, GSH-PX, CAT, and T-AOC were significantly decreased in lungs after CLP. The activity of NF-κB was significantly increased and subsequently, the mRNA expression of TNF-α, IL-1β and MMP2 and MMP9 were significantly increased after CLP. Those above injury parameters were more severe in Prdx 6 knockout mice than those in wildtype mice. Conclusions Prdx 6 knockout aggravated the CLP induced lung injury by augmenting oxidative stress, inflammation and matrix degradation partially through NF-κB pathway.

Acute lung injury (ALI) is a severe inflammatory disease, for which no specific treatment exists. The decreased ratio of regulatory T cells (CD4+ CD25+ FoxP3 Tregs) and Th17 cells is implicated in ALI and inflammation. We here investigated whether maintaining the balance of CD4+ CD25+ Foxp3+ Tregs and Th17 cells can alleviate lung injury. For CD4+ CD25+ FoxP3 Treg depletion, 200 μg of an anti-CD25 antibody was administered intraperitoneally per mouse on days -3 and -1 before lipopolysaccharide (LPS) instillation. And 150 μg of TGF-β was administered intraperitoneally per mouse on day 0 after LPS instillation. To down-regulate of Th17 cells, 200 μg per mouse of isotype, IL-17 or IL-22 antibodies were injected intraperitoneally into mice at days 0 after LPS instillation. We detected lung morphology; lung wet-to-dry weight ratio; protein concentration, the count of total cells, neutrophils and macrophages, and cytokines in bronchoalveolar lavage fluid (BALF). And we also evaluated the percentage of CD4+ CD25+ Foxp3+ Tregs in lung, and Th17 cells in lung. CD4+ CD25+ Foxp3+ Tregs depletion via anti-CD25 treatment or TGF-β neutralization delayed recovery of ALI. The prolonged inflammation was mainly dominated by neutrophils, macrophages and Th17 cells. Furthermore, inhibition of Th17 cells via monoclonal antibodies against IL-17 and IL-22 alleviated ALI inflammation by inhibiting the recruitment of neutrophils and macrophages, increasing the number of CD4+ CD25+ Foxp3+ Tregs. Our findings support a critical role for CD4+ CD25+ Foxp3+ Tregs in regulating from ALI pathophysiology, and a potential therapeutic role for the inhibition of Th17 cells in ALI treatment. These findings provide a rationale for treating patients with ALI by modulating CD4+ CD25+ Foxp3+ Tregs and Th17 cells.

Background Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, including gefitinib, are first-line drugs against advanced non-small cell lung cancer with activating EGFR mutations. However, the development of resistance to such drugs is a major clinical challenge. Methods The role of annexin A5 in resistance to EGFR tyrosine kinase inhibitors was investigated by qPCR and western blot of relevant molecules, by CCK8 and EdU assay of cell proliferation and viability, by annexin V/propidium iodide assay of apoptosis and cell cycle distribution, by JC-1 assay of mitochondrial integrity, and by xenograft assay of tumorigenicity. Results We found that annexin A5 is upregulated in gefitinib-resistant cell lines, as well as in clinical specimens resistant to EGFR tyrosine kinase inhibitors. Accordingly, knockdown of the gene from gefitinib-resistant cells restores gefitinib sensitivity in vitro and in vivo by downregulating polo-like kinase 1 signal pathway, thereby inducing mitochondrial damage, caspase activation, cell cycle arrest at G2/M, and, finally, apoptosis. Conclusions The data indicate that annexin A5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and G2/M cell cycle arrest, and is thus a potential therapeutic target in non-small cell lung cancers resistant to EGFR tyrosine kinase inhibitors.

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31+ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

BACKGROUND This study applied a combined cancer biomarker panel to clinically identify small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC) in a high-risk population. METHODS The serum levels of 4 biomarkers (progastrin-releasing peptide [ProGRP], carcinoembryonic antigen [CEA], squamous cell carcinoma antigen [SCC], and cytokeratin 19 fragment [CYFRA21-1]) were determined in 153 patients with a high risk of lung cancer (12 with a new diagnosis of SCLC, 52 with NSCLC, and 89 without lung cancer). Information about diagnosis delays was collected through interviews of all participants. RESULTS Significantly higher serum levels of ProGRP (P

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury.

Cryptococcal infection primarily affects the lung or the central nervous system and rare cases have been reported involving the gastrointestinal tract. However, among patients with HIV/AIDS, the gastrointestinal involvement is increasing. According to the PubMed search results, there were seven cases reported involving duodenal cryptococcosis combined with AIDS in five reports. Here, we report the case of a patient found to have AIDS combined with duodenal, pulmonary, and subsequent neurological cryptococcal infection simultaneously. The duodenal cryptococcosis was diagnosed on the basis of PET/computed tomography, which showed intense captation of glucose metabolism in duodenum (maximum standardized uptake value 16.53); a positive serum cryptococcal latex agglutination test; and upper gastrointestinal endoscopy-guided duodenal biopsy that confirmed Cryptococcus neoformans yeast. The patient's HIV screen test was positive. Because of refusal of lumbar puncture and the difficulty of performing transbronchial lung biopsy, the pulmonary and neurological involvements were the only clinical diagnoses. This case indicates that when cryptococcosis exists in a rare location, AIDS should be considered and when cryptococcosis occurs in the HIV-infected patient, disseminated disease is more common.

Prospective epidemiological studies, observational cross-sectional studies and some randomised prevention trials have demonstrated inconsistent findings of the impact of vitamin E on asthma risk. The goals of this study were to explore whether this differing association of vitamin E on asthma risk is due to an interaction of vitamin E isoforms. To address this question, in a population-based asthma incidence study we assessed the interaction between the plasma concentrations of vitamin E isoforms α-tocopherol and γ-tocopherol on asthma risk. Second, to understand the mechanisms of any interaction of these isoforms, we conducted experimental supplementation of α-tocopherol and γ-tocopherol isoforms in mice on the outcome of allergic airway inflammation. We found that in the highest γ-tocopherol tertile, low levels of α-tocopherol were associated with increased asthma risk, while highest tertile α-tocopherol levels trended to be protective. Similarly, in a mouse model of asthma, diet supplementation with α-tocopherol decreased lung inflammation in response to house dust mite (HDM) challenge. In contrast, diet supplementation with γ-tocopherol increased lung inflammation in response to HDM. These human and animal studies provide evidence for the competing effects of the vitamin E isoforms, in physiological concentrations, on asthma and allergic airway disease.

Background and objective Chemotherapy drugs, such as cisplatin (DDP), improve the survival of patients with lung cancer by inducing apoptosis in cancer cells, which quickly develop resistance to DDP through uncharacterized mechanisms. Glioma Pathogenesis-Related Protein 1 (GLIPR1) plays an important role in cell proliferation, migration and apoptosis. However, the expression and function of GLIPR1 in mediating DDP resistance in human lung adenocarcinoma A549/DDP and human large cell lung cancer H460/DDP cells has not yet been reported. Methods In this study, real-time PCR (RT-PCR) and western blot were used to examine the mRNA and protein expression of GLIPR1, respectively. Bright-field microscopy, the cell counting kit-8 (CCK-8) assay, flow cytometry analysis and JC-1 dye were used to measure the cellular morphology, proliferation, apoptosis and mitochondrial membrane potential, respectively. Results Compared to human lung adenocarcinoma A549 cells, the mRNA and protein expression of GLIPR1 were significantly increased in DDP-resistant A549/DDP cells (p < 0.05). Similarly, the mRNA level of GLIPR1 in DDP-resistant H460/DDP cells was also significantly higher than that in DDP-sensitive H460 cells (p < 0.05). Silencing of GLIPR1 in A549/DDP and H460/DDP cells led to increased apoptosis via a mitochondrial signaling pathway following incubation with various concentrations of DDP. Furthermore, GLIPR1 downregulation markedly reduced the protein expression of Bcl-2, and increased the cleaved Poly (ADP-Ribose) Polymerase (PARP) and cleaved caspase-3 in DDP-resistant A549/DDP cells. Conclusion In this study, we demonstrated for the first time that GLIPR1 could modulate the response of DDP-resistant A549/DDP and H460/DDP cells to cisplatin. Therefore, GLIPR1 deserves further investigation in the context of none-small lung cancer (NSCLC).

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. Keratinocyte growth factor-2 (KGF-2), a potent epithelial cell mitogen, may have a role in preventing ventilator-induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF-2 in VILI in rats. Forty healthy adult male Sprague-Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high-volume zero positive end-expiratory pressure) and HVZP+KGF-2 were given intratracheally equal PBS and 5 mg/kg KGF-2 72 hrs before 4 hrs HVZP ventilation (20 ml/kg), respectively, while PBS and KGF-2 were administered in the same manner in Groups Control and KGF-2, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis factor-α, macrophage inflammatory protein 2), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre-treatment with KGF-2 showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF-2 might be considered a promising prevention for human VILI or other acute lung injury diseases.

Blocking PcrV, an essential component of the Type III secretion system (TTSS), has demonstrated efficacy against Pseudomonas aeruginosa infections. However, most of the results came from laboratory strains. Whether it is applicable to clinically isolated multi-drug resistant (MDR) strains is unknown. In this study we investigated the expression level of TTSS in clinically isolated MDR P. aeruginosa strains and the effects of anti-PcrV antibody on MDR isolate induced acute lung injury (ALI). The expression level of TTSS was quantified in 53 isolates including 25 MDR strains and 28 susceptible strains. We investigated the effect of anti-PcrV antibody through a murine model induced by instillation of a MDR strain into the left lung through trachea. Our results showed that the expression level of TTSS in MDR strains is comparable to susceptible strains. Anti-PcrV ensured the survival of challenged mice, reduced the bacteria numbers and attenuated lung inflammation and injury. This study proved that anti-PcrV may be a potentially effective strategy against MDR P. aeruginosa induced ALI.

Chronic intermittent hypoxia (CIH) is the main pathophysiological feature of obstructive sleep apnea (OSA). It destroys the normal sleep rhythm and affects many basic physiological processes, leading to repeated arousal from sleep. Orexin, a very potent arousal neuromodulator found in the brain, has been shown to play a significant role in the promotion and maintenance of arousal. These findings may indicate that orexin participates in the arousal-promoted processes induced by CIH. However, so far, little is known about alterations in orexin and its receptors during CIH. In the present study, the influence of CIH and re-oxygenation on the expression of prepro-orexin (PPO) in the hypothalamus and orexin receptors in the hypothalamus and the medulla of rats were evaluated. The expression of PPO, OX1R, and OX2R mRNA was evaluated by RT-PCR after the rats had been exposed to intermittent hypoxia (IH) for either 1 week or 5 weeks. The control group was exposed to intermittent air (IA). The results showed the levels of expression of PPO, OX1R, and OX2R mRNA to be significantly higher in the brains of rats exposed to IH for 5 weeks than in the brains of control rats. This effect was reversed by 5 weeks of re-oxygenation. These findings indicated that CIH could increase the expression of orexin and its receptors in the brain. This may contribute to the sleep fragmentation experienced by patients with OSA, which is induced by repeated arousal.

In addition to protein-coding genes, the human genome makes a large amount of noncoding RNAs, including microRNAs and long noncoding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to have a critical role in the regulation of cellular processes such as cell growth and apoptosis, as well as cancer progression and metastasis. Although it is well known that microRNAs can target a large number of protein-coding genes, little is known whether microRNAs can also target lncRNAs. In the present study, we determine whether miR-21 can regulate lncRNA expression. Using the lncRNA RT-PCR (reverse transcription-polymerase chain reaction) array carrying 83 human disease-related lncRNAs, we show that miR-21 is capable of suppressing the lncRNA growth arrest-specific 5 (GAS5). This negative correlation between miR-21 and GAS5 is also seen in breast tumor specimens. Of interest, GAS5 can also repress miR-21 expression. Whereas ectopic expression of GAS5 suppresses, GAS5–siRNA increases miR-21 expression. Importantly, there is a putative miR-21-binding site in exon 4 of GAS5; deletion of the miR-21-binding site abolishes this activity. Experiments with in vitro cell culture and xenograft mouse model suggest that GAS5 functions as a tumor suppressor. We further show that the biotin-labeled GAS5–RNA probe is able to pull down the key component (AGO2) of the RNA-induced silencing complex (RISC) and we subsequently identify miR-21 in this GAS5–RISC complex, implying that miR-21 and GAS5 may regulate each other in a way similar to the microRNA-mediated silencing of target mRNAs. Together, these results suggest that miR-21 targets not only tumor-suppressive protein-coding genes but also lncRNA GAS5.

Background and objective Alveolar fluid clearance (AFC) is important for the resolution of acute lung injury (ALI). The role of cystic fibrosis transmembrane conductance regulator (CFTR) in AFC has not been entirely elucidated in animal models of ALI. The aim of this study was to investigate the role of CFTR and its mechanisms in AFC in normal and ALI mice. Methods Seventy mice were randomly divided into 14 groups and ALI was established by intratracheal instillation of lipopolysaccharide (LPS). After 48 h, CFTR activator CFTRact-16 or inhibitor CFinh-172 with or without β-agonist was instillated intratracheally and AFC was measured with radioisotopic tracer. Results Although there was no effects of CFTRact-16 on AFC in mice with or without isoproterenol, CFinh-172 markedly decreased isoproterenol-stimulated AFC in both normal (P

Telocytes (TCs) are interstitial cells with telopodes – very long prolongations that establish intercellular contacts with various types of cells. Telocytes have been found in many organs and various species and have been characterized ultrastructurally, immunophenotypically and electrophysiologically (http://www.telocytes.com). Telocytes are distributed through organ stroma forming a three-dimensional network in close contacts with blood vessels, nerve bundles and cells of the local immune system. Moreover, it has been shown that TCs express a broad range of microRNAs, such as pro-angiogenic and stromal-specific miRs. In this study, the gene expression profile of murine lung TCs is compared with other differentiated interstitial cells (fibroblasts) and with stromal stem/progenitor cells. More than 2000 and 4000 genes were found up- or down-regulated, respectively, in TCs as compared with either MSCs or fibroblasts. Several components or regulators of the vascular basement membrane are highly expressed in TCs, such as Nidogen, Collagen type IV and Tissue Inhibitor of Metalloproteinase 3 (TIMP3). Given that TCs locate in close vicinity of small vessels and capillaries, the data suggest the implication of TCs in vascular branching. Telocytes express also matrix metalloproteases Mmp3 and Mmp10, and thus could regulate extracellular matrix during vascular branching and de novo vessel formation. In conclusion, our data show that TCs are not fibroblasts, as the ultrastructure, immunocytochemistry and microRNA assay previously indicated. Gene expression profile demonstrates that TCs are functionally distinct interstitial cells with specific roles in cell signalling, tissue remodelling and angiogenesis.

Aquaporins (AQPs ) are water channel proteins supposed to facilitating fluid transport in alveolar space, airway humidification, pleural fluid absorption, and submucosal gland secretion . In this chapter, we mainly focus on the expression of 4 AQPs in the lungs which include AQP1, AQP2 , AQP4 and AQP5 in normal and disease status, and the experience of AQPs function from various model and transgenic mice were summarized in detail to improve our understanding of the role of AQPs in fluid balance of respiratory system. It has been suggested that AQPs play important roles in various physiology and pathophysiology conditions of different lung diseases. There still remains unclear the exact role of AQPs in lung diseases, and thus continuous efforts on elucidating the roles of AQPs in lung physiological and pathophysilogical processes are warranted.

Nucleotide excision repair (NER) pathway plays critical roles in repairing DNA disorders caused by platinum. To comprehensively understand the association between variants of NER and clinical outcomes of platinum-based chemotherapy, 173 SNPs in 27 genes were selected to evaluate association with toxicities and efficiency in 1004 patients with advanced non-small cell lung cancer. The results showed that consecutive significant signals were observed in XPA, RPA1, POLD1, POLD3. Further subgroup analysis showed that GTF2H4 presented consecutive significant signals in clinical benefit among adenocarcimoma. In squamous cell carcinoma, rs4150558, rs2290280, rs8067195 were significantly associated with anemia, rs3786136 was significantly related to thrombocytopenia, ERCC5 presented consecutive significant signals in response rate. In patients receiving TP regimen, significant association presented in neutropenia, thrombocytopenia and gastrointestinal toxicity. Association with anemia and neutropenia were found in GP regimen. rs4150558 showed significant association with anemia in NP regimen. In patients > 58, ERCC5 showed consecutive significant signals in gastrointestinal toxicity. Survival analysis showed SNPs in POLD2, XPA, ERCC6 and POLE were significantly associated with progression free survival, SNPs in GTF2H4, ERCC6, GTF2HA, MAT1, POLD1 were significantly associated with overall survival. This study suggests SNPs in NER pathway could be potential predictors for clinical outcomes of platinum-based chemotherapy among NSCLC.

Modulation of inflammation is a crucial component of the development of acute lung injury. A20, a ubiquitin editing enzyme, may regulate cellular inflammatory reactions, particularly those involving the signaling pathway of nuclear factor NF-κB (NF‑κB). The present study investigated the mechanism by which A20 downregulated NF‑κB and further contributed to macrophage polarization from the M1 to M2 phenotypes in lipopolysaccharide (LPS)‑induced lung injury. Sprague‑Dawley rats injected with LPS were used in the present study. Bronchoalveolar lavage fluid and lung tissue were collected from each experimental rat. A macrophage cell line was used to test the expression levels of A20. Tumor necrosis factor‑α (TNF‑α), interleukin‑1 beta (IL‑1β) and NF‑κB activities were assessed by ELISA and polymerase chain reaction. Macrophage phenotypes were assayed using fluorescence‑activated cell sorting. Elevated levels of TNF‑α, IL‑1β, NF‑κB and A20 were observed in the macrophages of rats treated with LPS. Furthermore, A20 overexpression inhibited NF‑κB DNA binding activity and increased macrophage polarization from the M1 to M2 phenotype in lung macrophages of the NR8383 cell line. It was concluded that the A20 protein in macrophages modulates lung injury induced by LPS. The overexpression of A20 in macrophages may be involved in modulating macrophage polarization. The mechanisms and molecular identification of macrophage polarization activation may provide a basis for the treatment of inflammation in lung injury.

Integrin αvβ5 mediates pulmonary endothelial barrier function and acute lung injury (LI), but its roles in cell apoptosis and autophagy are unclear. Thus, the aims of this study were to investigate the significance of αvβ5 in ischemia-reperfusion (I/R)-induced apoptosis and LI and to explore the relationship between αvβ5 and autophagy. Human pulmonary microvascular endothelial cells (HPMVECs) were pretreated with an αvβ5-blocking antibody (ALULA) and challenged with oxygen-glucose deprivation/oxygen-glucose restoration, which mimics I/R; then, cellular autophagy and apoptosis were detected, and cell permeability was assessed. In vivo, mice were pretreated with the autophagy inhibitor chloroquine (CLQ), followed by treatment with ALULA. The mice then underwent operative lung I/R. LI was assessed by performing a pathological examination, calculating the wet/dry lung weight ratio and detecting the bronchial alveolar lavage fluid (BALF) protein concentration. αvβ5 inhibition promoted HPMVEC autophagy under I/R in vitro, alleviated cell permeability, decreased the apoptosis ratio, and activated caspase-3 expression. These outcomes were significantly diminished when autophagy was inhibited with a small-interfering RNA construct targeting autophagy-related gene 7 (si ATG7). Moreover, ALULA pretreatment alleviated I/R-induced LI (I/R-LI), which manifested as a decreased wet/dry lung weight ratio, an altered BALF protein concentration, and lung edema. Preinhibiting autophagy with CLQ, however, eliminated the protective effects of ALULA on I/R-LI. Therefore, inhibiting αvβ5 effectively ameliorated I/R-induced endothelial cell apoptosis and I/R-LI. This process was dependent on improved autophagy and its inhibitory effects on activated caspase-3.

Specific changes in immune repertoires at genetic level responding to the lethal H7N9 virus are still poorly understood. We performed deep sequencing on the T and B cells from patients recently infected with H7N9 to explore the correlation between clinical outcomes and immune repertoire alterations. T and B cell repertoires display highly dynamic yet distinct clonotype alterations. During infection, T cell beta chain repertoire continues to contract while the diversity of immunoglobulin heavy chain repertoire recovers. Patient recovery is correlated to the diversity of T cell and B cell repertoires in different ways – higher B cell diversity and lower T cell diversity are found in survivors. The sequences clonally related to known antibodies with binding affinity to H7 hemagglutinin could be identified from survivors. These findings suggest that utilizing deep sequencing may improve prognostication during influenza infection and could help in development of antibody discovery methodologies for the treatment of virus infection.

Objectives: To investigate the role of CHD4 in the development of non-small-cell lung cancer(NSCLC) and determine the potential mechanisms of action. Methods: Immunohistochemistry was used to detect gene expression in 146 formalin-fixed, paraffin-embedded surgical lung specimens. The correlation of gene expression levels and clinical parameters in NSCLC patients was analyzed. In vitro experiments were performed to confirm the functional roles of the genes. Gene ontology (GO) enrichment analysis, western blot and co-immunoprecipitation assays were used to identify the mechanism by which those genes contribute to NSCLC development. Measurements and Main Results: Compared with non-cancerous tissues, CHD4 was overexpressed in cancer tissues, and its expression level correlated with the malignant progression of NSCLC. CHD4 could be used as an independent factor for NSCLC patient prognosis. Inhibition of CHD4 was observed to reduce the proliferative ability as well as promote the apoptosis rate of the NSCLC cells. Meanwhile, reduced CHD4 expression could induce the cell cycle arrest at G1/S phase transition. We identified that down-regulation of CHD4 could deactivate the RhoA/ROCK signaling pathway, leading to the hypothesis that the CHD4 might promote the development of NSCLC through the regulation of the RhoA/ROCK signaling pathway. Conclusions: These findings highlight an important role of CHD4 in the regulation of proliferative abilities of NSCLC and suggest a potential application of CHD4 in cancer treatment.

Influenza A (H7N9) virus induced high mortality since 2013. It is important to elucidate the potential genetic variations that contribute to virus infection susceptibilities. In order to identify genetic mutations that might increase host susceptibility to infection, we performed exon sequencing and validated the SNPS by Sanger sequencing on 18 H7N9 patients. Blood samples were collected from 18 confirmed H7N9 patients. The genomic DNA was captured with the Agilent SureSelect Human All Exon kit, sequenced on the Illumina Hiseq 2000, and the resulting data processed and annotated with Genome analysis Tool. SNPs were verified by independent Sanger sequencing. The DAVID database and the DAPPLE database were used to do bioinformatics analysis. Through exon sequencing and Sanger sequencing, we identified 21 genes that were highly associated with H7N9 influenza infection. Protein-protein interaction analysis showed that direct interactions among genetic products were significantly higher than expected (p = 0.004), and DAVID analysis confirmed the defense-related functions of these genes. Gene mutation profiles of survived and non-survived patients were similar, suggesting some of genes identified in this study may be associated with H7N9 influenza susceptibility. Host specific genetic determinants of disease severity identified by this approach may provide new targets for the treatment of H7N9 influenza.

High altitude pulmonary oedema (HAPE) severely affects non-acclimatized individuals and is characterized by alveolar flooding with protein- rich oedema as a consequence of blood-gas barrier disruption. Limited choice for prophylactic treatment warrants effective therapy against HAPE. Keratinocyte growth factor-2 (KGF-2) has shown efficiency in preventing alveolar epithelial cell DNA damages in vitro. In the current study, the effects of KGF-2 intratracheal instillation on mortality, lung liquid balance and lung histology were evaluated in our previously developed rat model of HAPE. We found that pre-treatment with KGF-2 (5 mg/kg) significantly decreased mortality, improved oxygenation and reduced lung wet-to-dry weight ratio by preventing alveolar-capillary barrier disruption demonstrated by histological examination and increasing alveolar fluid clearance up to 150%. In addition, KGF-2 significantly inhibited decrease of transendothelial permeability after exposure to hypoxia, accompanied by a 10-fold increase of Akt activity and inhibited apoptosis in human pulmonary microvascular endothelial cells, demonstrating attenuated endothelial apoptosis might contribute to reduction of endothelial permeability. These results showed the efficacy of KGF-2 on inhibition of endothelial cell apoptosis, preservation of alveolar-capillary barrier integrity and promotion of pulmonary oedema absorption in HAPE. Thus, KGF-2 may represent a potential drug candidate for the prevention of HAPE.

Background and objective: The aim of this study was to investigate the changes in expression of aquaporins (AQP) during differentiation of human bronchial epithelial cells and the role of lipopolysaccharide (LPS) in AQP expression. Methods: The levels of AQP3, AQP4 and AQP5 transcripts in human primary cultured bronchial epithelial cells were evaluated by real-time polymerase chain reaction at different time points before and after treatment with LPS. Western blotting was performed to assess the effects of LPS on AQP3, AQP4 and AQP5 expressions in normal human bronchial epithelial cells. Using pharmacological tools, the pathways involved in the regulation of LPS-induced changes in AQP5 were further explored. Results: The levels of AQP3, AQP4 and AQP5 transcripts were increased during differentiation of human bronchial epithelial cells. Expression of AQP5, but not AQP3 or AQP4, was downregulated by LPS. LPS-induced downregulation of AQP5 was inhibited by p38 and c-Jun N-terminal kinase (JNK) inhibitors. Conclusions: This study demonstrated that LPS decreases AQP5, but not AQP3 or AQP4, expression in human primary bronchial epithelial cells. The downregulation of AQP5 expression is mediated through a p38/JNK signalling pathway.

Hongbing Shen and colleagues identify three new susceptibility loci for lung cancer in the Chinese population. Their follow-up analyses suggest that two of these loci interact multiplicatively with smoking dose to influence lung cancer risk. To find additional susceptibility loci for lung cancer, we tested promising associations from our previous genome-wide association study (GWAS)1 of lung cancer in the Chinese population in an extended validation sample size of 7,436 individuals with lung cancer (cases) and 7,483 controls. We found genome-wide significant (P < 5.0 × 10−8) evidence for three additional lung cancer susceptibility loci at 10p14 (rs1663689, close to GATA3, P = 2.84 × 10−10), 5q32 (rs2895680 in PPP2R2B-STK32A-DPYSL3, P = 6.60 × 10−9) and 20q13.2 (rs4809957 in CYP24A1, P = 1.20 × 10−8). We also found consistent associations for rs247008 at 5q31.1 (IL3-CSF2-P4HA2, P = 7.68 × 10−8) and rs9439519 at 1p36.32 (AJAP1-NPHP4, P = 3.65 × 10−6). Four of these loci showed evidence for interactions with smoking dose (P = 1.72 × 10−10, P = 5.07 × 10−3, P = 6.77 × 10−3 and P = 4.49 × 10−2 for rs2895680, rs4809957, rs247008 and rs9439519, respectively). These results advance our understanding of lung cancer susceptibility and highlight potential pathways that integrate genetic variants and smoking in the development of lung cancer.

Ovalbumin (OVA)-induced asthma in mouse lungs causes changes in the mRNA and protein levels of aquaporins (AQPs). AQP expression was examined in the presence of various anti-asthmatic agents, including dexamethasone, ambroxol, and terbutaline. The influence of these agents on OVA-induced airway inflammation was also evaluated. The mRNA expression levels of AQP1, 4, and 5 were significantly reduced and that of AQP3 was significantly increased 24h after the last OVA exposure. The protein levels of AQP1, 3, and 5 mirrored the mRNA expression profiles, but AQP4 did not exhibit any changes. Only the mRNA and protein expression levels of AQP1 and AQP5 were significantly increased by these three anti-asthmatic agents. Dexamethasone and ambroxol improved the eosinophil infiltration, mucus secretion, and pulmonary edema caused by OVA, but terbutaline only alleviated pulmonary edema. These results indicate that AQP1 and AQP5 are closely related to pulmonary edema but not to eosinophil infiltration or mucus secretion during asthma. Anti-asthmatic agents could alleviate pulmonary edema through upregulating the expression of AQP1 and AQP5 in mouse lungs that have OVA-induced asthma.

Multiple cells play critical roles in the pathogenesis of lung diseases, even though the exact mechanisms are still not clear. Telocytes are characterized by telopodes, which are thin and long prolongations, and a small amount of cytoplasm rich with mitochondria, as shown by immune-positive staining against CD34, c-kit and vimentin. Telocytes have been found in many organs of mammals, including the trachea and lung. This report summarizes the latest findings associated with telocytes, with a special focus on the lung, and demonstrates that telocytes exist in the smooth muscle layer under cartilage and bronchiole in the lung, and also in the interstitial space of alveoli. Telocytes have a mediate connection with epithelial cells and direct connection with smooth muscle cells both in blood vessels and bronchiole in the lung. Telocytes also have a close relationship with other cell types, such as immune cells and stem cells. Telopodes appear with dichotomous branching and alternation of podom and podomer, forming a 3D network structure with complex homo- and hetero-cellular junctions. All characteristics of telocytes in lung tissue indicate that telocytes may play a potential, but important, role in the pathogenesis of lung diseases.

Insufficiency of tissue repair by pulmonary fibroblasts may contribute to the decrease in elastic fibres in chronic obstructive pulmonary disease (COPD). In this study, the repair function of COPD fibroblasts was assessed by examining the response to transforming growth factor (TGF)-β1. Primary pulmonary fibroblasts were cultured from lung tissue of COPD patients and smoking control subjects. Cellular proliferation was measured with Alamar Blue reduction method. Levels of tropoelastin mRNA and soluble elastin was measured using real-time RT-PCR and Fastin elastin assay respectively. The percentage of increase in proliferation and elastin production after TGF-β1 (1 ng/ml) treatment was calculated for fibroblasts from each subject. COPD fibroblasts showed slower proliferation than control fibroblasts, and a reduced response to TGF-β1 stimulation. The promotive effect of TGF-β1 on elastin synthesis in control fibroblasts was significantly diminished in fibroblasts from COPD patients. Our findings indicate that COPD lung fibroblasts have a significantly decreased response to TGF-β1 in terms of proliferation and elastin production.

Background Aquaporin 5 (AQP5) is widely expressed in various organ and tissues. In light of the novel oncogenic properties of aquaporins (AQPs), here we investigated the effect of AQP5 knockdown by RNAi on transmembrane osmotic water permeability, cell migration potential and cell volume regulation ability. Methods AQP5 expression was inhibited by short hairpin RNA in SPC-A1 cells, a lung adenocarcinoma cell line. Cells loaded with a fluoroprobe (calcein-AM) were immersed in either isosmotic, hyperosmotic or hyposmotic solutions, and fluorescence intensity was recorded using confocal microscopy. These measurements were used to calculate osmotic water permeability coefficients (Pf) and to monitor regulated volume decrease (RVD). Tumor cell migration and invasion assays were performed in a modified Boyden chamber. Wound healing and colony forming ability were also tested. Results Although self-quenching was not found in SPC-A1 cells, we observed a linear relationship between fluorescence intensity and cell water volume, suggesting that this method is a sensitive and reproducible way to measure single-cell transmembrane water permeability. Cells in which the AQP5 gene was silenced showed a 49.4% decrease in osmotic water permeability, a 55.3% decrease in migration and a 28.4% decrease in invasion potential. In addition, RVD decreased remarkably with reduced osmotic water permeability. Conclusion Our results suggest that AQP5, which mediates water permeability and thus regulates cell shape and volume, is a potentially important determinant in cell migration.

To investigate the role and signaling pathway of peroxiredoxin 6, a newly identified peroxidase, in lipopolysaccharide-induced acute lung injury.Prospective, randomized, controlled study.Research laboratory.Peroxiredoxin 6 (-/-) and wild-type C57BL/6 mice.Wild-type or peroxiredoxin 6 (-/-) mice were challenged by intratracheal instillation of lipopolysaccharide (5 mg/kg) for 4 hrs or 24 hrs for lung injury measurement. In other studies, peritoneal macrophages, isolated from wild-type and peroxiredoxin 6 (-/-) mice, were preincubated in presence or absence of mitogen-activated protein kinases inhibitors for 30 mins before being stimulated with lipopolysaccharide (1 μg/mL) for 4 hrs.Bronchoalveolar lavage myeloperoxidase activity and the lung injury score were significantly increased in peroxiredoxin 6 (-/-) mice compared with wild-type mice after lipopolysaccharide instillation at both 4 hrs and 24 hrs. Hydrogen peroxide and malondialdehyde levels, as well as nuclear factor-κB activities, tumor necrosis factor-α, interleukin-1β, and matrix metalloproteinase-9 messenger RNA, protein concentration, and activities were significantly increased whereas total antioxidative capability was markedly decreased in lungs of peroxiredoxin 6 (-/-) mice compared with wild-type mice. In vitro studies showed intracellular reactive oxygen species levels and release of tumor necrosis factor-α, interleukin-1, and matrix metalloproteinase-9 were significantly increased in macrophages from peroxiredoxin 6 (-/-) mice compared with that from wild-type mice after lipopolysaccharide stimulation. Cytokines release was partially suppressed by extracellular signal-regulated kinase and c-Jun N-terminal kinase inhibitors, but not by the p38 mitogen-activated protein kinase inhibitor.Deletion of peroxiredoxin 6 exaggerates lipopolysaccharide-induced acute lung injury and inflammation with increased oxidative stress, inflammatory responses, and matrix degradation, all of which were partially dependent on nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun N-terminal kinase pathways.

Hypoxia down-regulates the expression of the growth arrest-specific homeobox (Gax) in pulmonary arterial smooth muscle cells (PASMCs), resulting in increased cell proliferation and decreased apoptosis, but the mechanism for this response remains unclear. The present study investigated the role of Gax in the development of hypoxia-induced pulmonary hypertension (PH). We found that hypoxia suppressed the expression of endogenous Gax in rats, but not in those pretreated intratracheally with a Gax construct (Ad-Gax). Hypoxic rats pretreated with Ad-Gax were resistant to hypoxia-induced PH, right ventricular hypertrophy, increased wall thickness, and the muscularization of pulmonary arterioles. Hypoxia-induced PASMC proliferation and suppression of Gax expression were blocked by the Mitogen-activated protein kinase (MEK) inhibitor U0126. The PASMCs with Ad-Gax transfection exhibited hyperexpression of the Bcl-2-associated X protein (Bax) and hypoexpression of B-cell lymphoma 2 (Bcl-2), leading to cell apoptosis. Thus, our data indicate that the enhanced expression of Gax inhibits hypoxia-induced PASMC proliferation, probably via the extracellular-signal-regulated kinase (ERK) 1/2 pathway, and induces the apoptosis of hypoxic PASMCs via the Bcl-2/Bax pathway. Gax may be a potential new therapeutic target for pulmonary hypertension.

Airway inflammation and mucus hyperproduction play the central role in the development of asthma, although the mechanisms remain unclear. The aquaporin (AQP)-5 may be involved in the process due to its contribution to the volume of liquid secreted from the airways. The present study firstly found the overexpression of AQP5 in the airway epithelium and submucosal glands of asthmatics. Furthermore, we aimed at evaluating the role of AQP5 in airway inflammation and mucous hyperproductions during chronic allergic responses to house dust mite (HDM). Bronchoalveolar lavage levels of interleukin (IL)-2, IL-4, IL-10, interferon-γ and Mucin 5AC (MUC5AC), and number of peribronchial and perivascular cells were measured in AQP5 wild-type and AQP5 knockout (KO) mice. We found that HDM induced airway inflammation, lung Th2 cell accumulation and mucin hypersecretion in C57BL/6 mice rather than AQP5 KO mice. Expression of MUC5AC and MUC5B proteins and genes in the lung tissue was significantly lower in AQP5 KO mice. Thus, our results implicate involvement of AQP5 in the development of airway inflammation and mucous hyperproduction during chronic asthma.

Water channel aquaporin 5 (AQP5) is highly expressed at the apical membrane of alveolar type I epithelial cells and confers high osmotic water permeability. AQP5 is also expressed in lung cancer tissue. Previous studies showed there was an up-regulation of AQP5 expression in cancer tissue compared to surrounding normal tissue. In addition, expression of AQP5 in lung cancer tissue was associated with poor prognosis. Herein, we tested the role of AQP5 in lung cancer oncogenesis and development. Lung cancer cells with different expression of AQP5 were used to study cell proliferation and migration, two important parameters for tumour cell biology. We found enhanced proliferation and migration potential in cancer cells with high AQP5 expression, while reduced proliferation and metastasis potential in cancer cells with low AQP5 expression. Oncogene analysis showed significantly increased PCNA and c-myc expression in AQP5 transfected cells. AQP5 transfected cells also showed significant increased MUC5AC mucin expression, which might contribute to the enhanced metastasis potential of lung cancer. AQP5 overexpression resulted in enhanced activation of the epidermal growth factor receptor (EGFR), extracellular receptor kinase (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) pathway in cancer cells. Moreover, deletion of AQP5 demonstrated decreased activation of the EGFR/ERK/p38 MAPK pathway in AQP5 knockout mice lungs, while deletion of AQP1 or AQP3 did not exhibit significant changes on activation of the EGFR/ERK/p38 MAPK pathway in lung tissue. In conclusion, our results provide evidence for AQP5-facilitated lung cancer cell proliferation and migration, possibly through activation of the EGFR/ERK/p38 MAPK signalling pathway, but why AQP5 but not other aquaporin expression affects the EGFR/ERK/p38 MAPK pathway still needs further exploration.

There is growing evidence that the signal pathway between hepatocyte growth factor (HGF) and its receptor c-Met plays an important role in the development of lung cancer, although the specificity of such role is to be clarified. It seems clear that the HGF/c-Met signal contributes to the metastasis of cancer cells to the lung by stimulating the hyperproduction and overactivation of cytokines and enzymes, e.g. HGF, vascular endothelial growth factor and matrix metalloproteases. The HGF/c-Met signal may act as the candidate responsible for the development of epidermal growth factor receptor (EGFR) kinase inhibitor resistance. Experimental evidence showed that the combination of both EGFR and c-Met inhibitors had synergetic or additive therapeutic effects on lung cancer. Although the mechanism of interaction between HGF/c-Met and transforming growth factor-a/EGFR remains unclear, the cross-talk and balance between those two signal pathways are critical and necessary in the development of new therapies for lung cancer.

Objective Lung cancer has emerged as a leading cause of cancer death in the world. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. RNA interference (RNAi) has shown promise in gene silencing in vitro, the potential of which in developing new methods for the therapy of non-small-cell lung cancer (NSCLC) needs to be further tested in vivo. In this study, chemically synthesized double-stranded RNA (dsRNA) targeting epidermal growth factor receptor (EGFR) was transfected into NSCLC cell line SPC-A1 cells and established the tumor burdened athymic nude mice model to investigate whether dsRNA could induce gene silencing in NSCLC cells in vivo.

Increasing evidence suggests that the chemokine system coordinates leukocyte migration in immunity and in- flammation, involving in the pathogenesis of many pulmonary diseases. Chemokines are small proteins which interact with specific receptors to exert their chemotactic functions for inflammatory cells and constitutive cells. Among the com- plex system, CC chemokine receptors (CCRs) represent a subfamily of chemokine receptor, which is considered to play an important role in the pathogenesis of diseases characterized by disordered inflammation and immunity, among which some respiratory diseases, such as asthma, chronic obstructive pulmonary disease, acute lung injury, comprise one impor- tant part. The present paper reviewed the research of possible relationship and progress between this system and human lung diseases. Chemokines and their receptors are essential components of Th1- and Th2-mediated responses via the re- cruitment of lymphocytes and macrophages. The local expression of homeostatic chemokines in pulmonary also plays an important role in protective immune responses. Thus, it is important to understand the potential role of CCRs in respira- tory diseases and it may provide exciting new targets for therapeutic intervention.

5-Lipoxygenase (5-LO) is an important enzyme that dominates the metabolism of arachidonic acid and the synthesis of leukotrienes, of which leukotriene B4 (LTB4) is suggested to be one of the most potent chemoattractants and activators of leukocytes. 5-LO is normally limited to hematopoetic cells, e.g. granulocytes and macrophages, where LTB4 is produced. Leukotrienes play an important role in the innate defense system, in the antimicrobial defense system, and in intercellular signaling. Under pathophysiological conditions, overproduction of LTB4 was noted in multiple cells. It is known that 5-LO and its production of LTB4 are involved in the maintenance of normal host defense, initiation of inflammation, migration of leukocytes, development of endothelial barrier dysfunction, hyperresponsiveness of smooth muscle cells, stimulation of mucus secretion, reduction of mucociliary clearance, and tissue remodeling. In order to understand the role of 5-LO and its production in functions of the innate host defense...

Diffuse panbronchiolitis (DPB) is a pulmonary disease of unknown etiology that predominantly affects East Asians, particularly Japanese with a prevalence of 0.00028. Research has shown that HLA-B54 and HLA-A11 are positively associated with this disease. HLA-A, -B, and -DR loci were analyzed using an oligonucleotide microarray in both Chinese patients with DPB and normal control subjects. The most marked difference between the patients and the controls was the decreased frequency of HLA-A2 ( p = 0.001, OR = 0.12), which showed a negative association with the disease; however, there was no significant contribution of HLA-B loci. Interestingly, the frequency of HLA-A11 was increased ( p = 0.007, OR = 3.9), in accordance with previous reports on Japanese and Koreans. In addition, the frequency of HLA-DRB5*010/020 slightly increased ( p = 0.049). The HLA-associated genes for DPB are located between the HLA-A and HLA-B loci. Differences in HLA-associated genes may partially explain differences in the incidence of DPB among different populations.