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1.  Evaluation of the Effects of Quercetin on Damaged Salivary Secretion 
PLoS ONE  2015;10(1):e0116008.
With the aim of discovering an effective method to treat dry mouth, we analyzed the effects of quercetin on salivary secretion and its mechanism of action. We created a mouse model with impaired salivary secretion by exposure to radiation and found that impaired secretion is suppressed by quercetin intake. Moreover, secretion levels were enhanced in quercetin-fed normal mice. To elucidate the mechanisms of these effects on salivary secretion, we conducted an analysis using mouse submandibular gland tissues, a human salivary gland epithelial cell line (HSY), and mouse aortic endothelial cells (MAECs). The results showed that quercetin augments aquaporin 5 (AQP5) expression and calcium uptake, and suppresses oxidative stress and inflammatory responses induced by radiation exposure, suggesting that quercetin intake may be an effective method to treat impaired salivary secretion.
PMCID: PMC4309588  PMID: 25629520
2.  Synergistic Effect of Bolus Exposure to Zinc Oxide Nanoparticles on Bleomycin-Induced Secretion of Pro-Fibrotic Cytokines without Lasting Fibrotic Changes in Murine Lungs 
Zinc oxide (ZnO) nanoparticles are widely used in various products, and the safety evaluation of this manufactured material is important. The present study investigated the inflammatory and fibrotic effects of pulmonary exposure to ZnO nanoparticles in a mouse model of pulmonary fibrosis. Pulmonary fibrosis was induced by constant subcutaneous infusion of bleomycin (BLM). Female C57BL/6Jcl mice were divided into BLM-treated and non-treated groups. In each treatment group, 0, 10, 20 or 30 µg of ZnO nanoparticles were delivered into the lungs through pharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and the lungs were sampled at Day 10 or 14 after administration. Pulmonary exposure by a single bolus of ZnO nanoparticles resulted in severe, but transient inflammatory infiltration and thickening of the alveolar septa in the lungs, along with the increase of total and differential cell counts in BLAF. The BALF level of interleukin (IL)-1β and transforming growth factor (TGF)-β was increased at Day 10 and 14, respectively. At Day 10, the synergistic effect of BLM and ZnO exposure was detected on IL-1β and monocyte chemotactic protein (MCP)-1 in BALF. The present study demonstrated the synergistic effect of pulmonary exposure to ZnO nanoparticles and subcutaneous infusion of BLM on the secretion of pro-fibrotic cytokines in the lungs.
PMCID: PMC4307267  PMID: 25561223
zinc oxide nanoparticles; metal fume fever; pulmonary fibrosis; bleomycin; animal model
3.  Lobar analysis of collapsibility indices to assess functional lung volumes in COPD patients 
We investigated correlations between lung volume collapsibility indices and pulmonary function test (PFT) results and assessed lobar differences in chronic obstructive pulmonary disease (COPD) patients, using paired inspiratory and expiratory three dimensional (3D) computed tomography (CT) images.
We retrospectively assessed 28 COPD patients who underwent paired inspiratory and expiratory CT and PFT exams on the same day. A computer-aided diagnostic system calculated total lobar volume and emphysematous lobar volume (ELV). Normal lobar volume (NLV) was determined by subtracting ELV from total lobar volume, both for inspiratory phase (NLVI) and for expiratory phase (NLVE). We also determined lobar collapsibility indices: NLV collapsibility ratio (NLVCR) (%) = (1 − NLVE/NLVI) × 100%. Associations between lobar volumes and PFT results, and collapsibility indices and PFT results were determined by Pearson correlation analysis.
NLVCR values were significantly correlated with PFT results. Forced expiratory volume in 1 second, measured as percent of predicted results (FEV1%P) was significantly correlated with NLVCR values for the lower lobes (P<0.01), whereas this correlation was not significant for the upper lobes (P=0.05). FEV1%P results were also moderately correlated with inspiratory, expiratory ELV (ELVI,E) for the lower lobes (P<0.05). In contrast, the ratio of the diffusion capacity for carbon monoxide to alveolar gas volume, measured as percent of predicted (DLCO/VA%P) results were strongly correlated with ELVI for the upper lobes (P<0.001), whereas this correlation with NLVCR values was weaker for upper lobes (P<0.01) and was not significant for the lower lobes (P=0.26).
FEV1%P results were correlated with NLV collapsibility indices for lower lobes, whereas DLCO/VA%P results were correlated with NLV collapsibility indices and ELV for upper lobes. Thus, evaluating lobar NLV collapsibility might be useful for estimating pulmonary function in COPD patients.
PMCID: PMC4266257  PMID: 25525352
segmentation; emphysema; computed tomography; pulmonary function
4.  Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury 
Respiratory Research  2014;15(1):150.
Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin’s suppressive effects.
Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells.
Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects.
Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.
PMCID: PMC4276052  PMID: 25413579
Quercetin; Heme oxygenase 1; Intratracheal administration; Alveolar macrophage; Acute lung injury
5.  Significance of Pulmonary Arterial Pressure as a Prognostic Indicator in Lung-Dominant Connective Tissue Disease 
PLoS ONE  2014;9(9):e108339.
Lung-dominant connective tissue disease (LD-CTD) is a new concept for classifying the subset of patients with interstitial pneumonia who have clinical features suggesting an associated CTD, but whose features fall short of a clear diagnosis of CTD under the current rheumatologic classification systems. The impact of mean pulmonary arterial pressure (MPAP) in LD-CTD has not been sufficiently elucidated.
To evaluate the survival impact of MPAP measured during the initial evaluation in patients with LD-CTD.
We retrospectively analyzed the initial evaluation data of 100 LD-CTD patients undergoing pulmonary function test, 6-min walk test (6MWT), and right heart catheterization (RHC).
The mean MPAP was 16.2±4.4 mm Hg, and 18 patients had MPAP≥20 mm Hg. A univariate Cox proportional hazard model showed that MPAP and several variables have a statistically significant impact on survival. With stepwise, multivariate Cox proportional analysis, MPAP (HR  = 1.293; 95% CI 1.130–1.480; p<0.001) and mean forced vital capacity (FVC) % predicted (HR = 0.958; 95% CI 0.930–0.986; p = 0.004) were shown to be independent determinants of survival.
Higher MPAP and lower %FVC at the initial evaluation were significant independent prognostic factors of LD-CTD. MPAP evaluation provides additional information of disease status and will help physicians to predict mortality in LD-CTD.
PMCID: PMC4182458  PMID: 25268705
6.  Phase II study of erlotinib for previously treated patients with EGFR wild-type non-small-cell lung cancer, following EGFR mutation status reevaluation with the Scorpion Amplified Refractory Mutation System 
Molecular and Clinical Oncology  2014;2(6):991-996.
While assessing the efficacy of erlotinib in patients with epidermal growth factor receptor (EGFR) wild-type (WT) non-small-cell lung cancer (NSCLC), the sensitivity of the method used for the EGFR mutation analysis may affect the evaluation of the efficacy. We conducted a phase II study of erlotinib for previously treated patients with EGFR WT NSCLC screened by the peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) clamp method, which is known to be highly sensitive. The primary endpoint was the objective response rate (ORR). Preplanned reevaluation of the EGFR genotype as an exploratory endpoint was performed using the Scorpion Amplification Refractory Mutation System (S-ARMS) assay. Erlotinib was administered daily until disease progression or development of unacceptable toxicity. A total of 53 evaluable patients were enrolled. The histological subtypes were adenocarcinoma in 40 patients, squamous cell carcinoma in 9 patients and not otherwise specified NSCLC in 4 patients. Partial response (PR) was achieved in 6 patients (4 with adenocarcinoma and 2 with squamous cell carcinoma). The ORR was 11.3% [95% confidence interval (CI): 4.3–23.0]. The median progression-free survival (PFS) was 1.8 months (95% CI: 1.2–2.3). Samples from 26 of the 53 patients (49.0%) were available for EGFR mutation reanalysis with the S-ARMS assay. Of these 26 samples, only 1 sample of adenocarcinoma was found to be EGFR mutation-positive (exon 19 deletion) and the patient achieved a PR. The EGFR WT genotype was reconfirmed by the S-ARMS assay in the remaining 25 patients and 2 of these patients exhibited a PR. This study did not meet the primary endpoint, although erlotinib was found to be moderately effective in pretreated patients with EGFR WT NSCLC, even when the EGFR mutational status was confirmed by the highly sensitive PNA-LNA clamp PCR method.
PMCID: PMC4179810  PMID: 25279187
erlotinib; non-small-cell lung cancer; epidermal growth factor receptor wild-type
8.  Endobronchial ultrasound echoic image of pulmonary hamartoma 
Respirology Case Reports  2014;2(2):85-86.
A 62-year-old man with an indicated chest radiographic abnormality was referred to our hospital for more thorough examinations. Endobronchial ultrasound-guided transbronchial needle aspiration was performed because of a mass at the left hilum. Endobronchial ultrasound images showed scattered high-density spots in a low echoic and mosaic density. The pathological findings revealed pulmonary hamartoma. Subsequently, the mass was resected and comparison of ultrasound findings and pathological findings indicated that the scattered high echoic spots appeared to reflect cartilaginous tissues and bronchial epithelium inside the tumor.
PMCID: PMC4184515  PMID: 25473576
EBUS-TBNA; hamartoma; ultrasound image
9.  Changes in plasma levels of B-type natriuretic peptide with acute exacerbations of chronic obstructive pulmonary disease 
Elevated plasma B-type natriuretic peptide (BNP) levels and their association with heart failure have been reported in subjects with acute exacerbations of chronic obstructive pulmonary disease (AECOPD).
To examine and compare plasma BNP levels and diastolic and systolic dysfunction in subjects with AECOPD and stable chronic obstructive pulmonary disease (COPD).
In all, 87 unselected consecutive hospitalizations due to AECOPD in 61 subjects and a total of 190 consecutive subjects with stable COPD were recruited. Plasma BNP levels were compared cross-sectionally and longitudinally. Transthoracic echocardiographic examinations were also performed in the hospitalized subjects.
In the hospitalized subjects, the median plasma BNP level (interquartile range) was 55.4 (26.9–129.3) pg/mL and was higher than that of patients with stable COPD: 18.3 (10.0–45.3) for Global Initiative for Chronic Obstructive Lung Disease grade I; 25.8 (11.0–53.7) for grade II; 22.1 (9.1–52.6) for grade III; and 17.2 (9.6–22.9) pg/mL for grade I V, all P<0.001. In 15 subjects studied prospectively, the median plasma BNP level was 19.4 (9.8–32.2) pg/mL before AECOPD, 72.7 (27.7–146.3) pg/mL during AECOPD, and 14.6 (12.9–39.0) pg/mL after AECOPD (P<0.0033 and P<0.0013, respectively). Median plasma BNP levels during AECOPD were significantly higher in ten unsuccessfully discharged subjects 260.5 (59.4–555.0) than in 48 successfully discharged subjects 48.5 (24.2–104.0) pg/mL (P=0.0066). Only 5.6% of AECOPD subjects were associated with systolic dysfunction defined as a left ventricular ejection fraction (LVEF) <50%; a further 7.4% were considered to have impaired relaxation defined as an E/A wave velocity ratio <0.8 and a deceleration time of E >240 ms. BNP levels were weakly correlated with the E/peak early diastolic velocity of the mitral annulus (Ea) ratio (Spearman’s rank correlation coefficient =0.353, P=0.018), but they were not correlated with the LVEF (Spearman’s rank correlation coefficient =−0.221, P=0.108).
A modest elevation of plasma BNP is observed during AECOPD. It appears that AECOPD may have an impact on plasma BNP levels that is not attributable to heart failure.
PMCID: PMC3921082  PMID: 24523584
chronic obstructive pulmonary disease; acute exacerbations of chronic obstructive pulmonary disease; B-type natriuretic peptide; heart failure
10.  Clinical impact of prevalence and severity of COPD on the decision-making process for therapeutic management of lung cancer patients 
Recent studies suggest that coexistence of chronic obstructive pulmonary disease (COPD) might be independently related to a worse prognosis for lung cancer. However, because data on the substantial prevalence of COPD and its severity in Asian lung cancer patients remain limited, clinical impact of prevalence and severity of COPD among the population has not been fully evaluated. Furthermore, patients with COPD often have comorbidities. Thus, whether the decision-making process for therapeutic management of lung cancer patients might be independently affected by COPD remains elusive.
Clinical impact of prevalence and severity of COPD were evaluated in 270 Japanese patients with newly diagnosed lung cancer who were sequentially registered and underwent bronchoscopy from August 2010 to July 2012 at Nagoya University hospital. Furthermore, to explore whether or not the severity of airflow obstruction might affect the decision to propose thoracic surgery with curative intent, we evaluated data from patients with lung cancer at stage 1A to 3A who underwent spirometry and bronchoscopy.
The prevalence rate of COPD was 54.4% among Japanese patients with lung cancer who underwent bronchoscopy. The incidence of Global Initiative for Chronic Obstructive Lung Disease (GOLD) grades 1 and 2 was significantly higher than that of GOLD grade 3. Although COPD-related comorbidities were not independent factors for proposing thoracic surgery, the number of thoracic surgeries performed was significantly less in the COPD group than the non-COPD group. Multivariate analysis showed that more severe airway obstruction, advanced clinical staging, and higher age, were independent factors associated with the decision on thoracic surgery.
We demonstrated a high prevalence of COPD among Japanese lung cancer patients. Based on the knowledge that severity of COPD is one of the most important factors in the therapeutic decision, comprehensive assessment of COPD at bronchoscopy might allow us to implement the optimum management for lung cancer patients.
PMCID: PMC3922111  PMID: 24498965
Chronic obstructive lung disease; Bronchoscopy; Spirometry screening assessment; Thoracic surgery; Japanese population
11.  The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma 
Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21WAF1/CIP1 and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.
PMCID: PMC3479344  PMID: 22510946
apoptosis; BMAL1; mesothelioma; targeted therapy; mitotic catastrophe
12.  Involvement of TGFβ-Induced Phosphorylation of the PTEN C-Terminus on TGFβ-Induced Acquisition of Malignant Phenotypes in Lung Cancer Cells 
PLoS ONE  2013;8(11):e81133.
Transforming growth factor β (TGFβ) derived from the tumor microenvironment induces malignant phenotypes such as epithelial-mesenchymal transition (EMT) and aberrant cell motility in lung cancers. TGFβ-induced translocation of β-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is known to exert phosphatase activity by binding to E-cadherin complexes via β-catenin, and recent studies suggest that phosphorylation of the PTEN C-terminus tail might cause loss of this PTEN phosphatase activity. However, whether TGFβ can modulate both β-catenin translocation and PTEN phosphatase activity via phosphorylation of the PTEN C-terminus remains elusive. Furthermore, the role of phosphorylation of the PTEN C-terminus in TGFβ-induced malignant phenotypes has not been evaluated. To investigate whether modulation of phosphorylation of the PTEN C-terminus can regulate malignant phenotypes, here we established lung cancer cells expressing PTEN protein with mutation of phosphorylation sites in the PTEN C-terminus (PTEN4A). We found that TGFβ stimulation yielded a two-fold increase in the phosphorylated -PTEN/PTEN ratio. Expression of PTEN4A repressed TGFβ-induced EMT and cell motility even after snail expression. Our data showed that PTEN4A might repress EMT through complete blockade of β-catenin translocation into the cytoplasm, besides the inhibitory effect of PTEN4A on TGFβ-induced activation of smad-independent signaling pathways. In a xenograft model, the tumor growth ratio was repressed in cells expressing PTEN4A. Taken together, these data suggest that phosphorylation sites in the PTEN C-terminus might be a therapeutic target for TGFβ-induced malignant phenotypes in lung cancer cells.
PMCID: PMC3838341  PMID: 24278390
13.  Influence of cheek support on respiratory impedance measured by forced oscillation technique 
SpringerPlus  2013;2:342.
The forced oscillation technique (FOT) is a useful tool to assess respiratory resistance and reactance during tidal breathing in patients with respiratory diseases, specifically asthma and chronic obstructive pulmonary disease. Although the FOT has been clinically used, results of respiratory impedance can be affected by various factors such as upper airway artifact. We investigated the effects of cheek support on respiratory resistance and reactance measured by a commercially available FOT equipment MostGraph-01. Respiratory resistance at 20 Hz (R20) with support of the cheeks was significantly higher than those without the cheek support in healthy subjects. Two different cheek support protocols, support of the cheeks by subjects themselves and an operator, were compared in healthy volunteers and patients with respiratory diseases. The cheek support protocols significantly affected respiratory resistance at 5 Hz (R5) and reactance at 5 Hz (X5) in the patient group but not in the healthy subjects. Moreover, for X5, there was a significant interaction between cheek support protocols (by a subject or operator) and groups (healthy or diseased). In conclusion, during impedance measurements using the FOT, application of cheek support either by subjects or the operator is recommended to reduce upper airway artifacts, however, results obtained by two protocols may be different in patients with respiratory diseases. Contribution of the chest wall and position of the arms to the mechanical properties should be carefully considered in physiological studies in which the FOT is attempted.
PMCID: PMC3733071  PMID: 23961407
Asthma; Cheek support; COPD; Forced oscillation technique; MostGraph; Respiratory resistance; Reactance; Upper airway shunt
14.  COPD and disease-specific health status in a working population 
Respiratory Research  2013;14(1):61.
It has been debated whether treatment should be started early in subjects with mild to moderate COPD. An impaired health status score was associated with a higher probability of being diagnosed with COPD as compared with undiagnosed COPD.
To investigate the health status in a healthy working population, to determine reference scores for healthy non-smoking subjects, and to investigate the relationship between their health status and airflow limitation.
A total of 1333 healthy industrial workers aged ≥40 years performed spirometry and completed the St. George’s Respiratory Questionnaire (SGRQ) and the COPD Assessment Test (CAT).
The prevalence of COPD defined by the fixed ratio of the forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) was 10.9%, and the prevalence defined by the Lower Limit of Normal was 5.0%. All SGRQ and CAT scores were skewed to the milder end. In 512 non-smoking subjects with normal spirometry, the mean SGRQ score was 5.7, and the mean CAT score was 5.8. In 145 people with COPD defined by the fixed ratio, the mean SGRQ score was 7.9, with a zero score in 6.9% of the subjects. Using the CAT, the mean score was 7.3, with 7.6% of the scores being zero. The scores in patients identified using the Lower Limit of Normal approach were: SGRQ 8.4 (13.4% had a score of zero) and CAT 7.4 (13.4% had a score of zero). Although the 95th percentiles of the Total, Symptoms, Activity, and Impact scores of the SGRQ and CAT sores were 13.8, 34.0, 23.4, 7.2 and 13.6 in the 512 healthy non-smoking subjects, respectively, they were also distributed under their upper limits in over 80% of the COPD subjects.
The COPD-specific health status scores in a working population were good, even in those with spirometrically diagnosed COPD. All scores were widely distributed in both healthy non-smoking subjects and in subjects with COPD, and the score distribution overlapped remarkably between these two groups. This suggests that symptom-based methods are not suitable screening tools in a healthy general population.
PMCID: PMC3679860  PMID: 23725096
Chronic obstructive pulmonary disease; St. George’s Respiratory Questionnaire; Symptoms and COPD; Smoking and health; Health related quality of life
15.  Inspiratory capacity as a preoperative assessment of patients undergoing thoracic surgery 
Although inspiratory capacity (IC) is strongly associated with the disease severity of chronic obstructive pulmonary disease, there was no appropriate equation to compute predicted values for IC. Furthermore, whether assessment of IC can identify the risk of prolonged postoperative stay (PPS) in patients undergoing thoracic surgery also remains unclear. To evaluate whether %IC predicted, for which the new equation to compute the predicted values for IC was utilized, could be applied to identify the risk of PPS, we retrospectively analysed the cases of 412 patients who underwent thoracic surgery in Nagoya University Hospital. The multivariate analysis demonstrated that %IC predicted < 85% was one of the most critical risk predictors for PPS (odds ratio, 1.65; 95% confidence intervals, 1.03–2.648) and, in particular, was independent of percentage predicted forced expiratory volume in 1 s (%FEV1) < 80%. A combined assessment of ICFEV1 Low, defined as %IC predicted <85% or %FEV1 <80%, was able to identify more than double the number of patients with PPS, compared with %FEV1 <80% alone (65.9 vs. 28.5%, respectively). This is the first study to demonstrate the significance of %IC predicted in screening for the risk for PPS in patients undergoing thoracic surgery.
PMCID: PMC3329291  PMID: 22307392
Chronic obstructive lung disease; Inspiratory capacity; Lung cancer; Screening assessment; Surgical complications
16.  STIM1 Regulates Platelet-Derived Growth Factor-Induced Migration and Ca2+ Influx in Human Airway Smooth Muscle Cells 
PLoS ONE  2012;7(9):e45056.
It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca2+ concentrations ([Ca2+]i) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca2+ sensor that activates Orai1, the Ca2+ channel responsible for store-operated Ca2+ entry (SOCE). We investigated the role of STIM1 in [Ca2+]i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca2+-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca2+]i followed by sustained [Ca2+]i elevation. Sustained increases in [Ca2+]i due to PDGF-BB were significantly inhibited by a Ca2+ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca2+]i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca2+ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.
PMCID: PMC3439366  PMID: 22984609
17.  Transient but Not Stable ZEB1 Knockdown Dramatically Inhibits Growth of Malignant Pleural Mesothelioma Cells 
Annals of surgical oncology  2011;19(Suppl 3):634-645.
The role of ZEB1, a master epithelial-tomesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear.
The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference–mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity.
Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1 knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent.
RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.
PMCID: PMC3413790  PMID: 22086445
18.  Pivotal role of epithelial cell adhesion molecule in the survival of lung cancer cells 
Cancer Science  2011;102(8):1493-1500.
Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers including lung cancer, and its contribution to increased proliferation through upregulation of cell cycle accelerators such as cyclins A and E has been well established in breast and gastric cancers. Nevertheless, very little is known about its role in supporting the survival of cancer cells. In addition, the functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. In this study, we show that RNAi-mediated knockdown of EpCAM suppresses proliferation and clonogenic growth of three EpCAM-expressing lung cancer cell lines (H3255, H358, and HCC827), but does not induce cell cycle arrest in any of these. In addition, EpCAM knockdown inhibits invasion in the highly invasive H358 but not in less invasive H3255 cells in a Transwell assay. Of note, the EpCAM knockdown induces massive apoptosis in the three cell lines as well as in another EpCAM-expressing lung cancer cell line, HCC2279, but to a much lesser extent in a cdk4/hTERT immortalized normal human bronchial epithelial cell line, HBEC4, suggesting that EpCAM could be a therapeutic target for lung cancer. Finally, EpCAM knockdown partially restores contact inhibition in HCC827, in association with p27Kip1 upregulation. These results indicate that EpCAM could contribute substantially to the pathogenesis of lung cancer, especially cancer cell survival, and suggest that EpCAM targeted therapy for lung cancer may have potential.
PMCID: PMC3381954  PMID: 21535318
19.  ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility 
Molecular Biology of the Cell  2011;22(20):3840-3852.
Using a library of siRNAs, we found that ARHGAP18 was essential for the organization of actin stress fibers and focal adhesion. ARHGAP18 is one of the crucial factors for the regulation of RhoA in order to control cell motility and spreading.
Rho GTPases are molecular switches that transmit biochemical signals in response to extracellular stimuli to elicit changes in the actin cytoskeleton. Rho GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. These states are regulated by two distinct families of proteins—guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We studied the role of a previously uncharacterized GAP, ARHGAP18 (MacGAP). Overexpression of ARHGAP18 suppressed the activity of RhoA and disrupted stress fiber formation. Conversely, silencing of ARHGAP18 by small interfering RNA transfection–enhanced stress fiber formation and induced rounding of cells. We examined the role of ARHGAP18 in cell spreading and migration. Immunofluorescence analysis revealed that ARHGAP18 was localized to the leading edge during cell spreading and migration. ARHGAP18-knockdown cells showed impaired spreading, premature formation of stress fibers, and sustained activation of RhoA upon cell attachment. In addition, knockdown and overexpression of ARHGAP18 resulted in the inhibition and promotion of cell migration, respectively. Furthermore, ARHGAP18 was required for the polarization of cells for migration. Our results define ARHGAP18 as one of the crucial factors for the regulation of RhoA for the control of cell shape, spreading, and migration.
PMCID: PMC3192863  PMID: 21865595
20.  Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells 
PLoS ONE  2011;6(10):e26384.
Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.
PMCID: PMC3195692  PMID: 22022610
21.  Endothelial–Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis 
The pathological hallmark lesions in idiopathic pulmonary fibrosis are the fibroblastic foci, in which fibroblasts are thought to be involved in the tissue remodeling, matrix deposition, and cross-talk with alveolar epithelium. Recent evidence indicates that some fibroblasts in fibrosis may be derived from bone marrow progenitors as well as from epithelial cells through epithelial–mesenchymal transition. To evaluate whether endothelial cells could represent an additional source for fibroblasts, bleomycin-induced lung fibrosis was established in Tie2-Cre/CAG-CAT-LacZ double-transgenic mice, in which LacZ was stably expressed in pan-endothelial cells. Combined X-gal staining and immunocytochemical staining for type I collagen and α-smooth muscle actin revealed the presence of X-gal–positive cells in lung fibroblast cultures from bleomycin-treated mice. To explore the underlying mechanisms, by which loss of endothelial-specific markers and gain of mesenchymal phenotypes could be involved in microvascular endothelial cells, the effects of activated Ras and TGF-β on the microvascular endothelial cell line MS1 were analyzed. Combined treatment with activated Ras and TGF-β caused a significant loss of endothelial-specific markers, while inducing de novo mesenchymal phenotypes. The altered expression of these markers in MS1 cells with activated Ras persisted after withdrawal of TGF-β in vitro and in vivo. These findings are the first to show that lung capillary endothelial cells could give rise to significant numbers of fibroblasts through an endothelial–mesenchymal transition in bleomycin-induced lung fibrosis model.
PMCID: PMC2937229  PMID: 19767450
fibroblasts; myofibroblasts; endothelial cells; LacZ; fibrosis
22.  Actin Cytoskeleton Regulates Stretch-Activated Ca2+ Influx in Human Pulmonary Microvascular Endothelial Cells 
During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), regions of the lung are exposed to excessive stretch, causing inflammatory responses and further lung damage. In this study, the effects of mechanical stretch on intracellular Ca2+ concentration ([Ca2+]i), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uniaxial stretching, using a cell-stretching apparatus. After stretching and subsequent unloading, [Ca2+]i, as measured by fura-2 fluorescence, was transiently increased in a strain amplitude–dependent manner. The elevation of [Ca2+]i induced by stretch was not evident in the Ca2+-free solution and was blocked by Gd3+, a stretch-activated channel inhibitor, or ruthenium red, a transient receptor potential vanilloid inhibitor. The disruption of actin polymerization with cytochalasin D inhibited the stretch-induced elevation of [Ca2+]i. In contrast, increases in [Ca2+]i induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced elevation of [Ca2+]i. A simple network model of the cytoskeleton was also developed in support of the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca2+ channels. In conclusion, mechanical stretch activates Ca2+ influx via stretch-activated channels which are tightly regulated by the actin cytoskeleton different from other Ca2+ influx pathways such as receptor-operated and store-operated Ca2+ entries in HPMVECs. These results suggest that abnormal Ca2+ homeostasis because of excessive mechanical stretch during mechanical ventilation may play a role in the progression of ALI/ARDS.
PMCID: PMC2911568  PMID: 19648475
Ca2+ channels; F-actin; mechanotransduction; mechanical stress; stretch-activated channel
23.  A New Microarray System to Detect Streptococcus pneumoniae Serotypes 
Streptococcus pneumoniae, one of the most common gram-positive pathogens to colonize the human upper respiratory tract, is responsible for many severe infections, including meningitis and bacteremia. A 23-valent pneumococcal vaccine is available to protect against the 23 S. pneumoniae serotypes responsible for 90% of reported bacteremic infections. Unfortunately, current S. pneumoniae serotype testing requires a large panel of expensive antisera, assay results may be subjective, and serotype cross-reactions are common. For this study, we designed an oligonucleotide-based DNA microarray to identify glycosyltransferase gene sequences specific to each vaccine-related serotype. Out of 56 isolates representing different serotypes, only one isolate, representing serotype 23A, was not detected correctly as it could not be distinguished from serotype 23F. Our data suggest that the microarray provides a more cost-effective and reliable way of monitoring pneumococcal capsular types.
PMCID: PMC3118663  PMID: 21716703
24.  Knockdown of ZEB1, a master epithelial-to-mesenchymal transition (EMT) gene, suppresses anchorage-independent cell growth of lung cancer cells 
Cancer letters  2010;296(2):216-224.
We found that among four master epithelial-to-mesenchymal transition (EMT)-inducing genes (ZEB1, SIP1, Snail, and Slug) ZEB1expression was most significantly correlated with the mesenchymal phenotype (high Vimentin and low E-cadherin expression) in non-small cell lung cancer (NSCLC) cell lines and tumors. Furthermore, ZEB1 knockdown with RNA interference in three NSCLC cell lines with high ZEB1 expression suppressed to varying degrees mass culture growth and liquid colony formation but in all cases dramatically suppressed soft agar colony formation. In addition, ZEB1 knockdown induced apoptosis in one of the three lines, indicating that the growth inhibitory effects of ZEB1 knockdown occurs in part through the activation of the apoptosis pathway. These results suggest that inhibiting ZEB1 function may be an attractive target for NSCLC therapeutic development.
PMCID: PMC3110825  PMID: 20452118
Lung cancer; Epidermal growth factor receptor; Anchorage-independent growth; EMT; MicroRNA; RNA interference
25.  Clinical characteristics of Pneumocystis pneumonia in non-HIV patients and prognostic factors including microbiological genotypes 
The number of patients with non-HIV Pneumocystis pneumonia (PCP) is increasing with widespread immunosuppressive treatment. We investigated the clinical characteristics of non-HIV PCP and its association with microbiological genotypes.
Between January 2005 and March 2010, all patients in 2 university hospitals who had been diagnosed with PCP by PCR were enrolled in this study. Retrospective chart review of patients, microbiological genotypes, and association with 30-day mortality were examined.
Of the 82 adult patients investigated, 50 patients (61%) had inflammatory diseases, 17 (21%) had solid malignancies, 12 (15%) had hematological malignancies, and 6 (7%) had received transplantations. All patients received immunosuppressive agents or antitumor chemotherapeutic drugs. Plasma (1→3) β-D-glucan levels were elevated in 80% of patients, and were significantly reduced after treatment in both survivors and non-survivors. However, β-D-glucan increased in 18% of survivors and was normal in only 33% after treatment. Concomitant invasive pulmonary aspergillosis was detected in 5 patients. Fifty-six respiratory samples were stored for genotyping. A dihydropteroate synthase mutation associated with trimethoprim-sulfamethoxazole resistance was found in only 1 of the 53 patients. The most prevalent genotype of mitochondrial large-subunit rRNA was genotype 1, followed by genotype 4. The most prevalent genotype of internal transcribed spacers of the nuclear rRNA operon was Eb, followed by Eg and Bi. Thirty-day mortality was 24%, in which logistic regression analysis revealed association with serum albumin and mechanical ventilation, but no association with genotypes.
In non-HIV PCP, poorer general and respiratory conditions at diagnosis were independent predictors of mortality. β-D-glucan may not be useful for monitoring the response to treatment, and genotypes were not associated with mortality.
PMCID: PMC3073915  PMID: 21439061

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