Effect of hyperhomocysteinemia on a murine model of smoke-induced pulmonary emphysema.

Hyperhomocysteinemia was reported to enhance endoplasmic reticulum (ER) stress and subsequent apoptosis in several cells. However, the precise mechanisms of smoking susceptibility associated with hyperhomocysteinemia has not been fully elucidated. This study included 7- to 9-week-old C57BL6 male mice induced with hyperhomocysteinemia and were exposed to cigarette smoke (CS). A549 cells (human alveolar epithelial cell line) were cultured with homocysteine and were exposed to cigarette smoke extract (CSE) to observe cell viability and expression of proteins related to the ER stress. After 6 months of CS exposure, pulmonary emphysema was more severely induced in the group under the condition of hyperhomocysteinemia compared to that in the control group. The apoptotic A549 cells increased as homocysteine concentration increased and that was enhanced by CSE. Protein expression levels of ER stress markers were significantly increased after simultaneous stimulation. Notably, vitamin B12 and folate supplementation improved ER stress after simultaneous stimulation of A549 cells. In this study, we showed that hyperhomocysteinemia exacerbates CS exposure-induced emphysema in mice, suggesting that hyperhomocysteinemia and CS stimulation enhance ER stress and subsequent induced apoptosis in alveolar epithelial cells. It was suggested that there is a synergistic effect between homocysteine and CS.

Effect of Iron Deficiency on a Murine Model of Smoke-induced Emphysema.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD). Smoking susceptibility is important for the onset and development of COPD. We previously reported an association between serum iron concentrations and pulmonary function in male smokers. However, the mechanism governing smoking susceptibility in relation to iron deficiency is unclear; this study aimed to elucidate this mechanism. C57BL/6 male mice were fed an iron-deficient or normal diet and then exposed to cigarette smoke. BAL, histological analysis, and pulmonary function tests were performed after cigarette smoke exposure. Human alveolar type II epithelial A549 cells were treated with an iron chelator. Subsequently, A549 cells were exposed to cigarette smoke extract. In mice exposed to cigarette smoke for 2 weeks, the concentration of alveolar macrophages in the BAL fluid recovered from iron-deficient mice was significantly higher than that in normal diet mice. IL-6 and MCP-1 (monocyte chemotactic protein 1) concentrations in the BAL fluid increased significantly from baseline in iron-deficient mice, but not in normal diet mice. In mice exposed to cigarette smoke for 8 weeks, the pathological mean linear intercepts, physiological total lung capacity, and functional residual capacity in the lungs of iron-deficient mice were significantly greater than in normal diet mice. Phosphorylation of NF-κB was enhanced in the lungs of iron-deficient mice exposed to cigarette smoke and in the iron-chelating A549 cells exposed to cigarette smoke extract. Iron deficiency exaggerated cigarette smoke-induced pulmonary inflammation, suggesting that it may accelerate COPD development.

Impact of cigarette smoking on decline in forced expiratory volume in 1s relative to severity of airflow obstruction in a Japanese general population: The Yamagata-Takahata study.

BACKGROUND: Few studies are available regarding the annual decline of forced expiratory volume in 1s (FEV1) in chronic obstructive pulmonary disease patients with mild airflow obstruction. This study sought to clarify to what extent cigarette-smoking individuals with mild airflow obstruction lose pulmonary function annually. METHODS: From 2004 to 2006, pulmonary function tests were performed on people >40 years of age, during the annual health checkup held in Takahata, Yamagata, Japan (initial study population, n=3253). In 2011, pulmonary function tests were performed again on participants who agreed to undergo reexamination (follow-up study population, n=838). RESULTS: Smokers have decreased pulmonary function in terms of percent forced vital capacity (FVC), %FEV1, and FEV1/FVC; the stages of airflow obstruction were also more severe in smokers than never-smokers. The annual decline in FEV1 was significantly greater in smokers than in never-smokers. The median annual decline in FEV1 was most significant in individuals with mild airflow obstruction. The annual decline in FEV1 was greater in smokers with mild airflow obstruction than in smokers with moderate airflow obstruction. In analyzing the decline in %FEV1, the annual change in smokers with mild airflow obstruction was greater than that in smokers with normal spirometric values. CONCLUSION: The annual decline in FEV1 was most significant in smokers with mild airflow obstruction in a Japanese general population. This highlights the importance of early detection of chronic obstructive pulmonary disease patients among the general population in order to prevent disease progression in undiagnosed patients.

MafB enhances efferocytosis in RAW264.7 macrophages by regulating Axl expression.

The transcription factor MafB is involved in cellular differentiation and phagocytosis in macrophages. Macrophages phagocytose apoptotic cells in vivo; this process, which is known as efferocytosis, requires Axl receptor tyrosine kinase (Axl) activity. However, the association between MafB and efferocytosis, as well as that between MafB and Axl, in macrophages is unknown. We hypothesized that MafB modulates macrophage efferocytosis by regulating Axl expression. Fluorescent-labeled apoptotic thymocytes were added to RAW264.7-MafB-shRNA and control cells, and the proportion of phagocytosis-positivey fluorescence microscopy and flow cytometry. In addition, Axl mRNA and protein were quantified by real-time PCR and western blotting in each group. RAW264.7-MafB-shRNA cells were transfected with a plasmid expressing green fluorescent protein (GFP)-tagged Axl or a control empty plasmid expressing only GFP. The capacity for phagocytosis of apoptotic cells was assessed in GFP-positive cells gated based on fluorescence intensity. In RAW264.7-MafB-shRNA cells, capacity for phagocytosis of apoptotic thymocytes was significantly reduced compared with that of control cells, as determined by fluorescence microscope and flow cytometry. Axl mRNA and protein expression was significantly reduced in RAW264.7-MafB-shRNA cells relative to control cells. Furthermore, the capacity of RAW264.7-MafB-shRNA cells, transfected with an Axl-expressing plasmid, for phagocytosis of apoptotic thymocytes was significantly greater than that of cells transfected with the control plasmid. Collectively, the present findings indicate that MafB enhances efferocytosis by regulating Axl expression in RAW264.7 macrophages.

MafB silencing in macrophages does not influence the initiation and growth of lung cancer induced by urethane.

An increased number of tumor-associated macrophages (TAMs) that exhibit the M2 macrophage phenotype is related to poorer prognosis in cancer patients. MafB is a transcription factor regulating the differentiation of macrophages. However, involvement of MafB for the development of TAMs is unknown. This study was designed to investigate the role of MafB in a murine urethane-induced lung cancer model. Urethane was injected intraperitoneally into wild-type and dominant-negative MafB transgenic mice. Twenty-four weeks later, mice were sacrificed and their lungs removed for pathological analysis. The numbers and mean areas of lung cancer were evaluated. In addition, the numbers of Mac-3-positive macrophages were evaluated in each tumor. The numbers and mean areas of lung cancer induced by urethane administration were not significantly different between wild-type and dominant-negative MafB transgenic mice. The numbers of TAMs in lung cancer tissue were not significantly different between the two groups. MafB silencing using dominant-negative MafB did not influence the initiation and growth of lung cancer in mice exposed to urethane. These data suggest that MafB may not be related to the development of TAMs.

Role of chemokine C-C motif ligand-1 in acute and chronic pulmonary inflammations.

BACKGROUND: Chemokine C-C motif ligand 1 (CCL1) accumulates C-C motif chemokine receptor 8 positive leukocytes to the inflammatory sites. Single-nucleotide polymorphisms in the chemokine CCL1 gene are associated with exacerbation of chronic obstructive lung disease. However, it is unclear whether CCL1 has immunomodulatory functions during pulmonary inflammation. This study aimed to elucidate this issue using newly generated transgenic mice that express CCL1 in the lungs (SPC-CCL1 mice). METHODS: To evaluate the phenotypes of these mice, lung section and bronchoalveolar lavage (BAL) fluid analyses were performed. We intratracheally administered lipopolysaccharide (LPS) or Mycobacterium bovis as a model of acute or chronic lung inflammation, respectively. RESULTS: No histological differences were observed between lung tissue from SPC-CCL1 Tg and wild-type mice in the resting condition and after LPS administration. In the resting condition, the total BAL cell concentration was lower in SPC-CCL1 Tg mice than in wild-type mice (P = 0.0097). Flow cytometric analyses showed that SPC-CCL1 Tg mice had fewer F4/80-positive cells than wild-type mice (P = 0.0278). After intratracheal LPS administration, CCL1 overexpression changed neither the total numbers nor population of BAL cells. After mycobacterial administration, pulmonary granuloma formation was significantly enhanced. The degree of Immunostaining for endoplasmic reticulum to nucleus signaling 1, a molecule associated with granuloma formation and endoplasmic reticulum stress, was significantly enhanced in the granuloma regions of SPC-CCL1 mice treated with Mycobacterium, compared to those of wild-type mice. CONCLUSIONS: CCL1 overexpression in the lungs did not change the acute inflammatory response induced by LPS, but enhanced granuloma formation after mycobacterial treatment, possibly through enhancing endoplasmic reticulum stress.

Impact of cigarette smoking on maximal expiratory flows in a general population: the Takahata study.

BACKGROUND: Maximal expiratory flows (MEFs) depend on the elastic recoil pressure in the alveoli, airway resistance and bronchial collapsibility. MEFs at lower levels of vital capacity [MEFs at x% FVC (MEF(x))] would indicate the patency of peripheral airways. In Japan, a ratio of MEF(50) to MEF(25) (MEF(50)/MEF(25)) greater than 4.0 is used as an index of injury to the small airways in subjects without airflow limitation. However, to date there have been no epidemiological investigations relating to this index. The aim of this study was to evaluate the impact of cigarette smoking on MEFs in a general population, and to assess the validity of using this index to evaluate injury to the small airways. METHODS: Subjects aged 40 years or older (n=2,917), who had participated in a community-based annual health-check in Takahata, Japan, were enrolled in the study. MEF(75), MEF(50) and MEF(25) were measured in these subjects. RESULTS: In smokers, as compared with never-smokers, the percentage predicted MEFs (%MEFs) decreased according to the aging of the population, except in the case of %MEF(25) in females. In males, but not in females, %MEFs decreased significantly with an increase in cigarette consumption. In both genders, MEF(50)/MEF(25) was slightly, but significantly, elevated with aging of the population. In addition, 36.5% of subjects who participated in this health-check had MEF(50)/MEF(25) values greater than 4.0. No difference in MEF(50)/MEF(25) was observed between smokers and never-smokers. CONCLUSION: Cigarette smoking enhanced the age-related decline in MEFs. Since many healthy subjects aged 40 years or older have MEF(50)/MEF(25) values greater than 4.0, the use of this criterion may over-estimate the presence of small airway disease.

Impairment of pulmonary function is an independent risk factor for atrial fibrillation: the Takahata study.

BACKGROUND: Chronic pulmonary disorders, such as chronic obstructive pulmonary disease (COPD) and fibrosing lung diseases, and atrial fibrillation (AF), are prevalent in elderly people. The impact of cardiac co-morbidities in the elderly, where pulmonary function is impaired, cannot be ignored as they influence mortality. The relationship between the prevalence of AF and pulmonary function is unclear. The aim of this study was to evaluate this relationship in participants in a health check. METHODS: Subjects aged 40 or older (n = 2,917) who participated in a community-based annual health check in Takahata, Japan, from 2004 through to 2005, were enrolled in the study. We performed blood pressure measurements, blood sampling, electrocardiograms, and spirometry on these subjects. RESULTS: The mean FEV(1) % predicted and FVC % predicted in AF subjects was significantly lower than in non-AF subjects. The prevalence of AF was higher in those subjects with airflow limitation or lung restriction than in those without. Furthermore, AF prevalence was higher in those subjects with severe airflow obstruction (FEV(1) %predicted < 50) than in those who had mild or moderate airflow obstruction (FEV(1) %predicted ≥ 50), although there was no difference between the prevalence of AF in subjects with 70≤ FVC %predicted <80 lung restriction and those with FVC %predicted <70. Multiple logistic regression analysis revealed that FEV(1) %predicted and FVC %predicted are independent risk factors for AF (independent of age, gender, left ventricular hypertrophy, and serum levels of B-type natriuretic peptide). CONCLUSION: Impaired pulmonary function is an independent risk factor for AF in the Japanese general population.

The relationship between serum uric acid and spirometric values in participants in a health check: the Takahata study.

BACKGROUND: Tissue hypoxia induces the degradation of adenosine triphosphate, resulting in the production of uric acid (UA). Patients with chronic obstructive pulmonary disease (COPD) have been reported to have high serum levels of UA (sUA), compared with control subjects. However, the relationship between sUA levels and spirometric measures has not been investigated in detail in a general population. METHODS: Subjects aged 40 years or older (n = 2,917), who had participated in a community-based annual health check in Takahata, Japan, in 2004 and 2005, were enrolled in the study. These subjects performed spirometry, their blood pressure was measured, and a blood sample was taken. RESULTS: sUA levels were significantly higher in males than in females. Percent predicted forced vital capacity [FVC %predicted] (r = -0.13) and forced expiratory volume in 1 s [FEV(1) %predicted] (r = -0.118) were inversely correlated with sUA levels in females but not in males. Univariate regression analysis indicated that age, body mass index (BMI), ethanol intake, mean blood pressure (BP), and serum creatinine (sCr) were significantly associated with sUA levels in males. In females, age, BMI, mean BP, hemoglobin A1c, sCr, FVC %predicted, and FEV(1) %predicted were significantly associated with sUA levels. Multiple linear regression analysis showed that for both genders, FVC %predicted and FEV(1) %predicted were predictive for sUA levels, independently of the other clinical parameters. Subjects with lung restriction had higher sUA levels than subjects without lung restriction. In addition, subjects with moderate and severe airflow limitation had higher sUA levels than subjects without airflow limitation or those with mild airflow limitation. CONCLUSION: FVC %predicted and FEV(1) %predicted were significantly associated with sUA levels in a general population.

FEV6 and FEV1/FEV6 in Japanese participants of the community-based annual health check: the Takahata study.

BACKGROUND: Forced expiratory volume in 6 seconds (FEV(6)) is becoming a substitute of forced vital capacity (FVC). However, the Japanese predictive equation for FEV(6) has not been established, and the validity for the use of FEV(1)/FEV(6) for diagnosing airflow limitation in Japanese has not been confirmed. METHODS: Subjects aged 40 or older, who had participated in a community-based health check in Takahata, Japan, from 2004 through 2005, were enrolled. The smoking histories of these subjects were investigated using a self-reporting questionnaire. FVC, FEV(1), and FEV(6) were measured using spirometric machines. Predictive equations of FEV(6) were obtained from never-smoking subjects without history of pulmonary diseases by multiple linear regression assay. RESULTS: FEV(6) and FEV(1)/FEV(6) were significantly correlated with FVC (r=0.998, p<0.001) and FEV(1)/FVC (r=0.989, p<0.001), respectively. The cutoff values of percent predicted (%) FEV(6) and FEV(1)/FEV(6) for discrimination of having the restrictive lung disorder determined by %FVC <0.8 and having the airflow limitation determined by FEV(1)/FVC <0.7 were 0.80 and 0.72, respectively (%FEV(6): sensitivity=0.995, specificity=0.983, positive predictive value =0.832, negative predictive value =1.000; FEV(1)/FEV(6): sensitivity=0.942; specificity=0.971; PPV=0.787; NPV=0.993). When the 5th percentile the lower limit of normal values was used as criterion for discrimination of having airflow limitation, sensitivity, specificity, PPV, and NPV of FEV(1)/FEV(6) were 0.932, 0.985, 0.808, and 0.995, respectively. CONCLUSION: The results of the present study suggest that %FEV(6) and FEV(1)/FEV(6) are excellent substitutes for %FVC and FEV(1)/FVC, respectively. We confirmed the validity of the use of FEV(6) and FEV(1)/FEV(6) for identifying pulmonary diseases in Japanese individuals.

Relationship between habit of cigarette smoking and airflow limitation in healthy Japanese individuals: the Takahata study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation. The prevalence of airflow limitation in Japan is 10.9% (16.4% of males and 5.0% of females). Cigarette smoking is well known as a major cause of COPD. However, few epidemiological studies have evaluated the effects of cigarette smoking on pulmonary function in healthy subjects. METHODS: Subjects aged 40 years or older (n=2,917), who had participated in a community-based annual health check in Takahata, Japan, from 2004 through 2005, were enrolled in the study. The smoking histories of these subjects were investigated using a self-reported questionnaire. Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV(1)), and forced expiratory flow at 25-75% of FVC (FEF(25-75)) were measured by standard procedures using spirometric machines. RESULTS: There were 554 current smokers (18.6%) and 403 former smokers (13.8%). The prevalence of airflow limitation defined by FEV(1)/FVC <0.7 in this population was 10.6%, and prevalence of airflow limitation defined by 5th percentile lower limit of normal was 6.4%. In smokers, percent predicted values of measured spirometric parameters (%FVC, %FEV(1) and %FEF(25-75)) decreased significantly with age, except for male %FVC. Also, percent predicted values of measured spirometric parameters decreased significantly with increasing pack-years, except for female %FEF(25-75). CONCLUSION: Cigarette smoking increased the prevalence and severity of airflow limitation. It is concluded that cigarette smoking increases the risk of airflow limitation in a healthy Japanese population.

A novel polymorphism in CDC6 is associated with the decline in lung function of ex-smokers in COPD.

The effect of smoking cessation on the rate of decline in lung function in patients with advanced stages of chronic obstructive pulmonary disease (COPD) has not been clarified. Saccharomyces cerevisiae cell division cycle 6 homolog (CDC6) protein possesses the pro-apoptotic properties. We tested our hypothesis that the individual susceptibility to rapid decline in lung function despite smoking cessation in patients with advanced stages of COPD is attributed to the genetic variants in the CDC6 gene. We prospectively followed 82 patients (ex-smokers) during 30months and evaluated the differences among the genotypes in the annual rate of decline in FEV(1.0) (%predicted) with ten single nucleotide polymorphisms (SNPs) in and around the CDC6 gene. We found significant differences in SNP5 (National Center for Biotechnology Information SNP reference: rs2077464), SNP6 (rs13706), SNP7 (rs7217852), and SNP8 (rs9904270) with a gene-dosage effect (ANOVA overall-P=0.029-0.030). The individual allele of SNP5G, SNP6A, SNP7G, and SNP8T were associated with rapid decline in FEV(1.0) (%predicted) [odds ratio (95% confidence interval)=2.35 (1.19-4.65), P=0.014]. The SNP5G/SNP6A/SNP7G/SNP8T haplotype was associated with an increased risk of deterioration of FEV(1.0) (%predicted) (P=0.017). Importantly, SNP6 caused a change in amino acids in CDC6 protein (Val441Ile), immediately upstream of the caspase-3-dependent cleavage site of CDC6 (Asp442) during apoptosis. These results suggest that CDC6 may be one of the susceptibility genes that contribute to rapid decline in lung function despite smoking cessation in these patients with COPD.

Enhanced immediate inflammatory response to Streptococcus pneumoniae in the lungs of mice with pulmonary emphysema.

BACKGROUND AND OBJECTIVE: Pulmonary emphysema is associated with frequent respiratory infections but little is known about the reasons for this susceptibility to bacterial infection. We previously demonstrated an impaired inflammatory response to Streptococcus pneumoniae in an experimental emphysema mouse model at 24 h, or longer following bacterial inoculation. Toll-like receptors (TLR) have been recognized as regulators in the inflammatory response. We examined the expression of TLR on alveolar macrophages in experimental emphysema mice and evaluated the immediate inflammatory response of the emphysematous lung to streptococcal infection. METHODS: Elastase was administered once into mice trachea to induce pulmonary emphysema. Three weeks later, expression of TLR-2 and TLR-4 in the BAL cells was examined by immunostaining. Following the intratracheal inoculation of Streptococcus pneumoniae, pro-inflammatory cytokine concentrations were measured in the BAL fluids of the control and emphysema mice. RESULTS: The expression of TLR-2 and TLR-4 was significantly elevated in the alveolar macrophages of emphysema mice. Six hours after infection, neutrophils in the BAL fluid of emphysema mice were significantly increased, and the levels of tumour necrosis factor-alpha, IL-1beta and IL-6 were significantly elevated, compared with the control mice. At 3 h post inoculation, macrophage inflammatory protein-2 levels were significantly elevated. CONCLUSIONS: The immediate inflammatory response in the emphysematous lung is significantly enhanced in response to streptococcal infection. This may be partly attributed to the increased expression of TLR in the alveolar macrophages of emphysema mice.

Clarithromycin suppresses invasiveness of human lung adenocarcinoma cells.

BACKGROUND: It has been speculated that clarithromycin (CAM), a 14-membered ring macrolide, possesses antitumor effects besides antimicrobial and anti-inflammatory effects. METHOD: We evaluated the effects of CAM on the growth and invasiveness of A549 lung adenocarcinoma cells. RESULTS: Although CAM did not affect the growth of A549 cells, the Matrigel invasion assay showed that the potential of invasion was diminished by CAM treatment. When analyzed by flow cytometry, CAM suppressed alpha(2)- and beta(1)-integrin expression. Furthermore, thymidine phosphorylase (TP) expression was diminished by CAM treatment in a dose-dependent manner. A specific TP inhibitor also suppressed beta(1)-integrin expression in flow cytometric analysis. CONCLUSIONS: These results suggest that CAM may suppress invasive activity of A549 cells in part by diminishing the expression of TP, alpha(2)- and beta(1)-integrin, which may be a downstream signal of the TP pathway, and that CAM could be useful in the treatment of lung adenocarcinoma.