Lysophospholipid Acyltransferase 9 Promotes Emphysema Formation via Platelet-activating Factor.

Cigarette smoking is known to be the leading cause of chronic obstructive pulmonary disease (COPD). However, the detailed mechanisms have not been elucidated. Platelet-activating factor (PAF), a potent inflammatory mediator, is involved in the pathogenesis of various respiratory diseases, such as bronchial asthma or COPD. We focused on lysophospholipid acyltransferase 9 (LPLAT9), a biosynthetic enzyme of PAF, in the pathogenesis of COPD. LPLAT9 gene expression was observed in excised COPD lungs and single-cell RNA sequencing data of alveolar macrophage (AM). LPLAT9 was predominant and upregulated in AM, particularly monocyte-derived AM, in patients with COPD. To identify the function of LPLAT9/PAF in AM on the pathogenesis of COPD, we exposed cigarette smoke (CS) to systemic LPLAT9 knockout (LPALT9-/-) mice. CS increased the number of AM, especially monocyte-derived fraction, which secreted matrix metalloprotease 12 (MMP12). Also, CS augmented LPLAT9 phosphorylation/activation on macrophage and, subsequently, PAF synthesis in mice lung. LPLAT9-/- mice lung reduced PAF production after CS exposure. Intratracheal PAF administration accumulated AM by increasing monocyte chemoattractant protein-1 (MCP1). After CS exposure, AM accumulation and subsequent pulmonary emphysema, a primary pathologic change of COPD, were reduced in LPALT9-/- mice than in LPLAT9+/+ mice. Notably, these phenotypes got worsened again by LPLAT9+/+ bone marrow transplantation in LPALT9-/- mice. Thus, CS-induced LPLAT9 activation in monocyte-derived AM aggravated pulmonary emphysema via PAF-induced further AM accumulation. These results suggest that PAF synthesized by LPLAT9 has an important role in the pathogenesis of COPD.

Participant Selection from the General Japanese Population for Pulmonary Function Tests Using a Questionnaire on Symptoms and Smoking Habits during Annual Health Checkups: The Yamagata-Takahata Study.

Objective Pulmonary function tests are essential for diagnosing respiratory diseases, such as chronic obstructive pulmonary disease (COPD), but are typically not performed in Japan during annual health checkups, which hinders the early diagnosis of respiratory diseases. Methods Individuals who agreed to participate in the Yamagata-Takahata study during medical checkups in Takahata (Yamagata Prefecture, Japan) in 2011 were examined. We interviewed 669 participants (49.0% men; mean age, 67.7 years old) regarding their respiratory symptoms and smoking habits and performed pulmonary function tests during the study. Results Based on pulmonary function test results, 141 participants had pulmonary dysfunction, and 115 had obstructive pulmonary dysfunction. The risk of respiratory dysfunction, particularly obstructive respiratory dysfunction, was examined by referring to a questionnaire tool for an early COPD diagnosis. The associations between age, the smoking history, respiratory symptoms, and obstructive respiratory dysfunction were evaluated. Obstructive respiratory dysfunction was found in 17.6% of participants ≥50 years old and 19.5% ≥60 years old, 30.3% had a smoking history, and 32.8% had respiratory symptoms. Furthermore, the participants with multiple factors had a higher probability of obstructive respiratory dysfunction. Conclusion Subjects with obstructive pulmonary dysfunction are expected to be efficiently identified by extracting individuals by age and smoking habit and through a respiratory symptom questionnaire, although pulmonary function tests cannot be performed for all individuals during health checkups.

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.

Smaller erector spinae muscle size is associated with inability to recover activities of daily living after pneumonia treatment.

BACKGROUND: Elderly patients who are hospitalized due to pneumonia experience deterioration of their activities of daily living (ADL) during this period; in some cases, this loss of ADL is not recovered at the end of antibiotic treatment. In this study, we examined whether erector spinae muscle cross-sectional area (ESMCSA) measured by computed tomography (CT) could predict a low level of ADL at the end of antibiotic treatment for pneumonia. METHODS: Eighty patients (mean age 74.8 years) with pneumonia, who were admitted to Yamagata university hospital between 2015 and 2016, were analyzed retrospectively. In all cases, chest CT was performed on admission and ESMCSA was measured at the level of the 12th thoracic vertebra. Patient levels of ADL were also measured, both on admission and at the end of treatment, using the Barthel Index. RESULTS: Patients with lower levels of ADL at the end of treatment were significantly older and tended to have a lower body mass index, poorer nutritional status, and more severe pneumonia than did patients who were self-reliant. Significantly smaller ESMCSAs were noted in patients who required assistance at the end of treatment than in those who were self-reliant. In multivariate logistic regression analysis, smaller ESMCSA was significantly associated with a lower level of ADL at the end of treatment, independent of age, sex, severity of pneumonia, nutritional status, or dehydration status. CONCLUSION: These results suggest that ESMCSA can predict ADL level after antibiotic treatment of pneumonia.

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.

Heart-type fatty acid binding protein as a prognostic factor in patients with exacerbated chronic obstructive pulmonary disease.

BACKGROUND: The designation of some cardiac-specific proteins as prognostic biomarkers in chronic obstructive pulmonary disease (COPD) exacerbations suggest that the process of exacerbation involves cardiomyocyte injury. Among these cardiac biomarkers, heart-type fatty acid binding protein (h-FABP) is considered a very sensitive diagnostic marker for cardiomyocyte injury and a prognostic marker in chronic heart failure. However, the prognostic usefulness of h-FABP in patients with COPD remains unclear. METHODS: Sixty-six patients were enrolled in this study. Subjects who recovered from COPD exacerbation and were discharged without needing home oxygen therapy were defined as the improved group. Those who died of the COPD exacerbations, were discharged but needed home oxygen therapy, or were transferred to a rehabilitation hospital for respiratory failure and the remaining aftereffects of exacerbation were defined as the unimproved group. RESULTS: The improved and unimproved groups included 54 and 12 subjects, respectively. Compared with the improved group, the unimproved group had significantly higher white blood cell counts and alanine aminotransferase, lactate dehydrogenase, blood urea nitrogen (BUN), uric acid, potassium, and h-FABP levels, and significantly lower total protein and total cholesterol levels and estimated glomerular filtration rates, either at admission or during the early morning within 24h after admission. A multivariate analysis revealed that higher serum h-FABP and potassium levels were independently predictive of a poor prognosis following a COPD exacerbation, and a receiver operating characteristic curve analysis yielded a cutoff of 4.5ng/ml for predicting lack of improvement. CONCLUSION: H-FABP may predict the outcomes of COPD exacerbation.

Prevalence of diabetes mellitus in individuals with airflow obstruction in a Japanese general population: The Yamagata-Takahata Study.

BACKGROUND: Diabetes has been reported as a comorbidity of chronic obstructive pulmonary disease (COPD) in Western countries, but it has not been demonstrated in epidemiological reports in Japan. The purpose of this study was to clarify whether the relationship between airflow obstruction and diabetes can be confirmed in a Japanese general population. METHODS: From 2004 to 2006, blood sampling and pulmonary function tests were performed on 3045 people over the age of 40 years in annual health check-ups held in Takahata, Yamagata Prefecture, Japan. Pulmonary function was re-evaluated in 2009 and 2011. RESULTS: The prevalence of diabetes did not differ between subjects with and without airflow obstruction. Furthermore, although body mass index decreased, no increase in the prevalence of diabetes was observed with the progression of airflow obstruction. The annual changes in forced expiration volume in 1s (FEV1) did not differ depending on the presence or absence of diabetes in the study population. CONCLUSION: There was no difference in the prevalence of diabetes between subjects with airflow obstruction and those without. As patients with COPD in Japan are thinner than in the West, diabetes may not be a common comorbidity in Japanese patients with COPD.

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.