Takao Tsuji

Alveolar cell senescence exacerbates pulmonary inflammation in patients with chronic obstructive pulmonary disease.

Background: Alveolar cell senescence is accelerated in patients with chronic obstructive pulmonary disease (COPD). Objectives: We tested the hypothesis that alveolar cell senescence contributes to the chronic inflammation that affects the lungs of COPD patients. Methods: We exposed alveolar type II-like epithelial (A549) cells to a G-quadruplex-interacting telomerase inhibitor in vitro to induce cellular senescence and analyzed the production of proinflammatory cytokines and the activation of NF-ĸB. Human dermal microvascular endothelial cells (HDMECs) were serially passaged to induce replicative senescence. We also immunostained human lung tissue sections obtained from COPD patients, asymptomatic smokers and asymptomatic nonsmokers and examined correlations between type II cell senescence and inflammation. Results: Senescent A549 cells and HDMECs, whether stimulated with lipopolysaccharide or not, produced greater amounts of IL-6, IL-8 and TNF-α, which paralleled NF-ĸB activation, than did presenescent cells. There were positive correlations between the percentages of senescent type II cells that expressed p16INK4a and the percentages of type II cells that expressed phosphorylated NF-ĸB. The lung tissue of the COPD patients contained higher percentages of proinflammatory senescent type II cells that co-expressed p16INK4a and phosphorylated NF-ĸB than the tissue from asymptomatic smokers and asymptomatic nonsmokers. Higher percentages of p16INK4a-positive senescent type II cells than of p16INK4a-negative presenescent type II cells were positive for phosphorylated NF-ĸB. Conclusions: Senescence of alveolar epithelial cells is associated with functional alterations of the cells to a proinflammatory phenotype and may contribute to the pathogenesis of COPD.

Therapeutic effects of erythropoietin in murine models of endotoxin shock

Objective:Erythropoietin has recently emerged as a cytoprotective cytokine, which possesses the ability to protect many tissues, including the brain, heart, and kidneys, against ischemia or traumatic injury. We investigated the therapeutic effects of erythropoietin in a murine model of endotoxin shock. Design:Prospective, randomized study. Setting:University-based research laboratory. Subjects:Male BALB/c mice. Interventions:Mice intraperitoneally received either lipopolysaccharide (LPS) from Escherichia coli or vehicle. Erythropoietin was administered at a dose of 1000 IU/kg subcutaneously at different time points after LPS administration. We also investigated the effect of erythropoietin on the development of septic shock caused by cecal perforation. Measurements and Main Results:Treatment of mice with erythropoietin, within 2 hours after LPS administration, improved the mortality rate. Treatment of cecal perforated mice with erythropoietin extended survival by 12 hours, but all animals died by 72 hours in both groups. Erythropoietin attenuated apoptosis in the lungs, liver, small intestine, thymus, and spleen, as assessed by terminal deoxynucleotidyl transferase-mediated nucleotide nick-end labeling staining, active caspase-3 immunostaining and immunoblotting, and measurements of caspase-3/7 activity. Erythropoietin also reduced inducible nitric oxide synthase expression, nitric oxide production, peroxynitrite formation, and tissue hypoxia. In contrast, erythropoietin did not affect the degree of LPS-induced inflammation, as assessed by measurements of blood levels of interleukin-1&bgr;, interleukin-6, tumor necrosis factor-&agr;, growth-related oncogene/keratinocyte-derived cytokine, and high mobility group box 1, the phosphorylation levels of nuclear factor &kgr;B, and the number of neutrophils infiltrating the lungs and the liver. Conclusions:The results of the study demonstrate that administration of a large dose of erythropoietin after induction of experimental endotoxemia improved survival and that the beneficial effects of erythropoietin were associated with inhibition of apoptosis, nitric oxide production, and tissue hypoxia, without alterations in inflammatory responses.

DNA damage as a molecular link in the pathogenesis of COPD in smokers

In this study, we investigated whether DNA double-strand breaks (DSBs) contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). We immunofluorescence-stained lung tissue samples obtained from COPD patients, asymptomatic smokers and nonsmokers for markers of DSBs. The numbers of DSB foci (phosphorylated histone 2AX (&ggr;H2AX), phosphorylated ATM (ataxia telangiectasia mutated) substrate and phosphorylated p53-binding protein-1 foci) per cell in alveolar type I and II cells and endothelial cells were higher in the COPD patients than in the asymptomatic smokers and nonsmokers. The lung tissue in which type II cells contained higher numbers of &ggr;H2AX foci per cell had higher percentages of type II cells that expressed p16INK4a (p16), phosphorylated nuclear factor (NF)-&kgr;B and interleukin (IL)-6, and of alveolar wall cells that expressed active caspase-3. The type II cells that contained higher numbers of &ggr;H2AX foci per cell had higher rates of expression of p16, phosphorylated NF-&kgr;B, and IL-6. Half of the alveolar wall cells that expressed active-caspase-3 contained &ggr;H2AX foci. Type II cells that stained positive for 8-hydroxy-2-deoxyguanosine contained a higher number of &ggr;H2AX foci per cell than the type II cells that stained negative. In conclusion, DSBs, at least in part caused by oxidative stress, appear to contribute to the pathogenesis of COPD by inducing apoptosis, cell senescence and pro-inflammatory responses.

Senescence-associated secretory phenotype in a mouse model of bleomycin-induced lung injury

Bleomycin produces DNA damage, apoptosis and senescence, all of which play crucial roles in the development of pulmonary fibrosis. Recently, close attention has been paid to a DNA damage-induced phenotypic change (senescence-associated secretory phenotype; SASP) as a trigger for the secretion of various mediators which modify the processes of tissue injury, inflammation, repair and fibrosis. We characterized the SASP in a murine model of bleomycin-induced lung injury. Mice were intratracheally administered bleomycin or control saline, and the lungs were obtained on days 7, 14 and 21. The occurrence of DNA damage and the SASP in the lungs was examined by immunostaining. γH2AX immunostaining of the bleomycin-treated lungs revealed double-strand breaks (DSBs), largely within E-cadherin-positive, β4-integirn-positive alveolar epithelial cells. The DSBs were associated with phosphorylation of ATM/ATR, a central signal transducer mediating the DNA damage response, and upregulation of the cyclin-dependent kinase inhibitor p21(CIP1). The DSBs persisted for at least 21 days after the bleomycin exposure, although it began to wane after 7 days. A subpopulation of the γH2AX-positive, DNA-damaged cells exhibited the SASP, characterized by overexpression of IL-6, TNFα, MMP-2 and MMP-9, in association with the phosphorylation of IKKα/β and p38 MAPK. Persistent DNA damage and the SASP are induced in the process of bleomycin-induced lung injury and repair, suggesting that these events play an important role in the regulation of inflammation and tissue remodeling in bleomycin-induced pneumopathy.

Undernutrition in Patients with COPD and Its Treatment

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disorder of the lung and whole body caused mainly by tobacco smoking. Patients with advanced COPD are in a state of undernutrition, referred to as pulmonary cachexia; the exercise performance and quality of life (QOL) of these patients are deteriorated, the vital prognosis is unfavorable, and the medico-economic burden posed by poorly nourished COPD patients is high. The mainstays of COPD treatment are pharmacotherapy, mainly with bronchodilators, and non-pharmacotherapeutic approaches such as respiratory rehabilitation and nutrition counseling. Nutritional supplement therapy, consisting primarily of high calorie intake, has been demonstrated to be effective for maintaining and improving the muscle strength and exercise tolerance in poorly nourished COPD patients. The efficacy of intake of various nutrients, besides a high calorie intake, for amelioration of the disease state of COPD has also been reported. The roles of adipokines in the pathophysiology of COPD have begun to receive attention recently, and not only their regulatory effects on appetite and nutritional status, but also their influence on systemic inflammation have been increasingly clarified. We review the papers on COPD and nutrition and discuss the role of nutritional supplement therapy in the treatment of COPD.

Systemic effects of acute cigarette smoke exposure in mice

Abstract Context: Cigarette smoke (CS) causes both pulmonary and extrapulmonary disorders. Objective: To determine the pulmonary and extrapulmonary effects of acute CS exposure in regard to inflammation, oxidative stress and DNA damage. Materials and methods: Mice were exposed to CS for 10 days and then their lungs, heart, liver, pancreas, kidneys, gastrocnemius muscle and subcutaneous (inguinal and flank) and visceral (retroperitoneum and periuterus) adipose tissues were excised. Bronchoalveolar lavage fluid samples were obtained for differential cell analysis. Inflammatory cell infiltration of the tissues was assessed by immunohistochemistry for Mac-3+ cells, F4/80+ cells and CD45+ cells. Oxidative stress was determined by immunohistochemistry for thymidine glycol (a marker of DNA peroxidation) and 4-hydroxy hexenal (a marker of lipid peroxidation), by enzyme-linked immunosorbent assay for protein carbonyls (a marker of protein peroxidation) and by measurements of enzyme activities of glutathione peroxidase, superoxide dismutase and catalase. DNA double-strand breaks were assessed by immunohistochemistry for γH2AX. Results: CS exposure-induced inflammatory cell infiltration, oxidative stress and DNA damage in the lung. Neither inflammatory cell infiltration nor DNA damage was observed in any extrapulmonary organs. However, oxidative stress was increased in the heart and inguinal adipose tissue. Discussions: Induction of inflammatory cell infiltration and DNA damage by acute CS exposure was confined to the lung. However, an increased oxidative burden occurred in the heart and some adipose tissue, as well as in the lung. Conclusions: Although extrapulmonary effects of CS are relatively modest compared with the pulmonary effects, some extrapulmonary organs are vulnerable to CS-induced oxidative stress.

The danger signal plus DNA damage two-hit hypothesis for chronic inflammation in COPD

Inflammation in chronic obstructive pulmonary disease (COPD) is thought to originate from the activation of innate immunity by a danger signal (first hit), although this mechanism does not readily explain why the inflammation becomes chronic. Here, we propose a two-hit hypothesis explaining why inflammation becomes chronic in patients with COPD. A more severe degree of inflammation exists in the lungs of patients who develop COPD than in the lungs of healthy smokers, and the large amounts of reactive oxygen species and reactive nitrogen species released from inflammatory cells are likely to induce DNA double-strand breaks (second hit) in the airways and pulmonary alveolar cells, causing apoptosis and cell senescence. The DNA damage response and senescence-associated secretory phenotype (SASP) are also likely to be activated, resulting in the production of pro-inflammatory cytokines. These pro-inflammatory cytokines further stimulate inflammatory cell infiltration, intensifying cell senescence and SASP through a positive-feedback mechanism. This vicious cycle, characterised by mutually reinforcing inflammation and DNA damage, may cause the inflammation in COPD patients to become chronic. Our hypothesis helps explain why COPD tends to occur in the elderly, why the inflammation worsens progressively, why inflammation continues even after smoking cessation, and why COPD is associated with lung cancer. A vicious cycle, involving mutually reinforcing inflammation and DNA damage, may cause chronic inflammation in COPD http://ow.ly/nYW4u

A systemically administered EP2 receptor agonist stimulates pulmonary angiogenesis in a murine model of emphysema

We investigated whether systemically administered EP2 receptor agonists would stimulate angiogenesis in the emphysematous lungs of mice. Saline or porcine pancreatic elastase was intratracheally administered to C57BL/6J mice to induce the formation of emphysematous lesions, and 4 weeks later the mice were intraperitoneally injected with an EP2 receptor agonist, ONO-AE1-259 or PGE2, or saline on days 1-5 each week for 3 weeks. Intraperitoneal ONO-AE1-259 in the mice in the intratracheal saline group increased pulmonary capillary volume to 114.9% of the control value (P<0.05), and when administered to the intratracheal elastase group, ONO-AE1-259 partially restored pulmonary capillary volume, from 70.9% of the control value to 88.3% of the control value (P<0.05). Intraperitoneal PGE2 tended to increase pulmonary capillary volume in the mice in the intratracheal saline group (P=0.07) but not in the intratracheal elastase group. Intraperitoneal ONO-AE1-259 and PGE2 each increased the concentration of vascular endothelial growth factor (VEGF) and the number of endothelial progenitor cells (EPCs) in circulating blood. These results suggest that systemically administered ONO-AE1-259 stimulates pulmonary angiogenesis in an elastase-induced murine model of emphysema.

A murine model of airway fibrosis induced by repeated naphthalene exposure

The airway epithelium serves as a biological barrier essential for host defense against inhaled pollutants. While chronic epithelial injury, commonly associated with chronic obstructive pulmonary disease and bronchiolitis obliterans syndrome, often results in airway fibrosis, limited animal models of airway fibrosis have been established. Club cells (Clara cells) in the small airways represent an important population of epithelial progenitor cells and also the principal site of localization of the cytochrome P-450 monooxygenase system, which metabolically activates xenobiotic chemicals such as naphthalene by converting them to toxic epoxide intermediates. We hypothesized that repeated exposure to naphthalene may cause prolonged loss of club cells, triggering aberrant local epithelial repair mechanisms that lead to peribronchial fibrosis. We administered intraperitoneal injections of naphthalene to C57/BL6J mice once a week for 14 consecutive weeks. Repeated club cell injury caused by naphthalene triggered regional hyperproliferation of epithelial progenitor cells, while other regions remained denuded or squamated, resulting in fibroblast proliferation and peribronchial collagen deposition associated with upregulation of the fibrogenic cytokines transforming growth factor-β and connective tissue growth factor. The total collagen content of the lung assessed by measurement of the hydroxyproline content was also increased after repeated exposure to naphthalene. These results lend support to the relevance of repeated injury of airway epithelial cells as a trigger for resting fibroblast proliferation and airway fibrosis. This model of airway fibrosis is simple and easy to reproduce, and may be expected to advance our understanding of the pathogenesis and potential treatment of airway fibrotic disorders.

Estimation of parasympathetic nerve function during sleep in patients with obstructive sleep apnea by instantaneous time–frequency analysis

BACKGROUND AND OBJECTIVES The pathophysiologic aspects of parasympathetic nerve (PN) function during sleep in patients with obstructive sleep apnea (OSA) studied by classical power spectrum analysis on heart rate variability (HRV) are highly controversial. The controversy is attributed to methodologic concerns, such as poor time resolution involved in power spectrum analysis. We aimed to establish the appropriate method for the investigation of PN function in OSA patients with apneas and hypopneas using instantaneous time-frequency analysis with complex demodulation (CD) and sufficient time resolution. METHODS A total of 30 patients with PSG-confirmed mild to severe OSA were recruited for the analysis of frequency spectra contained in R-R intervals (RRI) of overnight electrocardiograph (ECG) tracings. High-frequency (HF) domains ranging between 0.15 and 0.40 Hz were selected for analysis. Among these domains, the HF domain with the maximum instantaneous amplitude was defined as the main HF peak and was used as the surrogate marker of PN discharge. Based on density spectrum array (DSA) map for main HF peak constructed with a time scale of 1s and a frequency resolution of 0.002 Hz (HF-DSA map), the shift in central frequency (CF) of main HF peak over time was continuously monitored. When the main HF peak with the same CF lasted for more than 20 s or 5 min on HF-DSA map, the PN function was considered to be stable or very stable. The measurements were then repeated after continuous positive airway pressure (CPAP) treatment. RESULTS The extent of PN-evoked modulation of RRI was enhanced in nonrapid eye movement (NREM) sleep, though the stability was reduced in both NREM and rapid eye movement (REM) sleep. These peculiar behaviors of PN function were reversed by CPAP treatment. CONCLUSION We found that instantaneous time-frequency analysis allowed estimation of transitional changes in PN function during sleep in OSA patients.