PubMed Health⌕ Search

Biomedical subjects

Kazutetsu Aoshiba

Publications and source records attributed to Kazutetsu Aoshiba.

17 recordsLinked to original sources
PubMed2006

Alveolar cell senescence in patients with pulmonary emphysema.

RATIONALE AND OBJECTIVES: The prevalence of chronic obstructive pulmonary disease (COPD) is age-dependent, suggesting an intimate relationship between the pathogenesis of COPD and aging. In this study we investigated whether the senescence of alveolar epithelial and endothelial cells is accelerated in emphysematous lungs. METHODS: Samples of lung tissue were obtained from patients with emphysema, asymptomatic smokers, and asymptomatic nonsmokers. Paraffin-embedded lung tissue sections were evaluated for cellular senescence by quantitative fluorescence in situ hybridization to assess telomere shortening, and by immunohistochemistry to assess the expression of senescence-associated cyclin-dependent kinase inhibitors. Tissue sections were also immunostained for proliferating cell nuclear antigen (PCNA), surfactant protein A, and CD31. MAIN RESULTS: The patients with emphysema had significantly higher percentages of type II cells positive for p16INK4a and p21CIP1/WAF1/Sdi1 than the asymptomatic smokers and nonsmokers. They had also significantly higher percentages of endothelial cells positive for p16INK4a than the asymptomatic smokers and nonsmokers, and higher percentages of endothelial cells positive for p21CIP1/WAF1/Sdi1 than the asymptomatic nonsmokers. Telomere length in alveolar type II cells and endothelial cells was significantly shorter in the patients with emphysema than in the asymptomatic nonsmokers. The level of p16INK4a expression was negatively correlated with the level of PCNA expression. The level of alveolar cell senescence was positively correlated with airflow limitation. CONCLUSIONS: These results suggest that the senescence of alveolar epithelial and endothelial cells is accelerated in patients with emphysema. Cellular senescence may explain the abnormal cell turnover that promotes the loss of alveolar cells in emphysematous lungs.

Cell Proliferation↗
PubMed2006

Oxidative stress increases Fas ligand expression in endothelial cells.

BACKGROUND: Fas ligand (FasL) induces apoptosis in Fas-bearing target cells, such as leukocytes, and up-regulation of FasL expression on the endothelium may contribute to anti-inflammatory reactions that attenuate leukocyte extravasation during inflammation. Since oxidants generated during inflammation and cigarette smoking may modulate endothelial function, we examined the effect of H2O2 and cigarette smoke on endothelial FasL expression. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to nontoxic concentrations of H2O2 and cigarette smoke extracts (CSE). Membrane FasL expression was assessed by immunostaining with anti-FasL antibody followed by either monolayer-cell-based spectrofluorimetry or flow cytometry. Soluble FasL in culture supernatants was measured by enzyme-linked immunosorbent assay. For the cytotoxic assay, HUVECs were exposed to H2O2 and co-cultured with neutrophils. Neutrophils were stained by a peroxidase/diaminobenzidine-based reaction, and apoptosis was evaluated on the basis of nuclear morphology after Giemsa staining. To analyze in vitro FasL expression in arteries, rat thoracic aortas were incubated with H2O2, and paraffin-embedded sections were prepared for immunohistochemistry with anti-FasL antibody. RESULTS: Exposure of HUVECs to H2O2 dose-dependently increased their levels of both membrane and soluble forms of FasL expression. CSE exposure also caused increased levels of FasL expression, but the increase was partially inhibited by the addition of catalase. When co-cultured with neutrophils, HUVECs exposed to H2O2 significantly promoted neutrophil apoptosis. Rat thoracic aortas incubated with H2O2 exhibited increased FasL expression on their endothelium. CONCLUSION: Low levels of oxidative stress increase FasL expression on endothelial cells, thereby potentially reducing leukocyte extravasation and tissue damage.

Journal Article↗
PubMed2006

Macrophage colony-stimulating factor aggravates rather than regenerates emphysematous lungs in mice.

BACKGROUND: Lung regeneration is an innovative strategy that may cure pulmonary emphysema. The bone marrow (BM) harbors pulmonary stem cells. Hematopoietic cytokine-driven mobilization of BM cells may thus support lung regeneration. OBJECTIVES: The aim of this study was to determine whether systemic administration of macrophage colony-stimulating factor (M-CSF) leads to the regeneration of lungs in a murine model of elastase-induced emphysema. METHODS: C57BL/6J mice were administered elastase intratracheally. Four weeks later, in the absence or presence of elastase treatment, mice were intraperitoneally given either M-CSF or saline on days 1-5 each week for 3 weeks. Lung tissue was harvested 24 h after the last injection. RESULTS: M-CSF administration without prior elastase did not affect the mean linear intercept, surface area, or surface area/lung volume. In contrast, M-CSF administration following elastase injury caused a greater increase in the mean linear intercept and greater decreases in surface area and surface area/lung volume than saline administration following elastase, indicating that M-CSF aggravated emphysema. This aggravation of emphysema was accompanied by accumulation of pulmonary alveolar macrophages (AMs) expressing metalloproteinase (MMP)-9 and MMP-12. M-CSF stimulated AMs to express MMPs in vitro. CONCLUSIONS: These results suggest that M-CSF administration does not support lung regeneration but rather aggravates the lung destruction associated with elastase injury.

Animals↗
PubMed2006

C-reactive protein products generated by neutrophil elastase promote neutrophil apoptosis.

BACKGROUND: C-reactive protein (CRP), a prototypical acute phase protein, is increased as much as 1000-fold during acute inflammation, suggesting its biological role in that process. CRP can be modified at sites of inflammation where proteases have been released by neutrophils migrating to tissues. In this study, we investigated whether native CRP and neutrophil elastase-digested products of CRP modulate the rate of neutrophil apoptosis. METHODS: Neutrophils were isolated from venous blood of healthy volunteers and incubated with either native CRP (1, 10, or 30 microg/mL) or CRP digested with neutrophil elastase (1 U/mL). After 6 and 24 h of incubation, neutrophils were harvested and examined for apoptosis under light microscopy. RESULTS: We found that CRP degradation products generated by neutrophil elastase, but not native CRP, promoted neutrophil apoptosis and cell death. The rate of apoptosis was not affected by the addition of elastase that was not incubated with CRP. CONCLUSIONS: These results suggest that CRP degradation products generated by neutrophil elastase promote neutrophil apoptosis. Cleavage of CRP by neutrophil elastase may offer protection from inflammatory injury.

Apoptosis↗
PubMed2005

Activation of nuclear factor-kappaB in airway epithelial cells in patients with chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Nuclear factor-kappaB (NF-kappaB) is a transcription factor that mediates proinflammatory gene expression. NF-kappaB is activated when its inhibitor, IkappaBalpha, is phosphorylated and degraded. OBJECTIVES: The aims of this study were to compare the number of phosphorylated IkappaBalpha-immunopositive airway epithelial cells (AECs) in the peripheral airways of patients with COPD, asymptomatic smokers and asymptomatic nonsmokers. METHODS: We examined lung tissues obtained from 10 smokers with COPD, 7 asymptomatic smokers and 10 asymptomatic nonsmokers. Paraffin-embedded sections and immunohistochemical techniques were used to assess the number of phosphorylated IkappaBalpha-positive AECs as well as the numbers of tumor necrosis factor (TNF)alpha-positive, 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive and 4-hydroxy-2-nonenal (4-HNE)-positive AECs in the peripheral airway specimens. RESULTS: The percentages of phosphorylated IkappaBalpha-positive AECs and TNFalpha-positive AECs out of the total number of AECs were significantly higher in patients with COPD than in asymptomatic nonsmokers (p < 0.05). The percentage of phosphorylated IkappaBalpha-positive AECs was positively correlated with the percentage of TNFalpha-positive AECs (r = 0.82, p < 0.01), but not with the percentage of 8-OHdG-positive AECs or the percentage of 4-HNE-positive AECs. CONCLUSIONS: The activation of NF-kappaB, which was evaluated by measuring the level of phosphorylated IkappaBalpha, was enhanced in the peripheral airway epithelia of the COPD patients in this study. The activation of NF-kappaB in the peripheral airway epithelium is associated with an elevated level of TNFalpha and seems to occur through a mechanism independent of oxidative stress.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2004

Cigarette smoke induces senescence in alveolar epithelial cells.

Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or by telomere-independent signals (stress-induced senescence). The alveolar epithelium is often injured by a variety of inhaled toxins, including cigarette smoke (CS). In the present study, we investigated whether exposure to CS induces senescence of alveolar epithelial cells. In vitro experiments showed that exposure of A549 cells or normal human alveolar epithelial cells to sublethal concentrations of aqueous CS extracts induced cellular senescence. The senescence was characterized by a dose- and time-dependent increase in senescence-associated beta-galactosidase activity, senescence-associated changes in cell morphology, an increase in cell size and lysosomal mass, accumulation of lipofuscin, overexpression of p21(CIP1/WAF1/Sdi1) protein, and irreversible growth arrest. In vivo experiments in Institute for Cancer Research mice showed that inhalation of CS for 2 wk induced increases in senescence-associated beta-galactosidase activity, lipofuscin accumulation, and p21(CIP1/WAF1/Sdi1) protein expression in alveolar epithelial cells. These results suggest that CS induces a phenotype that is indistinguishable from that of senescence in alveolar epithelial cells. The induction of cellular senescence by CS may contribute to impaired re-epithelialization, leading to CS-related chronic lung diseases.

Animals↗
PubMed2004

Increased levels of cell death and proliferation in alveolar wall cells in patients with pulmonary emphysema.

BACKGROUND: Pulmonary emphysema, a major component of COPD, is pathologically characterized by destructive alterations in pulmonary architectures as a result of persistent inflammation. However, alterations in the turnover of pulmonary cells are less well understood. This study was designed to examine whether the turnover of alveolar wall cells is altered in patients with emphysema. PATIENTS AND MEASUREMENTS: We obtained lung tissue specimens from patients with emphysema who had undergone lung volume reduction surgery (13 patients) as well as asymptomatic smokers (7 patients) and nonsmokers (9 patients) undergoing lung resections for solitary lung cancers. Paraffin-embedded lung tissue sections were evaluated for apoptosis and proliferation using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) or immunohistochemistry for Bax, proliferation cell nuclear antigen (PCNA), and topoisomerase IIalpha. Tissue sections were also immunostained for epithelial membrane antigen, surfactant protein A, and CD31. RESULTS: The percentages of alveolar wall cells undergoing apoptosis and proliferation of the total number of alveolar wall cells were significantly higher in patients with emphysema than in asymptomatic smokers and nonsmokers (p < 0.05). The percentage of TUNEL-positive alveolar wall cells was positively correlated with the percentage of PCNA-positive alveolar wall cells. Most of the TUNEL-positive and PCNA-positive cells were alveolar epithelial cells. CONCLUSIONS: These results suggest that the turnover of alveolar wall cells is enhanced in emphysematous lungs, compared to healthy lungs. Emphysema may be a dynamic disease process in which alveolar wall cell death and proliferation are repeated.

Aged↗
PubMed2004

Differences in airway remodeling between asthma and chronic obstructive pulmonary disease.

The functional consequence of asthma and chronic obstructive pulmonary disease (COPD)is airflow limitation, which is mostly reversible in asthma and not fully reversible in COPD. In both diseases, inflammatory conditions are associated with cellular and structural changes,referred to as remodeling, and these structural changes may lead to thickening of the airway wall, thereby promoting airway narrowing and airflow limitation. However, the pattern of infiltrated cells and the pattern of structural changes occur differently in the two diseases. In asthma, CD4+, T lymphocytes, eosinophils, and mast cells are the predominant cells involved,whereas in COPD, CD8+, T lymphocytes, and macrophages are predominantly involved. In severe cases of asthma and COPD, neutrophil infiltration becomes evident. Regarding structural changes, epithelial injury and early thickening of reticular basement membrane are highly characteristic of the airway wall of asthmatics. Increases in airway smooth muscle mass occur in large airways of severe asthmatics and in small airways of patients with COPD. Thickening of the airway wall, goblet cell hyperplasia, mucous gland hypertrophy, and the luminal obstruction caused by inflammatory exudates and mucous are features of both asthma and COPD. Squamous epithelial metaplasia and airway wall fibrosis are commonly observed characteristics of COPD. Destruction and fibrosis of the alveolar wall occur in COPD but not in asthma. The remodeling processes accompanied by chronic inflammatory infiltrates interact in a complex fashion and contribute to the development of airflow limitation in both asthma and COPD.

Airway Obstruction↗
PubMed2003

Epidermal growth factor receptor tyrosine kinase inhibition augments a murine model of pulmonary fibrosis.

The inappropriate regeneration of sequentially injured epithelium is an important process leading to pulmonary fibrosis. Previous studies have shown that the epithelial expression of epidermal growth factor receptor (EGFR) is increased in fibrotic lung tissue, compared with normal lung tissue, suggesting that EGFR-mediated signaling is involved in epithelial regeneration in fibrotic lung diseases. We examined the effect of EGFR inhibition using ZD1839, a selective EGFR tyrosine kinase inhibitor (TKI), on bleomycin-induced pulmonary fibrosis in mice. ICR mice were administered a single intratracheal injection of bleomycin (5 units/kg) on day 1. ZD1839 (200 mg/kg) or vehicle alone were administered p.o. 1 h before this injection and on days 1-5 each week for 3 weeks. Lung tissue was harvested on day 21. Lung histology and collagen analysis performed on day 21 showed more severe fibrosis in the mice receiving both bleomycin and the EGFR-TKI than in the mice receiving bleomycin and the vehicle. An immunohistochemistry analysis showed that phosphorylated EGFR and proliferation cell nuclear antigen were highly expressed by the regenerated epithelial cells in the mice treated with bleomycin and the vehicle. In contrast, the expression of these antigens was attenuated in the mice treated with bleomycin and the EGFR-TKI. In vitro studies also demonstrated that the addition of ZD1839 at a concentration of < or =1 microM suppressed the proliferation of type II-like epithelial cells (A549) but not that of lung fibroblasts (IMR90). These results suggest that the inhibition of EGFR phosphorylation augments bleomycin-induced pulmonary fibrosis by reducing regenerative epithelial proliferation. Our data suggest that EGFR-TKIs should be used with caution in cancer patients with pulmonary fibrosis.

Animals↗
PubMed2003

Immunohistochemical evaluation of oxidative stress in murine lungs after cigarette smoke exposure.

Cigarette smoking generates an oxidative stress in the lung, which may contribute to the pathogenesis of chronic obstructive pulmonary disease. We performed an immunohistochemical study to evaluate oxidative stress in the lung after acute cigarette smoke (CS) exposure in mice. Paraffin-embedded lung tissue sections were prepared from mice exposed and unexposed to CS for 1 h. The sections were immunostained with antibodies against 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative DNA adduct, and 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation product. The bronchiolar and alveolar epithelium of mice unexposed to CS exhibited weak signals for 8-OHdG and 4-HNE, whereas by 1 h after CS exposure the signals in the bronchiolar epithelial cells and the alveolar epithelial cells, particularly type II cells, had increased dramatically. The increases in both were associated with increased 8-OHdG levels in bronchoalveolar lavage fluid as determined by enzyme-linked immunoassay. These results suggest that acute CS exposure imposes oxidative stress predominantly on bronchiolar epithelial and alveolar type II cells, confirming that cigarette smoking causes oxidative damage to the respiratory epithelium.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2003

Alveolar wall apoptosis causes lung destruction and emphysematous changes.

Pulmonary emphysema is characterized by alveolar wall destruction and airspace enlargement. Recent evidence indicates that epithelial or endothelial apoptosis may be involved in the pathogenesis of emphysema. Here, we describe the induction of emphysematous changes, including airspace enlargement, alveolar wall destruction, and enhanced lung distensibility, in mice receiving a single intratracheal injection of active caspase-3 and Chariot, a newly developed protein transfection reagent. Epithelial apoptosis and enhanced elastolytic activity (optimal at pH 5.5) in bronchoalveolar lavage were noted. Emphysematous changes were also generated in mice receiving an intratracheal injection of nodularin, a proapoptotic serine/threonine kinase inhibitor. This murine model provides direct evidence that confirms that alveolar wall apoptosis causes emphysematous changes. Furthermore, this simple technique for protein transfection of lung tissue can be used in a variety of future applications.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Apoptosis of wound fibroblasts induced by oxidative stress.

Irreversible lung parenchymal injury is usually healed by fibrosis, which depends on the abilities of fibroblasts to proliferate, migrate into the wound, and survive. Because the lung is frequently exposed to increased oxidative stress, which is thought to mediate apoptosis, we examined whether oxidative stress induces apoptosis in fibroblasts during wound healing. We performed an in vitro scratch wound assay where cultured fibroblast monolayers were exposed to H2O2 (10-500 microM) after artificial wounding. Apoptosis was evaluated by nuclear staining with Hoechst33342 or terminal deoxynucleotidyl transferase (TdT)-mediated nucleotide nick end-labeling (TUNEL). Intracellular oxidants were assessed with the peroxide-sensitive fluorochrome carboxydichlorodihydrofluorescein (CDCF). We found that repopulating fibroblasts at the wound margin, but not quiescent fibroblasts at the intact site, selectively underwent oxidant accumulation and apoptosis in response to H2O2 exposure. Some of the apoptotic cells had incorporated bromodeoxyuridine (BrdU), an indicator of proliferating cells. These results suggest that oxidative stress selectively induces apoptosis in fibroblasts that are stimulated to proliferate and/or migrate into the wound. Fibroblast apoptosis induced by oxidative stress during wound repopulation may be relevant to intractable wound healing.

Apoptosis↗
PubMedDate not supplied

Transforming growth factor-beta promotes fibroblast apoptosis induced by H2O2.

Transforming growth factor-beta (TGF-beta) is believed to play a central role in fibroblastic wound repair and subsequent scar formation. Fibroblasts undergo apoptosis as granulation tissue evolves into a scar. To determine whether TGF-beta influences fibroblast apoptosis, human lung fibroblasts (IMR-90) were treated with TGF-beta (2 to 10 ng/mL) and then exposed to H(2)O(2) (50 to 150 microM), an inducer of fibroblast apoptosis. Apoptosis was evaluated by nuclear staining with Hoechst33342 and terminal deoxynucleotidyl transferase (TdT)-mediated nucleotide nick-end labeling. TGF-beta alone did not induce fibroblast apoptosis, but it dose-dependently augmented the apoptosis induced by exposure to H(2)O(2). TGF-beta also increased the intracellular level of peroxides measured with carboxydichlorodihydrofluorescein, but it did not affect the protein levels of Bcl-2, Bax, Bad, or p53 proteins. These results suggest that TGF-beta promotes lung fibroblast apoptosis induced by H(2)O(2), probably by increasing intracellular peroxides. Thus, TGF-beta may promote the elimination of fibroblasts from wounds, particularly under conditions of exposure to enhanced oxidative stress in the lung.

Apoptosis↗
PubMedDate not supplied

Differences in the distribution of CD4+ and CD8+ T cells in emphysematous lungs.

BACKGROUND: Recent studies have shown that T cells are the predominant cells infiltrating the alveolar wall of patients with emphysema. Since CD4+ and CD8+ cells have dissimilar function, they may have different distributions in emphysematous lungs. OBJECTIVES: The aim of this study was to investigate whether CD4+ and CD8+ are differently distributed in the lungs of patients with emphysema. METHODS: We evaluated CD4+ and CD8+ cell infiltration in lung tissue sections obtained from smokers with pulmonary emphysema who had undergone lung volume reduction surgery (n = 10). Paraffin-embedded lung tissue sections were immunostained using antibodies against CD4+ or CD8+, and distributions of CD4+ and CD8+ cells were evaluated using a morphometric analysis. We determined the degree of emphysema using the area fraction of the alveolar wall (A(A,aw)) and examined its relation to the number of cells per unit area of the alveolar wall (NA). RESULTS: The majority of CD4+ cells were located in the lung parenchyma of severe emphysematous lesions characterized by the lowest alveolar wall fraction (A(A,aw)). In contrast, more CD8+ cells were located in mild emphysematous lesions than in severe lesions. CONCLUSIONS: These results suggest that CD4+ and CD8+ cells are differently distributed according to the severity of emphysema. CD4+ T cells are located preferentially in severe emphysematous lesions whereas CD8+ T cells occur preferentially in mild emphysematous lesions.

Aged↗