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.

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.

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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.

Alveolar macrophages (AMs) may play a critical role in cigarette smoke (CS)-related pulmonary diseases. This study was designed to determine whether CS induces apoptosis of AMs. In in vitro studies, mouse, rat, and human AMs and human blood monocyte-derived macrophages cultured with aqueous whole CS extracts underwent apoptosis that was detected by light and electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. The gas phase of CSE did not cause apoptosis. The CS-induced apoptosis was associated with increased oxidative stress, Bax protein accumulation, mitochondrial dysfunction, and mitochondrial cytochrome c release but was independent of p53, Fas, and caspase activation. This apoptosis was inhibited by antioxidants such as glutathione, ascorbic acid, and alpha-tocopherol. In in vivo studies where rats were exposed to the smoke from 10 cigarettes over 5 h in an exposure chamber, approximately 3% of AMs obtained by bronchoalveolar lavage after 24 h showed apoptosis. These results suggest that acute CS exposure is capable of inducing apoptosis of AMs.

Cellular senescence is a state of irreversible growth arrest. In this paper the authors examined whether bleomycin, an agent that causes pulmonary fibrosis, induces the senescence of alveolar epithelial cells.Type II-like alveolar epithelial (A549) cells or rat primary type II cells were exposed to bleomycin and then evaluated for markers of cellular senescence. Bleomycin was also administered intratracheally in C57BL/6 mice.The authors found that exposure to bleomycin induced cellular senescence in A549 cells and rat primary type II cells. The senescence was characterised by a dose-and time-dependent increase in senescence-associated b-galactosidase activity, senescenceassociated changes in cell morphology, an increase in cell size and lysosomal mass, the overexpression of p21 protein, and irreversible growth arrest. The intratracheal injection of bleomycin in mice induced an increase in senescence-associated b-galactosidase activity in type II epithelial cells, reaching a maximum at day 7.These results suggest that bleomycin induces a phenotype that is indistinguishable from that of senescence in alveolar epithelial cells. The induction of epithelial senescence by bleomycin may contribute to the pathway of impaired re-epithelialisation leading to pulmonary fibrosis. Eur Respir J 2003; 22: 436-443.

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 p16^INK4a 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 p16^INK4a and phosphorylated NF-ĸB than the tissue from asymptomatic smokers and asymptomatic nonsmokers. Higher percentages of p16^INK4a-positive senescent type II cells than of p16^INK4a-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.

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 (cH2AX), 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 cH2AX foci per cell had higher percentages of type II cells that expressed p16 INK4a(p16), phosphorylated nuclear factor (NF)-kB and interleukin (IL)-6, and of alveolar wall cells that expressed active caspase-3. The type II cells that contained higher numbers of cH2AX foci per cell had higher rates of expression of p16, phosphorylated NF-kB, and IL-6. Half of the alveolar wall cells that expressed active-caspase-3 contained cH2AX foci. Type II cells that stained positive for 8-hydroxy-2-deoxyguanosine contained a higher number of cH2AX 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.