The effect of smoking cessation and steroid treatment on emphysema in guinea pigs

BACKGROUND: Emphysema induced by cigarette smoking is characterized by an inflammatory process, which is resistant to steroid and remains active in lung tissue long after smoking has stopped. Latent adenoviral infection (Ad5) increases emphysema development and the inflammatory response to cigarette smoke and, in allergic lung inflammation, suppresses anti-inflammatory effects of steroids. OBJECTIVES: The present study was designed to examine the effect of smoking cessation and steroid treatment on lung emphysema and inflammation in a guinea pig model of emphysema and to determine if latent adenoviral infection induces resistance to the inflammatory effects of steroid. METHODS: Latent adenovirus or sham infected animals exposed to room air or cigarette smoke for 16 weeks were either sacrificed immediately or treated with dexamethasone or diluent for an additional 5 weeks without smoke exposure. Lung morphometry, inflammatory cells and mediators were studied. RESULTS: Smoking cessation was associated with an increase in lung surface area and surface area to volume ratio. Smoking cessation was also associated with decreases in lung neutrophils, CD4 cells, and IL-8, RANTES and IFN-gamma mRNAs to control levels. Steroid treatment significantly lowered neutrophils, eosinophils and IFN-gamma mRNA and, while adenoviral infection did not alter these steroid-induced changes, it independently increased airway wall neutrophils and CD8 cells. CONCLUSION: Smoking cessation decreases lung inflammation and latent adenoviral infection does not induce steroid resistance in this animal model.     

Acute effects of smoke exposure on the cellular and cytokine profile in isolated perfused lungs

The aim of this study was to assess the acute effects of cigarette smoke exposure on cellular and cytokine profile in BAL fluids in an isolated perfused rabbit assay. The experimental animals were categorized into four groups: (1) unexposed controls and (2) cigarette smoke-exposed animals perfused with autologous whole blood; (3) unexposed controls and cigarette smoke-exposed; (4) cigarette smoke-exposed animals perfused with Krebs' Ringer solution containing 5% bovine serum albumin and glucose. Cigarette smoke induced an increase in total cell numbers (mainly alveolar macrophages in BAL fluids) and an increase in the permeability index of BAL. Levels of interleukin 8 were also significantly decreased in BAL fluids due to acute effects of cigarette smoke exposure. The most likely explanation for cigarette smoke-induced increase of inflammatory cells in BAL in lungs is because of the release of pre-existing cells from reservoirs within the lungs. The acute effects of cigarette smoke-induced increase of pulmonary epithelial permeability may also play an important role in the cellular recruitment into airspaces from the lung reservoirs.     

Macrophage metalloelastase mediates acute cigarette smoke-induced inflammation via tumor necrosis factor-alpha release

The cells and proteases that mediate cigarette smoke-induced emphysema are controversial, with evidence favoring either neutrophils and neutrophil-derived serine proteases or macrophages and macrophage-derived metalloproteases as the important effectors. We recently reported that both macrophage metalloelastase (MMP-12) and neutrophils are required for acute cigarette smoke-induced connective tissue breakdown, the precursor of emphysema. Here we show how these disparate observations can be linked. Both wild-type (MMP-12 +/+) mice and mice lacking MMP-12 (MMP-12 -/-) demonstrated rapid increases in whole-lung nuclear factor-kappaB activation and gene expression of proinflammatory cytokines after cigarette smoke exposure, indicating that a lack of MMP-12 does not produce a global failure to upregulate inflammatory mediators. However, only MMP-12 +/+ mice demonstrated increased whole-lung tumor necrosis factor-alpha (TNF-alpha) protein or release of TNF-alpha from cultured alveolar macrophages exposed to smoke in vitro. Levels of whole-lung E-selectin, an endothelial activation marker, were increased in only MMP-12 +/+ mice. These findings suggest that, acutely, MMP-12 mediates smoke-induced inflammation by releasing TNF-alpha from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil-derived proteases. TNF-alpha release may be a general mechanism whereby metalloproteases drive cigarette smoke-induced inflammation.     

Antioxidant diet protects against emphysema, but increases mortality in cigarette smoke-exposed mice

Oxidative stress plays an important role in cigarette smoke-induced lung inflammation and emphysema. We produced an enriched diet by adding freeze-dried fruits and vegetables and additional supplements to the 8604 Teklad Rodent Diet, a standard rodent diet. In this study, we examined the effects of the antioxidant-enriched diet on cigarette smoke-induced lung inflammation and emphysema. CH3/HeN mice were fed either a regular diet or the supplemented diet. These mice were exposed to filtered air, a low concentration of cigarette smoke (total particulate matter: 100 mg/m3) or a high concentration of cigarette smoke (total particulate matter: 250 mg/m3) for 6 h/day, 5 days/week for total 16 weeks. Surprisingly, increased mortality (53%) was observed in the high concentration of cigarette smoke-exposed mice fed the antioxidant diet compared to the high concentration of cigarette smoke-exposed mice that were fed a regular diet (13%). The necropsy analysis revealed nasal passage obstruction due to mucous plugging in cigarette smoke-exposed mice on the antioxidant diet. However, the antioxidant diet significantly reduced neutrophilic inflammation and emphysema in the high concentration of cigarette smoke-exposed mice as compared to the regular diet /high concentration of cigarette smoke controls. The antioxidant capacity in the bronchoalveolar fluid or oxidative damage to the lung tissue was not affected by the antioxidant diet. Pro-MMP-2, MMP-2, and MMP-9 activity did not correlate with the protective effects of AOD on cigarette smoke-induced emphysema. These data suggest that the antioxidant diet reduced cigarette smoke-induced inflammation and emphysema, but increased mortality in the obligate nose-breathing mice.     

Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.     

Humic acid enhances cigarette smoke-induced lung emphysema in mice and IL-8 release of human monocytes

Tobacco smoke is the main factor in the etiology of lung emphysema. Generally prolonged, substantial exposure is required to develop the disease. Humic acid is a major component of cigarette smoke that accumulates in smokers’ lungs over time and induces tissue damage.

Objectives

To investigate whether humic acid pre-loading potentiates the development of cigarette smoke-induced lung emphysema in mice and increases IL-8 release by human monocytes.

Methods

C57BL/6J mice received humic acid or aqueous vehicle by tracheal installation on day 0 and day 7. From day 21 to day 84, the mice were exposed to cigarette smoke or clean air for 5 days/week. Twenty-four hours after the last exposure we determined leukocytes in lung lavage, heart hypertrophy and alveolar wall destruction. Human monocytes were incubated with cigarette smoke extract (CSE), humic acid or the combination overnight.

Results

Humic acid nor cigarette smoke caused alveolar wall destruction within two months. Interestingly, the combination did induce lung emphysema. Humic acid, cigarette smoke or the combination did not change leukocyte types and numbers in lung lavage fluid, but the combination caused peribronchiolar and perivascular lymphocyte infiltration. Humic acid treatment resulted in a high proportion of alveolar macrophages heavily loaded with intracellular granula. Humic acid also induces the release of IL-8 from human monocytes and enhances the CSE-induced IL-8 release.

Conclusions

Humic acid deposition in the lungs potentiates the development of cigarette smoke-induced interstitial inflammation and lung emphysema. Moreover, humic acid promotes IL-8 release from human monocytes. Since humic acid accumulates steadily in the lungs of smokers, this may provide an explanation for the natural history on late onset of this disease. The model described here offers a novel way to study emphysema and may direct the search for new therapeutic approaches.     

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.     

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.     

A novel antiapoptotic role for alpha1-antitrypsin in the prevention of pulmonary emphysema

RATIONALE: There is growing evidence that alveolar cell apoptosis plays an important role in emphysema pathogenesis, a chronic inflammatory lung disease characterized by alveolar destruction. The association of alpha1-antitrypsin deficiency with the development of emphysema has supported the concept that protease/antiprotease imbalance mediates cigarette smoke-induced emphysema. OBJECTIVES: We propose that, in addition to its antielastolytic effects, alpha1-antitrypsin may have broader biological effects in the lung, preventing emphysema through inhibition of alveolar cells apoptosis. METHODS, MEASUREMENTS, AND MAIN RESULTS: Transduction of human alpha1-antitrypsin via replication-deficient adeno-associated virus attenuated airspace enlargement and emphysema caused by inhibition of vascular endothelial growth factor (VEGF) receptors with SU5416 in mice, a model of apoptosis-dependent emphysema lacking neutrophilic inflammation. The overexpressed human serine protease inhibitor accumulated in lung cells and suppressed caspase-3 activation and oxidative stress in lungs treated with the VEGF blocker or with VEGF receptor-1 and -2 antibodies. Similar results were obtained in SU5416-treated rats given human alpha1-antitrypsin intravenously. CONCLUSIONS: Our findings suggest that inhibition of structural alveolar cell apoptosis by alpha1-antitrypsin represents a novel protective mechanism of the serpin against emphysema. Further elucidation of this mechanism may extend the therapeutic options for emphysema caused by reduced level or loss of function of alpha1-antitrypsin.     

辛伐他汀对烟雾暴露大鼠肺泡上皮细胞的作用

目的 探讨辛伐他汀对烟雾暴露大鼠肺气肿肺泡上皮细胞及血管内皮生长因子(VEGF)表达的影响.方法 24只12周龄健康雌雄各半Wistar大鼠随机数字表法分为对照组、烟雾组、药物组和烟雾+药物组(烟+药组),每组6只,同步饲养16周,雌雄大鼠同步、分箱饲养16周.分别采用逆转录PCR法测定肺组织中VEGF的mRNA表达,酶联免疫吸附法测定BALF和组织中VEGF含量,免疫组织化学二步法检测VEGF和增殖细胞核抗原(PCNA)表达水平.多组间比较采用单因素方差分析,两两比较采用LSD-t检验.结果 烟+药组大鼠肺泡上皮细胞PCNA阳性细胞构成比[(10.3±2.0)%]明显高于烟雾组[(4.8±0.8)%];BALF和组织匀浆中VEGF水平[(187±15)ng/L和(6782±50)ng/L]接近于对照组[(200±20)ng/L和(7558±330)ng/L],明显高于烟雾组[(71±16)ng/L和(4149±110)ng/L];肺泡和支气管上皮细胞中VEGF表达[(67.7±5.0)%和(49.0±3.0)%]近似于对照组[(68.3±3.3)%和(51.3±2.9)%],明显高于烟雾组[(27.0±5.9)%和(16.3±2.7)%].烟+药组与烟雾组比较,差异均有统计学意义(t值为1.117～12.001,均P＜0.01).结论 辛伐他汀可部分促进肺泡上皮细胞增殖,提高肺组织中VEGF含量及肺泡上皮细胞表达,延缓烟雾暴露所致大鼠肺气肿的进展.     

Antioxidant Diet Protects Against Emphysema,but Increases Mortality in Cigarette Smoke-Exposed Mice

Oxidative stress plays an important role in cigarette smoke-induced lung inflammation and emphysema. We produced an enriched diet by adding freeze-dried fruits and vegetables and additional supplements to the 8604 Teklad Rodent Diet, a standard rodent diet. In this study, we examined the effects of the antioxidant-enriched diet on cigarette smoke-induced lung inflammation and emphysema. CH3/HeN mice were fed either a regular diet or the supplemented diet. These mice were exposed to filtered air, a low concentration of cigarette smoke (total particulate matter: 100 mg/m³) or a high concentration of cigarette smoke (total particulate matter: 250 mg/m³) for 6 h/day, 5 days/week for total 16 weeks. Surprisingly, increased mortality (53%) was observed in the high concentration of cigarette smoke-exposed mice fed the antioxidant diet compared to the high concentration of cigarette smoke-exposed mice that were fed a regular diet (13%). The necropsy analysis revealed nasal passage obstruction due to mucous plugging in cigarette smoke-exposed mice on the antioxidant diet. However, the antioxidant diet significantly reduced neutrophilic inflammation and emphysema in the high concentration of cigarette smoke-exposed mice as compared to the regular diet /high concentration of cigarette smoke controls. The antioxidant capacity in the bronchoalveolar fluid or oxidative damage to the lung tissue was not affected by the antioxidant diet. Pro-MMP-2, MMP-2, and MMP-9 activity did not correlate with the protective effects of AOD on cigarette smoke-induced emphysema. These data suggest that the antioxidant diet reduced cigarette smoke-induced inflammation and emphysema, but increased mortality in the obligate nose-breathing mice.     

Time course of cigarette smoke-induced pulmonary inflammation in mice.

Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease. In the present study a murine model of tobacco smoke-induced emphysema was used to investigate the time course of airway and pulmonary inflammatory response, with a special emphasis on pulmonary dendritic cell (DC) populations. Groups of mice were exposed to either cigarette smoke or to control air for up to 24 weeks. In response to cigarette smoke, inflammatory cells (i.e. neutrophils, macrophages and lymphocytes) progressively accumulated both in the airways and lung parenchyma of mice. Furthermore, a clear infiltration of DCs was observed in airways (10-fold increase) and lung parenchyma (1.5-fold increase) of cigarette-exposed mice at 24 weeks. Flow cytometric analysis of bronchoalveolar lavage (BAL) DCs of smoke-exposed mice showed upregulation of major histocompatability complex II molecules and costimulatory molecules CD40 and CD86, compared with BAL DCs of air-exposed mice. Morphometric analysis of lung histology demonstrated a significant increase in mean linear intercept and alveolar wall destruction after 24 weeks of smoke exposure. In conclusion, the time course of the changes in inflammatory and dendritic cells in both bronchoalveolar lavage and the pulmonary compartment of cigarette smoke-exposed mice was carefully characterised.     

The Importance of Ultramicroscopic Emphysema in Cigarette Smoke-Induced Lung Disease

To determine the role of the alveolar pores in cigarette smoke-induced lung disease, we examined the alveolar pores of guinea pigs exposed to cigarette smoke for 12 months, and compared these data to those obtained from sham-smoked animals, correlating the data with airspace size and lung function. We found that the smoke-exposed animals had a larger mean number of pores per alveolus (p < 0.001), and the distributions of pore size and shape were significantly shifted to indicate a larger and more irregular pore configuration (p < 0.001, 01 respectively). In the smoke exposed group, there was a significant correlation of pore number with total lung capacity (TLC) (0.68 p < 0.05), RV (0.70, p < 0.05), and FEV(0.1)/FVC(-0.77, p < 0.02). No correlations were identified between pore size or shape and the lung function tests. We conclude that cigarette smoke exposure produces an increase in the number of alveolar pores, a process which we believe represents ultramicroscopic emphysema. These alterations appear to precede any increase in airspace size, and may help to explain abnormal lung function in cigarette smokers without macroscopic emphysema or small airway disease. This is the first study to clearly document an increased number of alveolar pores, with a significant number of either/or large and irregular pores, after chronic smoke exposure, but in the absence of gross emphysema.     

Latent adenoviral infection in the pathogenesis of chronic airways obstruction.

Childhood infection of the respiratory tract has been proposed as an independent risk factor in the pathogenesis of the chronic obstructive pulmonary disease (COPD) that develops in cigarette smokers. The present study examines adult lung tissue for latent adenoviral DNA because many of its 41 serotypes cause childhood respiratory disease and the virus is known to persist in other tissues in a latent form. Lung tissue resected for solitary nodules from 20 patients with airways obstruction and 20 patients without airways obstruction, matched for age, sex, and smoking history, were compared to determine whether adenoviral DNA is more commonly found in patients with COPD. The polymerase chain reaction (PCR) was used to examine two widely separated segments of the adenoviral genome. In situ hybridization (ISH) was performed using a probe covering the entire viral genome to determine the types of cell infected by the virus. The PCR analysis showed that a 675 base pair target sequence of the E1A region of the adenovirus was present in most of the lungs studied with greater copy numbers in the smokers with airways obstruction in both paraffin-embedded (p less than 0.002) and frozen lung tissue (p less than 0.016), whereas the E3/19K region showed no difference between the groups. When sufficient copy numbers were present to localize the DNA by ISH it was found in epithelial cells of the smokers who had airways obstruction. These data are consistent with a current model of adenoviral integration into host DNA and suggest that the E1A region of the adenovirus may contribute to the pathogenesis of COPD.     

An animal model of autoimmune emphysema

Although cigarette smoking is implicated in the pathogenesis of emphysema, the precise mechanisms of chronic progressive alveolar septal destruction are not well understood. We show, in a novel animal model, that immunocompetent, but not athymic, nude rats injected intraperitoneally with xenogeneic endothelial cells (ECs) produce antibodies against ECs and develop emphysema. Immunization with ECs also leads to alveolar septal cell apoptosis and activation of matrix metalloproteases MMP-9 and MMP-2. Anti-EC antibodies cause EC apoptosis in vitro and emphysema in passively immunized mice. Moreover, immunization also causes accumulation of CD4+ T cells in the lung. Adoptive transfer of pathogenic, spleen-derived CD4+ cells into naive immunocompetent animal also results in emphysema. This study shows for the first time that humoral- and CD4+ cell-dependent mechanisms are sufficient to trigger the development of emphysema, suggesting that alveolar septal cell destruction might result from immune mechanisms.     

Autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by chronic exposure to cigarette smoke (CS), which involves airway obstruction and alveolar loss (i.e., emphysema). The mechanisms of COPD pathogenesis remain unclear. Our previous studies demonstrated elevated autophagy in human COPD lung, and as a cellular and tissue response to CS exposure in an experimental model of emphysema in vivo. We identified the autophagic protein microtubule-associated protein 1 light chain-3B (LC3B) as a positive regulator of CS-induced lung epithelial cell death. We now extend these initial observations to explore the mechanism by which LC3B mediates CS-induced apoptosis and emphysema development in vivo. Here, we observed that LC3B(-/-) mice had significantly decreased levels of apoptosis in the lungs after CS exposure, and displayed resistance to CS-induced airspace enlargement, relative to WT littermate mice. We found that LC3B associated with the extrinsic apoptotic factor Fas in lipid rafts in an interaction mediated by caveolin-1 (Cav-1). The siRNA-dependent knockdown of Cav-1 sensitized epithelial cells to CS-induced apoptosis, as evidenced by enhanced death-inducing signaling complex formation and caspase activation. Furthermore, Cav-1(-/-) mice exhibited higher levels of autophagy and apoptosis in the lung in response to chronic CS exposure in vivo. In conclusion, we demonstrate a pivotal role for the autophagic protein LC3B in CS-induced apoptosis and emphysema, suggestive of novel therapeutic targets for COPD treatment. This study also introduces a mechanism by which LC3B, through interactions with Cav-1 and Fas, can regulate apoptosis.     

Retinoid treatment of Emphysema in Patients on the Alpha-1 International Registry. The REPAIR study: study design, methodology and quality control of study assessments

Emphysema is characterized by the destruction of alveolar wall and enlargement of alveolar airspaces, resulting in a reduction of the total lung gas exchange area, loss of lung elastic recoil and hyperinflation. The REPAIR study (Retinoid treatment of Emphysema in Patients on the Alpha-1 International Registry) is the first proof-of-concept study of a new potential disease-modifying drug, Palovarotene?, an orally active, gamma selective retinoid agonist in patients with emphysema secondary to alpha-1-antitrypsin deficiency (AATD) as a model population for the general smoke-induced emphysema population. This article describes the study design as well as the effectiveness of the quality control that was implemented on the key efficacy endpoints, based on data derived from the placebo-treated subjects. In this multicentre, multinational study the implementation of standardized procedures included: careful site selection, use of trained staff, regular monitoring and machine calibration, use of biological controls and regular feedback to sites by an independent quality control centre. All of these procedures resulted in high-quality measurements of lung density, spirometry, static lung volumes and gas transfer. It was also confirmed that CT lung density was the most sensitive endpoint followed by TLco, FEV(1) and RV measured by body box.     

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.