Comparison of direct and indirect models of early induced acute lung injury.

BACKGROUND: The animal experimental counterpart of human acute respiratory distress syndrome (ARDS) is acute lung injury (ALI). Most models of ALI involve reproducing the clinical risk factors associated with human ARDS, such as sepsis or acid aspiration; however, none of these models fully replicates human ARDS. AIM: To compare different experimental animal models of ALI, based on direct or indirect mechanisms of lung injury, to characterize a model which more closely could reproduce the acute phase of human ARDS. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to intratracheal instillations of (1) HCl to mimic aspiration of gastric contents; (2) lipopolysaccharide (LPS) to mimic bacterial infection; (3) HCl followed by LPS to mimic aspiration of gastric contents with bacterial superinfection; or (4) cecal ligation and puncture (CLP) to induce peritonitis and mimic sepsis. Rats were sacrificed 24 h after instillations or 24 h after CLP. RESULTS: At 24 h, rats instilled with LPS or HCl-LPS had increased lung permeability, alveolar neutrophilic recruitment and inflammatory markers (GRO/KC, TNF-α, MCP-1, IL-1ß, IL-6). Rats receiving only HCl or subjected to CLP had no evidence of lung injury. CONCLUSIONS: Rat models of ALI induced directly by LPS or HCl-LPS more closely reproduced the acute phase of human ARDS than the CLP model of indirectly induced ALI.

Nebulized Heparin Attenuates Pulmonary Coagulopathy and Inflammation through Alveolar Macrophages in a Rat Model of Acute Lung Injury.

Objective Alveolar macrophages play a key role in the development and resolution of acute respiratory distress syndrome (ARDS), modulating the inflammatory response and the coagulation cascade in lungs. Anti-coagulants may be helpful in the treatment of ARDS. This study investigated the effects of nebulized heparin on the role of alveolar macrophages in limiting lung coagulation and inflammatory response in an animal model of acute lung injury (ALI). Methods Rats were randomized to four experimental groups. In three groups, ALI was induced by intratracheal instillation of lipopolysaccharide (LPS) and heparin was nebulized at constant oxygen flow: the LPS/Hep group received nebulized heparin 4 and 8 hours after injury; the Hep/LPS/Hep group received nebulized heparin 30 minutes before and 4 and 8 hours after LPS-induced injury; the LPS/Sal group received nebulized saline 4 and 8 hours after injury. The control group received only saline. Animals were exsanguinated 24 hours after LPS instillation. Lung tissue, bronchoalveolar lavage fluid (BALF) and alveolar macrophages isolated from BALF were analysed. Results LPS increased protein concentration, oedema and neutrophils in BALF as well as procoagulant and proinflammatory mediators in lung tissue and alveolar macrophages. In lung tissue, nebulized heparin attenuated ALI through decreasing procoagulant (tissue factor, thrombin-anti-thrombin complexes, fibrin degradation products) and proinflammatory (interleukin 6, tumour necrosis factor alpha) pathways. In alveolar macrophages, nebulized heparin reduced expression of procoagulant genes and the effectors of transforming growth factor beta (Smad 2, Smad 3) and nuclear factor kappa B (p-selectin, CCL-2). Pre-treatment resulted in more pronounced attenuation. Conclusion Nebulized heparin reduced pulmonary coagulopathy and inflammation without producing systemic bleeding, partly by modulating alveolar macrophages.

Role of heparin in pulmonary cell populations in an in-vitro model of acute lung injury.

BACKGROUND: In the early stages of acute respiratory distress syndrome (ARDS), pro-inflammatory mediators inhibit natural anticoagulant factors and initiate an increase in procoagulant activity. Previous studies proved the beneficial effects of heparin in pulmonary coagulopathy, which derive from its anticoagulant and anti-inflammatory activities, although it is uncertain whether heparin works. Understanding the specific effect of unfractioned heparin on cell lung populations would be of interest to increase our knowledge about heparin pathways and to treat ARDS. METHODS: In the current study, the effect of heparin was assessed in primary human alveolar macrophages (hAM), alveolar type II cells (hATII), and fibroblasts (hF) that had been injured with LPS. RESULTS: Heparin did not produce any changes in the Smad/TGFß pathway, in any of the cell types evaluated. Heparin reduced the expression of pro-inflammatory markers (TNF-α and IL-6) in hAM and deactivated the NF-kß pathway in hATII, diminishing the expression of IRAK1 and MyD88 and their effectors, IL-6, MCP-1 and IL-8. CONCLUSIONS: The current study demonstrated that heparin significantly ameliorated the cells lung injury induced by LPS through the inhibition of pro-inflammatory cytokine expression in macrophages and the NF-kß pathway in alveolar cells. Our results suggested that a local pulmonary administration of heparin through nebulization may be able to reduce inflammation in the lung; however, further studies are needed to confirm this hypothesis.

Obesity and intermittent hypoxia increase tumor growth in a mouse model of sleep apnea.

BACKGROUND: Intermittent hypoxia and obesity which are two pathological conditions commonly found in patients with obstructive sleep apnea (OSA), potentially enhance cancer progression. OBJECTIVE: To investigate whether obesity and/or intermittent hypoxia (IH) mimicking OSA affect tumor growth. METHODS: A subcutaneous melanoma was induced in 40 mice [22 obese (40-45g) and 18 lean (20-25g)] by injecting 10(6) B16F10 cells in the flank. Nineteen mice (10 obese/9 lean) were subjected to IH (6h/day for 17days). A group of 21 mice (12 obese/9 lean) were kept under normoxia. At day 17, tumors were excised, weighed and processed to quantify necrosis and endothelial expression of vascular endothelial growth factor (VEGF) and CD-31. VEGF in plasma was also assessed. RESULTS: In lean animals, IH enhanced tumor growth from 0.81±0.17 to 1.95±0.32g. In obese animals, a similar increase in tumor growth (1.94±0.18g) was observed under normoxia, while adding IH had no further effect (1.69±0.23g). IH only promoted an increase in tumoral necrosis in lean animals. However, obesity under normoxic conditions increased necrosis, VEGF and CD-31 expression in tumoral tissue. Plasma VEGF strongly correlated with tumor weight (ρ=0.76, p<0.001) in the whole sample; it increased in lean IH-treated animals from 66.40±3.47 to 108.37±9.48pg/mL, p<0.001), while the high baseline value in obese mice (106.90±4.32pg/mL) was unaffected by IH. CONCLUSIONS: Obesity and IH increased tumor growth, but did not appear to exert any synergistic effects. Circulating VEGF appeared as a crucial mediator of tumor growth in both situations.

Pre-treatment with mesenchymal stem cells reduces ventilator-induced lung injury.

Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injury in animals challenged by bleomycin or bacterial lipopolysaccaride. It is not known, however, whether MSCs protect from ventilator-induced lung injury (VILI). This study investigated whether MSCs have a potential role in preventing or modulating VILI in healthy rats subjected to high-volume ventilation. 24 Sprague-Dawley rats (250-300 g) were subjected to high-volume mechanical ventilation (25 mL·kg(-1)). MSCs (5 × 10(6)) were intravenously or intratracheally administered (n=8 each) 30 min before starting over-ventilation and eight rats were MSC-untreated. Spontaneously breathing anesthetised rats (n=8) served as controls. After 3 h of over-ventilation or control the animals were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were sampled for further analysis. When compared with controls, MSC-untreated over-ventilated rats exhibited typical VILI features. Lung oedema, histological lung injury index, concentrations of total protein, interleukin-1ß, macrophage inflammatory protein-2 and number of neutrophils in BALF and vascular cell adhesion protein-1 in lung tissue significantly increased in over-ventilated rats. All these indices of VILI moved significantly towards normalisation in the rats treated with MSCs, whether intravenously or intratracheally. Both local and systemic pre-treatment with MSCs reduced VILI in a rat model.

Effects of exercise training on airway responsiveness and airway cells in healthy subjects.

Airway responsiveness to methacholine (Mch) in the absence of deep inspirations (DIs) is lower in athletes compared with sedentary individuals. In this prospective study, we tested the hypothesis that a training exercise program reduces the bronchoconstrictive effect of Mch. Ten healthy sedentary subjects (M/F: 3/7; mean + or - SD age: 22 + or - 3 yr) entered a 10-wk indoor rowing exercise program on rowing ergometer and underwent Mch bronchoprovocation in the absence of DIs at baseline, at weeks 5 and 10, as well as 4-6 wk after the training program was completed. Exercise-induced changes on airway cells and markers of airway inflammation were also assessed by sputum induction and venous blood samples. Mean power output during the 1,000 m test was 169 + or - 49 W/stroke at baseline, 174 + or - 49 W/stroke at 5 wk, and 200 + or - 60 W/stroke at 10 wk of training (P < 0.05). The median Mch dose used at baseline was 50 mg/ml (range 25-75 mg/ml) and remained constant per study design. At the pretraining evaluation, the percent reduction in the primary outcome, the inspiratory vital capacity (IVC) after inhalation of Mch in the absence of DIs was 31 +/- 13%; at week 5, the Mch-induced reduction in IVC was 22 + or - 19%, P = 0.01, and it further decreased to 15 + or - 11% at week 10 (P = 0.0008). The percent fall in IVC 4-6 wk after the end of training was 15 + or - 11% (P = 0.87 vs. end of training). Changes in airway cells were not associated with changes in airway responsiveness. Our data show that a course of exercise training can attenuate airway responsiveness against Mch inhaled in the absence of DIs in healthy subjects and suggest that a sedentary lifestyle may favor development of airways hyperresponsiveness.

Hemopoietic and angiogenetic progenitors in healthy athletes: different responses to endurance and maximal exercise.

The effects of endurance or maximal exercise on mobilization of bone marrow-derived hemopoietic and angiogenetic progenitors in healthy subjects are poorly defined. In 10 healthy amateur runners, we collected venous blood before, at the end of, and the day after a marathon race (n = 9), and before and at the end of a 1.5-km field test (n = 8), and measured hemopoietic and angiogenetic progenitors by flow cytometry and culture assays, as well as plasma or serum concentrations of several cytokines/growth factors. After the marathon, CD34(+) cells were unchanged, whereas clonogenetic assays showed decreased number of colonies for both erythropoietic (BFU-E) and granulocyte-monocyte (CFU-GM) series, returning to baseline the morning post-race. Conversely, CD34(+) cells, BFU-E, and CFU-GM increased after the field test. Angiogenetic progenitors, assessed as CD34(+)KDR(+) and CD133(+)VE-cadherin(+) cells or as adherent cells in culture expressing endothelial markers, increased after both endurance and maximal exercise but showed a different pattern between protocols. Interleukin-6 increased more after the marathon than after the field test, whereas hepatocyte growth factor and stem cell factor increased similarly in both protocols. Plasma levels of angiopoietin (Ang) 1 and 2 increased after both types of exercise, whereas the Ang-1-to-Ang-2 ratio or vascular endothelial growth factor-A were little affected. These data suggest that circulating hemopoietic progenitors may be utilized in peripheral tissues during prolonged endurance exercise. Endothelial progenitor mobilization after exercise in healthy trained subjects appears modulated by the type of exercise. Exercise-induced increase in growth factors suggests a physiological trophic effect of exercise on the bone marrow.

Bronchial epithelial damage after a half-marathon in nonasthmatic amateur runners.

High neutrophil counts in induced sputum have been found in nonasthmatic amateur runners at rest and after a marathon, but the pathogenesis of airway neutrophilia in athletes is still poorly understood. Bronchial epithelial damage may occur during intense exercise, as suggested by investigations conducted in endurance-trained mice and competitive human athletes studied under resting conditions. To gain further information on airway changes acutely induced by exercise, airway cell composition, apoptosis, IL-8 concentration in induced sputum, and serum CC-16 level were measured in 15 male amateur runners at rest (baseline) and shortly after a half-marathon. Different from results obtained after a marathon, neutrophil absolute counts were unchanged, whereas bronchial epithelial cell absolute counts and their apoptosis increased significantly (P < 0.01). IL-8 in induced sputum supernatants almost doubled postrace compared with baseline (P < 0.01) and correlated positively with bronchial epithelial cell absolute counts (R(2) = 0.373, P < 0.01). Serum CC-16 significantly increased after all races (P < 0.01). These data show mild bronchial epithelial cell injury acutely induced by intense endurance exercise in humans, extending to large airways the data obtained in peripheral airways of endurance-trained mice. Therefore, neutrophil influx into the airways of athletes may be secondary to bronchial epithelial damage associated with intense exercise.

Environmental conditions, air pollutants, and airway cells in runners: a longitudinal field study.

Runners have increased numbers of neutrophils in the airways at rest and after exercise compared with sedentary individuals. The aim of this study was to determine whether Mediterranean seasonal changes in temperature, humidity or airborne pollutants affect the airway cells of runners training outdoors in an urban environment. In nine male amateur runners, cell composition, apoptosis, and inflammatory mediators were measured in induced sputum collected at rest (baseline) and the morning after races held in the fall (21 km), winter (12 km), and summer (10 km). Concentrations of air pollutants were below the alert threshold at all times. Neutrophil differential counts tended to increase after all races (P = 0.055). Apoptosis of neutrophils increased with ozone (P < 0.005) and particulate matter <10 microm (PM10) (P < 0.05) exposure. Bronchial epithelial cell counts were low at all times and weakly correlated with ozone and PM10 concentrations. Apoptotic bronchial epithelial cells increased after all races (P < 0.05). Inflammatory mediators in induced sputum were low at baseline and after the races, and correlated with neutrophil differential counts only at rest. In conclusion, apoptosis of airway cells in runners appears to be affected by both exercise and environmental conditions. Apoptosis of neutrophils increased with exposure to environmental pollutants while apoptosis of bronchial epithelial cells increased after intense exercise. Since no relationship was observed between neutrophil counts and inflammatory mediators 20 h after races, airways inflammation at this time point appears blunted in healthy runners and little affected by exposure to mild seasonal changes and airborne pollutants.

Endurance training damages small airway epithelium in mice.

RATIONALE: In athletes, airway inflammatory cells were found to be increased in induced sputum or bronchial biopsies. Most data were obtained after exposure to cold and dry air at rest or during exercise. Whether training affects epithelial and inflammatory cells in small airways is unknown. OBJECTIVES: To test whether endurance training under standard environmental conditions causes epithelial damage and inflammation in the small airways of mice. METHODS AND MEASUREMENTS: Formalin-fixed, paraffin-embedded lung sections were obtained in sedentary (n = 14) and endurance-trained (n = 16) Swiss mice at baseline and after 15, 30, and 45 days of training. The following variables were assessed (morphometry and immunohistochemistry) in small airways (basement membrane length < 1 mm): (1) integrity, proliferation, and apoptosis of bronchiolar epithelium; and (2) infiltration, activation, and apoptosis of inflammatory cells. MAIN RESULTS: Compared with sedentary mice, bronchiolar epithelium of trained mice showed progressive loss of ciliated cells, slightly increased thickness, unchanged goblet cell number and appearance, and increased apoptosis and proliferation (proliferating cell nuclear antigen) (p < 0.001 for all variables). Leukocytes (CD45(+) cells) infiltrated airway walls (p < 0.0001) and accumulated within the lumen (p < 0.001); however, apoptosis of CD45(+) cells did not differ between trained and sedentary mice. Nuclear factor-kappaB translocation and inhibitor-alpha of NF-kappaB (IkappaBalpha) phosphorylation were not increased in trained compared with sedentary mice. CONCLUSIONS: Bronchiolar epithelium showed damage and repair associated with endurance training. Training increased inflammatory cells in small airways, but inflammatory activation was not increased. These changes may represent an adaptive response to increased ventilation during exercise.

Airway cell composition at rest and after an all-out test in competitive rowers.

PURPOSES: This study was designed to assess: a) whether rowing affects airway cell composition, and b) the possible relationship between the degree of ventilation during exercise and airway cells. SUBJECTS AND METHODS: In nine young, nonasthmatic competitive rowers (mean age +/- SD: 16.2 +/- 1.0 yr), induced sputum samples were obtained at rest and shortly after an all-out rowing test over 1000 m (mean duration: 200 +/- 14 s), during which ventilatory and metabolic variables were recorded breath-by-breath (Cosmed K4b, Italy). RESULTS: At rest, induced sputum showed prevalence of neutrophils (60%) over macrophages (40%); after exercise, total cell and bronchial epithelial cell (BEC) counts tended to increase. In the last minute of exercise, mean VE was 158.0 +/- 41.5 L x min(-1), and VO2 x kg(-1) 62 +/- 11 mL x min(-1). Exercise VE correlated directly with postexercise total cell (Spearman rho: 0.75, P < 0.05) an dmacrophage (rho: 0.82, P < 0.05) counts. A similar trend was observed for exercise VE and changes in BEC counts from baseline to postexercise (rho: 0.64, P = 0.11). Exercise VE did not correlate with airway neutrophil counts at rest or after exercise. Expression of adhesion molecules by airway neutrophils, macrophages, and eosinophils decreased after the all-out test. CONCLUSION: Similar to endurance athletes, nonasthmatic competitive rowers showed increased neutrophils in induced sputum compared with values found in sedentary subjects. The trend toward increased BEC postexercise possibly reflected the effects of high airflows on airway epithelium. Airway macrophages postexercise were highest in rowers showing tile most intense exercise hyperpnea, suggesting early involvement of these cells during exercise. However, the low expression of adhesion molecules by all airway cell types suggests that intense short-lived exercise may be associated with a blunted response of airway cells in nonasthmatic well-trained rowers.

Airway cells after swimming outdoors or in the sea in nonasthmatic athletes.

BACKGROUND: Marathon runners and elite swimmers showed increased inflammatory cells in the airways at baseline. Although airway neutrophils increase further after a marathon race, the airway response to swimming is unknown. The aim of this study was to assess the effects of swimming on airway cells. To avoid the concomitant effects of chronic exposure to chlorine, the study was conducted in seven nonasthmatic swimmers [mean age (SD): 23.3 +/- 7.7 yr, training: 32 +/- 15 km.wk-1] habitually training in an outdoor pool (OP), i.e., a low-chlorine environment. METHODS: Spirometry, exhaled nitric oxide (NO), induced sputum, and peripheral blood samples were obtained at baseline, after a 5-km trial in OP, and after a 5-km race in the sea (S), i.e., hypertonic airway exposure. RESULTS: Airway neutrophil differential counts at baseline were higher in swimmers than in sedentary controls (N = 10), but cell counts, neutrophil elastase, and eosinophil cationic protein were unaffected by 5-km swimming. After swimming, L-selectin expression on airway cells decreased, suggesting exercise-induced cell mobilization into the airways and/or direct effects of hyperventilation on airway cells. After S, airway eosinophil differential counts increased slightly. Exhaled NO concentration was 19 +/- 6 ppb at baseline, 8 +/- 4 ppb after OP, and 21 +/- 7 ppb after S (P < 0.005 for OP vs baseline and S). CONCLUSIONS: In swimmers not chronically exposed to high chlorine concentrations, data obtained at baseline suggest a direct relationship between airway neutrophilia and endurance training. The low L-selectin expression by airway cells postexercise suggests hyperventilation-induced cell recruitment or modulation of cell function. Hypertonic exposure of airways during exercise may slightly increase airway eosinophils and exhaled NO. Overall, 5-km swimming exerted smaller effects on airway cells than running a marathon.