Elisabet Ferrer

Cigarette Smoke-induced Oxidative Stress A Role in Chronic Obstructive Pulmonary Disease Skeletal Muscle Dysfunction

RATIONALE Inflammation and oxidative stress contribute to muscle dysfunction in patients with chronic obstructive pulmonary disease (COPD). Oxidants contained in cigarette smoke (CS) induce adverse effects on tissues through oxidative phenomena. OBJECTIVES To explore oxidative stress and inflammation in quadriceps of human smokers and in diaphragm and limb muscles of guinea pigs chronically exposed to CS. METHODS Muscle function, protein oxidation and nitration, antioxidants, oxidized proteins, inflammation, creatine kinase activity, and lung and muscle structures were investigated in vastus lateralis of smokers, patients with COPD, and healthy control subjects and in diaphragm and gastrocnemius of CS-exposed guinea pigs at 3, 4, and 6 months. MEASUREMENTS AND MAIN RESULTS Compared with control subjects, quadriceps muscle force was mildly but significantly reduced in smokers; protein oxidation levels were increased in quadriceps of smokers and patients with COPD, and in respiratory and limb muscles of CS-exposed animals; glycolytic enzymes, creatine kinase, carbonic anydrase-3, and contractile proteins were significantly more carbonylated in quadriceps of smokers and patients with COPD, and in respiratory and limb muscles of CS-exposed guinea pigs. Chronic CS exposure induced no significant rise in muscle inflammation in either smokers or rodents. Muscle creatine kinase activity was reduced only in patients with COPD and in both diaphragm and gastrocnemius of CS-exposed animals. Guinea pigs developed bronchiolar abnormalities at 4 months of exposure and thereafter. CONCLUSIONS CS exerts direct oxidative modifications on muscle proteins, without inducing any significant rise in muscle inflammation. The oxidative damage to muscle proteins, which precedes the characteristic respiratory changes, may contribute to muscle loss and dysfunction in smokers and patients with COPD.

Cigarette smoking exacerbates nonalcoholic fatty liver disease in obese rats

The prevalence of cigarette smoking (CS) is increased among obese subjects, who are susceptible to develop nonalcoholic fatty liver disease (NAFLD). We investigated the hepatic effects of CS in control and obese rats. Control and obese Zucker rats were divided into smokers and nonsmokers (n = 12 per group). Smoker rats were exposed to 2 cigarettes/day, 5 days/week for 4 weeks. The effects of CS were assessed by biochemical analysis, hepatic histological examination, immunohistochemistry, and gene expression analysis. Phosphorylation of AKT and extracellular signal‐regulated kinase (ERK) and quantification of carbonylated proteins were assessed by western blotting. As expected, obese rats showed hypercholesterolemia, insulin resistance, and histological features of NAFLD. Smoking did not modify the lipidic or glucidic serum profiles. Smoking increased alanine aminotransferase serum levels and the degree of liver injury in obese rats, whereas it only induced minor changes in control rats. Importantly, CS increased the histological severity of NAFLD in obese rats. We also explored the potential mechanisms involved in the deleterious effects of CS. Smoking increased the degree of oxidative stress and hepatocellular apoptosis in obese rats, but not in controls. Similarly, smoking increased the hepatic expression of tissue inhibitor of metalloproteinase‐1 and procollagen‐alpha2(I) in obese rats, but not in controls. Finally, smoking regulated ERK and AKT phosphorylation. The deleterious effects of CS were not observed after a short exposure (5 days). Conclusion: CS causes oxidative stress and worsens the severity of NAFLD in obese rats. Further studies should assess whether this finding also occurs in patients with obesity and NAFLD. (HEPATOLOGY 2010.)

Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig

BackgroundCigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by altering the structure and function of pulmonary vessels at early disease stages. The objectives of this study were to evaluate the effects of long-term exposure to cigarette smoke on endothelial function and smooth muscle-cell proliferation in pulmonary arteries of guinea pigs.Methods19 male Hartley guinea pigs were exposed to the smoke of 7 cigarettes/day, 5 days/week, for 3 and 6 months. 17 control guinea pigs were sham-exposed for the same periods. Endothelial function was evaluated in rings of pulmonary artery and aorta as the relaxation induced by ADP. The proliferation of smooth muscle cells and their phenotype in small pulmonary vessels were evaluated by immunohistochemical expression of α-actin and desmin. Vessel wall thickness, arteriolar muscularization and emphysema were assessed morphometrically. The expression of endothelial nitric oxide synthase (eNOS) was evaluated by Real Time-PCR.ResultsExposure to cigarette smoke reduced endothelium-dependent vasodilatation in pulmonary arteries (ANOVA p < 0.05) but not in the aorta. Endothelial dysfunction was apparent at 3 months of exposure and did not increase further after 6 months of exposure. Smoke-exposed animals showed proliferation of poorly differentiated smooth muscle cells in small vessels (p < 0.05) after 3 months of exposure. Prolonged exposure resulted in full muscularization of small pulmonary vessels (p < 0.05), wall thickening (p < 0.01) and increased contractility of the main pulmonary artery (p < 0.05), and enlargement of the alveolar spaces. Lung expression of eNOS was decreased in animals exposed to cigarette smoke.ConclusionIn the guinea pig, exposure to cigarette smoke induces selective endothelial dysfunction in pulmonary arteries, smooth muscle cell proliferation in small pulmonary vessels and reduced lung expression of eNOS. These changes appear after 3 months of exposure and precede the development of pulmonary emphysema.

Stimulation of soluble guanylate cyclase prevents cigarette smoke-induced pulmonary hypertension and emphysema.

RATIONALE Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. No therapy stopping progress of the disease is available. OBJECTIVES To investigate the role of the soluble guanylate cyclase (sGC)-cGMP axis in development of lung emphysema and pulmonary hypertension (PH) and to test whether the sGC-cGMP axis is a treatment target for these conditions. METHODS Investigations were performed in human lung tissue from patients with COPD, healthy donors, mice, and guinea pigs. Mice were exposed to cigarette smoke (CS) for 6 hours per day, 5 days per week for up to 6 months and treated with BAY 63-2521. Guinea pigs were exposed to CS from six cigarettes per day for 3 months, 5 days per week and treated with BAY 41-2272. Both BAY compounds are sGC stimulators. Gene and protein expression analysis were performed by quantitative real-time polymerase chain reaction and Western blotting. Lung compliance, hemodynamics, right ventricular heart mass alterations, and alveolar and vascular morphometry were performed, as well as inflammatory cell infiltrate assessment. In vitro assays of cell adhesion, proliferation, and apoptosis have been done. MEASUREMENTS AND MAIN RESULTS The functionally essential sGC β1-subunit was down-regulated in patients with COPD and in CS-exposed mice. sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma, and inhibited adhesion of CS-stimulated neutrophils. CONCLUSIONS The sGC-cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.

Effects of Aclidinium Bromide in a Cigarette Smoke–Exposed Guinea Pig Model of Chronic Obstructive Pulmonary Disease

Long-acting muscarinic antagonists are widely used to treat chronic obstructive pulmonary disease (COPD). In addition to bronchodilation, muscarinic antagonism may affect pulmonary histopathological changes. The effects of long-acting muscarinic antagonists have not been thoroughly evaluated in experimental models of COPD induced by chronic exposure to cigarette smoke (CS). We investigated the effects of aclidinium bromide on pulmonary function, airway remodeling, and lung inflammation in a CS-exposed model of COPD. A total of 36 guinea pigs were exposed to CS and 22 were sham exposed for 24 weeks. Animals were nebulized daily with vehicle, 10 μg/ml, or 30 μg/ml aclidinium, resulting in six experimental groups. Pulmonary function was assessed weekly by whole-body plethysmography, determining the enhanced pause (Penh) at baseline, after treatment, and after CS/sham exposure. Lung changes were evaluated by morphometry and immunohistochemistry. CS exposure increased Penh in all conditions. CS-exposed animals treated with aclidinium showed lower baseline Penh than untreated animals (P = 0.02). CS induced thickening of all bronchial wall layers, airspace enlargement, and inflammatory cell infiltrate in airways and septa. Treatment with aclidinium abrogated the CS-induced smooth muscle enlargement in small airways (P = 0.001), and tended to reduce airspace enlargement (P = 0.054). Aclidinium also attenuated CS-induced neutrophilia in alveolar septa (P = 0.04). We conclude that, in guinea pigs chronically exposed to CS, aclidinium has an antiremodeling effect on small airways, which is associated with improved respiratory function, and attenuates neutrophilic infiltration in alveolar septa. These results indicate that, in COPD, aclidinium may exert beneficial effects on lung structure in addition to its bronchodilator action.

Mesenchymal stem cells restore lung function by recruiting resident and nonresident proteins.

Because human lungs are unlikely to repair or regenerate beyond the cellular level, cell therapy has not previously been considered for chronic irreversible obstructive lung diseases. To explore whether cell therapy can restore lung function, we administered allogenic intratracheal mesenchymal stem cells (MSCs) in the trachea of rats with chronic thromboembolic pulmonary hypertension (CTEPH), a disease characterized by single or recurrent pulmonary thromboembolic obliteration and progressive pulmonary vascular remodeling. MSCs were retrieved only in high pressure-exposed lungs recruited via a homing stromal derived factor-1α/ CXCR4 pathway. After MSC administration, a marked and long-lasting improvement of all clinical parameters and a significant change of the proteome level were detected. Beside a variation of liver proteome, such as caspase-3, NF-κB, collagen1A1, and α-SMA, we also identified more than 300 resident and nonresident lung proteins [e.g., myosin light chain 3 (P16409) or mitochondrial ATP synthase subunit alpha (P15999)]. These results suggest that cell therapy restores lung function and the therapeutic effects of MSCs may be related to protein-based tissue reconstituting effects.

Pulmonary inflammatory reaction and structural changes induced by cigarette smoke exposure in the Guinea pig.

Abstract Cigarette smoke (CS) induces an inflammatory process in the lung that may underlie the development of chronic obstructive pulmonary disease (COPD). The nature and characteristics of this process have not been fully established in animal models. We aimed to evaluate the pulmonary inflammatory reaction and its involvement in structural changes in guinea pigs chronically exposed to CS. 19 Hartley guinea pigs were exposed to 7 cigarettes/day, during 3 or 6 months. 18 control guinea pigs were sham-exposed. Numbers of neutrophils, macrophages and eosinophils and lymphoid follicles were assessed in different lung structures. Airway and vessel morphometry, alveolar space size and collagen deposition were also quantified. After 6 months of exposure, CS-exposed guinea pigs showed increased numbers of neutrophils, macrophages and eosinophils in the airways, intrapulmonary vessels and alveolar septa, as well as lymphoid follicles. Increased numbers of muscularized intrapulmonary vessels were apparent at 3 months. After 6 months of exposure, the airway wall thickened and the alveolar space size increased. Collagen deposition was also apparent in airway walls and alveolar septa after 6 months’ exposure. The magnitude of airway wall-thickening correlated with the number of infiltrating inflammatory cells, and the extension of collagen deposition correlated with alveolar space size. We conclude that in the guinea pig, 6 months of CS exposure induces inflammatory cell infiltrate in lung structures, at an intensity that correlates with airway remodelling. These changes resemble those observed in COPD, thus endorsing the pathogenic role of CS and the usefulness of this animal model for its study.

A systems biology approach reveals a link between systemic cytokines and skeletal muscle energy metabolism in a rodent smoking model and human COPD

BackgroundA relatively large percentage of patients with chronic obstructive pulmonary disease (COPD) develop systemic co-morbidities that affect prognosis, among which muscle wasting is particularly debilitating. Despite significant research effort, the pathophysiology of this important extrapulmonary manifestation is still unclear. A key question that remains unanswered is to what extent systemic inflammatory mediators might play a role in this pathology.Cigarette smoke (CS) is the main risk factor for developing COPD and therefore animal models chronically exposed to CS have been proposed for mechanistic studies and biomarker discovery. Although mice have been successfully used as a pre-clinical in vivo model to study the pulmonary effects of acute and chronic CS exposure, data suggest that they may be inadequate models for studying the effects of CS on peripheral muscle function. In contrast, recent findings indicate that the guinea pig model (Cavia porcellus) may better mimic muscle wasting.MethodsWe have used a systems biology approach to compare the transcriptional profile of hindlimb skeletal muscles from a Guinea pig rodent model exposed to CS and/or chronic hypoxia to COPD patients with muscle wasting.ResultsWe show that guinea pigs exposed to long-term CS accurately reflect most of the transcriptional changes observed in dysfunctional limb muscle of severe COPD patients when compared to matched controls. Using network inference, we could then show that the expression profile in whole lung of genes encoding for soluble inflammatory mediators is informative of the molecular state of skeletal muscles in the guinea pig smoking model. Finally, we show that CXCL10 and CXCL9, two of the candidate systemic cytokines identified using this pre-clinical model, are indeed detected at significantly higher levels in serum of COPD patients, and that their serum protein level is inversely correlated with the expression of aerobic energy metabolism genes in skeletal muscle.ConclusionsWe conclude that CXCL10 and CXCL9 are promising candidate inflammatory signals linked to the regulation of central metabolism genes in skeletal muscles. On a methodological level, our work also shows that a system level analysis of animal models of diseases can be very effective to generate clinically relevant hypothesis.

Effects of cigarette smoke and chronic hypoxia on airways remodeling and resistance. Clinical significance.

Previously we have reported that association of cigarette smoke (CS) and chronic hypoxia (CH) interact positively to physiopathologically remodel pulmonary circulation. In present study we have exposed guinea pigs to CS smoke (four cigarettes/day; 3 months; CS) and to chronic hypoxia (12% O(2), 15 days; CH) alone or in combination (CSCH animals) and evaluated airways remodeling and resistance assessed as Penh (enhance pause). We measured Penh while animals breathe air, 10% O(2) and 5% CO(2) and found that CS and CH animals have higher Penh than controls; Penh was even larger in CSCH animals. A rough parallelism between Penh and thickness of bronchiolar wall and muscular layer and Goblet cell number was noticed. We conclude that CS and CH association accelerates CS-induced respiratory system damage, evidenced by augmented airway resistance, bronchial wall thickness and muscularization and Goblet cell number. Our findings would suggest that appearance of hypoxia would aggravate any preexisting pulmonary pathology by increasing airways resistance and reactivity.

Insignificance of the anomalous magnetic moment of charged fermions for the equation of state of a magnetized and dense medium

We investigate the effects of the anomalous magnetic moment (AMM) in the equation of state (EoS) of a system of charged fermions at finite density in the presence of a magnetic field. In the region of strong magnetic fields (eB>m^2) the AMM is found from the one-loop fermion self-energy. In contrast to the weak-field AMM found by Schwinger, in the strong magnetic field region the AMM depends on the Landau level and decreases with it. The effects of the AMM in the EoS of a dense medium are investigated at strong and weak fields using the appropriate AMM expression for each case. In contrast with what has been reported in other works, we find that the AMM of charged fermions makes no significant contribution to the EoS at any field value.