Carme Casadevall

Upregulation of pro-inflammatory cytokines in the intercostal muscles of COPD patients

Muscle dysfunction is a characteristic feature of chronic obstructive pulmonary disease (COPD). Recent studies suggest that cytokines may operate as local regulators of both muscle function and regeneration. The aim of the present study was to characterise the expression of different cytokines in the external intercostal muscle of COPD. Muscle biopsies were obtained from 25 stable COPD patients and eight healthy controls. Local tumour necrosis factor (TNF)-α, interleukin (IL)-1β, -6 and -10 expressions (real-time PCR and ELISA), sarcolemmal damage (immunohistochemistry), and the transcript levels of CD18 were assessed. Muscle TNF-α and IL-6 transcripts were significantly higher in COPD patients compared with controls, and IL-1β and sarcolemmal damage showed a strong tendency in the same direction. Similar results were observed at protein level. The CD18 panleukocyte marker was similar in COPD and controls. Respiratory muscle function was impaired in COPD patients and it correlated to both the severity of lung function impairment and TNF-α muscle expression. Chronic obstructive pulmonary disease is associated with the upregulation of pro-inflammatory cytokines in the intercostal muscles. This phenomenon might be involved in respiratory muscle dysfunction.

Respiratory diseases and muscle dysfunction

Many respiratory diseases lead to impaired function of skeletal muscles, influencing quality of life and patient survival. Dysfunction of both respiratory and limb muscles in chronic obstructive pulmonary disease has been studied in depth, and seems to be caused by the complex interaction of general (inflammation, impaired gas exchange, malnutrition, comorbidity, drugs) and local factors (changes in respiratory mechanics and muscle activity, and molecular events). Some of these factors are also present in cystic fibrosis and asthma. In obstructive sleep apnea syndrome, repeated exposure to hypoxia and the absence of reparative rest are believed to be the main causes of muscle dysfunction. Deconditioning appears to be crucial for the functional impairment observed in scoliosis. Finally, cachexia seems to be the main mechanism of muscle dysfunction in advanced lung cancer. A multidimensional therapeutic approach is recommended, including pulmonary rehabilitation, an adequate level of physical activity, ventilatory support and nutritional interventions.

Muscle dysfunction in chronic obstructive pulmonary disease: update on causes and biological findings

Respiratory and/or limb muscle dysfunction, which are frequently observed in chronic obstructive pulmonary disease (COPD) patients, contribute to their disease prognosis irrespective of the lung function. Muscle dysfunction is caused by the interaction of local and systemic factors. The key deleterious etiologic factors are pulmonary hyperinflation for the respiratory muscles and deconditioning secondary to reduced physical activity for limb muscles. Nonetheless, cigarette smoke, systemic inflammation, nutritional abnormalities, exercise, exacerbations, anabolic insufficiency, drugs and comorbidities also seem to play a relevant role. All these factors modify the phenotype of the muscles, through the induction of several biological phenomena in patients with COPD. While respiratory muscles improve their aerobic phenotype (percentage of oxidative fibers, capillarization, mitochondrial density, enzyme activity in the aerobic pathways, etc.), limb muscles exhibit the opposite phenotype. In addition, both muscle groups show oxidative stress, signs of damage and epigenetic changes. However, fiber atrophy, increased number of inflammatory cells, altered regenerative capacity; signs of apoptosis and autophagy, and an imbalance between protein synthesis and breakdown are rather characteristic features of the limb muscles, mostly in patients with reduced body weight. Despite that significant progress has been achieved in the last decades, full elucidation of the specific roles of the target biological mechanisms involved in COPD muscle dysfunction is still required. Such an achievement will be crucial to adequately tackle with this relevant clinical problem of COPD patients in the near-future.

Activation of Satellite Cells in the Intercostal Muscles of Patients With Chronic Obstructive Pulmonary Disease

OBJECTIVE The respiratory muscles of patients with chronic obstructive pulmonary disease (COPD) display evidence of structural damage in parallel with signs of adaptation. We hypothesized that this can only be explained by the simultaneous activation of satellite cells. The aim of this study was to analyze the number and activation of those cells along with the expression of markers of microstructural damage that are frequently associated with regeneration. PATIENTS AND METHODS The study included 8 patients with severe COPD (mean [SD] forced expiratory volume in 1 second, 33% [9%] of predicted) and 7 control subjects in whom biopsies were performed of the external intercostal muscle. The samples were analyzed by light microscopy to assess muscle fiber phenotype, electron microscopy to identify satellite cells, and real-time polymerase chain reaction to analyze the expression of the following markers: insulin-like growth factor 1, mechano growth factor, and embryonic and perinatal myosin heavy chains (MHC) as markers of microstructural damage; Pax-7 and m-cadherin as markers of the presence and activation of satellite cells, respectively; and MHC-I, IIa, and IIx as determinants of muscle fiber phenotype. RESULTS The patients had larger fibers than healthy subjects (54 [6] vs 42 [4] microm(2); P< .01) with a similar or slightly increased proportion of satellite cells, as measured by ultrastructural analysis (4.3% [1%] vs 3.7% [3.5%]; P>.05) or expression of Pax-7 (5.5 [4.1] vs 1.6 [0.8] arbitrary units [AU]; P< .05). In addition, there was greater activation of satellite cells in the patients, as indicated by increased expression of m-cadherin (3.8 [2.1] vs 1.0 [1.2] AU; P=.05). This was associated with increased expression of markers of microstructural damage: insulin-like growth factor 1, 0.35 (0.34) vs 0.09 (0.08) AU (P< .05); mechano growth factor, 0.45 (0.55) vs 0.13 (0.17) AU (P=.05). CONCLUSIONS The intercostal muscles of patients with severe COPD show indirect signs of microstructural damage accompanied by satellite cell activation. This suggests the presence of ongoing cycles of lesion and repair that could partially explain the maintenance of the structural properties of the muscle.

Inflamacion y estres oxidativo en los musculos respiratorios y perifericos de pacientes con sepsis grave

BACKGROUND AND OBJECTIVE Oxidative stress and inflammation contribute to the diaphragm contractile dysfunction observed in animal models of sepsis and endotoxemia. In septic patients, molecular events have never been explored in their respiratory muscles. Levels of oxidative stress and inflammation were evaluated in a respiratory muscle, the external intercostal, and a limb muscle, the vastus lateralis, of patients with sepsis. PATIENTS AND METHODS Levels of oxidized and nitrated proteins, protein adducts of malondialdehyde and hydroxinonenal, antioxidant enzymes catalase and Mn-superoxide dismutase, tumor necrosis factor (TNF)-α, TNF-α receptors i and ii, interleukin (IL)-1 and IL-6, the panleukocyte marker CD18, and fiber type composition were explored using immunoblotting, real time-polymerase chain reaction, and immunohistochemistry in the external intercostal and vastus lateralis of patients with severe sepsis and/or septic shock. RESULTS Compared to the controls, in septic patients, levels of oxidized and nitrated proteins were increased in the vastus lateralis, but not in the external intercostal, while those of the antioxidant enzymes did not differ, and the proportions and sizes of the muscle fibers were not significantly different in any muscle between patients and controls. CONCLUSIONS Differences in activity between the respiratory and limb muscles may account for the differential pattern of oxidative stress and inflammation observed among patients with severe sepsis. These findings may have relevant implications for the clinical and therapeutic management of these patients.

Inflammatory Cytokines and Repair Factors in the Intercostal Muscles of Patients With Severe COPD

a b s t r a c t Objective: There is disagreement regarding the local action of cytokines in the respiratory muscles of patients with chronic obstructive pulmonary disease (COPD). The objective of this study was to analyze the relationships between cytokine expression and genetic activation of the mechanisms of muscle repair. Patients and Methods: Twenty-five patients with severe COPD and in stable condition were enrolled in the study. We performed a biopsy of the external intercostal muscle of the patients and analyzed the specimen for signs of muscle lesion (morphometry), infiltration of inflammatory cells (immunohistochemistry), and expression of selected genes (real-time polymerase chain reaction technique) corresponding to the cytokines (tumor necrosis factor α (TNF-α) and its type 1 and 2 receptors (TNFR1 and TNFR2), and interleukin (IL) 1β, IL-6, and IL-10), a pan-leukocyte marker (CD18), and key molecules in the repair- myogenesis pathways (Pax7, M-cadherin, and MyoD). Results: Expression of TNFR2 is directly related to inspiratory muscle function (represented by maximum sustainable inspiratory pressure; r=0.496; P<.05), whereas expression of CD18 is inversely related (r=0.462; P<.05). Moreover, expression of the 2 TNF-α receptors was directly related to that of the key molecules of the repair pathways analyzed (TNFR1 to Pax7 (r=0.650; P<.001) and M-cadherin (r=0.678; P<.001); TNFR2 to Pax7 (r=0.395; P<.05), M-cadherin (r=0.409; P<.05), and MyoD (r=0.418; P<.05)). Conclusions: Expression of TNF-α receptors bears a close relationship both to activation of the myogenesis programs and to inspiratory muscle function. This reinforces our hypothesis that some local cytokines take part in the repair of respiratory muscles in patients with COPD.

Citocinas inflamatorias y factores de reparacion en los musculos intercostales de pacientes con EPOC grave

OBJECTIVE There is disagreement regarding the local action of cytokines in the respiratory muscles of patients with chronic obstructive pulmonary disease (COPD). The objective of this study was to analyze the relationships between cytokine expression and genetic activation of the mechanisms of muscle repair. PATIENTS AND METHODS Twenty-five patients with severe COPD and in stable condition were enrolled in the study. We performed a biopsy of the external intercostal muscle of the patients and analyzed the specimen for signs of muscle lesion (morphometry), infiltration of inflammatory cells (immunohistochemistry), and expression of selected genes (real-time polymerase chain reaction technique) corresponding to the cytokines (tumor necrosis factor alpha [TNF-alpha] and its type 1 and 2 receptors [TNFR1 and TNFR2], and interleukin [IL] 1beta, IL-6, and IL-10), a pan-leukocyte marker (CD18), and key molecules in the repair-myogenesis pathways (Pax7, M-cadherin, and MyoD). RESULTS Expression of TNFR2 is directly related to inspiratory muscle function (represented by maximum sustainable inspiratory pressure; r=0.496; P<.05), whereas expression of CD18 is inversely related (r=0.462; P<.05). Moreover, expression of the 2 TNF-alpha receptors was directly related to that of the key molecules of the repair pathways analyzed (TNFR1 to Pax7 [r=0.650; P<.001] and M-cadherin [r=0.678; P<.001]; TNFR2 to Pax7 [r=0.395; P<.05], M-cadherin [r=0.409; P<.05], and MyoD [r=0.418; P<.05]). CONCLUSIONS Expression of TNF-alpha receptors bears a close relationship both to activation of the myogenesis programs and to inspiratory muscle function. This reinforces our hypothesis that some local cytokines take part in the repair of respiratory muscles in patients with COPD.

Inflamación y estrés oxidativo en los músculos respiratorios y periféricos de pacientes con sepsis grave

BACKGROUND AND OBJECTIVE Oxidative stress and inflammation contribute to the diaphragm contractile dysfunction observed in animal models of sepsis and endotoxemia. In septic patients, molecular events have never been explored in their respiratory muscles. Levels of oxidative stress and inflammation were evaluated in a respiratory muscle, the external intercostal, and a limb muscle, the vastus lateralis, of patients with sepsis. PATIENTS AND METHODS Levels of oxidized and nitrated proteins, protein adducts of malondialdehyde and hydroxinonenal, antioxidant enzymes catalase and Mn-superoxide dismutase, tumor necrosis factor (TNF)-α, TNF-α receptors i and ii, interleukin (IL)-1 and IL-6, the panleukocyte marker CD18, and fiber type composition were explored using immunoblotting, real time-polymerase chain reaction, and immunohistochemistry in the external intercostal and vastus lateralis of patients with severe sepsis and/or septic shock. RESULTS Compared to the controls, in septic patients, levels of oxidized and nitrated proteins were increased in the vastus lateralis, but not in the external intercostal, while those of the antioxidant enzymes did not differ, and the proportions and sizes of the muscle fibers were not significantly different in any muscle between patients and controls. CONCLUSIONS Differences in activity between the respiratory and limb muscles may account for the differential pattern of oxidative stress and inflammation observed among patients with severe sepsis. These findings may have relevant implications for the clinical and therapeutic management of these patients.

Expresión de los genes de la interleucina 10 (IL-10) y del factor de necrosis tumoral alfa (TNF-α) en músculos respiratorios y periféricos. Relación con el daño sarcolémico

Antecedentes El factor de necrosis tumoral alfa (TNF-a) se ha implicado en la perdida de masa muscular en la enfermedad pulmonar obstructiva cronica (EPOC) y otras enfermedades consumtivas. Su produccion estaria en relacion con la inflamacion derivada de la propia enfermedad pulmonar o el habito tabaquico, llegando al musculo por el torrente sanguineo. Sin embargo, tambien se ha sugerido que el TNF-α podria expresarse directamente en el tejido muscular. Sea cual fuere el lugar de produccion, la relacion del TNF-α con el dano muscular subsiguiente no es clara Objetivo Hemos estudiado la expresion del TNF-α y una interleucina inhibidora de su produccion (IL-10) en los musculos respiratorios principales y en un musculo periferico del perro Metodos Se han incluido nueve animales machos, adultos jovenes de raza Beagle. En todos ellos se tomaron biopsias del diafragma (Df), del intercostal externo (IExt) y de un musculo de las extremidades (vasto interno del cuadriceps o VInt). Se analizo la expresion de los genes de TNF-α e IL-10, a traves de la determinacion de sus ARNm corres-pondientes, utilizando la transcripcion reversa y reaccion en cadena de la polimerasa (RT-PCR). Tambien se evaluo el dano sarcolemico mediante deteccion de fibronectina intra-celular (inmunohistoquimica) Resultados Los valores de expresion de ambas citocinas evidenciaron una amplia dispersion interindividual. Por un lado, la expresion de TNF-α fue minima en Df y IExt (respectivamente, 0,02 ± 0,03 y 0,05 ± 0,06 u.a.), con valores relativamente elevados en VInt (0,14 ± 0,08 u.a.). A su vez, la IL-10 evidenciaba valores bajos en DF (0,6 ± 0,5 u.a.), que eran algo superiores en IExt (2,7 ± 1,9 u.a.; p Conclusiones a) TNF-α e IL-10 parecen expresarse constitutivamente en el musculo esqueletico canino; b) los valores basales de expresion del TNF-α son menores en los musculos repiratorios que en los de las extremidades mos-trando relacion en estos ultimos con el dano de membrana

Analysis of Respiratory Muscle Structure and Tumor Necrosis and Insulin-Like Growth Factor Expression in Chronic Obstructive Pulmonary Disease: Are Samples Valid if Obtained During Thoracotomy Performed Because of Localized Pulmonary Neoplasia?

OBJECTIVE Various methods have been used to obtain samples to study the structure of human respiratory muscles and the expression of diverse substances in them. Samples are most often obtained from autopsies, from muscle biopsies during thoracotomy performed because of a localized pulmonary lesion (TLL), and from ambulatory thoracoscopic biopsy in patients free of comorbidity (AT). The disadvantage of the first 2 of these methods lies in the possibility of interference from factors related to the patients death in the first case or from the disease that necessitated surgery in the second. Although AT is free from the disadvantages of the other 2 methods, it is impossible to obtain samples of the diaphragm the principal respiratory muscle with this procedure. The objective of this study was to analyze the fibrous structure of the external intercostal muscle of patients with chronic obstructive pulmonary disease and to quantify the expression of the principal inflammatory cytokine tumor necrosis factor alpha (TNF-alpha)- and of insulin-like growth factor (IGF-1) in the same muscle, comparing the results obtained with TLL and AT samples. METHODS Prospective and consecutive samples were taken of the external intercostal muscle (fifth space, anterior axillary line) in 15 patients with chronic obstructive pulmonary disease (mean [SD] age 66 [6] years; forced expiratory volume in 1 second 49% [9%] of predicted; PaO2 75 [9] mm Hg). Samples were taken during TLL (8 patients, all with pulmonary neoplasms but carefully selected in order to rule out systemic effects) or TA (7 patients). Patients with serious comorbidity were excluded from the second group. Samples were processed for structural analysis of fibers (immunohistochemical and enzymatic histochemical) and genetic expression of TNF-alpha and IGF-1 (real-time polymerase chain reaction). RESULTS No differences in the structure of fibers were found between the 2 groups. No differences were observed in the expression of TNF-alpha or IGF-1. CONCLUSIONS Using rigorous criteria, the TLL method appears to be suitable for studying the structural characteristics and expression of inflammatory cytokines and growth factors in the external intercostal muscle. Moreover, it can also be inferred that TLL is probably also useful for obtaining samples of the diaphragm, a muscle which cannot currently be sampled by any alternative method.