RESUMEN
The etiology of chronic obstructive pulmonary disease (COPD) is complex and involves an aberrant inflammatory response. Prostaglandin (PG)E2 is elevated in COPD, is a key modulator of lung fibroblast functions, and may influence COPD progression. Most studies evaluating the effects of PGE2 on lung fibroblasts have used acute exposures. The current study evaluated whether longer-term exposure would induce attenuation of PGE2 signaling as part of an autoregulatory pathway. Human fetal lung fibroblasts were pretreated with PGE2 for 24 hours, and migration and cAMP accumulation in response to acute stimulation with PGE2 were assessed. Fibroblasts from adults with and without COPD were pretreated, and migration was assessed. PGE2 pretreatment attenuated subsequent PGE2-mediated inhibition of chemotaxis and cAMP stimulation. This attenuation was predominantly due to an increase in phosphodiesterase (PDE)4-mediated degradation of cAMP rather than to decreased activation of PGE2 receptors (receptor desensitization). Albuterol- and iloprost-mediated signaling were also attenuated after PGE2 pretreatment, suggesting that activation of PDE4 was able to broadly modulate multiple cAMP-coupled pathways. Lung fibroblasts from adult control subjects pretreated with PGE2 also developed attenuation of PGE2-mediated inhibition of chemotaxis. In contrast, fibroblasts obtained from patients with COPD maintained inhibitory PGE2 signaling after PGE2 pretreatment. These data identify a PDE4-mediated attenuation of PGE2 inhibitory signaling in normal fibroblasts that appears to be altered in COPD fibroblasts. These alterations may contribute to COPD pathogenesis and could provide novel therapeutic targets.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Pulmón/patología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Albuterol/farmacología , Aminopiridinas/farmacología , Benzamidas/farmacología , Células Cultivadas , Quimiotaxis , Colforsina/farmacología , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Ciclopropanos/farmacología , Dinoprostona/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibronectinas/fisiología , Expresión Génica , Humanos , Iloprost/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Inhibidores de Fosfodiesterasa 4/farmacología , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Receptores de Epoprostenol , Receptores de Prostaglandina/agonistas , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Rolipram/farmacología , Sistemas de Mensajero SecundarioRESUMEN
Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E(2) (PGE(2)) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE(2) of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbecco's modified Eagle's medium, with or without PGE(2), and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE(2) and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE(2) was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase-A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE(2). The increased release of VEGF induced by PGE(2) was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE(2) can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall.
Asunto(s)
Dinoprostona/fisiología , Pulmón/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Pulmón/citología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Smoking is known to be linked to skin ageing and there is evidence for premature senescence of parenchymal lung fibroblasts in emphysema. To reveal whether the emphysema-related changes in cellular phenotype extend beyond the lung, we compared the proliferation characteristics of lung and skin fibroblasts between patients with and without emphysema. Parenchymal lung fibroblasts and skin fibroblasts from the upper torso (thus limiting sun exposure bias) were obtained from patients without, or with mild, or with moderate to severe emphysema undergoing lung surgery. We analysed proliferation rate, population doublings (PD), staining for senescence-associated beta-galactosidase (beta-gal) and gene expression of IGFBP-3 and IGFBP-rP1. Population doubling time of lung fibroblasts differed between control, mild, and moderate to severe emphysema (median (IQR) 29.7(10.0), 33.4(6.1), 44.4(21.2) h; p=0.012) and staining for beta-gal was elevated in moderate to severe emphysema. Compared to control subjects, skin fibroblasts from patients with emphysema did not differ with respect to proliferation rate, PD and beta-gal staining, and showed a lower abundance of mRNA for IGFBP-3 and -rP1 (p<0.05, each). These results suggest that the induction of a senescent fibroblast phenotype by cigarette smoke, as observed in emphysema, primarily occurs in the lung.
Asunto(s)
Senescencia Celular , Fibroblastos/fisiología , Pulmón/patología , Enfisema Pulmonar/patología , Piel/patología , Anciano , Proliferación Celular , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Enfisema Pulmonar/metabolismo , Piel/metabolismo , Fumar/metabolismo , Fumar/patología , beta-Galactosidasa/metabolismoRESUMEN
OBJECTIVE AND DESIGN: Reduced expression of histone deacetylase 2 (HDAC2) in alveolar macrophages and epithelial cells may account for reduced response of chronic obstructive pulmonary disease (COPD) patients to glucocorticoids. HDAC2 expression and its role in mediating glucocorticoid effects on fibroblast functions, however, has not been fully studied. This study was designed to investigate whether HDAC2 mediates glucocorticoid effects on release of inflammatory cytokines and matrix metalloproteinases (MMPs) from human lung fibroblasts. METHODS: Human lung fibroblasts (HFL-1 cells) were stimulated with interleukin (IL)-1 ß plus tumor necrosis factor (TNF)-α in the presence or absence of the glucocorticoid budesonide. Cytokines (IL-6 and IL-8) were quantified by enzyme linked immunosorbent assay (ELISA) and MMPs (MMP-1 and MMP-3) by immunoblotting in culture medium. The role of HDAC2 was investigated using a pharmacologic inhibitor as well as a small interfering ribonucleic acid (siRNA) targeting HDAC2. RESULTS: We have demonstrated that budesonide concentration-dependently (10(-10)-10(-7) M) inhibited IL-6, IL-8, MMP-1, and MMP-3 release by HFL-1 cells in response to IL-1ß plus TNF-α. While an HDAC inhibitor significantly blocked the inhibitory effect of budesonide on human bronchial epithelial cells (HBECs) and monocytes (THP-1 cells), it did not block the inhibitory effect of budesonide on release of cytokines and MMPs from HFL-1 cells. Similarly, an HDAC2-siRNA blocked budesonide inhibition of cytokine release in HBECs, but it did not block the inhibitory effect of budesonide on HFL-1 cytokine and MMP release. Furthermore, budesonide significantly blocked release of cytokines and MMPs to a similar degree in normal and COPD lung fibroblasts as well as in HFL-1 cells exposed or not exposed to cigarette smoke extract. CONCLUSION: These findings suggest that, in contrast to airway epithelial cells and monocytes/macrophages, HDAC2 is not required for budesonide to inhibit MMP and cytokine release by lung fibroblasts and this inhibitory pathway appears to be intact in cultured fibroblasts from COPD patients. These results also suggest that budesonide has the potential to modulate fibroblast-mediated tissue remodeling following airway inflammation in COPD, which is mediated via an HDAC2 independent pathway.
RESUMEN
The identification and validation of biomarkers to support the assessment of novel therapeutics for COPD continues to be an important area of research. The aim of the current study was to identify systemic protein biomarkers correlated with measures of COPD severity, as well as specific protein signatures associated with comorbidities such as metabolic syndrome. 142 protein analytes were measured in serum of 140 patients with stable COPD, 15 smokers without COPD and 30 non-smoking controls. Seven analytes (sRAGE, EN-RAGE, NGAL, Fibrinogen, MPO, TGF-α and HB-EGF) showed significant differences between severe/very severe COPD, mild/moderate COPD, smoking and non-smoking control groups. Within the COPD subjects, univariate and multivariate analyses identified analytes significantly associated with FEV(1), FEV(1)/FVC and DLCO. Most notably, a set of 5 analytes (HB-EGF, Fibrinogen, MCP-4, sRAGE and Sortilin) predicted 21% of the variability in DLCO values. To determine common functions/pathways, analytes were clustered in a correlation network by similarity of expression profile. While analytes related to neutrophil function (EN-RAGE, NGAL, MPO) grouped together to form a cluster associated with FEV(1) related parameters, analytes related to the EGFR pathway (HB-EGF, TGF-α) formed another cluster associated with both DLCO and FEV(1) related parameters. Associations of Fibrinogen with DLCO and MPO with FEV(1)/FVC were stronger in patients without metabolic syndrome (r â=â -0.52, p â=â 0.005 and r â=â -0.61, p =â 0.023, respectively) compared to patients with coexisting metabolic syndrome (r â=â -0.25, p â= â0.47 and r â=â -0.15, p â= â0.96, respectively), and may be driving overall associations in the general cohort. In summary, our study has identified known and novel serum protein biomarkers and has demonstrated specific associations with COPD disease severity, FEV(1), FEV(1)/FVC and DLCO. These data highlight systemic inflammatory pathways, neutrophil activation and epithelial tissue injury/repair processes as key pathways associated with COPD.
Asunto(s)
Biomarcadores/sangre , Síndrome Metabólico/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Proteínas de Fase Aguda , Anciano , Estudios de Cohortes , Comorbilidad , Femenino , Fibrinógeno/análisis , Volumen Espiratorio Forzado/fisiología , Factor Estimulante de Colonias de Granulocitos/sangre , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/sangre , Interleucina-3/sangre , Lipocalina 2 , Lipocalinas/sangre , Masculino , Síndrome Metabólico/sangre , Análisis Multivariante , Proteínas Proto-Oncogénicas/sangre , Capacidad de Difusión Pulmonar/fisiología , Enfermedad Pulmonar Obstructiva Crónica/sangre , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/sangre , Proteínas Recombinantes de Fusión/sangre , Análisis de Regresión , Proteínas S100/sangre , Proteína S100A12 , Fumar , Factor de Crecimiento Transformador alfa/sangreRESUMEN
BACKGROUND: Systemic effects of COPD are incompletely reflected by established prognostic assessments. We determined the prognostic value of objectively measured physical activity in comparison with established predictors of mortality and evaluated the prognostic value of noninvasive assessments of cardiovascular status, biomarkers of systemic inflammation, and adipokines. METHODS: In a prospective cohort study of 170 outpatients with stable COPD (mean FEV(1), 56% predicted), we assessed lung function by spirometry and body plethysmography; physical activity level (PAL) by a multisensory armband; exercise capacity by 6-min walk distance test; cardiovascular status by echocardiography, vascular Doppler sonography (ankle-brachial index [ABI]), and N-terminal pro-B-type natriuretic peptide level; nutritional and muscular status by BMI and fat-free mass index; biomarkers by levels of high-sensitivity C-reactive protein, IL-6, fibrinogen, adiponectin, and leptin; and health status, dyspnea, and depressive symptoms by questionnaire. Established prognostic indices were calculated. The median follow-up was 48 months (range, 10-53 months). RESULTS: All-cause mortality was 15.4%. After adjustments, each 0.14 increase in PAL was associated with a lower risk of death (hazard ratio [HR], 0.46; 95% CI, 0.33-0.64; P < .001). Compared with established predictors, PAL showed the best discriminative properties for 4-year survival (C statistic, 0.81) and was associated with the highest relative risk of death per standardized decrease. Novel predictors of mortality were adiponectin level (HR, 1.34; 95% CI, 1.06-1.71; P = .017), leptin level (HR, 0.81; 95% CI, 0.65-0.99; P = .042), right ventricular function (Tei-index) (HR, 1.26; 95% CI, 1.04-1.54; P = .020), and ABI < 1.00 (HR, 3.87; 95% CI, 1.44-10.40; P = .007). A stepwise Cox regression revealed that the best model of independent predictors was PAL, adiponectin level, and ABI. The composite of these factors further improved the discriminative properties (C statistic, 0.85). CONCLUSIONS: We found that objectively measured physical activity is the strongest predictor of all-cause mortality in patients with COPD. In addition, adiponectin level and vascular status provide independent prognostic information in our cohort.
Asunto(s)
Actividad Motora , Enfermedad Pulmonar Obstructiva Crónica/mortalidad , Adipoquinas/sangre , Anciano , Estudios de Cohortes , Ecocardiografía , Metabolismo Energético , Tolerancia al Ejercicio , Femenino , Humanos , Leptina/sangre , Masculino , Persona de Mediana Edad , Estado Nutricional , Pronóstico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Pruebas de Función Respiratoria , Espirometría , Análisis de Supervivencia , Tasa de SupervivenciaRESUMEN
BACKGROUND: The metabolic syndrome is a condition frequently found among individuals > 60 years of age. It predisposes affected individuals to systemic inflammation and physical inactivity. Systemic inflammation and physical inactivity are relevant extrapulmonary markers of morbidity and mortality in patients with COPD. Here, we studied the following: (1) the frequency of the coexisting metabolic syndrome in patients with chronic bronchitis (CB) and COPD of different severities; and (2) its association with systemic inflammation and physical inactivity. METHODS: In 30 patients with CB (normal spirometry finding) and in 170 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stages I to IV), we measured the characteristics of the metabolic syndrome, systemic inflammation (high-sensitivity C-reactive protein [hs-CRP], interleukin-6, fibrinogen), and the physical activity level. RESULTS: The frequencies of the metabolic syndrome in patients with CB, GOLD stages I, II, III, and IV, were 53%, 50%, 53%, 37%, and 44%, respectively (average, 47.5%). The levels of hs-CRP and interleukin-6 were significantly increased in patients with the metabolic syndrome, while the physical activity level was significantly decreased. Multivariate linear regression analyses revealed metabolic syndrome, physical activity level, and CB/GOLD stages to be independent predictors of hs-CRP and interleukin-6 levels, and physical activity level to be a predictor of fibrinogen levels. CONCLUSIONS: In our study, almost one-half of the patients with CB/COPD had coexisting metabolic syndrome, with a slightly lower frequency in patients with severe COPD. The coexisting metabolic syndrome is associated with an increase in the levels of some systemic inflammatory markers and physical inactivity, independent of lung function impairment.