RESUMO
Electronic cigarettes (e-cigs) are often promoted as safe alternatives to smoking based on the faulty perception that inhaling nicotine is safe until other harmful chemicals in cigarette smoke are absent. Previously, others and we have reported that, similar to cigarette smoke, e-cig aerosols decrease CFTR-mediated ion transport across airway epithelium. However, it is unclear whether such defective epithelial ion transport by e-cig aerosols occurs in vivo and what the singular contribution of inhaled nicotine is to impairments in mucociliary clearance (MCC), the primary physiologic defense of the airways. Here, we tested the effects of nicotine aerosols from e-cigs in primary human bronchial epithelial (HBE) cells and two animal models, rats and ferrets, known for their increasing physiologic complexity and potential for clinical translation, followed by in vitro and in vivo electrophysiologic assays for CFTR activity and micro-optical coherence tomography (µOCT) image analyses for alterations in airway mucus physiology. Data presented in this report indicate nicotine in e-cig aerosols causes 1) reduced CFTR and epithelial Na+ channel (ENaC)-mediated ion transport, 2) delayed MCC, and 3) diminished airway surface hydration, as determined by periciliary liquid depth analysis. Interestingly, the common e-cig vehicles vegetable glycerin and propylene glycol did not affect CFTR function or MCC in vivo despite their significant adverse effects in vitro. Overall, our studies contribute to an improved understanding of inhaled nicotine effects on lung health among e-cig users and inform pathologic mechanisms involved in altered host defense and increased risk for tobacco-associated lung diseases.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Nicotina , Animais , Humanos , Ratos , Nicotina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística , Depuração Mucociliar , Furões , Aerossóis e Gotículas Respiratórios , Pulmão , AerossóisRESUMO
RATIONALE: The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure. OBJECTIVES: To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis. METHODS: Ferrets were exposed for 6â months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1â month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes. MEASUREMENTS AND MAIN RESULTS: Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy. CONCLUSIONS: The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.
Assuntos
Bronquite Crônica , Doença Pulmonar Obstrutiva Crônica , Animais , Bronquite Crônica/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Furões/metabolismo , Hipertrofia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Microtomografia por Raio-XRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive lung disease with poor treatment options. However, most mouse models of COPD produce a primarily emphysematous disease not recapitulating clinically meaningful COPD features like chronic bronchitis. METHODS: Wild-type ferrets (Mustela putorius furo) were divided randomly into two groups: whole body cigarette smoke exposure and air controls. Ferrets were exposed to smoke from 1R6F research cigarettes, twice daily for six months. RNA-sequencing was performed on RNA isolated from lung tissue. Comparative transcriptomics analyses of COPD in ferrets, mice, and humans were done to find the uniquely expressed genes. Further, Real-time PCR was performed to confirmed RNA-Seq data on multiple selected genes. RESULTS: RNA-sequence analysis identified 420 differentially expressed genes (DEGs) that were associated with the development of COPD in ferrets. By comparative analysis, we identified 25 DEGs that are uniquely expressed in ferrets and humans, but not mice. Among DEGs, a number were related to mucociliary clearance (NEK-6, HAS1, and KL), while others have been correlated with abnormal lung function (IL-18), inflammation (TREM1, CTSB), or oxidative stress (SRX1, AHRR). Multiple cellular pathways were aberrantly altered in the COPD ferret model, including pathways associated with COPD pathogenesis in humans. Validation of these selected unique DEGs using real-time PCR demonstrated > absolute 2-fold changes in mRNA versus air controls, consistent with RNA-seq analysis. CONCLUSION: Cigarette smoke-induced COPD in ferrets modulates gene expression consistent with human COPD and suggests that the ferret model may be uniquely well suited for the study of aspects of the disease.
Assuntos
Furões , Doença Pulmonar Obstrutiva Crônica , Animais , Humanos , Camundongos , Furões/genética , Interleucina-18 , Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Transcriptoma , Receptor Gatilho 1 Expresso em Células Mieloides/genética , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismoRESUMO
Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by decreased alveolar-capillary barrier function, pulmonary edema consisting of proteinaceous fluid, and inhibition of net alveolar fluid transport responsible for resolution of pulmonary edema. There is currently no pharmacotherapy that has proven useful to prevent or treat ARDS, and two trials using beta-agonist therapy to treat ARDS demonstrated no effect. Prior studies indicated that IL-8-induced heterologous desensitization of the beta2-adrenergic receptor (ß2 -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP). Interestingly, phosphodiesterase (PDE) 4 inhibitors have been used in human airway diseases characterized by low intracellular cAMP levels and increases in specific cAMP hydrolyzing activity. Therefore, we hypothesized that PDE4 would mediate IL-8-induced heterologous internalization of the ß2 -AR and that PDE4 inhibition would restore beta-agonist-induced functions. We determined that CINC-1 (a functional IL-8 analog in rats) induces internalization of ß2 -AR from the cell surface, and arrestin-2, PDE4, and ß2 -AR form a complex during this process. Furthermore, we determined that cAMP associated with the plasma membrane was adversely affected by ß2 -AR heterologous desensitization. Additionally, we determined that rolipram, a PDE4 inhibitor, reversed CINC-1-induced derangements of cAMP and also caused ß2 -AR to successfully recycle back to the cell surface. Finally, we demonstrated that rolipram could reverse CINC-1-mediated inhibition of beta-agonist-induced alveolar fluid clearance in a murine model of trauma-shock. These results indicate that PDE4 plays a role in CINC-1-induced heterologous internalization of the ß2 -AR; PDE4 inhibition reverses these effects and may be a useful adjunct in particular ARDS patients.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Interleucina-8/imunologia , Receptores Adrenérgicos beta 2/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CXCL1/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/farmacologia , Regulação para Baixo/efeitos dos fármacos , Masculino , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , beta-Arrestina 1/metabolismoRESUMO
Structural changes to airway morphology, such as increased bronchial wall thickness (BWT) and airway wall area, are cardinal features of chronic obstructive pulmonary disease (COPD). Ferrets are a recently established animal model uniquely exhibiting similar clinical and pathological characteristics of COPD as humans, including chronic bronchitis. Our objective was to develop a microcomputed tomography (µCT) method for evaluating structural changes to the airways in ferrets and assess whether the effects of smoking induce changes consistent with chronic bronchitis in humans. Ferrets were exposed to mainstream cigarette smoke or air control twice daily for 6 mo. µCT was conducted in vivo at 6 mo; a longitudinal cohort was imaged monthly. Manual measurements of BWT, luminal diameter (LD), and BWT-to-LD ratio (BWT/LD) were conducted and confirmed by a semiautomated algorithm. The square root of bronchial wall area (âWA) versus luminal perimeter was determined on an individual ferret basis. Smoke-exposed ferrets reproducibly demonstrated 34% increased BWT (P < 0.001) along with increased LD and BWT/LD versus air controls. Regression indicated that the effect of smoking on BWT persisted despite controlling for covariates. Semiautomated measurements replicated findings. âWA for the theoretical median airway luminal perimeter of 4 mm (Pi4) was elevated 4.4% in smoke-exposed ferrets (P = 0.015). Increased BWT and Pi4 developed steadily over time. µCT-based airway measurements in ferrets are feasible and reproducible. Smoke-exposed ferrets develop increased BWT and Pi4, changes similar to humans with chronic bronchitis. µCT can be used as a significant translational platform to measure dynamic airway morphological changes.
RESUMO
The mechanisms by which cigarette smoking impairs airway mucus clearance are not well understood. We recently established a ferret model of cigarette smoke-induced chronic obstructive pulmonary disease (COPD) exhibiting chronic bronchitis. We investigated the effects of cigarette smoke on mucociliary transport (MCT).Adult ferrets were exposed to cigarette smoke for 6â months, with in vivo mucociliary clearance measured by technetium-labelled DTPA retention. Excised tracheae were imaged with micro-optical coherence tomography. Mucus changes in primary human airway epithelial cells and ex vivo ferret airways were assessed by histology and particle tracking microrheology. Linear mixed models for repeated measures identified key determinants of MCT.Compared to air controls, cigarette smoke-exposed ferrets exhibited mucus hypersecretion, delayed mucociliary clearance (-89.0%, p<0.01) and impaired tracheal MCT (-29.4%, p<0.05). Cholinergic stimulus augmented airway surface liquid (ASL) depth (5.8±0.3 to 7.3±0.6â µm, p<0.0001) and restored MCT (6.8±0.8 to 12.9±1.2â mm·min-1, p<0.0001). Mixed model analysis controlling for covariates indicated smoking exposure, mucus hydration (ASL) and ciliary beat frequency were important predictors of MCT. Ferret mucus was hyperviscous following smoke exposure in vivo or in vitro, and contributed to diminished MCT. Primary cells from smokers with and without COPD recapitulated these findings, which persisted despite the absence of continued smoke exposure.Cigarette smoke impairs MCT by inducing airway dehydration and increased mucus viscosity, and can be partially abrogated by cholinergic secretion of fluid secretion. These data elucidate the detrimental effects of cigarette smoke exposure on mucus clearance and suggest additional avenues for therapeutic intervention.
Assuntos
Desidratação , Doença Pulmonar Obstrutiva Crônica , Adulto , Humanos , Depuração Mucociliar , Muco , Fumar/efeitos adversos , ViscosidadeRESUMO
Transforming growth factor ß (TGF-ß), signaling induced by cigarette smoke (CS), plays an important role in the progression of airway diseases, like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD), and in smokers. Chronic bronchitis is characterized by reduced mucociliary clearance (MCC). Cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in normal MCC. TGF-ß and CS (via TGF-ß) promote acquired CFTR dysfunction by suppressing CFTR biogenesis and function. Understanding the mechanism by which CS promotes CFTR dysfunction can identify therapeutic leads to reverse CFTR suppression and rescue MCC. TGF-ß alters the microRNAome of primary human bronchial epithelium. TGF-ß and CS upregulate miR-145-5p expression to suppress CFTR and the CFTR modifier, SLC26A9. miR-145-5p upregulation with a concomitant CFTR and SLC26A9 suppression was validated in CS-exposed mouse models. While miR-145-5p antagonism rescued the effects of TGF-ß in bronchial epithelial cells following transfection, an aptamer to block TGF-ß signaling rescues CS- and TGF-ß-mediated suppression of CFTR biogenesis and function in the absence of any transfection reagent. These results demonstrate that miR-145-5p plays a significant role in acquired CFTR dysfunction by CS, and they validate a clinically feasible strategy for delivery by inhalation to locally modulate TGF-ß signaling in the airway and rescue CFTR biogenesis and function.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , MicroRNAs/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Fumar/efeitos adversos , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Camundongos , Camundongos Mutantes , MicroRNAs/genética , Doença Pulmonar Obstrutiva Crônica/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Cigarette smoking is associated with chronic obstructive pulmonary disease and chronic bronchitis. Acquired ion transport abnormalities, including cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, caused by cigarette smoking have been proposed as potential mechanisms for mucus obstruction in chronic bronchitis. Although e-cigarette use is popular and perceived to be safe, whether it harms the airways via mechanisms altering ion transport remains unclear. In the present study, we sought to determine if e-cigarette vapor, like cigarette smoke, has the potential to induce acquired CFTR dysfunction, and to what degree. Electrophysiological methods demonstrated reduced chloride transport caused by vaporized e-cigarette liquid or vegetable glycerin at various exposures (30 min, 57.2% and 14.4% respectively, vs. control; P < 0.0001), but not by unvaporized liquid (60 min, 17.6% vs. untreated), indicating that thermal degradation of these products is required to induce the observed defects. We also observed reduced ATP-dependent responses (-10.8 ± 3.0 vs. -18.8 ± 5.1 µA/cm2 control) and epithelial sodium channel activity (95.8% reduction) in primary human bronchial epithelial cells after 5 minutes, suggesting that exposures dramatically inhibit epithelial ion transport beyond CFTR, even without diminished transepithelial resistance or cytotoxicity. Vaporizing e-cigarette liquid produced reactive aldehydes, including acrolein (shown to induce acquired CFTR dysfunction), as quantified by mass spectrometry, demonstrating that respiratory toxicants in cigarette smoke can also be found in e-cigarette vapor (30 min air, 224.5 ± 15.99; unvaporized liquid, 284.8 ± 35.03; vapor, 54,468 ± 3,908 ng/ml; P < 0.0001). E-cigarettes can induce ion channel dysfunction in airway epithelial cells, partly through acrolein production. These findings indicate a heretofore unknown toxicity of e-cigarette use known to be associated with chronic bronchitis onset and progression, as well as with chronic obstructive pulmonary disease severity.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Células Epiteliais/efeitos dos fármacos , Glicerol/efeitos adversos , Transporte de Íons , Fumaça/efeitos adversos , Fumar/efeitos adversos , Acroleína/química , Trifosfato de Adenosina/metabolismo , Brônquios/metabolismo , Bronquite Crônica/fisiopatologia , Sobrevivência Celular , Fumar Cigarros , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Progressão da Doença , Eletrofisiologia , Células Epiteliais/metabolismo , Glicerol/metabolismo , Humanos , Espectrometria de Massas , Muco/metabolismo , Nebulizadores e Vaporizadores , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Sistema Respiratório/efeitos dos fármacos , Fatores de TempoRESUMO
Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-µm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P < 0.05) and human bronchi (P < 0.05) within minutes of exposure. The mechanism involved CSE-induced reduction of CFTR gating, decreasing CFTR open-channel probability by approximately 75% immediately after exposure (P < 0.05), whereas surface CFTR expression was partially reduced with chronic exposure, but was stable acutely. CSE treatment of purified CFTR resulted in acrolein modifications on lysine and cysteine residues that likely disrupt CFTR gating. In primary HBE cells, CSE reduced airway surface liquid depth (P < 0.05) and ciliary beat frequency (P < 0.05) within 60 minutes that was restored by coadministration with ivacaftor (P < 0.005). Cigarette smoking transmits acute reductions in CFTR activity, adversely affecting the airway surface. These effects are reversible by a CFTR potentiator in vitro, representing a potential therapeutic strategy in patients with chronic obstructive pulmonary disease with chronic bronchitis.
Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Depuração Mucociliar/efeitos dos fármacos , Quinolonas/farmacologia , Fumar/efeitos adversos , Acroleína/farmacologia , Sequência de Aminoácidos , Brônquios/patologia , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Mucosa/patologia , Tomografia de Coerência Óptica , Traqueia/patologiaRESUMO
BACKGROUND: Dysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) can be elicited by cigarette smoke and is observed in patients with chronic bronchitis. We have previously demonstrated in human airway epithelial cell monolayers that roflumilast, a clinically approved phosphodiesterase 4 inhibitor that reduces the risk of exacerbations in chronic obstructive pulmonary disease patients with chronic bronchitis and a history of exacerbations, activates CFTR-dependent chloride secretion via a cAMP-mediated pathway, partially restores the detrimental effects of cigarette smoke on CFTR-mediated ion transport, and increases CFTR-dependent gastrointestinal fluid secretion in isolated murine intestine segments. Based on these findings, we hypothesized that roflumilast could improve CFTR-mediated chloride transport and induce secretory diarrhea in mice exhibiting cigarette smoke-induced CFTR dysfunction. METHODS: A/J mice expressing wild type CFTR (+/+) were exposed to cigarette smoke or air with or without roflumilast and the effect of treatment on CFTR-dependent chloride transport was quantified using nasal potential difference (NPD) measurements in vivo and short-circuit current (Isc) analysis of trachea ex vivo. Stool specimen were collected and the wet/dry ratio measured to assess the effect of roflumilast on secretory diarrhea. RESULTS: Acute roflumilast treatment increased CFTR-dependent chloride transport in both smoke- and air-exposed mice (smoke, -2.0 ± 0.4 mV, 131.3 ± 29.3 µA/cm2, P < 0.01 and air, 3.9 ± 0.8 mV, 147.7 ± 38.0 µA/cm2, P < 0.01 vs. vehicle -0.3 ± 0.7 mV, 10.4 ± 7.0 µA/cm2). Oral administration of roflumilast over five weeks completely reversed the deleterious effects of cigarette smoke on CFTR function in smoke-exposed animals, in which CFTR-dependent chloride transport was 64% that of air controls (roflumilast, -15.22 ± 2.7 mV vs. air, -14.45 ± 1.4 mV, P < 0.05). Smoke exposure increased the wet/dry ratio of stool specimen to a level beyond which roflumilast had little additional effect. CONCLUSIONS: Roflumilast effectively rescues CFTR-mediated chloride transport in vivo, further implicating CFTR activation as a mechanism through which roflumilast benefits patients with bronchitis.
Assuntos
Aminopiridinas/uso terapêutico , Benzamidas/uso terapêutico , Fumar Cigarros/tratamento farmacológico , Fumar Cigarros/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Inibidores da Fosfodiesterase 4/uso terapêutico , Aminopiridinas/farmacologia , Animais , Benzamidas/farmacologia , Ciclopropanos/farmacologia , Ciclopropanos/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Feminino , Exposição por Inalação/efeitos adversos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Inibidores da Fosfodiesterase 4/farmacologiaAssuntos
Acetilcisteína/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Humanos , CamundongosRESUMO
Cigarette smoking causes acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction and is associated with delayed mucociliary clearance and chronic bronchitis. Roflumilast is a clinically approved phosphodiesterase 4 inhibitor that improves lung function in patients with chronic bronchitis. We hypothesized that its therapeutic benefit was related in part to activation of CFTR. Primary human bronchial epithelial (HBE) cells, Calu-3, and T84 monolayers were exposed to whole cigarette smoke (WCS) or air with or without roflumilast treatment. CFTR-dependent ion transport was measured in modified Ussing chambers. Airway surface liquid (ASL) was determined by confocal microscopy. Intestinal fluid secretion of ligated murine intestine was monitored ex vivo. Roflumilast activated CFTR-dependent anion transport in normal HBE cells with a half maximal effective concentration of 2.9 nM. Roflumilast partially restored CFTR activity in WCS-exposed HBE cells (5.3 ± 1.1 µA/cm(2) vs. 1.2 ± 0.2 µA/cm(2) [control]; P < 0.05) and was additive with ivacaftor, a specific CFTR potentiator approved for the treatment of CF. Roflumilast improved the depleted ASL depth of HBE monolayers exposed to WCS (9.0 ± 3.1 µm vs. 5.6 ± 2.0 µm [control]; P < 0.05), achieving 79% of that observed in air controls. CFTR activation by roflumilast also induced CFTR-dependent fluid secretion in murine intestine, increasing the wet:dry ratio and the diameter of ligated murine segments. Roflumilast activates CFTR-mediated anion transport in airway and intestinal epithelia via a cyclic adenosine monophosphate-dependent pathway and partially reverses the deleterious effects of WCS, resulting in augmented ASL depth. Roflumilast may benefit patients with chronic obstructive pulmonary disease with chronic bronchitis by activating CFTR, which may also underlie noninfectious diarrhea caused by roflumilast.
Assuntos
Aminopiridinas/farmacologia , Benzamidas/farmacologia , Brônquios/efeitos dos fármacos , Bronquite Crônica/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Células Epiteliais/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Aminofenóis/farmacologia , Aminopiridinas/toxicidade , Animais , Benzamidas/toxicidade , Brônquios/metabolismo , Brônquios/fisiopatologia , Bronquite Crônica/metabolismo , Bronquite Crônica/fisiopatologia , Células Cultivadas , AMP Cíclico , Ciclopropanos/farmacologia , Ciclopropanos/toxicidade , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/induzido quimicamente , Diarreia/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Secreções Intestinais/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Potenciais da Membrana , Camundongos , Depuração Mucociliar/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/toxicidade , Quinolonas/farmacologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Fatores de TempoRESUMO
BACKGROUND: Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population. METHODS: Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV1/FVC < 0.70 and FEV1 < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network's Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations. RESULTS: Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS). CONCLUSIONS: The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.
Assuntos
Bronquite Crônica/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Triagem de Portadores Genéticos , Mutação/genética , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Bronquite Crônica/diagnóstico , Bronquite Crônica/epidemiologia , Estudos de Casos e Controles , Feminino , Humanos , Exposição por Inalação/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Fumar/epidemiologiaAssuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Fumar/efeitos adversos , Animais , Animais Recém-Nascidos , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Feminino , Mães , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Ratos , Ratos Sprague-Dawley , Traqueia/metabolismo , Traqueia/fisiopatologiaRESUMO
RATIONALE: Several extrapulmonary disorders have been linked to cigarette smoking. Smoking is reported to cause cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the airway, and is also associated with pancreatitis, male infertility, and cachexia, features characteristic of cystic fibrosis and suggestive of an etiological role for CFTR. OBJECTIVES: To study the effect of cigarette smoke on extrapulmonary CFTR function. METHODS: Demographics, spirometry, exercise tolerance, symptom questionnaires, CFTR genetics, and sweat chloride analysis were obtained in smokers with and without chronic obstructive pulmonary disease (COPD). CFTR activity was measured by nasal potential difference in mice and by Ussing chamber electrophysiology in vitro. Serum acrolein levels were estimated with mass spectroscopy. MEASUREMENTS AND MAIN RESULTS: Healthy smokers (29.45 ± 13.90 mEq), smokers with COPD (31.89 ± 13.9 mEq), and former smokers with COPD (25.07 ± 10.92 mEq) had elevated sweat chloride levels compared with normal control subjects (14.5 ± 7.77 mEq), indicating reduced CFTR activity in a nonrespiratory organ. Intestinal current measurements also demonstrated a 65% decrease in CFTR function in smokers compared with never smokers. CFTR activity was decreased by 68% in normal human bronchial epithelial cells exposed to plasma from smokers, suggesting that one or more circulating agents could confer CFTR dysfunction. Cigarette smoke-exposed mice had decreased CFTR activity in intestinal epithelium (84.3 and 45%, after 5 and 17 wk, respectively). Acrolein, a component of cigarette smoke, was higher in smokers, blocked CFTR by inhibiting channel gating, and was attenuated by antioxidant N-acetylcysteine, a known scavenger of acrolein. CONCLUSIONS: Smoking causes systemic CFTR dysfunction. Acrolein present in cigarette smoke mediates CFTR defects in extrapulmonary tissues in smokers.
Assuntos
Acroleína/sangue , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumar/efeitos adversos , Suor/química , Idoso , Animais , Cloretos/sangue , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Tolerância ao Exercício/efeitos dos fármacos , Tolerância ao Exercício/fisiologia , Feminino , Humanos , Mucosa Intestinal/química , Masculino , Camundongos , Pessoa de Meia-Idade , Mucosa Nasal/química , Fumar/metabolismo , Fumar/fisiopatologia , Sódio/sangue , EspirometriaRESUMO
BACKGROUND: Acrolein, an aldehyde in smoke from tobacco products, inhibits CFTR function in vitro. Ivacaftor is an FDA-approved potentiator that improves mutant CFTR function. This human clinical study investigated the relationship between two urinary markers of tobacco smoke exposure - the acrolein metabolite 3-HPMA and the nicotine metabolite NNAL - and sweat chloride response to ivacaftor in the G551D Observational Trial (GOAL). METHODS: 3-HPMA (low: <50th centile; moderate: 50-75th centile; high: >75th centile) and NNAL (detectable/undetectable) in GOAL samples was quantified with LC-MS/MS. Self-report of tobacco smoke exposure (Y/N) served as a subjective measure. Change in sweat chloride from pre- to 6 months post-ivacaftor treatment (ΔSC) was the primary CFTR-dependent readout. RESULTS: The sample included 151 individuals, mean age 20.7 (SD 11.4) years, range 6-59 years. Smoke exposure prevalence was 15 % per self-reports but 27 % based on detectable NNAL. 3-HPMA was increased in those reporting tobacco smoke exposure (607 vs 354 ng/ml, p = 0.008), with a higher proportion of smoke-exposed in the high- vs low-acrolein group (31 % vs 9 %, p=0.040). Compared to low-acrolein counterparts, high-acrolein participants experienced less decrease in sweat chloride (-35.2 vs -48.2 mmol/L; p = 0.020) and had higher sweat chloride values (50.6 vs 37.6 mmol/L; p = 0.020) 6 months post-ivacaftor. The odds of ivacaftor-mediated potentiation to near normative CFTR function (defined as SC6mo <40 mmol/L) was more than twice as high in the low-acrolein cohort (OR: 2.51, p = 0.026). CONCLUSIONS: Increased urinary 3-HPMA, an acrolein metabolite of tobacco smoke, is associated with a diminished sweat chloride response to ivacaftor potentiation of CFTR function.
RESUMO
The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex (EMC), which was previously found to modulate ribosome collisions during "preemptive quality control" of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.
RESUMO
"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.