Evaluation of a novel CFTR potentiator in COPD ferrets with acquired CFTR dysfunction.

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 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 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.

Comparative transcriptomics in human COPD reveals dysregulated genes uniquely expressed in ferrets.

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.

Airway remodeling in ferrets with cigarette smoke-induced COPD using µCT imaging.

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 (√WA) 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. √WA 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.

Excess mucus viscosity and airway dehydration impact COPD airway clearance.

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 6 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±0.6 µm, p<0.0001) and restored MCT (6.8±0.8 to 12.9±1.2 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.

Regional citrate anticoagulation for hemorrhage experiments in rats.

INTRODUCTION: Hemorrhage alone without concomitant trauma often results in a hypercoagulable state that makes it difficult to prevent clotting within the blood withdrawal catheters. Although systemic administration of heparin can ameliorate this problem, heparin use has many additional actions that may confound interpretation of the hemorrhage experiments. The problem can be resolved by the use of a dual lumen catheter that anticoagulates only the blood within the withdrawal circuit. We describe the design of such a catheter and evaluate its function in studies of hemorrhagic shock in rats. MATERIALS AND METHODS: Construction directions are provided for the dual lumen catheter along with a commercial source. The catheters were connected to computer controllable infusion syringes. Either citrate or heparin was used for regional extracorporeal anticoagulation. Rats were anesthetized and hemorrhaged to 40mmHg for more than 15min through the use of a computer program written in Labview. Ionized calcium measurements were obtained pre- and posthemorrhage. RESULTS: The catheters remained patent throughout the experiments. There was no significant difference in the ionized calcium whether citrate or heparin was used for extracorporeal anticoagulation. CONCLUSION: The dual lumen catheters are suitable for the study of hemorrhagic shock in rats without the need for systemic anticoagulation. The catheters can be used with computer-controlled hemorrhage procedures.

A ferret model of COPD-related chronic bronchitis.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US. The majority of COPD patients have symptoms of chronic bronchitis, which lacks specific therapies. A major impediment to therapeutic development has been the absence of animal models that recapitulate key clinical and pathologic features of human disease. Ferrets are well suited for the investigation of the significance of respiratory diseases, given prior data indicating similarities to human airway physiology and submucosal gland distribution. Here, we exposed ferrets to chronic cigarette smoke and found them to approximate complex clinical features of human COPD. Unlike mice, which develop solely emphysema, smoke-exposed ferrets exhibited markedly higher numbers of early-morning spontaneous coughs and sporadic infectious exacerbations as well as a higher level of airway obstruction accompanied by goblet cell metaplasia/hyperplasia and increased mucus expression in small airways, indicative of chronic bronchitis and bronchiolitis. Overall, we demonstrate the first COPD animal model exhibiting clinical and pathologic features of chronic bronchitis to our knowledge, providing a key advance that will greatly facilitate the preclinical development of novel treatments for this disease.