Final Activity Report Summary - AIRWAY DISEASE (Pathogenesis and treatment of chronic airway disease: novel animal models for studies on modifier genes, lung repair mechanisms and novel therapeutic)
Chronic obstructive lung diseases belong to the most common chronic diseases in children and adults and constitute the fourth leading cause of death worldwide. Despite their medical and socio-economical importance, the pathogenesis of chronic obstructive lung diseases remains poorly understood and there are currently no therapies available that target these diseases at their root cause. The overall goal of this project was to use of transgenic mouse models to advance our current understanding of the pathogenesis and the preclinical development of more effective therapies for common chronic airway diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD).
First, we undertook a preclinical evaluation of drugs designed to improve airway surface hydration, including the classical Na+ channel blocker amiloride and novel more potent and long-acting analogs in ENaC-transgenic mice as a model of chronic obstructive lung disease. In these studies we showed for the first time that preventive inhibition of Na+ hyperabsorption with amiloride has significant therapeutic effects and reduces pulmonary mortality and morbidity in this murine model and established a novel preventive treatment paradigm for chronic obstructive lung diseases.
Further, because the short-acting compound amiloride had no effects when treatment was started in ENaC-transgenic mice with established lung disease, we evaluated novel more potent and long-acting ENaC blockers in adult mice with chronic lung disease. These studies identified a long acting ENaC blocker with significant therapeutic effects in adult ENaC-transgenic mice demonstrating that late intervention with this class of compounds has the capacity to reverse mucus obstruction in established lung disease. These results in a murine model suggest that long acting ENaC blockers may have therapeutic effects in patients with chronic lung diseases.
Second, we used quantitative phenotype analyses in combination with transcript expression analyses by whole genome cDNA microarrays to identify disease modifier genes for chronic airway disease in ENaC-transgenic mice with severe versus mild lung disease. From a number of candidate genes that were identified using this approach, and based the observation that ENaC-transgenic mice develop emphysema and macrophage activation in addition to chronic airway disease we focussed on the functional elucidation of the macrophage elastase (MMP12) as a modifier of disease severity in ENaC-transgenic mice.
Using genetic and pharmacologic approaches and FRET-based activity assays, we were able to demonstrate that MMP12 plays and important role in pulmonary inflammation and emphysema formation in ENaC-transgenic mice as well as other murine models of pulmonary inflammation.
Further, these studies suggest that MMP12 activity, as determined by novel highly sensitive and quantitative FRET based assays, may be a useful novel biomarker to monitor disease activity and severity of chronic obstructive lung disease in vivo.
Third, we used the tetracycline regulated (tet-on) system and generated novel mouse model with conditional obstructive airway disease, in which the lung disease phenotype can be turned on and off in the living animal at any age to circumvent age-specific and developmental issues associated with constitutive ENaC overexpression, and for studies of lung repair when transgene expression is switched off. We expect that this novel mouse model will allow us to control the onset and severity of lung disease and will therefore be a highly valuable novel in vivo model for studies related to the role of chronic exposure to environmental stimuli (e.g. allergens, particulates, or pathogens) in the pathogenesis of chronic airway disease, and for studies on repair mechanisms of the diseased lung. Taken together, we expect that the results driven from this project will lead to the identification of novel therapeutic targets and have important implications for the development of effective therapies and the design of future clinical trials in patients with chronic obstructive airway diseases including cystic fibrosis.
First, we undertook a preclinical evaluation of drugs designed to improve airway surface hydration, including the classical Na+ channel blocker amiloride and novel more potent and long-acting analogs in ENaC-transgenic mice as a model of chronic obstructive lung disease. In these studies we showed for the first time that preventive inhibition of Na+ hyperabsorption with amiloride has significant therapeutic effects and reduces pulmonary mortality and morbidity in this murine model and established a novel preventive treatment paradigm for chronic obstructive lung diseases.
Further, because the short-acting compound amiloride had no effects when treatment was started in ENaC-transgenic mice with established lung disease, we evaluated novel more potent and long-acting ENaC blockers in adult mice with chronic lung disease. These studies identified a long acting ENaC blocker with significant therapeutic effects in adult ENaC-transgenic mice demonstrating that late intervention with this class of compounds has the capacity to reverse mucus obstruction in established lung disease. These results in a murine model suggest that long acting ENaC blockers may have therapeutic effects in patients with chronic lung diseases.
Second, we used quantitative phenotype analyses in combination with transcript expression analyses by whole genome cDNA microarrays to identify disease modifier genes for chronic airway disease in ENaC-transgenic mice with severe versus mild lung disease. From a number of candidate genes that were identified using this approach, and based the observation that ENaC-transgenic mice develop emphysema and macrophage activation in addition to chronic airway disease we focussed on the functional elucidation of the macrophage elastase (MMP12) as a modifier of disease severity in ENaC-transgenic mice.
Using genetic and pharmacologic approaches and FRET-based activity assays, we were able to demonstrate that MMP12 plays and important role in pulmonary inflammation and emphysema formation in ENaC-transgenic mice as well as other murine models of pulmonary inflammation.
Further, these studies suggest that MMP12 activity, as determined by novel highly sensitive and quantitative FRET based assays, may be a useful novel biomarker to monitor disease activity and severity of chronic obstructive lung disease in vivo.
Third, we used the tetracycline regulated (tet-on) system and generated novel mouse model with conditional obstructive airway disease, in which the lung disease phenotype can be turned on and off in the living animal at any age to circumvent age-specific and developmental issues associated with constitutive ENaC overexpression, and for studies of lung repair when transgene expression is switched off. We expect that this novel mouse model will allow us to control the onset and severity of lung disease and will therefore be a highly valuable novel in vivo model for studies related to the role of chronic exposure to environmental stimuli (e.g. allergens, particulates, or pathogens) in the pathogenesis of chronic airway disease, and for studies on repair mechanisms of the diseased lung. Taken together, we expect that the results driven from this project will lead to the identification of novel therapeutic targets and have important implications for the development of effective therapies and the design of future clinical trials in patients with chronic obstructive airway diseases including cystic fibrosis.