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The natural history of COPD is complex, and the disease is best understood as a syndrome resulting from numerous interacting factors throughout the life cycle with smoking being the strongest inciting feature. Unfortunately, diagnosis is often delayed with several longitudinal cohort studies shedding light on the long 'preclinical' period of COPD. It is now accepted that individuals presenting with different COPD phenotypes may experience varying natural history of their disease. This includes its inception, early stages and progression to established disease. Several scenarios regarding lung function course are possible, but it may conceptually be helpful to distinguish between individuals with normal maximally attained lung function in their early adulthood who thereafter experience faster than normal FEV1 decline, and those who may achieve a lower than normal maximally attained lung function. This may be the main mechanism behind COPD in the latter group, as the decline in FEV1 during their adult life may be normal or only slightly faster than normal. Regardless of the FEV1 trajectory, continuous smoking is strongly associated with disease progression, development of structural lung disease and poor prognosis. In developing countries, factors such as exposure to biomass and sequelae after tuberculosis may lead to a more airway-centred COPD phenotype than seen in smokers. Mechanistically, COPD is characterized by a combination of structural and inflammatory changes. It is unlikely that all patients share the same individual or combined mechanisms given the heterogeneity of resultant phenotypes. Lung explants, bronchial biopsies and other tissue studies have revealed important features. At the small airway level, progression of COPD is clinically imperceptible, and the pathological course of the disease is poorly described. Asthmatic features can further add confusion. However, the small airway epithelium is likely to represent a key focus of the disease, combin

The natural history of COPD is complex, and the disease is best understood as a syndrome resulting from numerous interacting factors throughout the life cycle with smoking being the strongest inciting feature. Unfortunately, diagnosis is often delayed with several longitudinal cohort studies shedding light on the long 'preclinical' period of COPD. It is now accepted that individuals presenting with different COPD phenotypes may experience varying natural history of their disease. This includes its inception, early stages and progression to established disease. Several scenarios regarding lung function course are possible, but it may conceptually be helpful to distinguish between individuals with normal maximally attained lung function in their early adulthood who thereafter experience faster than normal FEV1 decline, and those who may achieve a lower than normal maximally attained lung function. This may be the main mechanism behind COPD in the latter group, as the decline in FEV1 during their adult life may be normal or only slightly faster than normal. Regardless of the FEV1 trajectory, continuous smoking is strongly associated with disease progression, development of structural lung disease and poor prognosis. In developing countries, factors such as exposure to biomass and sequelae after tuberculosis may lead to a more airway-centred COPD phenotype than seen in smokers. Mechanistically, COPD is characterized by a combination of structural and inflammatory changes. It is unlikely that all patients share the same individual or combined mechanisms given the heterogeneity of resultant phenotypes. Lung explants, bronchial biopsies and other tissue studies have revealed important features. At the small airway level, progression of COPD is clinically imperceptible, and the pathological course of the disease is poorly described. Asthmatic features can further add confusion. However, the small airway epithelium is likely to represent a key focus of the disease, combin

The airway epithelium is both a physical barrier protecting the airways from environmental insults and a significant component of the innate immune response. There is growing evidence that exposure of the airway epithelium to environmental insults in early life may lead to permanent changes in structure and function that underlie the development of asthma. Here we review the current published evidence concerning the link between asthma and epithelial damage within the airways and identify gaps in knowledge for future studies.

The aim of the present study was to investigate the role of 12-lipoxygenase (12-LOX) on platelet-induced airway smooth muscle cell (ASMC) proliferation. Co-incubation of platelets and ASMC caused platelet activation as determined by morphological changes. Simultaneously, reactive oxygen species (ROS)-generation was detected and ASMC proliferation (measured by using the MTS assay) increased significantly. Furthermore, we found that the 12-LOX inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and Baicalein prevented platelet activation in a co-cultures of platelets and ASMC. The inhibitory effect of CDC and Baicalein on platelets was also registered in a pure platelet preparation. Specifically, the 12-LOX inhibitors reduced collagen-induced platelet aggregation both in the presence and absence of external added fibrinogen. Importantly, platelet-induced ASMC proliferation and ROS production generated during the platelet/ASMC interaction was significantly inhibited in the presence of 12-LOX inhibitors. In conclusion, our findings reveal that 12-LOX is crucial for the observed enhancement of ASMC proliferation in co-cultures of platelets and ASMC. The present result suggests that 12-LOX activity is important in the initial step of platelet/ASMC interaction and platelet activation. Such action of 12-LOX represents a potential important mechanism that may contribute to platelet-induced airway remodelling.

Chlamydia infections are associated with exacerbations of asthma however the mechanisms are poorly understood. In this thesis we infected dendritic cells from healthy controls and asthmatic patients to determine if the immune response to chlamydial infection by these key immune cells could explain this association of chlamydial infection with asthma attacks. Infected dendritic cells from asthmatic patients showed increased expression of multiple inflammatory cytokine genes and genes for several tissue remodelling proteins, suggesting that infected dendritic cells play a central role in driving the airways damage associated with asthma. The findings provide a greater understanding of the role of infections in asthma and may provide a basis for new therapies to treat this important disease.

The aim of the present study was to investigate the role of 12-lipoxygenase (12-LOX) on platelet-induced airway smooth muscle cell (ASMC) proliferation. Co-incubation of platelets and ASMC caused platelet activation as determined by morphological changes. Simultaneously, reactive oxygen species (ROS)-generation was detected and ASMC proliferation (measured by using the MTS assay) increased significantly. Furthermore, we found that the 12-LOX inhibitors cinnamyl-3,4-dihydroxy-a-cyanocinnamate (CDC) and Baicalein prevented platelet activation in a co-cultures of platelets and ASMC. The inhibitory effect of CDC and Baicalein on platelets was also registered in a pure platelet preparation. Specifically, the 12-LOX inhibitors reduced collagen-induced platelet aggregation both in the presence and absence of external added fibrinogen. Importantly, platelet-induced ASMC proliferation and ROS production generated during the platelet/ASMC interaction was significantly inhibited in the presence of 12-LOX inhibitors. In conclusion, our findings reveal that 12-LOX is crucial for the observed enhancement of ASMC proliferation in co-cultures of platelets and ASMC. The present result suggests that 12-LOX activity is important in the initial step of platelet/ASMC interaction and platelet activation. Such action of 12-LOX represents a potential important mechanism that may contribute to platelet-induced airway remodelling., Funding Agency:Heart- and Lung foundation County of Östergotlands LänSwedish Research Council

The airway epithelium is the barrier to inhaled allergens and other agents and thus plays a central role in the restriction of, and response to, such insults. In asthma, ineffective barrier function and aberrant epithelial signalling are believed to be important in both the development and exacerbation of asthma. A review of the literature suggests that rodent models of asthma might not accurately represent the healthy or consequently the diseased airway epithelium of humans. Furthermore, little is known about small airway epithelial remodelling or the long-term effects of allergen exposure in the absence of continued antigenic stimulation. This thesis sought to firstly characterise epithelial remodelling following house dust mite (HDM) allergen challenge in a sheep model of experimental asthma (Chapters 3 and 4). In this model, chronic allergen challenge resulted in the induction of asthmatic features including goblet cell hyperplasia, epithelial hypertrophy, increased EGFR expression and allergen-induced proliferation of airway epithelial cells. Also provided is the first evidence for allergen induced goblet cell degranulation in a non-rodent system, and for degranulation induced via a relevant human allergen (HDM). The long-term effects of allergen exposure on the epithelium were also examined. Epithelial remodelling induced through chronic exposure to HDM remained for three months following the cessation of direct allergen challenge (Chapter 4). Goblet cell hyperplasia, epithelial hypertrophy and epithelial growth factor receptor (EGFR) expression remained in the absence of continued direct allergen challenge, indicative that the epithelium does not require continued allergen exposure to retain an asthmatic phenotype. Microarray analysis of epithelial brushing biopsies (Chapter 5), again demonstrated allergen driven epithelial cell proliferation and a general asthmatic phenotype including the down regulation of cellular tight junction mRNA. Subsequent qPCR valid

Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling. (C) 2011 Elsevier Inc. All rights reserved., Funding Agencies:strategic areas Cardiovascular Inflammation Research Centre (CIRC)County of Östergötlands Län STIFUD Olle ESwedish Research Council

Background: Few studies have directly compared airway remodelling assessed by computed tomography (CT) between asthma and chronic obstructive pulmonary disease (COPD). The present study was conducted to determine whether there are any differences between the two diseases with similar levels of airflow limitation under clinically stable conditions. Methods: Subjects included older male asthmatic patients (n = 19) showing FEV1/FVC <70% with smoking history less than 5-pack/year. Age- and sex-matched COPD patients (n = 28) who demonstrated similar airflow limitation as asthmatic patients and age-matched healthy non-smokers (n = 13) were recruited. Using proprietary software, eight airways were selected in the right lung, and wall area percent (WA%) and airway luminal area (Ai) were measured at the mid-portion of the 3rd to 6th generation of each airway. For comparison, the average of eight measurements per generation was recorded. Results: FEV1% predicted and FEV1/FVC was similar between asthma and COPD (82.3 ± 3.3% vs. 77.6 ± 1.8% and 57.7 ± 1.6% vs. 57.9 ± 1.4%). At any generation, WA% was larger and Ai was smaller in asthma, both followed by COPD and then controls. Significant differences were observed between asthma and controls in WA% of the 3rd to 5th generation and Ai of any generation airway, while no differences were seen between COPD and controls. There were significant differences in Ai of any generation between asthma and COPD. Conclusions: Airway remodelling assessed by CT is more prominent in asthma compared with age- and sex-matched COPD subjects in the 3rd- to 6th-generation airways when airflow limitations were similar under stable clinical conditions.

Until the relationship between symptoms, lung function tests, airway inflammation, airway hyper-responsiveness (AHR), exacerbations and remodelling is clarified, regular treatment seems to enable a greater disease control than on-demand therapy in most patients with mild persistent asthma. Current guideline classification based on disease severity remains a cornerstone in asthma management. However, the heterogeneity of asthma, the growing emphasis on subphenotypes, including molecular phenotypes identified by -omics technologies, and their possible implications in terms of different asthma severity, progression and therapeutic response, are changing current asthma treatment mainly based on disease severity classification to a pharmacological strategy more focused on the individual patient., Until the relationship between symptoms, lung function tests, airway inflammation, airway hyper-responsiveness (AHR), exacerbations and remodelling is clarified, regular treatment seems to enable a greater disease control than on-demand therapy in most patients with mild persistent asthma. Current guideline classification based on disease severity remains a cornerstone in asthma management. However, the heterogeneity of asthma, the growing emphasis on subphenotypes, including molecular phenotypes identified by -omics technologies, and their possible implications in terms of different asthma severity, progression and therapeutic response, are changing current asthma treatment mainly based on disease severity classification to a pharmacological strategy more focused on the individual patient.

Background: Few studies have directly compared airway remodelling assessed by computed tomography (CT) between asthma and chronic obstructive pulmonary disease (COPD). The present study was conducted to determine whether there are any differences between the two diseases with similar levels of airflow limitation under clinically stable conditions. Methods: Subjects included older male asthmatic patients (n = 19) showing FEV1/FVC <70% with smoking history less than 5-pack/year. Age- and sex-matched COPD patients (n = 28) who demonstrated similar airflow limitation as asthmatic patients and age-matched healthy non-smokers (n = 13) were recruited. Using proprietary software, eight airways were selected in the right lung, and wall area percent (WA%) and airway luminal area (Ai) were measured at the mid-portion of the 3rd to 6th generation of each airway. For comparison, the average of eight measurements per generation was recorded. Results: FEV1% predicted and FEV1/FVC was similar between asthma and COPD (82.3 ± 3.3% vs. 77.6 ± 1.8% and 57.7 ± 1.6% vs. 57.9 ± 1.4%). At any generation, WA% was larger and Ai was smaller in asthma, both followed by COPD and then controls. Significant differences were observed between asthma and controls in WA% of the 3rd to 5th generation and Ai of any generation airway, while no differences were seen between COPD and controls. There were significant differences in Ai of any generation between asthma and COPD. Conclusions: Airway remodelling assessed by CT is more prominent in asthma compared with age- and sex-matched COPD subjects in the 3rd- to 6th-generation airways when airflow limitations were similar under stable clinical conditions.

The urokinase plasminogen activator receptor (PLAUR) is a membrane anchored receptor that has been associated with a number of disease states. In these diseases, elevated receptor levels were associated with increased disease aggressiveness and higher mortality rates. Through genetic studies PLAUR has been identified as an asthma susceptibility gene. In these studies, coding and untranslated region single nucleotide polymorphisms showed association with asthma diagnosis and decline in lung function. In addition, association with baseline lung function in smokers and PLAUR SNPs was identified. This suggests that PLAUR plays a role in respiratory disease. Work presented in this thesis aimed to i) identify whether serum levels of PLAUR are associated with obstructive lung disease and lung function parameters, ii) identify novel regulatory mechanisms determining PLAUR levels, both at the genetic level in primary bronchial epithelial cells and at the protein level for serum PLAUR, iii) explore a role for the different isoforms in asthma and COPD and iv) determine novel variation in the PLAUR gene and 5` and 3` distal regions through next generation sequencing. Levels of the soluble cleaved form of PLAUR in serum were determined to be significantly elevated in COPD and asthma subjects compared to a control population. This identified an association between the soluble cleaved receptor and disease per se. However, PLAUR levels in serum could not be related to lung function parameters. With regards to receptor regulation, a genome-wide association study identified a novel post-translational PLAUR regulatory mechanism. This involved key SNPs in the human plasma kallikrein gene promoter that directed human plasma kallikrein enzymatic activity to cleave PLAUR in a post-translational mechanism. PLAUR gene regulation was also investigated via molecular biology, identifying that in primary bronchial epithelial cells, PLAUR regulation involves the gene’s 5 prime and 3 prime untran

Bronchial thermoplasty is a treatment for asthma. Whether during thermoplasty the airway wall fraction exposed to temperatures necessary to affect cells is sufficient to explain its histopathological impact is unclear. Airway smooth muscle and bronchial epithelial cells were exposed to media (37-70°C) for 10 seconds to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution following thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 severe asthmatics. In vitro airway smooth muscle and epithelial cell number decreased significantly following addition of media heated to ≥65°C. In silico simulations showed heterogeneous heat distribution; amplified in larger airways, with <10% of the airway wall heated >60°C for airways with an inner radius ~4mm. In vivo 6 weeks post-thermoplasty asthma control (ACQ6) improved (mean difference: 0.7 [95%-CI 0.1-1.3]; p=0.03), airway smooth muscle mass decreased (absolute median reduction: 5 [IQR 0-10]%; p=0.03) and epithelial integrity increased (14 [6-29]%; p=0.007); neither of which were related to improved asthma control. Integrated in vitro and in silico modelling suggested that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating; nor did this reduction confer a greater improvement in asthma control.

Bronchial thermoplasty is a treatment for asthma. Whether during thermoplasty the airway wall fraction exposed to temperatures necessary to affect cells is sufficient to explain its histopathological impact is unclear. Airway smooth muscle and bronchial epithelial cells were exposed to media (37-70°C) for 10 seconds to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution following thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 severe asthmatics. In vitro airway smooth muscle and epithelial cell number decreased significantly following addition of media heated to ≥65°C. In silico simulations showed heterogeneous heat distribution; amplified in larger airways, with <10% of the airway wall heated >60°C for airways with an inner radius ~4mm. In vivo 6 weeks post-thermoplasty asthma control (ACQ6) improved (mean difference: 0.7 [95%-CI 0.1-1.3]; p=0.03), airway smooth muscle mass decreased (absolute median reduction: 5 [IQR 0-10]%; p=0.03) and epithelial integrity increased (14 [6-29]%; p=0.007); neither of which were related to improved asthma control. Integrated in vitro and in silico modelling suggested that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating; nor did this reduction confer a greater improvement in asthma control.

Bronchial thermoplasty is a treatment for asthma. Whether during thermoplasty the airway wall fraction exposed to temperatures necessary to affect cells is sufficient to explain its histopathological impact is unclear. Airway smooth muscle and bronchial epithelial cells were exposed to media (37-70°C) for 10 seconds to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution following thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 severe asthmatics. In vitro airway smooth muscle and epithelial cell number decreased significantly following addition of media heated to ≥65°C. In silico simulations showed heterogeneous heat distribution; amplified in larger airways, with <10% of the airway wall heated >60°C for airways with an inner radius ~4mm. In vivo 6 weeks post-thermoplasty asthma control (ACQ6) improved (mean difference: 0.7 [95%-CI 0.1-1.3]; p=0.03), airway smooth muscle mass decreased (absolute median reduction: 5 [IQR 0-10]%; p=0.03) and epithelial integrity increased (14 [6-29]%; p=0.007); neither of which were related to improved asthma control. Integrated in vitro and in silico modelling suggested that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating; nor did this reduction confer a greater improvement in asthma control.

The urokinase plasminogen activator receptor (PLAUR) is a membrane anchored receptor that has been associated with a number of disease states. In these diseases, elevated receptor levels were associated with increased disease aggressiveness and higher mortality rates. Through genetic studies PLAUR has been identified as an asthma susceptibility gene. In these studies, coding and untranslated region single nucleotide polymorphisms showed association with asthma diagnosis and decline in lung function. In addition, association with baseline lung function in smokers and PLAUR SNPs was identified. This suggests that PLAUR plays a role in respiratory disease. Work presented in this thesis aimed to i) identify whether serum levels of PLAUR are associated with obstructive lung disease and lung function parameters, ii) identify novel regulatory mechanisms determining PLAUR levels, both at the genetic level in primary bronchial epithelial cells and at the protein level for serum PLAUR, iii) explore a role for the different isoforms in asthma and COPD and iv) determine novel variation in the PLAUR gene and 5` and 3` distal regions through next generation sequencing. Levels of the soluble cleaved form of PLAUR in serum were determined to be significantly elevated in COPD and asthma subjects compared to a control population. This identified an association between the soluble cleaved receptor and disease per se. However, PLAUR levels in serum could not be related to lung function parameters. With regards to receptor regulation, a genome-wide association study identified a novel post-translational PLAUR regulatory mechanism. This involved key SNPs in the human plasma kallikrein gene promoter that directed human plasma kallikrein enzymatic activity to cleave PLAUR in a post-translational mechanism. PLAUR gene regulation was also investigated via molecular biology, identifying that in primary bronchial epithelial cells, PLAUR regulation involves the gene’s 5 prime and 3 prime untran