The pathophysiology of asthma is characterized by airway inflammation, remodeling and hyper-responsiveness. Phenotypic changes in airway smooth muscle cells (ASM) play a pivotal role in the pathogenesis of asthma. ASM cells promote inflammation and are key drivers of airway remodeling. While airway hyper responsiveness and inflammation can be managed by bronchodilators and anti-inflammatory drugs, ASM remodeling is poorly managed by existing therapies. Therefore, targeting ASM remodeling remains a challenge, and a deeper understanding of the molecular mechanism that regulates ASM phenotypes in asthma pathogenesis will facilitate the search for next-generation asthma therapy. MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell proliferation and inflammation - two phenotypes that are often altered in asthmatic ASM. We thus hypothesized that microRNAs regulate ASM phenotypes in asthma and represent new targets for future therapy. In this thesis, we used a genomic approach that combined next-generation sequencing with functional cellular assays to characterize the role of microRNAs in regulating airway smooth muscle function and drug response to conventional therapies. In Chapter 2, we identified miR-10a as the most abundant microRNA expressed in the primary human airway smooth muscle (HASM) cells. Using an unbiased target identification approach, we identified several novel potential targets of miR-10a, including the catalytic subunit alpha of PI3 kinase (PIK3CA)--the central component of the PI3K pathway. We demonstrated that miR-10a directly suppresses PIK3CA expression by targeting its 3' Untranslated region (3'-UTR). Inhibition of PIK3CA by miR-10a reduced AKT phosphorylation and blunted the expression of cyclins and cyclin-dependent kinases that are required for HASM proliferation. In Chapter 3, we examined the effect of conventional asthma therapies on miRNA expression. While we did not find significant changes in miRNA levels, it remains to be determined whether microRNAs play a role in ASM tissue response to asthma therapy. Our study is the first to examine the role of microRNAs in ASM proliferation. Results from our study identified a novel microRNA-mediated regulatory mechanism of PI3K signaling and ASM proliferation. They suggest further that miR-10a is a potential therapeutic target to treat airway remodeling in asthma.

Aim. To assess associations between occupational air pollution and respiratory health, especially in miners. Background. Indications of associations between occupational exposure or social economic status and respiratory health have been found in several population-based studies. However, there have been few longitudinal studies of the putative correlations, the effects of environmental and genetic factors have seldom been simultaneously studied, and studies of miners have generated conflicting results. Material and methods. Population-based Obstructive Lung Disease in Northern Sweden (OLIN) cohorts surveyed in 1986, 1992 and 1996, and two industry-based materials, were used in cross-sectional and longitudinal studies. Inflammatory markers were compared in sputa from miners after a vacation of at least four weeks, after repeated occupational exposures for at least three months, and controls. The mortality from silicosis was studied in 7729 miners with at least 1 year of exposure. Multivariate analyses were used to adjust for confounders. Results. Up to about 30-40% (etiologic fraction) of incident symptoms in persons both with and without a family history of asthma (FHA) could be explained by exposure to occupational air pollution. Low socio-economic status (SES) was associated with impaired respiratory health. Population attributable risks for most examined disorders were about 10%. Current and ex-miners had increased prevalence of recurrent wheeze, longstanding cough, physician-diagnosed chronic bronchitis, and a trend for increased sputum production. For physician-diagnosed chronic bronchitis a multiplicative interaction was found between exposure and smoking habits. Ex-miners that had been exposed for on average 13 years and whose exposure had ceased 16 years before the study had an increased prevalence of physician-diagnosed chronic bronchitis and chronic productive cough and a trend to increased use of asthma medicines. Miners exposed underground for 18 years, on average, to diesel exhaust (with 0.28 mg/m3 nitrogen dioxide and 27 μg/m3 elemental carbon on average, EC) and particles (3.2 mg/m3 inhalable dust on average) had signs of higher inflammatory activity in their airways, i.e. significantly higher frequencies of macrophages, neutrophils, and total cells compared with referents. The activity in miners was similar after a vacation of at least four weeks and after repeated exposures for three months. There were 58 deaths from silicosis (underlying and contributing cause of death) and a clear dose-response relationship. The data indicated an increased risk of severe silicosis after long-term exposure to 0.1 mg/m3 respirable quartz, the current maximum allowable concentration (MAC) in Sweden and many other countries. Conclusion. Occupational exposure to dust, gases, and fumes impaired respiratory health, accounting for up to 30-40% of some respiratory symptoms in the general population. Low socio-economic status was associated with impaired respiratory health. The complex profiles of dust and diesel exhaust substances found in mines may cause inflammatory reactions in their lungs and persistent respiratory symptoms in occupationally exposed miners. Long-term exposure to quartz at the present MAC level may cause severe silicosis.

Although asthma is primarily thought to be an inflammatory disease of the airways, it has recently been hypothesized that the altered mechanical environment of an asthmatic airway may contribute to the development of the disease through changes in cellular phenotype. In regards to this hypothesis, the effects of stretch on airway smooth muscle (ASM) have previously been investigated using 2D cell culture. However, over the last few years there has been an increasing appreciation to the importance of the role of the 3D extracellular matrix in the regulation of cellular response. For this reason, the work presented in this thesis covers the development of a device capable of high-throughput investigations into the effects of acute or chronic, uniaxial, oscillatory mechanical strain on an array of miniature, 3D, multi-cell, tissue-engineered constructs.

Objective: We aim to improve our understanding of sleep physiology by describing the changes in mandibular position during sleep in normal subjects. Methods: We developed a novel method for mapping mandibular position simultaneously in three dimensions (anteroposterior, vertical and lateral) using magneto-resistive sensors strategically placed around 3 different moving joints on an external apparatus attached to the head and mandible. Spherical coordinates derived from these sensors provided information of jaw position in each of the three measurement planes. We assessed changes in jaw position in twelve healthy subjects (6 male, 6 female) aged (mean ± SD) 23 ± 7 years, Body Mass Index 22.5 ± 3.4 kg/m2, and with nasal resistance 3.24 ± 0.67 cmH2O/L/s by recording mandibular position simultaneously with overnight sleep polysomnography. Results: Jaw position was significantly influenced by sleep stage (p<0.001). The transition from wake to light sleep (stage one) was accompanied by significant jaw closure and jaw protrusion (p<0.05). As non-rapid-eye-movement (NREM) sleep deepened from stages 1 through slow wave sleep (SWS), vertical jaw opening (p<0.05) and posterior jaw movement progressively increased (p<0.05). REM sleep was associated with the greatest degree of jaw opening of all sleep stages (p<0.05). Lateral jaw position was not significantly different between sleep stages. Conclusion: This study describes, for the first time, an accurate method of measuring changes in mandibular position during sleep in all three dimensions. The observed changes during sleep in healthy subjects suggest a simultaneous modulation of upper airway muscular tone, which may be important in the understanding of upper airway occlusion in Obstructive Sleep Apnea. / Thesis (Master, Physiology) -- Queen's University, 2008-08-29 14:27:57.726

No description available.

Smooth muscle is a primary determinant of physiology as its ability to contract affords dynamic control of diameter of the hollow organs it encircles including the airways. Mature airway smooth muscle (ASM) cells are phenotypically plastic, enabling them to subserve contractile, proliferative, migratory and secretory roles that relates to its function in health and disease. ASM cells can control airway diameter both acutely, via reversible contraction, and chronically, by driving fixed changes in structure and function properties of the airway wall. However, the scope of research on ASM biology and function has broadened greatly in the past two decades, embracing the now recognized dynamic and multifunctional behavior, but there is always a need to investigate the role of new proteins regulating ASM phenotype in vitro and lung function in vivo. The multimeric dystrophin-glycoprotein complex (DGC) links the extracellular matrix (ECM) and actin cytoskeleton while caveolae form membrane arrays on ASM cells. Using ASM cells and tissues from human and canine and intact mouse for lung physiology, we investigated the role of DGC in phenotype maturation. We also investigated the mechanism for the organization of DGC with caveolae and further tested whether this is functionally important in mobilizing intracellular calcium in ASM cells, contraction of ASM tissue and finally its role in airway physiology. Our data demonstrate that the expression of DGC is an integral feature and a key determinant for phenotype maturation of human ASM cells. Our new data reveals an interaction between caveolin-1 and DGC and indicate that this association, in concert with anchoring to the actin cytoskeleton, underpins the spatial organization of caveolae on the membrane and has a functional role in receptor-mediated calcium release in ASM in vitro, ASM contraction ex vivo and lung function in vivo. Collectively our study indicates that the organization of caveolae and DGC, and its link from ECM to the actin cytoskeleton with in caveolae are a determinant of phenotype and functional properties of ASM, which underpins its role in physiology and pathophysiology of chronic airway diseases such as asthma.

Smooth muscle is a primary determinant of physiology as its ability to contract affords dynamic control of diameter of the hollow organs it encircles including the airways. Mature airway smooth muscle (ASM) cells are phenotypically plastic, enabling them to subserve contractile, proliferative, migratory and secretory roles that relates to its function in health and disease. ASM cells can control airway diameter both acutely, via reversible contraction, and chronically, by driving fixed changes in structure and function properties of the airway wall. However, the scope of research on ASM biology and function has broadened greatly in the past two decades, embracing the now recognized dynamic and multifunctional behavior, but there is always a need to investigate the role of new proteins regulating ASM phenotype in vitro and lung function in vivo. The multimeric dystrophin-glycoprotein complex (DGC) links the extracellular matrix (ECM) and actin cytoskeleton while caveolae form membrane arrays on ASM cells. Using ASM cells and tissues from human and canine and intact mouse for lung physiology, we investigated the role of DGC in phenotype maturation. We also investigated the mechanism for the organization of DGC with caveolae and further tested whether this is functionally important in mobilizing intracellular calcium in ASM cells, contraction of ASM tissue and finally its role in airway physiology. Our data demonstrate that the expression of DGC is an integral feature and a key determinant for phenotype maturation of human ASM cells. Our new data reveals an interaction between caveolin-1 and DGC and indicate that this association, in concert with anchoring to the actin cytoskeleton, underpins the spatial organization of caveolae on the membrane and has a functional role in receptor-mediated calcium release in ASM in vitro, ASM contraction ex vivo and lung function in vivo. Collectively our study indicates that the organization of caveolae and DGC, and its link from ECM to the actin cytoskeleton with in caveolae are a determinant of phenotype and functional properties of ASM, which underpins its role in physiology and pathophysiology of chronic airway diseases such as asthma.

Allergic airway disease, i.e. allergic rhinitis (AR) and asthma, is a common health problem. The prevalence is increasing in most countries of the world. Traffic-related air pollution has been found to induce and enhance allergic airway disease, but the underlying mechanisms are not known. Oxylipins are fatty acid metabolites, of which several have been linked to asthmatic airway inflammation. Oxylipin profiles have previously been investigated in bronchoalveolar lavage (BAL), mainly reflecting the peripheral lung, but not in bronchial wash (BW), which better reflect the proximal airways. The airway epithelium is covered by a respiratory tract lining fluid (RTLF) The RTLF contains antioxidants to protect from oxidative stress, which may be caused by exposure to air pollution. Previous studies have reported diminished levels of the antioxidant ascorbate (vitamin C) in the RTLF of patients with asthma. Little is known about the regulation of vitamin C in the lung. The aim of this thesis was to investigate airway inflammatory responses to diesel exhaust exposure in patients with AR and allergic asthma; to evaluate oxylipin profiles in different regions of the lung in patients with allergic asthma; and to study the distribution of vitamin C transporters in the airways of patients with allergic asthma. Diesel exhaust (PM10 100 μg/m3 for 2 h) induced a neutrophilic airway inflammation in healthy individuals evaluated 18 h after exposure. Patients with AR and asthma did not respond with an enhanced airway inflammation. However, a small increase in myeloperoxidase was found in BAL from patients with AR, as well as decreases in epithelial tryptase and BW stem cell factor. This indicates that other mechanisms than classical inflammation are responsible for the increased sensitivity to traffic-related air pollution in patients with allergic airway disease. Oxylipin baseline profiles differed between peripheral and proximal airways in both allergic asthmatics and healthy individuals. Total oxylipin concentrations, and five individual oxylipins, primarily from the lipoxygenase (LOX) pathway, were elevated in BW from asthmatics compared to healthy controls, supported by immunohistochemical staining of 15-LOX-1 in the bronchial epithelium. This suggests that lung compartment-specific sampling should be considered in future studies. Sodium dependent vitamin C transporter 2 (SVCT2) was, for the first time, found present in the human lung epithelium, localised mainly within goblet cells. A negative correlation between SVCT2+ goblet cells and vitamin C suggests that these cells may play a hitherto unknown function in ascorbate re-uptake and recycling at the air-lung interface.

Astma yra lėtinė kvėpavimo takų uždegimo liga. Mokslininkai neabejoja, jog 2-o tipo T limfocitai pagalbininkai bei eozinofilinis kvėpavimo takų uždegimas yra astmos patogenezės pagrindas. Tačiau šis mechanizmas ne visuomet gali paaiškinti astmos metu esančio kvėpavimo takų uždegimo bei klinikinių simptomų įvairumą, eigos ypatumus ir net skirtingą atsaką į gydymą. Eksperimentiniai tyrimai su gyvūnais parodė 17-o tipo T limfocitų pagalbininkų (Th17) svarbą alerginės astmos vystymuisi. Todėl šio mokslinio tyrimo tikslas buvo įvertinti Th17 limfocitų vaidmenį sergant alergine astma. Tyrimo metu sergantiesiems alergine astma nustatytas didesnis Th17 limfocitų kiekis periferiniame kraujyje bei didesnė interleukino (IL)-17 koncentracija serume ir indukuotuose skrepliuose, lyginant su sveikais asmenimis. Be to, bronchų provokacija su Dermatophagoides pteronyssinus alergenu sukėlė Th17 limfocitų ir IL-17 kiekio padidėjimą praėjus 7 ir 24 val.po jos, ypatingai ryškų sergantiems alergine astma su ankstyva ir vėlyva bronchų obstrukcija. Atlikto tyrimo rezultatai parodė, kad Dermatophagoides pteronyssinus alergenas sukelia vietinį ir sisteminį Th17 limfocitų imuninį atsaką kuris yra susiję su vėlyvos fazės kvėpavimo takų uždegimu. / Allergic asthma is a chronic inflammatory disease of the airways associated with the response of predominant type 2 helper T lymphocytes to an inhaled allergen. However, differences in inflammation and clinical symptoms of this disease not always can be explained by this mechanism. Recent animal model studies have shown the importance of type 17 helper T lymphocytes (Th17) in the development of allergic asthma. The role of these cells in causing allergen-induced airway inflammation as well as systemic inflammatory response in human is still not well defined. Therefore, we investigated the peripheral blood Th17 lymphocyte response to inhaled Dermatophagoides pteronyssinus (D. pteronyssinu) allergen in patients with allergic asthma. The present study has shown that patients with allergic asthma have a higher percentage of peripheral blood Th17 lymphocytes and elevated serum as well as induced sputum interleukin-17 levels compared with healthy subjects. Moreover, all studied allergic asthma patients, especially with early- and late-phase asthmatic reaction, showed an enhanced airway and systemic Th17 lymphocyte response 7 h and 24 h after bronchial challenge. We documented an enhanced local and systemic Th17 lymphocyte response to inhaled D. pteronyssinus in association with late-phase allergen-induced airway inflammation in patients with allergic asthma.

The role of dopamine in directly modulating somatic motoneuron excitability and hence muscle tone is unknown. We investigated whether dopamine influences the trigeminal motor pool (MoV) that innervates the masseter and tensor palatini muscles, both of which function to maintain upper airway patency. We hypothesized that dopamine facilitates motor outflow at the MoV. We focally applied apomorphine (nonspecific dopamine receptor agonist) at the MoV in anaesthetized rats. We also applied receptor-specific agonists and antagonists to determine the receptor subtype mediating dopaminergic mechanisms of action. We demonstrated that dopaminergic transmission at the MoV potently increased motor outflow via the D1-like receptor and facilitated masseter and tensor palatini muscle tone. It is unknown whether endogenous dopamine release on to airway motoneurons influences their activity to regulate muscle tone in natural sleep-wake behaviours. This issue warrants investigation because the neurochemical basis of upper airway motor dysfunction (e.g. obstructive sleep apnea) remains poorly characterized.