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Browsing Open Theses and Dissertations by Department "Cardiovascular & Respiratory Sciences"
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Item Open Access A Pharmacodynamics Analysis of Glucocorticoid Receptor-Mediated Gene Expression in BEAS-2B Human Airway Epithelial Cells(2016) Joshi, Taruna; Giembycz, Mark; Newton, Robert; McKay, Derek; von der Weid, Pierre; Hirota, Simon; Cameron, LisaInternational treatment guidelines recommend that inhaled glucocorticoids be used as a monotherapy to control mild-to-moderate asthma. However, a combination of an inhaled glucocorticoid with a long-acting β2-adrenoceptor agonist is recommended for managing moderate-to-severe asthma, which cannot be controlled by glucocorticoids alone. Increasing evidence supports the idea that glucocorticoids acting through the glucocorticoid receptor (GR) can attenuate inflammatory responses either by inducing anti-inflammatory genes (a process called transactivation) or by suppressing pro-inflammatory genes (a process called transrepression). Gene transactivation by glucocorticoids was initially thought to be responsible for causing the metabolic side-effects and, hence, is relatively understudied when compared to transrepression. However, it has become increasingly clear that transactivation plays an important role in the anti-inflammatory actions of glucocorticoids. Moreover, the extent to which clinically-relevant glucocorticoids are equivalent in their ability to promote gene expression is unclear. This thesis describes the first pharmacodynamic approach to evaluate the transactivation potential of a panel of glucocorticoids alone and in combination with indacaterol (Ind), a long-acting β2-adrenoceptor agonist. Pharmacodynamic analyses showed that magnitude of luciferase gene induction was agonist dependent (i.e. seven different glucocorticoids tested displayed varying degrees of agonism). In addition, there were significant differences in agonist potency and, more importantly, the relationship between GR occupancy and response. To complement the reporter studies, similar analyses were performed on four glucocorticoid-inducible candidate genes. Three of these (GILZ, p57kip2 and CRISPLD2) are genes with potential anti-inflammatory activity and a fourth gene, PDK4 is predicted to promote metabolic side-effects. Similar to the luciferase reporter system, the expression of these genes was agonist-dependent and displayed markedly different GR occupancy-response relationships. Furthermore, Ind, when combined with the seven GR agonists tested, synergistically enhanced transactivation, the magnitude of which was agonist and gene dependent. These studies demonstrate that when gene transactivation is used as a functional output, glucocorticoids used to treat asthma are not biologically-equivalent. It is proposed that these differences may be exploited to therapeutic advantage. Thus, the generation of gene expression ‘fingerprints’ in target and off-target human tissues may allow new GR agonists to be rationally designed for asthma with an improved therapeutic index.Item Open Access Acquired Mechanisms of Bicuspid Aortic Valve-Associated Aortopathy(2018-07-05) Guzzardi, David G.; Fedak, Paul; O'Brien, Edward; Di Martino, Elena S.Bicuspid aortic valve (BAV)-associated aortopathy is characterized by progressive aortic extracellular matrix (ECM) remodeling leading to aneurysm, dissection or rupture. The cause of this aortopathy is unclear; a genetically-driven basis has been favoured, but recent studies implicating an acquired valve-mediated hemodynamic mechanism have challenged this long-standing view despite no clear link between aortic hemodynamics and ECM remodeling having been delineated. We hypothesized that aortopathy in human BAV patients is influenced by valve-mediated wall shear stress (WSS) in a regionally-dependent manner. Aortic tissue specimens from BAV patients that received pre-operative 3-dimensional time-resolved phase-contrast magnetic resonance imaging (4D flow MRI) to compute regional WSS were assessed quantitatively for their expression of aortopathy. Compared to aortic tissue subjected to normal WSS, adjacent tissue from the same BAV aortas subjected to regionally-elevated WSS exhibited demonstrably worse elastic fiber histopathology, increased protease expression and elevated levels of transforming growth factor β-1 (TGFβ-1) consistent with maladaptive aortic ECM remodeling. We also observed that incremental increases in aortic WSS in the human BAV aorta correlate with increased severity of elastic fiber histopathology, and that this association is most strongly observed in BAV patients with primary stenosis and in mildly-dilated (< 4.5 cm) aortas in the earlier stages of disease. These novel data support a critical role for valve-mediated hemodynamics in coordinating the expression of BAV-associated aortopathy and dispute the assumption that aortic pathology in these patients is primarily driven by genetics. Fluoroquinolone (FQ) antibiotic use constitutes another acquired mechanism of aortopathy that may place BAV patients with pre-existing aortic pathology at risk of disease exacerbation. However, no cellular mechanism has been provided underlying this association. We hypothesized that in aortic myofibroblast cells from BAV-associated aortopathy patients, FQ exposure would alter the proteolytic profile favouring ECM dysregulation and modulate collagen expression. We observed that FQ exposure generates a functional increase in ECM degradation driven by reduced tissue inhibitors of matrix metalloproteinases (TIMPs) alongside impaired compensatory collagen-1 expression. These findings may explain the increased incidence of acquired FQ-associated aortopathy and encourage judicious use of FQ in BAV patients with pre-existing aortic pathology.Item Open Access Association between glycemic load and cognitive function in community-dwelling older adults: results from the Brain in Motion study(2017) Garber, Anna; Poulin, Marc; Friedenreich, Christine; Csizmadi, Ilona; Longman, Richard S.; Sajobi, Tolulope; Shearer, JaneBackground: Impaired glucose tolerance is a risk factor for non-age-related cognitive decline and is also associated with measures of physical activity (PA) and cardiorespiratory fitness (CRF). A low glycemic load (GL) diet can aid in the management of blood glucose levels, but little is known about its effect on cognition with poor glucoregulation. Objective: The aim of this thesis was to assess the relation between GL and cognitive function by glucoregulation, and possible mediatory effects by CRF and PA, in older adults. Design: A cross-sectional analysis of 194 cognitively healthy adults aged ≥55 years (mean=65.7, SD=6.1) was conducted. GL was assessed using a quantitative food frequency questionnaire, and glucoregulation was characterized on the HOMA-IR index. Subjects also completed a cognitive assessment, CRF testing, a validated self-reported PA questionnaire, and a blood draw. Multiple linear regression models adjusted for significant covariates were used to evaluate the relation between GL and cognition, and mediation analysis was used to assess potential mediatory effects by CRF and PA. Results: GL was inversely associated with global cognition (β=-0.014; 95% CI -0.024, -0.0036) and figural memory (β =-0.035; 95% CI -0.052, -0.018) in subjects with poor glucoregulation. Neither CRF nor PA mediated these relations. In subjects with good glucoregulation, no association was found between GL and cognitive function (p>0.05). Conclusions: A low GL diet is associated with better cognitive function in older adults with poor glucoregulation. This study provides supportive evidence for the role of GL in maintaining better cognitive function during the aging process.Item Open Access Bioinductive Effects of Acellular Biologic Scaffolds Promote Adaptive Cardiac Repair Following Myocardial Infarction(2018-09-17) Svystonyuk, Daniyil A.; Fedak, Paul; Tibbles, Lee Anne; Duff, Henry J.; Giles, Wayne R.Ischemic injury may lead to structural remodeling and progressive loss of function, resulting in eventual decompensation to heart failure. Acellular biologic ECM scaffolds retain their native 3-D architecture along with a profile of bioactive constituents that may be leveraged surgically to support myocardial healing. In a proof of concept study, we have previously identified FGF-2 bound to the acellular ECM scaffolds. As such, we hypothesized that FGF-2-dependent bioinductive signaling from surgically implanted acellular scaffolds may attenuate maladaptive structural remodeling and improve functional recovery post-myocardial infarction (MI). First, we observed that FGF-2 has potent anti-fibrotic properties that limited human cardiac fibroblast activation and cell-mediated ECM dysregulation in an in vitro 3-D model. Biochemical characterization showed that ECM scaffolds intact with bioactive constituents released FGF-2 under passive conditions. In a rodent model of myocardial infarction, animals that received intact ECM scaffolds following ischemic injury showed improved functional recovery with evidence of new blood vessel assembly underlying the implantation site. The functional benefits and neovascularization processes were absent in animals that received inactivated scaffolds where FGF-2 bioavailability was limited. The FGF-2-dependent bioinductive effect favorably targeted cardiac fibroblasts, who demonstrated phenotypic plasticity away from a pro-fibrotic phenotype and towards a pro-reparative vasculogenic phenotype. The anti-fibrotic effects of acellular ECM scaffold-derived FGF-2 were consistent with our in vitro studies, however the phenotypic change was unexpected. The redirection in fibroblast phenotype was associated with a modified cardiac scar characterized by a pro-vasculogenic paracrine microenvironment capable of supporting new blood vessel formation and attenuating fibrotic processes. Once again, limiting FGF-2 bioavailability from the ECM scaffolds or blocking FGF receptors in cardiac fibroblasts abolished the induced vasculogenic phenotype. We extended our observations to human subjects where biologic scaffolds were surgically implanted at the site of ischemic injury as an adjunct to standard surgical revascularization. In patients with severe microvascular obstruction and concomitant cardiac dysfunction, acellular biologic scaffolds improved global scar volume and stimulated regional recovery of resting myocardial perfusion. In summary, acellular biologic scaffolds stimulate myocardial healing following ischemic injury through FGF-2-dependent bioinductive signaling that modifies the ischemic scar to support neovascularization, adaptive remodeling, and functional recovery.Item Open Access Cardiovascular Magnetic Resonance Imaging in Cardiometabolic Disease(2016-01-26) Schmidt, Anna; Anderson, Todd; Friedrich, Matthias; White, James; Pacaud, Danièle; Lau, DavidThe unrelenting incidence of obesity and type 2 diabetes has become a global public health concern. Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in these cohorts. Cardiovascular Magnetic Resonance (CMR) imaging is a robust imaging modality with techniques to provide sensitive detection of early cardiovascular changes in obesity and diabetes. In a prospective study of obese adolescents (n=11, 10 to 20 years old) and a healthy weight control group (n=14), we found obesity was associated with increases in Left ventricular (LV) mass, LV wall thickness and mass:volume ratio. In this “early stage” of remodeling, these structural changes were not associated with elevations in Native T1, a surrogate marker of tissue fibrosis. Strain analysis, by novel CMR tissue tracking analysis, revealed a hyper-contractile state, likely representing a combined contribution of elevated systolic blood pressure with compensatory myocyte hypertrophy. Obese subjects were additionally enrolled in a comprehensive 6-month lifestyle intervention; subjects did not experience weight loss or any change in cardiac parameters after 6 months. Further along the natural history of CVD as related to metabolic disease, the second study looked to examine possible sub-clinical changes in the hearts of otherwise healthy diabetic patients. This study examined twenty-eight healthy type 2 diabetic patients without any observable vascular complications. The complementary use of CMR-based T1 mapping and 3-dimensional strain analysis demonstrated expansion of the extracellular matrix and a reduction in global longitudinal strain. In the endpoint of the cardiometabolic disease spectrum, we analyzed individuals with known or suspected Coronary Artery Disease. This study explored the relationship between intra-thoracic fat volume (ITFV) and 4D myocardial strain-based markers of adverse remodeling. In non-infarcted myocardium, ITFV is associated with reductions in myocardial strain. These findings suggest ITFV to be a potentially important marker of adverse ventricular remodeling. The findings of these three studies suggest a capacity of contemporary CMR to identify early changes in cardiometabolic disease and also lend insight into the progression of diabetic heart disease. CMR provides a non-invasive, accurate and reproducible imaging modality with the potential to be useful in screening and CVD risk-stratification.Item Open Access CaV3.2 Channels and BKCa-Mediated Feedback in Vascular Smooth Muscle(2018-06-28) Hashad, Ahmed Mohamed; Welsh, Donald Gordon; Chen, Sui Rong; Altier, Christophe; von der Weid, Pierre-YvesThe vascular T-type Ca2+ channel, CaV3.2, regulates arterial tone by triggering Ca2+ sparks and activating large conductance Ca2+-activated K+ (BKCa) channels. Despite being an integral element of an arterial feedback loop, little is known of its regulation and how key receptors and signaling pathways use this channel to influence tissue perfusion. This thesis will begin to fill key knowledge gaps, undertaking experiments that progress from individual smooth muscle cells to whole arteries, and which entail the use of patch clamp electrophysiology, Ca2+ imaging, pressure myography, immunohistochemistry, quantitative polymerase chain reaction (qPCR) and computational modeling. In initial work, perforated patch electrophysiology was used in concert with Ca2+ imaging to illustrate the coordinated interplay between CaV3.2 and two other Ca2+ permeable conductances in setting voltage-dependent Ca2+ spark production and BKCa channel activation. A second layer of experiments subsequently revealed that caveolae help couple CaV3.2 to Ca2+ sparks generation by placing this T-type Ca2+ channel in close proximity to its intracellular target, ryanodine receptors (RyR). Disruption of the structural arrangement impaired the ability of CaV3.2 to mediate BKCa-mediated feedback in intact resistance arteries. Final experiments revealed that CaV3.2 channels are targeted by common vasoactive stimuli through unique signaling pathways. Of note, was the ability of Angiotensin II to suppress CaV3.2 channel activity through the generation of reactive oxygen species (ROS) by NADPH oxidase (Nox). In summary, this thesis advances our knowledge of Ca2+ handling in vascular smooth muscle by providing new regulatory insight into CaV3.2, a T-type Ca2+ channel involved in optimizing arterial tone and tissue perfusion.Item Open Access Cerebrovascular and Ventilatory Function in Chronic Obstructive Pulmonary Disease(2015-02-03) Hartmann, Sara E.; Poulin, MarcChronic Obstructive Pulmonary Disease (COPD) is a disease primarily affecting the pulmonary system, most commonly resulting from prolonged exposure to cigarette smoke. While systemic respiratory disturbances in COPD are apparent, the chemical regulation within the brainstem and at the carotid bodies is unclear. Furthermore, the role that cerebral blood flow contributes to respiratory disturbances is poorly understood. Recent studies have demonstrated peripheral vascular impairments in COPD, which may have further implications for exercise –hyperemia. Oxidative stress serves as a plausible mechanism leading to vascular impairments, and the overall increased risk of stroke and cardiovascular disease. This collection of studies set out to define the ventilatory and cerebrovascular responses to acute alterations in PCO2 and PO2 in COPD patients, and the relationship to oxidative stress. A secondary focus was to characterize both cerebral and peripheral blood flow during exercise. The thesis begins by investigating the cerebrovascular and ventilatory responses to acute euoxic-hypercapnia in mild-moderate COPD patients, and the relationship between these physiological parameters, and markers systemic of oxidative stress. Second, the cerebrovascular responses during moderate cycling exercise are defined. The final three studies collectively investigate the effect of the antioxidant, vitamin C, on the cerebrovascular and ventilatory responses to acute hyperoxic-hypercapnia, isocapnic-hypoxia, and on forearm blood flow during handgrip exercise, in COPD patients and healthy controls. This thesis demonstrates that the cerebrovascular and ventilatory responses to hypercapnia, but not hypoxia, are impaired in COPD. Vitamin C was found to augment the ventilatory response to hyperoxic-hypercapnia suggesting oxidative stress contributes to the overall ventilatory limitations in COPD. Dynamic cycling exercise (at a matched relative intensity), evoked a similar cerebrovascular response between COPD and controls, however, a modest increase in workload increased cerebral blood flow in COPD to levels greater than controls, reducing the cerebrovascular reserve capacity. Lastly, forearm blood flow in COPD patients during exercise was similar to controls, and was not affected by vitamin C. Overall, this series of experiments provides a better understanding of the complex systemic consequences of COPD in an integrated nature, thereby advancing knowledge in this important area of study.Item Open Access Electrocardiographic Parameters in the Assessment of Myocardial Fibrosis and Left Ventricular Systolic Function(2016) Narous, Mariam; Exner, Derek V.; Anderson, Todd J.; Fine, Nowell M.; White, James A.Assessment of cardiac structure and function is central to the care of patients with heart disease. Cardiac magnetic resonance (CMR) is the gold standard for such assessment, however it is expensive and oftentimes not readily accessible. We sought to evaluate the utility of electrocardiographic (ECG) and impedance-based parameters in estimating the amount of myocardial scar, left ventricular (LV) systolic function and myocardial deformation. Consecutive patients (n = 241; 42% female; mean age 55 years) undergoing clinical CMR and ECG assessments were recruited. ECG analysis was performed manually, using both the Modified Selvester Score (MSS) and the presence of fractionated QRS (fQRS) signals, and impedance testing using the Non-Invasive Cardiac System (NICaS). While MCS was of value, neither fQRS nor NICaS meaningfully predicted scar extent, LV systolic function. Or the amount of myocardial deformation. These results support additional investigation of the utility of the MSS in estimating cardiac structure and function among patients in whom cardiac imaging is clinically indicated.Item Open Access Expression and Role of Proteoglycan 4/Lubricin in the Human Pericardium: Implications as a Therapeutic to Prevent Post-Operative Pericardial Adhesions(2017) Park, Daniel; Fedak, PaulRetrosternal pericardial adhesions form post-cardiac surgery and are associated with greater mortality risks for patients undergoing re-sternotomies. Fibrous adhesion development is mediated by cardiac myofibroblasts. Expression of proteoglycan 4 (PRG4) in the human pericardial anatomy and the biological functions of recombinant human PRG4 on human cardiac myofibroblasts was investigated. Expression of PRG4 in the human pericardial fluid and localized expression of PRG4 by pericardial mesothelial cells lining the pericardial cavity was observed. Recombinant human PRG4 prevented human cardiac myofibroblast adhesion to coated surfaces, attenuated transforming growth factor-β1 mediated collagen matrix gel contraction, decreased collagen fiber alignment by activated cardiac myofibroblasts and increased cell roundness. Replacing the lost endogenous PRG4 found in pericardial fluid with recombinant human PRG4 could attenuate the formation of post-operative pericardial adhesions and increase the safety and feasibility of re-sternotomies.Item Open Access Facilitating Access and Adherence to Exercise in People with Type 2 Diabetes(2015-02-03) Armstrong, Marni; Sigal, RonaldExercise is a key component in the management of type 2 diabetes. It improves blood glucose control and quality of life, and reduces cardiovascular events and mortality. Despite the strong evidence demonstrating protective effects of exercise in type 2 diabetes, much of this population remains inactive, and many of those who begin an exercise program do not continue long-term. Finding strategies to support the adoption and long-term adherence to exercise is a valuable endeavor. The aim of this thesis was to examine the effectiveness of motivational interviewing, a potential facilitator to behaviour change and maintenance, together with an examination of pre-exercise stress testing which has been identified as a plausible barrier to supervised exercise programs. This was accomplished through a variety of methods within three distinct studies. These included a systematic review and meta-analysis of motivational interviewing in weight loss interventions, a randomized controlled trial on the effectiveness of motivational interviewing-based counselling to promote maintenance of exercise after the completion of a supervised exercise program, and finally a data linkage study examining the utility of pre-exercise stress testing within a retrospective cohort from a real-world supervised exercise program. The results of the first two studies gave support to use of motivational interviewing as a strategy to improve the effectiveness of weight loss interventions and as an approach to help promote the maintenance of physical activity after a supervised exercise program. In the examination of the use of pre-exercise stress testing, we discovered that the rate of cardiovascular outcomes was low, and that in the few cases where referral for pre-exercise stress testing resulted in a change in care, the patients had multiple cardiac risk factors. These results suggest that improved pre-exercise risk stratification might help identify the small subset of patients with type 2 diabetes likely to benefit from pre-exercise stress testing, thereby streamlining the process prior to the initiation of a supervised exercise program. Within supervised exercise programs, implementing strategies that employ effective facilitators, as well as reduce barriers such as unnecessary testing, may help improve participation and long-term adherence to exercise in people with type 2 diabetes.Item Open Access Human Rhinovirus Infection of Airway Epithelial Cells Modulates Airway Smooth Muscle Migration(2015-12-22) Shariff, Sami; Leigh, Richard; Proud, David; Kubes, Paul; Giembycz, MarkThe traditional paradigm of airway remodeling in asthma has held that remodeling occurs after many years of chronic inflammation. However, studies have confirmed that remodeling changes are observed in children even before the clinical diagnosis of asthma is established. There is now robust evidence to indicate that children with recurrent human rhinovirus (HRV)-induced wheezing episodes are at significantly increased risk of developing subsequent asthma. A feature of airway remodeling is increased airway smooth muscle (ASM) mass with a greater proximity of the ASM to the subepithelial region, and we interrogated the hypothesis that HRV-induced alterations of airway epithelial cell biology might regulate ASM migration. We demonstrated that ASM chemotaxis is GPCR dependent, can be regulated by cAMP and is dependent upon CCL5 release by the epithelium post-HRV infection. These observations substantiate the growing body of evidence that links HRV infections to the subsequent development of asthma.Item Open Access Identification of Novel Mechanisms for Myogenic Control of Cerebral Arterial Diameter(2013-04-29) Zhong, Xi; Cole, WilliamMyogenic control of cerebral arterial diameter plays a fundamental role in the maintenance of normal vascular resistance and blood flow in the brain. Myogenic control of cerebral artery diameter is achieved by inherent pressure-dependent mechanisms of Ca2+-CaM-MLCK activation, ROK-mediated Ca2+ sensitization and cytoskeleton reorganization. The findings presented in this thesis identify novel elements in these molecular mechanisms, including: (1) Kv9.3 subunits that co-assemble with Kv2.1 subunits to form ScTx1-sensitive channels that regulate Em, (2) Kv7.4-containing channels that also regulate Em, and (3) a cytoskeleton protein, VASP, that is involved in a dynamic process of actin polymerization in response to pressure elevation. Both Kv2.1/9.3 channels and Kv7.4-containing channels of cerebral myocytes were shown to contribute to native Kv currents in myogenic control of cerebral arterial diameter. Regulation of VASP phosphorylation in the process of cytoskeleton reorganization was shown to participate in the pressure-dependent myogenic response and NO-mediated vasodilation of cerebral arteries. In summary, these findings provide novel understanding of the basic molecular mechanisms that contribute to the precise control of vascular smooth muscle contractility, and provide potentially important insights for identification of dysfunctional mechanisms leading to abnormal arterial regulation in pathological conditions, as well as the development of therapeutic strategies to treat arterial dysfunction.Item Open Access Impact of Intermittent Hypoxia on Human Cardiorespiratory and Cerebrovascular Function(2016) Beaudin, Andrew Edward; Poulin, Marc J.; Hanly, Patrick J.; Wilson, Richard J. A.; Anderson, Todd J.; Thompson, Roger J.; Horner, Richard L.Obstructive sleep apnoea (OSA) is a chronic sleep disorder characterized by intermittent hypoxia (IH) exposure during sleep and is an independent risk factor for cardiovascular and cerebrovascular disease. IH in untreated OSA is advanced as the principal pathway leading to the greater risk of vascular disease associated with OSA. Additionally, IH is implicated in the propagation of OSA severity by increasing ventilatory instability, in part, by enhancing ventilatory chemosensitivity. Therefore, the focus of this thesis was to investigate the mechanisms through which IH functions and the role of IH in disrupting vascular and ventilatory regulation in OSA. The molecular pathways through which IH disrupts vascular and ventilatory regulation are poorly understood, but IH-induced inflammation is believed to be a primary contributor. Using a human experimental model of IH during wakefulness and a clinical population of untreated OSA patients, Study 1 investigated the role of cyclooxygenase (COX)-1 and COX-2 derived prostanoids (mediators of the inflammatory response and vascular regulation) in IH-induced alterations in cardiovascular and cerebrovascular regulation. Additionally, Study 2 examined the role of inflammation in IH-induced respiratory plasticity. Study 3 investigated the effects of nocturnal oxygen therapy (to remove IH) and continuous positive airway pressure (CPAP; gold standard OSA treatment) on cardiorespiratory and cerebrovascular responses to hypoxia in newly diagnosed OSA patients. Finally, Study 4 assessed the feasibility of adapting our human IH model to sleep while incorporating the ability to assess cardiovascular and cerebrovascular responses to hypoxia and hypercapnia during sleep. Studies 1-3 add substantial knowledge to this important area of research. Specifically, they reveal that 1) cyclooxygenase (COX)-1 and COX-2 differentially regulate blood pressure and cerebrovascular responses to acute and chronic IH; 2) inflammation does not contribute to IH-induced respiratory plasticity following an acute (6h) IH exposure; and 3) both nocturnal oxygen and CPAP treatment of OSA may lower blood pressure during isocapnic-euoxia and the hypoxic ventilatory response, but neither modality effects vascular responses to hypoxia. Lastly, Study 4 showed it is feasible to apply our human IH model to sleep and to concurrently assess vascular responses to hypoxia and hypercapnia during sleep.Item Open Access Interaction of Silica Nanoparticles with Primary Alveolar Epithelial Cells(2015-09-01) Bogari, Nawaf N.; Amrein, MatthiasThe fate of nanoparticles reaching the alveolar lung is not completely understood. Clearance of these particles has been ascribed to the alveolar macrophages scavenging this region. In contrast, in my thesis, I show in vitro, that particles interact more strongly with the alveolar epithelial cells (AECs) than alveolar macrophages (AMs), are then taken up efficiently and transported across the cells and released on the basolateral side. First, I developed a protocol for obtaining a pure culture of rat AEC. Single cell force spectroscopy showed the AEC to respond strongly and in a clathrin-independent manner to the particles, unlike a host of control cells, including alveolar macrophages. Fluorescence light microscopy and total internal reflection fluorescence light microscopy (TIRF) demonstrated the transport to be novel, actin-dependent and microtubule independent. In summary, my thesis provides evidence of a second clearance mechanism in addition to AMs.Item Open Access Investigation into the Bronchoprotective and Anti-inflammatory Properties of RGS2(2016) George, Tresa; Newton, Robert; Giembycz, Mark; Kelly, MargaretThe clinical management of asthma involves treatment with inhaled corticosteroids (ICS) and long-acting β2-adrenoceptor agonists (LABAs). Regulator of G-protein signalling (RGS) 2 inhibits signalling from Gq protein-coupled receptors. In humans, combinations of a glucocorticoid and a LABA synergistically enhanced the expression of RGS2. In mice, 3 weeks of HDM challenge or 30 min of LPS challenge reduced lung function and induced airways inflammation. Compared to wild-type, Rgs2-/- mice showed significantly increased airways resistance and reduced compliance, in both HDM- and LPS-challenges. There was no difference between wild-type and Rgs2-/- mice in HDM-induced and LPS-induced inflammation. There was a trend towards increased inflammatory cell counts in the BALF of Rgs2-/- compared to wild-type. There was a trend towards increased expression for many HDM-induced cytokines/chemokines in Rgs2-/-. There was no difference between wild-type and Rgs2-/- mice in LPS-induced expression of cytokines/chemokines. These data show Rgs2 is bronchoprotective in HDM-induced inflammation and suggest a modest anti-inflammatory role. Rgs2 is bronchoprotective in LPS-induced inflammation, but more studies are required to address a possible anti-inflammatory role. If applicable to humans, these data suggest that therapeutics, for example ICS/LABA combination therapies, which are designed to maximize RGS2 expression, will be beneficial for asthma control and management.Item Open Access Investigation of Depolarization as the Trigger for Lubricin and Hyaluronan Secretion from Non-Excitable Human Synovial Fibroblasts(2017) Narsingani, Karim; Giles, Wayne; Schmidt, Tannin; Benseler, Susanne; Braun, AndrewHyaluronan (HA) and Lubricin are major components of synovial fluid that are produced by synovial fibroblasts. Their secretion pathway remains unclear. However, calcium has been shown to play a major role. Depolarization may increase intracellular calcium. This study hypothesized that depolarization may lead to an increase in HA and Lubricin. Human synovial fibroblasts (HSF) from two donors were depolarized with 100mM extracellular K+. The media was analyzed for HA and Lubricin using and HA ELISA and AlphaLISA, respectively. Calcium fluorescence determined intracellular calcium concentration ([Ca2+]i). A generalized estimating equation (GEE) was used for statistical analysis. In the high K+ group HA concentration decreased (p=0.000); however, the Lubricin concentration did not change (p=1.000) and [Ca2+]i decreased. Depolarization with 100mM K+ for 12h did not increase [Ca2+]i, Lubricin levels and decreased HA levels in the HSF conditioned media. Results may indicate K+ is not the ion responsible for the resting membrane potential.Item Open Access Modulation Of Fibroblast Function By Human Rhinovirus Infected Airway Epithelium(2015-12-23) Shelfoon, Christopher; Proud, David; Leigh, Richard; Kelly, Margaret; Patel, KamalaThickening of the lamina reticularis – a feature of airway remodeling in asthma – can be found in the airways of young children with wheeze. In association, wheezing illness due to human rhinovirus infection is an important risk factor for asthma development. We hypothesize that rhinovirus infection may be involved in the pathogenesis of lamina reticularis thickening and, by extension, airway remodeling. We show that upon infection with human rhinovirus the airway epithelium releases chemoattractants that are able to recruit human bronchial fibroblasts – the cell associated with lamina reticularis thickening. CXCL10 and CXCL8 were revealed as key players in this response, broadening their role in asthma pathogenesis. Additionally, fibroblast migration was experimentally abrogated using formoterol and other cAMP elevating agents. Taken together, the work in this thesis supports the hypothesis that rhinovirus infection is involved in the pathogenesis of airway remodeling.Item Open Access Modulation of glucocorticoid-inducible gene expression: Effects of inflammatory stimuli and long-acting β2-adrenoceptor agonists(2014-09-15) Rider, Christopher Francis; Newton, Robert; Giembycz, Mark AnthonyGlucocorticoids, acting on the glucocorticoid receptor (GR; NR3C1), are the most effective medication for controlling inflammation in the majority of asthmatics. However, some asthmatics, particularly those with severe disease, have reduced responses to glucocorticoids, a condition known as glucocorticoid resistance. Likewise, glucocorticoids have reduced effectiveness in asthmatics who smoke and during exacerbations, which are frequently induced by human rhinovirus (HRV) infection. Glucocorticoids reduce inflammatory protein production by directly inhibiting inflammatory transcription factors, including nuclear factor-kappa B (NF-κB) and by enhancing the expression of potentially anti-inflammatory genes (transactivation). The importance of transactivation in glucocorticoid activity is increasingly apparent, but the impact of inflammatory mediators on anti-inflammatory gene expression is understudied. Human bronchial epithelial, BEAS-2B, cells stably transfected with a 2×glucocorticoid response element (GRE) reporter system, which models glucocorticoid-inducible gene expression, demonstrate concentration-dependent activation by glucocorticoids. However, dexamethasone-induced 2×GRE activation was time-dependently reduced by pre-treatment with inflammatory mediators, including tumor necrosis factor (TNF), interleukin-1β (IL1B) and cigarette smoke extract (CSE). Furthermore, TNF pre-treatment decreased dexamethasone-induced mRNA expression of genes with potentially anti-inflammatory activity, including cyclin dependent kinase inhibitor 1C (CDKN1C) and TSC22 domain family protein 3 (TSC22D3/GILZ), in bronchial epithelial and airway smooth muscle cells. Likewise, pre-incubations with HRV or the synthetic double-stranded viral RNA mimetic polyinosinic:polycytidylic acid (poly(I:C)) reduced dexamethasone-induced 2×GRE activation. Poly(I:C) also reduced dexamethasone-induced CDKN1C expression. Approaches to reverse TNF-induced glucocorticoid hyporesponsiveness were evaluated, including addition of long-acting β2-adrenoceptor agonists (LABAs), use of novel GR agonists and inflammatory signalling pathway inhibition. LABAs, such as formoterol, potentiated 2×GRE reporter activation and CDKN1C expression through a time- and PKA-dependent mechanism that did not enhance GR expression, agonist affinity or translocation, but instead allows for gene specific control. Therefore, LABA addition functionally reversed glucocorticoid hyporesponsiveness induced by TNF or poly(I:C). However, novel GR agonist-induced 2×GRE activation was repressed by TNF or poly(I:C) treatment, with the degree of repression correlating with agonist efficacy. Finally, inhibition of the NF-κB and c-Jun N-terminal kinase (JNK) mitogen activated protein kinase pathways partially reversed TNF-induced glucocorticoid hyporesponsiveness. These results may contribute to the development of improved treatments for combating glucocorticoid hyporesponsiveness during exacerbations and in severe asthma.Item Open Access Molecular Basis and Regulation of Ca2+ Release Termination and its Role in Cardiomyopathies(2017) Liu, Yingjie; Chen, S. R. Wayne; Braun, Andrew P.; French, Robert JohnIt is known that sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle is initiated via cardiac ryanodine receptor (RyR2) through a mechanism called Ca2+-induced Ca2+ release. However, how the SR Ca2+ release is terminated is undetermined. The objective of the current study is to understand the molecular basis and regulation of RyR2-mediated Ca2+ release termination and its role in the pathogenesis of cardiac diseases. Based on recent 3D structural analyses, the NH2-terminal region of RyR2 interacts with the channel domain via the central domain and undergoes dynamic conformational changes during channel gating. It has also been discovered that the NH2-terminal region consists of three distinct domains. HEK293 cell studies on domain deletions and disease mutations demonstrate that the different domains play different roles in RyR2 function. The NH2-terminal region is a major determinant of Ca2+ release activation and termination. Enhanced luminal Ca2+ activation of RyR2 has been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). However, in addition to CPVT, many RyR2 mutations can also cause cardiomyopathies. Knock-in mouse models harboring cardiomyopathy- associated RyR2 mutations have been generated to investigate the causal mechanisms of cardiomyopathies. The exon-3 deletion mouse model exhibited markedly reduced RyR2 expression level and no characteristic phenotype. The RyR2-R420W mouse model showed enhanced susceptibility to CPVT and altered cytosolic Ca2+ transient properties, suggesting that the abnormal cytosolic Ca2+ transient may be a key factor in the pathogenesis of cardiomyopathies. Calmodulin (CaM) is a regulatory protein that binds and inhibits RyR2 in the presence of cytosolic Ca2+. The inhibitory effect depends on the affinity of CaM for Ca2+ and RyR2. CaM mutations affecting either aspect may result in aberrant regulation of RyR2 activity, hence abnormal Ca2+ release termination. Indeed, arrhythmogenic mutations and most newly discovered CaM variants delayed Ca2+ release termination while others enhanced it, indicating that CaM is a major modulator of RyR2-mediated Ca2+ release termination. Overall, Ca2+ release termination is an intrinsic property of RyR2 that can be regulated by modulators such as CaM. Altered Ca2+ release termination is critically involved in the pathogenesis of cardiac diseases.Item Open Access Molecular Mechanism of Calcium Release Activation and Termination(2013-04-15) Tian, Xixi; Chen, S. R. WayneIt is now well established that sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle is triggered via a mechanism termed Ca2+-induced Ca2+ release (CICR). It is unknown, however, how the SR Ca2+ release is terminated. The overall objective of the current study is to understand the mechanisms of activation and termination of SR Ca2+ release mediated by the cardiac ryanodine receptor (RyR2) and their roles in the pathogenesis of cardiac disease. A simple HEK293 cell expression system was established to assess the impact of RyR2 mutations on both Ca2+ release activation and termination. We found that mutations in the pore-forming region of RyR2 affected either the activation or the termination of Ca2+ release or both, indicating that the pore-forming region is a major determinant of Ca2+ release termination. Two additional regions, the N-terminal region and the calmodulin binding domain (CaMBD) of RyR2, were also found to be important for the termination of Ca2+ release. These results demonstrate that RyR2 itself controls the termination of Ca2+ release. RyR2 and its numerous modulators together form a macromolecular complex. Whether these modulators regulate the activation or termination of Ca2+ release is largely unknown. Our results show that both the 12.6 kDa FK506 binding protein (FKBP12.6) and calmodulin (CaM) facilitate the termination of Ca2+ release, but have little effect on Ca2+ release activation. On the other hand, cytosolic Ca2+ affects both the activation and termination of Ca2+ release, whereas CaM-dependent protein kinase II (CaMKII) only alters the activation of Ca2+ release. These data indicate that Ca2+ release termination is a common target of RyR2 regulation. RyR2 modulators are believed to exert their impact on channel function by inducing conformational changes in the channel, but these ligand-induced conformational changes and their functional correlation have yet to be demonstrated. Using a novel fluorescence resonance energy transfer (FRET)-based conformational probe, we assessed conformational changes in the “clamp” region near the corners of the square-shaped three-dimensional structure of RyR2 upon activation by a number of ligands. Our data demonstrate that conformational changes in the clamp region of RyR2 are ligand dependent, and suggest that RyR2 possesses multiple ligand-dependent gating mechanisms associated with distinct conformational changes. Enhanced luminal Ca2+ activation has been recognized as a common defect of RyR2 mutations linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). However, why some CPVT mutations are also associated with cardiomyopathies is unknown. Single cell luminal Ca2+ imaging revealed that RyR2 mutations that are associated with dilated cardiomyopathy (DCM) or arrhythmogenic right ventricular dysplasia type 2 (ARVD2) markedly reduced the threshold for Ca2+ release termination and increased the fractional Ca2+ release. In contrast, a RyR2 mutation associated with hypertrophic cardiomyopathy (HCM) increased the threshold for Ca2+ release termination and reduced the fractional Ca2+ release. These results provide the first evidence that abnormal fractional Ca2+ release attributable to aberrant termination of Ca2+ release is a common defect in RyR2-associated cardiomyopathies. Overall, these findings provide novel and important insights into the molecular basis and regulation of Ca2+ release activation and termination, and their roles in the genesis of cardiac arrhythmias and cardiomyopathies.
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