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.

Asthma is a respiratory disease that affects 2.5-3 million Canadians. This condition is characterized by a Th2-driven immune response that implicates the infiltration of eosinophils and remodelling of the airways. In the last decade, airway smooth muscle cells (ASMC) have became the subject of intense research in the field of inflammatory lung diseases including asthma. It is known that ASMC respond to a wide variety of inflammatory mediators such as cytokines and chemokines. Function of ASMC in the context of asthma has extended beyond its traditional role of a structural cell. Indeed, it is believed that they can participate in the initiation and the perpetuation of the inflammatory response that takes place in the airway of asthmatic subjects. The general aim of this work was to investigate the role of ASMC in the pathogenesis of asthma. More specifically, we studied the expression of two C-C chemokine receptors, CCR3 and CCR1 in the context of asthma. / For the first time, this work describes the expression of chemokine receptors by ASMC. We have shown that eotaxin, an important chemokine in asthma, induces migration of ASMC through the activation of CCR3. Although CCR3 expression is not regulated by Th2 cytokines in vitro, ASMC isolated from asthmatic patients expressed intrinsically higher levels of the surface receptor when compared to controls. We also describe the expression of CCR1 by ASMC, a receptor involved in airway remodelling in an animal model of asthma. We reported the expression of CCR1 mRNA in biopsies obtained from mild, moderate and severe asthmatics and showed that mild group express the highest level of CCR1. We also confirmed that ASMC express the receptor in vivo and showed that stimulation of this receptor with its ligands induces intra-cellular calcium mobilization, which confirms its functionality. Regulation of CCR1 on ASMC was also assessed using proinflammatory, Th1 and Th2 cytokines. We found that TNF-alpha and to a lesser extent, IFN-gamma, upregulated CCR1 mRNA and protein expression, while Th2 cytokines had no effect. The effect of these two cytokines was totally suppressed by either dexamethasone or mithramycin. / Collectively, our results demonstrate the expression of functional C-C chemokine receptors by ASMC. Interestingly, we have shown that CCR3 activation mediates ASMC migration and provides a new possible mechanism for the increased smooth muscle mass observed in asthmatic patients. Although the exact function of the CCR1 expressed by ASMC is unknown, our results suggest an involvement in asthma pathogenesis, possibly through airway remodelling.

Collagens are abundant components of the extracellular matrix in the atherosclerotic plaque. In addition to contributing to lesion volume and mechanical stability, collagens can influence the behavior of macrophages and smooth muscle cells (SMCs) and have profound effects on both inflammation and fibrosis during lesion development. The aim of this thesis was to define a functional role for the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, in murine models of atherogenesis. In our first study, using Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice fed a high fat diet, we identified DDR1 as a novel positive regulator of atherogenesis. Targeted deletion of DDR1 attenuated atherosclerotic plaque development by limiting inflammation and accelerating matrix accumulation and resulted in the formation of macrophage poor, matrix rich lesions. In the second study, we used bone marrow transplantation to generate chimeric mice with a deficiency of DDR1 in bone marrow derived cells and reveal a central role for macrophage DDR1 in atherogenesis. Deficiency of DDR1 in bone marrow derived cells reduced lesion size by limiting macrophage accumulation in the developing plaque. Moreover using BrdU pulse labeling, we demonstrated reduced monocyte recruitment into the early fatty streak lesions of Ddr1-/-;Ldlr-/- mice. In our third study, we again utilized bone marrow transplantation to generate mice with deficiency of DDR1 in the host derived tissues such as the vessel wall and uncovered a distinct role for DDR1 expressed on resident vessel wall smooth muscle cells in the regulation of matrix accumulation and fibrous cap formation during atherogenesis. Deficiency of DDR1 in vessel wall cells resulted in robust accumulation of collagen and elastin and resulted in the formation of larger atherosclerotic plaques, with thick fibrous caps. Taken together, these studies support a critical role for DDR1 in the development of the atherosclerotic plaque. We demonstrate that DDR1 exerts distinct and opposing effects on lesion size by regulating both monocyte recruitment and matrix accumulation. These studies underscore the importance of collagen signaling during atherogenesis, and identify DDR1 as a key transducer; providing signals that regulate both inflammation and fibrosis during atherogenesis.

No description available.

An increase in coronary disease prevalence and mortality highlights the growing need for therapies to treat atherosclerotic vessels. While current bypass procedures utilize autologous vessels for small caliber grafts, there is a big push towards the use of engineered tissues to bypass diseased portions of arteries. Cardiovascular tissue engineering is the emerging discipline that aims to create a functional substitute. Ideally, a tissue engineered blood vessel would possess the relevant cells and matrix proteins that interact in a physiologic manner and will respond to the environmental cues of the host. A particular obstacle to achieving appropriate vessel structure is the inclusion of elastin in a tissue engineered media equivalent. Rat arterial smooth muscle cells that were retrovirally mediated to overexpress versican V3 have been shown to have an enhanced expression of tropoelastin in vitro as well as in injury models. The unique tropoelastin expression by these adult cells was studied in the context of tissue engineered media equivalents. Changes to the extracellular matrix architecture and composition, stimulation with medium additives, and cyclic distension, were shown to increase tropoelastin synthesis in V3 versican overexpressing cells. This study not only expanded the characterization of V3 versican overexpressing smooth muscle cells, it also explored the novel use of these cells as a tropoelastin source in a tissue engineered media equivalent.

Arterial remodeling is a process by which arteries respond to sustained changes in their mechanical environment. This process occurs in a way such that an artery's local mechanical environment (circumferential, shear, and axial stress) is maintained at a homeostatic level. However, most studies utilize a methodology that controls the global parameters (pressure, flow rate, and axial stretch). This approach often confounds the results since the actual drivers of remodeling are not independently isolated. This research involved developing a methodology and system capable of independently controlling each of the local parameters and examining the effect of axial stress on remodeling. An organ culture system capable of monitoring and controlling the three global parameters and calculating the cross sectional geometry was developed. This combination of hardware was incorporated into LabVIEW which afforded the user the ability to define desired values for the local mechanical parameters. Porcine common carotid arteries were cultured for seven days in this system under physiologically normal circumferential and shear stresses and a constant axial stress of either 150 kPa or 300 kPa. Material response, general arterial morphology, tissue viability, and collagen synthesis were examined in order to gauge the effectiveness of the organ culture system and assess any arterial remodeling. The results of this study demonstrate the ability of the organ culture system in achieving and maintaining target values of stress throughout the culture period. Cell viability, general arterial morphology, and collagen synthesis rates were maintained for all arteries. The elevated axial stress appeared to cause a softening of the artery in both the axial and circumferential direction. It was hypothesized that this softening was the result of a changing collagen structure. Additional softening seen in arteries was attributed to the effects of the culture system.

Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

The syndrome known as Gastric Dilation Air Sacculitis (GDAS) has recently been described by Lumsden et al. (2002) for Chinook salmon (Oncorhynchus tshawytscha, Walbaum), in seawater (SW) culture in New Zealand. The syndrome is characterised by distended abdomens, gastric dilation and air sacculitis, increased feed conversion ratios (FCR) and mortality. Consequently, financial returns on affected stocks are greatly reduced. A study into the epidemiology and physiology of the syndrome was initiated, working with the major aquaculture company, The New Zealand King Salmon Company (NZKS). The study revealed causative factors of GDAS. GDAS was experimentally induced only in saltwater by feeding a commercially manufactured low-cohesion pelleted diet. Control groups were fed a different diet with high physical cohesion. Low-cohesion pellets have previously been associated with a high incidence of GDAS in commercial sea cages. These data implicated osmoregulatory stress and physical properties of the feed in GDAS development. In addition, gastrointestinal (GI) physiology in GDAS -affected and -control fish was characterised. The process of GDAS development in O. tshawytscha is characterised by a loss of smooth muscle tone of the stomach as it distends. Laplace's law (P= 2T/r, where P is the distending pressure, T is the tension in the wall and r is the radius of the cylinder) predicts that unless muscle mass increases, the ability of the stomach wall to contract will be lost and consequently a loss of GI motor function will result. Therefore, GI circular smooth muscle integrity in terms of (1) stimulated and maximal contractility, (2) osmoregulatory ability of the intestine and the (3) control of the GI system was studied in pathologically affected (+ve) and unaffected (-ve) smolt. Affected fish showed changes in GI circular smooth muscle function and osmoregulatory dysfunction. Feeding different diets induced distinct gastric evacuation patterns. The intestinal brake hypothesis is presented and argued to be the probable mechanism for GDAS development. GDAS (+ve) serum showed the presence of factors capable of contracting gut smooth muscle. In addition, potential humoral mediators of the intestinal brake in fish were investigated.

The current study aims to investigate the relative contributions of each of the processes that govern airway smooth muscle mechanical behaviour. Studies have shown that breathing dynamics have a substantial effect on airway constriction in healthy and diseased subjects, yet little is known about the dynamic response of the main instigator of airway constriction, Airway Smooth Muscle (ASM). In this work several models are developed to further the understanding of ASM dynamics, particularly the roles and interactions of the three dominant processes in the muscle: contractile dynamics, length adaptation and passive dynamics. Three individual models have been developed, each describing a distinct process or structure within the muscle. The first is a contractile model which describes the contractile process and the influence of external excitation on contractile behaviour. The second model incorporates the contractile model to describe length adaptation, which includes the reorganisation and polymerisation of contractile elements in response to length changes. The third model describes the passive behaviour of the muscle, which entails the mechanical behaviour of all non-contractile components and processes. As little data on the passive dynamics of the muscle was available in the literature, a number of experiments were conducted to investigate relaxed ASM dynamics. The experimental data and mathematical modelling showed that passive dynamics plays not only a dominant role in relaxed ASM, but contributes considerably to the dynamics of contracted muscle as well. A novel theory of sequential multiplication in passive ASM is proposed and implemented in a mathematical model. Experiments and literature validated the model simulations. Further integration of the models and improved force control modelling of length adaptation is proposed for future study. It is likely that the coupling of the models presented here with models describing other airway wall components will provide a more complete picture of airway dynamics, which will be invaluable for understanding respiratory disease.