Caveolin-1 is a marker protein for caveolae and can be a regulator of intracellular signaling pathways that contribute to the pathogenesis of human diseases. In the present study, the structural and functional changes of the lung in caveolin-1 null mice (Cav-1-/-) were assessed. Respiratory mechanics, measured using a small animal ventilator, revealed heightened central airway resistance (Rn), tissue resistance (G) and tissue elastance (H) in response to inhaled methacholine. The respiratory hyperreactivity is associated with increased collagen deposition around central and peripheral airways in Cav-1-/- mice; however, no difference was found in smooth muscle α-actin quantity between mouse strains. Similar to our in vivo findings, tracheal rings from Cav-1-/- mice mounted on an isometric wire myograph exhibited enhanced maximum active contractile force without a change in sensitivity (EC50) to methacholine. Rho kinase (ROCK1/2), protein kinase C (PKC) and extracellular signal regulated kinase 1/2 (ERK1/2) signaling were assessed as possible sources of the enhanced airway reactivity observed in Cav-1-/- mice. Inhibition of Rho kinase markedly blunted in vivo lung function responses (Rn) and (G) and ex vivo smooth muscle responses to methacholine. In fact, inhibition of Rho kinase completely eliminated any difference in response between mouse strains. Thus, our data indicate that Cav-1 may regulate mechanisms, such as Rho/Rho kinase signaling, that determine airway smooth muscle contraction and airway fibrosis; thus, it could be an important regulator of airway biology and physiology in health and disease.

Caveolin-1 is a marker protein for caveolae and can be a regulator of intracellular signaling pathways that contribute to the pathogenesis of human diseases. In the present study, the structural and functional changes of the lung in caveolin-1 null mice (Cav-1-/-) were assessed. Respiratory mechanics, measured using a small animal ventilator, revealed heightened central airway resistance (Rn), tissue resistance (G) and tissue elastance (H) in response to inhaled methacholine. The respiratory hyperreactivity is associated with increased collagen deposition around central and peripheral airways in Cav-1-/- mice; however, no difference was found in smooth muscle α-actin quantity between mouse strains. Similar to our in vivo findings, tracheal rings from Cav-1-/- mice mounted on an isometric wire myograph exhibited enhanced maximum active contractile force without a change in sensitivity (EC50) to methacholine. Rho kinase (ROCK1/2), protein kinase C (PKC) and extracellular signal regulated kinase 1/2 (ERK1/2) signaling were assessed as possible sources of the enhanced airway reactivity observed in Cav-1-/- mice. Inhibition of Rho kinase markedly blunted in vivo lung function responses (Rn) and (G) and ex vivo smooth muscle responses to methacholine. In fact, inhibition of Rho kinase completely eliminated any difference in response between mouse strains. Thus, our data indicate that Cav-1 may regulate mechanisms, such as Rho/Rho kinase signaling, that determine airway smooth muscle contraction and airway fibrosis; thus, it could be an important regulator of airway biology and physiology in health and disease.

No description available.

Aberrant phenotypes of airway smooth muscle cells are central to the pathophysiology of asthma. The hypercontractile nature of these cells and hypertrophy are the key reasons for the excessive narrowing of the airways observed in allergic asthma. Although previous studies have indicated a role of enhanced content of smMLCK in modulating the contractile reactivity, as well as an indication of hypertrophy of HASM cells in asthmatic conditions, the effect of IgE on the expression of smMLCK in HASM cells is not fully understood. In this study, we demonstrate that IgE augments the expression of smMLCK at the mRNA and protein level. Inhibition of IgE binding with anti-FcεRI blocking antibody, Syk silencing, pharmacological inhibitors to MAPK (ERK1/2, p38, and JNK) and PI3K significantly diminished the IgE-mediated smMLCK expression in HASM cells. Finally, we found that IgE, similar to metacholine induces the contraction of HASM cells grown on collagen gel matrix. Our data suggest that IgE stimulates the phosphorylation of ERK, P38, STAT3 and induces the dephosphorylation of smMLCK to phosphorylate myosin regulatory light chain in HASM cells. Taken together, our data suggest a modulatory role of IgE in regulating the contractile machinery and hypertrophic phenotype of HASM cells.

Aberrant phenotypes of airway smooth muscle cells are central to the pathophysiology of asthma. The hypercontractile nature of these cells and hypertrophy are the key reasons for the excessive narrowing of the airways observed in allergic asthma. Although previous studies have indicated a role of enhanced content of smMLCK in modulating the contractile reactivity, as well as an indication of hypertrophy of HASM cells in asthmatic conditions, the effect of IgE on the expression of smMLCK in HASM cells is not fully understood. In this study, we demonstrate that IgE augments the expression of smMLCK at the mRNA and protein level. Inhibition of IgE binding with anti-FcεRI blocking antibody, Syk silencing, pharmacological inhibitors to MAPK (ERK1/2, p38, and JNK) and PI3K significantly diminished the IgE-mediated smMLCK expression in HASM cells. Finally, we found that IgE, similar to metacholine induces the contraction of HASM cells grown on collagen gel matrix. Our data suggest that IgE stimulates the phosphorylation of ERK, P38, STAT3 and induces the dephosphorylation of smMLCK to phosphorylate myosin regulatory light chain in HASM cells. Taken together, our data suggest a modulatory role of IgE in regulating the contractile machinery and hypertrophic phenotype of HASM cells.

<p><strong>Introduction: </strong>Obesity is associated with asthma and airway hyperresponsiveness, though the mechanisms behind this relationship remain unclear. It is unlikely to be due to a direct effect of leptin on human airway smooth muscle cells (ASMC) (Nair, <em>et al.</em>, 2009). Since adipocytes are known to produce a wide array of mediators, we hypothesized that adipocytes may directly modulate human ASMC biology.</p> <p><strong>Objectives:</strong> To determine and compare the effects of intra and extrathoracic adipocyte secretions on ASMC proliferation, chemotaxis, contractility and cytokine synthesis.</p> <p><strong>Methods:</strong> Human ASMC and human adipocytes were cultured from primary samples (intrathoracic or extrathoracic). Adipocyte-conditioned media was used as a treatment in proliferation cell count assays, Transwell migrations, muscle bath experiments and to induce interleukin (IL)-6, tumor necrosis factor (TNF)-α and eotaxin production (as measured with a Bioplex). The effects of adipocyte-myocyte co-culture were also investigated on the proliferation, migration and cytokine synthesis of the ASMC.</p> <p><strong>Results: </strong>Adipocyte supernatants and co-culture did not significantly affect the growth of ASMC in the presence of 10% fetal calf serum. The adipocyte supernatants were not chemotactic, and did not affect the migration of ASMC towards platelet derived growth factor (PDGF). Similarly, co-culture did not have any effect on ASMC chemotaxis. Cytokine synthesis was also unchanged by adipocytes. Adipocyte supernatants did not have any effect on the contractile or relaxant responses of bovine tracheal smooth muscle strips. There was no significant difference between adipocyte depot location, with intrathoracic and extrathoracic adipocytes having a similar effect.</p> <p><strong>Conclusion:</strong> Human adipocytes do not directly modulate airway smooth muscle proliferation, migration, contractility and cytokine synthesis. These data point to some other cause for the association between obesity and asthma, though the role of other cells present in the adipose tissue of obese individuals cannot be ruled out.</p> / Master of Science (MSc)

Increased bulk of smooth muscle mass around the airways is a typical feature of asthma. Several mediators act in concert or antagonistically to regulate airway smooth muscle (ASM) cell proliferation. This thesis focuses on fibroblast growth factor (FGF)2 and transforming growth factor (TGF)[béta]1 which are known to be sequentially upregulated in the lung following allergic challenge and have recently been shown to synergize together in ASM cell proliferation. Emphasis is put toward the conflicting studies documenting the mitogenic effect of TGF[béta]1 in vitro and to its seemingly potent effect in vivo. Thereafter, different asthma mediators, such as IL-4 and IL-13, are introduced and how their mitogenic potential toward ASM cells could be altered by FGF2 is presented. Finally, how the controversial issue between in vitro and in vivo data regarding the mitogenic effect of leukotrienes could be reconciliated and how it could be related to FGF2 and TGF[béta]1 proliferative synergism is discussed.

Doctor of Philosophy (PhD) / It is well recognised that both genetic and environmental factors determine an individual’s predisposition to asthma. In recent years, the airway smooth muscle (ASM) cell has come to the attention of researchers to, not merely be a contractile cell of the airway, but one that orchestrates events affecting airway remodelling and proliferation. Experiments described in this thesis have, for the first time, examined genes that are associated with various aspects of the pathogenesis of asthma by using the candidate gene approach and a genome wide search. Genes have not only been identified to be differentially expressed in ASM cells derived from asthmatic and non-asthmatic participants, but have also been linked with a functional consequence of asthma. The three genes found to be differentially regulated between ASM cells derived from asthmatic and non-asthmatic participants were Peroxisome Proliferator-Activated Receptor- gamma (PPARγ), mimecan and fibulin-1. Expression of the anti-proliferative transcriptional factor PPARγ, found by the candidate gene approach, was elevated in ASM cells derived from asthmatic participants. Whilst elevated, the anti-proliferative effect of PPARγ was absent in ASM cells derived from asthmatic participants. By microarray analysis, mimecan, an anti-proliferative agent was identified. Mimecan levels, although not different basally in ASM cells, were upregulated by transforming growth factor β (TGFβ) only in asthmatic derived ASM cells. Silencing mimecan, by the use of specific oligonucleotides, increased proliferation of ASM cells. This suggested that by increasing mimecan expression, the proliferation of ASM cells may be halted. Fibulin-1, also found by microarray analysis and the final gene examined in this thesis, was found in elevated levels in BAL fluid, serum and ASM cells obtained from asthmatic participants. In addition, ASM cells derived from asthmatic participants, for the first time were shown to have faster wound healing rates compared with nonasthmatics. The elevated fibulin-1 levels in ASM cells derived from asthmatic participants, in the presence of TGFβ, were demonstrated to contribute to this increased wound healing. Specifically, fibulin-1 was found to affect wound healing by increasing proliferation rather than migration. The current available treatments for asthma, target the contractility and inflammatory conditions in the airway. Through this thesis, novel genes discovered to be associated with proliferation may be potential therapeutic targets to treat asthma. In particular, the fibulin-1 gene is outstandingly promising, as it was shown that silencing fibulin-1 resulted in slower wound healing rates through decreased cell proliferation, to possibly inhibit the airway remodelling observed in asthma, and furthermore, corticosteroid therapy was demonstrated not to affect to this gene.

The prevalence and economic burden of chronic airway disorders such as asthma is on the rise annually. Allergic asthma is characterized by chronic airway inflammation, airway hyper-responsiveness (AHR), and structural airway remodeling due to increased smooth muscle mass. Most allergic asthma occurs due to the overproduction of immunoglobulin E (IgE) antibodies against common allergens. Classically, IgE has been shown to modulate airway smooth muscle (ASM) contraction/relaxation which is believed to be the underlying cause of airway hyperreactivity. However, the molecular mechanisms underlying IgE effects on ASM cell are not established. Recently, the high-affinity Fc receptor for IgE (FcεRI) has been identified in human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthma patients. However, it is unknown whether FcεRI activation on ASM can modulate the immune response within the airways. We hypothesized that the IgE-FcεRI interaction plays a key role in inducing phenotypic and functional changes in ASM cells that eventually contributes to the establishment of airway inflammation, AHR, and remodeling. We sought to investigate the regulation, effector functions, and underlying mechanisms of FcεRI activation in ASM cells. Our work shows that the proinflammatory tumor necrosis factor (TNF) and T helper type 2 (Th2) cytokine interleukin (IL)-4 enhanced the FcεRI abundance and amplified the IgE-induced chemokine (eotaxin-1/CCL11, RANTES/CCL5, IL-8/CXCL8, and IP-10/CXCL10) release in ASM cells via transcriptional mechanisms. Both TNF and IgE induced a novel, Th2-favoring cytokine thymic stromal lymphopoietin (TSLP) through the activation of spleen tyrosine kinase (Syk), and nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). In addition, IgE induced de novo DNA synthesis and ASM cell proliferation via mitogen-activated protein kinases (MAPKs) and signal-transducer and activator of transcription 3 (STAT3) activation. Collectively, our data suggest that the IgE-induced FcεRI activation leads to the expression of multiple chemokines in ASM which may indirectly recruit inflammatory cells and promote allergic airway inflammation; IgE induces TSLP which can promote the Th2 immune responses within the airways; and IgE may potentially induce airway remodeling by directly inducing ASM cell proliferation. Therefore, targeting the IgE-FcεRI network on ASM may offer a novel therapeutic strategy in allergic asthma.

The prevalence and economic burden of chronic airway disorders such as asthma is on the rise annually. Allergic asthma is characterized by chronic airway inflammation, airway hyper-responsiveness (AHR), and structural airway remodeling due to increased smooth muscle mass. Most allergic asthma occurs due to the overproduction of immunoglobulin E (IgE) antibodies against common allergens. Classically, IgE has been shown to modulate airway smooth muscle (ASM) contraction/relaxation which is believed to be the underlying cause of airway hyperreactivity. However, the molecular mechanisms underlying IgE effects on ASM cell are not established. Recently, the high-affinity Fc receptor for IgE (FcεRI) has been identified in human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthma patients. However, it is unknown whether FcεRI activation on ASM can modulate the immune response within the airways. We hypothesized that the IgE-FcεRI interaction plays a key role in inducing phenotypic and functional changes in ASM cells that eventually contributes to the establishment of airway inflammation, AHR, and remodeling. We sought to investigate the regulation, effector functions, and underlying mechanisms of FcεRI activation in ASM cells. Our work shows that the proinflammatory tumor necrosis factor (TNF) and T helper type 2 (Th2) cytokine interleukin (IL)-4 enhanced the FcεRI abundance and amplified the IgE-induced chemokine (eotaxin-1/CCL11, RANTES/CCL5, IL-8/CXCL8, and IP-10/CXCL10) release in ASM cells via transcriptional mechanisms. Both TNF and IgE induced a novel, Th2-favoring cytokine thymic stromal lymphopoietin (TSLP) through the activation of spleen tyrosine kinase (Syk), and nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). In addition, IgE induced de novo DNA synthesis and ASM cell proliferation via mitogen-activated protein kinases (MAPKs) and signal-transducer and activator of transcription 3 (STAT3) activation. Collectively, our data suggest that the IgE-induced FcεRI activation leads to the expression of multiple chemokines in ASM which may indirectly recruit inflammatory cells and promote allergic airway inflammation; IgE induces TSLP which can promote the Th2 immune responses within the airways; and IgE may potentially induce airway remodeling by directly inducing ASM cell proliferation. Therefore, targeting the IgE-FcεRI network on ASM may offer a novel therapeutic strategy in allergic asthma.