Increased airway smooth muscle (ASM) mass contributes to airway hyperresponsiveness (AHR) in asthma and is orchestrated by growth factors, cytokines and chemokines. Airway contractile responses are influenced by neuromediators, such as acetylcholine, and glutamate released by parasympathetic and sympathetic airway nerves. Hyperactivity of these neural elements, termed neurogenic inflammation, is linked with hypercontractility and AHR. Glutamate is a non-essential amino acid derivative, and its physiological role is traditionally considered with respect to its being the primary excitatory neurotransmitter in brain, and regulation of neuronal development and memory. In allergic inflammation, immune cells including dendritic cells, neutrophils and eosinophils, constitutively synthesize and release glutamate, which signals through activation of glutamate receptors, most important among which are ionotrophic N-methyl D-aspartate receptors (NMDA-R). We hypothesized that glutamatergic signaling mediated through NMDA-Rs plays an important role in inducing functional Ca2+ responses in human (H) ASM cells that can underpin airway hypercontractility. We investigated the expression and function of NMDA-Rs in HASM cells, and assessed the effects of pro-inflammatory cytokines on NMDA-R expression and functional responses. Moreover, we measured airway responses to NMDA in mice, murine thin cut lung slice preparations, and floating collagen gels seeded with HASMs. Our data reveal that airway myocytes express multi-subunit NMDA-R complexes that function as receptor-operated calcium channels (ROCCs), mobilizing intracellular Ca2+ in ASM in vitro and airway contraction ex vivo. Individual airway myocytes treated with NMDA-R agonist exhibit disparate temporal patterns of intercellular Ca2+ flux that can be partitioned into four discrete function sub-groups. Further we show that tumor necrosis factor (TNF) exposure modulates NMDA-R subunit expression, and these changes are associated with a shift in the distribution of myocytes in individual Ca2+-mobilization sub-groups in vitro. Further, post-TNF exposure, NMDA-R agonists’ treatment induced Ca2+-dependent airway dilation in murine lung slice preparations, an effect that was prevented by co-treatment with inhibitors of nitric oxide synthase (NOS) or cyclooxygenase (COX). Taken together, we conclude that NMDA-R regulate HASM-mediated airway contraction and their role can be affected upon exposure to asthma-associated inflammatory mediators. Thus, NMDA-Rs are of relevance to mechanisms that determine airway narrowing and AHR associated with chronic respiratory diseases. / October 2015

共著者あり。共著者名:Munakata Mitsuru, Sato Atsuko, Amishima Masaru, Homma Yukihiko, Kawakami Yoshikazu. / Hokkaido University (北海道大学) / 博士 / 医学

Morbidity and mortality attributable to asthma arise mainly from contraction of airway smooth muscle (ASM) and resulting bronchospasm. Bronchospasm that is induced in the laboratory is easily reversed by a spontaneous deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthma is not. In response to a spontaneous DI, contracted ASM fluidizes rapidly and then resolidifies slowly, but molecular mechanisms accounting for these salutary bronchodilatory responses -and their dramatic breakdown in asthma- are unknown. Using a multi-scale approach, I show here that both the baseline contractile force and the fluidization response of ASM are independent of the cytoskeletal protein zyxin, whereas the resolidification response is zyxin-dependent. At the levels of the stress fiber, the isolated cell, and the integrated airway, zyxin acts to stabilize the contractile apparatus and promote the resolidification response. More than just the motor of contraction, ASM is thus viewed in the broader context of a self-healing active material wherein resolidification and its molecular determinants contribute to the biology of bronchospasm.

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.

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.

Asthma is a chronic disorder highlighted by intermittent airway inflammation and characterized by paroxysmal dyspnea and airway hyperresponsiveness (AHR). A key feature of severe asthma is the development of airway wall remodeling, which is thought to occur through repeated rounds of inflammation and tissue repair. Remodeling includes structural changes such as increased mass of airway smooth muscle (ASM), and excessive collagen deposition. ASM cells contribute to airway remodeling via the expression and secretion of extracellular matrix (ECM) proteins. This is particularly driven by inflammatory processes, which include mediators such as transforming growth factor (TGF)-β1 and damage associated molecular pattern (DAMP) proteins, such as high mobility group box 1 (HMGB1). HMGB1 is ubiquitously expressed as a non-histone DNA-binding protein that can regulate gene expression, but can also be released in response to stress to underpin inflammation and tissue repair. In this study we tested the hypothesis that extracellular HMGB1 induces signaling pathways that control responses linked to progression of airway inflammation, remodeling and hyperresponsiveness in human ASM cells. We used primary cultured ASM cells as well as hTERT-immortalized human ASM cells. With immunoblotting we demonstrate that exogenous HMGB1 (10 ng/mL) can induce rapid and sustained phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) that is comparable to that induced by a potent mitogen, platelet derived growth factor (PDGF-BB, 10 ng/mL). We also found that TGF-β1 (2.5 ng/mL) promotes the accumulation of secreted HMGB1 in culture medium in a time line concomitant with expression of ECM proteins, collagen and fibronectin, suggesting a role for HMGB1 in pro-fibrotic effects of TGF-β1. By lentiviral delivery, we induced stable expression of short hairpin RNA (shRNA) that silenced expression of endogenous HMGB1 or mammalian diaphanous 1 (mDia1), a cytoplasmic scaffold protein that is required for HMGB1-induced cell responses through one of its receptors, receptor for advanced glycation end products (RAGE). Immunoblot analyses revealed that silencing of mDia1 was associated with markedly decreased induction of p42/p44 MAPK phosphorylation by exogenous HMGB1. In HMGB1-silenced human ASM cells, we observed significantly reduced synthesis and secretion of collagen A1 and fibronectin in response to TGF-β1 (2.5 ng/mL, 0-48 hrs). However, exogenous HMGB1 was not sufficient to rescue ECM synthesis in response to TGF-β1 in HMGB1-silenced cells - this suggests that intracellular, but not necessarily secreted HMGB1, regulates ECM expression and secretion in response to TGF-β1. Consistent with this interpretation, exogenous HMGB1 alone was not sufficient to induce ECM synthesis or secretion in primary cultured ASM cells. In conclusion, we show that though in human ASM cells extracellular HMGB1 alone can activate MAPK signaling, likely via mDia1-dependent pathways involving RAGE. it is not capable of prompting ECM protein expression. Recombinanat exogenous HMGB1 does not appear to directly affect ECM synthesis, rather intracellular (nuclear) HMGB1 likely modulates activity of genes that are affected by TGF-β1. Overall, HMGB1 has potential to regulate tissue repair processes involving ASM through intracellular and extracellular mechanisms, thus our findings support further work to elucidate the role of HMGB1 in pathogenesis of obstructive airway disease.

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.

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.

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.