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The implication of tone on airway responsiveness in

vivo in mice and on the contractile capacity of airway

smooth muscle

Mémoire

Audrey Lee-Gosselin

Maîtrise en médecine expérimentale Maître ès sciences (M.Sc.)

Québec, Canada

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Résumé

Comprendre et mieux définir la pathophysiologie de l’asthme est essentiel au développement de traitements plus efficaces. L’hyperréactivité bronchique et le tonus élevé du muscle lisse en-tourant les voies respiratoires sont deux caractéristiques majeures de l’asthme. Si une causalité existe entre ces caractéristiques, elle demeure encore inconnue. Le travail présenté dans ce Mé-moire décrit comment un tonus, induit par un spasmogène, affecte la réactivité bronchique à une bronchoprovocation in vivo chez la souris. La capacité contractile de trachées murines excisées a aussi été mesurée afin d’évaluer si la réponse obtenue in vivo implique le muscle lisse. Les résultats présentés dans ce mémoire démontrent qu’in vivo, les souris exposées à un tonus voient leur réactivité bronchique augmenter en réponse à une dose d’un spasmogène, comparées aux souris contrôles. Les résultats démontrent également que la réponse obtenue est causée, du moins en partie, par une augmentation de la capacité contractile du muscle lisse. Suite à ces résultats, les mécanismes moléculaires possiblement impliqués dans le gain de force induit par le tonus ont été investigués. L’hypothèse est que les voies de signalisation en aval de l’activation des récepteurs couplés aux protéines G sont responsables de l’augmentation de la capacité contractile du muscle lisse. L’inhibition de la polymérisation de l’actine, l’activa-tion de la chaîne légère de myosine, l’actival’activa-tion de protéines G et l’inhibil’activa-tion des protéines kinases activées par les mitogènes ont donc été évaluées. Les résultats démontrent qu’aucune des voies de signalisation étudiées est impliquée dans le gain de force du muscle lisse provoqué par un tonus induit par la présence continue d’un spasmogène. Ces résultats démontrent la complexité que représente la recherche des mécanismes moléculaires du gain de force et que cette recherche doit être plus approfondie.

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Abstract

To understand and better define the pathophysiology of asthma is essential for the development of more effective treatments. Airway hyperresponsiveness and an elevated airway smooth muscle tone are two common features of asthma. Whether causality exists between these two characteristics is unknown. The work presented in this Master’s thesis describes how a tone induced by a spasmogen affects airway responsiveness in vivo in mice to a spasmogenic challenge. The contractile capacity of excised murine tracheas was also measured to evaluate whether the obtained response in vivo involved airway smooth muscle. The results presented in this Master’s thesis demonstrate that mice exposed to tone in vivo have an increased response to a high dose of a spasmogen, compared to control mice. The results also show that this response is caused, at least partly, by an increase in airway smooth muscle contractile capacity. Following these results, molecular mechanisms possibly involved in the gain in force induced by tone were investigated. It was hypothesized that signaling pathways downstream of G protein-coupled receptors were responsible for the increase in airway smooth muscle contractile capacity. Therefore, the inhibition of actin polymerization, the activation of myosin light-chain, the activation of G proteins, and the inhibition of mitogen-activated protein kinases were evaluated to assess whether they mediate the gain in force induced by tone. The results show that none of the pathways studied were implicated in the gain in force induced by tone elicited by the continuous presence of a spasmogen. These latter results demonstrate that the mechanisms leading to a gain in airway smooth muscle force following an induced tone are complex and will require further investigation.

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Contents

Résumé iii

Abstract v

Contents vii

List of Tables xi

List of Figures xiii

List of Abbreviations xv

Remerciements xix

Foreword xxi

Introduction 1

Hypotheses and objectives . . . 2

Hypotheses . . . 3 Objectives . . . 3 1 Asthma 5 1.1 Definition of Asthma . . . 5 1.2 Risk Factors . . . 6 1.2.1 Host Factors . . . 6 1.2.2 Environmental Factors . . . 7 1.3 Physiopathology . . . 7 1.3.1 Airway Inflammation. . . 8 1.3.2 Airway Remodeling . . . 9 1.3.3 Airway Obstruction . . . 10

Airway Smooth Muscle Tone . . . 11

1.3.4 Airway Hyperresponsiveness. . . 12

2 Intracellular Pathways Governing Airway Smooth Muscle Contraction 15 2.1 Spasmogens . . . 16

2.1.1 Methacholine . . . 16

2.1.2 Thromboxane A2 . . . 17

2.1.3 Potassium Chloride. . . 19

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2.1.5 Sodium Fluoride . . . 19

2.2 Actin Polymerization . . . 20

2.3 RhoA/Rho-associated, Coiled Coil-containing Kinase Pathway . . . 21

2.4 Mitogen-Activated Protein Kinase Pathways . . . 21

2.4.1 p38 Signaling Pathway . . . 22

2.4.2 Extracellular Signal-Regulated Kinase Signaling Pathway . . . 23

2.4.3 c-Jun N-terminal Kinase Signaling Pathway . . . 24

3 The Gain of Smooth Muscle’s Contractile Capacity Induced by Tone on in vivo Airway Responsiveness in Mice 25 Résumé . . . 26

Abstract . . . 27

Introduction. . . 28

Material and Methods . . . 28

Animals . . . 28

In vivo Airway Responsiveness . . . 28

The Contractility of Excised Murine Tracheas . . . 29

Statistical Analyses . . . 31

Results . . . 31

In vivo Airway Reactivity . . . 31

The Contractility of Excised Murine Tracheas . . . 32

Discussion . . . 35

New Findings in Relation to Previous Results . . . 35

Rapid Change in ASM Contractile Capacity . . . 37

The Effect Seems Greater In Vivo than Ex Vivo . . . 38

Limitations . . . 39

Conclusion . . . 40

Grants . . . 40

Disclosures . . . 41

4 Investigation of Signaling Pathways Involved in the Gain in Force In-duced by Tone 43 4.1 Introduction. . . 43

4.2 Material and Methods . . . 44

4.2.1 Animals . . . 44

4.2.2 Spasmogens . . . 44

4.2.3 Inhibitors . . . 45

4.2.4 Western Blot Analyses . . . 45

4.2.5 Statistical analyses . . . 46

4.3 Results. . . 46

4.3.1 Spasmogens . . . 46

4.3.2 Inhibitors . . . 46

4.3.3 Western Blot Analyses . . . 50

4.4 Discussion . . . 51

4.5 Conclusion . . . 53

Conclusion 55 Perspectives . . . 56

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A Other Publications 59

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List of Tables

1.1 Asthma prevalence around the world. Data from Graham-Rowe (2011) [42]. . . 5

4.1 List of tested spasmogens . . . 44

4.2 List of tested inhibitors . . . 45

4.3 Percentage of increase (%) in ASM contractile capacity following an exposure

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List of Figures

1.1 Comparison of normal and asthmatic airways. Reproduced from The National

Heart, Lung, and Blood Institute [85]. . . 8

1.2 Pulmonary function testing using spirometry. Reproduced from Gildea and

McCarthy (2003) [40]. . . 10

1.3 Lung volumes and lung capacities. Reproduced from Gildea and McCarthy

(2003) [40]. . . 11

1.4 Bronchoprovocation tests with methacholine in asthmatic and normal subjects.

Reproduced from Woolcock (2015) [109]. . . 12

2.1 The terminal molecular stages of the signaling pathways leading to ASM

con-traction. Reproduced from Kuo et al. (2015) [67].. . . 16

2.2 Muscarinic receptor signaling pathways that regulate contraction in smooth

muscle. Reproduced from Gerthoffer (2005) [39]. . . 18

2.3 Signaling pathways downstream of RhoA/ROCK that regulate smooth muscle

contraction. Modified from SABiosciences; QIAGEN Company (2015) [27]. . . 22

2.4 Mitogen-activated protein kinase signaling pathways in mammalian cells.

Re-produced from Bennett (2006) [8]. . . 23

3.1 Dosing regimen for MCh delivery in control and experimental groups. . . 29

3.2 An elevated tone increased airway responsiveness in vivo in mice. . . 32

3.3 A tone increased the absolute contractile capacity of excised murine tracheas

regardless of the spasmogen used to elicit tone. . . 33

3.4 The kinetics of the gain in force induced by tone. . . 34

3.5 The reversibility of the gain in force induced by tone elicited by MCh and U46619. 35

4.1 Tone induced by SOV did not increase the contractile capacity of excised murine

tracheas.. . . 47

4.2 Tone induced by NaF did not increase the contractile capacity of excised murine

tracheas.. . . 47

4.3 Actin polymerization inhibitors latrunculin B and imatinib did not inhibit the

gain in force induced by tone. . . 48

4.4 ROCK inhibitor Y-27632 did not inhibit the gain in force induced by tone.. . . 49

4.5 MAPKs inhibitors, SB203580, U0126, and SP600125, did not inhibit the gain

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List of Abbreviations

ACh Acetylcholine

AHR Airway hyperresponsiveness Arp2/3 Actin-related protein 2 and 3 ASM Airway smooth muscle ADP Adenosine diphosphate ATP Adenosine triphosphate BIM I Bisindolylmaleimide I Ca2+ Calcium ions

CaCl2 Calcium chloride

CaMKII calmodulin-dependent protein kinase II cAMP 3’-5’-cyclic adenosine monophosphate

CCh Carbachol

DAG Diacyl-glycerol DI Deep inspiration DMSO Dimethyl sulfoxide DSP Dual-specific phosphatase

EC30 Concentration of a spasmogen that gives 30% of the maximal response EFS Electrical field stimulation

ERK Extracellular signal-regulated kinase

EtOH Ethanol

FEV1 Forced expiratory volume in 1 second

FRC Functional residual capacity FVC Forced vital capacity

GDP Guanosine diphosphate

GM-CSF Granulocyte macrophage colony stimulating factor GPCR G protein-coupled receptor

GTP Guanosine triphosphate

HBSMc Human bronchial smooth muscle cells HRV Human rhinovirus

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IFN Interferon

IL Interleukin

IP3 Inositol trisphosphate

IUCPQ Institut universitaire de cardiologie et de pneumologie de Québec JNK c-Jun N-terminal kinase

KCl Potassium chloride

Kh2PO4 Potassium dihydrogen phosphate

LIMK LIM-kinase

MAPK Mitogen-activated protein kinase

MAPKK MAPK kinase

MAPKKK MAPK kinase kinase

MCh Methacholine

MEK MAPK kinase

MgSO4 Magnesium sulfate

min Minute

MIP-1α Macrophage inflammatory protein MLC Myosin light-chain

MLCK Myosin light-chain kinase MLCP Myosin light-chain phosphatase mRNA Messenger ribonucleic acid

ms Milliseconde

MYPT1 Myosin phosphatase target subunit 1 NaCl Sodium chloride

NaF Sodium fluoride NaHCO3 Sodium bicarbonate

N-WASp neuronal Wiskott-Aldrich syndrome protein PAF Platelet activating factor

PDGF Platelet-derived growth factor PEEP Positive end-expiratory pressure

Pfn-1 Profilin 1

PGH2 Prostaglandin H2

PIP2 Phosphatidylinositol 4,5-bisphosphate

PKC Protein kinase C pMLC Phosphorylated MLC

PTP Protein tyrosine phosphatase

RANTES Regulated upon activation, normal T cell expressed and secreted ROCK Rho-associated, coiled coil-containing kinase

Rrs Respiratory system resistance RSV Respiratory syncytial virus

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RV Residual volume

s Second

SOV Sodium orthovanadate TGF Transforming growth factor TNF Tumor necrosis factor TP Thromboxane receptor TXA2 Thromboxane A2

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Remerciements

Pour commencer, je tiens à remercier sincèrement mon directeur de recherche Ynuk Bossé pour son encadrement, ses conseils et son soutient tout au long de ma maîtrise. Sa grande expertise dans le domaine, sa rigueur scientifique et son leadership ont grandement contribué au développement de mon intérêt grandissant pour la recherche scientifique et pour ce projet de maîtrise. Merci d’avoir cru en moi et de la belle opportunité qu’était de travailler dans son laboratoire.

Je voudrais également remercier les collaborateurs du projet, David Marsolais et Marie-Renée Blanchet, pour leur grande motivation et leur support dans ce projet de recherche.

Un merci spécial à Cyndi pour toute son aide et son écoute lors des longues journées passées au laboratoire. Merci également à David, Marie-Josée, Anick, Katherine, Mélissa et Anne-Marie pour leur support technique et leurs précieux conseils qui m’ont grandement aidé à avancer lors des moments plus difficiles.

Je profite de cette occasion pour remercier les organismes subventionnaires qui ont contribué au financement de ce projet de recherche, soit la Fondation de l’IUCPQ, le Réseau en Santé Respiratoire du Fonds de recherche du Québec - Santé, et Merck.

Je voudrais exprimer ma plus profonde gratitude à mes parents et à ma soeur pour leur amour inconditionnel, leur support constant et leur encouragement tout au long de mes études. Enfin, je voudrais remercier mon copain Jean-Christophe pour toute sa patience, son amour et ses encouragements afin que je puisse passer à travers cette étape. Merci également pour toute l’aide qu’il m’a apporté durant ces deux dernières années.

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Foreword

This Master’s thesis presents my work, over the last two years, on the effect of airway smooth muscle tone on airway responsiveness and on airway smooth muscle’s contractile capacity, and its putative involvement in asthma and airway hyperresponsiveness.

To begin with, theintroductionpresents general knowledge on the subject, and is followed by a more in-depth presentation inchapter 1. Theintroductionalso presents the hypotheses and objectives of the project. Chapter 2then describes the signaling pathways of smooth muscle contraction that have been studied within the framework of my project.

Chapter 3 focuses on an article entitled The gain of smooth muscle’s contractile capacity induced by tone on in vivo airway responsiveness in mice. I participated in the discussion leading to the design of the protocols and performed all the experiments and analyses for both the in vivo airway respiratory system resistance measurements and the ex vivo tracheal contractile measurements. I also prepared the figures and actively participated to the writing and editing of every stages of the manuscript. David Gendron helped me to understand the FlexiVent device as well as the data obtained from the software. He also participated to our discussions and contributed to the revisions of the paper along with Dr. Marie-Renée Blanchet, Dr. David Marsolais, and Dr. Ynuk Bossé. Dr. Bossé supervised my work throughout the entire process. This manuscript was published in the Journal of Applied Physiology.

Chapter 4 relates to the work on signaling pathways that were suspected to be involved in the increase of airway smooth muscle’s contractile capacity induced by tone elicited by the continuous presence of a spasmogen. I helped design the experiments, performed all the laboratory work, and analyzed the data in concert with Dr. Bossé.

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Introduction

Asthma is a respiratory inflammatory chronic disorder that affects people of all ages through-out the world. It has become a serious global health issue, touching approximately 300 million people [6,37]. In some countries, the prevalence of asthma is increasing among children, but is decreasing or remaining stable in other age groups. Asthma is a burden by its healthcare costs, lost of productivity at school or at work, and reduced participation in family life. When uncontrolled, daily life activities can be a struggle. Asthma can even be fatal, estimating to be the cause of premature death of 250,000 people each year [6].

In the last two decades, many scientific advances have improved our understanding of asthma and our ability to control it [6]. However, many patients remain untreatable with today’s available treatments, and many aspects of the disorder remain ill-defined. It is clear for experts in the field that the substantial global burden of asthma can be drastically reduce through scientific research. Indeed, a better understanding will help us improve asthma control with new approaches or by improving actual treatments [6,37].

This Master’s thesis focuses on understanding the role of airway smooth muscle (ASM) tone in airway responsiveness, and its implications in airway hyperresponsiveness (AHR). Tone in asthmatic airways is elevated compared to normal airways [82]. This means that airway smooth muscle in asthmatic airways is continuously activated to a greater extant than normal airways, presumably due to a higher release of spasmogens [73].

AHR is another major feature of asthma, and is used as a clinical diagnostic. It is an exagger-ated decline of lung function in response to the activation of ASM by a spasmogenic challenge [12, 19, 108]. These observations suggest that targeting airway smooth muscle in asthma is critical for successful treatments. Actual therapies for asthma include airway smooth muscle bronchodilators, usually in combination with corticosteroids. However, to some patients, these treatments have no effect. In addition, some asthmatic patients may develop tachyphylaxis to β2-agonists, tolerance to their bronchoprotective effects, and adverse effects [73].

The first chapter of this Master’s thesis consist of a general description of asthma, its risk factors, and its physiopathologies. The common features observed in asthmatics are airway inflammation, airway remodeling, airway obstruction, elevated tone, and airway

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hyperrespon-siveness. Their importance in this respiratory condition is briefly explained with emphasis on the role of airway smooth muscle.

The second chapter focuses on airway smooth muscle contraction pathways. The different spasmogens used throughout this project and their mechanisms of action are discussed. Ad-ditionally, the effect and the signaling pathways targeted by the inhibitors used in the exper-iments are explained.

Thethird chapterpresents an article published in the Journal of Applied Physiology entitled The gain of smooth muscle’s contractile capacity induced by tone on in vivo airway respon-siveness in mice. The methods used to measure airway responsiveness in vivo in mice and the contractile capacity of isolated murine tracheas with and without pre-exposure to tone are thoroughly explained. Further, the results obtained are presented and discussed.

The fourth chapter of this Master’s thesis consists of the methods used to target several molecules involved in different signaling pathways. Myosin light-chain phosphorylation, actin polymerization, RhoA/Rho-associated, coiled coil-containing kinase pathway, G proteins, and mitogen-activated protein kinases are studied. The results are presented and then discussed. To end this Master’s thesis, a briefconclusion summarizing the main concepts and the main findings of this research project is presented. Also, suggestions concerning the next steps to follow in order to pursue the project are formulated.

Hypotheses and objectives

AHR and airway obstruction due to a sustained contractile activation of ASM are two impor-tant features of asthma [19]. However, it is unclear whether or not causality exists between these two characteristics. The principal player in these characteristics is ASM, where excessive changes of lung function occurs by changing the level of ASM activation with either bron-chodilators or spasmogens. This phenotype is believed to be caused by ASM’s environment that is modified in asthma [13]. Understanding how the effect of ASM is altered in asthmatic lung will greatly improve our understanding of AHR and asthma symptoms [14,71].

Recent data demonstrate that asthmatic ASM alone, taken out of its normal environment, is not stronger than non-asthmatic ASM [53]. This suggest that ASM cells are not themselves altered. This lead to the proposition that ASM force is not fixed, but is adaptable. The amount of force generated by ASM depends on the amount of spasmogens that stimulate it. A greater release of spasmogens by inflammatory or structural cells present in asthmatic airways could lead to more airway narrowing. Indeed, many spasmogens are overly expressed in asthmatic lungs. The most studied are histamine, leukotrienes, endothelin-1, bradykinin, prostaglandin D2, and thromboxane A2 (TXA2) [12]. Previously, Bossé et al. (2009) [14] demonstrated, in

ovine muscle strips, that a short pre-exposure to tone (30 min) induced by the continuous 2

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presence of a spasmogen (acetylcholine (ACh)) augments ASM force over time. So in addition to stimulate ASM, the continuous presence of spasmogen increases the contractile capacity of ASM over time. This suggest that the contractile capacity of normal ASM can be altered by its surrounding environment. This phenomenon could possibly play a role in AHR, but has not yet been studied in vivo.

Hypotheses

Two hypotheses have been formulated in this Master’s thesis. The first one is in regard to the effect of tone on airway responsiveness, and the other one is specific to the molecular mechanisms involved. They are presented as followed:

– An elevated tone augments airway responsiveness in mice in vivo by increasing the contractile capacity of ASM;

– The gain in force following an exposure to tone involves signaling pathways indepen-dent from the level of myosin light-chain (MLC) phosphorylation but that lead to actin polymerization.

Objectives

The specific objectives of this research project are:

– Measure the effect of an elevated tone on airway responsiveness in mice in vivo;

– Measure the contractile capacity of ASM in murine tracheas pre-exposed or not to an elevated tone;

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Chapter 1

Asthma

1.1

Definition of Asthma

Asthma is a respiratory disorder that affects around 300 million individuals worldwide. Its prevalence ranges from 1% to 18% of the population in different countries (Table 1.1) [19,37]. People of all age can develop asthma, and it is the most common chronic condition in children [19].

Table 1.1: Asthma prevalence around the world. Data from Graham-Rowe (2011) [42]. Country Prevalence (%) Australia 14.7 Canada 14.1 China 2.1 England 15.3 Indonesia 1.1 Mexico 3.3 New Zeland 15.1 Russia 2.2 Scotland 18.4 United States 10.9

In asthma, several cell types play different roles, such as mast cells, eosinophils, T lympho-cytes, macrophages, neutrophils, epithelial cells, and smooth muscle cells. Patients suffering from asthma will often have recurrent episodes of wheezing, breathlessness, chest tightness and coughing, all of which are associated with airway obstruction. Airflow obstruction in the lung is reversible spontaneously or with the use of a treatment (i.e. bronchodilators and corticos-teroids). Indeed, in most cases, asthma can be controlled, resulting in no more than occasional episodes of airway obstruction and cases severe exacerbations become rare. However, for some people, the medication is inefficient. It then becomes a problem not only for the individual, but also for the society. The cost of asthma is substantial and has to take into account the

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direct medical costs (hospital admissions and medications) and the indirect non-medical costs (time lost from work and school, and premature death). Yet, not properly treating asthma results in higher monetary costs. Approximately 50% of health care charges is allocated to patients that do not respond to treatments, known as having severe asthma [19, 37, 56]. It is believed that the economic burden of this respiratory disorder could be greatly reduced by improving asthma control through research [37].

1.2

Risk Factors

Factors that influence the risk of asthma are numerous and are not all well understood. They can be divided into two categories: the host factors and the environmental factors. Having said that, it is difficult to categorize specifically asthma risk factors as they are complex and interact with each other [37].

1.2.1 Host Factors

Genetic. Many genes have been involved in the development of asthma [18]. Current re-search focuses on genes linked to predisposition to atopy (elevated levels of allergen-specific immunoglobulin E (IgE) antibodies), to predisposition to airway hyperresponsiveness, to the production of inflammatory mediators (cytokines, chemokines, and growth factors), and to the ratio between T helper lymphocytes; i.e. Th1 versus Th2 [37].

There are also some evidence that certain genes are associated with response to asthma treat-ments. For example, variations in the encoding of β-adrenoreceptor have shown different re-sponses in subjects to β2-adrenergic agonists, a class of bronchodilators used to treat asthma.

In addition, the responsiveness to glucocorticosteroids and leukotriene modifiers treatments may be modified by certain genes [37].

Obesity. Leptin, a satiety hormone produced by fat cells could have an effect on airway function and increase the possibility of developing asthma [37]. Leptin could possibly induce naïve T cells proliferation and modulate cytokine production to favour Th1. However, the association between asthma and obesity, and its effect on airway function is still unknown [36]. Gender. Prior to the age of 14, boys are more likely to develop asthma than girls, with a prevalence twice as high. As age increases, the prevalence of asthma becomes greater in women. However, it is not quite clear why there is such a relationship between age and sex [37].

Infections. Several viruses are known to influence the development of asthma, either to pre-vent or to cause exacerbations. On one hand, an infection during infancy by the respiratory syncytial virus (RSV), the human rhinovirus (HRV), or the parainfluenza virus will produce 6

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symptoms that resembles those of childhood asthma. Moreover, approximately 40% of the children will continue to wheeze or have asthma later in their childhood [37]. One the other hand, respiratory infections early in life with measles and RSV can be protective in the devel-opment of asthma. Several common bacterias also seem to be associated with the develdevel-opment and exacerbations of asthma, such as streptococcus pneumoniae, hemophilus influenzae, and moraxella catarrhalis [37,63].

1.2.2 Environmental Factors

Allergens. Many allergens trigger symptoms and exacerbation in asthma patients. A wide variety of allergens, for instance domestic mites, furred animals, cockroaches and fungi, are known to cause theses adverse effects and are commonly present in human homes. Outdoor allergens such as pollens and molds are also important causes of asthma symptoms. Allergens are a major problem for asthma management as it is impossible to eradicate or to avoid them completely. It is likely that any intervention will be costly and will not achieve sufficient benefits [37].

Occupational sensitizers. More than 350 substances encountered in the work environment have been linked to the development of asthma [99]. These substances can be highly reactive small molecules, irritants, immunogens, and complex plant and animal biological products [37].

Pollutants. Passive and active smoking is an important pollutant. Parents of children with asthma are advised not to smoke in proximity to them and to avoid smoking in rooms used by their children. Smoking also reduces the benefits of glucocorticosteroids and increases the decline in lung function. Other major air pollutants are nitrogen oxides, carbon monoxide, carbon dioxide, sulfur dioxide, formaldehyde, ozone, acidic aerosols, molds and biomass fuels [37].

Medications. Some medications such as aspirin and other nonsteroidal anti-inflammatory drugs can cause exacerbations in asthma patients [37].

1.3

Physiopathology

Figure 1.1 illustrates the difference between normal and asthmatic airways. The main ob-servation is that asthmatic airways have a thickened airway wall. The thickening is caused by airway inflammation, airway remodeling, and mucus accumulation [19,37, 85]. The tick-ened airway wall causes a reduction in the luminal area, which impedes the flow of air when breathing [78].

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Figure 1.1: Comparison of normal and asthmatic airways. Section A shows the position of the lungs and the airways in the body. Section B and C depicts the longitudinal and the cross-sectional composition of normal and asthmatic airways, respectively. Reproduced from The National Heart, Lung, and Blood Institute [85].

1.3.1 Airway Inflammation

Asthma is a chronic inflammatory disorder of the airways that implicates predominately mast cells, macrophages, eosinophils, neutrophils, and Th2 lymphocytes, which infiltrate the bronchial mucosa. This respiratory disorder may be the result of persistent inflammatory pro-cess through which several and cumulative changes in the airway structure occur (also known as airway remodeling). More than 100 different mediators are recognized to be involved in the inflammation that characterize asthma [19, 37]. The inflammation is normally present in the conductive airways, but becomes present in the smaller airways when asthma severity increases [19].

Macrophages. Alveolar macrophages are located in the bronchial and alveolar lumen. They play an important role in the innate defense system by means of phagocytosis of inhaled par-ticules and elimination of infectious agents. Macrophages secrete leukotrienes, prostaglandins, oxygen free radicals, interleukin (IL)-6, IL-8, IL-10, IL-12, IL-13, GM-CSF, Tumor Necrosis Factor (TNF-α), interferon (INF)-γ, and Platelet-Derived Growth Factor (PDGF) [19]. Mast cells. Mast cells are present in the bronchial mucosa and in the peribronchovascular space. They are mostly implicated in allergic asthma, as they possess high affinity recep-tors for IgE (FcRI). These cells produce and release various inflammatory mediarecep-tors such as proteinases, IL-3, IL-4, IL-5, TNF, Granulocyte Macrophage Colony Stimulating Factor 8

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(GM-CSF), Platelet Activating Factor (PAF) which can act as a bronchoconstrictor, and other bronchoconstrictors (histamine, thromboxane, leukotriene) [19]. The increased number of these cells in asthmatic lung, especially in ASM tissue, may be linked to airway hyperre-sponsiveness [37,20].

Eosinophils. Eosinophils are also particularly implicated in allergic asthma. These cells are altered in the bronchial mucosa by the chemokine Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES), Macrophage Inflammatory Protein (MIP-1α), eotaxins, and metabolites of arachidonic acid. They exert their pro-inflammatory effects by releasing IL-3, IL-4, IL-5, IL-1IL-3, basic proteins, chemokines, growth factors, enzymes, and lipid mediators [19]. They may also play a role in airway remodeling [37,52]. Eosinophils activate monocytes, fibroblasts, and B lymphocytes [19].

Neutrophils. Neutrophils are located in the bronchial mucosa of patients suffering from severe asthma. They play a major role in bacterial defense by producing proteinases, oxidative agents, TNF-α, IL-1α, IL-6, IL-8, α-defensins, and leukotriene B4 [19].

Dentritic cells. Dentritic cells recognize allergens from the airway surface. Next, they migrate to regional lymph nodes permitting an interaction between these cells and naïve T cells. This stimulates the adaptive immunity against the allergens [37].

T lymphocytes. T lymphocytes are present in a high number in asthmatic airways. They release IL-3, IL-4, IL-5, IL-9, and IL-13 that mediate eosinophilic inflammation and the pro-duction of IgE by B lymphocytes [19, 37]. IL-4, in particular, stimulates B lymphocyte-class switching to IgE. IL-3 and IL-5 mediate eosinophilic inflammation. There is a predominance of Th2 in lungs of light to moderate asthma [19]. This could be due, in part, to a reduction in regulatory T cells that normally inhibit Th2 cells [37]. In more severe asthma, Th1 and CD8+ T cells are more predominant. These latter produce TNF-α and IFN-γ [19].

1.3.2 Airway Remodeling

Airway remodeling is described as changes in the airway’s structure. It can be a change of content, of relative composition, or quantity of the structural elements, which then lead to mechanical and geometrical alterations of the airways. For example, the epithelium is altered. Indeed, the asthmatic epithelium is desquamated which increase its fragility and permeability. This can lead to higher exposition of the airways to gas and particles. Its role as a protection barrier to the external environment is thus impaired. The epithelium can be damaged by pollutants and allergens. Following the exposition, eosinophils liberate toxic factors that are harmful to the epithelium. Asthmatic epithelium is also characterized by goblet cell hyperplasia, which can lead to mucus hypersecretion. Additionally, the epithelium

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may play a role in the development of airway remodeling. The bronchial epithelium produces many cytokines and growth factors implicated in structural changes [19,35].

The ASM content is also changed [57]. There is an increase in the number of ASM cells (hyperplasia), plus these cells undergo hypertrophy. The ASM enlargement plays major roles in airway obstruction and AHR [69]. Indeed, the airways become more obstructed due to the augmentation in the thickness of the airway wall. Furthermore, a higher number of ASM cells are available to contract when stimulated by a spasmogen, thus promoting AHR [19].

Other features of airway remodeling in asthma include a subepithelial fibrosis, an increase in the number of blood vessels due to the vascular endothelial growth factor, and a deposition of proteoglycans in the bronchial wall [19].

1.3.3 Airway Obstruction

Figure 1.2: Pulmonary function testing using spirometry. The patient inhales maximally from FRC to TLC, and then exhales rapidly to RV. This maneuver generates the FVC. The FEV1

is the volume exhaled during the first second of a forced expiratory maneuver from TLC to RV. Reproduced from Gildea and McCarthy (2003) [40].

Airway obstruction in asthmatic patients is variable and reversible. It is reversible sponta-neously or by treatment. The obstruction is evaluated with a spirometer, a device measuring lung function, as shown in Figure 1.2. Figure 1.3 presents the different lung volumes and capacities to help understand the changes observed in spirometry. In airways where obstruc-tion is present, the airway lumen is decreased, which impedes the flow of air in and from the alveoli. When asthma is stable, sometimes pulmonary function tests are normal. However, when the obstruction is severe, the following changes can be noted [19]:

– A lower forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) ratio;

– An increase in airway resistance as assessed by the peak expiratory flow or by the forced expiratory flow between 25-75% of vital capacity (VC);

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– A reduction in VC;

– An increase in residual volume (RV) and functional residual capacity (FRC), showing a certain degree of gas retention.

Figure 1.3: Lung volumes and lung capacities. Reproduced from Gildea and McCarthy (2003) [40].

Airway obstruction is caused by airway remodeling, an excessive mucus accumulation, and an elevated tone [19, 14]. The effect of an elevated tone is the main focus of this thesis, and is explained in the following section.

Airway Smooth Muscle Tone

Tone is defined as a sustained contractile activation of ASM elicited by the continuous presence of one or several spasmogens. Normal airways are not completely relaxed. They maintain a tone, commonly named the baseline airway tone [10, 23]. ASM is innervated by the vagus nerve of the parasympathetic nervous system, which releases ACh [89]. It has been reported that inhalation of terbutaline, a bronchodilator, increases FEV1 of 1.8% in normal adults

[10]. Another study demonstrated a mean change of 4.3% in FEV1 after inhalation of a

bronchodilator [48]. Nonetheless, baseline tone in normal subjects does not fulfill the criteria for obstructive airway disorder [10]. However, asthmatic people present a baseline ASM tone higher than normal, referred to as an elevated tone. Indeed, FEV1 increases at least by 200

mL or by 12% to a bronchodilator [33].

An elevated tone is allegedly caused by airway inflammation. Indeed, inflammatory cells re-lease several mediators that are overexpressed in asthmatic airways. Some of them affect the contraction of ASM directly. These include histamine, leukotrienes, TXA2, bradykinin, and

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lysophosphatidic acid, sphingosine-1-phosphate, and some cytokines. For example, IL-1β af-fects ASM contractile capacity to bradykinin, des-Arg9-bradykinin, substance P, serotonin,

neurokinin A, ACh, and methacholine (MCh) [12].

1.3.4 Airway Hyperresponsiveness

AHR is a major feature of asthma. It is defined as an excessive decline in lung function in response to ASM activation to a bronchoprovocation by a spasmogen. In comparison to normal, asthmatic react to lower doses of a spasmogen (increase sensitivity) and more forcefully to a given dose (increase reactivity). Lung specialists use this parameter to assess asthma severity in patients. They measure the level of airway responsiveness by calculating the % fall of FEV1 caused by the inhalation of serial incremental doses of a spasmogen (Figure 1.4). The

greater the % fall is, the higher the airflow limitation. Furthermore, severe asthmatic airways fail to reach a plateau. Indeed, they can attain a level of airway responsiveness incompatible with life (almost or completely close). In normal individuals, a plateau is normally reached. This means that at a certain dose, ASM contraction becomes maximal, and it cannot contract further even at higher doses. It is possible that asthmatics lack the mechanisms that inhibit severe airway narrowing and closure during a spasmogenic challenge [19,108].

Figure 1.4: Bronchoprovocation tests with methacholine in asthmatic and normal subjects. Curve A is a dose-response curve from normal subjects. Curve B demonstrates a dose-response curve from a mild asthmatic patient. Airway sensitivity and reactivity are increased, but the maximal response reaches a plateau at the highest doses. Curve C illustrates severe hyperresponsiveness. A small dose of 1 µmoles is responsible for a drop of 18% of FEV1.

Moreover, severe AHR is characterized by the lack of a plateau. Reproduced from Woolcock (2015) [109].

The mechanisms leading to AHR are not yet well understood. They are probably due to airway 12

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inflammation (variable component) and to airway remodeling (fixed component). The altered ASM mass can possibly have modified contractile proprieties and thereby react differently to inflammatory mediators [19].

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Chapter 2

Intracellular Pathways Governing

Airway Smooth Muscle Contraction

Smooth muscle contraction relies on indirect signaling pathways. In all muscle types, the contractile machinery is composed of actin and myosin, and has to be activated to induce contraction [67]. The terminal stages of the conical contraction pathway is shown inFigure 2.1. The Ca2+-calmodulin complex activates the cross-bridge cycling by activating myosin

light-chain kinase (MLCK). MLCK can then phosphorylate the regulatory 20-kDa MLC at serine-19 or threonine-18 [67, 93], and form cross-bridges with actin, producing phosphorylated actomyosin [67].

Once MLC is activated, it changes conformation to increase the angle in the neck domain of the myosin heavy chain. This step is required for the binding of myosin head on the actin filament. This interaction is the cross-bridge. The hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate by ATPase on the myosin head is also required for cross-bridge formation. Upon binding, the inorganic phosphate is released, which reinforces the binding between myosin and actin. The activated myosin head then pivots following the release of ADP, resulting in a power stroke where filaments of actin and myosin slide pass each other. The cross-bridge detaches when new ATP binds to myosin [67,105]. MLC phosphorylation alone is not enough to enable smooth muscle contraction. Work from Wang et al. (2014) [106] showed that without actin polymerization, smooth muscle contraction is inhibited even though MLC is phosphorylated. Therefore, actin filamentogenesis is an essential process in contraction. Actin filaments enhance the transmission of force between the contractile units and the extracellular matrix, and possibly the number of actin and myosin filaments working in parallel [67,106].

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Figure 2.1: The terminal molecular stages of the signaling pathways leading to ASM con-traction. Ca2+ binds to calmodulin, which activates MLCK. Phosphorylated MLC can bind

to actin, and form phosphorylated actomyosin. This enables the cross-bridge cycle to start. Reproduced from Kuo et al. (2015) [67].

2.1

Spasmogens

A spasmogen is any mediator that contracts ASM. They are known as a bronchoconstrictor. In this study, several spasmogens were used to test the hypotheses. In this section, the mechanisms by which they contract ASM is explained.

2.1.1 Methacholine

MCh is a synthetic choline ester, non-selective muscarinic receptor agonist that simulates ACh of the parasympathetic nervous system. ACh is a very important neurotransmitter released by the vagus nerve to activate muscarinic receptors on smooth muscle [4]. Compared to ACh, MCh has a greater duration of action, and is widely used in bronchoprovocative challenges for diagnosing asthma [19].

So far, five distinct muscarinic receptor genes have been identified (M1, M2, M3, M4, M5). Not

all subtypes of receptors have been recognized in the human lung, but M1, M2, M3 are present.

Many evidences suggest that these latter play different physiological roles in the airways [4]. M1-receptor. Studies show that M1-receptors are abundant in human lung, and are

local-ized to the alveolar walls. However, their physiological role is still to be determined [4, 5]. It has been suggested that M1-receptor inhibits M3-receptor mediated bronchoconstriction by

stimulating the release of bronchorelaxing agents from the epithelia and nerves [64].

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M2-receptor. Binding studies demonstrate that in lung membrane preparation, M2-receptors population is very low, although messenger ribonucleic acid (mRNA) have been detected in cultured human airway smooth muscle cells. These receptors play an important role in the regulation of cholinergic neurotransmission. In fact, pre-junctional muscarinic receptors on post-ganglionic airway cholinergic nerves inhibit the release of ACh, thus inhibiting airway smooth muscle contraction by parasympathetic nerves [4,5].

It has been suggested that M2-receptors could be dysfunctional in asthmatic airways [4]. An

inhalation test of pilocarpine, a selective pre-junctional receptor stimulator, was given to non-asthmatic and non-asthmatic subjects. In non-non-asthmatic people, pilocarpine had an inhibitory effect on cholinergic reflex bronchoconstriction. Contrary to non-asthmatic individuals, pi-locarpine had no effect in asthmatic patients. Another evidence explaining why asthmatic patients could have a functional defect of M2-receptors is that there is an increase in

choliner-gic tone following the blockade of the inhibitory β-adrenercholiner-gic receptors, which is presumably due to a greater release of ACh that is normally switched off by the activation of M2-receptors

on the nerves [4]. In agreement with these findings, M2-receptor deficient mice present

en-hanced bronchoconstriction responses to vagal stimulation [64]. Inflammation could be the reason why these receptors become dysfunctional as it may lead to their down-regulation [4]. In fact, a study [5] demonstrated that the cytokine transforming growth factor (TGF)-β1 down-regulates M2-receptor protein and gene expression in human embryonic lung fibroblasts

by reducing the rate of gene transcription. TNF-α and IL-1β had the same repercussions [5]. M3-receptor. M3-receptors have been identified in ASM of large and small human airways. They are also expressed on submucosal glands, which augments mucus secretion, and slightly expressed on airway epithelial cells [4].

After being activated, M3-receptors trigger rapid phosphoinositide hydrolysis, and the

forma-tion of inositol trisphosphate (IP3). IP3 then allows the release of calcium (Ca2+) ions from

intracellular stores, which leads to ASM contraction [4]. Due to its bronchoconstricting effect, M3-receptors are targets for therapeutic treatments in asthma. To date, muscarinic receptor

antagonists used as bronchodilating drugs are not selective to any subtype of receptors. It is clear that blocking the effect of M2-receptors (and perhaps M1-receptors) will increase

bron-choconstriction [64]. The signaling pathways, as illustrated in Figure 2.2, are initiated by the binding of MCh to muscarinic receptors [39].

2.1.2 Thromboxane A2

TXA2 is a prostanoid that initiates vascular and ASM contraction. It is synthesized by

oxida-tion of arachidonic acid by cyclooxygenase 1 into prostaglandin H2 (PGH2). Thereafter, PGH2

is metabolized into TXA2 by thromboxane synthase. In addition to ASM, the epithelium and

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com-Figure 2.2: Muscarinic receptor signaling pathways that regulate contraction in smooth muscle. Muscarinic receptors are coupled to G proteins. When activated, they produce secondary messengers to increase the intracellular concentration of Ca2+. These ions bind to calmodulin

and send an activation signal to MLCK to promote contraction. Reproduced from Gerthoffer (2005) [39].

munication [1]. In asthmatic airways, levels of TXA2 and TXB2 (stable metabolite of TXA2)

and airway sensitivities to these mediators are greatly elevated compared to normal airways [1,72].

TXA2binds to a specific thromboxane receptor (TP) that is coupled to a G protein of the Gq/11

family. The downstream signaling is similar to the M3-receptor. Briefly, phospholipase Cβ

is activated by the G protein to catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into a diacyl-glycerol (DAG) and IP3. IP3 binds to a ligand-gated Ca2+ channel on

the sarcoplasmic reticulum membrane to release Ca2+ [1,72]. It has also been demonstrated

that the entry of extracellular Ca2+ into the cell by L-type voltage-gated calcium channel is

necessary for TXA2to induce a contraction [72]. In addition, binding of TXA2 to TP on nerves

or nerve terminals can constrict ASM indirectly by increasing ACh release. This mechanism is dependent of the M3-receptor [1].

To assess the response of TXA2 in contractile studies, such as the one performed in this

Master’s thesis, U46619 is normally used. U46619 is a stable chemical that acts as a TP receptor agonist [72].

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2.1.3 Potassium Chloride

Potassium chloride (KCl) is a well known stimulus used to bypass G protein-coupled receptors (GPCR). KCl-induced contraction is produced by depolarizing the cell membrane by clamp-ing the membrane potential at a higher value than the restclamp-ing level. This causes Ca2+ entry

through voltage-operated Ca2+ channels (VOCCs), more precisely L-type voltage-gated Ca2+

channels [83]. The entry of Ca2+ increases intracellular concentration and activates the

down-stream signaling events that lead to ASM contraction (Figure 2.1). In addition, KCl changes Ca2+ sensitivity by modifying MLCP activity [93]. The decreased activity of MLCP also leads

to an increase in MLC phosphorylation [83,93].

Naturally, the entry of extracellular Ca2+into the cell augments the intracellular concentration

of Ca2+, but that entry also amplifies the increase by the release of Ca2+ from intracellular

stores. This entry is also required to optimally refill the depleted internal stores of Ca2+ [83].

2.1.4 Sodium Orthovanadate

The mode of action of sodium orthovanadate (SOV) on ASM contraction is not well known [103]. What is known is that SOV inhibits protein tyrosine phosphatases (PTPs), dual-specific phosphatases (DSPs), ATPases, and alkaline phosphatases. Phosphatases remove phosphate groups from phosphorylated residues. Consequently, when phosphatases are inhibited, the level of protein phosphorylation is augmented leading to smooth muscle contraction [118]. It is also known that SOV inhibits DSPs. DSPs act upon tyrosine and/or serine/threonine residues. MLC20is phosphorylated on serine-19 or on threonine-18 residue by MLCK [67,54].

The dephosphorylation of MLC20 is performed by MLCP, a serine/threonine-specific protein

phosphatase [22,100]. SOV could deactivate MLCP, thus increasing MLC phosphorylation.

2.1.5 Sodium Fluoride

The mechanism of action of sodium fluoride (NaF) on ASM contraction is not well understood. The contraction induced by NaF seems attributable to several pathways [47]. One of these is the adenylate cyclase system. Indeed, NaF is known to be a G protein activator important in the adenylate cyclase system [41,94,95]. Adenylate cyclase catalyzes the conversion of ATP into 3’-5’-cyclic adenosine monophosphate (cAMP) and pyrophosphate. cAMP is a second messenger essential for intracellular signal transduction. Several receptors can trigger the stimulation or inhibition of adenylate cyclase. For example, muscarinic receptors exert an inhibitory control. These receptors are coupled to a protein G, termed Gi, that inhibits the

enzymatic activity of adenylate cyclase. In smooth muscle, decrease activity of cAMP results in smooth muscle contraction. Protein Gs stimulates the production of cAMP by adenylate

cyclase, which translates to ASM relaxation [41].

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histamine and bradykinin, are increased in asthmatic airways. The importance of adenylate cyclase in asthma is emphasized by the salutary effect of β-adrenoceptor agonists, medica-tions used by asthmatic patients to relax their ASM. The effector targets of β-adrenoceptor stimulation are the adenylate cyclases. The medication intends to augment cAMP cellular level, enabling ASM relaxation. However, some asthmatic patients show reduced relaxation in response to the medication. It is possible that pathways leading to ASM relaxation in response to agonists that increase cAMP may be impaired in asthmatic airways [34].

The effect of NaF on protein Gq/11 in ASM contraction is not well understood. Studies

in vascular smooth muscle show that NaF activates a protein G, perhaps Gq, that in turn

activates phospholipase C [47]. Phospholipase C hydrolyzes PIP2 into IP3, which releases

Ca2+ from the sarcoplasmic reticulum. Indeed, NaF increases the formation of IP

3 in aortic

myocytes, rat tail artery, and rabbit femoral artery [47]. DAG, which is also a by product of PLC hydrolysis activates protein kinase C (PKC). This results in the activation of L-type Ca2+ channels and ultimately in ASM contraction [47].

2.2

Actin Polymerization

Actin is present in the cell under two forms: G-actin, the free globular monomeric form, and F-actin, the polymeric filamentous form. To generate force, G-actin has to polymerize into the F-actin form to create microfilaments. These microfilaments are essential in smooth muscle contraction, as they form the contractile units with myosin. Additionally, actin polymer-ization facilitates contraction by stabilizing the contractile machinery inside the cell to the extracellular matrix. It enhances the linkage of actin filaments to integrins [106].

Actin filamentogenesis requires a protein called c-Abl. It is a non-receptor tyrosin kinase that regulates actin dynamics. Contractile stimuli induce c-Abl phosphorylation. The activated c-Abl phosphorylates cortactin, resulting in its association with profilin-1 (Pfn-1), a protein that promotes actin polymerization, leading to ASM contraction. The disruption of the in-teraction between cortactin and Pfn-1 attenuates actin polymerization and force development [106]. c-Abl is also involved in another important downstream cascade for actin polymeriza-tion. It activates Abi1, an adapter protein which in turn activates neuronal Wiskott-Aldrich syndrome protein (N-WASp). N-WASp then binds to actin-related protein 2 and 3 (Arp2/3) complex, thus forming a template for the addition of G-actin to existing F-actin filaments. This is necessary for the regulation of actin polymerization and tension development [106,116]. Because of its importance in actin dynamics, c-Abl has been targeted in this study to evaluate whether actin polymerization is involved in the gain in force induced by tone. Imatinib was used as the inhibitor of c-Abl. It inactivates the enzyme by binding to the clif between the N-and C-lobes of the kinase domain [84].

Latrunculin B is also a well-known inhibitor of actin polymerization. More specifically, la-20

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trunculin B sequesters G-actin by forming a 1:1 complex with G-actin, which then prevents F-actin assembly [62].

2.3

RhoA/Rho-associated, Coiled Coil-containing Kinase

Pathway

The RhoA/Rho-associated, coiled coil-containing kinase (ROCK) pathway play a central role in many biological processes, including smooth muscle contraction. Figure 2.3 shows the different signaling pathways influenced by RhoA/ROCK. The ones that are colored are of interest in this Master’s thesis.

RhoA is a member of the Ras superfamily of small GTP-binding proteins. It has been shown that the RhoA/ROCK pathway is involved in many pathophysiologies of asthma (i.e. AHR, airway remodeling, β2-adrenergic desensitization, and eosinophil recruitment). Importantly,

ROCK contributes to Ca2+ sensitization by altering the phosphorylation status of MLCP.

ROCK activated by RhoA inactivates MLCP by stimulating the phosphorylation of threonine-696 and -853 of myosin phosphatase target subunit 1 (MYPT1), the regulatory subunit of MLCP. This inhibition leads to increased levels of MLC phosphorylation, resulting in ASM contraction. Furthermore, ROCK can increase MLC phosphorylation by phosphorylating directly MLC [66,67].

In addition, ROCK plays a role on actin stabilization by inhibiting actin filament depoly-merization. ROCK phosphorylates LIM-kinase (LIMK), which in turn deactivates cofilin by phosphorylation. Since cofilin is essential for actin disassembly, its inhibition favors actin stabilization [25,55,76].

To assess the function of ROCK in cells, specific inhibitors have been developed, such as: Y-27632. Y-27632 inhibits the phosphorylation of MYPT1 in a concentration-dependent manner [66]. It competes with ATP for the binding to the catalytic site [66]. The application of the inhibitor affects the force of contraction induced by a spasmogen without reducing intracellular concentration of Ca2+ [66]. Y-27632 also reverses G protein sensitization, causing, ASM

relaxation as well [90].

2.4

Mitogen-Activated Protein Kinase Pathways

MAPKs are a group of serine/threonine kinases that transduce important signals in the cell to external stimuli. In pulmonary inflammatory disorder, deregulation of MAPKs contribute to airway inflammation and AHR. In ASM, GPCR (e.g. muscarinic and TP receptors) strongly regulate MAPKs [8, 72]. The signaling pathways described below are the three main MAPK cascades named after the terminal kinases [8]. They are activated by a three-tiered sequen-tial phosphorylation. The sequence starts by MAPK kinase kinase (MAPKKK), followed

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Figure 2.3: Signaling pathways downstream of RhoA/ROCK that regulate smooth muscle contraction. Pathways of interest are highlighted in color. When activated by RhoA, ROCK can follow two downstream cascades important in smooth muscle contraction; MLC phospho-rylation and actin polymerization. ROCK can directly phosphorylate MLC, and can indirectly increase MLC phosphorylation by inhibiting MLCP. ROCK inhibits actin depolymerization by activating LIMK, which in turn phosphorylates and inactivates the actin-severing protein cofilin. Modified from SABiosciences; QIAGEN Company (2015) [27].

by MAPK kinase (MAPKK), and then by MAPK, as shown in Figure 2.4. The signaling pathways are counter-regulated by MAPK phosphatase [32].

2.4.1 p38 Signaling Pathway

There are four isoforms of p38 kinase: α, β, γ, and δ. The expression of the α-isoform has been found in canine tracheal, colon, and vascular smooth muscle [39].

p38 kinase participates in smooth muscle contraction, cell migration, oxidative stress signaling, and cytokine synthesis [39]. This kinase is involved in IL-1β-induced rat tracheal smooth

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Figure 2.4: A summary presenting the interactions between protein kinases of the three main MAPK signaling cascades: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. Reproduced from Bennett (2006) [8].

muscle cell proliferation. p38 is also involved in the production of IL-6 by ASM stimulated by bradykinin [32]. p38 kinase can be activated by multiple GPCRs. It has been shown that p38 regulates muscarinic M2-receptor, but not M3, ACh-induced contraction, and angiotensin

II-induced contraction. The mechanisms by which p38 kinase contributes to ASM contraction are unclear. It is possible that p38 promotes production of reactive oxygen species, activates phospholipases and/or phosphorylates HSP27. HSP27 function is also unclear. It has been suggested that this protein binds to contractile proteins to facilitate contraction, acts as a chaperon for Rho and PKC, and regulates actin filament structure [39].

p38 kinase α and β-isoforms are inhibited by pyridinyl imidazoles SB203580, SB239063, and SB202190 [32, 39]. The three inhibitors exert their effect by competing with ATP for the ATP-binding pocket [44,65,112].

2.4.2 Extracellular Signal-Regulated Kinase Signaling Pathway

Extracellular signal-regulated kinase (ERK) are critical effectors of growth factor stimulation, tissue development, and smooth muscle contraction. There exist 3 isoforms of ERK: ERK1, ERK2, and ERK5. ERK2 has been shown to be predominant in the lung [8]. Studies in

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canine colon smooth muscle demonstrate that muscarinic M2-receptors couples preferentially

to ERK. Several effector proteins that contribute to smooth muscle contraction receive signals from activated ERK. Evidences suggest that one of them is caldesmon, an actin binding pro-tein which represses contraction. ERK phosphorylates caldesmon on serine-789 to partially dissociate it from actin, leaving the myosin-binding sites exposed for actomyosin interactions. Other sites of action of ERK related to smooth muscle contraction could possibly be phospho-lipases activation, regulation of myoplasmic Ca2+ levels, and activation of MLCK [31,39]. It

has also been proposed that ERK is activated by Ca2+/calmodulin-dependent protein kinase

II (CaMKII). The activated ERK could also phosphorylate MLCK [93].

To date, there are no selective inhibitors of ERK. To inhibit this signaling cascade, mitogen-activated protein kinase kinase (MEK) have been targeted [8]. U0126 inhibits selectively the phosphorylation of MEK1 and MEK2.

2.4.3 c-Jun N-terminal Kinase Signaling Pathway

The c-Jun N-terminal kinases (JNK) (JNK1, JNK2, and JNK3) are stress-activated protein kinases. They can be activated by environmental shock, inflammatory stimuli, and growth factors. All of these are present in several pulmonary disorders such as asthma, acute respi-ratory distress syndrome, and chronic obstructive pulmonary disease, which is why they are important research targets [8]. For instance, it has been demonstrated that JNK signaling has a regulatory effect on TP receptors related to airway inflammation and airway remodeling in asthma [72]. JNK1 and JNK2 are expressed in the lung. They phosphorylate serines on targeted proteins including transcription factors, adaptor proteins, cytoskeletal proteins, and apoptosis-regulating proteins [8].

The involvement of JNK on ASM contraction are not well understood. It has been observed in allergen-challenged rats and mice that a JNK inhibitor causes a significant decrease in ASM proliferation and goblet cells hyperplasia [8]. ASM stimulated by TNF-α, 1β, 4, or IL-13 will show an increase in JNK phosphorylation, meaning that this pathway is present and activated by inflammatory mediators in ASM [8,32].

SP600125 is often used as a JNK inhibitor. This ATP-competitive inhibitor decreases JNK activity in a concentration-dependent manner [8]. However, it is not an entirely selective protein kinase inhibitor [8]. This means that its effect could potentially spread to other molecules. Caution must be used while interpreting data using SP600125 [8].

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Chapter 3

The Gain of Smooth Muscle’s

Contractile Capacity Induced by Tone

on in vivo Airway Responsiveness in

Mice

Audrey Lee-Gosselin, David Gendron, Marie-Renée Blanchet, David Marsolais, and Ynuk Bossé

Institut Universitaire de Cardiologie et de Pneumologie de Québec (IUCPQ), Laval Univer-sity, Quebec, Canada

Correspondence to: Ynuk Bossé

IUCPQ

Pavillon Marguerite-d’Youville, Y4186 2725, chemin Sainte-Foy

Québec, Qc, G1V 4G5

Phone: 418-656-8711 (ext. 3489) E-mail: ynuk.bosse@criucpq.ulaval.ca

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Résumé

L’hyperréactivité bronchique à une bronchoprovocation par un spasmogène, telle que la MCh, et l’augmentation du tonus de base mesurée par la reversibilité de l’obstruction bronchique suite à l’inhalation d’un bronchodilatateur, sont deux caractéristiques de l’asthme. Cependant, l’influence qu’a le tonus sur le degré de réactivité bronchique est encore inconnue. L’hypothèse est qu’un tonus élevé augmente la réactivité bronchique in vivo en augmentant la capacité contractile du muscle lisse. Des souris anesthésiées, trachéotomisées, paralysées et ventilées mécaniquement ont été exposées (groupe expérimental) ou non (groupe contrôle) à un tonus élevé pendant 20 min, induit par la nébulisation en série de petites doses de MCh. La résistance du système respiratoire était mesurée durant cette période, et par la suite, la réponse maximale à une dose cumulative élevée de MCh était mesurée et comparée entre les groupes. Afin de confirmer l’implication directe du muscle lisse, la capacité contractile de trachées murines a été mesurée avec et sans pré-exposition à un tonus induit soit par la MCh ou l’analogue du TXA2 (U46619). Deux spasmogènes différents ont été testés, car il est probable que divers

spasmogènes soient impliqués dans l’augmentation du tonus dans l’asthme. Les résultats démontrent qu’une pré-exposition à un tonus augmente la réactivité bronchique in vivo de 126 ± 37 % et augmente la capacité contracile de trachées excisées ex vivo de 23 ± 4 % pour la MCh et 160 ± 63 % pour le U46619. En conclusion, un tonus élevé, qu’il soit provoqué par un agoniste muscarinique ou par l’analogue du TXA2, peut contribuer à l’hyperréactivité

bronchique en augmentant la capacité contracile du muscle lisse des voies respiratoires.

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Abstract

Airway hyperresponsiveness to a spasmogenic challenge, such as MCh, and an increased base-line tone, measured by the reversibility of airway obstruction with a bronchodilator, are two common features of asthma. However, whether the increased tone influences the degree of airway responsiveness to a spasmogen is unclear. Herein, it was hypothesized that the in-creased tone augments airway responsiveness in vivo by increasing the contractile capacity of ASM. Anesthetized, tracheotomized, paralyzed and mechanically ventilated mice were either exposed (experimental group) or not (control group) to tone for 20 min, which was elicited by nebulizing serial small doses of MCh. Respiratory system resistance was monitored during this period and the peak response to a large cumulative dose of MCh was then measured at the end of 20 min to assess and compare the level of airway responsiveness between groups. To confirm direct ASM involvement, the contractile capacity of excised murine tracheas was measured with and without pre-exposure to tone elicited by either MCh or a TXA2 mimetic

(U46619). Distinct spasmogens were tested because the spasmogens liable for the increased tone in asthma are likely to differ. The results indicate that pre-exposure to tone increases airway responsiveness in vivo by 126 ± 37 % and increases the contractile capacity of excised tracheas ex vivo by 23 ± 4 % for MCh and 160 ± 63 % for U46619. It was concluded that an increased tone, regardless of whether it is elicited by a muscarinic agonist or a TXA2 mimetic,

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Introduction

AHR to an inhaled spasmogen and an exaggerated response to an inhaled bronchodilator are two typical features of asthma; so much, that they both became objective criteria to diag-nose asthma [91]. The exaggerated response to bronchodilator testifies that tone is elevated in asthma [82]. It is also established that the extent of tone correlates with the degree of airway responsiveness, both in healthy individuals [10] and in asthmatic patients [9]. How-ever, whether a link of causality exists between the extent of tone and the degree of airway responsiveness has never been ascertained.

We recently demonstrated that tone elicited by the continuous presence of a spasmogen in-creases the contractile capacity of ovine tracheal strips within a timescale of minutes [14,15,

89]. This gain of ASM contractile capacity caused by tone is relevant to the understanding of AHR. In fact, it supports tonic activation of ASM as an underlying contributor of AHR in asthma. Yet, whether the gain of contractile capacity caused by an increased tone occurs in vivo is unknown. This matter represents the gist of the present study [71].

Material and Methods

Animals

Nine to 14-week-old male and female C57BL/6J mice (Jackson Laboratory, Bar Harbor, USA) were used for both the in vivo and ex vivo experiments. The protocols were approved by the Committee of Animal Care of Laval University in accordance with the guidelines of the Canadian Council on Animal Care.

In vivo Airway Responsiveness

In vivo airway responsiveness was assessed by measuring the changes in respiratory system resistance (Rrs) induced by intratracheal nebulization of MCh in live, anesthetized, tra-cheotomized, paralyzed and mechanically ventilated mice. Specifically, mice anesthetized with Ketamine-Xylazine (100 and 10 mg/kg, respectively) were tracheotomized and connected to a computer-controlled ventilator (FlexiVent, SCIREQ, Montreal, Canada) at a respiratory frequency of 150 breaths/min, a tidal volume of 10 ml/kg, and a positive end-expiratory pres-sure (PEEP) of 3 cmH2O. Once the ventilation was established, they were paralyzed with 0.1

mg/kg of pancuronium bromide injected intra-muscularly. Rrs was measured by the FlexiVent using the Snapshot-150 perturbations. The heart rate was monitored continuously by electro-cardiography throughout the experiment to ensure proper anesthesia.

The dosing regimen for MCh delivery is illustrated inFigure 3.1. Age and sex-matched mice were either exposed (experimental group) or not (control group) to tone for 20 min before being administered with a final large dose of MCh. The MCh was delivered by nebulization 28

(51)

during tidal breathing, and deep inspiration (DI) was omitted throughout the entire protocol. The tone in the experimental group was induced with 4 small doses of MCh administered at 5 min intervals; an initial dose of 10 mg/ml followed by 3 doses of 5 mg/ml. A final dose of 75 mg/ml was then delivered 5 min after the last dose of 5 mg/mL. At each MCh dose, Rrs was measured 3 times before and 12 times after the administration of MCh at 9 s intervals. The same perturbations that were required to measure Rrs were performed in mice of the control group not exposed to tone. However, only one single dose of 100 mg/mL of MCh was delivered at the end in the control group. The purpose of the single 100 mg/mL dose was to equate the total quantity of MCh delivered in the experimental group (100 mg/mL = 10 mg/mL + ( 3 × 5 mg/mL ) + 75 mg/mL). Rrs in response to the final dose was then compared between the control and the experimental groups. In an additional set of experiments, it was demonstrated that the diluent alone (i.e. saline) neither induced tone nor affected the responsiveness to 100 mg/mL of MCh (data not shown).

mg/mL mg/mL

0

10

mg/mL

0

mg/mL

0

mg/mL

0

100

mg/mL mg/mL

5

mg/mL

5

mg/mL

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mg/mL

75

Control group

Experimental group

20 min

Figure 3.1: Dosing regimen for MCh delivery in control and experimental groups. Protocol for measuring whether exposure to an elevated tone increases airway responsiveness in mice in vivo. Mice were either exposed (experimental group) or not (control group) to an elevated tone induced by 4 small doses of MCh administered 5 min apart. A final large dose (75 mg/mL or 100 mg/mL) was then administered and the respiratory system resistance that was obtained was compared between groups of sex- and age-matched mice.

The Contractility of Excised Murine Tracheas

Mice were euthanized with Ketamine-Xylazine (200 mg/kg and 10 mg/kg, respectively) and the tracheas were placed into Krebs solution (pH 7.4, 111.9 mM NaCl, 5.0 mM KCl, 1.0 mM KH2PO4, 2.1 mM MgSO4, 29.8 mM NaHCO3, 11.5 mM glucose, 2.9 mM CaCl2). The whole

trachea was then mounted in a 40 mL organ bath containing Krebs solution maintained at 37°C between 2 platinum electrodes (2 mm wide × 50 mm long). A distending force of 5 mN

Figure

Table 1.1: Asthma prevalence around the world. Data from Graham-Rowe (2011) [42].
Figure 1.1: Comparison of normal and asthmatic airways. Section A shows the position of the lungs and the airways in the body
Figure 1.2: Pulmonary function testing using spirometry. The patient inhales maximally from FRC to TLC, and then exhales rapidly to RV
Figure 1.3: Lung volumes and lung capacities. Reproduced from Gildea and McCarthy (2003) [40].
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