University of Groningen
Developmental and pathological roles of BMP/follistatin-like 1 in the lung
Tania, Navessa
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Tania, N. (2017). Developmental and pathological roles of BMP/follistatin-like 1 in the lung. University of Groningen.
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GENERAL
INTRODUCTION
Preface
The primary aim of this thesis is to explore the functional roles of an endogenous bone morphogenetic protein (BMP) antagonist, Follistatin-like 1 (Fstl1), in lung development and in the pathological processes of chronic obstructive pulmonary disease (COPD). BMP signaling plays active roles in lung development as evident in embryonic and
perinatal lethality and lung developmental defect of BMP-deficient mice.1 It is now
apparent that BMP signaling also plays crucial regulatory roles in adult lung homeostasis
by modulating inflammation and repair in the lung.2,3 However, the involvement of
BMP signaling has not been previously explored in the pathophysiology of COPD. We hypothesized that the imbalance between BMP and its antagonist Fstl1 contributes to dysregulation of early postnatal lung development, airway inflammation, and adult airway epithelial cell differentiation similar to that observed in COPD. This thesis sheds light on Fstl1 as a novel mediator of airway epithelial-driven inflammation in the lung and potentially of goblet cell metaplasia and the loss of club cells in COPD epithelium. Therefore, targeting Fstl1 is worth pursuing for the treatment of airway inflammation and epithelial remodeling in COPD.The first part of this chapter provides an overview of COPD pathophysiology, including chronic airway inflammation, airway epithelial remodeling, vascular remodeling, and currently available treatment. The second part of this chapter covers the current understanding of BMP pathway in lung development and chronic respiratory diseases. The third part of this chapter focuses on the latest discoveries on developmental and pathological roles of Fstl1. Finally, the scope of this thesis and the specific aims of our studies are described in the last part of this chapter.
1. Chronic Obstructive Pulmonary Disease (COPD)
The Global Initiative for Chronic Obstructive Lung Diseases (GOLD) guidelines stated COPD as “a common preventable and treatable disease, characterized by persistent
airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. Exacerbations and comorbidities contribute to the overall severity in individual patients’’.4 According to the World Health Organization, COPD will be the third major cause of death worldwide by 2020, surpassing stroke.5 Cigarette smoking accounts for 85-90% of COPD cases and is the best-known etiological factor for the development of COPD. However, 10-15% of COPD patients are never smokers and only about 15-20% of smokers manifest COPD,6 suggesting a role for environmental factors and susceptibility-associated (epi)genetic make-up. This has led to the concept that impaired inflammatory responses to cigarettes smoke-induced injury manifest to COPD in susceptible persons.7 Accumulating evidence from genome-wide (epi)genetic association studies discovered several loci that are strongly associated with COPD susceptibility.8–12 The major loci that associated with COPD susceptibility include HHIP (hedgehog interacting protein) and FAM13A (family with sequence similarity 13 member A) on chromosome 4 as well as CHRNA3/5 (cholinergic nicotinic acetylcholine receptor) on chromosome 15.8,9,13,14 The common symptoms of COPD include breathlessness, chronic and progressive dyspnea, chronic cough, and excessive sputum production.4 The episodes of acute worsening
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of COPD symptoms, the-so-called exacerbations, aggravate primarily in patients with
severe disease, and approximately 78% is linked with respiratory infections.4 The
molecular and cellular mechanisms underlying the symptoms and pathobiology of COPD are not completely understood. The current understanding from histopathology and current available treatment of COPD is summarized below.
1.1. The pathology of COPD
The pathology of COPD displays highly heterogeneous phenotypes involving abnormal airway wall architectures, peribronchial fibrosis, destruction of alveolar septa (emphysema), and altered airway epithelial composition, including goblet cell
metaplasia and loss of club cells.15,16 Major remodeling throughout the respiratory
tracts (including proximal, distal and peripheral airways, lung parenchyma and pulmonary vasculature) and changes in the cellular differentiation profile of the airway
epithelium collectively manifest in a progressive decline of lung function.17 The staging
of COPD that reflects disease severity is typically determined by spirometry.4 A new
hypothesis addresses the potential involvement of developmental pathways in COPD based on accumulating data from genome-wide association studies and microarray profiling, indicating dysregulation of developmental factors originally known for their
roles in embryonic development.18 BMP signaling is pivotal during lung development,
nevertheless, it has been largely unexplored in adult lung as well as the pathogenesis of COPD. BMP signaling is found decreased in the lung of pulmonary fibrosis patients and
following bleomycin injury in mice.19,20 Accordingly, accumulating evidence indicates
a marked increase of the endogenous BMP antagonist Fstl1 in the lung of patients
suffer from respiratory diseases.21–25 This thus raises the possibility that an imbalance between BMP and its endogenous antagonists may lead to airway inflammation and airway epithelial remodeling in COPD. 1.2. Persistent inflammation of the airways in COPD Chronic airway inflammation is the primary pathophysiological feature of COPD and is believed to be the major contributor of structural changes in COPD airways.17 The number of neutrophils and macrophages is positively correlated with COPD severity.26
Airway inflammation in COPD is associated with infiltration of various innate and adaptive immune cells with prolonged retention time in the respiratory tracts, primarily
in peripheral airways (bronchioles), lung parenchyma, and pulmonary arteries.27,28
Previously, infiltrating leukocytes were considered to be the key player of airway inflammation and the major factor in COPD development. However, latest discoveries show that the structural cells, such as bronchial epithelial cells, lung fibroblasts, and airway smooth muscle cells also express and secrete molecules that regulates innate
As a part of the normal repair mechanism to injury, airway epithelium of healthy individuals mediate the activation of innate immune responses via pattern recognition receptors (PRRs) to release “danger signals” which subsequently activate
Toll-like receptor (TLRs).32 Activation of TLR receptor promotes release of cytokines to
recruit inflammatory cells to the site of injured epithelium in the lung. Resolution of inflammation is responsible for switching “off” the inflammatory response when the toxic insults are successfully cleared out. However, chronic exposure to inhaled toxic compounds, such as cigarette smoke, activates airway epithelial cells to secrete a myriad of pro-inflammatory cytokines and chemokines, including interleukin-6 (IL-6), CXC-chemokine ligand 1 (CXCL1), CXCL8, CC-chemokine ligand 2 (CCL2), C-X-C-Motif ligand 8, IL-1β, tumor necrosis factor (TNF)-α and granulocyte macrophage
colony-stimulating factor (GM-CSF).33–35 Airway inflammation in COPD is associated with
persistent and exaggerated innate and adaptive inflammatory responses involving
recruitment of CD8+ T-helper-1 (Th1) cells, neutrophils, monocytes, and lymphocytes.
Inflammatory mediators released by bronchial epithelial cells following cigarette smoke-induced injury have a paracrine effect on neighboring mesenchymal cells, such as lung fibroblasts and airway smooth muscle cells.30,36 Several studies have shown that these mesenchymal cells are more than just structural cells as they express and release inflammatory mediators, like IL-6, IL-8, TNF-α, which contribute to the neutrophil and monocyte infiltration to the lung.30,31 The number of inflammatory cells, such as macrophages, neutrophils and CD8+ T cells, is increased in sputum and bronchoalveoalar lavage (BAL) of COPD patients.37,38 Neutrophil chemotactic proteins including leukotriene B4 (LTB4), IL-8, CXCL1 and CXCL8, GRO-a (growth-related oncogene-a), and ENA-78 (epithelial neutrophil-activating
protein of 78 kDa) are increased in the sputum and BAL fluid of COPD patients.39–41
Macrophages also induce release of LTB4, IL-8, TNF-α, MCP-1, and reactive oxygen species upon cigarette smoke-induced injury. These secreted factors activate and recruit neutrophils to the lung, leading to increase secretion of proteases (neutrophil elastase, proteinase-3, matrix metalloproteinases (MMP)-8, MMP-9, cathepsin
G) which is associated with emphysema.42,43 In addition, GM-CSF and granulocyte
colony-stimulating factor (G-CSF) promote neutrophil survival, proliferation, and
differentiation.44,45 The transcription factor nuclear factor-κB (NF-κB) is known to be
the central regulator of inflammatory protein expression.46 In addition, E-selectin which
mediates neutrophil adhesion to endothelial cells is increased on endothelial cells of
bronchial mucosa of COPD patients.47 Macrophages secrete chemokines interferon-c
inducible protein (IP-10), interferon-inducible T-cell chemoattractant (I-TAC), and
monokine-induced by interferon-c (Mig).48 The increased numbers of macrophages in
COPD patients can be a result of increased recruitment of circulatory monocytes and/ or increased cell proliferation and survival. Collectively, accumulation of inflammatory cells in the lung aggravates a chronic inflammatory state and results in airway structural
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Disruption of epithelial-driven innate immune functions is also strongly affected by impaired airway epithelial structure and composition following epithelial injury which will be described on the next section. Persistent airway inflammation leading to recruitment and activation of neutrophils and macrophages in COPD is illustrated in Figure 1. IL-8 IL-6 neutrophils macrophages
Recruitment and ac�va�on of innate immune cells
? Fstl1
airway smooth muscle cells lung fibroblasts
? Healthy epithelium
ciliated cells
goblet cells club cells
basal cells Chapter 5 Chapter 5 Chapter 3 ? COPD epithelium
goblet cell metaplasia
?
loss of club cells Chapter 7 BMP4
Figure 1. Persistent airway inflammation is a major hallmark of COPD pathogenesis. The healthy epithelium lining of adult airways is composed of a mixture balance of goblet cells, ciliated cells, basal cells and club cells. Cigarette smoke and inhaled toxic pollutants promotes airway epithelial remodeling, including goblet cell metaplasia, mucus hypersecretion, and loss of secretory club cells. In addition, cigarette smoke and inhaled toxic pollutants induce release of inflammatory mediators from epithelial cells with autocrine and paracrine effects on neighboring mesenchymal cells, such as fibroblasts and airway smooth muscle cells. Pulmonary mesenchymal cells secrete IL-6 and IL-8 that contributes to an infiltration of neutrophils and macrophages to the lungs. The abnormal accumulation of inflammatory cells aggravates airway inflammation in the lungs and leads to the development of COPD. The expression of Fstl1 in COPD compared to non-COPD lungs is investigated in Chapter 3. The role of Fstl1 in inflammatory cytokine release from pulmonary mesenchymal cells is shown in Chapter 5.The effect of BMP4 on adult airway epithelial cells is examined in Chapter 7.
1.3. Remodeling of airway epithelium in COPD Airway epithelium acts as the first protective barrier between the external environment and the internal lung tissue and the rest of the body. The healthy epithelium lining of adult large cartilaginous airways is made up of a layer of basal cells that are attached to the basal lamina underneath a layer of airway lumen columnar cells with a balanced mixture of goblet cells, ciliated cells and club cells. This epithelial structure is important for the mucociliary clearance function.16 In trachea, up to 60% of the total cell number
are ciliated cells and approximately 20% are mucus secreting goblet cells.51,52 The
number of ciliated cells falls to approximately 15% by the 5th airway generation. The distal airways predominantly consist of serous and secretory club cells.29 In the proximal bronchioles, the epithelial cells become more cuboidal and, in addition to ciliated cells, contain secretory club cells. Finally, in the most distal bronchioles, only club cells are identified.53 The cellular composition and function of COPD airway epithelium was found to be altered as evident by the loss of tight junctions, basal and goblet cell hyperplasia, and loss of secretory club cells and ciliated cells, resulting in loss of its barrier function and excessive mucus production.16,54 Cigarette smoke causes the goblet cell hyperplasia and the loss of ciliated cells, resulting in a reduction of mucociliary clearance and mucus plug formation. In COPD, increased numbers of goblet cells are also found in the small bronchi and bronchioles, where there are normally very few.55 The cellular events in epithelial repair after injury have been described.49,56,57 The airway epithelium is more than just a physical barrier as evidenced by the fact that bronchial epithelial cell transcribe and secretes numerous soluble factors, such as cytokines, chemokines and growth factors. Club cells are important in controlling lung inflammation by secreting a number of anti-inflammatory agents, such as secretoglobin (SCGB3A) and uroplakin (UPK3A).58 Surfactant proteins (SP-A, SP-B, SP-D), also called collectins, are produced by club cells and alveolar epithelial cells and play an important role in airway epithelial defense mechanism. At the bronchiolar level, the club cell secretory protein (CCSP), also called CC10, is able to modulate lung inflammatory and immune responses. However, club cells are reduced in bronchial biopsies from COPD
patients59,60 and in peripheral and central airways in lung tissue of COPD patients,61
which could contribute to the aggravated inflammatory process. The air-liquid culture provides a valuable system to mimic a well differentiated epithelial cells in vitro and
permits the study of the re-epithelialization after injury.62,63 Therefore, this is an
excellent tool to study epithelial cell differentiation under various condition. However, there are limited studies addressing the functional roles of BMP signaling in the adult lung epithelial cells which may enlighten the consequence of aberrant BMP signaling in the context of COPD.
1.4. Remodeling of extracellular matrix in COPD
In healthy lung, non-cartilaginous small airways (<2 mm) contribute to only ~25% to the total airway resistance, whereas in COPD lungs, it becomes the major site of increased airway resistance as a result of increased deposition of extracellular matrix,
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infiltrating inflammatory cells release inflammatory mediators that promote production of extracellular matrix from pulmonary mesenchymal cells which contribute to tissue
remodeling and respiratory dysfunction.30 Multiple studies have reported increased
extracellular matrix protein expression, such as fibronectin, collagen I, hyaluronan, and laminin in COPD airway and lung parenchyme.67–70 Airway smooth muscle cell hyperplasia and hypertrophy cause thickening of smooth muscle bundles surrounding COPD airways, whereas airway fibroblasts contribute to sub-epithelial fibrosis in the airway wall. The primary roles of pulmonary fibroblasts are tissue repair and remodeling by producing diverse extracellular matrix proteins, such as fibronectin, collagen, and airway smooth muscle actin, whereas airway smooth muscle cells are mainly responsible for airway contraction. Transforming growth factor (TGF)-β1 produced by epithelial cells in the small airways is a key player in tissue remodeling and is found to be markedly increased in COPD. It is associated with increased deposition of extracellular matrix within and surrounding the smooth muscle bundles of COPD, which results in thickened airway walls and increased in stiffness.71 An imbalance between the production and degradation of extracellular matrix could possibly explain excessive matrix deposition and may lead to airway fibrosis in COPD in addition to proliferating airway smooth muscle cells induced by cigarette smoking.72 In COPD, the number of myofibroblasts (airway fibroblasts with
a more contractile phenotype) is increased and contributes to reduction of airway
elasticity.73 Fibroblasts from individuals with COPD have reduced capacity for tissue
repair due to increased prostaglandin E (PGE) and TGF-β1 expression.74 The thickness
of small airway wall is associated with COPD severity. The obstructed small airways are characterized by abnormal architecture of epithelium and smooth muscle layers and
the presence of fibrosis.65,75
1.5. Remodeling of the pulmonary vasculature in COPD
Patients with COPD also frequently suffer from pulmonary arterial hypertension (PAH), one of the common comorbidities of COPD. PAH is characterized by increased arterial pressure in the lungs as a result of narrowing of vessel lumen due to vascular
remodeling and increased vasoconstriction.76,77 Endothelial cells are important for
vascular homeostasis by maintaining vascular tone and remodeling.78 Cigarette smoking
disrupts endothelial functions of pulmonary arteries in COPD.79,80 Endothelial cells are
key players in regulation of cell and vessel growth, thus it is believed that endothelial dysfunction initiates vascular remodeling in COPD. Endothelial dysfunction leads to uncontrolled intimal endothelial cell proliferation consequently resulting in thickened
pulmonary muscular arteries observed in smokers and patients with mild COPD.79,80
Inflamed lung tissue is hypoxic and promotes angiogenesis by increasing growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and
epidermal growth factor (EGF).77,81 These growth factors are involved in tissue repair
and vascular remodeling which eventually contribute to microvascular remodeling observed in COPD. The opposing effects of angiogenic and angiostatic factors such as chemokines (CXC family) that induce or inhibit neovascularization, respectively,
for endothelial and epithelial cell survival following injury. Numerous studies showed increased expression of VEGF and VEGF receptors (Flk-1 and Flt-1) in pulmonary muscular arteries of patients with mild and moderate COPD and in current smokers
without airway obstruction.81,83–85 An increase of angiogenic factors are also associated
with thickening of the pulmonary arterial walls.76,81,83–85 However, VEGF expression is
low in total lung homogenates of severe emphysema patients.86 It has been proposed
that low VEGF may be involved in the emphysema process through cell apoptotic mechanisms, as the lung microvascular endothelial cells and alveolar septal capillary
cells needs VEGF for cell survival.86,87 Endothelial cell death leads to loss of capillaries
may be a major mechanism of emphysema.86 Macrophages, CD8+ T lymphocytes,
neutrophils, fibroblasts that involves in immune response can promotes angiogenesis in many ways by secreting TNF-α which also is an angiogenic factor. Additionally, the protein expression of endothelin-1 (ET-1) and ET-1 receptor ETA that mediate vascular smooth muscle conctration is increased in PAH.88,89 1.6. Current treatments Currently, there is no effective cure for COPD. Inhaled corticosteroids (e.g. fluticasone, budesonide), phosphodiesterase 4 inhibitors (roflumilast), short-acting (e.g. albuterol) or long-acting b2-adrenoreceptor agonists (e.g. salmeterol, formoterol), and long-acting anti-cholinergics (e.g. tiotropium, ipratropium, glycopyrronium bromide, umeclidinium) are used alone or in combination to relieve the symptoms and inflammation.90 However, these therapies do not change the course of the disease.90 Therefore, understanding
the molecular mechanism of COPD is undoubtedly important to identify a novel molecular target that can decelerate the progression of COPD. A better understanding of impaired repair process which contributes to COPD may lead to the identification of new molecular targets and more effective therapy options in COPD treatment.
2. Bone morphogenetic protein (BMP) signaling in the lung
In 1965, Marshall R. Urist discovered osteogenic factors present in bone matrix extracts capable of promoting de novo cartilage formation and bone formation in vivo,
namely bone morphogenetic protein (BMP).91,92 BMP signaling belongs to the TGF-β
superfamily, which is phylogenetically conserved.91 Genetic studies in mice demonstrate
the importance of BMP signaling in embryonic dorsal-ventral paterning93–95 and
embryonic lung development.3,96,97 It is now evident that BMPs govern diverse cellular
effects including stem cell maintenance, migration, differentiation, proliferation, and
programmed cell death.98 A growing body of evidence indicates the involvement of BMP
signaling in normal lung physiology and pathology conditions in adulthood, strongly suggesting that BMP signaling also plays a pivotal role in adult lung homeostasis,
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2.1. Players in BMP signaling 2.1.1. BMP ligands
BMPs (which are also referred to as growth and differentiation factors (GDFs)) are
secreted glycosylated proteins, with 20 different members identified and characterized.98
BMP polypeptides consist of an N-terminal secretory signal peptide, a prodomain that ensures proper folding, and a C-terminal mature peptide. Mature BMPs are derived from cleavage of the BMP precursors by serine endoproteases in the trans-Golgi network and subsequently secreted. Homo or heterodimerization of BMP ligands forms an active BMP agonist. Secreted BMPs bind either to the extracellular matrix, extracellular antagonists, co-receptors or to transmembrane receptors. Activation of BMP signal transduction leads to either transcriptional responses (e.g. Smad-dependent signaling) or non-transcriptional responses (e.g. Smad-independent signaling, cytoskeletal
rearrangements).98
2.1.2. BMP receptors and co-receptors
The BMP receptors are composed of two types of transmembrane serine/threonine kinase receptors, type I and type II receptors, which are highly homologous but not redundant, and are capable of activating both dependent and Smad-independent signaling. The receptors encompass an extracellular ligand binding domain and a cytoplasmic serine/threonine kinase domain. The type I receptor has two additional motifs in its kinase domain, a glycine/serine-rich region prior the GS-box kinase domain and a L45 loop composed of eight amino acids.99 Type I receptors include activin receptor-like kinase 1 (ALK1), activin receptor type Ia (ActRIa or ALK2), BMP receptor type Ia (BMPRIa or ALK3) and BMP receptor type Ib (BMPRIb or ALK6). These receptors form heteromeric complexes with the type II receptors, including BMP receptor type II (BMPRII) and activin receptors type IIA (ACVRIIA also known as ActRIIA)
and activin receptors type IIB (ACVRIIB also known as ActRIIB).100 The specificity of
BMP signaling is tightly controlled by selective extracellular regulators, receptors, co-receptors, and cytosolic receptor-associated proteins to exert specific BMP functions which will be described in more detail below. Activation of BMP signaling is preceded by oligomerization of BMP ligands and type I receptor kinase. Activated type I receptor undergo transphosphorylation of its GS-box via constitutively active type II BMP kinase receptors.98,99 After the formation of a heterotetrameric complex, the signal propagates
to the nucleus either in intracellular regulator Smad-dependent (canonical) or Smad-independent (non-canonical) manner.98 Co-receptors that positively regulates BMP
signaling are the repulsive guidance molecules (RGM)-a and -c, Dragon (RGMb), the receptor tyrosine kinase (RTK) c-Kit, and the TGF-β1/BMP type III receptors Endoglin and Betaglycan. In contrast, BMP signaling is negatively regulated by the pseudoreceptor
2.1.3. Downstream BMP/Smad-dependent signaling
In the canonical pathway, the transduction of extracellular signals to regulation of
gene transcription inside the nucleus is mediated by Smad proteins.99 There are three
types of Smad proteins, BMP receptor-regulated Smads (R-Smads), common mediator Smad (co-Smad), and inhibitory Smads (I-Smads). BMP ligands bind to the type I BMP receptors resulting in phosphorylation of R-Smads (Smad1/5/8), followed by trimeric complex formation with co-Smad (Smad4), and translocation of the complex into the
nucleus,98,100,101 which subsequently induces transcription of BMP downstream target
genes,102 presumably in collaboration with other co-repressors and co–activators.103
I-Smads (Smad6 and Smad7) negatively regulate BMP/Smad signaling by preventing phosphorylation of R-Smads, instead targeting R-Smads and type I receptor for ubiquitin– proteasome degradation. Two conserved domains of R-Smads are the N-terminal DNA and protein binding MH1 (mad homology 1) domain and the C-terminal MH2 domain which are connected by a variable proline-rich linker region.104,105 2.1.4. Downstream BMP/Smad-independent signaling In addition to canonical BMP signaling, BMP ligands activate gene transcription in a Smad-independent manner. BMP ligands can activate the MAPK pathway by recruitment of TAK1, TAB1, and XIAP to type I BMP receptors, which subsequently phosphorylate p38 MAPK or JNK.106 Additionally, ERK mediates transcription of BMP2-regulated genes.107
BMP regulates sets of genes which are involved in lung and vascular homeostasis, including epithelial and vascular remodeling. BMP regulated genes, including jagged1,
GATA2, and endoglin, have been identified in endothelial cells.102 BMPs can also activate
TGF-β activated kinase 1 (TAK1) leading to p38/JNK/Erk MAP kinase activation.108,109
TAK1 mediates phosphorylation of Smad1 at C-terminal serine residues, suggesting it functions as an upstream activating kinase for Smad1/5/8 in BMP signaling.109 2.1.5. Fine tuning of BMP signaling BMP functions are tightly modulated by BMP antagonists, co-receptors, and intracellular regulatory proteins. BMP antagonists function through direct binding with BMP ligands and prevent BMPs from binding to their transmembrane receptors. Pseudo-receptors sequester BMP ligands at the cell surface. Co-receptors and intracellular regulatory proteins interact with the receptors to regulate downstream BMP functioning. The spatiotemporal interplay between BMP and its regulators govern developmental and cellular processes. BMP antagonists are cysteine knot containing motif which form a functional homodimer. BMP antagonists bind selectively to distinct BMP ligands in a context- and concentration-dependent manner to maintain BMP gradients, which have been extensively studied during embryonic lung development. BMP antagonists include Noggin, gremlin, twisted gastrulation (Tsg), and Fstl1. Antagonists are categorized based on their cysteine knot motif: the Chordin/Noggin family contains a ten-membered cysteine ring, Tsg contains a nine-membered cysteine ring and the DAN/Cerberus family
contains an eight-membered cysteine ring.110 Molecular regulation of BMP signaling
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2.2. BMP signaling in lung development and adult lung homeostasis
Lung development is orchestrated by precise coordination of several molecular pathways, including BMP signaling. The canonical BMP signaling is important in lung Extracellular Cytoplasm JNK MAPK TAK1 Nucleus ON Smad-dependent Smad-independent Erk p38 Smad1/5P Smad1/5 Smad4 P Smad1/5P Smad1/5 Smad4 P ON
type III/ co-receptor (e.g.: Endoglin) BMP Gremlin type I type II ALK1 ALK2 BMPRII ALK3 ACVRIIA ACVRIIB P P ALK6 Extracellular regulators (e.g.: Fstl1)
Progenitor cell maintenance Differentiation
BRE BRE
Proliferation
Figure 2. Molecular regulation of BMP signaling. Activation of BMP signaling is initiated by binding of BMP ligands to the type I BMP receptors (ALK1, 2, 3, 6) followed by recruitment of constitutively active type II BMP receptor receptors (BMPRII, ACVRIIA, ACVRIIB) to form a signal-activating complex. In cytoplasm, the signal propagates either via Smad-dependent or Smad-independent manner. In a Smad-dependent signaling, the transduction of BMP signals is mediated by Smad1/5 proteins. Phosphorylated Smad1/5 proteins form a complex with Smad4 protein which subsequently translocate to nucleus and bind to a 52 base-pair BMP response element (BRE) on the promoter region of the target genes. In a non-Smad signaling, BMP ligands can activate the MAPK pathway by recruitment of TAK1 to type I BMP receptors, which subsequently phosphorylate Erk/JNK/p38. Thereby, phosphorylated Erk/JNK/p38 can translocate into the nucleus. Among others, BMP functions are tightly regulated by co-receptors (e.g. Endoglin) and extracellular regulators (e.g. Fstl1).
development.3 Fundamental roles of BMP signaling in lung development are evident from BMP mutations associated with congenital lung defects in human and genetic studies in transgenic mice and familial mutation in PAH. Deletion of lung epithelial-specific Smad1 resulted in impaired airway branching morphogenesis, decreased sacculation, disruption of distal lung epithelial cell proliferation and differentiation which contribute to severe postnatal respiratory distress. Wu et al. also revealed that BMP4/Smad1 signaling modulates other molecular pathways including Wnt/β-catenin by expressing Wnt inhibitory factor 1 (Wif1) in the developing fetal mouse lung.111 Overexpression of BMP4 in the epithelium, on the other hand, leads to smaller
lungs and to a marked decrease in epithelial cell proliferation.97 In adults, lung tissue homeostasis is required for continuous regeneration and subsequent differentiation of stem cells in order to maintain lung architecture and proper functions. BMP signaling maintains an undifferentiated state of the airway and alveolar epithelial progenitors for lung epithelial homeostasis and tissue injury repair.3 A tight and precise regulation of pulmonary vascular development is required for normal lung development.112 Canonical BMP signaling plays key roles during embryonic development of the pulmonary vasculature.3 Activation of BMPRII promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. BMPRII is a novel mediator of endothelial nitric-oxide synthase activation.113 2.3. BMP signaling in lung diseases Dysregulation of BMP signaling has been implicated in several lung diseases. Although in adult lung tissue, active canonical BMP signaling is hardly detectable.3 Reactivation of canonical BMP signaling is observed after lung injury with naphthalene and bleomycin in the same pattern as early lung development in bronchial and alveolar epithelial
cells, respectively, presumably as part of repair processes.3 In addition, BMP ligands
expression and BMP/Smad activity are upregulated inflamed bronchial epithelium of
allergic asthma.114,115 It has been demonstrated that activation of BMP signaling induces
expression of its own antagonist, implying a negative feedback loop in the regulation of BMP signaling. The expression of the BMP4 antagonist gremlin was found to be
upregulated in patients with idiopathic pulmonary fibrosis (IPH).116 Additionally, the
expression of BAMBI is increased in plasma and lung of COPD patients.117,118 The role
of BMP in lung cancer is contradictive. In one hand, BMP2 is increased along with activation of Smad1/5 and its downstream target Id1 and promote tumor growth in
human lung cancer.119 On the other hand, low levels of BMP7 in lung cancer tissues
are correlated with lymph node metastasis. In vitro, BMP7 negatively regulates cell
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2.4. BMP signaling in pulmonary vascular diseases
BMP signaling is important in embryonic vascular development and adult vascular homeostasis. Consequently, dysregulation of BMP signaling has been strongly associated
with the pathogenesis of hereditary vascular diseases, including familial PAH.121 BMP
signaling regulates vascular smooth muscle maturation, endothelial cell proliferation and
angiogenesis.122 Endothelial cells are a major player in vascular homeostasis. Endothelial
dysfunction, including abnormal proliferation, impaired endothelial mesenchymal communication, decreased endothelial nitric-oxide synthase (eNOS) activity, and loss
of bioactive nitric oxide (NO), play a prominent role in the development of PAH.113,123
Accumulating studies from genetic association study in hereditary vascular diseases and genetic studies in transgenic mice pinpoint the mutation of BMP components to
be strongly associated with PAH.124,125 Most importantly, BMP2 and BMP4 failed to
stimulate eNOS phosphorylation in PAECs derived from patients carry mutations in the
BMPRII gene.113 This is supported by animal studies of lung endothelial-specific BMPR2
KO mice resulting in spontaneous PAH.126 Interestingly, impaired BMP signaling was
also observed in common forms of PAH, including hypoxic PAH.127 Mechanistically, the negative regulator of vascular smooth muscle cell proliferation and pro-apoptotic, BMP2 and its downstream non-canonical BMP signaling are upregulated after acute hypoxia exposure. In contrast, prolonged hypoxia exposure results in downregulation of BMPRII expression and inactivation of its downstream signaling in pulmonary vasculature.127 In addition, the expression of BMPRIb, BMPRII, and Smad4, 5, 6, and 8 was decreased in accordance with reduced levels of active Smad1 and Smad-regulated genes id1 and id3 in lungs of monocrotaline model of PAH.128 Furthermore, the expression of the BMP antagonist gremlin 1 is increased in small pulmonary vessel walls of hypoxic PAH mouse models.129 Taken together, these studies demonstrate a central role for BMP and its inhibitors in pulmonary vascular remodeling and the pulmonary vascular resistance in hypoxic PAH.129 3. Follistatin-like 1 During lung development and adult homeostasis processes, the functions of BMPs are tightly controlled for proper morphogenesis. Although the expression of BMPs and their receptors is not strictly limited to certain areas, the expression of BMP antagonists is spatiotemporally tightly controlled, indicating that BMP antagonists fine tune the level of BMP functioning and act as molecular switches of BMP signaling.1 The endogenous
antagonist of BMP, Fstl1 (also known as TGF-β-stimulated clone 36 (TSC-36)), was originally identified as a TGF-β inducible gene in a mouse osteoblastic cell line with two isoforms.130,131Fstl1 is a secreted cysteine-rich glycosylated protein of 38 kDa containing an follistatin (FS)-like domain containing 10 conserved cysteine residues and a strong similiarity with BM-40/osteonectin/SPARC family.130 Human Fstl1 is highly conserved with >92% amino acid sequence identity with rat and mouse.132 The Fstl1 orthologues have been identified and sequenced from human, rat, mouse, avian, xenopus, macaques, and fish.132–136 The crystal structure of a truncated form of recombinant Fstl1 containing follistatin-like domain has been resolved.137 Fstl1 is composed of three distinct domains,
including follistatin N-terminal domain-like (FOLN), extracellular calcium (EC)-binding domain containing two EF-hand motifs, and a von Willebrand factor type C domain (VWC) with cell type specific glycosylation. However, unlike other family members, the calcium-binding domains of Fstl1 are non-functional.131 3.1. Cellular expression of Fstl1 During embryonic development, Fstl1 mRNA is detected in different organs, but the highest expression is found in the lung, predominantly in mesenchymal cells, including vascular and airway smooth muscle cells,138,139 but also in endothelial cells and goblet cells of airway epithelium. In adult tissue, Fstl1 has been proposed as a cardiokine,140–143
myokine,144,145 adipokine,146 and, osteogenic factor147 due to it is ubiquitous expression
and functions in the heart, muscle, bone, skeletal muscle tissue,144,145,148 neurons,149
and trophoblasts.150 The effects of Fstl1 on its downstream targets and the upstream
factors known to regulate Fstl1 expression has been reviewed.151 In response to divers
factors, such as TGF-β1, IL-1β, TNF-α and IL-6, the expression of Fstl1 gene was found to be highly expressed in mesenchymal lineages, including fibroblasts, cardiomyocytes,
chondrocytes, adipocytes, and osteocytes.151–153However, recent studies showed
that Fstl1 is also expressed by hematopoietic lineages, such as bone
marrow-derived osteoclasts147 and lung alveolar macrophages.24IL-13 triggers lung alveolar
macrophages to express a significant increase of Fstl1 mRNA, whereas TGF-β and
Fstl1 increase Fstl1 expression in lung epithelial cells.22,24 Furthermore, Fstl1 triggers
inflammatory cells, ranging from monocytes, macrophages, and T-cells to express and release pro-inflammatory cytokines and chemokines, including IL-1β, TNF-α, IL-6,
IFN-γ, IL-8, MCP-1, and IP-10.151,154,155 Among others, expression of Fstl1 is negatively
regulated by microRNAs (miRs), including miR-198,156 miR-27a,157 miR-21,158
and miR-206.159Although Fstl1 is primarily known as a BMP antagonist,21,160,161 current findings suggest that Fstl1 may also regulate various physiological processes by interacting with different receptors, including disco-interacting protein 2 homolog A (DIP2A),162–164 toll-like receptor 4 (TLR4),165,166 and the sodium-potassium pump.149 3.2. Fstl1 signaling in lung and pulmonary vascular development Fstl1 is expressed in the developing lung.25,138 Embryonic knockout of Fstl1 in mice results in an extensive distortion in lung morphology and neonatal lethality due to respiratory
failure, demonstrating the functional importance of Fstl1 in lung development.160,161
Whole body knockout of Fstl1 exhibits smaller and abnormal lung morphology. Histological analysis demonstrated thickening of alveolar walls and reduction in airspaces and impaired alveolar epithelial differentiation as seen in reduction in mature
surfactant protein level.160,161 In addition to embryonic lung morphogenesis,25,138,151,160,161
the crucial roles of Fstl1 in organogenesis have also been shown in other organs,
including brain,167 heart,168 and dorsal/ventral axis patterning.169 However, the role of
1
3.3. Physiological roles of Fstl1
Several studies reported that Fstl1 has diverse biological functions, including regulating
cell proliferation,170–172 differentiation,171 migration , invasion,157 wound healing,156
tissue repair,143,173,174 cell fate determination,139 and innate immune responses.172 The
roles of Fstl1 in tissue repair in response to injury have been most widely studied in the cardiovascular system. Fstl1 was found to be increased in models of acute and chronic heart injury, including myocardial infarction, pressure overload-induced
hyperthrophy, and ischemia/reperfusion injury.140,173,175,176 Systemic administration of
Fstl1 or overexpression of Fstl1 showed to be protective in the cardiovascular system by reducing cell apoptosis and inflammatory responses, and promoting endothelial cell survival and trigger a revascularization process and restore tissue function.140,148,176,177 Recent studies demonstrated that Fstl1 expression is also induced in response to lung injury.21,23,158 Further investigation is needed to unravel the impact of upregulation of Fstl1 in the lung. Collectively, these data suggest that the injury-induced upregulation of Fstl1 play a clinically relevant role in the modulation of pathological processes. Furthermore, previous studies demonstrate paracrine signaling of Fstl1 in cell and
tissue communication.141,142,144,148,172 However, the roles of secreted Fstl1 in cell-cell
communication in the lung have not been previously studied. 3.4. The pathological roles of Fstl1 in inflammatory diseases
The function of Fstl1 has been most extensively studied in inflammatory diseases and a dual role of Fstl1 in inflammatory responses has been reported. It is clear that Fstl1 modulates inflammatory cytokine expression and release. However, the precise effect of Fstl1 in inflammatory responses, whether as a pro-inflammatory or an anti-inflammatory mediator remain controversial. A great body of evidence identifies Fstl1 as a pro-inflammatory cytokine that is induced by inflammatory cytokines, in turn Fstl1 triggers inflammatory responses from target cells, particularly inflammatory
cells.178 Several studies have shown that Fstl1 is markedly upregulated in inflammatory
conditions.179–181 Furthermore, silencing of Fst1 decreases the production of TNF-α,
IL-6, and IL-8 from human arterial endothelial cells induced by oxidized low-density
lipoprotein.166 Additionally, high Fstl1 is observed in response to bacterial infection182
and low degree of inflammation in obese individuals.183
An anti-inflammatory role for Fstl1 is supported by other studies. Mice treated with recombinant human Fstl1 show a decreased expression of IL-6, MMP3, and MMP9 in animal models of arthritis.184,185 Immuno suppressant roles of Fstl1 have been also demonstrated in organ transplantation, and tissue injury.166,186,187 These discrepancies may be explained by the differences in the experimental conditions and different organs. However, the inflammatory roles of Fstl1 in COPD have not been previously explored. Furthermore, Fstl1 has been proposed as a biomarker in several diseases, including
systemic-onset juvenile rheumatoid arthritis,153 osteoarthritis,188 systemic juvenile
idiopathic arthritis,181 chronic systolic heart failure,189 acute coronary syndrome,190
3.5. The pathological roles of Fstl1 in airway remodeling in the lung
Recent studies showed upregulation of Fstl1 expression in the lungs patients with severe
asthma and idiopathic pulmonary fibrosis.22,23 Further in vivo studies demonstrated
that Fstl1 haploinsufficient mice and specific inactivation of Fstl1 in myeloid cell lineages attenuate airway remodeling in animal models of asthma and lung fibrosis,
respectively.21,22 Studies from the same group demonstrated that recombinant Fstl1
induces lung macrophages to express pro-remodeling factor MMP9 mRNA and protein
through TLR4 receptor activation.24 Conversely, the other study showed that Fstl1
attenuates MMP9 expression in animal model of arthritis.185
3.6. The pathological roles of Fstl1 in airway remodeling in cancer
Fstl1 has been implicated in several cancer, including lung cancer,192–194 pancreatic
cancer,195 bone metastasis,196,197 ovarian cancer and endometrial carcinogenesis,198,199
nasopharyngeal carcinoma,172 glioblastoma,200 and prostate cancer.201 However, the role
of Fstl1 in cancer is complex and controversial.
On one hand, Fstl1 has been reported as a pro-cancer metastasis. On the other hand, Fstl1 shows an anti-cancer effect. High expression of Fstl1 is associated with poor
prognosis of glioblastoma200 and favors the progression of metastatic prostate cancer.201
Therefore, Fstl1 has been proposed as therapeutic target for prostate cancer.202
Targeting Fstl1 prevents bone metastasis by regulating the immune function, suggesting that Fstl1 can mediate cancer cell invasion.197,203 Fstl1 is significantly downregulated in metastatic kidney cancer. Fstl1 mRNA is significantly lower in cancerous tissue compared to adjacent normal tissue.204 A recent study demonstrated an association between Fstl1 polymorphisms and renal cell carcinoma (RCC) risk and prognosis, suggesting that the variant genotype CC of rs1259293 is associated with RCC development by suppressing
Fstl1 expression.205 Fstl1 has been suggested as putative tumor suppressor gene as
decreased Fstl1 has been reported,206 including in kidney cancer,204 lung cancer,192,194
ovarian and endometrial cancer,198,199 nasopharyngeal carcinoma,172and bone metastasis
of prostate cancer.196 Hypermethylation of the Fstl1 promoter is observed in cancer, leading to cell proliferation and invasion.172 Re-expression of Fstl1 and Fstl1 treatment negatively regulates lung cancer cell invasion and metastasis193,207 and result in growth inhibition of human lung cancer cell lines. Fstl1 transcripts are not detectable in highly aggressive and proliferative lung cancer cells and overexpression of Fstl1 shows an anti-proliferative effect.192 Tumor suppressant effects of Fstl1 in ovarian and endometrial
carcinogenesis has also been suggested.198 Knockdown of Fstl1 induces apoptosis
through a mitotic arrest and caspase-dependent cell death, suggesting that Fstl1 plays
1
Scope of the thesis
The notion that developmental signaling pathways are reactivated during chronic lung disease attracted scientists to dissect the roles of these pathways related with the pathophysiology of COPD. However, little is known about the involvement of BMP signaling
in COPD. BMP/Fstl1 signaling is a key player in embryonic lung morphogenesis,85,86 with
diverse physiological roles including cellular communication.139,142,172 Recent studies
demonstrate that aberrant Fstl1 expression is associated with several pathological
lung conditions21–24,158 and inflammatory disorders,154,155,180 underscoring its great
importance in adult tissue homeostasis and repair. However, the involvement of Fstl1 in COPD remains unexplored. This thesis will delineate the role of the endogenous BMP antagonist Fstl1 in lung development and shed light on its potential contribution to the pathogenesis of COPD. The specific aims of this thesis are to investigate the role of Fstl1 in pulmonary vasculature development and COPD, particularly in airway epithelial-mesenchymal cell communication and adult tracheal epithelial cell differentiation. To this aim, the developmental roles of Fstl1 were studied in conditional endothelial-specific Fstl1 knockout mice and in vitro study using pulmonary cell lines, including bronchial epithelial cells, lung fibroblasts, and airway smooth muscle cells.
In Chapter 2, the role of endothelial Fstl1 in the postnatal development of the
pulmonary vasculature was investigated. The changes in molecular signaling, lung physiology, and morphology of pulmonary vessels were dissected in endothelial-specific Fstl1 knockout mice compared to littermate controls.
Our investigation of Fstl1 expression and BMP/Smad activation in lung tissue of
COPD patients is presented in Chapter 3, providing evidences of dysregulation of Fstl1
expression in different parts of COPD lung tissue.
Chapter 4 provides a perspective on the expression levels and contribution of
the TGF-β superfamily members, activin-A and its endogenous follistatin antagonist in smoking-induced airway inflammation in COPD both in vitro and in vivo.
The role of Fstl1 in inflammatory cytokine release from lung mesenchymal cells
was explored in Chapter 5, which provides evidences on pro-inflammatory roles of Fstl1
1 in coordinating epithelial-mesenchymal communication in inflammatory cytokine release in the lung. We identified epithelial-derived factors in culture supernatants of Fstl1 overexpressing bronchial epithelial cells which mediate Fstl1-conditioned media-induced IL-6 and IL-8 release from lung fibroblasts.
Chapter 6 describes the latest understanding of the contribution of pulmonary
mesenchymal cells in modulating airway inflammation in various lung diseases.
Furthermore, the effect of BMP4 in differentiation of primary adult tracheobronchial
epithelialcells using air-liquid interface culture was studied in Chapter 7. The potential
implications of aberrant BMP signaling related with airway epithelial remodeling in COPD lungs was discussed.
References
1. Yanagita M. BMP antagonists: their roles in development and involvement in pathophysiology. Cytokine Growth Factor Rev. 2005;16(3):309-317. doi:10.1016/j.cytogfr.2005.02.007.
2. Rock J, Königshoff M. Endogenous lung regeneration: potential and limitations. Am J Respir Crit Care Med. 2012;186(12):1213-1219. doi:10.1164/rccm.201207-1151PP.
3. Sountoulidis A, Stavropoulos A, Giaglis S, et al. Activation of the canonical bone morphogenetic protein (BMP) pathway during lung morphogenesis and adult lung tissue repair. PloS One. 2012;7(8):e41460. doi:10.1371/journal.pone.0041460.
4. Vestbo J, Hurd SS, Agustí AG, et al. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med. 2013;187(4):347-365. doi:10.1164/rccm.201204-0596PP.
5. Lopez AD, Murray CCJL. The global burden of disease, 1990–2020. Nat Med. 1998;4(11):1241-1243. doi:10.1038/3218.
6. Corhay J-L, Frusch N, Louis R. [COPD: genetics and environmental interactions]. Rev Médicale Liège. 2012;67(5-6):292-297.
7. Rovina N, Koutsoukou A, Koulouris NG. Inflammation and Immune Response in COPD: Where Do We Stand? Mediators Inflamm. 2013;2013:e413735. doi:10.1155/2013/413735.
8. Pillai SG, Kong X, Edwards LD, et al. Loci identified by genome-wide association studies influence different disease-related phenotypes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010;182(12):1498-1505. doi:10.1164/rccm.201002-0151OC.
9. van der Plaat DA, de Jong K, Lahousse L, et al. Genome-wide association study on the FEV1/FVC-ratio in never-smokers identifies HHIP and FAM13A. J Allergy Clin Immunol. September 2016. doi:10.1016/j.jaci.2016.06.062.
10. Morrow JD, Cho MH, Hersh CP, et al. DNA methylation profiling in human lung tissue identifies genes associated with COPD. Epigenetics. August 2016:0. doi:10.1080/15592294.2016.122645.
11. Hobbs BD, Parker MM, Chen H, et al. Exome Array Analysis Identifies a Common Variant in IL27 Associated with Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med. 2016;194(1):48-57. doi:10.1164/rccm.201510-2053OC.
12. Bruse S, Moreau M, Bromberg Y, et al. Whole exome sequencing identifies novel candidate genes that modify chronic obstructive pulmonary disease susceptibility. Hum Genomics. 2016;10:1. doi:10.1186/s40246-015-0058-7.
13. Cho MH, Boutaoui N, Klanderman BJ, et al. Variants in FAM13A are associated with chronic obstructive pulmonary disease. Nat Genet. 2010;42(3):200-202. doi:10.1038/ng.535.
14. Pillai SG, Ge D, Zhu G, et al. A genome-wide association study in chronic obstructive pulmonary disease (COPD): identification of two major susceptibility loci. PLoS Genet. 2009;5(3):e1000421. 15. Barnes PJ. Chronic Obstructive Pulmonary Disease. N Engl J Med. 2000;343(4):269-280. doi:10.1056/NEJM200007273430407.
16. Shaykhiev R, Crystal RG. Early events in the pathogenesis of chronic obstructive pulmonary disease. Smoking-induced reprogramming of airway epithelial basal progenitor cells. Ann Am Thorac Soc. 2014;11 Suppl 5:S252-258. doi:10.1513/AnnalsATS.201402-049AW.
17. Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 2008;8(3):183-192. doi:10.1038/nri2254.
18. Shi W, Chen F, Cardoso WV. Mechanisms of lung development: contribution to adult lung disease and relevance to chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2009;6(7):558-563. doi:10.1513/pats.200905-031RM.
19. Myllärniemi M, Lindholm P, Ryynänen MJ, et al. Gremlin-mediated decrease in bone morphogenetic protein signaling promotes pulmonary fibrosis. Am J Respir Crit Care Med. 2008;177(3):321-329. doi:10.1164/rccm.200706-945OC.
20. De Langhe E, Cailotto F, De Vooght V, et al. Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis. Respir Res. 2015;16:38. doi:10.1186/s12931-015-0202-x.
21. Dong Y, Geng Y, Li L, et al. Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice. J Exp Med. 2015;212(2):235-252. doi:10.1084/jem.20121878.
22. Miller M, Beppu A, Rosenthal P, et al. Fstl1 Promotes Asthmatic Airway Remodeling by Inducing Oncostatin M. J Immunol Baltim Md 1950. September 2015. doi:10.4049/jimmunol.1501105.
1
23. Murphy N, Gaynor KU, Rowan SC, et al. Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis. Am J Pathol. 2016;186(3):600-615. doi:10.1016/j.ajpath.2015.10.032.
24. Miller M, Esnault S, Kurten RC, et al. Segmental allergen challenge increases levels of airway follistatin-like 1 in patients with asthma. J Allergy Clin Immunol. 2016;138(2):596-599.e4. doi:10.1016/j. jaci.2016.01.019.
25. Li X, Fang Y, Li X, et al. Apical Secretion of FSTL1 in the Respiratory Epithelium for Normal Lung Development. PloS One. 2016;11(6):e0158385. doi:10.1371/journal.pone.0158385.
26. Di Stefano A, Capelli A, Lusuardi M, et al. Severity of airflow limitation is associated with severity of airway inflammation in smokers. Am J Respir Crit Care Med. 1998;158(4):1277-1285. doi:10.1164/ ajrccm.158.4.9802078.
27. Saetta M, Di Stefano A, Turato G, et al. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1998;157(3 Pt 1):822-826. doi:10.1164/ajrccm.157.3.9709027.
28. Saetta M, Baraldo S, Corbino L, et al. CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1999;160(2):711-717. doi:10.1164/ ajrccm.160.2.9812020.
29. Thorley AJ, Tetley TD. Pulmonary epithelium, cigarette smoke, and chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2007;2(4):409-428.
30. Alkhouri H, Poppinga WJ, Tania NP, Ammit A, Schuliga M. Regulation of pulmonary inflammation by mesenchymal cells. Pulm Pharmacol Ther. 2014;29(2):156-165. doi:10.1016/j.pupt.2014.03.001. 31.
Gosens R, Rieks D, Meurs H, et al. Muscarinic M3 receptor stimulation increases cigarette smoke-induced IL-8 secretion by human airway smooth muscle cells. Eur Respir J. 2009;34(6):1436-1443. doi:10.1183/09031936.00045209.
32. Beg AA. Endogenous ligands of Toll-like receptors: implications for regulating inflammatory and immune responses. Trends Immunol. 2002;23(11):509-512. doi:10.1016/S1471-4906(02)02317. 33. Mio T, Romberger DJ, Thompson AB, Robbins RA, Heires A, Rennard SI. Cigarette smoke
induces interleukin-8 release from human bronchial epithelial cells. Am J Respir Crit Care Med. 1997;155(5):1770-1776. doi:10.1164/ajrccm.155.5.9154890.
34. Mortaz E, Henricks P a. J, Kraneveld AD, Givi ME, Garssen J, Folkerts G. Cigarette smoke induces the release of CXCL-8 from human bronchial epithelial cells via TLRs and induction of the inflammasome. Biochim Biophys Acta. 2011;1812(9):1104-1110. doi:10.1016/j.bbadis.2011.06.002.
35. Hellermann GR, Nagy SB, Kong X, Lockey RF, Mohapatra SS. Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells. Respir Res. 2002;3:22.
36. Osei ET, Brandsma C-A, Noordhoek JA, Timens W, Postma D, Heijink I. Crosstalk between epithelium and fibroblasts; implications for COPD. Eur Respir J. 2014;44(Suppl 58):P3899.
37. Keatings VM, Barnes PJ. Granulocyte activation markers in induced sputum: comparison between chronic obstructive pulmonary disease, asthma, and normal subjects. Am J Respir Crit Care Med. 1997;155(2):449-453. doi:10.1164/ajrccm.155.2.9032177.
38. Pesci A, Balbi B, Majori M, et al. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur Respir J. 1998;12(2):380-386.
39. Traves S, Culpitt S, Russell R, Barnes P, Donnelly L. Increased levels of the chemokines GROα and MCP-1 in sputum samples from patients with COPD. Thorax. 2002;57(7):590-595. doi:10.1136/ thorax.57.7.590.
40. Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med. 1996;153(2):530-534. doi:10.1164/ajrccm.153.2.8564092.
41. Nocker RE, Schoonbrood DF, van de Graaf EA, et al. Interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease. Int Arch Allergy Immunol. 1996;109(2):183-191.
42. Beeh KM, Beier J. Handle with care: targeting neutrophils in chronic obstructive pulmonary disease and severe asthma? Clin Exp Allergy J Br Soc Allergy Clin Immunol. 2006;36(2):142-157. doi:10.1111/ j.1365-2222.2006.02418.x.
functions in lung chronic inflammatory diseases (cystic fibrosis and chronic obstructive pulmonary disease). J Aerosol Med Pulm Drug Deliv. 2008;21(1):125-144. doi:10.1089/jamp.2007.0653. 44. Gomez-Cambronero J, Horn J, Paul CC, Baumann MA. Granulocyte-macrophage colony-stimulating
factor is a chemoattractant cytokine for human neutrophils: involvement of the ribosomal p70 S6 kinase signaling pathway. J Immunol Baltim Md 1950. 2003;171(12):6846-6855.
45. Hamilton JA. GM-CSF in inflammation and autoimmunity. Trends Immunol. 2002;23(8):403-408. doi:10.1016/S1471-4906(02)02260-3.
46. Di Stefano A, Caramori G, Oates T, et al. Increased expression of nuclear factor-kappaB in bronchial biopsies from smokers and patients with COPD. Eur Respir J. 2002;20(3):556-563.
47. Di Stefano A, Maestrelli P, Roggeri A, et al. Upregulation of adhesion molecules in the bronchial mucosa of subjects with chronic obstructive bronchitis. Am J Respir Crit Care Med. 1994;149(3 Pt 1):803-810. doi:10.1164/ajrccm.149.3.7509705.
48. Saetta M, Mariani M, Panina-Bordignon P, et al. Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2002;165(10):1404-1409.
49. Puchelle E, Zahm J-M, Tournier J-M, Coraux C. Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2006;3(8):726-733. doi:10.1513/pats.200605-126SF.
50. Yoshida T, Tuder RM. Pathobiology of cigarette smoke-induced chronic obstructive pulmonary disease. Physiol Rev. 2007;87(3):1047-1082. doi:10.1152/physrev.00048.2006.
51. Wanner A, Salathé M, O’Riordan TG. Mucociliary clearance in the airways. Am J Respir Crit Care Med. 1996;154(6 Pt 1):1868-1902. doi:10.1164/ajrccm.154.6.8970383.
52. Lozewicz S, Wells C, Gomez E, et al. Morphological integrity of the bronchial epithelium in mild asthma. Thorax. 1990;45(1):12-15.
53. Kotton DN, Morrisey EE. Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat Med. 2014;20(8):822-832. doi:10.1038/nm.3642.
54. Brune K, Frank J, Schwingshackl A, Finigan J, Sidhaye VK. Pulmonary epithelial barrier function: some new players and mechanisms. Am J Physiol - Lung Cell Mol Physiol. 2015;308(8):L731-L745. doi:10.1152/ajplung.00309.2014.
55. Cosio MG, Hale KA, Niewoehner DE. Morphologic and morphometric effects of prolonged cigarette smoking on the small airways. Am Rev Respir Dis. 1980;122(2):265-221. doi:10.1164/ arrd.1980.122.2.265.
56. Coraux C, Hajj R, Lesimple P, Puchelle E. In vivo models of human airway epithelium repair and regeneration. Eur Respir Rev. 2005;14(97):131-136. doi:10.1183/09059180.05.00009702.
57. Yahaya B. Understanding Cellular Mechanisms Underlying Airway Epithelial Repair: Selecting the Most Appropriate Animal Models. Sci World J. 2012;2012:e961684. doi:10.1100/2012/961684. 58. Rokicki W, Rokicki M, Wojtacha J, Dżeljijli A. The role and importance of club cells (Clara cells) in the
pathogenesis of some respiratory diseases. Kardiochirurgia Torakochirurgia Pol Pol J Cardio-Thorac Surg. 2016;13(1):26-30. doi:10.5114/kitp.2016.58961.
59. Gamez AS, Gras D, Petit A, et al. Supplementing Defect in Club Cell Secretory Protein Attenuates Airway Inflammation in COPD. Chest. 2015;147(6):1467-1476. doi:10.1378/chest.14-1174.
60. Park HY, Churg A, Wright JL, et al. Club cell protein 16 and disease progression in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2013;188(12):1413-1419. doi:10.1164/rccm.201305-0892OC.
61. Pilette C, Godding V, Kiss R, et al. Reduced epithelial expression of secretory component in small airways correlates with airflow obstruction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2001;163(1):185-194. doi:10.1164/ajrccm.163.1.9912137.
62. Whitcutt MJ, Adler KB, Wu R. A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. Vitro Cell Dev Biol J Tissue Cult Assoc. 1988;24(5):420-428.
63. Kaartinen L, Nettesheim P, Adler KB, Randell SH. Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell Dev Biol Anim. 1993;29A(6):481-492.
64. Hogg JC, Macklem PT, Thurlbeck WM. Site and nature of airway obstruction in chronic obstructive lung disease. N Engl J Med. 1968;278(25):1355-1360. doi:10.1056/NEJM196806202782501. 65. Hogg JC, Chu F, Utokaparch S, et al. The Nature of Small-Airway Obstruction in Chronic Obstructive
1
Pulmonary Disease. N Engl J Med. 2004;350(26):2645-2653. doi:10.1056/NEJMoa032158.
66. Hogg JC, Timens W. The Pathology of Chronic Obstructive Pulmonary Disease. Annu Rev Pathol Mech Dis. 2009;4(1):435-459. doi:10.1146/annurev.pathol.4.110807.092145.
67. Eurlings IMJ, Dentener MA, Cleutjens JPM, et al. Similar matrix alterations in alveolar and small airway walls of COPD patients. BMC Pulm Med. 2014;14:90. doi:10.1186/1471-2466-14-90. 68. Kranenburg AR, Willems-Widyastuti A, Moori WJ, et al. Enhanced bronchial expression of extracellular
matrix proteins in chronic obstructive pulmonary disease. Am J Clin Pathol. 2006;126(5):725-735. 69. Annoni R, Lanças T, Yukimatsu Tanigawa R, et al. Extracellular matrix composition in COPD. Eur
Respir J. 2012;40(6):1362-1373. doi:10.1183/09031936.00192611. 70. Dentener MA, Vernooy JHJ, Hendriks S, Wouters EFM. Enhanced levels of hyaluronan in lungs of patients with COPD: relationship with lung function and local inflammation. Thorax. 2005;60(2):114-119. doi:10.1136/thx.2003.020842. 71. Barnes PJ, Shapiro SD, Pauwels RA. Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J. 2003;22(4):672-688. 72. Pera T, Gosens R, Lesterhuis AH, et al. Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype. Respir Res. 2010;11(1):48. doi:10.1186/1465-9921-11-48. 73. Evanko SP, Potter-Perigo S, Bollyky PL, Nepom GT, Wight TN. Hyaluronan and versican in the control
of human T-lymphocyte adhesion and migration. Matrix Biol J Int Soc Matrix Biol. 2012;31(2):90-100. doi:10.1016/j.matbio.2011.10.004.
74. Togo S, Holz O, Liu X, et al. Lung Fibroblast Repair Functions in Patients with Chronic Obstructive Pulmonary Disease Are Altered by Multiple Mechanisms. Am J Respir Crit Care Med. 2008;178(3):248-260. doi:10.1164/rccm.200706-929OC. 75. van den Berge M, ten Hacken NHT, Cohen J, Douma WR, Postma DS. Small airway disease in asthma and copd: Clinical implications. Chest. 2011;139(2):412-423. doi:10.1378/chest.10-1210. 76. Hashimoto M, Tanaka H, Abe S. Quantitative analysis of bronchial wall vascularity in the medium and small airways of patients with asthma and COPD. Chest. 2005;127(3):965-972. doi:10.1378/ chest.127.3.965. 77. Siafakas NM, Antoniou KM, Tzortzaki EG. Role of angiogenesis and vascular remodeling in chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2007;2(4):453-462.
78. Green CE, Turner AM. The role of the endothelium in asthma and chronic obstructive pulmonary disease (COPD). Respir Res. 2017;18:20. doi:10.1186/s12931-017-0505-1. 79. Santos S, Peinado VI, Ramírez J, et al. Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J. 2002;19(4):632-638. 80. Ferrer E, Peinado VI, Díez M, et al. Effects of cigarette smoke on endothelial function of pulmonary arteries in the guinea pig. Respir Res. 2009;10(1):76. doi:10.1186/1465-9921-10-76. 81. Santos S, Peinado VI, Ramirez J, et al. Enhanced expression of vascular endothelial growth factor in pulmonary arteries of smokers and patients with moderate chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2003;167(9):1250-1256. doi:10.1164/rccm.200210-1233OC.
82. Nilsson I, Shibuya M, Wennström S. Differential activation of vascular genes by hypoxia in primary endothelial cells. Exp Cell Res. 2004;299(2):476-485. doi:10.1016/j.yexcr.2004.06.005.
83. Kranenburg AR, de Boer WI, Alagappan VKT, Sterk PJ, Sharma HS. Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease. Thorax. 2005;60(2):106-113. doi:10.1136/thx.2004.023986.
84. Calabrese C, Bocchino V, Vatrella A, et al. Evidence of angiogenesis in bronchial biopsies of smokers with and without airway obstruction. Respir Med. 2006;100(8):1415-1422. doi:10.1016/j. rmed.2005.11.009.
85. Peinado VI, Ramírez J, Roca J, Rodriguez-Roisin R, Barberà JA. Identification of vascular progenitor cells in pulmonary arteries of patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2006;34(3):257-263. doi:10.1165/rcmb.2005-0255OC.
86. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, et al. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest. 2000;106(11):1311-1319. doi:10.1172/JCI10259.
87. Voelkel NF, Gomez-Arroyo J, Mizuno S. COPD/emphysema: The vascular story. Pulm Circ. 2011;1(3):320-326. doi:10.4103/2045-8932.87295.
88. White DG, Cannon TR, Garratt H, Mundin JW, Sumner MJ, Watts IS. Endothelin ETA and ETB receptors mediate vascular smooth-muscle contraction. J Cardiovasc Pharmacol. 1993;22 Suppl 8:S144-148.
