**3. Smooth muscle cells in disease**

Structural and functional changes to smooth muscle cells and their microenvironment contributes to tissue remodeling in diseases such as lung obstructive and vascular diseases [2-5], which are the focus of this chapter. Tissue remodeling involving smooth muscle cells is likely to be a result of injury and dys-regulated repair processes linked to inflammation and extravascular coagulation and fibrinolysis [20-23].

#### **3.1. Obstructive lung diseases**

**Keywords:** Extracellular matrix (ECM), Coagulation, Collagen, Fibrinolysis, Integ‐

Smooth muscle cells function by contracting following activation of actin and myosin fila‐ ments, in a process involving myosin light chain phosphorylation, mediated by Ca2+-depend‐ ent pathways [1]. In many diseases, alterations in smooth muscle cell function including contractile responses, growth and phenotype, contribute to tissue remodeling. In obstructive lung diseases such as severe asthma and chronic obstructive pulmonary disease (COPD), increases in the stiffness and mass of the airway smooth muscle (ASM) bundle contribute to fixed airway obstruction and hyper-responsiveness [2, 3]. In vascular injury and diseases such as atherosclerosis and pulmonary arterial hypertension (PAH), the migration, stiffening, proliferation and growth of vascular smooth muscle (VSM) cells contribute to the enlargement of the blood vessel wall, which in effect reduces lumen size, thus an increase in vascular resistance [4, 5]. Alterations in the microenvironment of the smooth muscle cell, particularly in the composition and structure of the ECM, accompany changes in smooth muscle biology in disease. Smooth muscle cells have a large role in modifying their microenvironment in disease by producing ECM protein (*e.g.,* collagen, fibrin, fibronectin and proteoglycans [6]) and factors which regulate ECM formation (*e.g.,* tissue factor in fibrin formation [7]). Further‐ more, smooth muscle cells secrete proteases (*e.g.,* urokinase plasminogen activator or uPA [8]) and crosslinking enzymes (*e.g.,* lysyl oxidases [9]) which modulate ECM structure and form. The ECM in turn regulates smooth muscle function by the provision of both biochemical and biomechanical cues, in a process involving complexes formed between integrins, focal adhesion (FA) proteins and the actin-cytoskeleton. Both biochemical and mechano-transduc‐ tion signaling in smooth muscle cells are mutually interdependent. In disease, the altered ECM may perpetuate tissue remodeling by augmenting smooth muscle growth, migration, cytokine production, cell stiffness and proliferation in a detrimental feed forward mechanism. Aside from important biomechanical contributions in tissue remodelling, smooth muscle cells are also potent producers of an array of inflammatory mediators, including cytokines, chemokines and cell adhesion molecules (CAMs) [10-13]. These inflammatory mediators, as well as the ECM produced by smooth muscle cells, influence the type and quantity of inflammatory cells

Smooth muscle cells are phenotypically-plastic stromal cells, which are very capable of differentiating in response to injury and inflammation in disease. Whilst myogenic, the structure, mechanical properties, contractility and function of smooth muscle cells are different

rins, Proteases

that infiltrate damaged tissue in disease [14, 15].

**2. Smooth muscle cells**

**1. Introduction**

360 Muscle Cell and Tissue

Obstructive lung diseases, including asthma, COPD, bronchitis and bronchiectasis, are characterized by airway obstruction. The latter is a limitation of airflow, caused by the narrowing of bronchioles. The mechano-contractile properties of the ASM cell make it the primary effecter of bronchospasm, which is an acute contraction of the airways that occurs in asthma and bronchitis. ASM cells are also involved in the array of persistent tissue structural changes of the airway wall that contribute to airway obstruction in asthma and COPD [2, 24, 25]. In airway wall remodeling (AWR), ASM cell hyperplasia, hypertrophy and changes in production of ECM protein contribute to an increase in airway wall thickness and reduction in airway wall distensibility [26]. ASM cells in disease are major contributors to the increases in production of ECM components, including the wound type collagens, I and III, and fibronectin. ASM cells also have an important inflammatory role in airway obstructive diseases, being potent producers of growth factors, cytokines and other pro-inflammatory mediators, including granulocyte macrophage-colony stimulating factor (GM-CSF), intracel‐ lular adhesion molecule (ICAM), interleukin-1α (IL-1 α), eotaxin, leukaemia inhibitory (LIF), fractalkine and vascular cell adhesion molecule (VCAM) [10]. Pro-inflammatory mediator expression in ASM cells is stimulated primarily by cytokines and growth factors produced by inflammatory cells and the epithelium [27]. The ECM in the microenvironment of the ASM cell can modulate cytokine expression by ASM cells [28]. Furthermore, cyclical changes in mechanical strain associated with breathing also modulate cytokine expression by ASM cells [29]. As a consequence of their pivotal role in AWR, therapies that target the ASM cell are continually being explored as possible new treatments for obstructive lung diseases [30]. Although eliminating ASM by bronchial thermoplasty is effective in reducing airway obstruc‐ tion, less invasive and costly therapies are urgently needed. An effective muscle relaxing, antiremodeling treatment that specifically targets ASM cells is expected to reduce airway reactivity and symptoms in patients with asthma or COPD [31, 32].

#### **3.2. Vascular diseases**

VSM cells reside in the medial layer of blood vessels, mediating changes in vascular tone by contracting or relaxing in response to vasoconstrictors and vasodilators respectively. In vascular injury and disease, VSM cells switch from a quiescent, contractile phenotype to a synthetic phenotype, which produces cytokines and ECM protein (*e.g.,* fibronectin and sulphated proteoglycans), undergoes hyperplasia (*i.e.,* increased proliferation) and/or hyper‐ trophy (*i.e.,* increased cell size). In atherosclerosis, a cause of stenosis and blood vessel occlusion, synthetic VSM cells migrate from the media to the intima where they proliferate and contribute to intimal thickening [22, 23]. Furthermore, within the intima, VSM can alter the composition of the ECM. For example, VSM cells produce increased amounts of LDLaccumulating sulphated proteoglycans, which increase lipid loading, a feature of athero‐ thrombotic disease [33]. In hypertension, VSM cells undergo hypertrophic remodeling in the media of blood vessels, contributing to excessive arterial vascular contractile tone, hence high blood pressure [34]. Hypoxia, inflammation and shear stress contribute to vasoconstriction and pulmonary vascular remodeling in PAH [35, 36]. Medial thickening in proximal pulmo‐ nary vessels is caused by the hypertrophy, hyperplasia and ECM production of resident VSM cells, whereas in pre-capillary arterioles, the VSM cells that contribute to medial thickening are derived from intermediate cells in blood vessels or adventitial fibroblasts, which differen‐ tiate into VSM. Like ASM cells, VSMs contribute to inflammation in vascular disease by producing pro-inflammatory mediators such as the platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), interferon-γ (IFN- γ), and monocyte chemotactic protein-1 (MCP-1). Furthermore, VSM cells express CAMs (*e.g.,* VCAM-1 and ICAM-1), which have a role in retaining monocytes and macrophages within lesions such as those formed in the intima in atherosclerosis. Aberrant Akt/PI3kinase signaling resulting from a defect in PTEN phosphatase activity is thought to contribute to the pro-inflammatory phenotype of VSM cells in PAH [37]. In a murine model of PAH, the targeting of the PTEN gene specifically in VSM cells augments an inflammatory response and vascular remodeling [38].

### **4. ECM of smooth muscle**

The ECM of the smooth muscle cell microenvironment changes in injury, disease and with ageing. ECM production and modification by smooth muscle cell is regulated by intrinsic (*e.g.,* epigenetics) and extrinsic (*e.g.,* oxidative stress, growth factors and cytokines) factors [15]. Abnormalities of the ECM are a key feature of tissue remodeling in obstructive lung [39] and vascular disease [34].

#### **4.1. Collagen**

inflammatory cells and the epithelium [27]. The ECM in the microenvironment of the ASM cell can modulate cytokine expression by ASM cells [28]. Furthermore, cyclical changes in mechanical strain associated with breathing also modulate cytokine expression by ASM cells [29]. As a consequence of their pivotal role in AWR, therapies that target the ASM cell are continually being explored as possible new treatments for obstructive lung diseases [30]. Although eliminating ASM by bronchial thermoplasty is effective in reducing airway obstruc‐ tion, less invasive and costly therapies are urgently needed. An effective muscle relaxing, antiremodeling treatment that specifically targets ASM cells is expected to reduce airway reactivity

VSM cells reside in the medial layer of blood vessels, mediating changes in vascular tone by contracting or relaxing in response to vasoconstrictors and vasodilators respectively. In vascular injury and disease, VSM cells switch from a quiescent, contractile phenotype to a synthetic phenotype, which produces cytokines and ECM protein (*e.g.,* fibronectin and sulphated proteoglycans), undergoes hyperplasia (*i.e.,* increased proliferation) and/or hyper‐ trophy (*i.e.,* increased cell size). In atherosclerosis, a cause of stenosis and blood vessel occlusion, synthetic VSM cells migrate from the media to the intima where they proliferate and contribute to intimal thickening [22, 23]. Furthermore, within the intima, VSM can alter the composition of the ECM. For example, VSM cells produce increased amounts of LDLaccumulating sulphated proteoglycans, which increase lipid loading, a feature of athero‐ thrombotic disease [33]. In hypertension, VSM cells undergo hypertrophic remodeling in the media of blood vessels, contributing to excessive arterial vascular contractile tone, hence high blood pressure [34]. Hypoxia, inflammation and shear stress contribute to vasoconstriction and pulmonary vascular remodeling in PAH [35, 36]. Medial thickening in proximal pulmo‐ nary vessels is caused by the hypertrophy, hyperplasia and ECM production of resident VSM cells, whereas in pre-capillary arterioles, the VSM cells that contribute to medial thickening are derived from intermediate cells in blood vessels or adventitial fibroblasts, which differen‐ tiate into VSM. Like ASM cells, VSMs contribute to inflammation in vascular disease by producing pro-inflammatory mediators such as the platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), interferon-γ (IFN- γ), and monocyte chemotactic protein-1 (MCP-1). Furthermore, VSM cells express CAMs (*e.g.,* VCAM-1 and ICAM-1), which have a role in retaining monocytes and macrophages within lesions such as those formed in the intima in atherosclerosis. Aberrant Akt/PI3kinase signaling resulting from a defect in PTEN phosphatase activity is thought to contribute to the pro-inflammatory phenotype of VSM cells in PAH [37]. In a murine model of PAH, the targeting of the PTEN gene specifically in VSM

and symptoms in patients with asthma or COPD [31, 32].

cells augments an inflammatory response and vascular remodeling [38].

The ECM of the smooth muscle cell microenvironment changes in injury, disease and with ageing. ECM production and modification by smooth muscle cell is regulated by intrinsic

**4. ECM of smooth muscle**

**3.2. Vascular diseases**

362 Muscle Cell and Tissue

Collagen is the most prominent structural protein of connective tissue. Native collagen I and III form fibrils comprising supra-molecular aggregates of collagen that are stabilized by interactions between their helical domains [40]. These fibrils have a high tensile strength that maintains the structural integrity of tissue by counter-balancing distending forces, such as those evoked by breathing on the airway wall [41]. In the airways of patients with asthma and COPD, the ECM is expanded by fibrils of collagen I and III, including around and within smooth muscle bundles, reducing airway wall distensibility [26]. Increased collagen, as well as ASM hyperplasia and hypertrophy, also contribute to airway wall thickening that is linked to increased airway-reactivity [2]. In the media of the healthy blood vessel wall, collagen I and III are the main components of the ECM, with elastin also being abundant in the media of larger arterioles [42, 43]. Collagen fibrils formed by VSM cells also have an important role in fibrous cap stabilization, a stage of atherogenesis [44]. Whilst the native forms of collagen impose mechanical strain on cells, it is the denatured forms of collagen, which perhaps are more biochemically-reactive. Increases in the activities of plasmin and MMPs, proteases which denature native collagen, occur in both respiratory [45] and vascular [34] disease. The dena‐ turation of collagen fibrils immediately adjacent to and surrounding smooth muscle cells by pericellular proteolysis may have an important role in regulating phenotype and function in disease.

#### **4.2. Fibronectin**

Fibronectin is a high molecular weight glycoprotein and constituent of the ECM. Fibronectin binds to the α5β1 integrin on cells to transmit biochemical and mechanical signals, and is a nucleator of fibrillogenesis *(i.e.,* collagen fibril assembly). In asthma, levels of fibronectin, along with collagen I and III, are increased within the smooth muscle bundle of the airway wall [46]. Additionally, ASM cells from asthmatics, when compared to non-asthmatics, secrete more fibronectin, which in turn augments increased ASM cell proliferation and cytokine production [47]. Rhinovirus-infection [48], cigarette smoke extract [49] and the pro-fibrogenic mediator TGF-β [50], all stimulate ASM cells to produce fibronectin. VSM cells in disease also produce increased amounts of fibronectin. During atherogenesis, fibronectin synthesized by VSM cells becomes more abundant in the blood vessel wall, which augments fibrillogenesis [44] and stimulates smooth muscle cell proliferation and cytokine production [42, 43].

#### **4.3. Fibrin**

Extravascular accumulation of fibrin, formed by the coagulation cascade, occurs in a number of diseases, including respiratory and vascular diseases [21, 51-55]. Increased vascular permeability allows blood-circulating hemostatic factors such as factor VII (FVII), factor X (FX) and plasminogen to enter damaged and inflamed tissue to become activated and participate in coagulation and fibrinolysis (Figure 1).

**Figure 1.** Extravascular coagulation and fibrinolysis. In inflamed tissue such as the airway wall in asthma, plasma con‐ taining factor VII (FVII) and FX leak into the extravascular compartment. FVII, combined with tissue factor (TF), formed by epithelial, stromal (e.g., smooth muscle cells) and inflammatory cells, transforms FX into the serine pro‐ tease, FXa. The latter, combined with FV, activates thrombin, which in turn converts fibrinogen into fibrin. Whilst fi‐ brin has an important role in physiological wound-repair, it also serves as a scaffold for migrating stromal cells, and can be converted into fibrin degradation products (FDPs), which stimulate smooth muscle cell migration and cytokine production. Fibrinolysis (i.e., the break-down of fibrin) is catalysed by plasmin, formed by the activation of plasmaderived plasminogen. The latter is catalysed by tissue-type plasminogen activator (tPA).

Abundant extravascular fibrin is a specific hallmark of lung disease including asthma, and is thought to be a result of hyper-coagulation and suppressed fibrinolysis (*i.e.,* fibrin degrada‐ tion). The key mediator of fibrinolysis is plasmin, which can be formed by the proteolytic activation of plasminogen by either the tissue-type or urokinase type plasminogen activators (tPA or uPA respectively) [56, 57]. However, only tPA has fibrin-dependent amplification activity, facilitating its role as the primary mediator of fibrinolysis. Levels of tPA are much higher than uPA in the airway lumen [58-60]. The reduced AWR observed in plasminogen activator inhibitor-1 (PAI-1) gene knockout mice challenged with aerosolized allergen is likely to be a consequence of less airway fibrin [61, 62]. PAI-1 inhibits fibrinolysis by blocking the actions tPA in plasmin formation.

The medial layer of blood vessels, where VSM cells are prominent in number, is exposed to plasma exudate, even under normal physiological conditions. Whilst fibrin is present in normal arterial intima, its levels are increased in atherosclerotic lesions, particularly early proliferative, gelatinous-lesions [21]. Again, hypercoaulation and suppressed fibrinolysis are likely to contribute to increased fibrin formation in the vasculature in disease. The coagulant, tissue factor (TF), is highly expressed in VSM cells during atherogenesis [7], being rapidly induced by growth factors and cytokines [7]. Hypoxia, a driver of PAH, induces the upregulation of PAI-1 in pulmonary artery VSM cells [63]. In injury and disease, the deposition of fibrin into the ECM serves as a scaffold to support smooth muscle cell migration [64]. Furthermore, fibrin degradation products (FDPs) formed by fibrinolysis, stimulate production of pro-inflammatory mediators (*e.g.,* C-reactive protein in VSM cells [65]).

#### **4.4. Proteolglycans**

and plasminogen to enter damaged and inflamed tissue to become activated and participate

**Figure 1.** Extravascular coagulation and fibrinolysis. In inflamed tissue such as the airway wall in asthma, plasma con‐ taining factor VII (FVII) and FX leak into the extravascular compartment. FVII, combined with tissue factor (TF), formed by epithelial, stromal (e.g., smooth muscle cells) and inflammatory cells, transforms FX into the serine pro‐ tease, FXa. The latter, combined with FV, activates thrombin, which in turn converts fibrinogen into fibrin. Whilst fi‐ brin has an important role in physiological wound-repair, it also serves as a scaffold for migrating stromal cells, and can be converted into fibrin degradation products (FDPs), which stimulate smooth muscle cell migration and cytokine production. Fibrinolysis (i.e., the break-down of fibrin) is catalysed by plasmin, formed by the activation of plasma-

Abundant extravascular fibrin is a specific hallmark of lung disease including asthma, and is thought to be a result of hyper-coagulation and suppressed fibrinolysis (*i.e.,* fibrin degrada‐ tion). The key mediator of fibrinolysis is plasmin, which can be formed by the proteolytic activation of plasminogen by either the tissue-type or urokinase type plasminogen activators (tPA or uPA respectively) [56, 57]. However, only tPA has fibrin-dependent amplification activity, facilitating its role as the primary mediator of fibrinolysis. Levels of tPA are much higher than uPA in the airway lumen [58-60]. The reduced AWR observed in plasminogen activator inhibitor-1 (PAI-1) gene knockout mice challenged with aerosolized allergen is likely to be a consequence of less airway fibrin [61, 62]. PAI-1 inhibits fibrinolysis by blocking the

The medial layer of blood vessels, where VSM cells are prominent in number, is exposed to plasma exudate, even under normal physiological conditions. Whilst fibrin is present in normal arterial intima, its levels are increased in atherosclerotic lesions, particularly early proliferative, gelatinous-lesions [21]. Again, hypercoaulation and suppressed fibrinolysis are likely to contribute to increased fibrin formation in the vasculature in disease. The coagulant, tissue factor (TF), is highly expressed in VSM cells during atherogenesis [7], being rapidly induced by growth factors and cytokines [7]. Hypoxia, a driver of PAH, induces the up-

derived plasminogen. The latter is catalysed by tissue-type plasminogen activator (tPA).

actions tPA in plasmin formation.

in coagulation and fibrinolysis (Figure 1).

364 Muscle Cell and Tissue

Proteoglycans are another important component of the ECM synthesized by smooth muscle cells in disease including VSM cells during atherogenesis [42, 43] and ASM cells in asthma [66]. In atherogenesis, sulphated proteoglycans, via ionic interactions with ApoB100 and ApoE, entrap low-density lipoprotein (LDL) within the vessel wall. Bound oxidized-LDL augments macrophage lipid uptake and foam cell formation, and stimulates VSM cells to secrete greater amounts of sulphated proteoglycans [34].
