**7. Targeting smooth muscle cell biology as therapy**

Strategies that target smooth muscle cell biology, including ECM-smooth muscle cell interac‐ tions, may be beneficial in the treatment of tissue remodeling diseases. Approaches that target proteases, which modulate the ECM or ECM-signaling of smooth muscle cells in disease, will be of particular focus in this chapter section.

#### **7.1. Contractile, hypertrophic and hyperplasic regulators**

Targeting factors which regulate smooth muscle contractility, hyperplasia and/or hypertrophy are currently used in the treatment of airway and vascular diseases. β2-Adrenergic receptor agonists, which increase the intracellular second messenger, cyclic AMP (cAMP), in ASM cells, are used pharmacologically to dampen bronchoconstriction in asthma and COPD [135]. Blockade of endothelin (ET) receptors are an effective treatment for PAH. Endothelin-1 (ET-1) is both a vasoconstrictor and mitogen, evoking its effects on VSM cells through binding to ETA or ETB receptors. Another treatment of PAH is prostacyclin (or its analogs), an eicosanoid, which like the prostaglandins and thromboxane, is vasodilatory [136]. Eicosanoids, which are released by endothelial cells, stimulate increases in the levels of intracellular cAMP in VSM cells via binding prostanoid/G-protein coupled receptors. Increases in cAMP inhibit myosin light chain phosphorylation, causing relaxation, as well as inhibiting proliferation [137]. Similarly, the endogenous vasodilator nitric oxide (NO), which is released by endothelial cells, also inhibits both myosin light chain phosphorylation (via increases in cGMP) and proliferation of VSM cells [36]. Various NO donors such as glycerine trinitrate and sodium nitroprusside (SNP) have been used in the treatment of vascular disease and endothelial dysfunction (*e.g.,* angina pectoris and hypertension) [138].

#### **7.2. Growth factors**

Growth factors such as PDGF, TGF-β and VEGF which regulate smooth muscle proliferation and size, may also be potential targets for the treatment of vascular and lung obstructive diseases [15]. Imatinib, a receptor tyrosine kinase inhibitor which inhibits PDGF signaling, attenuates both VSM cell hyperplasia and hypertrophy in pre-clinical models of vascular disease [139]. However, imatinib was withdrawn from clinical trials for the treatment of advanced PAH, because of serious side effects and increased morbidity [140]. Inhibiting specific aspects of TGF-β signaling may be another growth factor-targeting strategy to treat tissue remodeling in disease. Aberrant TGF-β signaling, important in regulating smooth muscle function including cell stiffness and proliferation [141, 142], contributes to tissue remodeling in vascular and respiratory diseases [143-145]. The TGF-β superfamily member, activin A, is linked with the progression of PAH, stimulating VSM cell proliferation [141]. Administration of follistatin, an endogenous inhibitor of activin A, attenuates inflammation and remodeling in experimental models of lung injury and disease [146].

#### **7.3. MMPs**

which lacks a transmembrane or intracellular domain [78]. The interaction of uPA with uPAR to activate co-receptors including integrins, is likely to be influenced by the engagement of integrins with the ECM. In support, fibulin 5 [127] and vitronectin [41] are integrin binding ECM proteins, which modulate uPA-uPAR signaling [134]. uPAR-independent activation of αvβ1-integrin by uPA in smooth muscle cell may also be similarly affected by the ECM and

Strategies that target smooth muscle cell biology, including ECM-smooth muscle cell interac‐ tions, may be beneficial in the treatment of tissue remodeling diseases. Approaches that target proteases, which modulate the ECM or ECM-signaling of smooth muscle cells in disease, will

Targeting factors which regulate smooth muscle contractility, hyperplasia and/or hypertrophy are currently used in the treatment of airway and vascular diseases. β2-Adrenergic receptor agonists, which increase the intracellular second messenger, cyclic AMP (cAMP), in ASM cells, are used pharmacologically to dampen bronchoconstriction in asthma and COPD [135]. Blockade of endothelin (ET) receptors are an effective treatment for PAH. Endothelin-1 (ET-1) is both a vasoconstrictor and mitogen, evoking its effects on VSM cells through binding to ETA or ETB receptors. Another treatment of PAH is prostacyclin (or its analogs), an eicosanoid, which like the prostaglandins and thromboxane, is vasodilatory [136]. Eicosanoids, which are released by endothelial cells, stimulate increases in the levels of intracellular cAMP in VSM cells via binding prostanoid/G-protein coupled receptors. Increases in cAMP inhibit myosin light chain phosphorylation, causing relaxation, as well as inhibiting proliferation [137]. Similarly, the endogenous vasodilator nitric oxide (NO), which is released by endothelial cells, also inhibits both myosin light chain phosphorylation (via increases in cGMP) and proliferation of VSM cells [36]. Various NO donors such as glycerine trinitrate and sodium nitroprusside (SNP) have been used in the treatment of vascular disease and endothelial dysfunction (*e.g.,*

Growth factors such as PDGF, TGF-β and VEGF which regulate smooth muscle proliferation and size, may also be potential targets for the treatment of vascular and lung obstructive diseases [15]. Imatinib, a receptor tyrosine kinase inhibitor which inhibits PDGF signaling, attenuates both VSM cell hyperplasia and hypertrophy in pre-clinical models of vascular disease [139]. However, imatinib was withdrawn from clinical trials for the treatment of advanced PAH, because of serious side effects and increased morbidity [140]. Inhibiting specific aspects of TGF-β signaling may be another growth factor-targeting strategy to treat tissue remodeling in disease. Aberrant TGF-β signaling, important in regulating smooth

**7. Targeting smooth muscle cell biology as therapy**

**7.1. Contractile, hypertrophic and hyperplasic regulators**

be of particular focus in this chapter section.

angina pectoris and hypertension) [138].

**7.2. Growth factors**

biomechanical forces [79].

370 Muscle Cell and Tissue

Targeting proteases that modify smooth muscle ECM structure and its biomechanical properties are another strategy to treat vascular and obstructive lung disease. Inhibition of pericellular collagenolysis *in situ* may reduce ASM cell hyperplasia in AWR by maintain‐ ing the anti-proliferative effects of fibrillar pericellular collagen. Administration of the MMP inhibitory antibiotic doxycycline reduces airway hyper-responsiveness in allergen-chal‐ lenged mice [147], supporting the concept that interventions with selective MMP inhibi‐ tors could be beneficial in the treatment of diseases such as asthma (see Table 1). In preclinical models of vascular disease and injury, doxycycline has been shown to attenuate acute pulmonary thromboembolism (APT)-evoked pulmonary hypertension and right ventricular dysfunction [148].



**The effects of targeting ECM proteases in experimental models of lung and vascular disease**


**The effects of targeting ECM proteases in experimental models of lung and vascular disease**

**Table 1.** A summary of findings from studies that have targeted ECM proteases and receptors in experimental models of lung and vascular disease. The animal models used were: (i) Acute pulmonary thromboembolism (APT), Which is induced with autologous blood clots; (ii) Allergen, A model of allergic airway inflammation which has characteristics of asthma pathology. In the model, antigen sensitized animals are repeatedly challenged with aerosolized antigen (e.g., ovalbumin, house dust mite extract); (iii) Balloon angioplasty, A vascular intervention procedure which causes restenosis; (iv) Bleomycin, Intranasal administration of bleomycin induces acute lung injury and subsequent pulmonary inflammation and fibrosis. This model also features extensive remodeling in the upper airways involving ASM cell hypertrophy and hyperplasia; (v) Endotoxin, Intranasal administration of endotoxin (i.e., lipopolysaccharide or LPS) also induces acute lung injury and pulmonary inflammation; and (vi) Hypoxia, A model of pulmonary arterial hypertension (PAH).

#### **7.4. Coagulants**

**The effects of targeting ECM proteases in experimental models of lung and vascular disease**

(tranexamic acid) and genetic (plasminogen knockout)

(plasminogen knockout)

(plasminogen knockout)

(plasminogen knockout)

Plasminogen Genetic

372 Muscle Cell and Tissue

Plasminogen Genetic

Plasminogen Genetic

PAR-1 Pharmacological

(ER-112780-06)

**Target Approach Model (challenge) Outcome (as compared to wild type or**

PAI-1 Pharmacological (IMD-1622) Arterial injury Reduced arterial neointimal formation,

PAI-1 Genetic (PAI-1 knockout) Allergen Reduced AWR including sub-epithelial

PAI-1 Genetic (PAI-1 knockout) Endotoxin Reduced neutrophil recruitment to the

PAI-1 Genetic (PAI-1 knockout) Arterial injury Reduced arterial neointimal formation

PAR-1 Genetic (PAR-1 knockout) Bleomycin Reduced collagen accumulation in lung

PAR-1 Pharmacological (Atopaxar) APT Reduced neointimal thickening in arterial

tPA Therapeutic (nebulized tPA) Allergen Reduced airway hyper-responsiveness [52]

PAR-1 Pharmacological (F16618) Balloon angioplasty Reduced restenosis [158]

PAI-1 Genetic (PAI-1 knockout) Bleomycin Reduced lung collagen [152].

PAI-1 Pharmacological (Tiplaxtinin) Allergen Reduced AWR [153]

**vehicle control treated mice following**

pulmonary vascular remodeling [75].

increases in adhesion molecules, fibrinogen

lungs [154] and fibrin deposition and AWR

and pulmnonary inflammation [102].

**challenge or injury)**

Bleomycin Reduced alveolar macrophage / Increased lung collagen [150].

Hypoxia Reduced pulmonary hypertension and

accumulation [151]

fibrosis [61, 62].

in the airways [155].

blood vessels [157]

[156].

Allergen Reduced AWR [98].

Hypoxia No effect [75].

Reducing the accumulation of extravascular fibrin in damaged tissue would be expected to reduce tissue remodeling in disease. Anti-coagulants are already used in the treatment of vascular disease to reduce thrombosis by disrupting hemostasis. Whilst clinical asthma trials with nebulized heparin have provided mixed results [161], systemic administration of *fondaparinux,* a selective inhibitor of FXa, attenuates AWR in experimental asthma [149] (table 1). The success of anticoagulant therapy in treating asthma and other respiratory diseases may depend on the anticoagulant, which differ in potency for coagulation-dependent and independent targets. Selective small molecule FXa inhibitors (*e.g., apixaban*) are used as treatment for venous thromboembolism and stroke prevention in patients with atrial fibrilla‐ tion [162], but still have anti-coagulant related bleeding risks, which can be potentially fatal.

The coagulants FXa and thrombin may also contribute to tissue remodeling via their activation of PAR-1 on stromal cells including ASM and VSM cells. PAR-1 antagonists reduce AWR in a murine model of chronic allergic airway inflammation [98] and pulmonary inflammation and fibrosis in mice following bleomycin-induced lung injury [102, 103] (table 1). Similarly PAR-1 antagonists reduce restenosis [158] and intimal thickening [157] in the vascular wall following balloon injury *in vivo* (table 1). Orally administered *Vorapaxar*, a PAR-1 inhibitor, is used as anti-platelet therapy for prevention of secondary thrombotic cardiovascular events in patients with a prior myocardial infarction [163]. However, it is because of the ability to suppress thrombin-stimulated platelet aggregation, thus disrupting hemostasis, that PAR-1 inhibitors such as *Vorapaxar* are associated with potentially fatal bleeding complications [164]. For PAR-1 inhibitors to be used as treatment for tissue remodeling disease, they will need to be modified to selectively target the extravascular cell-mediated actions of coagulant proteases without disrupting hemostasis. Taking advantage of integrin co-receptors and adaptors that differen‐ tiate PAR-1 responses in platelet and endothelial cells as compared to inflammatory and extravascular structural cells (*e.g.* smooth muscle cells) may be one approach to achieve selectively in regards to PAR-1-drug targeting.

#### **7.5. Fibrinolytic agents**

Potential therapies that reduce fibrin by using fibrinolytic agents, have been considered in the treatment of lung disease for some time [165]. Thrombolytic therapy using tPA-mimetics are already used for stroke and myocardial infarction, despite a higher risk of bleeding compli‐ cations [166]. A potential strategy to treat asthma is to augment airspace fibrinolysis. Tiplax‐ tinin, a small molecule inhibitor of PAI-1 [153], or inhaled tPA [52], attenuate AWR or reactivity in allergen-challenged mice (table 1). In animal studies of vascular injury and disease, PAI-1 inhibitors [151] or PAI-1 gene deletion [156] reduced neointima formation.

#### **7.6. uPA and annexin A2**

Both uPA and annexin A2 may be potential novel drug targets in the treatment of chronic respiratory disease [167]. Both uPA and annexin A2 gene-deletion reduces pulmonary inflammation in various murine models [84, 150, 160], and uPA antibodies reduce inflamma‐ tion and edema in a mouse model of acute lung injury [159] (table 1). However, further preclinical characterization of these inhibitors as therapy for tissue remodeling diseases is required. The roles of uPA and annexin A2 in cancer are well established, and their targeting by either pharmacological or antibody-based therapies have been shown to reduce tumour growth and/or metastasis in a number of pre-clinical cancer models [86-90]. Furthermore, clinical trials for cancer have shown uPA inhibitors are well tolerated in humans and have generated promising results [87].

#### **7.7. Cross-linking enzymes**

As the expression of lysyl oxidases are dysregulated in PAH, modulation of lung matrix crosslinking may limit pulmonary vascular remodeling associated with PAH. In support, βaminopropionitrile, an inhibitor of lysyl oxidase, attenuates the effect of hypoxia on vascular remodeling in experimental PAH [9] (table 1).

#### **7.8. Actin-cytoskeleton**

The coagulants FXa and thrombin may also contribute to tissue remodeling via their activation of PAR-1 on stromal cells including ASM and VSM cells. PAR-1 antagonists reduce AWR in a murine model of chronic allergic airway inflammation [98] and pulmonary inflammation and fibrosis in mice following bleomycin-induced lung injury [102, 103] (table 1). Similarly PAR-1 antagonists reduce restenosis [158] and intimal thickening [157] in the vascular wall following balloon injury *in vivo* (table 1). Orally administered *Vorapaxar*, a PAR-1 inhibitor, is used as anti-platelet therapy for prevention of secondary thrombotic cardiovascular events in patients with a prior myocardial infarction [163]. However, it is because of the ability to suppress thrombin-stimulated platelet aggregation, thus disrupting hemostasis, that PAR-1 inhibitors such as *Vorapaxar* are associated with potentially fatal bleeding complications [164]. For PAR-1 inhibitors to be used as treatment for tissue remodeling disease, they will need to be modified to selectively target the extravascular cell-mediated actions of coagulant proteases without disrupting hemostasis. Taking advantage of integrin co-receptors and adaptors that differen‐ tiate PAR-1 responses in platelet and endothelial cells as compared to inflammatory and extravascular structural cells (*e.g.* smooth muscle cells) may be one approach to achieve

Potential therapies that reduce fibrin by using fibrinolytic agents, have been considered in the treatment of lung disease for some time [165]. Thrombolytic therapy using tPA-mimetics are already used for stroke and myocardial infarction, despite a higher risk of bleeding compli‐ cations [166]. A potential strategy to treat asthma is to augment airspace fibrinolysis. Tiplax‐ tinin, a small molecule inhibitor of PAI-1 [153], or inhaled tPA [52], attenuate AWR or reactivity in allergen-challenged mice (table 1). In animal studies of vascular injury and disease, PAI-1

Both uPA and annexin A2 may be potential novel drug targets in the treatment of chronic respiratory disease [167]. Both uPA and annexin A2 gene-deletion reduces pulmonary inflammation in various murine models [84, 150, 160], and uPA antibodies reduce inflamma‐ tion and edema in a mouse model of acute lung injury [159] (table 1). However, further preclinical characterization of these inhibitors as therapy for tissue remodeling diseases is required. The roles of uPA and annexin A2 in cancer are well established, and their targeting by either pharmacological or antibody-based therapies have been shown to reduce tumour growth and/or metastasis in a number of pre-clinical cancer models [86-90]. Furthermore, clinical trials for cancer have shown uPA inhibitors are well tolerated in humans and have

As the expression of lysyl oxidases are dysregulated in PAH, modulation of lung matrix crosslinking may limit pulmonary vascular remodeling associated with PAH. In support, β-

inhibitors [151] or PAI-1 gene deletion [156] reduced neointima formation.

selectively in regards to PAR-1-drug targeting.

**7.5. Fibrinolytic agents**

374 Muscle Cell and Tissue

**7.6. uPA and annexin A2**

generated promising results [87].

**7.7. Cross-linking enzymes**

Cytoskeletal changes in smooth muscle cells occur in association with increases in stiffness (*i.e.,* elastic modulus), size (hypertrophy) and migration [168-170]. A strategy that softens cells by regulating the formation or depolymerisation of the actin-cytoskeleton may be beneficial in the treatment of vascular and lung obstructive diseases. However, there are yet no actintargeting drugs used clinically [171]. Cytochalasins and latrunculins are cytotoxic as they nonselectively disrupt actin microfilaments. However, there are different populations of actin filaments, spatially organised into distinct cellular compartments with unique functions. Their organization is regulated by associated-actin binding proteins. Therapeutically, actin filament populations need to be targeted specifically, by modulating the binding of specific actinbinding proteins. One approach maybe to target the assembly of FA complexes. Phosphory‐ lation of FAK leads to disassembly of FA complexes-actin filaments [172]. ECM proteins (*e.g.,* collagen and fibronectin) that engage integrins regulate FAK activity and actin-cytoske‐ leton organization [173]. FAK regulates actin-cytoskeleton organisation by modulating the association of actin with actin-capping proteins such as gelsolin [174]. Complexes between FAK, gelsolin and the actin-cytoskeleton also regulate gene expression of contractile proteins (*e.g.,* α-smooth muscle actin, calponin and transgelin [SM22]) via the actin-filament-dependent transcriptional co-activator, MRTF-A (myocardin-related transcription factor-A) [174].
