**5. ECM remodeling by smooth muscle cells**

Asides from producing ECM protein, smooth muscle cells also modulate the structure and form of the ECM. Extracellular modification of ECM protein involves integrins and enzymes which proteolyze and cross-link collagen. The serine protease, plasmin, also has the ability to directly activate smooth muscle cells via receptor based mechanisms. Major modifications of ECM protein by smooth muscle cells are described below:

#### **5.1. Fibrillogenesis**

Smooth muscle cells including VSM [67] and ASM [68] cells polymerize collagen fibrils into larger supramolecular collagen assemblies. Collagen fibrils can vary greatly in diameter (20-500 nm) and form different supramolecular structures such as bundles, weaves, and layers to suit differing roles and functions. The ability of smooth muscle cells to increase the diameter of fibrils would increase the tensile strength and resistance of the fibrils to collagenolysis by MMPs. Collagen fibrillogenesis involves integrin binding (*e.g.,* α2β1, α11β1) and is integrated with fibronectin fibril formation [69]. Lipid accumulation by VSM cells inhibits collagen fibrillogenesis [70], contributing to the destabilization of fibrous caps, hence rupture of atherosclerotic plaques [44].

#### **5.2. Proteolysis**

Matrix metalloproteases (MMPs) and plasmin are proteases involved in the degradation of ECM proteins such as fibronectin and denatured collagen. Plasmin also cleaves and activates the zymogen forms of MMPs including the MMP collagenases (MMP-1, MMP-13 and MMP-14), which denature collagen fibrils [71]. MMPs including MMP-1, MMP-2, MMP-12 and MMP-14 are expressed by human ASM cells *in vivo* [72] and/or *in vitro* [68] and conversion of plasminogen into plasmin by ASM cells is associated with MMP activation and increases in collageneolytic activity [73]. MMPs and components of the plasminogen activation system are co-expressed during remodeling in the airways following allergen challenge [74]. VSM cells also produce and secrete MMPs including MMP-2, MMP-3, MMP-7 and MMP-9 [34]. MMPs formed by VSM cells are considered to contribute to the thinning, thus destabilization of the atherosclerotic fibrous cap in atherogenesis [44]. Like ASM cells, VSM cells do not produce plasminogen *de novo*, but express urokinase plasminogen activator (uPA), which readily activates plasminogen convected from plasma through the blood vessel wall [22]. Hypoxia induces uPA expression in VSM cells, in association with increased cell migration and matrix invasion, suggesting a role of plasmin proteolysis in PAH [8].

Interestingly uPA and plasminogen gene deletion (but not tPA gene deletion) also reduces hypoxia-induced PAH and pulmonary vascular remodeling in mice [75]. These effects are likely to be independent of fibrinolysis, as uPA does not bind fibrin with the same high affinity as tPA. Plasmin formed by uPA has pro-inflammatory and -remodeling activities which are not associated with the break-down of fibrin. Furthermore, uPA has plasmin(ogen)-independ‐ ent effects. The amino-terminal fragment of uPA interacts with its receptor, uPAR to activate other receptors such as the formyl-peptide receptor 2 (FPR2) [76] and the epidermal growth factor receptor (EGFR) [77], to regulate migration, chemotaxis and cytokine production. uPA binding also regulates the affinity of uPAR for the α3β1-integrin [78], to regulate cell adhesion and cell signaling. Furthermore, the kringle domain of uPA interacts with the αvβ1-integrin in an uPAR-independent manner [79]. Increases in the levels of uPA are associated with a number of pathologies, including asthma, COPD and PAH [59, 60, 80-82]. For smooth muscle cells, plasminogen activation by uPA is accelerated by the annexin A2 hetero-tetramer (AIIt) [83], an extracellular protein complex comprised of annexin A2 and S100A10 (p11). The AIIt also serves as a signal transducer for plasmin in mediating its pro-inflammatory effects on ASM cells [84] and macrophages [85]. The importance of annexin A2 in cancer is becoming increasingly recognised, however, little is still known about the role of annexin A2 in respira‐ tory disease [86-90].

The proteolytic activity of plasmin releases the otherwise latent forms of growth factors such as epidermal growth factor (EGF) and TGF-β [91, 92]. Plasminogen activation by smooth muscle cells is associated with MMP activation [73] and targeting the EGFreceptor (EGFR) or MMPs attenuates plasmin(ogen)-stimulated proliferation [83]. The effects of plasmin(ogen) on EGFR signaling are contributed by heparan sulphate proteoglycan binding EGF (HB-EGF), an EGFR ligand, which is released from heparin by MMP-mediat‐ ed proteolysis (Figure 2). Like EGFR transactivation, plasmin-stimulated mobilization of matrix-bound TGF-β contributes to collagen synthesis in smooth muscle cells in a manner involving TGF-β receptor signaling [91]. TGF-β also stimulates ASM cell proliferation [93], in an indirect manner involving autocrine bFGF production [94].

Plasmin also activates the protease-activated receptor-1 (PAR-1), the proto-typical receptor of thrombin (and FXa). PAR-1 is expressed in inflammatory cells including, macrophages, mast cells and eosinophils [96-99], and extravascular structural cells including, smooth muscle cells [100]. Activation of PAR-1 in stromal cells is considered to evoke pro-tissue remodeling activities, which contributes to disease pathology. Levels of PARs are increased in structural cells in tissue remodeling lung diseases [101], and targeting PAR-1 reduces pulmonary

**Figure 2.** Plasmin-evoked EGFR transactivation. HB-EGF is an important ligand of EGFR, and is released from hepar‐ an sulphate proteoglycan by the proteolytic actions of MMPs. HB-EGF is expressed in the airway epithelium and smooth muscle in situ [95].

inflammation and tissue remodeling in mouse models of lung injury and disease, including asthma [98, 102, 103]. Furthermore, PAR-1 activation elicits increased cytokine and collagen expression [103-105] and proliferation [106] of lung stromal cells, including ASM cells [107]. The up-regulation of both PAR-1 and PAR-2 in VSM cells following injury and their subse‐ quent activation by hemostatic proteases is considered to contribute to the pathogenesis of atherosclerosis [5]. Whilst plasmin has ~10 times less affinity for PAR-1 than thrombin [108], integrin co-receptors augment plasmin-evoked PAR-1 activation [109]. Binding to α9β<sup>1</sup> integrin localizes plasmin to the cell surface and protects it from α2-antiplasmin inhibition, increasing PAR-1 activation, whilst also activating pathways downstream of α9β1 integrin through integrin-linked kinase (ILK) [109]. Additionally to plasmin, the plasmin-activated MMP-1 and MMP-13 also cleave the N-terminal exodomain of PAR-1, but at sites alternative to those of thrombin, FXa and plasmin, eliciting distinct cellular responses [110].

#### **5.3. Cross-linking**

co-expressed during remodeling in the airways following allergen challenge [74]. VSM cells also produce and secrete MMPs including MMP-2, MMP-3, MMP-7 and MMP-9 [34]. MMPs formed by VSM cells are considered to contribute to the thinning, thus destabilization of the atherosclerotic fibrous cap in atherogenesis [44]. Like ASM cells, VSM cells do not produce plasminogen *de novo*, but express urokinase plasminogen activator (uPA), which readily activates plasminogen convected from plasma through the blood vessel wall [22]. Hypoxia induces uPA expression in VSM cells, in association with increased cell migration and matrix

Interestingly uPA and plasminogen gene deletion (but not tPA gene deletion) also reduces hypoxia-induced PAH and pulmonary vascular remodeling in mice [75]. These effects are likely to be independent of fibrinolysis, as uPA does not bind fibrin with the same high affinity as tPA. Plasmin formed by uPA has pro-inflammatory and -remodeling activities which are not associated with the break-down of fibrin. Furthermore, uPA has plasmin(ogen)-independ‐ ent effects. The amino-terminal fragment of uPA interacts with its receptor, uPAR to activate other receptors such as the formyl-peptide receptor 2 (FPR2) [76] and the epidermal growth factor receptor (EGFR) [77], to regulate migration, chemotaxis and cytokine production. uPA binding also regulates the affinity of uPAR for the α3β1-integrin [78], to regulate cell adhesion and cell signaling. Furthermore, the kringle domain of uPA interacts with the αvβ1-integrin in an uPAR-independent manner [79]. Increases in the levels of uPA are associated with a number of pathologies, including asthma, COPD and PAH [59, 60, 80-82]. For smooth muscle cells, plasminogen activation by uPA is accelerated by the annexin A2 hetero-tetramer (AIIt) [83], an extracellular protein complex comprised of annexin A2 and S100A10 (p11). The AIIt also serves as a signal transducer for plasmin in mediating its pro-inflammatory effects on ASM cells [84] and macrophages [85]. The importance of annexin A2 in cancer is becoming increasingly recognised, however, little is still known about the role of annexin A2 in respira‐

The proteolytic activity of plasmin releases the otherwise latent forms of growth factors such as epidermal growth factor (EGF) and TGF-β [91, 92]. Plasminogen activation by smooth muscle cells is associated with MMP activation [73] and targeting the EGFreceptor (EGFR) or MMPs attenuates plasmin(ogen)-stimulated proliferation [83]. The effects of plasmin(ogen) on EGFR signaling are contributed by heparan sulphate proteoglycan binding EGF (HB-EGF), an EGFR ligand, which is released from heparin by MMP-mediat‐ ed proteolysis (Figure 2). Like EGFR transactivation, plasmin-stimulated mobilization of matrix-bound TGF-β contributes to collagen synthesis in smooth muscle cells in a manner involving TGF-β receptor signaling [91]. TGF-β also stimulates ASM cell proliferation [93],

Plasmin also activates the protease-activated receptor-1 (PAR-1), the proto-typical receptor of thrombin (and FXa). PAR-1 is expressed in inflammatory cells including, macrophages, mast cells and eosinophils [96-99], and extravascular structural cells including, smooth muscle cells [100]. Activation of PAR-1 in stromal cells is considered to evoke pro-tissue remodeling activities, which contributes to disease pathology. Levels of PARs are increased in structural cells in tissue remodeling lung diseases [101], and targeting PAR-1 reduces pulmonary

in an indirect manner involving autocrine bFGF production [94].

invasion, suggesting a role of plasmin proteolysis in PAH [8].

tory disease [86-90].

366 Muscle Cell and Tissue

Dysregulated matrix cross-linking and stability contributes to tissue remodeling in vascular disease and ageing. Lysyl oxidases cross-link collagen fibrils by modification of the ε-amino group in the side chain of lysines. Lysyl oxidase activity is increased in the vascular lesions of patients with PAH [9]. The expression of lysyl oxidases in VSM cells is responsive to hypoxia and increases in the lungs of mice in experimental PAH [9]. Glycation of collagens also induces covalent bridges between collagen fibrils, an age-related modification that contributes to arterial stiffness [34]. Similarly, vascular calcification, which involves transglutaminase-2 induced collagen crosslinking and dimerization of osteopontin, is a degenerative and endstage process of atherosclerosis, which also contributes to the stiffness of ageing arteries [111]. Vascular calcification involves the de-differentiation of the VSM cell to a pro-calcificatory phenotype, where expression of the matrix Gla protein (MGP) and bone morphogenetic protein-2 (BMP-2) are decreased and increased respectively.
