**6. Smooth muscle cell and ECM interactions**

Smooth muscle cells are highly sensitive to the biochemical and mechanical state of the surrounding ECM. Signal transduction, and the transmission and distribution of mechani‐ cal forces within the cell are dependent on the actin-cytoskeleton. This deformable polymer network also has important roles in maintaining cell shape (size) and cell migration [112]. The interaction of the actin-cytoskeleton with the contractile apparatus and the ECM involves integrins and focal adhesion (FA) complexes, and is a key determinant of the biochemical and mechano-sensing properties of smooth muscle cells. The highly mallea‐ ble actin-cytoskeleton constantly rearranges, in a process involving assembly and disassem‐ bly, in response to both bio-physical and -chemical stimuli [113]. Alterations in smooth muscle stiffness in health and disease involve changes to actin-cytoskeleton organization, which is influenced by the ECM [114].

#### **6.1. Biochemical transduction**

Biochemically, ECM protein signal through integrin receptors and the dystrophin-glycopro‐ tein complex in smooth muscle cells [115]. The native fibrillar and denatured non-fibrillar forms of collagen regulate smooth muscle cell function differentially. The anti-proliferative effect of fibrillar collagen type I on VSM [116] and ASM [68] cells, which arrest cells in the G1 phase of the cell cycle, involves α2β1 integrin binding. Although, the non-fibrillar forms of collagen types I and III do not contribute structurally to tissue integrity, they retain cell signaling activity. The GFOGER motif of the characteristic triple helix of non-fibrillar type I collagen binds with high affinity to the integrins α1β1, α2β1, α10β1, and α11β1 [117]. Fur‐ thermore, proteolytic cleavage of the helix by MMPs and plasmin reveals the 'matricryptic' RGD integrin-binding site [118]. Non-fibrillar type I collagen stimulates smooth muscle cell proliferation [119], survival [120], and cytokine release [28] in an integrin-dependent manner. Similarly, fibronectin, which binds α5β1 integrin via its RGD motif, also stimulates smooth muscle cell proliferation [119] and augments cytokine production in response to stimuli such as IL-1β [28]. Fibrin degradation products (FDPs) also regulate smooth muscle cell migration via binding the α5β3 integrin [64], as well as stimulating smooth muscle cell cytokine produc‐ tion and proliferation via binding TLR-4 [121].

#### **6.2. Mechano-transduction**

Connections between the ECM and the actin cytoskeleton of smooth muscle cells allows for an efficient transfer of force between the contractile apparatus and the extracellular environ‐ ment, which is important in myogenic force generation [122]. These same connections, which involve integrins and FA complexes, also regulate smooth muscle cell stiffness and phenotype. Mechanical forces, whether external or internal to tissue in which smooth muscle cells reside, stretch and strain protein-cell surface integrin FA complexes, activating downstream intracel‐ lular signaling pathways. This process is termed mechano-transduction, and often elicits mechanically sensitive ion channels to open or close, changing the polarity of the membrane potential, which then activates voltage-gated channels. Mechanical strain in smooth muscle cells activates RhoA, a pivotal regulator of actin adhesion organization [123]. The β1-integrin is an important mechanoreceptor, regulating cell function and viability via the inositol 3-kinase (PI3K)/Akt signaling pathway, in a process involving FA kinase (FAK) and integrin-linked kinase (ILK) phosphorylation [124, 125].

**6. Smooth muscle cell and ECM interactions**

which is influenced by the ECM [114].

tion and proliferation via binding TLR-4 [121].

**6.2. Mechano-transduction**

**6.1. Biochemical transduction**

368 Muscle Cell and Tissue

Smooth muscle cells are highly sensitive to the biochemical and mechanical state of the surrounding ECM. Signal transduction, and the transmission and distribution of mechani‐ cal forces within the cell are dependent on the actin-cytoskeleton. This deformable polymer network also has important roles in maintaining cell shape (size) and cell migration [112]. The interaction of the actin-cytoskeleton with the contractile apparatus and the ECM involves integrins and focal adhesion (FA) complexes, and is a key determinant of the biochemical and mechano-sensing properties of smooth muscle cells. The highly mallea‐ ble actin-cytoskeleton constantly rearranges, in a process involving assembly and disassem‐ bly, in response to both bio-physical and -chemical stimuli [113]. Alterations in smooth muscle stiffness in health and disease involve changes to actin-cytoskeleton organization,

Biochemically, ECM protein signal through integrin receptors and the dystrophin-glycopro‐ tein complex in smooth muscle cells [115]. The native fibrillar and denatured non-fibrillar forms of collagen regulate smooth muscle cell function differentially. The anti-proliferative effect of fibrillar collagen type I on VSM [116] and ASM [68] cells, which arrest cells in the G1 phase of the cell cycle, involves α2β1 integrin binding. Although, the non-fibrillar forms of collagen types I and III do not contribute structurally to tissue integrity, they retain cell signaling activity. The GFOGER motif of the characteristic triple helix of non-fibrillar type I collagen binds with high affinity to the integrins α1β1, α2β1, α10β1, and α11β1 [117]. Fur‐ thermore, proteolytic cleavage of the helix by MMPs and plasmin reveals the 'matricryptic' RGD integrin-binding site [118]. Non-fibrillar type I collagen stimulates smooth muscle cell proliferation [119], survival [120], and cytokine release [28] in an integrin-dependent manner. Similarly, fibronectin, which binds α5β1 integrin via its RGD motif, also stimulates smooth muscle cell proliferation [119] and augments cytokine production in response to stimuli such as IL-1β [28]. Fibrin degradation products (FDPs) also regulate smooth muscle cell migration via binding the α5β3 integrin [64], as well as stimulating smooth muscle cell cytokine produc‐

Connections between the ECM and the actin cytoskeleton of smooth muscle cells allows for an efficient transfer of force between the contractile apparatus and the extracellular environ‐ ment, which is important in myogenic force generation [122]. These same connections, which involve integrins and FA complexes, also regulate smooth muscle cell stiffness and phenotype. Mechanical forces, whether external or internal to tissue in which smooth muscle cells reside, stretch and strain protein-cell surface integrin FA complexes, activating downstream intracel‐ lular signaling pathways. This process is termed mechano-transduction, and often elicits mechanically sensitive ion channels to open or close, changing the polarity of the membrane potential, which then activates voltage-gated channels. Mechanical strain in smooth muscle

In arterial blood vessels, integrin-mediated mechano-transduction triggers intracellular Ca2+ mobilization in VSM cells to evoke myogenic responses [126]. The integrin-mediated adhesion of VSM cells to the ECM is dynamically regulated in opposing directions by vasoconstrictors (*e.g.,* angiotensin II) and vasodilators (*e.g.,* nitric oxide) to control cell contractility, hence blood vessel tone [127]. VSM cells are in turn subject to varying mechanical strain (*i.e.,* intraluminal pressure) generated by moving blood. In the airways, the ASM cell regulates bronchomotor tone, contracting in response to various contractile agonists including acetylcholine, serotonin, histamine and endothelin-1 [128]. The ASM cell is also subject to mechanical forces resulting from cyclical expansion of airway diameter and cyclical lengthening of the airway wall due to breathing [122]. The ECM modulates the effects of these forces on cell function and phenotype. Cyclical strain (4% elongation) that is equivalent to normal breathing inhibits ASM cell proliferation when grown on laminin, but not collagen type I (denatured) *in* vitro [129]. The ratio of laminin to collagen is higher in the airways of non-asthmatics than asthmatics. When ASM cells grown on type I collagen are subject to increased cyclical strain (17-18% elongation), equivalent to those generated by bronchospasm and hyperinflation, an increase in migration and proliferation occurs in a MMP-dependent manner [130].

In disease, smooth muscle cell biology is affected by changes in the ECM, including increases in substrate stiffness and the generation of isometric forces in tethered collagen lattices, which influence mechano-transduction. In severe asthma, the "strait jacket" effect of ASM cells being embedded in a rigid microenvironment may reduce the amplitude of the oscillatory forces associated with breathing. This is likely to have potential consequences on smooth muscle biology, as has been proposed for lung fibroblasts in fibrotic foci (FF) lesions in idiopathic pulmonary fibrosis (IPF) [131]. Tensile collagen fibrils within FF form a rigid environment that reduces the magnitude of cyclical strain the fibroblasts are normally subjected to in healthy tissue upon breathing. A reduction in the amplitude of force of breathing-associated cyclical strain is thought to increase lung fibroblast fibrogenic activity and stiffness.

#### **6.3. Interconnection of biochemical- and mechanical-evoked signaling**

ECM biochemistry and mechanics are very much interconnected in their regulation of smooth muscle behavior. The influence of mechanics on smooth muscle cell biology is modulated by ligand biochemistry. For example, smooth muscle cells grown on fibronectin, as compared to laminin, exhibit increased cell adhesion and cytoskeletal polymerization in response to increasing stiffness of the substrata, *i.e.,* the 'stiffness by ligand' effect [132]. In VSM cells, FAs formed by cell contact with different ECM proteins, exhibit different mechanical characteristics resulting in distinct force-generating reactions [133]. VSM cells attach more strongly to fibronectin than collagen type I, vitronectin and laminin, to evoke greater myogenic forcegeneration [133]. Integrin co-receptors may also modulate PAR-1 and uPA signaling in response to biomechanical forces. uPA elicits cellular responses via binding its receptor, uPAR, 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 biomechanical forces [79].
