The myosin regulatory system is more important in smooth muscles than striated muscles. The myosin light chain (MLC) phosphorylation state that determines actin polymerization associated with smooth muscle contraction is tightly controlled by the relative activities of antagonist enzymes myosin light chain kinase(MLCK) and phosphatase(MLCP; Fig. 8.1).

Myosin light chain kinase activated by calcium–calmodulin increases sensitivity of myosin to calcium and regulates actin–myosin interactions. Phosphorylation of MLC leads to cross-bridge formation between myosin heads and actin filaments, and hence to smooth muscle contraction. Dephosphorylation of myosin light chains by myosin light chain phosphatase causes smooth muscle relaxation.

The RhoA–RoCK pathway inhibits myosin phosphatase. Stimulation of GPCRs generates smooth myocyte contraction via Ca^2 +–calmodulin-dependent activation of MLCK and subsequent myosin phosphorylation. Kinase RoCK also regulates MLC phosphorylation. Myosin phosphatase- and RhoA-interacting protein (MPRIP)⁵ is detected withprotein kinase-G and myosin phosphatase and colocalize with myofibrils in smooth myocytes.

In humans, airway smooth myocytes are endowed with multiple types ( > 350 [721]) ofG-protein-coupled receptors that participate in the intra-, auto-, juxta-, and paracrine regulation. Furthermore, alternative splicing diversifies the recepterome and augments the number of GPCR types encountered on their surface, hence heterogeneity in GPCR signaling. More than 190 GPCRs have, on average, 5 different receptor isoforms that result from various types of alternative splicing of pre-mRNAs, such as alternative splice donors and acceptors, novel introns, intron retentions, exon(s) skips, and novel exons [721].

In airways,GPCRs expressed on smooth myocytes contribute to the regulation of contraction and relaxation state. Histamine, leukotriene, prostaglandin, and muscarinic Gq-coupled receptors support contraction, whereas Gs-coupled β2-adrenergic receptors foster relaxation.

In addition, in airway smooth myocytes, G-protein-coupled receptors modulate secretion of growth factors and other signaling mediators such as eicosanoids that intervene in airway inflammation (Table 8.6).

Rapid desensitization results from receptor phosphorylation byG-protein- coupled receptor kinases (GRK). Arrestin binding mediated by GRKs initiates receptorendocytosis for resensitization after recycling to the plasma membrane. In addition, activated intracellular kinases, such as protein kinases PKA and PKC also phosphorylate GPCRs and lead to GPCR–G-protein decoupling.

Plasmalemmal CD44 glycoprotein, or epican, a heparan sulfate proteoglycan, is also synthesized by airway smooth myocytes. This molecule interacts with hyaluronic acid,collagens, andmatrix metallopeptidases; its glycosylated form with fibrinogen and fibrin; its sialyfucosylated glycoforms with selectins. Matrix metallopeptidase MMP2 is an autocrine factor required for airway smooth myocyte proliferation.

Mural smooth myocytes have a large functioning length range, in opposition to striated myocytes that works at most at 20% of their resting length. The malleability of the myofilament lattice of smooth myocytes results from remodeling of contractile filaments of the cytoskeleton in response to stress and strain exerted on these cells [742].

Smooth muscle length is determined not only by the balance between active force that it generates and passive reaction developed by the external load, but also, due to the breathing time scale, by an outside-equilibrium behavior [743]. In normal conditions, tidal fluctuations of muscle length suffice to avoid bronchoconstriction.

The malleable cytoskeleton behavior is similar to that of soft, colloidal materials [743].²⁷ The elastic modulus ranges from the order of 1 Pa to 1 kPa. The stiffness over a wide range of frequencies does not exhibit any characteristic molecular relaxation time or resonant frequency. Frictional stress within the network is typically smaller than elastic stress. Both stress types are scale-free, as material rheological responses are not associated with any particular relaxation time and rise with frequency. On application of shear of sufficient magnitude, these materials become fluid-like bodies. When shear is stopped, they return to their original solid-like state. Colloidal dispersions can actually exhibit both fluid-like and solid-like behavior, with various types of transition between these two states. Colloidal particles with an attractive interparticle energy display a fluid-to-solid transformation due to gelation, at a critical volume fraction that depends on the magnitude of attractive interparticle energy on contact. Gelation occurs when interconnected colloid-rich regions solidify. Other features of soft, glassy materials include the rheological pattern that results from the competition between the spontaneous restructuration (slow scale-free recovery of rheological properties, the so-called aging) and destruction of the internal structure by shear (“shear rejuvenation”). Intermittent motions result from nanoscale rearrangements. Sub- and superdiffusive displacements rely on interactions between material constituents. Material transfer creates to low- and high-density regions of given types of constituents. Slowing of dynamics and more prominent intermittency of structural rearrangements indicate approach of a transition and kinetic arrest of colloidal particles, when crowded particles are trapped by their nearest neighbors (gelation).

Smooth myocytes adapt to a wide range of cell lengths and maintain optimal contractility. Assembly and disassembly of cytoskeletal filaments in smooth myocytes enable adaptation to large changes in cell geometry. The ability of actomyosin filaments to form and dissolve within the myofilament lattice at adequate sites and suitable instants yields the basic mechanism of cell adaptation [742]. Formation of myosin filaments is promoted by various filament-stabilizing proteins such as caldesmon. Many proteins may be assigned, removed, or reassigned in the context of reversible reorganization of the cytoskeleton. Myosin filament length is determined by the size of the actin filament network [742].

In airway smooth myocytes, the amount of contractile filaments can change rapidly to accommodate the context. Actin filament content actually increases during contractile activation. Myosin filament content also varies. In relaxed smooth myocytes, the density of myosin thick filaments is low, although of significant number. Myosin filaments indeed undergo rapid cycles of assembly and disassembly. Upon contractile stimulation, the content of myosin filaments in airway smooth myocytes rises strongly [742]. In solution, assembly of non-phosphorylated myosin monomers into filaments depends on monomer concentration. Once a critical concentration is reached, self-assembly occurs. The critical concentration increases when ATP is available and decreases when the myosin regulatory light chain is phosphorylated. In addition, C-terminal isoforms SM1 and SM2 of smooth muscle myosin heavy chain influence stability of filament assembly. Isoform SM1 confers a greater stability [742]. Yet, myosin filament lability is required for adaptive cell remodeling. Synthesis of SM1 and SM2 isoforms differs among different types of smooth myocytes as well as during development. Isoform SM2 may enhance SMC adaptation to large changes in length. In the presence of ATP^Mg, phosphorylation of the myosin regulatory light chain as well as other facilitating factors in smooth myocytes may regulate thick filament assembly.

Upon large-amplitude length oscillation applied to relaxed trachealis muscle strips, myosin filaments can partially disassemble [742]. These filaments reassemble when smooth muscle is repeatedly activated isometrically, following phosphorylation of regulatory myosin light chains. Moreover, myosin filament formation increases in the presence of actin filaments. Caldesmon that is detected interspersed in the actin filament network where myosin filaments are located can also provoke reassembly of long myosin filaments.

Every cell exerts traction on its matrix and experiences more or less large time-dependent stretch exerted by its environment. The cytoskeleton can stiffen, hence increasing traction (reinforcement response), or can soften (fluidization response) [744]. Therefore, cells respond to imposed stretch using 2 procedures. (1) Reinforcement refers to rapid actin polymerization and increased focal adhesion assembly that cause heightened cytoskeletal stiffness and traction forces. Actin polymerization and action of nanomotors can indeed generate a stress field and traction forces. However, in cells that bear cyclically significant stretch (e.g., cells of heart and lung), cell reinforcement that causes cell stiffening thus progressively impedes normal organ deformation during the functioning cycle. Hence, the reinforcement response corresponds to an inadequate strategy. These cell types then select the opposing compensatory mechanism. Fluidization means prompt reduction in cytoskeleton stiffness and elevation in molecular mobility.

When they are subjected to repetitive homogeneous stretch, human airway smooth myocytes rapidly soften, fluidize, and decrease traction force, with subsequent slow recoveries [744]. After completion of a transient stretch–unstretch maneuver of 4-s duration, the traction field quickly decays and slowly recovers. In addition, in substrates of different stiffness, the greater the matrix stiffness, the larger the static traction. On the other hand, cell response to dynamic traction is similar, whatever the matrix stiffness [744]. Therefore, dynamic responses to stretch are governed by a bulk mechanism with a time scale of the order of 1 s that does not consider changes in substrate stiffness. In addition, uni- and biaxial, isotropic stretches cause identical fluidization responses. Whereas homogeneous stretch induces fluidization, heterogeneous stretch induces reinforcement. Although fluidization response does not depend on stretch homogeneity. subsequent recoveries markedly differ [744]. Cell response is influenced by the duration of transient stretch. Traction recovery after a homogeneous biaxial stretch of 4-s duration never exceeds the prestretch value, whereas traction recovery following a homogeneous stretch of 30-s duration overtakes it. However, this reinforcement response disappears when homogeneous biaxial transient stretches are applied repetitively. On the other hand, repetitive, heterogeneous, transient stretches generate upon load removal a rapid fluidization followed by reinforcement [744]. Other smooth myocyte types as well as human umbilical vein endothelial cells exhibit the same behavior.

In summary, in repetitive homogeneous cell stretch, whether isotropic or not, the fluidization response prevails. The reinforcement response to repetitive load transients in human airway smooth myocytes occurs in the case of heterogeneous cell deformations. Transient stretch causes [744]: (1) a quick detachment of the myosin nanomotor from actin; (2) a transient decline in ^Factin concentration; and (3) a change in interactions among cytoskeletal proteins.

Regulation of cell functions, such as proliferation and differentiation, relies not only on chemical compounds such as growth factors, but also on physical andmechanical signals, in particular geometrical and rheological features of the extracellular matrix and eventual anisotropy that influence cell spreading and curvature.³⁴

Deformations of the plasma membrane activates mechanosensitive immersed proteins, such as mechanically gated ion channels, receptors, and cell adhesion molecules. In particular, integrins that cluster to build focal adhesions tether the intracellular actin cytoskeleton to the extracellular matrix. Focal adhesions are signalling hubs that contain kinases, such as FAK, MAPKs, and Src, and small GTPases such as Ras. The actin cytoskeleton also connects to the nucleus and its nucleoskeleton with intermediate filaments and lamins, via nestins (Vol. 1 – Chap. 4. Cell Structure and Function). Nuclear strains and stresses exerted on nuclear lamins and other nucleoskeletal constituents and changes in interactions of nuclear structural proteins with chromatin may modulate the chromatin organization and interplays with DNA-binding proteins, thus controlling gene expression patterns. Furthermore, mechanical stresses are converted into chemical signals that can be transmitted down to to the nucleus to modify gene expression.

In particular, effectors of theHippo pathway, or kinase (STK3 and STK4) cassette, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ; or WWTR1) relay mechanical cues to the nucleus [750]. The regulation of YAP and TAZ mediators requiresRhoA GTPase and actin–myosin contractility. Transmitter YAP precludes expression of the contractile phenotype of smooth myocytes, as it impedes activity of the myocardin–SRF complex.

Chromatin remodeling enables SMC differentiation, in particular demethylation of dimethylated Lys9 of histone-3 (H3K₉me²) and Lys20 of histone-4 (H4K₂₀me²) that allows decompaction of SMC marker gene chromatin in SMC precursors [748]. Differentiated vascular smooth myocytes possess several specific histone tail modifications (acetylation [H3K₉ac, H3K₁₄ac, and H4ac) and methylation (H3K₄me², H3K₇₉me²) at SMC marker gene CC(A/T-rich)6GG (CArG) boxes. These histone modifications enable access of themyocardin–SRF complex and coactivatorP300 histone acetyltransferase to SMC-specific CArGs, thereby activating gene transcription. Dedifferentiation of vascular smooth myocytes (phenotype change) results from removal of these histone modifications from chromatin at SMC marker gene CArG boxes, except H3K₄me² that may help maintain SMC marker genes as euchromatin and permit redifferentiation under adequate conditions [748].

During embryo- and fetogenesis, vascular smooth myocytes have various sources (e.g., neural crest, somites, and proepicardium, in particular mesoangioblasts of the dorsal aorta in mammals, in addition to endothelial cells upon endothelial–mesenchymal differentiation), according to the vascular compartment, in addition to a common population of progenitor cells, such as TIE1 + , PECAM1^low, Cdh5 − , PTPRc − precursors [751]. These precursors also express smA and homeodomain-containing factor Msx2.

Notch and Wnt control vSMC differentiation and modulate their phenotype according to exerted stimuli via transcription factors and cofactors that determine gene expression program.

Notch signaling in TIE1 + precursor cells is involved in developing arteries, whatever the vascular compartment, but not in mature vascular smooth myocytes, i.e., once the artery is stabilized. Notch upregulates transcription of the mesenchymal markers smA and PDGFRβ; it supports endothelial–mesenchymal transdifferentiation.

Smooth myocytes of major arteries arise from several cell lineages. Smooth myocytes of the developing arch artery, ductus arteriosus, and proximal regions of the major aortic arch branches derive from the cardiac neural crest; those of the root of the aorta and pulmonary artery from the second heart field; those of the the descending aorta from somites [752].

Smooth myocytes of organ arteries have also diverse origins. The neural crest gives rise to smooth myocytes and pericytes of most brain vessels; the mesothelium and proepicardium generate mesenteric and coronary vessels, respectively [752].

Notch stimulates various aspects of vSMC specification, differentiation, and maturation during development. Notch positively regulates differentiation of cardiac neural crest precursor cells into smooth myocytes. Notch is also required in the second heart field for proper patterning of the outflow tract [752]. Notch controls the differentiation of epicardium-derived cells into coronary smooth myocytes.

In classical Notch signaling, the extracellular domain of Notch receptor on the surface of signal-receiving cells interacts with the extracellular domain of Notch ligand on apposed signal-sending cells (Vol. 3 – Chap. 10. Morphogen Receptors). Four Notch receptors (Notch-1–Notch-4) on signal-receiving smooth myocytes tether to 5 transmembrane Delta-like and Jagged ligands (DLL1, DLL3–DLL4, and Jag1–Jag2) on apposed signal-sending endothelial and smooth muscle cells. Notch-1 and Notch-4 reside primarily in endothelial cells; Notch-1 pulmonary artery smooth myocytes during development; Notch-2 in mesenchymal cells and aorta and pulmonary artery smooth myocytes; Notch-3 in smooth myocytes and pericytes of the microvasculature of the central nervous system [752]. Ligands DLL1, DLL4, and Jag2 lodge on endothelial cells; Jag1 on both endothelial and smooth muscle cells. In addition, Notch receptors can interact with other ligand types.³⁵

Notch receptor is transported to the cell surface as a heterodimer. Notch receptor precursor is actually cleaved by furin during transport to the plasma membrane into a large ectodomain (Notch^ECD) and a membrane-tethered intracellular domain (Notch^MTIC) that remain connected by a juxtamembrane heterodimerization domain.

Upon ligand binding, Notch is successively cleaved byadamlysins such as ADAM17 at the extracellular juxtamembrane region and by the γ-secretase complex that liberates the Notch intracellular domain (Notch^ICD) from the plasma membrane. On the one hand, Notch^ECD isendocytosed into the signal-sending cell. On the other, Notch^ICD translocates to the nucleus, where it forms an active transcriptional complex with RBPJκ transcription factor and coactivators such as Mastermind-like coactivator (MamL). In the absence of Notch, RBPJκ represses transcription, as it recruits histone deacetylases and other corepressors. Nuclear Notch^ICD displaces the histone deacetylase–corepressor complex from RBPJκ, enabling transcriptional activation of Notch target genes such as class-B basic helix–loop–helix proteins Hairy enhancer of Split (HES1–HES7 or bHLHb37–bHLHb43) and HES-related transcriptional regulators (HRT1–HRT3). In addition, other Notch target includes PDGFRβ, the microRNA cluster miR143–miR145, and components of the Notch pathway (e.g., Notch-3 and Jag1).

Intimal hyperplasia is characterized by a temporospatial change in Notch-1 to Notch-3 activity [752]. In smooth myocytes, Notch-1, but not Notch-3, mediates proliferation, migration, and survival, hence neointima formation.

Pulmonary arterial hypertension results from a remodeling of small pulmonary arteries with proliferation of smooth muscle and endothelial cells that causes wall thickening and lumen narrowing. The Notch-3-HES5 pathway is involved [752].

Platelet-derived growth factor-BB and transforming growth factor-β mediate vSMC phenotype switching. The former is involved in the repression of vSMC-specific gene expression; the latter promotes the contractile phenotype. Notch cooperates with PDGF and TGFβ to regulate vSMC migration and differentiation [745]. On the other hand,Wnt inhibitory factor-1 hampers PDGFbb-induced vSMC proliferation.

Secreted, cysteine-rich Wnt glycoproteins that are encoded by 19 genes regulate SMC proliferation, differentiation, migration, and survival. They interact with 10 Frizzled receptors as well as coreceptors, such as low-density lipoprotein-related proteins LRP5 and LRP6 for the canonical, β-catenin dependent Wnt pathway and receptor protein Tyr kinase-like orphan receptor ROR2 for the non-canonical, β-catenin independent Wnt axis.

Wnt proteins regulate the synthesis of several matrix constituents, such as fibronectin and versican, as well as matrix metallopeptidases (MMP2, MMP3, MMP7, MMP9, MMP13, MMP14, and MMP26).

In the conventional pathway, β-catenin translocates to the nucleus, where it assists in the TCF–LEF-mediated transcription of target genes. In arterial and venous smooth myocytes, the β Ctn–TCF axis upregulates cyclin-D1 and downregulates CKI1a cyclin-dependent kinase inhibitor, thus increasing the proliferative rate. Wnt1 and Wnt3a can trigger cyclin-D1 production in arterial smooth myocytes in vitro. Under growth factor stimulation, Wnt4 supports arterial SMC proliferation in vitro; it also contributes to pathological intimal thickening in vivo [753].

In unconventional pathways, Wnts initiate the PLC–Ca^2 + axis, thereby activating via calmodulin CamK2 and PP3 that stimulate NFκ B and NFAT transcription factors on the one hand and PKC that stimulates NFκ B and CREB transcription factors on the other. Furthermore, Wnt can bind to ROR2 in the absence of Frizzled to release Ca^2 + from intracellular stores and activate JNK kinase. In addition, Wnt can also activate Rho and Rac GTPases, hence RoCK on the one hand, and PKA, thus CREB on the other. Factor NFAT1 promotes cyclin-D1 gene expression, hence arterial SMC proliferation [753]. In addition, CamK2 favors arterial SMC proliferation. Moreover, JNK is activated in proliferating smooth myocytes.

The canonical Wnt pathway is controlled by Dickkopf inhibitors that preclude Wnt–LRP interaction. Both canonical and non-canonical Wnt pathways are inhibited by secreted Frizzled-related proteins and Wnt inhibitory factor-1 that prevent Wnt-Fz linkage. Nevertheless, Dickkopf-1 promotes SMC proliferation in rat mesenteric microvasculature [753].

Differentiation of venous and arterial smooth myocytes may also be regulated by Wnt proteins. Wnt3a promotes the expression of the early myofibroblast marker SM22α; Wnt7b may intervene during differentiation of pulmonary smooth myocytes [753].

MicroRNAs contribute to the regulation of vascular smooth myocyte fate. The microRNA cluster miR143–miR145 as well as miR21 and miR1 support the contractile phenotype. On the other hand, miR221, miR146a, miR26a, and miR24 promote cell proliferation after vascular injury [745]. The microRNA cluster miR143–miR145 may be activated by Jagged-1–Notch andmyocardin–SRF axes. In young hypertensive patients, the expression of angiotensin AT₁receptor is negatively correlated with miR155 agent.

Krüpple-like factors KLF4 and KLF5,ETS-like transcription factor ELk1, HES-related repressor protein HERP1,FoxO4, and RelA subunit ofNFκ B hinder vSMC-specific gene expression. These mediators propagate effects ofPDGFbb,TGFβ, and Notch, at least partly by disrupting SRF–myocardin interaction, thereby supporting the synthetic and proliferative phenotype of vascular smooth myocytes (Table 8.9) [745]. MicroRNA-143 and -145 are targets of SRF and myocardin, which maintain the contractile phenotype of vascular smooth myocytes and target KLF4 and ELk1 transcription factors.

MicroRNA-146a targets the 3′-untranslated region of the Krüppel-like factor Klf4 transcript, thereby promoting vSMC proliferation in vitro andintimal hyperplasia in vivo [754].³⁶ Conversely, Krüppel-like factor KLF4 binds to and impedes activity of the miR146a promoter (mutual inhibition withfeedback loop), in competition with its KLF5 antagonist that promotes vSMC proliferation via miR146a regulator.³⁷