Characterization of Vascular Smooth Muscle Cell Phenotypes

Given the multiple origins and distinct subpopulations of VSMCs, a compelling central question for understanding VSMC biology is how cells from these diverse embryonic origins, initially expressing lineage-specific pathways, differentiate to express the same marker genes specifically characteristic of VSMCs.^8,10 Another question is how these same VSMCs, responding to both extrinsic and intrinsic cues, can alter expression of these genes (and thus molecular pathways), leading to diverse phenotypes with distinct and diverse functions. VSMC phenotypes can be loosely divided into three types: contractile/differentiated, synthetic/dedifferentiated, and inflammatory.

Contractile, differentiated vascular smooth muscle cells

Contractile or differentiated VSMCs are characterized by a repertoire of contractile proteins, contractile-regulating proteins, contractile agonist receptors, and signaling proteins responsible for contraction and maintenance of vascular tone.^3,11,12 Of the VSMC “marker” proteins expressed in the contractile phenotype repertoire (Fig. 3-1), the most discriminating markers are smooth muscle myosin heavy chain (SMMHC) in conjunction with alpha-smooth muscle actin (αSMA), smoothelin, SM-22α, h1-calponin, and h-caldesmon.² In addition to expressing these proteins associated with contractile function, contractile VSMCs exhibit differential levels of ECM components (increased collagen types 1 and IV) and matrix-modifying enzymes (decreased matrix metalloproteinases [MMPs] and increased tissue inhibitors of matrix metalloproteinases [TIMPs]). Contractile VSMCs are further characterized by an elongated spindle-shaped morphology in culture, a low proliferative rate, and expression of α1β1, α7β1 integrins and the dystrophin-glycoprotein complex (DGPC).^3,13

Figure 3-1 Summary of VSMC phenotype characteristics along the phenotypic continuum between contractile, differentiated phenotype on left and synthetic, dedifferentiated phenotype on right, with some of the environmental cues that modulate this continuum.

Col, collagen; ECM, extracellular matrix; FN, fibronectin; LN, laminin; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitors of MMPs.

(Adapted from Beamish JA, He P, Kottke-Marchant K, et al: Molecular regulation of contractile smooth muscle cell phenotype: implications for vascular tissue engineering. Tissue Eng Part B Rev 16:467–491, 2010; Moiseeva EP: Adhesion receptors of vascular smooth muscle cells and their functions. Cardiovasc Res 52:372–386, 2001; Rensen SS, Doevendans PA, van Eys GJ: Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J 15:100–108, 2007; and Raines EW, Bornfeldt KE: Integrin α7β1 COMPels smooth muscle cells to maintain their quiescence. Circ Res 106:427–429, 2010.)

Upstream Mediators of Phenotypic Modulation

Growth-inducing factors

Soluble factors that include growth factors, hormones, and reactive oxygen species (ROS) serve as upstream mediators of the phenotypic switch from contractile to synthetic VSMCs, which results in large part from coordinate activation/repression of VSMC marker genes important in the contractile response^2,3,15,16 (Fig. 3-2). Some of the most important growth-inducing factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), and basic fibroblast growth factor (bFGF). Growth factors bind to surface membrane receptor tyrosine kinases (RTKs), triggering sequential downstream signaling pathways mediated through complex formation of activated RTKs with adaptor and signaling proteins Grb2/Shc/Sos, and activation of intracellular kinases, including phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs: extracellular signal regulated kinase, ERK1/2, p38MAPK, and c-jun NH2-terminal kinase, JNK), Akt, MAPKAPK2, and p70^S6 kinase (p70^S6K). These signals not only transcriptionally mediate the switch to the synthetic phenotype, but also serve to promote growth and survival. In addition, ROS such as hydrogen peroxide (H₂O₂) produced by activation of NADPH oxidases, multimeric enzymes containing p22phox and other subunits depending upon the specific isoform, can act as second messengers for canonical G protein–coupled receptor (GPCR) and RTK pathways.¹⁷

Figure 3-2 Summary of multiple soluble extracellular factors, their receptors, their interacting signaling pathways, and various transcription factors responsible for expression of the synthetic/dedifferentiated VSMC phenotype, characterized by growth/survival pathways and ECM formation.

Details are outlined in text.

(Adapted from Owens GK, Kumar MS, Wamhoff BR: Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84:767–801, 2004; Berk BC: Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 81:999–1030, 2001; Griendling K, Harrison D, Alexander R: Biology of the vessel wall. In Fuster V, Walsh R, O’Rourke R, Poole-Wilson P, editors: Hurst’s the heart. 12th ed. New York, 2008, McGraw-Hill, pp 135–154; Mehta PK, Griendling KK: Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 292:C82–C97, 2007; and Hilenski L, Griendling K, Alexander R: Angiotensin AT1 receptors. In Re R, DiPette D, Schiffrin E, Sowers J, editors. Molecular mechanisms in hypertension. London, 2006, Taylor and Francis, pp 25–40.)

Differentiation-inducing factors

In contrast to growth factor–stimulated proliferation, the cytokine transforming growth factor β (TGF-β) and members of the bone morphogenetic protein (BMP) subgroup of this family promote the differentiated, contractile phenotype in VSMCs by inducing expression of the VSMC contractile genes αSMA and calponin (Fig. 3-3). Transforming growth factor β binds to a tetrameric complex consisting of two type I and two type II receptors, resulting in phosphorylation of Smads, transcription factors named for Caenorhabditis elegans Sma and Drosophila Mad (mothers against decapentaplegic).¹⁸ Within the TGF-β signaling pathway itself, different Smads control expression of different markers. For example, Smad3 transactivates the SM22α promoter, while Smad2 activates the αSMA gene. Other soluble factors that inhibit proliferation and increase differentiation include heparin and retinoic acid.⁹ Most smooth muscle differentiation markers share additional common transcriptional pathways, discussed in more detail later. For example, both TGF-β-induced phosphorylated Smads and ECM-induced activation of integrins, mediated through focal adhesion components vinculin, talin, and tensin, in concert with changes in cytoskeletal F/G actin dynamics, result in myocardin-related transcription factor (MRTF) induction of cytoskeletal/contractile genes (see Fig. 3-3).

Figure 3-3 Summary of soluble and insoluble extracellular factors, their receptors, their interacting signaling pathways, and transcription factors responsible for expression of the contractile/differentiated VSMC phenotype.

Details are outlined in text.

(Adapted from Owens GK, Kumar MS, Wamhoff BR: Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84:767–801, 2004; Berk BC: Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 81:999–1030, 2001; Griendling K, Harrison D, Alexander R: Biology of the vessel wall. In Fuster V, Walsh R, O’Rourke R, Poole-Wilson P, editors. Hurst’s the heart. 12th ed. New York, 2008, McGraw-Hill, pp 135–154; Mehta PK, Griendling KK: Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 292:C82–C97, 2007; and Hilenski L, Griendling K, Alexander R: Angiotensin AT1 receptors. In Re R, DiPette D, Schiffrin E, Sowers J, editors. Molecular mechanisms in hypertension. London, 2006, Taylor and Francis, pp 25–40.)

Transcriptional Regulation of Vascular Smooth Muscle Cell Diversity

The complex web of signaling pathways induced by these external signals—whether they are soluble, insoluble, structural, or mechanical—converge on a network of transcription factors (TFs) that coordinately regulate gene expression and act as “master switches” for growth and differentiation²⁷ (Fig. 3-4). Transcription of VSMC-specific differentiation or proliferative genes is regulated by cooperative interaction of TFs and their coregulators, including SRF,²⁸ myocardin and myocardin-related TFs (MRTF-A and -B),²⁹ Ets domain transcription factors known as ternary complex factors (TCFs),³⁰ zinc finger factors GATA6³⁰ and PRISM/PRDM6,³¹ and Krüppel-like factors (KLFs).^32,33

Figure 3-4 Model for opposing roles of transcription factors, their coregulators, and chromatin remodeling enzymes in control of vascular smooth muscle cell (VSMC) growth or differentiation.

Differentiation-inducing extracellular cues such as G protein–coupled receptor (GPCR) or integrin activation, which increase myocardin or modulate Rho-mediated actin dynamics, respectively, stimulate signaling pathways leading to the transcription factor serum response factor (SRF). SRF binds to a CArG deoxyribonucleic acid (DNA) sequence found in promoters of many cytocontractile genes and interacts with myocardin/MRTF/p300 histone acetylase to promote VSMC marker gene expression. Growth factor signaling through the mitogen extracellular signal regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway represses VSMC marker genes by phosphorylation of the ternary complex factor (TCF) Elk-1 and by increasing KLF4 expression. Phospho-Elk-1 inhibits SRF interaction with myocardin and KLF4, which binds to G/C-rich elements located in regulatory elements controlling expression of VSMC contractile genes, recruits histone deacetylase (HDAC), and reduces SRF binding to CArG elements. Ang II, angiotensin II; MRTF, myocardin-related transcription factor; PDGF, platelet-derived growth factor; RTK, receptor tyrosine kinase.

(Adapted from Wang D-Z, Olson EN: Control of smooth muscle development by the myocardin family of transcriptional coactivators. Curr Opin Genet Dev 14:558–566, 2004; and Pipes GC, Creemers EE, Olson EN: The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes Dev 20:1545–1556, 2006.)

Posttranscriptional Regulation of Vascular Smooth Muscle Cell Diversity: Noncoding microRNAs

Upstream signaling and downstream transcriptional pathways in VSMCs are intertwined with a multitude of micro ribonucleic acid (miRNAs) that act as “rheostats” and “switches” in regulating protein activity in development, function, and disease.⁴⁴ miRNAs are small, noncoding RNAs (20-25 nucleotides in length) that associate with a miRNA-induced silencing complex (miRISC) of regulatory proteins, including Argonaute family proteins, Argonaute interacting proteins of the GW182 family, eukaryotic initiation factors (eIFs), polyA-binding complexes, decapping enzymes/ activators, and deadenylases, to induce posttranscriptional silencing of their target genes.⁴⁵ These multiple components are assembled and interact in a multistep process with components of the translational machinery to inhibit translation initiation, mark mRNAs for degradation through deadenylation, and sequester targets into cytoplasmic P bodies.⁴⁴ Multiple mechanistic models for miRNA-induced gene silencing have been proposed that provide insights into the molecular mechanisms of translational inhibition, deadenylation, and mRNA decay, but questions remain concerning the kinetics and ordering of these translational events and whether they are coupled or independent.⁴⁵ A recent unifying model for miRNA-regulated gene repression is an attempt to reconcile the often conflicting existing data. It proposes that recruitment of Argonaute and associated GW182 proteins to miRNA induces binding to the mRNA 5′m⁷ Gcap, thus blocking translation initiation, potentially by mRNA deadenylation. Subsequent to miRNA-mediated deadenylation, mRNA is degraded through recruitment of decapping proteins.⁴⁶ In this model, inhibition of translation initiation is linked to subsequent rapid mRNA decay in a coupled process. Because miRNAs—which in general are negative regulators of gene expression—may be almost as important as transcription factors in controlling gene expression in the pathogenesis of human diseases,⁴⁷ insights into the functions of this class of noncoding RNAs are important in evaluating their potential use as therapeutic targets.⁴⁵

Cardiovascular-specific, highly conserved miRNAs miR-143 and miR-145, the most abundant miRNA in the vascular wall,⁴⁸ are key players in programming VSMC fate from multipotent progenitors in embryonic development and in reprogramming VSMCs during phenotypic modulation in the adult^44,49 (Fig. 3-5). miR-143 and miR-145 have distinct sequences but are clustered together and transcribed as a bicistronic unit. Upstream in the genomic sequence of miR-143/145 is a conserved SRF-binding CArG box site, indicating control by SRF and myocardin.^49,50 These miRNAs cooperatively feed back to modulate the actions of SRF by targeting a network of transcription factors/coactivators/corepressors. This network includes miR-145-induced repression of KLF4, a positive regulator of proliferation and myocardin repressor; miR-143-induced repression of Elk-1, a myocardin competitor and positive regulator of proliferation; and, contrary to the usual inhibitory role of miRNA, miR-145-induced stimulation of myocardin, a positive regulator of differentiation. Thus, miR-145 is necessary and sufficient for VSMC differentiation, and the miR-143/miR-145 cluster acts as an integrated signaling node to promote differentiation while concurrently repressing proliferation.⁴⁹ Although mice with genetic deletions for miR-143/145 show no obvious abnormalities in early development, VSMCs in the adult exhibit both structural and phenotypic differences in injury- or stress-induced vascular remodeling. Ultrastructural analysis of arteries from miR-143/145 knockout mice shows reduced numbers of medial VSMCs with a contractile appearance, and an increase in synthetic VSMCs.⁵¹ These results suggest that miR-143 and miR-145 modulate cytoskeletal structure, actin dynamics, and modulation to a dedifferentiated phenotype⁵⁰ (see Fig. 3-5). Importantly, miR-143/145 knockout mice with increased synthetic VSMCs develop spontaneous neointimal lesions in the femoral artery in the absence of hyperlipidemia and inflammation, supporting a key role for phenotypically altered VSMCs in the pathogenesis of lesion formation.⁵¹

Figure 3-5 Model for regulation of vascular smooth muscle cell (VSMC) phenotypes by cardiovascular-specific micro ribonucleic acids (microRNAs) miR-143 and miR-145.

These miRNAs act as signaling nodes to modulate serum response factor (SRF)-dependent transcription by regulating coactivators and co-repressors to control VSMC proliferation or differentiation. miR-145 represses proliferation by repressing KLF4 and promotes differentiation by stimulating myocardin; miR-143 represses proliferation by repressing Elk-1. miR-143/145 also controls actin/cytoskeletal remodeling by repressing KLF4/5 and regulators of actin dynamics, including MTRF/SRF activity.

(Adapted from Liu N, Olson EN: MicroRNA regulatory networks in cardiovascular development. Dev Cell 18:510–525, 2010; Cordes KR, Sheehy NT, White MP, et al: MiR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 460:705–710, 2009; and Xin M, Small EM, Sutherland LB, et al. MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury. Genes Dev 23:2166–2178, 2009.)

While miR-143 and miR-145 play keys roles in the contractile phenotype of VSMCs and the response to injury,⁵² miR-221 and miR-222 are modulators of VSMC proliferation, although largely by affecting growth-related signaling pathways rather than by controlling VSMC phenotype. miR-221 and miR-222, encoded by a gene cluster on the X chromosome, are up-regulated in VSMCs in neointimal lesions and in proliferating cultured VSMCs stimulated by PDGF-BB.⁵³ Studies show that two CDKIs, p27^KIP1 and p57^KIP2, have miR-221 and miR-222 binding sites and are gene targets for miR-221 and miR-222 in the rat carotid artery in vivo.⁵³ Thus, miR-221 and miR-222 are pro-proliferative because they repress two CDKIs, p27^KIP1 and p57^KIP2. Furthermore, PDGF, via miR-221 induction, inhibits VSMC differentiation via c-kit-induced inhibition of myocardin.⁵⁴

Posttranslational Regulation of Vascular Smooth Muscle Cell Diversity: Epigenetics

The “epigenetic landscape” controls gene expression by chemical modifications that mark regions of chromosomes either by methylation of promoter CpG sequences in the DNA itself, or by covalent modification of histone proteins that package DNA by posttranslational addition of methyl, acetyl, phosphoryl, ubiquityl, or sumoyl groups, leading to expression/repression of transcription (reviewed in⁵⁵). In VSMCs, multiple levels of epigenetic controls exist for gene expression leading to differentiation or dedifferentiation programs in healthy cells and for dysregulated gene expression in vascular disease. These epigenetic changes in VSMCs involve both DNA and histone methylation as well as histone acetylation/deacetylation. Methylation of histones, catalyzed by histone methyltransferases (HMTs), results in a tight, stable epigenetic mark between methylated histones and chromatin that can be passed to daughter cells, thus providing “epigenetic memory” that defines cell lineage and identity by controlling SRF access to VSMC-specific marker genes.⁵⁵ Acetylation is controlled by HATs, which promote gene transcription by destabilizing chromatin structure to an “open,” transcriptionally active conformation, and HDACs, which promote chromatin condensation to a “closed,” transcriptionally silent conformation with restricted access to DNA. Histone acetylation/deacetylation thus serves to regulate transcription in a rapid and “on/off” manner in response to dynamic environmental changes and links the cell’s genome with new extrinsic signals.⁵⁵

In VSMCs, SRF binding to CArG boxes in the promoters of SMC marker genes to promote the VSMC differentiated phenotype depends upon alterations of chromatin structure, including histone methylation and acetylation. In a model for epigenetic regulation of VSMC phenotype,⁵⁶ SRF binding to CArG boxes in VSMC marker gene promoters is blocked by conditions such as PDGF-BB exposure or vascular injury. Such conditions promote KLF4-induced myocardin suppression as well as KLF4-induced recruitment of HDACs, resulting in “closed” deacetylated chromatin and transcriptional repression of VSMC marker genes. Histone methylation, in contrast, is not affected by PDGF-BB and may serve as a permanent “memory” for VSMC identity during repression of SRF-dependent transcription and can, once repressive signals are terminated, reactivate the differentiation program by recruiting myocardin/SRF complexes or HATs to VSMC marker genes for reexpression. In the absence of KLF4 activation, SRF/myocardin can bind to HAT-induced acetylated “open” chromatin at CArG boxes for transcriptional activation of VSMC marker genes, thus promoting VSMC differentiation. In addition, myocardin induces acetylation of histones in the vicinity of SRF-binding promoters in VSMC marker genes by association with p300, a ubiquitous transcriptional coactivator with its own intrinsic HAT activity, leading to synergistic activation of VSMC marker gene expression. This pro-myogenic program is antagonized and repressed by myocardin binding to class II HDACs, which strongly inhibits expression of marker genes αSMA, SM22α, SMMLCK and SMMHC. These opposing actions of HATs and HDACs on SRF/myocardin function to activate or repress, respectively, VSMC differentiation and serve to regulate transcription in a rapid and reversible manner in response to dynamic changes in the environment.⁵⁵

Often, transcription mediators play roles in both classic signal transduction pathways and epigenetic programming.⁵⁷ Smad proteins, for example, transmit TGF-β signals from the membrane to the nucleus to mediate gene transcription and VSMC differentiation. The balance between Smad-induced recruitment of corepressors or coactivators to TGF-β-responsive genes is associated with activation of HDAC or HAT (p300), which then alters histone acetylation. Transforming growth factor β induces histone hyperacetylation at the VSMC marker gene SM22 promoter through recruitment of HATs, Smad3, SRF, and myocardin, demonstrating a role for HATs and HDACs in TGF-β activation of VSMC differentiation.⁵⁸

A proposed example of metabolic memory stored in the histone code of VSMCs is found in the dysregulation of histone H3 methylation, an epigenetic mark usually associated with transcriptional repression in type 2 diabetes.⁵⁹ In VSMCs derived from type 2 diabetic db/db mice, levels of H3K9me3 (H3 lysine-9 trimethylation), as well as its HMT, are both reduced at the promoters of inflammatory genes. This loss of repressive histone marks, leading to increased inflammatory gene expression, is sustained in VSMCs from db/db mice cultured in vitro, suggesting persistence of metabolic memory. These results suggest that dysregulation in the histone code in VSMCs is a potential mechanism for increased and sustained inflammatory response in diabetic patients who continue to exhibit “metabolic memory” and vascular complications after glucose normalization.⁶⁰

Cell-Cell Adhesion Molecules: Cadherins and Gap Junction Connexins

After investment of VSMCs to the EC layer of nascent vessels, vascular stabilization, also known as maturation,⁶¹ is regulated by the sphingosine 1-phosphate (S1P) receptor S1P1, a GPCR on ECs. S1P1 activates the cell-cell adhesion molecule N-cadherin in ECs and induces formation of direct N-cadherin-based junctions between ECs and VSMCs required for vessel stabilization.⁶¹ To maintain VSMC quiescence within the vascular wall, cadherin-mediated cell-cell adherens-type junctions between VSMCs inhibit VSMC proliferation, possibly by inhibiting the transcriptional activity of β-catenin, a component of the Wnt signaling pathway, which interacts with the intracellular domain of cadherins.⁶² Inhibition of β-catenin or stabilization of cadherin junctions in VSMCs may be useful in treating vascular disease or injury.

Another type of direct intercellular junction between cells in the vasculature is the gap junction.⁶³ Gap junctions, formed by connexin proteins between ECs and VSMCs and between VSMCs, are intercellular channels that allow movement of metabolites, small signaling molecules, and ions between cells.^63–65 Of the four connexin proteins expressed in VSMCs (Cx37, Cx40, Cx43, and Cx45), Cx45 is exclusively found in VSMCs, while Cx43 is the most prominent and is essential for coordination of proliferation and migration.⁶³ Homotypic gap junctions between VSMCs coordinate changes in membrane potential and intracellular Ca^2 +, and heterotypic contacts between ECs and VSMCs at the myoendothelial junction control vascular tone by EC-mediated VSMC hyperpolarization. Notably, expression and/or activity of vascular connexins are altered in vascular diseases such as hypertension, atherosclerosis, or restenosis⁶⁴ and in diabetes.⁶³

Cell-Matrix Adhesion Molecules: Integrins and Syndecans

Contraction

The primary function of differentiated VSMCs is to maintain vascular tone. This is an active process requiring significant energy expenditure, especially in resistance arterioles. A number of hormones and peptides regulate VSMC contraction, including catecholamines, Ang II, and endothelin-1. Contractions can be phasic, lasting only minutes, or tonic, depending on the stimulus.

In nearly all cases, stimulation of VSMC with contractile agents results in activation of a specific GPCR (Fig. 3-6). The immediate response is activation of PLC, which cleaves the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) to release inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃, in turn, binds to its receptor (a channel) on the sarcoplasmic reticulum (SR), creating an open conformation and translocating Ca^2 + to the cytoplasm. Simultaneously, receptor activation depolarizes the plasma membrane by altering the activity of pumps such as the sodium/potassium–adenosine triphosphate (Na⁺/K⁺-ATPase), and channels that include Ca^2 +-sensitive K⁺ channels and TRP channels.⁹⁹ Membrane depolarization leads to activation of voltage-dependent L-type Ca^2 + channels, calcium influx, and a more sustained but less robust elevation of cytosolic calcium. Moreover, Ca^2 + entry through these channels activates ryanodine receptors on the SR, further increasing Ca^2 + release into the cytosol.

Figure 3-6 Model for contraction cascade in vascular smooth muscle cell (VSMC).

Binding of contractile agonists to G protein-coupled receptors (GPCRs) activates phospholipase C (PLC) and subsequent PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to release inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), leading to increased mobilization of Ca^2 +. Ca^2 + combines with calmodulin (CaM) and activates myosin light chain kinase (MLCK)-induced phosphorylation of myosin light chain (MLC), which, together with actin, initiates contraction. In addition, guanine nucleotide exchange factor (GEF) activation of Rho leads to Rho kinase stimulation and inhibition of myosin light chain phosphatase (MLCP), resulting in enhancement of contraction. ADP, adenosine diphosphate; ATP, adenosine triphosphate; GTP, guanosine triphosphate.

(Adapted from Griendling K, Harrison D, Alexander R: Biology of the vessel wall. In Fuster V, Walsh R, O’Rourke R, Poole-Wilson P, editors. Hurst’s the heart. 12th ed. New York, 2008, McGraw-Hill, pp 135–154.)

The increased cytoplasmic calcium binds to calmodulin (CaM) at a ratio of four calcium ions to one CaM molecule. Calmodulin then undergoes a conformational change, and binds to and activates myosin light chain kinase (MLCK), the enzyme responsible for phosphorylation of the 20-kD regulatory myosin light chain (LC20) on serine 19. Activated LC20 facilitates actin-mediated myosin adenosine triphosphate (ATPase) activity and cyclic interaction of myosin and actin,¹⁰⁰ leading to contraction. Contraction is maintained even when calcium drops, suggesting that LC20 becomes sensitized to calcium, likely by inhibition of myosin phosphatase (see later discussion).¹⁰¹

Because the increase in intracellular calcium caused by vasoconstrictors is largely responsible for activation of the contractile apparatus, essential mechanisms exist to limit Ca^2 + entry and clear Ca^2 + from the cytosol. Ryanodine receptors cluster to release calcium sparks, which in turn stimulate Ca^2 +-activated large conductance K channels (BK channels) to cause hyperpolarization and limit L-type calcium channel activity.¹⁰² Additionally, the sarcoplasmic reticulum Ca^2 +-ATPase (SERCA) mediates Ca^2 + reuptake into the SR and serves to maximize Ca^2 + extrusion from the cell because the newly taken-up SR Ca^2 + is released in a directed manner towards the plasma membrane, where a plasma membrane Ca^2 +-ATPase extrudes Ca^2 + from the cell. Importantly, SERCA is inhibited by CaM kinase II–mediated phosphorylation.¹⁰³

Recently, ROS and reactive nitrogen species (RNS) have emerged as effective modulators of contractile signaling.¹⁰⁴ Specifically, high levels of ROS oxidize SERCA, thereby inhibiting its activity. Hydrogen peroxide applied externally increases IP₃ receptor-mediated release of Ca^2 + into the cytosol, and activation of NADPH oxidases by contractile agonists sensitizes the IP₃ receptor to IP₃. Ryanodine receptors are also redox-sensitive. S-nitrosylation activates them, and exposure to endogenous levels of ROS and RNS can protect these receptors from inhibition by CaM at high concentrations of calcium. Both hydrogen peroxide and superoxide can stimulate Ca^2 + entry via L-type or T-type calcium channels (including TRP channels), but S-nitrosylation by nitric oxide (NO) is inhibitory. Thus, in general, ROS and RNS inhibit Ca^2 + pumps and activate Ca^2 + entry and release, resulting in an increase in intracellular Ca^2 + concentration.

Myosin light chain phosphatase (MLCP) is also a vital regulator of vascular contraction. It is a multimeric enzyme composed of a regulatory myosin-binding subunit (MYPT1), a catalytic subunit (PP1c), and a 20-kD protein (M20). The activity of MLCP is largely regulated by Rho kinase-mediated phosphorylation of MYPT1 on Thr695, either directly or via Rho-kinase activation of ZIP kinase.¹⁰¹ Myosin light chain phosphatase activity can also be inhibited by CPI-17 (PKC-potentiated PP1 inhibitory protein of 17 kD), which when phosphorylated by PKC, acts as a pseudosubstrate, binds to PP1c, and competes with LC20 for phosphorylation. Inhibition of MLCP activity enhances contraction, as mentioned, by inducing Ca^2 + sensitization of the contractile apparatus.¹⁰⁵

Rho kinase has thus emerged as an important part of the contraction cascade.¹⁰⁶ In addition to its role in enhancing contraction, such as in response to Ang II, it is a major regulator of relaxation. Its activator, the small-molecular-weight GTPase RhoA, is a target of NO, which by activating protein kinase G (PKG), inactivates Rho, thus indirectly inhibiting Rho kinase, increasing MLCP activity, and inhibiting contraction.

It is noteworthy that paracrine factors such as NO secreted by neighboring ECs represent the major mechanism of vasorelaxation. Shear stress forces and hormones such as acetylcholine or bradykinin stimulate ECs to secrete NO, which in turn initiates VSMC relaxation.^3,107,108 Nitric oxide induces relaxation of smooth muscle potentially via a number of pathways, the most important of which depend on its ability to release cyclic guanosine monophosphate (cGMP). It can directly (via S-nitrosylation of cysteine residues) or indirectly (through PKG) activate BK channels,¹⁰⁹ thus causing membrane hyperpolarization and reducing influx through L-type Ca^2 + channels. In addition, PKG phosphorylates IP₃ receptor-associated PKG-I substrate (IRAG), which inhibits Ca^2 + release from IP₃ receptors. Nitric oxide also increases Ca^2 + uptake via S-glutathionylation of SERCA and decreases the Ca^2 + sensitivity of contractile proteins. This pathway is perturbed in diabetic animal models, in which high levels of ROS derived from NADPH oxidase 4 irreversibly oxidize SERCA, rendering it insensitive to NO.¹¹⁰ In addition to regulating Ca^2 + levels, NO-mediated activation of PKG can phosphorylate PP1c and/or MYPT1 to block vasoconstrictor-mediated inhibition of MLCP.

Other relaxing factors secreted by endothelial cells include hydrogen peroxide, prostaglandins, and epoxyeicosatrienoic acids (EETs). In addition, perivascular adventitial adipocytes (PVAs) have also been shown to secrete factors that influence contractility (reviewed in¹¹¹). These cytokines, collectively known as adipokines, are both vasoactive and pro- and antiinflammatory, and include cytokines TNF-α, IL-6, chemokines (IL-8 and monocyte chemoattractant protein [MCP-1]) and hormones (leptin, resistin, and adiponectin).^111,112

Proliferation

Vascular smooth muscle cell proliferation is important in early vascular development and in repair mechanisms in response to injury. However, excessive VSMC proliferation contributes to pathology, not only in vascular proliferative diseases such as atherosclerosis but also, ironically, as a consequence of the intervention procedures used to treat these occlusive atherosclerotic diseases and their complications, including postangioplasty restenosis, vein bypass graft failure, and transplant failure.¹¹³

Vascular smooth muscle cell proliferation can be regulated by myriad soluble and insoluble factors that activate a variety of intracellular signaling pathways such as MAPK or Janus kinase/signal transducers, tyrosine phosphorylation, and mitogen-activated proteins.^114,115 Regardless of the initial proliferative stimulus, these signaling pathways ultimately converge onto the cell cycle¹¹⁶ (Fig. 3-7). The four distinct phases of the cell cycle are: (1) Gap 1 (G1) in which factors necessary for DNA replication are assembled; (2) DNA replication or S phase; (3) Gap 2 (G2) in preparation for mitosis; and (4) mitosis or M phase. Restriction points in the cell cycle exist at transitions between G1/S and G2/M. Progression through the cell cycle phases is regulated by cyclin-dependent kinases (CDKs) and their regulatory cyclin subunits. Cyclins D/E and CDK2, 4, and 5 control G1, cyclin A and CDK2 control the S phase along with the DNA polymerase cofactor PCNA, and cyclins A/B and CDK1 control the M phase. Cyclin-dependent kinases such as p27^KIP1 and p21^CIP1 bind to and inhibit the activation of cyclin-CDK complexes (see Fig. 3-7). Activities of these enzymes depend upon phosphorylation status of CDKs, levels of expression of cyclins, and nuclear translocation of cyclin-CDK complexes. One regulatory protein is survivin, which competitively interacts with the CDK4/p16^INK4a complex to form a CDK4/survivin complex, thus inducing CDK2/cyclin E activation and S-phase entry and cell cycle progression.¹¹⁷ Transcription factors that transactivate CDKs and CDKIs also mediate cell cycle progression. It is known that p53, GAX, and GATA-6 induce p21^CIP1 expression, leading to G1 phase arrest, and E2F transcription factors control the G1/S transition regulated by the retinoblastoma protein Rb, the product of the rb tumor suppressor gene. Rb exerts its negative regulation on the cell cycle by binding to E2F transcription factors, rendering them ineffective as transcription factors. When the Rb/E2F complex is phosphorylated by CDKs in early G1, the complex is dissociated, leaving E2F available to activate genes required for S-phase DNA synthesis.¹¹⁶ It is worth noting that the HDAC inhibitor trichostatin A blocks proliferation by induction of the cell cycle inhibitor p21^CIP1 and suppression of Rb protein phosphorylation, leading to subsequent cell cycle arrest at the G1/S phase.¹¹⁷

Figure 3-7 Model for cell cycle regulation in Vascular smooth muscle cells (VSMCs).

Mitogens activate growth factor receptor tyrosine kinase (RTKs), G protein-coupled receptors (GPCRs), NADPH oxidase, and integrins to stimulate extracellular signal regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K) and Rho/Rac pathways, which converge onto cell cycle components, especially cyclin D, to regulate proliferation. Cyclin regulatory subunits and cyclin-dependent kinases (CDKs) catalytic subunits form holoenzymes that are phase-specific for the four phases of the cell cycle: G1, deoxyribonucleic acid (DNA) replication or S phase, G2, and mitosis or M phase. Endogenous cyclin-dependent kinase inhibitors (CDKIs), including p21, p27, and p57, inactivate cyclin/CDKs and therefore block cell cycle progression and proliferation. Other cell cycle regulators include the tumor suppressor p53 and the transcription factors GAX and GATA-6 that stimulate CDKI p21CIP₁ and induce cell cycle arrest. Cooperating with cyclin/CDKs is proliferating cell nuclear antigen (PCNA) for transition through G1 and S phases. Hyperphosphorylation of the retinoblastoma protein (pRb) releases elongation factor 2 F (E2F), allowing cell cycle progression through the G1 phase restriction point and expression of genes required for DNA synthesis. Activation of p53 or Rb pathways results in cell cycle arrest and senescence.

(Adapted from Fuster JJ, Fernandez P, Gonzalez-Navarro H, et al: Control of cell proliferation in atherosclerosis: insights from animal models and human studies. Cardiovasc Res 86:254–264, 2010; and Dzau VJ, Braun-Dullaeus RC, Sedding DG: Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med 8:1249–1256, 2002.)

In addition to cell cycle regulatory proteins, telomerase activity is required for VSMC proliferation. Telomeres are noncoding DNA TTAGGG repeat sequences at the ends of chromosomes that cap and stabilize chromosomes against degradation, recombination, or fusion.¹¹⁸ Associated with telomeric DNA are protein complexes, including telomerase, that synthesize new telomeric DNA in cells with high proliferative potential. Telomerase consists of an RNA component and two protein components, one of which is telomerase reverse transcriptase (TERT), the catalytic component and limiting factor for telomerase activation. When telomerase expression is low, telomere attrition with each mitotic cycle results in chromosome shortening and instability, replicative senescence, and growth arrest. In VSMCs, posttranslational phosphorylation of TERT is linked to telomerase activation, and levels of telomerase expression and activity correlate with proliferation.¹¹⁸ Importantly, telomerase activation and telomere maintenance have been associated with excessive VSMC proliferation in both animal and human vascular injury and disease;¹¹⁸ disruption of telomerase activity reduces this proliferative response.

Growth of VSMC is initiated by exposure of the cells to pro-proliferative signals. Classical growth factors activate RTKs, either directly or via GPCR-mediated transactivation.^116,117 Growth factors in VSMCs binding to RTKs include PDGF, bFGF, IGF-1, TGF-β, EGF, and hypoxia-inducible factor (HIF), and mitogens that activate GPCRs include hormones such as Ang II,¹⁵ endothelin, or oxidized LDL. Activation of these receptors stimulates sequential signaling cascades mediated by Ras, p70^S6K, Rac/NADPH/ROS, PI3K/Akt, MEK/ERK, or MAPKK/p38MAPK, which induce cyclin D1 expression.¹¹⁵ Src homology 2–containing protein tyrosine phosphatase 2 (SHP2), a member of the non-receptor protein tyrosine phosphatase family, dephosphorylates tyrosine residues on target proteins in response to growth factors, hormones, and cytokines.¹¹⁹ In VSMCs, SHP2 is a positive mediator of IGF-1- and LPA-induced MAPK signaling pathways; SHP2 has negative effects on EGF- and Ang II-induced Akt signaling, implicating SHP2 in modulating cell cycle progression, growth, and migration.

An important integration point in growth factor signaling is mTOR (mammalian target of rapamycin), which regulates protein synthesis, cell cycle progression, and proliferation.¹¹⁷ Mammalian target of rapamycin is a protein kinase that regulates translation initiation through effectors p70^S6K and eIF4E, leading to protein synthesis necessary for cell division. Rapamycin, an immunosuppressive macrolide antibiotic, inhibits mTOR downstream signaling cascades, with reductions in protein synthesis leading to cell cycle arrest.¹¹⁶ In VSMCs, rapamycin inhibits the mTOR/p70^S6K signaling axis, promotes a VSMC differentiated, contractile phenotype by regulating transcription of contractile proteins, and induces expression of the antiproliferative CDKIs p21^CIP and p27^KIP to inhibit cell cycle progression.¹¹⁷ Use of rapamycin (sirolimus)-coated coronary stents is highly effective in reducing the postangioplasty restenosis rate in interventional cardiology.¹²⁰

Ion channels for Ca^2 +, Mg^2 +, and K⁺ are also activated by growth factors and mediate proliferation. Transient increases in Ca^2 + concentration, together with subsequent Ca^2 + binding to its intracellular receptor CaM, are universally required for proliferation.¹²¹ The mechanism for the Ca^2 + sensitivity of this G1-to-S transition involves the Ca^2 +-dependent binding of CaM to cyclin E and activation of CDK2 to promote G1/S transition and VSMC proliferation (reviewed in^122,123). Elevated levels of Mg^2 + increase expression of cyclin D1 and CDK4 and decrease activation of p21^CIP1 and p27^KIP1 through an ERK1/2-dependent, p38 MAPK-independent pathway.¹²⁴ Changes in VSMC K⁺ channel expression profiles and activity are linked to cell cycle progression, implicating these ion channels as “internal timers” of VSMC cell division.¹²⁵ Growth factor–induced release of Ca^2 + from intracellular Ca^2 + storage organelle activates and up-regulates intermediate-conductance Ca^2 +-activated K⁺ (IK_Ca)-type K⁺ channels, the predominant Ca^2 +-sensitive K⁺ channel in proliferating VSMCs.¹²⁶ In addition, voltage-gated K⁺ channels K_V1.3¹²⁷ and K_V3.4¹²⁸ are up-regulated in proliferating VSMCs. Blockade of these Ca^2 +-activated and voltage-gated K⁺ channels inhibits proliferation and attenuates vascular disease/injury–induced remodeling in rodents.¹²⁹

Signals from insoluble ECM-activated integrins and from soluble growth factor mitogens converge and jointly regulate upstream cytoplasmic signaling networks to mediate expression of cyclin D1 and cyclin E and associated CDK4/6 and CDK2 in the G1 phase, the part of the cell cycle most affected by extracellular stimuli.¹³⁰ In addition, joint RTK/integrin complex signaling networks impact G1 phase regulation by inhibiting p21^CIP1 and p27^KIP1, resulting in Rb phosphorylation and induction of E2F-dependent genes, with progression to autonomous stages of the cell cycle (S, G2, and M) that are independent of external stimuli.

As noted previously, Notch proteins are also important regulators of VSMC proliferation (reviewed in²²). Notch4/HRT-induced repression of p27^KIP1 and Notch3/HRT1-induced repression of p21^CIP1, as well as up-regulation of Akt signaling, an anti-apoptosis pathway, result in promotion of VSMC proliferation. Furthermore, Notch1 is critical in mediating neointimal formation and remodeling after vascular injury.

Peroxisome proliferator-activated receptors (PPARs), nuclear hormone receptors with regulatory roles in lipid and glucose metabolism, are beneficial in VSMCs by targeting genes for cell cycle progression, cellular senescence, and apoptosis to inhibit proliferation and neointimal formation in atherosclerosis and postangioplasty restenosis (reviewed in¹³¹). Activation of PPARα suppresses G1-to-S progression by inducing expression of p16^INK4a (a CDKI), thereby inhibiting phosphorylation of Rb.¹³² This antiproliferative effect is mediated by repression of telomerase activity by inhibiting E2F binding sites in the TERT promoter.¹³³ Another PPAR isotype, PPARγ, also blocks G1-to-S cell cycle transition by preventing degradation of p27^KIP1, resulting in inhibition of pRb phosphorylation and suppression of E2F-regulated genes responsible for DNA replication.¹³¹ Similar to PPARα, PPARγ also inhibits telomerase activity in VSMCs by inhibition of early response gene Ets-1-dependent transactivation of the TERT promoter.¹³¹ Thiazolidinediones (TZD), PPARγ agonists used clinically in the treatment of type 2 diabetes mellitus, decrease VSMC proliferation and prevent atherosclerosis in murine models of the disease.¹³¹

Cyclic adenosine 3′,5′-monophosphate (cAMP) and cGMP are second messengers in myriad signal transduction pathways.¹³⁴ In VSMCs, cAMP serves as an antagonist both to mitogenic signaling pathways (by inhibiting MAPK, PI3 kinase, and mTOR signaling axes) and to cell cycle progression (by down-regulating cyclins or up-regulating CDKI p27^KIP1). An additional antiproliferative effect is due to down-regulation of S-phase kinase-associated protein-2 (Skp2) mediated by inhibition of FAK phosphorylation and adhesion-dependent signaling. Skp2 is a ubiquitin ligase subunit that targets p27^KIP1 for proteasomal degradation, thus promoting VSMC proliferation.¹³⁵

A more recently appreciated pathway that controls VSMC growth involves miRNAs. The potential involvement of these molecules was first noted in balloon-injured rat carotid arteries, where several miRNAs, including miR-21, are up-regulated compared with control arteries (reviewed in¹³⁶). Cell culture models show that miR-21 is a pro-proliferative and anti-apoptotic regulator of VSMCs, with target genes phosphatase and tensin homology deleted from chromosome 10 (PTEN), programmed cell death 4 (PDCD4), and Bcl-2. miR-21 has opposite effects on PTEN and Bcl-2: overexpression down-regulates PTEN and up-regulates Bcl-2. PTEN modulates VSMCs through PI3K and Akt signaling pathways, while Bcl-2 mediates its downstream signaling through AP-1.

Finally, cell-cell junctions, as described above for cadherins and gap junction connexins, and cell-matrix contacts can greatly influence VSMC proliferation (reviewed in¹¹⁵). Normally, resident VSMCs, surrounded by and binding to polymerized collagen type 1 fibrils through α2β1 integrins, exhibit low proliferation indices, are arrested in the G1 phase of the cell cycle, and are refractory to mitogenic stimuli. In this quiescent state, levels of cell cycle regulatory proteins are modulated to inhibit the G1/S transition: cyclin E and CDK2 phosphorylation is inhibited, while CDKIs are up-regulated and suppress cyclin E/CDK2 activity. Additionally, p70^S6K, a potent stimulator of mitogenesis and a regulator of p27^KIP1, is suppressed. In contrast, VSMCs on monomeric collagen matrices are responsive to growth factor signals which result in increased cyclin E–associated kinase activity and cell proliferation. These differential responses of VSMCs to structurally distinct forms of collagen type 1 are reflected in the differential regulation of cell cycle proteins and the differential response to mitogenic stimuli. Therefore, perturbations or degradation of the collagen matrix, as found in sites of monomeric collagen in vascular lesions, result in altered VSMC proliferation, response to mitogens, and neointimal formation.⁷⁶

Migration

Smooth muscle migration is an essential element of wound repair, but unchecked migration and proliferation can contribute to neointimal thickening and development of atherosclerotic plaques. A number of promigratory and antimigratory molecules regulate VSMC migration, including peptide growth factors, ECM components, and cytokines.¹³⁷ The extent of migration is also influenced by physical factors such as shear stress, stretch, and matrix stiffness. PDGF-BB, bFGF, and S1P are among the most potent pro-migratory stimuli in the vascular system. Intracellular signaling cascades initiated by these growth factors act in concert with those activated by integrin receptor interaction with matrix to mediate the migratory response. Matrix surrounding the migrating cell must be degraded by MMPs to allow a pathway into which the cell can protrude. Important promigratory matrix components include collagen I and IV, osteopontin, and laminin. Matrix interactions can also be antimigratory, as with the formation of stable focal adhesions, activation of TIMPs, and heparin.

When a cell begins to migrate, a number of coordinated events must take place in a cyclic fashion¹³⁸ (Fig. 3-8). Signaling mechanisms that regulate migration have mostly been studied in fibroblasts, but recently many have been confirmed in VSMCs. Migration requires specialized signaling domains at the front and rear of the cell. When confronted with a migratory stimulus, the cell senses the gradient and establishes polarity. Plasma membrane in the form of lamellipodia is then extended in the direction of movement. This process is controlled by reorganization of the actin cytoskeleton just under the protruding membrane. New focal complexes are formed in the lamellipodia via cytoskeletal remodeling and integrin interaction with the matrix. The cell body begins to contract, powered by engagement and phosphorylation of myosin II, and focal adhesions in the rear of the cell become detached, leading to retraction of the “tail” of the cell. Finally, adhesion receptors are recycled by endocytosis and vesicular transport. Successful migration is thus dependent on proper temporal and spatial activation of many molecules, most of which are related to cytoskeletal elements.

Figure 3-8 Summary of signaling and effectors molecules leading to remodeling of actin cytoskeleton at the leading edge and in focal contacts in migrating vascular smooth muscle cells (VSMCs).

In response to promigratory stimuli and activation of multiple intracellular signaling pathways (details given in text), cells extend lamellipodia and form new focal contacts, areas of dynamic actin turnover. Coordination of actin dynamics depends upon multiple actin binding and associated proteins for actin filament nucleation and extension (actin-related protein [Arp2/3], WAVE, Wiskott-Aldrich’s syndrome [WASP], mDia, profilin) and actin filament depolymerization (cofilin) and filament capping and severing (gelsolin), remodeling events regulated by small G-proteins Rho, Rac, and cdc42 and Rho-activated protein kinase (ROCK). Myosin II activation by Ca^2 +/calmodulin (CaM)/myosin light chain kinase (MLCK) and p21-activated kinase (PAK) generates traction forces on the matrix to move the cell forward. In turn, matrix components exert tractile forces by matrix/integrin binding-induced phosphorylation of focal contact components such as paxillin, focal adhesion kinase (FAK) and c-Src, which induce actomyosin motor protein interaction to move the cell forward.

(Adapted from Gerthoffer WT: Mechanisms of vascular smooth muscle cell migration. Circ Res 100:607–621, 2007.)

Much is known or inferred about the signaling mechanisms activated by PDGF in migrating cells.¹³⁷ When PDGF-BB binds to PDGFRs, receptor autophosphorylation creates binding sites for phospholipase Cγ, which mobilizes calcium; PI3K, which forms the membrane-targeting lipid PIP₂; and Ras, which activates MAPKs. Nucleation of new actin filaments at the leading edge is initiated by binding of nucleation promoting factors verprolin-homologous protein (WAVE) and Wiskott-Aldrich’s syndrome protein (WASP) to actin-related protein ARP2/3; phosphorylation of the actin binding coronin; and dissociation of actin capping proteins, many of which are regulated by PIP₂. Extension of new actin filaments is promoted by formins (mDia1 and mDia2), which act on the plus end of actin filaments in coordination with profilin. Regulation of mDia proteins is largely via conformational changes induced by the small G-proteins RhoA and cdc42. Profilin increases nucleotide exchange on G-actin monomers, thus enhancing actin polymerization. Severing of existing actin filaments is a consequence of activation of gelsolin and cofilin, which limit filament length and initiate turnover of existing filaments. Rac also regulates actin reorganization in the lamellipodium, perhaps by activation of p21-activated kinase (PAK)-mediated phosphorylation of actin binding proteins. The result of these complicated, coordinated events is protrusion of lamellipodia in the direction of the detected migratory stimulus (see Fig. 3-8).

Once lamellipodial protrusion has occurred, it is necessary for the cell to create new contacts with the matrix and dissolve ones no longer needed. These nascent focal contacts provide traction for eventual contraction of the cell body and propulsion of the cell forward.¹³⁷ Very little is known about focal adhesion composition in VSMCs, but signaling at focal adhesions is coordinated by integrin interaction with the matrix, integrin clustering, activation of a series of protein tyrosine kinases including integrin-linked kinase (ILK), FAK and Src, and interaction with the cortical F-actin cytoskeleton. Phosphorylation of focal adhesion components including FAK and paxillin occurs during VSMC migration, as does turnover of focal adhesion proteins by membrane-type metalloproteinases. Regulation of focal adhesion turnover is also intimately related to the microtubular network.

The final major event in cell migration is contraction of the cell body. Similar to contraction in differentiated cells, cell body contraction is initiated through calcium-mediated activation of MLCK and MLC phosphorylation following matrix interaction. RhoA and Rho kinase may also play a role because pharmacological inhibition of Rho kinase blocks migration of VSMCs.¹³⁹ Current theory suggests that myosin II generates traction forces on the matrix, and the matrix in turn regulates myosin II activation.¹³⁷

Much research remains to fully understand the mechanisms underlying VSMC migration, but the potential for identifying new targets for prevention of restenosis and plaque formation is obvious.

Inflammation

As noted earlier, VSMCs can assume an inflammatory phenotype that is found primarily in atherosclerotic lesions. These cells are found in the media of the vessel wall and express both markers of differentiation and inflammatory genes such as VCAM-1 and exhibit activated NF-κB signaling.¹⁴⁰ One of the primary stimuli for development of this inflammatory phenotype is oxidized LDL, but ECs activated by disturbed flow also contribute to inflammatory changes in VSMC by secreting proinflammatory cytokines.¹⁴

Oxidized LDL and other cytokines like IL-1β and TNF-α stimulate VSMC expression of chemokines such as MCP-1, TNF-α, and chemokine (C-X-C motif) ligand 1 (CXCL1), as well as adhesion molecules such as VCAM-1, ICAM-1, and CCR-2, the receptor for MCP-1. Because many of these molecules activate NF-κB, exposure to one of them often induces the expression of others, resulting in propagation of a positive feedback signaling mechanism to enhance the local inflammatory response. The end result is recruitment and adhesion of T cells and monocytes to smooth muscle cells (SMCs) in the vessel wall.

Proinflammatory gene expression in VSMC, as in other cell types, is largely a consequence of posttranscriptional regulation of inflammatory gene expression by the stress-activated protein kinase p38MAPK and transcriptional regulation by proinflammatory transcription factors such as NF-κB and STAT1/3. Both of these pathways are activated by ROS, which have been shown to be increased in inflammatory regions of plaques as a result of macrophage infiltration as well as direct stimulation of VSMCs by cytokines. Stimulation of cytokine receptors activates p38MAPK, which controls proinflammatory protein levels by MAPKAPK-2 mediated phosphorylation of adenylate uridylate–rich elements (AREs) binding proteins such as tristetraprolin (TTP), thus promoting mRNA stability of TNF-α.¹⁴¹ Many other inflammatory gene mRNAs, including MCP-1, IL-1β, IL-8, intercellular adhesion molecule 1 (ICAM-1), and VCAM-1, also contain AREs. It should be noted that ARE binding proteins can both stabilize and destabilize mRNA: HuR protects ARE-containing transcripts from degradation, but AUF1 destabilizes its targets. p38MAPK can also regulate inflammatory protein expression by translational regulation via activation of MAPK signal-integrating kinase-1 (Mnk-1), which phosphorylates the translation initiation factor eIF-4E and enhances its affinity for the mRNA cap.¹⁴² Transcriptional regulation of proinflammatory gene expression is largely a consequence of activation of the NF-κB pathway. Commonly, the p65-p50 heterodimer is the transactivating factor that binds to NF-κB-containing elements to increase proinflammatory gene transcription. Regulation of gene expression by STATs is a consequence of the canonical tyrosine kinase receptor activation of JAK, and subsequent phosphorylation of STAT followed by translocation to the nucleus.

Another major environmental factor that contributes to maintenance of the VSMC proinflammatory phenotype is the matrix milieu in which cells exist. In atherosclerotic plaques, VSMCs begin to secrete collagen I and collagen III, but also, as a result of NF-κB activation, express MMP-1, MMP-3, and MMP-9, which degrade collagen fibrils to the monomeric form, thus promoting an inflammatory phenotype, as evidenced by an increase in VCAM-1 expression.⁷⁵ A similar response is seen to osteopontin, which is also increased in atherosclerosis.¹⁴³ The effects of these matrix proteins on VSMCs are mediated by binding to specific integrins, most likely α5β1 or αvβ3.¹⁴ The nonintegrin matrix receptor CD44, which binds to hyaluronic acid in the matrix, has also been implicated in the transition to the proinflammatory phenotype, as shown by its ability to stimulate VCAM-1 expression.¹⁴⁴

Senescence, Apoptosis, and Autophagy

In response to aging and oxidative stress, cells that have accumulated damaged organelles/proteins/DNA due to limitations in DNA repair or antioxidant mechanisms rely on two processes to avoid replication and passing the damage to daughter cells: permanently arresting the cell cycle (senescence), or programmed cell death, including apoptosis (self-killing) or autophagy (self-eating).¹⁴⁵

Senescent cells are permanently arrested in the G1 phase of the cell cycle and exhibit specific senescence-associated markers such as β-galactosidase, heterochromatin foci, and accumulation of lipofuscin granules. Unlike quiescent cells, senescent cells are not responsive to growth factors.¹⁴⁶ Multiple stresses, including DNA-damaging radiation or chemicals, mitochondrial dysfunction, and oxidant stress, can invoke two types of senescence programs: stress-induced premature senescence (SIPS) and replicative senescence associated with accelerated telomere uncapping or shortening.¹⁴⁷ These diverse stimulatory pathways converge onto two effector pathways: the tumor suppressor protein p53 and the Rb pathways; p53 is normally targeted to proteasome-mediated degradation by mouse double minute 2 MDM2). Mitogenic stress or DNA damage suppresses MDM2 activity, resulting in p53-mediated activation of the CDKI p21 and cell cycle arrest.¹⁴⁵ In the second pathway, stress or damage activates Rb, which then binds to and inhibits E2F, a transcription factor required for the G1 phase/S phase transition to cell cycle progression (see Fig. 3-7). These two senescence pathways exhibit cross-talk at the level of p53 and can overlap death pathways. Senescent cells release degradative proteases, growth factors, and inflammatory cytokines, which impact on neighboring cells.

In VSMCs, DNA damage caused by ROS (e.g., superoxide, hydrogen peroxide, hydroxyl radicals) incites rapid (within days) SIPS. There are increased levels of ROS in all diseased layers of an atherosclerotic lesion, particularly in the plaque itself,¹⁴⁷ and senescent VSMCs have been identified in injured arteries and in the intima of atherosclerotic plaques.¹⁴⁸

Many of the changes in senescent VSMCs are reminiscent of changes indicative in age-related vascular disease, implicating cellular senescence in vascular pathologies.¹⁴⁸ Therefore, a model for how senescence contributes to vascular disease emerges. Atherogenic stimuli such as Ang II initially stimulate proliferation, followed by mitogen-induced SIPS or replicative senescence via telomere uncapping. Inflammatory cytokine/chemokine release by senescent VSMCs results in ECM degradation. The decreased cellularity and increased inflammation contribute to plaque instability.¹⁴⁸

Senescent VSMCs are also implicated in vascular calcification. They exhibit enhanced expression of osteoblastic genes such as alkaline phosphatase (ALP), type 1 collagen, and RUNX-2, while expression of matrix Gla protein (MGP), an anticalcification factor, is down-regulated.¹⁴⁹

Apoptosis, the controlled activation of proteases and hydrolases within an intact cell’s plasma membrane boundary so that neighboring cells are not affected and an immune response is not triggered,¹⁵⁰ is an important mechanism for blood vessel remodeling during proliferative vascular disease and after therapeutic interventions (e.g., angioplasty/stenting of arteries, vein bypass graft surgery).¹⁵¹ Mitogens such as thrombin or PDGF can induce proliferative episodes in VSMCs within atherosclerotic lesions (reviewed in¹⁵²). Proliferation is counterbalanced by death-inducing VSMC apoptosis triggered by a variety of proinflammatory mediators, cytokines, oxidized lipids, and free radicals produced by immune cells within the plaque. These proinflammatory mediators activate caspases, components of the extrinsic death receptor pathway (e.g., Fas/CD95 TRAIL [TNF-related apoptosis-inducing ligand]), and/or cause intrinsic mitochondrial dysfunction in VSMCs under the control of Bcl family members (reviewed in¹⁵³).

Interactions among mitogenic, apoptotic, and survival signals produce a variety of lesion characteristics and determine whether there is a fragile fibrous cap poised for rupture, a lipid-rich necrotic core, or a fibrotic and calcified core (reviewed in¹⁵²). High percentages of apoptotic VSMCs within atherosclerotic plaques are one of the major causes of plaque rupture due to decreased cellularity in the media and thinning of the fibrous cap. In addition, reduced phagocytotic clearance of apoptotic VSMCs, resulting in necrotic VSMCs, and low levels of VSMC apoptosis over extended periods of hyperlipidemia induce viable VSMC release of IL-6 and MCP-1 to produce chronic inflammation.¹⁵⁴ Apoptotic VSMCs also generate thrombin, promoting coagulation.¹⁵²

Vascular smooth muscle cell apoptosis has also been associated with other lesion characteristics including inflammation, calcification, thrombosis, and aneurysms (reviewed in¹⁵⁶). In vivo, VSMC apoptosis causes release of cytokines and MCP-1, recruiting macrophages. Vascular calcification has been associated with inorganic phosphate–induced VSMC apoptosis and subsequent generation of VSMC-derived matrix vesicles that serve as the nidus for calcification (reviewed in¹⁵⁶). Statins restore the Gas6-mediated survival pathway and inhibit VSMC calcification by preventing apoptosis.

In addition to apoptosis, autophagy, a survival process by which the cell degrades its own components, such as damaged organelles or long-lived aberrant or aggregated proteins,¹⁴⁵ contributes to pathology in atherosclerotic plaques. Ultrastructural analysis of VSMCs in the fibrous cap of advanced plaques reveals characteristics of cells undergoing autophagic degradation.¹⁵⁷ Because autophagy is a survival mechanism and not a death pathway, VSMC autophagy in the fibrous cap may function in plaque stability and protection from oxidative stress.¹⁵⁷ If oxidative stress damages lysosomal membranes, lysosome/autophagic vacuole fusion is impaired and apoptosis ensues.