Smooth Muscle Cell Signal Transduction: Implications of vascular biology for vascular surgeons

Abstract

Vascular smooth muscle cells (SMC) exhibit varied responses after vessel injury and surgical interventions, including phenotypic switching, migration, proliferation, protein synthesis, and apoptosis. Although the source of the SMC that accumulate in the vascular wall is controversial, possibly reflecting migration from the adventitia, from the circulating blood, or in situ differentiation, the intracellular signal transduction pathways that control these processes are being defined. Some of these pathways include the Ras-MAPK, PI3K-Akt, Rho, death receptor-caspase, and nitric oxide pathways. Signal transduction pathways provide amplification, redundancy, and control points within the cell and culminate in biological responses. We review some of the signaling pathways activated within SMC that contribute to SMC heterogeneity and development of pathology such as restenosis and neointimal hyperplasia.

Smooth muscle cell diversity under normal and pathological conditions

Under normal homeostatic conditions found in healthy vascular physiology, vascular smooth muscle cells (SMC) ordinarily have a low turnover rate, with low baseline levels of both proliferation and apoptosis.^1,2 Acceleration of the rate of SMC turnover, with increases in the rates of both proliferation and apoptosis, is thought to be involved in the pathogenesis of atherosclerotic lesions, restenosis after interventional therapy, and vein graft arterialization.^1–6 For example, one theory suggests that the combined action of growth factors, proteolytic agents, and extracellular matrix proteins that are produced by a dysfunctional endothelium and/or inflammatory cells, induce migration of resident SMC from the media into the neointima, as well as subsequent neointimal SMC proliferation, to form atherosclerotic lesions.^3,7,8

However, even in normal vascular tissue SMC demonstrate a heterogeneous population of cells. Spindle-shaped differentiated SMC show contractile properties, with a low frequency of proliferation, and are induced into this phenotype by heparin and transforming growth factor-β (TGF-β). Rhomboid-shape dedifferentiated SMC show a high degree of protein synthesis, proliferation and migratory activity, and are induced into this phenotype by basic fibroblast growth factor (bFGF) and platelet derived growth factor-BB (PDGF-BB).⁷

The source of these different SMC phenotypes is controversial, and has been studied most extensively after vascular interventions. Some of the potential sources of heterogeneous SMC populations that contribute to vascular remodeling include migration of cells from the adventitia, in situ differentiation and expansion, or accumulation from distant sources such as the bone marrow. The “myofibroblast” SMC phenotype is thought to be a marker of SMC that are involved in and accumulate during pathological interactions such as restenosis, with increased expression of several markers of SMC differentiation, as well as increased proliferation, migration, and production of extracellular matrix proteins, cytokines, and chemokines.^8–11 Several groups have reported that myofibroblasts are derived from the adventitia, and are involved in neointimal formation as well as vein graft arterialization.^8,12–16

In addition to the potential adventitial source of myofibroblasts that may contribute to SMC remodeling, bone marrow-derived progenitor (stem) cells may also contribute to the heterogeneity of cells involved in vascular wall remodeling. Bone marrow-derived progenitor cells are known to be released into the circulation following mechanical, immunological, and/or humoral stimulation after vascular injury.^17,18 Although the mechanisms by which SMC participate in formation of atherosclerotic lesions are becoming established, similar mechanisms of progenitor cell activation during homing and contribution to restenosis may be also be active in SMC after vascular interventions or during vein graft arterialization.^19,20

Although most vascular structures develop from the mesoderm, SMC develop from several embryologically distinct origins. For example, SMC in the branchial arch-derived vessels derive from the neural crest, and coronary artery SMC derive from the overlying endothelium.^7,21 In addition, recent studies suggest that bone marrow-derived progenitor cells have the ability to develop into SMC with the typical phenotype of neointimal SMC, hypercholesterolemia-induced neointimal cells, and SMC associated with transplant arteriopathy.^1,22–25 During SMC development, the myogenic process requires expression of smooth muscle cell-specific genes such as vimentin, α-actin, SM-22, caldesmon, calponin, SM-myosin heavy chain (MHC), and smoothelin.^7,8,26,27 These SMC-specific genes are characterized by their stimulated expression upon activation of their promoter CArG box element by the transcriptional factor serum response factor (SRF), with the co-factor myocardin.^26,28,29 Control of this process is thought to be mediated by phosphorylated Elk-1, which is activated by the extracellular signal-regulated kinase 1/2 (ERK1/2) and/or phosphatodylinositol 3-kinase (PI3K)-Akt pathways, resulting in inhibition of SRF-myocardin activation of SMC gene expression.

Although the contractile differentiated SMC phenotype is the typical SMC phenotype that comprises the vascular wall under most normal physiological conditions, and the synthetic dedifferentiated SMC phenotype exists during developmental or pathological conditions, the molecular mechanisms involved in the regulation of SMC phenotype, as well as the ability of SMC to change phenotype, are not well established.^7,30,31 Some of the many factors that may influence SMC phenotype include: mechanical forces, contact agonists, reactive oxygen species, endothelial-SMC interactions, thrombin, neuronal factors, TGF-β1, and extracellular matrix components such as laminin and type I and IV collagens.^1,19,32–39 As such, it is difficult to study the “in vivo” SMC contractile phenotype, because of phenotype switching rapidly upon SMC isolation and culture in vitro. This paradoxical but reversible phenotype switching is one of the interesting unique characteristics of SMC.

The pathological accumulation of different SMC populations is of crucial importance to vascular surgeons. After vascular wall injury, whether accumulating from disease pathophysiology or after surgical interventions, ligand molecules such as growth factors, inflammatory factors, and reactive oxygen and nitrogen species are induced in the vessel or graft wall. These upstream induction factors stimulate SMC intracellular signal transduction pathways, culminating in SMC gene expression that leads to the lesions of restenosis and neointimal hyperplasia. In this review, we focus on SMC signal transduction that contributes to their heterogeneity and development of pathology. These SMC intracellular signal transduction pathways may be attractive points of control to potentially limit vascular disease due to SMC, and prolong the value of surgical interventions.

SMC pathophysiology and signal transduction

Signal transduction pathways provide amplification, redundancy, and control points within the SMC, and culminate in biological responses. The discovery of the G-protein families led to the concept of signal transduction cascades as we know the field today, but was preceded by years of discovery of how signals were transmitted by hormones, hormone receptors, and second messengers such as calcium and cyclic nucleotides, that established the field of classical signal transduction. Although these signal cascades seem to be complex, biological stimuli are often present in low concentration; both extracellular signals and intracellular responses must be amplified to induce a cell response. Importantly, the complexity of these pathways may serve to both integrate the signal, ensuring that the cell’s response – which may be significant and even induce cell death – is appropriate for that signal, and allow for signal propagation with fidelity in the face of damaged cell machinery, such as may occur during normal aging. Importantly, understanding these pathways may allow control, perhaps stimulating desirable responses and limiting undesirable ones.

1) Protein kinases play an important role in signal transduction

One way to rapidly accomplish intracellular signal transduction is to add phosphate groups to other proteins, a process known as phosphorylation and that is accomplished by enzymes called kinases. In particular, two classes of kinases, the serine/threonine kinases and the tyrosine kinases, play critical roles in mammalian biology. In general, serine/threonine kinases have broad substrate specificity even though they bind to a limited number of amino acid sequences. These kinases include the well known protein kinase A (PKA), protein kinase B (PKB) (also known as Akt), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and TGF-β superfamilies. On the other hand, since only 0.1% of tyrosine exists as a potential site of phosphorylation by kinases, tyrosine kinases also play critical regulatory and signaling roles.

Tyrosine kinases exist in two different families. The first broad class of these kinases interact with membrane bound receptor tyrosine kinases (RTK) such as PDGF receptor, epidermal growth factor (EGF) receptor, insulin-like growth factor-I (IGF-I) receptor, and the Scf1/c-kit receptor. second class of tyrosine kinases is the non-receptor type kinases that interact with downstream targets of RTK, and include c-Src, Jak, and Fak. As compared with serine/threonine kinases that are typically activated by cAMP, cGMP, Ca²⁺, calmodulin and diacylglycerol, tyrosine kinases are activated by dimerization and auto-phosphorylation. Further propagation of the RTK-transduced signal is via several pathways, including the Grb-Sos-Ras-MAPK, PI3K-Akt, phospholipase Cγ–PKC/Ca²⁺ and janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways; these signaling pathways culminate in modification of several cell behaviors such as protein synthesis, cell proliferation, cell survival, and migration, as well as gene transcription.

Signal transduction from RTK is controlled at several points, including negative feedback signals. In addition, signals are directly and selectively inhibited by protein tyrosine phosphatases, phospholipid dephosphorylases such as phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and src-homology 2-containing inositol 5’ phosphatase (SHIP). The kinases are also deactivated by ubiquitination.

2) Regulation of SMC phenotypic switching

One of the most interesting features of SMC is that they are not terminally differentiated in mature vascular tissue, allowing modulation of their phenotype under certain conditions. SMC dedifferentiation and phenotype change is thought to be an important aspect of vascular wall remodeling during atherosclerosis and neointimal hyperplasia. Differentiated SMC have a spindle shape, low proliferation rate and physiological contractile functions; dedifferentiated SMC have a rhomboid or epithelioid shape, high proliferation and migration activity, increased proteolytic activity, lower levels of cytoskeletal and contractile proteins, and high sensitivity to apoptotic stimuli.^7,26

Despite the well known and important role that SMC dedifferentiationand phenotype switching plays in repair of vascular wall injury, very few factors and pathways have been identified that modulate these phenotypic changes (Figure 1).^26,40 PDGF-BB is one of the few factors implicated in SMC phenotype switching; PDGF-BB is the only factor yet described that can induce a profound suppression of the SMC marker genes SM α-actin, SM-MHC and SM22α.^26,40–46 Kawai and Owens recently reviewed several pathways that may be involved in SMC phenotype modulation.⁴⁰ Some of these pathways include Krüppel-like factor 4 (KLF4), phosphorylated Elk-1, HERP1, FOXO4, YY1, FHL2 and several homeobox proteins. For example, KLF4 is induced by PDGF-BB and potently represses the expression of multiple differentiated SMC marker genes through a combination of effects including suppression of myocardin expression, inhibition of SRF binding to intact chromatin, and suppression of myocardin-induced gene activation.^10,47,48 Another transcriptional factor, Elk-1 (a ternary complex of Ets domain proteins), can induce PDGF-BB and suppress SMC marker genes including SM22α and SM α-actin. This is accomplished through suppression of CArG promoter element-SRF-myocardin dependent transcription.^28,41 Elk-1 is phosphorylated by PDGF-BB and signals downstream though MAPK/ERK kinase1/2 (MEK1/2) -ERK1/2, ultimately cleaving SRF-myocardin.

Figure 1.

Signaling during SMC phenotype switching. The left side of the figure denotes pathways involved in signaling during SMC differentiation to the contractile phenotype. IGF-I causes expression of genes associated with the contractile, differentiated phenotype through the PI3K-Akt pathway, while at the same time blocks the Ras-MAPK pathway with the IPS-I/SHP2 complex. The right side of the figure denotes pathways involved in signaling during SMC phenotype switching to the synthetic phenotype. Several growth factors stimulate SMC phenotype switching by stimulating MAPK directly as well as by cleaving the IPS-I/SHP2 complex. MAPK transposition to the nucleus inhibits transcription of genes associated with the contractile phenotype and stimulates expression of genes associated with growth. Signals from each cascade inhibit the opposite cascade. IGF-I: insulin like growth factor-I; PDGF: platelet derived growth factor; EGF: epidermal growth factor; FGF: fibroblast growth factor; PI3K: phosphoinositide 3-kinase; IRS-I: insulin receptor substrate-I; SHP2: Scr homology protein 2; MAPK: mitogen-activated protein kinase. p: phosphorylation

IGF-I maintains the differentiated SMC phenotype by triggering the PI3K and PKB/Akt pathways. This cascade also blocks dedifferentiation depending on the recruitment of src homology protein 2 (SHP2) by insulin receptor substrate-1 (IRS-I).^49,50 SHP2/IRS-I cleavage is induced by PDGF, FGF or EGF, and results in activation of the MEK-ERK1/2 and MAPK kinase 6 (MKK6)-p38MAPK pathways, mediated by the intermediate Grb2/Sox complex and Ras activation (Figure 1). Transcriptional and splicing factors then induce cell migration, proliferation and ECM synthesis.^49,50 Although there is some evidence that IGF-I may not maintain SMC differentiation,^51–55 it is likely that IGF-I secretion plays an important role in SMC differentiation, proliferation and migration.

3) Regulation of SMC proliferation and migration

After switching phenotype, SMC migrate and proliferate in the vascular wall to promote healing of vessel injury; proliferation and migration of dedifferentiated SMC results in accumulation of cells and formation of the lesions of atherosclerosis, restenosis, or neointimal hyperplasia. This phenomenon is thought to be stimulated by growth factors within the injured vascular wall that regulate downstream signal transduction in SMC, such as PDGF-BB and bFGF (Figure 2). These factors stimulate SMC pathways such as the Ras-MAPK and PI3K-Akt pathways that stimulate cell proliferation, and the Rho kinase family monomer G proteins Cdc42, Rac, and Rho that stimulate cell migration. These pathways promote not only phenotype switching, migration and proliferation but also regulate extracellular matrix synthesis.⁵⁶

Figure 2.

Proliferative signaling during SMC response to injury. The figure shows convergent signaling pathways resulting in protein synthesis and cell proliferation, leading to restenosis and neointimal hyperplasia. Implications of vascular intervention may include inducing growth factors that are SMC mitogens and chemoattractants. These factors stimulate SMC signal transduction pathways including the Ras-MAPK and the PI3K-Akt-mTOR pathways for growth gene transcription. PDGF-BB: platelet derived growth factor-BB; bFGF: basic fibroblast growth factor; PI3K: phosphoinositide 3-kinase; PDK1: 3-phosphoinositide-dependent kinase 1; mTOR: mammalian target of rapamycin ; MAPK: mitogen-activated protein kinase.

The cellular source of these factors that stimulate SMC is not clear, as vascular wall injury can induce humoral, autocrine and paracrine growth factors such as PDGF-BB, bFGF, and HB-EGF from endothelial cells, SMC, and invasive cells such as macrophages and platelets.^6,57–61 We have previously shown that nonlaminar shear stress, such as might be present after vessel injury, results in ERK1/2 activation from both PDGF-BB and interleukin−1αand results in SMC chemotaxis and proliferation.^62,63

The MAPK/ERK cascade is one of the well known signal transduction pathways for SMC proliferation and induction of additional growth factor secretion.^64–68 A protein-tyrosine kinase (PTK) receptor is activated by a growth factor, resulting in receptor phosphorylation and binding of adaptor proteins such as Grb2 and Shc to the activated receptor. Adaptor protein binding leads to Ras activation of the GTP-binding protein family, via the mammalian son-of-sevenless (mSOS; guanine nucleotide exchange factor), activating Raf MAPKK/MEK and the downstream molecules, p44 MAPK/p42 MAPK (ERK1/2). Phosphorylated MAPK enters the nucleus to form a complex with the transcriptional factors Elk-1 and Sap1 (an Ets family member), inducing transcription by binding to the SRE promoter of genes such as c-fos. This mechanism is thought to be critical in the regulation of gene expression for proliferation, migration, differentiation and phenotypic switching (Figure 2).^58,69–75

It is typical for many stimuli to activate multiple signal transduction cascades. For example, PTK receptors also activate the PI3K-Akt pathway, in addition to the MAPK pathway, when stimulated by signals that induce cell proliferation and cell survival. The phosphorylated PTK receptor activates PI3K, phosphorylating PI(3,4)P₂ to form PI(3,4,5)P₃. The PH domain of Akt recruits PI(3,4,5)P₃ on the cell membrane, and 3-Phosphatidylinositol-dependent kinase (PDK) 1/2 then phosphorylate Akt on either Thr308 or Ser473. Phosphorylated Akt activates the target of rapamycin (mTOR)-raptor complex to trigger cell growth, cell survival, NO production, cell proliferation and cell cycling and delaying G1/S exit.^76–83 Interestingly, these pathways all interact with each other. For example, activated PKA can have negative crosstalk with the Ras-MAPK and PI3K-Akt pathways as well as promote cell proliferation at the genetic level.⁶⁴ Negative control of these pathways is also via phosphatases such as PTEN, which controls Akt pathway activation by inhibition of PI(3,4,5)P₃ phosphorylation and inhibition of PI3K activity.⁸⁴

The Rho small GTP-binding protein family induces SMC migration by control of the intracellular cytoskeletal architecture. The G-protein-coupled receptor (GPCR) can be activated by angiotensin II, thrombin, endothelin-1 or Ras that is recruited by activated PTK receptors and can induce Rho family (Rho, Rac, Cdc42) activation. This event stimulates Rho kinase or mDia, and reconstruction of the actin filament or F actin, resulting in cell migration.^85,86 Activated Rho also induces MEK1/MLKs/MEK4 activation, upstream of c-Jun N-terminal kinase (JNK), and induces cell differentiation, survival and apoptosis. There is evidence of Rho activation in vascular injury and transplantation, and that this pathway induces cell mitogenesis, actin polymerization, cell migration, and neointimal formation.^87–93

4) Regulation of ECM accumulation

During SMC migration successful production and remodeling of the extracellular matrix (ECM) is necessary to form the neointima.⁹⁴ Interestingly, the converse is also true, i.e. degradation of the ECM during vascular injury induces SMC migration and proliferation. PDGF, angiotensin-II, and TGF-β have the ability to control ECM stability by changing the ECM components synthesized and secreted by SMC.^95–97 bFGF decreases SMC elastin production through ERK1/2 pathway activation;⁹⁸ elastin is abundant in the normal media, but a decrease in aortic elastin can induce plaque fragility in atherosclerosis and be associated with vessel weakening such as occurs during aneurysm formation.⁹⁹ ECM remodeling is the net result of the balance of matrix production and degradation; ECM synthesis is generally regulated by the matrix metalloproteinases (MMP), whereas ECM degradation is regulated by MMP inhibitors such as TIMP and RECK. In vascular remodeling, MMPs not only synthesize matrix, but also induce ECM degradation and remodeling, in addition to stimulating cell proliferation and migration.^40,100 The interactions between the MMP-TIMP/RECK systems are not well described and are the subject of active investigation.

Induction of the SMC dedifferentiated phenotype during ECM degradation is strongly related to cell-to-matrix and cell-to-cell adhesion molecules. Growth factors interact with these adhesion molecules to induce SMC migration and proliferation.¹⁰¹ For example, nectin is not only found in cell-to-cell junctions but can also activate Cdc42 and Rac, a small GTP-binding protein with PTK receptor activating properties.^102–104 Cadherin can also activate Rac. Cdc42 and Rac can then each modulate reorganization of the actin cytoskeleton and activate additional signal transduction cascades such as JNK.¹⁰⁵

5) Bone marrow derived progenitor cell differentiation

It has been recently described that bone marrow-derived progenitor cells (BMC) may play an important role in vascular wall remodeling. Mouse embryonic stem cells can develop a SMC phenotype when activated by TGF-β1 through Smad−2/3.⁴⁷ Pluripotent 10T1/2 cells demonstrate modulation of this response via bFGF-induced MEK signaling and suppression of SRF transcription.⁴⁰ PDGF-BB induces TR-BME2 cells, a murine bone marrow-derived endothelial progenitor cell line, to differentiate into contractile- and synthetic-type SMC.⁹⁷ ECM and adhesion molecules have been reported to participate in the differentiation of BMC progenitor cells into SMC; smooth muscle progenitor cells have been found to display large numbers of α1β1 and α5β1 integrins, more than endothelial cells, and which can capture fibronectin. This observation leads to the hypothesis that specific integrin expression in progenitor cells may provide key signaling in cell development.^19,106,107 The exciting discovery of these BMC stem cells offers vascular surgeons the possibility not only to improve post-interventional vascular or vein graft patency but also may provide for the creation of novel conduits in vascular surgery such as tissue engineered vascular autografts.^108,109 However, despite mounting evidence of a role for stem cells in the response to vascular injury, the details of downstream signaling pathways are not yet well-defined.

6) Regulation of SMC apoptosis

Apoptosis is widely known as programmed cell death that is a part of normal development, senescence and other diverse biological processes. Accumulation of normal and abnormal tissue depends on the delicate balance between cell proliferation and apoptosis; as such it is difficult to assess the importance of apoptosis without careful measurements of both proliferative and apoptotic components. Neointimal hyperplasia and restenosis after vascular interventions also involve the SMC apoptotic pathway during vascular wall remodeling. In the rat carotid artery balloon injury model, approximately 70% of medial SMC showed evidence of apoptosis after only 1 hour post injury; despite this early decrease in SMC cell number, neointima formation continues for several weeks.¹¹⁰ Another biological stimulant of SMC apoptosis is laminar shear stress, suppressing SMC proliferation and restenosis after an endothelial-denuding injury such as angioplasty (Figure 3).¹¹¹

Figure 3.

Apoptotic signaling during SMC response to injury. The figure shows divergent pathways resulting in control of apoptosis, leading to different outcomes. The extrinsic apoptosis pathway is stimulated by signals external to the SMC, whereas the intrinsic apoptosis pathway is stimulated by signals internal to the SMC nucleus and /or mitochondria. In the extrinsic pathway, apoptosis ligands activate caspases −8 and −10 or JNK. The intrinsic pathway is activated by DNA damage or genetic programs and induces the Apaf1-caspase−9 complex directly or through the release of cytochrome c. Both the internal and external pathways activate caspases−3, −5, and −7 to effect apoptosis. TNFα: tumor necrosis factorα; JNK: c-Jun N-terminal kinase; PKA: protein kinase A ; PI3K: phosphoinositide 3-kinase ; ERK: extracellular signal-regulated kinase; Cyto c: cytochrome c; Apaf1: apoptosis activating factor 1

Known triggers of vascular cell apoptosis include OxLDL, oxysteroles, reactive oxygen or nitrogen species, radiation, cytokines, and viral/bacterial products.¹⁰⁰ Suppressors of apoptosis include turbulent shear stress, low levels of nitric oxide (NO), growth factors, the IAP protein family, vitamins C and E, and other antioxidants. Several apoptotic ligands such as INFγ, FasL, TNFα, IL-1, reactive oxygen or nitrogen species, and radiation can indirectly induce SMC apoptosis via activated T-cell antigen presentation, pro-inflammatory mediators or activated macrophage immune priming or phagocytosis.

Signal transduction to transmit the apoptotic death signal is carefully regulated. There are two well defined upstream pathways, the extrinsic and the intrinsic pathways (Figure 3). In the extrinsic pathway, ligands activate the apoptotic pathways through receptors such as Fas, TNFR and the DR3/4/5 receptors, leading to downstream caspase activation and mitochondrial dysfunction. Fas activation due to FasL expression on T-cells, macrophages and monocytes induces polymerization of Fas-associated death domain (FADD) and activation of pro-caspase-8. Caspase-8 cleaves a member of the Bcl-2 family, Bid, inducing cytochrome c release from the mitochondria, and activating caspase-9; apoptosis is accomplished by caspase-3 activation.^112,113 The TNFR similarly stimulates apoptosis by activation of the caspase-8 pathway, and also simultaneously activates the regulatory NF-κB pathway, protecting the cell from apoptosis.^114,115 The Bcl-2 protein family also regulates the apoptosis pathways to influence cell survival; this family forms heterodimers between apoptosis-inhibiting proteins such as Bcl-2, Bcl-X_L, and A1, and inducing proteins such as Bax, Bad, Bid, regulating cell survival.¹¹⁶ Cell survival factors inhibit Bad activity via several pathways, including by PKA, ERK1/2, PKC, and PI3K-Akt pathway activation (Figure 3).

In contrast, the intrinsic apoptotic pathway is directly stimulated by gene transcription, DNA damage, mitochondrial stress and endoplasmic reticulum stress; these stimuli directly activate caspases−1, −2, and −9, or indirectly via p53 or cytochrome c (Figure 3). These cascades induce Apaf1 polymerization to activate caspase−9. Finally, as with the extrinsic pathway, caspase−3 is activated by caspase−9-Apaf1 to induce apoptosis.

7) Signal transduction in vascular pathology

Diabetes mellitus is one of the major risk factors for vascular disease.^117,118 Diabetic patients with hyperglycemia often have accelerated neointimal formation, and correlates with increased PDGF-β receptor and TGF-β receptor expression by SMC.^119–121 Downstream of the PTK receptors, hyperglycemia induces p38 MAPK, but not ERK1/2, and activates both PKC-dependent and –independent pathways in rat aortic SMC.¹²² There is also evidence that IGF binding protein is degraded in hyperglycemic conditions, inducing IGF-I levels that stimulate the differentiated SMC phenotype.¹²³ Since IGF-I is also associated with inhibition of the synthetic SMC phenotype, and SMC proliferation and migration (Figure 1), IGF-I may also have potential as an anti-proliferative therapeutic agent. Other proteins in this pathway may also have potential as therapeutic agents; for example, pregnancy-associated plasma protein-A (PAPP-A), one of the metalloproteinases associated with IGF binding protein-4 (IGFBP-4), significantly decreases neointimal formation.¹²⁴ On the other hand, the complexity of the signal transduction pathways precludes easy translation of in vitro and animal studies to the treatment of human patients. For example, diabetic patients may also induce ECM/cytoskeletal production through G-protein-coupled receptors; if so, then this additional mechanism of signaling produces IP₃ and Ca²⁺ release as well.¹²⁵

Restenosis after vascular interventions is associated with activation of SMC signal transduction pathways; accumulation of SMC in restenotic lesions may be a source of symptoms.¹²⁶ Identification of these SMC signal transduction pathways has led to identification of potential points of control For example, rapamycin is an established inhibitor of vascular remodeling with use in drug eluting stents in both the coronary and periphery beds.^127,128 Rapamycin inhibits neointimal formation after carotid artery balloon angioplasty in animal models, and may be useful in human patients with advanced disease. For example, rapamycin inhibited neointima formation even in conditions of low flow, such as present in patients with extensive runoff disease (Figure 4). Interestingly, rapamycin does not affect low flow-induced inward remodeling (Figure 4), suggesting that its effects on signal transduction pathways and inhibition of neointimal hyperplasia are specific.

Figure 4.

Relevance of mTOR signal transduction pathway in the response to vascular injury. The right carotid artery of New Zealand white rabbits was subjected to sham operation (control), balloon injury (B), outflow branch ligation to reduce flow (LF), or both balloon injury and reduction in flow (B+LF), and harvested after 21 days. Either rapamycin (5 mg/kg) or saline was orally administered daily from 48 hours prior to the procedure until harvest; in animals given rapamycin, serum levels (day 7) were therapeutic (mean 10.9 ± 0.5 ng/mL; therapeutic range 4–12 ng/mL; n=21). Animals treated with rapamycin demonstrated significant inhibition of neointimal thickening in balloon-injured arteries (B), including arteries treated with low flow (B+LF; p<0.0001). Negative remodeling was evident in all vessels in the low flow groups (LF, B+LF), and rapamycin did not affect this reduction in vessel size due to low flow. A, low power magnification; B, high power magnification. Reprinted from Vascular Pharmacology, Vol number, Paszkowiak et al, Evidence supporting changes in Nogo-B levels as a marker of neointimal expansion but not adaptive arterial remodeling, Pages No., Copyright (Year), with permission from Elsevier.

The intracellular target of rapamycin is the mTOR protein, which is normally phosphorylated by Akt (Figure 2). Rapamycin inhibits mTOR downstream activation, including JNK activation, and induces apoptosis.¹²⁹ mTOR has two important downstream pathways, stimulation of S6K1/2 and suppression of 4E-BP1. c-Jun phosphorylation is required for activating 4E-BP1, but not S6K1/2, and takes place in a relative deficiency of p53, suggesting a role for rapamycin not only in molecular targeting for cancer, but also in targeting immature SMC as might be found in vascular injury. Accordingly, rapamycin inhibits SMC migration.¹³⁰

Conclusion

SMC are complex cells, capable of existing in heterogeneous populations and switching phenotypes upon various stimuli. The signal transduction pathways controlling SMC activation and phenotype switching are becoming established and may suggest additional points of control. Additional pathways are present as well. For example, caveolin-1 signaling has been implicated in SMC proliferation and migration through a Ca²⁺ mediated pathway.¹³¹ NO is implicated in SMC vasorelaxation through cytosolic soluble guanylate cyclase signaling. NO signaling is also linked to the Akt pathway, and thus involved in cell migration, proliferation, and apoptosis. Intraluminal eNOS gene delivery may reduce the response to vascular injury and may be another application of modulation of signal transduction pathways.¹³² The SMC signal transduction pathway control points are gradually appearing in new therapeutic modalities such as drug eluting stents and local gene transduction.^{127,128,133,134} As mechanisms of SMC signal transduction continue to be elucidated and understood, we will hopefully be able to provide improved care and quality of life for our patients.