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The effects of stretch on vascular smooth muscle cell phenotype in vitro
Cardiovascular Pathology 17 (2008) 98 – 102
Review Article
The effects of stretch on vascular smooth muscle cell phenotype in vitro Anastassi T. Halkaa,b,c, Neill J. Turnerb,c, Andrew Cartera, Jonathan Ghosha,b,c, Michael O. Murphya,b,c, John P. Kirtonb,c, Cay M. Kieltyb,c, Michael G. Walkera,b,4 a
Department of Vascular Surgery, Manchester Royal Infirmary, Manchester, UK UK Centre for Tissue Engineering, University of Manchester, Manchester, UK c Faculty of Life Sciences, University of Manchester, Manchester, UK
b
Received 29 January 2007; received in revised form 6 March 2007; accepted 13 March 2007
Abstract Vascular smooth muscle cells (VSMC) situated in the tunica media of veins and arteries are central to maintaining conduit integrity in the face of mechanical forces inherent within the cardiovascular system. The predominant mechanical force influencing VSMC structural organisation and signalling is cyclic stretch. VSMC phenotype is manipulated by externally applied stretch which regulates the activity of their contractile apparatus. Stretch modulates cell shape, cytoplasmic organisation, and intracellular processes leading to migration, proliferation, or contraction. Drug therapy directed at the components of the signalling pathways influenced by stretch may ultimately prevent cardiovascular pathology such as myointimal hyperplasia. D 2008 Elsevier Inc. All rights reserved. Keywords: Vascular smooth muscle cells; Stretch; Phenotype; Signalling; RhoA; FAK
1. Introduction Vascular smooth muscle cells (VSMC), in the tunica media of arteries and veins, are mechanosensors of the cardiovascular system. Through modulating blood vessel tone and diameter, VSMC influence blood pressure, regulating tissue perfusion. VSMC also contribute to the development of cardiovascular pathology, including atherosclerosis and myointimal hyperplasia (MIH). VSMC are inherently plastic, never attaining a terminal phenotype. Instead, a phenotype is adopted in accordance with environmental signals, like stretch, functional requirements such as repair after vessel injury and the maintenance of blood pressure [1,2]. Mature VSMCs express alpha smooth muscle actin (aSMA), smooth muscle myosin heavy chain, h1-calponin, and SM22a, protein markers indicating enhanced contractility. Immature VSMC are prone to 4 Corresponding author. Department of Vascular Surgery, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK. Tel.: +44 0 161 2764525; fax: +44 0 161 2768014. E-mail address: [email protected] (M.G. Walker). 1054-8807/08/$ – see front matter D 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2007.03.001
proliferation, migration, and synthesis of extracellular matrix (ECM) components [1]. Blood vessels in vivo are exposed to radial, axial, and circumferential strain generated by pulse pressure. This strain is counterbalanced by the anisotropic generation of vessel wall tone induced by VSMC contraction, elastin, and collagen [3]. Circumferentially orientated VSMC in the tunica media are not directly exposed to these stretching forces. Instead, stretch deforms the surrounding ECM in which the VSMC are embedded, indirectly stimulating their cell membrane receptors. As the VSMC membrane, cytoskeleton, and the contractile apparatus are structurally and functionally associated, they are all distorted by stretch [4]. Receptor activation and morphological changes alter intracellular signalling, modifying gene expression which ultimately determines VSMC phenotype. This review focuses on how stretch influences VSMC phenotype via the RhoA/Rho-kinase (RhoA/ROCK) and the focal adhesion kinase (FAK)/c-Src signalling complexes (Fig. 1). Investigating the effects of stretch on VSMC surface receptors and signalling pathways involves the use of in vitro models [5]. In most cases, cells are cultured on a
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Fig. 1. Rho and FAK are influenced by stretch and manipulate VSMC phenotype. CARG: CC(A/T)6GG-box motif; SRE: serum response element.
silicone membrane which is stretched, thereby directly inducing stimulation [2,5,6]. Although stretch is exerted in only two dimensions by these systems they remain effective models for examining the effects of external mechanical forces on individual cells. A model in which cells are seeded into three-dimensional scaffolds and then exposed to an external force is preferred by some groups [7]. Regardless of the model used, in vitro studies do not allow easy distinction between stretch effects due to transmembrane force transfer and stretch effects due to a global change in cell morphology which causes generalised deformation of the plasma membrane and the cytoskeleton [8].
2. Mechanical stretching globally affects vascular smooth muscle cell morphology The cell membrane, regarded as an extension of the cytoskeleton, forms an interface between the ECM and the contractile apparatus [4]. In culture, integrins cluster at sites of cell attachment and tension and in response to stretch [9]. Integrins associate with transmembrane proteins like syndecans, adhesion plaques (APs) to which actin binds and a sub-membrane network of structural and signalling proteins including FAK and c-Src [10]. The accumulation of the aforementioned structural and signalling components constitutes focal adhesions (FAs). FAs are integral to cell structure and are also a locus for mechanotransduction, i.e., the conversion of a mechanical stimulus into an intracellular signal altering gene expression [10,11]. In static conditions, zyxin, a mechanotransducing protein, binds to actin in the cytoskeleton and also predominates in VSMC FAs along with vinculin [9]. After stretching, whilst vinculin remains
in the FAs, zyxin rapidly translocates to the nucleus and modulates the expression of mechanosensitive genes such as endothelin-B receptor, a contributor to hypertensioninduced arterial remodelling [9]. An organised system of cross-talk exists between specific ECM components and the terminal filaments of the actin cytoskeleton [12]. The ECM influences the response of VSMC to the application of stretch [13]. In static conditions, the expression of contractile markers is influenced by the substrate on which VSMC are cultured with aSMA levels raised in cells cultured on laminin but not fibronectin [14]. VSMC cultured on fibronectin and collagen and then exposed to stretch demonstrate augmented DNA synthesis typical of a synthetic phenotype. The opposite is true for VSMC cultured on laminin and elastin and exposed to the same magnitude of stretch. These effects are blocked by anti-h3-integrin and anti-avh5-integrin but not by anti-h1integrin indicating intimate integrin involvement [15]. The VSMC cytoskeleton, a complex biological circuit, resists mechanical strain and operates as a spatial regulator and integrator of signal transduction pathways which may be simultaneously influenced by stimuli such as stretch and growth factors [16,17]. Cytoskeletal arrangement has been described in terms of tensegrity implying balanced tension and compression stabilising a structure constituted of rigid elements held together by elastic members [18]. Tensegrity assumes that all cells have an inherent cytoskeletal tension or prestress which develops secondary to the establishment of FAs [18]. As increasing stress is applied locally the cytoskeleton stiffens proportionally. VSMCs’ prestressed system of molecular connections consists of the contractile apparatus and intermediate filaments, balanced by compression-bearing microtubules and traction at sites of cellular
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attachment [19]. The prestressed system enables discrete mechanical signal transfer from the cell surface attachment sites via the cytoskeleton to the nucleus. Specific signals may even be transferred to distinct structures in the cell, such as nuclear transcription apparatus. In vitro, the rheological properties of SMC are thought to be influenced by their FAs, the activity of their contractile apparatus and the application of mechanical stretch [6]. It is theorised that increased SMC distension stiffens its cytoskeleton maintaining its shape. Simultaneously, the friction between the cell and its surroundings amplifies, reducing its ability to move. Conversely, cells exposed to less mechanical strain deform more easily maintaining their motility [6]. Stretch-induced rheology depends on VSMC phenotype since mature and immature VSMC have distinct cytoskeletal structures and functions.
3. Signalling pathways influenced by stretch that manipulate VSMC phenotype RhoA, Rac and Cdc42 are members of the Rho family of small GTPases, which contribute to coordinated cell behaviour by modulating transcription and the actin cytoskeleton [20]. Mechanical stretch stimulates RhoA via an unknown mechanism, initiating signalling pathways that enhance VSMC proliferation and contractility [21]. RhoA expression in freshly plated VSMC is biphasic, with an initial peak 1 h after seeding and a higher peak when a contractile phenotype is adopted [22]. During the first peak, temporary cytoskeletal reorganisation initiates cell migration and proliferation whilst the second increase instigates characteristics that define the contractile phenotype. RhoA has distinct effects as it mediates different downstream effectors including the mitogen-activated protein kinases (MAPKs) [21,23]. The primary genes encoding SMC contractile proteins are regulated by the stretch-induced RhoA/ROCK pathway and associated transcription factors, most importantly the serum response factor (SRF) [24,25]. The promoters of these genes all possess a pair of CArG-box motifs, CC(A/T)6GG, which are necessary for their expression [26]. SRF binds to the serum response element region containing the 10-bp CArGbox sequence facilitating activation of this motif alone or as a macromolecule bound to myocardin, its specific coactivator [27]. Myocardin increases the promoter activity of the CArG-dependent VSMC contractile markers by up to 60fold [28]. Mutations of these promoter regions abolished aSMA and SM22a transcription in transiently transfected SMC [29]. In vitro, RhoA positively regulates SRF-mediated transcription of aSMA and SM22a in VSMC via enhanced actin polymerisation, stimulating the SRF homodimer binding to their CArG boxes [30]. With RhoA overexpression, VSMC adopt the characteristics of a contractile phenotype, which are lost when RhoA is inhibited [23]. Although RhoA activation does induce a contractile phenotype, it does not
completely organise actin and myosin, suggesting the involvement of other pathways [23]. Synthetic VSMC migrate, proliferate, and do not express contractile markers [1]. The sudden inability of a VSMC to express a contractile marker during vessel injury is hypothesised to occur secondary to the presence of degenerate CArG-box motifs which contain a single G or C substitution in their central A/T-rich region [31]. Degenerate CArG boxes are also modulated by the SRF/ myocardin complex therefore implying a common mechanism for VSMC phenotype control [31,32]. Cell migration requires FAs to be in a constant flux of assembly and disassembly, assisted by reciprocal and cyclical RhoA and Rac/Cdc42 regulation inducing lamellipodia and filopodia [33]. FAK, a cytoplasmic soluble protein tyrosine kinase, is a biomechanical sensor and a signalling bswitchQ stimulated by stretch in vitro [13,34,35]. Stretch-induced integrin clustering recruits FAK to FAs where it binds to talin [10,36]. FAK is auto-phosphorylated at Tyr397, activating an attachment site for c-Src which activates other autophosphorylation sites [37,38]. The FAK/c-Src pathway has Rho and Rac as its downstream targets, contributing to their reciprocal regulation which characterises motility [33,35]. Rearrangement of actin is necessary for stretch-induced cell migration. Protein-kinase-C-delta translocates to the SMC cytoskeleton in response to stretch and is hypothesised to mediate actin reorganisation [39]. The FAK/c-Src complex can also stimulate MAPKs, such as extracellular receptor kinase 1 and 2 (ERK1/2) and p38-MAPK [40]. ERK1/2 encourages proliferation of VSMC exposed to stretch, whilst p38-MAPK increases apoptosis [41]. ERK1/2 is upregulated by both pulsatile and sustained stretch, contributing to MIH and atherosclerosis [38]. The pathway mediating ERK1/2-induced VSMC proliferation in the aorta is determined by the type of stretch exerted. Physiological pulsatile stretch in the aorta activates ERK1/2 in a FAKindependent manner, which may be part of normal vessel maintenance [38]. In the latter model, ERK1/2 is also activated by sustained vascular stretch, but in this instance, FAK acts as an upstream mediator induced by ECM– integrin interaction and c-Src activation. In the presence of stretch, ERK1/2 activation in VSMC requires an intact actin cytoskeleton as well as a functioning Rho/ROCK pathway. Disrupting the actin cytoskeleton using cytochalasin-D, inhibiting RhoA using botulinum-C3 exoenzyme and inhibiting ROCK using PD98058 attenuated ERK1/2 activity despite VSMC stimulation using stretch [21]. Preventing ERK1/2 activation by inhibiting FAK phosphorylation or Rho/ROCK activity may reduce cell proliferation and could be a therapeutic target to prevent graft stenosis.
4. Discussion Therapies targeting the components of the aforementioned signalling pathways in VSMC may combat cardio-
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vascular disease. A good example are the statins that maintain and enhance SRF-mediated transcription of contractile markers ensuring that VSMC regain or preserve a contractile phenotype [23,42]. Statins inhibit 3-hydroxy-3-methylglutaryl-CoA (HMGCoA)-reductase, stopping isoprenylation of small GTPases and preventing their translocation from the cytosol to the cell membrane, thereby halting their signalling [43,44]. RhoA/ROCK together with the ERK1/2 and phosphatidylinositol-3-kinase/Akt pathways is thought to be essential for stretch-induced venous SMC proliferation [45]. Inhibition of the RhoA/ROCK pathway, for example by statins, could therefore be a therapeutic target to reduce the incidence of graft occlusion secondary to MIH [46]. Future research is required to develop improved in vitro models, essential to investigating mechanotransduction in VSMC, which better replicate the in vivo environment. This process will facilitate the identification of the intracellular mechanisms at key signalling pathways and will also direct the development of more effective clinical therapies.
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protein kinase through interactions with and activation of c-Src. J Biol Chem 1997;272:13189 – 95. [41] Goldman J, Zhong L, Liu SQ. Degradation of alpha-actin filaments in venous smooth muscle cells in response to mechanical stretch. Am J Physiol Heart Circ Physiol 2003;284:H1839 – 47. [42] Munoz-Garcia B, et al. Fn14 is upregulated in cytokine-stimulated vascular smooth muscle cells and is expressed in human carotid atherosclerotic plaques: modulation by atorvastatin. Stroke 2006;37:2044 – 53. [43] Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990;343:425 – 30.
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Review Article
The effects of stretch on vascular smooth muscle cell phenotype in vitro Anastassi T. Halkaa,b,c, Neill J. Turnerb,c, Andrew Cartera, Jonathan Ghosha,b,c, Michael O. Murphya,b,c, John P. Kirtonb,c, Cay M. Kieltyb,c, Michael G. Walkera,b,4 a
Department of Vascular Surgery, Manchester Royal Infirmary, Manchester, UK UK Centre for Tissue Engineering, University of Manchester, Manchester, UK c Faculty of Life Sciences, University of Manchester, Manchester, UK
b
Received 29 January 2007; received in revised form 6 March 2007; accepted 13 March 2007
Abstract Vascular smooth muscle cells (VSMC) situated in the tunica media of veins and arteries are central to maintaining conduit integrity in the face of mechanical forces inherent within the cardiovascular system. The predominant mechanical force influencing VSMC structural organisation and signalling is cyclic stretch. VSMC phenotype is manipulated by externally applied stretch which regulates the activity of their contractile apparatus. Stretch modulates cell shape, cytoplasmic organisation, and intracellular processes leading to migration, proliferation, or contraction. Drug therapy directed at the components of the signalling pathways influenced by stretch may ultimately prevent cardiovascular pathology such as myointimal hyperplasia. D 2008 Elsevier Inc. All rights reserved. Keywords: Vascular smooth muscle cells; Stretch; Phenotype; Signalling; RhoA; FAK
1. Introduction Vascular smooth muscle cells (VSMC), in the tunica media of arteries and veins, are mechanosensors of the cardiovascular system. Through modulating blood vessel tone and diameter, VSMC influence blood pressure, regulating tissue perfusion. VSMC also contribute to the development of cardiovascular pathology, including atherosclerosis and myointimal hyperplasia (MIH). VSMC are inherently plastic, never attaining a terminal phenotype. Instead, a phenotype is adopted in accordance with environmental signals, like stretch, functional requirements such as repair after vessel injury and the maintenance of blood pressure [1,2]. Mature VSMCs express alpha smooth muscle actin (aSMA), smooth muscle myosin heavy chain, h1-calponin, and SM22a, protein markers indicating enhanced contractility. Immature VSMC are prone to 4 Corresponding author. Department of Vascular Surgery, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK. Tel.: +44 0 161 2764525; fax: +44 0 161 2768014. E-mail address: [email protected] (M.G. Walker). 1054-8807/08/$ – see front matter D 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2007.03.001
proliferation, migration, and synthesis of extracellular matrix (ECM) components [1]. Blood vessels in vivo are exposed to radial, axial, and circumferential strain generated by pulse pressure. This strain is counterbalanced by the anisotropic generation of vessel wall tone induced by VSMC contraction, elastin, and collagen [3]. Circumferentially orientated VSMC in the tunica media are not directly exposed to these stretching forces. Instead, stretch deforms the surrounding ECM in which the VSMC are embedded, indirectly stimulating their cell membrane receptors. As the VSMC membrane, cytoskeleton, and the contractile apparatus are structurally and functionally associated, they are all distorted by stretch [4]. Receptor activation and morphological changes alter intracellular signalling, modifying gene expression which ultimately determines VSMC phenotype. This review focuses on how stretch influences VSMC phenotype via the RhoA/Rho-kinase (RhoA/ROCK) and the focal adhesion kinase (FAK)/c-Src signalling complexes (Fig. 1). Investigating the effects of stretch on VSMC surface receptors and signalling pathways involves the use of in vitro models [5]. In most cases, cells are cultured on a
A.T. Halka et al. / Cardiovascular Pathology 17 (2008) 98 – 102
99
Fig. 1. Rho and FAK are influenced by stretch and manipulate VSMC phenotype. CARG: CC(A/T)6GG-box motif; SRE: serum response element.
silicone membrane which is stretched, thereby directly inducing stimulation [2,5,6]. Although stretch is exerted in only two dimensions by these systems they remain effective models for examining the effects of external mechanical forces on individual cells. A model in which cells are seeded into three-dimensional scaffolds and then exposed to an external force is preferred by some groups [7]. Regardless of the model used, in vitro studies do not allow easy distinction between stretch effects due to transmembrane force transfer and stretch effects due to a global change in cell morphology which causes generalised deformation of the plasma membrane and the cytoskeleton [8].
2. Mechanical stretching globally affects vascular smooth muscle cell morphology The cell membrane, regarded as an extension of the cytoskeleton, forms an interface between the ECM and the contractile apparatus [4]. In culture, integrins cluster at sites of cell attachment and tension and in response to stretch [9]. Integrins associate with transmembrane proteins like syndecans, adhesion plaques (APs) to which actin binds and a sub-membrane network of structural and signalling proteins including FAK and c-Src [10]. The accumulation of the aforementioned structural and signalling components constitutes focal adhesions (FAs). FAs are integral to cell structure and are also a locus for mechanotransduction, i.e., the conversion of a mechanical stimulus into an intracellular signal altering gene expression [10,11]. In static conditions, zyxin, a mechanotransducing protein, binds to actin in the cytoskeleton and also predominates in VSMC FAs along with vinculin [9]. After stretching, whilst vinculin remains
in the FAs, zyxin rapidly translocates to the nucleus and modulates the expression of mechanosensitive genes such as endothelin-B receptor, a contributor to hypertensioninduced arterial remodelling [9]. An organised system of cross-talk exists between specific ECM components and the terminal filaments of the actin cytoskeleton [12]. The ECM influences the response of VSMC to the application of stretch [13]. In static conditions, the expression of contractile markers is influenced by the substrate on which VSMC are cultured with aSMA levels raised in cells cultured on laminin but not fibronectin [14]. VSMC cultured on fibronectin and collagen and then exposed to stretch demonstrate augmented DNA synthesis typical of a synthetic phenotype. The opposite is true for VSMC cultured on laminin and elastin and exposed to the same magnitude of stretch. These effects are blocked by anti-h3-integrin and anti-avh5-integrin but not by anti-h1integrin indicating intimate integrin involvement [15]. The VSMC cytoskeleton, a complex biological circuit, resists mechanical strain and operates as a spatial regulator and integrator of signal transduction pathways which may be simultaneously influenced by stimuli such as stretch and growth factors [16,17]. Cytoskeletal arrangement has been described in terms of tensegrity implying balanced tension and compression stabilising a structure constituted of rigid elements held together by elastic members [18]. Tensegrity assumes that all cells have an inherent cytoskeletal tension or prestress which develops secondary to the establishment of FAs [18]. As increasing stress is applied locally the cytoskeleton stiffens proportionally. VSMCs’ prestressed system of molecular connections consists of the contractile apparatus and intermediate filaments, balanced by compression-bearing microtubules and traction at sites of cellular
100
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attachment [19]. The prestressed system enables discrete mechanical signal transfer from the cell surface attachment sites via the cytoskeleton to the nucleus. Specific signals may even be transferred to distinct structures in the cell, such as nuclear transcription apparatus. In vitro, the rheological properties of SMC are thought to be influenced by their FAs, the activity of their contractile apparatus and the application of mechanical stretch [6]. It is theorised that increased SMC distension stiffens its cytoskeleton maintaining its shape. Simultaneously, the friction between the cell and its surroundings amplifies, reducing its ability to move. Conversely, cells exposed to less mechanical strain deform more easily maintaining their motility [6]. Stretch-induced rheology depends on VSMC phenotype since mature and immature VSMC have distinct cytoskeletal structures and functions.
3. Signalling pathways influenced by stretch that manipulate VSMC phenotype RhoA, Rac and Cdc42 are members of the Rho family of small GTPases, which contribute to coordinated cell behaviour by modulating transcription and the actin cytoskeleton [20]. Mechanical stretch stimulates RhoA via an unknown mechanism, initiating signalling pathways that enhance VSMC proliferation and contractility [21]. RhoA expression in freshly plated VSMC is biphasic, with an initial peak 1 h after seeding and a higher peak when a contractile phenotype is adopted [22]. During the first peak, temporary cytoskeletal reorganisation initiates cell migration and proliferation whilst the second increase instigates characteristics that define the contractile phenotype. RhoA has distinct effects as it mediates different downstream effectors including the mitogen-activated protein kinases (MAPKs) [21,23]. The primary genes encoding SMC contractile proteins are regulated by the stretch-induced RhoA/ROCK pathway and associated transcription factors, most importantly the serum response factor (SRF) [24,25]. The promoters of these genes all possess a pair of CArG-box motifs, CC(A/T)6GG, which are necessary for their expression [26]. SRF binds to the serum response element region containing the 10-bp CArGbox sequence facilitating activation of this motif alone or as a macromolecule bound to myocardin, its specific coactivator [27]. Myocardin increases the promoter activity of the CArG-dependent VSMC contractile markers by up to 60fold [28]. Mutations of these promoter regions abolished aSMA and SM22a transcription in transiently transfected SMC [29]. In vitro, RhoA positively regulates SRF-mediated transcription of aSMA and SM22a in VSMC via enhanced actin polymerisation, stimulating the SRF homodimer binding to their CArG boxes [30]. With RhoA overexpression, VSMC adopt the characteristics of a contractile phenotype, which are lost when RhoA is inhibited [23]. Although RhoA activation does induce a contractile phenotype, it does not
completely organise actin and myosin, suggesting the involvement of other pathways [23]. Synthetic VSMC migrate, proliferate, and do not express contractile markers [1]. The sudden inability of a VSMC to express a contractile marker during vessel injury is hypothesised to occur secondary to the presence of degenerate CArG-box motifs which contain a single G or C substitution in their central A/T-rich region [31]. Degenerate CArG boxes are also modulated by the SRF/ myocardin complex therefore implying a common mechanism for VSMC phenotype control [31,32]. Cell migration requires FAs to be in a constant flux of assembly and disassembly, assisted by reciprocal and cyclical RhoA and Rac/Cdc42 regulation inducing lamellipodia and filopodia [33]. FAK, a cytoplasmic soluble protein tyrosine kinase, is a biomechanical sensor and a signalling bswitchQ stimulated by stretch in vitro [13,34,35]. Stretch-induced integrin clustering recruits FAK to FAs where it binds to talin [10,36]. FAK is auto-phosphorylated at Tyr397, activating an attachment site for c-Src which activates other autophosphorylation sites [37,38]. The FAK/c-Src pathway has Rho and Rac as its downstream targets, contributing to their reciprocal regulation which characterises motility [33,35]. Rearrangement of actin is necessary for stretch-induced cell migration. Protein-kinase-C-delta translocates to the SMC cytoskeleton in response to stretch and is hypothesised to mediate actin reorganisation [39]. The FAK/c-Src complex can also stimulate MAPKs, such as extracellular receptor kinase 1 and 2 (ERK1/2) and p38-MAPK [40]. ERK1/2 encourages proliferation of VSMC exposed to stretch, whilst p38-MAPK increases apoptosis [41]. ERK1/2 is upregulated by both pulsatile and sustained stretch, contributing to MIH and atherosclerosis [38]. The pathway mediating ERK1/2-induced VSMC proliferation in the aorta is determined by the type of stretch exerted. Physiological pulsatile stretch in the aorta activates ERK1/2 in a FAKindependent manner, which may be part of normal vessel maintenance [38]. In the latter model, ERK1/2 is also activated by sustained vascular stretch, but in this instance, FAK acts as an upstream mediator induced by ECM– integrin interaction and c-Src activation. In the presence of stretch, ERK1/2 activation in VSMC requires an intact actin cytoskeleton as well as a functioning Rho/ROCK pathway. Disrupting the actin cytoskeleton using cytochalasin-D, inhibiting RhoA using botulinum-C3 exoenzyme and inhibiting ROCK using PD98058 attenuated ERK1/2 activity despite VSMC stimulation using stretch [21]. Preventing ERK1/2 activation by inhibiting FAK phosphorylation or Rho/ROCK activity may reduce cell proliferation and could be a therapeutic target to prevent graft stenosis.
4. Discussion Therapies targeting the components of the aforementioned signalling pathways in VSMC may combat cardio-
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vascular disease. A good example are the statins that maintain and enhance SRF-mediated transcription of contractile markers ensuring that VSMC regain or preserve a contractile phenotype [23,42]. Statins inhibit 3-hydroxy-3-methylglutaryl-CoA (HMGCoA)-reductase, stopping isoprenylation of small GTPases and preventing their translocation from the cytosol to the cell membrane, thereby halting their signalling [43,44]. RhoA/ROCK together with the ERK1/2 and phosphatidylinositol-3-kinase/Akt pathways is thought to be essential for stretch-induced venous SMC proliferation [45]. Inhibition of the RhoA/ROCK pathway, for example by statins, could therefore be a therapeutic target to reduce the incidence of graft occlusion secondary to MIH [46]. Future research is required to develop improved in vitro models, essential to investigating mechanotransduction in VSMC, which better replicate the in vivo environment. This process will facilitate the identification of the intracellular mechanisms at key signalling pathways and will also direct the development of more effective clinical therapies.
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