Vascular smooth muscle cell in atherosclerosis

Acta Physiol 2015, 214, 33–50

REVIEW

Vascular smooth muscle cell in atherosclerosis D. A. Chistiakov,1,2 A. N. Orekhov3,4,5 and Y. V. Bobryshev3,6,7 1 Research Center for Children’s Health, Moscow, Russia 2 The Mount Sinai Community Clinical Oncology Program, Mount Sinai Comprehensive Cancer Center, Mount Sinai Medical Center, Miami Beach, FL, USA 3 Institute for Atherosclerosis, Skolkovo Innovative Center, Moscow, Russia 4 Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, Moscow, Russia 5 Department of Biophysics, Biological Faculty, Moscow State University, Moscow, Russia 6 Faculty of Medicine, School of Medical Sciences, University of New South Wales, Kensington, Sydney, NSW, Australia 7 School of Medicine, University of Western Sydney, Campbelltown, NSW, Australia

Received 9 January 2015, revision requested 2 February 2015, revision received 5 February 2015, accepted 9 February 2015 Correspondence: Y. V. Bobryshev, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia. E-mail: [email protected]

Abstract Vascular smooth muscle cells (VSMCs) exhibit phenotypic and functional plasticity in order to respond to vascular injury. In case of the vessel damage, VSMCs are able to switch from the quiescent ‘contractile’ phenotype to the ‘proinflammatory’ phenotype. This change is accompanied by decrease in expression of smooth muscle (SM)-specific markers responsible for SM contraction and production of proinflammatory mediators that modulate induction of proliferation and chemotaxis. Indeed, activated VSMCs could efficiently proliferate and migrate contributing to the vascular wall repair. However, in chronic inflammation that occurs in atherosclerosis, arterial VSMCs become aberrantly regulated and this leads to increased VSMC dedifferentiation and extracellular matrix formation in plaque areas. Proatherosclerotic switch in VSMC phenotype is a complex and multistep mechanism that may be induced by a variety of proinflammatory stimuli and hemodynamic alterations. Disturbances in hemodynamic forces could initiate the proinflammatory switch in VSMC phenotype even in pre-clinical stages of atherosclerosis. Proinflammatory signals play a crucial role in further dedifferentiation of VSMCs in affected vessels and propagation of pathological vascular remodelling. Keywords arteries, artery wall, atherogenesis, atherosclerosis, vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) are involved in the control of vascular tone and diameter through the mechanism of contraction. Mature VSMC is quiescent and has a ‘contractile’ phenotype associated with the stable production of smooth muscle (SM) contractile proteins. Those include SM a-actin, SM22a, SM myosin heavy chain (MHC), H1-calponin and smoothelin, all are recognized as selective VSMC markers (Owens et al. 2004). In steady state, VSMCs very rarely proliferate and exhibit a low synthetic activity.

Vascular smooth muscle cells are not terminally differentiated and hence exhibit phenotypic plasticity (Fig. 1) (Babaev et al. 1990, 1993, Bobryshev & Lord 1996, Gomez & Owens 2012). This feature discriminates VSMCs from skeletal muscle cells and cardiomyocytes that are terminally differentiated (Perry & Rudnick 2000). The capacity of VSMCs to switch the phenotype is regulated by external signals and accompanied by induction of expression of VSMC markers related to proliferation and motility and decrease in expression of selective

© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12466

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Smooth muscle cells in atherosclerosis

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· D A Chistiakov et al.

(b)

Figure 1 Electron microscopic images showing of the typical appearance of so-called contractile (a) and synthetic (b) phenotypes of smooth muscle cells that reside in the intima of large human arteries. In contrast to ‘contractile’ phenotype, the cytoplasm of which is filled with myofilaments (a), the cytoplasm of ‘synthetic’ phenotype contains a well-developed rough endoplasmic reticulum (b). In (a, b): N – nucleus. In (a), arrows show ‘dense bodies’, located in the cytoplasm along the basal membrane. In (b), arrows show cisterns of endoplasmic reticulum. In (b), note that the cell is surrounded by a large number of collagen fibres. Scale bars = 2 lm (a, b).

VSMC markers (Gomez & Owens 2012). Generally, phenotypic changes in VSMCs are required during embryonic angiogenesis, neovascularization, vascular remodelling and repair of vessel injury (Fischer et al. 2006). In atherosclerosis, VSMCs are involved in remodelling of arterial wall in order to maintain blood flow in affected vessels due to atherosclerotic changes. The ability of VSMCs to transdifferentiate to myeloid cells such as macrophage-like cells was shown in vitro and in vivo. After cholesterol load, Rong et al. (2003) reported formation of macrophage-like cells from cultured murine VSMCs associated with induction of macrophage-specific markers, phagocytosis and ability to present antigens. Andreeva et al. (1997) observed colocalization of expression of CD68, a macrophage marker, and SM a-actin, a SMC marker, in human aortic cells located in lipid-rich regions of atherosclerotic vessels such as fatty streaks and plaque shoulders. In atherosclerotic lesions, SMCs can also transdifferentiate to chondrocyte-like cells (Bobryshev 2005) or cells expressing osteoblast-specific transcription factor Cbfa1 (Bobryshev et al. 2008b). On the other hand, myeloid cells such as macrophages and bone marrow-derived myeloid progenitors were shown to be capable to transform into SMC-like cells. Cultured macrophages treated with transforming growth factor-b (TGF-b) or thrombin start to express SM a-actin (Martin et al. 2009, Stewart et al. 2009). In atherosclerotic lesions of apolipoprotein E (apoE)34

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deficient mice, Iwata et al. (2010) showed that some SM a-actin-positive cells are of myeloid origin, but they never differentiate to mature SMCs as they do not express SM MHC, a marker of mature VSMCs. Other researchers also showed that most SMCs in plaques are of local origin but not of hematopoietic origin (Caplice et al. 2003; Bentzon et al. 2006, 2007, Daniel et al. 2010). In this review, we consider phenotypic plasticity of VSMCs in atherosclerosis and molecular mechanisms that regulate transition in the VSMC phenotype in the plaque.

Hemodynamic forces induce the proinflammatory switch in the VSMC phenotype in atheroprone arterial regions Local susceptibility to atherosclerosis is regulated by blood flow patterns. Arterial regions exposed to unidirectional laminar flow are resistant to atherosclerosis. However, there are some regions (branch points, bifurcations and curvatures), which are exposed to disturbed flow and therefore become vulnerable to plaque development (Davies et al. 2010). The laminar flow generates steady or pulsative patterns of shear stress that induce production of nitric oxide (NO) and prostaglandin E2 (PGE2) in endothelial cells that in turn initiate atheroprotective responses in VSMCs such as vasodilatation and anti-inflammation (Qiu et al. 2013). In VSMCs, laminar flow leads to the downregulation of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signalling associated with subsequent inhibition of production of matrix metalloproteinase (MMP)-2, TGF-b and plateletderived growth factor receptors (PDGFRs), for example factors that stimulate VSMC proliferation and migration (Goldman et al. 2007). By stimulation of endothelial NO synthase, laminar flow also increases bioavailability of NO, which then induces dilatation of VSMCs. Although both laminar flow and disturbed flow induce production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) involved in signal transduction, the laminar flow also causes induction of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (Takabe et al. 2011). Nrf2 is a transcription factor that upregulates expression of many antioxidant and detoxifying enzymes and therefore prevents the induction of proatherogenic oxidative stress (Warabi et al. 2007). In addition, regular shear stress induces Kr€ uppel-like factor 2 (Klf2), an atheroprotective transcription factor that controls expression of many vasoactive endothelial genes involved in the regulation of normal constriction/dilation of VSMCs (Lee et al. 2006b).


Acta Physiol 2015, 214, 33–50

D A Chistiakov et al.

In contrast, disturbed flow creates highly unstable patterns of shear stress that induces proinflammatory activation of the vascular endothelium associated with secretion of proinflammatory chemokines such as chemokine (C-C motif) ligand 2 [CCL2 or monocyte chemotactic protein 1 (MCP-1)] and interleukin-8 (IL8) and adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) (Fig. 2) (Davies et al. 2013). Endothelial production of adhesion molecules attracts monocytes to the inflamed site that attach to activated

· Smooth muscle cells in atherosclerosis

endothelial cells (ECs) and start to produce inflammatory cytokines such as IL-1b and tumour necrosis factor-a (TNF-a), thereby further enhancing local inflammation (Malek et al. 1999). In a model of arterial wall, Fan et al. (2010) showed that monocytes preferentially attach to the endothelium in regions the flow was the slowest and wall shear stress was the lowest. In fact, disturbed hemodynamic forces play a critical role in endothelial dysfunction followed by induction of the inflammatory phenotype of SMCs

Monocyte

ARTERIAL LUMEN

oxLDL Cholesterol

Perturbed shear stress

MCP-1 IL-8

Endothelial cells

Adhesion

ICAM-1 VCAM -1

NF-kB

Fibronecn Osteoponn Syndecan-4

Collagen IV, laminin, perlecan IL-8

INTIMA MEDIA VCAM-1 ICAM-1 Collagen IV

Subendothelial invasion Macrophage

MCP-1 IL-8 CXCL1

MMP-1,-2, -3, -9 oxLDL Adhesion LOX1 NF-kB NFAT Integrin α5β 1 Integrin αvβ 3

VSMCs

IL-1β TNF-α

Collagen I Fibronecn

Figure 2 Proinflammatory activation of vascular smooth muscle cells (VSMCs) and ECs in atherosclerosis. Disturbed blood flow creates abnormal pattern of shear stress that could induce local production of proinflammatory molecules by endothelium in atheroprone regions of the arterial wall. Endothelial cells’ release of inflammatory chemokines (MCP-1 and IL-8) attracts monocytes that attach to activated ECs due increased production of adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and ICAM-1. Indeed, EC-mediated recruitment of monocytes promotes their subendothelial infiltration to intima media where they differentiate to macrophages and further contribute to the local inflammation through production of matrix metalloproteinas (MMPs) and various proinflammatory mediators. OxLDL and cholesterol play a key role in proinflammatory activation of ECs and VSMCs. OxLDL could bind to its receptor LOX-1 on the surface of VSMCs and upregulate transcription factor nuclear factor (NF)-jB that drives expression of many proinflammatory molecules including cytokines IL-1b and tumour necrosis factor-a (TNF-a). Proinflammatory switch in the VSMC phenotype is accompanied with the loss of expression of smooth muscle (SM) contractile proteins and induction of proliferation and migration. IL-1b and TNF-a also contribute to changes in the extracellular matrix (ECM) composition that surrounds VSMCs. Mainly, replacement of collagen 4, a characteristic of ECM of quiescent VSMCs, to collagen 1 and fibronectin supports VSMC dedifferentiation. Integrins a5b1 and aVb3 mediate proinflammatory stimuli from the ECM and upregulate transcription factors NF-jB and nuclear factor of activated T-cell (NFAT), which prime expression of inflammatory genes and further involvement of proinflammatory-activated VSMCs to neointima formation and proatherogenic vascular remodelling. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12466

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(Hastings et al. 2007, 2009). On the VSMC surface, mechanical stretch signals could be sensed by a variety of mechanoreceptors including integrins (Wernig et al. 2003), Notch receptors (Morrow et al. 2005, Zhu et al. 2011b), insulin-like growth factor-1 receptor (IGF1R;Liu et al. 2011), PDGFRs (Liu et al. 2003) and radiation-inducible immediate-early gene 3 (IER3) (Schulze et al. 2003). Integrins and Notch receptors mediate mechanotransduction to PTK2 protein tyrosine kinase 2 (PTK2) that further activate downstream Ras homolog A (RhoA)/rho-associated, coiledcoil-containing protein kinase 1 (ROCK1)-dependent signalling mechanisms (Gambillara et al. 2008). RhoA/ROCK1 signalling stimulates VSMC migration. Integrin activation also leads to the upregulation of Src/Ras/MEK1/2 (ERK1/2) signalling and stimulation of nuclear factor (NF)-jB, a proinflammatory transcription factor (Zampetaki et al. 2005). Integrindependent signalling as well as activation of PDGFR and IER3 also leads to the induction of expression of p53 through MEK1/2/p38MAPK- and PI3K/Akt-mediated signalling pathways respectively, and therefore stimulate proliferation and apoptosis (Mayr et al. 2002, Wernig et al. 2003). During plaque progression, different lesion regions are exposed to various patterns of shear stress. High shear stress predominates on the top of the plaque causing VSMC apoptosis through NO-mediated inhibition of the PDGF-survivin pathway (Yu et al. 2012) and integrin b1-dependent induction of p53, a proapoptotic regulator (Wernig et al. 2003). Fitzgerald et al. (2008) reported increased apoptosis of cultured bovine aortic SMCs after expose to increased laminar shear stress associated with significant downregulation of Akt signalling, activation of caspase-3 and increase in phosphorylation of a Bcl-2-associated X protein (Bax), a proapototic factor. Indeed, shear stressinduced apoptosis of VSMCs in the lesion cap could be a reason of increased plaque rupture in the upstream of the stenosis. In contrast, low (oscillatory) shear stress presented in the downstream lesion regions such as plaque shoulders activates VSMC proliferation through TGFb, IGF-1 and PDGF released by ECs (Qi et al. 2011, Wang et al. 2014). In VSMC-EC cocultures, implementation of low orbital or oscillatory stress was accompanied with increased VSMC proliferation through upregulation of both Akt- and ERK1/2-dependent signalling mechanisms (Haga et al. 2003, Asada et al. 2005), while high laminar shear stress inhibited VSMC proliferation and migration followed by decrease in expression of TGF-b and PDGFR-b and production of MMP-2 (Ueba et al. 1997, Palumbo et al. 2000, Garanich et al. 2005). In vascular grafts, disturbed flow with low shear stress was shown to 36

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support VSMC proliferation, an observation that helps to explain why neointima formation and plaques are preferentially formed in slow-flow regions (Fan & Karino 2010). Arterial regions with disturbed flow and low shear stress also provide favourable conditions for adhesion of proatherogenic low-density lipoprotein (LDL) particles to the endothelium (Fan et al. 2010).

Effects of oxidized LDL on the phenotype of VSMCs Oxidized LDL (oxLDL) is a well-established risk factor for atherosclerosis. Accumulation and subsequent oxidation of LDL in arterial intima is the initial event in atherogenesis (Pirillo et al. 2013). There are several scavenger receptors including lectin-type oxidized LDL receptor 1 (LOX-1) that mediate cell effects of oxLDL. LOX-1 was first identified in ECs as the major oxLDL receptor (Sawamura et al. 1997). However, other cells such as VSMCs and macrophages also express LOX-1 along with other scavenger receptors (Mehta et al. 2006). Expression of LOX-1 could be induced or upregulated by a variety of proinflammatory factors including lipopolysaccharide (LPS), TNF-a, interleukin (IL)-1b, interferon (IFN)-c, oxLDL itself and shear stress (Pirillo et al. 2013). OxLDL and LOX-1 colocalize with SMCs of human restenotic plaques, suggesting a role for LOX1 in oxLDL-induced SMC proliferation and restenosis (Eto et al. 2006). In VSMCs, oxLDL promotes phenotypic switch towards the proinflammatory phenotype associated with their dedifferentiation, proliferation and migration (Liu et al. 2014a). Treatment of human coronary artery SMCs with oxLDL resulted in increased cell proliferation and mobility in a dose-dependent manner and was accompanied with induction of MMP-9 through integrin avb3- and osteopontin-mediated mechanisms and activation of PTK2, MEK, ERK1/2 and NF-jB (Chen et al. 2009b). At high concentrations, oxLDL induces LOX-1mediated apoptosis of VSMCs by increasing expression of Bax and suppressing B lymphoma-2 (Bcl-2), an anti-apoptotic factor (Kataoka et al. 2001). Indeed, oxLDL-induced apoptosis of VSMCs could contribute to plaque destabilization. In support of this, Kataoka et al. (2001) reported colocalization of LOX-1 with Bax, especially in the ruptureprone shoulder region of human lesions suggesting for involvement of LOX-1 in plaque instability. There are also studies that focused on the effects of depletion of LOX-1 in VSMCs or atherosclerosis (Mehta et al. 2007, Dai et al. 2013). Deletion of LOX-1 reduces atherogenesis and is associated with reduction




(Hastings et al. 2007, 2009). On the VSMC surface, mechanical stretch signals could be sensed by a variety of mechanoreceptors including integrins (Wernig et al. 2003), Notch receptors (Morrow et al. 2005, Zhu et al. 2011b), insulin-like growth factor-1 receptor (IGF1R;Liu et al. 2011), PDGFRs (Liu et al. 2003) and radiation-inducible immediate-early gene 3 (IER3) (Schulze et al. 2003). Integrins and Notch receptors mediate mechanotransduction to PTK2 protein tyrosine kinase 2 (PTK2) that further activate downstream Ras homolog A (RhoA)/rho-associated, coiledcoil-containing protein kinase 1 (ROCK1)-dependent signalling mechanisms (Gambillara et al. 2008). RhoA/ROCK1 signalling stimulates VSMC migration. Integrin activation also leads to the upregulation of Src/Ras/MEK1/2 (ERK1/2) signalling and stimulation of nuclear factor (NF)-jB, a proinflammatory transcription factor (Zampetaki et al. 2005). Integrindependent signalling as well as activation of PDGFR and IER3 also leads to the induction of expression of p53 through MEK1/2/p38MAPK- and PI3K/Akt-mediated signalling pathways respectively, and therefore stimulate proliferation and apoptosis (Mayr et al. 2002, Wernig et al. 2003). During plaque progression, different lesion regions are exposed to various patterns of shear stress. High shear stress predominates on the top of the plaque causing VSMC apoptosis through NO-mediated inhibition of the PDGF-survivin pathway (Yu et al. 2012) and integrin b1-dependent induction of p53, a proapoptotic regulator (Wernig et al. 2003). Fitzgerald et al. (2008) reported increased apoptosis of cultured bovine aortic SMCs after expose to increased laminar shear stress associated with significant downregulation of Akt signalling, activation of caspase-3 and increase in phosphorylation of a Bcl-2-associated X protein (Bax), a proapototic factor. Indeed, shear stressinduced apoptosis of VSMCs in the lesion cap could be a reason of increased plaque rupture in the upstream of the stenosis. In contrast, low (oscillatory) shear stress presented in the downstream lesion regions such as plaque shoulders activates VSMC proliferation through TGFb, IGF-1 and PDGF released by ECs (Qi et al. 2011, Wang et al. 2014). In VSMC-EC cocultures, implementation of low orbital or oscillatory stress was accompanied with increased VSMC proliferation through upregulation of both Akt- and ERK1/2-dependent signalling mechanisms (Haga et al. 2003, Asada et al. 2005), while high laminar shear stress inhibited VSMC proliferation and migration followed by decrease in expression of TGF-b and PDGFR-b and production of MMP-2 (Ueba et al. 1997, Palumbo et al. 2000, Garanich et al. 2005). In vascular grafts, disturbed flow with low shear stress was shown to 36

Acta Physiol 2015, 214, 33–50

support VSMC proliferation, an observation that helps to explain why neointima formation and plaques are preferentially formed in slow-flow regions (Fan & Karino 2010). Arterial regions with disturbed flow and low shear stress also provide favourable conditions for adhesion of proatherogenic low-density lipoprotein (LDL) particles to the endothelium (Fan et al. 2010).

Effects of oxidized LDL on the phenotype of VSMCs Oxidized LDL (oxLDL) is a well-established risk factor for atherosclerosis. Accumulation and subsequent oxidation of LDL in arterial intima is the initial event in atherogenesis (Pirillo et al. 2013). There are several scavenger receptors including lectin-type oxidized LDL receptor 1 (LOX-1) that mediate cell effects of oxLDL. LOX-1 was first identified in ECs as the major oxLDL receptor (Sawamura et al. 1997). However, other cells such as VSMCs and macrophages also express LOX-1 along with other scavenger receptors (Mehta et al. 2006). Expression of LOX-1 could be induced or upregulated by a variety of proinflammatory factors including lipopolysaccharide (LPS), TNF-a, interleukin (IL)-1b, interferon (IFN)-c, oxLDL itself and shear stress (Pirillo et al. 2013). OxLDL and LOX-1 colocalize with SMCs of human restenotic plaques, suggesting a role for LOX1 in oxLDL-induced SMC proliferation and restenosis (Eto et al. 2006). In VSMCs, oxLDL promotes phenotypic switch towards the proinflammatory phenotype associated with their dedifferentiation, proliferation and migration (Liu et al. 2014a). Treatment of human coronary artery SMCs with oxLDL resulted in increased cell proliferation and mobility in a dose-dependent manner and was accompanied with induction of MMP-9 through integrin avb3- and osteopontin-mediated mechanisms and activation of PTK2, MEK, ERK1/2 and NF-jB (Chen et al. 2009b). At high concentrations, oxLDL induces LOX-1mediated apoptosis of VSMCs by increasing expression of Bax and suppressing B lymphoma-2 (Bcl-2), an anti-apoptotic factor (Kataoka et al. 2001). Indeed, oxLDL-induced apoptosis of VSMCs could contribute to plaque destabilization. In support of this, Kataoka et al. (2001) reported colocalization of LOX-1 with Bax, especially in the ruptureprone shoulder region of human lesions suggesting for involvement of LOX-1 in plaque instability. There are also studies that focused on the effects of depletion of LOX-1 in VSMCs or atherosclerosis (Mehta et al. 2007, Dai et al. 2013). Deletion of LOX-1 reduces atherogenesis and is associated with reduction




increased migration of VSMCs and enhanced adhesion of monocytes to ECs (Lee et al. 2013b). In VSMCs, TLR4 could be activated by LPS that in turn leads to the induction of the proinflammatory activation of VSMCs associated with secretion of IL-1a, IL-6 and MCP-1 (Yang et al. 2005). In VSMCs, TLR4 stimulation also upregulates NF-jB-dependent expression of MMP-9, a matrix metalloproteinase whose activation is related to plaque vulnerability and pathological vascular remodelling (Li et al. 2012). Proinflammatory cytokines such as TNF-a and IL1b were shown to contribute to the proinflammatory activation of VSMCs. TNF-a induces release of adhesion molecules such as ICAM-1 and VCAM-1 and MMP-9 production in VSMCs through p42/ p44MAPK–p38MAPK–Jnk–NF-jB signalling mechanism (Fig. 2) (Lee et al. 2006a, Lin et al. 2008). Similarly, IL-1b was shown to induce production of VCAM-1, ICAM-1 and MMP-9 in VSMCs through the same pathway (Wang et al. 2005, Liang et al. 2007). However, IL-4, a Th2-type cytokine, utilizes an NF-jB-independent mechanism to induce expression of VCAM-1, suggesting for an alternative proinflammatory signalling (Wright et al. 1999). ICAM-1 and VCAM-1 stimulate attachment of monocytes to VSMCs (Couffinhal et al. 1994, Wang et al. 1994). The adhesion between monocytes and VSMCs results in VSMC-dependent secretion of IL-6 and MCP-1 (Chen et al. 2009a), suggesting that cell adhesion molecule expression in VSMCs may enhance local inflammation. Surprisingly, IL-8 produced by proinflammatoryactivated ECs exhibits anti-inflammatory effects on VSMCs. Treatment of cultured VSMCs with IL-8 did induce production of VCAM-1, while cotreatment with IL-1b and IL-8 resulted in decreased expression of VCAM-1 compared to the treatment with IL-1b alone (Hastings et al. 2009). In VSMCs, IL-8 inhibits p38MAPK/Jnk-mediated signalling activated by IL-1b and upregulates Erk-dependent pathway that in turn leads to the downregulation of VCAM-1 (Wang et al. 2005, Zhang et al. 2011).

Molecular mechanisms that control phenotypic switch in VSMCs in atherosclerosis As already mentioned, change in the VSMC phenotype from the contractile type to the proinflammatory type is accompanied by decrease in expression of SM contractile proteins such as SM a-actin and SM MHC. The mechanism of transcriptional repression was found to be involved in changes of regulation of SMC marker genes. For example, the promoter of the SM22a gene contains a highly conserved G/C repres38

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sor element 50 to the proximal CaRG element [a binding site for myocardin, a critical factor for SMC differentiation (Chen et al. 2002, Yoshida et al. 2003)] that is shared between other SMC marker genes (Owens et al. 2004). The mutation in this element resulted in preventing suppression of expression of SM contractile genes in mouse models of vascular injury (Regan et al. 2000) and atherosclerosis (Wamhoff et al. 2004). Further studies showed that binding of Klf4, a transcriptional regulator, to the G/C element led to the inhibition of expression of SM22a and other SM marker genes. Furthermore, Klf4-dependent downregulation of SM markers was dependent on PDGF and oxidized phospholipids, both are factors that induce proatherogenic activation and phenotypic switching in VSMCs (Dandre & Owens 2004, Cherepanova et al. 2009, Salmon et al. 2012). PDGF-BB-mediated signalling was reported to activate a transcription factor Ets-1 (Dandre & Owens 2004) that in turn activates expression of the PDGF A-chain in VSMCs through cooperative interaction with Sp1 (Santiago & Khachigian 2004). Moreover, Sp1 is able to stimulate expression of Klf4, which is a major molecular regulator of the phenotypic switch in VSMC towards hyperplasia (Deaton et al. 2009). In fact, Klf4 is a transcription factor essential for maintenance of the self-renewal of embryonic stem cells and prevention of any differentiation (Jiang et al. 2008). Klf4 is normally not expressed in SMCs and inhibits myocardin-induced expression of SMC marker genes via downregulation of transcription of both the myocardin gene and myocardin target genes (Liu et al. 2005). For example, a cooperative interaction between Klf4, ETS domain-containing protein (ELK)-1 and histone deacetylase (HDAC)2 in the SM22a promoter is required to silence the expression of this gene (Salmon et al. 2012). Indeed, KLF4dependent recruitment of histone deacetylases leads to histone hypo-acetylation and epigenetic downregulation of the myocardin gene and SMC marker genes (Gomez & Owens 2012). In VSMCs, expression of Klf4 is upregulated in atherosclerosis (Cherepanova et al. 2009) and vascular wounds (Yoshida et al. 2008). Therefore, this factor is involved in VSMC dedifferentiation as a mechanism responsible for vessel healing through vascular remodelling. However, in cardiovascular proliferative diseases such as atherosclerosis, Klf4 activation becomes deregulated and poorly controlled in lesions, which in turn causes aberrant arterial wall remodelling in plaque regions and further proatherosclerotic progression (Zheng et al. 2010). Finally, Klf4 is involved in the upregulation of VSMC-mediated extracellular matrix (ECM) gene expression such as type collagen VIII


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induced by proatherogenic oxidized phospholipids (Cherepanova et al. 2009). NF-jB comprises a family of heterodimeric transcription factors that contribute to inflammation, proliferation and apoptosis (Bours et al. 1992). In quiescent VSMCs, NF-jB exists as an inactive p50p50 homodimer that is constitutively bound to DNA, thereby preventing expression of proinflammatory genes (Cao et al. 2006). However, inflammatory stimuli cause formation of the p65–p50 heterodimer that could direct transcription of proinflammatory genes (Bourcier et al. 1997). Activation of NF-jB in VSMCs plays a crucial role in induction of the proinflammatory phenotype because this transcription factor drives expression of many genes involved in inflammation. Specifically to VSMCs, those include IL-8 (Wang et al. 2002), MCP-1 (Landry et al. 1997), chemokine (C-XC motif) ligand 1 (CXCL1) (Kim et al. 2008), VCAM-1 (Landry et al. 1997), ICAM-1 (Cercek et al. 1997) and MMP-1, -2, -3 and -9 (Fig. 2) (Bond et al. 2001, Moon et al. 2004, Cui et al. 2014). CXCL1 could attract neutrophils to inflamed sites (Schumacher et al. 1992), while MMPs are required for ECM degradation and increasing migration of VSMCs (Chen et al. 2013). Although IL-8 has anti-inflammatory effects to VSMCs, this chemokine is primarily involved in the attraction of neutrophils and other granulocytes to the sites of inflammation. Neutrophils are the fastest responders to any proinflammatory stimuli and therefore could play a marked role in early stages of atherosclerosis-associated inflammation by releasing IL-8, elastase and MMP-8 that in turn contribute to pathological vascular remodelling and EC apoptosis (Dorweiler et al. 2008). The nuclear factor of activated T-cell (NFAT) family involves several transcription factors that contribute to the immune response. Typically, NFAT proteins are present in a cell in an inactive (dephosphorylated) sate outside of the nucleus (Nilsson et al. 2008). NFAT could be activated by proinflammatory and vasoactive stimuli (Pape et al. 2008, Min et al. 2009) through the mechanism involving ECM and adhesion proteins such as osteopontin (Tanabe et al. 2011), syndecan-4 (Finsen et al. 2011) and integrin b3 (Zhang et al. 2012). NFAT phosphorylation by Ca2+dependent protein kinase calcineurin results in its activation and translocation to the nucleus (Loh et al. 1996) where this transcription factor starts to drive the expression of target genes involved in VSMC proliferation and migration (Yellaturu et al. 2002, Liu et al. 2004). In VSMCs, NFAT was shown to also induce expression of IL-6 (Abbott et al. 2000). Another key player in VSMC dedifferentiation is a cluster of two microRNAs (miR): miR-221 and miR222. Both miRs are encoded by a single gene and have



more than 85% of similarity in the nucleotide sequence (di Leva et al. 2010). They share common signalling pathways and overlapping targets. Angiotensin II was shown to upregulate expression of miR-221/ miR-222 in both ECs and VSMCs (Zhu et al. 2011a). Angiotensin II-dependent induction of miR-221/miR222 is mediated by cAMP response element-binding protein (CREB), a transcription factor. On the other hand, miR-221/miR-222 suppresses expression of RAS p21 protein activator 1 (RASA1), a CREB inhibitor, thereby providing a positive feedback on its own expression (Jin et al. 2012). PDGF produced by proinflammatory-activated ECs could also induce expression of miR-221 in VSMCs (Davis et al. 2009). In VSMCs, miR-221/miR-222 inhibits a range of cell cycle regulators such as mast/stem cell growth factor receptor (SCFR), phosphatase and tensin homolog (PTEN), p21/Cip1, p27/Kip1 and p57/Kip2 (Liu et al. 2009). These regulators control expression of SM contractile proteins as well as expression of myocardin and MyoD, two transcriptional factors crucial for differentiation of VSMC (Reynaud et al. 2000, Wang et al. 2003). PTEN also inhibits production of the proinflammatory chemokine MCP-1 (Koide et al. 2007, Furgeson et al. 2010) and stromal cell-derived factor 1a (SDF-1a) (Nemenoff et al. 2011), which is involved in the activation of VSMC chemotaxis and mobility (Pan et al. 2012). Finally, the miR-221/miR222 cluster was shown to downregulate expression of p53 upregulated modulator of apoptosis (PUMA), thereby reducing VSMC apoptosis (Sarkar et al. 2013). Indeed, these data clearly suggest for critical role of miR-221/miR-222 in inducing changes in VSMCs from the contractile phenotype to proliferative. In fact, upregulation of miR-221/miR-222 occurs in sites of vascular injury and is required for the recruitment of VSMCs to heal the wound (Albinsson & Sessa 2011). However, in atherosclerosis, overexpression of miR-221/miR-222 may have detrimental effects through activation of VSMC hyperplasia, neointima formation and pathological vascular remodelling (Chistiakov et al. 2014).

Changes in ECM composition during VSMC phenotypic switching Normally, VSMCs are surrounded by ECM (basal lamina) composed of laminin, collagen IV and perlecan. In steady state, basal lamina provides signals that support VSMC ‘contractile’ phenotype and prevent dedifferentiation, growth and proliferation (Barnes & Farndale 1999). However, in case of vessel damage and vascular proliferative diseases, the composition of basal lamina becomes changed, with appearance of osteopontin, fibronectin and syndecan-4 (Fig. 2), for


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increased migration of VSMCs and enhanced adhesion of monocytes to ECs (Lee et al. 2013b). In VSMCs, TLR4 could be activated by LPS that in turn leads to the induction of the proinflammatory activation of VSMCs associated with secretion of IL-1a, IL-6 and MCP-1 (Yang et al. 2005). In VSMCs, TLR4 stimulation also upregulates NF-jB-dependent expression of MMP-9, a matrix metalloproteinase whose activation is related to plaque vulnerability and pathological vascular remodelling (Li et al. 2012). Proinflammatory cytokines such as TNF-a and IL1b were shown to contribute to the proinflammatory activation of VSMCs. TNF-a induces release of adhesion molecules such as ICAM-1 and VCAM-1 and MMP-9 production in VSMCs through p42/ p44MAPK–p38MAPK–Jnk–NF-jB signalling mechanism (Fig. 2) (Lee et al. 2006a, Lin et al. 2008). Similarly, IL-1b was shown to induce production of VCAM-1, ICAM-1 and MMP-9 in VSMCs through the same pathway (Wang et al. 2005, Liang et al. 2007). However, IL-4, a Th2-type cytokine, utilizes an NF-jB-independent mechanism to induce expression of VCAM-1, suggesting for an alternative proinflammatory signalling (Wright et al. 1999). ICAM-1 and VCAM-1 stimulate attachment of monocytes to VSMCs (Couffinhal et al. 1994, Wang et al. 1994). The adhesion between monocytes and VSMCs results in VSMC-dependent secretion of IL-6 and MCP-1 (Chen et al. 2009a), suggesting that cell adhesion molecule expression in VSMCs may enhance local inflammation. Surprisingly, IL-8 produced by proinflammatoryactivated ECs exhibits anti-inflammatory effects on VSMCs. Treatment of cultured VSMCs with IL-8 did induce production of VCAM-1, while cotreatment with IL-1b and IL-8 resulted in decreased expression of VCAM-1 compared to the treatment with IL-1b alone (Hastings et al. 2009). In VSMCs, IL-8 inhibits p38MAPK/Jnk-mediated signalling activated by IL-1b and upregulates Erk-dependent pathway that in turn leads to the downregulation of VCAM-1 (Wang et al. 2005, Zhang et al. 2011).

Molecular mechanisms that control phenotypic switch in VSMCs in atherosclerosis As already mentioned, change in the VSMC phenotype from the contractile type to the proinflammatory type is accompanied by decrease in expression of SM contractile proteins such as SM a-actin and SM MHC. The mechanism of transcriptional repression was found to be involved in changes of regulation of SMC marker genes. For example, the promoter of the SM22a gene contains a highly conserved G/C repres38

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sor element 50 to the proximal CaRG element [a binding site for myocardin, a critical factor for SMC differentiation (Chen et al. 2002, Yoshida et al. 2003)] that is shared between other SMC marker genes (Owens et al. 2004). The mutation in this element resulted in preventing suppression of expression of SM contractile genes in mouse models of vascular injury (Regan et al. 2000) and atherosclerosis (Wamhoff et al. 2004). Further studies showed that binding of Klf4, a transcriptional regulator, to the G/C element led to the inhibition of expression of SM22a and other SM marker genes. Furthermore, Klf4-dependent downregulation of SM markers was dependent on PDGF and oxidized phospholipids, both are factors that induce proatherogenic activation and phenotypic switching in VSMCs (Dandre & Owens 2004, Cherepanova et al. 2009, Salmon et al. 2012). PDGF-BB-mediated signalling was reported to activate a transcription factor Ets-1 (Dandre & Owens 2004) that in turn activates expression of the PDGF A-chain in VSMCs through cooperative interaction with Sp1 (Santiago & Khachigian 2004). Moreover, Sp1 is able to stimulate expression of Klf4, which is a major molecular regulator of the phenotypic switch in VSMC towards hyperplasia (Deaton et al. 2009). In fact, Klf4 is a transcription factor essential for maintenance of the self-renewal of embryonic stem cells and prevention of any differentiation (Jiang et al. 2008). Klf4 is normally not expressed in SMCs and inhibits myocardin-induced expression of SMC marker genes via downregulation of transcription of both the myocardin gene and myocardin target genes (Liu et al. 2005). For example, a cooperative interaction between Klf4, ETS domain-containing protein (ELK)-1 and histone deacetylase (HDAC)2 in the SM22a promoter is required to silence the expression of this gene (Salmon et al. 2012). Indeed, KLF4dependent recruitment of histone deacetylases leads to histone hypo-acetylation and epigenetic downregulation of the myocardin gene and SMC marker genes (Gomez & Owens 2012). In VSMCs, expression of Klf4 is upregulated in atherosclerosis (Cherepanova et al. 2009) and vascular wounds (Yoshida et al. 2008). Therefore, this factor is involved in VSMC dedifferentiation as a mechanism responsible for vessel healing through vascular remodelling. However, in cardiovascular proliferative diseases such as atherosclerosis, Klf4 activation becomes deregulated and poorly controlled in lesions, which in turn causes aberrant arterial wall remodelling in plaque regions and further proatherosclerotic progression (Zheng et al. 2010). Finally, Klf4 is involved in the upregulation of VSMC-mediated extracellular matrix (ECM) gene expression such as type collagen VIII


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(Steitz et al. 2001, McCormick et al. 2005, NakanoKurimoto et al. 2009). In fact, the capacity of lesion VSMCs to change towards CVCs is induced by the mechanism of replicative senescence due to the loss of ability to express SMC contractile proteins and because of increased apoptosis (Steitz et al. 2001, Nakano-Kurimoto et al. 2009). In senescent VSMCs, expression of runt-related transcription factor-2 (RUNX-2), a core transcriptional factor, which induces osteoblastic differentiation, is activated (Nakano-Kurimoto et al. 2009). This factor initiates a classical transcriptional programme of calcification via JAK2/STAT5B mechanism in human osteoblasts (Darvin et al. 2013). In lesion VSMCs, RUNX-2 could be induced with a variety of stimuli including oxLDL (Maziere et al. 2013), ROS (Byon et al. 2008), b-glycerophosphate (Bear et al. 2008) and fibroblast growth factor-2 (FGF-2; Nakahara et al. 2010). RUNX-2 drives expression of bone morphogenetic protein-2 (BMP-2), a member of TGFb protein superfamily that plays a key role in the development of bone and cartilage (Tanaka et al. 2008). In VSMCs, BMP-2 induction is followed with upregulation of Pit-1, a sodium-dependent inorganic phosphate cotransporter, and downregulation of SM22a and other SM markers (Li et al. 2006, 2008). Indeed, BMP-2 is critically involved in proatheroslerotic dedifferentiation and ossification of VSMCs. Vascular smooth muscle cell calcification is accompanied with activation of transglutaminase 2 (TG2), an enzyme that involved in arterial remodelling and vascular repair through catalysing formation of glutaminelysine cross-links between ECM proteins, which stabilizes ECM and promotes binding of signalling molecules such as osteopontin to the matrix proteins (Vanbavel & Bakker 2008). Johnson et al. (2008) showed a non-redundant role of TG2 in atherogenic calcification and switching of the ‘contractile’ VSMC phenotype to the chondro-osseous differentiation. Osteopontin-dependent signalling mediates TG2-induced mineralization of VSMCs associated with upregulation of calcification genes such as RUNX-2, Pit-1, muscle segment homeobox protein 2 (MSX-2), tissue-non-specific alkaline phosphatase (TNAP) and cartilage-specific proteoglycan core protein (CSPCP, or aggrecan) (Johnson et al. 2008). MSX-2 acts as a transcriptional repressor of calcification inhibitor genes such as osteopontin, osteoprogerin and MGP that are constitutively expressed in normal VSMCs to prevent ectopic calcification (Vanbavel & Bakker 2008). MSX-2 also activates expression of Osterix, a transcription factor involved in terminal osteoblastic differentiation (Taylor et al. 2011). Aggrecan is a proteoglycan that is normally expressed by chondrocytes and involved in chondro-



cyte–chondrocyte and chondrocyte–ECM interactions (Kiani et al. 2002). Aggrecan was shown to be expressed in calcified arteries, thereby suggesting for possibility of transformation of VSMCs not only to CVCs but also to chondrocyte-like cells (Tyson et al. 2003). TNAP is responsible for generation of inorganic phosphate, an essential component of hydroxyapatite, a major calcifying molecule. Normally, TNAP is not expressed in arterial SMCs. TNAP induction in VSMCs means irreversible modification towards CVCs as this enzyme is involved in enzymatic hydrolysis of inorganic pyrophosphate, a key suppressor of biomineralization (Bobryshev et al. 2014). PHOSPHO1, a soluble phosphatase that is an essential component of matrix vesicles (MVs), also contributes to generation of inorganic pyrophosphate from phosphatidylcholine and phosphocholine and therefore participates in proatherogenic calcification of VSMCs (Kiffer-Moreira & Narisawa 2013). Matrix vesicles produced by calcifying VSMCs promote vascular calcification as those particles contain all machinery such as TNAP, PHOSPHO1, nucleotide pyrophosphatase, adenosine triphosphatase, Pit-1 and Ca2+-binding molecules (phosphatidylcholine and annexon 1) essential to induce hydroxyapatite crystallization within the MVs (Anderson 2003). Structural aspects of the formation of MVs by VSMCs in the intima of human arteries have been intensely studied by means of electron microscopic analysis (Bobryshev et al. 1995, 2007, 2008a, Bobryshev 2005, McCormick et al. 2005). From biochemical point of view, the formation of first crystals leads to the constitutive release of crystals through the MV membrane due to increased intake of both Ca2+ and phosphate by calcifying VSMCs (Anderson 2003). Exposure of initial crystals to the extracellular fluid enriched with Ca2+ and phosphate propagates further calcification as those crystals serve as nuclei for deposition of new hydroxyapatite crystals (Anderson et al. 2005, McCormick et al. 2005). Apoptotic bodies released by dying VSMCs could also initiate formation of crystallization foci (London et al. 2005). In the normal arterial wall, apoptotic bodies are efficiently cleared mostly through the phagocytosis (Clarke & Bennett 2006). Healthy human VSMCs are potent phagocytes of apoptotic VSMCs (Clarke et al. 2010). However, in the atherosclerotic vessel wall, apoptotic bodies accumulate due to the absence or impairment of clearing mechanisms especially in case of chronic apoptosis. Inhibition of phagocytosis occurs through the IL-1mediated mechanism. Apoptotic VSMCs release both IL-1a and IL-1b that in turn initiate secretion of proinflammatory cytokines such as MCP-1, TNF-a and IL-6 in surrounding non-apoptotic VSMCs and induce


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an inflammatory response (Clarke et al. 2010). In an ApoE-deficient murine model of inducible VSMC apoptosis, Clarke et al. (2008) reported that chronic low-level apoptosis was able to enhance plaque growth by twofold, with advanced calcification, thickened fibrous lesion cap and enlarged necrotic core. Indeed, VSMC apoptosis could significantly contribute to plaque vulnerability and further rupture (Clarke et al. 2006). In cultured VSMCs, cell apoptosis was shown to also modulate phenotypic switch in VSMCs through activating cell proliferation, migration and enhanced collagen I synthesis (Yu et al. 2011).

Conclusion In conclusion, phenotypic switching of VSMCs from the contractile type towards proliferation and mobility is a physiological response to repair vessel damage. However, in atherosclerosis, the normal reaction of VSMCs to change the phenotype could be impaired by proinflammatory stimuli and oxidative stress. Indeed, blocking proinflammatory change in VSMC phenotype in affected vessels could be of clinical importance. Since p38MAPK-mediated signalling is crucially involved in mediating proinflammatory responses in VSMCs, inhibiting this pathway could have a therapeutic meaning to treat a chronic inflammatory disease such as atherosclerosis (Fisk et al. 2014). To date, several p38MAPK inhibitors are under clinical evaluation. For inhibitors such as SCIO469, BIRB-796 and VX-702, a clinical significance was reported in treatment of inflammatory diseases such as rheumatoid arthritis and Grohn’s disease (Orr et al. 2009). However, regarding cardiovascular disease, p38MAPK inhibitors are currently tested in pre-clinical studies only. For BIRB-796, improved survival and reduced cardiovascular damage were reported in rats with artificially induced angiotensin II-mediated hypertension (Park et al. 2007). VX-702-mediated p38MAPK inhibition was shown to preserve platelets from diverse alterations known as a platelet storage lesion (Skripchenko et al. 2013). These observations suggest for a clinical potential of p38MAPK inhibitors in the cardiovascular therapy. Although scientific findings comprehensively show that phenotypic changes in VSMCs to the proinflammatory type contribute to atherosclerosis, therapeutic targeting of these cells is still in its infancy. To date, no therapeutic tools are available to specifically treat atherosclerotic VSMCs. Indeed, more efforts are required to develop therapeutics that could directly influence VSMCs to prevent their phenotypic switching towards proatherosclerotic activation and hyperplasia. 42

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Conflict of interests There are no conflict of interests for the article. We wish to thank the Russian Scientific Foundation (grant 14-15-00112), Russian Federation for support of our work.

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example ECM proteins that could induce VSMC growth via Erk-dependent activation of cell cycle regulators (Moiseeva 2001). Proinflammatory cytokines such as IL-1b and TNF-a upregulate expression of fibronectin (Molossi et al. 1995). Cultured SMCs attached to fibronectin were found to produce three times more NF-kB compared to the cells attached to bare plastic (Qwarnstr€ om et al. 1994). Osteopontin could mediate angiotensin II-induced expression of IL1b in VSMCs through NF-kB- and AP-1-dependent mechanisms (Yin et al. 2009). In mice with experimental uraemia that was concerned as a strong risk factor for cardiovascular disease, depletion of osteopontin resulted in significantly decreased uraemic atherosclerosis and less advanced dedifferentiation of VSMCs (Pedersen et al. 2013). Syndecan-4 is a transmembrane heparan sulphate proteoglycan that works as a coreceptor for various growth factors (Shin et al. 2001). Ikesue et al. (2011) showed that deletion of syndecan-4 in mice led to limited neointima formation and reduced VSMC proliferation after vascular injury. Indeed, osteopontin, syndecan-4 and fibronectin could be induced in proinflammatory conditions and mediate proatherogenic dedifferentiation of VSMCs. Integrins transmit extracellular signals as receptors for ECM proteins. In VSMCs, integrins a5b1 and avb3 were shown to be primarily involved in interaction with proinflammatory-induced ECM proteins to mediate proatherosclerotic proliferation and neointima formation (Fig. 2) (Corjay et al. 1999, Kappert et al. 2001, Chen et al. 2009b). CD44, a cell surface glycoprotein, is also involved in the transmission of the proinflammatory signal to VSMC through the interaction with hyaluronic acid from the ECM. In response to CD44-mediated stimulus, VSMCs starts to release VCAM-1 and migrate (Cuff et al. 2001). ROS-dependent signalling associated with NADPH oxidase activation contributes to the activation of CD44 expression and hyaluronic acid synthesis (Vendrov et al. 2010). Expression of CD44 in VSMCs could be induced by thrombin, a serine protease essential for blood coagulation (Vendrov et al. 2006). CD44 plays the atherogenic role as its expression in VSMCs promotes atherosclerosis through hyaluronic aciddependent mechanism of activation of VSMC migration (Zhao et al. 2008). In atherosclerosis, VSMCs are able to express collagen I and collagen III (Barnes & Farndale 1999). Polymerized collagen I inhibits VSMC proliferation and migration (Koyama et al. 1996). However, proinflammatory-activated VSMCs produce MMP-1 and MMP9 that degrade collagen I and collagen IV respectively. Monomeric collagen I rapidly activates expression of VCAM-1 through NFAT-dependent mechanism 40

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(Minami et al. 2006, Orr et al. 2009). Indeed, collagen I monomers could stimulate migration of VSMCs. Effects of collagen I on the phenotypic switching in VSMCs probably mediated by integrin a2b1, which is preferentially binds to this type of collagen (Heino 2000). In addition, production of integrin a2b1 is upregulated in activated VSMCs (Skinner et al. 1994). Discoidin domain receptors DDR1 and DDR2 could also bind collagens and therefore mediate their effects on VSMCs (Franco et al. 2002). In mice, DDR1 knockout resulted in decreased VSMC migration to collagen I and reduced production of MMP-2 and MMP-9 (Hou et al. 2002), suggesting for an essential role of this receptor in the regulation of MMP expression and VSMC mobility. Lu et al. (2011) showed that collagen I-induced VSMC migration via DDR1 involves Src/MAPK signalling. However, precise molecular mechanisms of how collagen I could influence the phenotype of VSMCs are little known and should be further studied. Discussing the changes in ECM composition during VSMC phenotypic switching, it is necessary to mention also that not only MMP, but tissue inhibitor of MMP (TIMP) which regulates MMP activity, affects VSMC function (George 2000, Siasos et al. 2012, Roycik et al. 2013, Lin et al. 2014). Imbalance of MMP/TIMP plays an important role in the development of atherosclerosis, with VSMCs being crucially involved (Herman et al. 2001, Orbe et al. 2003, Kunz 2007, Raffetto & Khalil 2008).

VSMC calcification Arterial calcification is commonly occurs in advanced atherosclerosis. Calcification of the vascular wall decreases its elasticity and changes hemodynamics in affected arteries. Defects in arterial wall elasticity induced by calcium phosphate deposits cause systolic hypertension that leads to left ventricular hypertrophy, elevated oxidative stress, diastolic dysfunction and valve incompetence (Karwowski et al. 2012). Vascular smooth muscle cells stimulated towards the proinflammatory phenotype were shown to markedly contribute to atherogenic calcification giving rise to so-called calcified vascular cells (CVCs). Ectopic calcification is a hallmark of late atherosclerosis. This process involves mechanisms similar to those as in a normal bone biomineralization (Magne et al. 2005). In humans, specimens taken from the post-mortem atherosclerotic aortas exhibited higher expression of biomineralization markers such as osteopontin, bone morphogenetic protein-2 (BMP-2), osteonectin, collagen I, osteocalcin and S100A9, but less expression of calcification inhibitors including osteopontin, osteopregerin, fetuin-A and matrix Gla protein (MGP)




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