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Cardiovascular Research 1999 41(2):345-360; doi:10.1016/S0008-6363(98)00286-7
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Fibrous cap formation or destruction — the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation

Andrew C Newby* and Alla B Zaltsman

Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK

* Corresponding author. Tel.: +44-117-928-3582; fax: +44-117-928-3581; email: a.newby@bris.ac.uk

Received 24 June 1998; accepted 19 August 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Evolution of the...
 3 Role of the...
 References
 
Endothelial activation and infiltration of monocyte macrophages are essential prerequisites for fibrous cap formation, which comprises proliferation and migration of smooth muscle cells and net matrix deposition. Macrophage foam cells and endothelium act as a source of growth factors and chemoattractants for smooth muscle cells. However, growth factors alone do not stimulate smooth muscle cell proliferation or migration. This requires, in addition, the remodelling of the extracellular matrix, at least partly mediated by metalloproteinases. In particular, loss of basement membrane components and contact with the interstitial matrix appears to be required to release a brake on proliferation and migration exerted by the basement membrane. Unless there is a change in the phenotype of macrophages in advanced lesions, it is not clear why fibrous cap destruction rather than formation should take place in macrophage-rich shoulder regions of plaques. Impaired cap formation caused by smooth muscle senescence, mummification and propensity to apoptosis may be as important as increased cap destruction in promoting plaque rupture.

KEYWORDS Arteriosclerosis; Vascular smooth muscle; Extracellular matrix; Metalloproteinases


   1 Introduction
Top
 Abstract
 1 Introduction
2 Evolution of the...
 3 Role of the...
 References
 
The characteristic feature of the advanced atherosclerotic plaque is irregular thickening of the arterial intima by inflammatory cells, extracellular lipid (atheroma) and fibrous tissue (sclerosis) (Fig. 1). A large part of the lesions comprise seemingly inert and acellular fibrous tissue but there is often a distinct and highly cellular fibrous cap (Fig. 1). In eccentric lesions, such as the one shown in Fig. 1, there is also a leucocyte-rich ‘shoulder region’ at the limit between the plaque and the relatively normal tissue. The fibrous cap undoubtedly contributes something to luminal encroachment but its importance has recently been emphasised as a strong determinant of the likelihood of plaque rupture, which leads in turn to thrombosis followed either by occlusion or episodic plaque expansion [1–4]. Fibrous cap formation arises from the migration and proliferation of vascular smooth muscle cells and from matrix deposition [5]. The mechanisms underlying these processes have rightly therefore been the focus for an enormous body of molecular and cellular biology literature, itself the subject of authoritative reviews and regular updates [6–11]. The purpose of this article is to focus critically on the extent to which these processes might determine plaque stability.


Figure 1
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  		Fig. 1 Macrophage distribution in an advanced atherosclerotic plaque. A human carotid plaque was stained by immunocytochemistry of CD68 as a marker of macrophages (brown stain). fc=fibrous cap, lc=lipid core, sr=shoulder region. The scale bar shows 100 µm.

 

   2 Evolution of the fibrous cap
Top
 Abstract
 1 Introduction
 2 Evolution of the...
3 Role of the...
 References
 
2.1 Experimental approaches
The primary tool for investigating the evolution of atherosclerosis in humans has been the careful observation of necropsy material from individuals of widely different ages [12, 13]. The reasonable premise is that plaque age is somehow related to biological age. Alternatively, in fewer studies, the degree of stenosis has been taken as an indirect indicator of plaque age [14]. From observations of patients of different ages, fibrous cap formation appears to be a relatively late event in atherosclerosis, occurring after and, by implication, as an evolution from macrophage-rich fatty streak lesions [15]. This sequence of events is consistent with studies of cholesterol fed rabbits [16, 17]and primates [18–21]and with observations on genetically hyperlipidaemic rabbits [22, 23]and mice [24]. The animals used to model atherogenesis have a shorter life span than man and economic considerations preclude observations over the greater than 20 years that human lesions require to evolve into clinically apparent disease. The hope is that the evolution of lesions in these models mirrors that in man except in being conveniently accelerated. An additional difference between man and most of the animal models, except swine [25]and primates [20], is the presence of a significant population of mesenchymal cells, presumed to be vascular smooth muscle cells, in the normal arterial intima. This population and, hence, neointimal thickness is expanding throughout life, even in the absence of atherosclerosis. Studies by Smirnov's group (Orekhov et al. [26]) in which the intimal cell population in the aorta was counted showed that human aortic atherosclerotic plaques contained approximately twice as many smooth muscle cells as found in the neighboring normal areas. Pathological studies demonstrate that atherosclerotic plaques develop within preexisting areas of neointimal thickening in man [27], an observation confirmed in an experimental swine model [25].

2.2 Determinants of fibrous cap morphogenesis
Plaque morphology is generated by the organised motion of the constituent cell types, namely vascular smooth muscle cells and various blood leucocytes. As shown in Fig. 2a, the initial and most well established event is the movement of blood monocytes across the endothelium and their transformation to tissue macrophages in the subendothelial matrix [28–32]. Entry of further monocytes and lymphocytes leads to the fatty streak, which is regarded as a stable lesion, due perhaps to its low haemodynamic profile. Expression of endothelial leukocyte adhesion molecules and production of macrophage chemoattractants, including monocyte chemotactic peptide-1 (MCP-1), from smooth muscle cells [33]may orchestrate this motion. Oxidation of low-density lipoprotein (LDL) that has entered the vessel wall has a central role in stimulating adhesion molecule and MCP-1 expression [34]. Subsequently, there appears to be migration of smooth muscle from the media into the intima (Fig. 2a). This is clearly visible at early time points in both mice and rabbits (Fig. 3), where there is no pre-existing intimal population. In man and other animals where there is already an intimal smooth muscle cell population, it is possible that plaque smooth muscle cells derive solely from these cells [8]. Interestingly, smooth muscle cell proliferation has been observed in diffuse intimal thickenings [35], which implies that this population could expand to provide plaque smooth muscle cells. However, the frequent observation of large disruptions of the internal elastic lamina and wasting of the media at the base of human atherosclerotic plaques (see, for example, reference [36]) strongly suggests that at least some plaque smooth muscle cells are recruited from the media.


Figure 2
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  		Fig. 2 Scheme of cell movements in plaque morphogenesis. (a) LDL and monocytes move through the endothelium, where oxidised LDL is formed, stimulates production of MCP-1 and is taken up to generate macrophage-derived foam cells. (b) Macrophage-derived PDGF and other factors (not shown) encourage medial and pre-existing intimal SMC to migrate through and around foam cells. (c) Macrophage, endothelial and platelet-derived PDGF promote further migration and proliferation of SMCs to produce a distinct fibrous cap. Macrophages penetrate into the media attracted by MCP-1. FC=foam cell, MCP-1=monocyte chemotactic peptide-1, Mph=monocyte/macrophage, (ox)-LDL=(oxidised) low density lipoprotein, PDGF=platelet-derived growth factor, SMC=smooth muscle cell, T=T lymphocyte.

 

Figure 3
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  		Fig. 3 Smooth muscle and macrophage distribution in an early aortic atherosclerotic plaque from a cholesterol-fed rabbit. Rabbits were fed a 1% cholesterol diet for eight weeks [149]. Sections were stained for (A) macrophages (RAM-11 antibody) and (B) vascular smooth muscle cells ({alpha}-smooth muscle actin) [149]. Arrows denote the presence of smooth muscle cells both at the margin between the media (m) and plaque (p) and surrounded by macrophages. The scale bar represents 25 µm.

 
Data from human plaques of type III, before formation of a distinct fibrous cap [15], apoE-deficient mice on a Western-type diet [24]and cholesterol-fed rabbit aortas (e.g. Fig. 3) suggest that migration of smooth muscle cells then occurs through foam cell-rich areas, as illustrated schematically in Fig. 2b. Production of platelet-derived growth factor (PDGF) B chains from macrophages, which has been observed at all stages of atherogenesis in both human and experimental models [5, 37, 38], is probably relevant in recruiting smooth muscle cells to the intima. Heparin-binding epidermal growth-factor-like growth factor (HB-EGF) from macrophages also plays a role in smooth muscle cell (SMC) migration [39]. However, the expected outcome would be a mosaic of macrophages and smooth muscle cells, as seen in Fig. 3. Additional mechanisms must explain the formation of a distinct fibrous cap. Sorting of cells through cell surface cadherins may contribute [40], since smooth muscle cells, but not macrophages, express N-cadherin [41]. Homotypic binding between arrays of N-cadherin monomers could then lead to strong smooth muscle cell-to-cell interactions that would exclude macrophages. N-cadherin is also expressed together with VE-cadherin on endothelial cells [41]and this favours endothelial cell smooth muscle cell contacts [42].

Migration of smooth muscle cells along an endothelial to medial gradient of chemoattractant is another likely mechanism underlying cap formation (Fig. 2c). Activated endothelium [43]produces PDGFBB [44]and insulin-like growth factor-1 (IGF-1) [9]. The endothelium is essential for neointimal migration of smooth muscle cells in organ cultures of pig aorta [45]. The presence of endothelium [46]and PDGF [47]are also both necessary for neointimal migration of smooth muscle cells in human saphenous veins. A further possibility is PDGFAB production from platelets, where there is evidence for platelet adhesion to sub-endothelium in the late stage of atherogenesis (Fig. 2c) [21, 48]. An additional possibility is fibrin degradation products, which are known to arise from plasma fibrinogen and to adopt a subendothelial to medial concentration gradient in human plaques [49, 50]. Perhaps these factors act in concert.

Apart from the medial to intimal migration of smooth muscle cells described in Fig. 2, there is also circumferential motion, leading to expansion of the media. This accounts for the well accepted compensatory increase in arterial diameter with atherosclerosis [51], and for the initial increase in lumen diameter in vein grafts [52].

A further event may be penetration of macrophages into the media (Fig. 2c), for which there is good evidence in mouse [53]and Watanabe rabbit models [54]. In human coronary arteries and, particularly in aneurysmal dilatation of the abdominal aorta, there is wasting and inflammatory infiltration of the media, which may be analogous to the situation in ApoE–/– mice [53, 55]. Cholesterol-fed, genetically normal rabbits seem to have more superficially superimposed lesions (Fig. 3). Similarly, in human coronary vein grafts, lesions often show little penetration into the previously thickened neointima, which leads to the invidious, friable toothpaste-like lesions that are characteristic of failed grafts [56]. In the later stages of fibrous plaque morphogenesis (Fig. 2c), there may be migration of smooth muscle cells away from sites of cell proliferation. At the same time, penetration of fresh monocytes must be preferentially directed to the perimeter of the fibrous cap to explain the existence of leucocyte-rich shoulder regions. Mast cells are detected preferentially in the shoulder regions of later stage atheromas [57]. These might contribute to the fresh entry of macrophages and lymphocytes as well as to T lymphocytes growth by producing tumour necrosis factor {alpha} (TNF{alpha}) [58]. TNF{alpha} is a mediator of endothelium–leukocyte adhesion molecule-1 induction [59]and an enhancer of T lymphocyte growth [60]. Macrophages and endothelial-derived chemoattractants in these shoulder regions presumably attract further smooth muscle cells either from the subjacent media or from the edges of the existing fibrous cap.

2.3 What is the source of the neointimal connective tissue?
A pervasive concept, advanced by Chamley-Campbell and Campbell [61], is that normal contractile smooth muscle cells can undergo reversible modulation into a synthetic phenotype that is capable of both proliferation and enhanced matrix synthesis [62]. The response to vascular injury, such as balloon dilatation, clearly demonstrates that smooth muscle cells, as a population, possess the ability to change phenotype [63, 64]. However, cell proliferation can occur before and, hence, independently of phenotypic modulation [65, 66]. What remains uncertain is whether or not the sluggish proliferation in atherosclerotic plaques results from phenotypic modulation of typical contractile smooth muscle cells. Data from several species suggests the possibility that the smooth muscle cells in arteries represent several phenotypes in a lineage. Schwartz et al. [7]and Clowes and Schwartz [63]originally observed that adult rat arteries retain a population of smooth muscle cells with a neonatal epithelioid phenotype. Moreover, intimal smooth muscle cells share many aspects of this phenotype, suggesting that they may be derived selectively from it [67]. The epithelioid phenotype has not yet been identified in other species, including man. However, bovine pulmonary arteries contain four distinct cell phenotypes, at least two of which are clearly smooth muscle cells [68]. Observations of cell shape in alcoholic–alkaline etched preparations of human aorta also identify four populations (elongated, elongated with side process, irregular and stellate). The proportion of the stellate phenotype, possibly equivalent to the senescent phenotype in culture, increases in atherosclerotic tissue [12, 26]or in vitro in response to transforming growth factor-β (TGF-β) [69].

Some recent observations may cast doubt on whether the atherosclerotic neointimal mesenchymal cells derive from differentiated smooth muscle cells at all. Many lesional neointimal cells stain poorly for typical markers of smooth muscle cells, including desmin and smooth muscle cell {alpha}-actin or myosin [70–72]. Some authors suggest a fibroblast origin for these cells [73], while others suggest, more cautiously, an indeterminate origin [66, 68, 74]. Adventitial fibroblasts expressing smooth muscle cell {alpha}-actin have been identified migrating into the thickened neointima in experimental venous arterial interposition grafts [75]and in restenotic lesions after angioplasty [73]. Development of more selective immunocytochemical markers that differentiate between smooth muscle cells and fibroblasts is necessary to determine if, and to what extent, plaque caps may be derived from myofibroblasts.

2.4 The role of cell proliferation
Simplistically, the data from Smirnov's studies, alluded to above, imply that a single population doubling in 20 years, equivalent to a proliferative index of approximately 0.01%, is sufficient to account for the increase in smooth muscle cell numbers in atherosclerotic plaques. The large amounts of acellular connective tissue in plaques [14]may imply the net loss of a further one or two cell doublings through necrosis or apoptosis. Even so, the very low rates of cell proliferation that have been observed in plaques (0–1% PCNA proliferation index) [35]are entirely consistent with the extent of intimal enlargement. These very low rates cannot be used therefore to discount the importance of proliferation as a component of atherogenesis. Smooth muscle cells are capable of doubling within 48 h in tissue culture or immediately after balloon injury [65]. The most interesting question is therefore not so much why smooth muscle cells proliferate in atherosclerosis but how their proliferation is so effectively suppressed in the normal vessel wall.

A parameter more difficult to define is the number of population doublings undergone by individual intimal cells. Benditt and Benditt [76]were the first to observe that lesional smooth muscle cells appear in many cases to have derived by clonal expansion of a small number of cells (or perhaps just one). Although not strictly proven, more modern polymerase chain reaction (PCR)-based methods [77]have largely confirmed the early work. At the same time, lesion smooth muscle cells display properties of transformed cells; their DNA transforms cultured fibroblasts that produce tumours in nude mice [78, 79]. Furthermore, plaque smooth muscle cells, like many transformed cells, show greater susceptibility to apoptosis [80]. If all of the intimal smooth muscle cells in a typical coronary atherosclerotic plaque, perhaps 105 [26], were derived over 20 years from a single cell, this represents only one doubling per year, an index of proliferation of 0.1%, which is still consistent with experimentally measured values. As a result, such cells might be close to their Hayflick limit [81]and, hence, tending towards senescence, for which there is morphological evidence, as detailed above.

2.5 What regulates smooth muscle cell proliferation in atherogenesis?
We previously proposed objective criteria, which are updated and generalised in Table 1, to evaluate the role of individual factors in aspects of atherogenesis [82]. Growth factors for isolated human smooth muscle cells can be categorised according to the structure of their receptors. PDGF, basic and acidic fibroblast growth factors (bFGF, aFGF), IGF-1 and epidermal growth factor (EGF) all have receptors with intrinsic tyrosine kinase activity [9, 83]; angiotensin II, endothelin, 5-hydroxytryptamine and thrombin have serpentine G-protein-coupled receptors [82, 84, 85], TGF-β has receptors with intrinsic serine/threonine kinase activity [86]and interleukin-1 (IL-1) and TNF-{alpha} have receptors that couple indirectly to serine/threonine kinases [87]. All of these agents fit criterion 1, having been shown to promote smooth muscle cell proliferation (see [7, 82]for reviews) either directly, or indirectly by upregulating production of another growth factor or its receptor. TGF-β has biphasic stimulatory and inhibitory effects on proliferation, mediated respectively by type I and II receptors [86, 88]. Given their importance in atherogenesis, the role of normal and oxidised lipoproteins in regulating smooth muscle cell proliferation has been extensively studied (see reference [89]). Several normal lipoprotein fractions stimulate smooth muscle cell proliferation either by acting as a nutrient [90]or a specific mitogen [89, 91]. Oxidised lipoproteins generally cause a decrease in smooth muscle cell expansion rates but through promoting apoptosis [91].


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  		Table 1 Criteria to assess the involvement of factors in atherogenesis

 
Criterion 2, that the factor and its receptors are present in plaques at sufficient levels, has been met by fewer agents. One problem is that atherogenesis is both slow and episodic and, hence, the factor may only be present transiently or patchily. The concentration of PDGF A and B chains is increased in atherosclerotic tissue [5, 92, 93], although recent data demonstrated more PDGFA in normal arteries than in plaques [94]. PDGFB chain protein has been associated, by immunocytochemistry, with macrophages and by in situ hybridisation with neointimal mesenchymal cells and endothelial cells in human atherosclerotic plaques [71]. PDGF {alpha} and β receptors are also abundant on smooth muscle cells in human atherosclerotic lesions [37, 71, 95]. The concentrations of IGF-1 [96]and TGF-β [97]are also elevated in primary atherosclerotic and restenotic tissues. The type II TGF-β receptors, associated with inhibition of proliferation, appear to be downregulated in neointimal smooth muscle cells, perhaps leading to unmasking of proliferative action through the type I receptor or through other factors [88]. Acidic and basic FGF are both present in normal and plaque smooth muscle cells and endothelium, although mainly sequestered in cells or bound to the extracellular matrix [98, 99]. Hence, their availability at FGF receptors, other than as a result of injury and cell death, is uncertain. Thrombin activity is increased in neointima and thrombin receptors are overexpressed in human atherosclerotic plaques [100–102].

With the possible exception of endothelin-1 [103], no growth factor has so far met criterion 3, that an inhibitor should prevent atherosclerosis. In the balloon-injured rat carotid artery, anti-PDGF antibodies inhibit migration of smooth muscle cells into the intima [104]while anti-bFGF antibodies inhibit the early proliferative response but do not reduce final intimal thickness [105]. A combination of anti-PDGF and anti-bFGF antibodies greatly inhibits neointima formation [106]. Antibodies to TGF-β demonstrate inhibitory activity against neointima formation in the same rat model [107]. Angiotensin-converting enzyme inhibitors or angiotensin receptor-1 blockers and endothelin receptor antagonists also partly inhibit neointima formation [108, 109]. Antithrombin agents also have inhibitory effects on neointima formation [110].

From these studies, multiple growth factors appear to act together to stimulate neointima formation in balloon-injured arteries by cooperative and, in cases, synergistic interactions. To what extent these conclusions can be extrapolated to atherosclerosis formation remains uncertain, however. Recent experience with expensive and unsuccessful clinical trials of many inhibitors of smooth muscle cell proliferation in angioplasty restenosis graphically illustrate the potential pitfalls of such premature extrapolation [8].

2.6 Growth factors are not enough to stimulate smooth muscle cell proliferation
The mitogenic action of growth factors on smooth muscle cells is dramatically modified by their microenvironment within the vessel wall. This is shown clearly in organ culture experiments of rabbit and pig aorta and of human saphenous veins (see Fig. 4). Exposure to serum, which is mitogenic for the same vesicular smooth muscle cells (VSMCs) when isolated, either fails entirely to stimulate VSMC proliferation [111]or does so selectively in the subendothelial space (Fig. 4a, Fig. 4c) [45, 46]. Proliferation in the media occurs only when this is injured (Fig. 4c) [111, 112]. These observations have counterparts in the balloon-injured rat carotid model where medial proliferation and the extent of neointima thickening depends not primarily on the extent of endothelial injury and, hence, exposure to platelet-derived mitogens but on the degree of medial injury [113]. One possible explanation for these phenomena is that multiple growth factors (e.g. PDGF plus bFGF) need to be simultaneously released by a variety of stimuli [105], or additional factors (e.g. proteases) are needed to achieve access of growth factors to their receptors [114]. Alternatively, we have proposed that interactions with the extracellular matrix surrounding smooth muscle cells in the normal artery strongly suppress proliferative responses [115]. This hypothesis depends in part on data from Clowes’ and Campbell's groups, demonstrating, in a series of classic experiments, the inhibitory effects of heparin on smooth muscle proliferation [116, 117]The same effect is obtained with heparan sulphate proteoglycans, many of which (e.g. syndecans, perlecan) are prominent components of the basement membrane that normally surrounds smooth muscle cells. Heparin and, hence, presumably heparan sulphates act directly against proliferation by inhibiting the binding of the activator protein-1 (c-fos/c-jun) transcription factor to its binding sites in DNA [118]. This mechanism probably accounts also for the inhibitory effects of heparin on secretion of extracellular proteases including tissue plasminogen activator (t-PA) and matrix metalloproteinases (MMPs) [118–121]. Work by Hedin et al. [122, 123]and Thyberg and Hedin [124]also shows that the basement membrane component, laminin, inhibits while the interstitial matrix component, fibronectin, promotes phenotypic modulation of smooth muscle cells. Based on such evidence, we have developed a detailed hypothesis, as illustrated in Fig. 5a, proposing that the basement membrane provides a ‘brake’ on proliferation of smooth muscle cells [11, 83, 115, 125]. Important support for this hypothesis has come from Ross's group, who showed that defined extracellular matrix components can prevent smooth muscle cells from passing through the cell cycle by upregulating the p27 cyclin-dependent kinase inhibitor [126]. Furthermore, we proposed that extracellular proteolysis is the rate-limiting step in catabolism of all basement membrane components and is, hence, the regulated step in relieving the inhibitory constraints on proliferation [83]. The basement membrane-degrading metalloproteinase, MMP-2, is produced constitutively by smooth muscle cells [115, 127]and is activated either by membrane-type metalloproteinases [128], thrombin [129]or reactive oxygen species [130]. A second basement membrane-degrading metalloproteinase, MMP-9, is induced by inflammatory cytokines [127], especially in combination with peptide growth factors [131].


Figure 4
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  		Fig. 4 Cell proliferation in organ cultures of human saphenous vein. (a) A segment of undamaged ‘freshly isolated’ vein cultured in 30% serum and 1 µCi/ml of [3H]thymidine for 14 days. The section has been stained with haematoxylin and eosin and subjected to autoradiography to reveal the presence of cells that have undergone DNA synthesis. In this section, dividing cells are restricted to the neointima (above the arrows). (b) A serial section to (a), stained to reveal the internal elastic lamina (arrows). (c) A segment of damaged ‘surgically prepared vein’ treated as in (a). Note the presence of dividing cells in the media (below the arrows). (d) A serial section to (c), revealing the internal elastic lamina (arrows). The scale bar represents 25 µm. The figure is adapted from reference [112].

 

Figure 5
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  		Fig. 5 Matrix interactions in contractile and synthetic smooth muscle cells — the ‘brake’ hypothesis. (a) Contractile smooth muscle cells are surrounded with a basement membrane of type IV collagen and laminin that is rich in heparin sulphate proteoglycans, including syndecans. Binding to β1 integrins generates a signal that maintains the contractile status and puts a brake on migration and proliferation. Release of the brake requires turnover of matrix components that is initiated by extracellular proteases including metalloproteinases (MMP), urokinase plasminogen activator (u-PA), and disintegrin and metalloproteinase-containing proteins (ADAMs). Endocytosis followed by further proteolysis and glycolysis completes the breakdown of matrix components. (b) Synthetic smooth muscle cells have degraded their basement membrane and come into contact with interstitial matrix components, including monomeric and polymerised types I and II collagen and fibronectin. These also bind to integrin, including, importantly, {alpha}5β1 and {alpha}vβ3 integrins, which may provide the signals for phenotypic change. Integrins can also act as points for organisation of stress fibres, while fibronectin and other glycoproteins provide track ways for migration. Activation of smooth muscle cells is associated with upregulation of MMPs that can promote turnover of the interstitial matrix.

 
Activation of MMP-2 and induction of MMP-9 are early events in the vascular response to injury [132–134]. Experiments in the human organ culture model demonstrated a spatial and temporal correlation between the expression of MMP-9 and medial cell proliferation in response to injury [135]. High concentrations of synthetic MMP inhibitors have been shown to inhibit proliferation of smooth muscle cells, particularly when proliferation is measured within the intact tissue in vitro [115]or at early time points after vascular injury in vivo [136]. On the other hand, other studies with synthetic inhibitors [137]or gene transfer of TIMP-1 in vivo [138]or by adenovirus-mediated delivery to human saphenous vein [139]showed no effect on proliferation. Hence, the role of basement membrane-degrading MMPs in regulating smooth muscle cell proliferation is uncertain. Heparanases [140, 141]and other classes of proteases, for example plasmin, cathepsins [142]or the more recently described disintegrin and metalloproteinase-containing proteins (ADAMs) [143], might also have a role in regulating basement membrane integrity and cell proliferation.

In the context of atherosclerosis, there is strong evidence for the upregulation of interstitial stromelysin (MMP-3), collagenase (MMP-1), metalloelastase (MMP-12) and MMP-9 expression in human atherosclerotic plaques, in the cholesterol-fed rabbit and in genetically modified mice (criterion 2) [144–150]. Activity of these proteases, in particular MMP-2, is also increased [146, 149]. Increased expression has been correlated with morphological and mechanical determinants of plaque rupture [144, 145, 147, 148]. Indeed, a strong case has been made that macrophage-derived MMPs are a primary cause of plaque rupture [144, 145, 147, 148]. However, in the rabbit and mouse models, increased MMP expression and activity occurs early in the development of lesions [53, 146, 149], implying that they have a role in plaque morphogenesis (i.e. cap formation) as well as plaque rupture. As yet, no protease has been shown by inhibitor studies to be essential for plaque formation or rupture (criterion 3), although the existence of knockout mice for many of these proteases promises much for the near future.

2.7 What regulates smooth muscle cell migration?
As with proliferation, directed smooth muscle migration requires the combined action of chemoattractants and remodelling of the extracellular matrix [151]. Matrix remodelling may take two forms, namely removal of constraints to movement and the formation of productive cell matrix interactions that are necessary for movement [9]. This in turn probably requires both activation of cell surface receptors for matrix molecules, including integrins, and the reorganisation of the matrix itself. Some of the important changes are illustrated in Fig. 5. An interesting unresolved question is whether the basement membrane simply acts as a physical barrier to movement, ‘the cage hypothesis', or whether signalling events actively suppress movement, ‘the brake hypothesis’ (Fig. 5).

Migration is frequently measured with recently dispersed cells crossing a microporous membrane (Boyden chamber assay) so as to minimise the effects of matrix interactions. Alternatively, reconstituted basement or interstitial matrix can be coated onto membranes in the same assay system so that chemotaxis and invasion can be measured together. When migration is studied from explants, in a complex organ culture or in an animal model, direct effects on chemotaxis and on matrix remodelling occur together, as they do physiologically. Not surprisingly, more regulatory interactions emerge the more complex the experimental model.

Using the Boyden chamber assay, some, but not all, smooth muscle mitogens have been shown to possess direct chemotactic activity, which implies that the pathways for stimulating migration are overlapping but distinct from those mediating proliferation. For example, IGF-1 is a weak activator of mitogenic pathways but potently induces migration of human VSMCs [152]. One of the most potent chemoattractants for smooth muscle cells is PDGFBB [5, 9, 38, 153]. PDGFBB and PDGFAA are equally good activators of the intracellular mitogenic pathways, including the mitogen-activated protein kinase (MAP kinase) cascade and early response gene (e.g. c-fos) induction. However, PDGFAA either has no effect or actually antagonises the chemotactic actions of PDGFBB chains [154, 155]. Lakatta's group has provided persuasive evidence that Ca2+ mobilization and Ca2+–calmodulin-activated kinase II, rather than MAP kinase, mediates chemotaxis [156, 157]. Other mitogens, including bFGF [158]and HB-EGF have also been shown to promote migration. The effects of growth factors on migration may be direct, indirect or both. For example, PDGF-BB induces synthesis of thrombospondin, which potentiates chemoattraction by PDGF [159]. Moreover, PDGF and other mitogens, especially when combined with inflammatory mediators, increase expression of MMPs [131, 160].

When invasion through reconstituted matrix is added to the chemotaxis assay, clear evidence for the involvement of extracellular proteases, including t-PA [161]and urokinase plasminogen activator (u-PA) [162]emerges. MMPs are also centrally involved. Synthetic MMP inhibitors or overexpression of tissue inhibitors of metalloproteinases-1, -2 or -3 inhibit migration of smooth muscle cells through basement membrane [136, 138, 163, 164]. The involvement of both classes of protease may be explained either by independent effects on matrix components or because plasmin is an activator of MMPs-1, -3, -7, -9 and -12.

Using more complex organ culture models, the importance of PDGF acting in concert with MMPs is confirmed. Migration of SMCs out of baboon aortic depends on MMP expression, and this is mediated by endogenous PDGF and bFGF [165]. Both anti-PDGF antibodies [47]and adenovirus-mediated overexpression of TIMP-1 [139]also inhibit neointima formation in human saphenous vein organ cultures by a selective effect on cell migration.

In vivo experiments in balloon-injured rat carotid arteries also confirm the importance of growth factors working together with extracellular proteases in SMC migration. Expression of t-PA and u-PA increases after balloon injury [166]and tranexamic acid, an inhibitor of plasminogen activation, decreases rat SMC migration in this model [167]. Increased t-PA activity is partly mediated by PDGF [168]. Overexpression of plasminogen activator inhibitor-1 (PAI-1) results in suppression of arterial neointimal formation in PAI-1-deficient mice [169]. In the case of MMPs, both synthetic inhibitors [136, 137]and seeding of cells transfected with TIMP-1 [138]inhibit migration of SMCs and either delay [136, 137]or inhibit [138]neointima formation.

2.8 Deposition of extracellular matrix
Contractile smooth muscle cells have low levels of total messenger RNA, demonstrating their limited capacity for new protein synthesis. Not surprisingly, therefore, synthesis of extracellular matrix molecules by contractile cells is limited, although basement membrane proteoglycans, including syndecans and perlecan, may be an exception [119]. Phenotypic modulation, provoked by isolation of cells from the tissue by collagenase, leads to a profound alteration in both the pattern (see Fig. 5) and extent of matrix molecule expression [170, 171]. Increased synthesis of hyaluronic acid is an early event that is generally associated with the production of an emergency matrix after tissue injury [172, 173]. Basement membrane components, for example laminin, are downregulated [174], while synthesis of type 1 collagen, elastin and fibronectin is greatly increased. The components of this matrix resemble the normal interstitial matrix, however, the abundance of monomeric rather than polymerised collagen may be of key importance in allowing cells to proliferate [126]. Fibronectin may polymerise into fibres, providing trackways for migration [175, 176]. Osteopontin also has a migratory effect on SMCs, which is mediated by complexing with the {alpha}vβ3 integrin [177]. Interactions of both β1 and β3 integrins with the extracellular matrix are important for directed cell migration [178](see Fig. 5). Vitronectin is also known to promote migration [179, 180]. Coincident with confluency, isolated smooth muscle cells begin to re-express basement membrane components, consistent with their proposed role in promoting quiescence [174].

Given the slow and focal nature of proliferation in atherosclerotic plaques, the concomitant changes in matrix turnover would be expected to be subtle. What one observes in an overall comparison of diseased and normal tissue is likely to be a distant and more permanent record of the immediate changes. Plaques are characterised by increased levels of collagens I and III, and elastin, as well as chondroitin sulphate- and dermatan sulphate-containing proteoglycans [181], components of interstitial rather than basement membrane. At the same time, atherosclerotic plaques have decreased levels of heparan sulphates, known components of basement membrane and inhibitors of SMC proliferation [181–183]. Prominent areas of collagen synthesis have been demonstrated within the fibrous cap [184], where the collagen producing smooth muscle cell is not necessarily proliferating [185]. The factors likely to be responsible for increased collagen production include PDGF and TGF-β [171]. This implies that PDGF and TGF produced by macrophages upregulate collagen and fibronectin production from smooth muscle cells [186, 187]and, hence, further promote fibrous cap formation.

Many of these changes can be seen also in animal models of balloon injury [171]. Other matrix molecules that are upregulated in these models include the fibril-associated type VIII collagen [188]and the multiadhesive glycoproteins, osteopontin [189, 190]and thrombospondin [159, 191]. RGD-containing peptides, which block binding to integrins, inhibit neointimal formation after balloon injury [192]or stent injury [193].


   3 Role of the smooth muscle cell in plaque rupture
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 Abstract
 1 Introduction
 2 Evolution of the...
 3 Role of the...
References
 
3.1 Contrasting hypotheses
The histological appearance of rupture-prone plaques is dealt with thoroughly elsewhere in this volume. Put simply, rupture occurs in plaques with a large lipid core, a thin cap and where there are abundant macrophages in relation to smooth muscle cells [194, 195]. It is perhaps the prevailing view that rupture results from the destruction of an established fibrous cap, and that this is mediated by macrophages [32, 196, 197]. An alternative interpretation is that new areas of fatty streak at the edges of existing fibrous plaque simply fail to generate or have not yet generated an adequate plaque cap. Table 2 lists six different hypotheses that might account for plaque rupture. Each has merit but gives central importance to a different component of the plaque. Below we consider these hypotheses from the perspective of smooth muscle cell biology.


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  		Table 2 Hypotheses to explain plaque rupture

 
3.1.1 Smooth muscle senescence
The direct evidence that only a tiny minority (i.e. clones) of cells respond to mitogenic stimuli in the slowly evolving human atherosclerotic plaque is reviewed above. If plaques are derived by clonal expansion, senescence becomes a real possibility that is supported by both morphological measures and the behaviour of plaque smooth muscle cells in culture. The presence of mediators such as TGF-β in the plaque may hasten senescence. The true cause of plaque rupture may therefore be the exhaustion of the reparative power of smooth muscle cells. If so, then the appropriate treatment, surprisingly, might be locally to reinforce the stimuli for proliferation with a growth factor such as PDGF. In this sense, it may be analogous to the observation that endothelial regrowth over a large denuded area of rat aorta remains incomplete, implying a limit to the regenerative capacity of the endothelium. This can be overcome, nevertheless, by infusion of a mitogen, basic FGF [198]. The vigorous smooth muscle cell response to gross angioplasty injury, metal stent implantation or deposition of a mural thrombus further implies that the senescent phenotype can be rescued by a sufficiently large stimulus.

3.1.2 Mummification
The difficulty in provoking the typical smooth muscle cell into proliferation has been emphasised throughout this article, as has the importance of basement membrane as an inhibitor. It seems highly unlikely therefore that the smooth muscle cells surrounded by multiple layers of basement membrane frequently found in atherosclerotic plaques [13]could play any part in fibrous cap formation. The reasons for the generation of this phenotype are unclear, however, it might also be rescued by the right stimulus.

3.1.3 Breakdown in the chemotactic gradients that promote cap formation
Aberrant regulation of the production of endothelial derived mediators is one of the earliest events in the preclinical phase of atherosclerosis. Increased expression of leukocyte adhesion molecules is another marker of dysfunction [43]. Late on in atherogenesis, focal loss of endothelial cells occurs [21, 48, 194]. Given these changes, it is likely that formation of endothelium-derived PDGFB-chains and other chemoattractants for smooth muscle cells is impaired also.

3.1.4 Production of inhibitory mediators
Prostaglandins that elevate cAMP concentrations [199, 200], nitric oxide, that elevates cGMP concentration [201–203], interferon-{gamma} [204, 205]and TGF-β [86]all antagonise the effects of smooth muscle cell mitogens. For example, vascular endothelial growth factor (VEGF) gene transfer causes NO-mediated reduction of neointima formation after cuff-induced injury in carotid arteries [206]. The pathways are probably diverse; downregulation of early events, such as MAP kinase activity [207]and later events, including cyclin D1 [208]and c-myc [209, 210]expression, both contribute. Leucocytes are probably the most important source of all of these inhibitory agents. As shown by Hansson and colleagues, infusion of interferon-{gamma} reduces intimal lesions caused by balloon injury (criterion 1) [206]and these are promoted in lymphocyte-deficient rats (criterion 3) [211]. Similar considerations might apply in atherogenesis. If so, the presence of leucocytes at the shoulder regions of advanced plaque might favour inhibition rather than proliferation of smooth muscle cells. One of the most important antigens for plaque lymphocytes is modified LDL and immunisation of LDL receptor-deficient rabbits with homologous malondialdehyde (MDA)-modified LDL or hypercholesterolaemic rabbits with homologous and oxidized LDL reduces plaque progression [212, 213]. This apparently beneficial effect might be a disadvantage at the shoulders of an advanced plaque where fibrous cap formation is critical for stability.

3.1.5 Smooth muscle cell death
Work by Bennett et al. [80, 214]has identified that increased susceptibility to apoptosis is an intrinsic characteristic of plaque smooth muscle cells. Although the levels of smooth muscle apoptosis actually present in plaques are controversial [215–219], it probably does occur. Given the slow turnover of smooth muscle cells and the relative rapidity of apoptosis (2–4 h) compared to division (24–48 h), one would predict that the true instantaneous rates of apoptosis are less than those of proliferation (i.e. less that 0.1%), which may be below the noise level of existing methods of detection. These issues are fully discussed elsewhere in this focused issue.

3.1.6 Impaired matrix formation
A further consequence of inadequate smooth muscle cell numbers will be inadequate matrix deposition. Matrix deposition may also be a direct target for the inhibitory action of mediators such as prostaglandins [220]and nitric oxide [221]. Once the matrix becomes devoid of smooth muscle cells, only degradation rather than turnover is possible. Hence, even levels of proteolysis no greater than those present in the evolving plaque might lead to net destruction of the intracellular connective tissue

3.2 New treatments?
Based on our current limited knowledge, caution is needed in proposing new treatments. The debate will undoubtedly be between ‘blunderbuss’ and ‘magic bullet'. Lipid lowering is arguably a blunderbuss approach, affecting as it does most aspects of plaque biology. However, its action at the root of atherogenesis is probably the key to its success. The search for critical molecular mediators of plaque instability will rightly be the holy grail for researchers in this field in the coming years. This quest will be hampered by the lack of appropriate animal models of plaque rupture, although transgenic animals may again provide an answer. Clinical trials will need to be large, protracted and expensive. Nevertheless, recent trials have established that both primary and secondary prevention of myocardial infarction is possible. Greater understanding of the basic biology of fibrous cap formation and rupture promises further significant advances.

Time for primary review 34 days.


   Acknowledgements
 
We thank Dr Mark Bond and Mr Jason Johnson for some of the illustrations. The authors’ work is supported by the British Heart Foundation and the Medical Research Council of Great Britain.


   References
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 3 Role of the...
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N. Leitinger
"Obese" Smooth Muscle Cells Fail to Assemble Collagen Fibrils
Circ. Res., April 10, 2009; 104(7): 826 - 828.
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 Cardiovasc ResHome page
A. Dwivedi, S. C. Slater, and S. J. George
MMP-9 and -12 cause N-cadherin shedding and thereby {beta}-catenin signalling and vascular smooth muscle cell proliferation
Cardiovasc Res, January 1, 2009; 81(1): 178 - 186.
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 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Higashi, S. Sukhanov, S. Parthasarathy, and P. Delafontaine
The ubiquitin ligase Nedd4 mediates oxidized low-density lipoprotein-induced downregulation of insulin-like growth factor-1 receptor
Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1684 - H1689.
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 Arterioscler. Thromb. Vasc. Bio.Home page
A. C. Doran, N. Meller, and C. A. McNamara
Role of Smooth Muscle Cells in the Initiation and Early Progression of Atherosclerosis
Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 812 - 819.
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