Thin-cap fibroatheromas (TCFAs) or vulnerable atherosclerotic plaques are considered a high-risk phenotype for acute cardiovascular events. TCFAs are identified by a thin rupture-prone fibrous cap, a large necrotic core, and a high content of leucocytes. Atherogenesis is dependent upon complex patterns of blood flow. Slow-flowing blood imposing low shear stress on the arterial wall up-regulates inflammatory signalling in endothelial cells and leucocytes, and modulates microRNAs to promote inflammation and monocyte recruitment. Hence, low shear stress is believed to promote conditions conducive to vulnerable plaque development. In this review, we explore how biomechanical factors modulate macrophage phenotype and plaque stability.
The development of atherosclerotic plaques happens silently over a prolonged period of time. Yet the most common manifestations of atherosclerotic disease are acute.1 Early studies reported that plaque rupture and thrombosis cause acute complications of atherosclerosis, prompting the aim of identifying ‘precursor’ lesions vulnerable to rupture and thrombosis.2,3 Virmani et al.4 named such high-risk lesions thin-cap fibroatheromas (TCFAs) and the recent literature associated these lesions with a higher occurrence of acute coronary syndromes (ACS).5 Collectively, these studies suggest a large necrotic core, high macrophage content, and thin fibrous cap are the hallmarks of plaque vulnerability. Macrophages are frequently observed at plaque-rupture sites,6 suggesting they are decisive in the rupture of vulnerable plaques. However, up to 30% of clinical events result from the disruption of the endothelium overlying plaques low in macrophages and extracellular lipids but rich in smooth muscle cells (SMCs) and extracellular matrix.7 Thus, despite the crucial role of macrophages in vulnerable plaque development, rupture-prone lesions often lack the presence of inflammatory cells at the time of rupture.
The localized nature of atherosclerosis has been appreciated since the earliest days of research in the field. Predilection sites of atherosclerosis are normally branching or curved arteries, where blood flow is not linear and slow, exerting pro-atherogenic low or oscillatory shear stress.8 Fast-flowing blood in straight vessels generates protective high shear stress (HSS). Low shear stress (LSS) occurs in inner curvatures (e.g. in the aortic arch, myocardial aspect of coronary arteries) and upstream of stenoses, where blood flow is unidirectional but shear levels average at a lower magnitude than shear levels in a straight vessel.9 Emerging literature has linked LSS to the development of vulnerable plaques. Oscillatory shear stress (OSS) involves changes in both the magnitude of shear stress and direction of blood flow, normally at branch points, bifurcations or downstream of stenoses.9
Our first aim is to review the relationship between shear stress and macrophage-associated inflammatory signalling [including regulatory microRNAs (miRs)] in vulnerable plaque development. Our second aim is to discuss the relationship between biomechanical factors and rupture of vulnerable lesions (Figure 1 ).
Outline of review. Biomechanical factors, more in particular low and oscillatory shear stress, activate endothelial cells leading to accumulation of macrophages in the vascular intima. These macrophages participate in vulnerable plaque development by inducing foam-cell formation, fibrous cap thinning, and plaque necrosis. On the other hand, biomechanical factors, such as HSS and strain, are ultimate determinants of vulnerable plaque rupture and thrombus formation. TCFA, thin-cap fibroatheroma.
2. Biomechanical stress and macrophage activity during vulnerable plaque development
2.1 Shear stress, endothelial cell activation, and monocyte recruitment
Mechanoreceptors on the endothelial cell surface sense changes in flow patterns converting biomechanical forces to biochemical responses, hence LSS can up-regulate adhesion molecules [e.g. vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1)] promoting monocyte recruitment10 (Figures 2A and 3A). OSS also allows monocyte arrest on the endothelial cell surface, up-regulates P-selectin, ICAM-1, elevates transglutaminase activity, and subsequently the expression of monocyte chemoattractant protein (MCP-1 or CCL2) and monocyte recruitment.11,12 Following low-density lipoprotein (LDL) retention, LSS can stimulate the production of reactive oxygen species by endothelial cells, which oxidatively modify LDL into oxidized LDL (oxLDL)9 serving as a further monocytic chemoattractant13 (Figure 2A). Endothelial responses to shear stress are discussed elsewhere in this Spotlight issue.
Shear stress and the development of a vulnerable atherosclerotic plaque. (A) Oscillatory blood flow (where flow is multidirectional) occurs in the carotid bifurcation, imposing low and oscillatory shear stress (where the magnitude of shear fluctuates at a low time-average) on the vessel wall. Lipids then become concentrated in the lumen before diffusing into the intima. Endothelial cells detect the low shear-stress levels and up-regulate their expression of adhesion molecules enabling the recruitment of monocytes into the intima. The monocytes differentiate into macrophages and phagocytose subintimal lipids transforming them into foam-cell macrophages. (B) A mature plaque develops with further infiltration of inflammatory cells, secretion of inflammatory cytokines and chemokines, migration of smooth muscle cells from the media and collagen synthesis to form a fibrous cap just beneath the endothelium, and neovascularization leading to intraplaque haemorrhage. Advanced lesional macrophages contribute to the formation of the necrotic core and thinning of the fibrous cap, which characterize the vulnerable plaque prone to rupture (thin-cap fibroatheroma).
miRs, which are short non-coding RNA molecules that exert post-transcriptional effects on gene expression,14 are involved in shear-stress-induced endothelial cell dysfunction (Figure 3A). We can distinguish two groups of miRs linked to endothelial cell dysfunction: those directly targeting adhesion molecules and those promoting monocyte recruitment by targeting inflammatory mediators (Table 1A). The first group consists of miR-126 which directly inhibits VCAM-1,15 and miR-17–3p and miR-31 which directly inhibit E-selectin and ICAM-1.16 In contrast, miR-21 combined with OSS enhances the expression of VCAM-1 and MCP-1 indirectly by targeting the anti-inflammatory transcription factor peroxisome proliferator-activated receptor-α.17 The athero-susceptible regions of the inner aortic arch are also characterized by lower miR-10a levels, enhancing the expression of IκB/NFκB-mediated inflammatory adhesion molecules and cytokines by reduced targeting of two key regulators of IκBα degradation.18 Finally, miR-155 and miR-221/222 down-regulate the transcription factor v-ets erythroblastosis virus E26 oncogene homolog-1 and its downstream inflammatory molecules (e.g. VCAM-1, MCP-1),19 and miR-181b inhibits VCAM-1 and E-selectin indirectly by targeting importin-α3, which is required for nuclear translocation of NFκB20 (Figure 3A and Table 1A).
MicroRNAs regulate key macrophage processes during the development of vulnerable plaques. (A) Endothelial dysfunction, at sites with disturbed flow dynamics, is a critical event in the pathogenesis of atherosclerosis. miRs are involved in endothelial cell activation, which is characterized by increased production of pro-inflammatory adhesion molecules (e.g. VCAM-1, ICAM-1, E-selectin) and cytokines (e.g. MCP-1). (B) Various miRs are involved in monocyte differentiation and activation of plaque macrophages. (C) Scavenger receptor-mediated uptake of lipids by macrophages together with defective cholesterol and lipid efflux in these cells leads to foam-cell formation and is controlled by miRs. (D) MiRs also control apoptosis of lesional macrophages resulting in necrotic core formation. Black arrows between miRs and mechanistic processes indicate stimulation of the process by the corresponding miR. Blunted arrows indicate inhibition. Dotted lines indicate miRs with contradictory functions described in the literature.
Infiltration of monocytes may depend on their type. In humans, chemokine (C-X3-C motif) receptor-1 (CX3CR1) is highly expressed on classical CD14++CD16− monocytes while non-classical CD14+CD16++ monocytes express low CX3CR1 levels.21 CD14++CD16− monocytes efficiently adhere to activated endothelium and preferentially accumulate in lesions.22 On the other hand, mature murine monocytes have been classified into two subsets according to their expression of Ly-6C, and the chemokine receptors chemokine (C-C motif) receptor-2 (CCR2) and CX3CR1. Classical Ly-6ChighCCR2highCX3CR1low monocytes depend on CCR2 to mobilize from bone marrow, respond to MCP-1,23 and infiltrate atheromata by CCR2, CCR5, CX3CR1, and their cognate ligands.24,25 Non-classical Ly-6ClowCCR2lowCX3CR1high monocytes patrol the endothelium to maintain homeostasis, relying on the β2-integrin LFA-1 and CX3CR1 for crawling and extravasation,26 while using CCR5 to infiltrate lesions.25
2.2 Plaque macrophages and foam-cell formation
Once infiltrated, monocytes differentiate into macrophages. Studies of monocyte subsets in mouse models of the peritoneal infection Listeria monocytogenes and myocardial infarction suggest Ly-6Chigh monocytes differentiate into macrophages and dendritic cells with a pro-inflammatory transcriptional programme, while Ly-6Clow monocytes differentiate into macrophages exhibiting activity associated with tissue repair.26,27 miRs have also emerged as critical regulators of monocyte differentiation (Figure 3B and Table 1B). miR-155, miR-222, miR-424, and miR-503 synergistically promote monocyte differentiation.28 miR-9, miR-17, miR-20a, and miR-106a inhibit monocyte differentiation.28,29 miR-146a also inhibits the development of myeloid cells.30
Macrophages are believed to exist in a dichotomy mirroring Th1 and Th2 lymphocyte subsets. In response to bacterial motifs (e.g. lipopolysaccharide) and interferon-γ, macrophages are ‘classically’ activated, also termed M1.31 They produce cytokines such as interleukin (IL)-1, IL-23, tumour necrosis factor-α (TNF-α), and toxic mediators like nitric oxide.32 LSS can up-regulate pro-inflammatory mediators such as C-reactive protein, IL-6, GRO-α (or CXCL1), and IP-10 (or CXCL10) in atherosclerotic lesions,33,34 hence LSS could initiate inflammatory processes inducing M1 macrophage polarization. OSS could also facilitate M1 polarization as it enhances RelA (p65 subunit of NFκB) and c-Jun N-terminal kinase expression,35 which activate inflammatory mediators in atherosclerosis.36 Alternative macrophage polarization (or M2) was first identified upon exposure to IL-4.37 M2 polarizing signals inhibit pro-inflammatory chemokine expression by inhibiting STAT1 and the p65 subunit of NFκB38 while the p50 subunit of NFκB regulates M2-associated gene expression.39 M2 macrophages were shown to be the first to accumulate in murine atherosclerotic lesions, while lesion progression correlates with the predominance of M1 over M2 macrophages.40 Furthermore, a recently published study of human atherosclerotic plaques demonstrated that M1 macrophages were more frequently found in rupture-prone shoulder regions of the plaque, while M2 macrophages were more common in the adventitia.41 Hence this study suggests M1 macrophages may promote plaque vulnerability. Furthermore, the activation of macrophages is controlled by miRs (Figure 3B and Table 1C). miR-146a/b and miR-147 inhibit macrophage inflammatory responses.42,43 Leucocyte-specific miR-155 deficiency reduced plaque size and the number of lesional macrophages after partial carotid ligation in apolipoprotein E knockout (ApoE−/−) mice.44 In contrast, haematopoietic miR-155 deficiency enhanced atherosclerosis and decreased plaque stability in hyperlipidaemic mice.45
Lipids are also important activators of macrophage behaviour. The CD36 and SR-A scavenger receptors enable lipid uptake by macrophages,46 which is important for clearing oxLDL, but unregulated oxLDL uptake creates lipid-loaded foam cells. miR-155 significantly reduces oxLDL-induced lipid uptake by indirectly down-regulating scavenger receptors [oxLDL (lectin-like) receptor-1, CD36 and CD68].47 Similarly, miR-125a and miR-146a inhibit lipid uptake and decrease inflammatory cytokine secretion48,49 (Figure 3C and Table 1D). Defective ATP-binding cassette-A1 (ABCA1)-mediated cholesterol and lipid efflux causes unlimited lipid accumulation in macrophages. As macrophages cannot limit cholesterol uptake, they depend on cholesterol and lipid efflux pathways to prevent transforming into foam cells.50 Interestingly, miR-33 inhibits expression of ABCA151,52 and anti-miR-33 induces ABCA1 and cholesterol removal51,53 (Figure 3C and Table 1D).
2.3 Arterial remodelling, fibrous cap thinning, and plaque necrosis
Arterial sites where developing plaques are present undergo compensatory remodelling to maintain the luminal diameter.54 LSS may be the major stimulus for arterial remodelling, however, its associated inflammation stimulates elastase activity which can promote high-risk aneurysm-like expansion of the vessel wall.55 OSS increases matrix metalloproteinase (MMP)-2 and -9 activity which could promote arterial remodelling.56 Hence extensive arterial remodelling indicates the plaque is highly vulnerable, possibly due to a continuous cycle of rupture and healing. One study showed plaques at the early stages of remodelling are more prone to rupture,57 which could explain growth spurts of clinically silent plaques. This study concluded that plaque vulnerability must be judged from a combination of fibrous cap thickness, necrotic core thickness (rather than area), and the degree of arterial remodelling.57 A long-term (3-year) study showed arterial remodelling is a stronger predictor of clinical events than plaque rupture.58 A study of porcine coronary arteries showed that LSS positively correlates with the increasing severity of plaque characteristics associated with TCFAs.59 LSS associates with elevated collagenases and elastases, fibrous cap thinning, degradation of the elastic lamina, and arterial remodelling. In contrast, OSS up-regulates arginase-2 expression and SMC proliferation60 by activating the phosphoinositide 3-kinase - protein kinase B signalling pathway.61 Other factors stimulated by LSS and plaque macrophages include extracellular matrix degrading enzymes such as MMPs and cathepsins in the fibrous cap59 generating a vulnerable TCFA (Figure 2B). Macrophage-specific deletion of MMP13 in ApoE−/− mice increases plaque collagen content, which is independent of lesion size and macrophage content,62 and is M1 specific in murine macrophages,63 supporting a role for M1 macrophages in extracellular matrix degradation and hence fibrous cap thinning. Furthermore, circulating levels of MMP9 during ACS are increased compared with controls and are associated with a higher risk of cardiovascular death.64 Despite the crucial role of macrophage-derived MMPs in plaque destabilization, little is known about miRs regulating MMP production in atherosclerotic lesions. Nonetheless, an increasing number of reports have shown that miRs modulate MMP expression directly in other chronic inflammatory diseases (for review refer to Rutnam et al.65). The role of these miRs in matrix degradation and plaque rupture remains to be determined.
Ruptured plaques also have greater macrophage infiltration and apoptosis in the fibrous cap, with apoptosis being particularly high at rupture sites correlating with the presence of caspase-1.6 The increased inflammation activated by LSS activates the Fas ligand and hence macrophage apoptosis. Interestingly, miR-15a, miR-16, and miR-155 regulate apoptosis by targeting B-cell CLL/lymphoma28,66 (Figure 3D and Table 1E). In addition, excess inflammation means phagocytic clearance of apoptotic bodies—termed efferocytosis—becomes defective forming a necrotic core due to the accumulation of necrotic debris67 (Figure 2B). Inflammatory cytokines can activate caspases and SMC apoptosis reducing the SMC content of plaques and hence increasing their vulnerability.4 As SMCs can synthesize collagen, the loss of SMCs causes further thinning of the fibrous cap, and the loss of SMCs able to inhibit macrophage apoptosis serves to increase the size of the necrotic core.68
2.4 Macrophage-associated miRs as biomarkers for plaque vulnerability
Given the importance of macrophages in vulnerable plaque development, one could expect to detect specific miR signatures in patients' blood, which can serve as novel diagnostic and prognostic biomarkers for plaque vulnerability. Indeed, miR-146a in peripheral blood mononuclear cells is significantly increased in patients with ACS.69 In contrast, miR-146b is significantly decreased in blood monocytes of morbidly obese patients without clinical symptoms of coronary artery disease (CAD).70 Because obesity is an independent risk factor for CAD,71 miR-146b could be an early biomarker for later CAD-related events. Interestingly, miR-146 is involved in the regulation of several key macrophage-associated processes during atherosclerotic plaque progression, namely monocyte differentiation, macrophage activation, and foam-cell formation (Table 1). Furthermore, miR-146 modulates the expression of MMP16 gene in glioblastoma cells by targeting its 3′-UTR,72 suggesting an additional role of miR-146 in extracellular matrix thinning in atherosclerotic plaques.
Recent studies have also shown the existence of circulating miRs in human plasma, which are stable, reproducible, and correlate with various diseases, hence plasma miRs could become novel diagnostic biomarkers73,74. Endothelial cell-related miR-17 and miR-126 and inflammatory cell-related miR-155 are significantly down-regulated in plasma samples of patients with CAD.75 Interestingly, miR-155 functionally regulates monocyte infiltration and maturation, macrophage activation, foam-cell formation, and apoptosis in atherosclerotic lesions (Table 1), but represses MMP3 in synovial fibroblasts in rheumatoid arthritis.76 Plasma levels of miR-126, on the other hand, are already lower in patients with type 2 diabetes,77 suggesting its use as an early biomarker.
2.5 Modelling shear-stress-dependent vulnerable plaque development in mice
Cheng et al. developed a perivascular cast, which slows blood flow upstream of the cast creating LSS, HSS within the cast and OSS downstream.78 Initial studies using this cast showed that plaques developing in the LSS-modulated region of the carotid artery are larger with a high content of lipids, macrophages, larger necrotic cores, increased MMP activity, greater vascular remodelling, and intraplaque haemorrhage.33,79 LSS-modulated plaques up-regulate VCAM-1, ICAM-1, and the pro-inflammatory mediators C-reactive protein, IL-6, and vascular endothelial growth factor.33 In contrast, plaques developing in the OSS-modulated region have a much higher content of SMCs and collagen and lower expression of inflammatory mediators.33 Hence, this model is a valuable tool to study the development of stable and vulnerable atherosclerotic plaques simultaneously.
3. The relation between biomechanical stress and plaque rupture
3.1 Increasing stenosis modifies local flow patterns and inflammation
When arterial remodelling is exhausted, constrictive remodelling causes protrusion of the advanced plaque into the lumen creating a stenosis, which modifies surrounding shear-stress patterns. The surface of the lesion proximal to the point of maximal stenosis is subjected to HSS and high strain while the surface of the distal segment experiences LSS and OSS80 (Figure 4). Spatial localization of certain morphological features has been observed in advanced plaques.
Changes in biomechanical stress and plaque rupture. An exhaustion of compensatory remodelling causes the expanding plaque to change the local flow patterns. Hence, low and oscillatory shear stress only affects the distal portion of the plaque while fast linear blood flow from the common carotid artery imposes HSS and high strain on the fibrous cap. Greater smooth muscle cell proliferation and collagen synthesis in the distal segment stabilizes this region of the plaque. In contrast, inflammation is up-regulated in the upstream segment increasing macrophage accumulation. Therefore, HSS and strain at the upstream segment of the plaque are ultimate determinants of plaque rupture, which exposes the pro-thrombotic necrotic core to circulating coagulation factors activating the formation of a thrombus.
The upstream segment of plaques found at the carotid bifurcation have a higher expression of VCAM-1, greater monocyte adhesion, macrophage accumulation and apoptosis, intraplaque haemorrhage, thinner fibrous caps, and a greater incidence of plaque rupture and thrombosis.81 Plaque growth is more persistent in the distal region, which features higher SMC content81 (Figure 4). LSS also increases endothelin production, which stimulates SMC proliferation and their synthesis of extracellular matrix proteins.82 However, the distal segment is susceptible to thrombosis via endothelial erosion as this region displays higher luminal endothelial cell apoptosis.83
Mechanistically, monocyte recruitment and macrophages may be the primary mediators in developing plaque vulnerability in the upstream segment as the chemokines CCR2 and CCR6, MCP-1, and macrophage inflammatory protein-3α are all elevated in the upstream segment and in close association with areas of dense neovascularization,84 suggesting leucocytes may be recruited to the intima via microvessels. HSS can stimulate aggregation and activation of platelets, which release platelet-derived microparticles containing adhesion molecules such as P-selectin, up-regulating adhesion molecule expression by endothelial cells and monocytes.85 Hence, platelet-derived microparticles may enhance monocyte recruitment to the upstream segment. In addition, the upstream segment possesses increased T cells and dendritic cells (DCs).84 The majority of DCs in the upstream segment exhibit a mature phenotype expressing high levels of CD83, lysosomal-associated membrane protein-3,86 and HLA-DR, and are in frequent contact with T cells found in rupture-prone regions of the plaques,86 indicating DCs may contribute to destabilization of plaques by activating T cells. Furthermore, gelatinase activity and oxLDL accumulation is higher in the upstream segment,87 both in foamy macrophages and SMCs. MMPs—namely MMP2 and MMP9—may be primarily responsible for gelatinase activity in these plaques, breaking down extracellular matrix and collagen, thus thinning the fibrous cap.88
3.2 HSS and wall strain as ultimate determinants of plaque rupture
The arterial wall has to withstand various stresses and strains as a result of flowing blood. An ongoing debate exists on which blood flow conditions lead to plaque rupture. Some argue that HSS is responsible while evidence is now emerging that plaque rupture is associated with high mechanical strain in the fibrous cap. The main stresses experienced by the arterial wall include shear stress and wall (mechanical or tensile) stress where pulsatile blood flow circumferentially distends the arterial wall. With no blood pressure, a residual stress remains whose state depends on the thickness and composition of the arterial wall. A larger lipid core compresses against the more compliant fibrous cap increasing residual stress on the cap, which is highest at the thinnest points of the cap. Some areas of high residual stress at the rear of the lipid core co-localize with macrophages.89 This study proposes plaque rupture results from a balance between residual stress and external pressures from blood flow.89
In patients with CAD, arterial regions exposed to HSS co-localize with macrophages, rupture sites, nitric oxide, MMPs (primarily MMP9),90 and higher shear strain in the upstream region suggesting a mechanism whereby HSS increases strain and mechanically weakens the fibrous cap destabilizing the plaque (Figure 4). An MRI study of the human carotid bifurcation showed that while HSS associates with plaque rupture, the position of the stenosis where plaque wall stress is highest is a much better predictor of plaque rupture sites,91 tending to be in the upstream segment of these plaques. Symptomatic patients have much thinner fibrous caps and larger lipid cores, with plaque wall stress levels also much higher than asymptomatic patients, particularly at the shoulders of the lipid core,92 the highest risk site of plaque rupture. An irregular lumen shape is also associated with carotid plaque instability, where wall stress concentrates at the greatest curvature of the lumen increasing the risk of plaque rupture.93
Table 2 summarizes the discussed studies on inflammation in response to shear stress.
It is clear from these studies that there is a complex interaction between various biomechanical stresses and strains determining plaque vulnerability and the risk of rupture. LSS is a strong contributor to all the main features of vulnerable plaques. The mechanisms through which shear stress induces biological changes in the vasculature are complex and act (i) at the transcriptional level, by regulating pro-inflammatory and regulatory signalling pathways and (ii) at the post-transcriptional level through changes in miR expression patterns. However, after protrusion of the advanced plaque, HSS at the upstream segment of the plaque is associated with plaque rupture.
Existing methods used to study biomechanical stress in relation to vulnerable plaque development leave much to be desired. In silico modelling of haemodynamics gives a useful insight into the mechanical stresses experienced by the arterial wall, however, the results are merely predicted from arterial geometry as we currently do not have adequate tools to measure blood flow mechanics in vivo. Techniques used to image atherosclerotic plaques in vivo—particularly with the use of MRI imaging and two-photon excitation microscopy—enable the characterization of plaque morphology, inflammation, thrombus formation, the study of live recruitment, and ultimately the identification of potential biomarkers for diagnostic purposes.94,95 However, these developments have yet to be translated to the clinical setting and do not enable the study of biomechanics in relation to the development of atherosclerotic plaque vulnerability. Despite the development of a mouse model to study stable and vulnerable atherosclerotic plaques simultaneously, adequate mouse models to study plaque rupture are lacking. Therefore, more work is needed to develop mouse models, which can be used to extend our in vivo knowledge of macrophage-associated transcriptional programs leading to plaque rupture. Questions have also been raised on the definition and origin of myeloid cell subsets in recent studies.96 How these discoveries affect our understanding of pathogenic events leading to atherosclerosis and its complications is still uncertain. In addition, the majority of shear-stress-induced miR expression profiles have been generated in simplified in vitro experiments where cultured endothelial cells are exposed to different shear-stress regimes. Hence, the question arises to what extent these in vitro findings can be translated to the in vivo situation where endothelial cells are exposed to multiple mechanical and biological factors. Finally, further studies to determine the diagnostic and prognostic value of (i) deregulated miRs in circulating monocytes that can mirror plaque macrophage activation and (ii) circulating plasma miRs secreted by activated monocytes/macrophages aimed at identifying plaques at high risk of rupture before a serious cardiovascular event ensues, are warranted.
Conflict of interest: none declared.
A.S. is supported by a PhD studentship from the British Heart Foundation of Research Excellence, Imperial College London. M.H. is supported by a post-doctoral fellowship from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. P.H. has received funding from: Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (G.0548.08, G0846.11, and Vascular Biology Network), and by Interdisciplinair Ontwikkelingsfonds - Kennisplatform (KP/12/009). C.M. has received funding from: the British Heart Foundation; European Commission under the 6th Framework Programme through the SME call for ‘Life sciences, genomics and biotechnology for health’ (Contract number: LSHM-CT-2006-037400); European Collaborative Project on Inflammation and Vascular Wall Remodelling in Atherosclerosis Acronym: AtheroRemo; EU - HEALTH-2007-2.4.2-1. 2008; the Graham-Dixon Charitable Trust; the Kennedy Trustees. The Kennedy Institute of Rheumatology is funded by the Arthritis Research Campaign UK.
The article is part of the Spotlight Issue on: Biomechanical Factors in Cardiovascular Disease.
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