Proteinases and plaque rupture: unblocking the road to translation.

REVIEW URRENT C OPINION

Proteinases and plaque rupture: unblocking the road to translation Andrew C. Newby

Purpose of review To review progress over the past 5 years in relating extracellular proteinases to plaque rupture, the cause of most myocardial infarctions, and consider the most promising prospects for developing related treatments. Recent findings Cysteinyl cathepsins have been implicated in multiple macrophage functions that could promote plaque rupture. Cathepsin K is an attractive target because it is a collagenase and selective inhibitors are already being used in phase III clinical trials. Several serine proteinases clearly influence vascular remodelling and atherogenesis but important, unrelated actions limit their value as therapeutic targets. Among the metalloproteinases, new evidence supports roles for A Disintigrin and Metalloproteinases (ADAMs), including ADAM-10, ADAM-17 and ADAM-33, which suggest that selective inhibitors might be effective treatments. For ADAMs with ThromboSpondin domains (ADAMTSs), there are biological and genome-wide association data linking ADAMTS-7 to incidence of coronary heart disease but not increased risk of myocardial infarctions. In the case of matrix metalloproteinases (MMPs), selective inhibitors of MMP-12 and MMP-13 are available and may be appropriate for development as therapies. Novel targets, including MMP-8, MMP-10, MMP-14, MMP-19, MMP-25 and MMP-28, are also being considered. Summary New opportunities exist to exploit proteinases as therapeutic targets in plaque rupture. Keywords atherosclerosis, cathepsins, metalloproteinases, plaque rupture, serine proteinases

INTRODUCTION: THE EXTRACELLULAR MATRIX IN VASCULAR REMODELLING AND PLAQUE RUPTURE Extracellular proteinases were first implicated more than 20 years ago in vessel wall remodelling leading to intimal thickening. Studies soon followed suggesting that proteinases might have a contradictory role in advanced atherosclerosis, causing net destruction of the arterial extracellular matrix (ECM) leading to thinning and weakening of the plaque cap. Persuasive evidence was presented that proteinases might mediate plaque rupture, which leads to thrombus formation and underlies perhaps three-quarters of myocardial infarctions (MIs) and also many thrombotic strokes. Efforts are still going on to elucidate the basis for the opposing roles of proteinases in intima formation and plaque rupture. The objective is to develop new therapies to reduce the incidence of MI, which is the leading cause of death in developed countries. www.co-lipidology.com

As summarized in Fig. 1a, the normal vessel wall consists of an endothelium lying on a thin basement membrane rich in type IV collagen, laminin and heparan sulphate proteoglycans. Medial vascular smooth muscle cells (VSMCs) are also surrounded by a similar basement membrane. Between them the more abundant ECM is dominated by fibrillar type I and III collagens (CI, CIII), a multiplicity of glycoproteins and chondroitin or dermatan sulphate proteoglycans [1]. Elastin fibres are also prominent in many larger arteries that suffer from atherosclerosis. During vascular remodelling (Fig. 1b), VSMCs tend to lose their basement membranes [2], a process that University of Bristol and Bristol Heart Institute, Bristol, UK Correspondence to Prof Andrew C. Newby, Bristol Heart Institute, Research and Teaching Floor Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK. Tel: +44 1173423583; fax: +44 1179299737; e-mail: [email protected] Curr Opin Lipidol 2014, 25:358–366 DOI:10.1097/MOL.0000000000000111 Volume 25 Number 5 October 2014

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Proteinases and plaque rupture Newby

KEY POINTS Cysteinyl cathepsins, serine proteinases and metalloproteinases have all been implicated not only in intima formation but also in ECM destruction that could lead to plaque rupture. These opposing roles complicate the choice of drug targets for reducing plaque ruptures and hence MIs and strokes. Recent basic science identifies cathepsin K, neutrophil elastase, ADAMTS-7, MMP-12 and MMP-13 as favourable drug targets. Other proteases, including MMP-8, MMP-10, MMP-14, MMP-19 and MMP-25 merit further investigation.

requires the redundant action of more than one class of extracellular proteinase [3]. Matrix degradation removes barriers, whereas an altered pattern of cell surface integrins (Int) leads to new binding and signalling interactions that facilitate VSMC migration [4] and proliferation [5]. Cell-to-cell contacts mediated by cadherins (CAD) also need to be disassembled [6] and gap junctions may be dissociated into connexon hemi-channels (Cx) [7]. During vascular remodelling, VSMCs increase production of ECM components, and so there is no loss of vessel wall integrity but rather fibrosis [2]. In atherosclerosis by contrast (Fig. 1c), production of a greater range of proteinases at higher levels from invading macrophages as well as by overstimulation of resident VSMCs and endothelial cells promotes net destruction of structural ECM components [8], importantly collagens that provide most of the tensile strength [9]. Consistent with this, plaque rupture is associated with abundant foam cell macrophages and less collagen [9,10]. Ruptured plaques tend to have a large necrotic core, resulting from foam cell apoptosis, and relatively few viable VSMCs thereby reducing production of ECM. A thin fibrous cap and greater transference of tensile stress during the cardiac cycle because the large core is not braced by collagen can combine to cause plaque rupture. Some proteinases undoubtedly degrade ECM components directly or participate in activation cascades that lead to ECM degradation. However, many nonmatrix substrates are also cleaved as can be readily detected by proteomics [11]. Most importantly, proteinases can activate, inactivate or release cell-surface or sequestered mediators that may indirectly influence migration, proliferation and apoptosis of vascular cells. They may also have completely different functions, such as cathepsins in bacterial killing, the plasmin system in fibrinolysis and thrombin in coagulation and cell

signalling. The large number of neutral proteinases and their multiple effects complicate both basic understanding and the choice of potential drug targets to treat human plaque rupture.

RECENT INSIGHTS RELATING PROTEINASES TO PLAQUE RUPTURE Evidence for participation of proteinases has been based firstly on in-vitro expression and functional studies with vascular cells, especially macrophages and macrophage foam cells that are believed to provoke plaque rupture. To provide direct evidence in human, ex-vivo analysis of plaque tissues has been used to relate the presence of proteinases to histological features including large lipid core sizes, thin fibrous caps, abundance of macrophages and paucity of VSMCs as surrogates for plaque vulnerability. To gain further insight, many studies have relied on interventions in animals, although no model of plaque rupture has gained unequivocal acceptance [12]. Spontaneous plaque ruptures have been described in fat-fed ApoE mice [13], but their appearance is so different from that in humans that different mechanisms could well be involved [14]. Formation in the brachiocephalic artery of multilayered plaques that contain fibrin has been interpreted as evidence that they arise from past plaque ruptures [13], but this is also controversial [14]. Other mouse models have been developed that use extreme systemic or genetic interventions to increase the rate of plaque disruption [12]. Whether these replicate the human disease accurately and are therefore highly predictive when used for intervention studies is yet to be established. In the absence of plaque ruptures, most animal studies rely on the same histological surrogates used in human plaques: bigger plaques, larger lipid cores, less collagen, an increased ratio of macrophages to VSMCs and thinner plaque caps. Amongst these, low numbers of VSMCs and thin fibrous caps are the most worrisome because these could simply indicate less advanced plaques. All of these limitations need to be borne in mind when considering the effects of genetic manipulation of proteinases that have been reported. Another issue that is only rarely addressed is the different complement of proteases expressed by mice and men. For example, the substitution of matrix metalloproteinase (MMP)-13 for MMP-1 in mice compared with humans has been known for decades [15] and other examples are emerging (see below).

CATHEPSINS Proteinases with a neutral pH optimum are most likely to be active in the ECM. Hence, of the 11

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Atherosclerosis: cell biology and lipoproteins

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FIGURE 1. Vessel wall structure and proteolysis. (a) Endothelial cells sit on a thin basement membrane. Medial vascular smooth muscle cells (VSMCs) are also surrounded by a similar basement membrane connected to the cells by integrins (Int). Between them are elastin fibres, fibrillar collagens (CI, CIII), glycoproteins (GPs) and proteoglycans (not shown). Cadherins (CAD) and gap junctions also connect the cells. (b) Intima formation requires VSMCs to fragment or lose their basement membranes, cleave cadherins (CAD) and dissociate gap junctions into connexin hemi-channels (Cx) and alter the pattern of cell surface integrins (Int). Collagens and elastic fibres may be partially fragmented but are replaced by increased synthesis. (c) Atherosclerosis involves the recruitment of inflammatory macrophages (MF) and foam cells that promote extensive destruction of all extracellular matrix components, most importantly collagens, that provide most of the tensile strength. Apoptosis of VSMCs reduces production of new matrix components. Together, these contribute to plaque rupture.

cysteinyl cathepsins, only cathepsins B, K, L and S have been implicated in vascular biology and disease [16,17]. Although primarily active within intracellular vesicles, cathepsins are also secreted and can be associated with the cell surface by binding to specific receptors [17]. Expression of cathepsins is strongly related to proinflammatory activation of 360


macrophages [17]. The enzymes exert multiple effects on macrophages, VSMCs, endothelial cells and lipoproteins that could favour or ameliorate aspects of atherosclerosis progression and instability [17]. It has been known for some years that deletion of cathepsins S, K and L reduces plaque formation and complexity in mice, whereas deletion of Volume 25 Number 5 October 2014



cystatin C has the opposite effects [17]. This evidence points to a role in plaque rupture. However, counteracting effects of cathepsin S in promoting neointima formation as well as inflammation are well illustrated in a very recent study in mouse vein grafts [18]. Recent work also highlights possible influences of cathepsins on macrophage apoptosis, which can lead to proinflammatory secondary necrosis in the presence of defective efferocytosis. Conversely, autophagy can protect against apoptosis. Cathepsin L has been recognized as a mediator of apoptosis for some years [17]. Two discordant studies observed either inhibition [19] or stimulation [20] of autophagy by cathepsin L in mouse macrophages, which suggests that the effect may depend on the context of other activators. Recently, cathepsin G (a serine protease) was found to have adverse effects on mouse plaques possibly mediated by inhibition of efferocytosis of apoptotic cells leading to inflammation [21]. Studies also implicated both cathepsin B and L in cholesterol-crystal-induced inflammasome activation and interleukin-1a production in foam cell macrophages [22,23]. Looking toward clinical translation, Samokhin et al. [24] reported the beneficial effects of a selective cathepsin S inhibitor on plaque development and unstable histological features in mice. However, the role cathepsin S in antigen-presentation might frustrate the use of such inhibitors in humans [24]. Cathepsin K is a more attractive target because selective inhibitors are already showing promising effects in phase III clinical trials for osteoporosis [25]. Cathepsin K degrades collagen, the main strength-giving component of plaque caps, with a distinct cleavage pattern compared with the MMP collagenases [25]. A caveat may be the increased lipid uptake into foam cells that was observed in mouse knockout studies [26]. In a different approach, Shon et al. [27] took advantage of the accumulation of cathepsin B-positive macrophages in plaques to design a photodynamic therapy based on a cathepsin-B-activated theranostic compound that was activated after ultraviolet irradiation of mouse lesions. This type of photodynamic therapy might in principle be used as a local, invasive plaque stabilizing treatment in humans.

SERINE PROTEINASES Neutral serine proteinases with established roles in vascular biology include plasmin and plasminogen activators [28], thrombin, kallikrein [29], neutrophil elastase [30] and mast cell-derived chymase and tryptase [31,32]. Protective effects of various serine

protease inhibitors (SERPINs) in atherosclerosis models have provided an important part of the evidence for their participation. Recent protein expression studies in human plaques of SERPINA3 that targets elastase, cathepsin G and chymase confirm this concept [33]. The data were strengthened by studies showing preservation of elastin in plaques from mice overexpressing SERPINA3.

Plasmin system Plasmin and plasminogen activators were amongst the first proteases to be implicated in intima formation [34], but they also have profound effects on macrophage invasion and other functions linked to atherogenesis [28]. Not surprisingly, therefore, diverse overall effects on plaque development and stability have emerged from animal models [28]. Recent work shows that urokinase-type plasminogen activator expression specifically in macrophages increases atherosclerosis in two mouse models, at least in part by promoting S100A8/A9 expression [35]. Furthermore, studies have revealed that plasminogen/plasmin can promote foam cell formation by stimulating leukotriene B4 production and thereby increasing the expression of the scavenger receptor CD36 [36]. Although these investigations add to the justification for intervening, the prominent role of the plasmin system in fibrinolysis probably rules this out.

Human neutrophil elastase Interest in this protease as a target in atherosclerosis [30] was sparked by its detection in human plaque macrophages more than a decade ago [37]. Enthusiasm has been dampened, however, by the abandonment of clinical trials in acute lung injury, wherein the damaging effect of neutrophils is much clearer. Both lack of efficacy [38] and the possibility of side-effects that masked an effect on overall mortality have discouraged application of this therapy outside Japan [39]. Development of new classes of HNE inhibitors [40,41] may reopen this therapeutic avenue. A closely related elastase, PR3, could be an alternative target [42].

Chymase Mast cell chymase and tryptase have been implicated in atherogenesis, in part by their ability to activate MMPs. Consistent with this, Guo et al. [43 ] showed that lentivirus-mediated knockdown of chymase using small interfering RNA reduced MMP-9 pro-form activation and ameliorated a histologically defined ‘vulnerability index’ of plaques


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in a hamster model. Chymase, like several other extracellular proteinases, could also exert atherosclerosis-promoting effects by modifying lipoprotein particles [44].

METALLOPROTEINASES The metalloproteinases contain a Zn2þ ion at the catalytic site, and become active after cleavage of their pro-domain, in some cases within endosomes or by action of other extracellular proteinases. Of the 40 structurally related, cell surface A Disintegrin And Metalloproteinase (ADAM) proteins, only ADAMs 8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30 and 33 are catalytically active. By contrast, all of the 20 or so ADAMs with ThromboSpondin domains (ADAMTSs) are secreted enzymes. There are also at least 23 MMP enzymes, among which there are six cell surface-associated membrane-type MMPs (MTMMPs).

A disintegrin and metalloproteinases Evidence for the participation of ADAMs in atherogenesis through their functions as sheddases has been exhaustively reviewed recently [45]. ADAM-17 was the first to be implicated because of its role in releasing active tumour necrosis factor a. Furthermore, a protective effect against atherosclerosis is evident in ADAM-17 null mice. Recent literature expands the list of relevant substrates of ADAM-17, one particularly interesting example being Mer-TK, a receptor prominently involved in efferocytosis. ADAM-17 cleaves MerTK from inflammatory macrophages rendering them less effective at clearing apoptotic cells [46] and could therefore amplify plaque inflammation. Other recent literature provides human plaque expression and cell biological data supporting proinflammatory roles for ADAMs 8, 9, 10, 15 and 33 [45]. Further studies add genetic evidence implicating ADAM-8 [47] and ADAM-33 [48]. Interestingly, animal experiments indicate that ADAM-15 is protective against plaque instability [49] and the stimulatory effects of ADAM-12 on VSMC proliferation might also stabilize plaques [50]. Selective inhibition of specific ADAMs might therefore be needed to prevent plaque rupture in humans.

A disintegrin and metalloproteinases with ThromboSpondin domains ADAMTSs are relative newcomers to the list of proteinases implicated in atherosclerosis. In their review, Salter et al. [51] pointed out the potential importance for atherogenesis of proteoglycan 362


(especially versican) degradation by ADAMTSs. They also summarize expression and cell biology studies linking ADAMTS 1, 2, 4, 5, 7, 8 and 10 to inflammation and the biology of VSMCs and endothelial cells. The same group went on to clarify the regulation of ADAMTS-4 by proinflammatory and antiinflammatory pathways in human macrophages [52]. ADAMTS-7 is an interesting recent subject because there are both experimental and genetic data to support its importance. Initial experimental studies showed that small interfering RNA (si-RNA)mediated inhibition of ADAMTS-7 reduced proteolysis of its substrate thrombospondin-5 (also called COMP) and impaired VSMC migration in culture and intima formation in the rat carotid balloon injury model [53]. COMP was also shown to suppress phenotypic modulation of VSMC, also placing a brake on neointima formation [54]. It was subsequently shown that genetic variants of ADAMTS-7 are associated with incidence of symptomatic CHD but not MI in genome-wide association studies [55]. Interestingly, a protective allele of another ADAMTS-7 variant (rs3825807) was shown to confer reduced ability to become activated and to support less thrombospondin-5 cleavage and VSMC migration in culture [56 ]. This suggests that accumulation of VSMCs mediated by ADAMTS-7 promotes plaque growth and therefore CHD. However, more VSMC accumulation might be expected to increase cap thickness and protect plaques from rupture and hence MI. If the extrapolation is valid, a pharmacological inhibitor of ADAMTS-7 might be expected to give smaller plaques and reduce CHD but not MIs. A separate study showed association between ADAMTS-7 variant rs3825807 and aortic calcification [57 ], which is consistent with earlier data establishing a role for COMP in vascular calcification [58]. &&

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Matrix metalloproteinases Both adverse and protective effects of individual MMPs on plaque rupture have been deduced from animal models [59]. Moreover, broad-spectrum MMP inhibitors showed no effect in animal models, most likely because of the opposing actions of different MMPs [59]. Plaque rupture is difficult to quantify clinically, except by invasive imaging techniques such as intravascular ultrasound and optical coherence tomography, but its occurrence can be implied from the rates of clinical events. Using this approach, MMP-8 protein [60] and MMP-12-positive macrophages [61] measured in carotid atherosclerotic plaques ex vivo were shown to predict high incidence of adverse events following carotid Volume 25 Number 5 October 2014



endarterectomy. Furthermore, the abundance of MMP-8 or MMP-9 protein and MMP-12-positive macrophages was correlated with histological features of vulnerable plaques in the same material, whereas MMP-2 showed an inverse correlation [60–62]. Clinical trials with nonselective MMP inhibitors have so far been frustrated by a narrow therapeutic window [63], although pivotal trials of doxycycline, a pleiotropic agent that reduces expression and activity of several MMPs, are under way in the context of abdominal aortic aneurysms. Some problems might arise from off target effects and hence new chemistries to produce low-molecularweight MMP inhibitors are being pursued [64]. An alternative strategy is to define and target individual MMPs in the hope of maximizing therapeutic benefit and avoiding unwanted side-effects. A recent review highlighted the importance of collagenases in plaque rupture and pointed out that MMP-1, MMP-8 and MMP-13 [as well as MMP-14 (MT1-MMP) [15]] have the ability to degrade fibrillar collagen [9]. Human plaques express all four collagenase MMPs at their vulnerable shoulder regions [9,65]. MMP-1 is generally considered to be the predominant collagenase in human plaques, although the quantitative evidence for this is lacking. The wide distribution of MMP-1 in human tissues and its supposed role in physiological remodelling, for example in tendons, reduce its attractiveness as a drug target. The musculoskeletal syndrome, a dose-limiting side-effect of treatment with broad-spectrum MMP inhibitors, is thought to be at least in part owing to inhibition of MMP-1 [63]. Lenglet et al. [66] have argued that MMP-8, initially identified as neutrophil collagenase, is a good target to prevent plaque rupture based on its previously mentioned relationship to plaque instability in human carotid plaques [60,62]. There is also an excess risk associated with high circulating MMP-8 levels and specific gene polymorphisms [66], although Mendelian randomization studies would be welcome to establish causality. Laxton et al. [67] showed that MMP-8 knockout in ApoE null mice profoundly decreased atherosclerosis and improved the macrophage to VSMC ratio in plaques. Less atherosclerosis depended on reduced processing of angiotensin I to angiotensin II in MMP-8 null mice and also reduced recruitment of stem-like cells by an MMP-8, ADAM-10 cascade that promotes cadherin shedding [68 ]. However, the same MMP-8, ADAM-10 cascade also increases proliferation and migration of mouse VSMCs and promotes neointima formation [69 ]. MMP-8 is therefore another good example of the opposing role of proteinases in intima formation and plaque rupture. &&

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To add further uncertainty, Quillard et al. [70 ] very recently published contradictory findings that MMP-8 knockout produced no effect on atherosclerosis in ostensibly the same ApoE null mouse model. By contrast, Quillard et al. [70 ] showed that MMP-13 knockout significantly increased the collagen content of mouse plaques without affecting plaque size, in close agreement with their previous study [71] using a selective MMP-13 inhibitor. Triple knockouts of MMP-8, MMP-13 and ApoE had no additional phenotypes compared with MMP-13 ApoE double knockouts [70 ], which implies that MMP-13 is a more important collagenase than MMP-8 in mice [70 ]. The relevance of these findings to humans, which also express MMP-1, was addressed in their previous study [71] by showing that a selective MMP-13 inhibitor with no activity against MMP-1 or MMP-8 significantly reduced collagenase activity in human plaques using in-situ zymography. Although based on relatively small numbers of plaques (n ¼ 5), these data suggest that MMP-13 may be a significant collagenase in human plaques, consistent with previous histological findings [72]. Oddly, however, two independent in-vitro studies failed to detect MMP-13 mRNA in human macrophages [73,74] and so the source and stimuli for its production remains to be clarified. It is possible that plaque macrophages are a reservoir for MMP-13 synthesized by other vascular cells or that the complex microenvironment of the plaque is necessary to activate its production from macrophages. Otherwise, active secretion of MMP-13 seems to be restricted to human chondrocytes and certain tumours. Indeed circulating levels of MMP-13 are undetectable, in contrast to MMP-8 [75]. The membrane type-1 MMP, MMP-14, which mediates migration of both macrophages and VSMC, is upregulated under proinflammatory conditions [73]. It occurs in a subpopulation of plaque foam cells [65] the significance of which deserves further definition. Recent studies establish that GM-CSF can increase MMP-14 protein levels and activity in macrophages and foam cells by decreasing expression of micro-RNA-24 [76]. MMP-12 was initially described as a metalloelastase, although it has other defined substrates [15]. Johnson et al. showed that MMP-12 knockout reduced [77] and treatment of established plaques with a selective MMP-12 inhibitor [78] arrested plaque development in fat-fed ApoE null mice and ameliorated several surrogates of plaque instability including calcification. MMP-12 is more widespread histologically in mouse [78] than human [61] plaques, where it seems to be confined


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to deep macrophages surrounding the lipid core. Nevertheless, evidence that MMP-12 predicts adverse clinical events after endarterectomy [61] makes it an attractive drug target, and clinically applicable selective MMP-9/MMP-12 inhibitors have already proved well tolerated in clinical trials for chronic obstructive pulmonary disease [79]. More work is needed to define surrogate markers of efficacy for MMP-12 inhibitors, perhaps using imaging of MMP activity [80] before considering a clinical outcome trial in cardiovascular disease. Recent system-wide studies of MMPs and tissue inhibitors of MMPs (TIMPs) in human monocytes [81] and macrophages [73] provide a list of additional MMP candidates that have not been fully investigated. For example, MMP-10, which is upregulated in human macrophages under proinflammatory ‘M1’ conditions in vitro and in plaques [73], has been previously highlighted as a possible player in vascular remodelling [82]. It has also been shown to exert several effects on endothelial cells [83]. Detailed studies of its role in atherosclerosis and plaque rupture would be welcome. MMP-19 may also be a valid additional candidate for study based on relatively robust expression human macrophages [73] and in inflamed human arterial tissue [84]. MMP-25 (named leukolysin because high expression occurs selectively in leukocytes [85]) is also interesting, especially given the upregulation in human macrophages under M1 conditions [73]. MMP-28 (epilysin) appears to be expressed at very low levels in human macrophages [74]. Nevertheless, it has multiple roles in inflammatory mediator production, macrophage polarization and ECM production that on balance protected against ventricular rupture after MI in mice [86 ]. The effects on atherosclerosis formation and plaque rupture are therefore worthy of investigation, especially if the relevance to human disease can be established. Beneficial effects of systemic TIMP-1 and TIMP-2 gene therapy on plaque growth and implied stability were reported several years ago and confirmed recently for TIMP-1 in vein grafts [87]. TIMP-3 overexpression was also shown recently to reduce mouse atherosclerosis and the authors suggest this may be owing to inhibition of ADAM-17 as well as MMPs [88]. Interestingly, TIMP-3 reduces the inflammatory phenotype in mouse macrophages [89] and TIMP-3 expression is relatively increased in alternative compared to classically activated human macrophages [73]. TIMP-3 is expressed in only a subpopulation of human macrophages [65] and clinical studies relating TIMP-3 expression to plaque morphology and clinical outcomes would be welcome. &

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CONCLUSION AND CLINICAL PERSPECTIVE Cysteinyl cathepsins have multiple functions in plaque inflammation, but therapeutic targeting could be limited by their roles in other processes, including resistance to infection. Despite this, cathepsin K inhibitors have been used safely in elderly patients to prevent osteoporosis and future observational data might encourage controlled clinical trials in cardiovascular disease. Several serine proteinases clearly influence vascular biology and atherogenesis but feasible drug targets without effects on other biological processes are so far elusive. Neutrophil elastase may merit renewed interest. ADAMs and ADAMTSs have effects on both intima formation and by implication plaque cap development as well as surrogates of plaque rupture in mice. In future, individual ADAMs might need to be selectively targeted. Furthermore, inhibition of a subgroup of ADAMs and ADAMTSs might contribute to the beneficial effects of TIMP-1 [90] and TIMP-3 [91] gene therapy. For ADAMTS-7, there are genome-wide association data confirming its functional role in coronary heart disease but not MI, the basis for which requires further elucidation. The MMP system continues to stimulate pharmaceutical interest. MMP-13 appears an attractive drug target because human pathological findings and rodent experiments combine to imply a role in the destruction of collagen [9]. The availability of orally active selective inhibitors is a further reason for optimism and the restricted tissue distribution of MMP-13 could further recommend it because sideeffects should be few. However, definitive experiments to establish the relative importance of MMP-13 compared with MMP-1 in human plaques and to elucidate the mechanisms underlying MMP-13 production in plaque cells, perhaps using the methodology developed by Monaco and colleagues [92], seem warranted before progressing to clinical trials. MMP-12 is relatively amenable to development of selective low-molecular-weight inhibitors and could be a valid target if the activity in human plaques is sufficient to influence plaque rupture directly. Further efforts to establish causality for MMP-12 should be undertaken. There remain several other candidates, including MMP-10, MMP-14, MMP-19 and MMP-25, which urgently need to be investigated. In summary, efforts to develop an effective antiproteinase therapy for plaque rupture are far from exhausted and further exciting developments, perhaps including manipulation of miRNAs, seem likely. Acknowledgements None. Volume 25 Number 5 October 2014



Conflicts of interest The author receives research grants from the British Heart Foundation and the National Institute for Health Research.

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Neutrophil elastase in human atherosclerotic plaques: production by macrophages. Circulation 2003; 107:2829–2836. 38. Iwata K, Doi A, Ohji G, et al. Effect of neutrophil elastase inhibitor (sivelestat sodium) in the treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS): a systematic review and meta-analysis. Intern Med 2010; 49:2423–2432. 39. Lucas SD, Costa E, Guedes RC, Moreira R. Targeting COPD: advances on low-molecular-weight inhibitors of human neutrophil elastase. Med Res Rev 2013; 33 (Suppl 1):E73–E101. 40. Feng L, Liu X, Zhu W, et al. Inhibition of human neutrophil elastase by pentacyclic triterpenes. PloS One 2013; 8:e82794. 41. Madsen JL, Andersen TL, Santamaria S, et al. Synthesis and evaluation of silanediols as highly selective uncompetitive inhibitors of human neutrophil elastase. J Med Chem 2012; 55:7900–7908. 42. Korkmaz B, Jenne DE, Gauthier F. Relevance of the mouse model as a therapeutic approach for neutrophil proteinase 3-associated human diseases. Int Immunopharmacol 2013; 17:1198–1205. 43. Guo T, Wang J, Yang J, et al. Lentivirus-mediated RNA interference of & chymase increases the plaque stability in atherosclerosis in vivo. Exp Mol Pathol 2013; 95:51–56. The study uses an unusual hamster model in which mast cells were presensitized to demonstrate the effect of lentivirus-delivered small interfering RNA against chymase on plaque morphology. The results show an intriguing reduction in plaque vulnerability index that was associated with less pro-MMP-9 activation. A more detailed protocol for the delivery of the virus would have been useful. 44. Plihtari R, Hurt-Camejo E, Oorni K, Kovanen PT. Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase. J Lipid Res 2010; 51:1801–1809. 45. van der Vorst EP, Keijbeck AA, de Winther MP, Donners MM. A disintegrin and metalloproteases: molecular scissors in angiogenesis, inflammation and atherosclerosis. Atherosclerosis 2012; 224:302–308. 46. Thorp E, Vaisar T, Subramanian M, et al. Shedding of the MER tyrosine kinase receptor is mediated by ADAM17 through a pathway involving reactive oxygen species, protein kinase {delta}, and P38 map kinase. J Biol Chem 2011; 286:33335–33344. 47. Raitoharju E, Seppala I, Levula M, et al. Common variation in the ADAM8 gene affects serum sADAM8 concentrations and the risk of myocardial infarction in two independent cohorts. Atherosclerosis 2011; 218:127–133. 48. Figarska SM, Vonk JM, van Diemen CC, et al. ADAM33 gene polymorphisms and mortality. A prospective cohort study. PloS One 2013; 8:e67768. 49. Bultmann A, Li Z, Wagner S, et al. Loss of protease activity of ADAM15 abolishes protective effects on plaque progression in atherosclerosis. Int J Cardiol 2011; 152:382–385. 50. Smiljanic K, Dobutovic B, Obradovic M, et al. Involvement of the ADAM 12 in thrombin-induced rat’s VSMCs proliferation. Curr Med Chem 2011; 18:3382–3386. 51. Salter RC, Ashlin TG, Kwan AP, Ramji DP. ADAMTS proteases: key roles in atherosclerosis? J Mol Med 2010; 88:1203–1211. 52. Salter RC, Arnaoutakis K, Michael DR, et al. The expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 in human macrophages is inhibited by the antiatherogenic cytokine transforming growth factor-beta and requires Smads, p38 mitogen-activated protein kinase and c-Jun. Int J Biochem Cell Biol 2011; 43:805–811.



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This very elegant cell biological study established a convincing basis for the previously observed genetic association of ADAMTS-7 variants and cardiovascular disease but not acute events. In particular, the rs3825807 was shown to increase carotid atherosclerosis but not strokes was shown to code for a functional variant. The protective allele led to less proform activation and hence reduced degradation of a substrate thrombospondin-5. This in turn led to less VSMC migration in an in-vitro assay. These new genetic data on humans confirm concepts derived from experimental work that VSMCs can not only contribute to plaque growth but may also stabilize plaques against rupture. 57. van Setten J, Isgum I, Smolonska J, et al. Genome-wide association study & of coronary and aortic calcification implicates risk loci for coronary artery disease and myocardial infarction. Atherosclerosis 2013; 228:400– 405. This is a genome-wide study on heavy smokers who underwent no contrast computed tomography scans of the thorax. It establishes a relationship between the extent of aortic calcification and polymorphisms in three genes including the ADAMTS-7 variant rs3825807. The same variant was shown to be associated with increased carotid atherosclerosis but not acute coronary syndromes or strokes && [56 ]. 58. Du Y, Wang Y, Wang L, et al. Cartilage oligomeric matrix protein inhibits vascular smooth muscle calcification by interacting with bone morphogenetic protein-2. Circ Res 2011; 108:917–928. 59. Newby AC. Matrix metalloproteinase inhibition therapy for vascular diseases. Vasc Pharmacol 2012; 56:232–244. 60. Peeters W, Moll FL, Vink A, et al. Collagenase matrix metalloproteinase-8 expressed in atherosclerotic carotid plaques is associated with systemic cardiovascular outcome. Eur Heart J 2011; 32:2314–2325. 61. Scholtes VPW, Johnson JL, Jenkins N, et al. 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Low tissue inhibitor of metalloproteinases 3 and high matrix metalloproteinase 14 levels defines a subpopulation of highly invasive foam-cell macrophages. Arterioscler Thromb Vasc Biol 2008; 28:1647–1653. 66. Lenglet S, Mach F, Montecucco F. Role of matrix metalloproteinase-8 in atherosclerosis. Mediators of inflammation 2013; 2013:659282. 67. Laxton RC, Hu Y, Duchene J, et al. A role of matrix metalloproteinase-8 in atherosclerosis. Circ Res 2009; 105:921–929. 68. Xiao Q, Zhang F, Lin L, et al. Functional role of matrix metalloproteinase-8 in && stem/progenitor cell migration and their recruitment into atherosclerotic lesions. Circ Res 2013; 112:35–47. The stem cell contribution to human and mouse atherosclerosis may be controversial but this article presents clear data that cells bearing stem cell markers express MMP-8 and that this acts in a cascade with ADAM-10 to promote shedding of E-Cadherin and recruitment of these cells into mouse plaques. This pathway would be expected to favour formation of macrophage-rich vulnerable plaques. 69. Xiao Q, Zhang F, Grassia G, et al. Matrix metalloproteinase-8 promotes && vascular smooth muscle cell proliferation and neointima formation. Arterioscler Thromb Vasc Biol 2014; 34:90–98. The article characterizes an MMP-8, ADAM-10 cascade that mediates shedding of N-Cadherin and therefore promotes proliferation and migration of VSMCs. This would be expected to contribute to stabilization of plaques from rupture.

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70. Quillard T, Araujo HA, Franck G, et al. Matrix metalloproteinase-13 predominates over matrix metalloproteinase-8 as the functional interstitial collagenase in mouse atheromata. Arterioscler Thromb Vasc Biol 2014; 34:1179–1186. This study confirms the importance of MMP-13 as an endogenous collagenase in mouse atherosclerotic plaques, but acknowledges limitations in translating the results to human plaques that also contain MMP-1. 71. Quillard T, Tesmenitsky Y, Croce K, et al. Selective inhibition of matrix metalloproteinase-13 increases collagen content of established mouse atherosclerosis. Arterioscler Thromb Vasc Biol 2011; 31:2464–2472. 72. Sukhova GK, Schonbeck U, Rabkin E, et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation 1999; 99:2503–2509. 73. Huang WC, Sala-Newby GB, Susana A, et al. 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Volume 25 Number 5 October 2014


From prevention to plaque rupture and infarction.

Metalloproteinases promote plaque rupture and myocardial infarction: A persuasive concept waiting for clinical translation.

The Curious Road from Basic Pathogen Research to Clinical Translation.

Mechanisms of plaque formation and rupture.

Myocardial Bridge and Acute Plaque Rupture.

Atherosclerotic plaque rupture and acute ischemia.

Right atrial rupture after road traffic accident.

Acute Loss of miR-221 and miR-222 in the Atherosclerotic Plaque Shoulder Accompanies Plaque Rupture.

Unblocking beds.

Proteinases, their receptors and inflammatory signalling: the Oxford South Parks Road connection.

Plaque rupture in stable coronary artery disease.

Biomechanics and inflammation in atherosclerotic plaque erosion and plaque rupture: implications for cardiovascular events in women.

miRNAs in atherosclerotic plaque initiation, progression, and rupture.

Thrombosis formation on atherosclerotic lesions and plaque rupture.

Biofilms, lipoprotein aggregates, homocysteine, and arterial plaque rupture.

Orbitofrontal neurons acquire responses to 'valueless' Pavlovian cues during unblocking.

Aortoiliac occlusion secondary to atherosclerotic plaque rupture as the result of blunt trauma.

Letter: Unblocking of T lymphocytes.

Multimodality Intravascular Imaging Assessment of Plaque Erosion versus Plaque Rupture in Patients with Acute Coronary Syndrome.

Reply: plaque rupture in stable coronary artery disease.

Very late neoatherosclerotic plaque rupture in drug-eluting stent restenosis.

Circulating microRNAs as potential biomarkers for coronary plaque rupture.

Atherosclerosis and atheroma plaque rupture: imaging modalities in the visualization of vasa vasorum and atherosclerotic plaques.

The road to change.