TWO MAJOR MECHANISMS OF THE ACUTE THROMBOTIC COMPLICATIONS OF ATHEROSCLEROSIS
The acute coronary syndromes (ACS) persist as a leading cause of morbidity, mortality, and loss of quality life years. The epidemic of atherothrombosis today affects not only developed countries, but also those with emerging economies. Successes in acute care have markedly reduced in-hospital and short-term mortality in patients with ACS. Yet, many ACS survivors develop ischemic cardiomyopathy and the clinical syndrome of heart failure, a major limitation of quality of life, cause of mortality, and a burden to healthcare systems. Despite our very effective revascularization, lifestyle measures including cardiac rehabilitation programs, successful pharmacologic treatments including aspirin and other potent antiplatelet agents, β-adrenergic blocking drugs, and a variety of avenues to lower LDL levels, the residual burden of disease remains unacceptably high .
Much work on the mechanisms of ACS over the last several decades has focused on the so-called ‘vulnerable plaque’. These lesions have characteristically large lipid pools, many macrophages and other inflammatory leukocytes, thin fibrous caps depleted of interstitial collagen, and few arterial smooth muscle cells (Fig. 1, right). Research has made considerable inroads into understanding the pathophysiologic basis of development of such thin-capped atheromatous lesions . Overexpression of collagenases and reduced collagen synthesis signaled by inflammatory mediators can impair the biomechanical integrity of the plaque's fibrous cap. Pro-inflammatory cytokines can also augment the expression of the potent procoagulant tissue factor that triggers thrombosis in plaques that undergo rupture of the fibrous cap. Experimental and human studies have shown that lipid-lowering therapies can limit inflammation, reduce lipid accumulation, and reinforce the fibrous skeleton of plaques, changes that would render them less likely to rupture.
We have therefore hypothesized that some of the residual burden of risk in an era of extreme lipid lowering may result from superficial erosion, a mechanism of thrombotic complications of atherosclerotic plaques generally relegated to minority status as a cause of ACS. The lesions associated with superficial erosion in humans exhibit a morphology quite distinct from the thin-capped atheromata associated with plaque rupture. In stark contrast with ruptured plaques, they contain abundant smooth muscle cells and extracellular matrix (ECM), and exhibit scarce lipid and macrophage/foam cell accumulation (Fig. 1, left). Intravascular imaging using optical coherence tomography (OCT) readily identifies culprit lesions of ACS that have ruptured . Many nonruptured plaques that cause ACS may have provoked thrombus formation because of superficial erosion. OCT imaging indicates that up to one-third of ACS in the current era result from erosion rather than rupture [4,5].
We have argued that because of the increasing success of our LDL-lowering therapies we are currently witnessing a rise in the proportion of ACS caused by erosion. We further propose that this pathway to coronary thrombosis may persist in the current era of effective LDL-lowering therapy, as the relatively lipid-poor substrate may respond less well to such treatment than lipid-rich, thin-capped atheromatous plaques. Although we have achieved considerable mastery over the pathophysiology and prevention of the classical ‘vulnerable’ plaque, we have very little understanding of the pathophysiology and treatment of lesions prone to develop erosion. Hence, we need to understand better the mechanisms that underlie superficial erosion, a mechanism of thrombotic complications of plaques that appears on the rise in the era of effective LDL lowering.
EMERGING CONCEPTS OF THE MECHANISMS OF PLAQUE EROSION: ROLES FOR DISTURBED FLOW AND TOLL-LIKE RECEPTOR 2
Recent work has focused on the innate immune receptor toll-like receptor 2 (TLR2) as a contributor to altered endothelial function that can predispose to superficial erosion [6,7]. Observations on cultured human endothelial cells and on atherosclerotic mice support overexpression of TLR2 in regions of disturbed flow [8,9]. Endothelial cells in human atherosclerotic arteries distal to stenoses, regions that experience disturbed flow, show increased indices of cell death by apoptosis . We therefore hypothesized that TLR2 engagement could predispose towards endothelial cell desquamation, a likely trigger to thrombosis in eroded lesions. Increased TLR2 expression in regions of disturbed flow might lead to an increased activation of the receptor. Studies in atherosclerotic mice have furnished evidence that TLR2 actively contributes to experimental atherosclerosis [8,9]. Administration of Pam3CSK4 – a synthetic TLR2/TLR1 agonist – substantially increased atherosclerotic burden in LDLR−/− atherosclerotic mice. Mice with double deficiency of LDLR and TLR2, either globally or restricted to bone marrow-derived cells, did not show increased lesion size in response to TLR2 agonism. The atherogenic role of TLR2 seems therefore to depend on its direct action in cells of nonbone marrow origin . Using ApoE−/− atherosclerotic mice, other groups found that TLR2 activation by exogenous ligands increases atherosclerotic plaque size and neointima formation in femoral arteries, notably through promoting reactive oxygen species (ROS) production and smooth muscle cell migration in an interleukin (IL)-6-dependent manner [11–15].
Related to its ability to form heterodimers with other TLR family members, numerous exogenous and endogenous ligands can trigger TLR2 activation . TLR2 ligation to lipoproteins (i.e. oxLDL) with the scavenger receptor CD36, and high-mobility group box 1 protein (HMGB1) contributes to foam cell formation and inflammation, supporting its participation in atherogenesis [17–21].
TLR2 activation may derive from other endogenous ligands. Of particular interest in this context, TLR2 can bind low-molecular weight hyaluronic acid  via direct association with CD44  or with versican , two major components of ECM typically found in eroded lesions . Our recent work, and others’, showed that TLR2 agonists, lipoteichoic acid, or Pam3CSK4 suffice to promote a low-level, smoldering activation and dysfunction of endothelial cells with increased expression of E-Selectin and VCAM-1, endoplasmic reticulum stress, ROS production, and ultimately apoptosis. Treatment with hyaluronic acid, either in soluble form or coated on the culture dish, led to comparable results [6,26,27]. This observation further supports the involvement of hyaluronan as a disease-relevant endogenous TLR2 agonist. Combination of disturbed flow that associates with increased endothelial TLR2 expression and lesions with high glycosaminoglycan content might therefore set the stage for endothelial cells dysfunction and ultimately erosion.
A SPECIAL ROLE FOR GRANULOCYTES AND NEUTROPHIL EXTRACELLULAR TRAPS IN SUPERFICIAL EROSION?
Macrophages have traditionally commanded considerable attention as contributors to atherogenesis, because of their fundamental roles in every aspect of plaque formation, development, and rupture. Yet, other phagocytic and innate immune cells may also contribute to atherogenesis (e.g. mast cells, eosinophils, and notably neutrophils) . Our recent findings point to a particular place for polymorphonuclear leukocytes in propagating intimal injury in the context of superficial erosion.
Neutrophils furnish the first line of defence against pathogens and use an assortment of granular proteins, proteases, and ROS to attack microorganisms and amplify locally innate and adaptive immune responses. Neutrophils can also entrap infectious agents through the formation of neutrophil extracellular traps (NETs). NETs consist of web-like structures derived from decondensed chromatin with histones, antimicrobials, and proteins that capture and promote pathogen death. NET formation (NETosis) results from a controlled death program like apoptosis or necroptosis, even if in some rare cases netotic anucleate neutrophils remain motile and able to kill bacteria through phagocytosis and degranulation .
In addition to clearing microorganisms, neutrophils may also participate in the pathophysiology of cancer, autoimmunity, and chronic inflammatory diseases. Neutrophils can localize in complicated atherosclerotic lesions [6,29–31]. In apoE−/− mice, increased presence of NETs associated with higher expression of IFNα. Inhibition of NET formation by peptidyl-arginine deiminase 4 (PAD4) blockade reduced lesion size after 11 weeks of treatment and a high-fat diet (HFD), although the inhibitor used may lack specificity . Warnatsch et al. also reported that cholesterol crystals induced NETosis in vitro. NETosis occurred after 8 weeks on a HFD in cholesterol-rich areas of experimental atheromata, and depended on ROS, as diphenylene iodonium – an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or an inhibitor of the neutrophil-specific proteases neutrophil elastase (NEi) and proteinase 3 (PR3) – blocked this process. NET blockade using Apoe−/− mice lacking both neutrophil elastase and PR3 (Apoe−/−Elane−/−Prtn3−/−) or DNase injections (6 weeks) reduced lesions size in Apoe−/−Elane−/−Prtn3−/− animals after 8 weeks but not after 4 weeks of high-fat diet feeding . In contrast, others reported a similar protective effect in Apoe−/−Elane−/−Prtn3−/− mice, but only in early stages of the disease . In this latter study, DNase treatment for 4 weeks was insufficient to attenuate atherogenesis. This point, discussed by Döring et al. raises questions on the precise mechanisms implicated in the detrimental role of neutrophils on plaque progression, either via NETosis, or/and specific neutrophil proteases that affect inflammatory cytokines production and maturation.
Previous reports implied a direct role for neutrophils in superficial erosion and in endothelial desquamation. Ferrante et al. showed that systemic myeloperoxidase (MPO) levels – a heme protein most abundantly expressed in azurophilic granules in neutrophils – rose significantly in patients with eroded plaques compared with patients presenting with ruptured plaque, as determined by OCT. In vitro, ROS derived from MPO release can promote endothelial cell apoptosis,  and MPO-containing neutrophil microparticles damage vascular endothelial cells and disrupt the endothelial barrier . MPO can generate locally hypochlorous acid (HOCl), a potent pro-oxidant that can propagate damage in regions of arteries where granulocytes congregate. Activated endothelial cells can induce NETosis and reciprocally neutrophil and NETs directly induce endothelial dysfunction and damage of endothelial cells [39,40]. In Lupus erythematosis, NETosis associates with activation of endothelial MMP-2, impairment of endothelium-dependent vasorelaxation, and apoptosis [41,42].
In typical erosion-type lesions exposed to disturbed flow and rich in hyaluronic acid and versican, TLR2 activation can lead to endothelial cell stress, as stated previously. TLR2 ligation induces the production of IL-8, a chemokine that promotes neutrophil recruitment and adhesion . TLR2 can also interact with NETs and influence cytokine production, including IL-17 that further drives neutrophil chemotaxis . Such molecular interactions may sustain granulocyte recruitment, compensating for their short lifespan in tissue .
TLR2 activation impairs the ability of neighboring endothelial cells to repair breaches in the endothelial monolayer . Co-incubation of endothelial cells with TLR2 agonists and with neutrophils also promotes endothelial cell death in vitro. Such treatment further favors endothelial desquamation by inducing the nonfibrillar collagenases MMP2 and MMP9, capable of degrading components of the basement membrane to which endothelial cells attach.
To test the relevance of this hypothesis to human disease, we analyzed the association between TLR2 expression, luminal neutrophils, and NETs, and apoptotic endothelials in human endarterectomy specimens. We colocalized apoptotic endothelial cells with TLR2, neutrophils, and markers of NETosis in lesions complicated by superficial erosion, but not in plaques with characteristics considered ‘rupture-prone’ (Fig. 2) . ‘Stable’ lesions that exhibit little endothelial cell apoptosis express similar levels of TLR2 as do erosion-type lesions, but harbor few neutrophils. In contrast, typical ruptured lesions that contain far lower levels of putative endogenous agonists for TLR2 (e.g. hyaluronic acid, versican) contain similar neutrophil numbers, but lower TLR2 expression .
NETs also directly promote thrombosis through multiple mechanisms. Extracellular nucleic acids in NETs bind circulating platelets, coagulation factors, and VWF, acting as a solid-state reactor for the coagulation cascade and providing a scaffold for developing thrombi [44–46]. Neutrophil proteinases and extracellular histones activate platelets and accelerate thrombin formation [47,48]. In addition, histones, elastase, and MPO-generated HOCl each induce endothelial tissue factor production [37,49–51]. Supporting these in vitro studies, clinical data implicate NETs in coronary disease, venous thrombosis, and other thrombotic conditions [52–55]. Thrombi retrieved from the culprit arteries of patients with ACS show higher levels of NET markers. NETs isolated from culprit artery blood contain tissue factor. Furthermore, culprit arteries have higher blood levels of markers of NETs than nonculprit arteries . A separate study of patients with STEMI showed that a higher burden of NETs within thrombi correlated with greater infarct size and delayed resolution of ST-segment elevation . Collectively, these data indicate that the elevated levels of NETs observed at the intimal surface of eroded lesions may contribute causally to erosion-associated coronary thrombosis.
EXTENDING IN-VITRO STUDIES OF MECHANISMS OF SUPERFICIAL EROSION IN VIVO
Our in-vitro studies described above implicated disturbed flow, neutrophils, hyaluronan, and TLR2 ligation in endothelial damage, which we conjectured contribute to superficial erosion. Our recent study tested in mice the involvement of these factors in superficial intimal injury in vivo. We crafted intimal lesions in mouse carotid arteries that resemble the substrate of human eroded plaques by having high ECM content, but scant macrophages, intimal lesions, and lipid. This preparation used electrical injury to the adventitia, followed by 4 weeks of healing to form a neointima and allow re-endothelialization of the intimal surface. After this period of healing, we produced flow perturbation by means of periadventitial cuff. This intervention promoted downstream endothelial activation, neutrophil accumulation, death of endothelial cells, and their eventual desquamation. Mural thrombosis ensued. Inhibition of neutrophil function by two independent methods mitigated this endothelial damage and detachment. Administration of TLR2 agonists activated luminal endothelial cells, whereas TLR2 deficiency reduced intimal neutrophil content in zones of locally disturbed flow, and protected endothelial cells from injury and limited local thrombus formation. We do not affirm that this in vivo preparation represents a ‘model’ for superficial erosion, rather a tool to study under controlled experimental circumstances mechanisms that may pertain to plaque erosion in humans .
Bone marrow chimera experiments pointed to intrinsic vascular cells rather than leukocytes as the responders to TLR2 agonism. TLR2 localized mainly on the basal aspect of endothelial cells, strategically positioned to respond to intimal hyaluronate, a potential endogenous TLR2 ligand. These results in mice and parallel observations on human atheromata provided in vivo support for the notion that flow disturbance, neutrophils, and TLR2 signaling participate in the pathogenesis of superficial erosion.
EROSION MECHANISMS LIKELY INVOLVE MULTIPLE ‘HITS’
Given this ensemble of findings, we have proposed that sequential ‘hits’ promote endothelial cells desquamation and ultimately plaque erosion and thrombosis (Fig. 3). For example, TLR2 activation initially leads to chronic low-level endothelial activation and eventually neutrophil recruitment that jeopardizes intimal integrity by sloughing of endothelial cells. Then the tempo of mischief picks up, and from a low-level smoldering process, in a second, much more rapid phase, thrombosis ensues. NETs can amplify and propagate endothelial damage, widen the area of intimal damage, and potentiate local thrombosis. If the thrombus persists and grows to obstruct flow, the situation can cross the clinical threshold to produce ischemia, cause symptoms, and myocardial damage: an ACS.
CLINICAL IMPLICATIONS OF SUPERFICIAL EROSION
As noted above, we are beginning to unravel pathophysiologic pathways relevant to arterial thrombosis provoked by superficial erosion. This effort has merit not only from a basic science perspective, but may also provide a foundation for clinical advances. The triage and therapeutic pathways for patients with ACS depend on the clinical assessment, a surface electrocardiogram, and biomarkers of myocardial injury. None of these tools provides insight into the pathophysiologic mechanism of the ACS: erosion versus plaque rupture. Yet, the optimum management strategy for these two very different substrates for coronary artery thrombosis might very well vary considerably. OCT data suggest that the type of thrombus in erosion differs from that provoked by plaque rupture. Intimal erosion may give rise to platelet-rich, white thrombi, whereas clots that complicate plaque rupture have a more fibrin-rich ‘red’ character . Thus, intensive antiplatelet therapy might benefit particularly ACS caused by superficial erosion.
Indeed, a pilot clinical trial, EROSION, used OCT to gauge the resolution of coronary thrombi in patients with ST-segment elevation myocardial infarction (STEMI)-associated culprit lesions with the findings of superficial erosion versus rupture . The patients did not receive coronary artery stent deployment, the current standard of care for STEMI, but received intensive antiplatelet treatment. The study was not powered for events, but showed that most of the subjects had substantial resolution of the intra-coronary thrombosis after 30 days. Although this pilot study has many limitations, it does illustrate the enticing concept that we might tailor the treatment of ACS depending on the underlying mechanism that precipitated the thrombotic event. Performing OCT, an invasive procedure, will not prove practical for triage of ACS patients on a routine basis.
If we were to develop biomarkers based on pathophysiologic insights derived from mechanistic studies such as those described in this review that could distinguish ACS because of erosion from those arising from rupture it could revolutionize the triage and management of patients presenting with ACS. Such an advancement would represent a step towards a more personalized approach, and illustrate the value of translation of basic science insights to clinical practice.
Financial support and sponsorship
This work was supported by grants from the National Heart, Lung and Blood institute (NIH-R01HL080472) by the Leducq Foundation (Paris, France) and from the RRM charitable fund. G.F. was supported by the Harold M. English Fellowship Fund from Harvard Medical School (Boston, USA), the Fondation Bettencourt Schueller (Neuilly-sur-Seine, France), and the Philippe Foundation (New York, USA and Paris, France). T.M. received support from by the Sarnoff Cardiovascular Research Foundation (Great Falls, USA).
Conflicts of interest
There are no conflicts of interest.
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