Pathophysiology and clinical significance of plaque rupture

Giampaolo NICCOLI,MD, PhD
Institute of Cardiology
Catholic University of the Sacred Heart

Pathophysiology and clinical significance of plaque rupture

by G. Niccoli, F. Fracassi,
and F. Crea,

Experimental models of atherogenesis continue to contribute to elucidation of the molecular mechanisms behind plaque growth; however, the transition from coronary stability to instability is less well understood due to the lack of animal models reflective of human disease. The abrupt onset of acute coronary syndromes (ACS) is a strong indication of discontinuity in the progression of atherothrombosis. The causes of such discontinuity are complex, probably multiple, and still largely unknown. The complexity of postmortem and clinical observations suggests that it is unlikely that a common cause will be identified for the phenotype of ACS. To better understand the multiple causes of coronary instability, it would be desirable to construct a pathogenetic classification of ACS based on simple clinical descriptors. In this review, the multiple causes of coronary instability are discussed in 3 homogeneous groups of patients with a similar clinical presentation: (i) patients who have obstructive atherosclerosis and systemic inflammation, (ii) patients who have obstructive atherosclerosis without systemic inflammation, and (iii) patients with functional alterations of coronary circulation. Such classification of ACS provides a framework for understanding basic mechanisms responsible for coronary instability rather than a classification for immediate clinical use, such as that provided by the universal definition of myocardial infarction. However, our pathogenetic classification of ACS based on simple clinical descriptors might help in the search for new diagnostic algorithms and therapeutic targets

Medicographia. 2014;36:37-44 (see French abstract on page 44)

Atherosclerotic vascular disease is the leading cause of death in Western countries; among the different manifestations of disease, the acute coronary syndromes (ACS) play the major role. The pathogenesis of ACS is complex and not fully clarified, whereas mechanisms leading to atherosclerotic plaque growth have been better characterized, allowing an improvement in primary prevention. In particular, transition from coronary stability to instability should be better investigated with the aim of preventing acute events when primary prevention measures fail. Interestingly, clinical instability is heterogeneous from many points of view. Indeed, whereas some patients have 1 episode of ACS that will not recur during their lifetime, others have multiple (ie, recurrent) episodes. Additionally, some cases are associated with severe stenosis and others, with mild-to-moderate lesions; thus, plaque severity leading to acute coronary syndrome is heterogeneous, as are plaque morphology and composition. Indeed, postmortem studies and, recently, intravascular imaging have shown that ACS can be associated with plaque rupture, plaque erosion, or a smooth plaque.1-4 Taken together, all these observations suggest that the mechanisms of ACS are multiple. This review article is focused on the different pathogenetic mechanisms of ACS.

Pathophysiology of ACS

Plaque rupture associated with thrombosis is the most frequent plaque feature in patients with ACS.5-7 This observation has been obtained in the postmortem setting,4 but also in vivo by using intravascular ultrasound,8 angioscopy,9 and more recently, optical coherence tomography (OCT).1,10-11 In particular, OCT, with its high resolution, recently clarified that in vivo plaque rupture is associated with ACS in 75% of ST-elevation myocardial infarction (STEMI) patients.1 As suggested by postmortem4,12 and in vivo studies,13-14 the precursor of a ruptured plaque is a thin-cap fibroatheroma (TCFA), containing a large necrotic core covered by a thin, fibrous cap characterized by the presence of activated macrophages. TCFAs have been found to be associated with a higher risk of ACS using both noninvasive and invasive imaging modalities.15-16 It is worth noting, however, that rupture of TCFA may not necessarily lead to ACS, as asymptomatic plaque rupture is a frequent event leading to plaque progression.17-19 The reasons why some plaque ruptures lead to ACS and others do not are largely unknown. Mechanisms leading to plaque rupture may be classified according to levels of systemic markers of inflammation.20 Indeed, some patients exhibit marked elevation of inflammatory biomarkers, and widespread coronary microvascular and myocardial inflammation with activation of innate and adaptive immunity,21-22 while others have immeasurable levels of inflammatory biomarkers, suggesting mechanisms other than sustained inflammation.23

♦ Plaque rupture with activation of systemic inflammation
Several studies have shown that systemic inflammation, as assessed by C-reactive protein (CRP), plays a crucial role in the process of coronary instability.24-25 In particular, it has been found that inflammatory mechanisms regulate the fragility of the fibrous cap, as well as the thrombogenic potential of the lipid core.26-27 Ruptured plaques and vulnerable plaques, compared with intact plaques, have larger numbers of inflammatory cells, mostly monocyte-macrophages, but also T cells, eosinophils, and mast cells. These inflammatory cells, showing evidence of activation,28-29 are mostly located in the shoulders of the fibrous caps and in the lipid core as well as in the adventitia around areas of neovascularization.30-31 The latter might contribute to the recruitment of inflammatory cells in atherosclerotic plaques. Inflammatory activation is not confined to the culprit stenosis, as suggested by the observation of widespread neutrophil activation in the coronary circulation of patients with unstable angina32 and by the presence of activated T lymphocytes in remote unaffected myocardial regions in approximately two-thirds of patients with recent myocardial infarction (MI).33 Moreover, investigating all major coronary artery branches by intravascular ultrasound, Rioufol et al observed multiple ruptures in patients with a first ACS.8

Among cells of the innate immunity, neutrophils and macrophages play a major role. Interestingly, the activation of neutrophils in the coronary circulation is suggested by telomerase activation in neutrophils from the culprit coronary plaques of patients with ACS, but not in neutrophils from plaques of patients with stable angina.34 The reactivation of telomerase, demonstrated in the early phases of coronary instability, delays cell apoptosis, thus favoring persistence of inflammation.34 Neutrophil activation in ACS appears to be an early and short-lasting event.35

The predominant inflammatory cells in atherosclerotic plaques are macrophages recruited as monocytes from circulating blood. There are different subsets of monocytes with different gene expression patterns and, in particular, differential expression of CD14 and CD16.36-37 The amount of CD14+CD16+ monocytes in patients with coronary atherosclerosis is higher as compared with that in healthy subjects and peak levels of CD14hiCD16lo monocytes after acute MI correlate negatively with the recovery of left ventricular ejection fraction 6 months after MI.38 Moreover, macrophages are probably involved in the rupture of the fibrous caps as they produce larger amounts of matrix metalloproteinases (MMPs),39-41 enzymes thatdegrade all components of the extracellular matrix.42 The production and activation of MMPs are regulated at the level of gene transcription and by the cosecretion of tissue inhibitors of MMPs (TIMPS). Thus, increased gene transcription of MMPs or reduced activity of TIMPS can enhance matrix proteolysis. It has been shown that Toll-like receptors (TLR) may mediate the activation of monocytes-macrophages; indeed, monocytes accumulated within thrombi, obtained during primary percutaneous coronary interventions, specifically overexpress TLR4, together with specific patterns of locally expressed chemokines and cytokines, compared with circulating monocytes.43-44 Notably, Niessner et al45 have confirmed enhancedTLR4 in carotid plaques, probably mediated by interferon α released by plasmacytoid dendritic cells, specialized in sensing danger signals from bacteria and tissue breakdown.45 Thus, all components leading to activation of the MMP pathway are present in the atherosclerotic plaques. A recent study by Blair et al demonstrated that human platelets also express functional TLRs46; TLR2, in particular, promotes platelet-leukocyte interactions, amplifying platelet-derived inflammatory signals. In this prospective, platelets act as coprotagonists in plaque activation and as a major player in the thrombotic processes. Moreover, Beaulieu et al47 have shown TLR2 expression on megakaryocytes and suggested that inflammation through TLR2 stimulation can increase megakaryocyte maturation and modulate megakaryocyte phenotype, potentially influencing platelet function and thrombosis.

The involvement in coronary instability of cells of adaptive immunity has consistently been demonstrated with studies reporting enhanced activation of T cells in patients with ACS as compared with those with stable angina,21-22 and oligoclonal T-cell expansion in unstable coronary plaques compared with stable plaques.21,48 Based on the notion that the activation of T cells requires a specific antigenic stimulation mediated by antigen-presenting cells, some studies have identified candidate antigens, like Chlamydia pneumoniae, heat shock proteins, or oxidized low-density lipoproteins (oxLDLs), in plaques from patients with ACS.25 Accordingly, CD4+CD28 T cells, so called for the defective cell surface expression of CD28, a major costimulatory molecule critically involved in determining the outcome of antigen recognition by T cells, have been found to undergo clonal expansion in unstable coronary plaques21-22— where they release potent proinflammatory cytokines (mostly interferon γ), enhancing activation of innate immunity cells— and to have direct cytolytic effects on endothelial cells, amplified by high-sensitivity CRP (hs-CRP),49 and on vascular smooth muscle cells,50 thus promoting plaque rupture.51 Of note, both circulating and resident CD4+CD28 T cells spontaneously express interleukin (IL)-12 receptor and respond to IL-12 released by innate immunity cells with the up regulation of chemokine receptors; thus, IL-12 can favor tissue homing of CD4+CD28 T cells even in the absence of antigenic stimulation.52

Although their precise role in atherosclerosis remains controversial, recent studies have shown a proatherogenic role of another lymphocyte subset represented by T helper 17 (Th17) cells; they produce IL-17 involved in autoimmunity and allergic reactions.53 CD4+CD25+ regulatory T cells (Treg) also are profoundly perturbed in ACS. The normal functioning of Treg is essential to maintain the homeostasis of T-cell subsets involved in adaptive immunity. Accordingly, Treg expressing the forkhead/winged-helix transcription factor Foxp3 have been found to prevent atherosclerosis in mouse models.54 Consistently, a critical role for the anti-inflammatory cytokine IL-10 has been assumed in Treg-mediated atheroprotection, both in experimental models and in human atherosclerotic lesions, where Treg and IL-10 expression are colocalized. The balance between Th17 and Treg cells may be important in the development and prevention of inflammatory and autoimmune diseases.55 Recently published data provide evidence of a defective Treg compartment in ACS. The number and the suppression efficiency of Treg were reduced in patients with ACS compared with patients with stable angina and healthy controls.56 A parallel increase in the circulating levels of Th17 has also been observed.57 Taken together, these observations suggest that, at least in a subset of patients with ACS, the reduction of a counter regulatory response to the activation of aggressive effector T cells might play a key pathogenetic role and may become a potential therapeutic target.58

♦ Plaque rupture without activation of systemic inflammation
When plaque rupture occurs in the absence of systemic inflammatory activation, other mechanisms, including emotional and physical stress or changes in plaque composition may play a pathogenetic role.20 The ability of systemic stress to induce plaque rupture is related to sympathetic nervous system activation and catecholamine release associated with increase in heart rate, blood pressure, and coronary vasoconstriction favoring the rupture of vulnerable plaques59 and to platelet activation, hypercoagulability, and intense coronary microvascular constriction.60 It has been demonstrated that the highest shear stress is present in the shoulder region of the fibrous cap.7,61

Changes in plaque composition have been hypothesized by Abela et al62 as a possible cause of plaque rupture. Indeed, local changes in pH, temperature, cholesterol saturation, and hydration promote cholesterol crystallization in the lipid core, associated with quick volume expansion, potentially causing plaque fissure and thrombosis. This mechanism may be amplified by crystallization of free cholesterol from erythrocyte membranes when intraplaque hemorrhage occurs.63

♦ Plaque erosion
Plaque erosion is reported in at least one-third of patients dying of acute MI in postmortem histopathological studies.4 Coronary instability is assumed to be the result of plaque erosion if there is no continuity between the thrombus and the necrotic core and the thrombus is in direct contact with the fibrointimal plaque.64 On OCT analysis, plaque erosion consists of evidence of thrombi, an irregular luminal surface, and no evidence of cap rupture evaluated in multiple adjacent frames. Neutrophil activation seems to play a pivotal role in plaque erosion. We have recently showed that patients presenting with ACS associated with plaque erosion had higher systemic myeloperoxidase levels as compared with levels in patients exhibiting plaque rupture.65 Moreover, in postmortem coronary specimens, luminal thrombi superimposed on eroded plaques contained a much higher density of myeloperoxidase (MPO)-positive cells than thrombi superimposed on ruptured plaques.65 One study66 reported an intense immunostaining pattern for hyaluronan and its receptor, CD44, along the plaque/thrombus interface in eroded plaque, but not in fissured or stable plaque. Accumulation of hyaluronan and expression of CD44 along the plaque/thrombus interface of eroded plaques may promote de-endothelialization, resulting in CD44-dependent platelet adhesion and subsequent thrombus formation, in part mediated by a direct action of hyaluronan on fibrin polymerization. Furthermore, accumulation of hyaluronan in eroded plaques may promote CD44-dependent adhesion and accumulation of circulating neutrophils and MPO-expressing monocytes, which in turn may enhance endothelial cell death and promote thrombus formation. MPO, released by neutrophils, catalyzes the formation of MPO-derived reactive species (MDRS), such as hypochlorous acid (HOCl), using chloride, thiocyanate, or nitric oxide (NO) as the substrate and hydrogen peroxide as the cosubstrate. MDRS are responsible for consuming NO, which may result in impaired vasodilation, oxidation of LDL and high-density lipoprotein, activation of MMPs, oxidation of proteoglycans and glycosaminoglycans, and apoptosis of endothelial cells by activation of a specific pathway. Furthermore, activated neutrophils shed microparticles, which may transfer tissue factor into platelets, thus contributing to thrombosis. Tissue factor expression and activation is also induced by MDRS and oxLDL.66 MPO may also have a role in thrombus growth.67-68

Finally, calcified nodules, found to be more frequent in patients with diabetes,69 are a less common cause of coronary instability. They are lesions with the highest concentration of calcification relative to plaque area and can be a rare trigger for thrombosis.4

♦ Vasoconstriction of epicardial vessels or microcirculation
Epicardial coronary vasospasm is likely to play a key role in ACS, particularly in patients in whom coronary angiography fails to demonstrate the presence of an obstructive atherosclerotic plaque.6 In such patients, the incidence is about 50% as reported in the CASPAR study (Coronary Artery Spasm in Patients With Acute Coronary Syndrome)70 in patients with ACS and about 50% as reported by the ACOVA study (Abnormal COronary VAsomotion in patients with stable angina and unobstructed coronary arteries) in patients with stable angina.71 Coronary spasm is caused by vasoconstrictor stimuli acting on hyperreactive vascular smooth muscle cells, perhaps because of enhanced Rho-kinase activity.72 Spasm can occur at the site of an angiographically normal coronary segment or in the presence of a nonobstructive atherosclerotic plaque.73 Unpublished data by our research group show that ACS patients with smooth plaques without thrombus on OCT investigation have elevated cystatin C levels in agreement with data by Funayama et al74 showing elevated cystatin C levels in patients with vasospastic angina.

Vasoconstriction causing ACS also occurs at the microvascular level.75-76 In particular, intense coronary microvascular vasoconstriction plays an important role in the pathogenesis of Takotsubo syndrome,77 characterized by ischemic pain at rest, ST-segment elevation, cardiac enzyme release, and a characteristic regional akinesia more frequently affecting distal myocardial regions associated with hypercontractility of the remaining regions.

Finally, Mohri et al have described a clinical presentation of ACS characterized by chest pain, ST-segment elevation, normal epicardial coronary arteries, and normal myocardial function.78 As acetylcholine provocation testing reproduced the same symptoms and electrocardiographic alterations in the absence of epicardial vasospasm, the authors proposed that the causeof this syndrome was coronary microvascular spasm.

Clinical implications

Table I and Figure 1 summarize pathogenetic mechanisms of ACS along with diagnostic and therapeutic options tailored for individual mechanisms of coronary instability. Several studies have shown that patients with ACS in whom obstructive atherosclerosis is associated with elevated levels of CRP or other markers of inflammation have a worse outcome than patients with a similar severity of coronary atherosclerosis, but normal levels of inflammatory markers.14,32,79-82 Thus, in the former, reassessment of the inflammatory status after discharge may help in the identification of patients at higher risk of recurrence of coronary instability. Although the assessment of the inflammatory status is currently based on biomarkers only, recently developed imaging techniques able to monitor inflammatory cell activity in atherosclerotic plaques might prove to be more predictive than biomarkers.83 In addition, an unmet need in this patient subset is a specific anti-inflammatory treatment based on the modulation of both innate and adaptive immunity.24,84-85

In patients with ACS in whom plaque fissure is not associated with systemic inflammation, anatomic (more than functional) features of the atherosclerotic plaque are important in determining coronary instability. Because it is difficult to limit environmental, physical, or emotional triggers, an obvious target in this patient subset is plaque stabilization as achieved by intensive statin treatment.86 Inhibitors of phospholipase A2 represent another class of drugs that might help in plaque stabilization. Another important, but still elusive target to promote plaque stabilization is enhancement of cholesterol efflux.87 Among patients in whom plaque fissure is not associated with systemic inflammation and in whom ACS occurs in the absence of environmental, physical, or emotional triggers, more needs to be learned about the mechanisms modulating cholesterol crystallization, including the inflammasome pathway activated by cholesterol crystals, in order to identify new therapeutic targets.

Table I
Table I. Pathogenetic mechanisms, diagnosis and potential tailored therapy for acute coronary syndromes.

Abbreviations: IL-1β, interleukin 1β; IL-1Ra, interleukin 1 receptor a; MMPs, matrix metalloproteinases; MPO, myeloperoxidase; PLA2, phospholipase A2; TLRs,
Toll-like receptors; TNF-α, tumor necrosis factorα.

In patients with plaque erosion, the mechanism of inflammation is probably an intense local thrombogenic stimulus. Thus, in this subset of patients, a potent antithrombotic treatment perhaps based on double anti-aggregation and an oral anticoagulant might be the treatment of choice, but this approach needs to be tested in prospective studies.

Figure 1
Figure 1. Pathogenetic classification of acute coronary syndromes:
(i) patients with plaque rupture and systemic inflammation;
(ii) patients with plaque rupture without systemic inflammation;
(iii) patients with plaque erosion;
(iv) patients with smooth plaque.

Finally, epicardial and microvascular vasoconstriction is the key therapeutic target when ACS is not associated with obstructive atherosclerosis. Recently, data from clinical trials suggest that the outcome of these patients is, on average, better than that of patients with obstructive atherosclerosis; however, about 10% of patients presenting with ACS in the absence of coronary atherosclerosis had had a major cardiac event on 1-year follow-up.88 Although nitrates and calcium antagonists are helpful in patients with vasospastic angina, further efforts are needed to identify the molecular alterations responsible for smooth muscle cell hyperreactivity, because a sizeable proportion of patients with vasospastic angina are refractory to standard doses of vasodilators.73

It has been observed that fasudil, a specific Rho-kinase inhibitor, reduces the rate of coronary spasm episodes in patients with vasospastic angina.72 Similarly, further efforts are warranted to unravel the molecular mechanisms responsible for coronary microvascular dysfunction in Takotsubo syndrome and in unstable microvascular angina.♦

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Keywords: acute coronary syndrome; atherosclerosis; inflammation; microcirculation; plaque erosion; plaque rupture