Atherosclerosis 229 (2013) 124e129. Contents lists available at SciVerse ScienceDirect. Atherosclerosis

Atherosclerosis 229 (2013) 124e129 Contents lists available at SciVerse ScienceDirect Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis Coronary calcification identifies the vulnerable patient rather than the vulnerable Plaque Alessandro Mauriello a, *, Francesca Servadei a, Giuseppe Biondi Zoccai b, Erica Giacobbi a, Lucia Anemona a, Elena Bonanno a, Sara Casella a a Anatomic Pathology, University of Rome Tor Vergata, Italy b Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy article info abstract Article history: Received 21 September 2012 Received in revised form 11 March 2013 Accepted 11 March 2013 Available online 21 March 2013 Keywords: Coronary calcification Plaque vulnerability Vulnerable patient Histology Objective: Presence of coronary artery calcium (CAC) is associated with a high risk of adverse cardiovascular outcomes. Nevertheless, although CAC is a marker of atherosclerosis it is still uncertain whether CAC is a marker of plaque vulnerability. Therefore, the aim of this study was to verify if calcification identifies a vulnerable patient rather than the vulnerable plaque. Methods: A morphologic and morphometric study on 960 coronary segments (CS) of 2 groups of patients was performed: (i) 17 patients who died from AMI (510 CS); (ii) 15 age-matched control patients without cardiac history (CTRL, 450 CS). Results: Calcification was found in 47% CS of AMI and in 24.5% CS of CTRL. The area of calcification was significantly higher in AMI compared to CTRL (p ¼ 0.001). An inverse correlation was found between the extension of calcification and cap inflammation (r 2 ¼ 0.017; p ¼ 0.003). Multivariate regression analysis demonstrated that the calcification was not correlated with the presence of unstable plaques (p ¼ 0.65). Similarly, the distance of calcification from the lumen did not represent an instability factor (p ¼ 0.68). Conclusion: The present study suggests that CAC score evaluation represents a valid method to define the generic risk of acute coronary events in a population, but it is not useful to identify the vulnerable plaque that need to be treated in order to prevent an acute event. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction It has been widely demonstrated that acute coronary syndromes (ACS) are related to rupture and acute thrombosis over a mildly stenotic plaque, rather than to a slow growth with final occlusion of a plaque encroaching the lumen [1e3]. Several studies highlighted the role of inflammatory cells (macrophages and T lymphocytes), metalloproteases and cytokines in the transformation of a stable plaque into a vulnerable one [4e6]. It has been suggested that calcific content of a plaque is another key factor for plaque destabilization, potentially modifying mechanical plaque s characteristics and predisposing it to rupture [7] (Fig. 1). Indeed, calcification is the most frequent complication of atherosclerotic lesions [8]. There is a strong relationship between mortality and total coronary artery calcium (CAC) score evaluated * Corresponding author. Cattedra di Anatomia ed Istologia Patologica, Dipartimento di Biomedicina e Prevenzione, University of Rome Tor Vergata, Via Montpellier 1, 00133 Roma, Italy. Tel.: þ39 06 2023751; fax: þ39 06 20902209. E-mail address: alessandro.mauriello@uniroma2.it (A. Mauriello). by cardiac computed tomography (CT) [7,9,10]. Presence of CAC is a well-established marker of coronary plaque burden and is associated with a high risk of adverse cardiovascular outcomes [11,12]. Although coronary calcification is a marker of atherosclerosis, its effect on plaque instability seems to be less evident. It is still uncertain whether coronary calcification is a marker for plaque vulnerability. The recent introduction of intravascular ultrasound (IVUS) provided conflicting results compared to CT-based studies [13,14], showing minor calcification in culprit lesions of ACS in respect to patients with stable angina [15]. Moreover, it is worth noting that in the modified AHA classification of coronary plaques the frequent fibrocalcific plaques are considered as stable lesions [16]. These findings are difficult to reconcile with those derived by CT. In order to better define the role of calcification in coronary plaques destabilization we perform a detailed morphologic, morphometric and topographic study evaluating serial sections of the whole coronary tree of patients died from acute myocardial infarction (AMI) and non-cardiac causes. 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.03.010

A. Mauriello et al. / Atherosclerosis 229 (2013) 124e129 125 2. Materials and methods 2.1. Patient population We studied 960 coronary segments (CS) of 32 consecutive autopsies of 2 group of patients who have died at the Policlinico of the University of Rome Tor Vergata: 17 patients died from AMI (AMI group, 10 males/7 females, mean age 68.8 9.5 years) and 15 agematched control patients without positive cardiac history (CTRL group, 8 males/7 females, mean age 75.4 12.6 years) who died from non-cardiac causes and in whom at least one coronary showed a cross-sectional luminal stenosis >50%. In the AMI group, the time interval between symptom onset and death was less than or equal to 72 h for all cases. Clinical history, electrocardiographic findings and positive cardiac enzymes defined the presence and the localization of acute myocardial infarction. This diagnosis was then confirmed by the histological analysis. All autopsies were performed within 12e24 h of death. The study met the criteria of the code of proper use human tissue that is used in Italy for the use of human tissue. 2.2. Tissue handling and processing The three major epicardial coronary arteries (left anterior descending, left circumflex and right coronary arteries) were carefully dissected for the entire length from the origin and fixed with buffered formalin. All CS were cut transversely at 5 mm intervals. Ten segments were examined for each coronary artery, in particular 510 CS of patients who died from AMI and 450 CS of patients of the control group. Coronary segments from patients died from AMI were subdivided into two additional groups, (a) infarct related coronary arteries and (b) non-infarct related coronary arteries. The myocardium was macroscopically examined to detect the presence and extent of the infarcted area. In all cases, at least one complete transverse heart slice was sampled. Multiple myocardial samples were processed for histopathologic examination and the infarction confirmed by light microscopy. All samples were paraffin-embedded. For histopathologic examination, arterial sections were stained with hematoxylin and eosin and Movat s pentachrome stain. A immunohistochemical study was also performed in order to characterize and quantify inflammatory cells of the plaques using CD68 (anti-human macrophages; Dakopatts, Denmark) and CD3 (anti-human T cell; Dakopatts) monoclonal antibodies. 2.3. Histopathologic and morphometric studies Plaques were classified into three categories, according to the modified AHA atherosclerosis classification [16]: (1) unstable plaques, (2) stable ones and (3) pre-atherosclerotic lesions. Unstable lesions included both (a) culprit plaque characterized by the presence of an acute thrombus associated with plaque rupture or plaque erosion and (b) vulnerable plaques or thin fibrous cap atheromata, characterized by a lipid-rich core covered by a less than 65 mm thick fibrous cap containing many lipid-laden macrophage foam cells (>25 per high-power magnification). In this group we also included the calcified nodule corresponding to a lesion characterized by an eruptive, dense area of calcium protruding in the lumen. Stable plaque included both fibrous cap atheromata and fibrocalcific plaques. Fibrous cap atheromata was characterized by a large lipid-necrotic core containing extracellular lipid, cholesterol crystals and necrotic debris, covered by a thick fibrous cap with few inflammatory cells. Fibrocalcific plaques consisted mainly of fibrous tissue with large calcification. Pre-atherosclerotic lesions included the diffuse intimal thickening (DIT) and the pathological intimal thickening (PIT). Calcification was divided into (a) microcalcification if constituted only by spot <10 mm occupying <5% of cross-sectional plaque area and (b) macrocalcification if represented by large calcific plate 5% of plaque area. In each CS the following variables were recorded: (a) lumen area (L); (b) internal elastic lamina (IEL) area; (c) plaque area [IEL L]; (d) percentage of luminal stenosis [(IEL L)/IEL 100]; (e) crosssectional of calcification (CA) and necrotic lipidic core (LC); (f) the relative area of calcification (%CA) as [CA/plaque area 100] and that of necrotic lipidic core (%LC) as [LC/plaque area 100]; (g) the minimum thickness of the cap; (h) the minimum distance of calcification from the lumen. Cross-sectional images were acquired by a Nikon digital camera connected to a computer. Areas were measured by using the Scion Image program (Scion Corporation) for morphometric analysis. In order to determine hypertensive damage (irrespectively of type and amount of antihypertensive drug usage), arterial thickening was measured in the renal parenchyma [17]. Approximately 20 arteries 150e500 mm in diameter were analyzed from each kidney and the arterial histopathologic changes scored as following: 1: arteries and arterioles essentially free of intimal thickening; 2: focal mild intimal thickening; 3: concentric intimal thickening less than or equal to the thickness of the media; 4: concentric intimal thickening greater than the thickness of the media without concentric elastic duplication; 5: concentric intimal thickening greater than the thickness of the media with concentric elastic duplication in 3 or more vessels examined. Scores 4 and 5 were considered as indicator of chronic hypertensive status. The presence of other kidney diseases was also recorded. 2.4. Statistical analysis Data were analyzed by SPSS 14.0 (Statistical Package for the Social Sciences) software. Continuous and categorical variables are expressed as mean SD or SE and as frequency values and proportions, respectively. Pearson s chi-square test and Fisher s exact test were utilized to assess possible differences of dichotomous variables between plaques of the various groups examined. The means of normally distributed data were compared with Student t test. In the other cases the groups were compared with Manne Whitney s U test. Correlations between histologic measurements were made using a bivariate linear regression model. Multivariable linear regression analysis was performed to determine the morphological features associated to the presence of an unstable plaque and r 2 was computed using the unstable plaque as the only independent variable. A p-value of <0.05. 3. Results 3.1. General findings No differences were observed between AMI and CTRL groups for age, gender, distribution of major risk factors (hypertension, hyperlipidemia, smoking, diabetes) and renal pathology (Table 1). Myocardial histopathologic examination confirmed an acute transmural infarct as cause of death in all patients who died from AMI. Myocardium from CTRL group of patients showed neither infarct nor small necrosis in all cases. The cause of death in the CTRL group was bronchopneumonia in 8, bowel infarction in 2, pulmonary embolism in 4 and cerebral hemorrhage in 1 (Table 1). The 960 analyzed CS were constituted by 201 pre-atherosclerotic plaques (20.9% of cases), 705 stable plaques (73.4%) and 54 unstable plaques (5.7%). In the latter group, 37 plaques were vulnerable and

126 A. Mauriello et al. / Atherosclerosis 229 (2013) 124e129 Table 1 Baseline characteristics of patients. AMI group (17 patients) CTRL group (15 patients) Age, yrs (mean SD) 68.8 þ 9.5 75.4 þ 12.6 0.19 Sex e N (%): 0.75 Male 10 (58.8%) 8 (53.3%) Female 7 (41.2%) 7 (46.7%) Major risk factors e N (%): Hypertension 12 (70.6%) 9 (60.0%) 0.53 Diabetes 6 (35.3%) 4 (26.7%) 0.60 Smoke 10 (58.8%) 8 (53.3%) 0.75 Hypercholesterolemia 9 (52.9%) 7 (46.7%) 0.72 Kidney disease e N (%): Arterial thickening 0.53 Scores 1e2 3 (17.6%) 5 (33.4%) Score 3 4 (23.5%) 2 (13.3%) Scores 4e5 10 (58.9%) 8 (53.3%) Extensive glomerulosclerosis 2 (11.8%) 1 (6.7%) 0.62 Chronic pyelonephritis 14 (82.4%) 12 (80.0%) 0.86 Cause of death e N (%): 0.001 AMI 17 (100%) 0 Bronchopneumonia 0 8 (53.3%) Bowel infarction 0 2 (13.3%) Pulmonary embolism 0 4 (26.7%) Cerebral hemorrhage 0 1 (6.7%) 17 showed an acute thrombosis (1.8%), which was associated to a cap rupture in 14 cases and to an erosion in the remaining 3 cases. 32 out of 37 vulnerable plaques were observed in the coronary tree of patients who died from AMI, as well as the remaining 5 in the CTRL group (p ¼ 0.001) (Table 2). Only 1 plaque in the AMI group was constituted by a calcified nodule. The plaques of AMI, as compared to CTRL, showed a greater cross-sectional stenosis (73.8 14.9% vs. 52.1 13.3%, p ¼ 0.001), p were significantly more inflamed both in the cap and in the plaque shoulder (p ¼ 0.001) and had a greater lipidic-necrotic core (p ¼ 0.001) (Table 2). 3.2. Calcification Calcification was observed in 350 out of 960 CS (36.5%), in 107 cases (11.1%) as calcific spots (microcalcification) occupying less than 5% of the plaque area, while in the remaining 243 cases (25.4%) wide calcific plates were found (Table 2). In the AMI group 47% of CS showed calcification while in CTRL patients this feature was found only in 24.5% of analyzed segments (Table 2). The extension of calcific area was significantly higher in AMI patients compared to CTRL group (8.3 12.0% vs. 4.3 9.0%, p ¼ 0.001), while distance of calcification from the vascular lumen was similar (Table 2). Moreover, no significant differences were observed in calcification crosssectional area between coronary segments supplying the infarcted myocardium and those supplying normal myocardium (7.1 10.3% vs. 9.2 13.2%, p ¼ 0.08). A significant correlation was observed between the area of calcification and plaque area (r 2 ¼ 0.03; p ¼ 0.001). Similarly, the degree of lumen stenosis showed a positive correlation with the extension of calcification (r 2 ¼ 0.13; p ¼ 0.007), the latter being significantly larger in CS with >70% stenosis. 3.3. Plaque instability and calcification No statistical correlation was found between calcification and the presence of unstable plaques (thrombotic or vulnerable). In particular, calcification was present in 22 out of 54 unstable plaques (40.8%) and in 327 out of 705 stable plaques (46.3%; p ¼ 0.24). Similarly, the relative area of the plaque occupied by calcification was significantly greater in stable plaques than in unstable ones Table 2 Histological findings. AMI group (510 Coronary segments) CTRL group (450 Coronary segments) Plaques in the coronary segments (N,%) 0.001 Pre-atherosclerotic 47 (9.2%) 154 (34.2%) DIT 0 73 PIT 47 81 Stable 414 (81.2%) 291 (64.7%) Fibroatheromata 272 184 Fibrocalcific 142 107 Unstable 49 (9.6%) 5 (1.1%) Ruptured 14 0 Erosion 3 0 Vulnerable 32 5 Cross-sectional lumen 73.8 ± 14.9 52.1 ± 13.3 0.001 stenosis (% SD) Cross-sectional plaque 3.5 ± 1.7 2.8 ± 1.5 0.02 area (mm 2 SD) Calcification Cross-sectional area 0.6 ± 1.1 0.1 ± 0.3 0.001 (mm 2 SD) Relative cross-sectional 8.3 ± 12.0 4.3 ± 9.0 0.001 area (% SD) Distance from the 339.3 ± 239.5 235.4 ± 237.8 0.21 lumen (mm SD) Type (N,%) Microcalcification 67 (13.1%) 40 (8.9%) 0.001 Macrocalcification 173 (33.9%) 70 (15.6%) Lipidic-necrotic core (% SD) 38.3 ± 24.6 24.0 ± 17.9 0.001 Cap thickness (mm SD) 224.3 ± 185.9 215.6 ± 156.5 0.58 Plaque inflammation (cell mm 2 SD) In the cap 74.4 ± 43.6 29.3 ± 25.2 0.001 In the shoulder 137.1 ± 66.2 33.8 ± 60.1 0.001 P

A. Mauriello et al. / Atherosclerosis 229 (2013) 124e129 127 Fig. 1. Coronary plaques of a patient who died from acute myocardial infarction. Panel A: Vulnerable plaque ( thin fibrous cap atheromata ) characterized by a large lipidic-necrotic core associated with a thin inflamed fibrous cap. A small rupture was present in the shoulder of the plaque (arrow). No calcifications were observed (Movat, 2). Panel B: Plaque rupture with ulceration consisting of an excavated necrotic core with discontinuation of the fibrous cap and a luminal occluding thrombus (Movat, 2). Panel C: A stable fibrocalcific plaque mainly constituted by fibrous tissue with a large calcification (Movat, 2). Panel D: A stable healed lesion constituted by distinct layers of dense collagen interspersed with large calcifications (Movat, 2). (8.2 12.1% vs. 4.5 7.2%, p ¼ 0.03). In AMI patients the absolute and relative area of the plaque occupied by calcification was significantly smaller in culprit segments with acute thrombosis (ruptured or erosed) as compared to segments without rupture or erosion (p ¼ 0.03 and 0.004, respectively, Table in supplementary data). An inverse correlation was found between extension of calcification and cap inflammation (r 2 ¼ 0.017; p ¼ 0.003) and between calcification burden and cap thickness (r 2 ¼ 0.025; p ¼ 0.001). In plaques showing only microcalcifications, these were mainly localized close to the media (distance from the lumen: 604.9 57.5 mm). No statistical correlation has been observed between calcific area and extension of lipidic-necrotic core (r 2 ¼ 0.002; p ¼ 0.25). As reported in Table 3, the multivariate regression analysis demonstrated that the presence of a calcification was not correlated with the presence of an unstable plaque (p ¼ 0.65). Similarly also the distance of calcification from the lumen did not represent an instability factor (p ¼ 0.68). The only morphological features associated with the rupture, erosion or vulnerability of the plaques were a small cap thickness (p ¼ 0.001), a large lipidic-necrotic core (p ¼ 0.001) and the high inflammation of the cap (p ¼ 0.001). 4. Discussion The results of our morphologic study confirm that the severity of coronary calcification is closely related to atherosclerotic plaque burden, luminal stenosis and fatal ischemic cardiac events, as previously reported [7,10,11]. A possible explanation that calcification was more prevalent in patients with AMI than in controls could be that in the AMI group the stable or unstable plaques, as compared to pre-atherosclerotic lesions, are more common than in the CTRL group (90.8% vs. 65.8%) (Table 2). Nevertheless, in our study unstable plaques (vulnerable and ruptured ones) showed a significantly lower degree of calcification compared to stable Table 3 Multivariable analysis exploring the association between unstable plaques and plaque features. a Multivariate analysis Regression coefficient 95% CI p-value Plaque area (mm 2 ) 0.005 0.005 to 0.006 0.93 Luminal stenosis (%) 0.041 0.003 to 0.001 0.49 Cap thickness (microns) 0.259 0.001 to 0 0.001 Lipidic-necrotic core (%) 0.250 0.002e0.004 0.001 Calcification (mm 2 ) 0.032 0.040 to 0.025 0.65 Distance lumen-calcification (quartiles) 0.029 0.19 to 0.029 0.68 Cap inflammation (CD68 and CD3 positive cells/mm [2]) 0.240 0e0.001 0.001 Shoulder inflammation (CD68 and CD3 positive cells/mm [2]) 0.004 0e0 0.95 a Unstable plaques include vulnerable plaques, those with cap rupture, erosion and calcified nodule. Quartiles of distance lumen-calcification were: 1: <77 mm; 2: 78e 200 mm; 3: 201e440 mm; 4: >440 mm. r 2 was computed using as unstable plaque as the only independent variable; unstable plaque was coded as 2, and stable plaque as 1.

128 A. Mauriello et al. / Atherosclerosis 229 (2013) 124e129 plaques. An inverse correlation was also present between extension of calcification and degree of cap inflammation, the latter being considered the most important histological marker of coronary plaque instability. Therefore it could be speculated that coronary calcification, although able to identify vulnerable patients at risk of fatal cardiac events, should not be considered as a plaque vulnerability factor. Our data suggest that 2 types of atherosclerotic coronary disease can occur: one stable form not correlated with onset of symptoms, which grows slowly to form large plaques and determining a positive remodeling of the vessel, and a second unstable form at high risk of producing symptomatic rupture, the latter not necessarily being more stenotic. Calcification plays an indirect role in plaque instability and rupture. In particular, the presence of large calcifications in a severely stenotic coronary segment with stable (fibrocalcific) plaques should contribute to the development and the rupture of a vulnerable lesion in a less stenotic adjacent segment, as observed in our cases, inducing changes in mechanical properties and shear stress of the arterial wall [18]. In fact, luminal narrowing causes a modification of the laminar blood flow into a disturbed or oscillatory flow, determining an irregular distribution of wall shear stress in the region distal to stenosis. Various studies demonstrated that a non-laminar flow promotes changes in endothelial gene expression, leukocyte adhesion, enhanced oxidative stress and inflammatory state of the artery wall which together favor plaque rupture [19]. Similarly, vascular segments proximal to a severe stenosis are at risk of instability from a reduction of endothelial shear stress [20,21]. Low endothelial shear stress regulates multiple pathways within the atherosclerotic lesion, promoting an intense vascular inflammation, progressive lipid accumulation with formation and expansion of necrotic core and the development of a vulnerable plaque, that may undergo rupture with subsequent formation of an acute thrombus [20]. Moreover, changes in tensile strain that occur at the interface between calcified and adjacent non-calcified arterial segments could further promote the rupture of a vulnerable plaque [15]. All these factors could explain why plaque instability and calcification, although inversely correlated, are both related to acute cardiac clinical events. The present investigation indicates that although CAC correlates with coronary atherosclerotic plaque burden, CAC do not predict the segment that will undergo rupture. In fact, in patients with acute coronary syndromes plaque rupture frequently occurs in the presence of low-grade stenosis. At least 50% of patients with acute myocardial infarction and thrombosis have underlying plaques with insignificant luminal narrowing [1,2,22]. The greater deposition of calcium within an artery of patients with AMI makes the arterial wall more stiff and less expandable favoring at the same time the instability of segments mildly stenotic and with less or no calcification. Multivariate analysis showed that the only morphological features associated with an unstable plaque were a small cap thickness (p ¼ 0.001), a large lipidic-necrotic core (p ¼ 0.001) and the high inflammation of the cap (p ¼ 0.001), but not the calcification (Table 3). Previous studies by imaging methods demonstrated that small or spotty calcification, detected by IVUS or by optical coherence tomography (OCT) is a characteristic of vulnerable plaque [13,23]. Our morphological study does not confirm these observations because microcalcification were observed only in 13.0% (7/54) and macrocalcification in 27.8% (15/54) of vulnerable plaques. Current diagnostic methods to detect CAC are usually traditional coronary angiography, IVUS, electron beam computed tomography (EBCT) and multi-slice computed tomography (MSCT). Many studies based on these methods demonstrated a significant correlation between CAC score and coronary atherosclerotic burden [10,11,24]. Therefore, it is not surprising that numerous studies have shown that a high CAC score is a marker of increased risk of coronary events. Nevertheless, the correlation between CAC score and the presence of unstable plaques only occasionally has been evaluated in these studies. Since the onset of acute cardiac symptoms is due to the rupture of a vulnerable plaque, only the identification of vulnerable coronary lesions is the corner stone for the prevention of clinical events. The present study has the limit that it was performed in dead patients. Nevertheless a detailed histological study of the whole coronary tree can be made only in autoptic series. Moreover the small number of vulnerable lesions could have caused a possible underreporting limiting the scope of the multivariate analysis. In conclusion, the results of our study confirm that CAC score evaluation represents a valid method to define the generic risk of acute coronary clinical events in a population, but it is not useful to identify the vulnerable plaque that will potentially lead to symptoms onset and needs to be treated in order to prevent an acute event. To reach this goal it should be helpful to associate with the evaluation of CAC score new imaging techniques, such as spectroscopy, able to identify in vivo chemical composition of vulnerable plaques. Appendix A. 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