Relationship between Calcification and Vulnerability of the Carotid Plaques

Accepted Manuscript Relationship between calcification and vulnerability of the carotid plaques Rodolfo Pini, Gianluca Faggioli, Silvia Fittipaldi, Francesco Vasuri, Matteo Longhi, Enrico Gallitto, Gianandrea Pasquinelli, Mauro Gargiulo, Andrea Stella PII:

S0890-5096(16)31293-6

DOI:

10.1016/j.avsg.2017.04.017

Reference:

AVSG 3345

To appear in:

Annals of Vascular Surgery

Received Date: 28 November 2016 Revised Date:

4 March 2017

Accepted Date: 4 April 2017

Please cite this article as: Pini R, Faggioli G, Fittipaldi S, Vasuri F, Longhi M, Gallitto E, Pasquinelli G, Gargiulo M, Stella A, Relationship between calcification and vulnerability of the carotid plaques, Annals of Vascular Surgery (2017), doi: 10.1016/j.avsg.2017.04.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Relationship between calcification and vulnerability of the carotid plaques

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Authors: Rodolfo Pinia, Gianluca Faggiolia, Silvia Fittipaldi b, Francesco Vasuri b, Matteo Longhia, Enrico Gallittoa, Gianandrea Pasquinelli b, Mauro Gargiuloa, Andrea Stellaa.

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S. Orsola-Malpighi Hospital, Bologna University, Via Massarenti 9, I 40139 Bologna, Italy

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Corresponding author

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Rodolfo Pini

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: vascular surgery, Bologna University, “alma Mater Studiorum”

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: department of Experimental, Diagnostic and Specialty Medicine (DIMES);

[email protected]

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Tel.: +39 051 6364244; fax: +39 051 6363288.

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Policlinico S. Orsola, via Massarenti 9, 40138 Bologna, Italy

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No financial disclosure for any author

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No conflict of interest for any author

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Article Type: Original research

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Short title: calcification in carotid plaque

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Keywords: calcification, carotid plaque, stroke, specimen, histology

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Word count: 2281

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ACCEPTED MANUSCRIPT Introduction. Carotid plaques with a high degree of calcification are usually considered at low

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embolic risk. However, since a precise evaluation of the extent of calcification is not possible

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preoperatively through duplex ultrasound and postoperatively by conventional histological

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examination due to the de-calcification process, the relationship between the amount of calcium

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involvement and plaque vulnerability has not been evaluated yet. This study aims to correlate the

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extent of carotid plaque calcification with clinical, radiological and histological complications.

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Methods.

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endarterectomy between January to December 2014 were included in the study. The amount of

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carotid calcification was assessed at preoperative computed tomography (CT) through measurement

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of thickness and circumferential calcium extension and graded from 1 to 8 accordingly (Babiarz

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classification). Patients were then categorized into two groups (low-level: grade 1-5; high-level:

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grade 6-8) and correlated with clinical characteristics and ipsilateral cerebral ischemic lesions at

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CT. Vulnerability of the plaque was assessed histologically according with American Heart

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Association-AHA Classification. Results were overall blindly correlated.

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Results. One-hundred-five patients (81% male; age 73±8 years) were enrolled in the study. Forty

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(38%) were symptomatic and 43 (40%) had an ipsilateral focal lesion at preoperative cerebral CT.

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Thirty-six (38%) patients had high-level carotid calcification degree at CT scan. At histological

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analysis 56 (56%) plaques were considered complicated (AHA type VI). Patients with high-level

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and low-level carotid calcification had similar epidemiological risk factors, preoperative

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neurological symptoms and histological complications (17% vs. 15%, p=.76 and 50% vs. 55%,

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p=.62, respectively). The high-level calcification group showed a significantly higher incidence of

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ipsilateral cerebral lesions at preoperative CT (56% vs. 32%, p=.01).

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Conclusion. A high level of calcification of the carotid plaque is not necessarily associated with

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lower vulnerability: the incidence of preoperative neurological symptoms and histological

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complications is similar in patients with and without extensive carotid plaque calcification. Cerebral

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ischemic lesions may be even more frequent in presence of highly calcified plaques.

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ACCEPTED MANUSCRIPT Introduction

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Large randomized, controlled trials have demonstrated the benefit of carotid endarterectomy (CEA)

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in patients with hemodinamically significant stenosis of the bifurcation. Nevertheless, since most

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asymptomatic patients remain stroke-free with the sole medical therapy, many authors suggest

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carotid revascularization only when elements of vulnerability are evident in the carotid plaque1. As

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a matter of fact, morphological characteristics of the plaque may be more important than the degree

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of stenosis in the prediction of future strokes, thus identification of plaques at high embolic risk

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may improve treatment strategies2.

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Calcification is a prominent structural feature of advanced atherosclerotic plaques, and is readily

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detected with spiral CT3. Most studies have focused on the mechanisms involved in calcification of

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the coronary arteries4 and the correlation of the arterial wall calcification with plaque extension5,

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however, the role of calcification in plaque stability remains unclear. Although B-mode ultrasound

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is being increasingly used in order to identify rupture-prone unstable plaque, characterization of

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overall plaque calcification is imprecise with this technique 6-7.

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While definite histopathologic features, such as a large lipid-rich necrotic core with thin fibrous

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cap, extensive macrophage infiltration, and paucity of smooth muscle cells8 are associated with

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vulnerability, the role of calcification is less clear.

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The purpose of our study was therefore to quantify the carotid artery bifurcation calcification in

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symptomatic and asymptomatic patients by using multidetector-row CT and determine its

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relationship with clinical symptoms and histopathologic features of plaque instability.

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ACCEPTED MANUSCRIPT Methods

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Study design and setting

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Symptomatic and asymptomatic consecutive patients undergoing carotid endarterectomy between

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January to December 2014, at an academic center, were prospectively entered in the study. The

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inclusion criteria were: the availability of a computed tomography (CT) angiography of the neck

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and head performed no more than 1 month before the carotid intervention and the histological

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suitability of the atherosclerotic carotid specimens. The exclusion criteria were: non-atherosclerotic

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plaques, restenosis, and previous radiotherapy of the neck or head. All patients gave their informed

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consent for the procedure. The study was performed according to the rules of the Ethical Review

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Board of our institution.

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The primary end-point was to compare the clinical characteristics and histological elements of

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vulnerability (histological complications) in high and low calcified carotid plaque at preoperative

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CT.

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Patients

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Carotid revascularization was performed for asymptomatic (>60%) or symptomatic (>50%) carotid

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artery stenosis (according to the North American Symptomatic Carotid Endarterectomy Trial

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Collaborators [NASCET]9 criteria) fulfilling the requirements of Society for Vascular Surgery

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guidelines. The asymptomatic status was defined as absence of any cerebral or retinal events

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ipsilateral to the carotid stenosis in the past 6 months as in the ACST trial10 by clinical

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investigation, in cases of doubts an adjunctive evaluation by in-hospital neurologist was obtained.

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Clinical characteristics, technical aspects and perioperative (30-day) outcome were entered into a

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dedicated database as previously described11-12. The clinical characteristics included the following:

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age; sex; hypertension; dyslipidemia; diabetes mellitus; current smoking; coronary artery disease;

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ACCEPTED MANUSCRIPT chronic obstructive pulmonary disease; chronic renal failure (glomerular filtration rate <60 ml/min).

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Perioperative medical therapy was also considered: specifically, antiplatelet therapy, statins and oral

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anticoagulant therapy. All patients had a preoperative cerebral CT scan evaluation as a routinely

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standard procedure in our institution before carotid revascularization13-14; CEA was performed

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according to the Society for Vascular Surgery guidelines15. The CT angiography of the neck and

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head was at discretions of the physician requests in the preoperative period.

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Trained vascular surgeons performed all CEA procedures (under general anesthesia with routine

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shunting and Dacron patching). The 30-day stroke and death rate were collected.

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Carotid calcification evaluation at computed tomography

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The amount of carotid calcification was assessed at preoperative carotid plaque CT, performed at

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least 1 month before the carotid intervention, through measurement of thickness and circumferential

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calcium extension and graded from 0 to 4 respectively, accordingly with Babiarz classification16-17.

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The total amount of calcification (by the sum of the thickness and circumferential scores) ranged

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from 0 to 8 and patients were then categorized into two groups: low-carotid calcification (grade 0-5)

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and high-carotid calcification (grade 6-8), figure 1.

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Cerebral ischemic lesions evaluation at computed tomography

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Cerebral CT scan were evaluated by an experienced radiologist, as previously reported, and any

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ischemic damage was classified using the Stevens classification18, were considered for the analysis

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only the cerebral lesion ipsilateral to the stenosis and considered of embolic origin (type from 1 to

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4):

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1. (Embolic) Discrete subcortical were small, well-circumscribed hypodense lesions, usually only a little greater than 1 cm in size, adjacent to apparently non involved cerebral cortex in

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entire anterior and middle cerebral artery territories. 3. (Embolic) Small cortical infarcts had cortical distribution occupying usually substantially

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2. (Embolic) Large cortical infarcts had cortical distribution occupying usually >50% of the

<50% of the entire anterior and middle cerebral artery territories.

4. (Embolic) Basal ganglia infarcts were one or more circumscribed lesions in the basal ganglia or thalami, usually about 1 cm.

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the anterior and middle cerebral artery territory.

5. (Non-embolic) Watershed infarction involved cortical and subcortical regions at the

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periphery of the middle cerebral artery supply territory.

6. (Non-embolic) Diffuse white matter low-density changes were areas of poorly

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circumscribed low density in the cerebral white matter, usually bilaterally.

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7. (Non-embolic) Lacunar state was multiple hypodensities in the basal ganglia and thalami, bilaterally.

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Histological carotid plaque evaluation

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Histologic analysis was performed as previously reported. Carotid plaques were removed in full

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during surgery to preserve the plaque structure. Decalcification (up to 6 hours) was applied if

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requested, samples were cut in serial sections and the area with the highest percentage of stenosis

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was identified and defined as the area of interest for further analysis. Plaque tissue samples were

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fixed in formalin buffered 4% and embedded in paraffin; 5-µm-thick haematoxylin and eosin-

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stained sections were observed under a light microscope (LM, Olympus CX42). Carotid

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atherosclerotic lesions were defined according to the original American Heart Association (AHA)

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classification of 1995, and grouped as type I-VIII19,20,21. Histological features examined were: lipid

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core, neoangiogenesis and inflammation (scored from 0 to 5) and the maximum and minimum

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thickness of the fibrous cap. In according to AHA classification a vulnerable and rupture prone

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plaque was definite as AHA type VI, complicated with hemorrhage, surface defects and/or

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thrombus elements. An experienced pathologist performed all histopathological analysis.

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Statistical analysis

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Continuous data are expressed as mean ± standard deviation (SD) and categorical variables by

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relative and absolute frequencies. Analysis of differences between groups was performed with X2

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test for categorical variables and with unpaired Student’s t-test for continuous variables. Linear

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regression was performed to evaluate a possible correlation between the values of histological

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features and the calcification score at carotid CT. The sample size evaluation was performed to

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speculate a possible clinical benefit from the identification of calcific plaque as a protective factor

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from plaque vulnerability: with the hypothesis of 10% of vulnerability in “high calcific” plaques

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and 40% of vulnerability in “low calcific” plaques the whole sample – for an alpha error of 5% and

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beta error of 20% - was calculated in 80 specimens. The value of p < 0.05 was considered

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significant. Statistical tests were performed using Statistical Package for Social Sciences (SPSS

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22.0) for Windows computer software (SPSS, Chicago, IL, USA).

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Results

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In a one-year period out of 208 CEA procedures performed, 105 patients met the inclusion criteria

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for the present study. No postoperative complication (stroke or death) occurred in the present

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population. General clinical characteristics are reported in table I, in particular 80% of patients were

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male and the mean age of all population was 73±8 years; forty (38%) were symptomatic – stroke

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and TIA - and 42 (40%) had an ipsilateral pre-operative cerebral ischemic lesion at CT-scan.

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The calcium total score evaluated in the whole population was 4.5±2.5 and 36 (34%) patients were

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considered at high-level carotid calcification (calcium total score ≥ 6).

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The presence of high calcific plaque was not significantly associated with clinical characteristics or

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medical therapies. In the neurological status evaluation, the high-calcific plaque was not associated

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with preoperative symptoms (TIA or stroke), and time elapsed from surgery but the presence of

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preoperative ipsilateral cerebral ischemic lesion was significantly associated with the presence of

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high-calcific plaque (48% vs. 32%, P=.01), table I.

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Clinical characteristics and CT calcification evaluation

Histological characteristics and CT calcification

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All the 105 specimens were suitable for the histological analysis. Table II showed the distribution

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of histological plaque characteristics. In particular, 56 (53%) were considered complicated plaques

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(AHA type VI). The evaluation by linear regression of the carotid calcium total score by CT and the

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histological elements was associated with a trend to inverse correlation with lipid core extension

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(Fig. 2 and 3). No correlation was identified between the histological elements and the high and low

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calcific plaques, with the exception of the lipid core that was less extended in the high calcific

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plaques compared with low-calcific plaques (1.1±1.3 vs. 2.1±1.1, P=.01, respectively), table II. The

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distribution of histological complications (grade VI of AHA classification) was similar between

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high and low calcific plaques (50% vs. 55%, P=.62, respectively).

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ACCEPTED MANUSCRIPT Discussion

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This study aimed to correlate the extent of carotid plaque calcification with clinical, radiological

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and histological complications. For that reason, we used CT angiography to quantify the degree of

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carotid plaque calcification in 105 symptomatic and asymptomatic consecutive patients submitted

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to carotid endartectomy. The importance of the calcification scoring by the CT is due to the high

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sensibility of this technique to calcium; moreover the evaluation of calcification by CT imaging

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allows a more accurate histological evaluation of the element of plaque vulnerability because of the

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method of carotid specimens processing needs a de-calcification phase that not enable to a correct

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calcification evaluation20. The presence of high calcific plaque was not significantly associated with

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preoperative neurological symptoms or medical therapies. The distribution of histological

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complications (grade VI of AHA classification) was similar between high and low calcific plaques:

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50% in the high calcific and 56% in low calcific plaque. These values are very far from the

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hypothetical clinical significance speculated in the evaluation of the sample size: 10% in the high

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calcific and 40% in low calcific plaque.

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Calcification is frequently encountered in atherosclerotic disease, and has been proposed as

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a stabilizing factor rather than a risk factor for carotid plaque instability by some authors, however

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the results of the present study and of other authors lead to different conclusions. The relevance of

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atherosclerotic plaque calcification to plaque structural stability has been investigated most

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commonly in the coronary arteries22. Coronary calcific deposits detected with CT appear to be

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associated with an increased risk of coronary events23. However, the relationship between the

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amount of calcium involvement and plaque vulnerability has not been extensively evaluated yet in

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the carotid plaque. Kwee25 reported that clinically symptomatic plaques have a lower degree of

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calcification than asymptomatic plaques, however his systematic review was biased by a significant

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level of heterogeneity in the calcification evaluation. Shaalan et al.26 observed a strong inverse

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correlation between the extent of carotid plaque calcification and the intensity of plaque fibrous cap

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inflammation as determined by the degree of macrophage infiltration, and proposed the spiral CT

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as a quantitative marker for cerebrovascular ischemic event risk. Differently from these experiences, some other authors failed to identify an element of

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plaque stabilization in the carotid calcification, similarly to the present work. Collett and Canfield27

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analyzed the correlation of calcification with neoangiogenesis within the plaque, finding a direct

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correlation between spot calcification and micro-vascularization. Those authors suggested that

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angiogenic factors can influence the bone and cartilage formation through the cytokines released by

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endothelial cells, which can induce the differentiation of osteoprogenitor cells. New at al.28 in the

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analysis of 136 carotid plaques, correlated the amount of calcification with the presence of

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macrophage. Similarly, Eisenmenger et al.29 investigated the grade of carotid calcification by CT

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angiography and identified a high correlation with intraplaque hemorrhage. Other authors reported

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data from the Rotterdam study. Van den Bouwhuijsen et al.30 analysed 329 carotid specimens and

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identified a correlation between carotid calcification and intraplaque hemorrhage; the authors

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concluded that different types of calcification are possible and some of them are more frequently

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associated with plaque hemorrhage compared with others. Selwaness et al.31 by evaluating 1731

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carotid specimens, found no correlation between the extent of plaque calcification and neurological

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symptoms, similarly to the present work. Moreover, in the evaluation of two large atherosclerotic

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carotid plaque biobank, involving more than 1600 carotid plaques, no relationship between carotid

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calcification and new cerebral symptoms was found32.

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Giving all these premises, a high level of carotid calcification cannot be considered an

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element of plaque stability. Our work showed another interesting result, such as a correlation

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between highly calcified plaques and presence of cerebral ischemic lesion at CT (56% vs. 32%,

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p=.01) which is known to be associated with high risk for cerebral event in patients with carotid

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stenosis33.

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two different ways. In the first one there can be an evolution of an intraplaque hematoma

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determining a vulnerable plaque; in the second case the calcification is caused by the macrophage

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osteoblastic activity, which leads to a more stable plaque.

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The present study suffers from some limits. First, the population studied is heterogeneous,

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with both symptomatic and asymptomatic patients enrolled; however this heterogeneity can be

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useful to compare the specimens according to the previous neurological symptoms and to consider

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the clinical vulnerability of the plaque. Next, many of carotid revascularizations were performed

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after two weeks from symptoms; consequently the preoperative CT scan data may not be precisely

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representative of the actual carotid specimens.

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In conclusion, a high level of carotid plaque calcification is not necessarily associated with a

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low grade of vulnerability; as a matter of fact, the incidence of preoperative neurological symptoms

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and histological vulnerability is similar in patients with and without extensive carotid plaque

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calcification. At present, the simple evaluation of the extension of carotid plaque calcification

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cannot be considered a valid element to stratify the risk of vulnerability.

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disease: correlations between plaque composition and ipsilateral stroke risk. Stroke.

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2015;46(1):182-9.

348

33. Streifler JY, den Hartog AG, Pan S, Pan H et al.; ACST-1 trial collaborators. Ten-year risk

349

of stroke in patients with previous cerebral infarction and the impact of carotid surgery in

350

the Asymptomatic Carotid Surgery Trial. Int J Stroke. 2016;19.

351 352 353 354

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Table I: general clinical characteristic and comparison between High and Low CT calcium score patients

357 High CT calcium % (n:36)

Low CT calcium % (n: 69)

P

73±8

72±8

73±7

.34

Male sex

81 (85)

72 (26)

Hypertension

94 (99)

89 (32)

Dyslipidemia

71 (75)

78 (28)

Diabetes mellitus

33 (35)

28 (10)

Smoke

16 (17)

17 (6)

Coronary artery disease

32 (34)

28 (10)

COPD

.10

97 (67)

.08

68 (47)

.29

36 (25)

.38

16 (11)

.92

35 (24)

.46

0 (0)

3 (2)

.30

11 (4)

9 (6)

.68

61 (22)

62 (43)

.90

6 (2)

3 (2)

.50

96 (101)

100 (36)

95 (65)

.85

40 (42)

56 (20)

32 (22)

.01

38 (40)

39 (14)

38 (26)

.90

15 (16)

17 (6)

15 (10)

.76

23 (24)

22 (8)

23 (16)

.91

40 (15)

28 (4)

46 (12)

.28

2 (2)

Chronic renal failure

10 (10)

Medical therapy Statins

62 (65) 4 (4)

Anti-platelet therapy Cerebral status

Stroke TIA

AC C

Neurological symptom

EP

Cerebral ischemic lesion on CT

TE D

Oral anticoagulants

Timing before intervention

SC

85 (59)

M AN U

Age

≤2 weeks (between symt pts) High calcium score at CT

34 (36)

358 359 360

RI PT

Total: 105 % (n)

Clinical characteristics

COPD: chronic obstructive pulmonary disease; CT: computed tomography

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Table II: histological characteristics of carotid plaques and comparison between High and Low CT calcium score patients

363 High CT calcium % (n:36)

Low CT calcium % (n: 69)

P

53 (56)

50 (18)

55 (38)

.62

Max fibrous cap µm

1061±398

1190±325

Min fibrous cap µm

235±196

304±262

Lipid core

1.8±1.4

1.1±1.3

Inflammation

1.6±1.3

1.4±1.2

Neoangiognesis

1.2±1.2

1.0±1.2

365

M AN U

364

AHA: American heart association CT: computed tomography

366

AC C

EP

TE D

367

1001±417

.06

230±155

.14

2.1±1.1

.01

1.7±1.4

.20

1.3±1.1

.20

SC

AHA VI plaque

RI PT

Total: 105 %

Histological characteristics

ACCEPTED MANUSCRIPT Figure 1: example of highly calcified (panel A) and scarcely calcified carotid plaque evaluated at

369

CT angiography.

370

Figure 2: linear regression analysis of the calcium total score evaluated by computed tomography

371

and lipid core score (panel A), inflammation score (panel B) and neoangiogenesis score (panel C).

372

Figure 3: linear regression analysis of the calcium total score evaluated by computed tomography

373

and minimum (panel A) and maximum (panel B) thickens of the fibrous cap.

RI PT

368

AC C

EP

TE D

M AN U

SC

374

AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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