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\begin{document}

\title{The Role of Magnetic Resonance Imaging and the Expression of MMP-9
Protein in the Analysis of Carotid Atherosclerotic Plaques in Patients
Undergoing Carotid Endarterectomy: A Prospective Pilot Study}



\author[1]{Davorin Sef}%
\author[1]{Milan Milosevic}%
\author[1]{Marin Ostric}%
\author[1]{Tomislav Mestrovic}%
\author[1]{Bojan Jernej}%
\author[1]{Slavica Kovacic}%
\author[1]{Miljenko Kovacevic}%
\author[1]{Anita Skrtic}%
\author[1]{Vinko Vidjak}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Components of carotid atherosclerotic plaque can be analysed
preoperatively by non-invasive advanced imaging modalities such as
magnetic resonance imaging (MRI). The expression of matrix
metalloproteinase-9 protein (MMP-9), which has a potential role in
remodelling of atherosclerotic plaques, can be analysed
immunohistochemically. The aim of the present prospective pilot study is
to analyse histological characteristics and expression of MMP-9 in
carotid plaques of patients undergoing carotid endarterectomy (CEA) and
to investigate the correlation with preoperative clinical symptoms and
MRI features. Preoperative clinical assessment, MRI imaging,
postoperative histological and immunohistochemical analyses were
performed. Fifteen patients with symptomatic (7/15; 47\%) and
asymptomatic carotid artery stenosis undergoing CEA were included. Among
symptomatic patients, 5 (71\%) had recent stroke and 2 (29\%) had recent
transient ischaemic attack with a median timing of 6 weeks (IQR:1, 18)
before the surgery. Both groups did not significantly differ in respect
to preoperative characteristics. Prevalence of unstable plaque was
higher in symptomatic than asymptomatic patients, although it was not
significant (63\% vs. 37\%, \emph{p}=0.077). The expression of MMP-9 in
CD68 cells within the plaque by semiquantitative analysis was found to
be significantly higher in symptomatic as compared to asymptomatic
patients (86\% vs. 25\% with the highest expression, \emph{p}=0.014).
The average microvascular density was found to be higher and lipid core
area larger among both symptomatic patients and unstable carotid plaque
specimens, although this did not reach statistical significance
(\emph{p}=0.064 and \emph{p}=0.132, \emph{p}=0.360 and \emph{p}=0.569,
respectively). Our results demonstrate that MRI is reliable in
classifying carotid lesions and differentiating unstable from stable
plaques. We have also shown that the expression of MMP-9 is
significantly higher among symptomatic patients undergoing CEA.

~

Trial Registration: This study has been registered at the ISRCTN
registry (ID ISRCTN46536832), isrctn.org Identifier:
https://www.isrctn.com/ISRCTN46536832

~

Keywords: atherosclerosis, carotid endarterectomy, unstable plaque,
magnetic resonance imaging, matrix metalloproteinase.%
\end{abstract}%



\sloppy






\textbf{1. Introduction}

Carotid artery disease is the principal cause of ischemic stroke and the
benefit of carotid surgical revascularization in both symptomatic and
asymptomatic patients has been demonstrated by several trials {[}1-4{]}.
Atherosclerotic plaque formation is associated with endothelial cells
damage, which leads to lipid accumulation, immigration of
monocyte-derived macrophages, release of growth factors and activation
of vascular smooth muscle cells (VSMC) {[}5{]}. Atherosclerotic plaque
rupture leads to local platelet activation and thrombosis that may
result in embolization and the onset of clinical symptoms {[}6{]}.
Accumulating evidence confirm the key role of matrix metalloproteinase
(MMP) in plaque development and pathogenesis of atherosclerosis
{[}7-9{]}. MMP, among which is MMP-9, are enzymes that are produced by
infiltrating macrophages and VSMC and may have an important role in
plaque remodelling and destabilization by promoting the degradation of
extracellular matrix {[}8-10{]}. Increased serum levels of MMP-9 have
been detected in the coronary circulation among patients with acute
coronary syndrome, which suggested active process of plaque rupture and
increased risk of cardiovascular adverse events {[}11-13{]}. Similarly,
it was found that serum level of MMP-9 was significantly higher in
patients with carotid artery stenosis and unstable carotid plaques
undergoing carotid endarterectomy (CEA) {[}8, 14{]}.
However,\emph{Baroncini et al.} reported that MMP-9 is a part of
atherogenesis process but may not be a factor associated with acute
disruption events {[}15, 16{]}.

Several non-invasive imaging techniques have been evaluated for the
characterization of the carotid plaque because carotid angiography is
able to detect plaque ulceration but is unable to demonstrate plaque
morphology {[}17{]}. Despite the fact that duplex ultrasound is the
first-line imaging modality, magnetic resonance imaging (MRI) has the
advantage to image the aortic arch with supra-aortic arteries as well as
to detect the presence of specific plaque features (vulnerable plaque,
intra-plaque haemorrhage, lipid-necrotic core) suggesting higher stroke
risk {[}18-22{]}. Recent reports suggested that preoperative MRI may be
useful in the detection of the vulnerable plaque and can help in
identifying patients with high risk of cardiovascular events {[}19,
20{]}. Increased MMP-9 expression in plaques with increased neovascular
changes along with MRI scan of carotid arteries was studied but this
analysis was performed in an animal study model.{[}23{]}

The aim of the present prospective pilot study is to analyse
histological characteristics and expression of MMP-9 in carotid plaques
of patients undergoing CEA and to investigate the correlation with
preoperative clinical symptoms and MR features.

\textbf{2. Materials and methods}

\emph{2.1 Study design and patients}

This pilot study is a prospective observational single-blind study of
patients who underwent CEA at the Department of Cardiovascular Surgery
in the Magdalena - Clinic for Cardiovascular Diseases and at the
Department of Vascular Surgery in the University Hospital Merkur in
Croatia over a 3-year study period. Institutional ethic committee
approvals and Zagreb University School of Medicine ethic committee
approval were obtained, and all patients gave their written informed
consent. This study has been registered at the ISRCTN registry with
study ID ISRCTN46536832. The study was conducted as per the Helsinki
Declaration guidelines {[}24{]}. Identity of patients during data
collection was anonymised and protected using a unique patient number.

Clinical history taking and physical examination including a
neurological exam were performed by a surgeon. Clinical data including
history of cardiovascular disease, risk factors for atherosclerosis,
neurological event and timing since the most recent one, current medical
treatment and computed tomography (CT) scans of brain were recorded.
Stroke aetiology was determined based on a work-up including
neurological examination, duplex ultrasound, MRI, CT scan of brain and
echocardiography if required. Routine biochemical tests and highly
sensitive C-reactive protein (hsCRP) were performed preoperatively.
Before surgery, all patients underwent a duplex ultrasound plus MRI to
grade internal carotid artery (ICA) stenosis and assess the supra-aortic
and intracranial arterial system. The severity of carotid artery
stenosis was defined using the North American Symptomatic Carotid
Endarterectomy Trial Collaborators' (NASCET) criteria {[}25{]}.
Inclusion criteria were that the patient should be already scheduled for
CEA based on the indication for surgery with the consent to participate
in the study. CEA was indicated according to the ESVS guidelines and
recommendations of multicentric randomised controlled trials {[}1, 4,
25-27{]}. Exclusion criteria were contraindications to MRI (pacemaker,
metallic implant, claustrophobia), presence of suboptimal MRI
visualization of the carotid plaque or surgical specimen inadequate for
histological or immunohistochemical analysis.

Patients were divided into two groups based on the presence of symptoms
and the timing of the most recent symptom. Symptomatic patients were any
patients who suffered a carotid stenosis territory symptom within the
previous 6 months including ipsilateral stroke, transient ischaemic
attack (TIA) or amaurosis fugax. Patients without any history of recent
neurological symptoms or with nonspecific, non-hemispheric symptoms such
as dizziness or vertigo were considered as asymptomatic.

\emph{2.2. Preoperative magnetic resonance imaging}

MRI was performed using a 1.5-Tesla Vantage (Toshiba Medical Systems,
Tokyo, Japan) or Avanto Scanner (Siemens, Erlangen, Germany) with a head
and neck coil. Patient was placed in a standard dorsal position and the
coil was combined with a head holder to stabilize position and prevent
head rotation. A software \emph{syngo} MR B19 2017 was used. A
standardized MRI protocol was used to obtain four different
contrast-weighted images (time-of-flight {[}TOF{]} and T1-, PD- and
T2-wighted) of the carotid arteries 2 cm proximal and 2 cm distal to the
carotid bifurcation in axial and coronary planes {[}22, 28{]}. Fat
suppression was used to reduce signal from subcutaneous fat tissue. MR
angiography of carotid arteries was performed with 3-dimensional TOF
sequence (repetition time {[}TR{]}/time to echo {[}TE{]}/flip angle
{[}FA{]} = 30 ms/8 ms/20\selectlanguage{ngerman}°; matrix 160x256 mm).

Each carotid lesion was classified using the modified American Heart
Association (AHA) classification of carotid plaques identified by MRI
{[}28{]}. The Modified AHA classification was derived by \emph{Cai et
al.} from experience in reviewing MR images and extensive literature
review {[}28{]}. In this classification, we used: I-II) near-normal wall
thickness with no calcification (AHA classification types I and II were
merged into the type I-II due to a lack of the current resolution of
MRI), III) diffuse intimal thickening or small eccentric plaque with no
calcification, IV-V) plaque with a lipid or necrotic core surrounded by
fibrous tissue with possible calcification (for a similar reason, AHA
classification types IV and V were merged into the type IV-V), VI)
complex plaque with possible surface defect, haemorrhage or thrombus,
VII) calcified plaque, and VIII) fibrotic plaque without lipid core and
with possible small calcifications. According to this modified
classification, types IV-V and VI were classified as unstable plaques,
while the others were classified as stable plaques. MRI criteria for the
diagnosis of plaque vulnerability were thin fibrous cap, large
lipid-rich or necrotic core, and plaque disruption or intra-plaque
haemorrhage. MR images were stored in a digital archive as DICOM files
and independently analysed by two experienced investigators blinded to
the clinical history and histological findings. Any discrepancies were
resolved by discussion between the two investigators.

\emph{2.4. Histological analysis}

The carotid atherosclerotic plaque was excised during CEA using a
standard surgical technique with minimal and atraumatic manipulation of
the specimen previously described by \emph{Sef et al.} {[}29{]}. The
carotid plaque specimen was obtained immediately after the surgical
excision and then stored in fresh 4\% paraformaldehyde solution at 4°C.
The distal end of the plaque specimen was marked and processed by a
standard procedure to obtain pathohistological specimen that includes
fixation with 10\% buffered formalin. The specimen was embedded in
paraffin blocks, 4-\selectlanguage{greek}μ\selectlanguage{english}m-thick cross sections were dewaxed and prepared for
subsequent staining.

The sections were stained with standard haematoxylin-eosin (H\&E) and
with Masson method for detecting collagen tissue which will be
semiquantitatively analysed according to the study by \emph{Verhoeven et
al.} {[}30{]}. Each specimen was cut into 5-mm-long sections from the
central part of the plaque resulting in a total of 5 sections for
histological analysis. Plaque specimens were analysed for lipid, fibrous
tissue and calcium. Lipid core area was expressed as a percentage of the
total plaque area (TPA) as described previously using the following
semiquantitative scale: 0= \textless{}10\% of TPA, 1= 10-40\% of TPA and
2= \textgreater{}40\% of TPA {[}31, 32{]}. Histological examinations
were performed independently by two experienced investigators blinded to
the clinical history and MRI findings. Any discrepancies were resolved
by discussion between the two investigators.

\emph{2.5. Immunohistochemical analysis}

Immunohistochemical analysis of MMP-9 expression was performed in the
central section of the plaque and one of the adjacent sections. Primary
antibodies used in the study were: MMP-9 (Leica Mikrosysteme Vertieb
GMBH, UK, monoclonal, clone 15W2, 1:50 dilution), CD68 PG-M1
(DakoCytomation, Denmark, monoclonal, clone PG-M1, RTU), smooth mucle
actin (SMA) (DakoCytomation, Denmark, monoclonal, clone 1A4, RTU), CD34
(DakoCytomation, Denmark, monoclonal, clone QBend 10, RTU).

Specimen sections were deparaffinized and antigens MMP-9, CD68 PG-M1,
CD34 and SMA were unmasked in PT-module (DakoCytomation, Denmark) at
970\selectlanguage{english}C, 20 minutes in buffer derived from EnVision flex commercial ''3 in
1'' set (K8010, DakoCytomation, Denmark), and were then processed using
a standardised automated procedure in an Autostainer device
(DakoCytomation, Denmark), and stained with haematoxylin. Specimen
sections were used as positive controls for the investigated biomarkers
according to the manufacturer's recommendations. Semiquantitative
analysis of macrophages and SMA by staining with CD68 PG-M1 antibody and
SMA antibody, respectively, was performed according to the study
by\emph{Verhoeven et al.} {[}30{]}. Histological analysis of a type of
bleeding was performed by determining the average density of small blood
vessels by immunohistochemical staining of CD34 as described in the
study by \emph{Derksen et al.} {[}32{]}. The expression of MMP-9 protein
was semiquantitatively analysed on specimen sections in macrophages and
VSMC: 0=negative, 1=rare cells or one cluster of [?]20 cells, 2=less than
5 clusters of [?]20 cells, 3= [?]5 clusters consisted of [?]20 cells or one
cluster [?]100 cells {[}33{]}. Analysis of endothelial cells was performed
over 10 large areas with high magnification up to 400x, and the results
were demonstrated as: 0=negative, 1= \textless{}25\%, 2= 25-50\%, 3=
\textgreater{}50-75\% and 4= \textgreater{}75\% of stained endothelial
cells. The intensity of MMP-9 expression was demonstrated as:
0=negative, 1=visible using 400x magnification, 2=100x magnification and
3=40x magnification. Semiquantitative analysis of plaque specimen,
immunohistochemical staining of macrophages, VSMC, microvascular density
(MVD) and MMP-9 protein was performed using the software ''ToupTekView''
(Copyright \selectlanguage{ngerman}©, version x64 3.7.8481, www.touptek.com).
Immunohistochemical examinations were performed independently by two
experienced investigators blinded to the clinical history and MRI
findings. Any discrepancies were resolved by discussion between the two
investigators.

\emph{2.6. Statistical analysis}

Data were collected prospectively. Categorical variables were tabulated
as numbers with percentages. Continuous variables were shown as median
values, with corresponding IQR. Analysis of the normal distribution of
data (Kolmogorov-Smirnov test) was performed and, according to the
results, appropriate non-parametric statistical analysis was performed.
Differences among continuous variables \selectlanguage{english}\selectlanguage{english}were analysed using t-test and
Mann-Whitney U test. Differences in categorical variables were analysed
using Fisher's exact test. Difference between the groups was considered
significant at the level of 0.05. Statistical analysis was performed
using the licensed software IBM SPSS Statistica (version 25.0.
www.statsoft.com, StatSoft, Inc. 2017).

\textbf{3. Results}

In this prospective pilot study, 15 patients with symptomatic (7
patients, 47\%) and asymptomatic carotid artery stenosis undergoing CEA
were included. Among symptomatic patients, 5 (71\%) had recent stroke
and 2 (29\%) had recent TIA with a median timing of the most recent
event of 6 weeks (IQR: 1-18) before the surgery (Table 1). Demographic
and clinical characteristics including risk factors, NASCET degree of
stenosis, medications and preoperative laboratory results are summarized
in Table 1. Symptomatic and asymptomatic patients did not significantly
differ in respect to preoperative characteristics. Symptomatic patients
had significantly higher hsCRP and D-dimer levels (\emph{p} =0.016
and\emph{p} =0.036, respectively). Mean NASCET degree of stenosis was
higher in the symptomatic as compared to the asymptomatic group (95\%
{[}80-95{]} and 80\% {[}76.3-83.8{]}, \emph{p} =0.021, respectively).
There was no significant difference in the length of hospitalization and
perioperative complications between the two groups, and only one patient
in the symptomatic group had an episode of perioperative TIA which has
recovered after the surgery.

Carotid plaque features detected on preoperative MRI scan are summarized
in Tables 2 and 3. Prevalence of unstable plaque was higher in
symptomatic than asymptomatic patients, although this was not
statistically significant (63\% vs. 37\%, \emph{p} =0.077) (Table 2,
Fig. 1). The most common type of carotid plaque as per modified AHA
classification was type IV-V (10 patients, 65\%) (Table 3).

Differences between preoperative symptoms and histological and
immunohistochemistry features of carotid plaques are demonstrated in
Table 4. The expression of MMP-9 in CD68 cells within the carotid plaque
was found to be significantly higher in symptomatic as compared to
asymptomatic patients (86\% vs. 25\% with the highest
expression,\emph{p} =0.014). In the symptomatic group, 6 out of 7 plaque
specimens had the expression of MMP-9 in \selectlanguage{english}[?]5 clusters consisted of \selectlanguage{english}[?]20
cells or one cluster \selectlanguage{english}[?]100 cells, while this was found only in one
specimen in the asymptomatic group (Table 4, Fig. 2 and 3). It was also
higher in SMA cells in symptomatic patients, but this did not reach
statistical difference (\emph{p} =0.143). In addition, the average MVD
by immunohistochemical staining of CD34 was higher among the symptomatic
than asymptomatic patients (17 {[}6-23{]} vs. 6 {[}2.5-10{]}, although
this did not reach significance (\emph{p} =0.064). Similarly, lipid core
area was larger among the symptomatic than asymptomatic patients,
although it did not reach statistical significance (\emph{p} =0.132).

Differences between MRI features of carotid plaques and histological and
immunohistochemistry features of carotid plaques are demonstrated in
Table 5. There were no significant differences between several
histological features and expression of MMP-9 between the patients with
unstable or stable carotid plaque detected by preoperative MRI, although
both the average MVD by immunohistochemical staining of CD34 and lipid
core area were somewhat higher among patients with unstable than stable
plaque. However, this was not significant (\emph{p} =0.360 and\emph{p}
=0.569, respectively) (Table 5).

\textbf{4. Discussion}

The atherosclerotic plaque is a dynamic lesion that undergoes continuous
remodelling of the extracellular matrix on which its structural
stability depends. Previous studies demonstrate that acute changes
within the carotid plaque, such as IPH, fibrous cap rupture or
ulceration can lead to the onset of ischemic cardiovascular events
{[}6{]}. Recent reports suggest that MMP-9 may have a key role in all
stages of the atherosclerotic process during which are being secreted by
inflammatory cells and VSMC, although there is still lack of evidence
whether MMP-9 can affect the incidence of adverse events {[}7, 8, 34{]}.

The present pilot trial has demonstrated that the prevalence of unstable
plaque was higher in symptomatic than asymptomatic patients, although
this was not statistically significant (63\% vs. 37\%, \emph{p} =0.077).
Our main findings were that the expression of MMP-9 in CD68 cells within
the carotid plaque was found to be significantly higher in symptomatic
as compared to asymptomatic patients (86\% vs. 25\% with the highest
expression, \emph{p} =0.014). Importantly, the average MVD by
immunohistochemical staining of CD34 was found to be higher and lipid
core area larger among both symptomatic patients and unstable carotid
plaque specimens, although this did not reach statistical significance
(\emph{p} =0.064 and \emph{p} =0.132, \emph{p} =0.360 and \emph{p}
=0.569, respectively). Our study demonstrates the ability of MRI to
identify patients with high-risk carotid plaque and to differentiate
better which patients could benefit from early surgical
revascularization. These findings are in line with the results of other
studies and help in understanding that MRI can be the most accurate
advanced imaging modality in distinguishing vulnerable from stable
carotid plaques preoperatively, although further large prospective
comparative studies are still required {[}7, 19, 20, 28, 35, 36{]}.
Importantly, the most recent ESVS guidelines and recommendations of
multicentric randomised controlled trials highlight clinical/imaging
features in asymptomatic patients associated with an increased risk of
stroke despite the best medical treatment that indicate necessity for
carotid revascularisation {[}1, 4, 25-27{]}. A recent report comparing
carotid MRI, ultrasound and histological findings of carotid plaque
specimens has demonstrated that the findings of carotid MRI and
ultrasound are closely associated with specimen morphology, but
ultrasound alone is insufficient to detect the type of lesion
accurately.{[}36{]} In addition, we have found that the most common type
of carotid plaque as per modified AHA classification by MRI was type
IV-V (10 patients, 65\%), while type VI was the most common (89 out of
252 plaque specimens) in the report by \emph{Cai et al.}{[}28{]}.
Similarly, \emph{Million et al.} have reported the ability of MRI to
identify high-risk carotid plaque and to differentiate symptomatic from
asymptomatic patients, although their study was performed using a
3-Tesla system {[}19{]}.

Furthermore, our results showed that MMP-9 may play a key role in
remodelling and destabilization of the carotid plaque. As we
hypothesized, the expression of MMP-9 in CD68 cells was significantly
higher among symptomatic patients. Similarly, it was also higher in SMA
cells in symptomatic patients, but this did not reach statistical
difference (\emph{p} =0.143). Interestingly, symptomatic patients had
significantly higher preoperative hsCRP which is in accordance with the
inflammatory response and infiltration related to the destabilization of
the plaque and potential increased release of MMP-9. Similarly, recent
reports showed higher expression levels of MMP-9 obtained from
symptomatic when compared to asymptomatic patients {[}8, 37, 38{]}.
These findings highlighted MMP-9 as one of the most important enzymes
related to the plaque instability. On the other hand, \emph{Baroncini et
al.} reported higher MMP-9 concentration in normal tissues and in
asymptomatic patients suggesting that MMP-9 is a part of the
atherosclerotic process although not associated to acute plaque
disruption {[}15, 16{]}. Still, this finding remains part of debate
as\emph{Heo et al.} have reported that MMP-9 is significantly associated
with plaque rupture {[}38{]}. However, recent evidence suggests that an
increase in proteolytic activity may lead to an overall increase in
matrix degradation which could explain our results which demonstrate
that the average MVD could be higher and lipid core area larger among
both symptomatic patients and unstable carotid plaque specimens.

There are several limitations to our study. Our results showed a higher
prevalence of unstable plaque in symptomatic patients, and a higher
average MVD and larger lipid core area among both symptomatic patients
and unstable carotid plaque specimens. However, the differences did not
reach statistical significance probably because of the lack of power of
the study and smaller sample size which are limitations of the present
pilot study. \emph{Cappendijk et al.} also failed to demonstrate a
significant difference between symptomatic and asymptomatic patients
{[}39{]}. However, in times of constraints on resources, both additional
MRI analysis and detailed histologic work-up of plaques is costly and
time-consuming, thus explaining the necessity of conducting a pilot
trial. Although we obtained the best possible resolution for carotid
MRI, another limitation of the study could be related to the use of only
non-contrast MRI and motion artefacts. Further improvement in coil
design, use of 3-Tesla system and modification of imaging parameters
could be required, while the use of contrast-enhanced MRI would be less
justified in terms of routine preoperative diagnostic work-up. Another
limitation is that we have analysed our results semiquantitatively by
immunohistochemistry, and the results do not allow accurate
quantification. Nevertheless, our estimates were performed independently
by two investigators. In addition, it is also possible that we have
excluded some regions of plaques where inflammation was more advanced.

\textbf{5. Conclusions}

In this pilot study, we demonstrated that carotid MRI can be reliable in
classifying carotid lesions and differentiating unstable from stable
plaques. Our initial results showed that the expression of MMP-9 is
significantly higher among symptomatic patients undergoing CEA which
suggest that MMP-9 may affect remodelling and destabilization of the
carotid plaque among symptomatic patients. This study could help in
defining discriminative ability of preoperative MRI, and in improving
identification of patients with unstable plaque that have an indication
for CEA. However, further study will be anticipated with more
participants being recruited over a longer study period. Further
prospective and randomized studies are needed to provide better
understanding of the mechanisms by which MMP-9 can affect
atherosclerotic plaque structure and remodelling.

\textbf{Author contributions}

DS conceived and designed the research study, and prepared the original
draft; DS, MM, MO, BJ, SK, AS, VV provided help on investigation,
methodology, data collection, validation, and analyzed the data; MM
performed statistical analysis; DS, MM, MO, TM, BJ, SK, AS, VV reviewed
and edited the draft. All authors read and approved the final
manuscript.

\textbf{Ethics approval and consent to participate}

All patients gave written informed consent, and relevant Institutional
Review Boards approved this study.

\textbf{Acknowledgment}

We would like to express our gratitude to Dr. Juergen Falkensammer who
helped with designing the research study. Support from Inel -- Medical
Technologies company by providing antibodies useful for this research
project is greatly appreciated. Thanks to all the peer reviewers and
editors for their opinions and suggestions.

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\textbf{Tables}

\textbf{Table 1.} Patients' characteristics\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Characteristic} & \textbf{Symptomatic (N=7)} &
\textbf{Asymptomatic (N=8)} & p\tabularnewline
\midrule
\endhead
Age, years & 70 (60, 77; 48-77) & 69 (64.3, 72.8; 64-78) &
0.867\tabularnewline
Gender, male & 7 (100) & 4 (50) & 0.077\tabularnewline
Degree of Stenosis, \% & 95 (80, 95; 80-99) & 80 (76.3, 83.8; 70-85) &
0.021\tabularnewline
Timing of neurologic symptoms, weeks & 6 (1, 18; 1-20) & 0 &
\textless{}0.001\tabularnewline
Diabetes mellitus & 4 (57) & 3 (38) & 0.619\tabularnewline
Arterial hypertension & 6 (86) & 6 (88) & 0.733\tabularnewline
Ischemic heart disease & 3 (43) & 3 (38) & 0.622\tabularnewline
Dyslipidaemia & 4 (57) & 6 (75) & 0.608\tabularnewline
COPD & 0 (0) & 1 (13) & 0.533\tabularnewline
Chronic kidney disease & 2 (29) & 1 (13) & 0.569\tabularnewline
Smoking, active & 3 (43) & 1 (13) & 0.386\tabularnewline
Smoking, previous & 0 (0) & 2 (25) &\tabularnewline
Aspirin & 7 (100) & 7 (88) & 0.533\tabularnewline
Statins & 4 (57) & 6 (75) & 0.608\tabularnewline
Haemoglobin, g/L & 140 (117, 156; 114-162) & 136 (128.3, 142.8; 127-150)
& 0.955\tabularnewline
Leukocyte count, x109/\selectlanguage{english}L & 8.7 (7.3, 10.9; 6.2-11) & 7.95 (7, 9.6;
5.3-11.5) & 0.336\tabularnewline
C-reactive protein & 13.3 (5.2, 27; 1.5-36.4) & 0.8 (0.4, 1.5; 0.4-1.8)
& 0.016\tabularnewline
Serum glucose, mmol/L & 5.3 (4.6, 6.8; 4.4-6.8) & 5.45 (5.3, 8.5;
4.9-10.4) & 0.336\tabularnewline
PT & 0.805 (0.74, 0.91; 0.72-1) & 0.75 (0.56, 0.92; 0.56-0.92) &
0.548\tabularnewline
Fibrinogen, g/L & 5.65 (3.0, 7.5; 2.6-7.6) & 2.9 & 0.8\tabularnewline
D-dimer, mg/L FEU & 0.87 (0.61, 1.19; 0.55-1.49) & 0.4 (0.1, 0.46;
0.1-0.46) & 0.036\tabularnewline
Total Cholesterol, mmol/L & 4.25 (2.98, 4.7; 2.6-4.8) & 3.65 (2.55,
4.45; 2.2-4.7) & 0.486\tabularnewline
LDL Cholesterol, mmol/L & 2.55 (1.15. 3.13; 0.7-3.3) & 2 (1.1, 3.1;
1.1-3.1) & 0.857\tabularnewline
HDL Cholesterol, mmol/L & 0.92 (0.74, 1.07; 0.7-1.09) & 0.91 (0.9-1.2) &
0.629\tabularnewline
Triglycerides, mmol/L & 1.82 (1.2, 2.1; 1.04-2.2) & 0.8 (0.54, 1.29;
0.45-1.45) & 0.057\tabularnewline
Perioperative complications & 1 (14)* & 0 (0) & 0.467\tabularnewline
Length of hospitalization, days & 6 (3, 6; 2-8) & 3 (3, 6; 2-6) &
0.281\tabularnewline
\bottomrule
\end{longtable}

Data are median (quartiles; minimum-maximum) or count (percent).
*Transient ischaemic attack

aPTT, activated partial thromboplastin time; ICA, internal carotid
artery; FEU, fibrinogen equivalent units; HDL, high-density lipoprotein;
LDL, low-density lipoprotein; PT, prothrombin time;

\textbf{Table 2.} MRI features of carotid plaques among symptomatic and
asymptomatic patients\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{MR features} & \textbf{MR features} &\tabularnewline
\midrule
\endhead
& \textbf{Stable} & \textbf{Unstable} &
\emph{p}\selectlanguage{ngerman}\textbf{¹}\tabularnewline
Asymptomatic & 4 (100) & 4 (37) & 0.077\tabularnewline
Symptomatic & 0 & 7 (63) &\tabularnewline
\bottomrule
\end{longtable}

Data are count (percent). \selectlanguage{ngerman}¹Fisher's exact test

\textbf{Table 3.} MR features of carotid plaques by modified AHA
classification.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{AHA} & \textbf{n (\%)}\tabularnewline
\midrule
\endhead
Type I-II: near normal thickness, no calcification & 0
(0)\tabularnewline
Type III: diffuse intimal thickening or small eccentric plaque with no
calcification & 1 (7)\tabularnewline
Type IV-V: plaque with lipid or necrotic core surrounded by fibrous
tissue with possible calcification & 10 (65)\tabularnewline
Type VI: complex plaque with possible disruption, haemorrhage or
thrombus & 1 (7)\tabularnewline
Type VII: calcified plaque & 2 (14)\tabularnewline
Type VIII: fibrotic plaque without lipid core and with possible small
calcifications & 1 (7)\tabularnewline
\bottomrule
\end{longtable}

Data are count (percent).

\textbf{Table 4.} Differences between preoperative symptoms and
histological and immunohistochemistry features of carotid plaques.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& & \textbf{Symptoms} & \textbf{Symptoms} &
p\textsuperscript{1}\tabularnewline
\midrule
\endhead
& & \textbf{Asymptomatic (N=8)} & \textbf{Symptomatic (N=7)}
&\tabularnewline
& & \textbf{n (\%)} & \textbf{n (\%)} &\tabularnewline
MMP-9 CD34 & No expression & 3 (37.5) & 1 (14) & 0.273\tabularnewline
& 1-25\% & 0 (0) & 3 (43) &\tabularnewline
& 26-50\% & 0 (0) & 0 (0) &\tabularnewline
& 51-75\% & 2 (25) & 2 (29) &\tabularnewline
& \textgreater{}75\% & 3 (37.5) & 1 (14) &\tabularnewline
MMP-9 CD68 & No expression & 0 (0) & 1 (14) &
\textbf{0.014}\tabularnewline
& Rare or 1 cluster \textgreater{}20 cells & 4 (50) & 0 (0)
&\tabularnewline
& \textless{}5 clusters \textgreater{}20 cells & 2 (25) & 0 (0)
&\tabularnewline
& \textgreater{}5 clusters \textgreater{}20 cells or 1
cluster\textgreater{}100 cells & 2 (25) & 6 (86) &\tabularnewline
MMP-9 SMA & No expression & 0 (0) & 1 (13) & 0.143\tabularnewline
& Rare or 1 cluster \textgreater{}20 cells & 6 (75) & 2 (29)
&\tabularnewline
& \textless{}5 clusters \textgreater{}20 cells & 0 (0) & 2 (29)
&\tabularnewline
& \textgreater{}5 clusters \textgreater{}20 cells or 1
cluster\textgreater{}100 cells & 2 (25) & 2 (29) &\tabularnewline
MVD CD34 & n, median (interquartile ranges) & 6 (2.5, 10.0; 2, 15) & 17
(6, 23; 1, 34) & 0.064\textsuperscript{2}\tabularnewline
lipid core/TPA & \textless{}10\% & 0 (0) & 0 (0) & 0.132\tabularnewline
& 10-40\% & 6 (75) & 2 (29) &\tabularnewline
& \textgreater{}40\% & 2 (25) & 5 (71) &\tabularnewline
SMA cells & Low density and no circumference & 6 (75) & 2 (29) &
0.132\tabularnewline
& High density and 100\% circumference & 2 (25) & 5 (71)
&\tabularnewline
CD68 cells & Low density & 2 (25) & 1 (14) & 1.000\tabularnewline
& High density & 6 (75) & 6 (86) &\tabularnewline
\bottomrule
\end{longtable}

Data are count (percent) unless stated
otherwise.\textsuperscript{1}Fisher's exact test.
\textsuperscript{2}Mann Whitney test. MMP-9, matrix metalloproteinase 9;
MVD, microvascular density; SMA, smooth muscle cells; TPA, total plaque
area;

\textbf{Table 5.} Differences between MRI features of carotid plaques
and histological and immunohistochemistry findings.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& & \textbf{MR features} & \textbf{MR features} & p\selectlanguage{ngerman}¹\tabularnewline
\midrule
\endhead
& & \textbf{Stable} & &\tabularnewline
(N=4) & \textbf{Unstable} & & &\tabularnewline
(N=11) & & & &\tabularnewline
& & \textbf{n (\%)} & \textbf{n (\%)} &\tabularnewline
MMP-9 CD34 & No expression & 1 (25) & 3 (27) & 0.859\tabularnewline
& 1-25\% & 0 (0) & 3 (27) &\tabularnewline
& 26-50\% & 0 (0) & 0 (0) &\tabularnewline
& 51-75\% & 1 (25) & 3 (27) &\tabularnewline
& \textgreater{}75\% & 2 (50) & 2 (18) &\tabularnewline
MMP-9 CD68 & No expression & 0 (0) & 1 (9) & 1.000\tabularnewline
& Rare or 1 cluster \textgreater{}20 cells & 1 (25) & 3 (27)
&\tabularnewline
& \textless{}5 clusters \textgreater{}20 cells & 1 (25) & 1 (9)
&\tabularnewline
& \textgreater{}5 clusters \textgreater{}20 cells or 1
cluster\textgreater{}100 cells & 2 (50) & 6 (55) &\tabularnewline
MMP-9 SMA & No expression & 0 (0) & 1 (9) & 0.671\tabularnewline
& Rare or 1 cluster \textgreater{}20 cells & 2 (50) & 6 (55)
&\tabularnewline
& \textless{}5 clusters \textgreater{}20 cells & 0 (0) & 2 (18)
&\tabularnewline
& \textgreater{}5 clusters \textgreater{}20 cells or 1
cluster\textgreater{}100 cells & 2 (50) & 2 (18) &\tabularnewline
MVD CD34 & n, median (interquartile ranges) & 7 (2, 13.75; 2, 15) & 10
(5, 19; 1, 34) & 0.360\selectlanguage{ngerman}²\tabularnewline
lipid core/TPA & \textless{}10\% & 0 (0) & 0 (0) & 0.569\tabularnewline
& 10-40\% & 3 (75) & 5 (46) &\tabularnewline
& \textgreater{}40\% & 1 (25) & 6 (54) &\tabularnewline
SMA cells & Low density and no circumference & 2 (50) & 6 (54) &
1.000\tabularnewline
& High density and 100\% circumference & 2 (50) & 5 (46)
&\tabularnewline
CD68 cells & Low density & 1 (25) & 2 (18) & 1.000\tabularnewline
& High density & 3 (75) & 9 (82) &\tabularnewline
\bottomrule
\end{longtable}

Data are count (percent) unless stated otherwise. \selectlanguage{ngerman}¹Fisher's exact test.
²Mann Whitney test. MMP-9, matrix metalloproteinase 9; MVD,
microvascular density; SMA, smooth muscle cells; TPA, total plaque area;

\textbf{Figure legends}

\textbf{Figure 1.} Magnetic resonance imaging of unstable carotid plaque
(modified AHA classification type VI) in a patient with symptomatic left
internal carotid artery stenosis: A) and B) 3D-time of flight (TOF)
sequences; C) Proton density (PD) sequence.

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1A/Figure-1A}
\caption{{This is a caption
{\label{417601}}%
}}
\end{center}
\end{figure}

\textbf{Figure 2.} Immunohistochemical staining of MMP-9 positive cells
in an unstable plaque on 400x magnification field of a patient with
symptomatic carotid artery disease: A) MMP-9 positive endothelial cells,
B) MMP-9 positive vascular smooth muscle cells, C) MMP-9 positive
histiocytes.

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2C/Figure-2C}
\caption{{This is a caption
{\label{243930}}%
}}
\end{center}
\end{figure}

\textbf{Figure 3.} Immunohistochemical staining of MMP-9 negative cells
in a stable plaque on 400x magnification field of a patient with
asymptomatic carotid artery disease: A) MMP-9 negative endothelial
cells, B) MMP-9 negative vascular smooth muscle cells, C) MMP-9 negative
histiocytes.

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3A/Figure-3A}
\caption{{This is a caption
{\label{919812}}%
}}
\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

