Carotid artery disease: plaque

features and vulnerability

Fisnik Jashari

Department of Public Health and Clinical Medicine

Umeå, 2015

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) New Series No:1760 ISBN: 978-91-7601-364-9 ISSN: 0346-6612 Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Print & Media, Umeå University Umeå, Sweden, 2015

“Research is to see what everybody else has seen, and to think what nobody else has thought”

Albert Szent-Gyorgyi

To my family

 

  i  

Table of Contents

Abstract iv

List of Papers vi

Abbreviations vii

INTRODUCTION 1

Pathophysiology of atherosclerosis 1

Lesion types 2

Pathomechanisms of ischemic events 5

Risk factors 6

Epidemiology 6

CAROTID ARTERY STENOSIS 6

Who is at risk? 7

Degree of stenosis assessment 8

Carotid artery plaque features: the concept of plaque vulnerability 10

Imaging plaque features 10

Surface plaque morphology 10

Plaque composition 11

Thin cap fiboatheroma 12

Neovascularization and intraplaque hemorrhage 13

Arterial calcification 13

ASYMPTOMATIC CAROTID STENOSIS 14

Management of asymptomatic patients with carotid stenosis 15

Identifying high-risk asymptomatic patients 15

Bilateral carotid disease 16

“Silent” brain infarction 16

Multisite atherosclerosis disease 16

  ii  

Ultrasound markers of plaque vulnerability 17

Microembolic signals detection by transcranial Doppler 18

SYMPTOMATIC CAROTID STENOSIS 19

Management of symptomatic patients with carotid disease 20

Medical therapy 20

Interventional therapy 21

Identifying patients at high risk of symptom recurrence 23

Ultrasound imaging markers of vascular risk: subclinical atherosclerosis 24

Carotid artery wall measurements: intima-thickness and intima-media GSM 25

OBJECTIVES 28

METHODS 29

Preoperative patients evaluation 29

Definitions and clinical data of patients included in studies I, II and III 29

Carotid ultrasound examinations 30

Ultrasound data retrieval 30

Extraction of the carotid ultrasound examinations 31

Definitions of ultrasound derived plaque and intima-media complex features 31

Intima–media complex measurements 33

Cone Beam CT (CBCT) analysis 34

Carotid endarterectomy (CEA) procedure 34

Source of data 35

Inclusion/exclusion criteria 36

Statistical analysis 38

RESULTS 40

Study I 40

Study II 43

Study III 47

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Study IV 50

DISCUSSION 57

LIMITATIONS 64

CONCLUSIONS 66

Acknowledgement 68

References 70

                                                   

  iv  

Abstract

Background: Atherosclerosis is an important cause of stroke. Ultrasound offers the

convenience of real-time and detailed assessment of carotid plaque features as well as

arterial wall thickening and composition. Evaluation of these features is important for

determining patients’ risk of suffering vascular events and also contributes to selecting

the best treatment strategy.

Methods: Using ultrasound data analysis we have determined plaque features in the

bifurcation and internal carotid artery (ICA), including: surface plaque irregularities,

calcification, echogenicity (grey scale median-GSM) and other textural plaque features

(Juxtaluminal black area, entropy, coarseness). In addition, intima media thickens (IMT)

and its grey scale median (IM-GSM) was measured in common carotid artery (CCA).

Using Cone Beam CT (CBCT) we have quantified calcification volume of the carotid

plaques extracted after carotid endarterectomy procedure. For the meta-analysis we have

used comprehensive meta-analysis software version 3.

Study I: We have included 39 patients and we compared carotid plaque features of the

contralateral arteries with those located ipsilateral to symptomatic side and arteries of

asymptomatic patients.

Study II: The accuracy of US to detect atherosclerosis calcification was assessed against

CBCT in 88 patients.

Study III: Based on the previous vascular events in coronary, carotid and lower extremity

arterial system, 87 patients were divided into three groups: asymptomatic, symptoms in

one vascular system and symptoms in more that one vascular system. IMT, IM-GSM and

plaque features were compared between groups.

Study IV: We have meta-analyzed ten cohort prospective studies evaluating carotid

plaque echogenicity for cerebrovascular symptoms prediction.

Results: Study I. Plaques of the contralateral to symptomatic arteries had similar features to those

in symptomatic and more vulnerable than asymptomatic arteries.

Study II. Carotid ultrasound was accurate in detecting calcification volumes of ≥8mm3

with very high sensitivity but it was less accurate in detecting lower calcification volumes

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(<8mm3). Carotid calcification was not different between symptomatic and asymptomatic

patients.

Study III. Echogenicity of the intima-media complex (IM-GSM), but not its thickness

(IMT), was significantly decreased with increasing number of arterial systems affected by

atherosclerosis. IM-GSM was lower in patients with prior myocardial infarction and

stroke.

Study IV. Carotid plaque echogenicity evaluated by US could predict future

cerebrovascular events in patients with asymptomatic, relative risk RR 2.72 (95% CI,

1.86 to 3.96), and recurrent symptoms in symptomatic patients, RR 2.97 (95% CI, 1.85-

4.78).

Conclusion: Plaques located in the contralateral to symptomatic arteries have similar

features as symptomatic side and more vulnerable than asymptomatic arteries. Carotid

ultrasound could accurately detect larger but not smaller carotid plaque calcification

volumes (<8 mm3). Low IM-GSM could identify patients with multi-system

atherosclerosis disease, suggesting a better marker for determining systemic

atherosclerosis disease burden compared to conventional IMT. Finally, carotid plaque

echogenicity predicts future cerebrovascular events in patients with symptomatic and

asymptomatic carotid stenosis.

Keywords: Carotid atherosclerosis, ultrasound, plaque features, echogenicity, calcification, surface

plaque irregularities, subclinical atherosclerosis, cerebrovascular symptoms

  vi  

List of papers

I: Ibrahimi P, Jashari F, Johansson E, Gronlund C, Bajraktari G, Wester P, Henein

MY. Vulnerable plaques in contralateral carotid arteries in symptomatic patients:

A detailed ultrasound analysis. Atherosclerosis. 2014 Aug; 235(2):526-31.

II: Jashari F, Ibrahimi P, Johansson E, Ahlqvist J, Arnerlöv C, Garoff M,

Jäghagen EL, Wester P, Henein MY. Atherosclerotic Calcification Detection: A

Comparative Study of Carotid Ultrasound and Cone Beam CT. Int J Mol Sci. 2015

Aug 21;16(8):19978-88.

III: Jashari F, Ibrahimi P, Johansson E, Grönlund C, Bajraktari G, Wester P,

Henein MY. Carotid IM-GSM is more accurate than conventional IMT for

monitoring systemic nature of atherosclerosis. Manuscript.

IV: Jashari F, Ibrahimi P, Bajraktari G, Wester P, Henein MY. Carotid plaque

echogenicity predicts cerebrovascular symptoms: A systematic review and meta-

analysis. Manuscript submitted.

     

   

       

  vii  

Abbreviations    ACAS     Asymptomatic Carotid Atherosclerosis Study ACSRS Asymptomatic Carotid Stenosis and Risk of Stroke ACST Asymptomatic Carotid Surgery Trial ANSYSCAP Additional Neurological SYmptoms before Surgery of the Carotid Arteries ARIC Atherosclerosis Risk In Communities BMT Best Medical Treatment CAS Carotid Artery Stenting CAV Cardiovascular CBCT Cone Beam Computed Tomography CCA Common Carotid Artery CEA       Carotid  Endarterectomy  CEUS Contrast Enhanced Ultrasound CV Cerebrovascular CT Computed Tomography CTA Computed Tomography Angiography DSA Digital subtraction angiography ECST European Carotid Surgery Trial GSM Grey Scale Median HDL High Density Lipoprotein HR Hazard Ratios ICA Internal Carotid Artery IMT Intima-Media Thickness IM-GSM Intima Media-Gray Scale Median IPH Intraplaque Hemorrhage JBA Juxtaluminal Black Area LDL Low Density Lipoprotein MES Microembolic Signal MI Myocardial Infarction MRA Magnetic Resonance Angiography MRI Magnetic Resonance Imaging NASCET   North American Symptomatic Carotid Endarterectomy Trial NRI Net Reclassification Improvement OR Odds Ratios PET     Positron Emission Tomography PT Plaque Type TCD Transcranial Doppler US Ultrasound VA Veteran’s Affairs

  viii  

  1  

INTRODUCTION

Atherosclerosis is a progressive inflammatory disease characterized by accumulation of

lipids, fibrous elements and inflammatory cells in large and middle-sized arteries. It is the

main cause cardiovascular (CAV) and important cause of cerebrovascular (CV) events

(1). Epidemiologically, ischemic heart disease and cerebral ischemia represent the main

cause of death and premature disability in developed countries (2). Moreover, due to the

aging of the population, the global burden of atherosclerosis, and thereby its clinical

consequences will continue to rise in the coming decades (3). The underlying

pathogenesis of atherosclerosis involves an imbalance between lipid metabolism and

maladaptive immune response and consequently chronic inflammation of the arterial wall

(4). Although several risk factors associated with arterial wall disease have been

identified, disease burden and ischemic events could not be predicted based on their

identification alone, thus leaving space for other methods (e.g. arterial imaging and

biomarkers) to be employed for better patients risk stratification. Advances in medical

and interventional treatment strategies have emphasized the importance of identifying

vulnerable patients, their risks and optimum treatment. Also, developments of different

imaging techniques have aided considerably in this aspect by making non-invasive

evaluation and quantification of atherosclerosis in-vivo possible, in particular ultrasound,

which is an established imaging modality in atherosclerosis research.

Pathophysiology of atherosclerosis

The term atherosclerosis is derived from the Greek "athero," meaning gruel, or wax, and

"sclerosis" for hardening (induration), with the former referring to the corresponding lipid

pool (necrotic core) and the latter referring to the fibrous cap of the plaque's luminal

edge. Arterial wall is composed of three layers: tunica intima, tunica media and

adventitia. The intima is the inner lining of the vessel, directly adjacent to blood flow, a

very dynamic layer which is composed of a monolayer of endothelial cells. The initial

step of the atherosclerosis pathology is the dysfunction of these endothelial cells that

allows the accumulation of low-density lipoprotein particles (LDL) into the intima.

Subsequently LDL particles undergo oxidative modifications that may trigger a local

  2  

inflammatory response that signals the following steps in the lesion formation. This is

followed by monocytes migration, accumulation into the intima and their conversion to

macrophages, which characterizes the initiation of early atherosclerotic lesion formation.

Upon occupying the intimal space, macrophages will uptake lipoprotein particles by

receptor mediated endocytosis and become lipid-laden foam cells. This stage of the

disease become macroscopically apparent as fatty streaks, that could be visible in the

large arteries even in the first decade of life, as confirmed by autopsy studies. (4, 5)

Although formation of fatty streaks commonly precedes the development of a more

advanced atherosclerotic plaque, not all of them will progress to atheroma formation.

Also, not all mechanisms involved in atherosclerosis process are “pro-atherogenic”, in

fact high-density lipoprotein (HDL) particles and M2-like phenotype macrophages have

“anti-atherogenic” properties (4). Based on the above, atherosclerotic plaque could follow

different ways: a) it could have stable progression (in most cases), b) it could have abrupt

progression that leads to plaque rupture with superimposed thrombosis or c) it could

regress, as shown in longitudinal follow-up imaging studies.

Lesion types

The pathogenesis of atherosclerotic lesions has been analyzed at different stages of

disease and in different age groups. There are many histological classification of

atherosclerotic lesions, however, we hereby present a simple one, which emphasizes the

link between morphology and clinical manifestations (6). In addition, this classification

could be properly related to plaque imaging during different stages of the disease. Figure

1, shows all plaque types presented on a graph together with corresponding ultrasound

images.

A. Adaptive intimal thickening: Characterized by smooth muscle cells accumulation

in a proteoglycan-rich matrix in the absence of lipids and inflammatory cells. This

may provide a soil for lesion development, initially at the branching points but

later could spread into adjacent parts.

B. Intimal xanthoma (“fatty streak”): Characterized by LDL accumulation into the

intima. Subsequently, LDL particles will modify and undergo process of

  3  

oxidation and start to act as chronic stimulator for immune response. They induce

endothelial cells to express adhesion molecules, which interact with receptors on

the surface of the monocytes and stimulate their adhesion and migration into the

intima. Macrophages could express several different phenotypes. Some of them

attain pro-inflammatory (M1-like phenotype), and the others that have an M2-like

phenotype may secret factors that favor resolution of the inflammation. These

types of lesions could be fully reversible if the stimuli that caused them dissipate.

They are present already in the aorta of some infants in the first 6 months,

probably reflecting risk factors of the mothers, then their number decline in the

subsequent years.

C. Pathological intimal thickening: Pathological intimal thickening is sometimes

refereed to in the literature as an “intermediate lesion.” True necrosis is not

apparent. The fibrous cap overlying the areas of lipid is rich in smooth muscle

cells and proteoglycans. Macrophages and lymphocytes may also be present, but

these are usually sparse.

D. Fibroatheroma: this corresponds to the formation of necrotic core. The foam

cells, macrophages and smooth muscle cells accumulated in the intima over time

could undergo apoptosis and secondary necrosis. The reason why necrosis

happens in some but not the other lesions is not known. At this stage of the

disease neovascularization and intraplaque hemorrhage are often present.

E. Fibrocalcific plaque: Calcification is common in progressive atherosclerosis and

its content increases with age. Plaque components that undergo calcification are:

apoptotic cells, extracellular matrix and necrotic core. Sometimes these structures

undergo complete calcification; so calcification may constitute most of the plaque

volume. The last two phases of the disease progression (D and E) can interchange

between each other under different internal and external factors, e.g. statin therapy

could increase fibrotic composition within the soft plaques and in high doses can

aggravate plaque calcification.

  4  

Figure 1. Stages of atherosclerosis development and ultrasound imaging samples.

In each box; top left- a cross sectional view of the carotid artery animation, top

right- more detailed animation focused at the more active disease process, bottom

right- ultrasound plaque features attempt to correlate with the plaque features

represented in the animations.

A-adaptive intimal thickening, that can be accurately measured by ultrasound; B-

Intimal xanthoma, there is an assumption, but it is not yet confirmed by direct

histological studies if an echolucent intima media complex (low IM-GSM)

represents the intimal xanthoma, characterized by accumulation of LDL and

inflammatory cell particles; C- Pathological intimal thickening (intermediate

lesion), early non-stenotic lesion on ultrasound; D-Fibroatheroma with typical

features vulnerable plaques, echolucent plaque with juxtaluminal black area on

US; E-Fibrocalcific plaque.

  5  

Pathomechanism of ischemic events

The most dramatic complication of atherosclerotic plaque progression is thrombosis,

which may be caused by three different mechanisms: a) plaque rupture, b) plaque erosion

or c) calcified nodule (7). The first and most common one is plaque rupture, which is

defined as a fibrous cap disruption when the overlying blood contents are in continuity

with the underlying necrotic core. Plaque erosion is identified when the thrombus is

superimposed on a plaque substrate primarily composed of smooth muscle cells and

proteoglycans, and without a communication between blood contents and the necrotic

core. Calcified nodules are characterized by protrusion of the eruptive dense calcified

bodies into the luminal space and represent the least frequent morphology associated with

luminal thrombosis (8). Following plaque rupture, the pathomechanisms by which the

disease is caused differs in different arterial systems. While in coronary arteries

myocardial infarction (MI) is attributed to in-situ thrombosis, in carotid artery

embolization is the most common mechanism causing ischemic stroke (1) (figure 2).

Figure 2. Stable and unstable plaque features. Pathomechanisms of ischemic events in

coronary and carotid artery

LUMEN

STABLE PLAQUE

UNSTABLEPLAQUE

Thin fibrous capLarge lipid pool

Neovascularization

Inflammatory cells

Neovascularization

LUMEN

In-situ thrombosis

Artery-to-artery embolization

Plaquerupture

Plaque thrombosisin coronary artery

Embolization aftercarotid plaque rupture

Thin fibrous cap

  6  

Risk factors

There are multiple factors that contribute to atherosclerotic plaque progression. As

described above, the processes involved in atherosclerosis include: endothelial cell injury,

lipid metabolism, inflammation, and smooth muscle cell proliferation. Factors affecting

these processes may inhibit or accelerate atherosclerosis. In general atherosclerosis risk

factors are divided into two groups: non-modifiable (family history, male gender) and

modifiable (hyperlipidemia, diabetes mellitus, cigarette smoking, hypertension, and

dietary deficiencies of antioxidants) (1).

Epidemiology

Atherosclerosis-related cardiovascular (CAV) and cerebrovascular (CV) events are the

most common causes of deaths in the West (2). The prevalence of atherosclerosis in

carotid and coronary arteries increases exponentially with age, and is more prevalent in

men than in women. According to the World Health Organization, 15 million people

suffer stroke annually worldwide and of them 5 million die. In Europe alone nearly

650.000 stroke deaths occur per year (9). Approximately 85% of strokes are ischemic and

15-20% of them are attributed to carotid artery disease (10).

CAROTID ARTERY STENOSIS

Carotid artery stenosis is an important cause of ischemic stroke (11). Atherosclerosis

could affect any segment of the carotid artery with a predilection at the point when it

bifurcates into internal and external artery. The arterial wall is normally highly dynamic

because its pulsations follow those caused by the cyclic heart contraction and relaxation.

Arterial expansion alters artery radius, shape and curvature, which together interact to

make the blood flow in the arteries (e.g. common carotid artery) laminar. Laminar blood

flow is characterized by concentric layers of blood moving in parallel down the length of

a blood vessel with the highest velocity in the center of the lumen. When blood separates

at an arterial bifurcation, a complex pattern of fluid velocities is created. The highest

  7  

velocities at the center of flow come in contact with the flow divider, and this flow

separation continues until some distance into the subsequent branches (figure 3). These

regions have a low wall shear stress that contributes to alterations of endothelial cells, by

inducing expression of endothelial leukocyte adhesion molecules that contribute to

leukocyte migration into the intima and hence the development of atherosclerosis (12).

Figure 3. Hemodynamics at the carotid artery bifurcation

Who is at risk?

Moderate to severe asymptomatic carotid stenosis is found in 2-5% of women and 2-8%

of men over the age of 60 years. However the risk is much higher in high-risk groups.

The prevalence of asymptomatic 50-90% carotid stenosis in patients over 60 years with

three CAV risk factors is estimated at 16%, and in those with concomitant coronary

artery disease and contralateral carotid disease is 15% and 6%, respectively. The

Framingham study showed that predictors of carotid stenosis over 12 years are (13):

  8  

Ø Age (odds ratio (OR) 1.6-1.7/10 year increase in age)

Ø Smoking (OR 1.3-1.5 per 10 cigarettes smoked/day)

Ø Systolic blood pressure (OR 1.2 per 10 mmHg increase)

Ø Total cholesterol (OR 1.1-1.2 per 0.5 mmol/L increase).

Clinical identification of carotid artery stenosis used to be based on auscultation skills

and the confirmation of a bruit. This clinical sign has recently been proven to be of low

accuracy in depicting 70-99% stenosis with a sensitivity of 57% and specificity of 70%

(14). This low accuracy of auscultation highlighted a need for other accurate imaging

screening modalities such as ultrasound. In addition, detection of calcification on

panorama radiograph or cone beam CT (CBCT), during patients’ evaluation for dental

procedures, has been found to be associated with significant carotid stenosis (15).

Recommending these patients for carotid US examination has made possible the

identification of those with significant stenosis and implementation of different treatment

strategies for stroke prevention.

Degree of Carotid stenosis assessment

The degree of carotid artery stenosis could be evaluated by different imaging techniques,

however the gold standard against which other methods accuracy is evaluated is

conventional angiography (16). Using angiography, the degree of stenosis could be

quantified by two methods: one suggested by North American Symptomatic Carotid

Endarterectomy Trial (NASCET) (17) and the other by European Carotid Surgery Trial

(ECST) (18). Both methods evaluate the degree of stenosis as the percentage reduction in

the linear diameter of the carotid artery. Because these two trials have used different

strategies to quantify the degree of stenosis, applying both of them in the same carotid

artery will demonstrate different values (e.g. 82% ECST usually corresponds to 70%

NASCET stenosis) (19). This difference between the trials degree of estimated stenosis

can be corrected using the formula: NASCET=(ESCET-40)/0.6 (20). Because

angiography is an invasive investigation and is also associated with 0.3-1% risk of peri-

procedural neurological events, a growing interest for other non-invasive modalities that

could accurately evaluate the degree of stenosis and identify other plaque features that

  9  

could be of importance for patients risk stratification has been developed (21). A meta-

analysis conducted by Koelemay et al. (22) reported a sensitivity of 97% and specificity

of 99% for CTA in characterizing the degree of carotid artery stenosis. In particular, CTA

was accurate for assessing 70-99% stenosis and for excluding total artery occlusions.

Carotid duplex ultrasound (US) has been shown to be accurately comparable with digital

subtraction angiography (DSA) and today it is the most widely used method for

evaluating carotid stenosis. Carotid US has the benefits of speed, safety, bedside and its

radiation free properties. Stenosis is measured using Doppler principles of blood velocity

at the tightest stenosis point, which could be translated to equivalent DSA measurements.

One of the methods recommended for grading stenosis is to use peak systolic velocity. A

velocity more than 125 cm/s corresponds to >50% stenosis and a velocity >230 mm/s

corresponds to greater than 70% stenosis (23).

A meta-analysis of individual patients data showed an inter-observer variability

agreement, for ultrasound, for 70-99% degree of stenosis of 86% (79%-91%) (24). In

clinical practice, an additional confirmatory investigation to ultrasound has been

recommended before carotid endartercomy (CEA); including another imaging modality

(CTA) or a repeat ultrasound examination (10). However, there are data that suggests that

ultrasound-only strategy is as effective as adding one more modality in patients with 70-

99% stenosis selected for surgical intervention within 14 days. Assessing moderate

symptomatic carotid stenosis by ultrasound has been shown to be less accurate (25). Also

the accuracy is limited in cases with moderate contralateral stenosis or occlusion as well

as in arteries with extensive calcification. In borderline cases, adding another modality,

CTA or contrast enhanced magnetic resonance angiography (MRA) can reduce

unnecessary CEA performed (26). It has been shown that symptomatic patients with

>70% stenosis benefit from CEA and is therefore very important to measure accurately

the degree of stenosis. However, it was later demonstrated that the risk of embolism and

thrombosis is not related only to the stenosis severity but also with its composition and

other plaque features.

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Carotid artery plaque features: the concept of plaque vulnerability

Based on several morphological and histological characteristics, carotid plaques could be

defined as stable and unstable. In general, plaques that have been associated with

increased risk of ischemic events are defined as unstable or vulnerable. Vulnerable

plaques are characterized by the presence of a thin fibroatheromatous cap that overlies a

large lipid pool (>40% of the plaque), large necrotic core, intimal spotty calcification,

high inflammatory cell concentration, neovascularization and intraplaque hemorrhage.

While historically plaque features are evaluated histologically in autopsy studies or in the

arterial segments removed after surgical procedures, advances in imaging techniques

have made in vivo plaque visualization and identification of different components inside

the plaque possible.

Imaging plaque features

A number of non-invasive imaging modalities exist for reliably determining carotid

artery plaque features, including: ultrasound, magnetic resonance, computed tomography,

and positron emission tomography (PET) (16). Because studies included in this thesis are

mainly based on ultrasound technology, this section will be mostly focused on ultrasound

characterization of carotid plaque features, and also its strength and weakness will be

weighted against other non-invasive imaging modalities.

Surface plaque morphology

The surface morphology of the carotid plaques classified as smooth, mildly/markedly

irregular and ulcerated, is an important determinant of vascular events. A smooth surface

configuration is characterized by a regular luminal morphology which indicates a stable

plaque. Irregular surface morphology, especially marked irregularity has been shown to

be associated with increased risk of ischemic events (27). The third type of surface

morphology –ulceration, is significantly associated with increased risk of cerebral events,

and this was demonstrated in NASCET study (28). Of the different imaging techniques

that have been used to assess surface plaque morphology, computed tomography

  11  

angiography (CTA) has the greatest accuracy. Compared to ultrasound, CTA sensitivity

in detecting plaque ulceration has been shown the highest (93% vs. 37.5%) (29). Such

sensitivity of US for identification of ulceration is higher (77%) in plaques causing >50%

stenosis (27). Recently introduced ultrasound based techniques such as: contrast

enhanced US (CEUS) and ultrasound 3D reconstructions have been shown to have very

high accuracy for detecting ulceration (30, 31).

Plaque composition

As mentioned before, carotid plaques are composed of different components and the

relative proportion of these components can vary from one plaque to another, which

could determine its stability. Histologically, different plaque components have been

described and their association with ischemic events was determined. Carotid plaque

components most commonly found in patients with stroke and transient ischemic attack

(TIA) are: lipid pool, necrotic core and hemorrhage. The presence of calcium seems to

have a protective role, since it is found mostly in asymptomatic plaques. Because of the

clinical importance identifying such plaque features in vivo, many imaging modalities

have been employed (32, 33). Ultrasound as well as computed tomography (CT) and

magnetic resonance imaging (MRI) offer important information about carotid plaque

composition. On CT carotid plaques are classified based on the Hounsfield Units (HU)

density as fatty (<60 HU), mixed (60-130 HU) and calcified (>130 HU) (34). Very low

values (<0 HU) have been found to be associated with hemorrhage. Also MRI can depict

carotid plaque composition, especially plaque hemorrhage (35, 36).

On ultrasound, plaque echogenicity is the main parameter that reflects composition.

Plaque echogenicity has traditionally been evaluated by visual (subjective) grading

plaques echo reflection (e.g. echogenicity and echolucency), and its echo pattern

(heterogenous and homogenous). However, visual evaluation of plaque echogenicity has

some limitations because it is subjective and also plaque echogenicity could be affected

by ultrasound machine settings during the patients’ examination. Nevertheless, this

limitation could be overcome by computer assisted plaque normalization using image

processing and 2 references (blood and adventitia). The blood is scaled to zero, while the

brightest area of the adventitia is normalized to a grey scale of 190. Following

  12  

normalization, the plaque is outlined and its overall brightness evaluated by means of

grey scale median-GSM (grey scale range: 0-to-255; 0=black and 255= white). The lower

the GSM the more echolucent is the plaque. Conversely, echogenic plaques will have

higher GSM. However, because different studies have used different cut-off values to

define plaque echolucency, its use in clinical practice could not be suggested yet. In

addition to GSM evaluation, plaques could be objectively classified into four groups

based on Gray-Weal classification modified by Geroulakos (37):

Type 1: uniformly echolucent with <15% of the pixels in the plaque area being occupied

by pixel values >25;

Type 2: mainly echolucent where pixels with grey scale values >25 that occupy 15-50%

of the plaque;

Type 3: mainly echogenic with pixels >25 occupying 50-85% of the plaque;

Type 4: uniformly echogenic with grey scale >25, occupying >85% of the plaque.

Studies correlating US findings with plaque histology have revealed that echolucent

plaques are associated with lipids, necrotic core or intraplaque hemorrhage, while

echogenic plaques are associated with fibrous and calcified tissue. In addition to GSM

and plaque types there were other parameters developed such as: Juxtaluminal black area,

plaque coarseness and entropy.

Juxtaluminal black area (JBA) of the plaque is defined as the area of the plaque

components having grey-scale <25 without a visible echogenic cap. After imaging

normalization this area is outlined and expressed as mm2 (38). JBA <8 mm2 has been

shown to be associated with a 0.6 annual risk of stroke compared to 4.6% in patients with

JBA >8 mm2 (39).

Thin cap fibroatheroma

Histologically, fibrous cap is formed of smooth muscle cells relied within a collagen-

proteoglycan matrix, associated with macrophages and T lymphocytes (40). A thin

fibrous cap plays a critical role in determining plaque vulnerability. Following fibrous

cap rupture, the exposure of thrombogenic plaque components to the luminal blood flow

  13  

represents a critical step that eventually leads to thromboembolic complications. The best

non-invasive imaging modality to assess the status of the fibrous cap in carotid arteries is

MRI, which could define the fibrous cap as normal, thin or fissured/ruptured (41).

Currently, there are no imaging studies that have used ultrasonography to assess fibrous

cap, and CT proved not accurate for this purpose.

Neovascularization and intraplaque hemorrhage

Adventitia of the normal middle and large size arteries has a vasculature network (vasa

vasorum) that supplies the outermost part of the artery with oxygen and nutrition, while

the intima part is supplied with oxygen from the lumen through diffusion. In pathological

conditions (atherosclerosis), because the oxygen cannot achieve the deeper parts of the

plaque, some small vessels will proliferate from the adventitia towards the intima as

neovessels. The wall of these vessels lack pericytes and is very fragile and hence is prone

to rupture. It has been suggested that rupture of the neovessels is the cause of production

and expansion of intraplaque hemorrhage (IPH) (42). The presence of neovascularization

and/or intraplaque hemorrhage is considered a feature of vulnerability (43). The best

modality for visualizing IPH in the carotid arteries is magnetic resonance, which can

recognize the age of the hemorrhage as well (35). Using ultrasound, neovascularization

can be evaluated using microbubles based contrast, known as contrast enhanced

ultrasound (CEUS) (42). A recent study by Staub et al. concluded that the presence of

neovascularization inside the carotid plaque is associated with morphological features of

plaque instability (44).

Arterial Calcification

Calcium formation may be found in any arterial bed as well as in the microvessels. It may

be present in the intima and/or media, the latter being common in chronic kidney disease

or type II diabetes (16). The initial plaque calcification is associated with apoptotic

smooth muscle cell and appears as micro-calcifications bound to membrane vesicles.

Following plaque progression, the micro-calcifications combine together to form larger

calcium deposits. In patients with carotid atherosclerosis calcification is detected in 50-

  14  

60% of cases (45) and even often in significantly stenotic lesions (>50%) (15) but its

association with CV events is uncertain. Controversies exist as to the effect of

calcification on plaque nature with some evidence suggesting more stability (45) and

another indicating vulnerability, irrespective of the degree of stenosis (46). A systematic

review has shown that symptomatic plaques have less calcification than asymptomatic

ones (47). However, studies included in this review used wide range of methods to

quantify carotid calcification, highlighting the need for a well-validated, accurate and

reproducible technique for calcium quantification in patients with carotid atherosclerosis.

Plaque calcification in carotid arteries can be assessed by a number of imaging modalities

including: CT, ultrasound and MRI. While ultrasound may detect the presence of

calcification, it does not have the accurate means for quantifying its extent. Computed

tomography on the other hand has the ability to identify and quantify calcification. Even

calcification volumes of 1 mm3 can be detected and quantified by cone beam CT

(CBCT). Furthermore, it has been shown that the amount of calcification quantified by

carotid CT correlated with respective histology sections (48). Despite, such uncertainty

the extent of carotid calcification adds more accurate assessment to the plaque features.

Carotid US is an easy and economically favorable method to study different plaque

features and their potential association with symptoms. It has been reported that extensive

calcified carotid plaques are associated with adverse outcome 30 days after carotid artery

stenting (CAS), hence CEA should be recommended in such cases (49). Because many

centers today rely mainly on carotid US information before intervention, identification of

calcification and its severity seems to be important.

ASYMPTOMATIC CAROTID STENOSIS

Asymptomatic carotid stenosis refers to narrowing of the carotid artery caused by

atherosclerosis in patients who have not experienced a stroke or transient ischemic attack

in the brain related to cerebral circulation insufficiency. With the introduction of vascular

ultrasound in 1980s, investigation of carotid artery disease became easy, and on the

following years a high number of patients were diagnosed with a so-called asymptomatic

  15  

carotid disease. However, the prognosis and management of such patients remains

uncertain. Both natural history studies, as well as several large randomized trials showed

a low incidence of stroke in asymptomatic patients with carotid disease, only 1-2% per

year and a rather high incidence of cardiovascular morbidity and mortality (5-10% per

year) (50, 51).

Management of asymptomatic patients with carotid stenosis

The Asymptomatic Carotid Atherosclerosis Study (ACAS) (52) and the Asymptomatic

Carotid Surgery Trial (ACST) (53) provided evidence that CEA offers a 50% risk

reduction for ipsilateral stroke in patients with moderate to severe asymptomatic carotid

artery stenosis compared to medical treatment alone (annual stroke risk: 1% vs. 2%).

However, at the time when these trials were performed, patients had received a treatment

that today would be considered as suboptimal medical treatment, e.g. in the ACST statins

were used in only <10% of patients, while 20% of population was not taking antiplatelet

therapy. Furthermore, dietary modification, blood pressure control and statin dosage were

not close to those that are currently used in practice.

It has been suggested that up to 94% of CEA performed on the basis of these trials

recommendations are unnecessary. As compared to CEA for symptomatic stenosis, with

a number needed to treat (NNT) of 6 (or 3 for patients above 75 years old), to prevent a

stroke in asymptomatic patients the NNT were 83 (54). The advancement of the best

medical treatment (BMT) has questioned the invasive intervention (CEA or CAS) for

asymptomatic patients. Recently, a low risk of ipsilateral stroke in patients with

asymptomatic carotid stenosis has shown range from 0.4 to 1.0% per year (55).

Nevertheless, for a subgroup of patients at higher risk of stroke, invasive intervention

may still be justified. Identification of such high-risk patients who could benefit from

invasive intervention in addition to BMT is thus of crucial importance.

Identifying high-risk patients with asymptomatic carotid disease

In the past three years, it has become increasingly clear that BMT is the best choice for

the majority of patients with asymptomatic carotid disease, and there has been an

  16  

increasing effort in identifying factors that could stratify patients who may benefit of

intervention therapy (56). Many clinical and imaging features have been reported that are

associated with increased risk of stroke, and some of them will be described in the

following sections.

Bilateral carotid disease

Patients with bilateral carotid disease are at higher risk from suffering CV symptoms. In

asymptomatic carotid stenosis, it has been reported that patients with a history of

contralateral TIA/stroke were associated with a 3.4% annual risk of stroke compared to

1.2% in those with no previous symptoms (57). It has also been concluded that medically

treated patients with a history of contralateral symptoms prior to randomization were

significantly more likely to develop CV symptoms within 5 years follow-up in the

territory ipsilateral to asymptomatic stenosis compared to those without a history of

contralateral symptoms, 29% vs. 22%, respectively (58). In addition, patients with

bilateral carotid disease “vulnerable” textural features were confirmed in carotid plaques

of both sides compared to more stable features in patients with unilateral disease (59).

“Silent” brain infarction

About 20% of asymptomatic patients with carotid stenosis will have the evidence of

silent infarction on CT/MRI scans. Patients with ipsilateral “silent” infarction have been

shown to have a significantly higher annual risk of ipsilateral stroke compared to those

without, 3.6% vs. 1.0%, respectively (60). The same was confirmed in an ACST

subgroup analysis. Compared to patients with no evidence of brain infarction or prior

symptoms, those with ipsilateral infarction or a history of prior CV symptoms had a 5.8%

absolute risk increase for stroke in the subsequent 10 years (61).

Multisite atherosclerosis disease

Reduction of Atherosclerosis for Continued Health (REACH) (62) study analyzing

prospectively a cohort of 68,236 patients showed that after one year follow-up the rate of

  17  

vascular events is increased with the number of symptomatic diseased locations, ranging

from 5.31% in asymptomatic patients to 12.58%, 21.41% and 26.27% in patients with 1,

2 and 3 symptomatic arterial disease locations, respectively. Based on these results it

seems that the presence of multi-focal atherosclerosis disease has major clinical and

prognostic impact. However, the pathomechanism behind widespread atherosclerosis at

the level of distinct territories is complex; multiple risk factors, genetic, inflammatory

markers and loco-regional hemodynamics, all may play an important role (63, 64). The

impact of different risk factors on the disease prevalence in various locations is important

in predicting the affected arterial system: smoking is three times more likely to cause

lower extremity artery disease than coronary artery disease, compared to diabetes in

coronary artery disease and hypertension in carotid atherosclerosis (1, 65).

Ultrasound markers of plaque vulnerability

Degree of stenosis: There is conflicting evidence with regards to any relationship

between stenosis severity and future ischemic events. Subgroup analyses from the ECST

and NASCET showed that stenosis severity (but not near occlusion) was associated with

increased risk of stroke in medically treated patients. Also, the Asymptomatic Carotid

Stenosis and Risk of Stroke (ACSRS) study (57) has reported that the risk of stroke is

proportionally increased with the increase in the degree of stenosis from 0.8% to 1.4%

and 2.4%, for 50-69% to 70-89% and 90-99% stenosis, respectively. On the other hand,

neither ACAS (52) nor ACST (61) found any evidence that increasing stenosis severity is

a predictive of increased risk of stroke in the medically treated asymptomatic patients.

Stenosis progression: It has been assumed that progression of the stenosis severity as

shown with US could be associated with increased risk of stroke. Sabeti et al. (66)

showed that stenosis progression was associated with two-fold increase of stroke risk in

the 3-year follow-up period (OR 2.0; 95% CI 1.02-4.11). The ACSRS study (57) in a

subgroup analysis on this issue showed that at follow up stenosis severity remained

unchanged in 76%, regression was observed in 4% and stenosis progression in 20% of

patients. The 8-year cumulative risk of ipsilateral stroke was 0% for patients with US

evidence of stenosis regression, 9% in the group of patients with unchanged stenosis and

  18  

16% in patients with stenosis progression. Despite suggestions that stenosis progression

was associated with doubling rate of stroke per annum, 68% of the ischemic strokes

occurring during the follow-up affected patients with no evidence of stenosis progression,

thus indicating a low accuracy of the method for identifying high-risk patients.

Textural plaque features: As stated above, there are many ultrasound-derived textural

features that could be considered as a sign of plaque vulnerability. Soft (echolucent)

carotid plaques today could be evaluated using different cut-offs of GSM values (low

GSM=echolucent plaque) or by classifying plaques into plaque types (PT) as

recommended by Gray-Weale and Geroulakos (PT 1 and 2=echolucent plaque). In the

ACSRS study, asymptomatic patients with a GSM >30 had a very low annual rate of

stroke (0.6%), the annual stroke rate increased to 1.6% for those with a GSM of 15-30

and to 3.6% for those with GSM <15. The annual rate of stroke was progressively

increased when comparing plaque Types from 4 to 1, with Type 4 having a 0.4% annual

risk of stroke, compared to 0.8% and 3.0% for Type 3 and Type 1 or 2. Most studies that

used this classification to determine patients’ symptoms have defined plaques Type 1 and

2 as echolucent and plaque Types 3 and 4 as echogenic.

Microembolic signals detection by transcranial Doppler

Microembolic signal (MES) detection by transcranial Doppler (TCD) is another

important imaging modality that was used to identify patients at increased risk of stroke;

accordingly, consensus criteria have been established for interpreting these high­intensity

transient signals (67). Several studies evaluated the predictive role of MES detected by

TCD ultrasonography in patients with asymptomatic stenosis. Asymptomatic Carotid

Emboli Study (ACES) showed that the presence of spontaneous embolization was

associated with a five-fold increase in the risk of ipsilateral stroke (68). In 2005, Spence

et al. (69) showed that asymptomatic carotid stenosis patients who had two or more

microemboli during an hour of monitoring had a 1-year stroke risk of 15.6% compared to

only 1% risk of stroke for patients with no microemboli. Furthermore, it was

demonstrated that an aggressive approach to medical therapy has significantly reduced

MES on TCD ultrasonography (70).

  19  

Figure 4. Proposed algorithm for the management of patients with asymptomatic carotid

stenosis, relied on clinical data and data acquired by ultrasound imaging. BMT - Best

Medical Treatment, CEA - Carotid Endarterectomy, CAS-Carotid Artery Stenting. Note:

To decide between CAS and CEA other clinical and imaging features acquired by

modalities other than US should be taken into consideration, e.g. intracranial extension

of carotid plaque, patients with previous irradiation of the cervical region, carotid artery

tortuosity, etc.

SYMPTOMATIC CAROTID STENOSIS

First descriptions of carotid stenosis related to cerebral ischemic events date back to T.

Willis (1621-1675) and J. Wepfer (1620-1695) (71). Symptomatic carotid stenosis is

defined as carotid artery disease associated with ischemic symptoms (Stroke, TIA or

amaurosis fugax) in the anterior brain circulation. After ischemic events, the presence of

carotid artery stenosis is confirmed during an US, CTA, MRA or conventional

Asymptomatic carotid stenosis

<60% stenosis

60-99%stenosis

BMT

One of the below:(1) Echolucent plaque (PT I or II),

(2) >2 MES on transcranial Doppler(3) markedly irregular/ulcerated

plaques

CEA + BMT CEA or CAS + BMT

One of the below:(1) >70 years old patient

(2) Echolucent plaque(3) Extensive calcification

Yes No

YesNo

  20  

angiography examination. During the US examination the degree of stenosis is measured

together with the description of exact plaque location and plaque morphology. After

confirmation of the carotid disease in most cases, unless contraindicated, patients will be

recommended carotid endarterectomy (CEA) intervention. CEA consists removal of the

carotid atheroma together with intima and media layers, leaving the adventitia

unattached.

Management of symptomatic patients with carotid disease

In patients with acute CV accidents associated with carotid stenosis the initial therapy

comprises antiplatelet, statins and risk factors management (72). In patients with carotid

stenosis an intervention should be performed within <2 weeks. Benefit of

revascularization is greater in men, in older patients, and in severe stenosis (70-99%)

(72). In addition, a subgroup of patients with moderate carotid stenosis (50-69%) might

also benefit of revascularization, particularly those with moderate carotid plaque

irregularities, ulceration and echolucency that are confirmed to be at higher risk for stroke

recurrence (73). In addition, it was recently shown that the incidence of stroke recurrence

is very high in the first 72 hours after initial symptoms and an emergency CEA

intervention would be of benefit in this subgroup of patients.

Medical therapy

Medical therapy of stroke or TIA related to a carotid stenosis consists of immediate

administration of secondary prevention with antiplatelet therapy. Either dipyridamole

plus aspirin (74), ���clopidogrel alone (75), or aspirin alone is recommended (76, 77). In

symptomatic patients with >50% carotid stenosis associated with microembolic signals

administration of dual antiplatelet therapy (aspirin + clopidogrel) resulted in decreased

risk of stroke recurrence (78). The Clopidogrel in High-risk patients with Acute Non-

disabling Cerebrovascular Events (CHANCE) trial showed that the combined treatment

of clopidogrel and aspirin for the first 21 days after TIA or stroke, followed by

clopidogrel alone decreases the 90-day risk of stroke [hazard ratio, 0.68, 95% (CI, 0.57–

  21  

0.81), p<0.001], without increasing the risk of hemorrhage in comparison with aspirin

alone (79).

In addition to antiplatelet therapy, statins have been found to be highly effective in stroke

prevention among medically managed patients with CAV disease (71). In a meta-analysis

the stabilizing effect of statin therapy on carotid plaque has been confirmed, and has been

proposed to be related to the non-cholesterol-lowering, so-called pleiotropic effects of

statins (80). Several studies have also demonstrated an independent benefit from statins

on outcomes before carotid endarterectomy (81). A three-fold reduction in the rate of

perioperative stroke, and five-fold reduction of perioperative mortality among 1,566

statin-treated patients undergoing carotid endarterectomy, has been shown (82). Stroke

Prevention by Aggressive Reduction in Cholesterol Levels (SPARCL) trial has confirmed

that in patients with recent stroke or TIA, 80 mg of atorvastatin daily reduced the overall

incidence of strokes and of CAV events, despite a small increase in the incidence of

hemorrhagic stroke (83).

Interventional therapy

It was not before 1950s when DeBakey and Estcot for the first time tried surgical

intervention in the carotid stenosis for secondary stroke prevention. Later on, several

large randomized studies demonstrated that CEA is associated with better clinical

outcome after ischemic stroke. Indeed, the results of ECST and NASCET trials showed

primarily that compared to medical treatment, CEA reduced the risk of stroke at 5 years

of 15% among patients with a 70–99% and of 7.8% among those with a 50–69% stenosis.

No benefit could be demonstrated in patients with 0-49% stenosis (17, 18). Also, surgery

conferred no benefit in patients with “near-occlusion”, defined as a 90-95% stenosis

when the distal internal carotid artery (ICA) lumen is collapsed, as shown on angiography

(84). In a pooled analysis including ECST and NASCET trials data, a higher benefit from

surgery in men has been reported (85). While CEA was clearly beneficial in women with

>70% symptomatic carotid stenosis, this was not the case for those with 50–69% stenosis

(85). Furthermore, women had a lower risk of ipsilateral ischemic stroke on medical

treatment than men. This observation is possibly a reflection of the differences in the

pathology of the atherosclerotic plaques, with men having unstable carotid plaques with

  22  

more pronounced features of inflammation compared to women (86). Because most of

the recurrences occur early after stroke, it was confirmed that earlier interventions are

associated with lower rate of adverse outcomes (87). In addition to CEA, carotid artery

stenting (CAS) is another optional treatment for patients with carotid artery disease.

Carotid Revascularization Endarterectomy Versus Stenting Trial (CREST) (88), showed

that CEA and CAS can be performed with a procedural risk of <3% by carefully

credentialed surgeons and interventionists. However, it was demonstrated that CAS is

associated with inferior outcomes compared with CEA for patients with symptomatic

carotid disease (89). CAS was associated with higher rate of stroke, recurrent carotid

stenosis and considerably higher cost compared with CEA (89). For the present, CAS is

generally indicated for small proportion of symptomatic patients, those with: distal

lesions where CEA is more difficult to perform; scarred or infected neck; recurrent

stenosis following CEA; possibly contralateral internal carotid occlusion (90). However,

improvements in CAS technical skill and technology (membrane covered stents, newer

protection strategies) and better patient’s selection may alter this situation in the future.

One example is the use of the flow-reversal or flow-cessation embolic protection. A

meta-analysis of 4747 patients in 24 CAS studies found a significant benefit from

protected distal procedures with a relative risk reduction of 0.59 (95% CI, 0.47–0.73)

(91). Another important strategy is the avoidance of CAS in patients with echolucent

carotid disease or extensive calcification and in patients aged >70 who were shown to

have worse results with CAS (92). Several studies have confirmed that echolucent

plaques are associated with higher rates of emboli signals and in-stent restenosis

compared to echogenic plaques. There are controversial data according to CAS

interventions on echolucent plaques and recurrent symptoms. On the basis of this, we

undertook a systematic search on PubMed for studies evaluating associations between

carotid plaque echolucency and adverse outcomes (stroke, microembolization and in stent

restenosis). Out of 412 studies found after the first medical databases search, we

identified five studies appropriate to be qualitatively and quantitatively analyzed, which

evaluated different adverse outcomes in patients with echolucent plaques after CAS.

Pooled analysis showed that CAS in echolucent carotid plaques is associated with higher

risk of stroke (OR 2.33; 95% CI 1.73-4.65, p=0.015), microembolization (OR 2.77; 95%

CI 1.40-5.45, p=0.003), and in stent restenosis (OR 3.8; 95% CI 1.93-7.44, p<0.001).

  23  

In general, pooled OR of adverse outcomes for CAS performed in echolucent compared

to echogenic plaques was 2.92 (95% CI 1.97-4.32), p<0.001 (92-96).

Identifying patients at high risk for stroke recurrence

An analysis of pooled data of randomized clinical trials showed that the risk of stroke

recurrence is 21% at five years after symptoms (20). However, the risk of stroke in the

early period after onset of symptoms seems to be much higher than it was previously

thought, with contemporary natural history studies suggesting that within 72 hours of the

presenting symptom 8-15% of patients with a 50-99% stenosis may suffer a recurrent

stroke (97, 98). Accordingly, it would be of clinical benefit to identify this subgroup of

patients at increased risk and to deliver interventions as soon as possible (emergency

CEA) after onset of symptoms, accepting that benefit must always be balanced against

the potential for perioperative stroke.

ABCD2 (99) and ABCD2-I scoring system (100) that integrate clinical and brain imaging

data has been shown to have predictive value for symptom recurrences after TIA. In

addition, there are several imaging carotid plaque features that could also assist in

identifying high risk patients, more frequently associated with stroke recurrence

including: stenosis severity, plaque irregularity and ulceration and plaque echolucency

and the presence of contralateral occlusion. Even though, significant benefit of CEA

procedures was determined for 50-99% stenosis degree, a meta-analysis including more

than 6000 patients randomized in ECST, NASCET, and the Veteran’s Affairs (VA)

Study, revealed that for 1000 CEAs intervention only 78 strokes would be prevented at 5

years in patients with 50-69% stenosis, compared with 156 strokes prevented at 5 years in

patients with 70-99% stenosis (20). Plaque irregularity was associated with significantly

higher rates of ipsilateral stroke in patients randomized to BMT, compared with smooth

stenosis. In patients with smooth carotid plaques, CEA prevented about 100 strokes/1000

interventions at 5 years compared to 200 strokes/1000 CEAs at 5 years in patients with an

irregular plaque producing 75% stenosis (20, 28). Salem et al. reported that the presence

of plaques with GSM <25 were predictive of a significantly increased risk of recurrent

neurological events in the early period, prior to CEA (101). Furthermore, the presence of

  24  

contralateral occlusion is associated with a significant increase in the risk of late stroke

ipsilateral to the treated ICA stenosis in medically treated patients (85). In a recent

systematic review, it has been found that MES were highly predictive of stroke

recurrence in patients with carotid stenosis having an OR of 9.57; (95% CI, 1.54–59.38)

(102).

Figure 5. Proposed algorithm for the management of symptomatic patients with carotid

stenosis; BMT - best medical treatment, DUS - Duplex ultrasound, CEA - carotid

endarterectomy, CAS - carotid artery stenting.

Ultrasound imaging markers of vascular risk: subclinical atherosclerosis

Many large studies have confirmed the predictive value of carotid intima-media thickness

(IMT) for cardiovascular risk. On 2010 American Heart Association/American College

of Cardiology guidelines designated carotid IMT a class IIa recommendation for

Symptomatic carotid stenosis

0-49% stenosis

70-99%stenosis

BMT and regular DUS

Control

One of the below:(1) Echolucent plaque,

(2) >2 MES on transcranial Doppler(3) markedly irregular/ulcerated

plaques

Consider CEA + BMT

Consider CEA or CAS + BMT

One of the below:(1) >70 years old patient

(2) Echolucent plaque(3) Extensive calcification

Yes No

YesNo

50-69% stenosis

  25  

cardiovascular risk assessment in asymptomatic adults with intermediate risk of

cardiovascular disease (103). However, a careful evaluation of carotid IMT studies

recently together with quantitative assessment of their results revealed discrepancies in

methods with which IMT was assessed and suggested that IMT measurement in common

carotid artery (CCA) have only a minimal predictive power beyond traditional risk

factors, with no clinical importance (104, 105). Carotid plaque assessment appears to be a

better predictor of CAV and CV risk compared to IMT alone (106). In addition,

quantitative assessment of plaques such as plaque thickness, echogenicity, area, and

volume appears to be progressively more sensitive in predicting CV risk than assessment

of plaque presence alone (106). Recently, a new measure of carotid artery wall - intima-

media grey scale median (IM-GSM), that attempt to quantify the echogenicity of the

intima-media complex seems to have a better predictive value compared to IMT (107).

Carotid artery wall measurements: intima-media thickness and intima-media GSM

Carotid IMT is measured between intima-lumen interfaces and media-adventitia

interfaces of the arterial wall represented as a double-line density on carotid ultrasound

image (figure 6). Its accuracy for the measurement of far wall IMT in common carotid

artery was validated against histological specimens representing the true biological

thickness of the vessel wall (108). Otherwise, near-all assessment of IMT was associated

with systematic measurement error, because of the echogenicity of the adventitia layer,

the boundary between media and adventitia in the near wall, is not easy to be defined

(109). The differences of the IMT values between 25th and 75th percentile are very small

(<1mm) and therefore, for the measurement of IMT a high precision is required,

including, proper training of the sonographers and implementation of protocols for the

measurement of IMT. In general, IMT measurement has been shown to have a good intra

and interobserver correlation coefficient of >0.90. Furthermore, advances of US

technology have made possible automatic measurement of IMT, obviating the need to

perform manual measurements, and this have improved the reproducibility of the

measurements (110). These systems for IMT measurements incorporate IMT datasets

from large clinical studies that allow generation of carotid IMT percentile values for

individual patients (111).

  26  

Figure 6. A. Intima-media thickness (IMT) measured in common carotid artery (CCA). B.

Intima-media and adventitia differentiation on US images and C. Relevant histological

structure of carotid artery wall. 1 - Lumen-intima interface, 2 - media-adventitia

interface. (a) tunica-intima, (b) tunica media, (c) adventitia. Internal elastic membrane

separates intima from media and external elastic membrane separates media from

adventitia

One of the reasons behind the limitation of IMT measurements was the different

definition between studies. Some have used a cut-off of ≥0.9 mm or ≥1 mm (112, 113),

others defined an abnormal IMT as >75th percentile for age, ethnicity and sex, which is

also recommended by American Society of Echocardiography (114).

Salonen et al. were the first to demonstrate the association of carotid IMT with future

coronary events on 1991 (115). They showed that each 0.1 mm increment of IMT was

associated with 11% increased risk of MI on follow up. Subsequently, there were several

other large clinical studies, including: ARIC (Atherosclerosis Risk in Communities)

study (113), the CAPS (Carotid Atherosclerosis Progression Study) (116) the CHS

(Cardiovascular Health Study) (117), the MDCS (Malmo Diet and Cancer Study) (118)

and the Rotterdam Study (119), all showed that carotid IMT can be used to assess

incident CV disease risk. However, studies that evaluated the predictability of the IMT

over and above traditional risk factors (Framingham risk score) have been in most cases

negative. In the Multi-Ethnic Study of Atherosclerosis (MESA) (120), IMT measurement

in the CCA did not predict either MI or ischemic stroke after adjusting for Framingham

1 2

1

2

(a)

(b)

(c)

LUMEN

A B C

  27  

risk score. Lau et al. showed that the IMT measurements have an area under the curve of

0.69 for predicting CV events compared to 0.66 for Framingham risk score (121). These

data showed that IMT is not a highly predictive marker as it was thought, highlighting the

need for finding another measure (marker) on the carotid artery wall that could be a better

predictor of future risk. Measurement of echogenicity of the intima-media complex (IM-

GSM) was recently introduced as a possible marker of subclinical atherosclerosis. This

could probably differentiate between: adaptive intimal thickening (representing a hyper

reactivity of the smooth muscle cells in the tunica media) from intimal xanthoma or

pathological intimal thickening of the arterial wall (representing the accumulation of

LDL particles and inflammatory cells into intima) (figure 1). An echolucent IM-complex

(low IM-GSM) was associated with different risk factors of atherosclerosis and increased

risk of vascular events and mortality rate (122).

It has been shown that adding plaque and IMT data improved risk prediction in

intermediate risk patients but plaque presence in carotid artery is more effective than IMT

in predicting future ischemic events (106). A meta-analysis showed that compared to

IMT, carotid plaque had a 35% better predictive value of future ischemic events, after

adjusting for Framingham risk factors. In the ARIC study the model that performed best

for prediction of ischemic events, death and revascularization, included traditional risk

factors plus IMT plus carotid plaque (113). For intermediate risk patients NRI was 21%

and it was higher for women than man, 25.4% vs. 16.4% respectively. It was suggested

that carotid plaques grow faster in their length than thickness, and based on this it seems

that assessment of carotid plaque area is potentially a better indicator of disease burden.

In multivariable linear regression models, traditional risk factors explained only 15-17%

of the IMT compared to 52% of the carotid total plaque area. In addition, advancement of

US techniques has made possible assessment of carotid plaque volume by 3D images.

Early results showed plaque volume assessment is a more reliable and sensitive to

changes than cross-sectional area, and could be a better marker to evaluate the effects of

different treatments on the carotid plaque (123).

  28  

OBJECTIVES

Study I

To evaluate the status of the plaques located in the contralateral to symptomatic carotid

artery and to compare those plaques with plaques located in the same patients ipsilateral

side and also plaques located in asymptomatic carotid arteries.

Study II

To evaluate the accuracy of ultrasound in detecting carotid artery calcification as

compared to cone beam CT and in addition to compares calcification presence and

volume between different subgroup of patients and different risk factors.

Study III

To evaluate the ability of two different carotid arterial wall measures, IMT and IM-GSM

in differentiation between patients with and without prior vascular events in different

arterial systems and also between numbers of arterial systems affected by symptomatic

atherosclerosis disease.

Study IV

To evaluate the association between carotid plaque echogenicity and future

cerebrovascular ischemic events in asymptomatic and symptomatic patients, in a

systematic review and meta-analysis study.

  29  

METHODS

Preoperative patients evaluation

In all new cases presented with ischemic stroke a brain CTA, MRA or rarely

conventional angiography is performed and analyzed by experienced neuro-radiologists,

either at the Radiology department in Umeå University hospital or it would have been

performed at the referring hospital. Ultrasound examination is performed at the

department of Clinical Physiology at the Umeå University Hospital by experienced

vascular sonographers. All carotid scans are reported and also the images are illustrated

as a graph describing the location and the degree of stenosis and plaque calcification.

Ultrasound carotid imaging follows the NASCET-type protocol for assessing carotid

stenosis. All patients undergo a detailed neurological examination by a neurologist, who

determines if the symptoms are related to the diseased circulatory area in the brain. Also,

an echocardiogram is performed in these patients. Finally, the decision for CEA is

undertaken after discussion among the neurologist, internal medicine specialist and

vascular surgeon. For more complicated cases, there are meetings regularly organized,

“carotid rounds”, where cases are presented and discussed in detail by a group of multi-

disciplinary specialists (internal medicine, radiologists and vascular surgeons), and after

careful evaluation of risk and benefit the decision for BMT or invasive strategy (CEA vs.

CAS) is made.

Definitions and clinical data of patients included in Study I, II and III

Symptomatic patients with carotid stenosis were defined as those who had an ipsilateral

ischemic CV event (stroke, TIA or retinal artery embolization) within the 6 months prior

to the carotid scan, whereas asymptomatic patients were those with carotid

atherosclerosis but with no CV event within the same period (6 months).

Asymptomatic patients were identified because of a) objective or subjective bruit during

auscultation, b) a suspicion of CV symptoms which was later confirmed to be not of CV

  30  

origin, c) follow-up of a known carotid stenosis, d) previous known CV symptoms > 6

months before, e) detection of calcification in the territory of carotid artery by panorama

imaging, f) CV symptoms related to posterior circulation, or g) carotid disease detection

during evaluation of the thyroid gland.

Patients clinical data were prospectively analyzed to establish; a) the number and

duration of ischemic events; b) time of last symptom; c) risk factors (smoking, diabetes,

hypertension and dyslipidemia) and d) the surgical information. Data for coronary artery

disease, ongoing condition or prior events, were also collected. Routine biochemical data

were collected from the patient's clinical notes including lipid profile and glycated

hemoglobin (HbA1c).

Carotid ultrasound examinations

All patients included in Study I, II and III underwent preoperative carotid Doppler

ultrasound examinations, using a Siemens Acuson Sequoia 512® system with an 8L5

linear transducer. Using conventional Doppler ultrasound criteria the degree of carotid

stenosis was assessed (124).

Ultrasound data retrieval

Studies included in this thesis are retrospective analysis of the patients who were engaged

into two prospective studies:

1) Panorama arm of the SPACE study (15),

2) Additional Neurological SYmptoms before Surgery of the Carotid Arteries — a

Prospective study (ANSYSCAP) (125).

In the study I and II we used data of the Panorama arm of the SPACE study and in study

III we used only a subgroup of asymptomatic patients of the ANSYSCAP study. Over the

course of these studies, the ultrasound imaging storage process was upgraded from

analog to digital.

  31  

In study I and III, we aimed to obtain a detailed carotid plaque features analysis, we

decided not to combine in the same study data stored in digital and those stored in analog.

So in study I, we included only carotid ultrasound images stored in analog and for study

III we used only images of asymptomatic patients stored in digital. Because in study II

our intention was to only determine carotid plaque calcification (presence/absence), and

not undergoing detailed plaque analysis we decided to use both, data stored in analog as

well as those in digital.

Extraction of the carotid ultrasound examinations

The ultrasound images stored in analog or digital system were exported to the EchoPac

software (General Electric, EchoPac version 8.0.1, Waukesha, WI) and the IMT was

measured and the morphological features (plaque irregularities and calcification) were

assessed. We performed imaging normalization and measurements of the plaque and

intima-media complex features by using an in-house custom developed research software

package (Department of Biomedical Engineering — R&D, Umeå University Hospital,

Umeå, Sweden).

Definitions of ultrasound derived plaque and intima-media complex features

Plaque definition was taken as a focal protrusion that proved to be more than 50% the

thickness of the adjacent wall thickness (126). Each plaque was analyzed for plaque type,

calcification, irregularity, GSM, JBA, GSM of the JBA, DWAs, coarseness and entropy.

In the arteries with only one plaque, the feature of the latter was taken to represent the

artery. Arteries with more than one plaque were treated in a way that the congregated

information/measurements from all plaques represented that artery. When it came to

irregularity measurements, the worst abnormality of irregularity in all plaques was taken

to represent the artery. The presence of either calcification, JBA and DWA was marked

in all studied arteries too. An area-weighted average was taken for continuous features

(GSM, area of JBA and entropy) and used in relation to the size of the artery. The overall

plaque feature was assessed as an averaged sum of individual features multiplied by

plaque area and divided by the total plaque area.

  32  

Plaque irregularity (Study I and III): The surface plaque irregularities were categorized

into three groups: (1) smooth, (2) mildly irregular (height variations 0.4 mm along the

contour of the lesion), or (3) markedly irregular (height variations >0.4 mm) (127).

Grey scale median (GSM) (Study I and III): It was measured as the median of the grey

values of all pixels within cropped plaque image after image normalization (figure 7).

Juxtaluminal black-area (JBA) Study (III): These black areas close to the lumen without

visible echogenic cap represent the JBA (38). The area was manually detected and

outlined, which together with the corresponding GSM were calculated by the software.

The larger value was used in cases with several JBAs within a plaque.

Plaque type (Study I and III): According to the modified Geroulakos classification, after

image normalization and plaque delineation, plaques were classified by the software into

the four types (128) (Fig. 3):

Type 1, uniformly echolucent (black), <15% of pixels in the plaque area with values >25;

Type 2, mainly echolucent, pixels with grey scale values >25 occupying 15-50% of the

plaque area;

Type 3, mainly echogenic, pixels with grey scale values >25 which occupy 50-85% of the

plaque area; and

Type 4, uniformly echogenic, pixels with grey scale values >25 which occupy >85% of

the plaque area.

Calcifications (Study I and II): Were defined as hyperechogenic spots that produced

posterior shadowing.

Discrete white areas (DWAs): Discrete white areas, when present, were defined as those

without posterior shadowing but with pixels of grey scale values >124, colored red in the

grey scale-based stratified color mapping of the plaque types 1 to 3 (129).

  33  

Entropy (Study I and III): Entropy was taken as a measure of the random nature of the

grey-tone values within the plaque (130), with low values for homogenous tissue and

high values for heterogeneous composition.

Coarseness (Study III): Coarseness quantifies the granularity of the plaque texture, which

is consistent with high degree of local uniformity in intensity for large areas. Coarseness

was calculated using the neighborhood grey-tone difference matrix method (131). It has

already been demonstrated that coarseness discriminates symptomatic from

asymptomatic plaques with low values in symptomatic plaques consistent with

heterogenic, more granular texture and less uniform composition (132).

Figure 7. Image normalization and plaque features assessment

Intima–media complex measurements (Study III)

Both IMT and IM-GSM were measured in the distal segment of the common carotid

artery within 1 cm (± 2 mm) distance starting from the carotid bifurcation at the right and

left carotid systems. When a plaque was present in this region, the IM-complex free-of-

  34  

plaque was selected, and if it was <8 mm long the artery was excluded from the study (n

= 8).

Carotid-intima media thickness (IMT)

IMT was conventionally defined as the distance between the media–adventitia interface

and the lumen–intima interface. One-centimeter long segments of the far wall were

measured and an average of three cycles was taken (133).

Intima–media grey scale median (IM-GSM)

The median of the grey values of all pixels within a one-centimeter length cropped intima

media complex image was measured to reflect IM-GSM. The distal intima-media

complex in the normalized and standardized image was manually outlined, prior to

feature calculation.

Cone Beam CT (CBCT) analysis

Excised plaques after CEA were examined by CBCT (Cone Beam Computed

Tomography, 3D Accuitomo 170, J Morita MFG Corporation, Kyoto, Japan; 60 kV, 1

mA, 360°). Volumes of 4 × 4 or 6 × 6 cm were used and they had a resolution of 0.08–

0.125 mm voxel size. The reconstructions were made with 0.5 mm slice thickness and 0.5

mm increment. A software program (General Electric Company, Barrington, IL, USA,

advantage workstation 4.3, Volume Viewer 2) for volume measurements was also used.

All calcification spots were measured in mm3, with resolution of the volume

reconstructions of 1 mm3. Plaques with multiple dispersed calcifications had the largest

calcification nodule volume used to represent the intra-plaque calcification.

Carotid endarterectomy (CEA) procedure

All patients were operated under general anesthesia. Common, internal and external

carotid arteries were exposed and a longitudinal incision was made in the common

carotid artery and then extended into the internal branch until a healthy area distal to the

plaque. The intimal thickening of the common carotid artery was divided and the plaque

was gently dissected from the artery, leaving the adventitial layer intact with careful

  35  

handling of the plaque. After excision, the plaque was immediately placed in a plastic

tube and stored in −20 °C, then transferred to a −80 °C freezer until later taken out for

CBCT analysis.

Source of data

Study I and II

This is a secondary analysis of the Ultrasound of the Panorama arm of the SPACE study

[15]. In the main study, 100 consecutive patients with symptomatic or asymptomatic

carotid stenosis (≥50%) who were eligible for CEA were included. All patients included

agreed to extensive preoperative evaluation and underwent CEA.

Study III

This is a secondary analysis of the ANSYSCAP study (125). In the main study

consecutive symptomatic and asymptomatic patients with 50-99% stenosis were included

between August 2007 and December 2009. In ANSYSCAP, consecutive patients with a

50-99% carotid stenosis eligible for CEA were prospectively included between August

2007 and December 2009.

Study IV

For the systematic review and meta-analysis we have systematically searched electronic

databases (PubMed, MEDLINE, EMBASE and Cochrane Center Register) up to January

2015 for studies evaluating the effect of carotid plaque echogenicity on CV symptoms.

The search terms used were: “carotid atherosclerosis”, “carotid plaque”, “echogenicity”

“ultrasound” “symptoms”, “Stroke”, “TIA” “amaurosis fugax” in various combinations.

Two researchers (FJ and PI) independently performed the literature search, study

selection and data extraction. There was no time or publication limit in the literature

search. The selected reports were manually searched, and relevant publications, obtained

from the reference lists, were retrieved.

  36  

Inclusion/exclusion criteria

Study I

Out of 47 cases with a preoperative ultrasound examination stored in the analog format

we analyzed 39. Eight patients were excluded because either the examinations could not

be retrieved and analyzed (n = 7) or the patient had an asymptomatic carotid stenosis but

a recent (<6 months) CV event on the contralateral side. Of the 39 patients included, 33

had symptomatic carotid stenosis (50-99%) and 6 had unilateral or bilateral

asymptomatic stenosis (50-99%). All patients had at least one atherosclerotic plaque on

both sides. We further excluded 12 arteries because of total occlusion (n=2) or because of

>50% of the plaque covered by shadow derived from extensive plaque calcification

(n=10). So, in total we analyzed 66 arteries (25 contralateral to symptomatic, 30

symptomatic and 11 asymptomatic). The contralateral arteries were compared with the

symptomatic and asymptomatic arteries.

Study II

Out of 101 carotid plaques (of 94 patients) we finally analyzed 94 plaques (from 88

patients). Seven patients were excluded, five because of suboptimal image quality and

two because there was no calcification in the internal carotid artery (ICA) plaque but a

distinct calcification in the external carotid artery (ECA). Since it was unclear whether

the ECA would be included in the extirpated plaque, it was impossible to make a

reasonable assessment without breaking the blinding.

Study III

Out of 87 asymptomatic patients stored in the digital system we finally analyzed 166

arteries. Seven arteries were excluded because the common carotid artery segment free of

plaque was <0.8mm.

  37  

Figure 8. Flowchart of the patients selections for study I, II and III

Study IV

Studies reporting results on US assessment of plaque echogenicity and their association

with CV symptoms (Stroke, TIA, amaurosis fugax) were included in our analysis.

Specific inclusion criteria for the systematic review were: (1) Cohort prospective studies;

(2) English language articles; (3) studies with ≥30 subjects, and (4) ultrasound based

characterization of carotid artery plaque, visually based on Gray-Weale, or using

computer-assisted analysis (Geroulakos classification or GSM). Studies that visually

categorized plaques in only two groups echogenic/echolucent, that evaluated plaque

heterogeneity, that used imaging techniques other than US (e.g. MRI, CT, IVUS, PET),

and those that analyzed plaque features other than echogenicity (volume, degree of

  38  

stenosis, plaque irregularities, ulceration, neovascularization) as predictors of CV

symptoms were excluded (figure 9).

Figure 9. Flowchart of the studies selection for the study IV

Statistical analysis

Study I, II and III. All statistical analyses were performed using IBM SPSS Statistics

22. Categorical variables were expressed as percentages and continuous variables as

mean ± SD (median). Mean values of the plaque texture features were compared between

groups, using independent samples t-test and Man-Whitney test. Baseline differences

between groups for each plaque texture feature were tested using one-way ANOVA, for

more than two groups in analysis. Post hoc analysis (Bonferroni) was also performed for

Records identified through databases searching

(n=1385)

Additional records identified through

related citations search(n=20)

Records after duplicates removed(n=1387)

Records screened(n=1387)

Full-text articles assessedfor eligibility

(n=30)

Recordsexcluded(n=1357)

Cross-sectional studies (n=12)Evaluated plaque hetero-genicity (n=6)Categorized carotid plaque echogenicity in two groups (n=2)

vio Iden

tifica

tion

Scre

enin

gEl

igib

ility

Incl

uded

Studies included in qualitative analysis (n=10)

Studies included in meta-analysis

(n=10)

  39  

continuous variables. If variables did not have normal distribution, the non-parametric

Kruskal-Wallis test was used. Fisher's exact probability test or the Chi-2-test was used

when comparing two sets of binary or categorical values, with a pre-selected significance

level of p < 0.05.

Quartile analyses were used for categorizing calcification volume into four groups.

Kappa values were calculated to determine the accuracy of US in detecting calcification.

Logistic regression analysis was used to determine the association of an echolucent

intima-media complex with multi-system atherosclerosis disease.

Study IV.

We have separately analyzed studies that evaluated asymptomatic and symptomatic

patients. I2 statistic was used to measure the heterogeneity. When there was a significant

variation between studies size, length and follow-up, as well as patient’s characteristics,

we used random-effect. Publication bias was examined using Begg–Mazumdar test. All

analyses were conducted using Comprehensive Meta Analysis version 3 software (Biostat

inc., Englewood, NJ, USA).

  40  

RESULTS

Study I

Clinical data

The severity of carotid artery stenosis and other risk factors are presented in figure 10 and

11. There was no difference between asymptomatic and symptomatic patients with

carotid stenosis.

Figure 10. Clinical data in symptomatic and asymptomatic patients. BP-blood pressure,

SD-standard deviation

0"

10"

20"

30"

40"

50"

60"

70"

80"

90"

Symptoma3c" Asymptoma3c"

Age,%mean%(SD)%

0"20"40"60"80"100"120"140"160"180"200"

Symptoma/c" Asymptoma/c"

Systolic)BP)(SD))

0"10"20"30"40"50"60"70"80"90"

100"

Symptoma3c" Asymptoma3c"

Diastolic)BP)(SD))

0"

1"

2"

3"

4"

5"

6"

7"

Symptoma1c" Asymptoma1c"

Total&cholesterol&(SD)&

0"

0.5"

1"

1.5"

2"

2.5"

Symptoma.c" Asymptoma.c"

HDL$cholesterol$(SD)$

0"

0.5"

1"

1.5"

2"

2.5"

3"

3.5"

4"

Symptoma0c" Asymptoma0c"

LDL#cholesterol#(SD)#mmol/L mmol/L

mmol/L

mmHgmmHg

mmHg

  41  

Figure 11. Distribution of the different risk factors, previous or ongoing ischemic events

and therapy in symptomatic and asymptomatic patients

Degree of stenosis

Based on the carotid Doppler velocities there was no difference in the degree of stenosis

between contralateral and asymptomatic arteries. In symptomatic arteries the degree of

stenosis was higher compared to contralateral (p<0.001) and asymptomatic arteries

(p=0.006).

Contralateral vs. Symptomatic

The GSM and JBA in the plaques located in the contralateral to symptomatic side had

similar features to those in the symptomatic side (26.2 ± 7.3 vs. 24.9 ± 7.8, p = 0.536)

(figure 12) and (5.0 ± 3.9 vs. 4.6 ± 3.0, p = 0.80), respectively. Also, the plaque

irregularities and PT were not different between these two groups. The only plaque

feature that differentiated between symptomatic and contralateral arteries was the

prevalence of JBA, which was higher in symptomatic arteries (p=0.001).

0"

10"

20"

30"

40"

50"

60"

70"

80"

90"

100"

Women"

Current"smoker"

Diabetes"

Previous"myocardial"infarcBon"

Current"angina"

Stroke"6"months"before"

ClaudicaBon"intermitent"

Previous"revascularizaBon"of"any"type"

Any"lipid"lowering"medicaBon"

33.3"

6.1"

30.3"

15.2"

3"

24.2"18.2"

27.3"

93.9"

33.3"

0"

33.3"

16.7"

0"

16.7"

33.3"

50"

100"

SymptomaBc" AsymptomaBc"%

  42  

Contralateral vs. Asymptomatic

Contralateral arteries had more vulnerable morphological and textural plaque features

compared to those in the asymptomatic arteries, with lower GSM (26.2 ± 7.3 vs. 49.4 ±

14.6, p < 0.001), lower GSM of the JBA (5.0 ± 3.9 vs. 11.4 ± 2.1, p = 0.001), higher

prevalence of plaque type 1 and 2 and lower prevalence of type 3 and 4 (p = 0.001) and

higher prevalence of mild (60% vs. 36%) and marked irregularities (28% vs. 9%), p =

0.03, (Figure 12).

Figure 12. Echogenicity (left) and surface plaque morphology (right) between

asymptomatic, contralateral to symptomatic and symptomatic arteries

Reproducibility analyses

Interobserver variability was expressed using intra-class correlation coefficient. For GSM

it was 0.928 (95% CI, 0.899-0.950) and for JBA it was 0.972 (95% CI, 0.848-0.965). The

interobserver agreement for JBA was (K=0.948, p<0.0001) and for PT was (0.843,

p<0.001).

  43  

Study II

Clinical data

Mean age of the patients included in this study was 70 ± 7 years and 33% were females.

Symptomatic carotid artery stenosis was observed in 73 patients and asymptomatic

carotid stenosis in the remaining 15 patients. Six of the symptomatic patients underwent

bilateral carotid endarterectomy (CEA). A stenosis severity of 50%–69% and 70-99%

was found in 6 and 85 cases, respectively, and near-occlusion in three arteries. Patient’s

baseline characteristics are presented in Table 1.

Baseline Study population (n=88) Age (years), mean±SD) Females, n (%)

70±7 29 (33)

Systolic blood pressure (mmHg), mean±SD 147±22.6 Diastolic blood pressure (mmHg), mean±SD 78±12 Total cholesterol (mmol/l), mean±SD 4.61±1.03 LDL (mmol/l), mean±SD 2.60±0.92 HDL (mmol/l), mean±SD 1.27±0.48 Creatinine (μmol/l), mean±SD 84±25 HBA1c (mmol/mol), mean±SD 52.5±12.7 Symptomatic carotid stenosis, n (%) 73 (83) Prior myocardial infarction, n (%) 14 (16) Current angina pectoris, n (%) 7 (8) Heart failure, n (%) 1 (1.1) Previous stroke (>6 months to the present evaluation), n (%) 14 (16) Claudication (lower extremity artery disease), n (%) 10 (11.4) Any previous revascularization for ischemia, n (%) 24 (27.3) Current smoker, n (%) 9 (10.2) Diabetes, n (%) 29 (33) Lipid lowering medicine, n (%) 82 (93.2) Platelet inhibiting or anticoagulation medicine, n (%) 88 (100) Blood pressure reducing medicine, n (%) 84 (95.5)

Table 1. Patients’ data

  44  

Ultrasound vs. CBCT

Carotid artery calcification was detected in 87.2% of cases in the preoperative ultrasound

examinations and in 98.9% in CBCT examinations after CEA. There was no difference

between the 50%–69% and the 70%–99% degree of stenosis groups (Table 2).

Furthermore, there was no difference in carotid calcification between different risk

factors (Table 2). In general, the sensitivity of carotid ultrasound in detecting the

presence of calcification was 88.2%. The calcification volumes acquired by CBCT were

divided into 4 groups using quartile analysis; <8, 8–35, 36–70 and >70 mm3 (Figure 13).

Calcification volumes ≥8 mm3 were accurately detectable by ultrasound with a higher

sensitivity of 96% compared to the sensitivity of 62% for the calcification volume of <8

mm3 (Table 2, Figure 13).

CBCT\Ultrasound Total

arteries, n

Calcification on CBCT

Calcification on US

US Sensitivity

Calcification 94 93 82 88.20% Calcification ≥8

mm3 72 72 69 96%

Calcification <8 mm3

22 21 13 62%

Table 2. Accuracy of Doppler ultrasound in detecting carotid calcification

  45  

Figure 13. Quartiles of calcification volumes detected and not detected by ultrasound

Calcification vs. Symptoms

The presence of calcification, evaluated by US was not different between symptomatic

and asymptomatic patients. In addition, we explored whether dispersed calcification

within the plaque could be associated with patient’s symptoms, but we didn’t find any

difference between groups. Even using quartiles of calcification volumes evaluated by

CBCT couldn’t discriminate between symptomatic and asymptomatic patients. In table 3

we have compared calcification volume (< and ≥ than 8 mm) between different risk

factors.

Reproducibility analyses

The inter-observer agreement for the presence of calcification in the carotid artery

plaques evaluated by US was good (K=0.905, p<0.001).

  46  

Subgroups Arteries n

<8mm3 calcification volumes on CBCT,

n (%) p

Age <75 68 18 (26.5) ≥75 26 4 (15.3) 0.21

Gender Female 30 7 (23.3) Male 64 15 (23.4) 0.99

Symptomatic 79 20 (25.3) Asymptomatic 15 2 (13.3) 0.31 Current smoker

Yes 10 4 (40.0) No 84 18 (21.4) 0.19

Diabetes Yes 31 8 (25.8) No 63 14 (22.2) 0.60

Previous stroke Yes 17 2 (11.8) No 77 20 (26.0) 0.21

Previous MI Yes 15 3 (20.0) No 79 19 (24.0) 0.73

Statin therapy Yes 87 21 (24.1) No 7 1 (14.3) 0.55

Degree of stenosis 50-69% 6 0 (0) 70-99% 85 20 (23.5)

near-occlusion 3 2 (66.7) 0.08 Table 3. Subgroup analysis

  47  

Study III

Clinical data

In this study we included 87 asymptomatic patients (166 carotid arteries). Mean age of

the patients was 69±6 year and 34.5% were females). 23% of patients had previous MI,

16% had angina, 21% had previous ischemic stroke, and 22% (n=19) had lower limb

atherosclerosis in the form of intermittent claudication (Table 4). 50% (n=43) of the

patients were asymptomatic, 34% (n=30) had previous disease in one arterial system and

16% (n=14) had previous disease in multi-arterial systems.

Age, years, mean±SD 69±6 Female, n (%) 30 (34.5) Current smoking, n (%) 18 (20.7) Diabetes, n (%) 25 (28.7) Previous MI, n (%) 20 (23) Current angina, n (%) 14 (16.1) Previous stroke, n (%) 18 (20.7) Intermittent claudication, n (%) 19 (21.8) SBP (mmHg), mean±SD 145.7±20 DBP (mmHg), mean±SD 77±12 Anti-platelet or anti-coagulation therapy, n (%) 84 (96.6) Blood pressure lowering therapy, n (%) 80 (92) Lipid lowering therapy, n (%) 78 (89.7) HbA1c (%), mean±SD 5.2±1.1 Total cholesterol (mmol/l), mean±SD 4.67±0.9 LDL (mmol/l), mean±SD 2.57±0.9 HDL (mmol/l), mean±SD 1.34±0.4

Table 4. Patients’ data

Intima-media measurements vs. previous vascular symptoms

In patients with previous MI the IMT was higher (1.06±0.2 vs. 0.95±0.2 mm, p=0.034)

and IM-GSM was lower (21±15 vs. 33±16, p=<0.001). Also, patients with previous

stroke had lower IM-GSM (24±12 vs. 33±16, p=0.007) but IMT was not different

between those with and without stroke (1.03±02 vs. 0.96±0.2 mm, p=0.195). IMT and

  48  

IM-GSM were not different in patients with and without previous atherosclerosis disease

in the lower extremity (Figure 14).

Figure 14. IM-GSM in patients with previous stroke, MI angina and claudication

intermittent

IMT and IM-GSM vs. multi-system atherosclerosis disease

IM-GSM was significantly different between groups, it decreased significantly with

increasing number of arterial systems affected by symptomatic atherosclerosis disease,

37.7±15.4 vs. 29.3±16.4 vs. 20.7±12.9, p<0.001, for asymptomatic, symptoms in one and

in multi-arterial system disease, respectively. When analyzing IM-GSM measured in the

artery-side with higher IMT the results were similar (Table 5). IMT was not significantly

different between groups, 0.95±0.2mm vs. 0.98±0.2mm vs. 1.02±0.02mm, p=0.49 (Table

5, Figure 15).

  49  

Asymptomatic (n=83)

Previous symptoms in one arterial system (n=58)

Previous symptoms in >one arterial system (n=25)

p

IM-GSM, mean±SD* IM-GSM, mean±SD#

37.7±15.4 35.0±15.7

29.3±16.4 29.5±18.6

20.7±12.9 17.4±13.9

<0.001 0.001

IMT, mean±SD 0.95±0.2 0.98±0.2 1.02±0.2 0.49 Plaque GSM, mean±SD 41.4±17.0 33.0±16.0 20.9±10.0 <0.001 Plaque Coarseness, mean±SD 13.7±4.4 11.4±5.1 7.6±3.9 <0.001 Plaque Entropy, mean±SD 3.5±0.2 3.6±0.2 3.6±0.2 0.13 Plaque JBA, n (%) 14 (19.4) 14 (28.6) 13 (72.2) <0.001 Plaque GSM of JBA 6.8±5.0 12.1±9.0 3.8±3.3 0.01

Table 5. Common carotid wall and bifurcation plaque measurements in different groups.

(*)IM-GSM mean of both sides. (#) IM-GSM measured on the side with higher IMT

Figure 15. IM-GSM, plaque GSM and plaque coarseness were significantly decreased

with increasing number of arterial systems affected by atherosclerosis

p<0.001p<0.001

p<0.001

p=0.137p=0.614

Asymptomatic Previous symptoms in one arterial system

Previous symptoms in >1 arterial system

0

10

20

30

40

50Plaque GSMIM-GSMPlaque coarsenessPlaque entropyIMT

Mea

n

  50  

Using ROC curve analysis we determined IM-GSM of 25 as the best cut-off value to

differentiate between numbers of arterial systems affected by the atherosclerosis disease.

Out of 25 arteries in the multi-system group, 16 (64%) had an IM-GSM <25, having an

OR of 3.23 (95%CI 1.33-7.85), p<0.01. Even after adjusting for IMT this association

remained significant with OR 2.65 (95%CI 1.56-6.67), p=0.03.

Reproducibility analyses

For intima-media complex and plaque measurements there was a good agreement

between the two observers. The interobserver variability for IMT measurements

expressed by intra-class correlation coefficient was 0.977 (95% CI, 0.963-0.988) and for

IM-GSM it was 0.934 (95% CI, 0.783-980). For plaque measurements intraclass

correlation was 0.921 (95% CI, 0.890-0.942) and for JBA it was 0.910 (95% CI, 0.871-

0.960). In addition we performed Bland-Altman test for the intima-media complex

measurements and there was not any proportional bias for IMT and IM-GSM

measurements, mean difference between the two observers measurements was not

significantly different from zero.

Study IV (Systematic review and meta-analysis)

Study selection

After the first searching of online medical databases we identified 1385 studies. In

addition ten studies were identified through related citations. After excluding duplicates,

1387 studies were screened (table 6). By reading the abstracts of the selected papers, we

first depicted 30 potentially eligible articles for further review, of these only 10 (101,

134-142) met the inclusion criteria for qualitative analysis. One of the studies (137) had

analyzed separately symptomatic and asymptomatic patients and we included both groups

in the meta-analysis. The remaining 20 studies were excluded (figure 9).

  51  

Qualitative assessment and study characteristics

There were considerable differences between reports in the degree of stenosis between

asymptomatic studies included in this analysis with 3 studies that have included patients

with mild to severe carotid stenosis (30-99%), 3 studies with moderate to severe stenosis

(50-99%) and 2 studies with severe carotid stenosis (≥70%) stenosis.

Author, year Number of subjects

No. of arteries

analyzed

Degree of carotid stenosis

Echolucency definition

Mean follow-up (months)

Outcome

1.Polak et al. 1998 (134) 4886 N/A 30-99% PT 1 and 2 39.6 Stroke, TIA 2.Mathiesen et al. 2001 (135) 223 N/A 35-99% PT 1 and 2 36 Stroke, TIA, AF 3.Liapis et al. 2001 (136) 332 442 30-95% PT 1 and 2 44 Stroke, TIA, AF 4a.Grønholdt et al. 2001 (137) 111 111 50-99% GSM<75 52.8 Stroke

4b.Grønholdt et al. 2001 135 135 50-99% GSM<75 52.8 Recurrent symptoms

5.Nicolaides et al. 2005 (138) 1115 N/A 50-99% PT 1, 2 ,3 37.1 Stroke, TIA, AF 6.Hashimoto et al. 2009 (139) 250 297 50-99% GSM<69 25 Stroke 7.Topakian et al. 2011 (140) 435 N/A 70-99% PT 1 and 2 24 Stroke, TIA

8.Salem et al. 2012 (101) 158 N/A 50-99% PT 1 and 2 2 weeks Recurrent symptoms

9. Silvestrini et al. 2013 (141) 621 N/A 70-99% PT 1 and 2 22 Stroke TIA

10. Singh et al. 2013 (142) 206 N/A 30-99% PT 1 and 2 6 Recurrent symptoms

Table 6. Studies included in the qualitative and qualitative analysis. GSM - grey scale

median, AF - amaurosis fugax, TIA - transient ischemic attack, PT - plaque type based on

Gray-Weale or Geroulakos classification

Four studies have visually (qualitatively) evaluated plaque echogenicity and four others

performed computer-assisted (quantitative) analysis of the plaque echogenicity. Two

studies defined plaque echolucency based on a cut-off value of GSM (<69 and <75) and

six studies defined plaque types (PT) 1 and 2 as echolucent and PT 3 and 4 as echogenic.

All studies have used longitudinal section US images for carotid echogenicity evaluation.

Patient’s clinical outcome was defined as stroke, TIA or amaurosis fugax (AF) in 8

studies and as recurrent CV accidents in 3 studies (Table 6).

Seven studies showed a higher risk of ipsilateral CV accidents in asymptomatic patients

with echolucent carotid plaques. Two studies showed higher risk of recurrent CV

  52  

accidents and one study showed that echolucent plaques could predict future events in

symptomatic but not asymptomatic patients.

Although, we observed a difference in the follow-up periods between different

symptomatic studies included in the analysis, the association between plaque

echolucency and recurrent ischemic events was determined even at only 2 weeks after

symptoms initiation. The degree of stenosis among symptomatic patients studies ranged

from 30-90%.

Meta-analysis results

We have meta-analyzed 8 studies with 7937 asymptomatic patients and 3 studies with

499 symptomatic patients. Among studies that met the set criteria for inclusion in the

meta-analysis, one has reported separately data for symptomatic and asymptomatic

patients and we included both of them in the analysis separately. Echolucent carotid

plaques predicted future CV accidents in asymptomatic patients with pooled relative risk

(RR) of 2.72 (95% CI, 1.86 to 3.96), p<0.001 (figure 16), and also recurrent symptoms in

symptomatic patients with pooled RR of 2.97 (95% CI, 1.85-4.78), p<0.001 (figure 17).

Figure 16. Forest plot of the studies evaluating the association between echolucent

plaques and future CV accidents in asymptomatic patinets. I2=39

Study name Statistics for each study Risk ratio and 95% CI

Risk Lower Upper ratio limit limit Z-Value p-Value

Polak et al. 1998 2.780 1.359 5.686 2.800 0.005Liapis et al. 2001 1.900 1.080 3.341 2.229 0.026Mathiesen et al. 2001 2.380 1.181 4.795 2.426 0.015Gronholdt et al 2001 1.010 0.430 2.371 0.023 0.982Nicolaides et al. 2005 4.800 2.256 10.215 4.071 0.000Hashimoto et al 2009 4.400 1.182 16.383 2.209 0.027Topakian et al. 2011 6.430 1.352 30.573 2.339 0.019Silvestrini et al. 2013 4.150 1.764 9.763 3.261 0.001

2.720 1.864 3.968 5.191 0.000

0.01 0.1 1 10 100

Favours A Favours B

  53  

Figure 17. Forest plot of the studies evaluating the association between echolucent

plaques and recurrent CV accidents in symptomatic patinets. I2=0, Kendall’s Tau=0.66,

p=0.29

Subgroup analysis

Based on the degree of carotid artery stenosis (mild-to-severe vs. moderate-to-severe vs.

severe), the time period during which patients were recruited in the studies and US

imaging was performed (before 2000 vs. since 2000) definition of the echolucent plaque

(GSM cut-off vs. PT 1 and 2), and the methods used for assessment of carotid plaque

types (visually vs. computer assisted), we have performed some comparison analyses

between subgroup (table 7).

There was clear association between echolucent plaques and future CV symptoms for all

degrees of stenosis categories, however, the highest RR was observed among studies with

severe stenosis 4.72 (95% CI, 1.86-12.00), p<0.001 (figure 18). Also, studies that

included US data collected after 2000 showed higher RR 4.65 (95% CI, 2.84-7.61) than

those before the year 2000, RR 1.97 (95% CI, 1.40-2.78) (figure 19). In addition,

analyzing separately only studies that have used a GSM cut-off to define carotid plaque

echolucency the prediction of future CV events was not significant (p=0.23), whereas RR

of the studies that defined echolucency as PT 1 and 2 was 3.01 (95% CI, 2.02-4.50),

p<0.001 and in cases when the PT was defined using computer-assisted Geroulakos

classification RR was even higher 3.56 (95% CI, 2.08-8.08), p<0.001 (table 7)

!

Study name Statistics for each study Risk ratio and 95% CI

Risk Lower Upper ratio limit limit Z-Value p-Value

Gronholdt et al 2001 3.100 1.308 7.346 2.570 0.010Salem et al. 2012 6.200 1.860 20.667 2.970 0.003Singh et al. 2013 2.360 1.240 4.491 2.616 0.009

2.977 1.853 4.782 4.509 0.000

0.01 0.1 1 10 100

Favours A Favours B

Meta Analysis

  54  

Figure 18. Subgroup analysis between different carotid stenosis

Figure 19. Subgroup analysis based on the year of patients’ recruitment and US image

analysis

Group byComparison

Study name Comparison Statistics for each study Risk ratio and 95% CI

Risk Lower Upper ratio limit limit Z-Value p-Value

Mild-severe Polak et al. 1998 Mild-severe 2.780 1.359 5.686 2.800 0.005

Mild-severe Liapis et al. 2001 Mild-severe 1.900 1.080 3.341 2.229 0.026

Mild-severe Mathiesen et al. 2001 Mild-severe 2.380 1.181 4.795 2.426 0.015

Mild-severe 2.292 1.311 4.006 2.911 0.004

Moderate-severe Gronholdt et al 2001 Moderate-severe 1.010 0.430 2.371 0.023 0.982

Moderate-severe Nicolaides et al. 2005 Moderate-severe 4.800 2.256 10.215 4.071 0.000

Moderate-severe Hashimoto et al 2009 Moderate-severe 4.400 1.182 16.383 2.209 0.027

Moderate-severe 2.647 1.347 5.201 2.824 0.005

Severe Topakian et al. 2011 Severe 6.430 1.352 30.573 2.339 0.019

Severe Silvestrini et al. 2013 Severe 4.150 1.764 9.763 3.261 0.001

Severe 4.725 1.860 12.003 3.264 0.001

Overall 2.801 1.756 4.469 4.321 0.000

0.01 0.1 1 10 100

Favours A Favours B

!

Stenosis

!

Group byOutcome

Study name Outcome Statistics for each study Risk ratio and 95% CI

Risk Lower Upper ratio limit limit Z-Value p-Value

Before 2000 Polak et al. 1998 Before 2000 2.780 1.359 5.686 2.800 0.005

Before 2000 Liapis et al. 2001 Before 2000 1.900 1.080 3.341 2.229 0.026

Before 2000 Mathiesen et al. 2001 Before 2000 2.380 1.181 4.795 2.426 0.015

Before 2000 Gronholdt et al 2001 Before 2000 1.010 0.430 2.371 0.023 0.982

Before 2000 1.976 1.402 2.785 3.893 0.000

Since 2000 Nicolaides et al. 2005 Since 2000 4.800 2.256 10.215 4.071 0.000

Since 2000 Hashimoto et al 2009 Since 2000 4.400 1.182 16.383 2.209 0.027

Since 2000 Topakian et al. 2011 Since 2000 6.430 1.352 30.573 2.339 0.019

Since 2000 Silvestrini et al. 2013 Since 2000 4.150 1.764 9.763 3.261 0.001

Since 2000 4.652 2.840 7.618 6.108 0.000

Overall 2.975 1.287 6.877 2.549 0.011

0.01 0.1 1 10 100

Favours A Favours B

Year

  55  

Relative risk

Lower limit

Upper limit Z value p value

Between subgroups

(p) Degree of stenosis Mild-to-severe 2.29 1.31 4 2.51 0.004 Moderate-to-Severe 2.64 1.34 5.2 2.82 0.005 Severe 4.72 1.86 12 3.26 <0.001 0.42 Period of ultrasound data collections Before 2000 1.97 1.4 2.78 3.89 <0.001 Since 2000 4.65 2.84 7.61 6.1 <0.001 0.005 Echolucency definition

GSM cut-off 1.66 0.71 3.92 1.19 0.23 PT 1 and 2 3.01 2.02 4.5 5.4 <0.001 0.21 PT assessment

Visual 2.56 1.64 4 4.15 <0.001 Computer assisted 3.56 2.08 6.08 4.65 <0.001 0.35

Table 7. Subgroup analysis of asymptomatic patients studies

In addition, we performed meta-regression analysis of the echolucent plaques and

different stenosis catogery on future ischemic events risk in asymptomatic patients.

Although there was an increasing trend of future events with increased stenosis severity,

the slope was not significant (p=0.19) (figure 20).

Figure 20. Meta-regression analysis. Regression of Log risk rato (RR) on carotid stenosis

severity in patients with echolucent plaques

!

!

!

Regression of Log risk ratio on grade

grade

0.5 0.8 1.0 1.3 1.5 1.8 2.0 2.3 2.5 2.8 3.0 3.3 3.5

Log

risk

rat

io

3.50

3.00

2.50

2.00

1.50

1.00

0.50

0.00

-0.50

-1.00

Mild to severe Moderate to severe Severe

Regression of Log RR on carotid stenosis severity

β=0.31 (95% CI, -0.16-0.80), p=0.19

Log

risk

ratio

  56  

Publication Bias

There was no publication bias reported for the studies included in the meta-analysis

(Figure 17 and 21).

Figure 21. Asymptomatic patients, funnel plot of the standard error by log risk ratio.

Kandall’s Tau=0.392, p=0.173

-0.5 0.0 0.5 1.5 2.0 2.5-1.0-1.5

0.0

0.2

0.4

0.6

0.8

Sta

ndar

d E

rror

Log Risk ratio

  57  

DISCUSION

Study I

Findings: This study shows that carotid plaques in the contralateral to symptomatic side

have similar features compared with the symptomatic ones but more vulnerable features

than the asymptomatic arteries. Contralateral plaques have lower GSM, lower GSM of

the JBA, higher prevalence of plaque type 1 and 2 compared with asymptomatic arteries.

In addition, they have more mild and marked irregular plaques, than the asymptomatic

ones. JBA presence was the only plaque features that showed difference between

contralateral and symptomatic arteries.

Data interpretation: It is generally believed that the pattern of carotid atherosclerosis is

unpredictable. The findings in this study show that symptomatic patients had more

aggressive disease, in both carotid sides, in the form of irregular contour and signs of

inconsistent texture, suggesting vulnerable plaque features compared to those seen in

asymptomatic patients. Even though the symptoms were unilateral, our results clearly

show that contralateral arteries have very similar features. However, the JBA was more

frequent in the ipsilateral carotid arteries and this could represent an additional feature for

plaque instability which could also help in better patient risk stratification. Previous

studies confirmed that vulnerable patients may have multiple unstable lesions, other than

the culprit ones, either distally or even in another territory (1, 143). These claims support

our results. However, despite our findings and the differences we identified between

symptomatic and asymptomatic arteries, it must be mentioned that identification of

vulnerable plaques remains a contentious issue in clinical practice. Quantitative

assessment of carotid plaque echogenicity and textural features analysis can identify

potentially unstable plaques, which are echolucent (low GSM) compared to echogenic

(high GSM) asymptomatic plaques. Again using histology as the gold standard, it has

been confirmed in imaging studies that echogenic plaques represent densely fibrotic and

calcific composition (144), whereas, Low GSM was associated with large hemorrhagic

areas and lower GSM values located in the juxtaluminal position that was associated with

predominant necrotic cores. Morphological plaque features (e.g. surface plaque

  58  

irregularities), GSM and JBA have been used in the prospective studies as an attempt to

predict future vascular events (145, 146). To our knowledge this is the first study that,

used computer based morphological and textural plaque features in providing information

on plaque vulnerability in contralateral carotid arteries.

Clinical implications: The findings of this study might have important clinical

implications, as it: (a) confirms the generalized nature of atherosclerosis in patients with

carotid disease, for not only involving more than one segment at the side of the symptoms

but also the contralateral side, (b) confirms the assessment of carotid plaque features by

computer assisted videodensitometric plaque features as a valuable analysis that could

reliably differentiate symptomatic from asymptomatic carotid disease, and (c) confirm

that the presence of JBA in carotid arteries could be of clinical relevance in addition to

plaque echogenicity, because it was the only feature that showed difference between the

two carotid sides of symptomatic patients. In addition, it may have therapeutic

implication since; being at high risk of recurrent events, symptomatic patients with

bilateral disease should receive aggressive secondary prevention treatment in addition to

revascularization.

Study II

Findings: The results of this study show the sensitivity of US for calcification detection

compared to CBCT images acquired in plaque specimens collected after CEA. US

sensitivity was high in identifying calcification volume ≥8 mm3 by CBCT, but,

calcification volumes <8 mm3 were inconsistently detected, with a sensitivity of only

62%. There was no difference for calcification between symptomatic and asymptomatic

patients, neither between other risk factors and degree of stenosis.

Data interpretation: The most accurate method for detection and quantification of carotid

artery calcification is computed tomography, which is highly correlated with the amount

of calcification evaluated by histology sections (48, 147). Because CT involves radiation

and is an expensive procedure, imaging plaque features, including calcification, by other

  59  

radiation free method that is more patient friendly such as US could have significant

clinical relevance. But, why is the detection of calcification important. Firstly, it has

been confirmed that the presence of calcification in carotid plaque is associated with

lower risk of future brain ischemic events, suggesting that the presence of calcification is

an indicator of plaque stability (47). Sometimes identifying vulnerability based on its

composition is time consuming, and demands expertise, in such circumstances detection

of calcium in the plaque could be a fast and important indicator of plaque stability.

However, it should be mentioned that calcification is not always a sign of stability. In the

contrary, intimal plaque microcalcification is an accepted sign of plaque vulnerability,

however detection of its presence is not possible with non-invasive imaging modalities,

but only with optical coherence tomography (OCT) (16). Secondly, detection of

calcification could be important in decision making for interventional treatment of carotid

atherosclerosis (CEA vs. CAS). It has been reported that implanting a stent in a calcified

carotid plaque is associated with increased risk of ipsilateral stroke, within days after

intervention (49). As many centers now rely mainly on the carotid US findings before

intervention, identification of calcification may guide towards better patient risk

stratification.

An attempt has been made to evaluate the accuracy of US in detecting plaque

calcification by quantifying the total plaque echogenicity (148), however, because the

carotid plaques are very frequently heterogenic, this method proved unreliable. We

believe that our study is the first to demonstrate the US accuracy for calcification

detection, defined as hyperechoic spots that produce posterior shadowing. Also, our

findings show that non-detectable calcifications are of lower volumes and usually

dispersed throughout the plaque. US was highly accurate in identifying carotid plaque

calcification volume of at least 8mm3. However, this volume seems to be relatively

significant when compared with normal intima-media thickness, which in most cases is

<1mm thick but yet reliably quantifiable by US. The same imaging method failed in

some cases to detect calcification volume of less than 8mm3 with reliable accuracy. In

addition, we did not find any difference in calcification presence and volume between

symptomatic and asymptomatic patients. It has been shown that statin therapy increases

carotid plaque echogenicity (149) and also coronary artery calcification (150) and we

  60  

wished to assess this effect in our study. However, we have been limited to undergo such

comparison since, 87.3% of our patients were already using statin therapy, limiting us in

carrying out a reliable comparison between groups.

Clinical implication: Our finding showed that calcification volumes of at least 8mm3 are

accurately detectable by US, that could be taken as a validation of this method to decide

for CAS intervention, since only extensive plaque calcification that was correlated with

adverse events after carotid stenting. US seems not well suited for the detection of

smaller calcium deposits, although they might play a role in plaque vulnerability.

Although the method we used to determine US accuracy is subjective in nature, it

establishes a potential foundation for the future development of quantitative models,

which could guide towards improved identification of plaque characteristics as a step

towards achieving plaque stabilization through optimum treatment. Determining a grey

scale media (GSM) cut-off of the hyperechoic spots that do not produce shadow but

otherwise represent calcification could enable detection of earlier stages of calcification

and reduce the need for advanced CT examinations and the resultant radiation for

patients.

Study III

Findings: This study demonstrates that common carotid artery measures, IMT and IM-

GSM are associated with significant differences in the severity of the disease and

distribution of other arterial systems. It was only IM-GSM, but not IMT that was able to

differentiate between the numbers of arterial systems affected by atherosclerosis. It was

proportionally decreased with increasing number of diseased arterial systems, starting

from asymptomatic to more than one vascular system. The conventional measure of

subclinical atherosclerosis, IMT, was less sensitive in showing similar differences

between groups.

Data interpretation: In general, patients suffering from atherosclerosis in any system are

at increased risk for recurrent events in the same arterial system or even symptomatic

  61  

disease in another territory. This is explained by the systemic inflammatory nature of

atherosclerosis. Patients with previous stroke have been shown to be at high risk of

suffering recurrent strokes but even higher risk of suffering a future MI (1). Patients with

symptomatic disease in one vascular system have been reported to have a 25% risk for

developing MI, stroke or even vascular death compared to >40% in those with multi-

system arterial disease (1). Based on these data patients with multisite atherosclerosis can

be described as vulnerable in the view of the high risk they carry for vascular events. It

was in the light of our findings, that IMT showed differences only when comparing

patients with and without previous MI. On the other hand IM-GSM proved much more

sensitive in showing significantly progressive lower values as patients went from

asymptomatic to symptomatic single arterial system and finally to symptomatic multi-

system disease. In addition IM-GSM was significantly different in patients with previous

stroke and MI, compared to those without.

Conventional carotid IMT has been widely used in many studies attempting to identify

patients at increased risk for future vascular events. On the other hand, measurements of

the composition of arterial wall free of plaque (IM-GSM) were only attempted in few

cases. The association between IM-GSM and future vascular events has recently been

demonstrated, as it was shown to be associated with a three-fold greater risk of all-cause

mortality and an eight-fold greater risk of cardiovascular mortality (122). Recently it has

been demonstrated that IMT represents an adaptive thickening of the arterial wall more

than real atherosclerosis pathology (16). It was confirmed that age and hypertension are

the dominant risk factors for increased IMT whereas low values of IM-GSM related to

low levels of HDL-C and increased level of CRP (151). Furthermore, it has been

suggested that IM-GSM could add incremental valued over and above IMT for patients

risk stratification, since they both were related to different plaque features in the distal

segments of the carotid artery (ICA and bifurcation) (59).

Clinical implication: The ability of the IM-GSM to differentiate between patients with

and without previous ischemic events suggests that it could also identify asymptomatic

patients at higher risk. Identifying such patients should warrant aggressive medical

management of modifiable risk factors in an attempt to reduce/prevent vascular events.

  62  

However our findings could find clinical implication once they are reproduced

prospectively and the reproducibility of the technique is confirmed in a number of

studies.

Study IV

Findings: Our analysis shows that atherosclerotic plaque echogenicity evaluated by

carotid ultrasound accurately predicts the development of CV symptoms in asymptomatic

and symptomatic patients. This prediction was preserved for all degrees of stenosis.

Data interpretation:

Asymptomatic patients

Although a small benefit of CEA was observed in asymptomatic carotid stenosis,

advances in medical therapies and the well-documented fall in stroke rates in such

patients over the last decade questioned the need of surgical treatment in these group of

patients (50, 52, 53, 152). However, there is a subgroup of high-risk asymptomatic

patients, which could benefit from CEA, and identification of such patients could have an

important clinical impact. The analysis we undertook in this study provides a strong

evidence for the additional value of plaque echogenicity in identifying asymptomatic

patients at higher risk of future CV events. To strengthen the argument further, such

accuracy for predicting future events, based on plaque echogenicity, stood firm even after

analyzing separately different categories of carotid artery stenosis, although

asymptomatic patients with severe stenosis again seem to be at a higher risk.

A higher association between plaque echolucency and future CV events was found

among studies that started patients’ recruitment and US data collection from 2000 and

thereafter, studies that defined plaques as PT 1 and 2 and when computer assisted

analysis was used to evaluate plaque echogenicity. Accordingly, more advanced US

devices and computer-assisted analysis of plaque echogenicity could help better

recognition of plaque echolucency and consequently, better prediction of future ischemic

events. Even if the cost-benefit of these findings is ignored, the clinical benefit to

patients cannot be over emphasized if our findings are implemented, particularly in

asymptomatic patients with severe stenosis. However, carotid plaque echogenicity

  63  

quantification is not real life analysis since it needs special software and expertise. It is

also an operator dependent technique with some potential technical difficulties in some

patients.

Symptomatic patients

Several large clinical randomized trials demonstrated a significant surgical benefit for

symptomatic patients, over and above medical treatment, particularly in those with 70-

99% stenosis (17, 153). However, there is a subgroup of patients with lower degree of

stenosis who carry a high risk of stroke recurrence. Although, our analysis showed that

plaque echogenicity analysis could predict recurrent ischemic events in symptomatic

patients with moderate to severe stenosis, the small number of studies and differences in

the follow-up periods between them limits us in providing a strong conclusion. A high

rate of stroke recurrence even within the first 72 hours of symptoms and within the

following two weeks has recently been reported (98). More studies are needed to confirm

the association between plaque echolucency and recurrent events in the early hours/days

after initial which could indicate a need for an emergency CEA in order to prevent

another stroke, as has been suggested (101).

Clinical implications: Quantification of carotid plaque echogenicity is a technique with

great potential in daily clinical service, which may result in better patient stratification

according to potential development of disease related complications. Such approach may

guide towards identifying high-risk patients with asymptomatic stenosis as well as

symptomatics with moderate stenosis and implement treatment strategy. Based on our

results plaque echolucency in asymptomatic patients, particularly in those with severe

stenosis, should be taken as a sign for vulnerability that supports the need for surgical

intervention.

  64  

LIMITATIONS

Study I

The small number of patients, particularly the asymptomatic and the retrospective nature

of the study are the most important limitations. When comparing different groups, the

small number of patients may carry the risk of false negative. In addition, lack of detailed

information for atherosclerosis burden in other arterial systems and lack of body mass

index data is another limitation. The images used for the analysis were slightly clouded,

however, applying standard image despeckle-filtering prior to the image analysis

minimized the noise (154). In general, the little noise that remained in the images for the

final analysis might influence the value of entropy and weaken its potential to

differentiate between groups.

Study II

The evaluation of US positive and negative predictive value was not possible; because

almost all (98.9%) carotid plaques were calcified in CBCT. During CEA procedure there

might be small-calcified material not excised, but we have been working on the

assumption that the largest plaque with the largest calcium volume was removed and

assessed by CBCT. Furthermore, we cannot be sure that the calcification detected and

quantified by CBCT was the same hyperechogenic nodule seen by US, particularly in

cases with multiple calcifications, however, we made a further assumption that the nodule

with the largest volume was the same that caused the posterior shadowing. In addition,

our subgroup comparisons were weak, since the number of asymptomatic patients was

small compared with symptomatic patients.

Study III

This is a retrospective study with known limitations. We have compared the two carotid

artery wall measures with previous symptoms in different arterial systems, an information

  65  

that was acquired from patients’ medical records. Another limitation is the lack of

imaging plaque features in other vascular territories (coronary and lower extremity

arteries). Assessing plaque/IMT and GSM in other arterial system, particularly the ones

related to the developed events, would have been of great interest but were not possible

with the retrospective nature of the study. In addition, information on the duration of

individual risk factors that could have influenced the results was not available.

Study IV

A systematic review based on relevant key words might have missed important

publications, but two investigators blinded to each other’s means of search checked the

search. We used only publications in the English language; other relevant ones in

different languages might have been missed. Studies included in the meta-analysis were

of different designs and different definitions to determine plaque echolucency were used,

however we tried to overcome this by using the random effect meta-analysis. Another

limitation is the low number of studies included in the meta-analysis, particularly those

with symptomatic patients.

  66  

CONCLUSIONS

Study I

Symptomatic patients with carotid atherosclerosis seem to have profound features of

plaque vulnerability in the contralateral arteries as well. Carotid plaques of asymptomatic

patients were more stable compared to symptomatic and contralateral to symptomatic

side. These findings, support the concept of generalized atherosclerotic pathology rather

than incidental unilateral disease, suggest increased relative vulnerability of the

contralateral plaques in symptomatic carotid stenosis and hence emphasize the need for

more aggressive treatment for secondary atherosclerosis prevention.

Study II

Carotid US accurately detected carotid artery calcification volumes of at least 8mm3, but

it was less accurate in detecting smaller volume calcified plaques, with relatively high

false negativity. We didn’t find any difference in calcification presence or volume

between symptomatic and asymptomatic patients. Since calcification is a dynamic

process, finding a method that can quantify atherosclerotic calcification by avoiding

radiation could be of importance for longitudinal assessment of its progression and

effects on plaque stability.

Study III

Our results showed that IM-GSM but not conventional IMT to differentiate between

patients with multiple system atherosclerosis disease. Because it was confirmed that

patients with multisite atherosclerosis are at higher risk of ischemic events in future,

measurement of intima-media complex echogenicity (IM-GSM) seems to be a promising

method to identify high-risk patients.

  67  

Study IV

There is a clear association between carotid plaque echolucency and future CV events in

asymptomatic and symptomatic patients. These findings if implemented, should result in

better management of vulnerable patients as well as early recruitment of those requiring

surgical intervention, who else would have been considered wrongly stable. Before

implementing such strategy for selecting asymptomatic patients for surgical intervention,

current results need to be confirmed in randomized clinical trials testing isolated medical

treatment in echogenic plaque against medical treatment plus CEA in asymptomatic

patients with echolucent plaque.

  68  

Acknowledgement

Though only my name appears on the cover of this dissertation, a great many people have

contributed to its production. I owe my gratitude to all those people who have made this

dissertation possible.

First and foremost I express my sincerest gratitude to my supervisor Professor Michael

Henein. I have been amazingly fortunate to have an advisor who gave me the freedom to

explore on my own, and at the same time the guidance to recover when my steps faltered.

Michael taught me how to question thoughts and express ideas. His patience and support

helped me overcome many difficult situations and complete this dissertation. I hope that

one day I would become as good an advisor to my students as Michael has been to me.

My co-supervisor Professor Per Wester deserve a special thanks for his help, very

professional comments that always made important changes in my scientific work and for

scholarly devotion to my work during realizing the thesis. So many things I have learned

from him will be very helpful for my academic career and also will help me for proper

diagnosis and treatment of stroke patients.

I would like to thank very much Elias Johansson who provided me the opportunity to

use the clinical patients’ data he collected very carefully making my scientific work much

easier, and also for his insightful discussions that helped me to do the best with the data

analysis and also to widen my knowledge in the carotid stenosis field.

This thesis couldn’t be possible without the help of Professor Christer Grönlund, who

made special software that I have used in my studies and also for his fruitful comments in

all of my studies.

Particularly, I would like to acknowledge, Professor Gani Bajraktari for helping me

during my entire scientific journey that started the time I was a medical student and

continued throughout my entire doctoral studies. I hope for a lifelong collaboration with

my tireless professor.

  69  

I would like to thank all the co-authors for their constructive and helpful criticism during

designing, analyzing and writing the papers for this thesis.

Special thanks to Kerstin Rosenqvist, for making all administrative issues during my

studies very easy and for making me feel at the Umeå University like my second home.

I would like to give a heartfelt thank to my grandfather Isak, my grandmother Taibe, my

parents Bedri and Shqipe and to my brothers Haki and Dardan. I am sure that I would

not have been able to complete this work without their help, encouragement, patience,

love and devotion for my work and myself.

And finally to my wife, Pranvera, for encouraging me to begin this scientific journey,

for being always by my side and help me to achieve what we intended, and to my

daughter Elena for her existence.

  70  

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