Director: Prof. Stefano Del Prato
CLINICAL/MOLECULAR FEATURES AND PROTEOMICS DATA
TO IDENTIFY DETERMINANTS OF CAROTID PLAQUE RISK
Relatore: Chiar.mo Prof. Mauro Ferrari
Correlatore: Dott.ssa Silvia Rocchiccioli
Candidato: Dott. Michele Marconi
INTRODUCTION 4
ATHEROSCLEROSIS 5
REVASCULARIZATION IN ASYMPTOMATIC CAROTID STENOSIS 7
"HIGH-RISK” ASYMPTOMATIC CAROTID ARTERY STENOSIS 9
Clinical Features 9
Stenosis severity and progression 11
Silent embolization 12
Reduced cerebrovascular reserve and contralateral carotid occlusion 12
Lipid-rich Necrotic Core (LRNC) and Fibrous Cap (FC) 14
Intraplaque hemorrhage 15
Biomarkers 16
PROTEOMIC ANALYSIS 19
ATEROMARK TRIAL 20
AIMS OF THE STUDY 21
METHODS 21
Patient clinical characterization 22
Histology and immunohistochemical characterization 25
Tissue processing and secretome preparation 26
Secretome analysis 26
Western blot analysis and ELISA assays 27
Statistical analysis 27
RESULTS 28
Morphological characterization 28
Secretome analysis of endarterectomy specimens 31
Comparative analysis between plaque and its distal counterpart secretomes 33
Validation by Western Blot and ELISA assay 36
Immunohistochemistry quantification 37
ELISA assay of plasma samples 39
DISCUSSION 40
CONCLUSIONS 43
INTRODUCTION
Carotid plaque stenosis is responsible of 15-20% of all ischemic strokes; despite that, asymptomatic
stenosis represents a therapeutic dilemma. The best medical treatment (BMT) alone, instead of
surgery, may be adequate to prevent the risk of ictus in the majority of patients with carotid stenosis
that will never encounter ischemic complications. Currently the surgical indication of asymptomatic
plaque is only based on the stenosis degree measured by imaging methods. Identification of
vulnerability markers of carotid plaques could help to select patients who really will take advantage
from carotid endarterectomy. In literature many factors are under investigation for identifying
plaques with a major risk of stroke; among these plaque morphology, intracerebral circulation
assessment, silent stroke identification and biomarkers are proposed as possible decision making
tools.
Regarding biomarkers, several proteomics approaches have been up to now used to elucidate the
molecular mechanisms involved in plaque formation and complication. In particular the analysis of
plaque secretome (proteins secreted by the plaque) in patients submitted to carotid endarterectomy
could bring more informations compared to proteome, because secretome mimics the in vivo
condition and presents a reduced complexity, allowing the detection of under-represented potential
biomarkers.
The aim of this thesis is: at first, to review the literature concerning the identification of all vulnerability factors in patients with asymptomatic carotid stenosis, and subsequently to characterize the plaque secretome in patients submitted to carotid endarterectomy.
ATHEROSCLEROSIS
Atherosclerosis, or hardening of the arteries, is a degenerative progressive disease that involves medium and large-sized arteries wall, resulting in plaques that consist of necrotic cells, lipids and cholesterol crystals. These plaques can cause stenosis, embolization and thrombosis, and for this
reason, atherosclerosis represents the main cause of morbidity and mortality in Western countries
(roughly 5 million people are affected in the U.S.), due to acute cardiovascular events secondary to partial or total thrombotic obstruction of vessel lumen 1-2.
The earliest descriptions of atherosclerotic lesions were focused on morphologies of fatty streaks
(accumulation of white blood cells). The first application of the term atheroma was made in 1755
by von Haller, who used this word to describe a common type of plaque secreting a pultaceous
content from its core on sectioning.
Atherosclerotic plaque is actually defined by a core containing lipids (including esterified
cholesterol and cholesterol crystals) and debris from dead cells. All over there is a fibrous cap,
containing smooth muscle cells and collagen fibres, which function is to stabilize the plaque.
Macrophages, T cells and mast cells populate the plaque, frequently in an activated state; these
immune cells are able to produce cytokines, pro-thrombotic molecules, proteases and vasoactive
substances, all responsible of plaque inflammation and affecting the real vascular function. An
intact endothelium covers the plaque.
Atherosclerotic plaques could give rise to complications and symptoms or could remain asymptomatic for many years or decades; usually, it is possible to define two types of plaques:
● a stable one, rich in some extracellular matrix components (ie Collagen type I) and smooth
● an unstable plaque, which is rich in macrophages and foam cells; in this type of lesion it is
possible to see that the extracellular matrix (separating the arterial lumen from the lesion,
also known as fibrous cap) is usually weak and proximal to rupture. Vulnerable
atherosclerotic plaques (high-risk or unstable plaque) is associated with an increased risk of disruption, distal embolization and vascular events.
In the mid 1990s the terminology used to define atheromatous plaques was refined by the American
Heart Association (AHA) Consensus Group headed by Dr. Stary 3-4.
The AHA classification consists of 6 different categories to include the following types:
● early lesions of initial type I, adaptive intimal thickening; ● type II, fatty streaks;
● type III, transitional or intermediate lesions;
● advanced plaques characterized as type IV, atheroma; ● type V, fibroatheroma or atheroma with thick fibrous cap;
● type VI, complicated plaques with surface defects, and/or hematoma-hemorrhage, and/or thrombosis.
Atherosclerosis could affect different vascular districts with different clinical features. Carotid
artery atherosclerosis predisposes patients to TIA (transient ischemic attack) and stroke, and the risk
for these events is proportional to the severity of the carotid disease. Despite recent reductions in incidence and related mortality, stroke remains the third leading cause of death in the United States.
Current estimates suggest that 87% of strokes are ischemic and 13% are hemorrhagic 5.
Symptomatic carotid stenosis presents a clear indication to the surgical treatment in order to reduce
the risk of subsequent major stroke; that was highlighted by landmark studies such as NASCET and
benefit-risk ratio.
REVASCULARIZATION IN ASYMPTOMATIC CAROTID STENOSIS
The overall prevalence of asymptomatic carotid artery stenosis in the general population is
estimated at 2%–9%.
Despite guidelines indicate surgical or endovascular revascularization in severe asymptomatic
carotid artery stenosis, best medical therapy (BMT) is progressively changed over the time and the
older randomized studies, on which current guidelines are based, could be outdated. Two main
randomized studies completed in the 1990s, the Asymptomatic Carotid Artery Study (ACAS) and
the Asymptomatic Carotid Surgery Trial (ACST), analysed role of carotid endarterectomy (CEA) in
asymptomatic carotid artery stenosis for stroke prevention 8-9. In the ACAS more than one thousand
patients were randomized to CEA or best medical therapy. The five-year projected rate of ipsilateral
stroke was 11% for the medical group versus 5.1% for the surgical group (a 47% relative risk
reduction [RRR]). The ACST randomized more than three thousand patients into immediate CEA
or delayed surgery for symptoms only. The 30-day risk of stroke or death was 3.1%. The five-year
rates were 6.4% for CEA and 11.7% for medical therapy alone.
In these trials, medical therapy did not reflect current optimal treatment, with few patients receiving lipid-lowering and antihypertensive therapy.
Recent studies show a different evolution of the natural history of asymptomatic carotid artery
stenosis: the stroke risk has reduced significantly, and the risk-benefit of revascularization should
be reconsidered.
In the ACST (dated 2004) the five-years risk of any stroke in medically treated subjects was 11.8%,
while in 2010 the five-years risk was 7.2% 10-11.
high-dose statin drugs, antiplatelet therapy, associated with optimal blood pressure (BP) control,
smoking cessation, optimal glycemic control and other lifestyle changes 12. Numerous meta-analysis
demonstrated a sustained trend towards a decline in the stroke risk 13-15.
New randomized studies are proceeding, with the aim of comparing best medical treatment with
carotid revascularization: the Carotid Revascularization Endarterectomy Stenting Trial 2 (CREST
2) is currently recruiting subjects with asymptomatic carotid artery stenosis and ≥70% stenosis, in
order to compare either BMT alone vs BMT plus CEA, or BMT alone vs BMT plus CAS16.
The Stent-Protected Angioplasty in Asymptomatic Carotid Artery Stenosis vs Endarterectomy
(SPACE- 2) study aims to recruit about 3,500 patients and randomly assign them to BMT alone or CEA or CAS17.
"HIGH-RISK” ASYMPTOMATIC CAROTID ARTERY STENOSIS
Although the guidelines indicate the importance of the surgical treatment of carotid stenosis in
asymptomatic patients, the majority of those stenosis will never become symptomatic and, for this
reason, surgical procedures, as CEA or CAS, could be unnecessary. On the other hand, even if the
ongoing studies would show no benefits from CEA or CAS over BMT, a not negligible percentage
of patients will present stroke.
Nowadays surgical indication is mainly based on degree stenosis despite it has been demonstrated
insufficient to identify patient with an high risk of symptoms.
Thus, the identification of “high risk of stroke” patients could help physicians to maximize the
benefits of revascularization: therefore, besides degree of stenosis, other factors should be considered to create a sort of score of plaque or patient risk. A lot of research in recent years has focused on the identification of such "higher-risk" subgroups.
In the paragraphs below are listed the actual features and risk factors that expose patients to a major risk of stroke; they were divided into clinical features, stenosis severity, progression of stenosis, characteristics of plaque vulnerability and biomarkers.
Clinical Features
Clinical characteristics, such as male sex, current smoking, poorly controlled hypertension and
history of contralateral transient ischemic attack/stroke increase stroke risk in asymptomatic carotid artery stenosis 18.
Male sex could be interpreted as predictor of risk, as explained by Ota H.. Analysing 131 patients
with asymptomatic carotid stenosis > 50% on MRI, men had higher-risk plaque features than
women. Particularly, men tend to have higher incidence of large lipid-rich necrotic core (LRNC)
compared with women. The higher prevalence of these potential vulnerable plaque features in men
as compared with women may be one reason that CEA was shown to be more effective in reducing
subsequent stroke in asymptomatic men with carotid artery stenosis than in women. Additional
studies have shown that these gender differences in high-risk carotid plaque features exist even in
patients with asymptomatic carotid stenosis <50%. Thus, sex-based management may be important
in patients with asymptomatic carotid atherosclerosis across all stages of carotid stenosis 19.
Those data were confirmed in a combined analysis of the ACAS and ACST trials: in fact, whereas
men with asymptomatic carotid artery stenosis had a 51% relative risk reduction of stroke, women
did not. This could be even attributable to the higher risk of perioperative events in women. Thus,
there is a concern that women may benefit less from revascularization than men and future clinical
trials focusing on women are needed 20.
In older patients (more than 80 years of age) the benefit of revascularization for asymptomatic
carotid artery stenosis is most controversial, because in randomized studies the advantages from
surgery were seen after five-years follow-up. In ACST, there was no benefit in those over 75 years.
However, age cannot be an absolute contraindication with increasing life expectancy of the overall
population. In regards to the modality of revascularization, based on the CREST data, overall CEA had more favorable outcomes for those over 70 years of age and CAS for those under 70 years of age 18,21.
Regarding levels of BP and medical treatment, on a multivariate analysis, as described in 2013 by
King et al., antiplatelets ( P=0.001) and lower mean blood pressure (P=0.002) are independent
predictors of reduced risk of ipsilateral stroke and transient ischemic attack 22. Antiplatelets
(P<0.0001) and antihypertensives (P<0.0001) are independent predictors of a lower risk of any stroke or cardiovascular death, even in patients with asymptomatic carotid stenosis.
In the Asymptomatic Carotid Stenosis and Risk of Stroke (ACSRS) study, more than one thousand
smoking history (more than 10 pack/years), and history of contralateral TIAs or stroke could predict an increased risk of ipsilateral ischemic events 18.
Even diabetes (p=0.002) showed a significant independent association with ipsilateral neurological
events 23.
Even dyslipidemia play a fundamental role in plaque progression and complication. For this reason
importance of statin therapy in plaque stability and cardiovascular events reduction has been largely evaluated .
Stenosis severity and progression
The randomized ACAS and ACST trials did not show differences as predictor of future events in
stenosis severity between 60 and 99% range. The ACSRS study found that those with 50-69%
stenosis had a lower risk compared to those with 70-89% and 90-99% stenosis (0.8% vs. 1.4% vs.
2.4%)18-24.
Otherwise, there is significantly stronger evidence in regard to the predictive value of progressively worsening stenosis despite optimal medical therapy. In an analysis of the ACST data, performed by
Hirt LS over 1469 subjects, the incidence of ipsilateral neurologic events was higher for stenosis
that had progressed faster. The annual incidence of progression in the cohort was 5.2%. Ipsilateral
events occurred in 17% of patients 23.
In 2014, Kakkos et al. studied more than one thousand patients with asymptomatic carotid artery
stenosis for overall 4 years. Of these patients, 9.8% showed stenosis progression and had twice the
risk of ipsilateral stroke (21% vs. 12%) 25. Balestrini et al. followed for 42 months 535 patients
affected by moderate asymptomatic carotid artery stenosis; during their study they found that 96.7%
of patients without progressive stenosis encountered no ischemic events, while between subjects with progressive stenosis, 27.1% had an ipsilateral stroke26.
Silent embolization
Presence of ipsilateral silent embolic infarcts on Computed Tomography (CT) or MRI may be
predictive of increased risk of ipsilateral stroke. In the ACSRS study, there is a significant
difference between the annual stroke rate when silent embolic infarcts are present and when they are not (3.6% vs 1%; p = 0.005) 27 .
Even detection of microemboli on TCD identifies patients at higher stroke risk 28. In the
Asymptomatic Carotid Emboli Study (ACES), 482 patients with asymptomatic carotid artery stenosis underwent six monthly TCDs. The authors enrolled patients with asymptomatic carotid
stenosis of at least 70% from 26 international centers and used transcranial Doppler ultrasound
(TCD) to detect the presence of embolic signals from the ipsilateral middle cerebral artery. The
annual risk of ipsilateral stroke was 3.62% in patients with embolic signals and 0.7% in those
without them (HR 5.57). The data was confirmed by a meta-analysis of six reports involving 1,144
patients. However, most patients with these signals did remain stroke free at three years, and thus,
this test lacks the specificity for stand alone clinical use. However, this study suggests that a
noninvasive technique, emboli detection via TCD, can be used to stratify those patients with clinical
asymptomatic carotid stenosis into those who are at high and low risk of developing TIA and
ipsilateral stroke 29-30.
Reduced cerebrovascular reserve and contralateral carotid occlusion
The presence of an incomplete circle of Willis or of an intracranial or contralateral occlusive disease can cause a reduction in cerebral perfusion pressure.
Cerebrovascular reserve in such patients can be assessed using TCD or nuclear medicine techniques31-32. In a recent meta-analysis, impaired cerebrovascular reserve is strongly associated
Despite patients with asymptomatic carotid artery stenosis contralateral to an occluded carotid artery should be at higher risk of future stroke, a subgroup analysis of the ACAS study revealed that patients with asymptomatic stenosis contralateral to an occlusion may actually have a lower risk of ipsilateral (to the asymptomatic stenosis) stroke. The predicted 5-year rate of stroke in patients with
asymptomatic stenosis and contralateral occlusion enrolled in the medical arm was 3.5%, which
was significantly lower than the risk for patients without contralateral occlusion who were being treated with medical therapy (11.7%). Considering that for the same group with contralateral
occlusion, the 5-year rate of stroke for patients undergoing endarterectomy was 5.5%, it was
concluded that surgical intervention in this group was not beneficial and could even be harmful 34.
Plaque morphology
Numerous studies about natural history of atherosclerosis have led to the identification of some
features of an "unstable" plaque. Among these features, the presence of intraplaque hemorrhage, a
large lipid-rich necrotic core, and a thin fibrous cap, activated macrophages and fissured plaque are
included. With the use of imaging techniques such as ultrasonography and MRI, it is now possible to identify in vivo these high-risk plaques 35.
Multicontrast MRI of the carotid arteries has been validated with histology and it could identify and quantify various carotid plaque components, including the lipid-rich necrotic core (LRNC), the fibrous cap (FC) and the intraplaque hemorrhages (IPH). Further, multicontrast carotid plaque MRI
has been shown to be capable of quantifying the LRNC volume in the clinical setting of a
multicenter trial with low interscan variability.
Atherosclerotic plaques can also be studied using ultrasound, basing the characterization on their
echolucency and gray-scale value.
Lipid-rich Necrotic Core (LRNC) and Fibrous Cap (FC)
demonstrated in different studies. In a cross-sectional study of 97 consecutive patients with 50%–99% stenosis referred for carotid plaque study with MRI, there were significant associations
between recent ipsilateral carotid stroke or TIA and the presence of an LRNC as well as the
presence of a thin or ruptured FC. In that study, there was no correlation between carotid artery stenosis and symptoms 36-37.
A multivariate analysis of clinical and carotid plaque features in a study of 108 individuals with
50%–79% stenosis revealed that the LRNC size was the strongest predictor of new FC rupture or
ulceration after 3 years of follow-up 38.
Underhill HR et al proposed a grading system for plaque burden that they termed the "Carotid
Atherosclerosis Score" (CA score):
1. score 1, subjects with a maximum wall thickness ≤ 2 mm and no LRNC;
2. score 2, patients with a maximum wall thickness >2 mm, if they demonstrated < 20% maximum percentage area of LRNC;
3. score 3, if those patients had 20%–40% maximum percentage area of LRNC; 4. score 4 if their maximum percentage area of LRNC was > 40% (39).
The CA score was prospectively evaluated using an independent cohort of 73 asymptomatic
subjects who underwent serial carotid MRI over a 3-year period. Applied prospectively, the CA
score was associated with new FC rupture and greater lesion growth. The original AHA plaque
classification and these subsequent in vivo carotid plaque MRI trials suggest that the LRNC could
be a useful phenotype of atherosclerotic disease, and that may predict future plaque destabilization
and thromboembolic events.
Even ultrasounds have been used to study the plaque morphology and some authors, as Geroulakos,
set up an ultrasound classification of carotid plaque, afterwards adopted by the ACSRS study, in
order to correlate the type with the risk of ipsilateral ischemic events 18,40 . In this study, patients that
echolucent and therefore lipid rich; patients with Type 3 and Type 4 (mainly echogenic) plaques (0.8% and 0.4% per year, respectively) had a lower stroke risk.
A recent prospective study with 83 asymptomatic patients with carotid artery stenosis investigated
by Doppler ultrasonography and MR plaque imaging showed a higher risk of ischemic
cerebrovascular events in "high-risk lesions" characterized by intraplaque hemorrhage and a
lipid-rich necrotic core 41.
Furthermore, the size of an LRNC and its reduction with statin treatment have been evaluated with
MRI that could have a role in medical therapy surveillance 42-43.
Intraplaque hemorrhage
Intraplaque hemorrhage (IPH) can lead to a marked increase in the size of an LRNC and further plaque destabilization 44-45.
A recent prospective MRI study confirmed that carotid plaque IPH was associated with accelerated
plaque progression and the presence of IPH also suggests the possibility of other future IPH,
meaning that it is an important marker of evolution from stable to unstable plaque 46.
Intraplaque hemorrhage, alone or in combination with other plaque features, has been correlated
with the subsequent development of new ipsilateral carotid thromboembolic symptoms and a
progression in carotid plaque size. During a mean follow-up of 38.2 months in 154 patients with asymptomatic moderate carotid artery stenosis, 12 carotid cerebrovascular events occurred. Both the presence and size of IPH correlated with a new ipsilateral carotid stroke or TIA 47.
In a longitudinal study of 98 patients with asymptomatic moderate carotid artery stenosis, more than one-third of the patients demonstrated IPH, and all the six future ipsilateral carotid ischemic events occurred in these patients 48. Altaf et al. enrolled 64 recently symptomatic patients who showed a
carotid stenosis of 30%–69% degree in a prospective trial of carotid plaque MRI. On the initial carotid plaque MRI study, 39 patients (61%) demonstrated to have a IPH. After a mean follow-up
of 28 months, 14 new ipsilateral carotid events occurred, and 13 of them were in arteries with IPH on the initial carotid plaque MRI study 49.
In a meta-analysis of 8 longitudinal studies done by Saam et al., it has been demonstrated that the presence of IPH on MRI is associated with an approximately 5.6-fold higher risk for
cerebrovascular events, as compared with the risk in subjects without IPH 50.
Identification of morphologic features of vulnerability through in vivo imaging may help guide both medical therapy and surgical intervention. For instance, plaque with large LRNCs identified by
MRI could benefit the most from aggressive medical therapy or difference in presence, size,
location, and/or type of IPH can identify patients with carotid artery stenosis who are at an
increased risk of ipsilateral stroke and so who may benefit from surgical intervention.
Biomarkers
Many studies have been conducted to understand which biomarkers are involved in plaque
instability, and subsequently to monitor their expression trend in relation with instability and/or
symptoms.
In fact, serum biomarkers which reflect molecular processes related to plaque vulnerability
(inflammation, lipid accumulation, apoptosis, proteolysis, thrombosis and angiogenesis) could be
able to distinguish unstable from stable carotid artery stenosis, and for this reason they could be a powerful and not invasive tool in patient risk monitoring.
No serum biomarker has qualified for clinical use in carotid artery disease actually; nevertheless
some biomarkers, as the ones involved in inflammatory or proteolytic activity, seem to be
promising in the identification of vulnerable carotid plaques 51.
Regarding lipid accumulation, lipoprotein-associated phospholipase A2 (Lp-PLA2), a
pro-inflammatory enzyme, is highly expressed in symptomatic plaques 52. Well-known high-density
HDL levels are associated with reduced risk for cardiovascular disease. Furthermore, low HDL levels are related to ultrasound findings of carotid plaque vulnerability 53.
Many inflammatory markers have been related to atherosclerosis and plaque complications:
different cytokines (IL6, IL18) and metalloproteinase (MPO, PAPP-A) are studied in correlation
with cardiovascular risk. High-sensitivity CRP (hs-CRP) correlates with rapidly progressive morphological features of carotid plaque and with a greater risk for a stroke 54-55.
On the contrary the expression of anti-inflammatory cytokines, such as transforming growth
factor-b1 (TGF-b1), is increased in stable atherosclerotic carotid artery plaques compared with
unstable ones 56.
Despite research in this field is relatively limited and results are still conflicting, carotid
calcification may be a plaque-stabilizing factor and protective of acute neurologic events and, for
this reason, biomarkers of bone formation and bone structural proteins, that are expressed in
atherosclerotic plaques, such as bone morphogenetic protein-2 (BMP-2), osteopontin (OPN),
matrix-carboxyglutamic acid protein (MGP), and osteoprotegerin (OPG), have been studied in
relation to cardiovascular disease and plaque stability 57.
Even total serum gamma-glutamyltransferase (GGT) activity is an independent predictor for
cardiovascular events. Four GGT fractions have been identified in plasma and only one of them
(b-GGT) in atherosclerotic plaques. In a recent study of our group, we investigated the relationship
between plaque b-GGT activity and the histological features of plaque vulnerability. Intra-plaque
b-GGT activity correlates with the histological markers of vulnerable plaque and with plasma
b-GGT in human carotid atherosclerosis; these data support the possible role of b-GGT in clinically significant atherosclerotic disease 58.
Moreover, recent studies found a correlation between the carotid stenosis progression and some
biomarkers involved in this mechanism. These studies suggest that several microRNAs (miRNAs)
of stenosis is mostly unknown.
Biomarkers research is hampered by the fact that atherosclerosis is a systemic process, thus serum
biomarkers reflecting plaque instability may not be specific for carotid artery plaques; while, of
course, biomarkers should be highly specific and sensitive. Until now, biomarkers studied are not
enough valid indicator of increased risk and, at the moment, their role in clinical routine are not justified.
Biomarkers will not probably turn to be an independent factors in the identification of unstable
plaques, but they might provide additional information in a multimodal approach in the selection of patients for carotid artery surgery.
In the search for new molecules reflecting carotid plaque instability, new approaches such as
proteomic and genomic technique have been used.
Since proteins are involved in all cellular functions, control every biological mechanism and are
modified by diseases, proteomics prospect the possibility to study and identify key factors, which
PROTEOMIC ANALYSIS
The interest for novel biomarkers has grown due to the possibility to analyze proteins secreted by tissues into circulation 59.
The majority of proteomics studies were performed on plaque extracts analyzed by electrophoresis
followed by mass spectrometry, but with this approach, mainly constitutive proteins were identified,
while potential and usually low-expressed biomarkers could not be detected 60. Olson et al. used
two-dimensional differential gel electrophoresis in combination with tandem mass spectrometry, to
compare protein distribution in the longitudinal axis of severely atherosclerotic segments of internal carotid artery 61. In this way, the authors identified 19 proteins with a differential distribution along
the artery. Even Western array technology has been proposed to identify 7 differentially expressed
proteins in human carotid endarterectomy (CEA) specimens 62.
De Kleijn et al. found that osteopontin expression in carotid plaques and in blood is predictive for atherothrombotic events and, although it does not correlate with vulnerability features, it could be a frontrunner in use of tissue proteomics as clinically useful biomarker discovery 63.
Analysis of tissue secretome was suggested by Duran in 2003; this technique was very innovative
and useful, having the great advantage that secretome mimics the in vivo condition and implies a
reduced complexity compared to serum/plasma or entire tissue proteomics 64.
In a 2012 study were identified 22 proteins, obtained by the selection of nine plaque
secretome-specific antibodies and the analysis of their immuno purified antigens by mass
spectrometry. Among them, junction plakoglobin (and its isoforms) was suggested as a potential
biomarker of atherosclerosis 65-66.
distribution of mechanical forces (flow shear stress and plaque wall stress) and corresponding
morphological features (cell distribution and type) along the plaque and its distal side 67-69. In
particular, the low flow shear stress in downstream side is associated to atherosclerosis progression, with increased vascular smooth muscle cells (VSMCs) and macrophages; on the other hand, the high plaque wall stress in the upstream area is associated to cap rupture of vulnerable lesions and to
an increased expression of proteolysis and apoptosis markers. Thus, these reports support the
opinion that carotid plaque and its corresponding adjacent distal side may keep distinctive protein
signatures: therefore, differential expression of proteins released by plaque-containing upstream
segment (P) and by its downstream distal side (DS) segment can suggest markers related to
progression and others related to vulnerability.
ATEROMARK TRIAL
In 2012 Clinical Trials Ethics Committee of University of Pisa approved the ATEROMARK trial
“Studi proteomici e molecolari per la diagnosi della vulnerabilità della placca aterosclerotica e
correlazioni con tecniche di immagine” (proteomic and molecular analysis in vulnerability diagnosis of atherosclerotic plaque and imaging correlation).
The goal of the study was to perform a proteomic characterization of intra-plaque factors or factors released by atherosclerotic plaque.
Proteomic factors implicated in progression and vulnerability of plaque has been analyzed through
various morphological and imaging techniques, to understand their location and their expression in
the atherosclerotic plaque. Furthermore atherosclerotic plaques are analyzed by ex-vivo imaging techniques.
After all clinical test based on ELISA assay of blood sample of patients could validate plasma-secreted factors and identify potential biomarkers of disease.
Specific endpoint of the trial were:
1. analysis and identification of factors secreted by the plaque with proteomic techniques
2. morphological and imaging (in vivo and ex vivo) characterization of carotid plaque and
correlation with identified secreted factors regarding localization and vulnerability
3. validation of statistically significant differences in secretomic profile in different plaque categories
4. identification of secreted factors in blood sample
AIMS OF THE STUDY
In the first phase of the study the main objectives are :
1. to reconstruct a complete protein map of the overall atherosclerotic carotid secretion;
2. to evaluate the differences in secretomes from central plaque and corresponding distal side
segments, as putative areas of plaque complication and stable growth respectively;
3. to conduct a secretome-assisted plasma analysis of some differentially expressed proteins 70.
METHODS
Secretomes from carotid endarterectomy specimens were analyzed by a liquid chromatography
approach coupled with label free mass spectrometry. Differential expressions of proteins released
from plaques and from their downstream distal side segments were evaluated in each specimen.
Results were validated by Western blot analysis and ELISA assays. Histology and
immunohistochemistry were performed to characterize plaques and to localise the molecular factors
highlighted by proteomics.
A clear distinction in the distribution of cellular and extracellular- derived proteins, evidently
carotid endarterectomy samples. The expressions of thrombospondin-1, vitamin D binding protein,
and vinculin, as examples of extracellular and intracellular proteins, were immuno histologically
compared between adjacent segments and validated by antibody assays. ELISA assays of plasma
samples from patients and healthy volunteers confirmed a significantly higher concentration of
thrombospondin-1 and vitamin D binding protein in atherosclerotic subjects.
Taking advantage of the optimized workflow, a detailed protein profile related to carotid plaque
secretome has been produced which may assist and improve biomarker discovery of molecular
factors in blood.
Secretomes were analyzed by a liquid chromatography approach coupled with mass spectrometry
(LC-MS/MS). Label-free MS/MS based quantification was performed and validated by Western blot analysis and ELISA assay. Histology and immunohistochemistry were performed to
characterize CEA specimens and to localize the molecular factors highlighted by proteomics.
ELISA assays of thrombospondin-1 and vitamin D binding protein were performed in plasma
samples of 34 CEA candidates and 10 healthy controls.
Clinical data were derived from medical records, following data security guidelines and declaration of Helsinki.
All subjects gave written informed consent to participate to the study, in accordance with the Ethics
Committee requirements. The ethical approval for this study was granted by Clinical Trials Ethics
Committee of University of Pisa.
Patient clinical characterization
Human internal carotid artery (ICA) specimens and pre surgery plasma samples from 10 males and
4 females undergoing CEA for symptomatic or asymptomatic stenosis ≥70% were obtained from
Clinical Physiology. Additional 20 plasma samples of CEA candidates and 10 plasma samples of
healthy volunteers were collected at the Vascular Surgery Unit of Pisa University Hospital and at
the Institute of Clinical Physiology respectively.
Patient characteristics and database information on smoking habit, hypercholesterolemia and diabetes are summarized in Table 1.
Patients who presented with TIA or stroke within 6 months before surgery were classified as
symptomatic. No differences between symptomatic and asymptomatic patients were found for any of the reported parameters.
Stenosis on ICA was ≥ 70% at carotid color doppler ultrasound in all patients (mean 80±9%),
Feature Value (±SD) CEA patients for secretome (N=14)
Value (±SD) CEA patients for
plasma validation (N=34) Value (±SD) Healthy volunteers (N=10) Gender Females(n) 4 10 4 Males(n) 10 24 6 Age (years±SD) 72±9 74±7 70±5 Symptomatic (n) 6 11
-Type of event Stroke (n) 2 3
-TIA (n) 4 8 -Asymptomatic (n) 8 23 -Stenosis of ICA (%±SD) 80±9* 79±8* -Diabetes (n) 6 9 -Hypertension (n) 10 27 1 Hypercholesterolemia (n) 4 18 2 Smoking (n) 4 13 1 Statin treatment (n) 10 21 -Antiaggregant Therapy (n) 14 32
-Table 1 Clinical characteristics of population
[Categorical values are given as number of patients (n). Continuous variables are given as mean±SD.
TIA: Transient Ischemic Attack. ICA: Internal Carotid Artery. *Stenosis was measured by carotid EchoColor Doppler.]
Histology and immunohistochemical characterization
After 5 to 7 day fixation in 5% buffered formalin, P and DS samples were incubated in decalcifying
solution (Biooptica 05-M0300 ), washed and dehydrated in ascending alcohols series and embedded
in paraffin: transverse 5 μm thick consecutive sections were cut from each paraffin block by a
rotary microtome. Haematoxylin and Eosin (H&E) and Masson’s trichrome stains were used for histologic analysis.
For immunohistochemistry, sections were placed on positively charged slides, deparaffinized,
rehydrated and washed in distilled water; after incubation in H2O2 at room temperature, antigen
retrieval was accomplished and then sections incubated with diluted normal blocking serum.
Primary antibodies anti-αSMA (alpha smooth muscle actin) as a SMC phenotype marker,
anti-CD68 as a macrophage phenotype marker, anti-vinculin and anti-thrombospondin- 1 were
applied overnight to the slides in a 4°C humid chamber.
Following 30 min biotinylated secondary antibody incubation in peroxidase substrate solution
(DAB), slides were counterstained with Mayer’ s haematoxylin for 1 min. Antibody binding to cells and/or to extracellular components is visible as brown or dark brown stain (DAB), negative cells are stained blue (haematoxylin counterstain).
The same steps had been previously applied to a pool of CEA specimens (from symptomatic and
asymptomatic patients) which were processed en bloc and longitudinally, instead of transversely,
sectioned by microtome: microscopic examination of the obtained serial sections helped to guide
and standardise the splitting of fresh CEA samples, subsequently collected for secretome, into P and
DS segments.
Thrombospondin-1 and vinculin double immunostaining was performed. All sections from each
to Olympus Cell Sens Dimension software for image acquisition and morphometric analysis. Quantitative analysis of antibody staining within the lesional area of each sample was carried out on
several microscopic fields of consecutive sections, digitized at the same light source settings, by
double observer colour thresholding: reproducibility and statistical significance of results (expressed as % positive, dark brown pixels of the entire plaque area) as well as localization of different antibodies on the same region of adjacent sections was thus accomplished.
Carotid plaques were classified according to Stary’ s stages for atherosclerosis, American Heart
Association Committee on Vascular Lesions (3-4).
Tissue processing and secretome preparation
The arterial tissue was cultured according to de la Cuesta et al. with modifications. CEA specimens
(n=14) from surgery were transported in PBS on ice to the laboratory (65). Each specimen was
crosscut under macroscopic examination to separate P from DS segment. Samples were incubated
in 6-well plates in 2 ml of Eagle's Minimal Essential Medium (Sigma-Aldrich) supplemented with
penicillin and streptomycin. After 24 h the culture medium was harvested, centrifuged and stored at
− 80°C until analysis.
The secretome was submitted to reduction (dithiothreitol), alkylation (iodoacetamide) and digestion
(trypsin solution).
Secretome analysis
The solution was submitted to chromatographic separation of peptides that were directly processed
using mass spectrometer (LC-MS/MS analysis).
MS/MS data were processed with ProteinPilot™ Software (AB SCIEX, Toronto, Canada) in order
Western blot analysis and ELISA assays
Western blot analysis was performed using the following antibodies: anti-vinculin goat polyclonal antibody (1:500), antithrombospondin-1 mouse monoclonal antibody (1:200), and anti-α tubulin mouse polyclonal antibody (1:5000).
Dosage by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was performed on vitamin D binding protein and thrombospondin-1.
Statistical analysis
Principal component analysis (PCA) was conducted on mass spectrometric data of samples using
Marker View 1.2 software.
Student’s t Test was used as statistical parameter between the means of continuous variables to determine significant differences between categories of mass spectrometric data.
P value < 0.05 were considered significant to validate differences between categories and fold
change > 2.
Statistical analyses of other data were conducted using Origin 7.0 software. Data are expressed as
the mean ± SD. Differences between the means of the 2 continuous variables were evaluated by the Student’s t Test and results accepted when t Test>95% and P value<0.05. Paired t test was used for quantitative immunohistochemistry and ELISA assay.
RESULTS
Morphological characterization
Through microscopic examination of longitudinal sections of undivided CEA specimens is possible
to identify a plaque core containing area (P) comprised of typical components (lipid-rich necrotic
core, fibrous cap, cholesterol clefts, large calcium deposits, intraplaque hemorrhage/ fresh thrombi) and the distal side segment (DS) with fibromuscular tissue, small extracellular lipid and calcium
deposits. Fresh CEA specimens for secretome were subsequently divided to separate central plaque
P from its distal side DS, as depicted in Figure 1. Sections from all CEA specimens, each cut in two
(P and DS) were examined and classified for the presence of histologic hallmarks of atherosclerosis,
including lipid-rich necrotic core, cholesterol clefts, fibrosis, calcium, thin fibrous caps, macrophage infiltrate, neovessels, intraplaque haemorrhage/thrombi.
The DS segments showed small lesions at histologic examination, with atherosclerotic features
similar to those classified as Stary’ s types III and IV.
Proximal P segments, on the other hand, showed grossly evident fibrolipidic and/ or fibrocalcific,
mostly complicated plaques classified as types V and VI (Table 2).
Masson’ s trichrome stain, α SMA and CD68 immunostaining representative of P and DS segments
are shown in Figure 2; the quantitative analyses for α SMA and CD68 are also reported,
demonstrating a significantly higher content of VSMCs and CD68 positive macrophages in the
Figure 1 Longitudinal section of an undivided CEA specimen at artery center line level showing the
cutting line for P and DS segments separation. Prevalent complicated plaque features of P
(lipid-necrotic core, calcium deposits, fibrosis and haemorrage) and milder changes of its downstream
side DS (prevalent VSMCs and collagen component, small lipid and calcium deposits) are evidenced by Masson’s trichrome and α-SMA immunostaining. Original magnification 2×, insets 10×.
Table 2 Histologic characterization of P and DS samples
[(1) TFCA= thin fibrous cap, (2) Ca = calcification, (3) NC-Chol=necrotic core cholesterol clefts, (4) He = intraplaque hemorrhage/thrombus.]
Figure 2 Left: P and DS sections stained with (a) Masson’s trichrome stain, (b) α-SMA and (c) CD68
immunostain (original magnification 2×, insets 10×). P is a type VI plaque with a necrotic core,
calcifications, intraplaque thrombus and a thin fibrous cap composed of collagen and αSM-actin
positive cells; minor intraplaque CD68 positive staining is present and α-SMA -actin positive
contractile VSMCs are also visible on the outer border of the specimen (opposite to the lumen) due to
surgical cleavage of plaque from the intact tunica media. In DS, a much thinner lesion (equivalent to
Stary type III) with extensive α -SMA and CD68 cellular positivity is present. Right: From top to
bottom. Average values and SD of lesional area (mm2), intralesional α-SMA and CD68 stain positivity
(% of lesional area) of P and DS segments of all specimens’ sections: a smaller lesional area and a
markedly higher positivity of both cellular markers in the distal side of the plaque is evident (*p<0.05,
Secretome analysis of endarterectomy specimens
P and DS segments from the 14 enrolled patients were incubated for 24 h in serum free medium,
secretomes were collected, protein digested and peptides fractionated by reverse phase chromatography and analyzed by 5600 TripleTOF mass spectrometer.
Using this LC-MS/MS approach, 463 proteins were identified with a Protein Score (Confidence)
>95% and using local false discovery rate analysis >1% as stringent criterion to avoid false
positives.
The entire list of proteins reported in supplementary tables was analyzed in the context of the
published literature and firstly grouped as extracellular and intracellular proteins. Using Uniprot
database (www.uniprot.com), extracellular proteins were further subdivided in ECM proteins and
ECM associated proteins. The intracellular ones were grouped considering their localization in
cytosol, membrane, organelle, cytoskeleton and others (Figure 3A). The secretion potential of these
identified proteins was computed by submitting them to SecretomeP server that predicts protein
secretion route on the basis of the presence of the signal peptide, responsible for endoplasmic
reticulum addressing. The non-classical secretion through multi vesicular bodies was also determined by SecretomeP using specific databanks (Figure 3B).
Figure 3 Pie charts of the total identified proteins from P and DS. A) Identified proteins are divided on
the basis of their localization in intracellular or extracellular space based on Gene Ontology. B)
Identified proteins are evaluated with SecretomeP software to compute their secretion potential. They
Comparative analysis between plaque and its distal counterpart secretomes
In order to evaluate differentially released proteins between P and DS specimens, MarkerView 1.2
software was used and 31 proteins were found differentially secreted. These proteins were classified
into two categories: cellular (n=17) and extracellular proteins (n=14) (Table 3). The putative role of each factor in atherosclerosis and cardiovascular disease was reported in the table as suggested by the literature, in order to underline a possible correlation of their dis-regulation to the pathology. Nine out of the 14 extracellular proteins were found over-expressed in P secretome, whilst cellular proteins were found over-expressed in DS secretome, with the exception of smoothelin, which is a
recognized marker of VSMC contractile phenotype. Of these proteins eighteen have been already
reported in serum/plasma and listed in Human Protein Reference database (www. hprd.org). Origin
7.0 was used to represent the modulation of the differentially released proteins and the most
Table 3 Differentially released proteins by CEA specimens
[(1) Protein name is reported according with SwissProt 2011 database. (2) Role in atherosclerosis is
referred to data reported on the recent literature (3) ▼: down released in P compared with DS; ▲: up
released in P vs DS. (4) P values were generated using Marker View software. (5) Their presence in
Figure 4 Differential protein expression obtained by mass spectrometric analysis of P and DS. Four of
the 31 proteins reported in table 3 are chosen and comparative expression analysis is presented as Box
plots. Box plots show median value, standard deviation (SD), 50%, minimum and maximum intensity values and were exported from Marker View software.
Validation by Western Blot and ELISA assay
Western Blot analysis and also ELISA assay were applied to secretomes in order to validate the
differential release observed by mass spectrometric analysis. Vinculin and thrombospondin-1 were
selected for Western blot validation (Figure 5A), while vitamin D binding protein was selected for
ELISA assay (Figure 5B).
Figure 5 Left: Western blot analysis of P and DS secretomes. Densitometric quantification of vinculin
and thrombospondin-1 using Quantity One 1D analysis software. Representative images: 10 μg of
proteins were separated on SDS-PAGE and blotted on nitrocellulose membrane. Immunoblots were
probed with antibodies against vinculin and thrombospondin-1. Right: ELISA assay for vitamin D
Immunohistochemistry quantification
Double immunostaining of anti-vinculin and antithrombospondin-1 antibodies on P and DS sections
of a CEA specimen is shown in Figure 6. Anti-vinculin antibody selectively stains lesional cells
whereas thrombospondin-1 stain is confined to the plaque lipid-necrotic core and surrounding
fibrous cap.
Quantitative results derived from single immunostaining of both markers on sections of all CEA
specimens confirm the significantly higher (6.4±5.0 vs 3.4±3.0 P<0.02) vinculin positivity and the
much lower (0.8±0.8 vs 7.3±6.0 P<0.02) thrombospondin-1 positivity in the lesion area of DS as compared to corresponding P segments.
Figure 6 Top: Double immunostaining of thrombospondin-1 (red) and vinculin (brown) in P (A) and
DS (B) sections of a fibrocalcific type Vb plaque. Low magnification (2×, left) and high magnification
(10× to 40×) microscopic fields (insets) are shown. Neither cellular nor extracellular co-distribution of
the two antibodies is present. A selective cellular binding of vinculin both on P an DS sections is
evident, while thrombospondin-1 is almost exclusively located in the fibrocalcific core of P. Bottom:
Average values and SD of thrombospondin-1 and vinculin positivity (single immunostaining) in
lesional area of all CEA specimens showing a markedly greater stain in P as compared toDS for
ELISA assay of plasma samples
Dosage by double-antibody sandwich ELISA assays of thrombospondin-1 and vitamin D binding
protein were performed in plasma samples of 34 CEA patients and 10 age-matched healthy
controls. As reported in Table 1, 11 patients were symptomatic while 23 were asymptomatic.
Plasma levels of thrombospondin-1 and vitamin D binding protein were significantly higher in
atherosclerotic patients than in healthy subjects (thrombospondin-1: 71±20 vs 48±9 ng/mL P value=
0.02; Vitamin D binding protein: 205±133 vs 76±8 ng/mL P value= 0.05), while no significant
differences were observed between symptomatic and asymptomatic patients (thrombospondin-1:
67±14 vs 72±20 ng/mL P value=0.72; vitamin D binding protein: 234±137 vs 192±122 ng/mL P
value=0.69) (Figure 7).
Figure 7 ELISA assay for thrombospondin-1 and vitamin D binding protein expression in plasma
DISCUSSION
Several proteomics approaches have been carried out in order to clarify the mechanisms of
atherogenesis as well as to search for plaque presence and severity.
All these studies, due to technical limits as well as to low sensitive and low-throughput methods,
identified only a small number of proteins, restricting considerably the detection of novel low
abundant biomarkers 60-61.
In our study the secretome of human carotid plaques was tackled with a high performance, very
sensitive, geland label-free LC-MS/MS workflow. The unbiased approach, able to give a high
sensitive full screen overview of secreted proteins from human carotid artery, is presented here.
This method allowed the identification of 463 proteins thus producing a wide shot on proteins
released by atherosclerotic arteries.
In our procedure, CEA specimens were cut in P and downstream DS segments in order to compare
the secretomes of the upstream area of maximum stenosis to that of partially preserved downstream area, subjected to distinct flow shear stress and wall strain regimen. P segments were classified as
Stary’ s types V and VI, while DS segments displayed less severe wall changes. We considered this
approach useful to differentiate specific molecular factors of the central plaque area, where
complication features are present, from those of its distal side.
Indeed it has been recently recognized that the upstream side is associated with an increased
incidence of severe lesions with cap rupture, while the downstream side is associated with higher
VSMC content and it could thus be more representative of cellular growth 69.
According to this line of reasoning, we found that proteins involved in ECM organization are more
represented in P secretome while proteins concerning VSMC phenotype switch, cell contraction and
observations confirm and strengthen the role of VSMC activation in the early stages of plaque
development and, conversely, the role of ECM substrates and matrix remodeling in mature
plaques71.
Secretome expression of vinculin and thrombospondin-1 have been validated by western blot
analyses: the first one being an intracellular protein, involved in cell migration, the latter one as
representative of ECM-related proteins and reported to control atherosclerotic plaque maturation.
Single and double immunostaining confirmed western blot results of differential protein distribution
along CEA specimens, at the same time excluding significant tissue colocalization. A sandwich
ELISA assay was also used to confirm the differential secretion of vitamin D binding protein, an
extracellular protein reported as marker of acute events. Both western blots and ELISA tests
validated the mass data on secretomes.
Amongst the 463 identified proteins, 31 resulted differentially secreted by P and DS samples. Half
of them were extracellular factors, most of which already reported in plasma, suggesting that, once
released from tissue, they could be found in peripheral blood. For this reason, vitamin D binding
protein and thrombospondin-1 were measured in plasma samples of CEA candidates and found
significantly higher than in non atherosclerotic subjects. No significant association with symptomatology was evidenced. The correlation between secretome modulation and plasma levels
of these two proteins confirms that tissue-secreted protein analysis can be helpful to search for
novel disease-specific biomarkers in blood and improve non-invasive risk assessment.
The clinical impact of secretome approach to vascular disease and atherosclerosis arises from the
potential use of plaque-secreted molecules as useful predictors of disease outcome and/or of stroke
risk assessment. An untargeted secretome strategy can be able to differentiate progressing from
stable plaques as well as discriminating rupture-prone from non-vulnerable plaques, leading to a proper selection and optimal management of high risk patients.
for no more than 20% of strokes; (b) complicated ruptured plaques can be asymptomatic, limiting markers specificity in identification of stroke risk; (c) site-specificity of arterial VSMCs and extent
of plaque vascularization can influence type and quantity of proteins released in the blood. Despite
these restrictions, secretome analysis seems a most valuable tool to disclose the molecular basis of
atherosclerosis as well as of several other diseases, due to the unique combination of tissue
specificity of biomarkers and clinical feasibility of their detection in blood, aimed to improve patient diagnosis and monitoring.
Results of an untargeted secretome analysis are presented in this study: this unbiased strategy, as compared to targeted analysis, may complicate a prompt clinical output but it is surely the most adequate approach to unravel novel disease markers. The relatively small number of CEA
specimens analyzed represents a limit and the lack of sample stratification is the most relevant
consequence of this fact. Despite that, our high sensitive method evidences a very broad spectrum
of proteins in carotid plaque secretome and comparative analysis between P and its DS overrides
inter-patient variability. Two ECM proteins released by P are found significantly higher in plasma
from atherosclerotic patients without differences between symptomatic and asymptomatic ones,
supporting the mismatch between plaque-related and event-related factors, in agreement with other
findings.
A further clinical relevance of the present study relies on the evidence that a large number of CEA
secreted proteins are asymmetrically distributed along the atherosclerotic vessel, related to plaque
complication features: this implies that a broader cellular and extracellular protein profiling, rather
than isolated marker assays, may be required to increase specificity and predictive potentiality.
Future studies are needed to specify secretome-plasma proteins profiling at different stages of
CONCLUSIONS
Patients with asymptomatic carotid disease represent a therapeutic dilemma; surgical
revascularization could be an overtreatment in most of them while best medical treatment could
expose some of them to a risk of cerebral ischemic complications.
Stratification of cerebral ischemic risk is needed; many factors have been studied regarding clinical features, morphological characteristics of plaques and progression of stenosis.
Biomarkers are a promising field of research and together with previous mentioned factors could
help to create a risk score; that should afterwards be validated by a prospective observational study on a population of subjects with asymptomatic carotid stenosis.
In this study a secretomic approach has been used in order to identify different proteins secreted in different parts of the plaque; a large number of secreted proteins resulted asymmetrically expressed along the atherosclerotic vessel according to their cellular or extracellular origin, confirming that markers of stable plaque growth and plaque complication could be used as biomarkers of distinct and specific stages of disease. Despite that a significative difference of these proteins in the blood
of the asymptomatic patients and the symptomatic ones was not found supporting the fact that, at
the moment, these factors are plaque-related and not event-related.
Further studies should be done to complete specific secretome-plasma proteins profiles at different stages of carotid plaque growth and complication and its relation to clinical events.
REFERENCES
1) Libby P, Aikawa M, Schonbeck U: Cholesterol and atherosclerosis. Biochim Biophys Acta 1529:299, 2000.
2) Libby P. Inflammation in atherosclerosis. Nature. 2002; 420:868–874.
3) Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W Jr, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW: A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1994, 89:2462–2478.
4) Stary HC: Natural history and histological classification of atherosclerotic lesions: an update. Arterioscler Thromb Vasc Biol 2000, 20:1177–1178.
5) Richard Cambria et al. Rutherford’s vascular surgery. Jack L. Cronenwett, K. Wayne
Johnston; associate editors. 7th ed. 2010
6) North American Symptomatic Carotid Endarterectomy Trial Collaborators*Beneficial Effect of Carotid Endarterectomy in Symptomatic Patients with High-Grade Carotid Stenosis. N Engl J Med 1991; 325:445-453
7) European Carotid Surgery Trialists' Collaborative Group. Randomised trial of endarterectomy for recently symptomatic carotid stenosis: final results of the MRC European Carotid Surgery Trial (ECST). Lancet. 1998 May 9;351(9113):1379-87.
8) Executive Committee for the Asymptomatic Carotid Atherosclerosis Study. Endarterectomy for asymptomatic carotid artery stenosis. JAMA 1995;273:1421-8.
9) Halliday AW, Thomas D, Mansfield A. The Asymptomatic Carotid Surgery Trial (ACST). Rationale and design. Steering Committee. Eur J Vasc Surg 1994;8:703-10.
10) Halliday A, Harrison M, Hayter E, et al. 10-year stroke prevention after successful carotid endarterectomy for asymptomatic stenosis (ACST-1): a multicentre randomised trial. Lancet 2010;376:1074-84.
11) Marquardt L, Geraghty OC, Mehta Z, Rothwell PM. Low risk of ipsilateral in patients with asymptomatic carotid stenosis on best medical treatment: a prospective, population-based study. Stroke 2010;41:e11-7.
12) Constantinou, J, Jayia P, Hamilton G. Best evidence for medical therapy for carotid artery stenosis. J Vasc Surg 2013;58:1129-39.
13) Abbott AL. Medical (nonsurgical) intervention alone is now best for prevention of stroke associated with asymptomatic severe carotid stenosis: results of a systematic review and analysis. Stroke 2009;40:e573-83.
14) Hong KS, Yegiaian S, Lee M, Lee J, Saver JL. Declining stroke and vascular event
recurrence rates in secondary prevention trials over the past 50 years and consequences for current trial design. Circulation 2011;123:2111-9.
15) Naylor AR. What is the current status of invasive treatment of extracranial carotid artery disease? Stroke 2011;42:2080-5.
16) U.S. National Institutes of Health. Carotid Revascularization and Medical Management for Asymptomatic Carotid Stenosis Trial (CREST-2) (ClinicalTrials.gov website). 2014-2015. 17) Reff T, Eckstein HH, Amiri H, Hacke W, Ringleb PA; SPACE-2 Study Group Modification
of SPACE-2 study design. Int J Stroke 2014;9:E12-3.
18) Nicolaides AN, Kakkos SK, Kyriacou E, et al. Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification. J Vasc Surg 2010;52:1486-1496.e1-5.
19) Ota H, Reeves MJ, Zhu DC, Majid A, Collar A, Yuan C, DeMarco JK.Sex differences in patients with asymptomatic carotid atherosclerotic plaque: in vivo 3.0-T magnetic resonance study. Stroke. 2010 Aug;41(8):1630-5.
20) Rothwell PM, Goldstein LB. Carotid endarterectomy for asymptomatic carotid stenosis:
asymptomatic carotid surgery trial. Stroke 2004;35:2425-7.
21) Brott TG, Hobson RW, Howard G, et al., Stenting versus endarterectomy for treatment of carotid-artery stenosis. N Engl J Med 2010;363:11-23
22) King A, Shipley M, Markus H; ACES Investigators. The effect of medical treatments on stroke risk in asymptomatic carotid stenosis. Stroke. 2013 Feb;44(2):542-6.
23) Hirt LS.Progression rate and ipsilateral neurological events in asymptomatic carotid stenosis.Stroke. 2014 Mar;45(3):702-6.
24) Nicolaides AN, Kakkos SK, Griffin M, Sabetai M, Dhanjil S, Tegos T, Thomas DJ,
Giannoukas A, Geroulakos G, Georgiou N, Francis S, Ioannidou E, Doré CJ; Asymptomatic
Carotid Stenosis and Risk of Stroke (ACSRS) Study Group.Severity of asymptomatic
carotid stenosis and risk of ipsilateral hemispheric ischaemic events: results from the ACSRS study.Eur J Vasc Endovasc Surg. 2005 Sep;30(3):275-84.
25) Kakkos SK, Nicolaides AN, Charalambous I, et al. Predictors and clinical significance of progression or regression of asymptomatic carotid stenosis. J Vasc Surg 2014;59:956-67.e1. 26) Balestrini S, Lupidi F, Balucani C, et al. One-year progression of moderate asymptomatic
carotid stenosis predicts the risk of vascular events. Stroke 2013;44:792-4.
27) Kakkos SK, Sabetai M, Tegos T, et al. Silent embolic infarcts on computed tomography brain scans and risk of ipsilateral hemispheric events in patients with asymptomatic internal carotid artery stenosis. J Vasc Surg 2009;49:902-9.
