DOI: 10.1161/CIRCULATIONAHA.113.007603

1

Time Dependent Changes in Atherosclerotic Plaque Composition in Patients

Undergoing Carotid Surgery

Running title: van Lammeren et al.; Carotid plaque stabilisation

Guus W. van Lammeren, MD, PhD1,2*; Hester M. den Ruijter, PhD1,3*;

Joyce E. P. Vrijenhoek, MD1,2,4; Sander van der Laan, MSc1; Evelyn Velema, MSc1;

Jean-Paul P. M. de Vries, MD, PhD4; Dominique P.V. de Kleijn, PhD1,4,5; Aryan Vink, MD, PhD6;

Gert Jan de Borst, MD, PhD2; Frans L. Moll, MD, PhD2; Michiel L. Bots, MD, PhD3;

Gerard Pasterkamp, MD, PhD1

1Experimental Cardiology Laboratory, University Medical Center Utrecht; 2Dept of Vascular Surgery, University Medical Center Utrecht; 3Julius Center for Health Sciences and Primary

Care, University Medical Center Utrecht; 6Dept of Pathology, University Medical Center Utrecht; 4Interuniversity Cardiology Institute of the Netherlands, Utrecht, the Netherlands;

5Surgery NUS and CVRI NUHS, Singapore * Share first authorship

Address for Correspondence:

Gerard Pasterkamp, MD PhD

Experimental Cardiology Laboratory

University Medical Center Utrecht

Heidelberglaan 100, Room G02.523

3584 CX Utrecht, the Netherlands

Tel: (+31)887557155

Fax: (+31)302522693

E-mail: G.Pasterkamp@umcutrecht.nl

Journal Subject Codes: Atherosclerosis:[135] Risk factors, Stroke treatment - surgical:[76] Carotid endarterectomy, Etiology:[8] Epidemiology

Gert Jan de Borst, MD, PhD2; Frans L. Moll, MD, PhD2; Michiel L. Bots, MMD,D,D, PPhDhDhD33;;;

Gerard Pasterkamp, MD, PhD1

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DOI: 10.1161/CIRCULATIONAHA.113.007603

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Abstract

Background—Time dependent trends in the incidence of cardiovascular disease have been

reported in high-income countries. As atherosclerosis underlies the majority of cardiovascular

diseases, we investigated temporal changes in the composition of atherosclerotic plaques

removed from patients undergoing carotid endarterectomy.

Methods and Results—The Athero-Express study is an ongoing longitudinal vascular biobank

study that includes the collection of atherosclerotic plaques of patients undergoing primary

carotid endarterectomy in the province of Utrecht, the Netherlands from 2002 to 2011.

Histopathological features of plaques of 1583 patients were analysed in intervals of 2 years. The

analysis included quantification of collagen, calcifications, lipid cores, plaque thrombosis,

macrophages, smooth muscle cells, and microvessels. Large atheroma, plaque thrombosis,

macrophages and calcifications were less frequently observed over time with adjusted odds ratios

of 0·72 [0·650-0·789], 0·62 [0·569-0·679], 0·87 [0·800-0·940] and 0·75 (0·692-0·816) per 2 year

increase in time, respectively. These changes in plaque characteristics were consistently

observed in patient subgroups presenting with stroke, transient ischemic attack, ocular symptoms

and asymptomatic patients. Concomitantly, risk factor management and secondary prevention

strategies among vascular patients scheduled for carotid endarterectomy significantly improved

over the past decade.

Conclusions—In conclusion, over the past decade, atherosclerotic plaques harvested during

carotid endarterectomy show a time dependent change in plaque composition characterized by a

decrease in features currently believed to be causal for plaque instability. This seems to go hand

in hand with improvements in risk factor management.

Key words: atherosclerosis, stroke, vascular disease, carotid artery, macrophage, plaque haemorrhage, atheromatous, secular trend

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of 0·72 [0·650-0·789], 0·62 [0·569-0·679], 0·87 [0·800-0·940] and 0·75 (0·692-0·816) per 2 year

ncrease in time, , resps ectively. These changes in pplaquq e characteristics were consistently

obobbseseserrvrvede iin n pppatiienent t sus bgbgroroupups prpresene tiingg with h sttrorookkke, trranansisient isisschchc emmicic aattacck,k, oculaar sysympm toms

anndd d asa ymptommatatticic patatieientntnts.ss. CCCoononcocoommimitaaannttly, rissk ffacacactor r mmamannnaggegemmmenntnt aandnn ssececcononndadaryryry ppprereveveenntntioioionn n k

ttrarar tetetegigies amomomongngg vvvasasccuullaar paaatitienene tsts ssschchc ededdululededd ffforor cccaarrotottididid eennddaarartetererecctomommy ssigiggniniifificacanntntllyly iiimmpmproroveveved

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Introduction

Population-based studies demonstrate an age-adjusted decrease in manifestations of

atherosclerotic cardiovascular diseases such as stroke and coronary heart disease over the last

decades in high-income countries.1-5

The composition of atherosclerotic plaques is considered to reflect the local

atherosclerotic disease severity. Indeed, autopsy and clinical pathology studies have revealed that

thin capped atheromatous plaques are associated with rupture resulting in thrombotic occlusion

and a subsequent acute cardiovascular event.6 In addition, a minority of thrombotic events are

caused by plaque erosion in the presence of a fibrous lesion.7

Autopsy studies have provided many insights into the atherosclerotic plaque

characteristics that are associated with cardiovascular events. Yet, there is no report on temporal

changes in plaque phenotypes during the last decades coinciding with the decreased incidence of

cardiovascular disease reported in high-income countries. To investigate time dependent changes

in atherosclerotic plaque characteristics, we used the Athero-Express study, and analyzed

histological features of over 1500 plaques removed during carotid endarterectomy from patients

with similar symptomatology included from 2002 to 2011.

Methods

Athero-Express

Study population

Athero-Express is an ongoing longitudinal biobank study collecting carotid atherosclerotic

plaques from patients that undergo carotid endarterectomy in the University Medical Center

Utrecht or the St Antonius Hospital Nieuwegein, the Netherlands.8 Medical ethics committees of

Autopsy studies have provided many insights into the atherosclerotic plaqaqqueee

characteristics that are associated with cardiovascular events. Yet, there is no report on temporal

chhananangegegess ininin ppplalaquuuee e phphenotypes during the last dececcaaddees coinciding wwwith h thththeee decreased incidence of

caardddioi vascularar dddisi eaeasesee rrepepeporororteteteddd ininn hhhigighh-h-iincooommme cououountririieeses. ToToTo iinvnvveennn ststigigaaatee titimememe ddepeppenenndeded nttt cchahahanngnges

nn aaathththerererososclclclereerototicicc ppplalaqqquee e chchararracaccteteteririristssticiccs,s, wwwe e e usssedede tthhehe AAtAtheheherroro-E-EExpxpprereessssss ssstutudydyd , ananandd anananaaalyzyzzeddd

histological ffeaeaeatutut rereres s s ofofof oooveeerrr 1115000000 plpllaqaqueueu ss s rereremomomoveveved dd dudud ririingngng cccarararotototididi eeendndndarararteteterererectctomomomy y y frfrfromoo patients rr

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both participating centers approved the study and all included patients provided written informed

consent. All patients who underwent carotid endarterectomy from the start of the Athero-Express

in 2002 up to 2011 were included. Patients were excluded from the study if informed consent

was lacking. Indication for surgery was based on international guidelines for symptomatic and

asymptomatic carotid stenosis.9-11 All indications were reviewed by a multidisciplinary team of

vascular specialists before surgery. Patient data were obtained via standardized questionnaires

and pre-operative admission charts.

Plaque processing and assessment

Atherosclerotic plaques of patients were harvested during carotid endarterectomy according to a

standardized and previously reported protocol.8,12 Briefly, carotid plaques were divided into

segments of 5 mm thickness. The section with the largest plaque burden was classified as culprit

lesion and subjected to immunohistochemical staining. Plaques were stained for macrophages

(CD68), smooth muscle cells (alpha-actin), collagen (Picro-sirius Red), microvessels (CD34) and

presence of plaque thrombosis (consisting of the combination of luminal thrombi and/or

intraplaque hemorrhages-haematoxylin eosin and fibrin [Mallory's phosphotungstic acid-

hematoxylin]).8,12,13 Macrophages and smooth muscle cell stains were analysed quantitatively by

computerized analyses and expressed as percentage of plaque area. Plaque neovascularization

was identified by morphological structure with CD34+ staining that was counted in 3 hotspots

and subsequently averaged per cross-section. Collagen and calcifications were scored semi-

quantitatively at a 40X magnification, according to the following criteria: no (1) or minor (2)

staining along part of the luminal border of the plaque or a few scattered spots within the lesion,

moderate (3) or heavy (4) staining was scored when along the entire luminal border or evident

parts within the lesion. Also these categories were binned into no/minor and moderate/heavy for

tandardized and previously reported protocol.8,12 Briefly, carotid plaques were dddiivivididi ededed iiintntnto o o

egments of 5 mm thickness. The section with the largest plaque burden was classified as culprit

eesisiiononon aaandndnd sssubububjeectctcteeded to immunohistochemical stttaaiainniing. Plaques wewew re sstatataiinined for macrophages

CCCDD6D68), smoooththh mmmusscclc e e ceceelllllsss ((a(alplphahaha-a-actctiin), ccooollagggeenn (PPiPicrcroo--ssiiririuuus RRededd),)) mmmiciccroroovevessssselelels s (C(C( DD3D344)4 aand

prpresesesenenencecec ooff f plplp aqaqqueueue tthrhrromommbob sisisiss (((cococonsnnsisistitit ngngng ooof thththe e cococombmbbinnnatatatiiiononn ooff f luuumimim nnnall l ththrororommbmbii ananand//ooror

ntraplaque heheemomom rrrrrrhahah gegeges-s hahahaememematatatoxoxo ylyly innn eeososo ininin anannd dd fifif brbrb innn [[[MaMaMallllororory'yy s phphphosososphphphototo ununngsgsgstititicc c acaca id-

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logistic regression analyses. Size of the lipid core was visually assessed using polarized light and

cut off at 40% of the plaque area, based on the association with rupture prone plaques.14 Luminal

thrombus was defined as a solid mass formed from fibrin and platelets superimposed on the

plaque, within the vascular lumen.15 Loose erythrocytes in the lumen were scored negative for

luminal thrombi. Plaque thrombosis was defined as the combination of either presence of luminal

thrombus and/or intraplaque hemorrhage. Results are also shown separately. All histological

scorings were performed by the same dedicated technician during the entire study period. To

examine whether the assessment of plaque characteristics had changed over time, we performed

additional intraobserver analyses between different time intervals of our longitudinal study.

Definitions of risk factors and medical treatment

At the time of inclusion all patients were asked to complete an extensive questionnaire as

reported previously.5 Systolic blood pressure was measured before surgery in the outpatient

clinic, together with height and length to calculate the body mass index (BMI). Blood withdrawal

was performed at baseline to assess serum total cholesterol and creatinin levels. Creatinin was

used to calculate the estimated glomerular filtration rate (eGFR) with the

Modification of Diet in Renal Disease (MDRD) formula.16 Patients were considered

smokers if they reported to be smoking until the year of inclusion. Diabetic status was restricted

to those patients receiving medical treatment including insulin or oral glucose-lowering drugs.

Statin use at baseline was registered, as well as the use of antihypertensive drugs including

betablockers, ACE-inhibitors, angiotensin II antagonists and diuretics. Furthermore, prescription

of anti-thrombotic medication was registered including aspirin, oral anticoagulants, clopidogrel

and dipyridamol.

For patients undergoing carotid endarterectomy in Athero-Express, the index event was

Definitions of risk factors and medical treatment

At the time of inclusion all patients were asked to complete an extensive questionnaire as

eepopoportrtrtededed ppprererevivv ouuuslslslyy.y.5 Systolic blood pressure wasasas mmmeasured beforrre ee suurgrggeerery in the outpatient d

cllinnnicic, togetherer wwwittth h heheh igigghththt aaandndnd llenennggtgthh tooo calallcuuulateee ttthe bboododyyy mmamasss iinindedexxx ((BBMIMIMI).). BBBlololoododod wwititithdhdhdraraawaw

wawaass s pepeperfrforormememed d aatat bbbasaseeelininne e too aasssssesesessss seserururum mm totototaaall l chchhoololesssteteerororoll l annnd d d ccrcreaeaeatitit nnninn llevevvelells.s.s CCCrereeatiininininn wwwasas

used to calculullatatateee thththe e e esese titit mamaateteed d glglglomomomere ulululararar fffilili trtrtratata ioioi n nn rarar teee (((eGeGeGFRFRFR) ) ) wiwiiththth ttthehehe

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registered as transient ischemic attack, stroke, occular symptoms (including amaurosis fugax or

retinal infarction) or as an asymptomatic status. In addition, the time interval between the last

symptomatic event and surgery was registered.

Follow-up and outcome

The patients included in the Athero-Express study underwent a clinical follow-up that has been

described in detail previously.5 In short, patients were asked to fill in the follow-up questionnaire

after 1, 2 and 3 years. If the questionnaire was answered positively, further research was

performed to investigate the potential outcome of the event. If the patient did not respond, the

general practitioner was contacted. The events were assessed by two members of an outcome

assessment committee. The composite endpoint included any death of presumed vascular origin

(stroke, myocardial infarction, sudden death, other vascular death), non-fatal stroke, myocardial

infarction or any intervention not planned at the time of inclusion.

Statistical analyses

Statistical analyses were performed with SPSS 17.0 (Chicago, IL, USA). To avoid the limitation

of complete case analyses, multiple imputation was used to calculate missing values.17

Supplemental Table 1 gives information on the amount of missingness in our study. To

investigate time trends with sufficient sample sizes in different time periods, we studied plaque

and patient characteristics over 2 year intervals from 2002 to 2011 using Chi-square statistics.

Potential confounding variables were predefined based on observed baseline incomparability

over time (P-value for selection <0.20) and introduced in regression in a step-wise manner to

estimate their individual contribution in logistic regression models. For this analysis, plaque

characteristics were binned into no/minor and moderate/heavy.

assessment committee. The composite endpoint included any death of presumedd vvasasscuuulalalarr r orororiigigiin

stroke, myocardial infarction, sudden death, other vascular death), non-fatal stroke, myocardial

nnfafaarcrcrctititionon ooor r aany y y ininintet rvention not planned at the e ttitimmme of inclusion.n..

SStStatattisi tical annalalyyyseees

StStatatatisisstititicacall anananalalysyssesss wweeeree e pep rfrfforormememed d d wiwiiththh SPSPSPSSSS 11777.000 (C(CChihihicacacagogogo,,, ILILL, USUSU AA)A).. ToToo aaavovov ididid ttthehe limimmititaaatiioon

of complete cacaasesese aaanananalylyyseses s,, mmmuluu tititiplplp ee imimimpupuputatatatititiononon wwwasasa uuusesedd d tototo cccalalalcucuculaaatetete mmmisisissisisingngn vvvalalalueueues.s..171717

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Results

Patients

The demographics in the patient population scheduled for carotid endarterectomy are shown in

Table 1. The mean age at the time of surgery was 66·4 (±8·5) in 2002-2003 and 69·9 (±10·1) in

2010-2011. The proportion of asymptomatic patients in the same period of time was 21·2% for

2002-2003 and 9·2% for 2010-2011. For symptomatic patients, the median time between the last

event and surgery was 111 days [54-159] in 2002-2003 and 20 days [11-35] in 2010-2011

(Table 1). Prevalence of active smoking until the year of surgery was 45·6% in 2002-2003 and

32·1% in 2010-2011. Furthermore, total cholesterol levels at the time of inclusion were lower in

more recent years, concomitant with a higher frequency of statin prescription. The prescription

of angiotensin II antagonists and dipyridamol was also more frequent in more recent years

(Table 2). On the other hand, prescription of oral anticoagulants and clopidogrel were highest in

the early years of the study. These factors were introduced in the multivariate model to assess

their individual contribution to the association between plaque characteristics and year of

inclusion.

Plaque composition

During the past decade the prevalence of plaques with large lipid cores, covering >40% of the

plaque surface, were frequently observed in 2002-2003 (33·2%) and less frequently observed in

2010-2011 (14·4%, p<0·001) (Table 3). The occurrence of plaque thrombosis was more

prevalent in the early years (74·4%) as compared to more recent years (37·6%, p=0·001). There

were no differences in the number of intraplaque vessels over the years. The proportion of

heavily calcified plaques was also less prevalent in more recent years.

Strong fluctuations were observed in macrophage and smooth muscle cell counts between

more recent years, concomitant with a higher frequency of statin prescription. ThThhe prpp esesscrrcripipiptititionon

of angiotensin II antagonists and dipyridamol was also more frequent in more recent years

TaTaablblbleee 2)2)2).. OnOnOn thehee oootht er hand, prescription of oraaal ll aannticoagulants anana d clclclopopopidogrel were highest in

hhhe eae rly years s ofofo ttthee sstuuuddydy.. ThThTheseseee ffafacttoorrs wwwerrre innntrrooduuucceced d innn tthehee mmmululltititivaaaririatatatee e momoodededel l l totoo aaassssseessss

hheieieir r ininindidiviviidududualal cccononntrtriibbuututioion totot ttthehehe aaassssococociaiaiatititiononn bbbetettwweweeenen ppplalalaquququeee cchahaharararactctereriisistiticcscs aaandndd yyyeaearrr ooff

nclusion.

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2002 and 2011. For both cell types the highest values were observed in 2006-2007 and the

lowest values were observed in 2010-2011. The median macrophage percentages were 0·41 % in

2002-2003, and 0·09 % in 2011-2012. Figure 1 shows the semi-quantitative data on plaque

characteristics that are currently believed to be causally related with the vulnerability of the

plaque in two-year intervals. Panel A shows that the decrease in histologically identified plaque

thrombosis was mostly due to a decrease in both luminal thrombosis and intraplaque

hemorrhage. Also, the decrease in plaques with higher fat content coincided with an increase in

plaques with no detectable fat (Figure 1, panel B). For macrophages and calcifications (Figure

1, panel C and D), the heavy staining was less often observed over time whereas minor staining

prevailed in more recent years. Raw scatterplots of macrophages and smooth muscle cells as well

as semi quantitative data on collagen and smooth muscle cells are depicted in eFigure 1.

The association between plaque characteristics and year of surgery was assessed after

adjustment for patient and procedural characteristics that changed over time (age, index event,

delay between event and surgery, medication use) and traditional risk factors that are known to

be associated with atherosclerotic disease progression (presence of contralateral internal carotid

stenosis > 50%, history of peripheral arterial disease, history of coronary arterial disease,

smoking, mean arterial pressure, total cholesterol levels, kidney function (eGFR)). After

adjustment, all aforementioned plaque characteristics, except for smooth muscle cell content

remained significantly associated with year of surgery and indicated an decrease in features that

are considered to be associated with carotid plaque instability over time. Per 2 year increase, the

adjusted odds ratios for atheromatous plaques, plaque thrombosis and calcifications were 0·72

[0·650-0·789] (p<0·001), 0·62 [0·569-0·679] (p<0·001), 0·75 (0·692-0·816) respectively (Figure

2).

prevailed in more recent years. Raw scatterplots of macrophages and smooth musussclclle ee cececellllllss s asasas wwel

as semi quantitative data on collagen and smooth muscle cells are depicted in eFigure 1.

ThThTheee asasssoociciciaatation between plaque characterereriistttics and year oof f f surgrggererery y was assessed after

addjuuusts ment forr ppatatiientntt andndnd ppprororoceceduduurraral chchharacccteeeristtticcs thhhaatat cchahahangngeeded oveverrr ttiimemee (((agage,e, iindndndeexx eeeveventntnt,,

dedelalalay y y bebeb twtwweeeeeenn evevveennt t aananddd susurgrggererryyy, mmmededicici atatatioioion n usususe)e)e) aanandd d trtrradadadiiitioioonananal l riririsksks fffaccctotorsrsrs ttthahah tt t ararare kknknowowo nnn totoo

be associated d wiwiwiththh aaathththerererososclclclerererottticicic dddisisi eaaasesese ppprororogrgrgresesssisis onono ((prprpresesesenenencecece oof f f cococontntntrararalalalateterararal l l ininnteteternrnr al carotid

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Step-wise introduction of these patient characteristics and use of medication to the

regression model with either atheromatous plaques, plaque thrombosis and calcifications as an

outome revealed that none of these factors could be considered as confounding variables (change

in beta <10%). The Supplemental Table 2 shows the odds ratios of the association between

plaque and year of inclusion, following a step-wise correction for potential confounders.

Supplemental Table 3 shows the adjusted odds ratio for subgroups without statin use, blood

pressure medication and smoking. Table 4 depicts the time dependent changes in plaque

characteristics for each clinical presentation before surgery. The data reveal that the temporal

changes in plaque characteristics were consistent among these subgroups. During 3 years of

follow-up, event rates for composite cardiovascular endpoints remained similar. Of those

included in 2002-2003, 31·7% had experienced a major cardiovascular endpoint within 3 years

compared to 26·4% of those included in 2008 (Kaplan Meier plots are shown in eFigure2).

Discussion

The current study shows time dependent changes towards more plaques that show features

currently believed to be associated with plaque stability in carotid endarterectomy patients. This

coincided with more favorable cardiovascular risk factor levels and more prescription of

medications. Our data do not prove that more favorable risk factor levels or medication in the

last decade caused the time dependent changes in plaque characteristics. We can only conclude

that risk factors and plaque composition improved concurrently over the years.

Changes in cardiovascular disease morbidity and mortality in populations over decades

have been documented.1,2,4,5 For both, stroke and coronary heart disease favorable time trends in

incidence and mortality have been reported in high-income countries.1,2,4,5 These temporal

follow-up, event rates for composite cardiovascular endpoints remained similar. OfOfOf tthohohoseese

ncluded in 2002-2003, 31·7% had experienced a major cardiovascular endpoint within 3 years

coompmpmparararededed ttto oo 2226·4·44%%% of those included in 2008 (KKKapapa llan Meier plottsss are e shshshowo n in eFigure2).

DiDiiscscscususussisis ononn

The current ststtudududy y y shshshowowws ss tiiimememe ddepepepenenndeded ntntn ccchahahangngngeseses tototowaww rdrdrds s momomorerere ppplaaaquququeseses ttthahahat t shshhowowow fffeaeae tures

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changes are thought to be related to more favorable risk factor profile in populations both before

and after the event. Our study shows similar trends in risk factor levels and medication

prescription in carotid endarterectomy patients from 2002 to 2011. We observed a decrease in

total cholesterol, improved mean arterial pressure and a decrease in the prevalence of smoking in

more recent years. Also, an increase in the prescription of statins and angiotensin II receptor

antagonists in more recent years was observed. Controversially, the lower number of plaques that

show features currently believed to be associated with plaque instability together with a more

favorable risk factor level and increased medication did not coincide with a shift towards less

severe events before the patients were scheduled for carotid endarterectomy in our carotid

endarterectomy population. On the contrary, the inclusion of asymptomatic patients in our

cohort even decreased over time. In addition, the more recent included carotid endarterectomy

patients demonstrated similar event-free survival in the 3-year follow-up after surgery compared

to patients included in the early years of the Athero-Express study. These results show that

primary prevention measures may have improved the last decade coinciding with plaques that

show features currently believed to be associated with plaque stability. Yet, this did not translate

into a reduced event rate in patients with severe carotid artery disease. The time dependent

changes in plaque characteristics without subsequent reduction in inclusion rate or secondary

manifestations of atherosclerotic disease is of interest since it might challenge the current

concept that the unstable vulnerable plaque is the major determinant of outcome in patients

suffering from carotid artery disease.

Multiple studies suggest that the stroke population is changing in terms of severity of the

disease and stroke subtypes.18-21 A registry study spanning a 25-year time period shows that

stroke patients were less likely to suffer from large artery disease with and without significant

endarterectomy population. On the contrary, the inclusion of asymptomatic patitieeentststs iinn n ououourr r

cohort even decreased over time. In addition, the more recent included carotid endarterectomy

paatitiienenentststs dddemememooonststtrararateted similar event-free survivaaal l l ininn the 3-year foollllloww-u-uupp p after surgery compared

oo pppaatients inclcludududeedd iiinn n thhhe e eaeaearlrlrly y yeyeearararss ofoff the AAAtherroro--Expxpxpreresss sstutuddydy. ThThheesesee rereesusuultltss shshshowowo thahahat t

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stenosis.19 In the Netherlands, a remarkable decline in fatal ischemic stroke has been observed in

the last twenty years.18 This trend was not only observed following stroke in the Netherlands but

also for coronary heart disease.22

Our data support the idea that the underlying mechanism explaining cerebral

manifestations of atherosclerotic disease may be changing. Inevitably, our findings question the

validity of the vulnerable plaque hypothesis in this population. The carotid endarterectomy

patients in our study were being referred to the hospital with major events such as stroke, also in

the more recent years. Yet, their atherosclerotic plaque removed during surgery show features

currently believed to be associated with plaque stability. Although the symptomatic plaques

revealed more stable characteristics in recent years, we did not observe secular trends in the

proportion of patients with contralateral internal artery stenosis. This suggests that the observed

changes in composition of atherosclerotic plaques come without changes in the extent of

atherosclerotic disease.

Changes in patient demographics over time included increasing age, decreasing delay

between surgery and the last event, and changes in symptomatic status since we included less

asymptomatic patients. These changes in our population even strengthen the validity of our

findings. The older age of patients undergoing carotid endarterectomy during the inclusion

period might have several explanations. One might hypothesize that patients suffer from stroke at

an older age, as progression of atherosclerotic disease has been slowed down due to successful

improvements in primary prevention.23 On the other hand, overall prognosis and life expectancy

of individuals is improving, thereby allowing selection criteria for carotid revascularization to be

extended. Finally, elderly patients seem to have increased risks during carotid stenting as

compared with younger patients.24-27 These results from large randomized trials over the past

evealed more stable characteristics in recent years, we did not observe secular trtrrenenenddsd iin n thththeee

proportion of patients with contralateral internal artery stenosis. This suggests that the observed

chhananangegegess ininin ccooompopopossisitit on of atherosclerotic plaqueuees s cccome without ccchah ngnggeseses in the extent of t

attheeeror sclerotic c didd sseeasaseee.

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between surgrggererery y y ananandd d thththe ee laaaststs eeeveveentntn ,, anaa d d d chchhananangegeges s ininin sssymymy ptptptomomomatatticicic sstaaatututusss sisisincncnce e e wewewe iincncncluluuded less

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12

years, might have resulted in more frequent referral of elderly to undergo carotid

endarterectomy, instead of carotid stenting. Older age has been reported to be associated with

plaque characteristics currently believed to be associated with plaque instability.28,29

Controversially, in our study the plaques showed more features considered to reflect stable

plaques despite the fact that the patients became older at inclusion. Also, the delay between the

last event and surgery has decreased significantly, because timing of carotid endarterectomy is

essential in the prevention of secondary stroke.30 This has resulted in adjustments of guidelines

and, in our experience, shortening in referral delay. It has previously been shown that plaques

from patients operated on shortly after stroke reveal more inflammatory and thus unstable

characteristics, including macrophages and inflammatory proteins.31,32 Based on these previous

observations one would expect a higher prevalence of plaques with more features associated with

plaque instability in patients undergoing carotid surgery. Nevertheless, we observed a

stabilization of plaques in the last decade suggesting that the plaque differences over time are not

driven or confounded by shortening of the timing of surgery. Carotid endarterectomy for

asymptomatic individuals has been debated for long, and especially in recent years due to

improvements in medical treatment. Indications for surgery have likewise been changed over the

past decade, and a conservative approach for most asymptomatic patients is now proposed by

neurologists and vascular surgeons. Plaques from asymptomatic patients have a more fibrous and

lees inflammatory phenotype as compared with TIA and stroke patients.33 We observed more

plaques with more fibrotic and less inflammatory features whilst the proportion of asymptomatic

patients was lower. Furthermore, our findings is supported by our subgroup analyses that shows

that the time dependent changes in plaque characteristics were very consistent in the well-

defined 4 patient groups presenting with either a stroke, transient ischemic attack, ocular

characteristics, including macrophages and inflammatory proteins.31,32 Based on n thtthesee ee prprprevevevioioiousu

observations one would expect a higher prevalence of plaques with more features associated with

pllaqaqaqueueue iiinsnsstatatabibib litytyy iiinn patients undergoing carotid d susus rrrgery. Nevertheheelesss, , wwwe observed a

ttabbbili ization ofof ppplaaaquueees iiinn thththeee llalastst ddeececaadadee suuggggestiinngg thahahatt ththhe plplaaaquueue dddififi fefeererencncnceses ooovevever r r tititimememe aarrre not

drdrivivivenenn oorr cococonfnnfououundnddeded bbyy y shshororrtetenininingngng ooff f thhhee e tititimimimingngng ooff ssururu gegegeryryry. CCCaararotototididi eeenndndararrtetet rererectcttomomomy y fofoor r

asymptomaticicc iindndndivivivididi uauauals hhhasasas bbbeeeeeen n dedd bababateteted d d fofofor r r loloongngng, , , anannd d d esesespepepecicicialaa lyyy iin n n rererecececentntn yyyeaeaearsrsrs dddueu to

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13

symptoms or those who were asymptomatic. We have performed several analyses in which we

were unable to detect variables that confounded the relation between inclusion year and plaque

phenotype.

Limitations

Bias in the study of time dependent trends may occur when for example the diagnosis of the

disease has changed. Changes in detection of the disease may result in the phenomena like Will

Rogers (an increased incidence of the disease and lower rates of case-fatality).34 We did not

observe a decline in the total number of patients included or a shift towards less symptomatic

patients in the Athero-Express study. However, we cannot exclude that our observations over

time could be due to the differences in patient characteristics and indications for surgery that

have occurred over this same interval. The observational nature of this study merits careful

consideration and before making causal inferences this observation requires serial imaging

plaque characterization measurements in patients. In addition, the awareness among patients that

mild and transient symptoms may be due to a minor stroke or a transient ischemic attack may

have been improved. For example, as in many countries, repetitive educational campaigns were

raised in the Netherlands by the Dutch Heart Foundation in the late 1990s focusing on

recognition of early signs and symptoms of stroke and transient ischemic attacks. This may have

led to the aforementioned inclusion of less-severe diseased patients in our study with

concomitant different plaque characteristics. In part of the histological analyses we applied

arbitrary cut off values to categorise plaque phenotypes. This may have influenced our

observations. However, also computerized quantitative measurements such as CD68 revealed a

consistent change towards plaque stabilization over time. In addition, the interpretation of plaque

patients in the Athero-Express study. However, we cannot exclude that our obsererrvavavatitit ononns s ovovoverere t

ime could be due to the differences in patient characteristics and indications for surgery that

haaveveve ooocccccurururrerer ddd ovovvererer this same interval. The obsererrvavav ttional nature ofoff thiiss stststudu y merits careful rrr

coonnsnsideration anand d bebebefofoforeee mmmakakakiiingng cccauauausaal infeeereeencees tthisss oobobsseerrvavattiioonn rreqeqquuiireress s sseseririalalal iiimamam giginngng

plplaqaqaqueueue cchahaarararactctererizizatata ioioonn memem asssururu emememenenentsts iin nn papapattienenentsts.. IInn aaaddddditititiioion,n,n, ththhe awawawaararenennesessss amamamononongg g papaatiienentstss tthhahat

mild and transnssieieientntt sssymymymptptp ommms s s mamamay y y bebebe dddueueu ttto o o a a a mimim nononor r r sts rorookekeke ooor r a a a trtrt annnsisisienenent t t isisischchc emememicicic aaattttttaca k may

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characteristics has been performed by the same dedicated technician over the entire study period

to ensure continuity. The scoring system that was used has previously been validated by an

additional observer and an independent pathologist.12 Although we cannot fully exclude that

changes over time are caused by misinterpretations we feel that the strong consistency in the data

and different stains support our view that carotid symptomatic lesions show stabilization features

over the last decade.

In conclusion, over the past decade, atherosclerotic plaques harvested during carotid

endarterectomy show a time dependent change in plaque composition characterized by a

decrease in features currently believed to be causal for plaque instability. This seems to go hand

in hand with improvements in risk factor management.

Acknowledgments: Drs. Van Lammeren, Den Ruijter and Pasterkamp had full access to all of

the data in the study and take responsibility for the integrity of the data and the accuracy of the

data analysis. Role of the sponsor: The funding sponsor was not involved in the design and

conduct of the study; collection, management, analysis and interpretation of the data; and

preparation, review, or approval of the manuscript. Author Contributions: Drs Van Lammeren,

Den Ruijter and Pasterkamp had full access to all the data in the study and take responsibility for

the integrity of the data and the accuracy of the data analysis. Study concept and design: Van

Lammeren, de Kleijn, Moll, Pasterkamp. Acquisition of data:, Van Lammeren, Vrijenhoek, van

der Laan, De Borst, De Vries. Analysis and interpretation of the data: Van Lammeren, Den

Ruijter, van der Laan, Vrijenhoek, Velema, Vink. Drafting of the manuscript: Van Lammeren,

Den Ruijter. Critical revision of the manuscript for important intellectual content: Pasterkamp,

Moll, Vink, De Vries, de Kleijn, De Borst, Bots. Statistical analysis: Van Lammeren, Den

Ruijter, Bots. Obtained funding: Pasterkamp, Moll, de Kleijn. Administrative, technical or

material support: Vrijenhoek, Velema. Study supervision: Pasterkamp

Funding Sources: This project was funded by the University Medical Center Utrecht.

n hand with improvements in risk factor management.

Acknknnowowowleleledgdgdgmementntntss: Drs. Van Lammeren, Den RRRuiuiuijtjj er and Pasterkammppp hahh d full access to all of

hhhee dddatat in thhheee sttudududy y anannd d d tatatakekk rresesespopoponsnsibibililitty y y fofor ththe iinteegrgrgrititityy y of ttthhehe ddata aa aaandn tthehee aaaccccc ururacacyy y ofoff tttheheh

dadad taaa analysis. Roooleee of f ththhe sppononnsos r:: TTThehee fffundddinnng spopoponnsoror wwwaaas nnott innnvolvvvedd ininn ttthehe ddeeesigigi nnn aannd drr

coondndnducucuctt ofoff tttheheh ssttutuddydy;; cocolllleectitiiononon, mamamananagegegemememenntnt, , anana aalalyysysiisis aaandndnd intntntererrprprpretetetaatitiionnn oof f f ththhee dadadattata;; aanandd d

prprprepepeparararatatatioioionnn, rrrevevevieieiewww, ooorrr aaapppppprororovavavalll ofofof ttthehehe mmmanananusususcrcrcripipipttt. AuAuAuthththororor CCCononontrtrtribibibutututioioionsnsns::: DrDrDrsss VaVaVannn LaLaLammmmmmerererenenen,

DeDenn RuRuijijteterr anandd PaPaststererkakampmp hhadad ffululll acaccecessss ttoo alalll ththee dadatata iinn ththee ststududyy anandd tatakeke rresespoponsnsibibililitityy foforr

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Conflict of Interest Disclosures: None.

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2525555555.

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Table 1. Patient characteristics over time.

Patient characteristics 2002-2003 (n=250)

2004-2005 (n=408)

2006-2007 (n=352)

2008-2009 (n=332)

2010-2011 (n=271)

P value

Age, mean (±SD) 66·4 (8·50) 68·3 (9·3) 68·7 (9·2) 69·7 (8·9) 69·9 (10·1) <0·001 Gender, male, % 69·2 70·3 67·9 67·2 67·2 0·357 Smoking, % 45·6 33·1 33·8 30·4 32·1 0·003 Diabetes, % 21·2 22·1 25·6 19·3 22·1 0·893 Mean arterial pressure, mmHg (±SD) 107·9 (±14·7) 107·9 (±15·6) 107·2 (±16·3) 107·7 (±16·9) 104·5 (±15·1) 0·023 Total cholesterol, mmol/ml (±SD) 5·2 (±1·1) 4·7 (±1·2) 4·4 (±1·1) 4·4 (±1·2) 4·9 (±1·2) 0·002 eGFR, ml/min/1·73m2 (±SD) 71·5 (±18·6) 70·3 (±19·8) 72·7 (±22·8) 71·3 (±20·2) 73·5 (±19·9) 0·157 Body mass index, kg/m2 (±SD) 26·4 (±3·8) 26·2 (±3·9) 26·5 (±3·7) 26·1 (±3·4) 26·7 (±4·6) 0·525 Time since last cerebrovascular event, days, median [IQR] 111 [54-159] 65 [26-121] 44 [18-72] 30 [13-53] 20 [11-35] <0·001 Index event <0·001 Asymptomatic, % 21·2 17·2 13·9 10·8 9·2 Ocular, % 14·8 12·5 14·8 16·9 20·7 Transient ischemic attack, % 40·0 45·3 50·9 44·6 43·2 Stroke, % 24·0 25·0 20·5 27·7 26·9 Contralateral internal carotid stenosis > 50% 46·0 41·2 47·4 45·2 51·7 0·072 History of peripheral arterial disease 26·0 19·6 19·3 22·0 17·7 0·115 History of coronary arterial disease 37·2 28·7 31·5 30·7 29·2 0·188 Abbreviations: SD, standard deviation; IQR, interquartile range. P-value

cholesterol, mmol/ml (±SD) 5·2 (±1·1) 4·7 (±1·2) 4·4 (±1·1) 4·4 (±1 2·2))) 4·4·4 999 (±(±(±1·1·1 2)2)2), ml/min/1·73m2 (±SD) 71·5 (±18·6) 70·3 (±19·8) 72·7 (±22·8) 71·3 ±(± 0202 ·22) ) ) 73737 ·55·5 (((±1±1±19·9·9)9)mass index, kg/m2 (±SD) 26·4 (±3·8) 26·2 (±3·9) 26·5 (±3·7) 26·1 (±3±3 4·4·4))) 262626·77·7 ((±4±4± ·6·66) ) )since last cerebrovascular event, days, median [IQR] 111 [54-159] 65 [26-121] 44 [18-72] 30 [13-53] 20 [11-35] event ptomata icic, , % % 21·2 17·2 13·9 10·8 9·2 r, , % % % 14·8 12·5 14·8 16·9 20·7 ii nenent isisischchemic aattttacacack, %% 40·0·0 445·5 33 505 9·9 444·4·66 43·2 ee, % 2422 00·0 2225·5·5·00 0 202020·5·5 22277·7·777 262626·9·9 aala eeteraral internal carot didid stet nossisi >>> 50%0% 446·000 414141·2·2 447·4 4545·22 511·7 77rryyy ffof pperipheral arartett ri lala ddisi ea ees 2622 00·0 1119·9·9·66 19·3 222·2 0 17 77·7ryy ofofof ccorororononarary y ararartetet ririalal dd ssiseaeasese 3733 22·2 228·8·8 77 313131·5·5· 3330·0 77 2929 2·2·2viatiionons:s: SSSDDD, sstatat ndnd rard d ddede ivivi tatatioioi n;n; IIIQRQRQR, ininterqrquauartrtrtililile e e rarange. PP v-v-valalalueue

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Table 2. Preoperative medication prescription over time.

Prescribed preoperative medication 2002-2003 (n=250)

2004-2005(n=408)

2006-2007(n=352)

2008-2009(n=332)

2010-2011(n=271)

P value

Statins, % 64·8 73·3 81·2 78·6 79·3 <0·001 Betablockers, % 46·0 47·5 44·3 46·7 42·8 0·417 ACE-inhibitors, % 30·0 34·1 33·0 26·8 33·2 0·707 Angiotensin II antagonists, % 18·4 18·1 22·2 24·1 26·2 0·004 Diuretics, % 31·2 35·0 35·8 38·3 33·6 0·388 Aspirin, % 87·6 85·5 84·4 83·7 84·9 0·289 Oral anticoagulants, % 14·4 11·8 13·6 12·0 9·2 0·137

tics, % 31·2 35·0 35·8 38·3 33333·6·66 00·38in, % 87·6 85·5 84·4 83·7 84844·9·9 00·28anticoagulants, % 14·4 11·8 13·6 12·0 99·2·2 000·1·1333

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Table 3. Presence of atherosclerotic plaque components over time.

Plaque Composition 2002-2003 (n=250)

2004-2005(n=408)

2006-2007(n=352)

2008-2009(n=332)

2010-2011(n=271)

P value

Atheromatous plaque (lipid core > 40% of plaque surface), % 33·2 36·0 26·7 21·1 14·4 <0.001

Plaque thrombosis, % 74·4 75·5 62·5 49·1 37·6 <0.001

Intraplaque vessels, median [IQR] 6·7[4·3-10·4]

8·5[5·7-12·3]

7·7[4·7-11·9]

7·3[3·7-12·9]

6·3[2·3-11·7] 0.436

Collagen rich, % 73·6 85·8 80·1 76·8 78·6 0.643 Median % macrophage staining per plaque area [IQR]

0·41[0·09-1·30]

0·46[0·08-1·23]

1·04[0·46-1·96]

0·27[0·10-0·64]

0·09[0·01-0·51] <0.001

Median % smooth muscle cell staining per plaque area [IQR]

2·03[0·73-4·34]

1·08[0·34-2·65]

2·19[1·00-3·75]

1·23[0·46-2·21]

0·68[0·13-1·99] <0.001

Calcified plaque, % 52·0 62·0 59·7 51·2 24·0 <0.001 Abbreviations: IQR, interquartile range.

plaque vessels, median [IQR] [4·3-10·4] [5·7-12·3] [4·7-11·9] [3·7-12·9] [2[22·3·3·3-1-11·1·7]7] 0.43

gen rich, % 73·6 85·8 80·1 76·8 778·8·8 666 0.0 64an % macrophage staining per plaque area ]

0·41[0·09-1·30]

0·46[0·08-1·23]

1·04[0·46-1·96]

0·27[0·10-0·64]

0·0·0 09099[0·01-0·51] <00.0

an % smooth muscle cell staining per plaqueIQR]R]

2·03[0·73-4·34]]

1·08[0[0[0·34-2·65]

2·19[1·00-3·75]

1·23[0[0[0·46-2·21]

0·68[0·13-1·99] <0.0

ffieied d d plplplaqaque, %%% 52·0 62·0 59·9·7 77 51·2 24·0 <0.0vviaiai tionono s: IQR, intterqrqrquaartrtililileee rarangnge.e.e.

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Table 4A. Carotid plaque composition of asymptomatic patients

Plaque Composition 2002-2003 (n=53)

2004-2005(n=70)

2006-2007(n=49)

2008-2009(n=36)

2010-2011(n=25)

P value

Atheromatous plaque (lipid core > 40% of plaque surface), % 24·5 22·9 26·5 13·9 0·0 <0·001

Plaque thrombosis present % 62·3 75·7 53·1 41·7 16·0 <0·001

Intraplaque vessels (n/hotspot), median [IQR] 7·0[4·2-9·0]

8·7[6·6-12·8]

7·3[4·6-11·0]

6·5[2·6-14·9]

6·0[3·2-12·4] 0·255

Collagen rich plaques % 79·2 90·0 83·7 80·6 84·0 0·968 Median area of macrophages as % of plaque area [IQR]

0·41[0·12-1·22]

0·40[0·05-1·29]

0·91[0·41-1·65]

0·20[0·07-0·49]

0·04[0·00-0·19] 0·003

Median area of smooth muscle as % of plaque area [IQR]

2·96[0·94-5·26]

1·58[0·70-3·12]

2·14[1·09-3·81]

1·39[0·33-2·17]

2·06[0·98-3·68] 0·001

Calcified plaque, % 62·3 70·0 75·5 61·1 24·0 0·011

Table 4B. Carotid plaque composition of patients with ocular symptoms

Plaque Composition 2002-2003 (n=37)

2004-2005(n=51)

2006-2007(n=52)

2008-2009(n=56)

2010-2011(n=56)

P value

Atheromatous plaque (lipid core > 40% of plaque surface), % 21·6 31·4 25·0 19·6 5·4 0·007

Plaque thrombosis present % 83·8 66·7 53·8 44·6 37·5 <0·001

Intraplaque vessels (n/hotspot), median [IQR] 7·0[4·5-10·4]

8·7[5·0-10·7]

7·5[4·1-12·5]

9·0[2·3-13·6]

6·0[2·7-9·1] 0·660

Collagen rich, % 78·4 90·2 84·6 78·6 80·4 0·506 Median area of macrophages as % of plaque area [IQR]

0·49[0·05-1·36]

0·41[0·07-1·22]

0·84[0·39-1·56]

0·32[0·11-0·89]

0·11[0·01-0·44] 0·001

Median area of smooth muscle cells as % of plaque area [IQR]

3·03[0·80-5·98]

1·69[0·60-3·47]

3·05[1·44-4·93]

1·23[0·58-2·41]

0·59[0·07-2·16] <0·001

Calcified plaque, % 59·5 68·6 69·2 44·6 19·6 <0·001

rororomamamatototoususus ppplalalaquququeee (l(l(lipipipididid cccorororeee >> 404040%%% ofofof ppplalalaquququeee ce), %

ueue CComomo pop siititiononon 2200002-2-2 00200303 (n(n(n=3=3=37)7)7)

20202 4404 22-20000555(n(n( =5=5=51)1)1)

2020 6060 22-20000077(n(n=5=5=52)2)2)

[4 2-9 0] [6 6-12 8] [4 6-11 0] [2 6-14 9] [3[3 22-1-12 4]4]gen rich plaques % 79·2 90·0 83·7 80·6 8884·4·4·000 0·0 96an area of macrophages as % of plaque area ]

0·41[0·12-1·22]

0·40[0·05-1·29]

0·91[0·41-1·65]

0·20[0·07-0·49]

0·0·0 040404[0[00·000-0·0 191 ] 0·0·0 00000

an area of smooth muscle as % of plaque area ]

2·96[0·94-5·26]

1·58[0·70-3·12]

2·14[1·09-3·81]

1·39[0·33-2·17]

2·06[0·98-3·68] 0·00

fied d plplplaqaqaqueueue, , , %%% 62·3 70·0 75·5 61·1 24·0 0·01

e e BB4B.. Carotid plaqqueue comomo popositiitionon oof f f papap titienentststs www tith h ocoo ul rrar symyy tpt mmoms

20202 80808-2-200000 99(n(n( 5=5=56)6)6)

02020 010-2- 01010 11(n(nn=5=5=56)6)6)

PP P val

2121 6·6 331·1·4 4 2525·0·0 1919 6·6 55 4·4 00·0·000

ueue tthrhromombobosisiss prpresesenentt %% 8383·8·8 666·6·77 5353·8·8 4444·6·6 337·7·55 <0<0 0·0·0

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Table 4C. Carotid plaque composition of TIA patients

Plaque Composition 2002-2003 (n=100)

2004-2005(n=185)

2006-2007(n=179)

2008-2009(n=148)

2010-2011(n=117)

P value

Atheromatous plaque (lipid core > 40% of plaque surface), % 41·0 36·2 27·4 18·9 19·7 <0·001

Plaque thrombosis present, % 73·0 76·8 65·9 47·3 37·6 <0·001

Intraplaque vessels (n/hotspot), median [IQR] 6·7[4·3-11·0]

8·2[5·7-12·9]

8·0[4·7-12·0]

7·3[4·1-12·4]

6·0[2·3-12·2] 0·212

Collagen rich, % 66·0 84·9 77·1 75·0 77·8 0·664 Median area of macrophages as % of plaque area [IQR]

0·45[0·11-1·28]

0·48[0·09-1·15]

1·15[0·49-2·22]

0·26[0·10-0·62]

0·11[0·01-0·63] <0·001

Median area of smooth muscle cells as % of plaque area [IQR]

1·56[0·39-3·31]

0·89[0·30-2·34]

2·06[0·89-3·48]

1·21[0·47-2·50]

0·57[0·14-1·77] 0·008

Calcified plaque, % 48·0 58·9 53·6 47·3 23·9 <0·001

Table 4D. Carotid plaque composition of stroke patients

Plaque Composition 2002-2003 (n=60)

2004-2005(n=102)

2006-2007(n=72)

2008-2009(n=92)

2010-2011(n=73)

P value

Atheromatous plaque (lipid core > 40% of plaque surface), % 35·0 47·1 26·4 28·3 17·9 0·001

Plaque thrombosis, % 81·7 77·5 66·7 57·6 45·2 <0·001

Intraplaque vessels (n/hotspot), median [IQR] 6·8[5·0-12·4]

8·7[5·0-12·4]

7·3[3·0-11·5]

7·3[3·0-12·0]

6·7[1·7-12·0 0·704

Collagen rich % 78·3 82·4 81·9 77·2 76·7 0·479 Median area of macrophages as % of plaque area [IQR]

0·35[0·04-1·41]

0·44[0·08-1·38]

1·03[0·46-2·33]

0·33[0·10-0·81]

0·09[0·01-0·42] <0·001

Median area of smooth muscle as % of plaque area [IQR]

1·93[0·91-4·10]

0·96[0·21-2·46]

2·07[0·87-3·81]

1·24[0·37-1·97]

0·72[0·08-1·55] <0·001

Calcified plaque % 45·0 58·8 56·9 57·6 27·4 0·031

eueu CCComo positionon 2200 -2-2 002003 (n(n=6== 0)0)0

2022 044-2-2-2000000555(n( =1== 02020 ))

20 6060 22-2007nn(n 7=7=72)2))

2022 80808 22-20009(n(n=9== 2)2)2

0020 010-2- 0100 1

[4 3-11 0] [5 7-12 9] [4 7-12 0] [4 1-12 4] [22 33-1-122 2]2]gen rich, % 66·0 84·9 77·1 75·0 77777 ·8·88 000·66an area of macrophages as % of plaque area ]

0·45[0·11-1·28]

0·48[0·09-1·15]

1·15[0·49-2·22]

0·26[0·10-0·62]

0·0·0 111111[0[00·001-00·636 ] <0<0<0·0·00

an area of smooth muscle cells as % of plaqueIQR]

1·56[0·39-3·31]

0·89[0·30-2·34]

2·06[0·89-3·48]

1·21[0·47-2·50]

0·57[0·14-1·77] 0·00

fied d plplplaqaqaqueueue,,, %%% 48·0 58·9 53·6 47·3 23·9 <0·0

e e DD4D.. Carotid pplaalaququueee cococompmppososositititioioionn ofofo sstrtrtrokokoke e e papap titiienenentststs

(n( =7== 3)3)3P PP val

romamatototoususus ppplalalaquque (l(lipipipididid cccorororee e >>> 404040%% ofof ppplalalaququque eccce)e)e),, %%% 3535·0·0 447·7 11 626·4·4 2828·33 117·99 0·0·000

ue thrombosis, %%% 818181··777 7777·555 6666·6 7 77 575757·6 6 4544 ·2 <0·00066·88 88·77 77·33 77·33 66·77

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Figure Legends:

Figure 1. Semi-quantitative plaque information on intraplaque hemorrhage and luminal

thrombus (A), fat (B), macrophages (C) and calcification (D) based presence and/or degree of

staining.

Figure 2. Adjusted odds ratios for the presence of atherosclerotic plaque characteristics, per 2

year increase from 2002-2011. Odds ratios and p values were adjusted for age, gender, smoking,

total cholesterol, time since last event, index event, statin use, angiotensin II antagonist use, oral

anticoagulant use, clopidogrel use and dipyridamol use. anticoagulant use, clopidogrel use and dipyridamol use.

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Figure 1

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Figure 2

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L. Moll, Michiel L. Bots and Gerard PasterkampVelema, Jean-Paul P. M. de Vries, Dominique P. V. de Kleijn, Aryan Vink, Gert Jan de Borst, Frans

Guus W. van Lammeren, Hester M. den Ruijter, Joyce E. P. Vrijenhoek, Sander van der Laan, EvelynCarotid Surgery

Time Dependent Changes in Atherosclerotic Plaque Composition in Patients Undergoing

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2014 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation published online March 17, 2014;Circulation. 

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SUPPLEMENTAL MATERIAL

Supplemental Table 1

Variables used in this study % missingness

Age 0

Gender 0

Smoking 4.9

Diabetes 1.7

Mean arterial pressure 17.5

Total cholesterol 31.5

eGFR 4.3

Body mass index 8.7

Time since last cerebrovascular event 6.6

Index event 0.7

Contralateral stenosis 8.9

History of peripheral artery disease 2.0

History of coronary artery disease 2.0

Statins 3.1

Beta-blockers 3.1

ACE-inhibitors 3.3

Angiotensin II antagonists 3.4

Diuretics 3.1

Aspirin 3.2

Oral anticoagulants 3.1

Supplemental Table 2: Odds ratios between year of inclusion and plaque phenotype after addition of

potential confouders (bold).

Odds ratio (95% CI) between year

of inclusion (per 2 year increase in

time) and

Plaque

thrombosis

Atheromatous

plaque

Calcified

plaque

Univariate 0.64 (0.59-0.69) 0.76 (0.70-0.83) 0.76 (0.71-0.82)

Age 0.63 (0.58-0.69) 0.74 (0.68-0.81) 0.75 (0.69-0.81)

Age, gender 0.63 (0.58-0.69) 0.74 (0.68-0.81) 0.75 (0.69-0.81)

Age, gender, smoking 0.63 (0.58-0.69) 0.74 (0.68-0.81) 0.75 (0.69-0.81)

Age, gender, smoking, total

cholesterol

0.63 (0.58-0.69) 0.74 (0.67-0.81) 0.74 (0.68-0.80)

Age, gender, smoking, total

cholesterol, time since last event

0.63 (0.58-0.69) 0.73(0.67-0.80) 0.74 (0.69-0.80)

Age, gender, smoking, total

cholesterol, time since last event,

index event

0.63 (0.57-0.68) 0.73 (0.66-0.80) 0.74 (0.69-0.80)

Age, gender, smoking, total

cholesterol, time since last event,

index event, statin use

0.63 (0.58-0.68) 0.73 (0.67-0.80) 0.75 (0.69-0.81)

Age, gender, smoking, total

cholesterol, time since last event,

index event, statin use, angiotensin

II antagonist use

0.63 (0.58-0.69) 0.73 (0.67-0.80) 0.75 (0.69-0.81)

Age, gender, smoking, total

cholesterol, time since last event,

index event, statin use, angiotensin II

antagonist use, oral anticoagulant

use

0.63 (0.58-0.69) 0.73 (0.67-0.80) 0.75 (0.69-0.81)

Age, gender, smoking, total

cholesterol, time since last event,

index event, statin use, angiotensin II

antagonist use, oral anticoagulant

use, clopidogrel use

0.63 (0.58-0.69) 0.74 (0.67-0.81) 0.76 (0.70-0.82)

Age, gender, smoking, total

cholesterol, time since last event,

index event, statin use, angiotensin II

antagonist use, oral anticoagulant

use, clopidogrel use, dipyridamol

use

0.63 (0.57-0.69) 0.74 (0.67-0.81) 0.76 (0.70-0.82)

Supplemental Table 3. Subgroup analysis, stratified for statin use, blood pressure medication and

smoking status.

Adjusted odds ratios for the presence of atherosclerotic plaque characteristics, per 2 year increase from 2002-2011. Odds ratios and p

values were adjusted for age, gender, smoking, total cholesterol, time since last event, index event, statin use (expect in the subgroup

analyses), angiotensin II antagonist use, oral anticoagulant use, clopidogrel use and dipyridamol use.

Statin No statin

Plaque thrombosis, OR

(95% CI) 0.65 (0.58-0.72) 0.58 (0.48-0.70)

Atheromatous plaque,

OR (95% CI) 0.77 (0.69-0.86) 0.65 (0.54-0.79)

Calcified plaque

OR (95% CI) 0.75 (0.68-0.82) 0.82 (0.69-0.96)

Blood pressure

medication

No blood pressure medication

Plaque thrombosis,

OR (95% CI) 0.54 (0.44-0.66) 0.65 (0.59-0.72)

Atheromatous plaque,

OR (95% CI) 0.65 (0.51-0.82) 0.76 (0.69-0.85)

Calcified plaque

OR (95% CI) 0.61 (0.50-0.74) 0.81 (0.73-0.89)

Smoker Non-smoker

Plaque thrombosis,

OR (95% CI) 0.64 (0.55-0.74) 0.60 (0.50-0.74)

Atheromatous plaque,

OR (95% CI) 0.78 (0.66-0.92) 0.72 (0.64-0.81)

Calcified plaque

OR (95% CI)

0.76 (0.66-0.87) 0.71 (0.68-0.83)

Supplemental eFigure1.

Panel A and C show raw scatter plots of the % of macrophages and smooth muscle cells in plaques over the years. The % of plaques that fall

into categories of semi-quantitative staining (no, minor, moderate or heavy) are depicted in panel B for collagen and panel D for smooth

muscle cells.

Supplemental eFigure2.

Kaplan Meier survival curves of the patients included in the Athero-Express, stratified by inclusion year.