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Atherosclerosis 213 (2010) 21–29

Contents lists available at ScienceDirect

Atherosclerosis

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eview

arotid plaque formation and serum biomarkers

inda Hermusa, Joop D. Lefrandtb, René A. Tioc, Jan-Cees Breekd, Clark J. Zeebregtsa,∗

Department of Surgery (Division of Vascular Surgery), University Medical Center Groningen, University of Groningen, Groningen, The NetherlandsDepartment of Internal Medicine (Division of Vascular Medicine), University Medical Center Groningen, University of Groningen, Groningen, The NetherlandsDepartment of Cardiology, University Medical Center Groningen, University of Groningen, Groningen, The NetherlandsDepartment of Surgery, Division of Vascular Surgery, Martini Hospital Groningen, Groningen, The Netherlands

r t i c l e i n f o

rticle history:eceived 15 December 2009eceived in revised form 8 May 2010ccepted 10 May 2010vailable online 19 May 2010

eywords:arotid artery

a b s t r a c t

Treatment of carotid artery stenosis by endarterectomy or stenting can significantly reduce stroke risk,but is also associated with surgery related mortality and morbidity. At present it is neither possible toassess whether a carotid plaque will become symptomatic or not, nor to define the time when symptomswill occur. Identification of carotid plaques which confer excess risk of neurologic events is fundamentalin the selection of patients for vascular intervention.

Molecular processes such as inflammation, lipid accumulation, apoptosis, proteolysis, thrombosis andangiogenesis have shown to be highly related with plaque vulnerability. Serum biomarkers reflecting

ulnerable plaqueiomarker

nflammationroteolysisngiogenesis

these processes may distinguish unstable from stable carotid artery stenosis and thus be a powerful toolin the selection of patients for carotid surgery.

Until now, no serum biomarker has qualified for regular clinical use in carotid artery disease. However,several biomarkers, especially markers of inflammatory or proteolytic activity seem to be promising in theidentification of vulnerable carotid plaques. Therefore, it is anticipated that non-invasive risk assessmentin carotid artery disease by determination of serum biomarker levels may play an important future role

in clinical practice improving better selection criteria for vascular intervention.

© 2010 Elsevier Ireland Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221.1. Carotid artery stenosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221.2. Plaque vulnerability and serum biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2. Plaque development processes and serum biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.1. Lipid accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.2. Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.3. Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.4. Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.5. Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.6. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.7. Thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.8. Calcification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Discussion and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: ACS, acute coronary syndrome; AMI, acute myocardial infarction; Angendarterectomy; CCP, cathepsin cystein protease; CRP, C-reactive protein; hsCRP, high-growth factor-1; IL, interleukin; Lp-PLA2, lipoprotein-associated phospholipase A2; LDL, lmetallo-proteinase; MCP-1, monocyte chemo-attractant protein-1; BMP-2, morphogenfactor; PAI-1, plasminogen activator inhibitor-1; PF4, platelet factor 4; SAA, serum amylopathway inhibitor; t-PA, tissue plasminogen activator; TCD, trans cranial Doppler; TNF, tuVEGF, vascular endothelial growth factor; VLDL, very low density lipoprotein.∗ Corresponding author at: Department of Surgery (Division of Vascular Surgery), Univ

he Netherlands. Tel.: +31 503613382; fax: +31 503611745.

021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2010.05.013

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

-1, angiopoietin 1; Ang-2, angiopoietin 2; CAS, carotid artery stenosis; CEA, carotidsensitivity C-reactive protein; HDL, high density lipoprotein; IGF-1, insuline-likeow density lipoprotein; MGP, matrix-carboxyglutamic acid protein; MMP, matrixetic protein-2; OPN, osteopontin; OPG, osteoprotegerin; PlGF, placental growthid A; SA, stable angina; TM, thrombomodulin; TF, tissue factor; TFPI, tissue factormor necrosis factor; UA, unstable angina; u-PA, urokinase plasminogen activator;

ersity Medical Center Groningen, PO Box 30001, 9700 RB Groningen,

22 L. Hermus et al. / Atheroscler

Table 1Characteristics of the unstable plaque.

Unstable plaques Stable plaques

Atheromatous FibrousThin fibrous cap Thick fibrous capLarge lipid core Small lipid coreLess collagen Collagen richNon-calcified CalcifiedUlceration No ulcerationIntraplaque hemorrhage No intraplaque hemorrhage

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Infiltration of inflammatory cells(macrophages)

No infiltration of inflammatory cells

Proteolysis and remodelling No remodelling

. Introduction

.1. Carotid artery stenosis

Stroke is the third leading cause of death worldwide and strokeelated medical costs and disability are high. Approximately 15% oftroke and TIA’s are caused by an unstable carotid artery plaque.reatment of carotid artery stenosis by endarterectomy or stent-ng can significantly reduce stroke risk but is also accompanied byurgery related morbidity and mortality. In addition, not all carotidrtery plaques become symptomatic and result in stroke or TIA’s.herefore, the identification of carotid plaques which confer excessisk of neurologic events is fundamental to the selection of patientsor vascular intervention.

Current selection for carotid artery interventions is mainlyetermined by the grade of stenosis and symptomatology. It isroadly accepted to treat high-grade symptomatic carotid steno-is, but in lower grade (a)symptomatic stenosis interventions aretill a matter of debate. Nevertheless, there is growing awarenesshat stenosis severity alone is a poor guide as to whether the patienthould be treated with an intervention.

.2. Plaque vulnerability and serum biomarkers

Atherosclerotic plaques result from the interaction betweenodified lipids, extracellular matrix, monocyte-derivedacrophages and activated vascular smooth muscle cells that

ccumulate in the arterial wall. Plaque formation is often com-licated by conversion into an acute stage of vessel occlusion orhrombo-embolism evolving from the plaque, resulting in acutelinical complications of myocardial infarction and stroke.

During plaque development, various processes involved inhe progression of the atherosclerotic lesions have shown to bessociated with plaque vulnerability, such as inflammation, lipidccumulation, apoptosis, proteolysis, thrombosis and angiogen-sis. In addition, there are several morphological characteristicshat are related with plaque instability (Table 1) [1]. Advancedtherosclerotic plaques are characterized by a lipid core coveredy a fibrous cap composed of smooth muscle cells and extracel-

ular matrix. Ulcerative changes, cap disruption and intraplaqueemorrhage are preludes to acute atherosclerotic complications.owever, whereas some morphological plaque features may beetected by current imaging methods, further information onolecular processes is only available at post-operative or post-ortem examination of the plaque. In carotid artery plaques, in

ivo identification of processes related to plaque vulnerabilityould be of great advantage in the selection for vascular interven-

ion. Serum biomarkers representing processes of plaque instability

ay play an important role in this selection. In this review we

escribe the pathophysiological processes in carotid plaque forma-ion and summarize the serum biomarkers related to the variousrocesses.

osis 213 (2010) 21–29

2. Plaque development processes and serum biomarkers

2.1. Lipid accumulation

Accumulation of lipids by macrophages and smooth muscle cellsplays a key role in the early stages of atherogenesis. In advancedatherosclerotic plaques, lipid overload leads to cell death and con-tributes to the generation of a lipid core. In atherosclerotic plaques,unstable lesions have shown to have a much greater area occupiedby lipid [2]. In addition, treatment with lipid lowering statins inpatients with cardiovascular risk factors showed 25% proportionalreduction in the first event rate for stroke [3].

Serum markers of lipid metabolism and abnormal lipoproteinprofile are important predictors in atherosclerosis. For example,circulating levels of oxLDL have become a useful biochemical riskmarker for coronary heart disease [4]. In carotid artery disease,oxLDL has also shown to be related to carotid plaque instability[5].

A potential link between the effects of oxLDL and plaqueinstability is lipoprotein-associated phospholipase A2 (Lp-PLA2).Lp-PLA2 is produced by myeloid inflammatory cells and travelsalong with circulating LDL, hydrolyzing oxidized phospholipids inLDL. It is highly expressed in the diseased vessel and induces apro-inflammatory reaction. Epidemiological studies suggest thatLp-PLA2 is a potent predictor of future cardiovascular events [6,7].Recently, treatment with selective inhibitors of Lp-PLA2 in a swinemodel of coronary artery disease resulted in a decrease in plaquesize and necrotic core area and reduced medial destruction [8]. Thisindicates that Lp-PLA2 is a causative agent in the development ofatherosclerotic plaques and that Lp-PLA2 also may be related toplaque instability. In carotid artery disease, symptomatic carotidartery plaques showed a higher expression of Lp-PLA2 comparedto asymptomatic plaques [9]. However, serum values of Lp-PLA2were not determined is this study.

In contrast to LDL, high-density lipoprotein (HDL) plays animportant role in the mechanism of reversed cholesterol transportaway from the arterial wall. Accordingly, decreased HDL levels areassociated with increased risk for cardiovascular disease. In carotidartery disease, low HDL levels are related to ultrasound findingsassociated with plaque vulnerability [10].

Another lipoprotein that has been studied in atheroscleroticdisease is lipoprotein(a) (Lp(a)). High Lp(a) levels have been asso-ciated with risk for cardiovascular disease and a higher incidenceof ischemic stroke [11]. Lp(a) serum levels are strongly influ-enced by genetic background because of variants of the Lp(a) gene.Recently, it was reported that there are highly significant associa-tions between variants in the Lp(a) gene and risk for cardiovasculardisease [12]. In relation to carotid plaque vulnerability and strokerisk, serum Lp(a) levels are associated with ultrasound findings ofcarotid plaque instability [13]. In a small observational study, serumLp(a) was a significant independent predictor of carotid stenosisand carotid artery occlusion [14].

2.2. Inflammation

Inflammatory cells in atherosclerotic plaques are stimulatedto produce an extraordinary number of bioactive moleculessuch as growth agonists, growth antagonists, pro- and anti-inflammatory cytokines and chemokines. The balance betweenpro- and anti-inflammatory activities is important in progressionof atherosclerotic plaques, and inflammation can also elicit acute

plaque rupture resulting in acute coronary syndromes or stroke.As a consequence, the extent of inflammation as measured byspecific biomarkers likely reflects the activity of atherosclerotic dis-ease and thus may predict the individual’s risk for progression ofatherosclerosis and plaque instability. In this context, numerous

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tudies elucidated the association between inflammation and theccurrence of cardiovascular adverse events.

Another interesting phenomenon showing the relation betweennflammation and atherosclerosis is the increased risk of cardiovas-ular disease in patients with rheumatoid arthritis (RA) that is notxplained by traditional risk factors [15]. This also advocates anmportant role of inflammation in atherosclerosis and therefore,nflammatory markers may be of importance in the prediction oflaque vulnerability.

One of the first inflammatory biomarkers studied for predic-ion of atherosclerotic complications is C-reactive protein (CRP) andigh-sensitivity CRP (hs-CRP), measuring CRP levels in the lowerange. CRP is one of the acute-phase reactants indicating under-ying systemic inflammation, and has shown to have a predictivealue for cardiovascular disease or risk factors in healthy men [16]nd women [17]. Plasma CRP is also able to discriminate betweentable and unstable coronary artery disease; CRP levels are signif-cantly higher in patients with unstable coronary artery diseaseompared to stable patients [18]. In the JUPITER trial, statin therapyowered hs-CRP levels and achieved reduction of 62% in cardiovas-ular events in patients with successfully lowered serum levels ofs-CRP [19]. Finally, CRP significantly adds prognostic informationo the Framingham Risk Score (FRS) in risk prediction of cardiovas-ular events [20]. However, this was only true for patients in thentermediate risk group.

In carotid artery stenosis, hs-CRP correlates with morphologicaleatures of rapidly progressive carotid atherosclerosis defined byltrasound categorization [21]. Rost et al. [22] performed a com-unity based prospective study with a one-time measurement of

RP in 1462 stroke- and TIA-free men and women. Elevated plasmaevels of CRP could significantly predict a greater risk for a stroke.

Apart from (hs)-CRP as a marker of inflammation, several studiesn serum cytokine and chemokine levels have been performed toetermine potential biomarkers of unstable atherosclerosis. Stud-

es in carotid plaques comparing cytokine expression betweenymptomatic and asymptomatic plaques suggest cytokine milieuifferences between unstable and stable plaques. In addition,symptomatic patients with carotid artery stenosis have shownifferent intracellular cytokine expression in peripheral blood sam-les compared to patients with stroke, TIA or amaurosis fugax23].

Serum amyloid A (SAA) is an acute phase protein that canncrease monocyte and macrophage cytokine production and is ele-ated in atherosclerotic lesions. In patients with acute coronaryyndromes SAA is able to predict poor outcome [24] and a recenttudy showed that elevated SAA levels can identify patients withschemic stroke caused by atherothrombosis [25].

Another pro-inflammatory cytokine is IL-18, which is expressedy different cell types within the atherosclerotic plaque. IL-18romotes the Th-1 immune response and also enhances the pro-uction of matrix metallo-proteinases (MMP’s) [26]. In carotidrtery plaques, IL-18 expression is especially increased in unsta-le lesions [27]. Serum values of IL-18 have also shown to be arognostic factor for future coronary events [28], and positively cor-elate with carotid intima media thickness [29]. However, Koenigt al. [30] found no significant association between higher IL-18oncentrations and the risk for coronary events.

IL-6 is a pro-inflammatory cytokine that also has pro-therogenic properties. As with IL-18, IL-6 is produced by differentell types in the atherosclerotic plaque where it amplifies thenflammatory cascade and is also a pro-coagulant cytokine [31].

gain, increased expression of IL-6 was shown especially in unsta-le plaque regions [32]. This increased local expression of IL-6 mayesult in elevated systemic levels, but in carotid artery stenosishis has not yet been assessed. In healthy subjects, IL-6 predictshe risk of cardiovascular complications [30,33]. In patients with

sis 213 (2010) 21–29 23

acute coronary syndromes, baseline IL-6 levels are associated witha greater risk for a stroke [34]. The same holds true for hs-CRP andSAA.

Another cytokine that has been linked to atherosclerosis ismyeloperoxidase (MPO). MPO is an enzyme linked to both inflam-mation and oxidative stress. In atherogenesis, MPO is involvedin the oxidation process of LDL and thereby promoting foam cellformation in the vascular wall. In a number of epidemiological stud-ies MPO has shown to be associated with cardiovascular disease.However, in the setting of plaque instability and acute coronaryor carotid symptomatology, MPO data are conflicting. In carotidartery stenosis MPO has not proven to be of additional value in theidentification of high-risk plaques [35].

PAPP-A is a metallo-proteinase acting as a biomarker of inflam-mation by regulation of insulin-like growth factor (IGF). The exactfunction of PAPP-A is poorly defined. However, in coronary andcarotid plaque studies, expression of PAPP-A is elevated in specificmacrophage rich plaque accumulations at the shoulder region andsurrounding the lipid core [36,37]. This suggests a relation betweenPAPP-A and plaque vulnerability and serum PAPP-A levels may alsobe used as a valuable biomarker for the detection of vulnerableplaques. Serum PAPP-A has shown to have predictive value in acutecoronary syndromes [36,38]. However, data are conflicting and it isa subject of debate which molecular form of PAPP-A is most relevantin atherosclerosis; the PAPP-A/proMPB or free PAPP-A. In carotidartery disease, serum PAPP-A levels showed a positive predictivevalue for the presence of unstable plaques. However, asymptomaticpatients had significantly higher PAPP-A values compared to symp-tomatic patients [39].

Anti-inflammatory cytokines also play an important role inatherosclerosis and a balance between pro- and anti-inflammatorystimuli determines plaque progression. Cipollone et al. [40]found that stable atherosclerotic carotid artery plaques exhibitedincreased expression of the anti-inflammatory cytokine transform-ing growth factor-b1 (TGF-b1) compared with unstable carotidartery plaques.

Other cytokines that have been studied in atherosclerosis areIL-8, interferon � (IFN-�), IL-4, IL-12 and TNF-�. However, thesecytokines have not been studied as serum markers to predict strokerisk or the presence of unstable carotid plaques but serum TGF-b1levels were not assessed.

2.3. Proteolysis

Proteolysis is involved in the early stages of carotid plaquedevelopment as well as in the later stages of plaque destabilization.Release of proteolytic enzymes such as matrix metallo-proteinases(MMP’s) and cathepsin cystein proteases (CCP’s) is an importantcause of cap erosion, resulting in cap rupture and thus acute neuro-logic events. The presence or activity of proteolytic enzymes withina plaque is not necessarily associated with instability, but an imbal-ance between these enzymes and their inhibitors (tissue inhibitorsof metallo-proteinases (TIMP’s)) may lead to matrix degradationand plaque destabilization.

Several studies in carotid artery plaques have shown proteolyticactivity in relation to plaque instability. In unstable plaques thereis a local increase in active MMP-9 concentration [41]. In anotherstudy, transcript levels of MMP-1 and MMP-12 were found to behigher in patients with amaurosis fugax compared to asymptomaticpatients [42]. However, patients with stroke or transient ischemicattack did not show higher levels when compared to asymptomatic

patients [42].

Excessive expression of proteolytic enzymes in unstable carotidartery plaques may result in elevated serum levels. Therefore, sys-temic changes in MMP or CCP levels may identify patients withvulnerable plaques and help in targeting preventative intervention

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n those who are most at risk for acute events. Especially MMP’save shown promising results in this respect.

Kai et al. [43] were the first to study plasma MMP-2 and MMP-9evels in 33 patients with acute coronary syndrome (ACS) and 17atients with stable angina. Both, ACS and stable angina patients,ad higher plasma MMP-2 levels, but in ACS patients MMP-2 levelsere two times higher compared to healthy controls. In addition,MP-9 levels were elevated in ACS patients but not in stable angina

atients. MMP-9 levels sustained high values for three days andecreased in seven days after the acute coronary event to nor-al values. These findings indicate that MMP-2 plays a role in

therosclerosis in general but MMP-9 is an important factor in acutelaque rupture and thus a more interesting marker. Serum levelsf MMP-1 and MMP-3 have also shown to be significantly highern patients with ACS compared to controls and patients with stablengina [44].

In carotid artery disease, MMP-2 and MMP-9 levels wereetermined in a group of 27 symptomatic and 13 asymptomaticatients undergoing carotid endarterectomy. The symptomaticroup exhibited higher serum levels of MMP-2 and MMP-9, andMP-9 was strongly associated with the presence of macrophages

n the plaque [45].When determining serum markers in patients with symp-

omatic carotid artery stenosis, it is often hard to distinguishhether the increased values are a sign of plaque instability or

aused by cerebral injury after ischemia. For example, Rosembergt al. [46] demonstrated in animal models that MMP-9 activityncreases from the first 12 h after the cerebral ischemic event up tohe fifth day afterwards, whereas MMP-2 increases after the fifthay post-ischemia. In a necropsy study maximum MMP-9 activ-

ty was found between the second and fourth days after stroke,hereas MMP-2 activity persisted up to 4 months after the event

47].Even though multiple factors may play a role in serum MMP

alues, MMP’s are probably the most promising serum biomarkersf symptomatic carotid plaques to date.

Cathepsins also play a role in the remodeling of extracellularatrix by degrading of collagen and elastase, but less is known

bout cathepsins in carotid artery disease. In vivo knock-out stud-es revealed that deficiency of cathepsin K [48] and cathepsin

[49] significantly reduce atherosclerotic plaque size and pro-ression. Although the exact role of local cathepsin expression inlaque stability is unknown, cathepsins in atherosclerotic plaquesre mainly localized in macrophages. Therefore it is suggestedhat macrophage rich unstable plaques will show higher cathep-in expression. Only a few studies in serum cathepsin levels haveeen performed. Serum levels of cathepsin L [50] and cathepsin S51] were elevated in patients with coronary atherosclerosis com-ared to human subjects without atherosclerotic disease. In carotidrtery disease, serum cathepsin levels have not been reportedo have discriminating properties between stable and unstableisease.

.4. Angiogenesis

Microvessels are present in the normal arterial adventitia,here they supply the vessel wall with oxygen and nutrients. In

therosclerotic plaques, the formation of microvessels has beenecognized as a contributing factor to plaque destabilization andupture. In highly neovascularized plaques, microvessel densitys associated with macrophage infiltration, inflammation, intra-

laque hemorrhage and plaque rupture [52]. In carotid arterylaques, microvessel content is related to intra-plaque haemor-hage, plaque vulnerability and symptomatology [53].

The mechanisms underlying plaque angiogenesis are thoughto be driven by hypoxia, reactive oxygen species and inflamma-

osis 213 (2010) 21–29

tion [54]. Various cell types are activated by these mechanismsand stimulate expression of vascular growth factors such as vas-cular endothelial growth factor (VEGF) and placental growth factor(PlGF) [55]. Extravasation of inflammatory cells and red blood cellsthrough microvessel leakage are important contributors to plaqueprogression resulting in a high risk of intraplaque hemorrhage.Damage to fragile blood vessels causes adhesion of various celltypes resulting in inflammatory and proteolytic activity.

The balance between pro- and anti-angiogenic factors deter-mines initiation of neoangiogenesis and evaluation of these pro-or anti-angiogenic stimuli may be helpful in identifying vulner-able atherosclerotic plaques. Platelet factor 4 (PF4), a chemokinereleased by activated platelets, is an example of a pro-angiogenicfactor. PF4 levels in carotid plaques correlated between lesionseverity and symptomatology [56]. Other angiogenic factors areangiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2). Whereas Ang-1is known to stabilize vessels by maximizing interactions betweenendothelial cells and their surrounding, Ang-2 leads to looseningof these interactions. Vulnerable plaques with high vessel densityhave higher Ang-2 expression, making them more susceptible forintra-plaque hemorrhage [57].

Systemic anti-angiogenic therapy in tumor angiogenesis, forexample anti-VEGF therapy, has shown promising effects, but sys-temic determination of angiogenetic serum markers has never beenperformed.

Secondary to angiogenesis is intraplaque hemorrhage fromruptured neovessels. This is also a common feature of atheroscle-rotic plaques and is considered one of the identifying features ofthe unstable plaque. Hemorrhage into an atherosclerotic plaqueresults in the deposition of blood products into the extravascu-lar space such as red blood cells and hemoglobin. Hemolysis ofextravasated red blood cells leads to accumulation of hemoglobin,which exerts potent toxic effect through the generation of reac-tive oxygen species. This initiates a cascade of events leading toinflammation and plaque instability. The major defense mechanismagainst the toxic effects of hemoglobin is the protein haptoglobin,which makes haptoglobin–hemoglobin complexes that are clearedby macrophages. This clearance by the macrophage is mediatedby the scavenger receptor CD163. Serum levels of CD163 weredetermined by Aristoteli et al. [58] in patients with and with-out significant coronary artery disease. Increased levels of sCD163were found in patients with coronary artery disease and CD163levels were a significant predictor of the extent of disease. How-ever, Willemsen et al. [59] compared serum CD163 levels in aseries of 526 patients presenting with cardiac and non-cardiac chestpain, but did not find differences in CD163 levels between the twogroups.

2.5. Hypoxia

Hypoxia is one of the mechanisms involved in the regulationof neo-angiogenesis in physiological and pathological conditionsincluding atherosclerosis. In atherosclerotic plaques, hypoxia canoccur due to diminished oxygen diffusion capacity through calcifiedand thickened vessel walls or higher oxygen consumption due toactivated immune cells. The exact role of hypoxia in the progressionof atherosclerosis is still unknown. Besides angiogenetic influences,hypoxia may also be pro-inflammatory and anti-fibrotic. In the clin-ical setting, there are correlations found between hypoxia in carotidartery plaques and the presence of macrophages, angiogenesis andthrombus formation [54]. In vitro studies in macrophages and other

immune cells have shown that hypoxia increases the production ofcytokines and MMP’s. Hypoxic conditions in vivo with decreasedoxygen delivery, such as septic or haemorrhagic shock, have shownto activate the inflammatory response. This may also be true incarotid plaque formation. As a consequence, indirect markers of

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ypoxia in atherosclerosis may be cytokines, MMP’s or other mark-rs. However, serum biomarkers directly related to hypoxia haveot been studied in atherosclerotic plaques to date.

.6. Apoptosis

Apoptosis plays an important role in the initiation androgression of atherosclerosis and has been recognized as a fea-ure of advanced human atherosclerotic plaques. The advancedtherosclerotic plaque contains a necrotic core of dead cells andebris. Both, smooth muscle cells and inflammatory cells dieecause of the process of programmed cell death or apoptosis.

The significance of apoptosis can have different pathophysi-logical outcomes. On one side, apoptosis of inflammatory cellsay slow down the inflammatory reaction because the number of

ytokine producing immune cells is decreasing. This way, the unsta-le cellular-rich plaque can change into a more stable hypocellularlaque with possibly less collagen breakdown. On the other hand,ccumulation of dead cells results in enlargement of the necroticore of the plaque. In addition, when vascular smooth muscle cellsie, this results in weakening of the fibrous cap creating an unsta-le plaque that is prone to plaque rupture. The combination ofn increased necrotic core and a thin fibrous cap is an importanteterminant in plaque instability.

Only few studies have identified apoptotic markers in serum andtherosclerotic plaques. Annexin 5 is an example of a biomarker ofpoptosis that has been studied in atherosclerotic lesions. Exoge-eous radiolabelled annexin 5 was detected in symptomatic carotidrtery plaques [60]. In patients with coronary artery stenosis,lasma endogeneous annexin 5 levels were decreased comparedo healthy controls [61].

Whether apoptosis is an ongoing process in development of antherosclerotic plaque or if it is associated with the acute com-lications of atherosclerosis through plaque rupture still remainso be determined. A few preliminary results show an associationetween apoptotic activity and plaque rupture, but a clinical impli-ation of biomarkers of apoptosis in serum or plaques remainsnclear.

.7. Thrombosis

Thrombomodulatory factors have been implicated in carotidlaque instability. Rupture and thrombosis of coronary plaquesre important causes of myocardial infarction and in carotid arterylaques thrombotic activity is associated with stroke and TIA’s.

Unstable carotid artery plaques express a wide array ofhrombomodulatory factors such as tissue plasminogen activa-or (t-PA), urokinase plasminogen activator (u-PA), plasminogenctivator inhibitor-1 (PAI-1), tissue factor (TF), tissue factor path-ay inhibitor (TFPI), and thrombomodulin (TM) [62]. Expression

f thrombomodulatory factors is higher in unstable plaques com-ared to stable plaques in the acute stage [62].

In a large series of carotid endarterectomy specimens, throm-otic activity was seen in 74% and 35% of patients with ischemictroke and TIA’s respectively, and only in 14% of asymptomaticatients. In stroke patients, thrombotic activity was seen untileveral months after the first cerebrovascular event [63]. Thesendings suggest that thrombotic activity plays a crucial role inlaque rupture and the pathogenesis of stroke. In addition, patientsith thrombotically active plaques stay at risk for future neuro-

ogic events caused by micro-emboli of the thrombotically active

laque.

Thrombotic activity in carotid artery plaques seems to pre-ede acute neurologic events, and serum biomarkers reflectinghis activity may be of great help in selecting high risk patients.owever, thrombomodulatory factors have only been studied in

sis 213 (2010) 21–29 25

prediction of cardiovascular disease in general, but not for riskassessment in patients with carotid artery disease.

2.8. Calcification

Vascular calcification is an important manifestation ofatherosclerosis, which is tightly regulated by promoters andinhibitors. Many key regulators of bone formation and bonestructural proteins are expressed in atherosclerotic plaques, suchas bone morphogenetic protein-2 (BMP-2), osteopontin (OPN),matrix-carboxyglutamic acid protein (MGP), and osteoprotegerin(OPG) [64–67].

Approximately 15% of the carotid artery plaques contain calci-fications [68], but the influence of calcification in plaque stabilityis controversial. In coronary atherosclerosis, calcification assessedby computed tomography scanning predicted acute coronary syn-dromes [69]. However, others showed that more calcification wasseen in patients with stable angina compared to unstable angina[70].

In carotid artery stenosis, the presence of calcification in theplaque has shown to be associated with fewer symptoms ofstroke and transient ischemic attacks [68]. It has therefore beensuggested that calcification in carotid artery plaques may be aplaque-stabilizing factor and protective of acute neurologic events.

Serum levels of vascular calcification inhibitors have been stud-ied in relation to cardiovascular disease and plaque stability. Highlevels of OPG have shown to be an independent risk factor for car-diovascular disease [71]. In addition, serum OPN levels correlatedwith carotid artery intima thickness [72] and extend of coronarycalcification [73]. In a recent study, OPG and OPN levels in patientswith stable and unstable carotid artery stenosis and healthy sub-jects were compared [74]. Symptomatic patients had higher OPGand OPN levels compared to asymptomatic patients and healthysubjects.

Despite the clinical relevance of the mechanism of calcificationin atherosclerosis and plaque stability, research has been limitedand results are still conflicting [75].

3. Discussion and conclusion

Various pathophysiological processes are importantly involvedin carotid plaque instability, such as lipid accumulation, inflam-mation, proteolysis, angiogenesis, thrombosis, hypoxia and calcifi-cation. Molecules involved in these pathogenetic processes maybe present as serum biomarkers reflecting activity of processesin plaque vulnerability, and thus are potential serum biomarkersin the identification of vulnerable carotid artery plaques. Variousstudies have been performed to identify serum biomarkers forprediction of stroke risk or identification of vulnerable plaques(Tables 2 and 3). Serum biomarkers that have shown to be highlyassociated with the presence of vulnerable carotid artery plaquesare mainly inflammatory and proteolytic markers such as hs-CRP,SAA, IL-6, MMP-9, MMP-2, TIMP-1 and TIMP-2. This non-invasiveway to identify high-risk patients may be a very promising tool inthe future selection of patients for carotid surgery.

However, there are multiple sites in the body where atheroscle-rotic plaque development and instability may occur. Many serumbiomarkers in cardiovascular disease have been studied in coro-nary artery disease and not (yet) in patients with carotid arterystenosis. It might be argued that biomarkers established for coro-

nary plaque instability are less applicable in carotid artery diseasebecause pathophysiological mechanisms of stroke and myocar-dial infarction and carotid and coronary plaque development maybe different. Symptoms occurring from carotid artery plaquesappear to result mainly from plaque rupture or embolization

26 L. Hermus et al. / Atherosclerosis 213 (2010) 21–29

Table 2Serum biomarkers in carotid artery stenosis.

Author n Patients Marker Results

Mocco et al. [79] 59 25 symptomatic CAS sICAM-1 Symptomatic > asymptomatic > controls29 asymptomatic CAS5 controls

Loftus et al. [80] 70 CEA patients: MMP-1,2,3, 9 MMP-9: embolization > non-embolization21 spontaneous embolization TIMP-1, 2 MMP-1,2,3 and TIMP-1,2: no difference49 non-embolization (determined by TCD)

Alvarez Garcia et al. [81] 62 36 symptomatic CAS Hs-CRP symptomatic > asymptomatic26 asymptomatic CAS

Alvarez et al. [45] 40 27 symptomatic CAS MMP-2 MMP-2: symptomatic > asymptomatic13 asymptomatic CAS MMP-9 MMP-9: symptomatic > asymptomatic

Sapienza et al. [82] 68 29 unstable carotid artery plaque MMP-1,2,3,9 MMP-1,2,3,9: unstable > stable andcontrols

24 stable carotid artery plaque TIMP-1,2 TIMP-1,2: unstable < stable and controls15 Controls

Adabbo et al. [83] 108 75 Chronic kidney disease patients MMP-9 MMP-9, IL-6, IL-8, NT-ProBNP, t-PAI-1 andVEGF: significantly related to severity ofcarotid stenosis

33 Healthy individuals t-PAI-1IL-6, IL-8NT-proBNPVEGF

Taurino et al. [84] 23 8 symptomatic CAS MMP-9 MMP-9: higher in symptomatic patientscompared to controls

7 asymptomatic CAS MMP-2 MMP-2 and TIMP-2: no significance8 controls TIMP-2

Kadoglou et al. [74] 164 60 CAS and cerebral ischemia on CT OPN OPN and OPG: CAS with ischemia onCT > CAS without ischemia on CT

54 CAS no cerebral ischemia on CT OPG50 healthy controls

Yu et al. [85] 59 dialysis (CAPD) patients Adiponectin With carotid stenosis < no carotid stenosis20 patients with carotid artery stenosis25 patients without carotid artery stenosis

Debing et al. [86] 360 101 symptomatic CAS Hs-CRP Hs-CRP, sVCAM-1 and IL-6: CAS > controls79 asymptomatic CAS sICAM-1 sICAM-1: not significant180 controls sVCAM-1 No significant difference between

symptomatic vs asymptomatic CAS in allmarkers

IL-6

Koutouzis et al. [87] 119 62 symptomatic CAS IL-6 IL-6: symptomatic > asymptomatic57 asymptomatic CAS TNF-� TNF-�, IL-1b, SAA, hsCRP: no significant

differenceIL-1bSAAhsCRP

Sapienza et al. [88] 52 Re-stenosis after CEA: MMP-2,9 MMP-2,9: all re-stenosis CAS > controls,MMP-2,9: predictive value for re-stenosis

23 symptomatic CAS TIMP-1,2 TIMP-1,2: all re-stenosis CAS < controls29 asymptomatic CAS20 controls

Handberg et al. [93] 62 31 symptomatic CAS: sCD163 sCD163: symptomatic within 2months > asymptomatic or symptomaticlonger than 2 months ago

16 symptoms < 2 months15 symptoms 2–6 months31 symptomatic CAS

Heider et al. [94] 145 76 asymptomatic CAS MMP-7, 8, 9 Symptomatic > asymptomatic69 symptomatic CAS TIMP-1

Pelisek et al. [95] 101 37 Stable carotid artery plaques (histology) MMP-1, 2, 3, 7, 8, 9 MMP-1, MMP-7, TIMP-1, TNF-�, and IL-8:patients with unstable plaques > patientswith stable plaques

L. Hermus et al. / Atherosclerosis 213 (2010) 21–29 27

Table 2 (Continued)

Author n Patients Marker Results

64 Unstable carotid artery plaques (histology) TIMP-1,2TNF-�IL-1�, 6, 8, 10, 12

Olson et al. [96] 255 255 symptomatic CAS: suPAR(I-III) suPAR(I-III), suPAR(II-III): stroke andTIA > amaurosis fugax

121 Stroke suPAR(II-III)76 TIA58 AF

Sugioka et al. [97] 102 Patients with stable coronary artery disease: Neopterin Complex carotid arteryplaque > non-complex or no carotid arteryplaque

25 no carotid artery plaque43 non-complex carotid artery plaque34 complex carotid artery plaque

C tomy;i lar adT eopon

ftt

cfrondfpsia

f

TS

ht

AS, Carotid artery stenosis; TCD, trans cranial Doppler; CEA, carotid endarterecnterleukin; sICAM, soluble intracellular adhesion molecule, sVCAM, soluble vascuIMP, tissue inhibitors of metallo-proteinases; TNF, tumor necrosis factor; OPN, ost

rom advanced unstable plaques and diffusion of thrombi dis-ally, whereas in coronary artery disease plaque erosion and statichrombotic occlusion are more common [76].

Development of morphologically different plaques are likelyaused by the presence of important flow rate and shear stress dif-erences between the carotid and coronary arterial wall. High flowates in the carotid bifurcation result in less frequent occurrencef total occlusion of the carotid artery compared to the coro-ary circulation in which incidence of total occlusion in patientsying suddenly is 40% [77]. High flow rates also result in morerequent intraplaque hemorrhage in carotid artery plaques com-ared to coronary artery plaques [76]. Intraplaque hemorrhage has

hown to be related to larger necrotic cores and greater macrophagenfiltration into the plaque [78] resulting in advanced instabletherosclerotic lesions.

However, whereas the coronary artery circulation is indeed dif-erent from the carotid artery circulation, it must be pointed out

able 3erum biomarkers in atherosclerosis with carotid artery stenosis or stroke as outcome va

Author (year) N Patients Marker

Rost et al. [22] 1462 Community based cohort,non-stroke/TIA subjects

CRP

Elkind et al. [89] 279 Community based TNF-re

TNF-re

Chapman et al. [90] 1111 Community based IL-6

Hs-CRP

Ambrosius et al. [91] 76 Carotid ultrasound patientsin patients withatherosclerotic risk factors

IL-10

TFG-b1

IL-6

hsCRP

Halvorsen et al. [92] 5341 Population based: allpatients undergoingcarotid artery ultrasound

CRP

sCRP, High sensitivity C-reactive protein; TNF tumor necrosis factor; MCPT, maximum caransforming growth factor beta-1.

hsCRP, high sensitivity C-reactive protein; MMP, matrix metallo-proteinase, IL,hesion molecule; suPAR, soluble urokinase-type plasminogen activator receptor;tin; OPG, osteopontegerin.

that atherosclerosis is a systemic process and pathophysiologi-cal processes in coronary artery plaques and peripheral vasculardisease have many features in common with development andinstability of carotid artery plaques. This also indicates that serumbiomarkers reflecting plaque instability may not be specific forcarotid artery plaques, and it may be hard to distinguish whetherelevated biomarker levels indicate stroke risk or risk for myocar-dial infarction. In addition, for a biomarker to be useful in selectinghigh-risk plaques in a population with an overall low risk for stroke,it needs to be highly specific and sensitive. In biomarkers studiedso far, the increased relative risk is probably not high enough toqualify them for use in clinical routine.

In the near future, new potential biomarker molecules willbe discovered. Especially new approaches such as proteomic andgenomic technique will be used in the search for new moleculesreflecting carotid plaque instability. These biomarkers may notyet be independent discriminating factors in the identification of

riables.

End point Comments

12–14 year follow-up, riskfor development ofstroke/TIA

Higher risk when increasedCRP (RR = 1.4–2.0)

ceptor 1 Measurement maximumcarotid artery plaquethickness (MCPT)

TNF-receptors 1 and 2:MCPT elevation in highest50% group of TNF-receptorlevels

ceptor 2

Prediction of the presenceof carotid artery stenosis

IL-6 and hsCRP: plaquepresence > no plaquepresence

Carotid intima-mediathickness (IMT)

IL-10: lower levels,increased IMT

IL-6, and hsCRP: higherlevels, increased IMTTFG-b1: no significantassociation

Presence of carotid arteryplaque

CRP: no significantassociation presence ofcarotid artery plaque

rotid artery plaque thickness; IL, interleukin; IMT, intima media thickness; TGF-�1,

2 oscler

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8 L. Hermus et al. / Ather

nstable plaques, but will provide additional information on theatients risk for plaque instability. Therefore, biomarkers will cer-ainly play an important future role in a multimodal approach inhe selection of patients for carotid artery surgery.

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