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Atherosclerosis 225 (2012) 99e104

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Atherosclerosis

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Coronary perivascular adipose tissue characteristics are related to atheroscleroticplaque size and composition. A post-mortem study

Sandra N. Verhagen a, Aryan Vink b, Yolanda van der Graaf c, Frank L.J. Visseren a,*

aDepartment of Vascular Medicine, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The NetherlandsbDepartment of Pathology, University Medical Center, Utrecht, The NetherlandscDepartment of Epidemiology, Julius Center for Health Sciences and Primary Care, Utrecht, The Netherlands

a r t i c l e i n f o

Article history:Received 7 February 2012Received in revised form8 June 2012Accepted 24 August 2012Available online 10 September 2012

Keywords:PerivascularAdipose tissueAtherosclerosisInflammationPlaque composition

* Corresponding author. Tel.: þ31 30 2509111; fax:E-mail address: f.l.j.visseren@umcutrecht.nl (F.L.J.

0021-9150/$ e see front matter � 2012 Elsevier Irelahttp://dx.doi.org/10.1016/j.atherosclerosis.2012.08.031

a b s t r a c t

Background: Perivascular adipose tissue (pvAT) may influence atherosclerotic plaque formation. We aimto determine the association between the local amount and inflammatory properties of pvAT and the sizeand composition of atherosclerotic plaque in the left anterior descending artery (LAD).Methods: Post-mortem, a total of 139 cross sections of the LAD were obtained from 16 patients. PvATquantity was measured within an area of 3 mm around the LAD (pvAT-area (%)). Furthermore, inflam-matory properties of pvAT were measured (macrophages/400� field and adipocyte size). From plaquearea (mm2), plaque/media-ratio was calculated and morphologic characteristics were scored (presence ofa lipid core and calcification; collagen and smooth muscle cell content; macrophage (<10, 10e50, >50/400� field) and lymphocyte infiltration (<10, 10e25, >25/400� field)).Results: Plaque/media-ratio increased with increasing pvAT-area (b0.02; 95%CI 0.01e0.03) and pvAT-macrophages (b 0.10; 95%CI 0.05e0.16), but not with adipocyte area (b �0.00; 95%CI �0.07e0.06).PvAT-area was related to the presence of a lipid core (OR 1.05; 95%CI 1.03e1.08) and with macrophageand lymphocyte infiltration of atherosclerotic plaque (per increase in category OR 1.05; 95%CI 1.02e1.07and OR 1.04; 95%CI 1.01e1.07 respectively). PvAT macrophage infiltration was correlated with adventitiaand plaque macrophages.Conclusion: PvAT quantity and macrophage infiltration are highly related to atherosclerotic plaque sizeand composition in patients with coronary atherosclerosis. These results indicate potential involvementof pvAT in coronary atherosclerotic plaque development, although the causality of the relation has yet tobe determined.

� 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Atherosclerotic vascular disease is characterized by focal lesionsand areas without atherosclerotic plaque [1]. The focal aspect ofatherosclerotic plaque formation cannot be explained by riskfactors with a generalized effect on the vasculature such as smokingor hypercholesterolaemia, but may be explained by local conditionssuch as non-laminar haemodynamic flow [2] and local inflamma-tion [3,4].

Obesity is characterized by a state of low grade inflammation,which is involved in the process of atherosclerosis [5,6]. Increasedvisceral adipose tissue volume induces adipose tissue dysfunction[7] and systemic inflammation [8] as enlarged adipocytes produce

þ31 30 2505488.Visseren).

nd Ltd. All rights reserved.

a wide range of adipocytokines and chemokines, attractingmacrophages to adipose tissue [9]. Crosstalk between adipocytesand macrophages leads to enhanced secretion of inflammatoryadipokines and cytokines inducing systemic inflammation thatcontributes to the development of vascular diseases, insulin resis-tance and type 2 diabetes [10].

Adipose tissue around arteries, perivascular adipose tissue(pvAT), has been hypothesized to stimulate and induce athero-sclerotic plaque formation from outside the artery (‘from outside toinside’) by induction of a local inflammatory environment [11,12].Coronary pvAT is part of epicardial adipose tissue and althoughthere is no anatomical border, epicardial adipose tissue ismorphologically and functionally different from pvAT [13]. Inpatients suspected of coronary artery disease, the volume of pvATon CT is higher near coronary artery segments with atheroscleroticplaque than near coronary artery segments without plaque [14].Furthermore, infiltrates in the adventitia are related to intima

S.N. Verhagen et al. / Atherosclerosis 225 (2012) 99e104100

inflammation [15] and there is an over-expression of pro-inflammatory genes in epicardial adipose tissue of patients withcoronary artery disease compared to controls [16]. However, todetermine whether there is an influence of local adipose tissue onthe vascular wall, it is important to delimitate the region of interestand to take both adipose tissue quantity and inflammation intoaccount.

The aim of the present post-mortem study was to investigatelocal properties of pvAT in relation to local atherosclerosis. Weinvestigated the relation between coronary pvAT quantity andinflammation and coronary plaque size and composition in crosssections of the left anterior descending coronary artery (LAD).

2. Methods

A total of 16 patients with coronary atherosclerosis wereincluded in the study between April 2009 and February 2011.Patients were referred for routine autopsy to the department ofPathology of the University Medical Centre in Utrecht theNetherlands. Eligible patients had a vascular cause of death or were>40 years of age. Moreover, they did not fulfil any of the exclusioncriteria (history of PTCA, CABG, kidney failure, recent sepsis, knownreduction of body weight (>5%) in the past 2 years, malignancy inthe past 2 years, thyroid disease or oral use of steroid hormones). Inthe majority of patients (75%) death was caused by acute myocar-dial infarction. In the other cases death was caused by aspirationpneumonia following ischaemic stroke, by haemorrhagic stroke,ruptured aortic aneurysm and a work-related accident. The studymet the criteria of the code of proper use human tissue that is usedin the Netherlands for the use of human tissue.

2.1. Processing of coronary artery specimens

Bodies were stored at 4 �C and autopsy was performed by theattending pathologist. The LAD was prepared from the heart withthe surrounding adipose tissue and underlying myocardium takenalong in a radius of 1e2 cm (Supplemental Fig. 1A). The coronaryarteries were fixed in formaldehyde 4% for one day. Subsequentlythe material was decalcified in ethylenediaminetetra-acetic acid(EDTA) solution during 2 to 5 days depending on the extent ofcalcification.

Coronary arteries were cross sectioned, starting at the origin ofthe LAD (bifurcation of LAD and left circumflex coronary artery), at5 mm intervals. Subsequently cross sections where embedded inparaffin. Only cross sections within 6 cm from the origin of the LAD(n¼ 143) were analysed, as LAD-specimens of more than 6 cm fromthe origin of the LAD were only available in 4 cases. Cross sectionswith an intra-myocardial localization of the LAD, referred to asmyocardial bridge, were excluded because of virtual absence ofadipose tissue. Mean distance to the origin of the LAD was 3 cm.Proximal cross sections were defined as cross sections within thefirst 3 cm and distal cross sections as within 3e6 cm from the originof the LAD.

2.2. Histology and immuno-histochemical staining

Thin sections of 4 mm were processed for histological stainingwith haematoxylin & eosin (H&E), Picro-Sirius red, and elastin vonGieson. In addition immuno-histochemistry stainings were per-formed to quantify macrophages (CD68), lymphocytes (CD3 andCD20) and smooth muscle cells (a-smooth muscle actin).

For measurement of perivascular adipose tissue and athero-sclerotic plaque areas, elastin von Gieson histology specimens weredigitalized and analysed using Image Pro plus software(Mediacybernetics).

2.3. Perivascular adipose tissue quantity and characteristics

To determine the quantitative exposure of the coronary arteriesto adipose tissue, pvAT-thickness (mm) was measured at the levelof the lateral vessel wall, from the myocardium to the pericardium.PvAT-area (%) was defined as the percentage of adipose tissuewithin the area around the vessel. The area was delimitated bya circle with the radius of the vessel including the adventitia and anadditional 3 mm (Supplemental Fig. 1A). The intra-class correlation(ICC) of the pvAT-area (%) measurement with a 3 mm circle andpvAT-area (%) with a 5 mm circle around the vessel was 0.85(p < 0.001).

Macrophage infiltration and adipocyte size were measured inpvAT in a region within 3 mm of the coronary artery. Macrophageinfiltration was scored by determining the mean number of CD68-positive cells in 10 high power fields (HPF; magnification 400�) inall quadrants of the coronary artery (Supplemental Fig. 1C). Theadipocyte size was determined, by calculating the mean adipocytearea of 100 adipocytes in 100 randomly selected fields at 200�magnification (Supplemental Fig. 1B [17].) The ICC’s for pvAT-macrophages and adipocyte size were 0.76 (p < 0.001) and 0.90(p < 0.001) respectively.

2.4. Atherosclerotic plaque quantity and characteristics

Atherosclerotic plaque was defined as the area (mm2) betweenthe lumen and the internal elastic lamina (IEL) and the media wasdefined as the area (mm2) between the IEL and external elasticlamina (EEL) [4]. To account for arterial tapering the percentage ofvessel area consisting of plaque was calculated (lumen stenosis). Toaccount for arterial remodelling the ratio of the plaque area to themedia was calculated (plaque/media-ratio).

The morphologic characteristics of the plaque were scored bythe first author and each case was subsequently reviewed by thesecond author (Supplemental Fig. 2). The final score was givenwithmutual agreement. Presence of a lipid rich core was scored on H&E-and Picro-Sirius red stainings and presence of plaque calcificationon H&E and on haematoxylin counterstaining of immuno-histochemistry slides. Collagen and smooth muscle cell content ofatherosclerotic plaques was determined semi-quantitatively onPicro-Sirius red and a-smooth muscle actin stainings respectively.High collagen or smooth muscle cell plaque content was defined aspresence of these determinants in >50% of the plaque area and lowcontentas <50%. Macrophages were examined on CD68 stains andfor lymphocytes the categorization was based on CD3 and CD20stains. Macrophages and lymphocytes in plaque and adventitiawere scored in 4 categories. Plaque macrophages in the categories:no macrophages, few scattered, groups of 10e50, and >50. Plaquelymphocytes in the categories: no lymphocytes, groups of <10, 10e25, and >25. Adventitia macrophages in the categories: nomacrophages, <10, 10e25, and >25 per HPF (400�). Adventitiallymphocytes in the categories: no lymphocytes, scattered, aggre-gates of >25, and >100 in at least 1 quadrant. In the analyses thefirst 2 categories were taken together, because cases in these 2categories did not differ in adipose tissue measures.

2.5. Data analyses

If normally distributed, continuous variables are displayed asmeanwith standard deviation. To explore the confounding effect ofdistance to the origin of the LAD on the relation between pvAT andplaque characteristics, differences between cross sections close tothe origin of the artery and more distal sections were tested. Nor-mally distributed variables were tested with independent samplest-tests. Variables with skewed distributions are presented as

Table 2Perivascular adipose tissue (pvAT) and plaque characteristics according to distanceto origin of the LAD.

Proximal LAD(0e3 cm)

Distal LAD(3e6 cm)

p-value

n ¼ 76 n ¼ 63pvAT-area (%) 83 (74e96) 80 (69e89) 0.03pvAT-macrophages (n/HPF) 4.7 (3.0e7.2) 3.5 (2.5e6.0) 0.07Adipocyte cell size (mm2) 4051 � 793 4342 � 727 0.03

EEL (mm2) 9.8 � 4.5 5.0 � 2.4 <0.001Lumen (mm2) 2.5 (1.3e3.7) 1.2 (0.5e1.8) <0.001Plaque area (mm2) 4.6 (2.2e6.7) 1.8 (0.8e3.8) <0.001Lumen stenosis (%) 63 � 19 59 � 24 0.43Plaque/media-ratio 2.2 (1.2e3.6) 1.4 (0.9e2.6) 0.01

Lipid core present (n) 25 (33%) 8 (13%) 0.01Calcification present (n) 29 (38%) 13 (21%) 0.03Collagen (>50%) (n) 70 (92%) 60 (95%) 0.46Smooth muscle cells

(>50%) (n)44 (58%) 46 (73%) 0.06

Plaque macrophages(>10/HPF) (n)

48 (63%) 24 (38%) 0.01

Plaque lymphocytes(>10/HPF) (n)

36 (47%) 18 (29%) 0.03

Adventitia macrophages(>10/HPF) (n)

45 (59%) 26 (41%) 0.04

Adventitia lymphocytes(presence of aggregatesof >25) (n)

32 (42%) 15 (24%) 0.02

S.N. Verhagen et al. / Atherosclerosis 225 (2012) 99e104 101

medianwith interquartile range (IQR) and KruskaleWallis analyseswere used to test for differences. Numbers with percentage ofordinal variables are displayed and differences were tested withChi-square.

The relations between pvAT characteristics and continuous,dichotomous and ordinal plaque characteristics were evaluatedwith regression analyses. In order to take clustering of character-istics within subjects into account, general estimated equationsanalysis with a compound symmetry structure was used in allregression analyses. Models were linear for relations betweencontinuous measures of adipose tissue and plaque size. Althoughvariables were skewed, residuals in linear regression analyses wereconsidered normally distributed and therefore no transformationswere made to the dependent and independent variables. Binarylogistic models were used for dichotomous plaque characteristicsand ordinal logistic models for categorical plaque characteristics.Relations were explored in a univariable model (model I) and inmodels with distance to the origin of the LAD (model II) as a cova-riable. Analyses with pvAT-area were adjusted for pvAT-macrophages and adipocyte size in additional analyses. Generalestimated equations analyses with pvAT-macrophages wererepeated excluding LAD cross sections with intra plaque haemor-rhage or thrombus. Further sensitivity analyses were performed byanalysing data from cases with AMI only (n ¼ 12; 105 crosssections).

Mean � SD of variables with normal distribution are presented and median(interquartile range) of variables with a skewed distribution. Categorical variablesare presented as number with percentage. HPF: high power field; EEL: externalelastic lamina.

3. Results

3.1. Patient characteristics

Characteristics of patients are outlined in Table 1. Mean age was63 � 13 years and 10 out of 16 subjects were male (63%). Perpatient, a mean of 9 � 2 cross sections from 0.5 to 6.0 cm from theorigin of the LADwere available. Four cross sections from 2 subjectswere not included in the analyses, because of presence ofa myocardial bridge, leaving 139 cross sections available for anal-yses. In 6 out of 12 subjects with AMI, the culprit lesionwas locatedin the LAD. In the other 6 cases, the culprit lesion was located in decircumflex or right coronary artery. A number of 9 cross sections of6 subjects showed intra plaque haemorrhage and 3 cross sections of3 subjects showed thrombus in the LAD.

3.2. Perivascular adipose tissue and atherosclerosis in relation todistance to origin of the LAD

PvAT-thickness was 9.5 � 2.6 mm in proximal and 6.9 � 2.4 mmin distal sections (Table 2). Also pvAT-area (%), a measure notaffected by tapering of the coronary artery, was increased inproximal cross sections (83; IQR 74e96 and 80; IQR 69e89respectively). Atherosclerotic plaque was more pronounced in

Table 1Characteristics of autopsy cases (n ¼ 16).

Age (years) 63 � 12Males, n 10 (63%)Ethnicity white, n 15 (94%)Weight (kg) 85 � 16Cause of death AMI, n 12 (75%)Type 2 diabetes, n 3 (19%)Smoking, n 8 (67%)Hypertension, n 7 (44%)Statin use, n 2 (13%)Occlusion LAD, n 6 (38%)

AMI; acute myocardial infarction; LAD: left anterior descendingcoronary artery.

proximal than in distal cross sections with a plaque/media-ratio 2.2(IQR 1.2e3.6) versus 1.4 (IQR 0.9e2.6) and lesions were moreadvanced with a lipid core of 33% versus 13%.

3.3. Perivascular adipose tissue in relation to plaque size

There was a relation between pvAT quantity expressed as pvAT-area and plaque area (mm2) (b pvAT-area (%) 0.07; 95%CI 0.04e0.10), also after adjustment for distance to the origin of the LAD(Table 3a). The relation was still present with plaque/media-ratio,a measure of plaque size taking outward remodelling intoaccount (b pvAT-area (%) 0.02; 95%CI 0.01e0.03). Inflammation ofpvAT expressed as pvAT-macrophages (n/HPF) was related toplaque/media-ratio (b 0.10; 95%CI 0.05e0.16), but adipocyte sizewas not (b �0.00; 95%CI �0.07e0.06).

3.4. Perivascular adipose tissue in relation to plaque composition

An increase in pvAT-area (%) was associated with morphologicplaque characteristics (Table 3b), although the relation betweenpvAT-area (%) and collagen content was not (OR high versus lowcontent 0.92; 95%CI 0.83e1.01). Increasing pvAT-area (%) wasassociated with an increase in plaque macrophages (per categoryincrease OR 1.05; 95%CI 1.02e1.07) and plaque lymphocytes (OR1.04; 95%CI 1.01e1.07) (Fig. 1).

With an increase of pvAT-macrophages (n/HPF), plaques moreoften showed a lipid core, calcification and a low collagen content(OR high versus low collagen content 0.84; 95%CI 0.78e0.90).Furthermore, pvAT-macrophages (n/HPF) were associated withmacrophage infiltration in plaque and in the adventitia (per cate-gory increase OR 1.16; 1.06e1.27 and OR 1.38; 95%CI 1.13e1.67respectively) (Fig. 1). pvAT-macrophages (n/HPF) were also associ-ated with plaque and adventitia lymphocytes (per categoryincrease OR 1.13; 95%CI 1.03e1.23 and OR 1.15; 95%CI 1.03e1.29

Table 3aPerivascular adipose tissue (pvAT) in relation to atherosclerotic plaque size.

Model Plaque area (mm2)b (95%CI)

Lumen stenosis (%)b (95%CI)

Plaque/media-ratiob (95%CI)

pvAT-area (%) I 0.07 (0.04e0.10)* 0.21 (0.05e0.38)* 0.02 (0.01e0.03)*II 0.04 (0.01e0.07)* 0.18 (�0.05e0.42)* 0.02 (0.00e0.03)*

pvAT-thickness (mm) I 0.54 (0.28e0.80)* 0.84 (�0.42e2.10) 0.13 (0.02e0.25)*II 0.38 (0.10e0.67)* 0.65 (�1.29e2.58) 0.15 (0.07e0.22)*

pvAT-macrophages (n/HPF) I 0.14 (�0.02e0.29) 0.99 (0.39e1.58)* 0.10 (0.05e0.16)*II 0.09 (�0.04e0.22) 1.06 (0.09e2.02)* 0.13 (0.03e0.22)*

Adipocyte cell size (per 100 mm2) I �0.08 (�0.14e�0.01)* 0.13 (�0.54e0.80) �0.00 (�0.07e0.06)II �0.06 (�0.16e0.04) 0.11 (�0.64e0.86) 0.01 (�0.05e0.07)

*P < 0.05; model I: unadjusted; model II: adjusted for distance to the origin of the LAD.pvAT-area: % of pvAT within the perivascular area: a circle of 3 mm drawn around the vessel; pvAT-macrophages: mean number of CD68þ cells in 10 high power fields (HPF);adipocyte cell size: mean adipocyte area of 100 pvAT adipocytes in 100 randomly selected fields.Data are analysed with generalized estimating models (GEE) taking clustering within subjects into account.

S.N. Verhagen et al. / Atherosclerosis 225 (2012) 99e104102

respectively). There was no relation between adipocyte cell sizeand plaque composition. Overall, adjustment for distance to theorigin of the LAD (model II) did not substantially change the pointestimates for the relations between pvAT and plaque composition.

Sensitivity analyses were performed by analysing data from the12 cases with AMI only. In AMI cases relations between pvAT andatherosclerotic plaque were comparable to the whole group.Analyses excluding the 12 cross sections with intra plaque hae-morrhage or thrombus did not change the relation between pvAT-macrophages (n/HPF) and plaque size and composition. Moreover,repeating the analyses with pvAT-area as independent variable,using pvAT-macrophages and adipocyte size as covariates, did notchange the point estimates of the relations.

4. Discussion

In this post-mortem study pvAT quantity, measured as pvAT-area (%), was related to atherosclerotic plaque size and composi-tion in the LAD. In addition pvAT-macrophages, but not adipocytecell size of pvAT, was related to atherosclerotic plaque size andcomposition.

In most studies the total amount of epicardial adipose tissue,instead of the adipose tissue directly around the vessel is investi-gated in relation to coronary atherosclerosis, because of difficultiesin delimitation of pvAT. In the present study adipose tissue directlyaround the coronary artery was studied. Measuring this pvAT is ofmost interest because of the close anatomical relation between thisadipose tissue and the coronary arteries.

We observed an association between the amount of pvAT andatherosclerotic plaque size in the adjacent vascular wall, which is inaccordance with previous results. In two studies with patientssuspected of coronary artery disease or acute coronary syndrome,local atherosclerosis was highly related to the local volume of pvATas measured with CT [14,18]. PvAT was 27 cm3 over the proximal

Table 3bPerivascular adipose tissue (pvAT) in relation to atherosclerotic plaque composition.

Model Lipid coreOR (95%CI)

pvAT-area (%) I 1.05 (1.03e1.08)*II 1.04 (1.03e1.06)*

pvAT-thickness (mm) I 1.29 (1.10e1.51)*II 1.22 (1.01e1.46)*

pvAT-macrophages (n/HPF) I 1.22 (1.09e1.37)*II 1.20 (1.07e1.37)*

Adipocyte cell size (per 100 mm2) I 0.97 (0.91e1.02)II 1.00 (0.95e1.05)

*P < 0.05; model I: unadjusted; model II: adjusted for distance to the origin of the LAD.pvAT-area: % of pvAT within the perivascular area: a circle of 3 mm drawn around the vesadipocyte cell size: mean adipocyte area of 100 pvAT adipocytes in 100 randomly selectData are analysed with generalized estimating models (GEE) taking clustering within su

4 cm in one study [14] and 0.5 cm3 per coronary artery segment inthe other study [18]. The wide range in pvAT volumes that wasobserved may be due to differences in definitions of pvAT.

Quantity of pvAT and atherosclerotic plaque decreases fromproximal to distal portions of the coronary artery [1,11]. This mayconfound the relationship between the two variables, although onecan argue that a relation of pvAT and plaques with distance to theorigin of the coronary artery supports the theory of a causal relationbetween local adipose tissue and atherosclerosis. To overcome thisconfounding effect, we adjusted for distance to the origin of theLAD.With this adjustment, the relation between adipose tissue andplaque quantity was not affected. In addition we used measures ofpvAT and plaque size that take tapering and remodelling of thecoronary artery into account [4,19].

To the best of our knowledge this is the first study where pvATcharacteristics are investigated in relation to plaque composition.Plaque characteristics such as presence of a lipid core, plaquemacrophages and a decrease in smooth muscle cells are markers ofplaque instability [20]. The presence of a lipid core and a highmacrophage content are more common in patients with rupturedplaque than in patients with thin cap fibro-atheroma [20]. Althoughreferred to as markers of plaque instability, low collagen and plaquecalcification content in atherosclerotic plaques are less establishedas risk factors of plaque rupture [20,21]. In the present study, anincrease in pvATwas related to the presence of a lipid core andwiththe presence of calcification in plaque. In congruence, the totalvolume of the epicardial adipose tissue depot and the thickness ofpvAT measured on CT images is higher in patients with athero-sclerotic plaques compared to patients without plaques in thecoronary arteries [22,23]. Patients with mixed plaques, consistentwith a fibro-atheromatous lesions, have higher epicardial adiposetissue volume than patients with calcified plaques or no plaques[22]. Furthermore, pvAT is higher around coronary arteries withmixed plaques compared to other plaque types [14]. In support of

CalcificationOR (95%CI)

CollagenOR (95%CI)

Smooth muscle cellsOR (95%CI)

1.04 (1.02e1.06)* 0.92 (0.83e1.01) 0.96 (0.94e0.98)*1.03 (1.01e1.04)* 0.92 (0.82e1.03) 0.97 (0.96e0.99)*1.30 (1.05e1.60)* 0.83 (0.67e1.04) 0.75 (0.63e0.88)*1.22 (1.00e1.48)* 0.92 (0.82e1.03) 0.90 (0.71e1.13)1.13 (1.02e1.24)* 0.84 (0.78e0.90)* 0.91 (0.82e1.02)1.12 (1.02e1.23)* 0.85 (0.78e0.92)* 0.91 (0.82e1.00)0.94 (0.88e1.00) 1.05 (0.93e1.19) 1.06 (1.00e1.12)0.99 (0.94e1.04) 1.02 (0.93e1.13) 1.00 (0.93e1.06)

sel; pvAT-macrophages: mean number of CD68þ cells in 10 high power fields (HPF);ed fields.bjects into account.

Fig. 1. Perivascular adipose tissue (pvAT) characteristics in relation to atherosclerotic plaque inflammation. Bars indicate means with SEM. *P < 0.05 Data are analysed withgeneralized estimating models (GEE) taking clustering within subjects into account. OR’s (95%CI) indicate the chance per unit of the pvAT characteristic of being in a higher categoryof plaque inflammation.

S.N. Verhagen et al. / Atherosclerosis 225 (2012) 99e104 103

the relation between pvAT characteristics and calcified plaques inthe current study, there is an association between epicardialadipose tissue volume and calcium score [22,23] and betweenexpansion of epicardial adipose tissue and an increase in thenumber of calcified plaques [24].

In the present study macrophage infiltration in pvAT wasassociated with plaque size and plaque composition. These resultsare in concordancewith findings in 4 patients [25]. In pvAT near theatherosclerotic aorta, macrophages were more abundant than inpvAT near non-atherosclerotic peripheral arteries of the samepatient. No information on plaque composition was mentioned inthis study. The association between macrophages and atheroscle-rotic plaque also holds true for epicardial adipose tissue. Mediumconditioned by human epicardial adipose tissue is able to inducemigration of monocytes in vitro [26]. Moreover, in a recent studybiopsies of epicardial fat were taken during cardiac surgery [27]. Inpatients with coronary atherosclerosis, the pro-inflammatory M1macrophages in epicardial adipose tissue were more prominentthan in patients without coronary atherosclerosis.

Furthermore, inflammatory cell invasion in the adventitia isassociated with the extent of local atherosclerosis in patients withsevere CAD [15]. Moreover, adventitial lymphocytic aggregates andan increase in adventitial macrophage content, are well correlatedwith markers of plaque instability and plaque size [15,28]. There-fore, inflammation of surrounding tissues may be a continuousprocess. Not only is adventitial inflammation associated withatherosclerosis in other studies [15,28], the inflammation in pvATwas correlated with adventitial inflammation in the present study.

In contrast with other studies, we did not observe a relationbetween adipocyte size and inflammation. Adipocyte enlargementis thought to result in a pro-inflammatory state in subcutaneousand visceral adipose tissue [29]. Adipocyte size in pvAT and plaquecharacteristics were not related in the present study. However,perivascular adipocytes differ from adipocytes in subcutaneousadipose tissue and therefore this may not apply for all adiposetissue depots. Perivascular adipocytes are smaller and more irreg-ular in shape than subcutaneous adipocytes [13]. In epicardialadipose tissue the expression of MCP-1 decreases with adipocyte

S.N. Verhagen et al. / Atherosclerosis 225 (2012) 99e104104

enlargement [30], indicating that small adipocytes have increasedpro-inflammatory properties compared to larger adipocytes. This isin congruence with the finding in the present study that adipocytesize is negatively related to plaque area, although there is noassociation with other plaque characteristics.

In this study we have evaluated different pvAT and plaque char-acteristics. The observed results all point in the same direction,thereby consistently supporting the hypothesis that pvATmay affectatherosclerosis. However, this is a cross sectional studyand thereforeit is not possible to make inferences about causality. It may well bepossible that the relation is the other way around. It can be specu-lated that signals from the vessel wall influence the surroundingadipose tissue. The studypopulationof patientswith coronaryarterydisease showed a distribution of atherosclerotic burden betweenandwithin subjects. Due to sample size itwas not possible to analysecharacteristics between subjects. Generalized estimating equationsanalysis was applied to overcome variance caused by clustering ofpvAT and plaque characteristics. Furthermore, it was not possible toanalyse plaque morphology in a quantitative fashion. The semi-quantitative measures of plaque characteristics that were used,have been shown to be well reproducible in other studies [31].Finallywedidnothavemeasures of obesityavailable in all patients. Itwould have been interesting to knowwhether the relation betweenpvAT and atherosclerotic plaque is partly driven by obesity.

In conclusion, pvAT quantity and macrophage infiltration arerelated to quantitative, morphologic and inflammatory plaqueproperties in patients with coronary artery disease. These resultssupport the concept that pvAT may play a role in the developmentof coronary atherosclerosis, although the causality of the relationhas yet to be determined.

Funding sources

This work was supported by the Leatare Foundation, Monacoand the Catharijne Foundation, the Netherlands.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.atherosclerosis.2012.08.031.

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