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Atherosclerosis 229 (2013) 338e347

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Atherosclerosis

journal homepage: www.elsevier .com/locate/atherosclerosis

Flow cytometry and gene expression profiling of immune cells of thecarotid plaque and peripheral blood

Zohara Sternberg a,*, Husam Ghanimb, Kristen M. Gillotti c, Joseph D. Tario Jr. c,Frederick Munschauer a, Richard Curl d, Sonya Noor d, Jihnhee Yu e, Julian L. Ambrus Sr. a,Paul Wallace c, Paresh Dandona b

a Stroke Center, Kaleida Medical Center, Buffalo, NY, USAbDiabetic Center of Western NY, Kaleida Medical Center, Buffalo, NY, USAcDepartment of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY, USAdDepartment of Vascular Surgery, Kaleida Medical Center, Buffalo, NY, USAeDepartment of Biostatistics, State University of NY at Buffalo, Buffalo, NY, USA

a r t i c l e i n f o

Article history:Received 15 January 2013Received in revised form8 April 2013Accepted 26 April 2013Available online 14 May 2013

Keywords:Carotid stenosisCell adhesionCell surface markersGene expressionImmunophenotypingMononuclear cellsThrombosis

Abbreviations: ATF, atrial fibrillation; BP, blood prcerebrovascular disease; CAD, coronary artery disease;healthy control; HF, heart failure; HMGB, high-mobimetalloproteinase; MAP, mitogen activated protein; Mripheral arterial disease; PMT, photomultipler tube;transforming growth factor; TF, tissue factor; TNF, tum* Corresponding author. Jacobs Neurological Institu

E-mail address: zs2@buffalo.edu (Z. Sternberg).

0021-9150/$ e see front matter Published by Elseviehttp://dx.doi.org/10.1016/j.atherosclerosis.2013.04.035

a b s t r a c t

Objectives: The relative contribution of the local vs. peripheral inflammation to the atherothromboticprocesses is unknown. We compared the inflammatory status of the immune cells of the carotid plaquewith similar cells in peripheral circulation of patients with advanced carotid disease (PCDs).Methods: Mononuclear cells (MNCs) were extracted from carotid endarterectomy (CEA) samples byenzymatic digestion and subsequent magnetic cell sorting. The cell surface antigenic expressions, andmRNA expression levels were compared between CEA MNCs and peripheral MNCs, using flow cytometryand RT-PCR techniques.Results: The percentages of resting MNCs were lower, and activated MNCs, particularly monocytes, werehigher in the CEAMNCs, as compared to the peripheral MNCs. The percentages of activated T cells and Bcells were higher in the peripheral MNCs of PCDs, than in healthy controls (HCs), but the percentages ofactivated monocytes did not differ between the two groups. The expression levels of both pro-inflammatory/pro-thrombotic (P38, JNKB-1, Egr-1 PAI-1, MCP-1, TF, MMP-9, HMGB-1, TNF-a, mTOR)and anti-inflammatory (PPAR-g, TGF-b) mediators were significantly higher in the CEA MNCs ascompared to the peripheral MNCs. Furthermore, MMP-9 and PPAR-g expression levels were higher in theperipheral MNCs of PCDs than HCs.Conclusion: The inflammatory status is higher in the immune cells of the carotid plaque, as compared tothose cells in the peripheral blood. The altered expression levels of both pro-inflammatory/pro-thrombotic and anti-inflammatory mediators in the milieu of the plaque suggest that the balance be-tween these various mediators may play a key role in carotid disease progression.

Published by Elsevier Ireland Ltd.

1. Introduction

Advanced carotid atherosclerosis accounts for 30e40% of casesof ischemic stroke in the general population, and it leads to func-tional impairment and death [1]. Immune-inflammatory and pro-

essure; BMI, body mass index; CVDM, diabetes mellitus; JNK, Jun N-lity group protein B; mTOR, mamNC, mononuclear cell; MCP, mon

PPAR, peroxisome proliferator-actor necrosis factor; VCAM, vascula

te, Buffalo, NY, USA. Tel.: þ1 716 8

r Ireland Ltd.

thrombotic mechanisms play key roles in the pathology ofatherosclerosis, including the progression of the plaque, and itsinstability leading to rupture [2].

Morphological studies of carotid atherosclerotic plaque showthe presence of a rich infiltrate, consisting of monocytes-

, cardiovascular; CEA, carotid endarterectomy; CDP, carotid disease patients; CVD,terminal kinase; Egr, early growth response; ERK, extracellular regulated kinase; HC,malian target of rapamycin; MRA, magnetic resonance angiography; MMP, matrixocyte chemotactic protein; NF-KB, nuclear factor kappa B; P, peripheral; PAD, pe-ivated receptor; PAI, plasminogen activator inhibitor; TLR, toll-like receptor; TGF,r cell adhesion molecule.59 4235; fax: þ1 716 859 2430, þ1 716 859 7573.

Z. Sternberg et al. / Atherosclerosis 229 (2013) 338e347 339

macrophages and lymphocytes, in the plaques of subjects whosubsequently developed stroke [3]. The infiltrated T cells are oftenautoreactive, capable of inducing the procoagulant TF in macro-phages [4].

Furthermore, the oxidized LDL-stimulated macrophages/monocytes of the plaque could activate signaling pathways, such asMAP kinases and transcription factors Egr-1 and AP-1, resulting inthe subsequent upregulation of the pro-inflammatory cytokineTNF-a [2]. In addition, the binding of the ligand, HMGB-1, to thepattern recognition receptors, TLR-2 or TLR-4, could also activatethe transcription factor NF-kB [5], promoting TNF-a release fromactivated monocytes [6,7]. TNF-a is capable of increasing theexpression levels of macrophage activation marker, MCP-1 [8], andmatrix proteinases [9], facilitating plaque destabilization andrupture. Blocking the activity of TNF-a, or disrupting the expressionof its genes, diminishes the development of atherosclerosis inapoE�/� mice [10,11]. In addition, the activation of serine/threoninekinase mTOR (the mammalian target for the antibiotic rapamycin),downstream the TLRs, also promotes atherosclerosis [12].

Unlike the detrimental effects of the activated T cells andmonocytes, the B type immune cells are atheroprotective. Areduction in B cells, via splenectomy, or the adoptive transfer ofspleen cells reduces atherosclerotic lesion development in apoE�/�

mice [13]. Nevertheless, a recent study shows that B cell depletionreduces atherosclerosis [14].

Immunohistochemical studies show the presence of B cells inthe endarterectomy samples, as well as the upregulation of anti-inflammatory mediators such as the cytokine TGF-b [15]. Viasuppressing MCP-1 expression, TGF-b is capable of limitingatherosclerosis [16]. In addition, the nuclear transcription factor,PPAR-g, also plays an anti-inflammatory role in the pathogenesisof atherosclerosis [17], via suppressing PAI-1transcription factor[18], and reducing TNF-a production in stimulated macrophages[19].

A gene array study reports dysregulation in the mRNA expres-sion levels of 48% genes in the smoothmuscles and endothelial cellsof the carotid plaque, as compared to normal arteries [20].Furthermore, microdissected carotid plaque samples demonstratedifferentially expressed patterns of genes in macrophage-rich re-gions as compared to the smooth muscle cell-containing regions ofthe plaque [21]. The inflammatory status of the plaque correlatedwith the histological and morphological features related to plaquevulnerability [21].

Patients with carotid disease also demonstrate a higher thannormal systemic inflammation, indicated by higher TNF-a andMCP-1 serum levels [22]. However, very few studies havemeasuredinflammation simultaneously in the carotid plaque and peripheralblood. These studies show higher intra plaque levels of the in-flammatory markers IL-6 [1] and MMP-9 [23], as compared to theirlevels in the blood.

Due to their critical role in inflammatory processes, we sepa-rated MNCs from endarterectomy samples of patients with carotiddisease, and compared their inflammatory status with MNCsderived from the peripheral blood, using flow cytometry and RT-PCR techniques.

2. Material and methods

2.1. Population

A total of forty (40) patients with advanced carotid disease(PCDs) (23 M, 17 F), ages 70.3 � 9.5 years, who were enlisted toundergo carotid endarterectomy (CEA) for extracranial high-gradeinternal carotid artery stenosis (75e99% stenosis indicated byDoppler sonography and/or MRA) were recruited from Buffalo

General Hospital, Buffalo, NY. The time delay between symptoms tothe carotid endarterectomy ranged between 1 and 10 daysdepending on the severity of the symptoms. For asymptomaticpatients, the average delay between diagnosis and the surgicalintervention was three months.

Patients’ sample was compared with 32 (18 M, 14 F) age-matched (68.3 � 9.2 years) healthy controls (HCs). The HCs wererecruited from the Buffalo General Hospital Volunteers office andamong the hospital staff. Exclusion criteria for HCs included reportsof any self and/or family history of vascular disease (CAD, CVD,PAD), renal disease, and DM.

All investigations were approved by the Institutional ReviewBoard of the University of Buffalo, and informed consent was ob-tained from PCDs before scheduled surgery, and from HCs.

2.2. Measurements

Due to the relatively limited number of MNCs in the CEA sam-ples, 16/40 CEA samples were devoted to flow cytometry studies,and 16/40 to gene expression profiling. The other 8 CEA sampleswere unsuitable for analysis for one reason or another. Among theHC blood samples, 12 MNCs samples were used for flow cytometry,and 20 others for gene profiling.

Using specific cell surface markers, the levels of resting (T cells,monocytes, B cells) and activated (HLADR, macrophages, plasmacells) MNCs were determined in 16 CEA samples, and comparedwith similar cells in the peripheral circulation of PCDs and HCs.

In addition, the mRNA expression levels of the pro-inflammatory/pro-thrombotic and anti-inflammatory mediatorswere compared between CEA MNCs and peripheral MNCs of PCDs.The pro-inflammatory/pro-thrombotic mediators included MAPkinases, P38, JNKb-1, ERK-1, and the serine/threonine kinase mTOR;the pattern recognition receptors, TLR-2 and TLR-4; the ligand tothe pattern recognition receptors, the transcription factor, HMGB-1,and the transcription factor Egr-1; the coagulation factors, PAI-1and TF; themarker of plaque vulnerability, MMP-9 and the cytokineTNF-a; and the chemokine, MCP-1. The anti-inflammatory media-tors included the transcription factor, PPAR-g and the cytokine,TGFb-1.

Furthermore, the expression levels of selected genes, includingHMGB-1, Egr-1, PAI-1, MMP-9, TNF-a, MCP-1, and PPAR-g werecompared between peripheral MNCs of 17 PCDs and 20 HCs.

2.3. Reagents

The following fluorochrome-conjugated anti-human mono-clonal antibodies to cell surface receptors were used to determinethe activation state of the MNC’s subpopulations. The CD45-APC(purchased from BD) was used to gate MNCs (T cells, monocytesand B cells). The CD3-FITC (purchased from R&D Systems), HLADR-PerCP (purchased from BD) were used to tag the resting and acti-vated Tcells respectively. The CD14-PerCP (purchased from BD) andCD68-FITC (purchased from Invitrogen) were used to tag theresting and activated monocytes/macrophages respectively. TheCD20-PE and CD69-FITC (purchased from BD) were used to tag theresting and activated B cells/plasma cells respectively. CD45-APCantibody-covered microbeads (Miltenyi Biotec) was used to sepa-rate CEAMNCs.

Additional reagents were Ficoll-Hypaque and human IgG (SigmaAldrich), endotoxin-free Blenzyme III (combination of collagenaseand thermolysin) and Porcine elastase II (Roche Applied Bio-sciences). Primers for the genes under study were commerciallyavailable.

Table 1Clinical characteristics of PCDs and HCs.

PCDs HCs

Clinical characteristicsNumber of subjects 40 (23 M, 17 F) 32 (18 M, 14 F)Age (years) 70.3 � 9.5 68.3 � 9.2BMI (kg/m2) 29.9 � 6.2 26.9 � 4.4Plasma glucose 132.5 � 44.2 NTSystolic BP/diastolic BP (mmHg) 146.2 � 24.9/74.42 � 10.1 NTSymptomatic carotid plaque (%) 32.5 (13/40) NA

CV drug usageAntihypertensives/diuretics (%) 100 41.6Antiplateletes/anticoagulants (%) 97.5 16.6Hypolipidemics (%) 75 16.6Antidiabetics (%) 42.5 (17/40) 0

Other CV diseasesCAD (%) 57.5 NTPAD (%) 30 NTHF (%) 10 NTATF (%) 5 NT

Abbreviations: ATF: atrial fibrillation, BMI: body mass index, CV: cardiovascular,CAD: coronary artery disease, HC: healthy control, HF: heart failure, PAD: peripheralartery disease, PCD: patient with carotid disease.

Z. Sternberg et al. / Atherosclerosis 229 (2013) 338e347340

2.4. Sample processing

Blood samples from both PCDs and HCs were drawn in EDTAtubes. Patients’ blood was drawn from an indwelling catheter onthe day of surgery and processed within an hour. Venous blood wasobtained from HCs and was processed similarly. CEA surgicalsamples were taken from patients and immediately transferred onice to the laboratory for processing. All samples were processed by asingle research scientist.

2.5. Immune cell isolation

2.5.1. Peripheral bloodThe peripheral blood was diluted 1:1 with PBS (pH ¼ 7.3) and

layered on Ficoll, spun at 400 � g for 30 min to obtain the pe-ripheral bloodMNCs. The cell pellets werewashed three times withthe Hanks Buffered Salt Solution (HBSS).

2.5.2. CEA samplesThe available ultrasound/MRA tests results were limited to the

degree of stenosis, but did not provide information on the plaquemorphology. Therefore, CEA samples were graded by the naked eyefor the degree of calcification, fattiness, and thrombosis. Thegrading ranged from 1 to 5 (1 being the lowest, and 5 being thehighest). Subsequently, CEA samples wereweighted, washed twice,and meticulously cleared of peripheral blood and thrombotic ma-terials. To release plaque infiltrating immune cells, the plaqueswere minced into paste and digested with 60 mg/ml Blenzyme IIIand 100 mg/ml elastase for an hour in 37 �C in a rotating incubator.The volume of the buffer was adjusted to the weight of the CEAsamples (10 ml buffer/g of tissue).

The digestion was stopped by the addition of HBSS, whichincluded 0.5% BSA and 2 mM EDTA. The resulting cell suspensionwas filtered through serial filters from coarse to fine 30 microns).The cell pellets was washed twice with the same solution and re-suspended in 1 ml of the buffer. Subsequently, MNCs were sepa-rated from endothelial and smooth muscle cells by a positive se-lection, using CD45þ antibody-covered microbeads, according tothe manufacturer’s instructions. In order to minimize MNCs’ acti-vation, the magnetic cell sorting was conducted in a cold room.

2.6. Quality control tests

1. Preliminary studies were conducted to determine whethermagnetic cell sorting activates MNCs, by receptor crosslinking.Ficoll-derived peripheral MNCs of 7 HCs were divided to twoequal parts. One part was subjected to magnetic cell sorting,similar to the protocol applied to separate MNCs from the CEAsamples, whereas the other part was not. The effect of magneticcell sorting on MNCs’ activation was assessed using cell surfaceantibodies to activated T cells and monocytes.

2. In order to exclude the possibility of digestive enzymes activatingMNCs, Ficoll-derived peripheral MNCs from two patients wererandomly selected and equally divided into two parts. One partwas treated with Blenzyme and elastase, similar to the protocolapplied to digest CEA samples, while the other part was not.

2.6.1. ImmunophenotypingThe protocol for immunophenotyping has been described in

details in Suppl I.

2.6.2. RT-PCR techniqueTotal RNA was isolated using a commercially available RNA-

queousÂ(R)-4PCR Kit (Ambion, Austin, TX). RT-PCR was performedusing Stratagene Mx3000P QPCR System (La Jolla, CA), Sybergreen

master mix (Qiagen, CA) and gene specific primers (Invitrogen). Allvalues were normalized to the expression of a group of house-keeping genes including actin, ubiquitin C and cyclophilin A.

2.7. Statistical analysis

All statistical analyses were performed using SPSS 14.0 for Win-dows (SPSS Institute, Chicago, Illinois, USA). Pairwise t-test was car-ried out to determine the differences in the percentages of restingand activated cells, and differences in gene expression levels, be-tween CEA MNCs and peripheral MNCs. Differences between PCDs/HCs were analyzed by independent t-test. The Holm’s sequentiallyrejective algorithmwas applied to correct for multiple comparisons[24], and P-values of �0.01 were considered significant.

Spearman correlation was used to investigate the associationbetween MNCs activation state, plaque morphology (thrombosis,calcification, fattiness), and patients’ clinical characteristics (age,BMI, systolic and diastolic BP, plasma glucose), and betweenexpression levels of inflammatory-related mediators, plaquemorphology, and patients’ characteristics.

3. Results

3.1. Patient’s characteristics

Table 1 presents the characteristics of the PCDs and HCs. Inaddition to the carotid disease, a significant number of PCDs pre-sented with other CV related diseases, including CAD, PAD, HF andATF. In addition, CV drugs were commonly used by PCDs. As manyas 100% of PCDs were on antihypertensive/diuretics, 97.5% were onantiplatelets/anticoagulants, and 75% were on hypolipidemicdrugs. Furthermore, 42.5% of PCDs were on anti-diabetic drugs.

However, a significantly lower percentage of HCs were on anti-hypertensives/diuretics (41.6%), antiplatelets/anticoagulants (16.6%),hypolipidemics (16.6%), and antidiabetic (0%) drugs. The carotid dis-ease was symptomatic in 32.5% (13/40) of PCDs. CEA samples weredefined as symptomatic if symptoms were consistent with stroke ortransient ischemic attack.

3.2. CEA characteristics

The mean � SD weight of the obtained CEA samples was0.92 � 0.4 g, ranging from 0.3 to 1.9 g. The amount of mRNA

Z. Sternberg et al. / Atherosclerosis 229 (2013) 338e347 341

extracted from plaque ranged from 0.4 to 2.0 mg depending on theweight and cellularity of the plaque.

3.3. Quality control tests

The magnetic cell sorting had no significant effect on either Tcells ormonocytes activation states. Approximately 13.4� 2% of thetotal T cells were activated after magnetic cell sorting, as comparedto 10.7 � 0.1% without this treatment. Similarly, 4.8 � 0.02% ofmonocytes were in activated states compared to 7.7 � 0.03%without this treatment. Furthermore, the percentage of activated Tcells, which comprise the majority of the cells in the plaque, wassimilar in the presence (13.6 � 0.6%) and absence (12.0 � 1.2%) ofBlenzyme and elastase treatments (P-values > 0.05). Our resultsagreewith those of an earlier study reporting no effect of enzymaticdigestion on cells’ antigenic properties [25].

3.4. Flow cytometry studies

Fig. 1 presents the mean � SEM percentages of resting (Fig. 1A)and activated (Fig. 1B) T cells, monocytes, and B cells in the CEAsamples, and in the peripheral blood of PCDs and HCs. Theresting cells are defined as: (‘Marker Positive Events’ e ‘ActivatedCells’/Marker Positive Events’)*100, whereas activated cells aredefined as: (‘Activated Cells’/‘Activated Cells’ þ ‘Resting Cells’)*100,as described in Suppl 1.

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Fig. 1. Comparison between resting and activated MNCs of the carotid plaque andperipheral circulation. The Figure presents the mean � SEM percentages of resting (A)and activated (B) T cells, monocytes, and B cells in the CEA MNCs and peripheral MNCsof PCDs and HCs. P � 0.01 was considered significant. *, Significant differences in thepercentages of MNC’s subpopulations between CEA samples and peripheral MNCs ofPCDs. y, Significant differences between the percentages of MNC’s subpopulations inthe peripheral MNCs of PCDs and HCs. Abbreviations: A: activated, CEA: carotid end-arterectomy, PCD: patients with carotid disease, HC: healthy control, R: resting.

The percentages of resting T cells were near-significantly lowerin the CEA MNCs, than in the peripheral MNCs of PCDs (70.5 � 4.5%vs. 80.9 � 3.2%, P ¼ 0.03) (P < 0.01 is considered statistically sig-nificant), but the percentages of peripheral resting T cells did notdiffer significantly between PCDs and HCs (85.0� 4.1%). In addition,the percentages of resting monocytes were significantly lower inthe CEA MNCs, than in the peripheral MNCs of PCDs (52.4 � 11.9%vs. 88.7� 3.7%, P¼ 0.006), but the percentages of peripheral restingmonocytes did not differ between PCDs and HCs (91.1 � 2.8%). Thepercentages of resting B cells were significantly lower in the CEAMNCs, than in the peripheral MNCs of PCDs (79.0 � 7.3% vs.98.8 � 0.2%, P < 0.001), but the percentages of peripheral resting Bcells did not differ between PCDs and HCs (99.5 � 0.09%) (Fig. 1A).

The percentages of activated T cells were near-significantlyhigher in the CEA MNCs, than in the peripheral MNCs of PCDs(30.4 � 5.1% vs. 20.8 � 3.4%, P ¼ 0.07), but the percentages of pe-ripheral activated T cells were not statistically different betweenPCDs andHCs (15.1�3.8%). In addition, the percentages of activatedmonocytes were significantly higher in the CEA MNCs, than in theperipheral MNCs of PCDs (37.75� 4.7% vs. 8.9� 2.3% P< 0.001), butthe percentages of peripheral activated monocytes were similarbetween PCDs and HCs (7.57 � 1.9%). Furthermore, the percentagesof activated B cells were higher in the CEA MNCs, than in the pe-ripheral MNCs of PCDs (17.9 � 5.2% vs.1.1 � 0.2%, P < 0.001). Inaddition, the percentages of the peripheral activated B cells werehigher for PCDs, than HCs (0.40 � 0.09%, P ¼ 0.004) (Fig. 1B).

Fig. 2 presents flow cytometric histograms of cells in the pe-ripheral blood (Fig. 2a) and the carotid plaques (Fig. 2b). The flowcytometric acquisition and analysis of leukocyte populations aredescribed in details in the legend to Fig. 2.

3.5. Gene profiling studies of CEA MNCs and peripheral MNCs ofPCD

Fig. 3 andTable2 compare (pair-wise comparison) themean� SEMof the mRNA expression levels of the pro-inflammatory/pro-thrombotic and anti-inflammatory mediators between CEA MNCsand peripheral MNCs of PCDs. The pro-inflammatory/pro-thromboticmediators included P38, JNKb-1, ERK-1 andmTOR (Fig. 3A); TLR-2 andTLR-4 (Fig. 3B); HMGB-1 and Egr-1 (Fig. 3C); PAI-1 and TF (Fig. 3D);MMP-9 and TNF-a (Fig. 3E); and MCP-1 (Fig. 3F). The anti-inflammatory mediators included PPAR-g (Fig. 3G) and TGFb-1(Fig. 3H).

The expression levels of P38 (0.184 � 0.02 vs. 0.116 � 0.01,P ¼ 0.004), JNKB-1 (0.239 � 0.02 vs. 0.122 � 0.01, P < 0.001), andmTOR (0.156 � 0.02 vs. 0.11 � 0.01, P ¼ 0.006) were significantlyhigher in the CEA MNCs, than in the peripheral MNCs, but afteradjusting for multiple comparisons (P values of �0.01 wasconsidered significant), the differences in ERK-1 expression levelswere not statistically significant (0.135 � 0.01 vs. 0.111 � 0.01,P ¼ 0.043) (Fig. 3A).

In addition, differences in the mRNA expression levels of TLR-2(0.089 � 0.01 vs. 0.07 � 0.02, P ¼ 0.34) and TLR-4 (0.218 � 0.02 vs.0.184 � 0.02, P ¼ 0.29) between CEA MNCs and peripheral MNCswere not statistically significant (Fig. 3B).

The expression levels of HMGB-1 (2.81 � 0.29 vs. 0.20 � 0.01,P < 0.001) and Egr-1 (1.03 � 0.13 vs. 0.13 � 0.03, P < 0.001) weresignificantly higher in the CEA MNCs, than in peripheral MNCs ofPCDs (Fig. 3C). Similarly, the expression levels of PAI-1 (2.48 � 0.4vs. 0.156 � 0.029, P < 0.001) and TF (6.15 � 1.2 vs. 0.177 � 0.07,P < 0.001) were significantly higher in the CEA MNCs, than in pe-ripheral MNCs of PCDs (Fig. 3D).

The expression levels of MMP-9 (3.97 � 1.0 vs. 0.165 � 0.05,P < 0.001) and TNF-a (0.8 � 0.14 vs. 0.10 � 0.01, P < 0.001)were significantly higher in the CEA MNCs, than in the

Fig. 2. Representative flow cytometric data from cells in the peripheral blood (a) and the carotid plaques (b). The acquisition and analysis of leukocyte populations in patient andnormal samples from peripheral blood and carotid plaques were performed by flow cytometry, using fluorescently-labeled antibodies with specificity for T cells (anti-CD3),monocytes (anti-CD14), and B cells (anti-CD20). Activation states for each of these subsets were determined by evaluation of HLA-DR on T cells, CD68 on monocytes, and CD69 on Bcells. To analyze the samples, the scatter characteristics of cellular populations were identified using a bivariate histogram of FSC vs. SSC (Panel A). An elliptical region (R1) was usedto circumscribe the leukocyte populations for both Peripheral (a) and CEA (b) samples, using the peripheral blood sample as a reference. Thereafter, scatter-inclusive cells (R1) weregated to a histogram of SSC vs. APC CD45 (Panel B). A rectangular region (R2) was used to discriminate CD45þ events from CD45� events. Cells that were scatter-inclusive andCD45þ were subsequently evaluated for the activation state of each respective measured population. For T cells, R2 events were gated to a bivariate histogram of CD3 vs. HLA-DR(Panel C); for monocytes, R2 events were gated to a bivariate histogram of CD14 vs. CD68 (Panel D), and for B cells, R2 events were gated to a bivariate histogram of CD20 vs. CD69(Panel E). Quadstat regions were placed on each of the aforementioned plots to distinguish between marker-positive events that were resting (R3), and marker-positive events thatwere activated (R4).

Z. Sternberg et al. / Atherosclerosis 229 (2013) 338e347342

peripheral MNCs of PCDs (Fig. 3E). Similarly the expressionlevels of MCP-1 were significantly higher in the CEA MNCs, thanin peripheral MNCs of PCDs (25.87 � 4.2 vs. 0.113 � 0.02,P < 0.001) (Fig. 3F).

Furthermore, the expression levels of PPAR-g (3.26 � 0.47 vs.0.156 � 0.03, P < 0.001) (Fig. 3G) and TGF-b (0.166 � 0.01 vs.0.123 � 0.01, P < 0.001) (Fig. 3H) were significantly higher in theCEA MNCs, than in peripheral MNCs of PCDs.

Fig. 3. Inflammatory-related mediators in the CEA MNCs and peripheral MNCs of PCDs. The Figure compares (pair-wise) the mean � SEM mRNA expression levels of the pro-inflammatory and anti-inflammatory mediators between CEA MNCs (n ¼ 16) and peripheral MNCs (n ¼ 16) of PCDs. The pro-inflammatory mediators included MAP kinases,P38, JNKb-1, ERK-1, and the serine/threonine kinase mTOR (A); the pattern recognition receptorsTLR-2 and TLR-4 (B); the ligand to the pattern recognition receptor, the transcriptionfactor HMGB-1, and the transcription factor Egr-1 (C); the coagulation factors PAI-1 and TF (D); the marker of plaque vulnerability, MMP-9, the pro-inflammatory cytokine TNF-a (E);and the pro-inflammatory chemokine, MCP-1 (F). The anti-inflammatory mediators included the transcription factor PPAR-g (G), and the cytokine TGF-b (H). The asterisks showsignificant differences in the mRNA expression levels between CEA MNCs and peripheral MNCs of PCDs. P � 0.01 was considered significant. Abbreviations: CEA: carotid endar-terectomy, HMGB: high-mobility group protein B, JNK: c-Jun N-terminal kinase, EGR: early growth response, ERK: extracellular regulated kinase, mTOR: mammalian target ofrapamycin, MAP: mitogen activated protein, MMP: matrix metalloproteinase, P38: mitogen-activated protein kinase, MNC: mononuclear cell, MCP: monocyte chemotactic protein,PCD: patient with carotid disease, P: peripheral, PPAR: peroxisome proliferator-activated receptor, PAI: plasminogen activator inhibitor, TLR: tall like receptor, TGF-b: transforminggrowth factor beta, TF: tissue factor, TNF: tumor necrosis factor.

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Regression analysis revealed an association for mTOR (r¼ 0.706,P ¼ 0.005) and TGF-b (r ¼ 0.583, P ¼ 0.002) expression levels, be-tween CEA MNCs and peripheral MNCs of PCDs.

3.6. Peripheral MNCs of PCDs and HCs

Fig. 4 and Table 3 compare the mean � SEM mRNA expressionlevels of the pro-inflammatory mediators HMGB-1 and Egr-1(Fig. 4A); PAI-1 and MMP9 (Fig. 4B); TNF-a, and MCP-1 (Fig. 4C);and the anti-inflammatory mediator, PPAR-g (Fig. 4D) betweenperipheral MNCs of PCDs and HCs.

The mRNA expression levels of HMGB-1 (0.236 � 0.01 vs.0.233 � 0.014) and Egr-1 (0.370 � 0.09 vs. 0.215 � 0.05) were notsignificantly different between peripheral MNCs of PCDs and HCs(Fig. 4A). However, MMP-9 expression levels were significantlyhigher in the peripheral MNCs of the PCDs than in HCs (0.394� 0.11vs. 0.08 � 0.01, P ¼ 0.002), but no significant group differences inPAI-1 expression levels were observed (0.313 � 0.07 vs.0.255 � 0.05) (Fig. 4B). The MCP-1 expression levels tended to be

lower in the peripheral MNCs of PCDs than in HCs (0.191 � 0.05 vs.0.243 � 0.03, P ¼ 0.08), but no group differences in TNF-a expres-sion levels were observed (0.119 � 0.02 vs. 0.120 � 0.028) (Fig. 4C).Furthermore, the expression levels of PPAR-g were higher in theperipheral MNCs of the PCDs than in HCs (0.183 � 0.03 vs.0.090 � 0.02, P ¼ 0.01) (Fig. 4D).

3.7. Correlation studies

A higher plaque weight was associated with the symptomaticplaque (r ¼ 0.62, P ¼ 0.010). Furthermore, a significant relationshipwas observed between plasma glucose levels, and MCP-1 expres-sion levels in the peripheral MNCs of PCDs (r¼ 0.568, P¼ 0.001). Noother significant associations between the expression levels of in-flammatory mediators (either in the CEA MNCs or peripheralMNCs), and patients’ clinical characteristics were observed. Inaddition, the percentages of resting and activated MNCs did notcorrelate with plaque’s characteristics or with patient’s clinical

Fig. 3. (Continued)

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characteristics. Values of lipid profile were not available for corre-lation studies.

4. Discussion

We separated MNCs from endarterectomy samples andcompared their inflammatory states with similar cells in the

Table 2Comparison of the mRNA expression levels of the pro-inflammatory and anti-inflammatory mediators between CEA MNCs and peripheral MNCs of PCDs.

CEA-MNC Peripheral-MNC P-value

Pro-inflammatory mediatorsP38 0.184 � 0.02 0.116 � 0.02 0.004JNK-1 0.239 � 0.02 0.122 � 0.01 <0.001ERK-1 0.135 � 0.01 0.111 � 0.01 0.043mTOR 0.156 � 0.02 0.110 � 0.01 0.006TLR-2 0.090 � 0.01 0.077 � 0.02 0.34TLR-4 0.218 � 0.02 0.184 � 0.02 0.29HMGB-1 2.81 � 0.29 0.205 � 0.01 <0.001Egr-1 1.03 � 0.13 0.130 � 0.03 <0.001PAI-1 2.488 � 0.45 0.156 � 0.03 <0.001TF 6.58 � 1.20 0.177 � 0.07 <0.001MMP-9 3.97 � 1.07 0.165 � 0.05 <0.001TNF-a 0.80 � 0.14 0.10 � 0.01 <0.001MCP-1 25.87 � 4.2 0.11 � 0.02 <0.001

Anti-inflammatory mediatorsPPAR-g 3.26 � 0.47 0.297 � 0.14 <0.001TGF-b 0.166 � 0.01 0.123 � 0.01 <0.001

P < 0.01 is statistically significant. Abbreviations as in Fig. 3.

peripheral blood of patients suffering from advanced carotid dis-ease, by flow cytometry and RT-PCR techniques.

Our results show that most MNCs in the CEA samples were Tcells and monocytes, whereas significantly smaller percentageswere B cells. These results are consistent with morphologicalstudies showing the presence of T cells, monocytes, and B cells inthe carotid plaque using CD3, CD14 and CD20 cell surface anti-bodies respectively [26]. However, a recent flow cytometry study,which used CD19 antibody as the B cells surface marker, did notobserve a significant B cell labeling in the carotid plaque [25]. Thisdiscrepancy may stem from the nature of CD19 being expressedmainly in the early pro-B cell stage, whereas the CD19 expression isdown-regulated upon B cell maturation to plasma cells.

In addition, we observed higher percentages of activated MNCs,especially activated monocytes, in the CEA MNCs, as compared tothe peripheral MNCs. These observations are consistent with theresults of our genetic study showing higher expression levels of thepro-inflammatory markers in the CEA MNCs, as compared to theirexpression levels in the peripheral MNCs. For a number of inflam-matory mediators, including TF and MMP-9, increases in the mRNAexpression levels in the CEA MNCs approximated 100 times that ofthe peripheral MNCs, suggesting heightened immune cells-derivedinflammatory responses in themilieu of the plaque [1]. Moreover, itsuggests that these mediators may play critical roles in the pro-gression of carotid disease, including plaque rupture andthrombosis.

The increases in the expression levels of the inflammatory me-diators were not limited to the pro-inflammatory/pro-thrombotic

HMGB-1 Egr-1

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Fig. 4. Inflammatory-related mediators in the peripheral MNCs of PCDs and HCs. The Figure compares the mean � SEM mRNA expression levels of the proinflammatory tran-scription factors, HMGB-1 and Egr-1 (A); the coagulation factor, PAI-1, and the marker of plaque vulnerability, MMP9 (B); the cytokine TNF-a and the chemokine MCP-1 (C); and theanti-inflammatory transcription factor, PPAR-g, between peripheral MNCs of PCDs (n ¼ 17) and HCs (n ¼ 20). The asterisks show the significance of the differences in the expressionlevels of inflammatory-related markers between peripheral MNCs of PCDs and HCs. P � 0.01 was considered significant. Abbreviations as in Fig. 3.

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ones. The anti-inflammatory mediators, such as PPAR-g and TGF-b,were also significantly elevated in the CEA MNCs, as compared totheir expression levels in the peripheral MNCs. The increase inPPAR-g in the CEA MNCs also approximated 100 times that of theperipheral MNCs. Our results collectively suggest that the balancebetween pro-inflammatory/pro-thrombotic mediators and anti-inflammatory mediators may modulate plaque progression.

Table 3Comparison of the mRNA expression levels of the pro-inflammatory and anti-inflammatory mediators between the peripheral (P) MNCs of PCDs and HCs.

PCD (P-MNCs) HC (P-MNCs) P-value

Pro-inflammatory mediatorsHMGB-1 0.236 � 0.01 0.233 � 0.01 0.79Egr-1 0.37 � 0.09 0.169 � 0.03 0.12MMP-9 0.394 � 0.11 0.08 � 0.01 0.002PAI-1 0.31 � 0.07 0.255 � 0.05 0.79MCP-1 0.191 � 0.05 0.243 � 0.03 0.089TNF-a 0.119 � 0.02 0.120 � 0.02 0.54

Anti-inflammatory mediatorPPAR-g 0.183 � 0.03 0.09 � 0.02 0.01

P < 0.01 is statistically significant. Abbreviations as in Fig. 3.

However, it should be noted that inflammatory mediators such asp38 [27,28] and PPAR-g [17,29] have the potential to exert both a pro-atherogenic and an anti-atherogenic role. Nevertheless, the increasein p38 and other MAP kinases has been shown to accompany theprogression of carotid atherosclerosis in humans [30].

Due to a relatively small sample size, this study did notanalyze results in patient’s subgroups. Therefore, future studies,involving a larger cohort, should examine immune inflammatorystatus in symptomatic/asymptomatic and in diabetic/non-diabeticpatients.

One of the interesting observations in this study was the sig-nificant increase in the expression levels of HMGB-1 in the immunecells of the carotid plaque. HMGB-1, also known as amphoterin, is atranscription factor, and a key cytokine with a potential role innucleosome formation [31]. However, upon release from activatedmacrophages, HMGB-1 has the potential to promote key processesinvolved in the initiation and progression of atherosclerosis,including inflammation [32] and smooth muscle cell proliferation[33]. HMGB-1expression levels are enhanced in the atheroscleroticlesions, as compared to the levels in the normal arteries [33]. Inaddition, HMGB-1 reduction is associated with a reduced athero-sclerotic plaque size and favorable changes in the plaque

Z. Sternberg et al. / Atherosclerosis 229 (2013) 338e347346

morphology in mice lacking RAGE, the receptor for advanced gly-cation end-products [34].

In addition to the RAGE, signaling through TLR-2, TLR-4 alsocontributes toHMGB-1-induced inflammatory responses [6].Wedidnotmeasure RAGE expression levels, but TLR-2 and TLR-4 are knownto be the main receptors mediating HMGB-1-induced pro-inflammatory signaling in activated monocytes [5]. However, thesetwo receptors were not upregulated in the CEA MNCs. It is possiblethat HMGB-1 signaling through TLR-2 and TLR-4, in MNCs, occurs inthe early stages of the carotid lesion development, whereas thesereceptors are downregulated in advanced carotid lesions [5].

In addition, the percentages of activated monocytes, known tobe critical in the pathology of atherosclerosis [35], were similarbetween peripheral MNCs, and except for MMP-9 and PPAR-g,which showed differences, the expression levels of other inflam-matory markers were not significantly different between PCDs andHCs.

One factor that could lower the levels of activated monocytes,and the expression levels of inflammatory markers in the periph-eral MNCs of PCDs is the aggressive treatment of this group with acombination of CV drugs, including antihypertensives, anti-platelets, and hypolipidemics. In addition, many patients were us-ing multiple classes of CV drugs. Many CV drugs possess anti-inflammatory properties, capable of reducing peripheral inflam-mation [36e38]. Similarly, anti-diabetic drugs reduce immune cellactivation and the production of inflammatory mediators [22,39].Nevertheless, despite the chronic use of CV drugs, these patientspresented with severe carotid stenosis, suggesting the complexityof factors, which are involved in the progression of the carotidplaque.

Whether CV drugs influence MNC’s subpopulations differentlywarrants further investigations. However, unlike their levels in theperipheral circulation, the significantly higher percentages of acti-vated monocytes in the CEA samples suggest that CV drugs may beless effective in reducing activated monocytes in the advancedcarotid plaque [40].

Furthermore, the relative contribution of the resting vs. acti-vated MNCs to the inflammatory processes of the carotid plaque isnot well known. The results of an in vitro study show [41] that thepro-inflammatory and plaque destabilizing cytokine, IL-17, is pro-duced by both resting T cells and B cells as well as by activatedmonocytes and activated B cells. Similarly, both resting and acti-vated T cells could stimulate endothelial cells, promoting therelease of pro-atherogenicmediators, such as E-selectin and VCAM-1 [42].

In addition, MNCs are heterogeneous in nature. For example,different subsets of T cells are found in atherosclerotic plaque,including Th1 cells, Th2 cells, dendritic cells, and T regulatorycells, to mention a few. Based on the nature of their cytokineproduction, Th1 cells are thought to promote atherogenesis,whereas Th2 cells inhibit it [43]. Furthermore, dendritic cells areknown to promote atherosclerosis through pathways involvingVCAM1 [44], whereas CD4þ regulatory T cells augment TGF-bproduction, exerting anti-inflammatory and anti-atherogenic ac-tivities [45].

Similarly, more than one monocyte subtype may be involved inatherogenesis. Monocytes’ heterogeneity depends on a set of che-mokine receptors expressed on these cells, which dictates theirmigration properties, their role in plaque formation, and theiraccumulation in the plaque in the form of macrophages [46,47].Furthermore, differences in B cell’s subpopulations have beenobserved, and their ability to either promote or suppress athero-genic processes have been documented [48,49].

Variations in MNC’s sub-populations and subtypes, the in-teractions among the different cells, and their derived mediators

are few of the complex factors contributing to the progression ofthe carotid plaque. Whether pharmacological treatment and thereduction of systemic inflammation prevents or slows down theprogression of the advanced carotid disease warrants further in-vestigations. This knowledge will have a significant impact onfuture patients’ treatment.

4.1. Study limitations

All our patients were using CV drugs; therefore, we could notexamine the possible effects of these drugs on immune inflam-matory cells. In addition, MRA or ultrasound datawere not availablefor HC group, and therefore, one could not exclude the existence ofcarotid and or coronary artery disease in this group.

Funding source

This study was supported in part by a grant from “Jog for theJake,” a local philanthropic Foundation.

Disclosure

Authors report no conflict of interest.

Acknowledgment

The authors thank the nurses in the surgical department ofBuffalo General Medical Center for their help in providing endar-terectomy samples.

Appendix A. Supplementary data

Supplementary data related to this article can be found online athttp://dx.doi.org/10.1016/j.atherosclerosis.2013.04.035.

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