autoimmunity, may 2011; 44(3) 1–10

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Atherosclerosis development in SLE patients is not determined by

monocytes ability to bind/endocytose Ox-LDL

LINA M. YASSIN1, JULIAN LONDONO2,3, GUILLERMO MONTOYA2,3,

JUAN B. DE SANCTIS4, MAURICIO ROJAS1, LUIS A. RAMIREZ5, LUIS F. GARCIA1, &

GLORIA VASQUEZ1,5

1Grupo de Inmunologa Celular e Inmunogenetica, Facultad de Medicina, Universidad de Antioquia, Medelln, Colombia,

2Grupo de Investigacion en Sustancias Bioactivas, Instituto de Qumica Farmaceutica, Universidad de Antioquia, Medelln,

Colombia,3

Grupo de Inmunomodulacion, Facultad de Medicina, Universidad de Antioquia, Medelln, Colombia,4

Instituto de

Imunologa, Universidad Central de Venezuela, Caracas, Venezuela, and5

Grupo de Reumatologa, Facultad de Medicina,

Universidad de Antioquia, Medelln, Colombia

(Submitted 6 April 2010; revised 30 September 2010; accepted 6 October 2010)

Abstract

Patients with systemic lupus erythematosus (SLE) have a high risk of developing cardiovascular disease; however, themechanisms involved in the early onset of atherosclerosis in these patients are not clear. Scavenger receptors, CD36 andCD163 are expressed by mononuclear phagocytes and participate in the binding and uptake of oxidized low-densitylipoproteins (Ox-LDL), contributing to foam-cells formation and atherosclerosis development. The aim of the present studywas to evaluate CD36 and CD163 expression and Ox-LDL removal by monocytes from SLE and atherosclerotic patients,compared to similar age-range healthy controls. Healthy controls, SLE, and atherosclerotic patients were evaluated for carotidintima media thickness (CIMT), lipid profile, and native LDL (N-LDL) and Ox-LDL binding/endocytosis. SLE patientspresented decreased high-density lipoproteins (HDL) and increased Triglyceride levels, and half of the SLE patientshad increased CIMT, compared to their healthy controls (HC

SLE). The number of CD14CD163 cells was increased

in atherosclerosis healthy controls (HCAtheros) compared to HCSLE, but there were no differences between SLE oratherosclerotic patients and their respective healthy controls. Clearance assays revealed a similar capacity to bind/endocytoseOx-LDL by monocytes from SLE patients and HCSLE, and an increased binding and endocytosis of Ox-LDL by monocytesfrom atherosclerotic patients, compared to HCAtheros. The decreased CD36 and CD163 expression observed inatherosclerotic and SLE patients, respectively, suggest that these inflammatory conditions modulate these receptorsdifferentially. The increased CIMTobserved in SLE patients cannot be explained by Ox-LDL binding/endocytosis, which wascomparable to their controls.

Keywords: Atherosclerosis, SLE, scavenger receptors, OxLDL, endocytosis

Introduction

Atherosclerosis is nowadays considered a chronic

inflammatory disease occurring in the arterial walls

[1,2]. The process is initiated when plasma levels of

very low-density lipoproteins (VLDL) and LDL rise,

diffuse into the artery wall to an extent that exceeds

the capacity for elimination, and are retained in the

extracellular matrix activating the endothelium [1].

The endothelial activation is characterized by an

increased expression of ICAM-1 and V-CAM-1,

production of the monocyte chemotactic protein-1

[1,3], important for recruiting monocytes [4], and

detaching of endothelial cells generating gaps between

them [2]. Increased intercellular space and activation

of the endothelium allows for the accumulation ofLDL, and the adherence and migration of lympho-

cytes and monocytes to the subendothelium and

intima [1].

LDL are oxidized by reactive molecular species

generated by the endothelial cells and activated

monocytes/macrophages [5]. Ox-LDL can be recog-

nized by scavenger receptors (SR), expressed on the

Correspondence: G. Vasquez, Grupo de Reumatologa, Facultad de Medicina, Universidad de Antioquia, Calle 64 51D-154, Bloque 6,Medelln, Colombia. Tel: 0574-2106453. Fax: 0574-2106450. E-mail: [email protected]

Autoimmunity, May 2011; 44(3): 110q Informa UK, Ltd.ISSN 0891-6934 print/1607-842X onlineDOI: 10.3109/08916934.2010.530626


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recruited monocytes and macrophages [6]. Mono-

cytes differentiate into macrophages in the presence of

macrophage colony stimulating factor produced by

endothelial cells (EC) and smooth muscle cells [7].

Macrophages up-regulate the SR expression, increas-

ing Ox-LDL uptake [6] and leading to the delivery of

Ox-LDL into the lysosomes, where they are hydro-

lyzed to protein and cholesteryl ester.Cholesterol is then released into the cytoplasm,

where it accumulates and suffers re-esterification,

transforming the macrophages into foam cells, the

hallmark of the atherosclerotic plaque [5,8]. The

interaction between EC, monocytes/macrophages,

and T cells within the arterial walls, results in the

activation and production of inflammatory cytokines

such as IL-1b, TNF-a, and IFN-g, increasing tissue

damage and promoting the formation of atherosclero-

tic plaques [1,2].

Systemic lupus erythematosus (SLE) is also a

systemic inflammatory disease, in which, among other

alterations, there is strong evidence of early develop-ment of atherosclerosis and its consequences, like

myocardial infarction [9 13]. Evidence of the link

between SLE and atherosclerosis was provided by a

mouse model of SLE-atherosclerosis (gld.apoE2/2).

These mice showed accelerated atherosclerosis

induced by the autoimmune phenotype, which was

associated with a decreased ability to remove

apoptotic cells [14]. In humans, comparisons between

SLE patients and healthy controls with similar

demographic characteristics and risk factors, showed

that the prevalence of atherosclerosis was higher in

SLE patients [10].Also, there is a statistically significant increase of

coronary heart disease and stroke in SLE patients that

cannot be fully explained by the traditional Framing-

ham risk factors [15]. Thus, the inflammatory

phenomenon occurring in SLE patients may trigger

the development of atherosclerosis [10].

In this respect, SRs such as CD36 and CD163,

besides their capacity to bind Ox-LDL, also bind

apoptotic cells [6], which are the main targets of the

autoimmune response in SLE [16]. We previously

observed a decreased apoptotic cells removal in SLE

patients associated with cellular activation regardless

of CD36 [17].To further explore possible mechanisms for athero-

sclerosis development in SLE, the present study

compared the carotid intima media thickness

(CIMT), the lipid profile and the expression of

CD36 and CD163 in SLE patients, healthy SLE

controls, atherosclerotic patients and healthy athero-

sclerosis controls, as well as the capacity of their

monocytes to bind/endocytose Ox-LDL or native

LDL (N-LDL). Even though more than half of SLE

patients had increased CIMT, these patients did not

show differences in the number of CD36 and

CD163 monocytes, nor in Ox-LDL binding and

endocytosis, compared to HCSLE.

However, HCAtheros showed increased number of

circulating CD163 monocytes, as compared to

HCSLE, but no differences were observed between

patients and their respective healthy controls. The

expression of CD163 was decreased in SLE patients

monocytes compared to HCSLE, meanwhile mono-

cytes from atherosclerotic patients presented

decreased expression of CD36 compared to HCAtheros.

Also, Ox-LDL binding and endocytosis was increased

in atherosclerotic patients as compared to HCAtheros.

Patients and methods

Reagents

RPMI-1640, PBS, and fetal calf serum (FCS) were

purchased from GIBCO-BRL (Grand Island, NY,

USA); penicillin, streptomycin, Ficoll-Hypaque,

pooled human serum AB (PHS), and Limulus

Amebocyte Lysate assay kits (LAL) from Biowhittaker

(Walkersville, MD, USA); trypan blue, sodium azide,

bovine serum albumin, and 1,10dioctadecyl-3,3,30,30-

tetramethylindocarbocyanine (DiI) from SIGMA (St

Louis, MO, USA) and Invitrogen (Carlsbad, CA,

USA). Dimethyl sulfoxide (DMSO) was purchased

from MERCK (Darmstadt, Germany). Anti-CD14-

FITC (clone M5E2), anti-CD36-PE (clone CB38),

anti-CD163-PE (clone GHI/61), anti-HLA-DR-Cy

(clone TU36) and isotype control antibodies IgG2ak-

FITC (clone G155-178), IgG1k-PE (MOPC-21),

IgMk-PE (clone G155-178), and IgG2bk-PE-Cy5

(clone 27-35) were purchased from BD Bioscience(San Jose, CA, USA). Anti-CD36-FITC (clone

FA6.152) was obtained from Beckman-Coulter (Brea,

CA, USA) and anti-CD163-FITC (clone ED2) from

AbD Serotec (Oxford, UK). CALTAG Cal-Lyse

Lysing Solution was obtained from Caltag Laboratories

Inc. (Burlingame, CA, USA). Quantification Kit-Rapid

was purchased from Fluka Chemika AG (Buchs,

Switzerland) and the kit to evaluate the lipid profile

(Architect/Aeroset system) was obtained from Abbott

Diagnostics (Chicago, IL, USA). All media were

negative for LPS as evaluated by LAL assay.

Patients and controls

Thirty-eight SLE patients diagnosed according to the

American College of Rheumatology Criteria (ACR)

[18] and 21 patients with a previous vascular event

explained by atherosclerosis and no autoimmune

associated disease were recruited at the Hospital

Universitario San Vicente de Paul, Medelln, Colom-

bia. Two groups of healthy controls were also

recruited: 29 healthy controls with similar age-range

to SLE patients (HCSLE) from medical and laboratory

personnel of the Universidad de Antioquia, and 10

L. M. Yassin et al.2


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healthy controls with similar age-range to athero-

sclerotic patients (HCAtheros) at the outpatient clinic of

the Universidad Hospital Caracas, Venezuela. Patients

with SLE were classified according to the systemic

lupus erythematosus disease activity index (SLEDAI)

[19]. Healthy controls were included if they had

normal blood pressure, were nonsmokers, and showed

normal results on a routine physical examination. All

patients and controls signed an informed consent

form previously approved by the Ethics Committee of

the Instituto de Inmunologa, Universidad Central

de Venezuela and by the Ethics Committee of the

Instituto de Investigaciones Medicas, Facultad de

Medicina, Universidad de Antioquia.

Clinical parameters

The presence of atherosclerosis was established by

measuring the CIMT. Seven HCSLE, 10 HCAtheros,

16 SLE patients, and 9 atherosclerotic patients were

evaluated for carotid duplex (Aloka Prosound 3500Vascular echography) in the vascular service of

Hospital Universitario San Vicente de Paul, Medelln.

The serum total cholesterol (TC), triglycerides

(TGC), high-density lipoproteins (HDL), and LDL

levels were measured using the spectrophotometric

technique at the Hospital Universitario San Vicente de

Paul clinical laboratory and the Cardiology outpatient

clinic of the Hospital Universitario de Caracas in 14

atherosclerotic patients, 21 SLE patients, 5 HCSLE,

and 10 HCAtheros.

LDL isolation, oxidation, and labelingFifty milliliters of sodium citrate-anticoagulated blood

were obtained from healthy nonsmoking normolipe-

mic donors (age 2025) and centrifuged at 400g for

20 min. LDL were purified in a discontinuous density

gradient according to the method of Wilson et al. [20].

Human plasma (3.2 ml) from healthy donors was

centrifuged at 105,000g(Beckman XL-100) at 58C for

12 h in the presence of 1.6 ml NaCl (1006 g/ml) to

obtain the mixture of chylomicrons and VLDL.

The remaining plasma was mixed with 1.6 ml of

KBr (1.182g/ml) and centrifuged at the same

conditions for 18h, in order to obtain the LDL

fraction at the upper phase and the HDL fraction in

the lower phase. Purity of these fractions was

determined by SDS-PAGE electrophoresis, as com-

pared to total plasma (Figure 1A). Proteins in the

LDL fraction were quantified by Bradford using a

commercial kit, recovering 350400mg/ml of protein.

To obtain Ox-LDL, the purified LDL fraction was

incubated with an oxidizing solution of CuSO4(100mM) for 12h at 378C [21], and the reaction

stopped by transferring the LDL solution to 48C.

Oxidation of the lipid fraction was quantitatively

determined by the thiobarbituric acid reactivesubstances method [22], with minor modifications,

using a solution of thiobarbituric acid (0.67%),

trichloroacetic acid (15%), and HCl (0.1N). Changes

in the net charge of the protein fraction (Apo B 100) of

LDL due to oxidation, were evaluated by agarose

(0.8%) gel electrophoresis in barbital buffer (0.05 M,

pH 8.6, 100 V) for 2 h and stained with sudan black

(0.1% in ethanol) for 10 h (Figure 1B).

LDL fluorescent labeling with DiI was performed as

previously described [23,24], with minor modifications.

Ox-LDL and N-LDL were incubated with 300mg

DiI/mg of protein for 12 h at 378C in the dark. Labeled

Ox-LDL (Ox-LDL-DiI) and N-LDL (N-LDL-DiI)were centrifuged for 10 min at 400g at 48C. Fluor-

escence efficiency was evaluated by flow-cytometry

(Figure 1C) and labeled LDL stored at 48C until use.

Figure 1. LDL isolation, oxidation, and labeling. (A) SDS-PAGE of lipoprotein fractions CM/VLDL (second lane), LDL fraction (third

lane),HDL fraction (fourth lane),and whole plasma (fifth lane).The first lane shows themolecular weight ladder. (B)Agarose gelshowing the

electrophoretic mobility differences between N-LDL and Ox-LDL. (C) Flow cytometry LDL-DiI labeling efficiency; nonlabeled Ox-LDL

(shaded histogram), DiI-labeled Ox-LDL (opened histogram).

Ox-LDL binding/endocytosis in SLE 3


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Evaluation of CD36 and CD163 expression by flow

cytometry

One hundred microliters of EDTA-anticoagulated

blood from patients and controls were stained with

anti-CD14-FITC (15ml) and anti-HLA-DR-Cy

(10ml), plus anti-CD163-PE (10ml), anti-CD163-

FITC (5ml), anti-CD36-PE (6ml), or anti-CD36-

FITC (5ml), or with their respective isotype controlantibodies and incubated for 20 min in the dark at

room temperature (RT). Then, 100ml of Caltag

buffer were added to each tube, incubated for 10 min

under the same conditions, followed by addition of

1 ml of distilled water and incubated for 15 min.

Thereafter, the cells were placed in washing buffer

(PBS, 0.1% NaN3, 2% PHS) and evaluated by flow-

cytometry (Coulter Epics XLe FlowCytometer,

Coulter International Corporation, Hialeah, FL,

USA) and analyzed using the software Windows

Multiple Document Interface 2.8, WinMDI (Scripts

Research Institute, La Jolla, CA, USA). Leukocytes

were counted in hemocytometer with 0.3% acetic acid.

Peripheral blood mononuclear cells (PBMC) isolation

PBMCs from patients and controls were isolated by

centrifugation on Ficoll-Hypaque gradient, placed in

freezing medium (90% heat inactivated FCS: 10%

DMSO) and frozen at 21968C unt il use for

endocytosis assays.

Ox-LDL and N-LDL binding/endocytosis assays

Five hundred thousand (5 105) PBMCs from

healthy controls, atherosclerotic patients, and SLE

patients were incubated with different concentrations

of Ox-LDL-DiI (0.2, 0.5, 1.0, 10mg/ml) or with

10mg/ml of N-LDL-DiI, in a final volume of 1 ml of

complete medium (RPMI-1640 plus 10% FCS,

penicilline-100 U/ml, Streptomycin-100mg/ml)

(CM) for 1 h at 378C. Thereafter, the cells were

centrifuged at 400g for 5 min and incubated with

washing buffer for 10 min at RT and washed again.

Cells were stained with 5ml of anti-CD14-FITC for

30 min at 48C and evaluated by flow cytometry.

Binding/endocytosis of Ox-LDL-DiI and N-LDL-

DiI was analyzed in the CD14 gate. Two parameters

were evaluated: binding/endocytosis (total) of LDL

and endocytosis, in the presence of 0.4% trypan blue

[25]. Binding of LDL was calculated (total LDL

endocytosed).

Statistical analysis

Unpaired t-test was used for comparisons of CD36

and CD163, CIMT, lipid profile parameters, and

N-LDL binding/endocytosis evaluation. Two-wayANOVA with Bonferroni post-test was used for Ox-

LDL binding/endocytosis assays. Pearson correlation

was used to correlate the number of cells expressing

CD36 and CD163 with the SLEDAI score. The data

was analyzed with GraphPad Prism version 5.0

(GraphPad Software, San Diego, CA, USA).

Results

Clinical and demographic characteristics of studied subjects

The mean age for HCSLE, SLE patients, HCAtheros,

and atherosclerotic patients were 28 ^ 8, 34 ^ 12,

65 ^ 5, and 61 ^ 9, respectively (Table I). SLE

patients were under different immunosuppression

Table I. Clinical characteristics of patients and controls.

HCSLE SLE HCAtheros Atherosclerosis

Number 29 38 10 21

Age 28 ^ 8* 34 ^ 12 65 ^ 5 61 ^ 9

Treatment PDN, CLQ,

Azathioprine,

Cyclophosphamide,

Mycophenolate mofetil

Captopril,

Metoprolol, Warfarin,

ASA and statins

CIMT 0.66 ^ 0.17

n

7

0.87 ^ 0.29

n

16

0.73^ 0.13

n

10

1.15 ^ 0.26

n

9TC levels (mg/dl) 191 ^ 24

n 5

218^ 63

n 21

163^ 22

n 10

214^ 69{

n 14

TGC levels (mg/dl) 86 ^ 12

n 5

248 ^ 177

n 20

87^ 9

n 10

181^ 130

n 13

LDL levels (mg/dl) 112 ^ 22

n 5

117^ 25

n 13

95^ 9

n 10

129^ 59

n 12

HDL levels (mg/dl) 61 ^ 6

n 5

43^ 17k

n 18

52 ^ 11

n 10

51 ^ 17

n 13

*Data show the mean ^ SD. HC, Healthy controls; HCSLE, Healthy controls with similar age-range to SLE patients; HCAtheros, Healthy

controls with similar age-range to atherosclerosis patients; PDN, Prednisolone; CQL, chloroquine; ASA, Acetylsalicylic acid; CIMT, Carotid

Intima Media Thickness; TC, Total Cholesterol; mg/dl, milligrams/deciliter; TGC, Triglycerides; LDL, Low Density Lipoproteins; and

HDL, High Density Lipoproteins. Results were analyzed by unpaired t-test; (p , 0.001) compared to HCAtheros; (p , 0.05) compared to

HCAtheros;{ (p , 0.05) compared to HCAtheros;

(p , 0.05) compared to HCAtheros;k (p , 0.05) compared to HCSLE.



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schedules with prednisone, chloroquine, cyclopho-

sphamide, mycophenolate mofetil, and azathioprine,

and their SLEDAI score was 12 ^ 9. Atherosclerotic

patients were also under different treatments, with

captopril, metoprolol, warfarin, acetylsalicylic acid

(ASA) and statins (Table I).

Among the clinical parameters, CIMT was found

increased in all nine atherosclerotic patients tested

(1.15 mm^ 0.26) compared to 10 HCAtheros evalu-

ated (0.73 mm ^ 0.13, p , 0.05). The 16 SLE

patients presented a CIMT of 0.87 mm^ 0.29,

which did not reach statistical significant difference

when compared with 7 HCSLE evaluated; however,

since normal values of CIMT range from 0.36 to

0.9 mm and CIMT higher than 0.9 mm is associated

with a high prevalence of cardiovascular disease [26],

it is noteworthy that 9/16 of the SLE patients (56%)

evaluated presented a CIMT higher than 0.9 mm.

TC levels were increased in all 14 atherosclerotic

patients (214 ^ 69 mg/dl) compared to 10 HCAtheros

tested (163^

22 mg/dl p,

0.05). LDL levels weresimilar between the patients and their respective

healthy controls. TGC levels showed a nonsignificant

trend to increased values in the 20 SLE patients

(248 ^ 177mg/dl) compared to 5 HCSLE evaluated

(86 ^ 12 mg/dl). HDL levels were decreased in all 18

SLE patients (43 ^ 17 mg/dl) as compared to the 5

HCSLE (61 ^ 6 mg/dl, p , 0.05) studied.

LDL isolation, oxidation, and labeling

The fractions obtained from plasma of healthy

donors by ultracentrifugation, in the presence of

salts, as described in patients and methods, werecompared with whole plasma by SDS-PAGE

(Figure 1A). The LDL fraction showed mainly one

band of high molecular weight (.170 kDa) that may

correspond to the protein ApoB100, according to the

molecular weight described for apolipoproteins [5].

Several bands were observed in the fractions that

according to the isolation process should contain

CM/VLDL and HDL. Since oxidation increases the

negative net charge of LDL [27], the oxidation and

purity of Ox-LDL was verified by agarose gel

electrophoresis, confirming that the Ox-LDL band

migrated to the cathode, compared with the N-LDL

band (Figure 1B). DiI labeling efficiency, asconfirmed by flow cytometry, showed that 8090%

of LDL were labeled, compared with nonlabeled

LDL (Figure 1C).

CD14

CD163

cells are increased in HCAtheros compared

to HCSLE , and CD36 and CD163 expression are

differentially altered in atherosclerotic and SLE patients

Since CD36 and CD163 are involved in Ox-LDL and

apoptotic cells removal [6], the number (Figure 2A,B)

and the expression (Figure 2C,D) of these receptors

were compared in monocytes from patients and

controls. There was no difference in the number of

circulating CD14CD36 cells/ml between SLE

(153.1^ 205.5) or atherosclerotic patients (251 ^157.6) and their respective controls (161.3 ^ 211.7

and 219.6 ^ 74.2) nor between HCSLE and HCAtheros(Figure 2A). However, the number of CD14CD163

cells was increased in HCAtheros (377.1 ^ 71.75)

compared to HCSLE (235.4 ^ 164.7, p , 0.05), but

no differences were observed between SLE patients

(283.6^ 248.2) and HCSLE or between atherosclero-

tic patients (289.2^ 126.8) and HCAtheros(Figure 2B).

Comparison of CD36 expression among the groups

studied showed that CD36 MFI on CD14 cells was

decreased in atherosclerotic patients (22.0 ^ 28)

compared with HCAtheros (43 ^ 23.3, p , 0.05)

(Figure 2C). Contrariwise, CD163 MFI was found

decreased in SLE patients (32 ^ 18.1) compared to

HCSLE (67.5 ^ 17.8, p , 0.0001) (Figure 2D), but

there were no differences between HCSLE andHCAtheros neither for CD36 MFI (Figure 2C) nor

for CD163 MFI (Figure 2D).

Of note, in SLE patien ts the nu mber o f

CD14CD36 or CD163 cells did not correlate

with the disease activity, as measured by SLEDAI

(data not shown). These results suggest that the

differences observed in the number of circulating

CD14CD163 cells may be due to the age difference

between SLE patients/HCSLE and atherosclerotic

patients/HCAtheros, rath er than due to SLE or

atherosclerosis activity.

SLE patients and HCSLE have similar Ox-LDL

binding/endocytosis

Binding/endocytosis of Ox-LDL are important pro-

cesses for atherosclerosis development [2], thus

PBMCs were incubated with N-LDL-DiI and Ox-

LDL-DiI, and analyzed by flow cytometry. Figure 3

shows the CD14 cells not incubated with Ox-LDL

(Figure 3A), total binding/endocytosis (Figure 3B)

and endocytosis of DiI-labeled Ox-LDL (Figure 3C)

performed in a representative healthy control. Binding

and endocytosis of Ox-LDL at concentrations ranging

from 0.2 to 10mg/ml, showed a dose-dependent

increase in patients and controls (Figure 4).Monocytes from SLE patients, compared to

HCSLE, showed no differences in Ox-LDL binding

to CD14 cells (15.7^ 13.5 vs. 11.5^ 11) at

10mg/ml. At 0.2mg/ml atherosclerotic patients pre-

sented increased Ox-LDL binding (4.99^ 4.94)

compared to HCAtheros (1.55 ^ 0.75, p , 0.05)

(Figure 4A). Ox-LDL endocytosis was increased in

atherosclerotic patients compared to HCAtheros at

1mg/ml (20.66 ^ 14.71 vs. 7.78 ^ 1.69, p , 0.05)

and at 10mg/ml (52.2^ 21.56 vs. 11.71 ^ 3.89,

p , 0.01). HCAtheros presented a decreased Ox-LDL



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endocytosis (11.71 ^ 3.4) compared to HCSLE(40.72^ 25.22, p , 0.001) at 10mg/ml, suggesting

that the age decreases the capacity of CD14 cells to

endocytose Ox-LDL (Figure 4B).

The percentage of bound N-LDL to CD14

cellswas decreased in atherosclerotic patients (8.03^ 4.68)

and in HCSLE (8.08^ 8.38, p , 0.05) compared to

HCAtheros (15.51 ^ 4.46, p , 0.01), but no differences

100

101

2.64% 24.3% 19.6%

Ox-L

DL-Dil

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

104

100

101

102

CD14-FITC

103

104

100

101

102

103

104

A B C

Figure 3. Dot plot of Ox-LDL binding/endocytosis by monocytes from a healthy control donor. (A) Representative dot plot from a healthy

control showing CD14 cells not exposed to Ox-LDL, (B) total Ox-LDL-DiI (10mg/ml) binding/endocytosis, and (C) Ox-LDL-DiI

endocytosis (with trypan blue) by CD14 cells. Monocytes were gated based on forward and side scatter light parameters. Ox-LDL-DiI

fluorescence is shown on the Y-axis and CD14-FITC on the X-axis.

1200

1000

800

600

Numberof

C

D14+CD36+cells/l

Numberof

C

D14+CD163+cells/l

400

200

0

100

C D

A B

*

*

***

80

60

40

CD36M

FIinCD14+cells

20

0

100

80

60

40

CD163MFIinCD14+cells

20

0

1200

1000

800

600

400

200

0

HCSL

E

SLE

HCAthe

ros

Athe

roscler

osis

HCSL

E

SLE

HCAthe

ros

Athe

roscler

osis

HCSL

E

SLE

HCAthe

ros

Athe

roscler

osis

HCSL

E

SLE

HCAthe

ros

Athe

roscler

osis

Figure 2. Number and expression of CD36 and CD163 on monocytes from healthy controls, atherosclerotic patients, and SLE patients.

(A) Number of CD14CD36 and (B) CD14CD163 cells per microliter. (C) CD36 and (D) CD163 MFI in CD14 cells from HCSLE(n 29), SLE patients (n 38), HCAtheros (n 10), and atherosclerotic patients (n 21), evaluated by flow cytometry. Results show

individual values and means ^ SD. *p , 0.05, ***p , 0.001. Results were analyzed by unpaired t-test.



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were observed between HCSLE and SLE patients

(9.64 ^ 6.08). N-LDL endocytosis by CD14 cells

was not different between the groups (data not shown).In addition, there was no difference in Ox-LDL

binding/endocytosis between SLE patients with high

and low CIMT (data not shown).

Discussion

The risk of cardiovascular disease is very high in SLE;

however, the exact nature of the underlying vascular

pathology is not clear [28]. According to the Framing-

hams study, dyslipidemia with low levels of HDL and

high levels of LDL is a risk factor for developing

atherosclerosis [29,30]. Our SLE patients presented

decreased HDL levels and a trend toward increasedTGC levels compared to HCSLE. This pattern is similar

to the reported lupus pattern of dyslipoproteinemia,

defined by elevated levels of VLDL and TGC, and low

HDL levels [31], which was associated with athero-

sclerosis presented in SLE patients [10].

Increased CIMT is a marker for atherosclerosis that

correlates with established coronary heart disease [32].

Because CIMTwas used as criterion for early diagnosis

of atherosclerosis, we measured CIMT in patients and

controls. In adults, CIMT normal values range from

0.36 to 0.9 mm and values higher than 0.9 mm are

associated with high cardiovascular disease prevalence

[26]. As expected, the highest CIMTs were observed inatherosclerotic patients. In SLE patients CIMT was not

statistically increased, but 56% of SLE patients

presented CIMT .0.9 mm, even though they were

much younger than the atherosclerotic patients,

supporting the previous reports of early atherosclerosis

development in SLE patients [33].

Ox-LDL removal by SRs is a well-established event

in lipid deposition, foam cells formation, and

atherosclerosis development [5,6], the latter being

increased in SLE [10]. However, the presence of these

receptors on the monocyte membrane does not seem

to be determinant for atherosclerosis, since a similar

number of CD36 and CD163 monocytes was

observed in SLE patients, compared to HCSLE and inatherosclerotic patients compared to HCAtheros.

The decreased CD36 and CD163 MFI observed,

respectively, in atherosclerotic and SLE patients could

be explained by Statins treatment [34] in athero-

sclerotic patients and by the inflammatory state [35]

present in SLE patients. Furthermore, CD36 and

CD163 expression differences could suggest that the

role of these receptors in atherosclerosis development

is different in atherosclerotic and SLE patients.

Even though there were notdifferences in the number

of CD14CD36 and CD14CD163 cells between

HCAtheros and atherosclerotic patients, HCAtheros pre-

sented an increased number of CD163 in monocytes,compared to HCSLE, suggesting that aging, besides

other undetermined factors, may influence the number

of circulating CD163 monocytes.

Aging has been previously reported to be

accompanied by increased C reactive protein, serum

amyloid-A protein, and alpha-1-acid glycoprotein

[36], suggesting an increased pro-inflammatory state

in elderly subjects [37]. Additionally, systemic inflam-

matory stimuli such as a coronary artery bypass graft

surgery with cardiopulmonary bypass, was reported to

increase CD163 expression on monocytes [38].

Nevertheless, the role of CD163 and CD36 in

atherosclerosis development is not clear yet, sincethere are conflicting findings about their positive or

negative contribution to atherosclerosis [39 44].

However, there is evidence suggesting that SR class

A family members, including CD163 [6], are

implicated in the pathological deposition of cholesterol

on arterial walls during atherogenesis, as a result of

receptor-mediated uptake of oxidized LDL [8,45,46].

Ox-LDL binding and endocytosis by monocytes

were increased in atherosclerotic patients compared to

HCAtheros; but N-LDL binding was decreased in

atherosclerotic patients compared to their controls.

40A B

30

20

Ox-LDLb

indingtoCD14+cells

Ox-LDLen

docyt.byCD14+cells

10 *

0

80

60

*

***

40

20

HCSLE

HCAtheros.

Atherosclerosis

SLE

0

0.2 0.5 1.0 10 0.2 0.5 1.0 10

Figure 4. Binding/endocytosis of Ox-LDL by monocytes from patients and controls. (A) Percentage of CD14 cells from HCSLE, SLE

patients, HCAtheros, and atherosclerotic patients that have bound and (B) endocytosed Ox-LDL-DiI (mg/ml), evaluated by flow cytometry as

described in materials and methods. Results show means ^ SD and were analyzed by two-way ANOVA with Bonferroni post-test. *p , 0.05,

***p , 0.001.



8/10

However, the differences in Ox-LDL binding and

endocytosis observed between atherosclerotic patients

and HCAtheros cannot be explained by the number of

CD36 nor CD163 monocytes, since they were not

different between them.

These findings suggest that SR expressed on

monocytes from atherosclerotic patients may present

functional alterations that could lead to increased

Ox-LDL removal or that other SR, such as MARCO

or LOX-1, were also contributing to atherosclerosis

development.

In SLE patients, binding and endocytosis of Ox-

LDL by monocytes were not different to HCSLE,

suggesting that the early atherosclerosis development

reported in SLE patients [2,10] is not related to

alterations in Ox-LDL removal. Besides, the similar

N-LDL uptake observed between SLE patients and

HCSLE is not in agreement with the previously

reported decreased LDL internalization by leukocytes

from SLE patients. However, these differences could

be explained by the different experimental conditionsused for the N-LDL uptake assays [47].

Ox-LDL binding/endocytosis by SR, besides parti-

cipating in foam cells formation and atherosclerosis

development, may lead to an inflammatory response

through the activation of MAPKs and NFkB,

resulting in the production of the inflammatory

cytokines IL-1b and TNF-a [48 50], which are

major players in atherosclerosis development [51].

In conclusion, our results support that aging

increases the number of circulating CD163 mono-

cytes, but does not seem to determine an increase in

Ox-LDL removal capacity. Even though SLE patients

presented decreased HDL levels and increased TGClevels, Ox-LDL binding/endocytosis was similar to

their controls and hence cannot explain the increased

CIMTobserved in a high percentage of SLE patients.

Thus, it would be possible to suggest that the

mechanisms involved in atherosclerosis development

are different in the presence of a concomitant

autoimmune disease, such as SLE, but further studies

are needed to explore this possibility.

Acknowledgements

Thanks to the Hospital San Vicente de Paul for

allowing the development of the project, to the Flow

Cytometry Unit at the SIU (Sede de Investigacion

Universitaria), Universidad de Antioquia, and to Libia

M. Rodrguez for her helpful input concerning the

statistical analysis. The authors also thank the patients

and the healthy volunteers.

Declaration of interest: This work was supported by

Colciencias, Grant 11150416348 and Proyecto de

Sostenibilidad, Universidad de Antioquia. LMY was

beneficiary of a doctoral fellowship from Colciencias

and received partial funding from Proyecto de

Sostenibilidad Universidad de Antioquia. Authors

salaries were paid either by Colciencias (LMY, JL and

GM), the Universidad de Antioquia (MR, LAR, LFG

and GV) or by Universidad Central de Venezuela

(JBS). None of the authors has any potential financial

conflict of interest related to this manuscript. The

authors alone are responsible for the content and

writing of the paper.

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