Vascular Pharmacology 71 (2015) 84–92

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Vascular Pharmacology

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

Treatment with sulphated galactan inhibits macrophage chemotaxis and

reduces intraplaque macrophage content in atherosclerotic mice
Ana Luíza Gomes Quinderé a,b, Norma Maria Barros Benevides c, Graziano Pelli d, Sébastien Lenglet d,
Fabienne Burger d, Federico Carbone d,e, Rodrigo A. Fraga-Silva f, Nikolaos Stergiopulos f, Sabrina Pagano g,
Maria Bertolotto e, Franco Dallegri e, Nicolas Vuilleumier g, François Mach d, Fabrizio Montecucco d,e,g,⁎
a
CAPES Foundation, Ministry of Education of Brazil, Setor Bancário Norte, Quadra 2, Bloco L, Lote 6, 70040-020 Brasília, Brazil
b
Department of Biochemistry and Molecular Biology, Federal University of Ceará, Avenida Humberto Monte s/n, 60455-760 Fortaleza, Brazil
c
Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
d
Division of Cardiology, Foundation for Medical Researches, University of Geneva, 64, avenue de la Roseraie, 1211 Geneva, Switzerland
e
First Clinic of Internal Medicine, Department of Internal Medicine, University of Genoa School of Medicine,
IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro, viale Benedetto XV, 16132 Genoa, Italy
f
Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
g
Division of Laboratory Medicine, Department of Genetics and Laboratory Medicine, Geneva University Hospitals, n4, Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Experimental data from animal models and clinical studies support connections between the haemostasis and
Received 22 October 2014 inflammation in atherogenesis. These interfaces among inflammation and thrombogenesis have been suggested
Received in revised form 16 January 2015 as targets for pharmacological intervention to reduce disease progression. We hypothesize that the recently dis-
Accepted 24 February 2015
covered antithrombotic drug Sulphated Galactan (SG) (isolated from the red marine alga Acanthophora
Available online 10 April 2015
muscoides) might reduce atherosclerotic plaque vulnerability and inflammatory gene expression in 10-week
Keywords:
aged apolipoprotein E deficient (ApoE−/−) mice under high-cholesterol diet for additional 11 weeks. Then,
Antithrombotic drug the underlying cellular mechanisms were investigated in vitro. SG (10 mg/kg) or Vehicle was subcutaneously
Atherosclerosis injected from week 6 until week 11 of the diet. Treatment with SG reduced intraplaque macrophage and Tissue
Inflammation Factor (TF) content as compared to Vehicle-treated animals. Intraplaque TF co-localized and positively correlated
Red alga with macrophage rich-areas. No changes on atherosclerotic plaque size, and other intraplaque features of vulner-
ability (such as lipid, neutrophil, MMP-9 and collagen contents) were observed. Moreover, mRNA expression of
MMPs, chemokines and genetic markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic arches
and spleens was not affected by SG treatment. In vitro, treatment with SG dose-dependently reduced macro-
phage chemotaxis without affecting TF production. Overall, the chronic SG treatment was well tolerated. In con-
clusion, our results indicate that SG treatment reduced intraplaque macrophage content (by impacting on cell
recruitment) and, concomitantly, intraplaque TF content of potential macrophage origin in atherosclerotic mice.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction 2]. Atherogenesis results from an inflammatory process initiated by

Atherosclerosis progresses silently for years before becoming clini-
cally evident. By causing acute thrombosis on terminal arteries, the dis-
ease leads to myocardial infarction and ischemic stroke, which
dominates death and disability statistics for all regions of the world [1,
Abbreviations: CCL, C–C motif ligand; CXCL, C–X–C motif ligand; CV, coefficients of
variation; HDL-c, High-density lipoprotein cholesterol; LDL, Low-density lipoprotein; LDL-
c, Low-density lipoprotein cholesterol; MMP, Matrix MetalloProteinase; NMR, Nuclear
Magnetic Resonance; PAR, Protease-Activated Receptor; SG, Sulphated Galactan; TF,
Tissue Factor.
⁎ Corresponding author at: Cardiology Division, Foundation for Medical Researches,
Department of Internal Medicine, University of Geneva, 64 Avenue Roseraie, 1211
Geneva, Switzerland. Tel.: +41 22 382 72 38; fax: +41 22 382 72 45.
E-mail address: fabrizio.montecucco@unige.ch (F. Montecucco).
the intramural retention of cholesterol-containing low-density lipopro-
tein cholesterol (LDL) that activates vascular cells. Endothelial cells se-
crete chemokines that promote monocytes recruitment into the
subendothelial space, where they differentiate into macrophages [2,3].
Macrophages are major contributors to the inflammatory response
through secretion of pro-inflammatory mediators, such as chemokines,
cytokines and reactive oxygen and nitrogen species, and matrix-
degrading proteases [3]. In addition, macrophages present pathogen-
associated antigens to T cells thereby stimulating T cell activation [4]
and ingest modified and native lipoproteins, leading to massive accu-
mulation of intracellular cholesterol, which transforms them into foam
cells [5]. Intraplaque macrophages can proliferate [6] and also die, re-
leasing their lipid contents and Tissue Factor (TF) with the final forma-
tion of a pro-thrombotic necrotic core, a key component of unstable

http://dx.doi.org/10.1016/j.vph.2015.02.015
1537-1891/© 2015 Elsevier Inc. All rights reserved.
 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92
plaques [3]. During plaque rupture, large amounts of TF are released,
leading to thrombus formation [7] and elevated TF plasma levels in pa-
tients with unstable angina and acute coronary syndromes, which are
predictive of cardiovascular mortality [8]. In atherosclerotic plaques,
the expression of TF, a key initiator of coagulation cascade, has been de-
tected in macrophages, foam cells, endothelial cells, smooth muscle
cells and within the necrotic core [9–11]. Furthermore, the expression
and activity of TF can be induced by various stimuli such as cytokines,
growth factors, and biogenic amines [7]. Treatment strategies selective-
ly reducing TF within mouse atherosclerotic plaques have been poorly
investigated. In particular, some antithrombotic drugs might beneficial-
ly affect coagulation, intraplaque parameters of vulnerability, as well as
inflammation. Among these drugs, the recently discovered antithrom-
botic drug Sulphated Galactan ([SG], derived from the red alga
Acanthophora muscoides) might improve atherogenesis [12,13]. There-
fore, we hypothesized that SG could be able to reduce intraplaque vul-
nerability (primary outcome, assessed by histological parameters) in
ApoE −/− mice under high-cholesterol diet. Then, we assessed if SG
treatment might affect inflammatory gene expression (secondary out-
comes) within serum, aortic arches and spleen of atherosclerotic mice.
Finally, the cellular mechanisms underlying SG-mediated effects were
tested in vitro.
2. Materials and methods
2.1. Sulphated Galactan (SG) purification
The SG was extracted from the red alga A. muscoides by protease
digestion and purified by anion-exchange chromatography, as previ-
ously described [12]. The purity of SG was checked by nuclear mag-
netic resonance (NMR) spectroscopy to exclude the presence of
protein or other impurities [13]. The compound was verified as
endotoxin-free (b 0.25 EU/ml, using the limulus amoebocyte lysate
Endochrome assay).
2.2. Mouse model of atherogenesis
10-week old ApoE−/− mice in C57Bl/6 background were submit-
ted to high-cholesterol diet (20.1% fat, 1.25% cholesterol, Research
Diets, Inc., New Brunswick, NJ) for 11 weeks [14]. Mice were randomly
assigned to receive subcutaneous treatments either with Vehicle (NaCl
0.9%) or with SG. This drug (10 mg/kg, diluted in 200 μl NaCl 0.9% per
injection) and Vehicle (equal volume of 200 μl NaCl 0.9%) were subcuta-
neously injected five times per week in the last six weeks before animal
euthanasia. Mice well tolerated the treatment in the atherosclerosis
protocol and no clinical adverse event was reported. This mouse proto-
col was approved by local ethics committee and Swiss authorities and
conformed to the “position of the American Heart Association on Re-
search Animal Use” and to Animal Research: Reporting of In Vivo Exper-
iments (ARRIVE) guidelines.
2.3. Detection of inflammatory mediators in mouse serum
Mouse serum levels of C–C motif ligand (CCL) 2 and C–X–C motif li-
gand (CXCL) 1 were measured by colorimetric enzyme-linked immuno-
sorbent assay (ELISA, R&D Systems, Minneapolis, MN), following
manufacturer's instructions. The limits of detection for ELISA were
3.9 pg/ml for CCL2 and 15.625 pg/ml for CXCL1. Mean intra- and inter-
assay coefficients of variation (CV) were below 8% for both markers.
Haematological parameters, serum triglycerides, total cholesterol, low-
density lipoprotein cholesterol (LDL-c) and high-density lipoprotein
cholesterol (HDL-c), free fatty acids and glucose were routinely mea-
sured and expressed in mmol/l.
85
2.4. Immunohistochemistry in atherosclerotic plaques
Aortic sinuses from all mice were serially cut in 5 μm transversal sec-
tions, as described previously [15]. Sections from mouse specimens
were fixed in acetone and immunostained with specific antibodies
anti-mouse CD68 (macrophages, ABD Serotec, Dusseldorf, Germany),
anti-mouse Ly-6G (neutrophils, BD Pharmingen™, San Jose, CA), anti-
mouse Matrix MetalloProteinase (MMP)-9 (R&D Systems) and anti-
mouse TF (American diagnostic GmbH, Pfungstadt, Germany). Quantifi-
cations were performed using the MetaMorph software. Results were
calculated as percentages of stained area on total lesion area or number
of infiltrating cells per mm2 of lesion area.
2.5. Oil Red O staining for lipid content
Five sections per mouse aortic sinus and thoraco-abdominal aortas
were stained with Oil Red O, as previously described [15]. Sections
were counterstained with Mayer's hemalun and rinsed in distilled
water. Quantifications were performed using the MetaMorph software.
Data were calculated as ratios of stained area on total lesion area.
2.6. Sirius red staining for collagen content
Five sections per mouse aortic sinus were rinsed with water and in-
cubated with 0.1% Sirius red (Sigma Chemical Co, St Louis, MO) in satu-
rated picric acid for 90 min. Sections were rinsed twice with 0.01 N HCl
for 1 min and then immersed in water. After dehydration with ethanol
for 30 s and cover-slipping, the sections were photographed with iden-
tical exposure settings under ordinary polychromatic or polarized light
microscopy. Total collagen content was evaluated under polychromatic
light. Interstitial collagen subtypes were evaluated using polarized light
illumination; under this condition thicker type I collagen fibres ap-
peared orange or red, whereas thinner type III collagen fibres were yel-
low or green [15]. Quantifications were performed with MetaMorph
software. Data were calculated as percentages of stained area on total
lesion area.
2.7. Real time RT-PCR
Total mRNA was isolated with Tri-reagent (MRC Inc.) from spleens
and aortic arches of ApoE −/− mice. Reverse transcription was per-
formed using the ImProm-II Reverse Transcription System (Promega,
Madison, WI) according to the manufacturer's instructions. Real-time
PCR (StepOne Plus, Applied Biosystems) was performed with the
ABsolute™ QPCR Mix (ABgene). Specific mouse primers and probes
(Table 1) were used to determine the mRNA expression of TF,
chemokines (Ccl2, Ccl3, Ccl5, Cxcl1), MMPs (Mmp-3, Mmp-8, Mmp-9,
Mmp-10, Mmp-12), and markers of different T helper (Th) lymphocyte
subsets (Th1: Tim3, Ifng; Th2: Gata-3, Il4; Treg: Foxp3, Il10; Th17: Rorc,
Il17), and Hprt (reference gene) [14,16,17]. The fold change of mRNA
levels was calculated by the comparative Ct method. The resultant Ct
values were first normalized to the internal control. This was achieved
by calculating a delta Ct (ΔCt) by subtracting the internal control Ct
values from the Hprt Ct value. A delta delta Ct (ΔΔCt) was calculated
by subtracting the designated control ΔCt value from the other ΔCt
values. The ΔΔCt was then plotted as a relative fold change with the fol-
lowing formula: 2 − ΔΔCt.
2.8. Human macrophage isolation, differentiation and tissue factor func-
tional assays
Human macrophages were obtained as previously described [18].
Human monocytes were isolated from peripheral blood mononuclear
cells of 3 healthy blood donors, after written informed consent. Mono-
cytes were differentiated into macrophages using 24 h incubation
with 500 U/ml interferon (IFN)-gamma in culture medium (10% FCS
86 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92

Table 1
Mouse primers and probes used for real-time PCR.

Gene Function Nucleotide sequence Size (bp) Accession number

Ccl2 Fw 5′-GAGCATCCACGTGTTGGCT-3′ 68 NM_011333

Rv 5′-TGGTGAATGAGTAGCAGCAGGT-3′
Probe 5′-AGCCAGATGCAGTTAACGCCCCACT-3′
Ccl3 Fw 5′-CAGCCAGGTGTCATTTTCCT-3′ 114 NM_011337
Rv 5′-CCAAGACTCTCAGGCATTCAG-3′
Probe 5′-AAGAGAAACCGGCAGATCTGCGCT-3′
Ccl5 Fw 5′-TCACCATATGGCTCGGACAC-3′ 131 NM_013653
Rv 5′-CGAGTGACAAACACGACTGC-3′
Probe 5′-ACTCCCTGCTGCTTTGCCTACCTCT-3′
Cd4 Fw 5′-AGATCACAGTCTTCACCTGGAAGTT-3′ 125 NM_013488
Rv 5′-TGCCCCTTTTTTGGAATCAA-3′
Probe 5′-TTAATTAGAGGAGGTTCGCC-3′
Cxcl1 Fw 5′-CATAGCCACACTCAAGAATGGT-3′ 60 NM_008176
Rv 5′-TGAACCAAGGGAGCTTCAG-3′
Probe 5′-CGCGAGGCTTGCCTTGACC-3′
Foxp3 Fw 5′-GGCCCTTCTCCAGGACAGA-3′ 112 NM_054039
Rv 5′-GCTGATCATGGCTGGGTTGT-3′
Probe 5′-ACTTCATGCATCAGCTCTCCACTGTGGAT-3′
F3 Fw 5′-GTGCAGGCATTCCAGAGAAA-3′ 90 NM_010171
(TF) Rv 5′-AGTTGGTGGGTTTGGGTTG-3′
Probe 5′-CAACTGATTTCAAGACAATTTTGGAGTGG-3′
Gata3 Fw 5′-CAGAACCGGCCCCTTATCA-3′ 115 NM_008091
Rv 5′-CATTAGCGTTCCTCCTCCAGA-3′
Probe 5′-CGAAGGCTGTCGGCA-3′
Havcr2 Fw 5′-GCCGGTGGACCTCAGTTTC-3′ 69 NM_134250
(Tim3) Rv 5′-TGGGAGCCAGCACAGATCA-3′
Probe 5′-TGGGAGCCAGCACAGATCA-3′
Hprt Fw 5′-GACCGGTCCCGTCATGC-3′ 66 NM_013556
Rv 5′-TCATAACCTGGTTCATCATCGC-3′
Probe 5′-ACCCGCAGTCCCAGCGTCGTG-3′
Ifng Fw 5′-TGAGTATTGCCAAGTTTGAGGTCA-3′ 77 NM_008337
Rv 5′-GTGGACCACTCGGATGAGCT-3′
Probe 5′-CCACAGGTCCAGCGCCAAGCA-3′
Il4 Fw 5′-CAACGAAGAACACCACAGAGAG-3′ 89 NM_021283
Rv 5′-GCATGGAGTTTTCCCATGTT-3′
Probe 5′-AGCTCGTCTGTAGGGCTTCCAAGGT-3′
Il10 Fw 5′-TTTGAATTCCCTGGGTGAGAA-3′ 73 NM_010548
Rv 5′-ACAGGGGAGAAATCGATGACA-3′
Probe 5′-TGAAGACCCTCAGGATGCGGCTG-3′
Il17a Fw 5′-CTGTGATCTGGGAAGCTCAGT-3′ 124 NM_010552
Rv 5′-CTCTCAGGCTCCCTCTTCAG-3′
Probe 5′-CTGTGTCAATGCGGAGGGAAAG-3′
Mmp-3 Fw 5′-TGCAGTTGGAGAACATGGAG-3′ 141 NM_010809
Rv 5′-GTACCAGTGACATCCTCTGTCC-3′
Probe 5′-TTTTGATGGGCCTGGAACAGTCTTG-3′
Mmp-8 Fw 5′-GCATTCAGACAATCTATGGACCT-3′ 101 NM_008611
Rv 5′-GGTAGTAGCATCAAATCTCAGGTG-3′
Probe 5′-TCAGACAACCCCATCCAACCTACTG-3′
Mmp-9 Fw 5′-GTATCTGTATGGTCGTGGCTCTAA-3′ 91 NM_013599
Rv 5′-ACACATAGTGGGAGGTGCTGT-3′
Probe 5′-CTCCAGCCACCACCACAACTGAA-3′
Mmp-10 Fw 5′-GCACGAAGAAGGATCGGTTT-3′ 132 NM_019471
Rv 5′-ATGCTTGGGTGGCCTTGT-3′
Probe 5′-CTGTCTGTATGGAGCCACTAGCCATCCT-3′
Mmp-12 Fw 5′-AAAGACTGGTTCTTCTGGTGGA-3′ 91 NM_008605
Rv 5′-TACCAGATGGGATGCTTGG-3′
Probe 5′-CTTCCTGGGAGTCCAGCCACCA-3′
Rorc Fw 5′-AGAAGACCCACACCTCACAAA-3′ 89 NM_011281
Rv 5′-ACAGGTGATAACCCCGTAGTG-3′
Probe 5′-CCCTTGCAAGATCTGTGGGGACA-3′

in RPMI plus 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mM L-gluta-
mine, 1 mM pyruvate). Human macrophages (5 × 104 cells/well in 96-
well plates) were then stimulated in the presence or absence of LPS
(500 ng/ml) for 24 h, as previously described [19]. One hour after cul-
ture medium or lipopolysaccharide (LPS) stimulation, cells were co-
incubated with different concentrations (up to 100 μg/ml) of SG. The
concentrations of SG were calculated on the basis of previous studies
[12,13], averages of mouse blood volume (about 2 ml) and weight
(around 30 g) [20] and the dose (10 mg/kg) used in the in vivo. After
stimulation, the cells were tested for tissue factor activity. Tissue factor
activity was measured through its ability to promote the activation of
factor X (150 nM) (Hyphen BioMed, Neuville sur Oise, France) by factor
VIIa (5 nM) (Novo Nordisk Pharma, Küsnacht, Switzerland), in the pres-
ence of 1 mM CaCl2. The reaction was allowed to proceed for 15 min at
37 °C and was stopped by the addition of an excess of EDTA (5 mM final
concentration). Chromogenic substrate for factor Xa (FXa), S-Xa-11
(Hyphen BioMed), was then added at a final concentration of 3.5 mM,
and the change in absorbance readings was performed at an optical
density (OD) of 405 nm in the kinetic mode (Vmax, Molecular Devices,
Sunnyvale, CA). The linear absorbance changes were converted to a con-
centration of FXa generated by reference to a standard curve made with
a known amount of FXa (Hyphen BioMed). Results were expressed as
 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92 87

pM of generated FXa per minute. The local ethical committee approved
the investigation protocol, and it was conformed to the principles
outlined in the Declaration of Helsinki.
2.9. Mouse macrophage isolation and in vitro migration assay
Mouse peritoneal macrophages were obtained as previously de-
scribed by Zeini and co-workers [21]. 11-week aged ApoE −/−
mice (n = 8) were intraperitoneally injected with 1 ml of sterile
10% thioglycollate (Becton-Dickinson) 4 days before euthanasia (by
neck dislocation of 4% isoflurane-anaesthetized mice). Peritoneal
macrophages were pooled from the eight animals sacrificed to
perform four different experiments. Euthanized mice were intraper-
itoneally injected with 5 ml of sterile DMEM. The peritoneal fluid
was carefully aspirated to avoid haemorrhage and kept at 4 °C to
prevent the adhesion of the macrophages to the plastic. After centri-
fugation at 200 ×g for 10 min at 4 °C, the cell pellet was washed twice
with 45 ml of ice-cold PBS. Then, the cells were resuspended at the
density of 106 cells/ml in the chemotaxis buffer (RPMI 1640/25 mM
HEPES/0.5% [w/v] BSA) [22] in the presence or the absence of differ-
ent concentrations (up to 200 μg/ml) of SG for 1 h. To assess in vitro
the potential direct effects of mouse serum from Vehicle-treated
(n = 4) or SG-treated mice (n = 4), mouse macrophages were resus-
pended in different dilutions of mouse serum (respectively, 1:1; 1:2
and 1:4) and incubated for 1 h before the chemotaxis assay. Mouse
macrophage chemotaxis was assessed in a 48-well microchemotaxis
chamber using a 8 μm pore size polyvinylpyrrolidone-free filters
(Neuro Probe, Gaithersburg, MD). 4 × 105 macrophages were seeded
in the upper well, while control medium or mouse recombinant CCL2
(10 nM, positive control, R&D Systems, Minneapolis, Minnesota,
USA) was added to the lower wells [22]. After incubation (240 min,
37 °C), the filters were removed from the chambers, washed and
stained with May-Grünwald Giemsa. The cells of five random
water-immersion fields were counted and the chemotaxis index
(C.I.) was calculated by dividing the number of cells migrated to-
wards chemoattractants through the number of cells migrated to
chemotaxis medium alone. This mouse protocol was approved by
local ethics committee and Swiss authorities and conformed to the
“position of the American Heart Association on Research Animal
Use” and to Animal Research: Reporting of In Vivo Experiments (AR-
RIVE) guidelines.
3. Results
3.1. Treatment with SG does not induce relevant modifications on haemato-
logical, metabolic and inflammatory systemic parameters in atherosclerotic
mice
In order to clinically assess the safety of the chronic treatment with
SG in atherosclerotic mice, we evaluated haematological, metabolic
and inflammatory parameters in ApoE−/− mice. SG-treated mice did
not present any skin reactions or clinical modifications except to a slight
reduction of body weight at sacrifice as compared to Vehicle-treated
controls (Table 2). No changes in haemoglobin concentrations, red
blood cell and white blood cell counts were shown between the study
groups (Table 2). A weak increase in platelet count in SG-treated as
compared to Vehicle-treated controls was detected (Table 2). Consider-
ing metabolic parameters, no changes in total cholesterol, LDL-c, free
fatty acids and glucose concentrations were observed in SG-treated
mice as compared to Vehicle-treated controls (Table 2). Conversely, a
modest increase in serum HDL-c and reduction in triglyceride levels
were shown in SG-treated mice as compared to Vehicle-treated controls
(Table 2). Finally, treatment with SG was not associated with any mod-
ification on serum levels of pro-atherosclerotic chemokines (i.e., CCL2
and CXCL1) (Table 2).
3.2. Treatment with SG reduces macrophage and TF content within mouse
aortic root plaques
In order to assess bio-efficacy to SG treatment in atherogenesis, we
evaluated atherosclerotic plaque size within thoraco-abdominal aortas
and intraplaque parameters of vulnerability, as previously described as
relevant endpoints of atheroprogression [14,15]. Treatment with SG
did not affect atherosclerotic plaque size as well as intraplaque lipid,
neutrophil, MMP-9 and collagen content as compared to Vehicle-
treated animals (Table 3, Figs. 1A–B and 2A–C). Conversely, treatment
with SG was associated with significant reduction in intraplaque macro-
phage and TF contents as compared to Vehicle-treated animals (Table 3,
Fig. 3A and B). Considering ApoE−/− mice treated with Vehicle or SG, a
significant positive correlation (Spearman's rank correlation, r = 0.693,
Table 2
Mouse body weight, haematology, metabolism and inflammation at sacrifice.
Mouse profile Vehicle-treated SGa-treated mice p
mice (n = 12) (n = 13) value

2.10. Apoptotic cell measurement within atherosclerotic plaques Body weight before H.D.b, g 24.25 (22.65–25.45) 22.60 (21.95–23.85) 0.1825
Body weight at sacrifice, g 32.25 (30.05–34.35) 28.00 (26.90–30.65) 0.0077

We performed staining of frozen mouse aortic root sections with the Haematology
Dead End™ colorimetric terminal deoxynucleotidyltransferase-mediat- Haemoglobin, g/dl 12.35 (11.75–12.65) 11.80 (11.05–12.35) 0.1825
ed dUTP nick end labelling (TUNEL) system (Promega, Madison, WI) Red blood cells, ×106/μl 8.24 (8.07–8.37) 7.95 (7.60–8.23) 0.1147
Platelets, ×103/μl 930 (768.5–976.5) 1060 (928.50–1168) 0.0296
using diaminobenzidine as the chromogenic substrate (according to
Total WBCc, ×103/μl 4.50 (3.85–4.90) 5.20 (4.30–5.90) 0.1147
the manufacturer's instructions). Results were expressed as percentages Lymphocytes, ×103/μl 3.65 (2.90–4.20) 4.3 (3.55–4.80) 0.2644
of stained area on total plaque area, as previously described [23].
Metabolism
Total cholesterol, mmol/l 16.79 (15.46–23.45) 17.38 (14.31–20.18) 0.8490
HDL-cd, mmol/l 3.09 (2.84–3.22) 3.52 (3.15–3.81) 0.0084
2.11. Statistical analysis LDL-ce, mmol/l 13.32 (11.84–18.65) 14.25 (11.63–16.08) 0.8918
Triglycerides, mmol/l 1.130 (0.91–1.385) 0.76 (0.63–0.91) 0.0077
The Mann–Whitney nonparametric test (the normality assumption Free fatty acids, mmol/l 0.90 (0.77–1.08) 0.92 (0.58–1.05) 0.7648
of the variables' distribution in both groups was violated) was used for Glucose, mmol/l 26.00 (21.95–27.15) 22.70 (21.90–26.80) 0.5865
comparisons of continuous variables. Spearman's rank correlation coef- Inflammation
ficients were used to assess correlations between intraplaque macro- CCL2, pg/ml 101.7 (94.21–121.5) 109.3 (89.93–118.0) 0.8918
phages and TF. Results were expressed as median (interquartile CXCL1, pg/ml 294.0 (259.5–395.0) 370.2 (244.5–403.9) 0.6833
range). In vitro results were expressed as mean ± SD (macrophage che- Data are expressed as medians (interquartile range) p value calculated according to U
motaxis assay and TF). One-way ANOVA was used for multiple group Mann–Whitney test.
a
comparison, while unpaired t test was used for two group comparison SG: Sulphated Galactan.
b
H.D.: High-cholesterol Diet.
for in vitro experiments. Values of p b 0.05 (two-tailed) were consid- c
WBC: white blood cells.
ered significant. All analyses were done with GraphPad Instat software d
DL-c: High Density Lipoprotein cholesterol.
version 3.05 (GraphPad Software). e
LDL-c: Low Density Lipoprotein cholesterol.
88 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92

Table 3
Parameters of mouse plaque vulnerability.

Parameters Vehicle-treated SGa-treated mice p

mice (n = 7–12) (n = 8–13) value

Thoraco-abdominal aorta
Oil-red-O, % 6.39 (5.57–10.34) 7.47 (6.05–9.62) 0.4303

Aortic roots
Oil-red-O, ×103 μm2 307.6 (246.7–409.0) 309.0 (254.9–353.2) 0.6439
Macrophage + area, % 24.09 (20.11–27.67) 18.74 (18.06–22.86) 0.0180
Neutrophils/mm2 13.62 (6.05–19.82) 12.95 (6.47–18.87) 0.7648
MMPb-9, % 1.89 (0.94–2.48) 1.47 (1.07–2.58) 0.8490
Total collagen, % 39.35 (35.09–48.83) 43.52 (39.72–45.81) 0.2889
TFc, % 15.72 (11.13–18.00) 6.384 (4.311–8.665) 0.0012

Data are expressed as medians (interquartile range).

a
SG: Sulphated Galactan.
b
MMP: Matrix MetalloProteinase.
c
TF: Tissue Factor.

p = 0.004) was shown between intraplaque levels of macrophages and

TF (Fig. 3A). Interestingly, in plaques from ApoE −/− mouse aortic
roots, TF particularly co-localized with macrophage rich-areas
(Fig. 3B). These results indicate that in vivo SG treatment was associated
with intraplaque reduction of the atherothrombotic molecule TF of po-
tential macrophage origin.

3.3. Treatment with SG does not affect the mRNA expression

of inflammatory genes within mouse atherosclerotic aorta and spleen

As secondary endpoints, we focused on potentially anti-

inflammatory effects of SG treatment in atherosclerotic mice. No effect
on the mRNA expression of chemokines and genetic markers of Th1/2/
reg/17 lymphocyte polarization within mouse aortic arches and spleens

Fig. 2. Representative microphotographs of staining for neutrophils, MMP-9 and Sirius red
in aortic root plaques. A,B. Aortic root sections of Vehicle- and SG-treated ApoE−/− mice
were immunostained for Ly-6G (neutrophils) (A) and MMP-9 (B). C. Bright-field sections
without polarization (total collagen) are shown as figures on the upper part and corre-
sponding sections under polarized light illumination (collagen I and III) are shown as fig-
ures on the lower part.

was shown between the study groups, except for an increase in the Rorc
expression in the spleens of SG-treated mice (Table 4). In addition, SG
treatment did modify mRNA expression of different MMPs (i.e., Mmp-
3, Mmp-8, Mmp-9, Mmp-10, Mmp-12) in mouse aortic arches as com-
pared to Vehicle-treated animals (Table 5).

3.4. Treatment with SG reduces in vitro macrophage chemotaxis without

affecting TF production and intraplaque macrophage apoptosis

In order to determine if SG-mediated in vivo reduction of

Fig. 1. Representative microphotographs of Oil Red O stained thoraco-abdominal aortas
and aortic root plaques. A. Thoraco-abdominal aortas of Vehicle- and SG-treated
ApoE−/− mice. B. Aortic root sections of Vehicle-treated and SG-treated ApoE−/− mice.
intraplaque macrophage and TF contents was potentially due to drug-
induced inhibition of macrophage intraplaque recruitment, drug-
mediated inhibition of TF production or drug-induced increase in cell
apoptosis, we first assessed in vitro the effects of SG treatment on
mouse macrophage chemotaxis to the pro-atherosclerotic chemokine
CCL2. The in vitro chemotaxis assay demonstrated that SG treatment
dose-dependently inhibited CCL2-induced macrophage migration as
compared to control medium (Table 6). A significant abrogation of mac-
rophage migration was obtained at the concentration of 100 μg/ml of SG
(Table 6). Accordingly, serum from SG-treated mice significantly re-
duced macrophage migration towards CCL2 as compared to control me-
dium or serum from Vehicle-treated mice (Table 7), indicating that
 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92 89

Table 4
Th cell polarization and chemokine expression in ApoE−/− mice.

Marker (mRNA fold Vehicle-treated mice (n SG-treated mice (n p

increase) = 12) = 13) value

Aortic arches
Cd4 0.84 (0.47–1.40) 1.30 (0.66–1.8) 0.4869
Th1
Tim3 0.94 (0.68–1.40) 0.87 (0.80–1.40) 0.8490
Ifng 0.95 (0.66–1.60) 1.30 (0.97–2.10) 0.2312
Th2
Gata3 1.10 (0.95–1.20) 0.92 (0.52–1.20) 0.2025
Il4 0.90 (0.74–1.40) 1.60 (1.00–2.50) 0.1004
Treg
Foxp3 0.98 (0.56–1.40) 0.76 (0.43–0.76) 0.7237
Il10 1.00 (0.86–1.23) 1.80 (0.95–3.30) 0.1469
Th17
Rorc 0.92 (0.66–1.60) 0.51 (0.27–1.35) 0.0939
Il17 1.30 (0.37–2.20) 2.00 (1.30–4.40) 0.0508
Ccl2 0.95 (0.73–1.60) 1.40 (1.00–1.50) 0.2423
Ccl3 0.93 (0.85–1.10) 1.00 (0.74–1.30) 0.8490
Ccl5 1.10 (0.81–1.30) 1.30 (0.82–1.50) 0.3695
Cxcl1 1.10 (0.60–1.40) 1.30 (0.87–1.60) 0.2466

Spleen
Cd4 0.92 (0.72–1.30) 0.98 (0.77–1.20) 0.9078
Th1
Tim3 0.89 (0.78–1.10) 0.85 (0.68–1.00) 0.2710
Ifng 1.10 (0.71–1.30) 0.96 (0.71–1.10) 0.3247
Th2
Gata3 1.00 (0.78–1.30) 0.92 (0.78–0.99) 0.2238
Il4 1.00 (0.75–1.40) 1.30 (0.84–4.00) 0.2238
Treg
Foxp3 0.99 (0.65–1.20) 0.97 (0.75–2.1) 0.6021
Il10 0.99 (0.68–1.40) 0.81 (0.61–1.40) 0.6430
Th17
Rorc 1.00 (0.70–1.40) 2.40 (2.00–3.00) 0.0003
Il17 0.86 (0.71–1.50) 1.60 (0.66–13) 0.2238

Data are expressed as median (interquartile range).

SG presents a low anticoagulant activity, acting mostly by serpin-

Fig. 3. TF co-localizes with macrophages in aortic root plaques. A. Correlation between
intraplaque levels of TF and macrophages. B. Representative microphotographs of consec-
utive cryosections from root plaques from Vehicle-treated and SG-treated ApoE−/− mice
stained for TF and CD68 (macrophages).
treatment with SG was associated with abrogation of in vitro macro-
phage migration towards CCL2, a chemokine known to be highly
expressed within human and mouse atherosclerotic plaques [24,25].
Then, we investigated if SG treatment might be associated with re-
duction in TF mRNA levels within atherosclerotic aortic arches. As
shown in Fig. 4A, treatment with SG did not affect mRNA TF levels in
mouse atherosclerotic aorta as compared to control Vehicle. According-
ly, SG treatment did not affect in vitro TF production from cultured mac-
rophages in response to LPS stimulation as compared to control
medium (Fig. 4B).
Finally, we investigated if SG treatment might be associated with an
increase in intraplaque macrophage apoptosis. No difference in
intraplaque apoptosis was detected between Vehicle- and SG-treated
mice in vivo (Fig. 5A and B). Moreover, apoptotic (TUNEL positive)
areas in atherosclerotic plaques were very small and did not co-
localize within the macrophage-rich area (Fig. 5B).
independent mechanism, but drastically reduces arterial thrombus for-
mation, being as active as heparin, and in venous model, almost total in-
hibition was achieved. In contrast to heparin, the SG does not present
bleeding tendency [13]. It is interesting to note that heparin has been
long suggested as a useful approach in the prophylaxis and therapy of
atherosclerosis due to its effect on lipoprotein metabolism and its
wide range of anti-inflammatory effects [26,27]. A direct anti-
atherosclerotic effect of heparin on LDL receptor deficient mice reducing
aortic lesion areas has been demonstrated [28]. However, the unwanted
risk of haemorrhage has prevented the use of heparin and low-
molecular-weight heparins as anti-inflammatory therapies [29]. The
main finding of our study is represented by the demonstration that
treatment with SG was safe, well-tolerated and effective in reducing
intraplaque macrophage content as compared to Vehicle-treated ani-
mals. No major changes in the haematological, metabolic and inflamma-
tory parameters were observed. The SG-treated group at sacrifice
showed a slight reduction of body weight, a weak increase in platelet
count, a modest increase in serum HDL-c and a reduction in triglyceride
levels. The higher platelet count in SG-treated animals might be an in-
triguing finding, since it might reflect a reduced platelet degradation
Table 5
mRNA expression of MMPs in ApoE−/− mouse aortic arches.

Marker Vehicle-treated SG-treated mice p

4. Discussion (mRNA fold change) mice (n = 12) (n = 13) value

Mmp-3 1.14 (0.67–1.47) 0.87 (1.02–1.38) 0.9134

The SG extracted from A. muscoides presents the characteristic back- Mmp-8 0.99 (0.78–1.25) 0.67 (0.45–1.01) 0.0689
bone structure of sulphated galactans from red algae, i.e., alternating 4- Mmp-9 1.37 (0.72–1.66) 1.04 (0.69–3.35) 0.8521
linked α-galactose and 3-linked β-galactose, but also a complex pattern Mmp-10 1.09 (0.75–1.64) 1.25 (0.79–2.17) 0.5269
of substitutions with sulphate esters and methyl ethers along with the Mmp-12 0.95 (0.86–1.22) 0.95 (0.57–1.20) 0.6409

occurrence of 3,6-anhydro-α-galactoses makes its structure unique. Data are expressed as median (interquartile range).
90 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92

Table 6
Effects of SG on CCL2-induced mouse macrophage migration in vitro.

Polycarbonate assay (Chemotaxis Index)

SG (μg/ml)

0 1 10 100

CCL2 (10 nM) 5.49 ± 1.81 5.66 ± 1.30 3.24 ± 0.49 2.30 ± 0.54⁎

Data are expressed as mean ± SD (n = 4).

⁎ p b 0.05 vs. migration towards CCL2 in the absence of SG (0 μg/ml, control).

due to a potential general reduction in TF levels. However the limited

volume of mouse blood samples did not allowed us to collect both
serum and plasma from animals at the same time. Since we decided to
collect only serum, we were not able to measure mouse TF levels or ac-
tivity that is usually measured in plasma. Therefore, this interesting dis-
covery has to be presented as a future perspective for a new study. In
addition, treatment with SG did not reduce atherosclerotic lesion size
or any other histological features of intraplaque vulnerability (primary
outcome). Furthermore, no effect on the mRNA expression of genetic
markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic
arches and spleens was also shown between the study groups. Consid-
ering that plaque composition rather than plaque size has been de-
scribed as critical determinant of plaque vulnerability and rupture
[30], SG-mediated selective abrogation of intraplaque macrophage con-
tent might have a potential therapeutic relevance. Mononuclear phago-
cytes are major players in diverse pathological conditions including
chronic inflammatory diseases, such as atherosclerosis [31]. Fully differ-
entiated macrophages respond to tissue microenvironment and macro-
phage intracellular milieu through cell surface receptors or intracellular
sensors and change their phenotypic and functional characteristics [32,
33]. Transcription factors, epigenetic mechanisms, and posttranscrip-
tional regulators also underlie the different forms of macrophage activa-
tion and polarization. Treatment with SG was also associated with Fig. 4. Treatment with SG does not affect TF production in vivo and in vitro. A. mRNA ex-
reduction of intraplaque TF content. Notably, TF particularly co- pression levels of TF in aortic arches from mice treated with control Vehicle or SG. Relative
localized with macrophage rich-areas in aortic roots plaques and posi- expression normalized to Hprt was calculated with the comparative Ct method and shown
as fold change of mRNA levels. Data are expressed as medians (interquartile range), n =
tive correlation between intraplaque levels of TF and macrophages 12–13 per group. n.s.: non-significant. B. Human primary macrophages were incubated
was shown. Hence, our results might suggest that SG treatment selec- in the absence (control [CTL]) or presence of 500 ng/ml LPS for 24 h. One hour later,
tively reduced intraplaque TF of macrophage origin. Our data are in cells were co-incubated with the indicated concentrations of SG. Finally, tissue factor activ-
line with previous reports indicating that macrophages are important ity was measured through its ability to promote the activation of factor X. Data are
expressed as mean ± SD (n = 3 per group). * p b 0.05; n.s.: non-significant.
TF sources within atherosclerotic plaques. Indeed, TF expression,
which is up regulated in the inflammatory environment of atheroscle-
rotic plaques, has been detected in monocytes, macrophages and foam and CCL5, and CXCL1), suggesting a potential interference of the drug
cells [7,9–11]. Moreover, it has been reported that TF expression is di- directly on macrophage functions (chemotaxis/proliferation/apoptosis)
minished by anticoagulants, including heparin and low-molecular- instead of an indirect effect on the vascular expression of chemokines
weight heparins [34,35]. In addition to its pro-haemostatic functions, mediating their recruitment. In order to investigate if SG treatment
TF plays important roles in cell signalling mechanisms involving: activa- might directly affect macrophage functions, we explored macrophage
tion of both protease-activated receptor (PAR)-1 and -2 by the ternary chemotaxis, TF production and intraplaque macrophage apoptosis by
complex TF/FVIIa/FXa, and the PAR-dependent pathways, via the TF cy- in vitro and in vivo approaches. These experiments suggested that treat-
toplasmic domain and by transactivation of receptor tyrosine kinases ment with SG was able to exclusively reduce in vitro macrophage che-
[36,37]. Importantly, treatment with SG did not affect both systemic or motaxis in response to the pro-atherosclerotic chemokine CCL2
intraplaque levels of monocyte chemoattractants (such as CCL2, CCL3 without affecting the two other cell functions. These results allowed

Table 7
Effects of serum from Vehicle- or SG-treated mice on mouse macrophage-migration in vitro.

Polycarbonate assay (chemotaxis index)

Upper well

CTL Vehicle-treated serum SG-treated serum

Lower well 1:4 1:2 1:1 1:4 1:2 1:1

CTL 1.00 ± 0.0 1.19 ± 0.12 1.22 ± 0.08 1.31 ± 0.12 1.18 ± 0.10 1.24 ± 0.5 1.21 ± 0.08
CCL2 (10 nM) 5.49 ± 1.81 6.51 ± 2.05 6.98 ± 1.19 7.10 ± 1.27 4.42 ± 1.32 4.05 ± 0.60 2.71 ± 0.41⁎,†

Data are expressed as mean ± SD. (n = 4).

⁎ p b 0.05 vs. control medium (CTL)-stimulated cell migration to CCL2.
†
p b 0.01 vs. Vehicle-treated serum (1:1)-stimulated cell migration to CCL2.
 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92 91

Fig. 5. SG treatment does not affect in vivo intraplaque apoptosis. A. Quantification of apoptotic intraplaque areas in Vehicle- and SG-treated animals. Data are expressed as mean ± SD
(n = 4–5 per group). B. Representative microphotographs of consecutive cryosections from aortic roots of Vehicle- and SG-treated mice stained for TUNEL (apoptotic cells) and immu-
nostained for CD68 (macrophages).

us to identify a potential cellular mechanism (diminished macrophage
recruitment within atherosclerotic plaques) underlying SG-mediated
effects in vivo. This study has some limitations due to the low number
of mice that allowed a limited amount of immunostainings. We were
also not able to investigate the potential role of SG treatment in vivo
on macrophage subsets or other selective macrophage products within
atherosclerotic plaques. These points need to be investigated and poten-
tially clarified in a future project.
In conclusion, treatment with a novel class of antithrombotic polysac-
charides of natural origin (i.e., SG) reduced intraplaque macrophage con-
tent within mouse atherosclerotic plaques. Accordingly, SG treatment and
serum from SG-treated mice dose-dependently reduced in vitro mouse
macrophage chemotaxis in response to the pro-atherosclerotic chemo-
kine CCL2. Considering that intraplaque macrophage-rich areas co-
localized with TF positive regions, SG-mediated reduction in macrophage
content was associated with intraplaque reduction of TF of potential mac-
rophage origin. In addition, SG treatment did not affect intraplaque mac-
rophage apoptosis and macrophage release of TF. These results support
further evaluation of antithrombotic agents in the atherosclerotic
inflammatory process as a possible pathway to clarify the interplay of
haemostatic system and atherosclerosis.
Disclosure/Conflict of interest
All authors declare that no conflict of interest exists.
Acknowledgements
This study was supported by the European Commission (FP7-
INNOVATION I HEALTH-F2-2013-602114; Athero-B-Cell: Targeting
and exploiting B cell function for treatment in cardiovascular disease).
This work was also supported by the Swiss National Science Foundation
Grants to Dr. F. Montecucco (#310030_152639/1), to Dr N. Vuilleumier
(#310030_140736) and to Prof. F. Mach (#310030_152912/1). This
work was furthermore supported by grants from the Leenaards Founda-
tion to Dr. F. Montecucco and Dr. N. Vuilleumier and from the Founda-
tion “Gustave and Simone Prévot” to Dr. F. Carbone.
92 A.L. Gomes Quinderé et al. / Vascular Pharmacology 71 (2015) 84–92
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