Supplementary Material
Methods
Mouse model of shear stress-induced atherogenesis and plaque vulnerability
Apolipoprotein E deficient mice (apoE–/–) in a C57BL/6J background were obtained from The
Charles River Laboratories. Animals at 15–20 weeks of age were randomly assigned to receive
either vehicle (n=6) or the selective CB2 agonist JWH-133 (n=7) treatment. During the
experimental period, all animals were fed a Western-type diet consisting of 15% (wt/wt) cocoa
butter and 0.25% (wt/wt) cholesterol (Diet W; abDiets). After a 2-week period of Western diet,
shear stress in the right common carotid artery was altered by cast placement, as previously
described by Cheng et al.1 The cast consists of 2 longitudinal halves of a cylinder of the
thermoplastic material polyetherketon with a cone-shaped lumen (Supplementary figure 1B).
The geometry of the cast was developed to produce vortices downstream of the cast when placed
around the common carotid artery. This downstream region will therefore be exposed to
oscillations in shear stress (oscillatory shear stress region [OSS]). The upstream inner diameter is
500 μm (non-constrictive) and gradually declines to 250 μm at the downstream side of the cast
(constrictive). This tapering induces a gradual increase in shear stress (high shear stress region
[HSS]). In addition, the constrictive stenosis decreases the blood flow, resulting in a low shear
stress region (LSS) upstream from the cast.
For surgical procedure of cast implantation, the animals were anesthetized by isoflurane
inhalation, and the anterior cervical triangles were accessed by a sagittal anterior neck incision.
Both halves of the cast were placed around the right common carotid artery and fixed with a
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suture. After closing the wounds, the animals were allowed to recover. Nine weeks after surgery,
the animals were euthanized to collect tissue and serum samples. During the last 3 weeks before
euthanizing (from week 6 to 9 of cast implantation), mice were intraperitoneally injected with
the selective CB2 agonist JWH-133 (5 mg/kg/day for 5 consecutive days per week, Tocris
Bioscience, Bristol, UK) or respective vehicle control (Tocrisolve™ 100, Tocris Bioscience). In
selective experiments, to assess the CB2 levels in mouse carotid and aortic root plaques, animals
(n=4) were intraperitoneally and daily injected with PBS (for 5 consecutive days per week from
week 6 to 9 with cast). This animal study was approved by local ethics committee and Swiss
authorities and conformed to the “position of the American Heart Association on Research
Animal Use”.
Immunohistochemistry in human carotid plaques, mouse carotid plaques and mouse aortic
sinus
Frozen upstream and downstream human carotid specimens were serially cut in 7 μm sections, as
shown in Supplementary figure 2A. Mouse carotid artery was macroscopically cut in three
portions: region upstream of the cast device (LSS), region of the cast device (HSS) and
downstream of the cast (OSS) and frozen in OCT. Since no plaque was expected in HSS
regions,1 these tissues were analyzed for the presence of CB2 protein only in mice treated with
PBS. On the other hand, LSS and OSS portions from all mice were serially cut in 5 m
transversal sections, as described in Supplementary figure 2B. Mouse aortic sinus was serially
cut in 5 m transversal sections through the heart and aortic roots, as described in Supplementary
figure 2C. Sections from human and mouse specimens were fixed in acetone or in 50% Acetone
plus 50% Methanol (for CB1 and CB2 staining) and immunostained with specific antibodies
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(Supplementary table 2). Quantifications were performed with MetaMorph software. CB2 results
in mouse samples were calculated as percentages of stained area on total intima and media
surface area. Results for other parameters in mice and humans were calculated as percentages of
stained area on total lesion area or number of infiltrating cells on mm2 of lesion area.
Oil Red O staining for lipid content
Eight sections per portion (upstream and downstream the blood flow) of human carotid plaques,
eleven sections per mouse carotid LSS and OSS and five sections per mouse aortic sinus were
stained with Oil Red O, as previously described.1 Sections and aortas were counterstained with
Mayer’s hemalun and rinsed in distilled water. Quantifications were performed with MetaMorph
software. Data were calculated as ratios of stained area on total lesion area.
Sirius red staining for collagen content
Eight sections per portion (upstream and downstream the blood flow) of human carotid plaques,
eleven sections per mouse carotid LSS and OSS and five sections per mouse aortic sinus were
rinsed with water and incubated with 0.1% Sirius red (Sigma Chemical Co, St Louis, MO) in
saturated 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 seconds and cover-slipping, the
sections were photographed with identical 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 fibers appeared orange or red, whereas thinner type III collagen
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fibers were yellow or green.3 Quantifications were performed with MetaMorph software. Data
were calculated as percentages of stained area on total lesion area.
Real time RT-PCR
Total mRNA was isolated with Tri-reagent (MRC Inc.) from upstream or downstream specimens
of human carotid plaques. Reverse transcription was performed 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 primers and probes (Supplementary table 3) were used to determine the mRNA
expression of CNR1 (CB1 receptor), fatty acid amide hydrolase (FAAH), monoacylglycerol
lipase (MAGL), matrix metallopreoteinase-8 (MMP-8) and GAPDH (housekeeping gene). The
fold change of mRNA levels was calculated by the comparative Ct method. The measured Ct
values were first normalized to the GADPH internal control, by calculating a delta C t (ΔCt). This
was achieved by subtracting the GADPH Ct values from the gene of interest Ct value. A delta
delta Ct (ΔΔCt) was calculated by subtracting the designated baseline control group ΔCt value
from the study group ΔCt values. The ΔΔCt was then plotted as a relative fold change with the
following formula: 2-ΔΔCt.
Quantification of endocannabinoids
After lipid extraction and pre-purification, endocannabinoid fractions from upstream and
downstream portions of human carotid plaques were subjected to isotope-dilution liquid
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chromatography-atmospheric pressure chemical ionization-mass spectrometric analysis. AEA, 2AG, PEA and OEA were quantified as previously described.4,5
Human primary neutrophil isolation and culture
Human neutrophils were obtained from 7 healthy volunteers after informed consent. The local
ethical committee approved the investigation protocol that was conformed to the principles
outlined in the Declaration of Helsinki. Neutrophils were isolated from heparinized venous blood
by dextran sedimentation followed by centrifugation on Ficoll-Hypaque (from Cedarlane
Laboratories Ltd. [Ontario, Canada]) density gradient, as previously described.2 Neutrophils
resuspended in culture medium (serum-free RPMI 1640 medium containing 25 mmol/L Hepes)
were >97% pure, as determined by morphologic analysis of Giemsa-stained cytopreparations
(from Merck [Darmstadt, Germany]). Then, human neutrophils (5x105 cells per well) were preincubated with medium alone, or different doses (0.1, 0.3, 1 M) of JWH-133 for 2 hours at
37ºC in a humidified atmosphere and 5% CO2. In selective experiments, before cannabinoid
agonist incubation, 10-min pre-treatment with 1 M AM630 (a selective CB2 antagonist from
Tocris) was performed. Without washing, cells were stimulated in the presence or absence of 10
ng/ml phorbol-12-myristate-13-acetate (PMA, positive control from Sigma-Aldrich, Buchs,
Switzerland),6 or 100 ng/ml human recombinant TNF- (R&D Systems) for 30 min at 37ºC in a
humidified atmosphere 5% CO2 in adherence to polystyrene dishes. In selective experiments,
before stimulation with TNF-, cells were pre-incubated in the presence or absence of different
concentrations (0.1, 1, 25 M) of PD98059 (a selective inhibitor of mitogen-activated protein
kinase kinase [MEK], a kinase directly activating extracellular signal-regulated kinase [ERK],
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(from Biomol Research Laboratories, Inc., Plymouth Meeting, PA). Supernatants were collected
and stored at minus 80ºC to be tested for MMP-9 levels and pro-MMP-9 activity.
Flow cytometry
After isolation, neutrophils were pre-incubated in polystyrene dishes for 2h in the presence or
absence of different concentrations (0.1-1 M) of JWH-133 in serum-free RPMI 1640 medium
containing 25 mmol/L Hepes. Then, cells were collected in PBS and immediately stained with
Phycoerythrine (PE)-labeled antibody anti-human TNF receptor I (TNFRI), or anti-human
TNFRII, as well as corresponding PE-conjugated isotype controls (all from R&D Systems
Europe Ltd). Then, cells were washed with PBS one time and analyzed on a FACSCalibur (BD
Biosciences, Heidelberg, Germany). CellQuest software was used for the acquisition and
analysis. Data were expressed as average of positive cells on total cells counted and mean
fluorescence intensity (MFI, compared with expression on cells stimulate with control medium
alone [defined as 100%]).
Detection of inflammatory mediators in human serum and cell supernatants
In human serum, levels of MMP-8, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2,
TIMP-3 were measured by colorimetric enzyme-linked immunosorbent assay (ELISA, R&D
Systems, Minneapolis, MN), following manufacturer’s instructions. Serum insulin levels were
measured by colorimetric enzyme-linked immunosorbent assay (Millipore, St. Charles, MO),
following manufacturer’s instructions. In serum and cell supernatants, MMP-9 levels were
measured by ELISA (R&D Systems). The limits of detection for ELISA were 0.156 ng/ml for
MMP-8, 0.156 ng/ml for TIMP-1, 0.156 ng/ml for TIMP-2, 62.5 pg/ml for TIMP-3, 2 U/ml for
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insulin, 0.312 ng/ml for MMP-9. Mean intra- and inter-assay coefficients of variation (CV) were
below 8% for all markers. Serum glucose, triglycerides, total cholesterol, low-density lipoprotein
(LDL) cholesterol and high-density lipoprotein (HDL) cholesterol were routinely measured and
expressed in mg/dl.
Pro-MMP-9 zymographic assay
Pro-MMP-9 zymographic activity was assessed in human serum and cell supernatants. 9% SDSpolyacrylamide gels were copolymerized with gelatin (Sigma). Equal amounts of serum (2 l),
cell supernatants (30 l) and 1 ng of recombinant pro-MMP-9 standard (Calbiochem, Lucern,
Switzerland) were loaded on gels in the absence of reducing agents. Then, gels were rinsed and
stained with Coomassie Blue R-250, as previously described.2 Zymographic results were
expressed as pro-MMP-9 proteolytic activity and calculated on the basis of the following
formula: Serum/supernatant pro-MMP-9 = (Iobs /Istd) x Wstd, where Iobs and Istd are intensities of
gelatinolytic areas produced in gels by samples and by standard pro-MMP-9, and Wstd is the
weight (1 ng) of standard pro-MMP-9 loaded onto the gel. Pro-MMP-9 activity was expressed as
ng/ml (serum or cell supernatant), respectively as previously reported.2 Gelatinolytic bands were
measured with a gel analysis system (GeneGenius, Syngene, Cambridge, UK).
Western blot analysis
Freshly isolated human neutrophils (1 x 107 cells/ml) from 3 healthy donors were pre-incubated
with control medium (serum-free RPMI 1640 medium containing 25 mmol/l Hepes), JWH-133
vehicle (0.01% DMSO in control medium), or different concentrations of JWH-133 (0.1-1 M)
for 2h at 37°C in a humidified atmosphere and 5% CO2. Without washing, cells were stimulated
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in presence or absence of TNF- (100 ng/ml for 7 minutes, from R&D Systems). The incubation
was stopped on ice and the cells were centrifuged at 4°C. After removing supernatants, the
pellets were lysed in 400 l of Nonidet P40 buffer (20 mM Tris-HCl pH 7.5, 0.15 M NaCl, 10
mM NaF, 1% Nonidet P40, 10 g/ml glycerol, 1 mM phenylmethanesulphonyl-fluoride [PMSF],
10 g/ml leupeptin, 10 g/ml aprotinin, 0.5 mM Na3VO4). Equal amounts of protein (30 g)
were boiled in loading buffer (62.5 mM Tris-HCl pH 6.8, 0.75% SDS, 3.75% 2-mercaptoethanol,
8.75% glycerol and 0.025% bromophenol blue) and resolved by 10% SDS-polyacrylamide
electrophoresis. Then, proteins were transferred on nitrocellulose membrane at 4°C for 45
minutes. After blocking 1 hour in 5% non-fat dry milk and washing with Tris-buffered
saline/Tween 20 (10 mM Tris-base pH 7.4, 154 mM NaCl and 0.05% Tween 20), membranes
were incubated with appropriate dilution of anti-phospho-ERK1/2 (R&D Systems) primary
antibody (Ab), as well as corresponding secondary Ab. Blots were developed using the ECL
system (Immobilion Western, Millipore, USA). Membranes were then stripped, blocked and
reprobed to detect total intracellular kinase (using anti-ERK 1/2 [R&D Systems] Ab).
Immunoblots were scanned and quantifications were carried out by Image Quant software
version 3.3 (Molecular Dynamics, Sunnyvale, USA). Values of phospho-ERK 1/2 from three
different experiments were normalized to corresponding total amounts of ERK 1/2 and expressed
as percentages of control medium (defined as 100%).
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References
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de Crom R. Atherosclerotic lesion size and vulnerability are determined by patterns of
fluid shear stress. Circulation. 2006;113:2744-2753.
2. Montecucco F, Vuilleumier N, Pagano S, Lenglet S, Bertolotto M, Braunersreuther V,
Pelli G, Kovari E, Pane B, Spinella G, Pende A, Palombo D, Dallegri F, Mach F, RouxLombard P. Anti-Apolipoprotein A-1 auto-antibodies are active mediators of
atherosclerotic plaque vulnerability. Eur Heart J. 2011;32:412-421.
3. Crisby M, Nordin-Fredriksson G, Shah PK, Yano J, Zhu J, Nilsson J. Pravastatin
treatment increases collagen content and decreases lipid content, inflammation,
metalloproteinases, and cell death in human carotid plaques: implications for plaque
stabilization. Circulation. 2001;103:926-933.
4. Montecucco F, Matias I, Lenglet S, Petrosino S, Burger F, Pelli G, Braunersreuther V,
Mach F, Steffens S, Di Marzo V. Regulation and possible role of endocannabinoids and
related mediators in hypercholesterolemic mice with atherosclerosis. Atherosclerosis.
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5. Di Marzo V, Goparaju SK, Wang L, Liu J, Bátkai S, Járai Z, Fezza F, Miura GI, Palmiter
RD, Sugiura T, Kunos G. Leptin-regulated endocannabinoids are involved in maintaining
food intake. Nature. 2001;410:822-825.
6. Montecucco F, Bertolotto M, Ottonello L, Pende A, Dapino P, Quercioli A, Mach F,
Dallegri F. Chlorhexidine prevents hypochlorous acid-induced inactivation of alpha1antitrypsin. Clin Exp Pharmacol Physiol. 2009;36:e72-77.
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Supplementary figure 1. Scheme of the sampling of human and mouse carotid plaques. A.
Human carotid plaque was cut perpendicular to the long axis through the point of maximum
stenosis to obtain two portions (upstream and downstream the blood flow). The potential
influence of shear stress in the upstream and downstream portions of human plaque was
indicated as low shear stress (LSS), high shear stress (HSS) and oscillatory shear stress (OSS). B.
Mouse carotid plaques were processed in three portions depending on cast device placement and
analysed in the LSS portion (upstream part) and OSS portion (downstream the cast).
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Supplementary figure 2. Scheme of the sectioning of human and mouse plaques. A. Each
upstream and downstream portion was divided in the middle in two sub-segments (one-half for
histology [Histo] and one-half for (endocannabinoid measurements [Endo] and mRNA isolation
[RNA]). Specimens for Histo were serially cut in 7 μm sections. Series of eight sections
(separated by 105 µm from each other) were used for immunostaining. B. HSS, LSS and OSS
portions were serially cut in 5 m transversal sections. Series of eight sections for HSS and
eleven sections for LSS and OSS (separated by 45 m from each other) were used for
immunostaining. C. Mouse aortic sinus was serially cut in 5 m transversal sections. Series of
five sections (separated by 50 m from each other) were used for each staining.
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Supplementary figure 3. Treatment with the selective CB2 receptor agonist JWH-133 does
not affect lipid and inflammatory cell infiltration in mouse carotid plaques, but reduces
neutrophils in aortic roots. Representative microphotographs of mouse carotid (LSS and OSS
regions) and aortic root plaques showing staining for (A) lipids, (B) macrophages, and (C)
neutrophils in mice treated with vehicle or JWH-133.
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Supplementary figure 4. Treatment with JWH-133 reduces MMP-9 content in mouse
carotid and aortic roots plaques. Representative microphotographs of mouse carotid (LSS and
OSS regions) and aortic root plaques showing staining for (A) collagen and (B) MMP-9 in mice
treated with vehicle or JWH-133. A, collagen specificity is shown under bright illumination
without polarization (upper part) and under polarized light illumination (lower part).
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Supplementary figure 5. Pre-treatment with JWH-133 does not affect TNFRI and TNFRII
expression on human neutrophil surface membrane. Quantifications of TNFRI (A) and
TNFRII (B) surface expression (flow cytometry analysis) on human neutrophils cultured for 2h
in the presence or absence of different concentrations of JWN-133 (n=2). Data are expressed as
mean ± SD of percentages of positive cells and mean fluorescence intensity (MFI).
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Supplementary figure 6. Selective inhibition of EKR 1/2 intracellular signalling pathway
abrogated TNF--induced MMP-9 release by human neutrophils. MMP-9 release and
activity in supernatants of cells pre-incubated in polystyrene dishes in the presence or absence
(2h at 37ºC) of different concentrations of PD98059 (Inhibitor of MEK, a kinase directly
activating EKR 1/2). Then, cells were stimulated (30 min at 37ºC) with control medium (CTL),
10 ng/ml phorbol-12-myristate-13-acetate (PMA, positive control) or 100 ng/ml TNF-. Data
are expressed as median (interquartile range). A. MMP-9 release (n=7). B. Representative gel of
MMP-9 zymography. White band (arrow) on the gel represents the pro-MMP-9 gelatinolytic
activity of standard recombinant pro-MMP-9 (ST), and supernatants of cells. C. results of
densitometric quantifications of 7 different zymography experiments.
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