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SUPPLEMENT MATERIAL

In vivo animal studies

Intravital microscopy

The cremaster muscle was dissected free of tissues and exteriorized onto an

optical clear viewing pedestal. The muscle was cut longitudinally with a cautery and

held flat against the pedestal by attaching silk sutures to the corners of the tissue. The

muscle was then perfused continuously with warmed bicarbonate-buffered saline (pH

7.4) at a rate of 1 ml/min.

Cremasteric microcirculation was then observed using an intravital microscope

(Nikon Optiphot-2, SMZ1, Badhoevedorp, Netherlands) equipped with a 50x objective

lens (Nikon SLDW, Badhoevedorp, The Netherlands) and a 10x eyepiece. A video camera

(Sony SSC-C350P, Koeln, Germany) mounted on the microscope projected the image

onto a color monitor and the images were CCD recorded for playback analysis. Single

unbranched cremasteric venules and arterioles (20-40 m in diameter) were selected for

study. Vessel diameter was measured on-line using a video caliper (Microcirculation

Research Institute, Texas A&M University, College Station, Texas). Centerline red blood

cell velocity (Vrbc) was also measured on-line by using an optical Doppler velocimeter

(Microcirculation Research Institute, Texas A&M University, College Station, Texas).

Vessel blood flow was calculated from the product of mean red blood cell velocity (Vmean

= Vrbc / 1.6) and cross sectional area, assuming cylindrical geometry. Wall shear rate ()

was calculated based on the Newtonian definition: = 8 x (Vmean/Dv) s-1

, in which Dv is

vessel diameter1.

The number of adherent and emigrated leukocytes was determined off-line

during playback of the recorded images. A leukocyte was defined as adherent, to the

arteriolar or venular endothelium, if it was stationary for at least 30 s. Leukocyte

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adhesion was expressed as the number per 100 m length of vessel per 5 min.

Leukocyte emigration was expressed as the number of white blood cells per

microscopic field surrounding the vessel. In each animal, the leukocyte responses in

three to five randomly selected arterioles or postcapillary venules were averaged.

Histology and Immunofluorescence

Once intravital microscopy determinations were performed, mice were sacrificed

and the cremaster muscle was isolated and fixed in 4 % paraformaldehyde for 10

minutes. The protocol followed was similar to that previously described2, briefly,

whole-mounted muscles were incubated for 2 h in 0.2% Triton X-100, 1% BSA and

0.5% horse serum in phosphate-buffered saline (PBS). They were then incubated

overnight at 4°C with a primary Ab rabbit anti-mouse CX3CL1 (1/100 dilution) or

eFluor 450-conjugated anti-mouse CD31 (PECAM-1) (1/100 dilution). Samples were

subsequently washed with PBS and incubated for 1.5 h at room temperature with Alexa

Fluor 488-conjugated donkey anti-rabbit secondary antibody (1/500 dilution). All

antibodies were diluted in 0.1% PBS/BSA. The muscles were then mounted with

Slowfade Gold Reagent (Invitrogen, Eugene, Oregon, USA). Images were acquired by

means of a fluorescence microscope (Axio Observer A1, Carl Zeiss, NY) equipped with a

40x objective lens and a 10x eyepiece.

Effect of losartan on atherosclerosis development and cell composition in

apoE-/-

mice on atherogenic diet

ApoE-/-

(C57BL/6J) female mice were obtained from Charles River laboratories

and kept on a low-fat standard diet (2.8% fat). At 6 weeks of age, mice were fed with

high fat atherogenic diet (10.8% total fat, 0.75% cholesterol) for 6 weeks alone

(atherogenic diet group) or treated with an angiotensin-II AT1 receptor antagonist

(losartan, 10mg/kg/day; atherogenic diet + losartan). Losartan was administered

3

through a subcutaneously implanted osmotic minipump for the same period of time as

previously reported3. The control mouse group was kept on a low-fat standard diet for 6

weeks. After treatments, hearts containing the aortic root were harvested from mice,

washed with PBS, fixed with 4% paraformaldehyde/PBS overnight and paraffin-

embedded for sectioning and analysis as previously described4. The atheroma-rich

aortic arch with bifurcations was snap frozen for RNA extraction and mRNA

expression analysis as reported4.

Atherosclerosis was evaluated as lesion area in at least 3-5 aortic root cross-

sections stained with hematoxilyn/eosin4. For macrophage quantification in lesions a rat

anti-Mac-3 monoclonal antibody (1/200 dilution) was used. After peroxidase

inactivation (H2O2 0.3%) and blockade with horse serum, samples were incubated

overnight (4ºC) with the primary antibody. Detection was performed with a biotin-

conjugated goat anti-rat secondary antibody (1/300 dilution) followed by HRP-

Streptavidin and DAB substrate incubation. Slides were counterstained with

hematoxilin and mounted with EUKITT. For T-cell detection within the lesion, aortic

root cross-sections were blocked as above described and incubated overnight with an

anti-CD3 antibody (1/75 dilution) followed by an incubation for 1h at room temperature

with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody. Slides were

also stained with a monoclonal anti-SMα-actin-Cy3™ conjugated antibody (1/100

dilution) to visualize the smooth muscle cells in the artery wall and mounted with Slow-

Fade Gold antifade reagent Reagent (Invitrogen, Eugene, Oregon, USA). Preparations

were analyzed by fluorescent microscopy with an inverted fluorescent microscope

(Axio Observer A1, Carl Zeiss, NY).

RNA from mouse aortas was obtained using TRIzol Reagent. Real time RT-PCR

was performed using standard protocols employing the following primers (Forward:

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Fw; Reverse: Rv) were used: mouse cyclophilin Fw: 5’

TGGAGAGCACCAAGACAGACA-3’ and Rv 5’-TGCCGGAGTCGACAATGAT-3’;

mouse CX3CL1 Fw: 5’-GCGAAATCATGTGCGACAAG-3’ and Rv 5-

GCTGATAGCGGATGAGCAAAG-3’. Reactions were run on a thermal Cycler 7900

Fast System and results were analyzed with the software provided by the manufacturer

(Applied Biosystems).

Human studies

RT-PCR

Reverse transcription was performed in 300 ng of total RNA with TaqMan

reverse transcription reagents kit. cDNA was amplified with specific primers for

fractalkine (CX3CL1), TNFα , Nox 2, Nox 4, Nox 5 and GAPDH (all pre-designed by

Applied Biosystems) in a 7900HT Fast Real-Time PCR System (Applied Biosystem,

Carlsbad, CA) using Universal Master Mix (Applied Biosystems). Relative

quantification of these different transcripts was determined following the 2−ΔΔCt

method,

using GAPDH as an endogenous control and normalizing with respect to control group.

Flow cytometry

Cells were washed and incubated for 1 h at 2 x 106 cells/ml with a PE-

conjugated mAb against human CX3CL1 (1.25 µg/ml) in PBS with 0.2% BSA and

0.05% NaN3 on ice. After 2 washes, cells were suspended in PBS containing 2%

paraformaldehyde. The fluorescence signal of the labeled cells was then analyzed by

flow cytometry (FACSCanto Flow cytometer, BD Biosciences, Frankiln Lakes, NJ).

The expression of CX3CL1 (PE-fluorescence) was expressed as the mean of

fluorescence intensity (MFI).

Leukocyte-endothelial cell interactions under flow conditions

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The Glycotech flow chamber was assembled and placed on an inverted

microscope stage. Freshly isolated mononuclear cells (1 × 106/ml) were then perfused

across the endothelial monolayers (HUVEC, HUAEC or transfected HUAEC). In all

experiments, leukocyte interactions were determined after 5 min at 0.5 dyn/cm2. Cells

interacting on the surface of the endothelium were visualized and recorded (×20

objective, ×10 eyepiece) using phase-contrast microscopy (Axio Observer A1, Carl

Zeiss microscope, NY).

Immunofluorescence

Confluent endothelial cells were grown on glass coverslips and stimulated with

1 μM Ang-II (with or without an Ang-II AT1 receptor antagonist, EXP3174, 100 µM)

or vehicle for 24h. The cells were fixed with 4% paraformaldehyde and blocked in a

PBS solution containing 1% BSA. They were then incubated overnight at 4°C with a

primary mouse mAb against human CX3CL1 (1:200 dilution) in a 0.1% BSA/PBS

solution, followed by incubation for 45 min at room temperature with a secondary

antibody Alexa Fluor 488-conjugated goat anti-mouse mAb (1/1000 dilution). Cell

nuclei were counterstained with 4'-6-diamidino-2-phenylindole (DAPI). Images were

captured with a confocal microscope (Leica TCS/SP2, Solms, Germany).

Western Blot

After treatment, cells were washed, detached, collected and centrifuged at

15,000 g at 4ºC for 30 min to yield the whole extract. Protein content was determined

according to the Bradford method. Samples were denatured, subjected to SDS-PAGE

using a 10% running gel, and transferred to nitrocellulose membrane. Nonspecific

binding sites were blocked with 3% BSA in TBS solution and were then incubated

overnight with rabbit polyclonal antibody against human CX3CL1 (0.2 µg/ml), a

mouse polyclonal antibody against human Nox 2 (0.2 µg/mL), rabbit polyclonal

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antibody against human Nox 4 (2 µg/ml), a rabbit polyclonal antibody against human

Nox 5 (2 µg/ml) or a goat polyclonal anti-human TNFα (0.1 µg/ml). Subsequently

they were washed and incubated for a further 1 h with the corresponding secondary

HRP-linked antibody: anti-rabbit IgG (1:2000 dilution), anti-goat IgG or anti-mouse

IgG (1:2000 dilution), developed using the ECL procedure. Signals were recorded

using a luminiscent analyser (FujiFilm image Reader LAS1000, Fuji, Tokyo, Japan)

and analyzed using the software ImageJ (Windows free version).

Transfection of TNFα, Nox 2, Nox 4 or Nox 5 siRNA

The transfection reagent Lipofectamine RNAiMAX was employed following the

manufacturer's instructions. The mRNA expression for transcripts was determined by

real time RT-PCR after 48 h post-silencing and compared with the siRNA control at the

respective time in order to determine silencing efficiency. In addition, cells were tested

for TNFα, Nox 2, Nox 4 or Nox 5 expression by western blot of cell lysates.

Experimental protocols

In a first set of experiments, HUAEC and HUVEC were grown to confluence

and stimulated with 1 μM Ang-II or TNFα (20 ng/ml) for 1, 4 or 24h. Some plates were

incubated in the presence of the Ang-II AT1 receptor antagonist EXP3174 (100 µM) 1 h

prior to Ang-II stimulation. CX3CL1 mRNA expression was determined by RT-PCR

and the protein by flow cytometry, immunoflorescence analysis and western blot.

Another group of HUAEC and HUVEC were stimulated with Ang-II for 24h.

Freshly isolated human mononuclear cells were perfused across endothelial cell

monolayers and leukocyte-endothelial cell interactions were determined under flow

conditions. To determine the effect of endothelial CX3CL1 expression on mononuclear

recruitment, endothelial cells were incubated with a monoclonal neutralizing antibody

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against human CX3CL1 (5 μg/ml) or an isotype-matched control antibody (MOPC-21, 5

μg/ml) 10 min prior to mononuclear cell superfusion.

To investigate the possible contribution of TNFα to Ang-II-induced CX3CL1

expression and mononuclear cell recruitment, HUAEC were transfected for 48 h with

control or TNF-α-specific siRNA prior to Ang-II stimulation. Ang-II-induced responses

were measured 24h later.

To evaluate the potential involvement of NADPH and xanthine oxidase (XO) on

Ang-II-induced effects, cells were incubated for 1 h with a NADPH oxidase inhibitor

(apocynin, 30 µM) or a XO inhibitor (allopurinol, 100 µM) and were then stimulated with

Ang-II for 24h. The doses employed of these compounds were as previously described5.

In subsequent experiments, HUAEC were transfected with either control or Nox 2, Nox

4 or Nox 5–specific siRNA to determine the Nox isoform involved in these responses.

Forty-eight hour post-transfection the HUAEC were stimulated with 1 µM Ang-II and

CX3CL1 expression and mononuclear cell arrest were evaluated.

To further elucidate the signalling pathways involved in Ang-II-induced

responses, endothelial cells were pretreated with the inhibitors of ERK1/2 (PD098059,

20 μM), p38MAPK (SB202190, 20 μM), JNK (SP600125, 20 μM) or NFkB (MOL294,

2.5 μM) 1 h prior to Ang-II stimulation. These concentrations have been employed in

previous studies to inhibit ERK1/2, p38MAPK, JNK or NFkB6-8

. Following 24h

stimulation with 1 µM Ang-II, both CX3CL1 expression and mononuclear cell arrest were

determined.

In another set of experiments, endothelial cells were stimulated for 24h with

Ang-II (1 µM), TNFα (20 ng/ml) or IFNγ (20 ng/ml), independently or in the following

combinations: Ang-II + TNFα, Ang-II+ IFNγ, TNFα + IFNγ or Ang-II+ TNFα + IFNγ.

The impact on CX3CL1 expression and mononuclear cell capture was also measured. To

8

determine the contribution of endothelial CX3CL1 expression induced by IFNγ, IFNγ +

Ang-II, IFNγ + TNFα or IFNγ + Ang-II+ TNFα stimulation on mononuclear cell

recruitment, endothelial cells were incubated with a monoclonal neutralizing antibody

against human CX3CL1 (5 μg/ml) or an isotype-matched control antibody (MOPC-21, 5

μg/ml) 10 min prior to mononuclear cell superfusion. Then, freshly isolated human

mononuclear cells were perfused across endothelial cell monolayers and leukocyte-

endothelial cell interactions were determined under flow conditions.

Finally, to elucidate the potential contribution of ADAM10 and ADAM 17, cells

were preincubated for 30 min with an ADAM-10 inhibitor (GI254023X, 10 μM) or for

10 min with an ADAM-17 inhibitor (TAPI-2 (100 μM). These concentrations have been

used in previous studies9, 10

. After 24h stimulation with Ang-II (1 µM) or Ang-II (1 µM)+

TNFα (20 ng/ml) + IFNγ (20 ng/ml) , both CX3CL1 expression and mononuclear cell

arrest were determined.

Determination of platelet contribution to the CX3CL1-dependent Ang-II-

induced mononuclear cell adhesion to HUAEC

Platelet-leukocyte co-aggregation was assessed by flow cytometric analysis of

platelet (CD41)-positive cells within the CD45-positive cells (leukocytes). Duplicate

samples were incubated in the dark for 30 min with saturated amounts (1:10 dilution) of

the PE-conjugated mAb against human CD41 and the APC-conjugated mAb against

human CD45. Samples were run in a Flow cytometer (FACSCanto Flow cytometer, BD

Biosciences, Frankiln Lakes, NJ) and the expression of CD41 (PE fluorescence) and

CD45 (APC fluorescence) in mononuclear cells was measured. In some experiments the

aggregates were incubated with EDTA (10 mM, for 15 min, 37ºC) to promote platelet

dissociation as previously described11

. In another set of experiments, human whole

blood from healthy volunteers was treated with TRAP-6 (15 μM, 30 min at 37ºC) in the

9

presence of recombinant Hirudin (2.9 μM). Samples were stained with the above

mentioned conjugated antibodies. Then red blood cells were lysed and leukocytes were

fixed using an automated EPICS Q-PREP system (Coulter Electronics, Hialeah,

Florida) before run in the flow cytometer.

To determine the contribution of endothelial CX3CL1 expression to mononuclear

recruitment, HUAEC were stimulated with Ang-II for 24h. EDTA treated or untreated

isolated human mononuclear cells were perfused across the endothelial cell monolayers

and leukocyte-endothelial cell interactions determined under flow conditions. HUAEC

were incubated with a monoclonal neutralizing antibody against human CX3CL1 (5

μg/ml) or an isotype-matched control antibody (MOPC-21, 5 μg/ml) 10 min prior to

mononuclear cell superfusion. A similar procedure was employed to analyze leukocyte

adhesion in TRAP-6-stimulated or un-stimulated whole blood. For this purpose, whole

blood was diluted (1/10 in HBSS) prior to its perfusion.

Determination of CX3CL1 receptor (CX3CR1) expression by flow cytometry

To determine the effect of Ang-II on CX3CL1 receptor (CX3CR1) expression in

circulating monocytes and lymphocytes from healthy volunteers, mononuclear cells were

isolated and incubated with 1 μM Ang-II for 1, 4 or 24h. Some cells were incubated in

the presence of the Ang-II AT1 receptor antagonist, EXP3174 (100 µM) 1 h prior to

Ang-II stimulation. Duplicate samples were incubated on ice in the dark for 20 min with

saturated amounts (10 μl) of the carboxyfluorescein (CFS)-conjugated mAb against human

CX3CR1. Samples were run in a Flow cytometer (FACSCanto Flow cytometer, BD

Biosciences, Frankiln Lakes, NJ). The expression of CX3CR1 (CFS fluorescence) in

monocytes and lymphocytes was measured according to size (forward scatter) and

granularity (side scatter) and expressed as the mean of fluorescence intensity (MFI).

Materials

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Endothelial basal medium-2 (EMB-2) supplemented with endothelial growth

medium-2 (EGM-2) was acquired from Lonza Iberica (Barcelona, Spain). Ketamine and

xylazine hydrochloride were supplied by ORION Pharma (Espoo, Finland). Apocynine,

allopurinol, hirudin, PD098059, SB202190, SP600125, the monoclonal anti-SMα-actin-

Cy3™ conjugated antibody, the mouse anti-human β-actin mAb (clone AC-15), the

mAb IgG1 (MOCPC21) and the rabbit polyclonal anti-human Nox 5 Ab were

purchased from Sigma-Aldrich (Madrid, Spain). The rabbit polyclonal anti-mouse

CX3CL1 and the PE-conjugated conjugated rat monoclonal anti-mouse CD31 (clone 390)

were provided by eBioscience (Hatfield, UK). Recombinant human TNFα, IFN and the

rabbit polyclonal anti-human CX3CL1 employed for western blotting were acquired

from Peprotech (London, UK). The PE-conjugated mouse monoclonal anti-human

CX3CL1 (clone 51637), the CFS-conjugated mouse monoclonal anti-human CX3CR1

(clone 528728) and the mouse monoclonal anti-human CX3CL1 (clone 81506) were

purchased from R&D Systems (Abingdon, UK). The rabbit polyclonal anti-human Nox

4 was supplied by Abcam (Cambridge, UK). The mouse monoclonal anti-human Nox 2

(clone NL7) Ab and the DAB substrate were purchased from Serotec (Oxford, UK).

The sodium heparin (5000 U/ml or 50 mg/ml) was supplied by Pharmaceutical

Laboratories Rovi SA (Madrid, Spain). Ficoll-Paque TM plus and ECL developer were

purchased from GE Healthcare (Chalfont St Giles, UK). DAPI, TRIzol isolation

reagent, and Alexa Fluor 488-conjugated secondary antibodies were from Molecular

Probes-Invitrogen (Carlsbad, CA). The secondary HRP-linked anti-rabbit IgG Ab was

supplied by Cell Signaling Technology (Grand Island, NY). The secondary Abs, HRP-

linked anti-goat IgG, HRP-linked anti-mouse IgG and the anti-mouse CD3 antibody

were purchased from Dako (Glostrup, Denmark). HRP-Streptavidin was from

LABVISION Corporation, Thermo Fisher Scientific Inc. ( Kalamazoo, MI). TNFα,

11

Nox2, Nox4 or Nox5–specific siRNA were purchased from Dharmacon (Lafayette,

CO). TaqMan reverse transcription reagents kit were acquired from Applied

Biosystems, (Perkin-Elmer Corporation, Carlsbad,CA). The low-fat standard diet was

from Panlab (Barcelona, Spain). The high fat atherogenic diet (10.8% total fat, 0.75%

cholesterol) was adquired from, Sniff, (Germany). The Alzet 2004 osmotic minipumps

were from Charles River (Barcelona, Spain). The anti-Mac-3 mAb (clone M3/84) and

the biotin-conjugated goat anti-rat secondary Ab were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). The PE-conjugated mouse monoclonal anti-human

CD41Ab (clone HIP8) and the APC-conjugated mouse monoclonal anti-human CD45

Ab (clone HI30) were from BioLegend ( San Diego, CA). TRAP-6 was from TOCRIS

bioscience (Bristol, UK). The EUKITT was provided by Deltalab (Barcelona, Spain).

TAPI-2 was from Enzo Life sciences (Lausen, Switzerland). MOL-294 was kindly

donated by Dr. Kahn (Department of Pathobiology, University Washington, Seattle,

WA. Losartan and EXP3174 were kindly donated by Merck Sharp & Dohme, Madrid,

Spain. The ADAM-10 inhibitor, GI254023X was kindly provided by Dr. Andreas

Ludwig (Institute of Pharmacology and Toxicology RWTH Aachen University, Aachen,

Germany).

References

1. House SD, Lipowsky HH. Leukocyte-endothelium adhesion:

microhemodynamics in mesentery of the cat. Microvasc Res. 1987;34:363-379.

2. Massena S, Christoffersson G, Hjertstrom E, Zcharia E, Vlodavsky I, Ausmees

N, Rolny C, Li JP, Phillipson M. A chemotactic gradient sequestered on

endothelial heparan sulfate induces directional intraluminal crawling of

neutrophils. Blood. 2010;116:1924-1931.

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3. Scalia R, Gong Y, Berzins B, Freund B, Feather D, Landesberg G, Mishra G. A

novel role for calpain in the endothelial dysfunction induced by activation of

angiotensin II type 1 receptor signaling. Circ Res. 2011;108:1102-1111.

4. Gonzalez-Navarro H, Abu Nabah YN, Vinue A, Andres-Manzano MJ, Collado

M, Serrano M, Andres V. p19(ARF) deficiency reduces macrophage and

vascular smooth muscle cell apoptosis and aggravates atherosclerosis. J Am Coll

Cardiol. 2010;55:2258-2268.

5. Beltran AE, Briones AM, Garcia-Redondo AB, Rodriguez C, Miguel M,

Alvarez Y, Alonso MJ, Martinez-Gonzalez J, Salaices M. p38 MAPK

contributes to angiotensin II-induced COX-2 expression in aortic fibroblasts

from normotensive and hypertensive rats. J Hypertens. 2009;27:142-154.

6. Goebeler M, Kilian K, Gillitzer R, Kunz M, Yoshimura T, Brocker EB, Rapp

UR, Ludwig S. The MKK6/p38 stress kinase cascade is critical for tumor

necrosis factor-alpha-induced expression of monocyte-chemoattractant protein-1

in endothelial cells. Blood. 1999;93:857-865.

7. Henderson WR, Jr., Chi EY, Teo JL, Nguyen C, Kahn M. A small molecule

inhibitor of redox-regulated NF-kappa B and activator protein-1 transcription

blocks allergic airway inflammation in a mouse asthma model. J Immunol.

2002;169:5294-5299.

8. Han C, Liu J, Liu X, Li M. Angiotensin II induces C-reactive protein expression

through ERK1/2 and JNK signaling in human aortic endothelial cells.

Atherosclerosis. 2010;212:206-212.

9. Rabie T, Strehl A, Ludwig A, Nieswandt B. Evidence for a role of ADAM17

(TACE) in the regulation of platelet glycoprotein V. J Biol Chem.

2005;280:14462-14468.

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10. Schulz B, Pruessmeyer J, Maretzky T, Ludwig A, Blobel CP, Saftig P, Reiss K.

ADAM10 regulates endothelial permeability and T-Cell transmigration by

proteolysis of vascular endothelial cadherin. Circ Res. 2008;102:1192-1201.

11. Postea O, Vasina EM, Cauwenberghs S, Projahn D, Liehn EA, Lievens D,

Theelen W, Kramp BK, Butoi ED, Soehnlein O, Heemskerk JW, Ludwig A,

Weber C, Koenen RR. Contribution of platelet CX(3)CR1 to platelet-monocyte

complex formation and vascular recruitment during hyperlipidemia. Arterioscler

Thromb Vasc Biol. 2012;32:1186-1193.

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SUPPLEMENTAL FIGURES

Figure I: Effect of losartan on atherosclerosis development and cell composition in

apoE-/-

mice on atherogenic diet. Mice were sacrificed at 12 weeks of age after 6

weeks subjected to a low fat standart diet (control diet), high fat atherogenic diet

(atherogenic diet) or high fat atherogenic diet treated with losartan (atherogenic diet +

losartan). Atheroma lesion (A) was determined in 3-5 histological sections per mice.

Macrophage (Mac-3+, B) and T-cell (CD3

+, C) content as well as CX3CL1 mRNA

expression (D) were also evaluated. Representative images of the whole aortic root

cross-sections (E), Mac-3+ stained area (F) and CD3

+ cells (G) for the atherogenic diet

fed mice treated or not with losartan are shown. Results are the mean ± SEM of n=5

animals per group. *p<0.05 or **p<0.01 relative to values animals subjected to a

control diet; +p<0.05 relative to values in animal subjected to an atherogenic diet.

15

Figure II: Ang-II does not induce CX3CL1 mRNA expression after HUAEC and

HUVEC 1 h stimulation (A and B) but causes increased protein expression after

24h stimulation (Western blot, C and D). Results are the mean ± SEM of the 2-Ct

values of n= 5-6 independent experiments (A and B). **p<0.01 relative to values in the

medium group. Protein expression was determined by Western blotting. Results are the

mean ± SEM of at least 4 independent experiments (C and D). *p<0.05 or **p<0.01

relative to values in the medium group; +p<0.05 or ++p<0.01 relative to values in the

Ang-II group.

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Figure III: Platelet adhesion to mononuclear cells do not contribute to the

CX3CL1-dependent Ang-II-induced mononuclear cell adhesion to HUAEC at low

shear. Isolated mononuclear cells were incubated or not with EDTA (A, B and C) and

whole blood was treated (red) or not (blue) with TRAP-6 (E and F). Then, the samples

were double stained with an anti-CD41-PE and anti-CD45-APC mAbs. Results are the

mean ± SEM of n= 5 independent experiments. *p<0.05 or **p<0.01 relative to values

in the EDTA or TRAP-6 untreated groups respectively (C and F). Effect of a

neutralizing antibody against CX3CL1 function in the flow chamber assay (D, F).

Results are the mean ± SEM of 5 independent experiments. **p<0.01 relative to values

in the medium group; ++p<0.01 relative to values in Ang-II + MOPC21 group.

17

Figure IV: Ang-II does not affect CX3CL1 receptor expression (CX3CR1) in

human monocytes and lymphocytes. Results are the mean ± SEM of n=6 independent

experiments and are expressed as MFI.

18

Figure V: Ang-II increases Nox 2, Nox 4 or Nox 5 expression in HUAEC, which is

abolished in HUAEC transfected with siRNAs targeting Nox 2, Nox 4 or Nox 5.

Results are the mean ± SEM of the 2-Ct

values of n= 4-6 independent experiments.

Protein expression of the different Nox isoforms was determined by western blot.

Results are mean ± SEM of at least 4 independent experiments. *p<0.05 or **p<0.01

relative to values in the medium group; +p< 0.05 relative to values in the Ang-II group.

19

Figure VI: A neutralizing antibody against CX3CL1 function inhibited the

recruitment of mononuclear leukocytes induced by the combined stimulation of

IFNγ with TNFα and/or Ang-II. Cells were stimulated with IFNγ (20 ng/mL), Ang-II

(1 µM) + IFNγ, TNFα (20 ng/mL) + IFNγ or IFNγ + Ang-II + TNFα for 24 h. The

effect of a neutralizing antibody against CX3CL1 function was evaluated in the flow

chamber assay. Results are the mean ± SEM of 5 independent experiments. *p<0.05 or

**p<0.01 relative to values in the medium group; +p<0.05 relative to the respective

MOPC21 value.

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Figure VII: CX3CL1 expression (A) and mononuclear cell arrest (B) induced by

Ang-II or Ang-II + TNFα + IFNγ are increased by ADAM 17 inhibition in

HUAEC. Cells were stimulated with 1µM Ang-II or Ang-II (1 µM)+ TNFα (20 ng/ml) +

IFNγ (20 ng/ml) for 24 h. Some cells were pretreated with an ADAM 10 inhibitor or an

ADAM 17 inhibitor 30 or 10 min prior to the stimulus. Results are the mean ± SEM of

n=5 independent experiments (A and B). *p<0.05 or **p<0.01 relative to values in the

medium group.