supplement material - arteriosclerosis, thrombosis,...
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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
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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
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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
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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,
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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
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3. Scalia R, Gong Y, Berzins B, Freund B, Feather D, Landesberg G, Mishra G. A
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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
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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.
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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.
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10. Schulz B, Pruessmeyer J, Maretzky T, Ludwig A, Blobel CP, Saftig P, Reiss K.
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Theelen W, Kramp BK, Butoi ED, Soehnlein O, Heemskerk JW, Ludwig A,
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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.
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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.
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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.
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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.
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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.