Caillon et al full Methods

The angiotensin II type 2 receptor activates flow-mediated outward remodeling through
T cells-dependent interleukin-17 production
Antoine Caillon, Céline Grenier, Linda Grimaud, Emilie Vessieres, Anne-Laure Guihot,
Simon Blanchard, Eric Lelievre, Marie Chabbert, Etienne D Foucher, Pascale Jeannin, Céline
Beauvillain, Pierre Abraham, Laurent Loufrani, Yves Delneste , Daniel Henrion.
Detailed Material and methods 1
1. Animal protocols
Three-month-old mice (Charles River, L’Arbresles, France) were submitted to diverse
surgical procedures in order to increase blood flow. The investigation was performed in
agreement with the guidelines from Directive 2010/63/EU of the European Parliament on the
protection of animals used for scientific purposes (authorization of the laboratory # 00577).
The protocol was approved by the Institutional Animal Care and Use Committee (IACUC):
Committee on the Ethics of Animal Experiments of “Pays de la Loire” (CEEA, permits #
CEEA PdL 2012.118 and 2012.141).
1.1 Chronic increase in blood flow in the mesenteric artery.
Three consecutive first-order arteries, equivalent in size were used. Ligatures were
applied to 4 second-order arterial branches, as shown below. The artery located between the
two ligated arteries was designed as high flow (HF) artery. This artery had then to supply
blood to the surrounding tissues initially irrigated by the ligated vessels thank to the presence
of an arcading artery. Arteries located at distance of the ligated arteries were used as controls
(normal flow, NF) 1; location of the ligations is shown below:
2
1.2 Skin-flap procedures
Skin ischemia model consists in a U-shaped peninsular skin incision on the dorsal
surface of the mouse 2. In brief, the skin flap was elevated dorsally from the underlying
muscular bed and immediately resutured. The vascular remodeling was evaluated to compare
the right and left vascular pedicles arising from the lateral thoracic arteries at day 0 and day
21 with the ImageJ software (NIH) (see scheme below).
1.3. Anesthesia and analgesia
In each protocol, animals were anesthetized with isoflurane (2.5%) and treated with
buprenorphine (Temgesic®, 0.1 mg/kg, s.c.) before and after surgery. They were housed in a
thermoregulated pre-warmed, humidified incubator allowing mouse surveillance.
2. Experimental groups:
a- Untreated mice (named wild type, WT): Swiss mice (control for the Nude mice) and
FVBN mice (WT littermate control for the AT2Ry/- mice).
b- Wistar rats treated were treated or not with the AT2R blocker PD123319 (30 mg/kg
per day, solvent H2O) using ALZET osmotic minipumps, as previously described 3, 4.
After one week, they were submitted to surgery, as described above, in order to
increase blood flow in one mesenteric artery.
3
c- WT (FVB/N) mice treated daily with an anti-mouse CD3ε monoclonal antibody
(mAb) (150 µg clone 145-2C11. BioXcell, West Lebanon, NH) IP injection at day 0,
1, 3 and 6).
d- WT (FVB/N) mice treated at day 0 and 4 after ligation with 200 µg (IP injection) of
the following antibodies (BioXcell):
- Mouse IgG1,
- Mouse anti–mouse IL-17A Ab (clone 17F3).
e- Nude mice Crl:NU(Ico)-Foxn1nu (from Charles River; Swiss background) treated or
not with IL-17 (5 ng/h, IP, solvent Phosphate Buffered Saline) during one week using
Alzet minipump (ALZET osmotic minipumps, ref 1002; Cupertino, CA). IL-17 was
purchased from eBioscience (San Diego, CA).
f- Male mice lacking the gene encoding for AT2R (AT2Ry/-). Mice were on the FVB/N
background 5.
g- AT2Ry/- mice treated or not with IL-17 (5 ng/h, 7 days, IP, Alzet osmotic minipump,
ref 1002, solvent Phosphate Buffered Saline).
h- Mice lacking the gene encoding for IL-17 (IL-17-/-) and their WT littermate control.
i- Swiss mice aged 12 months were treated chronically with the AT2R agonist C21 using
Alzet osmotic minipumps (0.3 mg/kg per day for 3 consecutive weeks, solvent H2O)
or with the vehicle (0.9% NaCl, pH 9). One week after implanting the minipump,
surgery was performed in order to change blood flow in one mesenteric artery.
3. Removal of the tissues for analysis:
For mice submitted to the ligation of the mesenteric arteries (paragraph 1.1.), the
mesentery was quickly removed after CO2 euthanasia and placed in an ice-cold physiological
salt solution (PSS) of the following composition (in mmol/L): 135.0, NaCl, 15.0, NaHCO3,
4.6 KCl, 1.5, CaCl2, 1.2, MgSO4, 11.0, glucose, 10.0, N-2-hydroxyethylpiperazine-N-2ethylsulfonic acid. The PSS was maintained at pH 7.4, PO2 160 mmHg, PCO2 37 mmHg 6.
Mesenteric arteries (HF and NF) were gently dissected and divided into two segments,
proximal for the functional study and distal for histological and biochemical studies. For
analysis of the mechanism segments were collected after 2 days and for measurements of the
remodeling segments were collected after one week
For mice submitted to the skin flap procedure (paragraph1.2.), animals were
anesthetized as described above and vascular remodeling evaluated to compare the right and
left vascular pedicles arising from the lateral thoracic arteries at day 0 and day 21 using
ImageJ software (NIH) (see the scheme above in 2.1.3).
4
4. Pressure-diameter relationship in mesenteric arteries in vitro
Arterial segments collected after one week were cannulated at both ends and mounted
in a video monitored perfusion system (Living System, LSI, Burlington, VT) as previously
described 7. Two glass cannulae were used to cannulate a 2-3 mm long arterial segment.
Arterial segments were bathed in a 5 ml organ bath containing a Ca2+-free PSS containing
ethylene-bis-(oxyethylenenitrolo) tetra-acetic acid (EGTA, 2 mmol/L) and sodium
nitroprusside (SNP, 10 µmol/L). Pressure steps (10 to 125 mmHg) were then performed in
order to determine passive arterial diameter. Pressure and diameter measurements were
collected continuously using a Biopac data acquisition system (Biopac MP100 and
Acqknowledge® software; La Jolla, CA) 1.
Glass
micropipette
artery
5. Quantitative Real-time PCR
The levels of mRNA encoding selected proteins (see Table 1) in HF mesenteric arteries
from wild type mice and AT2R-/- mice after 2 days of ligation were determined by real-time
quantitative polymerase chain reactions (qPCRs), as previously described 8. Mesenteric
arteries were dissected in ice-cold physiological salt solution (PSS) and kept in 100 µL
RNAlater (Sigma-Aldrich, St Louis, MO) at -20°C until RNA extraction using the RNeasy®
micro kit (Qiagen, Venlo, The Netherlands). Total RNA (200 ng) extracted from each artery
were used to synthesize cDNA for RT-PCR using the QuantiTect® Reverse Transcription kit
(Qiagen) according to the manufacturer instruction. The qPCRs were performed on a Light
Cycler 480 (Roche Life Science) using Power SYBR® Green PCR Master Mix (Applied
Biosystems, Illkirch, France). Gene-specific primers were designed using Primer3 online
software and validated by testing PCR efficiency.
5
Table 1. List of the genes analyzed and primers used
NCBI Reference
Gene
Protein
Sequence
forward sequence (5'-3')
reverse sequence (5'-3'
agtr1a
AT1R (a)
NM_177322.3 actcacagcaaccctccaag
ctcagacactgttcaaaatgcac
agtr1b
AT1R (b)
NM_175086.3 gtgacatgatcccctgacagt
agtgagtgaactgtctagctaaatgc
agtrap
ATRAP
NM_009642.4 acatgcaccgtgaacgag
tgatgagtcaattgtctggtagg
bdkrb1
Bradykinin b1 R NM_007539.2 ccggtattgcccagtagga
cttggctcaatgctgtttca
bdkrb2
Bradykinin b2 R NM_009747.2 cggctcgcttgagaaaag
accgacagaaacccgagtag
cav1
caveolin1
NM_007616.2 aacgacgacgtggtcaaga
cacagtgaaggtggtgaagc
esr1
ER alpha
MN_007956.4 gctcctaacttgctcctggac
cagcaacatgtcaaagatctcc
gapdh
GAPDH
NM_008084.2 ccggggctggcattgctctc
ggggtgggtggtccagggtt
gusb
GUSB
NM_010368.1 ctctggtggccttacctgat
cagttgttgtcaccttcacctc
hprt ter
HPRT
NM_013556.2 tgatagatccattcctatgactgtaga aagacattctttccagttaaagttgag
ICAM
icam1
NM_010493.2 gctaccatcaccgtgtattcg
aggtccttgcctacttgctg
IFN-γ R1
ifngr1
NM_010511.2 tcaaaagagttccttatgtgccta
tacgaggacggagagctgtt
IFN- γ R2
ifngr2
NM_008338.3 tcaaaagagttccttatgtgccta
tacgaggacggagagctgtt
il17ra
IL17a R
NM_008359.2 tgggatctgtcatcgtgct
atcaccatgtttctcttgatcg
il17rc
IL17c R
NM_134159.4 tactttaaaccacacagacctggtt ggctctagcgaccacacct
il23r
IL23 R
NM_144548.1 ccaagtatattgtgcatgtgaaga
agcttgaggcaagatattgttgt
itgam
CD11b
NM_001082960.1 caatagccagcctcagtgc
gagcccaggggagaagtg
itpr3
IP3 R
NM_080553.3 atcttccaggagagcattgg
tcaggttgtagaaagacttctgga
mmp2
MMP2
NM_008610.2 gtgggacaagaaccagatcac
gcatcatccacggtttcag
MMP9
mmp9
NM_013599.2 ttctggcacacgcctttc
ccatagtaagtggggatcacg
mtus1
AtIP
NM_001005865.2 tgtgagaagcttcagagcattt
ggcggcgtttaagttgtc
iNOS
nos2
NM_010927.3 ctttgccacggacgagac
tcattgtactctgagggctgac
nos3
eNOS
NM_008713.4 ccagtgccctgcttcatc
gcagggcaagttaggatcag
parp1
PARP1
NM_007415.2 ctcatgctaccacgcacaac
cctcgcgctctatcttgaa
sell
L-Selectin
NM_011346.2 cagtgtggagcatctggaaa
aaaggctcacactggaccac
thbs1
TSP1
NM_011580.3 ccccaaccttcccaactc
gggttgtaatggaatggacag
timp1
TIMP1
NM_011593.2 catggaaagcctctgtggat
gatgtgcaaatttccgttcc
VCAM1
vcam
NM_011693.2 tgattgggagagacaaagca
aacaaccgaatccccaactt
Gene expression was quantified comparative cycle threshold (Ct) method and Hprt, Gapdh
and Gusb was used as reference. Differences in gene expression between groups were
compared with M.E.V. software.
6. Cell culture and activation of immune cells
Immune cells were isolated from lymph nodes and spleen of WT mice by grinding and
washed by centrifugation. CD4+ T cells were isolated by positive selection before purification
of CD44high CD62low memory T cells and CD44low CD62Lhigh naive T cells by FACS; purity
was >99% (data not shown), as assessed by flow cytometry.
Naive or memory CD4+ T cells (1×105), cultured in X-VIVO 15 medium (Lonza,
Basel, Switzerland), were stimulated with immobilized anti-CD3 mAb (145-2C11) and 1
µg/ml anti-CD28 mAb (eBioscience). Cells were stimulated with Angiotensin II (AngII, 10-8
M), the non-peptidic AT2R agonist C21 (gift of Vicore-Pharma, Sweden, 10-8M) or the
6
petidic AT2R agonist CGP42112 (10-8M, Sigma-Aldrich). IL-6 plus TGFβ (respectively 200
and 2 ng/ml) and IL-23 (200 ng/ml) were used as positive controls to induce IL-17A
production by naïve and memory T cells, respectively. In some experiments, cells were
stimulated in the presence of 0.1 µM of candesartan (TCV, 10-8M, gift of Astra-Zeneca,
Sweden) or PD123319 (10-7M, Sigma-Aldrich).
7. Cytokine quantification
IL-17A was quantified by ELISA (eBioscience).
8. Western-blot analysis
After an overnight starvation, the mouse endothelial cell MS-1 was cultured without or
with IL-17A (10-1000 ng/ml) during 6 h in X-Vivo 15 medium (Lonza, Basel, Switzerland)
The mouse brain microvascular endothelial cell MBrMec was cultured with or without IL17A (100 ng/ml) during 6h in Optimem medium. The mouse aorta smooth muscle cell
MOVAS was cultured with or without IL-17A (100 ng/ml) during 6h in DMEM medium.
Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Membranes
were subsequently incubated overnight with the primary antibody before incubation with the
appropriate peroxidase-labeled secondary antibody (table shown below) for 1 h. The reaction
was visualized by ECL detection according to the manufacturer's instructions (Bio-Rad,
Marnes-la-Coquette). Membranes were stripped in 0.1 M glycine, pH 2.8, for 1 h before reblotting.
Target Protein
eNOS/NOS type III
Phospho-eNOS (Ser 1177)
β-actin
Reference
610297
612392
A5316
Phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204) 9101
ERK
610031
Phospho-p38 MAPK (Thr180/Tyr182) 9211
p38 MAPK
9212
IKB-β
sc-74452
c-Jun
sc-45
Phospho-NFκB p65 (Ser 276)
sc-101749
Goat anti-Rabbit IgG (H+L) HRP conjugate
Goat anti-Mouse IgG (H+L) HRP conjugate
31460
31430
Provider
BD Biosciences
BD Biosciences
Sigma-Aldrich
Cell Signaling
Technology
BD Biosciences
Cell Signaling
Technology
Cell Signaling
Technology
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Thermoscientific
Thermoscientific
9. Effect of IL-17 on arterial contractility and endothelium-dependent relaxation.
Segments of mesenteric arteries (2-mm long each) were dissected and mounted in a
wire myography (Danish Myo-technology, DMT, Copenhagen, DK) 9. Cumulative
concentration-response curves to phenylephrine (0.001 to 10 µmol/L) and acetylcholine (0.01
7
to 10 µmol/L) were performed after one-hour incubation with IL-17A (10 ng/mL) or with the
vehicle. Cumulative concentration-response curves to acetylcholine were obtained before and
after incubation (20 min) with the NO-synthase inhibitor L-NAME (10 µmol/L).
Acetylcholine-dependent relaxation was performed after precontraction of the arteries with
phenylephrine and serotonin to 70% of their maximal contractile response 10.
10. Zymogram assay
Mouse macrophages (RAW 264.7 cell line), previously starved over-night, were treated or not
with IL-17A (100 ng/ml) during 24 h in X-Vivo 15 medium (Lonza, Basel, Switzerland). The
mouse brain microvascular endothelial cell MBrMec was cultured with or without IL-17A
(10-1000 ng/ml) during 24h in Optimem medium. The mouse aorta smooth muscle cell
MOVAS was cultured with or without IL-17A (10-1000 ng/ml) during 24h in DMEM
medium. Supernatants were collected to evaluate MMPs activated in zymogram assay 6
8
11. Flow cytometry analysis
Arteries with the surrounding perivascular tissue (including mainly fat tissue) were
isolated and digested using 450 U/ml collagenase type I and 5 U/ml elastase for 1 h at 37°C,
with intermittent agitation. Cell suspensions were then passed through a 100 µm diameter
filter before washing by centrifugation (800×g). Cells were fixed with 1% (v:v) PFA. After
washing, cells were stained for 25 min at 4°C with anti-CD3, anti-CD45, anti-RORγt, anti-Tbet and antiCD4 monoclonal antibodies (eBioscience) or isotype control monoclonal
antibodies. Fluorescence was analyzed on a LSR-II flow cytometer with DIVA software
driving (Becton Dickinson Biosiences, Franklin Lakes, NJ). Data were analyzed using the
FlowJo software (Tree Star Inc., Ashland, OR).
The sorting strategy used is detailed in the following figures:
After gating on the SSC and FSC parameter, immune CD45+ cells were isolated among
mononuclear cells before sorting CD3+ T cells among CD45+ cells. Contour plots from one
representative experiment of five independent experiments are shown below:
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Immune cells (CD45 cells) and T cells (CD3+ cells) were quantified by flow
cytometry in the mesenteric fat tissue around the NF and the HF artery.
Contour plots from one experiment representative of five independent
experiments are shown.
9
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Flow cytometry sorting of CD44high CD62Llow CD4+ memory T cells and of naive CD44low
CD62Lhigh CD4+ naive T cells is shown below (contour plots from one representative
experiment of 12 independent experiments are shown):
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10
12. Confocal imaging
Confocal imaging was performed as previously shown 11 on NF and HF arteries with
perivascular fat tissue collected after 2 days of ligationand stained with anti-mouse CD45
Antigen PE (eBioscience, Ref 12-4801 Clone BM8 1µg/ml), anti-mouse CD31 (PECAM-1)
FITC, (eBioscience, Ref 11-0311 Clone 390 2.5µg/ml), anti-mouse Ly-6G (Gr-1) PE
(eBioscience, Ref 12-5931 Clone RB6-8C5 1µg/ml), anti-mouse F4/80 Antigen PE
(eBioscience, Ref 12-4801 Clone BM8 1µg/ml) and Dapi, Sigma-Aldrich (Ref D9542
25µg/ml). PE-labeled anti-rabbit IgG antibodies (eBioscience) were used to detect bound
rabbit antibodies. Positive staining was visualized using confocal microscopy and the QEDimage software (Solamere Technology). Image acquisition was performed using Histolab
(Microvision). Pixel quantification was executed as described previously using Histolab 11.
13. Statistical Analysis
Results were expressed as means ± SEM.
Significance of the differences between groups was determined using 2-way ANOVA for
consecutive measurements (pressure-diameter curves and for concentration response curves to
acetylcholine and phenylephrine) followed by Bonferroni’s test.
When comparing only 2 groups, Mann-Whitney test was used. For comparisons involving 3
or more groups, one-way ANOVA was performed followed by Bonferroni’s test.
Statistical analysis was performed under GraphPad®.
Probability values less than 0.05 were considered significant.
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