Supplemental Methods
Study population
Exclusion criteria were: age >60 years, history or clinical evidence of hypertension (blood
pressure > 140/90 mmHg), clinical or biochemical evidence of diabetes or endocrine (thyroid,
adrenal, or gonadal) dysfunction, sustained ethanol consumption (more than 60 g per day),
dyslipidemia, smoking habits, renal or liver impairment, menopausal status, and any documented
cardiovascular disease. Patients assuming any chronic pharmacologic therapy were also excluded.
Obese patients were consecutively recruited among patients referring to the Department of
Clinical and Experimental Medicine of the University of Pisa for a complete clinical evaluation
preceding laparoscopic bariatric surgery. Control subjects were recruited among consecutive
patients referring to the Department of Surgery of the University of Pisa to undergo elective
laparoscopic cholecystectomy due to gallbladder stones. Exclusion criteria were the same as those
adopted for obese patients, and a body mass index (BMI) > 27 Kg/m2. Sitting blood pressure
(average of three records) was measured with a mercury sphygmomanometer, by means of a
standard cuff and a large cuff in lean and obese individuals, respectively. Insulin sensitivity was
estimated by the homeostasis model assessment of insulin resistance (HOMA-IR) Index.
Preparation and mounting of small arteries
Arterial segments were considered viable and used for experiments if they constricted by more
than 50% of their resting lumen diameter in response to an extraluminal application of highpotassium (125 mM) physiological solution containing 10 µM noradrenaline (NA).
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Nitrite Levels
Small vessels were incubated in Dulbecco’s modified Eagle’s medium for 14 h at 37° C in an
atmosphere of 5% CO2. After 14-h incubation, the medium was removed and divided into
aliquots. After loading the plate with samples, conversion of nitrate to nitrite was performed and
total nitrites were measured with the Griess reagent. The absorbance at 540 nm was measured, and
the nitrite concentration was determined by using a calibration curve of standard sodium nitrite
concentrations versus absorbance. Nitrite levels were corrected by milligrams of wet weight.
Detection of vascular superoxide anion generation
The in situ production of superoxide anion was measured by means of the fluorescent dye
dihydroethidium (DHE; Sigma). Three slides per segment were analyzed simultaneously after
incubation with antagonists or Krebs solution at 37°C for 30 min. Krebs-HEPES buffer containing
2 μM DHE was then applied onto each section and evaluated under fluorescence microscopy. The
percentage of arterial wall area stained with the red signal was evaluated using an imaging
software (McBiophotonics Image J; National Institutes of Health, Bethesda, MD).
Western Blot Analysis
50 g of proteins extracted from artery wall were diluted in SDS-PAGE buffer and heated at
100°C for 5 min. Samples and molecular weight markers were electrophoresed on Any kD MiniProtean TGX gels and transferred to PVDF membrane. After a blocking step using BSA 3% in
TTBS (TBS and Tween-20 0.05%) for 1 h at room temperature, blots were washed three times in
TTBS and incubated overnight at 4°C with both primary antibodies diluted 1:100 and anti-β-actin
diluted 1:500. The bands detection was performed incubating the blot at the same time with
2
species-specific secondary antibody diluted 1:5000 for 1 h at room temperature, followed by
enzymatic chemiluminescence kit.
Drugs, reagents and Solutions
N-nitro-L-arginine methyl ester, BQ-123, sodium nitroprusside and HANKS’balanced salt
solution: Sigma Chemicals, St. Louis, Missouri.
Infliximab: Schering-Plough (Merck), Whitehouse Station, N
TNF-α ELISA: R&D systems Minneapolis, MN
RNasy lipid tissue mini kit: Qiagen, Milan, Italy
cDNA Reverse transcription kit; TNF-α: Hs00174128_m1; Adiponectin: Hs00605917_m1; CD68:
Hs0283616_g1; GAPDH: Hs02758991-g 1 (Applied Biosystems, Life Technologies, Monza,
Italy).
SuperfrostPlus slides and immunofluorescence antibodies: ET-1: PA3-067, ETA : PA3-065, ETB :
PA3-066, TNF-α: PA5-19810 (Thermo Scientific, Braunschweig, Dreieich, Germany); TNF-α
receptor 1: ab19139: CD68: ab125047 (Abcam, Cambridge, UK).
Antifade mounting medium : Vectashield, Vector Laboratories, Burlingame, CA
Any kD Mini-Protean TGX gels: Bio-Rad Laboratories, Segrate, Italy
PVDF membrane and Enzymatic chemiluminescence kit: Millipore, Billerica, MA
WB primary antibodies: NOS3: sc-654; pNOS3: sc-21871 (Santa Cruz Biotechnology, Santa Cruz
CA); JNK 9252; p-JNK 9251S; p38MAPK 9212; p-p38MAPK 9111S; ERK 9102; pERK 9101S
(Cell Signaling Technology, Danvers, MA).
Secondary antibodies: AP307P (anti-rabbit) and AP308P (anti-mouse): Chemicon International,
Temecula, CA.
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MJ mini thermocycler: Biorad, Hercules, CA
Eco-Real Time: Illumina Inc., San Diego, CA
Cryostat microtome and Laser scanning confocal microscope: Leica, Nussloch, Germany.
Fluorescence microscope: Olympus BX61
Light transmission microscope EVOS FL Life Technologies.
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A 0
Relaxation response (%)
*
10
20
†
30
IFX 1 µM
IFX 10 µM
IFX 100 µM
IFX 1 mM
40
50
0
Relaxation response (%)
B
10
20
30
Time (min)
40
0
10
20
30
†
40
BQ-123
BQ-123
BQ-123
BQ-123
50
0.01 µM
0.1 µM
1 µM
10 µM
60
0
Contracting response (%)
C
10
20
30
Time (min)
40
0
2
*
4
*
6
L-name 1 µM
L-name 10 µM
L-name100 µM
L-name 1 mM
8
10
0
10
20
30
Time (min)
5
40
Figure S1
P<0.01
Nitrite levels (nM/mg weight)
45
*
*
40
P<0.001
35
30
P<0.001
P<0.01
P<0.05
25
20
15
10
5
0
Basal
IFX
Basal
Control subjects
IFX
Obese patients
PVAT +
Basal
IFX
Basal
Control subjects
IFX
Obese patients
PVAT Figure S2
6
Contracting response (%)
0
10
P < 0.001
20
30
-10
-9
-8
Endothelin-1
(10x
-7
-6
mol/L)
Figure S3
7
CD68 GENE EXPRESSION
A)
B)
CD68 PROTEIN EXPRESSION
target/reference (AU)
25
CONTROLS
OBESE
15
42kDa
10
ß-actin
5
0
C)
P<0.01
20
CONTROLS
IMMUNOFLUORESCENCE
OBESE
D)
TRANSMITTED LIGHT
CONTROLS
CONTROLS
dimer
monomer
OBESE
NOS3
ß-actin
OBESE
Figure S4
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Figure S1. Time-dependent concentration response-curves to Infliximab (IFX, Panel A) and BQ123 (Panel B) in pre-contracted small vessels, and to L-name (Panel C) in quiescent small vessels,
from obese patients.
Data presented as means ± SEM (n=8). *P<0.05; † P<0.001 for the whole curve.
Figure S2. Levels of nitrite measured in culture medium of small arteries from control subjects
and obese patients, in basal conditions or in the presence of infliximab (IFX), either with intact or
removed PVAT. Data presented as means ± SEM (n=8).
Figure S3. Concentration response-curves to exogenous Endothelin-1 in quiescent small vessels
from Obese (○ ) and Controls (● ). Data presented as means ± SEM (n=6).
Figure S4. RNA (panel A) and protein (panel B) CD68 expression in the vascular wall of Obese
and Controls. Representative image (immunofluorescence and light transmission, panel C)
showing CD68 protein expression in untreated small arteries from 1 Ctrl and 1 Obese. Panel D:
protein expression of monomeric and dimeric eNOS in the vascular wall of Obese and Controls.
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