ONLINE SUPPLEMENT
ANGIOTENSIN-CONVERTING ENZYME 2 ACTIVATION IMPROVES
ENDOTHELIAL FUNCTION
Rodrigo A. Fraga-Silva
1,6
, Fabiana P. Costa-Fraga
2
, Tatiane M. Murça
3
, Patrícia L. Moraes
3
,
Augusto Martins Lima
1,2
, Roberto Q. Lautner
1,2
, Carlos H. Castro
1,4
, Célia Maria A. Soares
4
,
Clayton L. Borges
4
, Ana Paula Nadu
2
, Marilene L. Oliveira
2
, Vinayak Shenoy
5
, Michael J.
Katovich
5
, Robson A.S. Santos
1,2
, Mohan K. Raizada
6
, Anderson J. Ferreira
1,3
1
National Institute of Science and Technology in Nanobiopharmaceutics (NanoBiofar);
Departments of
2
Physiology and Biophysics and
3
Morphology, Federal University of Minas
Gerais, Brazil;
4
Department of Physiological Sciences, Federal University of Goiás, Brazil;
Departments of
5
Pharmacodynamics and
6
Physiology and Functional Genomics, University of
Florida, USA.
Short title: ACE2 Activation and Endothelial Function
Address for Correspondence: Anderson José Ferreira, PhD
Department of Morphology
Federal University of Minas Gerais
Av. Antônio Carlos, 6627
31.270-901, Belo Horizonte, MG, Brazil
FAX: (55-31) 3409-2810 - Phone: (55-31) 3409-2811
e-mail: anderson@icb.ufmg.br
2
METHODS
Experimental Model of Diabetes
Diabetes was induced in Wistar rats using streptozotocin (STZ; Sigma-Aldrich, USA;
Cat# S0130), as described elsewhere.
1
Briefly, anesthetized rats (100mg/kg ketamine and
10mg/kg xylazine) were injected with STZ (50mg/kg), intravenously. The animals were deprived
of food twelve hours before the STZ injection. After ten days, blood glucose levels were
measured using a glucometer and the treatment with XNT (1mg/kg per day, during four weeks,
gavage) was initiated. At the end of the XNT treatment, the measurement of the blood glucose
levels was repeated and the animals were sacrificed.
Isolated Aortic Rings Preparation
Isolated aortic rings were used to evaluate the acute and chronic (SHR and diabetic
animals) vascular effects of XNT. Aortic rings (4mm) from the descending thoracic aorta, free of
adipose and connective tissues, were set up in gassed (95%O
2
and 5%CO
2
) Krebs-Henseleit
solution (110.8mmol/L NaCl, 5.9mmol/L KCl, 25.0mmol/L NaHCO
3
, 1.1mmol/L MgSO
4
,
2.5mmol/L CaCl
2
, 2.3mmol/L NaH
2
PO
4
and 11.5mmol/L glucose) at 37
o
C under a tension of 1g
for 1 hour to equilibrate. The vessels from mice were stabilized with 0.5g of tension. Mechanical
activity was recorded isometrically using a force transducer (World Precision Instruments,
USA), amplified (Model TMB-4; World Precision Instruments, USA) and stored in a personal
computer equipped with an analogue-to-digital converter board (AD16JR; World Precision
Instruments, USA) utilizing the CVMS data acquisition/recording software (World Precision
Instruments, USA). The effects of XNT were evaluated in aortic rings pre-constricted with
phenylephrine (0.1µmol/L). In the acute protocols (Sprague-Dawley rats, n=8 to 10), XNT was
added into the bath in increasing cumulative concentrations (0.1nmol/L to 100µmol/L) or as an
unique submaximal concentration (10µmol/L) after the stabilization of the response to
phenylephrine. To evaluate the role of Mas, D-pro
7
-Ang-(1-7) (1µmol/L and 10µmol/L) or A-
779 (10µmol/L) was added into the bath associated to the XNT (10µmol/L). Aortic rings of
Mas
+/+
and Mas
-/-
mice were incubated with the submaximal concentration (10µmol/L) of XNT
in the absence or presence of losartan after the stabilization of the response to phenylephrine. In
addition, the acetylcholine (ACh, at 1nmol/L to 1µmol/L) and sodium nitroprusside (SNP, at
1nmol/L to 1µmol/L) vasorelaxant responses were used to evaluate the endothelial function of
chronically XNT-treated SHR (1mg/kg per day, for four weeks, osmotic minipumps, model
2ML4-Alzet
®
) and of chronically XNT-treated diabetic rats (Wistar rats, 1mg/kg per day, four
weeks, gavage). Also, the participation of the endothelium in the vasorelaxant effects of XNT
was examined by incubating this compound with aortic rings from normal Wistar rats with or
without intact endothelium.
Measurement of ACE2 Activity
The enzymatic activity (n=5) of human recombinant ACE2 (rhACE2; R&D systems,
USA; Cat# 933-ZN) and of aorta samples from normal and diabetic animals (n=6-8) was
determined using a fluorogenic substrate (fluorogenic peptide VI; R&D systems, USA; Cat#
ES007) in the presence or absence of XNT. Enzymatic activity was measured with a Spectra
Max Gemini EM Fluorescence Reader (Molecular Devices, USA), as previously described.
2,3
Samples were read every 30 seconds for, at least, 40 minutes immediately after the addition of
the fluorogenic peptide substrate at 37°C. The data obtained using tissue samples were
represented as the average of all readings.
3
Angiotensin-(1-7) Measurement
Aortas (n=5) were homogenized with 0.045 N HCl in ethanol (10 ml/g of tissue)
containing 0.90 µmol/l p-hydroxymercuribenzoate, 131.50 µmol/l of 1,10-phenanthroline, 0.90
µmol/l phenylmethylsulfonyl fluoride (PMSF), 1.75 µmol/l pepstatin A, 0.032% EDTA, and
0.0043% protease-free bovine serum albumin (BSA) and evaporated. After evaporation, the
samples were dissolved in 0.003% trifluoracetic acid (TFA). Blood samples (n=8) were collected
and transferred to polypropylene tubes containing 1 mmol/l p-hydroxymercuribenzoate, 30
mmol/l of 1,10-phenanthroline, 1 mmol/l PMSF, 1 mmol/l pepstatin A, and 7.5% EDTA (50
µl/ml of blood). After centrifugation, plasma samples were frozen in dry ice and stored at -80°C.
Ang II and Ang-(1-7) was extracted onto a BondElut phenylsilane cartridge (Varian). The
columns were preactivated by sequential washes with 10 ml of 99.9% acetonitrile/0.1%
heptafluorobutyric acid (HFBA) and 10 ml of 0.1% HFBA. Sequential washes with 10 ml of
99.9% acetonitrile/0.1% HFBA, 10 ml of 0.1% HFBA, 3 ml of 0.1% HFBA containing 0.1%
BSA, 10 ml of 10% acetonitrile/0.1% HFBA, and 3 ml of 0.1% HFBA were used to activate the
columns. After sample application, the columns were washed with 20 ml of 0.1% HFBA and 3
ml of 20% acetonitrile/0.1% HFBA. The adsorbed peptide was eluted with 3 ml of 99.9%
acetonitrile/0.1% HFBA into polypropylene tubes rinsed with 0.1% fat-free BSA. After
evaporation, Ang II and Ang-(1-7) levels were measured by radioimmunoassay (RIA), as
previously described.
4
Protein concentration in the crude homogenates was determined by the
Bradford method.
Western Blotting
Descending thoracic aorta of diabetic rats (Wistar rats, n=4 to 10) were collected and
homogenized in lysis buffer containing 9mol/l ureia and 2% CHAPS with freshly added protease
inhibitor mix (GE Healthcare, UK; Cat# 80-6501-23). Thirty micrograms of protein were
separated by electrophoresis on a 10% polyacrylamide gel and transferred to nitrocellulose
membranes. Non-specific bindings were blocked with TBS-T (Tris-base at 3%, Tween 20, pH
7.6) containing 5% non-fat skim milk. Membranes were probed with one of following specific
primary antibodies: anti-catalase (1:1000, Cell Signaling Technology, USA; Cat# 8841), anti-
SOD (1:1000, Cell Signaling Technology, USA; Cat# 2770), anti-NOX2 (1:250, Santa Cruz
Biotechnology, USA; Cat# SC-130549), anti-ACE2 (1:500, Gene Tex, CA, USA; Cat#
GTX15348) or anti-GAPDH (1:5000, Santa Cruz Biotechnology, USA; Cat# sc-166545)
followed by incubation with secondary antibodies. Immunoreactive bands were quantified by
densitometry.
Immunohistochemistry
Paraffin-embedded ventricular sections (6µm, n=4 to 6 sections) were first incubated with
0.3% H
2
O
2
in phosphate-buffered saline (PBS) for 15 minutes followed by incubation with 2%
BSA in PBS containing 0.3% Triton X100 for 1 hour. Sections were incubated overnight at 4
o
C
with the anti-ACE2 antibody (1:250, Gene Tex, CA, USA; Cat# GTX15348) diluted in PBS
containing 0.3% Triton X100 and 0.3% BSA. After four or five rinses in PBS, biotinylated goat
anti-rabbit IgG secondary antibody was added for 1 hour followed by incubation with avidin-
biotin-peroxidase complex reagents (Dako LSAB+System-HRP, Dako, Inc., Carpinteria, CA,
USA) for 1 hour. The sections were stained with diaminobenzidine solution for 4 minutes and
counterstained with hematoxylin. Each step was followed by washing the sections with PBS
4
containing 0.3% Triton X100. Sections incubated without primary antibodies were used as
negative controls. The sections were analyzed using an Olympus BX 41 microscope (Olympus,
Inc., Irving, TX, USA). Five fields of each section were sequentially photographed under 40x
objective. The strongest labeling area of the positive labeled tissue was measured using the
Image Pro-Plus software and the results were expressed in percentage of occupied area. The
segmentation was based in the pixels number of the strongest labeling area.
Detection of Reactive Oxygen Species
To detect ROS production in aorta of diabetic rats (Wistar rats, n=6 to 8), 30µm-
cryosections of the descending thoracic aorta were stained with dihydroethidium (DHE; Sigma-
Aldrich, USA, Cat# 37291) at 2µmol/L in PBS for 15 minutes at 37ºC.
5
The slices were washed
with PBS and examined on a fluorescence microscope equipped with a digital imaging system
(Carl Zeiss MicroImaging, USA). Furthermore, the intracellular levels of ROS (n=9 to 12
experiments) in human aortic endothelial cells (HAEC; Cascade Biologics, USA; Cat# C-006-
5C) were also measured using DHE, as described elsewhere.
6
Briefly, cells were grown in glass
slides in a humidified 5% CO
2
/95% O
2
atmosphere at 37
o
C. ROS production was stimulated by
Ang II at 0.1µmol/L in the presence or absence of XNT at 1µmol/L. After 20 minutes, the cells
were washed twice with PBS and loaded with DHE at 2µmol/L for 5 minutes. HAEC were
washed with PBS and examined on a fluorescence microscope (Carl Zeiss MicroImaging, USA).
DHE fluorescence intensity of acquired digital images was quantified by the NIH software
Image J.
REFERENCES
1.
Dall'Ago P, Fernandes TG, Machado UF, Belló AA, Irigoyen MC. Baroreflex and
chemoreflex dysfunction in streptozotocin-diabetic rats. Braz J Med Biol Res. 1997;30:119-124.
2.
Hernández Prada JA, Ferreira AJ, Katovich MJ, Shenoy V, Qi Y, Santos RAS, Castellano
RK, Lampkins AJ, Gubala V, Ostrov DA, Raizada MK. Structure-based identification of small-
molecule angiotensin-converting enzyme 2 activators as novel antihypertensive agents.
Hypertension. 2008;51:1312-1317.
3.
Huentelman MJ, Zubcevic J, Katovich MJ, Raizada MK. Cloning and characterization of a
secreted form of angiotensin-converting enzyme 2. Regul Pept. 2004;122:61-67.
4.
Botelho LMO, Block CH, Khosla MC, Santos RAS. Plasma angiotensin-(1-7)
immunoreactivity is increased by salt load, water deprivation, and hemorrhage. Peptides.
1994;15:723-729.
5.
Sukhanov S, Higashi Y, Shai SY, Vaughn C, Mohler J, Li Y, Song YH, Titterington J,
Delafontaine P. IGF-1 reduces inflammatory responses, suppresses oxidative stress, and
decreases atherosclerosis progression in ApoE-deficient mice. Arterioscler Thromb Vasc Biol.
2007;27:2684-2690.
6.
Cai S, Khoo J, Channon KM. Augmented BH4 by gene transfer restores nitric oxide synthase
function in hyperglycemic human endothelial cells. Cardiovasc Res. 2005;65:823-831.
5
RESULTS
10
20
30
40
0
1000
2000
3000
4000
5000
rhACE2+XNT, n=5
rhACE2, n=5
Negative Control, n=5
*
*
*
Time (minutes)
R
e
la
ti
ve
Fl
uo
re
s
c
enc
e
f
o
r
rh
A
C
E
2
A
c
ti
v
ity
(A
.U
.)
Supplemental Figure S1. XNT enhances the activity of recombinant human ACE2 (rhACE2).
The fluorescence resulted from the breakdown of the fluorogenic substrate by rhACE2 in the
presence or absence of XNT. *P<0.001 (Two-way ANOVA followed by the Bonferroni's
multiple comparison test). Each point represents the mean ± SEM (n=5) of relative fluorescence
in arbitrary unit (A.U.).
CTRL
CTRL+XNT
STZ
STZ+XNT
0
100
200
300
*
*
*
*
R
e
la
ti
ve Fl
u
o
resce
nce f
o
r
A
o
rt
ic
A
C
E
2
a
c
tiv
ity
(A
.U
.)
Supplemental Figure S2. XNT enhances the activity of aortic ACE2 of normal (CTRL) and
diabetic (STZ) rats. The fluorescence resulted from the breakdown of the fluorogenic substrate
by ACE2 in the presence or absence of XNT. *P<0.05 (Student t-test). The data represent the
average of all readings (n=6-8 in each group). A.U.: arbitrary unit (A.U.).
6
A
STZ
STZ+XNT
0
10
20
30
40
50
60
70
*
n=8
n=8
pg/
m
l pl
a
s
m
a
B
STZ
STZ+XNT
0
1
2
3
4
n=5
n=5
pg/
m
g p
ro
tei
n
Supplemental Figure S3. Ang-(1-7) levels in (A) plasma and (B) aorta of diabetic (STZ) rats
treated or not with XNT. ACE2 activation significantly increased the concentration of Ang-(1-7)
in the plasma of diabetic rats. *P<0.05 (Student t-test).
7
STZ
STZ+XNT
0
10
20
30
40
50
60
70
n=8
n=8
p
g
/m
l p
la
s
m
a
Supplemental Figure S4. Plasma Ang II levels in diabetic (STZ) rats treated or not with XNT.
No significant changes were observed between the groups (Student t-test).
Supplemental Figure S5. Angiotensin-converting enzyme 2 (ACE2) protein expression in aorta
of control (non-diabetic) and diabetic rats treated or not with XNT. Representative blot and
quantification of the expression. A total of 30µg of protein was applied to the gel. Data were
normalized using GAPDH. One-way ANOVA followed by the Bonferroni’s multiple
comparison test. The data are presented as mean ± SEM (n=4 to 5).
8
Supplemental Figure S6. Expression of Angiotensin-converting enzyme 2 (ACE2) in aorta of
control (non-diabetic) and diabetic rats treated or not with XNT. Representative
photomicrographs of (A) control, (B) diabetic rat treated with saline and (C) diabetic rat treated
with XNT. The negative control (inset) was obtained by omitting the primary antibody from the
incubation procedure. (D) Quantification of ACE2 in aorta of control (non-diabetic) and diabetic
rats treated or not with XNT. One-way ANOVA followed by the Bonferroni’s multiple
comparison test. The data are presented as mean ± SEM. Scale bar represents 50 µm.
9
A
B
C
Supplemental Figure S7. Catalase, superoxide dismutase (SOD) and NOX2 protein expression
in aorta of diabetic rats. Representative blots and quantification of the expression of (A) catalase,
(B) SOD and (C) NOX2 in aorta of control (non-diabetic) and diabetic rats treated or not with
XNT. For each blot a total of 30µg of protein was applied to the gel. Data were normalized using
GAPDH. One-way ANOVA followed by the Bonferroni’s multiple comparison test. The data are
presented as mean ± SEM (n=6 to 10).
10
Supplemental Figure S8. XNT attenuates the Ang II-induced reactive oxygen species (ROS)
production in human aortic endothelial cells (HAEC). The cells were incubated with Ang II
(0.1µmol/L) in the presence or absence of XNT (1µmol/L). ROS production was detected using
dihydroethidium (DHE; 2µmol/L). Representative photomicrographs of HAEC showing the
ROS production in control cells (A), Ang II-treated cells (B), XNT-treated cells (C) and Ang
II+XNT-treated cells (D). Quantification of ROS content (E). **p<0.01 and ***p<0.001 (One-
way ANOVA followed by the Bonferroni’s multiple comparison test). Each column represents
the mean ± SEM (n=9 to 12 experiments) of relative fluorescence in arbitrary unit (A.U.).
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