E ndothelial cells play a central role in maintaining vascular
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- Bu səhifədəki naviqasiya:
- Short title
- METHODS Experimental Model of Diabetes
- Isolated Aortic Rings Preparation
- Measurement of ACE2 Activity
- Angiotensin-(1-7) Measurement
- Detection of Reactive Oxygen Species
- RESULTS 10 20 30 40 0 1000 2000
- Supplemental Figure S6.
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 ,
1,2 , Roberto Q. Lautner 1,2 , Carlos H. Castro 1,4 , Célia Maria A. Soares 4 ,
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
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
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 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
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.
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.
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.
Paraffin-embedded ventricular sections (6µm, n=4 to 6 sections) were first incubated with 0.3% H 2
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.
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.
1.
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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.
immunoreactivity is increased by salt load, water deprivation, and hemorrhage. Peptides. 1994;15:723-729. 5.
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.
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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+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 .)
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.).
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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
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+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).
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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
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).
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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.). Yüklə 176,4 Kb. Dostları ilə paylaş:
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