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Online data supplement
Material and Methods
Animal Model of In-Stent Restenosis
Five-year-old male adult non-human primates (cynomolgus monkeys) weighing 4 to 5 kg were fed a high-cholesterol diet (0.5% cholesterol and 6% corn oil) for 4 weeks, as we previously described.1 The animals were then randomized to 3 groups as follows: (1) a no-treatment vehicle control group (0.5% carboxymethyl cellulose sodium salt); (2) an olmesartan (15 mg/kg per day, Sankyo Pharmaceutical Co., Tokyo, Japan) group; and (3) a valsartan (50 mg/kg per day, Novartis Inc.) group. This dose of olmesartan or valsartan was selected because it is reported that olmesartan at 15 mg/kg reportedly does not affect systemic arterial pressure and heart rate in monkeys2. Five days after grouping, all monkeys were anesthetized with ketamine hydrochloride (10 mg/kg IM) and sodium pentobarbital (30 mg/kg IV), and the femoral artery was surgically exposed. Then, a 15 mm-long Multilink stent mounted over the 3-mm balloon was implanted in the iliac artery (30-s inflation at 6 atm followed by 60-s inflation at 8-10 atm, resulting in stent-to-artery ratio of 1.2:1.0) as we previously described.1 The femoral artery was then ligated and the incision was closed. After the operation, all monkeys were fed the same high-cholesterol diet. All monkeys were killed with a lethal dose of anesthesia 28 days after stenting for morphometrical analysis. The animal breeding, care before and after the stenting, and analyses were performed in Gaoyao Kangda Laboratory Animal Science and Technology in China. Male Japanese white rabbits (KBT Oriental, Tokyo) weighing 3.0 to 3.5 kg were fed a high-cholesterol diet containing 1% cholesterol and 3% peanut oil for 2 weeks, as we previously described.1 The animals were then divided into untreated control and olmesartan groups (olmesartan 3 mg/day). Five days after grouping, they were then anesthetized by intramuscular injection of xylazine (5mg/kg) and ketamine (35mg/kg). The right common carotid artery was surgically exposed, and stent was implantated in the iliac artery under fluoroscopic guidance as described above. The carotid artery was then ligated and the incision was closed. After the operation, rabbits were fed the same high-cholesterol diet. Rabbits were killed with a lethal dose of anesthesia 7, 10, or 28 days after stenting, for biochemical, immunohistochemical, and morphometrical analyses. The animal care before and after the stenting, and analyses were performed at the Experimental Animal Research Center and the Station for Collaborative Research and the Morphology Core, Kyushu University Graduate School of Medical Sciences. All rabbits and monkeys received aspirin at 81 mg/day and ticlopidine 100 mg/day from 5 days before stenting until euthanasia.
Morphometric and Immunohistochemical Analysis
As we described1, stented arterial segments were excised from rabbits and monkeys and fixed for 24 hours with 95 % ethanol and 1% acetic acid. Each stented segment was divided into two parts at the center of the stent. The proximal part was embedded in methyl methacrylate mixed with n-butyl methacrylate to allow for sectioning through metal stent struts. Serial sections were stained with elastica van Gieson. To evaluate the stent area, lumen area, medial area, and neointimal area, the neointimal thickening (neointimal area/length of internal elastica lamina) were measured. The average of the 3 to 5 sections was reported for each animal. The distal part was used for immunohistochemical analysis. After stent struts were gently removed with micro forceps, the tissue was dehydrated, embedded in paraffin, and cut into 5-µm thick slices. In rabbits, antibodies against macrophage/monocytes (RAM11, Dako), endothelial cells (CD31, Dako, vWF, Dako), ACE (CHEMICON), Ang II (Peninsula laboratories), AT1R (Biogenesis), AT2R (Santa Cruz), MCP-1 (monocyte chemoattractant protein-1) (a gift from Dr.Matsukawa, Kumamoto University), PDGF-B (Mochida), or non-immune mouse IgG (Zymed) were used. Following avidin-biotin amplification, the slides were incubated with diaminobenzidine and counterstained with hematoxylin. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining (in situ apoptosis detection kit, Takara) and evaluated as described previously.1 In monkeys, antibodies against MCP-1 (R & D), CCR2 (Sigma), or non-immune mouse IgG (Dako) were used. Morphometric analysis was performed using a microscope with a computerized, digital image analysis system, by a single observer who was blind to the treatment protocol.3,4
Detection of Superoxide
The proximal edge of rabbit stented iliac arteries and contra-lateral non-stented control arteries were excised, and snap frozen with OCT-compounds. The unfixed enzymatically intact sections (30 µm thick) from the arterial segments were simultaneously incubated with dihydroethidium (DHE; 10µm/L) in PBS for 30 minutes at 37 ºC in a humidified chamber protected from light. DHE is oxidized to ethidium bromide in the presence of superoxide, which intercalates into nuclear DNA and fluoresces red. Fluorescence was detected with a 585-nm long-pass filter. To examine whether or not NADPH oxidase is involved in DHE fluorescence, serial sections were pre-incubated for 30 minutes with either PBS or the NADPH oxidase inhibitors [apocynin (4-hydroxy-3-methoxy acetophenone) (Aldrich) at 500 µmol/L and diphenyliodonium (Aldrich) at 100 µmol/L] before incubation with DHE as previously reported.5 As previously reported, both inhibitors reduced DHE fluorescence by more than 50 %, suggesting that NADPH oxidase are involved in the pathogenesis of increased superoxide generation in stented arteries.
Quantitative Real-Time Reverse Transcription-PCR
Separate experiments were performed to determine the effects of ARB on gene expression. Stented rabbit iliac arterial segments were excised and snap frozen immediately after stent struts were gently removed with micro forceps. Contra-lateral non-stented arterial segments were used as control. Real-time PCR amplification was performed with the rabbits’ cDNA using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) as described previously.3 The respective PCR primers and TaqMan probes were designed from GenBank databases using a software program (Applied Biosystems; Table I in this online supplement). Results were analyzed using Sequence Detection Software (Applied Biosystems), expressed in arbitrary units, and adjusted for GAPDH mRNA levels.
Isolation of Mononuclear Cells and Vascular Progenitor Cell Culture
Rabbit peripheral blood mononuclear cells (MNCs) were initially isolated from by Ficoll density-gradient centrifugation. MNCs were then resuspended in either endothelial basal medium (EGM-2, Cambrex) to stimulate endothelial cell differentiation, or in EGM-2 supplemented with PDGF-BB (10 ng/mL) and bFGF (10 ng/mL) to stimulate smooth muscle cell differentiation, and placed on plates coated with fibronectin for a final evaluation of colony areas at day 14. In another experiment to evaluate the response of MNCs to antiogensin II or olmesartan, MNCs were stimulated with various concentrations of angiotensin II (10 nM-100 µM) or olmesartan (10 nM-100 µM) every 48 h. Morphological appearance and immunofluorescence were used to define endothelial cell and smooth muscle cell phenotypes.6,7 Primary antibodies to vWF and VE-Cadherin (both from Santa Cruz Biotechnology) were used to evaluate EPC. The EPCs were also confirmed to be positive for antibodies with Flk-1(Santa Cruz Biotechnology) and CD31 (Dako Corp), and to uptake of LDL particles after incubation with DiI-Ac-LDL (10 µg/mL, Biomedical Technologies). Antibodies against a-smooth muscle actin (a SMA, HHF35, IA4, Dako), myosin heavy chain (Dako), and calponin (Dako) was used to evaluate SMPC. Human coronary vascular smooth muscle cells (hcVSMCs) were used as a positive control and Cos-7 cells as a negative control for the SMC-specific markers. In each immunofluorescence experiment, an isotype-matched IgG control was also used. The percentages of SMPC- or EPC-marker-positive area per well were calculated.
Biochemical Measurements
Rabbit and monkey plasma total cholesterol levels were determined with commercially available kits (Wako Pure Chemicals). In rabbits, plasma Ang II levels were measured. Serum MCP-1 was measured using a human MCP-1 ELISA kit (R & D).
Measurements of plasma ARB levels and arterial blood pressure
Separate experiments were performed to determine the effects of ARB on arterial blood pressure in rabbits under light anesthesia 28 days after stenting. Before sacrifice, arterial blood pressure was measured directly with a pressure manometer (Nihon Kohden) by connecting a sheath catheter introduced into the femoral artery.
On the last day of the drug treatment period, blood samples were drawn before and after drug administration, and plasma drug concentration was determined by gas chromatography-mass spectrometry.
Statistical Analysis
Data are expressed as the mean ± SE. Statistical analysis of differences was performed by ANOVA and Bonferroni's multiple comparison tests. Values of P <0.05 were considered statistically significant.
References
1. Ohtani K, Usui M, Nakano K, Kohjimoto Y, Kitajima S, Hirouchi Y, Li XH, Kitamoto S, Takeshita A, Egashira K. Antimonocyte chemoattractant protein-1 gene therapy reduces experimental in-stent restenosis in hypercholesterolemic rabbits and monkeys. Gene Ther. 2004;11:1273-1282.
2. Takai S, Kim S, Sakonjo H, Miyazaki M. Mechanisms of angiotensin II type 1 receptor blocker for anti-atherosclerotic effect in monkeys fed a high-cholesterol diet. J Hypertens. 2003;21:361-369.
3. Ohtani K, Egashira K, Hiasa K, Zhao Q, Kitamoto S, Ishibashi M, Usui M, Inoue S, Yonemitsu Y, Sueishi K, Sata M, Shibuya M, Sunagawa K. Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lineage cells. Circulation. 2004;110:2444-2452.
4. Mori E, Komori K, Yamaoka T, Tanii M, Kataoka C, Takeshita A, Usui M, Egashira K, Sugimachi K. Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointimal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic rabbits. Circulation. 2002;105:2905-2910.
5. Dayal S, Arning E, Bottiglieri T, Boger RH, Faraci FM, Lentz SR. Cerebral vascular dysfunction mediated by superoxide in hyperhomocysteinemic mice. Stroke. 2004;35:1957-1962.
6. Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002;8:403-409.
7. Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation. 2002;106:1199-1204.
Online Table I. Probes used for real time PCR
Assay Sequence Acc. No.
MCP-1
Forward 5’-TGAGCACGTTTCAGTGAGCAT-3’ M28883
Reverse 5’-ACCACACCTGCCTTTACACCTAA-3’
TaqMan Probe 5’-ATGAAGTCGTAGACCAGCAGCCCCC-3’
IL-1b
Forward 5’-TCAGCACCTCTCAGACAGAGTACAT-3’ M26295
Reverse 5’-AAGACACGAATTCCATGCTGAA-3’
TaqMan Probe 5’-AAACAACAGTGGCGGCCAAGACCTAA-3’
IL-6
Forward 5’-CATGGTGCTGAAGAACATCCAA-3’ AF169176
Reverse 5’-ACTGGTTTTTCTGCTGCAGGTT-3’
TaqMan Probe 5’-AATGAAGAAGCCACCCTCAAGCCAGC-3’
TNF-a
Forward 5’-CATGGTGCTGAAGAACATCCAA-3’ M12845
Reverse 5’-ACTGGTTTTTCTGCTGCAGGTT-3’
TaqMan Probe 5’-AATGAAGAAGCCACCCTCAAGCCAGC-3’
TGF-b1
Forward 5’-TGCTTCAGCTCCACAGAGAAGA-3’ AB020217
Reverse 5’-GGCAGAAGTTGGCGTGGTA-3’
TaqMan Probe 5’-TGTGCGGCAGCTGTACATTGACTTCC-3’
p22-phox
Forward 5’- CTGGTCATTGCAAGCATCATCT-3’ AF323787
Reverse 5’- TCGATGGGCGTCCACTG-3’
TaqMan Probe 5’- CCTGCTGGCTGCCGTCCGAG-3’
gp91-phox
Forward 5’- AACCCTGAAGGAGGCCTGTTAG-3’ AF323788
Reverse 5’- GATGATTTTGGTGGAGGATGTGAT-3’
TaqMan Probe 5’-AGTGACGCCTGCCAACCGCGT-3’
GAPDH
Forward 5’-CTCTGGCAAAGTGGATGTTGTC-3’ L23961
Reverse 5’-GGGTGGAATCATACTGGAACATG-3’
TaqMan Probe 5’-CCATCAATGATCCATTCATTGACCTCCA-3’
Acc.No. indicates the accession number in GenBank
Online Figure I. Inhibitory effects of ARB on markers of RAS (ACE, Angiotensin II, AT1R, and AT2R) and oxidative stress after stent implantation in rabbits. a, Immunohistochemistry of the rabbit stented iliac artery. Arterial cross-sections are stained immunohistochemically for ACE, Angiotensin II, AT1R, and AT2R. * indicates stent strut. The bar indicates 100 µm. b, Inhibitory effect of olmesartan on AT1R expression 7 days after stenting. * indicates stent strut. The bar indicates 100 µm.
c, Effects of olmesartan on superoxide production on day 10. Laser-scanning fluorescent confocal micrographs of cross-sections of stented arteries from normal, control, and olmesartan-treated rabbits. Red fluorescence in nuclei represents DHE staining for superoxide. The elastic laminae demonstrated autofluorescent properties. These DHE experiments were performed in five animals, all with similar results. Bar = 200 µm.