Experimental Procedures. Systolic, diastolic and mean BP levels and heart rate were measured using a

Transcription

1 Supplemental Data Experimental Procedures Measurements of Blood Pressure and Heart Rate Systolic, diastolic and mean BP levels and heart rate were measured using a noninvasive computerized tail-cuff system (BP98A Softron Corp., Tokyo, Japan) at 2 weeks after pump implantation. Unanesthetized mice from each group were placed in a holding device mounted on a thermostatically controlled warming plate, maintained at 37 C. BP and heart rate were measured on two consecutive days, and at least 10 readings were taken for each measurement, as previously described. 1 Measurements of Plasma Lipid and Glucose Levels Blood was drawn through a heart puncture and collected into a tube containing a 1/10 volume of 0.5M EDTA (ph 7.4) and then centrifuged at 2000 g for 20 minutes. Plasma was stored at 80 until use. Plasma levels of glucose were determined by an enzymatic method with Quickauto Neo GLU-HK Shino-Test Corporation, Japan using a Hitachi 7180 Clinical Analyzer (Hitachi, Tokyo, Japan). Plasma levels of lipids

2 were also analyzed by an enzymatic method with type Wako CHO H (Wako Pure Chemical Industries. Ltd., Tokyo, Japan) using the same analyzer as that for glucose. Echocardiographic Analysis Transthoracic echocardiography was performed using a 15-MHz imaging transducer (Aplio 80 Toshiba Medical Systems Co. Ltd., Japan). Mice from each group were anesthetized by intraperitoneal injection of 20 mg/kg of 2.5% pentobarbital and 8 mg/kg of 2.0% xylazine. The left hemithorax of each mouse was carefully shaved, and M-mode images of the left ventricle were recorded when heart rate was kept at beats per minute. Percent fractional shortening (FS), left ventricular mass (LVM), and relative wall thickness (RWT) were calculated as described previously. 1 The ratio of early to late filling wave (E/A) of transmitral pulse-wave Doppler velocity was measured. Histological Analysis and Immunohistochemistry At the end of pump infusion, mice from each group were given a lethal dose of

3 pentobarbital by intra-peritoneal injection and perfused first with physiological saline and then with 10% neutral buffered formalin at 100 mmhg. Finally, the hearts and kidneys were excised. The whole heart and kidneys were resected and placed in 10% neutral buffered formalin overnight. After fixation, samples were embedded in paraffin. Then 3-µm-thick sections of the heart were cut and stained with Azan. The cross-sectional areas of LV myocytes were measured on the mid free wall of the LV from sections. Suitable cross-sectional areas were defined as having nearly circular capillary profiles and nuclei. Approximately 100 cells were counted in each section, and the average area was used for analysis as described previously. 1 To calculate the ratio of the interstitial fibrosis area in the left ventricular area, excluding perivascular fibrosis, 10 fields of samples were randomly selected from 3 individual sections. Moreover, we measured the perivascular fibrosis area, outer vascular area and medial area of coronary arteries in each section. The index of perivascular fibrosis was calculated as the ratio of the pericoronary fibrosis area to the outer vascular area. The index of arterial medial thickening was calculated as the ratio of the medial area to the outer vascular area. Measurements of the areas were performed using ImageJ 1.29, a

4 free software program. Samples of hearts and kidneys after fixation with paraffin as mentioned above were stained using anti-tgf-β1 rabbit polyclonal antibody (sc-146) (Santa Cruz Biotechnology, Inc, USA) at a dilution 1:50. PAS staining and fibrin(ogen) staining in kidney tissues of enos -/- mice were performed as described previously. 2, 3 Analysis of Urinary Excretion of 8-hydroxy-2'-deoxyguanosine 8-OHdG The formation of reactive oxygen species is a critical event in cardiovascular remodeling because it promotes cell proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. Martinet et al. revealed that 7,8-dihydro-8-oxo-2 -deoxyguanosine can be a marker of oxidative DNA damage and that it is increased in human atherosclerotic plaques. 4 Therefore, increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels are thought to be an appropriate marker for Ang II-induced DNA damage during cardiovascular remodeling. In this study, urinary excretion of 8-OHdG was measured using an ELISA kit according to the manufacturer s instructions (New 8-OHdG Check ELISA Kit, Japan Institute for the Control of Aging, Nikken SEIL Corporation, Shizuoka, Japan). Mice were housed

5 individually in metabolic cages that provided free access to tap water and food for 24 hours. We measured the concentration of urinary 8-OHdG to determine the total oxidative stress and corrected those levels by daily urinary excretion volume and body weight. Western Blot Analysis TGF-β1 expression and Smad 2/3 activation were evaluated by Western blot analysis as described previously. 1, 3 We used antibodies against TGF-β1, phosphorylated Smad 2/3 and total Smad 2/3 (Santa Cruz Biotechnology, Inc., USA). For Western blot analysis, 50 µg protein extracts from the hearts of WT and enos KO mice and kidneys of enos KO mice were boiled for 5 min in Laemmli sample buffer and then run on SDS-PAGE. The protein extracts were then transferred to a nitrocellulose membrane (Hybond TM-ECL, Amersham Biosciences, Buckinghamshire, UK). The membrane was blocked for 1 h at room temperature with 5% non-fat skim milk in phosphate-buffered saline / Tween 20 (PBS-T). The blots were incubated overnight at 4 C with antibodies for phosphospecific Smad 2/3, followed by incubation for 1 h with

6 anti-rabbit secondary antibody (horseradish peroxidase-conjugate). Immunoreactive bands were visualized using enhanced chemiluminescence with ECL reagent (Amersham ECL Plus Western Blotting Detection System, GE Healthcare, Buckinghamshire, UK) treatment and exposure to Hyperfilm-ECL. The intensity of the bands was measured using ImageJ version Superoxide Detection in Myocardial and Renal Tissues We evaluated cardiorenal superoxide production with in situ dihydroethidium (DHE) staining. 5 In brief, hearts and kidneys from mice were excised, rinsed in physiological saline, and frozen in optimal cutting temperature compound (Tissue-Tek, Sakura Fine Chemical, Tokyo, Japan) until use. Transverse sections (10 µm in thickness) were cut with a cryostat and placed on silane-coated glass slides. The slide-glasses with heart and kidney tissues were incubated with dihydroethidium in PBS (10 mmol/l) in a dark, humidified container at room temperature for 30 minutes. DHE is oxidized upon reaction with superoxide to ethidium bromide, which binds to DNA in the nucleus and fluoresces red. After placing a cover glass over the heart and kidney tissues, the heart

7 and kidney tissues were observed using a laser scanning confocal microscope (Leica TCS-NT, mounted on a Leica DMRB light microscope; Leica, Mannheim, Germany). The excitation wavelength was 488 nm, and emission fluorescence was detected with a 568 nm long-pass filter. Evaluation of Urinary Albumin Excretion and Glomerular Filtration Rate Urinary albumin excretion and glomerular filtration rate were estimated using urine samples by procedures previously described. 6 Statistical Analysis All data are expressed as means ± S.E. For comparisons among groups, statistical significance was assessed using a one-way analysis of variance, and the significance of each difference was determined by post hoc testing using Tukey-Kramer's method. Comparison of survival was performed with Kaplan-Meier analysis. These analyses were performed on a Microsoft Windows computer with the use of Excel (Microsoft XP) and Stat View statistical package (Stat View 5.0, SAS Institute Inc.). Statistical significance was considered at p < 0.05.

8 References 1. Ikeda Y, Aihara K, Sato T, Akaike M, Yoshizumi M, Suzaki Y, Izawa Y, Fujimura M, Hashizume S, Kato M, Yagi S, Tamaki T, Kawano H, Matsumoto T, Azuma H, Kato S. Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis. J Biol Chem. 2005;280: Li Z, Ohno N, Terada N, Zhou D, Yoshimura A, Ohno S. Application of periodic acid-schiff fluorescence emission for immunohistochemistry of living mouse renal glomeruli by an "in vivo cryotechnique". Arch Histol Cytol. 2006;69: Aihara K, Azuma H, Akaike M, Ikeda Y, Yamashita M, Sudo T, Hayashi H, Yamada Y, Endoh F, Fujimura M, Yoshida T, Yamaguchi H, Hashizume S, Kato M, Yoshimura K, Yamamoto Y, Kato S, Matsumoto T. Disruption of nuclear vitamin D receptor gene causes enhanced thrombogenicity in mice. J Biol Chem. 2004;279: Martinet W, Knaapen MW, De Meyer GR, Herman AG, Kockx MM. Elevated levels of oxidative DNA damage and DNA repair enzymes in human atherosclerotic plaques. Circulation. 2002;106: Miller FJ, Jr., Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998;82: Segev Y, Eshet R, Rivkis I, Hayat C, Kachko L, Phillip M, Landau D. Comparison between somatostatin analogues and ACE inhibitor in the NOD mouse model of diabetic kidney disease. Nephrol Dial Transplant. 2004;19:

9 Online Table 1 Plasma concentration of pitavastatin C57BL6/J C57BL6/J enos -/- enos -/- Ang II (-) Ang II (+) Ang II (-) Ang II (+) Pitavastatin (ng/ml) 0.83± ± ± ±0.11 Mean±SE, Each group n=6

10 a b Online Figure 1 PAS Fibrin(ogen) TGF-β1 DHE 25 µm Urine volume (ml/day) Pit (-) (+) (-) (+) Ang II (-) (+) Glomerular filtration rate (ml/min/g weight) Pit Ang II (-) (+) (-) (+) (-) (+) Urinary excretion of albumin (g/g Cre) Pit 0 Ang II (-) (+) (-) (+) (-) (+) Pit (-) (+) (-) (+) Ang II (-) (+) a. Representative findings of the glomerulus in C57BL6/J mice with or without Ang II stimulation. n=8 in each group. b. Renal function (left: daily urinary output, middle: glomerular filtration rate, right: urinary excretion of albumin) in C57Bl6/J mice. n=8 in each group.