1. Hideaki Higashino, Atsuko Niwa, Masaki Tabuchi, Kana Ooshima, and Hiroshi Sakaue Department of Pharmacology, Kinki University School of Medicine, Osaka-Sayama, 589-8511, Japan. Long-term Voluntary Exercise Decreased the Incidence of Apoplexy and Elongated the Lifespan through Activation of eNOS and Inhibition of Inflammatory Signaling Pathway in Stroke-Prone SHR

2. Intervention Therapy for Hypertension 1. Guidance for Improvement of Life Style a. Food (low salt, low calorie, much fiber) b. Physical exercise c. Save the body weight d. To avoid much stress e. Enjoy the daily life 2. Drug therapy a. Early treatment b. Select the appropriate drugs for prevention of AS

3. Objective: Clinical evidences show that exercise exerts atheroprotective or beneficial effects on cardiovascular events. Precisely causative mechanisms, however, are still unknown. Therefore, the hypothesis that endurance voluntary exercise decreases the inflammatory signaling through eNOS induction and ROS inhibition was assessed in SHRSP.

4. Methods Animals:Male SHRSP aged 6-week-old at pre-hypertensive stage Groups: 1. Voluntary wheel-running (WR): 2 to 3 km running/day 2. sedentary control (SED): in the cage without running Duration:8 weeks Analyses: Thoracic Aortae: NOS expression, eNOS activity, oxidative stress Akt, eNOS, phosphorylated ones by western blotting NADPH oxidase mRNA by RT-PCR. Activities of eNOS by using [3H]l-arginine Blood: Superoxide (O 2- ) production by flow cytometer using DHE Plasma: sICAM-1, MCP-1, 8-iso-PGF2αby ELISA Observation of the occurrence of apoplexy: keeping them until the death

5. 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 Age (weeks) Systolic blood pressure 6 7 8 91011121314 15 16 (mmHg) ** P<0.01 * P<0.05 SEDENTARY EXERCISE Significant difference compared with SED mean + SEM (n=6-12) ～ ～ Changes of blood pressure in SEDENTARY and EXERCISED SHRSP

6. 050100150200 0 50 100 Age (Days) (%) SEDENTARY (n=15) EXERCISE (n=13) EX vs. SED; P=0.016 Stroke morbidity Start of exercise Incidents of Stroke determined by stroke scores in SED and WR SHRSP

7. 0.5 1 100 200 Age (days) Periods of Life-Span in Sedentary and Exercised SHRSP Surviving p<0.05 ; WR vs.SED Sedentary rats (n=9) Exercised Rats (n=10)

8. SEDENTARY EXERCISE

9. 0 0.1 0.2 0.3 0.4 SEDWRWR 0 5 10 15 20 25 30 SEDWR (mm) (%) Thickness of media Collagen area of vessel wall * * p<0.001 n=10129 Thickness of SM layers and Collagen area in Thoratic aortae after exercise in SHRSP (Mean ± SEM)

10. Expression of angiotensin (AT)1 receptors and AT2 receptors in the aortas of SED and WR SHRSP SEDENTARY AT1R EXERCISE AT1R SEDENTARY AT2REXERCISE AT2R

11. 0 0.5 1 1.5 2 SEDWR AT 1 R / AT 2 R n=66 * p<0.005 (arb. units) (Mean ± SEM) Levels of AT1 & AT2 receptors, and ACE in the aortae between SED and WR 0 0.2 0.4 0.6 0.8 1 1.2 1.4 WR ACE (arb. units) SED AT 1 R/AT 2 R ACE n=8 6

12. Nox1 Relative intensity (arb. units) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 mRNA/ 18S rRNA SEDWR 4 4 mRNA protein 0 0.2 0.4 0.6 0.8 1 1.2 SEDWR 58 P<0.01 * NAD(P)H oxidase Subunit (Nox1) RNA in Aortas of EX SHRSP 8

13. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Relative intensity (arb. units) p-Akt/Akt SED WR p<0.001 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.2 0.4 0.6 0.8 1 1.2 Ser 1177 -p-eNOS eNOS protein SED WR p<0.05 Levels of eNOS, p-eNOS and p-Akt/Akt in the Aortae (Mean ± SEM) 12 n=11 12 11 12

14. ROS production 0 500 1000 1500 2000 2500 SED WR 5 5 P<0.01 * 0 50 100 150 200 250 300 350 400 450 BasalACh 10 -5 M Insulin10 -6 M NO production 5 5 555 5 P<0.01 * P<0.05 * x10 4 SED EXERCISE ROS and NO Productions in the Aortae between SED and WR

15. 0 2 4 6 8 10 12 14 16 [ 3 H]l-citrulline (nmol/mg/min) 0 100 200 300 400 500 600 cGMP (fmol/mg protein) NOS activity SED WR cGMP Comparison of NOS activities and cGMP production in the Aortae SED WR p<0.05 711n=5 5 (Mean ± SEM)

16. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Nitrotyrosine (arb. units) 0 20 40 60 80 100 120 140 Dihydroethidium (MFI) Nitrotyrosine in Aortae SED WR MFI in the Blood SED WR Nitrotyrosine contents in Aortae and MFI by DHE in the Blood p<0.05 p<0.001 (Mean ± SEM) n=7845

17. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 SED WR 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 p-Akt/Akt p<0.001 n=12 12 SEDWR 8 7 * * p<0.05 p-ERK/ERK Changes of Phosphorylated Akt, and ERK1/2 levels in the Aortae between SED and WR p-Akt/Akt p-ERK/ERK (Mean ± SEM) (arb. units)

18. 0 1 2 3 4 5 6 SEDWR TGF-β(ng/ml) n=6 4 (Mean ± SEM) Changes of serum TGF-β levels after exercise

19. 0 100 200 300 400 500 600 SED WR high sensitive CRP  g/ml) p<0.05 7 6 * soluble ICAM-1 5 10 15 20 25 0 SED WR (pg/ml) 22 p<0.01 * Plasma concentration Comparison of inflammatory biomarkers in SED and EX

20. 0 1 2 3 4 5 6 7 WR Complete PAI-1 6 4 (ng/ml) 0 2 4 6 8 10 SED WR MCP-1 87 p<0.05 * SED p<0.05 * Plasma concentration Concentrations of PAI-1 and MCP-1 in the Plasma 4

21. Physical Exercise PI 3 K Akt eNOS NO cGMP Prevention of Cardio-vasculitis Ang II, TNF-α PLC, PLD NAD(P)H oxidase ROS MAP kinases ONOO - MCP-1 ICAM-1 Mφ AT1 Rs/AT2 Rs Vascular dilatation HypertensionApoplexy Shear stress Fibrosis

22. Conclusions: Data showed that exercise could protect oxidative stress-induced cell injury or inflammation by an interaction with signaling molecules such as ASK1 /JNK/ p38MAPK through NO production and inhibition of superoxide production. Then, voluntary exercise significantly attenuated the changes of vascular remodeling, delayed stroke events and elongated the lifespan in exercised rats.