Hideaki Higashino , Atsuko Niwa, Masaki Tabuchi, Kana Ooshima, and Hiroshi Sakaue

1. 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 Hideaki Higashino, Atsuko Niwa, Masaki Tabuchi, Kana Ooshima, and Hiroshi Sakaue Department of Pharmacology, Kinki University School of Medicine, Osaka-Sayama, 589-8511, Japan.

2. Intervention Therapy for Hypertension 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 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 (O2-) 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. Changes of blood pressure in SEDENTARY and EXERCISED SHRSP (mmHg) 280.0 SEDENTARY 260.0 240.0 EXERCISE 220.0 Systolic blood pressure * P<0.05 * P<0.05 200.0 ** P<0.01 180.0 mean + SEM (n=6-12) 160.0 Significant difference compared with SED ～ ～ 140.0 15 16 8 7 9 10 11 12 13 14 6 Age (weeks)

6. 100 Incidents of Stroke determined by stroke scores in SED and WR SHRSP SEDENTARY (n=15) (%) Stroke morbidity 50 EXERCISE (n=13) EX vs. SED; P=0.016 Start of exercise 0 0 50 100 150 200 Age (Days)

7. 0 Periods of Life-Span in Sedentary and Exercised SHRSP 1 Exercised Rats (n=10) Sedentary rats Surviving (n=9) 0.5 p<0.05 ; WR vs.SED 100 200 Age (days)

8. SEDENTARY EXERCISE

9. Thickness of SM layers and Collagen area in Thoratic aortae after exercise in SHRSP Collagen area of vessel wall Thickness of media (mm) (%) 0.4 30 25 0.3 20 p<0.001 * 0.2 15 p<0.001 10 * 0.1 5 12 n=10 12 9 0 0 SED WR SED WR (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 AT2R EXERCISE AT2R

11. Levels of AT1 & AT2 receptors, and ACE in the aortae between SED and WR ACE AT1R/AT2R 2 1.4 1.2 (arb. units) 1.5 1 ACE (arb. units) 0.8 1 AT1R / AT2R 0.6 p<0.005 * 0.4 0.5 0.2 n=8 n=6 6 6 0 0 SED SED WR WR (Mean ± SEM)

12. 1.2 1.4 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0 NAD(P)H oxidase Subunit (Nox1) RNA in Aortas of EX SHRSP Nox1 protein mRNA P<0.01 * mRNA/ 18S rRNA Relative intensity (arb. units) 5 8 8 4 4 SED WR SED WR

13. Levels of eNOS, p-eNOS and p-Akt/Akt in the Aortae eNOS protein p-Akt/Akt Ser1177-p-eNOS 1.2 1.4 1.8 p<0.001 p<0.05 1.6 1.2 1 1.4 1 0.8 1.2 0.8 1 Relative intensity (arb. units) 0.6 0.8 0.6 0.4 0.6 0.4 0.4 0.2 0.2 0.2 12 12 n=11 11 12 12 0 0 0 WR WR SED SED SED WR (Mean ± SEM)

14. ROS and NO Productions in the Aortae between SED and WR NO production ROS production x104 2500 450 P<0.05 P<0.01 * * 400 2000 350 300 1500 250 P<0.01 * 200 1000 150 EXERCISE SED 100 500 50 5 5 5 5 5 5 5 5 0 0 Insulin10-6M Basal ACh 10-5M SED WR

15. Comparison of NOS activities and cGMP production in the Aortae NOS activity cGMP 16 600 p<0.05 14 500 12 400 10 [3H]l-citrulline (nmol/mg/min) cGMP (fmol/mg protein) 300 8 6 200 4 100 2 5 n=5 7 11 0 0 WR WR SED SED (Mean ± SEM)

16. Nitrotyrosine contents in Aortae and MFI by DHE in the Blood Nitrotyrosine in Aortae MFI in the Blood 1.4 140 p<0.05 p<0.001 120 1.2 100 1 80 0.8 Nitrotyrosine (arb. units) Dihydroethidium (MFI) 60 0.6 40 0.4 20 0.2 n=7 8 4 5 0 0 WR WR SED SED (Mean ± SEM)

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

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

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

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

21. Physical Exercise MCP-1 ICAM-1 Shear stress Ang II, TNF-α AT1 Rs/AT2 Rs PI3K Mφ PLC, PLD Akt NAD(P)H oxidase eNOS ROS ONOO- NO MAP kinases Hypertension Apoplexy Fibrosis cGMP Prevention of Cardio-vasculitis Vascular dilatation

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.