1. Effect of garlic on inflammation processes and atherosclerosis under human-like conditions in APOE*3-Leiden mice Sonia M.S. Espirito Santo 1,2, Wim van Duyvenvoorde 1, Bart J.M. van Vlijmen 1,2, Louis M. Havekes 1,2,3 and Hans M.G. Princen 1 1 TNO Prevention and Health, Gaubius Laboratory, Leiden, The Netherlands and 2 Departments of Cardiology and 3 Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands.

2. Garlic has an historical record for its use in medicine. Some studies have suggested that garlic significantly reduces plasma lipid levels, systolic and diastolic blood pressure and that it shows a strong antibiotic activity. Kik C, Kahane R, Gebhardt R. Nutr Metab Cardiovasc Dis. 2001;11:57-65. In the last 15 years, garlic clinical trials and animal studies with well-characterized and standardized garlic material, with a consistent study design and dosage are poorly found. Garlic and health

3. Possible effects of garlic on lipid metabolism CHOLESTEROL SYNTHESIS CONVERSION CHOLESTEROL UPTAKE receptors STORAGE ESTERS DIETARY BILE ACIDS LDL receptor CHOLESTERYL BILE other BILIARY CHOLESTEROL SECRETION LIVER INTESTINE CHYLO- MICRONS CHYLO- MICRON REMNANTS LDL VLDL

4. Possible effects of garlic on atherosclerosis Lipid core Media Intima Endothelium LDL O2O2 oxLDL Thrombus Collagen- degrading proteinases Shear stress Fibrous cap Monocytes Inflammation Oxidation

5. Contradictory effects attributed to garlic Old human trials on healthy patients treated with garlic oil reported a decrease in plasma lipid levels. Bordia A, et al. Atherosclerosis.1975;21:15-19. Bordia A, et al. Prostaglandins, Leukotrienes and Essential Fatty Acids. 1998;58:257-263. Recent human trials on hypercholesterolemic patients showed no effects of garlic powder (Kwai) and garlic oil treatment on plasma lipid levels. Simmons LA, et al. Atherosclerosis.1994;113:219-225. Berthold, et al. JAMA. 1998;279:1900-02. McCrindle BW, et al. Arch Pediatr Adolesc Med. 1998;152:1089-94. Isaacsohn JL, et al. Arch Intern Med.1998;158:1189-94. Gardner CD, et al. Atherosclerosis. 2001;154:213-220. Ziaei S, et al. Eur J Obstetrics & Gynecology. 2001;99:201-6. Allicin showed no effects on plasma lipid levels and decreased aortic fatty streaks-like lesions in hypercholesterolemic C57Black/6 mice. D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519. Induced-atherosclerosis in rabbits fed garlic oil and aged garlic extract (Kyolic) showed a decrease of aortic fatty streaks lesions. Bordia A, et al. 1980:428-37. Mand JK, et al. Indian Heart J. 1985;37:183-88. Efendy JL, et al. Atherosclerosis. 1997:132:37-42. Campbell JH, et al. J Nutr. 2001;131:1006S-09S. Durak I, et al. Nutr Metab Cardiovasc Dis. 2002 ;12:141-7. Hypercholesterolemic pigs and rats fed garlic oil and garlic powder (Kwai) showed no benefical effects on atherosclerosis development. Heinle H,et al. Drug Res. 1994;44:614-17. Lee WM, et al. Exper Mol Pathol. 1980:33:345-8.

6. Lipid metabolism - No effects of garlic powders and isolated compounds on plasma lipid levels and markers of cholesterol synthesis and cholesterol absorption under hyperlipidemic conditions. - Morado 200 garlic powder showed an improved liver function, decreased plasma cholesterol levels and serum amyloid A levels under atherogenic conditions. Fast atherosclerosis development –No decreasing effect of garlic on atherosclerosis of the aortic root was observed for all treatments. Espirito Santo SMS, et al. Effect of garlic powders and garlic isolated compounds on lipid metabolism. 2002. Previous reported effects of garlic (LI111, Morado 200, Allicin and DADS) in APOE*3- Leiden mice

7. Study the effect of 2 different doses of Printanor garlic powder under mild human-like conditions on inflammation related parameters on atherosclerosis development by means of a hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice. Effect of garlic on inflammation and atherosclerosis under human-like conditions: Aim

8. APOE*3-Leiden transgenic mouse as a model for hyperlipidemia and atherosclerosis studies Transgenic mice carrying a mutant form of human APOE*3 gene. Dominant dysfunctional APOE*3-Leiden results in defect in chylomicron/ VLDL clearance and a more human-like lipoprotein profile. Highly responsive to feeding cholesterol-rich diets. Develops atherosclerosis varying from fatty streaks to more human-like complicated lesions.

9. Atherosclerosis in APOE*3-Leiden mice Adherence of monocytes to the activated vessel wall Monocytes

10. Atherosclerosis in APOE*3-Leiden mice foam cells intima media { Media not affected Aorta with regular fatty streak

11. Atherosclerosis in APOE*3-Leiden mice { media intima fibrosis cholesterol clefts necrosis Affected media Severe plaque in aorta

12. Printanor FR 2001 (0.5% Prin): 0.5% of dry garlic powder with low sulphur content. Printanor FR 2001 (5% Prin): 5% of dry garlic powder with high sulphur content. Kyolic (positive control): 0.16% of dry garlic powder rich in S- allylcysteine reported to result in 64% reduction of fatty streaks in New Zealand white rabbits. Campbell JH, Efendy JL, et al. J.Nutrition. 2001;131:1006S-1009S. Effect of garlic on inflammation and atherosclerosis under human-like conditions: Garlic powders

13. 16 female APOE*3-Leiden mice per group 0.2% cholesterol diet for 24 weeks General parameters: Body weight, food intake, marker of liver function, plasma lipid levels and blood pressure. Inflammation related parameters: Serum amyloid A, soluble ICAM, Von Willebrand factor, fibrinogen, tissue-type plasminogen activator and whole blood stimulation assay. Atherosclerosis related parameters: Aortic root lesions typing, total area, macrophage and smooth muscle cell lesions composition. Effect of garlic on inflammation and atherosclerosis under human-like conditions: Experimental design

14. Effect of garlic on body weight gain of APOE*3-Leiden mice P<0.01

15. Effect of garlic on food intake and body temperature of APOE*3-Leiden mice Food intake (g/mouse/day) 2.76 ± 0.13 2.76 ± 0.10 2.73 ± 0.14 2.75 ± 0.12 Body temperature (  C) 38.3 ± 0.2 38.3 ± 0.3 Control 0.5% Prin 5% Prin Kyolic

16. Effect of garlic on plasma free fatty acids and glucose levels of APOE*3-Leiden mice Freefattyacids (mM) Glucose (mM) Control0.73 ± 0.1310.1 ± 1.2 0.5%Prin0.79 ± 0.119.4 ± 1.1 5%Prin0.60 ± 0.109.7 ± 0.7 Kyolic0.62 ± 0.189.5 ± 1.1 * * P<0.01

17. Effect of garlic on fecal fatty acids excretion of APOE*3-Leiden mice Total fat excretion (µmol/100g mouse/day) 0 200 400 600 800 Control0.5% Prin 5% Prin Kyolic * P<0.05

18. Effect of garlic on liver function of APOE*3-Leiden mice

19. Effect of garlic on plasma cholesterol levels of APOE*3-Leiden mice

20. Effect of garlic on plasma triglyceride levels of APOE*3-Leiden mice

21. Effect of garlic on lipoprotein profile of APOE*3-Leiden mice

22. Effect of garlic on blood pressure of APOE*3-Leiden mice

23. After 20 weeks of mildly cholesterol elevating diets with incorporated garlic powders: 5% Printanor treated group shows a significant (P=0.001) lower body weight, which coincides with: –a slight but significant lower plasma free fatty acids levels; –a significant higher fecal fatty acids excretion; No effects of garlic on liver function, plasma lipid levels, plasma glucose levels and blood pressure were observed. Conclusions

24. Focus on inflammation markers and atherosclerosis evaluation. Follow up with a Human intervention study. Future perspectives