1. Effect of garlic on inflammation processes and atherosclerosis under human-like conditions in APOE*3-Leiden mice Sonia M.S. Espirito Santo TNO Prevention and Health Gaubius Laboratory The Netherlands L University Medical Center Department of Cardiology The Netherlands

2. Garlic has an history for its use in cardiovascular 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. Clinical trials and animal studies with well- characterized and standardized garlic material, with a consistent study design and dosage are scarcely found in the last 15 years. Garlic and health

3. Possible effects of garlic on lipid metabolism CHOLESTEROL SYNTHESIS CHOLESTEROL UPTAKE STORAGE ESTERS DIETARY BILE ACIDS CHOLESTERYL BILIARY CHOLESTEROL LIVER INTESTINE CM Remnants VLDL LDL

4. Possible effects of garlic on atherosclerosis SHEAR STRESS Lipid core O2O2 oxLDL Thrombus Collagen- degrading proteinases Fibrous cap Media Intima Endothelium LDL Monocytes INFLAMMATION OXIDATION

5. Contradictory effects in garlic studies Old human trials in hypercholesterolemic patients reported a decrease in plasma lipid levels. Jain et al, 1993. Silagy et al, 1994. Steiner et al, 1996. Bordia et al, 1997. Recent human trials in hypercholesterolemic patients showed no effects of garlic treatment on plasma lipids. Simmons et al,1994. Neil et al, 1996. Breithaupt et al, 1997. Berthold et al, 1998. McCrindle et al, 1998. Isaacsohn et al, 1998. Gardner et al, 2001. Ziaei et al, 2001. Phelps et al, 1998. Satitvipawee et al, 2003. Induced-atherosclerosis in rabbits kyolic showed a decrease in aortic fatty streaks-like lesions. Efendy JL, et al. Atherosclerosis. 1997:132:37-42. Campbell JH, et al. J Nutr. 2001;131:1006S-09S. Allicin treatment in hypercholesterolemic C57Black/6 mice showed no effects on plasma lipid levels and decreased aortic fatty streak-like lesions. D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519. Hypercholesterolemic pigs and rats fed garlic oil and garlic powder (Kwai) showed no effects on atherosclerosis. 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 Kwai, Morado, Allicin and DADS on plasma lipid levels and markers of cholesterol synthesis and absorption under hyperlipidemic conditions. Morado garlic powder showed an improved liver function, decreased plasma cholesterol levels and serum amyloid A levels under severe atherogenic conditions. Severe 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 (Kwai, Morado, 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 processes and atherosclerosis under human-like conditions: Aim

8. Printanor FR 2001: 0.5% of dry garlic powder (low sulfur content) Printanor FR 2001: 5% of dry garlic powder (low sulfur content) Kyolic (positive control): 0.16% Previously 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

9. 16 female APOE*3-Leiden mice per group 0.2% cholesterol diet for 28 weeks General parameters: Body weight, food intake, marker of liver function, plasma lipid levels, blood pressure, and fecal fatty acids. Inflammation related parameters: General and vessel wall inflammation markers (Serum amyloid A, soluble ICAM, and von Willebrand factor) and whole blood stimulation assay. Atherosclerosis related parameters: Aortic root lesions typing, total lesion area, and monocytes adhesion. Effect of garlic on inflammation and atherosclerosis under human-like conditions: Experimental design

10. * Control 0.5%Prin Control Kyolic Control 5% Prin Effect of garlic on body weight gain in APOE*3-Leiden mice Weeks Body weight gain (g) *P<0.01 0 2 4 6 8 10 04812162024 04812162024 0481216202428 * * * *

11. Effect of garlic on food intake and plasma free fatty acids in APOE*3-Leiden mice Food intake (g/mouse/day) 2.79 ± 0.08 2.87 ± 0.12 2.75 ± 0.16 2.88 ± 0.10 Control 0.5% Prin 5% Prin Kyolic Freefattyacids (mM) 0.73 ± 0.13 0.79 ± 0.11 0.60 ± 0.10 0.62 ± 0.18 * * *P<0.05

12. Effect of garlic on fecal fatty acids excretion in 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 0 1 2 3 4 5 Total dry feces (g/ day) Control0.5% Prin 5% Prin Kyolic *

13. Effect of garlic on liver function in APOE*3-Leiden mice Plasma ALAT levels (U/L) Weeks Control 0.5%Prin Control Kyolic Control 5% Prin 0 50 100 150 200 04812162024 04812162024 0481216202428

14. Effect of garlic on plasma cholesterol levels in APOE*3-Leiden mice Weeks Cholesterol levels (mM) Control 0.5%Prin Control Kyolic Control 5% Prin 0 5 10 15 20 04812162024 04812162024 0481216202428

15. Effect of garlic on plasma triglyceride levels in APOE*3-Leiden mice Weeks Triglycerides levels (mM) Control 0.5%Prin Control Kyolic Control 5% Prin 0 1 2 3 4 04812162024 04812162024 0481216202428

16. Effect of garlic on lipoprotein profile in APOE*3-Leiden mice Cholesterol Triglycerides Fractions

17. Effect of garlic on blood pressure in APOE*3- Leiden mice Blood pressure (mmHg) 0 40 80 120 160 Control0.5% Prin 5% Prin Kyolic 12 weeks16 weeks Control0.5% Prin 5% Prin Kyolic

18. Effect of garlic on serum SAA and ICAM levels in APOE*3-Leiden mice 0 5 10 15 20 04812162024 04812162024 0481216202428 Control 0.5%Prin Control 5% Prin Control Kyolic SAA (µg/ml) ICAM (µg/ml) 0 10 20 30 40 0481216202404812162024 0481216202428

19. Effect of garlic on plasma vWF levels in APOE*3-Leiden mice 0 50 100 150 200 250 04812162024 04812162024 0481216202428 vWF (% PP rat) Control 0.5%Prin Control 5% Prin Control Kyolic

20. Effect of garlic on whole blood TNF  levels in response to LPS in APOE*3-Leiden mice Standard diet Atherogenic diet Control 0.5% Prin 5% Prin Kyolic 0 10 20 050100 150 LPS (ng/ml) TNF  (pg/ml) 40 60 * * * * * * *P<0.05

21. Effect of garlic on atherosclerosis development in APOE*3-Leiden mice Lesion typing Cholesterol exposure 0 100 200 300 400 500 0 20 40 60 80 100 Control0.5% Prin 5% Prin KyolicControl0.5% Prin 5% Prin Kyolic % mM Type I-III Type IV-V

22. Effect of garlic on atherosclerosis development in APOE*3-Leiden mice Lesion area Control0.5% Prin 5% Prin Kyolic µm 2 x 1000 0 50 100 150 200 250 Monocytes adhesion 0 10 20 30 40 50 Control0.5% Prin 5% Prin Kyolic # monocytes

23. Dietary garlic treatment on APOE*3-Leiden mice results in: Decreased body weight gain (P=0.007) for the 5% Printanor treated group: – lower (P=0.032) plasma free fatty acids – higher (P=0.043) fecal fatty acids excretion No effects on liver function, plasma lipids and glucose levels, and on blood pressure. Conclusions (1)

24. Dietary garlic treatment in APOE*3-Leiden mice results in: No effects on the TNF  production by monocytes in response to incubation with LPS, in an ex vivo whole blood assay. Garlic did not affect the activation status of circulating monocytes. No effects on markers of general and vessel wall inflammation, and on atherosclerosis development. Conclusions (2)

25. Human intervention study Collaboration with CHDR-Leiden Future perspectives

26. Ackowledgements Sonia Espirito Santo Erik Offerman Ria van den Hoogen Louis Havekes Bart van Vlijmen Hans Princen Wim van Duyvenvoorde