1. Effects on lipid levels and lipid metabolism under more human-like conditions. –Lipid levels, cholesterol synthesis and cholesterol absorption. Effects on fast atherosclerosis development and other processes that might be related with atherosclerosis. –Inflammation, blood pressure, platelet activation and atherosclerosis analysis. Aim of the study: Effects of garlic on lipid metabolism and atherosclerosis

2. 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 more human-like lipoprotein profile. Highly responsive to feeding cholesterol-rich diets. Develops atherosclerosis varying from fatty streaks to more human-like complicated lesions.

3. Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: Selection of compounds Lichtwer LI111 garlic powder: Reference dry garlic powder. Morado de Cuenca 200 (Mor200): Garlic powder rich in organic sulfur-containing compounds. Diallyl disulfide (DADS): Isolated garlic compound rich in sulfur. Allicin: Reference isolated garlic compound reported to result in 43% reduction of fatty streaks in C57Black/6 mice (D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519)

4. Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: Experimental scheme -2 Matching and start of the study Weight and food intake Blood sampling/ plasma isolation Plasma lipid levels Cholesterol lipoprotein profile Chol. synthesis/ Intestinal absorption Platelet activation measurements Marker for liver function Liver lipid composition Atherosclerotic aorta lesions area Inflammation markers 2 4 6 81012 14 80 p p 60 p 80 p p 65 p p p 8065 p pp p 80 65 60 65 16 Regular dietWestern dietAtherogenic diet 60 p Blood pressure Compound biostability 65 pp 80 p 5 65 p 15 Run in 182022 65 25 p 65 80 65 p 0 Atheroscleroticaorta lesions typing60 5 General parameters Inflammation and atherosclerosis related parameters Lipid metabolism related parameters 80

5. Effect of garlic on plasma cholesterol levels under 3 dietary conditions *P< 0.05 Plasma cholesterol levels Regular dietWestern dietAtherogenic diet mM Control3.19.363 LI1112.99.3 57 Mor 2003.09.1 44 * DADS2.98.370 Allicin 3.5 * 9.273

6. Effect of garlic on markers of cholesterol synthesis and intestinal absorption under 3 dietary conditions Regular diet *P< 0.05 Markers of cholesterol synthesis Markers of intestinal absorption

7. Effect of garlic on liver function under 3 dietary conditions t P=0.051 Regular dietWestern dietAtherogenic diet Control 33.430.3175 LI111 33.234.4 104 Mor 200 32.930.3 94.1 DADS 29.941.8203 Allicin 25.228.2171 Plasma alanine amino transferase t t U/L

8. Effect of garlic on Serum Amyloid A and von Willebrand factor concentrations under fast atherogenic conditions SAA (µg/ ml) Control LI111Mor 200DADS Allicin 0 50 100 150 200 250 vWF (%PP) Control LI111Mor 200DADSAllicin 0 2 4 6 8 * * p=0.001 p<0.001 *P< 0.05

9. Control Primador FRANCE 2001: 0.64 g/kg b.w. of dry garlic powder with low sulphur content. Primador FRANCE 2001: 6.4 g/kg b.w. of dry garlic powder with high sulphur content. Kyolic (positive control): 200 mg/kg b.w. of 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 powders on atherosclerosis under more human-like conditions: Groups selection

10. Effect of garlic powders on atherosclerosis under more human-like conditions: Experimental design Matching and start of the study Weight and food intake Blood sampling/ plasma isolation Plasma lipid levels Cholesterol lipoprotein profile vWf, t-PA and soluble ICAM Marker for liver function Liver lipid composition (p.m.) Aortic root lesions composition Aortic root lesions typing and area 812 60 55 60 p 55 Diet T + 0.2% cholesterol 55 p SAA and Fibrinogen Compound biostability 60 p 5 Run in 26 28 60 0 Aortic arch area and lipids deposition55 5 General parameters Inflammation and atherosclerosis development related parameters Lipid metabolism related parameters In weeks 5 5 4 60 16 60 20 60 24 60 p 55 All blood stimulation assay 55 p pppppp p p Mice on standard chow diet8