1. Sonia M.S. Espirito Santo, Bart J.M. van Vlijmen, Rien Buytenhek, Wim van Duyvenvoorde, Erik H. Offerman, Louis M. Havekes and Hans M.G. Princen Gaubius Laboratory, TNO-PG, Leiden, the Netherlands EU project Effect of garlic powders and garlic active compounds on lipid metabolism and atherosclerosis in APOE*3-Leiden mice

2. 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

3. Lipid metabolism CHOLESTEROL SYNTHESIS CONVERSION CHOLESTEROL UPTAKE receptors HMG-CoA reductase STORAGE ESTERS DIETARY BILE ACIDS LDL receptor 7  -hydroxylase 27-hydroxylase CHOLESTERYL BILE other BILIARY CHOLESTEROL ACAT SECRETION LIVER INTESTINE CHYLOMICRONS CHYLOMICRON REMNANTS LDL VLDL

4. 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 Soluble garlic extracts inhibit cholesterol biosynthesis in primary rat hepatocytes and human HepG2 cells. R. Gebhardt, Lipids, 1993, 28(7):613-9. Lichtwer garlic powder reduces plasma lipid levels and blood pressure in hypercholesterolemic rats. M Ali et al., Prostaglandins Leukot Essent Fatty Acids, 2000, 62(4):253-9. Allicin showed no effects on plasma lipid levels and decreased aorta atherosclerotic lesions in C57Black/6 mice fed a high fat/ cholesterol diet. D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519. Garlic powder showed no effects on plasma lipid levels in patients with moderated hypercholesterolemia. B.W. McCrindle et al, Arch Pediatr Adolesc Med, 1998, 152(11): 1089-94 and C.D. Gardner et al, Atherosclerosis, 2001, 154(1):213-2. Garlic oil showed no effects on plasma lipid levels, cholesterol synthesis and cholesterol absorption in patients with moderated hypercholesterolemia. H.K. Berthold et al, JAMA, 1998, 17(23):1900-2.

6. Aim of the study: Effects of garlic on Aim of the study: Effects of garlic on lipid levels and lipid metabolism (1) Study the effect of 2 garlic powders and 2 garlic active compounds on plasma lipid levels, cholesterol synthesis and intestinal absorption under more human-like conditions using hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice.

7. 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.

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

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

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

11. 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)

12. 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

13. Effect of garlic on food intake under 3 dietary conditions Control 3.63.23.3 LI111 3.33.0 2.6 Mor 200 3.43.0 2.8 DADS 3.02.6 2.4 Allicin 3.63.33.0 * * * Food intake Regular dietWestern dietAtherogenic diet g/ mouse/ day *P< 0.05

14. Effect of garlic on body weight under 3 dietary conditions *P< 0.05 Control 20.923.323.2 LI111 21.022.022.6 Mor 200 21.523.222.1 DADS 20.3 21.020.6 Allicin 20.522.623.4 Body weight ** Regular dietWestern dietAtherogenic diet g

15. 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

16. 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

17. Regular dietWestern dietAtherogenic diet Effect of garlic on cholesterol lipoprotein distribution under 3 dietary conditions *P< 0.05 Fraction number Cholesterol (mM) 0 1 2 3 4 159131721 0 1 2 3 4 159131721 VLDL 0.0 0.1 0.2 0.3 0.4 159131721 0.0 0.1 0.2 0.3 0.4 159131721 0 4 8 12 159131721 0 4 8 12 159131721  Control  LI111  Morado 200 LDLHDL  Control  DADS  Allicin

18. 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

19. Liver lipid composition Free cholesterolTriglycerides mg lipid/ g protein Cholesteryl esters Control 19.4 43.7154 LI111 15.9 * 31.5 * 130 * Mor 200 15.3 * 35.0 * 127 * DADS 17.8 49.7 171 * Allicin 17.3 37.8146 Effect of garlic on liver lipid levels under fast atherogenic conditions *P< 0.05

20. of the study of the effects of garlic Conclusions of the study of the effects of garlic on lipid levels and lipid metabolism (1) Under more human-like hyperlipidemic conditions (8-12 mM): – No beneficial effects of garlic powders and garlic isolated compounds were observed on lipid levels, cholesterol synthesis and intestinal absorption in APOE*3-Leiden mice. Under fast atherosclerosis conditions (45-70 mM): – Morado 200 showed a plasma cholesterol-lowering effect in APOE*3-Leiden mice. – LI111 and Morado 200 had a cholesterol and triglyceride liver storage lowering effect in APOE*3-Leiden mice.

21. Aim of the study: Effects of garlic on Aim of the study: Effects of garlic on fast atherosclerosis development and other atherosclerosis related processes (2) Study the effect of 2 garlic powders and 2 garlic active compounds on inflammation processes, blood pressure, platelet activation and fast atherosclerosis development using hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice.

22. 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

23. Effect of garlic on blood pressure and platelet activation under fast atherogenic conditions No effects of garlic powders and garlic isolated compounds were observed on the percentage of activated platelets in blood under all dietary treatments. No influence of garlic powders and garlic isolated compounds were found on blood pressure under regular and Western dietary conditions by cuff-tail method.

24. Effect of garlic on plaque area of the aortic root under fast atherogenic conditions *P< 0.05

25. Under fast atherogenic conditions: LI111 and Morado 200 showed an improved liver function. Morado 200 had a lowering effect on plasma cholesterol levels. Morado 200 and Allicin showed a lowering effect on serum amyloid A concentration, suggesting a decreased liver specific inflammation. LI111 and DADS treated groups showed a larger plaque area. of the study of the effects of garlic on Conclusions of the study of the effects of garlic on fast atherosclerosis development and other atherosclerosis related processes (2)

26. Study the effect of 2 garlic powders of the same strain with a high and a low content of sulfur-containing compounds and allicin as a positive control on inflammation related parameters and on mild human-like atherosclerosis development conditions by means of a hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice. Future mild atherosclerosis development study under more human-like conditions

27. Future mild atherosclerosis development study under more human-like conditions: Experimental scheme -2 Matching and start of the study Weight and food intake Blood sampling/ plasma isolation Plasma lipid levels Cholesterol lipoprotein profile Fibrinogen, t-PA and soluble ICAM Marker for liver function Liver lipid composition Moncytes adherence Atherosclerotic aorta lesions typing 614 60 p 55 60 55 60 p p p Western diet 60 p SAA and vWf Compound biostability 60 p 5 Run in 22 24 60 0 Atherosclerotic aorta lesions area55 5 General parameters Inflammation and atherosclerosis development related parameters Lipid metabolism related parameters In weeks 5 60