Exogenous Pathway Endogenous pathway

1. Difference in lipid metabolism and atherosclerosis among human, rodents, and WHHL / WHHLMI rabbits

2. Bile acids + Cholesterol Dietary lipid LDL receptors LDL receptors Intestine Liver LDL Peripheral tissued apoB editing enzyme B-100 Remnant receptors Chylomicron remnants HDL VLDL IDL Chylomicron E E B-100 E A E C C E B-48 B-100 B-48 CETP Plasma CETP Capillary Capillary LCAT Lipoprotein lipase Lipoprotein lipase Lipoprotein metabolism in human and rabbits Exogenous Pathway Endogenous pathway (Modified after Goldstein et al. N Engl J Med 1983; 309:288-286)

3. VLDL IDL E C E B-48 B-48 Lipoprotein metabolism in mice and rats Exogenous pathway Endogenous pathway Bile acids + Cholesterol Dietary lipids LDL receptors LDL receptors Liver intestine LDL Peripheral tissues apoB editing enzyme apoB editing enzyme B-100 Remnant receptors HDL IDL VLDL Chylomicron remnants Chylomicron E A E B-100 E C E B-100 B-48 E B-48 C Plasma ☓ ☓ CETP CETP Capillary LCAT capillary Lipoprotein lipase Lipoprotein lipase

4. Lipoprotein metabolism of ApoE KO Exogenous pathway Endogenous pathway Bile acids + Cholesterol Dietary lipids LDL receptors LDL receptors Liver intestine LDL Peripheral tissues apoB editing enzyme apoB editing enzyme B-100 ☓ ☓ Remnant receptors HDL IDL VLDL IDL VLDL Chylomicron remnants Chylomicron ☓ ☓ E ☓ ☓ A E ☓ ☓ E B-100 ☓ E C E C E B-100 B-48 E B-48 B-48 C B-48 Plasma ☓ ☓ CETP CETP Capillary LCAT capillary Lipoprotein lipase Lipoprotein lipase

5. Lipoprotein profiles of human and mice with HPLC ApoE KO mice Wild mice LDLR-KOmice Human VLDL + Chylomicron remnants (mV) (mV) (mV) 240 190 140 90 40 -10 170 150 130 110 90 70 50 30 10 -10 1.25% Cholesterol-fed LDL HDL LDL Standard diet VLDL Chylomicron remnants HDL Free Glycerol HDL VLDL Free Glycerol Free Glycerol 0 10 20 30 Fraction Number 15 17 19 21 23 25 27 29 31 33 35 37 15 17 19 21 23 25 27 29 31 33 35 37 15 17 192123 2527 29 31 33 35 Elusion time (min) Elusion time (min) Elusion time (min) Ishibashi S, et al. (JCI 1994; 93:1885-93) Total cholesterol triglyceride Data from Skylight Biotech Inc.

6. Lipoprotein metabolism of WHHL / WHHLMI rabbits Exogenous pathway Endogenous pathway Bile acids + Cholesterol Dietary lipids LDL receptors LDL receptors Intestine LDL Liver Peripheral tissues apoB editing enzyme B-100 Remnant receptors Chylomicron remnants VLDL HDL IDL Chylomicron E E B-100 E A E C C E B-48 B-100 B-48 CETP Plasma CETP Capillary Capillary LCAT Lipoprotein lipase Lipoprotein lipase

7. WHHLMI rabbit LDL HDL VLDL Free Glycerol Elusion time (min) Lipoprotein profiles in the plasma of human apoE-KO mice, and WHHLMI rabbits Human ApoE KOmice (mV) (mV) 240 190 140 90 40 -10 VLDL LDL HDL HDL VLDL Free Glycerol Free Glycerol 1517 19212325 27 29 31 33 35 37 15 17 19 21 23 25 27 29 31 33 35 37 Elusion time (min) Elusion time (min) Total cholesterol triglyceride Data from Skylight Biotech Inc.

8. Plasma CETP activity of WHHL rabbits Son YC, et al. (Arteriosclerosis 1986; 6: 345-351)

9. Coronary lesions of mouse models were different from human coronary plaques. Coronary plaque of human (Falk E, Br Heart J 1983; 50: 127-134) Coronary lesion of ApoE-KO/ LDLR-KO mouse fed high fat diet（Masson’s Trichrome stain） Caligiuri G, et al. PNAS 1999; 96: 6920-6924 Coronary lesion of ApoE-KO /SRBI-KO mice fed standard chow（Masson’s Trichrome stain） Braun A, et al. Cir Res 2002; 90: 270-276 Coronary lesion of LDLR-KO mice fed high fat diet （H&E stain） Ishibashi S, et al. JCI 1994; 93: 1885-1893 40 mm

10. Low SMCs Vulnerable plaque Lipid deposits Thin fibrous cap Macrophage accumulation (modif. after Davies MJ: Circulation 94: 2013-2020, 1996) Vulnerable coronary plaque of WHHLMI rabbit Ruptured coronary plaque of human Elastic van Gieson stain 1A4 immunostaining (Falk E, Br Heart J 1983; 50: 127-134) RAM-11 immunostaining Azan-Mallory stain (modif. after Shiomi M et al, ATVB; 2003; 23: 1239-1244 )

11. Statin increases cholesterol synthesis in rats (A) Day 0 Pravastatin Pravastatin Fujioka T, et al. (BBA 1995; 1254: 7-12)

12. Statin up-regulates HMG-CA reductase activity and fatty acids synthesis in rats Fujioka T, et al. (Eur J Pharmacol 1997; 328:235-239)

13. Plasma Lipid Levels and Degree of Atherosclerosis of Mature WHHL Rabbits Treated with Pravastatin Whole plasma cholesterol LDL cholesterol －14% (vs.Placebo) P<0.05 －17% (vs Placebo) P<0.05 (mg/dl) (mg/dl) Control at the start (10 months old) Placebo treatment from 10 to 22 months old Pravastatin treatment from 10 to 22 months old (modif. after Shiomi M et al, ATVB 1995; 15: 1938-1944)

14. Summary of species difference in lipid metabolism and atherosclerosis Mice and Rats Human WHHLMI HDL, VLDL, chylomicron remnants LDL LDL Main lipoproteins ApoB of VLDL ApoB Editing enzyme CETP HTGL Dietary fats /cholesterol Atherosclerosis Inflammatory marker Hypolipidemic effects of statins apoB-100 apoB-48 & apoB-100apoB-100 Ilium Ilium and LiverIlium Yes noneYes Unbound to membraneBound to membrane Bound to membrane Spontaneous Resistance Susceptible Resistance Susceptible Lipid–rich/Collagen-poor Various lesions Spontaneous Various lesions Serum Amyloid P component SAP CRP CRP No effects or Increase in cholesterol Effective Effective

15. Different phenotypes manifested in mouse and rabbits after the same gene transfer Genes transferred Mice Rabbits Lecitin:cholesterol acyltransferase Hepatic lipase apoE3 15-lipoxygenase Apolipoprotein(a) Lipoprotein lipase CRP (after modified by Koike T & Fan J, Laboratory Animal Technology and Science 2005; 17: 91-96) Pro-atherogenicAnti-atherogenic Pro-atherogenicAnti-atherogenic Anti-atherogenicPro-atherogenic Pro-atherogenicAnti-atherogenic unboundto apoBBoundndto apoB No changesinDecreasein visceral fatvisceral fat No function Function

16. Difference in lipid metabolism, atherosclerosis, and the effects of statins among human, rodents, and WHHL / WHHLMI rabbits WHHL / WHHLMI rabbits Human MiceandRats (Including transgenic and KO animals) ≒ ≠

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