Dyslipoproteinemia and Atherosclerosis Daniel W. Stock MD Founding Diplomate, American Board of Clinical Lipidology Member, National Lipid Association and Midwest Lipid Association Former Adjunct Clinical Professor Indiana University School of Medicine

Disclosures Speakers Bureau: Liposcience Inc. Lilly Pharmaceuticals Inc. Warner-Chilcott Inc.

Framingham showed ↑’ing risk with ↑’ing LDL and ↓’ing HDL cholesterol CAD status LDL-C HDL-C AMCE ∆ 4S 2º 188 46 34% LIPID 150 36 CARE 139 39 22% WOSCOPS 1º 193 44 31% AFCAPS/TexCAPS 37% ASCOT-LLA 131 50 36% HPS 1º& 2º NA 17% JUPITER 108 49 44% VA-HIT 112 32

Lipid Analysis Shortcomings 50% of Framingham AMCE patients had LDL <130 mg/dl 75% of Framingham AMCE patients < 55 yo had LDL <130 mg/dl Majority of lipid trial patients did not benefit from therapy On-treatment lipids did not predict clinical outcome Forces the treatment of low-risk individuals in order to protect high-risk individuals

Number of patients 5 50 500 5000 500,000 Beta HCG level

Number of patients LDL Cholesterol

“Lipids” and Atherosclerosis Atherosclerosis occurs because of abnormal lipoproteins and/or lipoprotein kinetics, not abnormal lipid levels. “… all abnormalities in plasma lipid concentrations, or dyslipidemia, can be translated into dyslipoproteinemia.” “… the shift of emphasis to lipoproteins offers distinct advantages in the recognition and management of such disorders.” SLIDE 7 The second fundamental concept is that cholesterol and triglyceride values have utility in that they serve as surrogate markers for the lipoproteins in which they are carried. Since the 1960’s it has been understood that lipoproteins are central to cholesterol and triglyceride transport. As such, Fredrickson noted that abnormal lipid concentrations could be translated into dyslipoproteinemias, or abnormal levels of lipoproteins responsible for cholesterol and triglyceride transport. Given the more central relationship of lipoproteins to atherosclerosis, Fredrickson further noted that shifting emphasis from lipids to lipoproteins offered “… distinct advantages in recognition and management of such disorders .” Fredrickson et al., NEJM 1967; 276: 148

Total Cholesterol (+) VLDL-C + LDL-C + HDL-C (+) (+) (-) SLIDE 6 Each major class of plasma lipoprotein (VLDL, LDL, HDL) comprises subclass particles of varying size. This would have no impact on risk assessment if not for the fact that some subclasses differ in their associations with CHD. High concentrations of large VLDL, small LDL, and IDL are associated with enhanced risk (as indicated by the bigger plus signs). And not all HDL subclasses appear to be protective (negative association with CHD); the two smallest subclasses appear to have a positive association with CHD (ie., there is “bad” good cholesterol!). The relative proportions of the subclasses in each lipoprotein class vary widely from person to person for metabolic reasons. As a result, people with the same levels of LDL and HDL cholesterol can have very different risks. For example, at a given level of LDL cholesterol, a person with mostly small, dense LDL particles has a 3 to 5-fold greater risk of CHD than someone with large LDL. Lipoprotein subclass information, which would improve diagnosis and treatment of CHD risk, has been unavailable to clinicians for analytical reasons. Subfractionation of lipoproteins is far more difficult and time-consuming than simple fractionation of LDL and HDL, requiring methods such as density gradient ultracentrifugation and gradient gel electrophoresis IDL LDL + + + + + + + + + + - - - + + Association with CHD: Positive (+) or Negative (-)

Lipoprotein Particles Apolipoprotein Apolipoprotein POLAR SURFACE COAT Phospholipid Free cholesterol NON-POLAR LIPID CORE Cholesterol Ester Triglyceride Because cholesterol and triglycerides are hydrophobic lipids that are insoluable in plasma, intravascular transport cannot occur with these lipids in their free state. Consequently, cholesterol esters and triglycerides are packaged into spherical lipoprotein particles that span a wide range of particle size and density. Lipoprotein particles are configured so that the outer surface is polar and the inner core is non-polar. The surface of lipoprotein particles is composed of a phospholipid monolayer, non-esterified cholesterol, and various apolipoproteins. The core of lipoprotein particles contains variable amounts of cholesterol ester and triglycerides. Apolipoprotein 10

Among Individuals At The Same LDL-C Level, The Number of LDL Particles Vary At the same LDL cholesterol, with small versus large size LDL Particles Up to 70% More Particles Cholesterol Balance 100 mg/dL 100 mg/dL Because of variability in the amount of cholesterol carried inside LDL particles, LDL-C often does not reflect accurately LDL-P. Two processes are primarily responsible for this phenomenon. First, small sized LDL particles carry less cholesterol per particle than large sized particles. For LDL particles differing by 3 nm in diameter, there is approximately 40% less core cholesterol in the smaller versus larger particle. Thus, the person with the smaller LDL particles will require almost 70% more particles to carry the same amount of LDLC than the person with the larger particles. Otvos JD, et al. Am J Cardiol 2002;90(suppl):22i-29i Cromwell WC, et al. J Clin Lipidology. 2007;1(6):583-592 Large LDL Small LDL Otvos JD, et al. Am J Cardiol 2002;90(suppl):22i-29i Cromwell WC, et al. J Clin Lipidology. 2007;1(6):583-592. 11

Among Individuals At The Same LDL-C Level, The Number of LDL Particles Vary At the same LDL cholesterol, with the same size LDL Particles (at any triglyceride level) Up to 40% More Particles 100 mg/dL Second, even at the same LDL particle size, the cholesterol content of LDL particles is not consistent. Due to cholesterol ester transfer protein (CETP) mediated exchange of triglycerides (TG) and cholesterol ester between cholesterol-rich (LDL and HDL) and TG-rich (VLDL, intermediate-density lipoprotein, and remnants) particles, the amount of cholesterol carried per particle is highly variable. When TG levels are elevated, or when LDL levels are decreased (even modestly), this exchange reaction results in altered LDL particles that are partially depleted in core cholesterol and enriched in core TG. When particles are partially cholesterol depleted, up to 40% more LDL particles are needed to transport the same amount of cholesterol compared to similar sized particles with the expected amount of cholesterol per particle. Otvos JD, et al. Am J Cardiol 2002;90(suppl):22i-29i Cromwell WC, et al. J Clin Lipidology. 2007;1(6):583-592 Normal Cholesterol Carried Per Particle Less Cholesterol Carried Per Particle Cholesterol Balance Otvos JD, et al. Am J Cardiol 2002;90(suppl):22i-29i Cromwell WC, et al. J Clin Lipidology. 2007;1(6):583-592. 12

Otvos JD, Jayarajah E, Cromwell, WC. AJC 2002;90(8A):22i-29i LDL-C often fails to reflect the number of LDL particles and the CHD risk they confer The number of LDL particles (LDL-P™) varies widely among patients with similar LDL-C values. In this analysis, patients with an LDL-C between 95 mg/dL and 105 mg/dL have highly variable LDL particles, and thus variable CHD risk. Otvos JD, Jayarajah E, Cromwell, WC. AJC 2002;90(8A):22i-29i

LDL-C Often Fails to Reflect the Number of LDL Particles LDL particles can be large or small, and the amount of cholesterol contained within these particles varies widely. Otvos JD, Jayarajah E, Cromwell, WC. AJC 2002;90(8A):22i-29i

Pathophysiology of Atherosclerosis Particle Movement Gradient driven Enhanced Endothelial Dysfunction “The rate of passive diffusion is increased when the circulating levels of LDL are elevated.” 1 Monocyte Adhesion molecules MCP-1 Colony- stimulating factors Tissue factor PAI-1 Endothelial cells Mildly modified LDL Extensively modified LDL Particle Retention Lipoprotein particle binding to proteoglycans Oxidative Modification Enhanced Macrophage Uptake Modified / retained lipoproteins 1 Weissberg PL, Rudd JH. Textbook of Cardiovascular Medicine 2nd ed. 2002. p. 6.

Odds Ratio per 1-SD Increment of on-trial value Alternative Measures of LDL as Predictors of CHD Events in VA-HIT p<0.001 Odds Ratio per 1-SD Increment of on-trial value p=0.17 p=0.25 p=0.31 LDL-C Non-HDL-C ApoB LDL-P Adjusted for treatment, age, hypertension, smoking, BMI, and diabetes Circulation 2006;113:1556-63

Odds Ratio per 1-SD Decrement of on-trial value Alternative Measures of HDL as Predictors of CHD Events in VA-HIT p<0.001 Odds Ratio per 1-SD Decrement of on-trial value p=0.18 p=0.42 HDL-C ApoA-1 HDL-P Adjusted for treatment, age, hypertension, smoking, BMI, and diabetes Circulation 2006;113:1556-63

Fig. 2 Survival Curves for Framingham Subjects with Concordant LDL-C and LDL-P Years of Follow-up Event-Free Survival Low LDL-P & Low LDL-C (n=1,249) High LDL-P & High LDL-C (n=1,251)

Fig. 2 Survival Curves for Framingham Subjects with Discordant LDL-C and LDL-P Years of Follow-up Event-Free Survival Low LDL-C High LDL-P (n=282) High LDL-C Low LDL-P (n=284)

Fig. 2 Survival Curves for Framingham Subjects with Concordant and Discordant LDL-C and LDL-P Years of Follow-up Event-Free Survival Low LDL-C High LDL-P (n=282) High LDL-C Low LDL-P (n=284) Low LDL-P & Low LDL-C (n=1,249) High LDL-P & High LDL-C (n=1,251)

NMR LDL-P vs. Lipids NMR LDL-P significantly better risk predictor than all lipid panel parameters Framing. Offspring Study*† Cardiovascular Health Study* VA-HIT*† PLAC-1* Healthy Women Study* MESA*† NMR LDL-P significantly better risk predictor than all lipid panel parameters except TC/HDL-C Women’s Health Study*† LDL pattern not an independent predictor of risk after NMR LDL-P adjustment Framing. Offspring Study*† VA-HIT*† Women’s Health Study*† MESA*† * Independent of sex, age, smoking status and BP † Superior predictor to and independent of apo B-100

NMR HDL-P vs. HDL-C NMR HDL-P better risk predictor than HDL-C Framing. Offspring Study*† PLAC-1* VA-HIT*† MESA*† Women’s Health Study*† NMR HDL-P better predictor of therapy response that HDL-C PLAC-1* VA-HIT*† * Independent of sex, age, smoking status and BP, and all lipid panel parameters † Superior predictor to and independent of apo B-100

Treatment Summary LDL-P Small LDL-P LDL Size HDL-P Large VLDL-P Statins   or   or   any Ezetimibe   or   or   any Niacin    ? any BAS     Only  Fibrates/ pioglitazone     Only  Ω-3 FA     Only 

Patient Bradley 65 YO WM, 71” tall 198#, BP 110/70m, never smoker, neg FHx of premature atherosclerosis, Dx of BPH, Dyslipoproteinemia, Atrial fibrillation, ED Lipoprotein Therapy: Pravastatin 20 MG QD TC = 145 LDL-C = 93 (15th) HDL-C = 39 (20th) TG = 66 Non HDL-C = 106 (10th) LDL-P = 1560 (60th) HDL-P = 28.7 (40th) Small LDL-P = 1369 LDL Particle Size = 19.8 Pattern B = 20.5-18.0 Large HDL-P = 6.8 (50th) Large VLDL-P = 0.1

Patient Bradley 65 YO WM, 71” tall 198#, BP 110/70m, never smoker, neg FHx of premature atherosclerosis, Dx of BPH, Dyslipoproteinemia, Atrial fibrillation, ED Changed from pravastatin to rosuvastatin 10 MG QD. TC = 123 LDL-C = 68 (3rd) HDL-C = 42 (25th) TG = 63 Non HDL-C = 81 (2nd) LDL-P = 1045 (20th) HDL-P = 27.7 (38th) Small LDL-P = 722 LDL Particle Size = 20.9 Pattern A = 23.0-20.6 Large HDL-P = 9.0 (75th) Large VLDL-P = 0.2

Patient Gregory 43 YO WM, 68” tall 155#, BP 110/80m, never smoker, non-drinker, regular exercise,+ FHx of premature atherosclerosis, No medical diagnoses. Lipoprotein Therapy: None TC = 199 LDL-C = 132 (50th) HDL-C = 54 (75th) TG = 67 Non HDL-C = 145 (40th) CXR: Mild Aortic Calcification Carotid U/S: minimal non-calcifying plaque L CCA and bilat ICA LDL-P = 1147 (30th) HDL-P = 25.0 (18th) Small LDL-P = 517 LDL Particle Size = 21.6 Pattern A = 23.0-20.6 Large HDL-P = 9.3 (80th) Large VLDL-P = 0.7 (20th)

Patient Gregory 43 YO WM, 68” tall 155#, BP 110/80m, never smoker, non-drinker, regular exercise,+ FHx of premature atherosclerosis, No medical diagnoses. Lipoprotein Therapy: simvastatin 20 MG QD TC = 118 LDL-C = 69 (< 2nd) HDL-C = 42 (28th) TG = 33 Non HDL-C = 76 (< 2nd) LDL-P = 808 (3rd) HDL-P = 26.2 (21st) Small LDL-P = 508 LDL Particle Size = 21.1 Pattern A = 23.0-20.6 Large HDL-P = 8.3 (78th) Large VLDL-P = 0.0 (5th)

Patient Gregory 43 YO WM, 68” tall 155#, BP 110/80m, never smoker, non-drinker, regular exercise,+ FHx of premature atherosclerosis, No medical diagnoses. Lipoprotein Therapy: lovastatin/ER niacin 1000/40 MG QD TC = 152 LDL-C = 97 (19th) HDL-C = 50 (65th) TG = 25 Non HDL-C = 102 (10th) LDL-P = 907 (7th) HDL-P = 31.0 (60th) Small LDL-P = 563 LDL Particle Size = 21.1 Pattern A = 23.0-20.6 Large HDL-P = 12.4 (95th) Large VLDL-P = 0.1 (10th) Carotid U/S: No demostrable plaque either carotid artery

Patient Fancher TC = 187 LDL-C = 84 (4th) HDL-C = 55 (52th) TG = 242 53 YO WF, 64” tall, 158#, BP 120/80, Never smoker, Dx IFG (FBS 110), menopause, s/p Radical Urinary Cystectomy and continent colon reservoir for Transitional Bladder CA Current Meds: None Lipoprotein therapy: None TC = 187 LDL-C = 84 (4th) HDL-C = 55 (52th) TG = 242 Non HDL-C = 132 (30th) LDL-P = 763 (3rd) HDL-P = 40.6 (80th) Small LDL-P = 443 LDL Particle Size = 21.1 Pattern A = 20.5-18.0 Large HDL-P = 5.4 (60th) Large VLDL-P = 5.9

Patient Chew TC = 192 LDL-C = 73 (2nd) HDL-C = 58 (55th) TG = 303 46 YO WF, 67” tall, 202#, BP 120/76, no tobacco since 93, 16 pack-year hx previously, Dx of menopause, Dyslipoproteinemia, IFG, Hepatic steatosis Current Meds: levothyroxine 150 MCG QD, spironolactone 50 MG QD, CEE 0.625 QD, HCTZ 12.5 QD, escitalopram 20 MG QD, buproprion 150 MG QD Lipoprotein therapy: rosuvastatin 10 MG QD TC = 192 LDL-C = 73 (2nd) HDL-C = 58 (55th) TG = 303 Non HDL-C = 134 (30th) LDL-P = 1913 (85th) HDL-P = 39.7 (80th) Small LDL-P = 1404 LDL Particle Size = 20.4 Pattern B = 20.5-18.0 Large HDL-P = 12.3 (95th) Large VLDL-P = 13.7

Patient Chew TC = 146 LDL-C = 62 (1st) HDL-C = 48 (18th) TG = 181 46 YO WF, 67” tall, 202#, BP 120/76, no tobacco since 93, 16 pack-year hx previously, Dx of menopause, Dyslipoproteinemia, impaired fasting glucose, Hepatic steatosis Current Meds: levothyroxine 150 MCG, spironolactone 50 MG QD, CEE 0.625 QD, HCTZ 12.5 QD, lexapro 20 MG QD, buproprion 150 MG QD Added Omega-3 Fatty Acid Ethyl Esters 4 G QD to rosuvastatin 10 MG QD TC = 146 LDL-C = 62 (1st) HDL-C = 48 (18th) TG = 181 Non HDL-C = 98 (7th) LDL-P = 1434 (50th) HDL-P = 36.2 (70th) Small LDL-P = 1103 LDL Particle Size = 20.45 Pattern B = 20.5-18.0 Large HDL-P = 7.2 (60th) Large VLDL-P = 2.6

Patient Jones TC = 158 LDL-C = 80 (5th) HDL-C = 62 (75) TG = 79 60 YO BM, 72” tall, 194#, 150/70, never smoker, Dx HTN, Dyslipoproteinemia Current Meds: ramipril 10 MG QD, spironolactone/HCTZ 12.5/12.5 MG QD Lipoprotein Therapy: lovastatin/ER niacin 1000/40 MG QD TC = 158 LDL-C = 80 (5th) HDL-C = 62 (75) TG = 79 Non HDL-C = 96 (5th) LDL-P = 1459 (50th) Small LDL-P = 1067 LDL Particle Size = 20.7 Pattern A = 23.0 - 20.6 Large HDL-P = 6.8 (50th) Large VLDL-P = 0.1

Patient Jones TC = 135 LDL-C = 86 (10th) HDL-C = 63 (75th) TG = 62 60 YO BM, 72” tall, 194#, 150-70, never smoker, Dx HTN, Dyslipoproteinemia Current Meds: Altace 10 MG QD, spironolactone/HCTZ 12.5/12.5 MG QD Changed to rosuvastatin 10 MG QD and ER niacin 1000 MG QD TC = 135 LDL-C = 86 (10th) HDL-C = 63 (75th) TG = 62 Non HDL-C = 72 (1st) LDL-P = 953 (10th) Small LDL-P = 652 LDL Particle Size = 20.9 Pattern A = 23.0 - 20.6 Large HDL-P = 10.1 (90th) Large VLDL-P = 0.2