HDL Particle Number Is It a Key to Understanding HDL Cardiovascular Risk and Therapy? Jim Otvos, PhD Chief Scientific Officer LipoScience, Inc. Adjunct Professor of Molecular & Structural Biochemistry, NC State University Adjunct Professor of Medicine, University of North Carolina-Chapel Hill The data I want to share with you today will be published soon in Atherosclerosis and it comes from the MESA study. I want to use this slide to show you the collaborators in this effort and to acknowledge and express my gratitude to two people in particular, Samia Mora and Richey Sharrett, who led these analyses. I also wanted to show you the affiliations of the authors of this paper, since they have credentials in epidemiology that I don’t have. I’m trying to convince you to take seriously what I tell you, not because I'm telling you, but because these are the findings of very good epidemiologists.

Quest for HDL-Raising Therapies Driven by Epidemiologic Findings for HDL Cholesterol Framingham Heart Study Data for men aged 50–70 years Relative Risk of Coronary Heart Disease After 4 Years 25 45 65 HDL-C (mg/dL) 85 100 160 220 LDL-C (mg/dL) Castelli WP. Can J Cardiol. 1988;4(Suppl A):5A-10A.

Overview History, not biomarker performance, made HDL-C the standard measure of HDL and HDL-related CVD risk. Association of HDL-C with CVD risk is less strong than it appears, due to confounding by LDL particle number. Alternate measures of HDL quantity are clinically available: apoA-1, HDL particle number (HDL-P) Cholesterol content of HDL particles varies >2-fold between patients and can be altered by drug treatment. The result is frequent discordance between HDL-C and HDL-P. Evidence is accumulating that CVD risk tracks with HDL-P (not HDL-C) in patients with discordant levels. So, here are the take-aways for this part of talk dealing with LDL……….

HDL Quantification by Cholesterol Content (chemical/enzymatic assay) 0.95 VLDL Chylomicron Remnants 1.006 IDL Density (g/ml) 1.02 LDL LDL Cholesterol Triglycerides (mainly) 1.06 HDL2 1.10 HDL3 1.20 HDL Cholesterol 5 10 20 40 60 80 1000 Diameter (nm)

HDL Quantification by Apolipoprotein Content (immunoassay) 0.95 VLDL Chylomicron Remnants 1.006 IDL Density (g/ml) 1.02 LDL 1.06 HDL2 1.10 HDL3 Apo B 1.20 Apo A-1 5 10 20 40 60 80 1000 Diameter (nm)

HDL Quantification by Particle Number (NMR; ion mobility) 0.95 VLDL Chylomicron Remnants 1.006 IDL Density (g/ml) 1.02 LDL LDL-P VLDL-P 1.06 HDL2 1.10 HDL3 1.20 HDL-P 5 10 20 40 60 80 1000 Diameter (nm) Clin Lab Med 2006;26:847-70

Lipoprotein Particle Number Measurements by NMR are Automated and Rapid (~1 min)

NMR Lipoprotein Particle Analysis Lipoprotein Subclass Particle Numbers 2a 3b ~2b 3a 3c Large VLDL Medium VLDL Small VLDL IDL Large LDL Small LDL Large HDL Med HDL Small HDL >60 nm 35-60 29-35 23-29 20.5-23 18-20.5 9.4-14 8.2-9.4 7.3-8.2 nm units of nmol/L µmol/L VLDL Particle Number (VLDL-P) VLDL Size LDL Particle Number (LDL-P) LDL Size HDL Particle Number (HDL-P) HDL Size Clin Lab Med 2006;26:847-70

Cholesterol and ApoA-1 Content of HDL Increase with HDL Particle Size HDL-C ApoA-1 HDL-P = Cholesterol and ApoA-1 Content of HDL Increase with HDL Particle Size A-1 A-1 A-1 Cholesterol Ester Triglycerides A-1 A-1 A-1 Large HDL Small HDL

HDL-C ApoA-1 HDL-P = Cholesterol Composition of HDL Varies Independently of HDL Particle Size A-1 A-1 Cholesterol Ester Triglycerides A-1 A-1 More Cholesterol (Less Triglyceride) Less Cholesterol (More Triglyceride)

Framingham Offspring Study (low HDL-P subgroup) HDL-C ApoA-1 HDL-P = Framingham Offspring Study (low HDL-P subgroup) n=191 HDL-C 214 ApoA-1 61 178 n=143 ApoA-1 (mg/dL) HDL-C (mg/dL) n=158 143 44 151 n=188 110 36 127 117 31 110 HDL Size <8.9 8.9-9.1 9.2-9.5 >9.5 nm HDL-P 25.1 25.5 25.6 25.7 umol/L Adapted from Freedman et al. Clin Chem 2004;50:1189

HDL Particles Inhibit Atherogenesis (by promoting cholesterol efflux, inhibiting LDL oxidation, etc…) HDL Inhibit Adhesion Molecule Expression Monocyte LDL Vessel Lumen Adhesion Molecules Endothelium MCP-1 LDL HDL Inhibit Oxidation of LDL Modified LDL Cytokines Inhibition of adhesion molecules The cytokine-induced expression of adhesion molecules in endothelial cells has been shown in vitro and more recently in vivo to be inhibited by HDL, in a process that potentially blocks a very early inflammatory stage in the development of atherosclerosis. Reference: Cockerill GW, Rye K-A, Gamble JR, Vadas MA, Barter PJ. High-density lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb Vasc Biol 1995;15:1987-1994. Foam Cell Macrophage Intima HDL Promote Cholesterol Efflux Cockerill GW et al. Arterioscler Thromb Vasc Biol 1995;15:1987-1994.

Relations with Future CHD Events in Men with Low HDL-C (VA-HIT) * ** * * ** Adjusted for treatment, age, diabetes, hypertension, smoking, and BMI *p<0.01 **p<0.001 Circulation 2006;113:1556-63

HDL-C is Independent of LDL-C, but NOT Independent of LDL-P Framingham Offspring Study 1000 1200 1400 1800 180 LDL-P 1600 160 LDL Particles (nmol/L) LDL Cholesterol (mg/dL) 140 LDL-C 120 100 20 40 60 80 100 HDL Cholesterol (mg/dL) Amer J Cardiol 2002;90:22i-29i

HDL Particles in the Framingham Offspring Study Total HDL-P HDL Size HDL Particles (mol/L) Small Large A quick word now about HDL because there was a really good session on HDL Saturday morning. And there was mention made several times of the fact that large HDL is the protective component of HDL as shown by epidemiology, with small and medium sized HDL not so important in terms of atheroprotection. Let me just show you data from Framingham, simply to show you that when HDL-C levels go from mid-range to high, the large HDL subclass is what accounts for the increase in HDL-C. But there is a corresponding reduction in small HDL as large HDL particle number increases, so these two variables are inversely correlated - not as strongly as large and small LDL, but enough to confound each others true relationships with cardiovascular disease risk. Medium HDL Cholesterol (mg/dL) J Clin Lipidol 2007;1:57-64

HDL-P or HDL Size Quartiles Odds Ratios for Future CAD in EPIC-Norfolk HDL Size (p<0.001) Odds Ratios* HDL-P (p<0.001) HDL-P or HDL Size Quartiles *adjusted for age, gender, and smoking (HDL-P and HDL size in same model) El Harchaoui et al. Ann Intern Med 2009;150:84-93

Odds Ratios for Future CAD in EPIC-Norfolk adjusted for LDL-P (apoB) HDL Size (p=0.92) Odds Ratios* HDL-P (p<0.001) Quartiles *adjusted for age, gender, smoking, apoB, and TG (HDL-P and HDL size in same model) El Harchaoui et al. Ann Intern Med 2009;150:84-93

Carotid IMT* (microns) HDL-C Relations with Carotid IMT in MESA Adjusted for LDL-C Carotid IMT* (microns) <41 41-48 49-59 >59 mg/dL HDL-C Quartiles *adjusted for age, gender, race, hypertension, smoking, BMI, and diabetes unpublished data

Carotid IMT* (microns) HDL-C Relations with Carotid IMT in MESA Adjusted for LDL-C Carotid IMT* (microns) Adjusted for LDL-P <41 41-48 49-59 >59 mg/dL HDL-C Quartiles *adjusted for age, gender, race, hypertension, smoking, BMI, and diabetes unpublished data

Carotid IMT* (microns) HDL-C vs HDL-P Relations with Carotid IMT in MESA Adjusted for LDL-P HDL-P HDL-C Carotid IMT* (microns) HDL Quartiles *adjusted for age, gender, race, hypertension, smoking, BMI, diabetes, and LDL-P unpublished data

Discordance Between HDL-C and HDL-P Subgroup with High HDL-C (≥ 55 mg/dL) Large HDL n=84 991 Carotid IMT (µm) n=451 Concordant 916 n=1133 886 <28 28-33 >33 µmol/L HDL-P Tertiles Results from MESA adjusted for age, sex, race, smoking, hypertension, BMI, diabetes, and LDL-P unpublished data

Discordance Between HDL-C and HDL-P Subgroup with Low HDL-C (≤ 42 mg/dL) Concordant n=1204 n=418 Small HDL 980 965 Carotid IMT (µm) n=64 911 <28 28-33 >33 µmol/L HDL-P Tertiles Results from MESA adjusted for age, sex, race, smoking, hypertension, BMI, diabetes, and LDL-P unpublished data

Conclusions HDL-C is not “HDL” ! Part of the “independent” association of HDL-C with CVD risk is not actually independent of LDL (particles). Given LDL-P, HDL-P is more strongly related to atherosclerotic risk than HDL-C. Raising HDL-C without raising HDL-P may have little or no clinical benefit. So, the conclusion is that LDL size doesn’t matter once LDL particle number is taken into account. It’s not a quality issue here, it’s a quantity issue. Small LDL particles are not more atherogenic than large LDL particles. That’s what the epidemiology now tells us, once the effects of confounding are eliminated. What does matter is LDL particle number, more so than LDL cholesterol. I want to take the last few minutes to show you some more data from MESA that supports this contention.

Backup Slides

Lipoprotein Particle Quantification by Nuclear Magnetic Resonance (NMR) Enabled by the Invariant Physical-Chemical Linkage of Lipoprotein Diameter and NMR Signal Frequency

Lipoprotein Diameter Determines the NMR Signal Frequencies of its Constituent Lipids Rosenson RS et al. Clin Chem 2011;57:392-410.

Measured Amplitude of the Methyl Signal of Each Subclass Provides Its Particle Concentration To a close approximation, a given size particle has a constant number of terminal lipid methyl groups, irrespective of core lipid compositional variation. Clin Lab Med 2006;26:847-70

Relations with Carotid IMT in MESA HDL Subclasses Relations with Carotid IMT in MESA ΔIMT (in m) per 1-SD Subclass Separate Models Joint Model + LDL-P Large HDL-P -25.1** Medium HDL-P -20.8** Small HDL-P 5.8 most protective From linear regression analyses in MESA adjusted for age, sex, race, SBP, hypertension medication, and smoking, excluding subjects on lipid lowering drugs. *p<0.05; **p<0.0001 unpublished data

Relations with Carotid IMT in MESA HDL Subclasses Relations with Carotid IMT in MESA ΔIMT (in m) per 1-SD Subclass Separate Models Joint Model + LDL-P Large HDL-P -25.1** -23.9** Medium HDL-P -20.8** -25.8** Small HDL-P 5.8 -15.9* From linear regression analyses in MESA adjusted for age, sex, race, SBP, hypertension medication, and smoking, excluding subjects on lipid lowering drugs. *p<0.05; **p<0.0001 unpublished data

Relations with Carotid IMT in MESA HDL Subclasses Relations with Carotid IMT in MESA ΔIMT (in m) per 1-SD Subclass Separate Models Joint Model + LDL-P Large HDL-P -25.1** -23.9** -10.0* Medium HDL-P -20.8** -25.8** -22.8** Small HDL-P 5.8 -15.9* -17.2* most protective?? From linear regression analyses in MESA adjusted for age, sex, race, SBP, hypertension medication, and smoking, excluding subjects on lipid lowering drugs. *p<0.05; **p<0.0001 unpublished data

Relations with Incident CVD Events (n=319) in MESA HDL Subclasses Relations with Incident CVD Events (n=319) in MESA HR (95%CI) per 1-SD HDL Subclass Separate Models Same Model + LDL-P, TG Large HDL-P 0.71** (.61-.83) 0.73** (.63-.85) 0.86 (.73-1.02) Medium HDL-P 0.83* (.73-.94) (.70-.98) Small HDL-P 1.07 (.96-1.19) 0.90 (.78-1.05) (.74-1.00) From proportional hazards regression analyses adjusted for age, sex, race, SBP, hypertension medication, and smoking. *p<0.05; **p<0.0001 unpublished data