Is There a Dysfunctional HDL? How do we Test HDL Function? Sergio Fazio, MD, PhD Professor of Medicine and Pathology Co-Director, Atherosclerosis Research Unit Vanderbilt University Medical Center Nashville, Tennessee

Benefits of Intensive LDL-C Lowering 30 5 10 15 20 25 Statin Placebo HPS CARE LIPID 4S LDL cholesterol (mg/dL) 210 190 170 150 130 110 90 70 Event (%) TNT (10 mg) TNT (80 mg) LaRosa JC et al. N Engl J Med. 2005;352.

Relationship between ↓LDL-C and atheroma burden 6/12/2018 6:16:23 AM Data from recent IVUS trials 1.8 REVERSAL Pravastatin CAMELOT Placebo 1.2 Median Δ in percent atheroma volume (%) 0.6 REVERSAL Atorvastatin A-Plus Placebo –0.6 ASTEROID Rosuvastatin r2 = 0.97 P < 0.001 –1.2 60 70 80 90 100 110 120 Mean LDL-C (mg/dL) Nissen SE et al. JAMA. 2006;295:1556-65.

Towards Medical Therapy of Coronary Disease A. Stop Progression (and stabilize the plaque?): 1. Extreme LDL reductions 2. Aggressive RF and T2D management 3. Maybe direct effects of ACE-I/ARB, Statins, ASA B. Induce Regression (and stabilize the plaque?): 1. Stop progression 2. Maybe activation of HDL pathway 3. Maybe direct effects of PPAR or LXR agonists

Cardiovascular Disease and HDL-C Levels 160 140 Men Women 120 100 Rate Per 1000 80 60 40 20 <34 35-54 >55 <34 35-54 >55 HDL Cholesterol (mg/dL) Kannel WB, Am J Cardiol. 1983;52:9B-12B.

Bezafibrate Infarction Prevention (BIP): Outcome by HDL-C Response to Therapy 40 35 30 25 20 15 10 5 Placebo T1 and T2* T3* Total Mortality (%) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time (Yrs) *T1, T2, and T3 represent tertiles of HDL increases Goldenberg I, et al. Arch Intern Med. 2009;169(5):508-514.

The HDL Pathway Hepatocyte Forming HDL Mature HDL Peripheral Cell Figure 1. Synopsis of HDL metabolism and targets of therapy. Interventions on Stage I aim at activating mechanisms of cholesterol exit. Only minor changes in plasma HDLc levels are expected. Interventions on Stage II include maneuvers that improve HDL maturation and/or improve HDL’s anti-oxidant and anti-inflammatory effects The influence of these interventions on plasma HDLc levels may vary from increases to decreases. Interventions on Stage III include maneuvers that block or facilitate cholesterol loss from HDL, causing large increases or decreases, respectively, in plasma HDLc levels. LXR: liver X receptor, master regulator of cholesterol exit from the cell. ABCA1, ABCG1: ATP-dependent binding cassettes A1 and G1, major transporters of cellular cholesterol to apoAI and HDL, respectively (LXR sensitive genes). ACAT1: acyl:CoA-cholesterol acyltransferase, catalyzes formation of cholesteryl esters and reduces the pool of free cholesterol available for export. ApoE: apolipoprotein E, produced by the macrophage in response to cholesterol loading (LXR sensitive gene); similarly to apoAI, apoE can act as cholesterol acceptor. ApoAIs: this term is meant to represent a variety of interventions aimed at raising endogenous production of apoAI (LXR sensitive gene) or at introducing apoAI mutants or mimetics as agents of increased HDL functionality. LCAT: lecithin-cholesterol acyltransferase, esterifies cholesterol on HDL, expanding its core. SR-BI: scavenger receptor type BI, also known as the HDL receptor, captures mature HDL and facilitates transfer of cholesterol cargo into the liver. HL: hepatic lipase, removes triglycerides from HDL before they can be recognized by SR-BI. CETP: cholesteryl ester transfer protein, facilitates trasnfer of cholesterol from HDL to apoB-containing lipoproteins. Peripheral Cell Stage I: Intracellular Cholesterol Trafficking ApoB-Lipoproteins Stage II: Cholesterol Acquisition and HDL Maturation Stage III: Cholesterol Exchange and Delivery

Cholesterol Exit from the Arterial Macrophage From: Li & Glass. Nature Medicine 2002

A Revised Look at Reverse Cholesterol Transport Removal of excess cholesterol from peripheral tissues is essential for whole-body cholesterol homeostasis Removal of excess cholesterol from arterial macrophages, which poses a unique biological challenge, is essential for vascular health

Targets along the HDL Pathway LXR ACAT1 ABCA1 ABCG1 ApoE ApoAIs ABCA1 ApoE LCAT ApoAIs ABCG1 ApoE LCAT SR-BI HL Hepatocyte Forming HDL Mature HDL Figure 1. Synopsis of HDL metabolism and targets of therapy. Interventions on Stage I aim at activating mechanisms of cholesterol exit. Only minor changes in plasma HDLc levels are expected. Interventions on Stage II include maneuvers that improve HDL maturation and/or improve HDL’s anti-oxidant and anti-inflammatory effects The influence of these interventions on plasma HDLc levels may vary from increases to decreases. Interventions on Stage III include maneuvers that block or facilitate cholesterol loss from HDL, causing large increases or decreases, respectively, in plasma HDLc levels. LXR: liver X receptor, master regulator of cholesterol exit from the cell. ABCA1, ABCG1: ATP-dependent binding cassettes A1 and G1, major transporters of cellular cholesterol to apoAI and HDL, respectively (LXR sensitive genes). ACAT1: acyl:CoA-cholesterol acyltransferase, catalyzes formation of cholesteryl esters and reduces the pool of free cholesterol available for export. ApoE: apolipoprotein E, produced by the macrophage in response to cholesterol loading (LXR sensitive gene); similarly to apoAI, apoE can act as cholesterol acceptor. ApoAIs: this term is meant to represent a variety of interventions aimed at raising endogenous production of apoAI (LXR sensitive gene) or at introducing apoAI mutants or mimetics as agents of increased HDL functionality. LCAT: lecithin-cholesterol acyltransferase, esterifies cholesterol on HDL, expanding its core. SR-BI: scavenger receptor type BI, also known as the HDL receptor, captures mature HDL and facilitates transfer of cholesterol cargo into the liver. HL: hepatic lipase, removes triglycerides from HDL before they can be recognized by SR-BI. CETP: cholesteryl ester transfer protein, facilitates trasnfer of cholesterol from HDL to apoB-containing lipoproteins. CETP Peripheral Cell Stage I: Intracellular Cholesterol Trafficking ApoB-Lipoproteins Stage II: Cholesterol Acquisition and HDL Maturation Stage III: Cholesterol Exchange and Delivery

Genetic Studies Do Not Support the HDL Hypothesis ABCA1 mutations causing low HDL are not associated with increased CV risk Some apoAI mutations causing low HDL are beneficial for the plaque HL mutations raising HDL are not protective CETP mutations raising HDL may increase CV risk

IDEAL and EPIC-Norfolk 1. In IDEAL, after adjustment for apoAI, the risk of a major coronary event doubled in subjects with HDLc>70 versus those with HDLc<59 mg/dl 2. In EPIC-Norfolk, after adjustment for apoAI, the risk of a MCE tripled in subjects with HDL>10 nm versus those with HDL<9nm JACC, February 2008

The MESA Study KE Watson et al. Circulation. 2007;116:II_540

ILLUSTRATE Trial Statin Statin/Torcetrapib No. of Patients 446 464 Total-C mg/dL 157 + 31 167 + 37 LDL-C mg/dL 87 + 22 70 + 25 HDL-C mg/dL 43 + 12 72 + 24 Nissen SE et al; NEJM 2007; 356 (13): 1304-16

LDL and Atheroma Volume in IVUS Trials Nissen SE et al; NEJM 2007; 356 (13): 1304-16

S. B. At 51 she has an MI. The LAD is completely occluded, the RCA is At 50 she visits PCP: Fx: Father MI, CABG @ 47 Brother MI, CABG @ 47 She stopped smoking 3 yrs ago HTN for 3 years, controlled Premarin for 8 yrs No CVD symptoms or diagnoses Was a cheerleader in HS and College Exercises regularly Glucose 78 Nl LFT, TFT, KFT TG 90 LDL 104 HDL 118 At 51 she has an MI. The LAD is completely occluded, the RCA is 68% occluded

The Dysfunctional HDL 1. Cholesterol extraction from peripheral cells 2. Lipid exchange and maturation 3. Cholesterol delivery to the hepatocyte 4. Abnormal lipid or protein composition 5. Anti-inflammatory activity 6. Anti-oxidant activity

The Dysfunctional HDL 1. HDL of patients with CHD are pro-inflammatory 2. Statin therapy reduces but does not correct the pro-inflammatory index of HDL 3. Pro-oxidant HDL in SLE and RA 4. No strong correlation between plasma HDL cholesterol levels and functionality

Serum Cholesterol Efflux Capacity Protocol (Rothblat-Rader) 3H-Cholesterol labeled + ACAT Inhibitor J774 MACROPHAGES apoB-depleted sera % FC Efflux cAMP + 0.2% BSA AHA Scientific Sessions| Amit Khera | 11.17.2009

Cholesterol Efflux Capacity in Coronary Artery Disease Patients 393 CAD patients, 302 controls Serum efflux capacity was an independent predictor of coronary artery disease status even after inclusion of HDL-C into the model. HDL quality provides incremental information on cardiovascular risk. Khera AV, et al. Circulation 2009;120:S469.

Uremic HDL Increases Macrophage Synthesis of IL-1b, IL-6, and TNF-a 10 20 30 40 C (n=3) P (n=5) IL-6 TNF-a 10 20 30 40 C (n=3) P (n=7) 5 10 15 20 C (n=3) P (n=7) ApoE-/- peritoneal macrophages preloaded with acLDL and exposed to 50 ug/ml of HDL and 50 ng/ml of LPS for 4 hrs . mRNA expression by real-time PCR Yamamoto et al. Unpublished 21

Summary Increasing plasma HDL levels is a strategy supported by current guidelines,but direct correlation between HDLc levels and RCT efficiency has not been demonstrated Improving RCT may have therapeutic value even in the absence of measurable plasma HDL effects Diagnostic tests for HDL functionality may target cholesterol trafficking or other processes (oxidation, inflammation) An ideal test is automated, scalable, and priced within the range of common biochemistries