HDL Efflux Capacity and Incident Cardiovascular Events in the Dallas Heart Study A Rohatgi,* A Khera, JD Berry, EG Givens, CR Ayers, KE Wedin, IJ Neeland, IS Yuhanna, DJ Rader, JA de Lemos, PW Shaul *corresponding author Donald W. Reynolds Cardiovascular Clinical Research Center

HDL-C ≠ CHD 25% Drug Class Trial HDL-C change NIACIN AIM-HIGH 25% Placebo Drug Class Trial HDL-C change NIACIN AIM-HIGH 25% HPS2-THRIVE 15% CETPI Torcetrapib 72% Dalcetrapib 35% Niaspan 20% CETPI AIM-HIGH 15% HPS2 10% Dalcetrapib Torcetrapib 5% A H T D 0% HPS2-THRIVE. NEJM 2014;371:203-12 1 2 3 4 Schwartz GG, et al. NEJM 2012;367:2089-99 Years Boden WE, et al. NEJM 2011;365:2255-67 Barter PJ, et al. NEJM 2007;357:2109-22

HDL Function: Reverse Cholesterol Transport apoA-I Liver apoA-I ABCA1 Cholesterol efflux ABCA1 SRB1 apoA-I ABCG1 Macrophage VLDL Bile/Feces Adorni MP, et al. J Lipid Res 2007;48:2453-62 Khera AV, et al. NEJM 2011;364:127-35 Rader DJ, Hovingh GK. Lancet 2014;384:618-625

Study Objective Objective: Hypothesis: Perform a systematic evaluation of the epidemiology of cholesterol efflux capacity in a large unselected population free of cardiovascular disease . Hypothesis: Cholesterol efflux capacity varies by sex, race and adiposity and is associated with incident cardiovascular events

Methods Dallas Heart Study Baseline N = 2924 Age = 30-65 (median 42) Cholesterol Efflux Capacity Baseline Dallas Heart Study ACAT inhibitor cAMP Metabolic phenotype Cholesterol Efflux Capacity Lipoprotein composition 9.4 years N = 2924 Age = 30-65 (median 42) 57% Women 49% Black Follow up ApoB-depleted plasma ASCVD (n=132): MI/Stroke PCI/CABG CV Death % efflux Total Normalized efflux % effluxsample % effluxreference Sankaranarayanan S, et al. J Lipid Res 2011;52:2332-40

Cholesterol Efflux Capacity HDL-C and Efflux by Sex and Race   N HDL Cholesterol (mg/dL) Cholesterol Efflux Capacity (Normalized) Women 1657 51 [43-61] 0.99 [0.83-1.18] Men 1267 44 [37-52] 1.01 [0.84-1.20] p-value <0.0001 0.1 Black 1443 49 [42-60] 0.98 [0.81-1.18] Non-Black 1481 45 [38-55] 1.01 [0.85-1.19] 0.002 Cholesterol efflux capacity is expressed as a ratio of percent cholesterol efflux of the sample normalized to the percent cholesterol efflux of a reference sample.

Determinants of HDL-C and Efflux HDL Cholesterol   Cholesterol Efflux Capacity Model R2 = 0.35 Model R2 = 0.03 Variable Std beta estimate Total cholesterol 0.22 0.12 Alcohol intake 0.18 HDL-C 0.09 Age 0.13 Male sex 0.05 Exercise dose 0.08 Hypertension 0.04 Black race 0.07 0.03 Smoking -0.06 -0.07 Waist to hip ratio -0.08 Body mass index -0.17 -0.23 Log Triglyceride -0.39 Model R2 reflects the total contribution of the model to the variance in the dependent variable (range 0.0-1.0).

Correlations with Lipoprotein/Metabolic Variables   HDL Cholesterol Cholesterol Efflux Capacity 0.07* HDL Particle concentration 0.52* 0.15* HDL Particle size 0.72* 0.02 Total Cholesterol 0.06* LDL Cholesterol -0.15* 0.10* Triglyceride -0.45* 0.05* Body Mass Index -0.23* -0.02 Waist to Hip Ratio -0.38* Truncal Fat (Dexa) -0.20* 0.004 Visceral Fat (MRI) -0.41* 0.03 HOMA-IR -0.29* -0.05* HDL variables Lipids Adiposity Insulin Res Values are Spearman correlation coefficients. * p<0.05, otherwise p=NS CEC=cholesterol efflux capacity; HOMA-IR: Homeostatic model assessment

Cholesterol Efflux Capacity and ASCVD Events Low efflux Hi efflux 10% Q1 Q2 Q3 Q4 8% ASCVD Events: MI 6% Stroke PCI/CABG CV death 4% N=132 Log rank p=0.002 2% 0% 1 2 3 4 5 6 7 8 9 Years ASCVD: atherosclerotic cardiovascular disease

HDL-C, Efflux Capacity and ASCVD Events HDL cholesterol Hazard Ratio Q4 vs Q1 (95% CI) Unadjusted 0.64 (0.40-1.03) TRF-adjusted 0.80 (0.47-1.37) TRF + HDL-P 1.08 (0.59-1.99) Cholesterol Efflux Capacity Unadjusted 0.44 (0.27-0.73) TRF-adjusted 0.30 (0.18-0.50) TRF + HDL-C 0.31 (0.18-0.52) TRF + HDL-P 0.34 (0.20-0.56) TRF + HDL-C + HDL-P 0.33 (0.19-0.55) 0.1 1.0 10 Hazard Ratio N=132/2416 for atherosclerotic cardiovascular disease events. Traditional risk factors (TRF) = age, sex, ethnicity, diabetes, hypertension, current smoking, body- mass index, total cholesterol, log triglycerides, statin use. HDL-C = HDL cholesterol. HDL-P = HDL particle concentration

Efflux Capacity and Hard ASCVD Events Primary ASCVD 132/2416 Hazard Ratio (95% CI) Q1 Referent Q2 0.71 [0.46-1.10] Q3 0.42 [0.26-0.68] Q4 0.33 [0.19-0.55] Hard ASCVD 84/2402 Q1 Referent Q2 0.69 [0.39-1.21] Q3 0.46 [0.25-0.84] Q4 0.40 [0.21-0.74] 0.1 1.0 10 Hazard Ratio Primary ASCVD: MI, stroke, coronary revascularization, and CV death Hard ASCVD: MI, stroke, death from MI or stroke. Models adjusted for age, sex, ethnicity, diabetes, hypertension, current smoking, BMI, total cholesterol, log triglycerides, statin use, HDL-C, and HDL-P.

Continuous Efflux Capacity and Cardiovascular Events Model Primary ASCVD (N=132/2416) Total CVD (N=172/2416) HR (95% CI) Unadjusted 0.75 (0.61-0.91) 0.84 (0.71-0.98) TRF-adjusted 0.65 (0.53-0.80) 0.75 (0.63-0.89) TRF + HDL-C 0.66 (0.54-0.81) 0.77 (0.64-0.91) TRF + HDL-P 0.69 (0.56-0.84) 0.79 (0.67-0.94) TRF + HDL-C + HDL-P 0.68 (0.55-0.84) Hazard ratios and 95% CIs for 1 SD increase in efflux capacity. Primary ASCVD: MI, stroke, coronary revascularization, and CV death Total CVD: ASCVD + peripheral revascularization, CHF and afib hospitalization

Efflux Capacity Improves Risk Prediction of ASCVD Events Model C-statistic Integrated Discrimination Improvement (IDI) Net Reclassification Index (NRI) Traditional Risk Factors 0.827 0.02 0.37 Traditional Risk Factors + Efflux capacity 0.841 P value/ 95% CI p=0.02 p=0.0008 (0.18-0.56) Traditional Risk Factors: age, sex, ethnicity, diabetes, hypertension, smoking, BMI, total cholesterol, log triglyceride, statin use

Summary In contrast to HDL cholesterol, cholesterol efflux capacity (CEC) is minimally correlated with risk factors, lipoproteins, adiposity, or insulin resistance. CEC is inversely associated with incident atherosclerotic CV events in a population-based study free of CVD This association is not attenuated by traditional risk factors, HDL cholesterol, or HDL particle concentration CEC, as a measure of reverse cholesterol transport, may provide the ability to interrogate key mechanisms related to cardiovascular disease in humans

Other Disease Pathways Implications HDL Cholesterol HDL function Risk Prediction Coronary Disease Free Stable ACS Therapeutic Response LDL cholesterol Non-HDL cholesterol HDL cholesterol HDL Function/Flux Other Disease Pathways