Introduction

High-density lipoprotein cholesterol (HDL-C) has long been known to the medical community and beyond as "good cholesterol," with numerous studies showing an inverse association between HDL-C and the risk of atherosclerotic cardiovascular disease (ASCVD). Accordingly, HDL-C has been hypothesized to have a causal relationship with ASCVD, with higher levels assumed to be atheroprotective. However, more recent research has cast doubt on the HDL-C hypothesis, as the absolute level of HDL-C may be of less clinical significance compared to its functional capacity. Specifically, the cholesterol efflux capacity (CEC) of HDL particles is a measurable variable that has received attention due to its potential protective role to the pathogenesis of ASCVD. The goal of this brief review is to discuss the scientific rationale regarding CEC and its association with ASCVD risk as well as the potential to modify CEC, allowing it to serve as a therapeutic target to reduce the burden of ASCVD.

Potential Flaws in the HDL-C Hypothesis

Low HDL-C levels are often associated with the metabolic syndrome phenotype, which includes elevated triglycerides, elevated blood pressure, and increased waist circumference, all known risk factors for ASCVD. Additionally, a Mendelian randomization study demonstrated that individuals with genetically elevated HDL-C did not have a decreased risk of myocardial infarction as would be expected based on epidemiologic data.¹ A study applying the Systemic Coronary Risk Evaluation (SCORE) ASCVD risk prediction model to the Copenhagen General Population study showed that incorporating HDL-C levels into the SCORE model did not improve discrimination in predicting future fatal ASVCD.² Finally, randomized trials studying pharmacologic agents that increase HDL-C have not demonstrated a decrease in the rate of ASCVD events. Two large trials have shown no ASCVD benefit with use of niacin,^3,4 and, despite a 72% increase in HDL-C, a randomized trial evaluating a cholesteryl ester transfer protein inhibitor showed an unexpected increased risk for cardiovascular events (hazard ratio, 1.25; 95% confidence interval [CI], 1.09 to 1.44; P=0.001).⁵

The Function of HDL-C and Measurement CEC

The primary function of the HDL particle appears to be its involvement in reverse cholesterol transport, taking cholesterol from the vascular system and transporting it to the liver for biliary excretion. The initial step in reverse cholesterol transport involves efflux of cholesterol from macrophages in the arterial wall, which is mediated by HDL particles.⁶ While cholesterol efflux capacity is thought to be responsible for a minority of the overall reverse cholesterol transport process, it is likely important in atheroprotection and thus potentially in clinical prognosis.⁷

Cholesterol efflux is also thought have other atheroprotective functions. Cholesterol efflux has been shown to protect macrophages from low-density lipoprotein-induced apoptosis and to enhance endothelial function by activating nitric oxide synthase to promote endothelial repair and induce angiogenesis.^8-10 Decreased cholesterol efflux capacity, on the other hand, has been shown to correlate with increased platelet reactivity in vitro.⁹

While there is no industry standard method to measure the cholesterol efflux capacity, most studies have used similar methods to the protocol described by de la Llera-Moya et alusing J774 macrophages, apolipoprotein B-depleted serum and patient serum.¹⁰ However, a fluorescence labeled reagent (BODIPY cholesterol) enables easier testing of a large number of samples.¹¹ These values only moderately correlated (R=0.54) with the "traditional" radio-labeled method. However, the BODIPY cholesterol may be a more important measurement of cholesterol efflux as it primarily measures ATP-Binding Cassette Transporter A1 (ABCA1) mediated efflux, the lone transporter in macrophage-specific efflux in people with similar HDL-C levels.

Clinical Studies Analyzing the Relationship of CEC to ASCVD

In 2011, Khera et al studied 203 healthy non-smoking white subjects without coronary artery disease or diabetes to determine the association between HDL-C related biomarkers and subclinical atherosclerosis as measured by carotid intima-media thickness.¹² They found no significant relationship between HDL-C and carotid intima-media thickness in both unadjusted and adjusted models (P=0.37 and P=0.73 respectively). However, a significant inverse relationship was seen between CEC and carotid intima-media thickness (-0.04 Beta Coefficient per 1-SD increase, 95% CI -0.06 to -0.01, P value 0.005). In a second population, 442 white patients determined to have coronary artery disease (CAD) as defined by luminal stenosis of more than 50% by coronary angiography were compared with 351 white control patients without evidence of coronary artery disease by angiography or with previous history of MI. They found that the highest quartile of efflux capacity was associated with a significantly lower risk of CAD compared to the lowest quartile (odds ratio, 0.38; 95% CI, 0.25 to 0.58; P<0.001).¹²

In 2013, Li et al explored the relationship between CEC and CAD in two case-control cohorts, one cohort of 1,150 patients undergoing coronary angiography and a second cohort of 577 patients visiting outpatient clinics.¹³ These studies found an inverse relationship between CEC and CAD at baseline of the study, but this relationship only remained significant in the adjusted model for the outpatient cohort. Paradoxically, higher CEC appeared to be associated with a higher risk of incident myocardial infarction and stroke over three years of follow-up. However, it should be noted that the control group was significantly older than those with CAD (mean age 72 years compared to 61 years) and therefore the results may be confounded by age.

In 2014, Rohatgi et al studied CEC in 2,416 Dallas Heart Study patients free from cardiovascular disease at baseline over a median follow-up period of 9.4 years.¹⁴ This study assessed CEC using the novel BODIPY cholesterol method. They found that increasing levels of CEC were associated with a decrease in risk for ASCVD events even after adjustment for traditional risk (hazard ratio for the fourth vs. first quartiles of CEC, 0.33; 95% CI, 0.19 to 0.55). Additionally, increasing quartiles of efflux capacity were not associated with traditional cardiovascular risk factors (other than lipid levels) nor with prevalent coronary artery calcium.

A more recent case-control analysis from the EPIC-Norfolk study analyzed 1,745 individuals,¹⁵ aged 40-79 years and free of ASCVD at baseline with an incident CAD event over ~15 years of follow-up compared to 1,749 age and sex matched individuals who remained free from cardiovascular disease at final follow-up. This study again demonstrated a non-significant relationship between HDL-C and ASCVD after adjusting for traditional risk factors and CEC. However, there was a significant inverse association between CEC and ASCVD, even after multivariable adjustment (Odds Ratio 0.80; 95% CI, 0.70–0.90).

In 2015, Zhang et al studied 313 patients newly diagnosed with CAD (confirmed by coronary angiography; 98 with myocardial infarction, 116 with unstable angina pectoris, 99 with stable angina pectoris) without previous lipid-lowering medications.¹⁶ The control group was 116 individuals with no evidence of CAD on angiography. Cholesterol efflux capacity measured at the time of enrollment was found to be highest in the control group, lower in the stable angina pectoris group, and lowest in the ACS group. In the subsequent three years of follow up, after multivariable adjustment, lower CEC was associated with increased risk of MI, stroke and cardiovascular mortality.

Is CEC a Potential Therapeutic Target?

While CEC levels appear to have an inverse association with ASCVD risk based on observational data, research is now needed to determine methods to modify CEC and, more importantly, determine if such modification actually impacts ASCVD outcomes. Certain hypotheses, like increasing pre-B-HDL (the major ligand of the ABCA1 transporter that drives CEC) have already been tested with mixed results.¹⁷ Two trials, the Effect of rHDL on Atherosclerosis-Safety and Efficacy (ERASE) Trial and Selective Delipidation Trial demonstrated that increased pre-B-HDL (by infusion and delipidation, respectively) reduced coronary atherosclerosis confirmed by intravascular ultrasound.^18,19 However, there have not yet been further studies evaluating clinical outcomes.

Another potential target is apolipoprotein A-I, a protein found on HDL molecules. As a sub-study of the 2011 CEC study above, Khera et al. measured CEC after treatment with pioglitazone using samples from a randomized control trial previously completed by Szapari et al.^12,20 Khera et al. showed that treatment with pioglitazone for 12 weeks resulted in a significant increase in efflux capacity compared with placebo (11.8% change, 95% CI 1.8-20.8). This may be due to enhanced transcription of apolipoprotein A-I, thereby increasing the efficiency of the ABCA1 pathway.

Fenofibrate has been shown to be a PPAR-a agonist similar to pioglitazone and was used in a randomized control study by Franceschini et al that showed significantly enhanced cholesterol efflux after 8 weeks.²¹ Later Khera et al. separately investigated a more potent and specific PPAR-a agonist for 8 weeks in a randomized control trial that again showed increased cholesterol efflux from macrophages without any change in the overall HDL-C levels.²²

While these methods do show promise by up-regulating cholesterol efflux capacity, more investigation is needed as to whether cholesterol efflux capacity actually impacts hard ASCVD outcomes.

Conclusions

Recent research has demonstrated that CEC may play an important protective role in atherogenesis and clinical ASCVD. It appears that the CEC of HDL particles may be more important than the absolute HDL-C level for atheroprotection. This finding may help explain the overall negative results seen in the trials of pharmacologic agents aimed at increasing the absolute level of HDL-C. Multiple studies have demonstrated that CEC inversely correlates with adverse ASCVD events, even after adjustment for traditional cardiovascular risk factors, including HDL-C. Although CEC has shown promise as a biomarker, more studies are needed to determine if CEC has any clinical utility as a prognostic tool or as a therapeutic target to reduce ASCVD.

References

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Keywords: Angina, Stable, Angina, Unstable, Apolipoprotein A-I, Apoptosis, Atherosclerosis, Biomarkers, Blood Pressure, Cardiovascular Diseases, Cholesterol Ester Transfer Proteins, Cholesterol, HDL, Constriction, Pathologic, Coronary Angiography, Coronary Artery Disease, Diabetes Mellitus, Lipoproteins, HDL, Lipoproteins, LDL, Metabolic Syndrome, Myocardial Infarction, Niacin, Nitric Oxide Synthase, Peroxisome Proliferator-Activated Receptors, Risk Factors, Stroke, Thiazolidinediones, Triglycerides, Primary Prevention, Secondary Prevention

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