High-Density Lipoprotein (HDL) Phospholipid Content and Cholesterol Efflux Capacity Are Reduced in Patients With Very High HDL Cholesterol and Coronary Disease

Plasma high-density lipoprotein cholesterol (HDL-C) levels are strongly inversely correlated with the incidence of coronary artery disease (CAD).¹ It has been estimated that for each milligrams per deciliter increase in HDL-C, the risk of cardiovascular events is decreased by 2% to 3%.² Consequently, levels of HDL-C are factored into many cardiovascular risk assessments, and HDL has been intensively pursued as a secondary goal for risk reduction after low-density lipoprotein cholesterol (LDL-C) lowering. The belief that levels of HDL-C have a causal relationship to the prevention of CAD has been referred to as the HDL cholesterol hypothesis.³

There have been recent challenges to the HDL-C hypothesis. Common variations associated with small changes in HDL levels are not associated with protection from coronary disease, in contrast to variants that affect LDL-C and triglycerides.^4,5 Recently, several clinical trials using agents that raise HDL-C have failed to show any clinical benefit. In the dal-OUTCOMES trial of the cholesteryl ester transfer protein inhibitor dalcetrapib, patients received dalcetrapib in addition to other agents that lower LDL-C. Although a significant elevation in HDL-C levels was noted in patients treated with dalcetrapib, the trial was terminated because of futility of the study.⁶ The HPS2-THRIVE trial was designed to assess cardiovascular outcomes in patients treated with extended release niacin and laropiprant, an antiflushing agent, in addition to a statin. However, HPS2-THRIVE missed its primary end point of reducing the risk of myocardial infarction, stroke, or coronary revascularizations compared with statin therapy alone.⁷ These studies have fueled the debate about a causal role of HDL-C in heart disease, and whether raising HDL-C levels is a viable therapeutic strategy.

HDL has several properties that may offer protection against CAD, including its role in promoting cholesterol efflux and reverse cholesterol transport.⁸ Genetic and pharmacological manipulations of HDL that increase reverse cholesterol transport in animal models are generally protective against atherosclerosis.⁹ However, HDL-C concentration does not always reflect its functionality. For example, even after controlling for HDL-C the cholesterol efflux capacity of HDL was inversely associated with prevalent carotid and coronary atherosclerosis¹⁰ and with incident cardiovascular events.¹¹

Extremely high HDL-C levels are generally associated with reduced risk of CAD. However, an unusual phenotype is that of very high HDL-C with development of CAD in the absence of traditional risk factors. We hypothesized that these individuals have altered composition and reduced function of their HDL that may predispose them to increased risk of CAD. We systematically recruited individuals with very high HDL both with and without CAD and compared the composition and function of HDL. We found that the HDL from high HDL-C subjects with CAD had reduced phospholipid content and reduced cholesterol efflux capacity when compared with the HDL from high HDL-C subjects without CAD.

Materials and Methods are available in the online-only Data Supplement.

In this study, we investigated a paradoxical phenotype, extremely high HDL-C associated with CAD. We hypothesized that individuals with this phenotype have altered HDL composition and function that put them at greater risk for CAD in the setting of high HDL-C. We found that individuals with very high HDL-C and CAD have reduced levels of HDL phospholipids and reduced HDL cholesterol efflux capacity. These findings add to the growing body of data linking HDL composition and function to clinical cardiovascular disease as distinct from HDL-C concentrations.

Cholesterol efflux is the first step of the reverse cholesterol pathway that can be assessed ex vivo by a method first developed by Rothblat and colleagues.¹² Khera et al¹⁰ demonstrated that HDL cholesterol efflux capacity was inversely associated with prevalent carotid and coronary atherosclerosis even after adjusting for HDL-C, a finding confirmed by Li et al.¹³ Recently, Rohatgi et al¹¹ showed that HDL efflux capacity was inversely associated with incident cardiovascular events after adjusting for HDL-C. Similarly, Hafiane et al¹⁴ and Shao et al¹⁵ have demonstrated impaired cholesterol efflux capacity in acute coronary syndrome. In contrast, Li et al¹³ reported a positive association of cholesterol efflux capacity with incident cardiovascular events in an angiographic cohort. Thus, there remain questions about the relationship of efflux capacity to cardiovascular disease.

The HDL from subjects with high HDL-C and CAD was reduced in phospholipid content. An inverse relationship has been noted between HDL-PL and the CAD.¹⁶ Furthermore, HDL-PL composition has been positively associated with cholesterol efflux capacity.^17–21 In our study, HDL-PL was a significant predictor of total cholesterol efflux capacity. Taken together, the reduction in HDL-PL levels may play a causative role in the reduced cholesterol efflux capacity of HDL. The reduced HDL-PL was not associated with a difference in HDL subfraction particle numbers or size. A more thorough lipidomic analysis of the HDL particles is of interest but outside the scope of this study.

Lecithin-cholesterol acyltransferase hydrolyzes HDL phospholipids and could influence HDL-PL content as well as potentially cholesterol efflux capacity. We measured the cholesterol esterification rate as an assay of endogenous lecithin-cholesterol acyltransferase that is also influenced by the endogenous lipoproteins, but found no evidence of a difference between cases and controls. Prior studies have demonstrated an inverse association between phospholipid transfer protein activity and cholesterol efflux capacity.²² We measured phospholipid transfer protein activity and found no significant differences between the 2 groups. One limitation inherent in the phospholipid transfer protein activity assay is that it measures transfer between synthetic donor and acceptor particles, rather than between native lipoproteins where lipid and protein composition may influence activity. It is possible that increased phospholipase activity (ie, hepatic and endothelial lipase) may underlie the lower phospholipid content in the HDL of these patients, but measurement of these lipases requires postheparin plasma, which was not available.

A limitation of this study is incomplete data on medical history (such as date of onset of CAD) and lack of prospective data. To minimize this problem, we selected controls that were either the same age or older than the cases. Furthermore, prior studies have demonstrated that CAD can reduce cholesterol efflux capacity.^14,15 Another limitation in our study is the assessment of only one of the major functions of HDL. HDL is a heterogeneous particle that has additional antioxidant and anti-inflammatory functions, as well as unknown differences in lipidomic and proteomic compositions between the cases and controls, which when all considered together may give a more comprehensive picture of HDL functionality.

The significant differences in LDL-C and apoB levels between the cases and controls are attributed to higher use of statins in the cases than in the controls. Of the subjects who provided information on statin usage, 80% of the cases and 32% of controls reported being on statins. Recent studies have shown conflicting data on the effect of statins on cholesterol efflux capacity making it difficult to assess the nature of their effect on our study population.^10,23–26 However, it is unlikely that statins from the polyethylene glycol precipitated supernatants would have a substantial effect on the cells during a 2-hour efflux assay. Recently, Miyamoto-Sasaki et al²⁷ determined cholesterol efflux capacity in dyslipidemic patients before and after treatment with pitavastatin. The statin increased serum HDL-C levels, HDL-PL levels, and enhanced cholesterol efflux capacity, suggesting it is unlikely that statins decreased the HDL-PL content and efflux capacity of the cases. In fact, it is possible that the observed differences may actually have been greater if measurements were taken before statin therapy.

In conclusion, individuals with the paradoxical phenotype of very high HDL-C and CAD were found to have reduced HDL phospholipid and HDL cholesterol efflux capacity as compared with controls with very high HDL-C and no CAD.

We would like to thank Phyllis May and Debra Cromley for their valuable technical assistance as well as Dawn Marchadier, Megan Mucksavage, and Stephanie DerOhannessian for their help with the project and Wiliam Lagor for his suggestions on the manuscript.