High-density lipoprotein (HDL) is a spherical complex made up of a heterogenous array of proteins and lipids, as well as small RNA molecules (1–4). Throughout its lifecycle, HDL undergoes continuous recruitment, assembly, delivery and turnover of its various protein and lipid components (5). With such a diversity of cargo, HDL is believed to have functions in numerous biological processes, such as in oxidation, inflammation and diabetes (3). HDL also removes cholesterol deposited in the arterial wall by LDL by both active and passive efflux processes that are commonly measured as its cholesterol efflux capacity (CEC). Subsequently, cholesterol on HDL is either directly taken up by the liver or is indirectly removed by the liver after being transferred to LDL by cholesteryl-ester transfer protein (CETP) (6). Ultimately, cholesterol delivered to the liver is either reutilized or is excreted into the intestine in the bile either as free cholesterol or as bile salt after conversion. The final subsequent removal of cholesterol from the body completes a process called Reverse Cholesterol Transport (RCT) by John Glomset in the late 1960s (3, 7). Given that many of the individual steps in RCT are potentially atheroprotective, and supported by numerous epidemiologic studies demonstrating an inverse-relationship between HDL-C levels and the risk of developing coronary heart disease, cholesterol on HDL became commonly referred to as ‘good’ cholesterol (8, 9). Based on this evidence, numerous HDL level-boosting agents were tested therapeutically as an approach to reduce cardiovascular disease (CVD) [summarized in (5, 9)].

Despite the seeming biologic plausibility of the atheroprotective role of RCT, none of HDL- based therapies tested to date have translated to human benefit (5, 10). The most recent clinical trial (AEGIS-II) infused CSL112 (reconstituted HDL) or placebo control in a total of 18,219 patients with acute myocardial infarction, coronary artery disease and other cardiovascular risk factors with the 90-day composite end point being either myocardial infarction, stroke or death (11). CSL112 was formulated with ApoA-1 purified from plasma and combined with phospholipid to form a reconstituted form of HDL, which both in vitro and in vivo studies was demonstrated to increase RCT (11, 12). Unfortunately, this clinical trial found no significant differences in the primary outcome measures for cardiovascular events between the CSL112 and the placebo treated groups (11). While these accumulating trial failures have diminished enthusiasm for HDL-raising agents as a CVD therapeutic target, a number of possible explanations have been proposed for these negative findings, which should be addressed before the RCT hypothesis is abandoned (13, 14).

One important limitation of the AEGIS-II study is that patients lacked both baseline and follow-up measurements of CEC (11, 13). Though this information would have been valuable for this study, the lack of suitable technology for performing high-throughput large-scale measurements of CEC made this unfeasible. However, recent reports have described new approaches for performing CEC assays, which may point to a future where CEC measurements are a routine diagnostic tool in the clinical laboratory. This review provides a brief history on the variety of methods used to assess RCT in the laboratory (Section 1) and the new cell-free approaches (Section 4). We also provide an overview of how HDL function is affected by many human diseases and disorders (Section 2), in response to therapeutic intervention (Section 3) and the HDL proteome (Section 5).

RCT was first studied in human and animal in vivo studies via compartmental modeling of the distribution of i.v. injected radiocholesterol in plasma lipoproteins, red blood cells and the liver (15). Subsequently models involving cholesterol RCT measurement of 3H-cholesterol or cholesterol mass changes in macrophages implanted i.p. in mice were developed and provided evidence for HDL-mediated RCT from macrophages in vivo (16, 17).

It has been almost fifteen years since the first cell-based assays were developed to quantify the initial step of RCT (18, 19) (Figure 1), namely CEC. These early CEC assays of HDL function typically utilize mouse macrophage J774, Raw 264.7 cells or human Thp-1 cells, which are either radiolabeled with 3H- or fluorescently-labeled with BODIPY fluorophore (dipyrrometheneboron difluoride) and then incubated with apoB-depleted sera for a short time period, typically 4 h (Figure 1A). Additionally, specific CEC pathways related to ABCA1, ABCG1 and SRB1 may be revealed by CEC assays in Baby Hamster Kidney (BHK), Chinese Hamster Ovary (CHO) or HeLa cell lines with stable expression of these proteins that also affect the efflux of lipids from cells (20). An index of CEC is then calculated either by using a ratio to a pool of healthy control subjects or a percentage apoB-depleted serum of a cellular total of cholesterol label (21, 22).

Detailed methodological aspects of the cell-based assays are thoroughly discussed elsewhere (23, 24). It should be noted that these assays are technically challenging. They are not only affected by cell-density but also cell adherence, which given the semi-adherent nature of J774, Raw 264.7 and Thp-1 cells make experimental reproducibility a problem (24). The relatively large assay-to-assay variability may best be mitigated by comparison of a control pool of healthy subjects with each test sample. Another alternative is to measure CEC in radiolabeled cells to purified HDL, isolated by either ultracentrifugation or size-exclusion chromatography (25, 26), which is perhaps a more accurate measure of CEC but is time-consuming.

Numerous reports have shown that systemic and vascular inflammatory states can impair RCT via perturbed CEC function, which consequently promotes plaque progression (27, 28). The inter-relationship between RCT and both inflammatory and non-inflammatory states is highlighted by a detailed evaluation of the effect of human diseases and disorders on RCT (Table 1).

In brief, nearly 40 published articles, covering almost 30 different human conditions, have shown that HDL can become dysfunctional leading to decreased CEC. In the following few paragraphs, we highlight the several examples of this by describing HDL function in Inflammatory diseases, Atherosclerotic Cardiovascular Disease, Familial Hypercholesterolemia, Diabetes and Chronic Kidney Disease.

The number of human studies reporting changes in CEC upon therapeutic intervention remains relatively small and mostly been described in inflammatory states. We mostly discuss here changes in CEC upon anti-inflammatory treatments of two autoimmune diseases, namely psoriasis and SLE, but the effect of a therapeutic intervention on CEC has been examined for several other diseases and conditions including Familial Hypercholesterolemia and Rheumatoid Arthritis (77–81).

Recently, several cell-free assays that closely correlate to the classic CEC assays done with cells for measuring HDL function have been developed, which vary in their potential for translation into a routine diagnostic test (90). Several cell-free HDL functionality assays measure CEC (91) or cholesterol uptake capacity (CUC) (92–94) by monitoring the efflux of fluorescent analogs of cholesterol from a lipid-coated substrate to apoB-depleted serum (Figures 1B,C). Recently, a fully automated immunoassay for CUC to assess HDL function and CVD risk has been developed (95). The latter assay appears to be promising, however, it relies on a monoclonal apoA-I antibody, which potentially introduces a selection bias as it does not capture non-apoA-I-containing HDL particles that also likely modulate CUC. Nevertheless, the rapid, high-throughput fully-automated CUC immunoassay, which highly correlates with CEC [81], appears to be a potential suitable surrogate assay for CEC.

Other means of HDL functionality assessment involve measurement of the rate of exogenous apoA-I exchange onto serum HDL by electron paramagnetic resonance (96, 97) or by using NBD/Alexa647 double-labeled ApoA-I, whose NBD/Alexa647 emission ratio increased upon exchange into HDL (98, 99) (Figures 1D,E). Several exchangeable plasma HDL apolipoproteins have been shown, however, to mediate both cellular cholesterol and phospholipid efflux (100). Since the aforementioned ApoA-I exchange assays use only exogenous ApoA-I as a probe, they are not able to fully assess endogenous exchangeable HDL apolipoprotein functionality.

We have developed a relatively simple and sensitive cell-free, HDL-specific phospholipid efflux (HDL-SPE) assay that is based on active solubilization of a non-exchangeable, head group-tagged fluorescent phosphatidylethanolamine (PE) from donor particles by endogenous exchangeable plasma HDL-derived apolipoproteins (ApoA-I, ApoA-II, ApoA-IV and ApoC-III) [90]. Whole sample plasma or serum and a pooled human reference plasma, are incubated with the fluoresent PE donor particles in 96-well plates for I hr at 37 ^o C with shaking (Figure 1F). The plates are centrifuged to pellet the dense donor lipid particles and supernatant HDL-associated PE fluorescence is measured and normalized to the reference plasma fluorescence [90]. HDL-SPE predicts incident CVD independent of traditional risk factors better than HDL-C and ApoA-I and is amenable for automation into a clinical diagnostic test (101).

The functionalities of HDL that underlies this assay includes the ability of the HDL-associated exchangeable lipoproteins to (i) dissociate from HDL particles (ii) bind to the surface of the donor lipid particle (iii) solubilize donor particle lipids and (iv) dissociate from the lipid particle along with the solubilized lipids. HDL-mediated phospholipid efflux is solely an active apolipoprotein-mediated process, as opposed to cholesterol-mediated efflux, which is based on equilibration of cholesterol pools on donor particle and acceptor lipoprotein surfaces via monomeric diffusion of cholesterol. Efflux of a non-transferable phospholipid on the other hand is HDL-specific and thus can be measured in whole plasma, whereas cholesterol efflux is non-specific and requires the use of ApoB-depleted plasma/serum (101, 102).

The multiple mechanistic steps underlying the HDL-SPE assay may be the reason why it was more strongly inversely associated with CAD in a CVD cohort than the cell-based CEC assay and was also more significantly inversely correlated with non-calcified plaque burden, total plaque burden, fibro-fatty burden and fibrous burden in CAD subjects assessed by coronary CT angiography (101). These findings suggest that HDL-SPE may be able assess the ability of HDL to solubilize excess extracellular lipid present in advanced arterial wall lesions, which tends to be more abundant than intracellular lipid in late atherosclerotic lesions (103). In this cohort, HDL-C was matched in CAD and non-CAD subjects. It is important to note that the cell-based CEC assay has been analyzed in other cohorts with plaque and has also been linked to non-calcified plaque.

The wide variety of subspecies and variability of composition of the HDL proteome has been shown consistently to play a major role in its functions (1). Some studies have shown that specific proteins such as apolipoprotein ApoA-I, ApoC-II, ApoC-III, and ApoA-IV differentially correlate with CEC. For instance, ApoA-I and ApoC-III are positively associated with high CEC, while ApoA-IV shows an inverse relationship in certain populations (104). Additionally, proteins like complement C3, ApoE, and plasminogen are inversely associated with CEC (104).

HDL subspecies also been shown to have an effect in cholesterol efflux. In particular, studies have shown that HDL3b and HDL3c are the most efficient mediators of cholesterol efflux via the ABCA1 pathway (105). These small HDL particles are enriched in negatively charged phospholipids, which enhance their functionality in cholesterol efflux and other activities (106). Conversely, large HDL particles are more effective in cholesterol efflux via the SR-BI pathway (107). Taken together, these observations still suggest that measuring the HDL proteome is not enough to completely explain the determinants in CEC in all HDL subspecies, but it does highlight the protein modifications and subspecies of lipids might be playing a role in CEC variations.

Due to the now many clinical trials that have failed to show any benefit from raising HDL-C in lowering CVD risk, many investigators in the field are losing interest or even abandoning their research efforts in this area. Recent studies have provided convincing evidence for the existence of dysfunctional HDL (90, 101, 108), which has shifted interest from measuring HDL-C levels to measuring HDL functionality as a possible future metric of CVD risk. We have shown that the HDL-SPE assay, which can be readily translated into a relatively rapid automated diagnostic assay, is at least as good as CEC in assessing incident CVD risk. Other cell-free assays of HDL function, particularly the CUC [84] and exogenous apoA-I exchange rate [88] assays, also show great promise for being developed into new diagnostic tests. These cell-free assays apparently reflect complementary risk information distinct from cell-based CEC and may offer improved risk prediction.

There is presently a great need for a well standardized and fully automated assays to conduct large studies on the effects of new drugs on HDL function. It will also be important to perform baseline and follow-up measurements of HDL function after any type of therapeutic intervention. Ideally these types of studies on HDL function should be done throughout the course of a clinical trial and not just at baseline as has been done for most previous studies. Longer follow-up times are also needed before definitive conclusions can be made concerning their effectiveness.

Lipid-rich plaque with longer lipid length, wider lipid arc, and higher degree of stenosis has identified in patients at higher risk of future cardiac events (109). Insofar as HDL-SPE was significantly inversely correlated with noncalcified plaque and fibro-fatty plaque burden, it may also serve as an additional metric to assess the need for invasive cardiologic procedures, such as cardiac catheterization. Although the association of other cell-free assays with non-calcified plaque burden have yet to be determined, they may also prove to be useful for assessing CVD risk and thus aid cardiologists in the future.

In summary, the new cell-free HDL functionality assays, will likely quicken the pace of research for better understanding the mechanisms underlying HDL functionality, as well as factors that modulate lipid removal from arterial wall plaque. Such information may prove vital for the possible future development of HDL based drugs.