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. Author manuscript; available in PMC: 2026 Jun 1.
Published in final edited form as: Curr Opin Lipidol. 2024 Oct 25;36(3):111–118. doi: 10.1097/MOL.0000000000000959

Distinct roles of size-defined HDL subpopulations in cardiovascular disease

W Sean Davidson 1, Tomas Vaisar 2,3, Jay W Heinecke 2,3, Karin E Bornfeldt 2,3,4
PMCID: PMC12003705  NIHMSID: NIHMS2028764  PMID: 39450930

Abstract

Purpose of review:

Doubts about whether high-density lipoprotein-cholesterol (HDL-C) levels are causally related to atherosclerotic cardiovascular disease (CVD) risk have stimulated research on identifying HDL-related metrics that might better reflect its cardioprotective functions. HDL is made up of different types of particles that vary in size, protein and lipid composition, and function. This review focuses on recent findings on the specific roles of HDL subpopulations defined by size in CVD.

Recent findings:

Small HDL particles are more effective than larger particles at promoting cellular cholesterol efflux because apolipoprotein A-I on their surface better engages ABCA1 (ATP binding cassette subfamily A member 1). In contrast, large HDL particles bind more effectively to scavenger receptor class B type 1 on endothelial cells, which helps prevent LDL from moving into the artery wall. The specific role of medium-sized HDL particles, the most abundant subpopulation, is still unclear.

Summary:

HDL is made up of subpopulations of different sizes of particles, with selective functional roles for small and large HDLs. The function of HDL may depend more on the size and composition of its subpopulations than on HDL-C levels. Further research is required to understand how these different HDL subpopulations influence the development of CVD.

Keywords: cardiovascular risk, HDL-cholesterol, HDL subpopulations, HDL function

Introduction

Low HDL-C levels consistently correlate with increased atherosclerotic cardiovascular disease (CVD) risk in large epidemiological and cross-sectional studies (1). However, evidence from randomized clinical trials questions whether increasing HDL-C provides therapeutic benefit (2). The challenge lies in connecting HDL's presumed cardioprotective functions with HDL metrics used clinically (3). HDL's cardioprotective role was traditionally linked to reverse cholesterol transport, where HDL removes excess cholesterol from cells and transports it to the liver for excretion (4). HDL also has other proposed protective functions, like preventing low-density lipoprotein (LDL) endothelial transcytosis, reducing lipoprotein oxidation, and possessing anti-inflammatory properties (5-8). HDL particles vary greatly in size, charge, and composition, with over 200 lipid and 280 protein species associated with HDL (9, 10). This diversity suggests that HDL is a collection of particles with distinct compositions and roles, making HDL-C an inadequate marker for HDL function.

HDL is traditionally defined as lipid-containing particles within a specific density range (1.063 and 1.210 g/mL), but this definition has limitations (11). Some HDL-like particles, like pre-β1 HDL, are excluded from this definition despite their role in cholesterol transport (12-14). Additionally, density-based methods of isolating HDL can alter the protein composition of the particles (15-19). Defining HDL by the presence of apolipoprotein A-I (APOA1), a key protein in HDL, offers a partial solution but introduces new limitations since other proteins also form HDL-like particles (20-22). Classifying HDL based on charge proves difficult due to the wide charge variability among lipoprotein particles (23). Techniques like 2-D non-denaturing gel separations also demonstrate considerable heterogeneity within HDL particles (24, 25). While these methods provide more detail than HDL-C, they still fall short of capturing HDL's full diversity.

One powerful approach is to identify specific HDL subpopulations by the proteins that drive their function. We define these as true HDL subspecies. For example, subspecies carrying haptoglobin-related protein and apolipoprotein L1 fight parasites, while others with APOA1 and lipopolysaccharide-binding protein protect the liver (26, 27). Complex functions like cardioprotection are likely to reflect the synergistic action of multiple HDL subspecies, which remain to be fully characterized. While this field develops, work has progressed on the use of methods for analyzing HDL subpopulations, i.e., those distinguished by physico-chemical properties, that are currently suitable for evaluating HDL in large clinical studies. These include particle sizing and quantifying subpopulations by nuclear magnetic resonance (NMR) (28), ion mobility analysis (IMA) (29), and calibrated IMA (30).

One hypothesis is that measuring HDL particle concentration (HDL-P), including its four major size subpopulations—extra small HDL (~7.6 nm diameter), small HDL (~8.2 nm), medium HDL (~9.1 nm), and large HDL (~10.9 nm)— might supersede traditional cholesterol tests with respect to HDL-mediated cardioprotection (31). While proton NMR has been widely used to quantify HDL-P (HDL-NMR), interpreting these results is challenging due to uncertainties in the relationship between HDL-NMR profiles and actual HDL subpopulations (32, 33). IMA, although precise in measuring particle size, is only semi-quantitative due to various influencing factors (29, 30). Calibrated IMA improves accuracy by converting IMA signals into absolute concentrations (30), aligning well with other methods like gel electrophoresis and electron microscopy. Importantly, individuals with similar HDL-C levels can have vastly different HDL particle subpopulation profiles (Figure 1).

Figure 1. Individuals with similar HDL-C levels can exhibit very different HDL subpopulation profiles.

Figure 1.

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HDL particle distribution profiles of 3 subjects (A, B and C) with similar HDL-C levels. Particle distribution of HDL subpopulations measured by differential ion mobility analysis shows that the relative abundance of different subpopulations can differ markedly in people with similar HDL-C levels. Raw data (rugged line), summed fitted data (thick smooth line), and fitted data for individual subpopulations - XS-HDL, S-HDL, M-HDL and L-HDL (thinner dotted lines within the summed fitted data) - are shown. Data tables present HDL-C and relative abundance of each subspecies (% total particle count).

This review focuses on recent advances in our understanding of the roles and functionalities of extra-small HDL, small HDL, medium HDL and large HDL subpopulations, as defined by calibrated IMA (30), in atherogenesis and CVD risk.

Extra-small HDL (XS-HDL) and small HDL (S-HDL) are highly effective in promoting cholesterol efflux via ABCA1.

One proposed cardioprotective role of HDL is its ability to promote cholesterol removal from lipid-laden macrophages, which play a key role in all stages of atherogenesis (34). This process begins with the ATP-binding cassette transporters ABCA1 (ATP binding cassette subfamily A member 1) and ABCG1 (ATP binding cassette subfamily G member 1). ABCA1 first facilitates the transfer of cholesterol from macrophages or hepatocytes to lipid-poor apolipoproteins, like APOA1 and APOE, and small dense HDL particles (35-37). Lecithin-cholesterol acyltransferase (LCAT) then matures these HDL particles by converting free cholesterol into cholesteryl esters that form a hydrophobic core to create larger HDL particles. ABCG1 and SRB1 (scavenger receptor class B type 1) further transfers cholesterol to these larger HDL particles, which then carry it to the liver for bile excretion (38).

Rothblat, Rader, and colleagues demonstrated that serum HDL, after the removal of lipoproteins containing apolipoprotein B (APOB), enhances cholesterol efflux from a macrophage cell line, mimicking the early steps of reverse cholesterol transport (39, 40). The efficiency of this process, known as cholesterol efflux capacity (CEC), is largely independent of HDL-C levels (39, 40). Large clinical studies have shown that both total macrophage CEC and ABCA1-specific CEC of serum HDL are strongly and negatively associated with existing and future CVD (41). Importantly, CEC predicts CVD risk independently of HDL-C levels (40-42). Lipid efflux from a cell-free source to human plasma HDL is also highly correlated with CVD protection (43). These data indicate that lipid/cholesterol efflux is a key factor in HDL's proposed anti-atherogenic effects in humans. Furthermore, it appears that the driving factor is the functionality of the HDL particles themselves.

Small HDL particles are responsible for a large portion of the CEC in serum by promoting cholesterol efflux from cells by the ABCA1 pathway (36, 37). High levels of XS-HDL particles are strongly and negatively associated with a lower risk of developing CVD in a large study of participants with type 1 diabetes, with this effect being more pronounced in men than in women (44, 45). However, the mechanisms behind this association are not fully understood.

To explore these mechanisms, we conducted studies on reconstituted HDL particles and clinical samples from control and LCAT-deficient subjects, who exclusively have very small HDL (XS-HDL) particles (46). We showed that XS-HDL and S-HDL contribute significantly to macrophage and ABCA1-mediated CEC. Molecular modeling and chemical cross-linking experiments showed that in larger HDL particles, APOA1 is tightly bound to lipids, making it less accessible to ABCA1. In contrast, the C-terminus of APOA1 in smaller HDL particles has more flexibility, allowing it to interact with ABCA1, a crucial step in cholesterol export from cells (Figure 2A). This mechanism supports both effective cholesterol efflux from macrophages and HDL biogenesis, including the cholesterol-loading of preβ–1 particles through hepatic ABCA1 (Figure 2A). However, extreme cholesterol depletion by S-HDL can induce pro-inflammatory effects in macrophages, at least in vitro (47).

Figure 2. Different functions of HDL subpopulations.

Figure 2.

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(A) Small HDL particles engage more efficiently with ABCA1 than large HDL particles because the C-termini of APOA1 (shown as structures protruding from the particle) are flipped off the surface of these small particles, enabling interaction with ABCA1 and efflux of phospholipids (PL) and cholesterol. This mechanism is likely to i) play a role in HDL biogenesis, which includes cholesterol efflux from hepatocytes through ABCA1 and ii) facilitate cholesterol efflux from macrophages and other cells in lesions of atherosclerosis. (B) Large HDL particles have a higher affinity to SRB1 than small HDL particles. This mechanism could serve an atheroprotective role by preventing endothelial transcytosis of LDL and other atherogenic APOB lipoproteins, such as triglyceride-rich lipoproteins (TRLs). Created in BioRender. Bornfeldt, K. (2024) BioRender.com/h66b969.

The AEGIS-II (APOA1 Event Reducing in Ischemic Syndromes II; NCT03473223) trial has prompted discussion about whether increasing small HDL with effective CEC is therapeutically beneficial (48). The trial tested CSL112, a human APOA1 infusion that increases CEC, to investigate if it could reduce the risk of recurrent CVD events after acute myocardial infarction. CSL112 failed to lower the risk of myocardial infarction, stroke, or death from cardiovascular causes (48). A major limitation of this study was the duration of therapy (4 weekly doses) and short primary endpoint: 90 days. In contrast, most studies demonstrate that CVD risk begins to diverge between statin-treated subjects and untreated control subjects only after two years of intervention (49, 50). A post-hoc analysis of the AEGIS-II trial suggested that participants who received CSL112 infusions had lower rates of CVD death, type 1 myocardial infarction, and stent thrombosis-related myocardial infarction compared with placebo, but the differences were not significant or were borderline significant (51). Thus, additional trials are needed to address whether increasing small HDL and CEC could be therapeutically effective.

Despite the strong negative association of XS-HDL quantified by calibrated IMA with incident CVD (44, 45), association does not indicate causation. For example, elevated levels of small HDL particles could result from increased production or reduced metabolism of those particles, and cardioprotection could therefore be mediated by other underlying factors. In future studies it will clearly be important to confirm the observation that small HDLs associate with cardioprotection in other populations of people with type 1 diabetes and to extend these observations to individuals with type 2 diabetes and those without diabetes.

Whether medium HDL (M-HDL), the most abundant HDL subpopulation, has distinct functions is unclear.

NMR analysis suggests that M-HDL particles are less abundant than smaller HDL subpopulations (52). In contrast, calibrated IMA demonstrates that M-HDL is the most abundant HDL particle type in humans (30, 53) in agreement with other orthogonal methods like size-exclusion chromatography, non-denaturing gradient gel electrophoresis, and isopycnic ultracentrifugation that show M-HDL as the most abundant HDL subpopulation in humans and mice (11, 30, 53, 54).

The formation and metabolism of HDL particles, including M-HDL, are complex processes. M-HDL was traditionally thought to form from nascent discoidal particles through the action of LCAT and other remodeling enzymes like phospholipid transfer protein (PLTP) (55). However, different hypotheses exist regarding M-HDL formation. Perfused rat livers selectively produce small discoidal HDL particles (56). Kinetic studies in non-human primates suggest that smaller HDL particles convert to medium and large HDL in the extra-vascular space (57, 58) while other studies suggest formation of different sizes of HDL particles—driven by the availability of cholesterol and phospholipid as well as levels of ABCA1 and APOA1—which are perhaps further remodeled in a noncirculating extravascular compartment (59-61).

Many clinical studies link M-HDL levels with cardiovascular health. Low M-HDL levels are associated with endothelial dysfunction and impaired coronary blood flow (62), while high levels are linked to protection from vascular complications in women with long-duration type 1 diabetes (53). Large population-based studies using NMR analysis show that M-HDL is negatively associated with the risk of myocardial infarction (63). Moreover, a Mendelian randomization study found that M-HDL, defined by NMR, has protective effects against coronary artery disease (64).

Unlike S-HDL and L-HDL, M-HDL does not yet have a clearly defined selective function, despite its abundance. This may be due to its heterogeneity as both HDL particles carrying APOA1 without APOA2 and particles carrying APOA1 and APOA2 fall into this size range. However, the largest and the smallest HDL particles contain the most diverse proteomes (65). M-HDL, similar to L-HDL, can inhibit LDL endothelial transcytosis via SRB1 (8), and stimulate vasorelaxation through SRB1 and downstream effects on endothelial nitric oxide synthase (66). However, M-HDL is much less effective than XS-HDL and S-HDL in mediating cholesterol efflux (37, 46). Further studies are needed to better understand the functions of M-HDL.

Large HDL (L-HDL) and its role in preventing LDL endothelial transcytosis.

L-HDL particles are thought to form partly through the action of PLTP, which transfers phospholipids to HDL during the breakdown of triglyceride-rich lipoproteins (TRLs) (67). This process involves the fusion of smaller HDL particles to create larger, lipid-rich HDL particles and smaller, lipid-poor APOA1 (preβ HDL particles).

Larger HDL particles have a higher affinity for SRB1 compared with smaller HDL particles (68) (Figure 2B). This is logical because a major function of SRB1 is to mediate hepatic uptake of cholesteryl ester and other lipids from HDL in the reverse cholesterol transport pathway, leading to excretion of cholesterol via the bile (69, 70). SRB1 is also expressed on endothelial cells, and larger lipid-rich HDL2 particles have been proposed to be more effective than HDL3 particles at protecting the endothelium (71). SRB1 primarily facilitates lipid uptake without internalizing the HDL particle, though it can also mediate HDL particle uptake and recycling (72, 73).

LDL transcytosis through the endothelium is mediated by SRB1 and activin receptor-like kinase 1 (ALK1) (7, 74, 75). HDL competes with LDL for SRB1 binding, with HDL being more effective in this competition (7, 76). We recently showed that L-HDL, isolated by ultracentrifugation and size-exclusion chromatography, is more effective than S-HDL at preventing LDL endothelial transcytosis (8). This protective effect of L-HDL is mediated by SRB1 rather than ALK1 (Figure 2B). Further research is needed to determine whether L-HDL also prevents other APOB-containing lipoproteins, such as TRL remnants, from entering arterial lesions via SRB1 (77).

The higher binding affinity of larger HDL particles to SRB1 compared with smaller HDL particles is likely influenced by the conformation of HDL surface recognition sites, including APOA1 (78). Differences in the protein or lipid composition of small, medium and large HDL particles might also affect SRB1 binding (72). For instance, APOC3 associates more with L-HDL than with M-HDL or S-HDL and can bind to SRB1 (8, 65, 79).

As discussed above, small HDLs have a better CEC compared with large HDLs (46), and CEC predicts CVD risk (41). It is hypothesized that L-HDL might have cardioprotective functions beyond cholesterol efflux. For instance, L-HDL’s ability to compete with LDL for SRB1 binding on endothelial cells may reduce LDL entry into the arterial wall, potentially lowering CVD risk. This is supported by recent studies showing that the balance between L-HDL and APOB100 predicts CVD risk in type 1 diabetes (8) and that large HDL is associated with lower leukocyte counts, a CVD risk factor, independent of cholesterol efflux (80). Moreover, reduced levels of both M-HDL and L-HDL, but not S-HDL, are associated with endothelial dysfunction in individuals with non-obstructive coronary artery disease (62), and large HDL particles correlate inversely with adiposity and arterial stiffness in youth who are lean, obese or have type 2 diabetes (81). Consistent with the results above, lower levels of L-HDL particles may accelerate vascular dysfunction by allowing more LDL into the vessel wall. However, these associations do not establish causation, and more mechanistic work is required to define any causal effects of low L-HDL levels on CVD risk.

One way to raise levels of L-HDL is via cholesteryl ester transfer protein (CETP) inhibition. Although numerous clinical trials of CETP inhibitors have failed, anacetrapib successfully lowered adverse cardiovascular outcomes in a four-year trial (82). Although the benefits were mostly attributed to LDL lowering, it remains possible that effects on HDL metabolism could provide long term benefits. The next generation CETP inhibitor obicetrapib, aside from potent LDL lowering, raised L-HDL by up to 50% (83), potentially working against LDL retention in the vessel wall. In addition, via increases in pre-β1 HDL particle levels, obicetrapib increased ABCA1-mediated CEC compared to placebo (84). Thus, targeted CETP inhibition might promote an overall increased flux of cholesterol through the HDL-mediated reverse cholesterol transport pathway. Should any CETP inhibitor make it to clinical use, the contributions of HDL would need to be carefully studied.

Conclusions

Low levels of HDL-C are generally associated with an increased risk of atherosclerotic cardiovascular events, such as myocardial infarction. However, pharmacologically increasing generic HDL-C levels does not seem to have a therapeutic benefit in randomized clinical trials.

There are several potential explanations for these observations. One possibility is that low HDL-C levels may serve as a marker for high levels of TRLs or other atherogenic factors, rather than being a direct cause of CVD. In this view, HDL-C levels reflect underlying risk factors rather than actively mediating cardiovascular protection.

Another explanation is that HDL’s protective effects are likely not solely determined by its cholesterol content. Research shows that different HDL subpopulations have varying cardioprotective functions. For instance, small HDL particles are more effective than large HDL particles at inducing cholesterol efflux from cells via ABCA1. In contrast, large HDL particles are more effective at preventing LDL transcytosis through endothelial cells via SRB1.

To determine whether specific HDL subpopulations have direct causative effects on atherogenesis, further basic and translational research is needed. Such studies would help clarify the distinct functional roles of different HDL particles and assess their impact on cardiovascular health.

Key points:

  • Medications that increase HDL-C levels do not have a clear therapeutic benefit in randomized cardiovascular outcome trials of high risk subjects, indicating that the association between low HDL-C and increased CVD risk does not imply causation.

  • HDL is made up of different types of particles that vary in size, lipid content, protein composition, and function.

  • Different HDL subpopulations have varying cardioprotective functions, with small HDL particles promoting cholesterol efflux via ABCA1 and large HDL particles hampering LDL endothelial transcytosis via SRB1.

  • Further basic and translational research is needed to determine whether specific HDL subpopulations play causal roles in atherogenesis.

Acknowledgments

This work was supported in part by grants from the National Institutes of Health; R35HL150754, R01HL149685, R01HL161829, P01HL151328, P01HL128203.

Abbreviations:

ABCA1

ATP binding cassette subfamily A member 1

ABCG1

ATP binding cassette subfamily G member 1

ALK1

activin receptor-like kinase 1

APOA1

apolipoprotein A1

CEC

cholesterol efflux capacity

CETP

cholesteryl ester transfer protein

CVD

cardiovascular disease

HDL-C

HDL-cholesterol

HDL-P

HDL particle concentration

IMA

ion mobility analysis

LCAT

lecithin-cholesterol acyltransferase

L-HDL

large size HDL particle

M-HDL

medium size HDL particle

NMR

nuclear magnetic resonance

PLTP

phospholipid transfer protein

SBR1

scavenger receptor class B type 1

S-HDL

small size HDL particle

XS-HDL

extra-small size HDL particle

Footnotes

Conflicts of interest

K.E. Bornfeldt serves on the scientific advisory board of Esperion Therapeutics. For the remaining authors none were declared.

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