2025 Volume 32 Issue 11 Pages 1359-1367
High-density lipoprotein (HDL) levels have long been inversely associated with cardiovascular disease (CVD) and are traditionally evaluated by serum HDL-cholesterol (HDL-C) levels. However, recent studies have raised doubts regarding the causal role of HDL quantity (HDL-C), drawing attention to HDL functionality. Reverse cholesterol transport (RCT) is a major anti-atherosclerotic mechanism involving ATP-binding cassette A1 (ABCA1), ATP-binding cassette G1 (ABCG1), scavenger receptor class B type I (SRB1), and regulatory factors, such as liver X receptor (LXR) and peroxisome proliferator-activated receptor gamma (PPARγ). Notably, HDL-C levels do not necessarily reflect RCT efficiency, and novel regulatory factors, such as microRNAs, endothelial lipase, and ANGPTL3, have been implicated. HDL also exhibits vasoprotective functions by enhancing nitric oxide (NO) production and modulating sphingosine-1-phosphate (S1P) signaling. Furthermore, it exerts anti-inflammatory effects by suppressing adhesion molecules, proinflammatory cytokines, and innate immune activation while modulating adaptive immune responses and attenuating tissue fibrosis. In addition, HDL influences megakaryopoiesis and platelet activation, thereby contributing to its antithrombotic properties. Despite these broad functional spectra, clinical assessments remain largely limited to cholesterol efflux capacity, and other key functional aspects have not been adequately explored. A more comprehensive understanding of HDL’s pleiotropic roles, spanning lipid metabolism, vascular biology, inflammation, and hemostasis, is necessary from both the basic and clinical perspectives. Recent studies have further suggested potential roles of HDL in the central nervous system, expanding its relevance beyond cardiovascular prevention and toward broader therapeutic applications.
In 1966, Glomset first proposed the concept of reverse cholesterol transport (RCT), in which excess cholesterol from the peripheral tissues is transported back to the liver1, 2). The Framingham Heart Study demonstrated that low high-density lipoprotein (HDL)-cholesterol (HDL-C) levels are an independent risk factor for cardiovascular disease (CVD), leading to its recognition as a therapeutic target for cardiovascular prevention3). Although early research emphasized HDL-C as a biomarker, subsequent genetic and pharmacological studies have questioned its causal role. In 2017, Madsen et al. revealed a U-shaped association between HDL-C levels and cardiovascular mortality based on data from the Copenhagen City Heart Study and Copenhagen General Population Study4). Consistent with these findings, evidence for Cardiovascular Prevention from Observational Cohorts in Japan (EPOCH-JAPAN) also supports these findings in Japanese patients5). Collectively, these epidemiological observations challenge the notion that elevated HDL-C levels are intrinsically protective against CVD.
From a genetic perspective, Zanoni et al. reported that loss-of-function mutations (P376L) in SCARB1, which encodes scavenger receptor class B type I (SR-BI), are associated with elevated HDL-C levels but paradoxically linked to an increased risk of CVD6). In addition, pharmacological interventions for increasing HDL-C levels have failed to prevent cardiovascular events. For example, a randomized controlled trial of cholesteryl ester transfer protein (CETP) inhibitors significantly increased HDL-C levels but failed to improve cardiovascular outcomes7). Based on these negative results from HDL-C raising studies, obicetrapib, another CETP inhibitor, is currently being explored for its potential to reduce low-density lipoprotein (LDL)-C levels combined with ezetimibe, suggesting a promising direction distinct from its HDL-raising effects8). As a result, the focus of research has shifted from the quantity of HDL-C to its quality, namely its functionality.
Cholesterol efflux capacity (CEC) has emerged as a strong inverse predictor of CVD9). CEC represents the ability of HDL to extract cholesterol from lipid-laden macrophages, a first step in RCT that has been clinically evaluated10, 11). More recently, novel assays, such as the HDL-specific phospholipid efflux (HDL-SPE) assay, have been developed. This method evaluates the ability of ApoA-1 to remove phospholipids, mainly phosphatidylethanolamine, which shows a strong correlation with CEC and has been shown to be a better predictor of cardiovascular disease than CEC in subjects from the PREVEND study12). The HDL-SPE assay was also validated in patients with familial hypercholesterolemia13).
In this review, we aimed to provide a comprehensive overview of RCT by integrating findings from both basic and clinical research.
The development of atherosclerosis begins with the accumulation of LDL particles in the subendothelial space, where they are oxidatively modified, internalized by macrophages, and forming foam cells14). Although LDL-lowering therapy with statins reduces cardiovascular events by approximately 30%, the substantial residual risk has focused on HDL functionality as a potential therapeutic target15). Enhancing RCT, as previously described, is a key strategy for inhibiting the development of atherosclerosis. CEC is reportedly the best candidate target for CVD. In 2006, Rader et al. reported that ATP-binding cassette transporters, most notably ABCA1 and ABCG1 in macrophages, play a pivotal role in RCT in vivo16). In a clinical study, the same group showed that CEC was an independent negative predictor of CVD in both cross-sectional and prospective studies9, 17). Consistent with these findings, our group reported that CEC also serves as an independent negative predictor of coronary artery disease in Japanese patients18). CEC has also been validated in other atherogenicity-related diseases, including familial hypercholesterolemia19), ischemic stroke20), chronic kidney disease21), diabetes mellitus22), rheumatoid arthritis23), and obstructive sleep apnea syndrome24). Paradoxically, high CEC was positively associated with the incidence of CVD in patients with low-stage CKD in the Dallas Heart Study21), although CEC was a predictor of CVD, as shown previously in patients without CKD. In addition, another study showed that CEC was not associated with the incidence of ASCVD in diabetes mellitus patients on hemodialysis25). Thus, the functionality of HDL has been extensively studied across a wide spectrum of diseases.
Nuclear Receptor and RCTSeveral studies have highlighted the involvement of nuclear receptors as key regulatory factors in RCT. For example, the liver X receptor (LXR) is a key transcriptional regulator of cholesterol homeostasis that regulates the expression of apolipoproteins, including ApoE, ApoC1, ApoC2, ApoC4, PTLP, and CETP. It also upregulates the expression of genes involved in cholesterol efflux including ABCA1 and ABCG126). LXR activation has been shown to enhance RCT and reduce atherosclerotic lesion formation27, 28). Interestingly, tissue-specific activation of LXR, notably in the liver and intestine, facilitated RCT in vivo; however, such effects were abrogated in the absence of ABCG5/8, thereby providing compelling evidence for the critical role of ABCG5/8 in mediating RCT29). Recently, Nishida et al. demonstrated hepatic LXR inhibition through liver-specific overexpression of sulfotransferase family 2 B member 1(Sult2b1), which inactivates oxysterols and reduces endogenous LXR activation, resulting in a reduction in HDL-C and attenuation of RCT in mice fed a high-fat diet. Importantly, this effect was mediated by the suppression of LXR target genes, including ABCG5/8 and CYP7A1, thus highlighting the essential role of hepatic LXR activation in maintaining RCT. Sult2b1 overexpression promoted hepatic cholesterol accumulation and impaired fecal sterol excretion, further supporting the critical role of hepatobiliary cholesterol handling in the regulation of RCT30). In addition, peroxisome proliferator-activated receptor gamma (PPARγ) regulates the expression of ABCA1 and ABCG1, thereby promoting cholesterol efflux from macrophages31). Activation of PPARγ by natural or synthetic ligands can enhance RCT and lower the atherosclerotic risk32).
The farnesoid X receptor (FXR), similar to the LXR, forms a heterodimer with RXR and contributes to RCT by regulating bile acid synthesis33, 34). Hepatic FXR activation promotes the expression of ABCG5 and ABCG8, thereby facilitating cholesterol excretion into bile and serving as the final step of RCT35). Recently, hepatic FXR activation has also been used to maintain the maturation and long-term viability of human iPSC-derived hepatic organoids, suggesting its potential application in regenerative medicine36). In contrast, excessive activation of intestinal FXR induces epithelial cell injury through ferroptosis and suppresses IL-22 production, potentially exacerbating intestinal inflammation37). These findings highlight the importance of tissue-specific regulation of FXR activation for the prevention of atherosclerosis.
MicroRNA (miRNA) and RCTMiRNAs are another candidate for modulating RCT. For example, miR-33 is a well-characterized negative regulator of RCT that suppresses the expression of ABCA1 and ABCG1, thereby inhibiting cholesterol efflux38). Antisense inhibition of miR-33 has been shown to elevate HDL levels and attenuate atherosclerosis in animal models. Similarly, miR-144 has been identified as a novel regulator that targets ABCA1 and modulates cholesterol efflux39). Its inhibition may represent a potential therapeutic approach to enhance RCT and mitigate atherosclerosis.
LDL-C Lowering Therapy and RCTLDL-C-lowering therapy also affected RCT. For example, ezetimibe, an NPC1L1 inhibitor, enhances RCT activity40), and the metabolic product of probucol suppresses atherosclerosis progression through RCT activation41). Interestingly, HDL-C levels may not directly reflect or influence the efficacy of RCT. For example, our group reported that overexpression of endothelial lipase (EL) lowered HDL-C levels but maintained RCT42). Conversely, knockout of EL increased HDL-C but did not enhance macrophage-derived RCT43). Furthermore, antisense oligonucleotides targeting ANGPTL3, an endogenous inhibitor of EL, further enhanced RCT, emphasizing the therapeutic potential of modulating EL activity by inhibiting ANGPTL3 44). These findings highlight the dissociation between HDL-C and HDL functionality, underscoring the importance of identifying novel regulators of RCT as promising targets for the development of anti-atherosclerotic therapy. Finally, the details of hepatic processing of HDL-derived cholesterol following an SR-BI-mediated uptake have been increasingly clarified. Aster proteins (GRAMD1s) have emerged as key mediators of non-vesicular cholesterol transport from the plasma membrane to the endoplasmic reticulum45). Although their precise contribution to RCT remains to be fully elucidated, they are expected to serve as novel intracellular regulatory factors potentially involved in RCT regulation.
In addition to its role in RCT, HDL exerts vasoprotective effects, constituting another key mechanism underlying its antiatherosclerotic properties. Indeed, HDL exerts multiple vasoprotective effects, including vasodilation through the regulation of eNOS activity, anti-inflammatory effects by reducing adhesion molecules, such as VCAM-1 and ICAM-1, antioxidant effects, and anti-apoptotic effects.
The endothelial function is primarily determined by the bioavailability of nitric oxide (NO), and endothelial dysfunction is considered early atherosclerosis46). HDL exerts vasodilatory effects by stimulating NO production in endothelial cells. These mechanisms are primarily mediated by two pathways. The first involves cholesterol efflux via the SR-B1 receptor in endothelial cells47, 48). SR-B1 mediated cholesterol efflux activates the phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K)/protein kinase B (Akt) pathway, leading to an increase in NO production. The second pathway involves sphingolipids, particularly sphingosine-1-phosphate (SIP), which binds to HDL via ApoM and interacts with SIP receptors47, 49, 50). Activation of the SIP receptor pathway leads to the mobilization of intracellular calcium, which activates AMP-activated protein kinase (AMPK) and PI3K/Akt, thereby increasing NO production. These dual actions of HDL are thought to independently activate the AMPK/PI3K/Akt pathway because S1P does not affect cholesterol efflux, as demonstrated in a recombinant HDL infusion study51).
A decrease in NO activity, which leads to endothelial dysfunction, induces coronary vasospasm52). Lower HDL-C levels have been reported in patients with coronary vasospasm angina (VSA) than non-VSA patients53, 54). Furthermore, we have shown that the cholesterol uptake capacity (CUC), a cell-free assay reflecting HDL-mediated cholesterol efflux, was impaired in patients55), suggesting that the HDL function may play a role in the pathogenesis of coronary vasospasm.
Inflammation is essential in the pathogenesis of atherosclerosis, and chronic inflammation within plaques contributes significantly to its destabilization14). Inflammatory biomarkers such as high-sensitivity C-reactive protein (hsCRP) have been identified as predictors of atherosclerotic risk and disease progression56). Persistent inflammation, which is not adequately controlled by conventional therapies, plays a critical role in the progression of atherosclerosis. The CANTOS trial demonstrated that inhibition of inflammation through the administration of the interleukin-1β (IL-1β) antagonist canakinumab significantly reduced major adverse cardiovascular events, independent of lipid levels57). Similarly, colchicine, an orally administered anti-inflammatory agent, has been shown to reduce cardiovascular events in patients with coronary artery disease58). In addition to the regulation of nitric oxide (NO) production as noted earlier, which constitutes a key function of HDL, its anti-inflammatory capacity also plays a significant role in vascular protection. A well-characterized mechanism involves downregulation of adhesion molecules, including VCAM-1 and ICAM-1, as previously noted59).
The capacity of HDL to suppress tumor necrosis factor α (TNF-α)-induced VCAM-1 expression in human umbilical vein endothelial cells (HUVECs) was first demonstrated in 1995 59). In 2021, it was further shown that HDL’s anti-inflammatory capacity, assessed by an assay measuring the suppression of TNF-α-induced VCAM-1 mRNA expression, serves as an independent predictor of CVD, highlighting its clinical relevance60). Furthermore, HDL inhibits the activation of nuclear factor-kappa B (NF-κB), a master transcriptional regulator of pro-inflammatory gene expression, thereby contributing to its anti-inflammatory effects61). Notably, S1P is also implicated in the suppression of NF-κB activation61). In addition to these anti-inflammatory effects, HDL can bind and neutralize lipopolysaccharide (LPS), a component of gram-negative bacteria, and lipoteichoic acid (LTA), a component of Gram-positive bacteria62). Notably, HDL interaction with LTA has been shown to suppress macrophage activation, indicating that HDL plays a crucial role in regulating inflammation in sepsis63).
For clinical application, synthetic HDL also reduced mortality from CLP-induced death in mice, a sepsis in vivo model. In light of its anti-inflammatory and endotoxin-neutralizing properties, HDL is increasingly being recognized as a potential therapeutic agent for sepsis in the future (clinical applications)64).
To evaluate the anti-inflammatory properties of HDL, it is imperative to consider its broader immunomodulatory effects on various immune cell populations. Macrophages are pivotal effector cells within both innate and adaptive immune systems, and accumulating evidence indicates that HDL exerts a significant regulatory influence on their inflammatory phenotypes.
HDL has been shown to attenuate the production of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, through the upregulation of transcriptional regulator activating transcription factor 3 (ATF3), which negatively modulates Toll-like receptor (TLR)-induced signaling pathways65). Another study revealed that HDL exerts anti-inflammatory effects independent of its CEC, particularly through the suppression of type I interferon responses in LPS-stimulated macrophages via the inhibition of the TLR4-TRAM/TRIF signaling pathway65). Concurrently, HDL promotes cholesterol efflux from macrophages via ATP-binding cassette transporters, including ABCA1 and ABCG1, thereby inhibiting foam cell formation and modifying the lipid raft microdomain organization. These structural alterations within lipid rafts influence membrane-associated TLR signaling, providing an additional mechanistic basis for HDL’s anti-inflammatory function66). In contrast, HDL has also been reported to promote inflammation in macrophages via the PKC–NF-κB/STAT1–IRF1 axis67). However, a previous study suggested that its anti-inflammatory effects outweigh its pro-inflammatory properties68). This implies that HDL may act in a pro-inflammatory manner when inflammatory responses are necessary for host defense, which is a particularly intriguing aspect of its function. HDL plays a central role in modulating adaptive immunity by influencing T cells. It suppresses the differentiation of pro-inflammatory T helper 1 (Th1) and T helper 17 (Th17) cells while promoting the survival of regulatory T cells (Tregs)69). This effect is largely dependent on apolipoprotein E (apoE) carried by HDL, which regulates caspase-dependent apoptosis and lipid metabolism in effector memory Tregs70). Similar to its effects on macrophages, HDL modulates T cell receptor (TCR) signaling by altering cholesterol levels within lipid rafts—membrane microdomains essential for TCR clustering and activation. By extracting cholesterol from these domains, HDL disrupts raft integrity and attenuates T cell activation, contributing to its overall anti-inflammatory function71). Alteration of lipid rafts by HDL is known to affect B-cell receptors (BCRs) in B cells, similar to its effects on T cells72).
Several studies have demonstrated that HDL exerts direct effects on inflammatory cells. However, accumulating evidence suggests that HDL also influences tissue fibrosis associated with inflammation.
For example, HDL activates S1PR1 in endothelial cells, promoting vascular regeneration and attenuating fibrotic remodeling. For example, HDL has been reported to enhance hepatic regeneration and reduce perisinusoidal fibrosis in chronic liver injury73). In addition, HDL inhibits endothelial-to-mesenchymal transition (EndMT), a process triggered by TGF-β1 and associated with fibrogenesis, thereby preventing fibrotic changes in endothelial cells74).
HDL also exerts inhibitory effects on fibroblast activation by suppressing TGF-β1-induced collagen deposition and modulating the expression of fibrosis-related markers75).
Taken together, these observations not only reinforce the role of HDL in regulating inflammatory responses but also highlight its direct involvement in the attenuation of tissue fibrosis. Such multifaceted actions strongly support the notion that HDL serves as a critical biological protector in the context of chronic inflammation and fibrotic diseases.
HDL regulates platelet biogenesis, specifically at the megakaryopoiesis level. HDL modulates the differentiation and maturation of megakaryocyte progenitor cells (MKPs), thereby influencing circulating platelet counts. A key mechanism underlying this regulation involves cholesterol efflux mediated by ATP-binding cassette transporter G4 (ABCG4). In the absence of ABCG4, intracellular accumulation of free cholesterol occurs in MKPs, leading to enhanced platelet production, a process by which HDL counteracts the promotion of cholesterol efflux76). Beyond hematopoiesis, HDL directly modulates the platelet function. It interacts with receptors such as SR-B1 and ABCA1 on the platelet membrane, thereby altering membrane cholesterol composition. ApoA1, the major apolipoprotein component of HDL, suppresses the expression of key activation markers, including P-selectin and integrin αIIbβ3, thereby attenuating platelet aggregation77). Similar to the mechanisms described in lymphocytes, HDL is also capable of remodeling lipid raft microdomains in the platelet membrane, influencing the spatial organization and activity of signaling molecules, such as FcγRII and glycoprotein Ib (GpIb)78).
This review provides a comprehensive overview of the key functional properties of HDL, with particular emphasis on RCT, vasodilatory effects, anti-inflammatory activity, and antithrombotic function. These diverse functionalities underpin the atheroprotective effects of HDL. However, integrated analyses that encompass these multiple roles remain limited. Furthermore, there is a notable lack of clinically applicable assays capable of evaluating HDL function beyond CEC and CUC. Thorough elucidation of the mechanisms underlying HDL functionality from both basic and clinical research perspectives represents a major challenge for future investigations. In addition, recent studies have increasingly highlighted the potential roles of HDL in the field of neuroscience79), thereby opening a new avenue for future research.
The authors declare that they have no competing interests.
