INTRODUCTION

Over time, cholesterol has been a topic of intense research interest. Thirteen Nobel Prizes have been awarded to scientists who made discoveries about cholesterol [1]. In the first half of the 20th century, five Prizes were awarded for refinements to the structure of cholesterol. Later, Konrad Bloch received the Prize in Physiology or Medicine for delineating the mechanism for free cholesterol biosynthesis from acetate, and 1 year later, Robert B. Woodward received the Prize in Chemistry for the total synthesis of cholesterol from hydroquinone. In 1975, John W. Cornforth received the prize in chemistry for his studies on the biosynthesis of cholesterol. In 1985 Michael S. Brown and Joseph L. Goldstein were recognized with the Prize in Physiology or Medicine for work on the regulation of cholesterol metabolism.

Cholesterol, and its solvent, phospholipids, are essential to structure and function in mammalian biology [2]. Among mammals, the surface monolayers of the plasma lipoproteins and bilayers of the plasma membranes of cells contain a variety of structurally and functionally distinct lipids of which the most abundant are phospholipids, especially phosphatidylcholine and sphingomyelin, and free cholesterol [3,4]. The lipoprotein surface monolayer and each leaflet of the PM bilayer can be thought of as a two-dimensional solution in which phospholipid is the solvent and free cholesterol is the major solute. The functions of other molecules in the solvent, especially proteins, are expected to be determined, in part, by the qualities of the two-dimensional solvents. Within this context, the relative abundance of free cholesterol can be expressed as

mol% FC = molFC/(molFC + molPL) Equation 1

where molFC + molPL, respectively, are the number of moles of free cholesterol and phospholipid within the monolayers and bilayers.

Free cholesterol has a diversity of structural and functional roles in cells [5–7]. Nearly 90% of cell-free cholesterol, derived from lipoprotein-receptor-mediated uptake or de-novo synthesis [1,5,6,8], occurs in the plasma membrane [9], where it comprises 10–45 mol% of plasma membrane lipids [9,10]. This high level of cholesterol is essential for the structural and functional integrity of the plasma membrane and genetically associated derangements in cholesterol metabolism underlie several human diseases, notably familial hypercholesterolemia [11], Nieman-Pick type C disease [12,13], Smith-Lemli-Opitz syndrome [14], abetalipoproteinemia [15], and hypobetalipoproteinemia [16]. Another physiological site of free cholesterol is the plasma; the surface monolayers of plasma high- and low-density lipoproteins from normolipidemic individuals are nearly 25 and ∼40 mol% free cholesterol, respectively [3]. 

Box 1:

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FREE CHOLESTEROL DYNAMICS AND BIOAVAILABILITY

Although thought to be insoluble in water, the aqueous solubility of free cholesterol is simply low. Free cholesterol has a maximum aqueous solubility of 4.7 nmol/l and reversibly self-associates with a critical micelle concentration of nearly 30 nmol/l. The affinity of free cholesterol for phospholipid is determined, in part, by its low solubility in water so that when phospholipid and water coexist, free cholesterol partitions almost exclusively into the phospholipid. The consequences of this higher solubility in phospholipid vs. low solubility in water are important to free cholesterol trafficking among phospholipid surfaces. Similar to a water molecule evaporating from a rain drop, free cholesterol molecules desorb from phospholipid surfaces. The tendency of free cholesterol to desorb from a phospholipid surface has been expressed as “active cholesterol” and in the traditional physico-chemical term, as fugacity. Active cholesterol has been implicated in one model of reverse cholesterol transport [17▪]. As with other lipids [18], the spontaneous transfer rate follows Kelvin's law and increases with decreasing particle radius. For example, the halftimes for free cholesterol transfer from HDL (D = ∼10 nm) and LDL (D = ∼22 nm) to other lipid surfaces are 5 and 45 min respectively [19]. This process occurs by rate-limiting free cholesterol desorption from the lipoprotein surface followed by rapid diffusion controlled association with the acceptor surface. This mechanism, which also underlies free cholesterol transfer to cells [20], has been generalized to other lipids including phospholipids, which transfer between phospholipid surfaces, that is, other lipoproteins or the plasma membranes of cells, at rates that decrease with increasing acyl chain length and decreasing unsaturation [18]. In contrast, cholesteryl esters and triglycerides containing long acyl chains are insoluble in water and require a carrier, that is, cholesteryl ester transfer protein, to move among lipoproteins [21–23].

ACCESSIBLE CHOLESTEROL

The organization of free cholesterol, which comprises 35–40 mol% of total plasma membrane lipids in animal cells, has been studied using novel free cholesterol binding bacterial cytolysins [24]. These include domain 4 of Anthrolysin O (ALOD4) and the fungal toxin, Ostreolysin A (OlyA), both of which are soluble, nonlytic proteins. Together these cytolysins reveal three different plasma membrane-free cholesterol pools: The first is a mobile free cholesterol pool that accessible to ALOD4 and rapidly transfers free cholesterol between the plasma membrane and the endoplasmic reticulum. Another is a sphingomyelin-sequestered free cholesterol pool revealed by OlyA fluorescence. Finally, there is a fixed free cholesterol pool essential for membrane integrity that is inaccessible to ALOD4, and is not liberated by sphingomylinase. Accessible vs. sequestered free cholesterol in human erythrocytes has been distinguished using ALOD4 [25]. Variation in erythrocyte accessible free cholesterol was independent of erythrocyte free cholesterol content, but varied with erythrocyte-phospholipid composition. Free cholesterol accessibility is different from active/bioavailable free cholesterol. The former is accessible to an exogenous free cholesterol specific probe [26] and the latter is the tendency to escape from a phospholipid surface monolayer.

TRADITIONAL MODEL OF REVERSE CHOLESTEROL TRANSPORT

Atherosclerosis occurs in the arterial wall. In one model, blood monocytes enter the subendothelial space of the arterial wall where they become resident macrophages. In the context of high plasma cholesterol LDL concentrations, excess LDL crosses the endothelium where it is oxidized or otherwise modified. The modified LDL is taken up by macrophages which are transformed to foam cells, a key step in atherosclerosis. The excess cholesterol burden can be relieved by the transfer of macrophage cholesterol to HDL [27], esterified by lecithin:cholesterol acyltransferase (LCAT) and ultimately disposed of by the hepatic HDL receptor [28], scavenger receptor class B type 1, which is encoded by the human SCARB1 and mouse Scarb1 genes. The macrophage-to-liver cholesterol transfer, that is, reverse cholesterol transport, is a widely accepted antiatherogenic mechanism [29].

The rapid in-vitro transfer of HDL-FC and LDL-FC to other lipoproteins [19] portended profound pathophysiological consequences and the need to refine the traditional RCT mechanism. First, in humans HDL-FC has a plasma halftime of nearly 10 min [30], too fast for meaningful esterification by LCAT and far shorter than the lifetime of HDL-proteins (∼6 days) [31]. Thus, spontaneous transfer is responsible for much of the total cholesterol biodistribution and cytotoxicity, particularly in the state of high plasma free cholesterol [32,33,34▪]. Indeed, cellular free cholesterol accumulation underlies foam cell death [35]. Second, and similarly, in wild-type mice HDL-FC turnover is rapid, t1/2 = 3.2 min, faster in Scarb1 overexpressing mice, t1/2 = 1.8 min [36] and slower in Scarb1-/- mice [37]. As with humans, less than 1% of mouse HDL-FC is esterified within these halftimes so that nearly all mouse free cholesterol turnover occurs without esterification. Collectively, these data support the hypothesis that most hepatic extraction of HDL-FC occurs without esterification. Not all HDL-FC is hepatically extracted. Free cholesterol transfers to nearly all other tissues, especially erythrocytes (Fig. 1) [37,38], by diffusion, a process that is the mechanistic basis of transintestinal cholesterol efflux, whereby plasma free cholesterol transfers to the intestine independent of hepatic uptake [39]. This mechanism works for free cholesterol because it is sparingly soluble in water [40,41] but not for cholesteryl esters, which are absolutely water-insoluble.

FIGURE 1: Tissue distribution of HDL-associated FC, PL and APOA1 following infusion of nascent HDL containing [3H]FC, [14C]PL and [125I]APOA1. Previously published [34▪].PATHOPHYSIOLOGY OF PLASMA HDL

An early study comparing plasma HDL concentrations with incident atherosclerotic cardiovascular disease (ASCVD) revealed an inverse relationship [42], a finding that has been confirmed in other studies including the large multidecade Framingham Heart Study [43–45]. These observations provoked tests of the effects of drugs that increase plasma HDL-C concentrations on incident ASCVD in patients receiving a statin to lower plasma LDL-C concentrations. Niacin raised plasma HDL-C concentrations and reduced those of LDL, VLDL and Lp(a) [46], but its addition to a statin increased the risk of serious adverse events without reducing risk of major ASCVD events [47], produced no meaningful ASCVD benefit [48], and showed no efficacy with respect to total or cause-specific mortality [49]. Studies of the triglyceride-lowering agent gemfibrozil [50,51] showed that the concurrent increase in HDL-C reduced ASCVD events, with an effect that was smaller than that predicted by cross-sectional data. Fenofibrate and a statin failed to reduce ASCVD events among patients with type 2 diabetes [52]. Lastly, CETP inhibitors, which profoundly increase plasma HDL-C concentrations, elicited either nil or unimpressive effects on ASCVD events [53–56]. However, CETP inhibition by anacetrapib reduced plasma LDL-C and APOB concentrations by 17 and 18% respectively while reducing the number of major coronary events in ASCVD patients [57]. Moreover, a newer CETP inhibitor, obicetrapib (10 mg) reduces LDL-C by 44% and APOB by 27% when in combination with a high-intensity statin [58]. The results of an outcomes trial of obicetrapib will not be released until 2024. Endothelial lipase variants associated with high plasma HDL-C concentrations do not confer lower ASCVD risk [59].

Conversely, probucol lowers HDL-C concentrations but prevents ASCVD in preclinical models [60] and some human studies [61]. Thus, pharmacological and genetic [59] effectors of increased HDL concentrations do not reduce ASCVD, especially with statin co-therapy. Paradoxically, several recent studies have revealed increased all-cause but not ASCVD mortality at high plasma HDL-C concentration [62–65]. In contrast, another study found increased ASCVD mortality at the low and high extremes of plasma HDL-C concentrations [66▪▪]. However, the characteristics of the five study cohorts were different (Table 1). The study that found increased mortality at high HDL-C concentrations was composed of subjects with preexisting ASCVD whereas the other study cohorts were composed of a general population or patients with renal insufficiency. It is relevant, that patients with low plasma HDL-C concentrations frequently present with one or more atherogenic components of metabolic syndrome, the mechanistic link between high plasma HDL-C concentrations and excess disease, especially ASCVD is perplexing and unknown but may be related to higher free cholesterol bioavailability, which supports the pathological free cholesterol transfer to various tissues. Moreover, in the REVEAL Trial, the mean HDL-C of the treatment group, 85 mg/dl, is at the low end of the range above which all-cause and ASCVD mortality increase [62–65,66▪▪]. Studies of CETP inhibitors showed that brief elevations of plasma HDL-C concentrations are not pathological. At the midpoint of the REVEAL Trial, HDL-cholesterol concentrations in the group receiving anacetrapib were increased to 85 mg/dl (+104%) vs. no change for the placebo group; no adverse ASCVD effects due to greatly elevated HDL-C concentrations were observed [57]. This was likely was likely due to the short duration to the midpoint, nearly 2 years vs. a lifetime in the observational studies of high HDL-C concentrations [62–65,66▪▪].

Table 1 - Summary of studies correlating plasma HDL concentrations with mortality Source (reference) Number surveyed Mean age (years) %Female Mortality Cohort CANHEART [62] 631 762 57 55 All cause Primary prevention Copenhagen City Heart Study and Copenhagen General Population Study [63] 116 508 58 55 All cause General population Health Survey for England & Scottish Health Survey [64] 37 059 58 53 All cause General population Veterans Affairs/St. Louis Healthcare System [65] 1 764 986 64 0 All cause Renal insufficiency UK Biobank & Emory Cardiovascular Biobank [66▪▪] 14 478 62 24 All cause and ASCVD CAD present
PATHOPHYSIOLOGY OF EXCESS HDL-FC

HDL-FC bioavailability has been expressed as

HDL-FCBI = HDL-P x mol% HDL-FC Equation 2

in which HDL-P is the HDL particle number, which can be approximated as HDL-C concentration [67], and mol% as defined by Equation 1 [68]. Given that free cholesterol rapidly diffuses to many tissue sites, a state of excess HDL-FCBI would be expected to be associated with higher free cholesterol content. This state is observed in mice with HDL-receptor deficiency (Scarb1-/-). According to literature values for HDL-C concentration and mol% free cholesterol [69], values for HDL-FCBI for wild-type and Scarb1-/- mice respectively are 8.3 and 73. The high HDL-FCBI in Scarb1-/- mice is associated with increased free cholesterol in some but not all tissues (Fig. 2). Notably, many of the tissues with an elevated free cholesterol content present with a profound disease. Blood erythrocytes have altered morphologies and are dysfunctional [32,33]; female Scarb1-/- mice are infertile and Scarb1-/- mice are susceptible to diet-induced atherosclerosis [70]. Although Equation (2) shows that the determinants of FCBI are the HDL particle number and the HDL-mol% free cholesterol, there are other likely factors. Free cholesterol transfer rates increase with decreasing HDL size [19], so the smaller HDL would have greater bioavailability. Also, HDL-phospholipid composition is also a determinant of transfer rates, increasing as sphingomyelin < unsaturated phosphatidylcholine < saturated phosphatidylcholine [71,72]. The effect of HDL size would be a meaningful determinant of FCBI in patients with a high plasma HDL2 concentration.

FIGURE 2: Lipid compositions of various tissues from male and female wild-type and Scarb1-/- mice as labelled [33].REVERSING FREE CHOLESTEROL LINKED DISEASES

Probucol, a compound with potent antioxidant properties, has been used as an anti-ASCVD therapy. Preclinical support of probucol as an ASCVD therapeutic was impressive. Probucol is a simple molecule (Fig. 3) that is nearly isostructural with that of a dimeric butylated hydroxy toluene, a powerful antioxidant commonly used as a food additive to extend the shelf life of food. There is preclinical and clinical support for the use of probucol for treating ASCVD. In the athero-susceptible Watanabe rabbit, probucol inhibits athero-progression. This observation implicated oxidized LDL in atherogenesis (Reviewed) [60]. Among patients with heterozygotic familial hypercholesterolemia, probucol treatment nearly normalized serum cholesterol and reduced xanthomas [73,74], effects that were most profound in patients who experienced the greatest decline in HDL-C [74]. This observation would seem to indicate that a dysfunctional HDL underlies some forms of ASCVD, an effect that may be independent of the antioxidant effects of probucol. Notably, high-dose vitamin E, another antioxidant, did not regress xanthomas in homozygous familial hypercholesterolemic patients [75], and in clinical trials, addition of vitamin E to statin therapy did not reduce ASCVD events [76].

FIGURE 3:

Structures of (a) probucol and (b) butylated hydroxy toluene.

However, probucol tests in humans were less convincing, at least in the context of familial hypercholesterolemia. A randomized, prospective study compared probucol (500 mg/day) with placebo in dyslipidemic, ASCVD patients (LDL-C concentration ≥ 140 mg/dl); both groups received a statin [77,78]. At the end of the study, the difference in the incidence of the primary end points (ASCVD death, nonfatal myocardial infarction, nonfatal stroke, hospitalization for unstable angina, hospitalization for heart failure, or coronary revascularization) in the probucol vs. placebo groups but did not reach statistical significance. As in other studies, probucol reduced HDL-C concentration (∼30%) [78–80], and in an era of high-HDL-C-is-better, this finding contributed, in part, to the withdrawal of probucol from the USA market.

Probucol is inextricably tied to HDL metabolism. Probucol inhibits ABCA1 [81], a source of early forms of HDL and the initial step in reverse cholesterol transport, suggesting it would be pro-atherogenic. However, probucol increases expression of the hepatic HDL receptor, SR-B1 [82], which mediates the final step in reverse cholesterol transport, hepatic HDL-C uptake [83]. The net effect is the observed reduction of plasma HDL concentrations. Probucol also inhibits expression of IL-1 beta, which is associated with the early stages of atherogenesis [84]. These probucol-mediated effects as well as its prevention of lipid macrophage-lipid accretion [85] would be expected to be athero-protective. Thus, it is likely that probucol elicits its antiatherogenic effects via nonanti-oxidative mechanisms.

Despite the negative outcomes of probucol trials in hypercholesterolemic patients, there is a setting in which probucol could be athero-protective, very high plasma HDL concentrations, that is, hyperalphalipoproteinemia, which is sometimes due to known monogenic disorders [86]. Although the underlying cause of ASCVD [66▪▪] mortality among patients with very high, more than 80 mg/dl, HDL-C concentrations is not known, studies in humans and mice provide clues. Among humans, ASCVD is associated with impaired in-vitro macrophage-free cholesterol efflux to patient HDL [87–90]. The human data are scant, but a mouse model of high plasma concentrations of HDL that is free cholesterol rich, that is, the HDL receptor deficient mouse (Scarb1-/-) has nearly 10 times the free cholesterol of wild-type mice and is susceptible to diet-induced ASCVD. Whereas free cholesterol efflux from macrophages to HDL of Scarb1-/- and wild-type mice is similar, this process is reversible [20,91], and the in-vitro free cholesterol influx from Scarb1-/- vs. wild type HDL to macrophages is three times greater [37]. This would be expected to increase the in-vivo macrophage free cholesterol burden thereby promoting ASCVD. This remains to be shown.

All-cause mortality excluding ASCVD is more difficult to explain. These causes include cancer [63] and risk of infection [92]. Among Scarb1-/- mice, some but not all tissues are free cholesterol rich when comparted with wild-type mice. Notably, many of the tissues that have increased free cholesterol content in the Scarb1-/- vs. wild-type mice are associated with a disease, heart, erythrocyte and platelet morphologies, and infertility among female Scarb1-/- mice. The erythrocyte defects and infertility in Scarb1-/- mice are reversed by probucol and by serum opacity factor, both of which greatly reduce plasma HDL-total cholesterol concentrations [93▪▪,94]. In the latter case, this was shown to be due, in part, to a reduction in free cholesterol in both HDL and the affected tissues [93▪▪]. Tests correlating HDL-FC concentrations with human female infertility and ASCVD have not been reported.

That being the case, lowering HDL concentrations and thus HDL-FCBI (Equation 2) is, hypothetically, an effective strategy that could be implemented by probucol therapy. Although this has never been tested in humans, studies in mice are supportive and encouraging. Moreover, free cholesterol efflux from macrophages to the HDL from Scarb1-/- mice was similar to that of WT mice. In contrast, free cholesterol influx from Scarb1-/- mice was higher (>400%) than that to wild-type HDL [37], suggesting that high HDL-free cholesterol bioavailability supports increased free cholesterol macrophage influx, which drives atherogenesis. This is supported by the observation that the addition of probucol to an atherogenic diet of Scarb1-/- mice reduces HDL-C and prevents atherosclerosis [95]. A similar experiment has not been conducted in humans with ASCVD on a background of high HDL-C concentrations. Studies in men and mice, cited above, showed that pharmacological and genetic increases in HDL-C do not prevent ASCVD. Thus, reduction of plasma HDL-C concentrations might possibly be antiatherogenic in patients with high HDL-associated ASCVD. Additional evidence is needed to validate such a claim of clinical benefit.

CONCLUSION

Whereas several studies revealed an association between plasma HDL-C concentrations and all-cause [63–65] and ASCVD [66▪▪] mortality, to date, there have been no studies correlating HDL-FC with mortality. Such studies would likely inform about the appropriate management of patients with ASCVD due to too much plasma HDL-FC.

Acknowledgements

None.

Financial support and sponsorship

This review was supported by funds from the National Institutes of Health to H.J.P. and C.R. (R01-HL149804; R01-HL163535) and from the Houston Methodist Foundation (H.J. P).

Conflicts of interest

None.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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