High-density lipoprotein: gene-based approaches to the prevention of atherosclerosis

Although the atheroprotective role of high-density lipoprotein (HDL) has been well documented in epidemiological and animal studies, highly effective therapeutic approaches for the selective increase of plasma HDL levels or function are not yet available. Several mechanisms by which HDL exerts an atheroprotective effect have been proposed on the basis of experiments in vitro and in vivo. These mechanisms include directing excess cellular cholesterol from the peripheral tissues to the liver in 'reverse cholesterol transport', inhibiting oxidative modification or aggregation of LDL, and modulating inflammatory responses to favour vasoprotection. This review gives an overview of the genes regulating these mechanisms, such as those encoding apolipoprotein AI, 1ecithin:cholesterol acyltransferase (LCAT), scavenger receptor B 1 (SR-BI), and the ATP-binding cassette transporter 1 (ABCl), and the potential to exploit them to develop gene-based therapeutic approaches to increase the level or function of HDL.     

ABCG1 and HDL protect against endothelial dysfunction in mice fed a high-cholesterol diet

Plasma HDL levels are inversely related to the incidence of atherosclerotic disease. Some of the atheroprotective effects of HDL are likely mediated via preservation of EC function. Whether the beneficial effects of HDL on ECs depend on its involvement in cholesterol efflux via the ATP-binding cassette transporters ABCA1 and ABCG1, which promote efflux of cholesterol and oxysterols from macrophages, has not been investigated. To address this, we assessed endothelial function in Abca1(-/-), Abcg1(-/-), and Abca1(-/-)Abcg1(-/-) mice fed either a high-cholesterol diet (HCD) or a Western diet (WTD). Non-atherosclerotic arteries from WTD-fed Abcg1(-/-) and Abca1(-/-)Abcg1(-/-) mice exhibited a marked decrease in endothelium-dependent vasorelaxation, while Abca1(-/-) mice had a milder defect. In addition, eNOS activity was reduced in aortic homogenates generated from Abcg1(-/-) mice fed either a HCD or a WTD, and this correlated with decreased levels of the active dimeric form of eNOS. More detailed analysis indicated that ABCG1 was expressed primarily in ECs, and that these cells accumulated the oxysterol 7-ketocholesterol (7-KC) when Abcg1(-/-) mice were fed a WTD. Consistent with these data, ABCG1 had a major role in promoting efflux of cholesterol and 7-KC in cultured human aortic ECs (HAECs). Furthermore, HDL treatment of HAECs prevented 7-KC-induced ROS production and active eNOS dimer disruption in an ABCG1-dependent manner. Our data suggest that ABCG1 and HDL maintain EC function in HCD-fed mice by promoting efflux of cholesterol and 7-oxysterols and preserving active eNOS dimer levels.     

High-density lipoprotein: a fall from grace?

Based on a plethora of in-vitro and in-vivo research data, high-density lipoprotein cholesterol (HDL) has been regarded as universally atheroprotective. Consequently, pharmacologically mediated HDL increase has emerged as a potential means to improve prevention and treatment of patients with atherosclerotic vascular disease. In particular, inhibition of cholesteryl ester transfer protein (CETP) was considered a promising strategy. Recently, the unanticipated and disappointing results of four large clinical trials with the CETP inhibitor torcetrapib have necessitated refinement of the HDL hypothesis. In addition, the progressive insight that HDL may actually be predominantly a carrier molecule of a wide array of proteins rather than merely a cholesterol-transporter has resulted in the interest to look beyond HDL levels alone. Here we will discuss the impact of recent developments on the HDL hypothesis as well as the advent of even more recent therapeutic developments in the HDL field.     

Apolipoprotein mimetic peptides: Mechanisms of action as anti-atherogenic agents

Apolipoprotein mimetic peptides are short synthetic peptides that share structural, as well as biological features of native apolipoproteins. The early positive clinical trials of intravenous preparations of apoA-I, the main protein component of high density lipoproteins (HDL), have stimulated great interest in the use of apolipoprotein mimetic peptides as possible therapeutic agents. Currently, there are a wide variety of apolipoprotein mimetic peptides at various stages of drug development. These peptides typically have been designed to either promote cholesterol efflux or act as anti-oxidants, but they usually exert other biological effects, such as anti-inflammatory and anti-thrombotic effects. Uncertainty about which of these biological properties is the most important for explaining their anti-atherogenic effect is a major unresolved question in the field. Structure-function studies relating the in vitro properties of these peptides to their ability to reduce atherosclerosis in animal models may uncover the best rationale for the design of these peptides and may lead to a better understanding of the mechanisms behind the atheroprotective effect of HDL.     

Reverse cholesterol transport and cholesterol efflux in atherosclerosis

Reverse cholesterol transport (RCT) is a pathway by which accumulated cholesterol is transported from the vessel wall to the liver for excretion, thus preventing atherosclerosis. Major constituents of RCT include acceptors such as high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), and enzymes such as lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), hepatic lipase (HL) and cholesterol ester transfer protein (CETP). A critical part of RCT is cholesterol efflux, in which accumulated cholesterol is removed from macrophages in the subintima of the vessel wall by ATP-binding membrane cassette transporter A1 (ABCA1) or by other mechanisms, including passive diffusion, scavenger receptor B1 (SR-B1), caveolins and sterol 27-hydroxylase, and collected by HDL and apoA-I. Esterified cholesterol in the HDL is then delivered to the liver for excretion. In patients with mutated ABCA1 genes, RCT and cholesterol efflux are impaired and atherosclerosis is increased. In studies with transgenic mice, disruption of ABCA1 genes can induce atherosclerosis. Levels of HDL are inversely correlated with incidences of cardiovascular disease. Supplementation with HDL or apoA-I can reverse atherosclerosis by accelerating RCT and cholesterol efflux. On the other hand, pro-inflammatory factors such as interferon-gamma (IFN-gamma), endotoxin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), can be atherogenic by impairing RCT and cholesterol efflux, according to in vitro studies. RCT and cholesterol efflux play a major role in anti-atherogenesis, and modification of these processes may provide new therapeutic approaches to cardiovascular disease. Further research on new modifying factors for RCT and cholesterol efflux is warranted.     

Weight loss achieved by bariatric surgery modifies high-density lipoprotein subfractions and low-density lipoprotein oxidation towards atheroprotection

ObjectivesWeight loss achieved by laparoscopic adjustable gastric banding (LAGB) induces an increase in high-density lipoprotein cholesterol (HDLc) but a small effect on low-density lipoprotein (LDL), although changes in their quality (size and composition) are uncertain. Our aim was to study the impact of weight loss, achieved 13-months after LAGB, on inflammation and dyslipidemia, focusing on HDL and LDL subfractions, and oxidized LDL (oxLDL).Design & methodsWe evaluated standard lipid profile, HDL and LDL subfractions, oxLDL, interleukin (IL)-6 and C-reactive protein (CRP), in twenty obese patients, before (T0) and 13-months after LAGB (T1), and in seventeen healthy controls.ResultsAt T1, patients showed lower body weight (12% median weight loss) and anthropometric indices; reduction in TG, atherogenic indices, oxLDL, oxLDL/LDL ratio, CRP and IL-6, and enhancement in HDLc; an increase in large HDL and intermediate HDL subfractions, and a decrease in small HDL subfraction; LDL subfractions were not modified. Percentual change (%Δ) of oxLDL, from T0 to T1, correlated significantly and positively with %Δ of small HDL subfraction and with %Δ of body mass index.ConclusionsWeight loss induced atheroprotective changes on inflammation, and lipid profile, enhancing larger HDL, the more atheroprotective subfraction, reducing the less protective subclass, small HDL, and reducing oxLDL and oxLDL/LDL ratio. Quality of lipoproteins appears useful cardiovascular risk biomarkers, deserving further studies.     

HDL therapy today: from atherosclerosis,to stent compatibility to heart failure

Abstract

Epidemiologically, high-density lipoprotein (HDL) cholesterol levels have been inversely associated to cardiovascular (CV) events, although a Mendelian Randomisation Study had failed to establish a clear causal role. Numerous atheroprotective mechanisms have been attributed to HDL, the main being the ability to promote cholesterol efflux from arterial walls; anti-inflammatory effects related to HDL ligands such as S1P (sphingosine-1-phosphate), resolvins and others have been recently identified. Experimental studies and early clinical investigations have indicated the potential of HDL to slow progression or induce regression of atherosclerosis. More recently, the availability of different HDL formulations, with different phospholipid moieties, has allowed to test other indications for HDL therapy. Positive reports have come from studies on coronary stent biocompatibility, where the use of HDL from different sources reduced arterial cell proliferation and thrombogenicity. The observation that low HDL-C levels may be associated with an enhanced risk of heart failure (HF) has also suggested that HDL therapy may be applied to this condition. HDL infusions or apoA-I gene transfer were able to reverse heart abnormalities, reduce diastolic resistance and improve cardiac metabolism. HDL therapy may be effective not only in atherosclerosis, but also in other conditions, of relevant impact on human health.

• Key messages

• High-density lipoproteins have as a major activity that of removing excess cholesterol from tissues (particularly arteries).

• Knowledge on the activity of high-density lipoproteins on health have however significantly widened.

• HDL-therapy may help to improve stent biocompatibility and to reduce peripheral arterial resistance in heart failure.

     

HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3

HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca2+ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). All three elevated intracellular Ca2+ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S1P3 (also known as LPB3 and EDG3) abolished the vasodilatory effects of SPC, S1P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S1P3-deficient mice, Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S1P3-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.     

Discrete roles of apoA-I and apoE in the biogenesis of HDL species: lessons learned from gene transfer studies in different mouse models

Using adenovirus-mediated gene transfer in apolipoprotein A-I (apoA-I)-deficient mice, we have established that apoA-I mutations inhibit discrete steps in a pathway that leads to the biogenesis and remodeling of high-density lipoprotein (HDL). To this point, five discrete categories of apoA-I mutants have been characterized that may affect the interactions of apoA-I with ATP-binding cassette superfamily A, member 1 (ABCA1) or lecithin:cholesterol acyl transferase (LCAT) or may influence the plasma phospholipid transfer protein activity or may cause various forms of dyslipidemia. Biogenesis of HDL is not a unique property of apoA-I. Using adenovirus-mediated gene transfer of apoE in apoA-I- or ABCA1-deficient mice, we have established that apolipoprotein E (apoE) also participates in a novel pathway of biogenesis of apoE-containing HDL particles. This process requires the functions of the ABCA1 lipid transporter and LCAT, and it is promoted by substitution of hydrophobic residues in the 261 to 269 region of apoE by Ala. The apoE-containing HDL particles formed in the circulation may have atheroprotective properties. ApoE-containing HDL may also have important biological functions in the brain that confer protection from Alzheimer's disease.     

PON-dering differences in HDL function in coronary artery disease

HDL cholesterol activates endothelial cell production of the atheroprotective signaling molecule NO, and it promotes endothelial repair. In this issue of the JCI, Besler et al. provide new data indicating that HDL from stable coronary artery disease (CAD) or acute coronary syndrome patients inhibits rather than stimulates endothelial NO synthesis and endothelial repair. This may be related to decreased HDL-associated paraoxonase 1 (PON1) activity. These observations support the concept that the cardiovascular impact of HDL is not simply related to its abundance, and the translation of the present findings to prospective studies of CAD risk and to evaluations of HDL-targeted therapeutics is a logical future goal.     

Studies on human serum high-density lipoproteins. VI. Studies on a cholesterol ester-releasing reaction in vitro.

High-density lipoproteins (HDL) turn turbid during in vitro incubation, concomitant with the formation of a cholesterol ester-rich lipoprotein, designated HDL-sup. The increase in turbidity (A) formed in relation to incubation time (t) is an asymptotic function: A=Uo(1 - e-k1t), where Uo is the amount of HDL with the property of releasing HDL-sup and k1 the velocity constant of the reaction. The increase in turbidity and formation of HDL-sup was not related to cholesterol ester content of the incubated fraction nor to exogenous factors like bacterial growth. The in vitro incubation was accompanied by a cholesterol esterification with a mean production of 8 nmol cholesterol ester/mg HDL protein, but also by a more pronounced degradation of phosphatidyl choline, 148 nmol/mg HDL protein. These data indicate that the lipid changes are induced by a two-step lecithin:cholesterol acyltransfer (LCAT) reaction. This reaction caused in HDL lipids a consumption of surface material and an increase in 'lipid core', presumably leading to a weakening and disruption of the lipoprotein surface with a recombination of 'lipid core' material in the form of HDL-sup.     

Estrogens and lipid metabolism]

In the woman, lipoprotein plasma concentrations vary according to ovarian function. In general, menopause induces variations in lipoprotein plasma concentrations which are related to an increase in cardiovascular risk. Oestrogen therapy increases the synthesis of all lipoproteins including ApoB and increases the rate of their metabolism in various degrees depending on the type of lipoprotein. Globally, under oestrogen treatment, LDL cholesterol is decreased and HDL cholesterol is increased--more specifically HDL 2 and Lp A1 fractions. Oestrogens alter the lipoprotein metabolism through numerous mechanisms. New drugs such as Selective Oestrogen Receptor Modulators or phytoestrogens act on lipid metabolism: these effects could be useful in clinical practice.     

Myeloperoxidase,paraoxonase-1, and HDL form a functional ternary complex

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein–associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr₇₁), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other’s function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.     

Therapeutic elevation of HDL-cholesterol to prevent atherosclerosis and coronary heart disease

Innovative pharmacological approaches to raise anti-atherogenic high-density lipoprotein-cholesterol (HDL-C) are currently of considerable interest, particularly in atherogenic dyslipidemias characterized by low levels of HDL-C, such as type 2 diabetes, the metabolic syndrome, and mixed dyslipidemia, but equally among individuals with or at elevated risk for premature cardiovascular disease (CVD). Epidemiological and observational studies first demonstrated that HDL-C was a strong, independent predictor of coronary heart disease (CHD) risk, and suggested that raising HDL-C levels might afford clinical benefit. Accumulating data from clinical trials of pharmacological agents that raise HDL-C levels have supported this concept. In addition to the pivotal role that HDL-C plays in reverse cholesterol transport and cellular cholesterol efflux, HDL particles possess a spectrum of anti-inflammatory, anti-oxidative, anti-apoptotic, anti-thrombotic, vasodilatory and anti-infectious properties, all of which potentially contribute to their atheroprotective nature. Significantly, anti-atherogenic properties of HDL particles are attenuated in common metabolic diseases that are characterized by subnormal HDL-C levels, such as type 2 diabetes and metabolic syndrome. Inhibition of cholesteryl ester transfer protein (CETP), a key player in cholesterol metabolism and transport, constitutes an innovative target for HDL-C raising. In lipid efficacy trials, 2 CETP inhibitors-JTT-705 and torcetrapib-induced marked elevation in HDL-C levels, with torcetrapib displaying greater efficacy. Moreover, both agents attenuate aortic atherosclerosis in cholesterol-fed rabbits. Clinical trial data demonstrating the clinical benefits of these drugs on atherosclerosis and CHD are eagerly awaited.     

Recombinant apolipoprotein A-I(Milano): a novel agent for the induction of regression of atherosclerotic plaques

Apolipoprotein (apo) A-I, because of its anti-atherogenic properties, provides a potentially powerful approach to the management of vascular diseases. In the clinic, patients with low high density lipoproteins (HDL)/apoA-I are at dramatically increased risk of coronary disease, the opposite being true for individuals with high HDL Drug studies, e.g., the VA-HIT trial with gemfibrozil, clearly associated a reduced risk of events with raised HDL-cholesterolemia. A number of animal studies have shown that the infusion of apoA-I containing synthetic HDL can inhibit atherosclerosis progression in experimental animals, being also able to stimulate reverse cholesterol transport in humans. Recently, high interest has been devoted to a molecular variant of apoA-I, apoA-I(Milano) (apoA-I(M)), characterized by a Cys for Arg substitution and formation of apoA-I(M)/A-I(M) dimers. These latter are characterized by a prolonged permanence in plasma and a more effective cholesterol removing function, which may offer an improved approach to the therapeutic management of arterial disease. Aside from a number of clinical studies on human apoA-I(M) carriers, all indicating a clear protection from cardiovascular disease in spite of markedly reduced HDL levels, animal investigations have provided definite indication as to the potential of apoA-I(M) infusion to directly reduce the extent of atherosclerotic plaques. In addition to the well known powerful cholesterol effluxing capacity of apoA-I(M), fibrinolytic properties and possibly antioxidant/vasodilator mechanisms seem to be in play. Ongoing clinical studies will provide final indication as to the potential of this new therapeutic approach.     

Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr-/- mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.     

HDL metabolism in hypertriglyceridemic states: an overview.

Reduced plasma high-density lipoprotein (HDL) cholesterol levels have been recognized as a highly significant independent risk factor for atherosclerotic cardiovascular disease. HDL levels are also inversely related to plasma triglyceride levels and there is a dynamic interaction between HDL and triglyceride (TG) rich lipoproteins in vivo. The mechanisms underlying the lowering of HDL in hypertriglyceridemic states have not been fully elucidated, but there is evidence to suggest that triglyceride enrichment of HDL, a common metabolic consequence of hypertriglyceridemia, may play an important role in this process. There is accumulating evidence to suggest that the primary mechanisms leading to reduced plasma HDL cholesterol levels and HDL particle number in hypertriglyceridemic states may be due to any one or a combination of the following possibilities: (1) small HDL particles, which are the product of the intravascular lipolysis of triglyceride-enriched HDL, may be cleared more rapidly from the circulation, (2) triglyceride-enriched HDL may be intrinsically more unstable in the circulation, with apo A-I loosely bound, (3) the lipolytic process itself of triglyceride-enriched HDL may lower HDL particle number by causing apo A-I to be shed from the HDL particles and cleared from the circulation, (4) a dysfunctional lipoprotein lipase or reduced LPL activity may contribute to the lowering of HDL levels by reducing the availability of surface constituents of triglyceride-rich lipoproteins that are necessary for the formation of nascent HDL particles. This review summarizes the evidence that triglyceride-enrichment of HDL is an important factor determining the rate at which HDL is catabolized, a mechanism which could explain, at least in part, the reduced plasma HDL cholesterol levels and particle number frequently observed in hypertriglyceridemic states.     

HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoE- and ABCG1-dependent pathway

Genetic deficiency or inhibition of cholesteryl ester transfer protein (CETP) leads to a marked increase in plasma levels of large HDL-2 particles. However, there is concern that such particles may be dysfunctional in terms of their ability to promote cholesterol efflux from macrophages. Recently, the ATP-binding cassette transporter ABCG1, a macrophage liver X receptor (LXR) target, has been shown to stimulate cholesterol efflux to HDL. We have assessed the ability of HDL from subjects with homozygous deficiency of CETP (CETP-D) to promote cholesterol efflux from macrophages and have evaluated the role of ABCG1 and other factors in this process. CETP-D HDL-2 caused a 2- to 3-fold stimulation of net cholesterol efflux compared with control HDL-2 in LXR-activated macrophages, due primarily to an increase in lecithin:cholesterol acyltransferase-mediated (LCAT-mediated) cholesteryl ester formation in media. Genetic knockdown or overexpression of ABCG1 showed that increased cholesterol efflux to CETP-D HDL was ABCG1 dependent. LCAT and apoE contents of CETP-D HDL-2 were markedly increased compared with control HDL-2, and increased cholesterol esterification activity resided within the apoE-HDL fraction. Thus, CETP-D HDL has enhanced ability to promote cholesterol efflux from foam cells in an ABCG1-dependent pathway due to an increased content of LCAT and apoE.